T cell regulatory antigen-presenting polypeptide and method for using the same

Abstract

The present disclosure provides T cell regulatory antigen-presenting polypeptides, including single-chain antigen-presenting polypeptides and multimeric antigen-presenting polypeptides. The present disclosure also provides nucleic acids comprising nucleotide sequences encoding the T cell regulatory antigen-presenting polypeptides of the present disclosure, and cells genetically modified with the nucleic acids. The T cell regulatory antigen-presenting polypeptides of the present disclosure are useful for regulating T cell activity. Accordingly, the present disclosure provides compositions and methods for regulating T cell activity, as well as compositions and methods for treating individuals with autoimmune disorders. TIFF2022522404000343.tif21128

Description

Technical Field

[0001] Cross-reference This application claims the benefit of U.S. Provisional Patent Application No. 62 / 814,715, filed Mar. 6, 2019, which is hereby incorporated by reference in its entirety.

Background Art

[0002] Introduction Central to the proper functioning of the mammalian immune system is the coordinated activity and communication between two specialized cell types, antigen-presenting cells (“APCs”) and T cells. APCs capture proteins from foreign organisms or abnormal proteins (e.g., due to gene mutations in cancer cells) and break them down into small fragments suitable as signals for scrutiny by the larger immune system, including T cells. In particular, APCs break down proteins into small peptide fragments, which then pair with proteins of the major histocompatibility complex (“MHC”) and are presented on the cell surface. The cell surface presentation of MHC containing peptide fragments, also known as T cell epitopes, provides a fundamental foothold for T cell surveillance, thereby enabling specific recognition by T cells. The peptide fragments can be of pathogen origin, tumor origin, or natural host protein (self-protein) origin. Furthermore, APCs can recognize other foreign components such as bacterial toxins, viral proteins, viral DNA, viral RNA, whose presence implies an escalation of the threat level. APCs relay this information to T cells via additional co-stimulatory signals to bring about a more effective response.

[0003] T cells recognize peptide-major histocompatibility complex (pMHC) complexes via a specialized cell surface receptor, the T cell receptor (TCR). The TCR is unique to each T cell, and as a result, each T cell is highly specific for a particular pMHC target. To appropriately address a world teeming with potential threats, the human body contains a large number (about 10,000,000) of distinct T cells with clearly different TCRs. Furthermore, any given T cell that is specific for a particular T cell peptide initially represents only a very small fraction of the total T cell population. Normally in a dormant state and limited in number, T cells with a particular TCR can be readily activated and amplified by APCs, resulting in a very powerful T cell response involving millions of T cells. Such activated T cell responses can attack and eliminate other cellular threats, including viral infections, bacterial infections, and tumors, as illustrated below. Conversely, widespread nonspecific activation of overactive T cell responses against self-antigens or common antigens can generate T cells that inappropriately attack and destroy healthy tissues or cells.

[0004] In humans, MHC proteins are referred to as human leukocyte antigens (HLAs). The HLA class II locus contains HLA-DM (HLA-DMA and HLA-DMB, which encode the HLA-DM α-chain and HLA-DM β-chain, respectively), HLA-DO (HLA-DOA and HLA-DOB, which encode the HLA-DO α-chain and HLA-DO β-chain, respectively), HLA-DP (HLA-DPA and HLA-DPB, which encode the HLA-DP α-chain and HLA-DP β-chain, respectively), HLA-DQ (HLA-DQA and HLA-DQB, which encode the HLA-DQ α-chain and HLA-DQ β-chain, respectively), and HLA-DR (HLA-DRA and HLA-DRB, which encode the HLA-DR α-chain and HLA-DR β-chain, respectively). SUMMARY OF THE INVENTION

[0005] Summary The present disclosure provides T cell regulatory antigen-presenting polypeptides, including single-chain antigen-presenting polypeptides and multimeric antigen-presenting polypeptides. The present disclosure provides nucleic acids comprising nucleotide sequences encoding the T cell regulatory antigen-presenting polypeptides of the present disclosure, and cells genetically modified with the nucleic acids. The T cell regulatory antigen-presenting polypeptides of the present disclosure are useful for regulating the activity of T cells. Accordingly, the present disclosure provides compositions and methods for regulating the activity of T cells, as well as compositions and methods for treating a person having an autoimmune disorder. [The present invention 1001] A multimeric T cell regulatory antigen-presenting polypeptide, a) A first polypeptide, i) A peptide presenting a celiac disease-related or type 1 diabetes-related (T1D-related) epitope capable of binding to a T cell receptor (TCR), ii) A first major histocompatibility complex (MHC) class II polypeptide comprising the above-mentioned first polypeptide, and b) A second polypeptide, i) A second MHC class II polypeptide comprising the above-mentioned second polypeptide and comprising, wherein one or both polypeptides of the multimeric polypeptide comprise one or more immunomodulatory polypeptides, wherein the first and second MHC class II polypeptides comprise (i) an MHC class II α-chain polypeptide selected from polypeptides having at least 90% amino acid sequence identity to the DRA*0101 polypeptide, DQA1*05:01 polypeptide, and DQA1*03:01 polypeptide, and (ii) an MHC class II β-chain polypeptide selected from polypeptides having at least 90% amino acid sequence identity to the DRB1*04:01 polypeptide, DRB1*03:01 polypeptide, DRB1*04:02 polypeptide, DRB1*04:05 polypeptide, DQB1*02:01 polypeptide, and DQB1*03:02 polypeptide, wherein one or both polypeptides of the multimeric polypeptide optionally comprise an immunoglobulin (Ig) Fc polypeptide or a non-Ig backbone, the multimeric T cell regulatory antigen-presenting polypeptide. [The present invention 1002] a1) The first polypeptide, in order from the N-terminus to the C-terminus, i) the peptide epitope, ii) an MHC class II β1 polypeptide, and iii) an MHC class II β2 polypeptide comprising, and b1) The second polypeptide, in order from the N-terminus to the C-terminus, i) the one or more immunomodulatory polypeptides, ii) an MHC class II α1 polypeptide, iii) an MHC class II α2 polypeptide, and iv) an Ig Fc polypeptide comprising, or a2) The first polypeptide, in order from the N-terminus to the C-terminus, i) the peptide epitope, ii) an MHC class II β1 polypeptide, and iii) an MHC class II β2 polypeptide comprising, and b2) The second polypeptide, in order from the N-terminus to the C-terminus, i) MHC class II α1 polypeptide, ii) MHC class II α2 polypeptide, iii) Ig Fc polypeptide, and iv) said one or more immunomodulatory polypeptides comprising, or a3) said first polypeptide, in order from N-terminus to C-terminus, i) said peptide epitope, ii) MHC class II β1 polypeptide, and iii) MHC class II β2 polypeptide comprising, and b3) said second polypeptide, in order from N-terminus to C-terminus, i) MHC class II α1 polypeptide, ii) MHC class II α2 polypeptide, iii) said one or more immunomodulatory polypeptides, and iv) Ig Fc polypeptide comprising, or a4) said first polypeptide, in order from N-terminus to C-terminus, i) MHC class II α1 polypeptide, ii) MHC class II α2 polypeptide, and iii) Ig Fc polypeptide comprising, and b4) said second polypeptide, in order from N-terminus to C-terminus, i) said one or more immunomodulatory polypeptides, ii) said peptide epitope, iii) MHC class II β1 polypeptide, and iv) MHC class II β2 polypeptide comprising, or a5) said first polypeptide, in order from N-terminus to C-terminus, i) MHC class II α1 polypeptide, ii) MHC class II α2 polypeptide, and iii) Ig Fc polypeptide comprising, and b5) said second polypeptide, in order from N-terminus to C-terminus, i) said peptide epitope, ii) MHC class II β1 polypeptide, iii) MHC class II β2 polypeptide, and iv) said one or more immunomodulatory polypeptides comprising, the multimeric T cell regulatory antigen-presenting polypeptide of the present invention 1001. [The present invention 1003] a) said MHC class II α1 polypeptide comprises an amino acid sequence having at least 95% amino acid sequence identity to the DRA1*01:01 polypeptide, and said MHC class II β1 polypeptide comprises an amino acid sequence having at least 95% amino acid sequence identity to the DRB1*04:01 polypeptide, or b) the MHC class II α1 polypeptide comprises an amino acid sequence having at least 95% amino acid sequence identity to the DQA1*0501 polypeptide, and the MHC class II β1 polypeptide comprises an amino acid sequence having at least 95% amino acid sequence identity to the DQB1*0201 polypeptide, or c) the MHC class II α1 polypeptide comprises an amino acid sequence having at least 95% amino acid sequence identity to the DQA1*0301 polypeptide, and the MHC class II β1 polypeptide comprises an amino acid sequence having at least 95% amino acid sequence identity to the DQB1*0302 polypeptide, The multimeric T cell regulatory antigen-presenting polypeptide of the present invention 1001 or 1002. [The present invention 1004] wherein the immunomodulatory polypeptide is a) comprises the amino acid sequence of a natural immunomodulatory polypeptide, or b) is a variant immunomodulatory polypeptide comprising an amino acid sequence having 1 to 10 amino acid substitutions compared to the amino acid sequence of a natural immunomodulatory polypeptide, and the variant immunomodulatory polypeptide has a reduced affinity for the immunocoregulatory polypeptide compared to the affinity of the natural immunomodulatory polypeptide for the immunocoregulatory polypeptide, The T cell regulatory antigen-presenting polypeptide according to any one of the present invention 1001 to 1003. [The present invention 1005] The T cell regulatory antigen-presenting polypeptide according to any one of the present invention 1001 to 1004, wherein the immunomodulatory polypeptide is a PD-L1 polypeptide, a FasL polypeptide, or a TGF-β polypeptide. [The present invention 1006] The T cell regulatory antigen-presenting polypeptide according to any one of the present invention 1001 to 1004, wherein the immunomodulatory polypeptide is a PD-L1 polypeptide. [The present invention 1007] The T cell regulatory antigen-presenting polypeptide of the present invention 1001, wherein the T1D-related peptide or celiac disease-related peptide has a length of about 4 amino acids to about 25 amino acids. [The present invention 1008] The T cell regulatory antigen-presenting polypeptide according to any one of the present invention 1001 to 1007, wherein the peptide is a T1D-related peptide comprising the amino acid sequence SLQPLALEGSLQSRG (SEQ ID NO: 78). [The present invention 1009] The T cell regulatory antigen-presenting polypeptide according to any one of the present invention 1001 to 1007, wherein the peptide is a celiac disease-related peptide. [The present invention 1010] i) The first polypeptide comprises the amino acid sequence of the 3003 polypeptide shown in FIG. 40C, and ii) The first polypeptide comprises the amino acid sequence of the 2639 polypeptide shown in FIG. 40B, The T cell regulatory antigen-presenting polypeptide of the present invention 1001. [The present invention 1011] i) The first polypeptide comprises the amino acid sequence of the 3004 polypeptide shown in FIG. 40D, and ii) The first polypeptide comprises the amino acid sequence of the 2639 polypeptide shown in FIG. 40B, The T cell regulatory antigen-presenting polypeptide of the present invention 1001. [The present invention 1012] i) The first polypeptide comprises the amino acid sequence of the 3005 polypeptide shown in FIG. 40E, and ii) The first polypeptide comprises the amino acid sequence of the 2639 polypeptide shown in FIG. 40B, The T cell regulatory antigen-presenting polypeptide of the present invention 1001. [The present invention 1013] i) The first polypeptide comprises the amino acid sequence of the 2932 polypeptide shown in FIG. 40A, and ii) The first polypeptide comprises the amino acid sequence of the 3213 polypeptide shown in FIG. 40F, The T cell regulatory antigen-presenting polypeptide of the present invention 1001. [The present invention 1014] i) The first polypeptide comprises the amino acid sequence of the 2932 polypeptide shown in FIG. 40A, and ii) The first polypeptide comprises the amino acid sequence of the 3214 polypeptide shown in FIG. 40G, The T cell regulatory antigen-presenting polypeptide of the present invention 1001. [The present invention 1015] a) Any of the T cell regulatory antigen-presenting polypeptides of the present invention 1001 to 1014, and b) A pharmaceutically acceptable excipient A composition comprising. [The present invention 1016] One or more nucleic acids comprising a nucleotide sequence encoding any of the T cell regulatory antigen-presenting polypeptides of the present invention 1001 to 1014. [The present invention 1017] One or more expression vectors comprising one or more nucleic acids of the present invention 1016. [The present invention 1018] A host cell genetically modified by one or more nucleic acids of the present invention 1016 or one or more expression vectors of the present invention 1017. [The present invention 1019] A method for reducing the number and / or activity of CD4 + and / or CD8 + auto-reactive T cells specific for an individual's type 1 diabetes-related epitope or celiac disease-related epitope, wherein said CD4 + Contacting T cells with any one of the T cell regulatory antigen-presenting polypeptides of the present invention 1001 to 1014, wherein said contacting reduces the number and / or activity of said CD4 + and / or CD8 + T cells, said method. [Present Invention 1020] A method for reducing the number and / or activity of CD4 + T cells and / or CD8 + self-reactive T cells specific for type 1 diabetes-related epitopes or celiac disease-related epitopes in an individual, said method comprising contacting said CD4 + T cells with any one of the T cell regulatory antigen-presenting polypeptides of the present invention 1001 to 1014, wherein said contacting increases the number of CD4 + Treg cells, whereby the number and / or activity of said CD4 + T cells and / or CD8 + T cells is reduced, said method. [Present Invention 1021] A method for treating type 1 diabetes or celiac disease in an individual, said method comprising administering to an individual in need thereof an effective amount of any one of the T cell regulatory antigen-presenting polypeptides of the present invention 1001 to 1014, wherein said administering treats said type 1 diabetes or celiac disease in said individual, said method. [Present Invention 1022] The method of Present Invention 1021, wherein said peptide epitope is a T1D-related epitope and said administering treats T1D in said individual. [Present Invention 1023] The method of Present Invention 1021, wherein said peptide epitope is a celiac disease-related epitope and said administering treats celiac disease in said individual.

Brief Description of the Drawings

[0006]

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BEST MODE FOR CARRYING OUT THE INVENTION

[0007] Definitions The terms "polynucleotide" and "nucleic acid" are used interchangeably herein and refer to polymeric forms of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, the term includes, but is not limited to, single-stranded, double-stranded or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers comprising purine and pyrimidine bases or other natural, chemical or biochemical modifications, non-natural or derivatized nucleotide bases.

[0008] The terms "peptide", "polypeptide" and "protein" are used interchangeably herein and refer to polymeric forms of amino acids of any length, which may include coded and non-coded amino acids, chemically or biochemically modified amino acids, or derivatized amino acids, as well as polypeptides having modified peptide backbones.

[0009] When a polynucleotide or polypeptide is said to have a certain percentage of "sequence identity" to another polynucleotide or polypeptide, it means that when the two sequences are aligned and compared, that percentage of bases or amino acids is the same and in the same relative position. Sequence identity can be determined in a number of different ways. To determine sequence identity, the sequences can be aligned using a variety of convenient methods and computer programs (e.g., BLAST, T-COFFEE, MUSCLE, MAFFT, etc.), which are available through websites on the World Wide Web including ncbi.nlm.nili.gov / BLAST, ebi.ac.uk / Tools / msa / tcoffee / , ebi.ac.uk / Tools / msa / muscle / , mafft.cbrc.jp / alignment / software / . See, for example, Altschul et al. (1990), J. Mol. Bioi. 215:403-10.

[0010] The term "conservative amino acid substitution" refers to the interchangeability of amino acid residues in a protein that have similar side chains. For example, a group of amino acids having aliphatic side chains consisting of glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic hydroxyl side chains consisting 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 consisting of phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains consisting of lysine, arginine, and histidine; a group of amino acids having acidic side chains consisting of glutamic acid and aspartic acid; and a group of amino acids having sulfur-containing side chains consisting of cysteine and methionine. Exemplary conservative amino acid substituents are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine-glycine, and asparagine-glutamine.

[0011] As used herein (e.g., with respect to the binding of a T cell regulatory antigen-presenting polypeptide to a polypeptide on a T cell (e.g., a T cell receptor)), the term "binding" refers to a non-covalent interaction between two molecules. A non-covalent bond refers to a direct association between two molecules by, for example, electrostatic, hydrophobic, ionic, and / or hydrogen bond interactions (including interactions such as salt bridges and hydrogen bridges). Non-covalent interactions generally have a dissociation constant (K -6 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 and is characterized by a dissociation constant (K D ). "Affinity" refers to the strength of a non-covalent bond, and an increase in binding affinity correlates with a decrease in K D . "Specific binding" generally has a dissociation constant of at least about 10 -7 M or more, for example, 5×10 -7 M, 10 -8 M, 5×10 -8M, 10 -9 Refers to a bond having an affinity of M or more. "Non-specific binding" generally refers to a bond having an affinity of less than about 10 -7 M (for example, a bond having an affinity of 10 -6 M, 10 -5 M, 10 -4 M) (for example, the binding of a ligand to a part other than the designated binding site or receptor). However, in some contexts, for example, the "specific binding" which is the binding between a TCR and a peptide / MHC complex can be in the range of 1 μM to 100 μM, or 100 μM to 1 mM. "Covalent binding" or "covalent bond" when used herein refers to the formation of one or more covalent chemical bonds between two different molecules.

[0012] As used herein, the terms "immunological synapse" or "immune synapse" generally refer to the natural interface between two immune cells that interact in an adaptive immune response, for example, the interface between an antigen-presenting cell (APC) or a target cell and an effector cell such as a lymphocyte, an effector T cell, a natural killer cell, etc. The immunological synapse between an APC and a T cell is generally initiated by the interaction between a T cell antigen receptor and a major histocompatibility complex molecule as described, for example, in Bromley et al., Annu Rev Immunol. 2001;19:375 - 96 (the disclosure of which is incorporated herein by reference in its entirety).

[0013] "T cell" includes all types of immune cells expressing CD3, including helper T cells (CD4 + cells), cytotoxic T cells (CD8 + cells), regulatory T cells (Treg), and NK-T cells.

[0014] The term "immunomodulatory polypeptide" (also referred to herein as "costimulatory polypeptide"), as used herein, by specifically binding to a cognate immunocoregulatory polypeptide on a T cell, in addition to a primary signal (e.g., that provided by the binding of the TCR / CD3 complex to a major histocompatibility complex (MHC) polypeptide loaded with a peptide), provides a signal that mediates a T cell response (including, but not limited to, proliferation, activation, differentiation, etc.), and includes a polypeptide on an antigen-presenting cell (APC) (e.g., dendritic cell, B cell, etc.) or a portion of a polypeptide on an APC. Immunomodulatory polypeptides can include, but are 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, Toll ligand receptor agonists or antibodies that bind thereto, and ligands that specifically bind to B7-H3. Also, costimulatory polypeptides can include, among others, antibodies that specifically bind to cognate costimulatory molecules present on T cells, including, but not limited to, IL-2, CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, LIGHT, NKG2C, B7-H3, and ligands that specifically bind to CD83.

[0015] As noted above, the "immunomodulatory polypeptide" (also referred to herein as "MOD") specifically binds to a cognate immunocoregulatory polypeptide on a T cell.

[0016] The "immunomodulatory domain" ("MOD") of the TMAPP of the present disclosure binds to a cognate immunocoregulatory polypeptide that can be present on a target T cell.

[0017] As used herein, "heterologous" means a nucleotide or polypeptide not found in the native, unmodified nucleic acid or protein, respectively.

[0018] As used herein, "recombinant" means the product of various combinations of the processes of cloning, restriction, polymerase chain reaction (PCR) and / or ligation that result in a construct in which a particular nucleic acid (DNA or RNA) has a structural coding or non-coding sequence distinguishable from the endogenous nucleic acid found in nature. A DNA sequence encoding a polypeptide can be assembled from cDNA fragments or a series of synthetic oligonucleotides to provide a synthetic nucleic acid that can be expressed from a recombinant transcription unit contained in a cell or cell-free transcription / translation system.

[0019] The terms "recombinant expression vector" or "DNA construct" are used interchangeably herein to refer to a DNA molecule containing a vector and at least one insert. Recombinant expression vectors are usually made for the expression and / or propagation of 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 control sequences.

[0020] As used herein, the term "affinity" refers to the equilibrium constant for the reversible binding of two agents (e.g., an antibody and an antigen), the dissociation constant (K D) It is represented by. The affinity is at least more than 1-fold, at least more than 2-fold, at least more than 3-fold, at least more than 4-fold, at least more than 5-fold, at least more than 6-fold, at least more than 7-fold, at least more than 8-fold, at least more than 9-fold, at least more than 10-fold, at least more than 20-fold, at least more than 30-fold, at least more than 40-fold, at least more than 50-fold, at least more than 60-fold, at least more than 70-fold, at least more than 80-fold, at least more than 90-fold, at least more than 100-fold, or at least more than 1,000-fold, or more, compared to the affinity of the antibody for an unrelated amino acid sequence. The affinity of the antibody for the 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. As used herein, the term "avidity" refers to the dissociation resistance of a complex of two or more agents after dilution.

[0021] The term "binding" refers to the direct association between two molecules, for example, by covalent, electrostatic, hydrophobic, and ionic and / or hydrogen bond interactions (including interactions such as salt bridges and hydrogen bridges). "Specific binding" is at least about 10 -7 M or more, for example, 5×10 -7 M, 10 -8 M, 5×10 -8 M and refers to a binding having an affinity exceeding that. "Non-specific binding" refers to a binding having an affinity of less than about 10 -7 M, for example, 10 -6 M, 10 -5 M, 10 -4 M and refers to a binding having an affinity such as that.

[0022] The terms "treatment", "treating", and the like are used herein generally to mean obtaining a desired pharmacological and / or physiological effect. The effect can be prophylactic in terms of completely or partially preventing a disease or its symptoms, and / or therapeutic in terms of partially or completely curing a disease and / or adverse effects that may result therefrom. "Treatment" as used herein encompasses any treatment of a disease or condition in a mammal and includes (a) prevention of the onset of a disease or condition in a subject that may be predisposed to the disease or condition but has not yet been diagnosed as having it, (b) inhibition of a disease or condition, i.e., preventing its occurrence, and / or (c) alleviation of a disease, i.e., inducing regression of the disease. A therapeutic agent can be administered before, during, or after the occurrence of a disease or injury. Treatment of an ongoing disease is particularly targeted, in which case the treatment is one that stabilizes or reduces the undesirable clinical symptoms of the patient. Such treatment is preferably carried out before the function in the diseased tissue is completely lost. The treatment method of the subject is preferably administered during the symptomatic period of the disease and, in some cases, after the symptomatic period of the disease.

[0023] The terms "individual", "subject", "host", and "patient" are used interchangeably herein and refer to any mammalian subject for which a diagnosis, treatment, or therapy is desired. Mammals include, for example, humans, non-human primates, rodents (e.g., rats, mice), lagomorphs (e.g., rabbits), ungulates (e.g., cows, sheep, pigs, horses, goats, etc.).

[0024] Before further describing the present invention, it is to be understood that the invention is not limited to the specific embodiments described, which can, of course, be varied in themselves. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting, since the scope of the present invention is defined only by the appended claims.

[0025] When a range of values is recited, all values between the upper and lower limits of that range, to the extent of one tenth of the unit of the lower limit, unless otherwise explicitly stated, and any other specified value or values within that specified range are understood to be included within the invention. The upper and lower limits of these narrower ranges may, independently of each other, be included within the narrower range and also within the invention, if any particular limit value within the specified range is excluded. When the specified range includes one or both of the limit values, the range excluding one or both of the included limit values is also included herein.

[0026] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, but the preferred methods and materials are described below. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials related to that publication.

[0027] It should be noted that, as used in this specification and the appended claims, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "Treg" includes a plurality of such Tregs, and reference to "MHC class II alpha chain" includes one or more MHC class II alpha chains and equivalents thereof known to those skilled in the art, and so on. Further, it should be noted that the claims may be drafted to exclude any optional element. Thus, this description is intended to serve as a basis for the use of exclusive terms such as "only", "solely" and the like, or the use of "negative" limitations, in connection with the detailed description of the elements of the claims.

[0028] For the sake of clarity, some features of the present invention are described in the context of separate embodiments, but it is also recognized that they can be provided in combination in one embodiment. Conversely, to avoid complication, various features of the present invention are described in the context of one embodiment, but these may also be provided separately or in any suitable partial combination. All combinations of embodiments of the present invention are clearly encompassed by the present invention and are disclosed herein as if every combination were individually and explicitly disclosed. In addition, all partial combinations of various embodiments and their elements are also clearly encompassed by the present invention and are disclosed herein as if every such partial combination were individually and explicitly disclosed herein.

[0029] The publications discussed herein are described only with respect to disclosures prior to the filing date of the present application. Any statement in this specification does not constitute an admission that the present invention has no right to antedate such publications based on prior invention. Further, the publication dates recited may be different from the actual publication dates, which may need to be individually confirmed.

[0030] Detailed Description The present disclosure provides a T cell regulatory antigen-presenting polypeptide (TMAPP) comprising: (a) a first polypeptide comprising (i) a peptide epitope and (ii) a first MHC class II polypeptide; and (b) a second polypeptide comprising a second MHC class II polypeptide, wherein the first and / or second polypeptide comprises one or more immunomodulatory polypeptides. The present disclosure provides a nucleic acid comprising a nucleotide sequence encoding the TMAPP of the present disclosure, and a cell genetically modified with the nucleic acid. The TMAPP of the present disclosure is useful for regulating the activity of T cells. Accordingly, the present disclosure provides a method for regulating the activity of T cells.

[0031] The present disclosure provides an antigen-presenting polypeptide (APP), wherein the APP of the present disclosure does not contain an immunomodulatory polypeptide. The APP of the present disclosure can be a single-chain polypeptide or a multi-chain (multimeric) polypeptide. The APP of the present disclosure is useful for diagnostic and therapeutic applications.

[0032] T cell regulatory antigen-presenting polypeptide The present disclosure provides a T cell regulatory antigen-presenting polypeptide (TMAPP), including single-chain TMAPP and multimeric TMAPP. In some cases, the TMAPP of the present disclosure contains two polypeptide chains and may be referred to herein as a "multimeric T cell regulatory antigen-presenting polypeptide" in some cases. In some cases, the TMAPP of the present disclosure contains a single polypeptide chain. The TMAPP of the present disclosure is also referred to as a "synTac polypeptide".

[0033] The TMAPP of the present disclosure contains one or more immunomodulatory polypeptides. In some cases, the TMAPP of the present disclosure contains a single immunomodulatory polypeptide. In some cases, the TMAPP of the present disclosure contains two or more immunomodulatory polypeptides (e.g., 2, 3, 4, or 5 immunomodulatory polypeptides).

[0034] In some cases, the TMAPP of the present disclosure contains two or more immunomodulatory polypeptides. In some cases, when the TMAPP of the present disclosure contains a first polypeptide and a second polypeptide, two or more immunomodulatory polypeptides are present only in the first polypeptide chain. In some cases, when the TMAPP of the present disclosure contains a first polypeptide and a second polypeptide, two or more immunomodulatory polypeptides are present only in the second polypeptide chain. In some cases, when the TMAPP of the present disclosure contains a first polypeptide and a second polypeptide, at least one of the two or more immunomodulatory polypeptides is present in the first polypeptide chain, and at least one of the two or more immunomodulatory polypeptides is present in the second polypeptide chain.

[0035] In some cases, when the TMAPP of the present disclosure comprises two immunomodulatory polypeptides, the two immunomodulatory polypeptides have the same amino acid sequence. That is, the TMAPP comprises two copies of the immunomodulatory polypeptide. In some cases, when the TMAPP of the present disclosure comprises two immunomodulatory polypeptides, the two immunomodulatory polypeptides do not have the same amino acid sequence. For example, 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 second amino acid sequences are not identical. In some cases, the first and second amino acid sequences differ from each other by 1 to 10 amino acids, 10 to 25 amino acids, or more than 25 amino acids. In some cases, the first and 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 each other.

[0036] The TMAPP of the present disclosure regulates the activity of T cells. In some cases, the TMAPP of the present disclosure reduces the activity of autoreactive T cells and / or autoreactive B cells. In some cases, the TMAPP of the present disclosure increases the number and / or activity of regulatory T cells (Tregs), thereby resulting in a decrease in the activity of autoreactive T cells and / or autoreactive B cells.

[0037] Immune regulatory polypeptides suitable for inclusion in the TMAPPs of the present disclosure include, but are not limited to, IL-2, transforming growth factor beta (TGFβ), JAG1, CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L, Fas ligand (FasL), inducible co-stimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM), CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, HVEM, lymphotoxin beta receptor, 3 / TR6, ILT3, ILT4, HVEM. In some cases, an immune regulatory polypeptide suitable for inclusion in the TMAPPs of the present disclosure contains 1 to 10 amino acid substitutions compared to a wild-type or native immune regulatory polypeptide and exhibits a reduced affinity compared to the affinity of the wild-type or native immune regulatory polypeptide for a cognate co-immune regulatory polypeptide (e.g., a co-immune regulatory polypeptide present on the surface of a T cell).

[0038] Multimeric T cell regulatory antigen-presenting polypeptide The TMAPPs of the present disclosure include i) a peptide epitope (a peptide recognized and bound by a TCR), ii) an MHC class II α-chain polypeptide, iii) an MHC class II β-chain polypeptide, and iv) an immune regulatory polypeptide (also referred to herein as a "MOD polypeptide" or "MOD domain"). In some cases, a TMAPP contains two polypeptide chains, and such a TMAPP is referred to herein as a multimeric TMAPP. The TMAPPs of the present disclosure may further include a dimerization polypeptide and one or both of an immunoglobulin scaffold (e.g., an Ig Fc polypeptide) or a non-immunoglobulin scaffold. Non-limiting examples of the multimeric TMAPPs of the present disclosure are schematically shown in FIGS. 1A-1E, FIGS. 2A-2D, and FIGS. 3A-3C.

[0039] In some cases, the TMAPP of the present disclosure comprises a single immunomodulatory polypeptide. In some cases, the TMAPP of the present disclosure comprises 2 copies of an immunomodulatory polypeptide. In some cases, the TMAPP of the present disclosure comprises 3 copies of an immunomodulatory polypeptide. When the TMAPP of the present disclosure comprises 2 or 3 copies of an immunomodulatory polypeptide, in some cases, the 2 or 3 copies are arranged tandemly. When the 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 each other by a linker.

[0040] The TMAPP of the present disclosure may comprise one or more linkers, wherein the one or more linkers are between i) an MHC class II polypeptide and an Ig Fc polypeptide (such a linker is referred to herein as "L1"), ii) an immunomodulatory polypeptide and an MHC class II polypeptide (such a linker is referred to herein as "L2"), iii) a first immunomodulatory polypeptide and a second immunomodulatory polypeptide (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., the first or second member of a dimerization pair), and vi) a dimerization polypeptide (e.g., the first or second member of a dimerization pair) and an Ig Fc polypeptide. In some cases, the L1 linker comprises (GGGGS)n (SEQ ID NO: 1), where n is 1, 2, 3, 4, 5, 6, 7, or 8. In some cases, the L2 linker comprises (GGGGS)n (SEQ ID NO: 1), where n is 1, 2, 3, 4, 5, 6, 7, or 8. In some cases, the L3 linker comprises (GGGGS)n (SEQ ID NO: 1), where n is 1, 2, 3, 4, 5, 6, 7, or 8. In some cases, the linker comprises the amino acid sequence GGSAAAGG (SEQ ID NO: 2).

[0041] In some cases, the TMAPP of the present disclosure comprises: a) a first polypeptide comprising, in order from the N-terminus to the C-terminus, i) a peptide antigen (an "epitope") recognized by a TCR (e.g., capable of being recognized and bound), ii) an MHC class II β1 polypeptide, and iii) an MHC class II β2 polypeptide; and b) a second polypeptide comprising, i) an immunomodulatory polypeptide, ii) an MHC class II α1 polypeptide, and iii) an MHC class II α2 polypeptide. In some cases, the TMAPP of the present disclosure comprises: a) a first polypeptide comprising, in order from the N-terminus to the C-terminus, i) a peptide antigen (an "epitope") recognized by a TCR (e.g., capable of being recognized and bound), ii) an MHC class II β1 polypeptide, and iii) an MHC class II β2 polypeptide; and b) a second polypeptide comprising, i) an immunomodulatory polypeptide, ii) an MHC class II α1 polypeptide, iii) an MHC class II α2 polypeptide, and iv) an Ig Fc polypeptide. The sequences of the immunomodulatory polypeptide, the MHC class II α1 polypeptide, the MHC class II α2 polypeptide, and the Ig Fc polypeptide (in order from the N-terminus to the C-terminus) can vary. As an example of possible sequences, in some cases, the TMAPP of the present disclosure comprises: a) a first polypeptide comprising, in order from the N-terminus to the C-terminus, i) a peptide antigen (an "epitope") recognized by a TCR (e.g., capable of being recognized and bound), ii) an MHC class II β1 polypeptide, and iii) an MHC class II β2 polypeptide; and b) a second polypeptide comprising, in order from the N-terminus to the C-terminus, i) an immunomodulatory polypeptide, ii) an MHC class II α1 polypeptide, iii) an MHC class II α2 polypeptide, and iv) an Ig Fc polypeptide. This sequence is schematically shown in FIG. 1A.As another example of a possible arrangement, in some cases, the TMAPP of the present disclosure includes: a) a first polypeptide comprising, in order from the N-terminus to the C-terminus, i) a peptide antigen (an "epitope") recognized by a TCR (e.g., capable of being recognized and bound), ii) an MHC class II β1 polypeptide, and iii) an MHC class II β2 polypeptide; and b) a second polypeptide comprising, in order from the N-terminus to the C-terminus, i) an MHC class II α1 polypeptide, ii) an MHC class II α2 polypeptide, iii) an Ig Fc polypeptide, and iv) an immunomodulatory polypeptide. This arrangement is schematically shown in FIG. 1B. As another example of a possible arrangement, in some cases, the TMAPP of the present disclosure includes: a) a first polypeptide comprising, in order from the N-terminus to the C-terminus, i) a peptide antigen (an "epitope") recognized by a TCR (e.g., capable of being recognized and bound), ii) an MHC class II β1 polypeptide, and iii) an MHC class II β2 polypeptide; and b) a second polypeptide comprising, in order from the N-terminus to the C-terminus, i) an MHC class II α1 polypeptide, ii) an MHC class II α2 polypeptide, iii) an immunomodulatory polypeptide, and iv) an Ig Fc polypeptide. This arrangement is schematically shown in FIG. 1C. The immunomodulatory polypeptide(s) can be on the same polypeptide chain as the MHC class II α1 and α2 polypeptides, as schematically illustrated in FIGS. 1A - 1C. Alternatively, the peptide antigen (an "epitope") can be on the same polypeptide chain as the MHC class II β1 and β2 polypeptides, as schematically illustrated in FIGS. 1D and 1E. For example, in some cases, the TMAPP of the present disclosure includes: a) a first polypeptide comprising, in order from the N-terminus to the C-terminus, i) an immunomodulatory polypeptide, ii) a peptide antigen (an "epitope") recognized by a TCR (e.g., capable of being recognized and bound), iii) an MHC class II β1 polypeptide, and iv) an MHC class II β2 polypeptide; and b) a second polypeptide comprising, in order from the N-terminus to the C-terminus, i) an MHC class II α1 polypeptide, ii) an MHC class II α2 polypeptide, and iii) an Ig Fc polypeptide. This arrangement is schematically shown in FIG. 1D.As another example, in some cases, the TMAPP of the present disclosure comprises: a) a first polypeptide comprising, in order from the N-terminus to the C-terminus, i) a peptide antigen (an "epitope") recognized by a TCR (e.g., capable of being recognized and bound), ii) an MHC class II β1 polypeptide, iii) an MHC class II β2 polypeptide, and iv) an immunomodulatory polypeptide; and b) a second polypeptide comprising, in order from the N-terminus to the C-terminus, i) an MHC class II α1 polypeptide, ii) an MHC class II α2 polypeptide, and iii) an Ig Fc polypeptide. This arrangement is schematically shown in FIG. 1E. In any one of the above embodiments, the TMAPP may comprise a single immunomodulatory polypeptide. In any one of the above embodiments, the TMAPP may comprise two copies of the immunomodulatory polypeptide, and the two copies may be arranged in tandem or separated by a linker. In any one of the above embodiments, the TMAPP may comprise three copies of the immunomodulatory polypeptide, and the three copies may be arranged in tandem or separated by a linker. When the TMAPP of the present disclosure comprises two immunomodulatory polypeptides, in some cases, the first immunomodulatory polypeptide is linked to the second immunomodulatory polypeptide by a linker (the "L3" linker), e.g., a linker about 2 to 50 amino acids in length. A suitable L3 linker comprises (GGGGS)n (SEQ ID NO: 1), where n is 1, 2, 3, 4, 5, 6, 7, or 8. In some cases, the TMAPP comprises a linker (the "L1") between the MHC polypeptide and the Ig Fc polypeptide, where exemplary suitable linkers comprise (GGGGS)n (SEQ ID NO: 1), where n is 1, 2, 3, 4, 5, 6, 7, or 8. In some cases, the TMAPP comprises a linker (the "L2") between the immunomodulatory polypeptide and the MHC polypeptide, where exemplary suitable linkers comprise (GGGGS)n (SEQ ID NO: 1), where n is 1, 2, 3, 4, 5, 6, 7, or 8.In some cases, when the TMAPP comprises two immunomodulatory polypeptides, in some cases, the two immunomodulatory polypeptides are separated by a linker ("L3"), where exemplary suitable linkers include (GGGGS)n (SEQ ID NO: 1) (where n is 1, 2, 3, 4, 5, 6, 7, or 8). In some cases, the linker between any two components of the TMAPP comprises the amino acid sequence GGSAAAGG (SEQ ID NO: 2). In any of the above embodiments, in some cases, the Ig Fc is an IgG1 Fc polypeptide. In any of the above embodiments, in some cases, the Ig Fc is an IgG4 Fc polypeptide. In any of the above embodiments, in some cases, the immunomodulatory polypeptide is a PD-L1 polypeptide. In any of the above embodiments, in some cases, the immunomodulatory polypeptide is a TGF-β polypeptide. In any of the above embodiments, in some cases, the immunomodulatory polypeptide is a FasL polypeptide. In some cases, the epitope is an autoepitope (an epitope of one's own antigen). In some cases, the epitope is a T1D-related autoepitope. For example, in some cases, the peptide epitope is a proinsulin peptide. In some cases, the epitope is a celiac disease-related autoepitope.

[0042] The two polypeptide chains of the TMAPP of the present disclosure can be covalently linked, for example, via disulfide bonds. The two polypeptide chains of the TMAPP of the present disclosure can also associate non-covalently with each other. The two polypeptide chains of the TMAPP of the present disclosure can be linked through the interaction between a first dimerization domain present in the first polypeptide and a second dimerization domain present in the second polypeptide. For example, the first polypeptide chain of the TMAPP of the present disclosure can include an Ig CH1 polypeptide as the first dimerization domain, and the second polypeptide chain of the TMAPP of the present disclosure can include the constant region of the immunoglobulin κ chain as the second dimerization domain.

[0043] Suitable Ig CH1 polypeptides have a length of about 90 to about 120 amino acids (e.g., about 90 to about 95 amino acids, about 95 to about 100 amino acids, about 100 to about 105 amino acids, about 105 to about 110 amino acids, about 110 to about 115 amino acids, or about 110 to about 120 amino acids) and the following CH1 amino acid sequence: It may include an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF0007691927000001.tif11151.

[0044] Suitable Ig κ-chain constant region polypeptides have a length of about 90 to about 120 amino acids (e.g., about 90 to about 95 amino acids, about 95 to about 100 amino acids, about 100 to about 105 amino acids, about 105 to about 110 amino acids, about 110 to about 115 amino acids, or about 110 to about 120 amino acids) and the following κ-chain constant region amino acid sequence: It may include an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF0007691927000002.tif11151.

[0045] In some cases, the TMAPP of the present disclosure comprises: a) a first polypeptide comprising, in order from the N-terminus to the C-terminus, i) a peptide antigen (“epitope”) recognized by a TCR (e.g., capable of being recognized and bound), ii) an MHC class II β1 polypeptide, iii) an MHC class II β2 polypeptide, and iv) an Ig κ chain constant region polypeptide; and b) a second polypeptide comprising, i) an immunomodulatory polypeptide, ii) an MHC class II α1 polypeptide, iii) an MHC class II α2 polypeptide, and iv) a CH1 polypeptide. In some cases, the TMAPP of the present disclosure comprises: a) a first polypeptide comprising, in order from the N-terminus to the C-terminus, i) a peptide antigen (“epitope”) recognized by a TCR (e.g., capable of being recognized and bound), ii) an MHC class II β1 polypeptide, iii) an MHC class II β2 polypeptide, and iv) an Ig κ chain constant region polypeptide; and b) a second polypeptide comprising, i) an immunomodulatory polypeptide, ii) an MHC class II α1 polypeptide, iii) an MHC class II α2 polypeptide, iv) a CH1 polypeptide, and v) an Ig Fc polypeptide. In some cases, the TMAPP of the present disclosure comprises: a) a first polypeptide comprising, in order from the N-terminus to the C-terminus, i) a peptide antigen (“epitope”) recognized by a TCR (e.g., capable of being recognized and bound), ii) an MHC class II β1 polypeptide, iii) an MHC class II β2 polypeptide, and iv) an Ig κ chain constant region polypeptide; and b) a second polypeptide comprising, in order from the N-terminus to the C-terminus, i) an immunomodulatory polypeptide, ii) an MHC class II α1 polypeptide, iii) an MHC class II α2 polypeptide, iv) a CH1 polypeptide, and v) an Ig Fc polypeptide. An example of such a TMAPP is schematically shown in FIG. 2A.In some cases, the TMAPP of the present disclosure comprises: a) a first polypeptide comprising, in order from the N-terminus to the C-terminus, i) a peptide antigen (an "epitope") recognized by a TCR (e.g., capable of being recognized and bound), ii) an MHC class II β1 polypeptide, iii) an MHC class II β2 polypeptide, and iv) an Ig κ chain constant region polypeptide; and b) a second polypeptide comprising, in order from the N-terminus to the C-terminus, i) an MHC class II α1 polypeptide, ii) an MHC class II α2 polypeptide, iii) an immunomodulatory polypeptide, iv) a CH1 polypeptide, and v) an Ig Fc polypeptide. An example of such a TMAPP is schematically shown in FIG. 2B. As another example of a possible arrangement, in some cases, the TMAPP of the present disclosure comprises: a) a first polypeptide comprising, in order from the N-terminus to the C-terminus, i) a peptide antigen (an "epitope") recognized by a TCR (e.g., capable of being recognized and bound), ii) an MHC class II β1 polypeptide, iii) an MHC class II β2 polypeptide, and iv) an Ig κ chain constant region polypeptide; and b) a second polypeptide comprising, in order from the N-terminus to the C-terminus, i) an MHC class II α1 polypeptide, ii) an MHC class II α2 polypeptide, iii) a CH1 polypeptide, iv) an immunomodulatory polypeptide, and v) an Ig Fc polypeptide. This arrangement is schematically shown in FIG. 2C. In some cases, the TMAPP of the present disclosure comprises: a) a first polypeptide comprising, in order from the N-terminus to the C-terminus, i) a peptide antigen (an "epitope") recognized by a TCR (e.g., capable of being recognized and bound), ii) an MHC class II β1 polypeptide, iii) an MHC class II β2 polypeptide, and iv) an Ig κ chain constant region polypeptide; and b) a second polypeptide comprising, in order from the N-terminus to the C-terminus, i) an MHC class II α1 polypeptide, ii) an MHC class II α2 polypeptide, iii) a CH1 polypeptide, iv) an Ig Fc polypeptide, and v) an immunomodulatory polypeptide. An example of such a TMAPP is schematically shown in FIG. 2D. In any one of the above embodiments, the TMAPP may comprise a single immunomodulatory polypeptide.In any one of the above embodiments, TMAPP may include two copies of an immunomodulatory polypeptide, and the two copies may be arranged tandemly or may be separated by a linker. In any one of the above embodiments, TMAPP may include three copies of an immunomodulatory polypeptide, and the three copies may be arranged tandemly or may be separated by a linker. When the TMAPP of the present disclosure includes two immunomodulatory polypeptides, in some cases, the first immunomodulatory polypeptide is linked to the second immunomodulatory polypeptide by a linker (the "L3" linker), for example, a linker having a length of about 2 to 50 amino acids. A preferred L3 linker includes (GGGGS)n (SEQ ID NO: 1), where n is 1, 2, 3, 4, 5, 6, 7, or 8. In some cases, TMAPP includes a linker (the "L1") between the MHC polypeptide and the Ig Fc polypeptide, where exemplary preferred linkers include (GGGGS)n (SEQ ID NO: 1), where n is 1, 2, 3, 4, 5, 6, 7, or 8. In some cases, TMAPP includes a linker (the "L2") between the immunomodulatory polypeptide and the MHC polypeptide, where exemplary preferred linkers include (GGGGS)n (SEQ ID NO: 1), where n is 1, 2, 3, 4, 5, 6, 7, or 8. In some cases, when TMAPP includes two immunomodulatory polypeptides, in some cases, the two immunomodulatory polypeptides are separated by a linker (the "L3"), where exemplary preferred linkers include (GGGGS)n (SEQ ID NO: 1), where n is 1, 2, 3, 4, 5, 6, 7, or 8. In some cases, the linker between any two components of TMAPP includes the amino acid sequence GGSAAAGG (SEQ ID NO: 2). In any of the above embodiments, in some cases, the Ig Fc is an IgG1 Fc polypeptide. In any of the above embodiments, in some cases, the Ig Fc is an IgG4 Fc polypeptide. In any of the above embodiments, in some cases, the Ig Fc is an IgG1 Fc polypeptide.In any of the above embodiments, in some cases, the Ig Fc is an IgG4 Fc polypeptide. In any of the above embodiments, in some cases, the immunomodulatory polypeptide is a PD-L1 polypeptide. In any of the above embodiments, in some cases, the immunomodulatory polypeptide is a TGF-β polypeptide. In any of the above embodiments, in some cases, the immunomodulatory polypeptide is a FasL polypeptide. In some cases, the epitope is an autoepitope (epitope of one's own antigen). In some cases, the epitope is a T1D-related autoepitope. For example, in some cases, the peptide epitope is a proinsulin peptide. In some cases, the epitope is a celiac disease-related autoepitope.

[0046] In some cases, the TMAPP of the present disclosure comprises: a) a first polypeptide comprising i) a peptide antigen (an "epitope") recognized by a TCR (e.g., capable of being recognized and bound), ii) an MHC class II β1 polypeptide, iii) an MHC class II β2 polypeptide, iv) an immunomodulatory polypeptide, and v) an Ig κ chain constant region polypeptide; and b) a second polypeptide comprising i) an MHC class II α1 polypeptide, ii) an MHC class II α2 polypeptide, and iii) a CH1 polypeptide. In some cases, the TMAPP of the present disclosure comprises: a) a first polypeptide comprising i) a peptide antigen (an "epitope") recognized by a TCR (e.g., capable of being recognized and bound), ii) an MHC class II β1 polypeptide, iii) an MHC class II β2 polypeptide, iv) an immunomodulatory polypeptide, and v) an Ig κ chain constant region polypeptide; and b) a second polypeptide comprising i) an MHC class II α1 polypeptide, ii) an MHC class II α2 polypeptide, iii) a CH1 polypeptide, and v) an Ig Fc polypeptide. As an example, in some cases, the TMAPP of the present disclosure comprises, in order from the N-terminus to the C-terminus: a) i) an immunomodulatory polypeptide, ii) a peptide antigen (an "epitope") recognized by a TCR (e.g., capable of being recognized and bound), iii) an MHC class II β1 polypeptide, iv) an MHC class II β2 polypeptide, and v) an Ig κ chain constant region polypeptide; and b) a second polypeptide comprising, in order from the N-terminus to the C-terminus: i) an MHC class II α1 polypeptide, ii) an MHC class II α2 polypeptide, iii) a CH1 polypeptide, and v) an Ig Fc polypeptide. Such TMAPP is schematically shown in FIG. 3A.As another example, in some cases, the TMAPP of the present disclosure comprises, in order from the N-terminus to the C-terminus: i) a peptide antigen (“epitope”) recognized by a TCR (e.g., capable of being recognized and bound), ii) an MHC class II β1 polypeptide, iii) an MHC class II β2 polypeptide, iv) an immunomodulatory polypeptide, and v) an Ig κ chain constant region polypeptide, and b) a second polypeptide comprising, in order from the N-terminus to the C-terminus: i) an MHC class II α1 polypeptide, ii) an MHC class II α2 polypeptide, iii) a CH1 polypeptide, and v) an Ig Fc polypeptide. Such a TMAPP is schematically shown in FIG. 3B. As another example, in some cases, the TMAPP of the present disclosure comprises, in order from the N-terminus to the C-terminus: i) a peptide antigen (“epitope”) recognized by a TCR (e.g., capable of being recognized and bound), ii) an MHC class II β1 polypeptide, iii) an MHC class II β2 polypeptide, iv) an Ig κ chain constant region polypeptide, and v) an immunomodulatory polypeptide, and b) a second polypeptide comprising, in order from the N-terminus to the C-terminus: i) an MHC class II α1 polypeptide, ii) an MHC class II α2 polypeptide, iii) a CH1 polypeptide, and v) an Ig Fc polypeptide. Such a TMAPP is schematically shown in FIG. 3C. In any one of the above embodiments, the TMAPP may comprise a single immunomodulatory polypeptide. In any one of the above embodiments, the TMAPP may comprise two copies of the immunomodulatory polypeptide, and the two copies may be arranged tandemly or separated by a linker. In any one of the above embodiments, the TMAPP may comprise three copies of the immunomodulatory polypeptide, and the three copies may be arranged tandemly or separated by a linker. When the TMAPP of the present disclosure comprises two immunomodulatory polypeptides, in some cases, the first immunomodulatory polypeptide is linked to the second immunomodulatory polypeptide by a linker (“L3” linker), e.g., a linker about 2 to 50 amino acids in length. A suitable L3 linker comprises (GGGGS)n (SEQ ID NO: 1), where n is 1, 2, 3, 4, 5, 6, 7, or 8.In some cases, TMAPP includes a linker ("L1") between the MHC polypeptide and the Ig Fc polypeptide, where exemplary suitable linkers include (GGGGS)n (SEQ ID NO: 1) (where n is 1, 2, 3, 4, 5, 6, 7, or 8). In some cases, TMAPP includes a linker ("L2") between the immunomodulatory polypeptide and the MHC polypeptide, where exemplary suitable linkers include (GGGGS)n (SEQ ID NO: 1) (where n is 1, 2, 3, 4, 5, 6, 7, or 8). In some cases where TMAPP includes two immunomodulatory polypeptides, in some cases, the two immunomodulatory polypeptides are separated by a linker ("L3"), where exemplary suitable linkers include (GGGGS)n (SEQ ID NO: 1) (where n is 1, 2, 3, 4, 5, 6, 7, or 8). In some cases, the linker between any two components of TMAPP includes the amino acid sequence GGSAAAGG (SEQ ID NO: 2). In any of the above embodiments, in some cases, the Ig Fc is an IgG1 Fc polypeptide. In any of the above embodiments, in some cases, the Ig Fc is an IgG4 Fc polypeptide. In any of the above embodiments, in some cases, the immunomodulatory polypeptide is a PD-L1 polypeptide. In any of the above embodiments, in some cases, the immunomodulatory polypeptide is a TGF-β polypeptide. In any of the above embodiments, in some cases, the immunomodulatory polypeptide is a FasL polypeptide. In some cases, the epitope is an autoepitope (an epitope of one's own antigen). In some cases, the epitope is a T1D-related autoepitope. For example, in some cases, the peptide epitope is a proinsulin peptide. In some cases, the epitope is a celiac disease-related autoepitope.

[0047] In some cases, the TMAPP of the present disclosure comprises: a) a first polypeptide comprising, in order from the N-terminus to the C-terminus, i) a peptide antigen (an "epitope") recognized by a TCR (e.g., capable of being recognized and bound), ii) an MHC class II β1 polypeptide, iii) an MHC class II α1 polypeptide, and iv) an MHC class II α2 polypeptide; and b) a second polypeptide comprising, i) an immunomodulatory polypeptide, and ii) an MHC class II β2 polypeptide. In some cases, the second polypeptide comprises, in order from the N-terminus to the C-terminus, i) an immunomodulatory polypeptide, and ii) an MHC class II β2 polypeptide. In some cases, the TMAPP of the present disclosure comprises: a) a first polypeptide comprising, in order from the N-terminus to the C-terminus, i) a peptide antigen (an "epitope") recognized by a TCR (e.g., capable of being recognized and bound), ii) an MHC class II β1 polypeptide, iii) an MHC class II α1 polypeptide, iv) an MHC class II α2 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 β2 polypeptide. In some cases, the second polypeptide comprises, in order from the N-terminus to the C-terminus, i) an immunomodulatory polypeptide, and ii) an MHC class II β2 polypeptide. In some cases, the TMAPP of the present disclosure comprises: a) a first polypeptide comprising, in order from the N-terminus to the C-terminus, i) a peptide antigen (an "epitope") recognized by a TCR (e.g., capable of being recognized and bound), ii) an MHC class II β1 polypeptide, iii) an MHC class II α1 polypeptide, iv) an MHC class II α2 polypeptide, and v) an Ig Fc polypeptide; and b) a second polypeptide comprising, i) an immunomodulatory polypeptide, and ii) an MHC class II β2 polypeptide. In some cases, the second polypeptide comprises, in order from the N-terminus to the C-terminus, i) an immunomodulatory polypeptide, and ii) an MHC class II β2 polypeptide.In some cases, the TMAPP of the present disclosure comprises: a) a first polypeptide comprising, in order from the N-terminus to the C-terminus, i) a peptide antigen (an "epitope") recognized by a TCR (e.g., capable of being recognized and bound), ii) an MHC class II β1 polypeptide, iii) an MHC class II α1 polypeptide, iv) an MHC class II α2 polypeptide, and v) a first member of a dimerization pair; and b) a second polypeptide comprising, i) an immunomodulatory polypeptide, ii) an MHC class II β2 polypeptide, and iii) a second member of a dimerization pair. In some cases, the second polypeptide comprises, in order from the N-terminus to the C-terminus, i) an immunomodulatory polypeptide, ii) an MHC class II β2 polypeptide, and iii) a second member of a dimerization pair. In some cases, the TMAPP of the present disclosure comprises: a) a first polypeptide comprising, in order from the N-terminus to the C-terminus, i) a peptide antigen (an "epitope") recognized by a TCR (e.g., capable of being recognized and bound), ii) an MHC class II β1 polypeptide, iii) an MHC class II α1 polypeptide, iv) an MHC class II α2 polypeptide, and v) a first leucine zipper polypeptide; and b) a second polypeptide comprising, i) an immunomodulatory polypeptide, ii) an MHC class II β2 polypeptide, and iii) a second leucine zipper polypeptide. In some cases, the second polypeptide comprises, in order from the N-terminus to the C-terminus, i) an immunomodulatory polypeptide, ii) an MHC class II β2 polypeptide, and iii) a second leucine zipper polypeptide. In some cases, the TMAPP of the present disclosure comprises: a) a first polypeptide comprising, in order from the N-terminus to the C-terminus, i) a peptide antigen (an "epitope") recognized by a TCR (e.g., capable of being recognized and bound), ii) an MHC class II β1 polypeptide, iii) an MHC class II α1 polypeptide, iv) an MHC class II α2 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 β2 polypeptide, and iii) a second leucine zipper polypeptide.In some cases, the second polypeptide comprises, in order from the N-terminus to the C-terminus, i) an immunomodulatory polypeptide, ii) an MHC class II β2 polypeptide, and iii) a second leucine zipper polypeptide. In any one of the above embodiments, the TMAPP may comprise a single immunomodulatory polypeptide. In any one of the above embodiments, the TMAPP may comprise two copies of the immunomodulatory polypeptide, and the two copies may be arranged tandemly or separated by a linker. In any one of the above embodiments, the TMAPP may comprise three copies of the immunomodulatory polypeptide, and the three copies may be arranged tandemly or separated by a linker. For example, in some cases, the TMAPP of the present disclosure comprises: a) a first polypeptide comprising, in order from the N-terminus to the C-terminus, i) a peptide antigen (an "epitope") recognized by a TCR (e.g., capable of being recognized and bound), ii) an MHC class II β1 polypeptide, iii) an MHC class II α1 polypeptide, iv) an MHC class II α2 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 immunomodulatory polypeptide, iii) an MHC class II β2 polypeptide, and iv) a second leucine zipper polypeptide. In some cases, the second polypeptide comprises, in order from the N-terminus to the C-terminus, i) a first immunomodulatory polypeptide, ii) a second immunomodulatory polypeptide, iii) an MHC class II β2 polypeptide, and iv) a second leucine zipper polypeptide. In some cases, the first and second immunomodulatory polypeptides comprise the same amino acid sequence.As another example, in some cases, the TMAPP of the present disclosure includes: a) a first polypeptide comprising, in order from the N-terminus to the C-terminus, i) a peptide antigen (an "epitope") recognized by a TCR (e.g., capable of being recognized and bound), ii) an MHC class II β1 polypeptide, iii) an MHC class II α1 polypeptide, iv) an MHC class II α2 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 β2 polypeptide. In some cases, the second polypeptide comprises, in order from the N-terminus to the C-terminus, i) a first immunomodulatory polypeptide, ii) a second immunomodulatory polypeptide, and iii) an MHC class II β2 polypeptide. In some cases, the first and second immunomodulatory polypeptides comprise the same amino acid sequence. In some cases, the TMAPP of the present disclosure includes: a) a first polypeptide comprising, in order from the N-terminus to the C-terminus, i) a peptide antigen (an "epitope") recognized by a TCR (e.g., capable of being recognized and bound), ii) an MHC class II β1 polypeptide, iii) an MHC class II α1 polypeptide, and iv) an MHC class II α2 polypeptide; and b) a second polypeptide comprising, i) an immunomodulatory polypeptide, ii) an MHC class II β2 polypeptide, and iii) an Ig Fc polypeptide. In some cases, the second polypeptide comprises, in order from the N-terminus to the C-terminus, i) an immunomodulatory polypeptide, ii) an MHC class II β2 polypeptide, and iii) an Ig Fc polypeptide. In some cases, the TMAPP of the present disclosure includes: a) a first polypeptide comprising, in order from the N-terminus to the C-terminus, i) a peptide antigen (an "epitope") recognized by a TCR (e.g., capable of being recognized and bound), ii) an MHC class II β1 polypeptide, iii) an MHC class II α1 polypeptide, and iv) an MHC class II α2 polypeptide; and b) a second polypeptide comprising, i) a first immunomodulatory polypeptide, ii) a second immunomodulatory polypeptide, iii) an MHC class II β2 polypeptide, and iv) an Ig Fc polypeptide.In some cases, the second polypeptide comprises, in order from the N-terminus to the C-terminus, i) a first immunomodulatory polypeptide, ii) a second immunomodulatory polypeptide, iii) an MHC class II β2 polypeptide, and iv) an Ig Fc polypeptide. In some cases, the first and second immunomodulatory polypeptides comprise the same amino acid sequence. In some cases, the TMAPP of the present disclosure comprises a) a first polypeptide comprising, in order from the N-terminus to the C-terminus, i) an immunomodulatory polypeptide, ii) an MHC class II β1 polypeptide, iii) an MHC class II α1 polypeptide, and iv) an MHC class II α2 polypeptide, and b) a second polypeptide comprising i) a peptide antigen (an "epitope") recognized by a TCR (e.g., capable of being recognized and bound), ii) an MHC class II β2 polypeptide, and iii) an Ig Fc polypeptide. In some cases, the second polypeptide comprises, in order from the N-terminus to the C-terminus, i) a peptide antigen (an "epitope") recognized by a TCR (e.g., capable of being recognized and bound), ii) an MHC class II β2 polypeptide, and iii) an Ig Fc polypeptide. In some cases, the TMAPP of the present disclosure comprises a) a first polypeptide comprising, in order from the N-terminus to the C-terminus, i) a first immunomodulatory polypeptide, ii) a second immunomodulatory polypeptide, iii) an MHC class II β1 polypeptide, iv) an MHC class II α1 polypeptide, and v) an MHC class II α2 polypeptide, and b) a second polypeptide comprising, in order from the N-terminus to the C-terminus, i) a peptide antigen (an "epitope") recognized by a TCR (e.g., capable of being recognized and bound), ii) an MHC class II β2 polypeptide, and iii) an Ig Fc polypeptide. In some cases, the second polypeptide comprises, in order from the N-terminus to the C-terminus, i) a peptide antigen (an "epitope") recognized by a TCR (e.g., capable of being recognized and bound), ii) an MHC class II β2 polypeptide, and iii) an Ig Fc polypeptide. In some cases, the first and second immunomodulatory polypeptides comprise the same amino acid sequence.When the TMAPP of the present disclosure comprises two immunomodulatory polypeptides, in some cases, the first immunomodulatory polypeptide is linked to the second immunomodulatory polypeptide by a linker (the "L3" linker), for example, a linker having a length of about 2 to 50 amino acids. Suitable L3 linkers include (GGGGS)n (SEQ ID NO: 1), where n is 1, 2, 3, 4, 5, 6, 7, or 8. In some cases, the TMAPP comprises a linker (the "L1") between the MHC polypeptide and the Ig Fc polypeptide, where exemplary suitable linkers include (GGGGS)n (SEQ ID NO: 1), where n is 1, 2, 3, 4, 5, 6, 7, or 8. In some cases, the TMAPP comprises a linker (the "L2") between the immunomodulatory polypeptide and the MHC polypeptide, where exemplary suitable linkers include (GGGGS)n (SEQ ID NO: 1), where n is 1, 2, 3, 4, 5, 6, 7, or 8. In some cases, when the TMAPP comprises two immunomodulatory polypeptides, in some cases, the two immunomodulatory polypeptides are linked by a linker (the "L. separated by (3), wherein exemplary suitable linkers include (GGGGS)n (SEQ ID NO: 1) (where n is 1, 2, 3, 4, 5, 6, 7, or 8). In some cases, the linker between any two components of TMAPP includes the amino acid sequence GGSAAAGG (SEQ ID NO: 2). In any of the above embodiments, in some cases, the Ig Fc is an IgG1 Fc polypeptide. In any of the above embodiments, in some cases, the Ig Fc is an IgG4 Fc polypeptide. In any of the above embodiments, in some cases, the immunomodulatory polypeptide is a PD-L1 polypeptide. In any of the above embodiments, in some cases, the immunomodulatory polypeptide is a TGF-β polypeptide. In any of the above embodiments, in some cases, the immunomodulatory polypeptide is a FasL polypeptide. In some cases, the epitope is a self-epitope (epitope of its own antigen). In some cases, the epitope is a T1D-related self-epitope. For example, in some cases, the peptide epitope is a proinsulin peptide. In some cases, the epitope is a celiac disease-related self-epitope.

[0048] In some cases, the TMAPP of the present disclosure comprises: a) a first polypeptide comprising, in order from the N-terminus to the C-terminus, i) a peptide antigen (an "epitope") recognized by a TCR (e.g., capable of being recognized and bound), ii) an MHC class II α1 polypeptide, iii) an MHC class II α2 polypeptide, and iv) an immunoglobulin or non-immunoglobulin framework polypeptide; and b) a second polypeptide comprising, in order from the N-terminus to the C-terminus, i) an immunomodulatory polypeptide, ii) an MHC class II β1 polypeptide, and iii) an MHC class II β2 polypeptide. In some cases, the TMAPP of the present disclosure comprises: a) a first polypeptide comprising, in order from the N-terminus to the C-terminus, i) an immunomodulatory polypeptide, ii) an MHC class II α1 polypeptide, iii) an MHC class II α2 polypeptide, and iv) an immunoglobulin or non-immunoglobulin framework polypeptide; and b) a second polypeptide comprising, in order from the N-terminus to the C-terminus, i) a peptide antigen (an "epitope") recognized by a TCR (e.g., capable of being recognized and bound), ii) an MHC class II β1 polypeptide, and iii) an MHC class II β2 polypeptide. In some cases, the TMAPP of the present disclosure comprises: a) a first polypeptide comprising, in order from the N-terminus to the C-terminus, i) a peptide antigen (an "epitope") recognized by a TCR (e.g., capable of being recognized and bound), ii) an MHC class II α1 polypeptide, and iii) an MHC class II α2 polypeptide; and b) a second polypeptide comprising, in order from the N-terminus to the C-terminus, i) an immunomodulatory polypeptide, ii) an MHC class II β1 polypeptide, iii) an MHC class II β2 polypeptide, and iv) an immunoglobulin or non-immunoglobulin framework polypeptide.In some cases, the TMAPP of the present disclosure comprises: a) a first polypeptide comprising, in order from the N-terminus to the C-terminus, i) an immunomodulatory polypeptide, ii) an MHC class II α1 polypeptide, and iii) an MHC class II α2 polypeptide; and b) a second polypeptide comprising, in order from the N-terminus to the C-terminus, i) a peptide antigen (an "epitope") recognized by a TCR (e.g., capable of being recognized and bound), ii) an MHC class II β1 polypeptide, iii) an MHC class II β2 polypeptide, and iv) an immunoglobulin or non-immunoglobulin backbone polypeptide. In some cases, the TMAPP of the present disclosure comprises: a) a first polypeptide comprising, in order from the N-terminus to the C-terminus, i) a peptide antigen (an "epitope") recognized by a TCR (e.g., capable of being recognized and bound), ii) an MHC class II α1 polypeptide, iii) an MHC class II α2 polypeptide, iv) an immunoglobulin or non-immunoglobulin backbone polypeptide, and v) a first member of a dimerization pair (e.g., a first leucine zipper polypeptide); and b) a second polypeptide comprising, in order from the N-terminus to the C-terminus, i) an immunomodulatory polypeptide, ii) an MHC class II β1 polypeptide, iii) an MHC class II β2 polypeptide, and iv) a second member of a dimerization pair (e.g., a second leucine zipper polypeptide). In some cases, the TMAPP of the present disclosure comprises: a) a first polypeptide comprising, in order from the N-terminus to the C-terminus, i) an immunomodulatory polypeptide, ii) an MHC class II α1 polypeptide, iii) an MHC class II α2 polypeptide, iv) an immunoglobulin or non-immunoglobulin backbone polypeptide, and v) a first member of a dimerization pair (e.g., a first leucine zipper polypeptide); and b) a second polypeptide comprising, in order from the N-terminus to the C-terminus, i) a peptide antigen (an "epitope") recognized by a TCR (e.g., capable of being recognized and bound), ii) an MHC class II β1 polypeptide, iii) an MHC class II β2 polypeptide, and iv) a second member of a dimerization pair (e.g., a second leucine zipper polypeptide).In some cases, the TMAPP of the present disclosure comprises: a) a first polypeptide comprising, in order from the N-terminus to the C-terminus, i) a peptide antigen (an "epitope") recognized by a TCR (e.g., capable of being recognized and bound), ii) an MHC class II α1 polypeptide, iii) an MHC class II α2 polypeptide, and iv) a first member of a dimerization pair (e.g., a first leucine zipper polypeptide); and b) a second polypeptide comprising, in order from the N-terminus to the C-terminus, i) an immunomodulatory polypeptide, ii) an MHC class II β1 polypeptide, iii) an MHC class II β2 polypeptide, iv) an immunoglobulin or non-immunoglobulin scaffold polypeptide, and v) a second member of a dimerization pair (e.g., a second leucine zipper polypeptide). In some cases, the TMAPP of the present disclosure comprises: a) a first polypeptide comprising, in order from the N-terminus to the C-terminus, i) an immunomodulatory polypeptide, ii) an MHC class II α1 polypeptide, iii) an MHC class II α2 polypeptide, and iv) a first member of a dimerization pair (e.g., a first leucine zipper polypeptide); and b) a second polypeptide comprising, in order from the N-terminus to the C-terminus, i) a peptide antigen (an "epitope") recognized by a TCR (e.g., capable of being recognized and bound), ii) an MHC class II β1 polypeptide, iii) an MHC class II β2 polypeptide, iv) an immunoglobulin or non-immunoglobulin scaffold polypeptide, and v) a second member of a dimerization pair (e.g., a second leucine zipper polypeptide). In any one of the above embodiments, the TMAPP may comprise two copies of the immunomodulatory polypeptide, and the two copies may be arranged tandemly or separated by a linker. In any one of the above embodiments, the TMAPP may comprise three copies of the immunomodulatory polypeptide, and the three copies may be arranged tandemly or separated by a linker. In some cases, the TMAPP comprises a linker ("L1") between the MHC polypeptide and the Ig Fc polypeptide, where exemplary suitable linkers include (GGGGS)n (SEQ ID NO: 1), where n is 1, 2, 3, 4, 5, 6, 7, or 8.In some cases, TMAPP includes a linker (“L1”) between the MHC polypeptide and the Ig Fc polypeptide, where exemplary suitable linkers include (GGGGS)n (SEQ ID NO: 1), where n is 1, 2, 3, 4, 5, 6, 7, or 8. In some cases, TMAPP includes a linker (“L2”) between the immunomodulatory polypeptide and the MHC polypeptide, where exemplary suitable linkers include (GGGGS)n (SEQ ID NO: 1), where n is 1, 2, 3, 4, 5, 6, 7, or 8. In some cases where TMAPP includes two immunomodulatory polypeptides, in some cases, the two immunomodulatory polypeptides are separated by a linker (“L3”), where exemplary suitable linkers include (GGGGS)n (SEQ ID NO: 1), where n is 1, 2, 3, 4, 5, 6, 7, or 8. In some cases, the linker between any two components of TMAPP includes the amino acid sequence GGSAAAGG (SEQ ID NO: 2). In any of the above embodiments, in some cases, the Ig Fc is an IgG1 Fc polypeptide. In any of the above embodiments, in some cases, the Ig Fc is an IgG4 Fc polypeptide. In any of the above embodiments, in some cases, the immunomodulatory polypeptide is a PD-L1 polypeptide. In any of the above embodiments, in some cases, the immunomodulatory polypeptide is a TGF-β polypeptide. In any of the above embodiments, in some cases, the immunomodulatory polypeptide is a FasL polypeptide. In some cases, the epitope is a self-epitope (epitope of one's own antigen). In some cases, the epitope is a T1D-related self-epitope. For example, in some cases, the peptide epitope is a proinsulin peptide. In some cases, the epitope is a celiac disease-related self-epitope.

[0049] In some cases, the TMAPP of the present disclosure comprises: a) a first polypeptide comprising, in order from the N-terminus to the C-terminus, i) a peptide antigen (an "epitope") recognized by a TCR (e.g., capable of being recognized and bound), ii) an MHC class II β1 polypeptide, iii) an MHC class II β2 polypeptide, and iv) an immunomodulatory polypeptide; and b) a second polypeptide comprising, in order from the N-terminus to the C-terminus, i) an MHC class II α1 polypeptide and ii) an MHC class II α2 polypeptide. In some cases, the TMAPP of the present disclosure comprises: a) a first polypeptide comprising, in order from the N-terminus to the C-terminus, i) a peptide antigen (an "epitope") recognized by a TCR (e.g., capable of being recognized and bound), ii) an MHC class II β1 polypeptide, iii) an MHC class II β2 polypeptide, and iv) an immunomodulatory polypeptide; and b) a second polypeptide comprising, in order from the N-terminus to the C-terminus, i) an MHC class II α1 polypeptide, ii) an MHC class II α2 polypeptide, and iii) an immunoglobulin or non-immunoglobulin scaffold polypeptide. In some cases, the TMAPP of the present disclosure comprises: a) a first polypeptide comprising, in order from the N-terminus to the C-terminus, i) a peptide antigen (an "epitope") recognized by a TCR (e.g., capable of being recognized and bound), ii) an MHC class II β1 polypeptide, iii) an MHC class II β2 polypeptide, and iv) an immunomodulatory polypeptide; and b) a second polypeptide comprising, in order from the N-terminus to the C-terminus, i) an MHC class II α1 polypeptide, ii) an MHC class II α2 polypeptide, and iii) an Ig Fc polypeptide.In some cases, the TMAPP of the present disclosure comprises: a) a first polypeptide comprising, in order from the N-terminus to the C-terminus, i) a peptide antigen (an "epitope") recognized by a TCR (e.g., capable of being recognized and bound), ii) an MHC class II β1 polypeptide, iii) an MHC class II β2 polypeptide, iv) an immunomodulatory polypeptide, and v) a first member of a dimerization pair; and b) a second polypeptide comprising, in order from the N-terminus to the C-terminus, i) an MHC class II α1 polypeptide, ii) an MHC class II α2 polypeptide, and iii) a second member of a dimerization pair. In some cases, the TMAPP of the present disclosure comprises: a) a first polypeptide comprising, in order from the N-terminus to the C-terminus, i) a peptide antigen (an "epitope") recognized by a TCR (e.g., capable of being recognized and bound), ii) an MHC class II β1 polypeptide, iii) an MHC class II β2 polypeptide, iv) an immunomodulatory polypeptide, and v) a first leucine zipper polypeptide; and b) a second polypeptide comprising, in order from the N-terminus to the C-terminus, i) an MHC class II α1 polypeptide, ii) an MHC class II α2 polypeptide, and iii) a second leucine zipper polypeptide. In any one of the above embodiments, the TMAPP may comprise a single immunomodulatory polypeptide. In any one of the above embodiments, the TMAPP may comprise two copies of the immunomodulatory polypeptide, and the two copies may be arranged in tandem or separated by a linker. In any one of the above embodiments, the TMAPP may comprise three copies of the immunomodulatory polypeptide, and the three copies may be arranged in tandem or separated by a linker. In some cases, the TMAPP comprises a linker ("L1") between the MHC polypeptide and the Ig Fc polypeptide, where exemplary suitable linkers include (GGGGS)n (SEQ ID NO: 1), where n is 1, 2, 3, 4, 5, 6, 7, or 8.In some cases, TMAPP includes a linker ("L2") between the immunomodulatory polypeptide and the MHC polypeptide, where exemplary suitable linkers include (GGGGS)n (SEQ ID NO: 1) (where n is 1, 2, 3, 4, 5, 6, 7, or 8). In some cases, when TMAPP includes two immunomodulatory polypeptides, in some cases, the two immunomodulatory polypeptides are separated by a linker ("L3"), where exemplary suitable linkers include (GGGGS)n (SEQ ID NO: 1) (where n is 1, 2, 3, 4, 5, 6, 7, or 8). In some cases, the linker between any two components of TMAPP includes the amino acid sequence GGSAAAGG (SEQ ID NO: 2). In any of the above embodiments, in some cases, the Ig Fc is an IgG1 Fc polypeptide. In any of the above embodiments, in some cases, the Ig Fc is an IgG1 Fc polypeptide. In any of the above embodiments, in some cases, the Ig Fc is an IgG4 Fc polypeptide. In any of the above embodiments, in some cases, the immunomodulatory polypeptide is a PD-L1 polypeptide. In any of the above embodiments, in some cases, the immunomodulatory polypeptide is a TGF-β polypeptide. In any of the above embodiments, in some cases, the immunomodulatory polypeptide is a FasL polypeptide. In some cases, the epitope is an autoepitope (an epitope of one's own antigen). In some cases, the epitope is a T1D-related autoepitope. For example, in some cases, the peptide epitope is a proinsulin peptide. In some cases, the epitope is a celiac disease-related autoepitope.

[0050] In some cases, the TMAPP of the present disclosure comprises: a) a first polypeptide comprising, in order from the N-terminus to the C-terminus, i) a peptide antigen (an "epitope") recognized by a TCR (e.g., capable of being recognized and bound), ii) an MHC class II β1 polypeptide, and iii) an MHC class II β2 polypeptide; and b) a second polypeptide comprising, in order from the N-terminus to the C-terminus, i) an immunomodulatory polypeptide, ii) an MHC class II α1 polypeptide, and iii) an MHC class II α2 polypeptide. In some cases, the TMAPP of the present disclosure comprises: a) a first polypeptide comprising, in order from the N-terminus to the C-terminus, i) a peptide antigen (an "epitope") recognized by a TCR (e.g., capable of being recognized and bound), ii) an MHC class II β1 polypeptide, and iii) an MHC class II β2 polypeptide; and b) a second polypeptide comprising, in order from the N-terminus to the C-terminus, i) an immunomodulatory polypeptide, ii) an MHC class II α1 polypeptide, iii) an MHC class II α2 polypeptide, and iv) an immunoglobulin or non-immunoglobulin scaffold polypeptide. In some cases, the TMAPP of the present disclosure comprises: a) a first polypeptide comprising, in order from the N-terminus to the C-terminus, i) a peptide antigen (an "epitope") recognized by a TCR (e.g., capable of being recognized and bound), ii) an MHC class II β1 polypeptide, and iii) an MHC class II β2 polypeptide; and b) a second polypeptide comprising, in order from the N-terminus to the C-terminus, i) an immunomodulatory polypeptide, ii) an MHC class II α1 polypeptide, iii) an MHC class II α2 polypeptide, and iv) an Ig Fc polypeptide.In some cases, the TMAPP of the present disclosure comprises: a) a first polypeptide comprising, in order from the N-terminus to the C-terminus, i) a peptide antigen (an "epitope") recognized by a TCR (e.g., capable of being recognized and bound), ii) an MHC class II β1 polypeptide, iii) an MHC class II β2 polypeptide, and iv) a first member of a dimerization pair; and b) a second polypeptide comprising, in order from the N-terminus to the C-terminus, i) an immunomodulatory polypeptide, ii) an MHC class II α1 polypeptide, iii) an MHC class II α2 polypeptide, and iv) a second member of a dimerization pair. In some cases, the TMAPP of the present disclosure comprises: a) a first polypeptide comprising, in order from the N-terminus to the C-terminus, i) a peptide antigen (an "epitope") recognized by a TCR (e.g., capable of being recognized and bound), ii) an MHC class II β1 polypeptide, iii) an MHC class II β2 polypeptide, and iv) a first leucine zipper polypeptide; and b) a second polypeptide comprising, in order from the N-terminus to the C-terminus, i) an immunomodulatory polypeptide, ii) an MHC class II α1 polypeptide, iii) an MHC class II α2 polypeptide, and iv) a second leucine zipper polypeptide. In any one of the above embodiments, the TMAPP may comprise a single immunomodulatory polypeptide. In any one of the above embodiments, the TMAPP may comprise two copies of the immunomodulatory polypeptide, and the two copies may be arranged in tandem or separated by a linker. In any one of the above embodiments, the TMAPP may comprise three copies of the immunomodulatory polypeptide, and the three copies may be arranged in tandem or separated by a linker. In some cases, the TMAPP comprises a linker ("L1") between the MHC polypeptide and the Ig Fc polypeptide, where exemplary suitable linkers include (GGGGS)n (SEQ ID NO: 1) (where n is 1, 2, 3, 4, 5, 6, 7, or 8).In some cases, TMAPP includes a linker (“L2”) between the immunomodulatory polypeptide and the MHC polypeptide, where exemplary suitable linkers include (GGGGS)n (SEQ ID NO: 1), where n is 1, 2, 3, 4, 5, 6, 7, or 8. In some cases, when TMAPP includes two immunomodulatory polypeptides, in some cases, the two immunomodulatory polypeptides are separated by a linker (“L3”), where exemplary suitable linkers include (GGGGS)n (SEQ ID NO: 1), where n is 1, 2, 3, 4, 5, 6, 7, or 8. In some cases, the linker between any two components of TMAPP includes the amino acid sequence GGSAAAGG (SEQ ID NO: 2). In any of the above embodiments, in some cases, the Ig Fc is an IgG1 Fc polypeptide. In any of the above embodiments, in some cases, the Ig Fc is an IgG4 Fc polypeptide. In any of the above embodiments, in some cases, the immunomodulatory polypeptide is a PD-L1 polypeptide. In any of the above embodiments, in some cases, the immunomodulatory polypeptide is a TGF-β polypeptide. In any of the above embodiments, in some cases, the immunomodulatory polypeptide is a FasL polypeptide. In some cases, the epitope is an autoepitope (an epitope of one's own antigen). In some cases, the epitope is a T1D-related autoepitope. For example, in some cases, the peptide epitope is a proinsulin peptide. In some cases, the epitope is a celiac disease-related autoepitope.

[0051] In some cases, the TMAPP of the present disclosure comprises: a) a first polypeptide comprising, in order from the N-terminus to the C-terminus, i) a peptide antigen (an "epitope") recognized by a TCR (e.g., capable of being recognized and bound), ii) an MHC class II β1 polypeptide, iii) an MHC class II α1 polypeptide, and iv) an MHC class II α2 polypeptide; and b) a second polypeptide comprising, in order from the N-terminus to the C-terminus, i) an immunomodulatory polypeptide and ii) an MHC class II β2 polypeptide. In some cases, the TMAPP of the present disclosure comprises: a) a first polypeptide comprising, in order from the N-terminus to the C-terminus, i) a peptide antigen (an "epitope") recognized by a TCR (e.g., capable of being recognized and bound), ii) an MHC class II β1 polypeptide, iii) an MHC class II α1 polypeptide, iv) an MHC class II α2 polypeptide, and v) an immunoglobulin or non-immunoglobulin scaffold polypeptide; and b) a second polypeptide comprising, in order from the N-terminus to the C-terminus, i) an immunomodulatory polypeptide and ii) an MHC class II β2 polypeptide. In some cases, the TMAPP of the present disclosure comprises: a) a first polypeptide comprising, in order from the N-terminus to the C-terminus, i) a peptide antigen (an "epitope") recognized by a TCR (e.g., capable of being recognized and bound), ii) an MHC class II β1 polypeptide, iii) an MHC class II α1 polypeptide, iv) an MHC class II α2 polypeptide, and v) an Ig Fc polypeptide; and b) a second polypeptide comprising, in order from the N-terminus to the C-terminus, i) an immunomodulatory polypeptide and ii) an MHC class II β2 polypeptide.In some cases, the TMAPP of the present disclosure comprises: a) a first polypeptide comprising, in order from the N-terminus to the C-terminus, i) a peptide antigen (“epitope”) recognized by a TCR (e.g., capable of being recognized and bound), ii) an MHC class II β1 polypeptide, iii) an MHC class II α1 polypeptide, iv) an MHC class II α2 polypeptide, and v) a first member of a dimerization pair; and b) a second polypeptide comprising, in order from the N-terminus to the C-terminus, i) an immunomodulatory polypeptide, ii) an MHC class II β2 polypeptide, and iii) a second member of a dimerization pair. In some cases, the TMAPP of the present disclosure comprises: a) a first polypeptide comprising, in order from the N-terminus to the C-terminus, i) a peptide antigen (“epitope”) recognized by a TCR (e.g., capable of being recognized and bound), ii) an MHC class II β1 polypeptide, iii) an MHC class II α1 polypeptide, iv) an MHC class II α2 polypeptide, and v) a first leucine zipper polypeptide; and b) a second polypeptide comprising, in order from the N-terminus to the C-terminus, i) an immunomodulatory polypeptide, ii) an MHC class II β2 polypeptide, and iii) a second leucine zipper polypeptide. In any one of the above embodiments, the TMAPP may comprise a single immunomodulatory polypeptide. In any one of the above embodiments, the TMAPP may comprise two copies of the immunomodulatory polypeptide, and the two copies may be arranged tandemly or separated by a linker. In any one of the above embodiments, the TMAPP may comprise three copies of the immunomodulatory polypeptide, and the three copies may be arranged tandemly or separated by a linker. In some cases, the TMAPP comprises a linker (“L1”) between the MHC polypeptide and the Ig Fc polypeptide, where exemplary suitable linkers include (GGGGS)n (SEQ ID NO: 1), where n is 1, 2, 3, 4, 5, 6, 7, or 8.In some cases, TMAPP includes a linker ("L2") between the immunomodulatory polypeptide and the MHC polypeptide, where exemplary suitable linkers include (GGGGS)n (SEQ ID NO: 1) (where n is 1, 2, 3, 4, 5, 6, 7, or 8). In some cases, when TMAPP includes two immunomodulatory polypeptides, in some cases, the two immunomodulatory polypeptides are separated by a linker ("L3"), where exemplary suitable linkers include (GGGGS)n (SEQ ID NO: 1) (where n is 1, 2, 3, 4, 5, 6, 7, or 8). In some cases, the linker between any two components of TMAPP includes the amino acid sequence GGSAAAGG (SEQ ID NO: 2). In any of the above embodiments, in some cases, the Ig Fc is an IgG1 Fc polypeptide. In any of the above embodiments, in some cases, the Ig Fc is an IgG4 Fc polypeptide. In any of the above embodiments, in some cases, the immunomodulatory polypeptide is a PD-L1 polypeptide. In any of the above embodiments, in some cases, the immunomodulatory polypeptide is a TGF-β polypeptide. In any of the above embodiments, in some cases, the immunomodulatory polypeptide is a FasL polypeptide. In some cases, the epitope is a self-epitope (an epitope of one's own antigen). In some cases, the epitope is a T1D-related self-epitope. For example, in some cases, the peptide epitope is a proinsulin peptide. In some cases, the epitope is a celiac disease-related self-epitope.

[0052] Single-chain T cell regulatory antigen-presenting polypeptide As described above, in some cases, the TMAPPs of the present disclosure are single-chain (single polypeptide chain) TMAPPs. The single-chain TMAPPs of the present disclosure include: i) a peptide antigen ("epitope") recognized by a TCR (e.g., capable of being recognized and bound), ii) an MHC class II β1 polypeptide, iii) an MHC class II β2 polypeptide, iv) an MHC class II α1 polypeptide, v) an MHC class II α2 polypeptide, and vi) at least one immunomodulatory polypeptide. The single-chain TMAPPs of the present disclosure may also include an Ig Fc polypeptide. The single-chain TMAPPs of the present disclosure may include two or more immunomodulatory polypeptides, where the two or more immunomodulatory polypeptides may have the same amino acid sequence or different amino acid sequences. The sequences of the components of the single-chain TMAPPs of the present disclosure may vary, including the arrangement of the immunomodulatory polypeptides. Non-limiting examples are shown in FIGS. 4A-4C. For example, in some cases, the single-chain TMAPPs of the present disclosure may include, in order from the N-terminus to the C-terminus: i) a peptide antigen ("epitope") recognized by a TCR (e.g., capable of being recognized and bound), ii) an MHC class II α1 polypeptide, iii) an MHC class II α2 polypeptide, iv) an MHC class II β1 polypeptide, v) an MHC class II β2 polypeptide, and vi) an Ig Fc polypeptide, where the immunomodulatory polypeptides of the single-chain TMAPP are located at one or more of: i) the N-terminus (the N-terminus of the peptide antigen), ii) between the peptide antigen ("epitope") and the MHC class II α1 polypeptide, iii) between the MHC class II α2 polypeptide and the MHC class II β1 polypeptide, iv) between the MHC class II β2 polypeptide and the Ig Fc polypeptide, and v) the C-terminus of the Ig Fc polypeptide. Such an arrangement is schematically shown in FIG. 4A.As another example, in some cases, the single-chain TMAPP of the present disclosure may include, in order from the N-terminus to the C-terminus, i) a peptide antigen ("epitope") recognized by a TCR (e.g., capable of being recognized and bound), ii) an MHC class II β1 polypeptide, iii) an MHC class II β2 polypeptide, iv) an MHC class II α1 polypeptide, v) an MHC class II α2 polypeptide, and vi) an Ig Fc polypeptide, where the immunomodulatory polypeptide of the single-chain TMAPP is located at one or more of i) the N-terminus (the N-terminus of the peptide antigen), ii) between the peptide antigen ("epitope") and the MHC class II β1 polypeptide, iii) between the MHC class II β2 polypeptide and the MHC class II α1 polypeptide, iv) between the MHC class II α2 polypeptide and the Ig Fc polypeptide, and v) the C-terminus of the Ig Fc polypeptide. Such an arrangement is schematically shown in FIG. 4B. As another example, in some cases, the single-chain TMAPP of the present disclosure may include, in order from the N-terminus to the C-terminus, i) a peptide antigen ("epitope") recognized by a TCR (e.g., capable of being recognized and bound), ii) an MHC class II β1 polypeptide, iii) an MHC class II α1 polypeptide, iv) an MHC class II α2 polypeptide, v) an MHC class II β2 polypeptide, and vi) an Ig Fc polypeptide, where the immunomodulatory polypeptide of the single-chain TMAPP is located at one or more of i) the N-terminus (the N-terminus of the peptide antigen), ii) between the peptide antigen ("epitope") and the MHC class II β1 polypeptide, iii) between the MHC class II α1 polypeptide and the MHC class II α2 polypeptide, iv) between the MHC class II β2 polypeptide and the Ig Fc polypeptide, and v) the C-terminus of the Ig Fc polypeptide. Such an arrangement is schematically shown in FIG. 4C. In any one of the above embodiments, the TMAPP may include a single immunomodulatory polypeptide. In any one of the above embodiments, the TMAPP may include two copies of the immunomodulatory polypeptide, and the two copies may be arranged tandemly or separated by a linker.In any one of the above embodiments, TMAPP may include three copies of an immunomodulatory polypeptide, and the three copies may be arranged tandemly or separated by a linker. In some cases, TMAPP includes a linker ("L1") between the MHC polypeptide and the Ig Fc polypeptide, where an exemplary suitable linker includes (GGGGS)n (SEQ ID NO: 1) (where n is 1, 2, 3, 4, 5, 6, 7, or 8). In some cases, TMAPP includes a linker ("L2") between the immunomodulatory polypeptide and the MHC polypeptide, where an exemplary suitable linker includes (GGGGS)n (SEQ ID NO: 1) (where n is 1, 2, 3, 4, 5, 6, 7, or 8). In some cases, when TMAPP includes two immunomodulatory polypeptides, in some cases, the two immunomodulatory polypeptides are separated by a linker ("L3"), where an exemplary suitable linker includes (GGGGS)n (SEQ ID NO: 1) (where n is 1, 2, 3, 4, 5, 6, 7, or 8). In some cases, the linker between any two components of TMAPP includes the amino acid sequence GGSAAAGG (SEQ ID NO: 2). In some cases, the linker between any two components of TMAPP includes the amino acid sequence GGSAAAGG (SEQ ID NO: 2). In any of the above embodiments, in some cases, the Ig Fc is an IgG1 Fc polypeptide. In any of the above embodiments, in some cases, the Ig Fc is an IgG4 Fc polypeptide. In any of the above embodiments, in some cases, the immunomodulatory polypeptide is a PD-L1 polypeptide. In any of the above embodiments, in some cases, the immunomodulatory polypeptide is a TGF-β polypeptide. In any of the above embodiments, in some cases, the immunomodulatory polypeptide is a FasL polypeptide. In some cases, the epitope is a self-epitope (an epitope of one's own antigen), for example, in some cases, the peptide epitope is a proinsulin peptide.

[0053] Class II MHC polypeptide As described above, the TMAPP of the present disclosure includes a Class II MHC polypeptide.

[0054] Naturally occurring Class II MHC polypeptides include an α-chain and a β-chain. "Class II MHC polypeptide" includes the α-chain and β-chain of human leukocyte antigen (HLA). MHC Class II polypeptides include MCH Class II DP α and β polypeptides, DM α and β polypeptides, DOA α and β polypeptides, DOB α and β polypeptides, DQ α and β polypeptides, and DR α and β polypeptides. As used herein, "Class II MHC polypeptide" may include only a Class II MHC α-chain polypeptide, a Class II MHC β-chain polypeptide, or only a portion of a Class II MHC α- or β-chain polypeptide. For example, "Class II MHC polypeptide" can be a polypeptide that includes i) only the α1 domain of the Class II MHC α-chain polypeptide, ii) only the α2 domain of the Class II MHC α-chain, iii) only the α1 and α2 domains of the Class II MHC α-chain, iv) only the β1 domain of the Class II MHC β-chain, v) only the β2 domain of the Class II MHC β-chain, vi) only the β1 and β2 domains of the Class II MHC β-chain, vii) the α1 domain of the Class II MHC α-chain, the β1 domain of the Class II MHC β-chain, and the β2 domain of the Class II MHC, etc.

[0055] Class II MHC polypeptides include alleles. The HLA locus is essentially highly polymorphic. As disclosed in Nomenclature for Factors of the HLA System 2000 (Hum. Immunol.; 62(4):419-68, 2001), there are 221 HLA-DRB1 alleles, 19 DRB3 alleles, 89 DRB4 alleles, 14 DRB5 alleles, 19 DQA1 alleles and 39 DQB1 alleles, and new alleles are constantly being discovered. In the 2007 revision of the WHO nomenclature Committee for Factors of the HLA System (www.anthonynolan.com / HIG / ), it was shown that there are 3 DRA alleles, 494 DRB1 alleles, 1 DRB2 allele, 44 DRB3 alleles, 13 DRB4 alleles, 18 DRB5 alleles, 3 DRB6 alleles, 2 DRB7 alleles, 10 DRB8 alleles, 1 DRB9 allele, 34 DQA1 alleles, 83 DQB1 alleles, 23 DPA1, 126 DPB1 alleles, 4 DMA alleles, 7 DMB alleles, 12 DOA alleles and 9 DOB alleles. As used herein, the term "Class II MHC polypeptide" includes allelic forms of any known Class II MHC polypeptide.

[0056] In some cases, the TMAPPs of the present disclosure include Class II MHC α chains that do not include the leader, transmembrane, and intracellular portions (e.g., cytoplasmic tails) that may be present in native Class II MHC α chains. Thus, in some cases, the TMAPPs of the present disclosure include only the α1 and α2 portions of the Class II MHC α chain and do not include the leader, transmembrane, and intracellular portions (e.g., cytoplasmic tails) that may be present in native Class II MHC α chains.

[0057] In some cases, the TMAPP of the present disclosure comprises a class II MHC β chain that does not include a leader portion, a transmembrane portion, and an intracellular portion (e.g., cytoplasmic tail) that may be present in a native class II MHC β chain. Thus, in some cases, the TMAPP of the present disclosure comprises only the β1 and β2 portions of the class II MHC β chain and does not include a leader portion, a transmembrane portion, and an intracellular portion (e.g., cytoplasmic tail) that may be present in a native class II MHC β chain.

[0058] MHC class II alpha chain The MHC class II alpha chain comprises an α1 domain and an α2 domain. In some cases, the α1 domain and the α2 domain present in an antigen-presenting cell are derived from the same MHC class II α chain polypeptide. In some cases, the α1 domain and the α2 domain present in an antigen-presenting cell are derived from two different MHC class II α chain polypeptides.

[0059] The MHC class II alpha chains suitable for inclusion in the TMAPPs of the present disclosure (e.g., multimeric TMAPPs; single-chain TMAPPs) lack a signal peptide. The MHC class II alpha chains suitable for inclusion in the multimeric polypeptides of the present disclosure can have a length of about 60 amino acids to about 190 amino acids. For example, the MHC class II alpha chains suitable for inclusion in the TMAPPs of the present disclosure can have a length of about 60 amino acids to about 80 amino acids, about 80 amino acids to about 100 amino acids, about 100 amino acids to about 120 amino acids, about 120 amino acids to about 140 amino acids, about 140 amino acids to about 160 amino acids, about 160 amino acids to about 180 amino acids, or about 180 amino acids to about 200 amino acids. The MHC class II α1 domain suitable for inclusion in the TMAPPs of the present disclosure can have a length of about 30 amino acids to about 95 amino acids. For example, the MHC class II α1 domain suitable for inclusion in the TMAPPs of the present disclosure can have a length of about 30 amino acids to about 40 amino acids, about 40 amino acids to about 50 amino acids, about 50 amino acids to about 60 amino acids, about 60 amino acids to about 70 amino acids, about 70 amino acids to about 80 amino acids, about 80 amino acids to about 90 amino acids, or about 90 amino acids to about 95 amino acids. The MHC class II α2 domain suitable for inclusion in the TMAPPs of the present disclosure can have a length of about 30 amino acids to about 95 amino acids. For example, the MHC class II α2 domain suitable for inclusion in the TMAPPs of the present disclosure can have a length of about 30 amino acids to about 40 amino acids, about 40 amino acids to about 50 amino acids, about 50 amino acids to about 60 amino acids, about 60 amino acids to about 70 amino acids, about 70 amino acids to about 80 amino acids, about 80 amino acids to about 90 amino acids, or about 90 amino acids to about 95 amino acids.

[0060] DRA In some cases, a suitable MHC class II α-chain polypeptide is the DRA polypeptide. The DRA polypeptide can have at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity with amino acids 26 - 203 of the DRA amino acid sequence shown in Figure 6. In some cases, the DRA polypeptide has a length of about 178 amino acids (e.g., 175, 176, 177, 178, 179, or 180 amino acids).

[0061] The "DRA polypeptide" includes allelic variants, e.g., natural allelic variants. Thus, in some cases, a suitable DRA polypeptide has the following amino acid sequence: TIFF0007691927000003.tif23151 (amino acids 26 - 203 of SEQ ID NO: 5, DRA*01:02:01, see Figure 6), or an allelic variant thereof. In some cases, the allelic variant is the DRA*01:01:01:01 allelic variant that differs from DRA*01:02:01 by having valine instead of leucine at position 242 of the sequence in Figure 6.

[0062] A suitable DRA α1 domain has the following amino acid sequence: An amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF0007691927000004.tif17133, 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 α1 domain has the following amino acid sequence: TIFF0007691927000005.tif11128, or can include a natural allelic variant.

[0063] A suitable DRA α2 domain has the following amino acid sequence: For TIFF0007691927000006.tif17128, it includes an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity, and may have a length of about 94 amino acids (e.g., 90, 91, 92, 93, 94, 95, 96, 97, or 90 amino acids).

[0064] DMA In some cases, a suitable MHC class II α-chain polypeptide is a DMA polypeptide. The DMA polypeptide may have at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity with amino acids 27 - 217 of the DMA amino acid sequence shown in Figure 11. In some cases, the DMA polypeptide has a length of about 191 amino acids (e.g., 188, 189, 190, 191, 192, or 193 amino acids).

[0065] The "DMAA polypeptide" includes allelic variants, e.g., natural allelic variants. Thus, in some cases, a suitable DMAA polypeptide has the following amino acid sequence: TIFF0007691927000007.tif24130 (amino acids 27 - 217 of SEQ ID NO:8 DMA*01:01:01, see Figure 11), or an allelic variant thereof.

[0066] A suitable DMA α1 domain has the following amino acid sequence: For TIFF0007691927000008.tif18132, it includes an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity, and may have a length of about 98 amino acids (e.g., 94, 95, 96, 97, 98, 99, 100, or 101 amino acids). A suitable DMA α1 domain has the following amino acid sequence: TIFF0007691927000009.tif17132, or may include its natural allelic variant.

[0067] A suitable DMA α2 domain has the following amino acid sequence: TIFF0007691927000010.tif17132, and includes an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity thereto, and may have a length of about 93 amino acids (e.g., 90, 91, 92, 93, 94, 95, 96, or 97 amino acids). A suitable DMA α2 domain has the following amino acid sequence: TIFF0007691927000011.tif17129, or may include its natural allelic variant.

[0068] DOA In some cases, a suitable MHC class II α-chain polypeptide is a DOA polypeptide. The DOA polypeptide may have at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to amino acids 26-204 of the DOA amino acid sequence shown in FIG. 13. In some cases, the DOA polypeptide has a length of about 179 amino acids (e.g., 175, 176, 177, 178, 179, 180, 181, or 182 amino acids).

[0069] The "DOA polypeptide" includes allelic variants, e.g., natural allelic variants. Thus, in some cases, a suitable DOA polypeptide has the following amino acid sequence: TIFF0007691927000012.tif23150 (SEQ ID NO: 11; amino acids 26 - 204 of DOA*01:01:01:01, see Figure 13), or an allelic variant thereof. In some cases, the allelic variant may be DOA*01:02 by having arginine (R80C) instead of cysteine at position 80 or DOA*01:03 variant by having valine (L74V) instead of leucine at position 74, as compared to DOA*01:01:01:01.

[0070] A preferred DOA α1 domain has the following amino acid sequence: For TIFF0007691927000013.tif18131, it includes an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity, and may have a length of about 85 amino acids (e.g., 83, 84, 85, 86, 87, or 88 amino acids). Preferred α1 domain sequences can incorporate the L74V and / or R80C substitutions found in DOA*01:02 and DOA*01:03 (amino acids corresponding to L74 and R80 are shown in italics and bold). A preferred DOA α1 domain has the following amino acid sequence: TIFF0007691927000014.tif17130, or may include natural allelic variants.

[0071] A preferred DOA α2 domain has the following amino acid sequence: For TIFF0007691927000015.tif17128, it includes an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity, and may have a length of about 94 amino acids (e.g., 91, 92, 93, 94, 95, 96, or 97 amino acids). A preferred DOA α2 domain has the following amino acid sequence: TIFF0007691927000016.tif17131, or may include its natural allelic variant.

[0072] DPA1 In some cases, a suitable MHC class II α-chain polypeptide is a DPA1 polypeptide. The DPA1 polypeptide may have at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity with amino acids 29 - 209 of the DPA1 amino acid sequence shown in FIG. 15. In some cases, the DPA1 polypeptide has a length of about 181 amino acids (e.g., 178, 179, 180, 181, 182, 183, or 184 amino acids).

[0073] The "DPA1 polypeptide" includes allelic variants, e.g., natural allelic variants. Thus, in some cases, a suitable DPA1 polypeptide has the following amino acid sequence: TIFF0007691927000017.tif24141 (amino acids 29 - 209 of SEQ ID NO: 14, DPA1*01:03:01:01, see FIG. 15), or its allelic variant.

[0074] A suitable DPA1 α1 domain has the following amino acid sequence: TIFF0007691927000018.tif17133 may include an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity thereto, and may have a length of about 87 amino acids (e.g., 84, 85, 86, 87, 88, or 89 amino acids). A suitable DPA1 α1 domain has the following amino acid sequence: TIFF0007691927000019.tif11146, or may include a natural allelic variant.

[0075] A suitable DPA1 α2 domain has the following amino acid sequence: For TIFF0007691927000020.tif17129, it may include an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity, and may have a length of about 97 amino acids (e.g., 91, 92, 93, 94, 95, 96, or 97 amino acids). A preferred DPA1 α2 domain has the following amino acid sequence: It may include TIFF0007691927000021.tif17129, or its natural allelic variant.

[0076] Other DPA1 polypeptides have the sequence: TIFF0007691927000022.tif31151 (SEQ ID NO: 17; amino acids 29 - 209 of DPA1*02:01:01:01, see Figure 15), or a variant thereof having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity.

[0077] A preferred DPA1 α1 domain may include an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to amino acids 29 - 115 of DPA1*02:01:01:01, SEQ ID NO: 17, and may have a length of about 87 amino acids (e.g., 84, 85, 86, 87, 88, or 89 amino acids). A preferred DPA1 α2 domain may include an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to amino acids 116 - 209 of DPA1*02:01:01:01, SEQ ID NO: 17, and may have a length of about 97 amino acids (e.g., 91, 92, 93, 94, 95, 96, or 97 amino acids).

[0078] DQA1In some cases, a suitable MHC class II α-chain polypeptide is a DQA1 polypeptide. The DQA1 polypeptide can have at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity with amino acids 24 - 204 of any of the DQA1 amino acid sequences shown in FIG. 17. In some cases, the DQA1 polypeptide has a length of about 181 amino acids (e.g., 177, 178, 179, 180, 181, 182, or 183 amino acids). In one embodiment, the DQA1 α-chain polypeptide has at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity with amino acids 30 - 227 of the DQA1*01:01 α-chain amino acids of ImMunoGeneTics (「IMGT」) / HLA accession number: HLA00601 in FIG. 17. In one embodiment, the DQA1 α-chain polypeptide has at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity with amino acids 30 - 227 of the DQA1*01:02 α-chain amino acids of IMGT / HLA accession number: HLA00603, GenBank NP_002113 in FIG. 17. In one embodiment, the DQA1 α-chain polypeptide has at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity with amino acids 30 - 227 of the DQA1*02:01 α-chain amino acids of IMGT / HLA accession number: HLA00607 in FIG. 17. In one embodiment, the DQA1 α-chain polypeptide has at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity with amino acids 30 - 227 of the DQA1*03:01:α-chain amino acids of IMGT / HLA accession number: HLA00609 in FIG. 17.In one embodiment, the DQA1 α-chain polypeptide can have at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity with amino acids 30 to 227 of the DQA1*04:01 α-chain amino acids of IMGT / HLA accession number: HLA00612 in FIG. 17. In one embodiment, the DQA1 α-chain polypeptide can have at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity with amino acids 30 to 227 of the DQA1*05:01 α-chain amino acids of IMGT / HLA accession number: HLA00613 in FIG. 17. In one embodiment, the DQA1 α-chain polypeptide can have at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity with amino acids 30 to 227 of the DQA1*06:01 α-chain amino acids of IMGT / HLA accession number: HLA00620 in FIG. 17.

[0079] The "DQA1 polypeptide" includes allelic variants, such as natural allelic variants. Thus, in some cases, a suitable DQA1 polypeptide has the following amino acid sequence: TIFF0007691927000023.tif24150, or an allelic variant thereof.

[0080] A suitable DQA1 α1 domain has the following amino acid sequence: It includes an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF0007691927000024.tif17135 and can have a length of about 87 amino acids (e.g., 84, 85, 86, 87, 88, or 89 amino acids). A suitable DQA1 α1 domain has the following amino acid sequence: TIFF0007691927000025.tif may contain 17150 or a natural allele variant.

[0081] A suitable DQA1 α2 domain has the following amino acid sequence: TIFF0007691927000026.tif contains an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to 17150 and may have a length of about 94 amino acids (e.g., 91, 92, 93, 94, 95, 96, or 97 amino acids). A suitable DQA1 α2 domain has the following amino acid sequence: TIFF0007691927000027.tif may contain 17150 or its natural allele variant.

[0082] DQA2 In some cases, a suitable MHC class II α-chain polypeptide is a DQA2 polypeptide. The DQA2 polypeptide may have at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to amino acids 24 - 204 of the DQA2 amino acid sequence shown in FIG. 18. In some cases, the DQA2 polypeptide has a length of about 181 amino acids (e.g., 177, 178, 179, 180, 181, 182, or 183 amino acids).

[0083] The "DQA2 polypeptide" includes allele variants, e.g., natural allele variants. Thus, in some cases, a suitable DQA2 polypeptide has the following amino acid sequence: TIFF0007691927000028.tif contains 24150 or its allele variant.

[0084] A suitable DQA2 α1 domain has the following amino acid sequence: For TIFF0007691927000029.tif17132, it may include an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity, and may have a length of about 87 amino acids (e.g., 84, 85, 86, 87, 88, or 89 amino acids). A preferred DQA2 α1 domain has the following amino acid sequence: TIFF0007691927000030.tif17148, or may include a natural allele variant.

[0085] A preferred DQA2 α2 domain has the following amino acid sequence: For TIFF0007691927000031.tif17150, it may include an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity, and may have a length of about 94 amino acids (e.g., 91, 92, 93, 94, 95, 96, or 97 amino acids). A preferred DQA2 α2 domain has the following amino acid sequence: TIFF0007691927000032.tif17150, or may include its natural allele variant.

[0086] MHC class II beta chain The MHC class II beta chain includes a β1 domain and a β2 domain. In some cases, the β1 domain and the β2 domain present in an antigen-presenting cell are derived from the same MHC class II β-chain polypeptide. In some cases, the β1 domain and the β2 domain present in an antigen-presenting cell are derived from two different MHC class II β-chain polypeptides.

[0087] The MHC class II beta chains suitable for inclusion in the TMAPP of the present disclosure (e.g., multimeric TMAPP; single-chain TMAPP) lack a signal peptide. The MHC class II beta chains suitable for inclusion in the TMAPP of the present disclosure can have a length of about 60 amino acids to about 210 amino acids. For example, the MHC class II beta chains suitable for inclusion in the TMAPP of the present disclosure can have a length of about 60 amino acids to about 80 amino acids, about 80 amino acids to about 100 amino acids, about 100 amino acids to about 120 amino acids, about 120 amino acids to about 140 amino acids, about 140 amino acids to about 160 amino acids, about 160 amino acids to about 180 amino acids, about 180 amino acids to about 200 amino acids, or about 200 amino acids to about 210 amino acids. The MHC class II β1 domain suitable for inclusion in the TMAPP of the present disclosure can have a length of about 30 amino acids to about 105 amino acids. For example, the MHC class II β1 domain suitable for inclusion in the TMAPP of the present disclosure can have a length of about 30 amino acids to about 40 amino acids, about 40 amino acids to about 50 amino acids, about 50 amino acids to about 60 amino acids, about 60 amino acids to about 70 amino acids, about 70 amino acids to about 80 amino acids, about 80 amino acids to about 90 amino acids, about 90 amino acids to about 95 amino acids, about 95 amino acids to about 100 amino acids, or about 100 amino acids to about 105 amino acids. The MHC class II β2 domain suitable for inclusion in the TMAPP of the present disclosure can have a length of about 30 amino acids to about 105 amino acids. For example, the MHC class II β2 domain suitable for inclusion in the TMAPP of the present disclosure can have a length of about 30 amino acids to about 40 amino acids, about 40 amino acids to about 50 amino acids, about 50 amino acids to about 60 amino acids, about 60 amino acids to about 70 amino acids, about 70 amino acids to about 80 amino acids, about 80 amino acids to about 90 amino acids, about 90 amino acids to about 95 amino acids, about 95 amino acids to about 100 amino acids, or about 100 amino acids to about 105 amino acids.

[0088] DRB1 In some cases, a suitable MHC class II β-chain polypeptide is a DRB1 polypeptide. In one embodiment, the DRB1 polypeptide can have at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity with amino acids 30-227 of any of the DRB1 amino acid sequences shown in FIG. 7, which shows a DRB1 precursor protein, where amino acids 1-29 are a signal sequence (underlined), 30-124 form a β1 region (bold), 125-227 are a β2 region (bold and underlined), and 228-250 are a transmembrane region.

[0089] In one embodiment, the DRB1 β-chain polypeptide can have at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity with amino acids 30 to 227 of the DRB1-1 (DRB1*01:01) beta-chain amino acid sequence Swiss-Prot / UniProt reference sequence ("sp") P04229.2 in FIG. 7. In one embodiment, the DRB1 β-chain polypeptide can have at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity with amino acids 30 to 227 of the DRB1-3 (DRB1*03:01) beta-chain amino acid sequence sp P01912.2 in FIG. 7. In one embodiment, the DRB1 β-chain polypeptide can have at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity with amino acids 30 to 227 of the DRB1-4 (DRB1*04:01) beta-chain amino acid sequence sp P13760.1 in FIG. 7. In one embodiment, the DRB1 β-chain polypeptide can have at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity with amino acids 30 to 227 of the DRB1-7 (DRB1*07:01) beta-chain amino acid sequence sp P13761.1 in FIG. 7. In one embodiment, the DRB1 β-chain polypeptide can have at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity with amino acids 30 to 227 of the DRB1-8 (DRB1*08:01) beta-chain amino acid sequence sp Q30134.2 in FIG. 7.In one embodiment, the DRB1 β-chain polypeptide may have at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity with amino acids 30 to 227 of the DRB1-9 (DRB1*09:01) beta-chain amino acid sequence sp Q9TQE0.1 in Figure 7. In one embodiment, the DRB1 β-chain polypeptide may have at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity with amino acids 30 to 227 of the DRB1-10 (DRB1*10:01) beta-chain amino acid sequence sp Q30167.2 in Figure 7. In one embodiment, the DRB1 β-chain polypeptide may have at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity with amino acids 30 to 227 of the DRB1-11 (DRB1*11:01) beta-chain amino acid sequence sp P20039.1 in Figure 7. In one embodiment, the DRB1 β-chain polypeptide may have at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity with amino acids 30 to 227 of the DRB1-12 (DRB1*12:01) beta-chain amino acid sequence sp Q95IE3.1 in Figure 7. In one embodiment, the DRB1 β-chain polypeptide may have at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity with amino acids 30 to 227 of the DRB1-13 (DRB1*13:01) beta-chain amino acid sequence sp Q5Y7A7.1 in Figure 7.In one embodiment, the DRB1 β-chain polypeptide has at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity with amino acids 30-227 of the DRB1-14 (DRB1*14:01) beta-chain amino acid sequence sp Q9GIY3.1 in FIG. 7. In one embodiment, the DRB1 β-chain polypeptide has at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity with amino acids 30-227 of the DRB1-15 (DRB1*15:01) beta-chain amino acid sequence sp P01911 in FIG. 7. In one embodiment, the DRB1 β-chain polypeptide has at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity with amino acids 30-227 of the DRB1-16 (DRB1*16:01) beta-chain amino acid sequence sp Q29974.1 in FIG. 7. In some cases, the DRB1 β-chain polypeptide has a length of about 198 amino acids (e.g., 195, 196, 197, 198, 199, 200, 201, or 202 amino acids).

[0090] The "DRB1 polypeptide" includes allelic variants, e.g., natural allelic variants. Thus, in some cases, a suitable DRB1 polypeptide has the following amino acid sequence: TIFF0007691927000033.tif24151 (amino acids 31-227 of DRB1-4, see FIG. 7A), or an allelic variant thereof.

[0091] A suitable DRB1 β1 domain has the following amino acid sequence: It may include an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF0007691927000034.tif11149 and may have a length of about 95 amino acids (e.g., 92, 93, 94, 95, 96, 97, or 98 amino acids). A preferred DRB1 β1 domain has the following amino acid sequence: It may include TIFF0007691927000035.tif11149, or a natural allele variant.

[0092] A preferred DRB1 β2 domain has the following amino acid sequence: It may include an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF0007691927000036.tif11150 and may have a length of about 103 amino acids (e.g., 100, 101, 102, 103, 104, 105, or 106 amino acids). A preferred DRB1 β2 domain has the following amino acid sequence: It may include TIFF0007691927000037.tif11150, or its natural allele variant.

[0093] DRB3 In some cases, a suitable MHC class II β-chain polypeptide is a DRB3 polypeptide. In one embodiment, the DRB3 polypeptide can have at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity with amino acids 30 to 227 of any DRB3 amino acid sequence shown in FIG. 8, which shows a DRB3 precursor protein, where amino acids 1 to 29 are a signal sequence (underlined), 30 to 124 form a β1 region (shown in bold), 125 to 227 is a β2 region, and 228 to 250 is a transmembrane region. In one embodiment, the DRB3 β-chain polypeptide can have at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity with amino acids 30 to 227 of the DRB1-3 (DRB3*01:01) beta-chain amino acid sequence GenBank NP_072049.1 in FIG. 8. In one embodiment, the DRB3 β-chain polypeptide can have at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity with amino acids 30 to 227 of the DRB1-3 beta-chain amino acid sequence of GenBank accession EAX03632.1 in FIG. 8. In one embodiment, the DRB3 β-chain polypeptide can have at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity with amino acids 30 to 227 of the DRB1-3 (DRB3*02:01) beta-chain amino acid sequence GenBank CAA23781.1 in FIG. 8. In one embodiment, the DRB3 β-chain polypeptide can have at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity with amino acids 30 to 227 of the DRB1-3 (DRB3*03:01) beta-chain amino acid sequence GenBank AAN15205.1 in FIG. 8.

[0094] "DRB3 polypeptide" includes allelic variants, for example, natural allelic variants. Thus, in some cases, a suitable DRB3 polypeptide has the following amino acid sequence: TIFF0007691927000038.tif31148, or an allelic variant thereof.

[0095] A suitable DRB3 β1 domain has the following amino acid sequence: It includes an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF0007691927000039.tif17134, and may have a length of about 95 amino acids (for example, 93, 94, 95, 96, 97, or 98 amino acids). A suitable DRB3 β1 domain has the following amino acid sequence: TIFF0007691927000040.tif17135, or may include a natural allelic variant.

[0096] A suitable DRB3 β2 domain has the following amino acid sequence: It includes an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF0007691927000041.tif17133, and may have a length of about 103 amino acids (for example, 100, 101, 102, 103, 104, or 105 amino acids). A suitable DRB3 β2 domain has the following amino acid sequence: TIFF0007691927000042.tif17133, or may include its natural allelic variant.

[0097] DRB4 In some cases, a suitable MHC class II β-chain polypeptide is a DRB4 polypeptide. The DRB4 polypeptide can have at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity with amino acids 30-227 of the DRB4 amino acid sequence shown in Figure 9. In some cases, the DRB4 polypeptide has a length of about 198 amino acids (e.g., 195, 196, 197, 198, 199, 200, 201, or 202 amino acids).

[0098] "DRB4 polypeptide" includes allelic variants, such as natural allelic variants. Thus, in some cases, a suitable CDR4 polypeptide has the following amino acid sequence: TIFF0007691927000043.tif31135, or an allelic variant thereof.

[0099] A suitable DRB4 β1 domain has the following amino acid sequence: TIFF0007691927000044.tif17134, and includes an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity thereto, and can have a length of about 95 amino acids (e.g., 93, 94, 95, 96, 97, or 98 amino acids). A suitable DRB4 β1 domain has the following amino acid sequence: TIFF0007691927000045.tif17134, or can include a natural allelic variant.

[0100] A suitable DRB4 β2 domain has the following amino acid sequence: For TIFF0007691927000046.tif17134, it may include an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity, and may have a length of about 103 amino acids (e.g., 100, 101, 102, 103, 104, or 105 amino acids). A preferred DRB4 β2 domain has the following amino acid sequence: It may include TIFF0007691927000047.tif17134, or its natural allelic variant.

[0101] DRB5 In some cases, a preferred MHC class II β-chain polypeptide is a DRB5 polypeptide. The DRB5 polypeptide may have at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity with amino acids 30 - 227 of the DRB5 amino acid sequence shown in Figure 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).

[0102] "DRB5 polypeptide" includes allelic variants, e.g., natural allelic variants. Thus, in some cases, a preferred DRB5 polypeptide has the following amino acid sequence: It includes TIFF0007691927000048.tif30148, or its allelic variant.

[0103] A preferred DRB5 β1 domain has the following amino acid sequence: For TIFF0007691927000049, it may include an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity, and may have a length of about 95 amino acids (e.g., 93, 94, 95, 96, 97, or 98 amino acids). A preferred DRB5 β1 domain has the following amino acid sequence: TIFF0007691927000050.tif17149, or may include a natural allele variant.

[0104] A preferred DRB5 β2 domain has the following amino acid sequence: For TIFF0007691927000051.tif17133, it may include an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity, and may have a length of about 103 amino acids (e.g., 100, 101, 102, 103, 104, or 105 amino acids). A preferred DRB5 β2 domain has the following amino acid sequence: TIFF0007691927000052.tif17133, or its natural allele variant.

[0105] DMB In some cases, a preferred MHC class II β-chain polypeptide is a DMB polypeptide. The DMB polypeptide may have at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity with amino acids 19 - 207 of the DMB amino acid sequence shown in Figure 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).

[0106] "DMB polypeptide" includes allelic variants, such as natural allelic variants. Thus, in some cases, a suitable DMB polypeptide has the following amino acid sequence: TIFF0007691927000053.tif24139, or an allelic variant thereof.

[0107] A suitable DMB β1 domain has the following amino acid sequence: TIFF0007691927000054.tif17132, and includes an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity thereto, and may have a length of about 94 amino acids (e.g., 92, 93, 94, 95, 96, or 97 amino acids). A suitable DMB β1 domain has the following amino acid sequence: TIFF0007691927000055.tif17134, or may include a natural allelic variant.

[0108] A suitable DMB β2 domain has the following amino acid sequence: TIFF0007691927000056.tif17137, and includes an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity thereto, and may have a length of about 95 amino acids (e.g., 93, 94, 95, 96, 97, or 98 amino acids). A suitable DMB β2 domain has the following amino acid sequence: TIFF0007691927000057.tif17135, or may include its natural allelic variant.

[0109] DOB In some cases, a suitable MHC class II β-chain polypeptide is the DOB polypeptide. The DOB polypeptide can have at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity with amino acids 27-214 of the DOB amino acid sequence shown 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).

[0110] The "DOB polypeptide" includes allelic variants, such as natural allelic variants. Thus, in some cases, a suitable DOB polypeptide has the following amino acid sequence: TIFF0007691927000058.tif24142, or an allelic variant thereof.

[0111] A suitable DOB β1 domain has the following amino acid sequence: It includes an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF0007691927000059.tif17133 and can have a length of about 94 amino acids (e.g., 92, 93, 94, 95, 96, or 97 amino acids). A suitable DOB β1 domain has the following amino acid sequence: TIFF0007691927000060.tif17133, or a natural allelic variant.

[0112] A suitable DOB β2 domain has the following amino acid sequence: For TIFF0007691927000061, it may include an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity, and may have a length of about 94 amino acids (e.g., 92, 93, 94, 95, 96, or 97 amino acids). A preferred DOB β2 domain has the following amino acid sequence: It may include TIFF0007691927000062, or its natural allelic variant.

[0113] DPB1 In some cases, a suitable MHC class II β-chain polypeptide is a DPB1 polypeptide. The DPB1 polypeptide can have at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity with amino acids 30 to 215 of any of the DPB1 amino acid sequences shown 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). In one embodiment, the DRB3 β-chain polypeptide can have at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity with amino acids 30 to 227 of the DPB1*01:01 beta-chain amino acid sequence of IMGT / HLA accession number: HLA00514 in FIG. 16. In one embodiment, the DRB3 β-chain polypeptide can have at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity with amino acids 30 to 227 of the DPB1*01:01 beta-chain amino acid sequence of IMGT / HLA accession number: HLA00517 in FIG. 16. In one embodiment, the DRB3 β-chain polypeptide can have at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity with amino acids 30 to 227 of the DPB1*03:01 beta-chain amino acid sequence of IMGT / HLA accession number: HLA00520 in FIG. 16. In one embodiment, the DRB3 β-chain polypeptide can have at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity with amino acids 30 to 227 of the DPB1*04:01 beta-chain amino acid sequence of IMGT / HLA accession number: HLA00521, GenBank NP_002112.3 in FIG. 16.In one embodiment, the DRB3 β-chain polypeptide can have at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity with amino acids 30 to 227 of the DPB1*06:01 beta-chain amino acid sequence of IMGT / HLA accession number: HLA00524 in FIG. 16. In one embodiment, the DRB3 β-chain polypeptide can have at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity with amino acids 30 to 227 of the DPB1*11:01 beta-chain amino acid sequence of IMGT / HLA accession number: HLA00528 in FIG. 16. In one embodiment, the DRB3 β-chain polypeptide can have at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity with amino acids 30 to 227 of the DPB1*71:01 beta-chain amino acid sequence of IMGT / HLA accession number: HLA00590 in FIG. 16. In one embodiment, the DRB3 β-chain polypeptide can have at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity with amino acids 30 to 227 of the DPB1*104:01 beta-chain amino acid sequence of IMGT / HLA accession number: HLA02046 in FIG. 16. In one embodiment, the DRB3 β-chain polypeptide can have at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity with amino acids 30 to 227 of the DPB1*141:01 beta-chain amino acid sequence of IMGT / HLA accession number: HLA10364 in FIG. 16.

[0114] The "DPB1 polypeptide" includes allelic variants, such as natural allelic variants. Thus, in some cases, a suitable DPB1 polypeptide has the following amino acid sequence: TIFF0007691927000063.tif24135, or an allelic variant thereof.

[0115] A suitable DPB1 β1 domain has the following amino acid sequence: TIFF0007691927000064.tif17132, and has an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity thereto, and may have a length of about 92 amino acids (e.g., 90, 91, 92, 93, or 94 amino acids). A suitable DPB1 β1 domain has the following amino acid sequence: TIFF0007691927000065.tif17131, or may include a natural allelic variant.

[0116] A suitable DPB1 β2 domain has the following amino acid sequence: TIFF0007691927000066.tif17133, and has an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity thereto, and may have a length of about 94 amino acids (e.g., 92, 93, 94, 95, 96, or 97 amino acids). A suitable DPB1 β2 domain has the following amino acid sequence: TIFF0007691927000067.tif17134, or may include its natural allelic variant.

[0117] DQB1 In some cases, a suitable MHC class II β-chain polypeptide is a DQB1 polypeptide. The DQB1 polypeptide can have at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity with amino acids 33-220 of the DQB1 amino acid sequence shown in FIG. 19A or FIG. 19B or FIG. 19C. In some cases, the DQB1 polypeptide has a length of about 188 amino acids (e.g., 186, 187, 188, 190, 191, or 192 amino acids).

[0118] The "DQB1 polypeptide" includes allelic variants, e.g., natural allelic variants. Thus, in some cases, a suitable DQB1 polypeptide has the following amino acid sequence: TIFF0007691927000068.tif24135, or an allelic variant thereof.

[0119] A suitable DQB1 β1 domain has the following amino acid sequence: It includes an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF0007691927000069.tif17135 and can have a length of about 94 amino acids (e.g., 92, 93, 94, 95, or 96 amino acids). A suitable DQB1 β1 domain has the following amino acid sequence: TIFF0007691927000070.tif17134, or a natural allelic variant thereof.

[0120] A suitable DQB1 β2 domain has the following amino acid sequence: For TIFF0007691927000071, it may include an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity, and may have a length of about 94 amino acids (e.g., 92, 93, 94, 95, or 96 amino acids). A preferred DQB1 β2 domain has the following amino acid sequence: TIFF0007691927000072.tif17133, or its natural allelic variant may be included.

[0121] DQB2 In some cases, a preferred MHC class II β-chain polypeptide is a DQB2 polypeptide. The DQB2 polypeptide may have at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity with amino acids 33 - 215 of the DQB2 amino acid sequence shown in FIG. 20A or FIG. 20. In some cases, the DQB2 polypeptide has a length of about 182 amino acids (e.g., 175, 176, 177, 178, 179, 180, 181, or 182 amino acids).

[0122] The "DQB2 polypeptide" includes allelic variants, e.g., natural allelic variants. Thus, in some cases, a preferred DQB2 polypeptide has the following amino acid sequence: TIFF0007691927000073.tif24133, or its allelic variant is included.

[0123] A preferred DQB2 β1 domain has the following amino acid sequence: For TIFF0007691927000074, it may include an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity, and may have a length of about 94 amino acids (e.g., 92, 93, 94, 95, 96, or 97 amino acids). A preferred DQB2 β1 domain has the following amino acid sequence: TIFF0007691927000075.tif17135, or may include a natural allele variant.

[0124] A preferred DQB2 β2 domain has the following amino acid sequence: For TIFF0007691927000076.tif17130, it may include an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity, and may have a length of about 94 amino acids (e.g., 92, 93, 94, 95, 96, or 97 amino acids). A preferred DQB2 β2 domain has the following amino acid sequence: TIFF0007691927000077.tif17130, and may include its natural allele variant.

[0125] Alleles and haplotypes related to disease risk Certain alleles and haplotypes of MHC class II are associated with diseases, for example, increasing the risk of developing certain diseases. For example, Erlich et al. (2008) Diabetes 57:1084; Gough and Simmonds (2007) Curr. Genomics 8:453; Mitchell et al. (2007) Robbins Basic Pathology Philadelphia: Saunders, 8 thSee, e.g., Margaritte-Jeannin et al. (2004) Tissue Antigens 63:562; and Kurko et al. (2013) Clin. Rev. Allergy Immunol. 45:170.

[0126] MHC class II polypeptides in type 1 diabetes (T1D) T1D is associated with alleles belonging to the HLA-DR3 and HLA-DR4 haplotypes / serotypes, with the strongest risks associated with alleles of the HLA-DQ8 (e.g., HLA-DQB1*03:02) and HLA-DQ2 serotypes. The associations of several high- and medium-risk haplotypes with their various DR serotypes are shown in the following table adopted from Kantarova and Buc, Physiol. Res. 56:255-266 (2007). TIFF0007691927000078.tif90130

[0127] The DR3 and DR4 protein isoforms / haplotypes that are stereotypically defined for the DRB1 gene are associated with the increased risk of developing T1D in individuals expressing such alleles. The DR3 serotype includes alleles encoding the proteins of DRB1*03:01, *03:02, *03:03, and *03:04, and the HLA-DRB1*0301 allele has been widely recognized for its association with the predisposition to T1D. The DR4 serotype includes alleles encoding the proteins of DRB1*04:01, *04:02, *04:03, *04:04, *04:05, *04:06, *04:07, *04:08, *04:09, *04:10, *04:11, *04:12, and *04:13. Certain HLA-DR4 (e.g., HLA-DRB1*0401 and HLA-DRB1*0405) are risk factors for T1D in individuals, while the HLA-DRB1*04:03 allele / isoform may confer protection. DRB1*16:01 has also been shown to be more frequent in diabetic children compared to healthy controls (Deja, et al., Mediators of Inflammation 2006:1-7 (2006)). Alleles / isoforms showing high association with T1D are suitable sources of MHC II α1, α2, β1, and β2 polypeptide sequences.

[0128] DQ2 and DQ8 are serotypes within the HLA-DQ system and are determined by the recognition of the DQ β-chain. As mentioned above, T1D is associated with the DR3 and DR4 alleles, but the risk factors most strongly associated with T1D include the HLA-DQ8 serotype (e.g., the HLA-DQB1*03:02 isoform), especially the HLA-DQ8.1 serotype (HLA-DQA1*03:01 / DQB1*03:02) and alleles of the HLA-DQ2 serotype (e.g., DQB1*02 alleles such as DQB1*02:01, DQB1*02:02, or DQB1*02:03). Jones, et al., Nat. Rev. Immunol. 2006, 6:271-282. In contrast, individuals carrying the HLA DQB1*0602 allele are protected from type 1 diabetes. Ibid.

[0129] DQ2 is most prevalent in Western Europe, North Africa, and East Africa, and is frequently observed in parts of Spain and Ireland. The association of T1D with HLA-DR is not as strong as that with HLA-DQ, but the insulin-reactive T cells derived from lymph nodes that deplete the pancreas of T1D patients appear to be restricted by HLA-DR4.1 rather than HLA-DQ8 or HLA-DQ2 (Kent et al., Nature 2005 435:224-228). The crystal structure of HLA-DQ2 shows a unique P6 pocket with large volume and polar characteristics, defined by the presence of Ser30β instead of Tyr30β typically found in other HLA-DQ molecules (see, for example, Figure 19B, Ser, 62). This, like the presence of a positively charged lysine residue at 71β (see Figure 19B Lys 103), is a unique feature of HLA-DQ2, and when combined with the polarity of the P4 and P9 pockets, this MHC class II peptide-binding groove becomes the most suitable groove for accommodating peptides with negatively charged anchor residues (see, for example, Jones et al, Nat. Rev. Immunol. 2006, 6:271-282). This is an important factor enabling HLA-DQ2 to present gluten-derived peptides rich in proline and glutamic acid residues (generated by deamidation of glutamine). Ibid. In one embodiment, Ser30β of the DQ2 (e.g., DQB1*02:01) molecule can be replaced with cysteine (S30C) to enable conjugation to the position of a peptide epitope co-translated as part of a T cell regulatory antigen-presenting polypeptide (e.g., utilizing cysteine at position 6 of the peptide epitope).

[0130] Even when considering the DQB1 locus alone, an association with T1D has been reported when position β57 is a neutral residue such as Ala or Ser. Both serotypes DQ2 and DQ8, which are associated with T1D, do not have Asp at position 57β. Instead, they have Ala at that location, which confers T1D susceptibility (see Ala89 of HLA-DQB1*02:01 in Figure 19B and HLA-DQB1*03:02 in Figure 19C, respectively). In contrast, DQB1*06:02, which has Asp at position β57 (position 89 in Figure 19A) of DQB1, has been found to be associated with resistance to T1D. Jones, et al., Nat. Rev. Immunol. 2006, 6:271-282. Position β57 of the molecule forms an important residue in peptide-binding pocket 9 (P9) of DQB1 and is involved in antigen presentation and interaction with the T cell receptor (TCR).

[0131] Individuals with the HLA haplotype DQA1*03:01-DRB1*03:02 are extremely susceptible to T1D (a 10- to 20-fold increase), especially when combined with DQA1*05:01-DRB1*02:01. See Notkins, A.L., J. Biol. Chem., 2002, 277(46):43545-48. Groups showing susceptibility to classically defined T1D include HLA-DR4.1 (HLA-DRA1*01:01 / DRB1*04:01), HLA-DR4.5 (HLA-DRA1*01:01 / DRB1*04:05), HLA-DQ2.5 (HLA-DQA1*05:01 / DQB1*02:01), and HLA-DQ8.1 (HLA-DQA1*03:01 / DQB1*03:02). (See, for example, Jones et al, Nat. Rev. Immunol. 2006, 6:271-282). The DRβ1*04:05-DQβ1*04:01 / DRβ1*08:02-DQβ1*03:02 genotype has been shown to be associated with acute-onset and slowly progressive T1D. Fulminant diabetes was associated with the DRβ1*04:05-DQβ1*040:1 / DRβ1*04:05-DQβ1*04:01 genotype in a Japanese population study by Kawabata, et al., Diabetologia 2009, 52:2513-21.

[0132] The above alleles associated with an increased risk of T1D are suitable candidates that can be employed in the α1, α2, β1, and / or β2 polypeptide sequences present in the TMAPPs of the present disclosure. In one embodiment, the TMAPP is DQ2.5-like and comprises α1 and α2 polypeptides adopted from DQA1*0501 and β1 and β2 polypeptides adopted from DQB1*0201. In one embodiment, the TMAPP is DQ8.1-like and comprises α1 and α2 polypeptides adopted from DQA1*0301 and β1 and β2 polypeptides adopted from DQB1*0302.

[0133] MHC Class II Polypeptides and Celiac Disease The HLA haplotypes DQ2 and DQ8 are associated with a high risk of developing celiac disease in individuals expressing such HLA haplotypes. DQ2 is the second highest risk factor for celiac disease, with the highest risk being having a close relative with the disease. Approximately 95% of all celiac patients are estimated to carry at least one DQ2 allele, and about 30% of those individuals carry two copies of the DQ2 allele. DQ2 isoforms have different associations with celiac disease. There is a strong association with the DQ2.5 isoform (DQB1*02:01 / DQA1*05:01). DQB1*0201 is genetically associated with DQA1*05:01, forming the DQ2.5 haplotype. DQ2.5 is present at high levels in the northern island regions of Europe and the Basque region of Spain, and in parts of Ireland, it has a phenotypic frequency of over 50%.

[0134] The immunodominant region of DQ2.5 is on α2-gliadin, which has a protease-resistant 33mer with six overlapping DQ2.5-restricted epitopes. The multiple epitopes result in strong binding of T cells to the DQ2.5-33mer complex. DQ2.5 binds to gliadin, but its binding is susceptible to the effect of deamidation caused by tissue transglutaminase, and most of the most affinity sites / epitopes are generated by its action. 33mer TIFF0007691927000079.tif4128 or the 19mer described similarly All or part of TIFF0007691927000080.tif4128 (e.g., 8 or more, 9 or more, 10 or more, 12 or more, 14 or more, or 16 or more consecutive amino acids) can be used as peptide epitopes. See, for example, Bruun, et al. 2016, J. Diabetes Res. 2016, 2016:1-11 Article ID 2424306.

[0135] As described above, T1D is associated with the DQ2.5 phenotype and may be associated with gluten-sensitive enteropathy (GSE) and juvenile-onset male T1D. Recent studies have shown that the combination of DQ2.5 and DQ8 (both presenting acidic peptides) greatly increases the risk of adult-onset T1D. The presence of DQ2 with DR3 can reduce the age of onset and the severity of autoimmune disorders.

[0136] The DQ2.5 haplotype confers one of the highest known genetic risks for celiac disease, although similar risks can also arise from very similar alleles of different haplotypes (e.g., other DQA1*05 and DQB1*02 alleles). The DQ2.2 phenotype has the form of α2-β2 (e.g., DQA1*02:01:DQB1*0202) and is associated with the development of some cases of celiac disease. The associated DQA1* alleles of HLA DQB1*0202 and the DQ2.2 haplotype do not generate the DQA1*05 subunit (α5, e.g., DQA1*05:01), so the DQ2.2 heterodimer cannot effectively present α-2 gliadin, while presenting other gliadins. Thus, multimers or single-chain T cell regulatory antigen-presenting polypeptides containing the DQ2.2 polypeptide sequence (e.g., DQA1*02:01:DQB1*0202) can be used to present non-α-2 gliadin peptides.

[0137] The DQ2.2 / DQ7.5 phenotype, also called DQ2.5trans, is also associated with celiac disease. The DQ7.5 phenotype defined by serotype has the DQA1*0505:DQB1*0301 haplotype. The DQA1*0505 or DQA1*0501 gene products, when processed to the cell surface, become α5 and can assemble MHC class II molecules with either DQB1*0202 or DQB1*0201 of the DQ2.2 allele. As a result, the isoforms generated by the two haplotypes of the DQ2.2 / DQ7.5 phenotype include HLA DQ α 5 β 2 (DQ2.5), α 2 β 2 (DQ2.2), α 2 β 7 (DQ7.2, e.g., DQA1*0201:DQB1*0301), and α 5 β 7 (DQ7.5).

[0138] DQ8 is involved in celiac disease in people who do not have DQ2. The DQ8.1 haplotype encodes the DQA1*0301:DQB1*0302 haplotype. DQ8 is very high in Native Americans from Central America and ethnic groups of Eastern American origin.

[0139] Two class II HLA genotypes (DQA1*05:DQB1*02{α 5 β 2} and DQA1*03:DQB1*03:02{α 3 β 3}) have been shown to greatly contribute to the genetic risk of celiac disease in families and are virtually essential for celiac disease that occurs in white individuals (see Murry et al., Clin. Gastroenterol. Hepatol. 2007;5(12):1406 - 1412). Groups showing susceptibility to classically defined T1D and celiac disease include HLA - DQ2.5 (HLA - DQA1*05:01 / DQB1*02:01) and HLA - DQ8.1 (HLA - DQA1*03:01 / DQB1*03:02). (See, for example, Jones et al, Nat. Rev. Immunol. 2006, 6:271 - 282).

[0140] The alleles associated with the increased risk of celiac disease described above are suitable candidates that can be employed in the α1, α2, β1, and / or β2 polypeptide sequences of the TMAPP of the present disclosure. In one embodiment, TMAPP is DQ2.5 - like and includes α1 and α2 polypeptides adopted from DQA1*0501 and β1 and β2 polypeptides adopted from DQB1*0201. In one embodiment, TMAPP is DQ2.2 - like and includes α1 and α2 polypeptides adopted from DQA1*02:01 and β1 and β2 polypeptides adopted from DQB1*02:01. In one embodiment, TMAPP is DQ8.1 - like and includes α1 and α2 polypeptides adopted from DQA1*0301 and β1 and β2 polypeptides adopted from DQB1*0302. In one embodiment, TMAPP is HLA DQ α 5 β 2(DQ2.5), α 2 β 2 (DQ2.2), α 2 β 7 (DQ7.2, for example, DQA1*0201:DQB1*0301), and α 5 β 7 Comprising α1, α2, β1, and β2 polypeptides adopted from isoforms generated by the DQ2.2 / DQ7.5 haplotype, including the (DQ7.5) molecule.

[0141] DRB1*03:01 DRB1*0301 (the "DRB1*03:01" in Figure 7) is associated with an increased risk of developing T1D. Thus, in some cases, the TMAPP of the present disclosure comprises a DRB1*03:01 polypeptide comprising an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to amino acids 30 - 227 of the DRB1*03:01 amino acid sequence shown in Figure 7. In some cases, the TMAPP of the present disclosure comprises a DRB1*03:01 polypeptide comprising an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to amino acids 30 - 124 of the DRB1*03:01 amino acid sequence shown in Figure 7. In some cases, the TMAPP of the present disclosure comprises a DRB1*03:01 polypeptide comprising an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to amino acids 125 - 227 of the DRB1*03:01 amino acid sequence shown in Figure 7.

[0142] DRB1*04:01 DRB1*04:01 is associated with an increased risk of developing T1D. Thus, in some cases, the TMAPPs of the present disclosure include a DRB1*04:01 polypeptide comprising an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acids 30 - 227 of the DRB1*04:01 amino acid sequence shown in FIG. 7. In some cases, the TMAPPs of the present disclosure include a DRB1*04:01 polypeptide comprising an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acids 30 - 124 of the DRB1*04:01 amino acid sequence shown in FIG. 7. In some cases, the TMAPPs of the present disclosure include a DRB1*04:01 polypeptide comprising an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acids 125 - 227 of the DRB1*04:01 amino acid sequence shown in FIG. 7.

[0143] DRB1*04:02 DRB1*04:02 is associated with an increased risk of developing T1D. Thus, in some cases, the TMAPPs of the present disclosure include DRB1*04:02 polypeptides comprising amino acid sequences having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to amino acids 30 - 227 of the DRB1*04:02 amino acid sequence described below. In some cases, the TMAPPs of the present disclosure include DRB1*04:02 polypeptides comprising amino acid sequences having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to amino acids 30 - 124 of the DRB1*04:02 amino acid sequence described below. In some cases, the TMAPPs of the present disclosure include DRB1*04:02 polypeptides comprising amino acid sequences having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to amino acids 125 - 227 of the DRB1*04:02 amino acid sequence described below.

[0144] TIFF0007691927000081.tif38152

[0145] DRB1*04:05 DRB1*04:05 is associated with an increased risk of developing T1D. Thus, in some cases, the TMAPPs of the present disclosure include a DRB1*04:05 polypeptide comprising an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to amino acids 30 - 227 of the DRB1*04:05 amino acid sequence described below. In some cases, the TMAPPs of the present disclosure include a DRB1*04:05 polypeptide comprising an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to amino acids 30 - 124 of the DRB1*04:05 amino acid sequence described below. In some cases, the TMAPPs of the present disclosure include a DRB1*04:05 polypeptide comprising an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to amino acids 125 - 227 of the DRB1*04:05 amino acid sequence described below.

[0146] TIFF0007691927000082.tif38151

[0147] DQA1*05:01-DQB1*02:01 (DQ2) DQ2 (DQA1*05:01-DQB1*02:01) is associated with an increased risk of developing celiac disease.

[0148] Thus, in some cases, the TMAPP of the present disclosure comprises a DQA1*05:01 polypeptide comprising an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to amino acids 24-204 of the DQA1*05:01 amino acid sequence shown in FIG. 17. In some cases, the TMAPP of the present disclosure comprises a DQA1*05:01 polypeptide comprising an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to amino acids 24-110 of the DQA1*05:01 amino acid sequence shown in FIG. 17. In some cases, the TMAPP of the present disclosure comprises a DQA1*05:01 polypeptide comprising an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to amino acids 111-204 of the DQA1*05:01 amino acid sequence shown in FIG. 17.

[0149] In some cases, the TMAPP of the present disclosure includes a DQB1*02:01 polypeptide comprising an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to amino acids 33 to 220 of the DQB1*02:01 amino acid sequence described below. In some cases, the TMAPP of the present disclosure includes a DQB1*02:01 polypeptide comprising an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to amino acids 33 to 126 of the DQB1*02:01 amino acid sequence described below. In some cases, the TMAPP of the present disclosure includes a DQB1*02:01 polypeptide comprising an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to amino acids 127 to 220 of the DQB1*02:01 amino acid sequence described below.

[0150] TIFF0007691927000083.tif38151

[0151] DQA1*03:01-DQB1*03:02 (DQ8) DQA1*03:01-DQB1*03:02 (DQ8) is associated with an increased risk of developing celiac disease.

[0152] Accordingly, in some cases, the TMAPP of the present disclosure includes a DQA1*03:01 polypeptide comprising an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to amino acids 24 to 204 of the DQA1*03:02 amino acid sequence shown in FIG. 17. In some cases, the TMAPP of the present disclosure includes a DQA1*03:01 polypeptide comprising an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to amino acids 24 to 110 of the DQA1*03:01 amino acid sequence shown in FIG. 17. In some cases, the TMAPP of the present disclosure includes a DQA1*03:01 polypeptide comprising an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to amino acids 111 to 204 of the DQA1*03:01 amino acid sequence shown in FIG. 17.

[0153] In some cases, the TMAPP of the present disclosure comprises a DQB1*03:02 polypeptide comprising an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to amino acids 33 to 220 of the DQB1:03:02 amino acid sequence described below. In some cases, the TMAPP of the present disclosure comprises a DQB1*03:02 polypeptide comprising an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to amino acids 33 to 126 of the DQB1*03:02 amino acid sequence described below. In some cases, the TMAPP of the present disclosure comprises a DQB1*03:02 polypeptide comprising an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to amino acids 126 to 220 of the DQB1*03:02 amino acid sequence described below.

[0154] TIFF0007691927000084.tif38150

[0155] DRB1*0401 and DRA1*0101 In some cases, the TMAPP of the present disclosure is i) the following DRA1*0101 amino acid sequence: an MHC α-chain polypeptide comprising an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF0007691927000085.tif31151, and ii) the following DRB1*0401 amino acid sequence: An MHC β-chain polypeptide comprising an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF0007691927000086.tif11150. In some cases, the TMAPP of the present disclosure comprises: i) a DRA1*0101 α-chain polypeptide; and ii) a DRB1*0401 β-chain polypeptide.

[0156] DQA1*0501 and DQB1*0201 In some cases, the TMAPP of the present disclosure comprises: i) an MHC α-chain polypeptide comprising an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the DQA1*0501 α-chain polypeptide; and ii) an MHC β-chain polypeptide comprising an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the DQB1*0201 β-chain polypeptide. In some cases, the TMAPP of the present disclosure comprises: i) a DQA1*0501 α-chain polypeptide; and ii) a DQB1*0201 β-chain polypeptide.

[0157] DQA1*0301 and DQB1*0302 In some cases, the TMAPPs of the present disclosure include: i) an MHC α-chain polypeptide comprising an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the DQA1*0301 α-chain polypeptide; and ii) an MHC β-chain polypeptide comprising an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the DQB1*0302 β-chain polypeptide. In some cases, the TMAPPs of the present disclosure include: i) the DQA1*0301 α-chain polypeptide; and ii) the DQB1*0302 β-chain polypeptide.

[0158] In some cases, the TMAPPs of the present disclosure include MHC class II α and / or β-chain alleles associated with an increased risk of developing a disease (e.g., T1D and / or celiac disease). For example, an individual treated with a TMAPP expresses an MHC class II α-chain and / or β-chain allele.

[0159] Scaffold polypeptide The TMAPPs of the present disclosure can include an immunoglobulin or non-immunoglobulin scaffold, whether multimeric or monomeric. The TMAPP polypeptides of the present disclosure can include an Fc polypeptide or another suitable scaffold polypeptide, whether multimeric or monomeric.

[0160] Suitable scaffold polypeptides include antibody-based scaffold polypeptides and non-antibody-based scaffolds. Non-antibody-based scaffolds include, for example, albumin, XTEN (extended recombinant) polypeptides, transferrin, Fc receptor polypeptides, elastin-like polypeptides (see, e.g., Hassouneh et al. (2012) Methods Enzymol. 502:215; e.g., polypeptides containing pentapeptide repeat units of (Val-Pro-Gly-X-Gly; SEQ ID NO: 59) (wherein X is any amino acid other than proline)), albumin-binding polypeptides, silk-like polypeptides (see, e.g., Valluzzi et al. (2002) Philos Trans R Soc Lond B Biol Sci. 357:165), silk-elastin-like polypeptides (SELP; see, e.g., Megeed et al. (2002) Adv Drug Deliv Rev. 54:1075), and the like. Suitable XTEN polypeptides include, for example, those disclosed in WO2009 / 023270, WO2010 / 091122, WO2007 / 103515, US2010 / 0189682, and US2009 / 0092582. See also Schellenberger et al. (2009) Nat Biotechnol. 27:1186). Suitable albumin polypeptides include, for example, human serum albumin.

[0161] Suitable backbone polypeptides are, in some cases, half-life extended polypeptides. Thus, in some cases, suitable backbone polypeptides increase the in vivo half-life (e.g., serum half-life) of the multimeric polypeptide as compared to a control multimeric polypeptide that does not have a backbone polypeptide. For example, in some cases, the backbone polypeptide increases the in vivo half-life (e.g., serum half-life) of the multimeric 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 as compared to a control multimeric polypeptide that does not have a backbone polypeptide. As an example, in some cases, the Fc polypeptide increases the in vivo half-life (e.g., serum half-life) of the multimeric 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 as compared to a control multimeric polypeptide that does not have an Fc polypeptide.

[0162] Fc polypeptide In some cases, the first and / or second polypeptide chains of the TMAPP of the present disclosure include an Fc polypeptide. The Fc polypeptide of the TMAPP of the present disclosure can be human IgG1 Fc, human IgG2 Fc, human IgG3 Fc, human IgG4 Fc, etc. In some cases, the Fc polypeptide has 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 amino acid sequence of the Fc region shown in FIGS. 21A-21G. In some cases, the Fc region includes an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to the human IgG1 Fc polypeptide shown in FIG. 21A. In some cases, the Fc region includes an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to the human IgG1 Fc polypeptide shown in FIG. 21A and includes a substitution at N77. For example, the Fc polypeptide includes a substitution at N77A. In some cases, the Fc polypeptide has 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 shown in FIG. 21A. For example, the Fc polypeptide includes 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 shown in FIG. 21A.In some cases, 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 shown in FIG. 21A. For example, 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 shown in FIG. 21A. In some cases, 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 shown in FIG. 21B. For example, 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 of the human IgM Fc polypeptide shown in FIG. 21B. In some cases, 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 shown in FIG. 21C. For example, 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 of the human IgA Fc polypeptide shown in FIG. 21C.

[0163] In some cases, 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 shown in Figure 21C. In some cases, 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 shown in Figure 21C.

[0164] In some cases, the IgG4 Fc polypeptide has the following amino acid sequence: TIFF0007691927000087.tif24151.

[0165] In some cases, the Fc polypeptide present in TMAPP comprises the amino acid sequence shown in Figure 21A (human IgG1 Fc). In some cases, the Fc polypeptide present in TMAPP comprises the amino acid sequence shown in Figure 21A (human IgG1 Fc), except for substitution of the asparagine at N297 (N77 of the amino acid sequence shown in Figure 21A) with an amino acid other than asparagine. In some cases, the Fc polypeptide present in TMAPP comprises the amino acid sequence shown in Figure 21C (human IgG1 Fc with substitution of N297A where N77 is in the amino acid sequence shown in Figure 21A). In some cases, the Fc polypeptide present in TMAPP comprises the amino acid sequence shown in Figure 21A (human IgG1 Fc), except for substitution of the leucine at L234 (L14 of the amino acid sequence shown in Figure 21A) with an amino acid other than leucine. In some cases, the Fc polypeptide present in TMAPP comprises the amino acid sequence shown in Figure 21A (human IgG1 Fc), except for substitution of the leucine at L235 (L15 of the amino acid sequence shown in Figure 21A) with an amino acid other than leucine.

[0166] In some cases, the Fc polypeptide present in TMAPP contains the amino acid sequence shown in FIG. 21E. In some cases, the Fc polypeptide present in TMAPP contains the amino acid sequence shown in FIG. 21F. In some cases, the Fc polypeptide present in TMAPP contains the amino acid sequence shown in FIG. 21G (human IgG1 Fc containing substitutions of L234A and L235A corresponding to positions 14 and 15 in the amino acid sequence shown in FIG. 21G). In some cases, the Fc polypeptide present in TMAPP contains the amino acid sequence shown in FIG. 21A (human IgG1 Fc) except for substitution of the proline of P331 (P111 in the amino acid sequence shown in FIG. 21A) with an amino acid other than proline, and in some cases, the substitution is P331S. In some cases, the Fc polypeptide present in TMAPP contains the amino acid sequence shown in FIG. 21A (human IgG1 Fc) except for substitution of leucine of L234 and L235 (L14 and L15 in the amino acid sequence shown in FIG. 21A) with an amino acid other than leucine. In some cases, the Fc polypeptide present in TMAPP contains the amino acid sequence shown in FIG. 21A (human IgG1 Fc) except for substitution of leucine of L234 and L235 (L14 and L15 in the amino acid sequence shown in FIG. 21A) with an amino acid other than leucine, and substitution of the proline of P331 (P111 in the amino acid sequence shown in FIG. 21A) with an amino acid other than proline. In some cases, the Fc polypeptide present in TMAPP contains the amino acid sequence shown in FIG. 21E (human IgG1 Fc containing substitutions of L234F, L235E, and P331S corresponding to amino acid positions 14, 15, and 111 in the amino acid sequence shown in FIG. 21E). In some cases, the Fc polypeptide present in TMAPP is an IgG1 Fc polypeptide containing substitutions of L234A and L235A (substitution of Ala for L14 and L15 in the amino acid sequence shown in FIG. 21A) as shown in FIG. 21G.

[0167] Linker As described above, the TMAPP of the present disclosure may include a linker peptide inserted, for example, between an epitope and an MHC polypeptide, between an MHC polypeptide and an Ig Fc polypeptide, between a first MHC polypeptide and a second MHC polypeptide, and the like.

[0168] Suitable linkers (also referred to as "spacers") can be easily selected and can be any of a number of suitable lengths, including 1 to 25 amino acids, 3 to 20 amino acids, 2 to 15 amino acids, 3 to 12 amino acids, etc., as well as 4 to 10 amino acids, 5 to 9 amino acids, 6 to 8 amino acids, or 7 to 8 amino acids. Suitable linkers 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. Suitable linkers can be 25 to 35 amino acids in length. Suitable linkers can be 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 amino acids in length. Suitable linkers can be 35 to 45 amino acids in length. Suitable linkers can be 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 amino acids in length. Suitable linkers can be 45 to 50 amino acids in length. Suitable linkers can be 45, 46, 47, 48, 49, or 50 amino acids in length.

[0169] Exemplary linkers include glycine polymers (G) n , glycine-serine polymers (e.g., (GS) n , (GSGGS) n (SEQ ID NO: 61) and (GGGS) n(SEQ ID NO: 62) (where n is at least one integer), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers can be used, and since both Gly and Ser are relatively unstructured, they can function as intermediate tethers between components. Glycine polymers can be used because glycine can utilize much more Φ-Ψ space than alanine and is less restricted than residues with longer side chains (see Scheraga, Rev. Computational Chem. 11173-142 (1992)). Exemplary linkers can include amino acid sequences including, but not limited to, GGSG (SEQ ID NO: 63), GGSGG (SEQ ID NO: 64), GSGSG (SEQ ID NO: 65), GSGGG (SEQ ID NO: 66), GGGSG (SEQ ID NO: 67), GSSSG (SEQ ID NO: 68), etc. Exemplary linkers can be, for example, Gly(Ser 4)n, (SEQ ID NO: 69) (where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) may be included. In some cases, the linker includes the amino acid sequence (GSSSS)n (SEQ ID NO: 69) (where n is 4). In some cases, the linker includes the amino acid sequence (GSSSS)n (SEQ ID NO: 69) (where n is 5). Exemplary linkers may include, for example, (GlyGlyGlyGlySer)n (SEQ ID NO: 1) (where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). In some cases, the linker includes the amino acid sequence (GGGGS)n (SEQ ID NO: 1) (where n is 1). In some cases, the linker includes the amino acid sequence (GGGGS)n (SEQ ID NO: 1) (where n is 2). In some cases, the linker includes the amino acid sequence (GGGGS)n (SEQ ID NO: 1) (where n is 3). In some cases, the linker includes the amino acid sequence (GGGGS)n (SEQ ID NO: 1) (where n is 4). In some cases, the linker includes the amino acid sequence (GGGGS)n (SEQ ID NO: 1) (where n is 5). In some cases, the linker includes the amino acid sequence (GGGGS)n (SEQ ID NO: 1) (where n is 6). In some cases, the linker includes the amino acid sequence (GGGGS)n (SEQ ID NO: 1) (where n is 7). In some cases, the linker includes the amino acid sequence (GGGGS)n (SEQ ID NO: 1) (where n is 8). In some cases, the linker includes the amino acid sequence (GGGGS)n (SEQ ID NO: 1) (where n is 9). In some cases, the linker includes the amino acid sequence (GGGGS)n (SEQ ID NO: 1) (where n is 10). In some cases, the linker includes the amino acid sequence AAAGG (SEQ ID NO: 70).

[0170] In some cases, the linker polypeptide present in the TMAPP of the present disclosure comprises a cysteine residue capable of forming a disulfide bond with a cysteine residue present in the second polypeptide of the TMAPP. In some cases, for example, a suitable linker has the amino acid sequence TIFF0007691927000088.tif4128.

[0171] Epitope-presenting peptide The peptide epitopes (also referred to herein as "peptide antigens" or "epitope-presenting peptides" or "epitopes") present in the TMAPP of the present disclosure represent epitopes for TCRs on the surface of T cells. The epitope-presenting peptide can have a length of about 4 amino acids to about 25 amino acids. For example, the epitope can have a length of 4 amino acids (aa) to 10 aa, 10 aa to 15 aa, 15 aa to 20 aa, or 20 aa to 25 aa. For example, the epitopes present in the TMAPP 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. In some cases, the epitope-presenting peptides present in the TMAPP of the present disclosure have a length of 5 amino acids to 10 amino acids, for example, 5 aa, 6 aa, 7 aa, 8 aa, 9 aa, or 10 aa.

[0172] The epitope-presenting peptides present in the TMAPP of the present disclosure are specifically bound by T cells, i.e., the epitopes are specifically bound by epitope-specific T cells. Epitope-specific T cells bind to an epitope-presenting peptide having a reference amino acid sequence but do not substantially bind to an epitope different from the reference amino acid sequence. For example, an epitope-specific T cell binds to an epitope-presenting peptide having a reference amino acid sequence and binds to an epitope different from the reference amino acid sequence, if at all, by less than 10 -6 M, less than 10 -5 M, or less than 10 -4binds with an affinity of less than M. Epitope-specific T cells are at least 10 -7 M, at least 10 -8 M, at least 10 -9 M, or at least 10 -10 M and can bind to a specific epitope-presenting peptide.

[0173] Suitable epitope-presenting peptides include, but are not limited to, epitope-presenting peptides associated with or present in "self" antigens (autoantigens).

[0174] Antigens associated with type 1 diabetes (T1D) include, for example, preproinsulin, proinsulin, insulin, insulin B chain, insulin A chain, the 65 kDa isoform of glutamate decarboxylase (GAD65), the 67 kDa isoform of glutamate decarboxylase (GAD67), tyrosine phosphatase (IA-2), heat shock protein HSP65, islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP), islet antigen 2 (IA2), and zinc transporter (ZnT8). See, for example, Mallone et al. (2011) Clin. Dev. Immunol. 2011:513210, and U.S. Patent Application Publication No. 2017 / 0045529. An antigen “associated with” a particular autoimmune disorder is an antigen that is the target of autoantibodies and / or autoreactive T cells present in an individual having that autoimmune disorder. In this case, such autoantibodies and / or autoreactive T cells mediate the pathological conditions associated with the autoimmune disorder. Suitable epitope-presenting peptides for inclusion in the antigen-presenting polypeptides of the present disclosure can be epitope-presenting peptides that are from about 4 amino acids in length to about 25 amino acids in length of any one of the above-described T1D-associated antigens. As one non-limiting example, the epitope-presenting peptide is proinsulin 73-90 (GAGSLQPLALEGSLQKR; SEQ ID NO: 72). As another non-limiting example, the epitope-presenting peptide is the following insulin (InsA(1-15) peptide: GIVDQCCTSICSLYQ (SEQ ID NO: 73). As another non-limiting example, the epitope-presenting peptide is the following insulin (InsA(1-15;D4E) peptide: GIVEQCCTSICSLYQ (SEQ ID NO: 74). As another non-limiting example, the epitope-presenting peptide is the following GAD65(555-567) peptide; NFFRMVISNPAAT (SEQ ID NO: 75). As another non-limiting example, the epitope-presenting peptide is the following GAD65(555-567;F557I) peptide; NFIRMVISNPAAT (SEQ ID NO: 76). As another non-limiting example, the epitope-presenting peptide is the following islet antigen 2 (IA2) peptide: SFYLKNVQTQETRTLTQFHF (SEQ ID NO: 77).As another non-limiting example, the epitope-presenting peptide is the following proinsulin peptide: SLQPLALEGSLQSRG (SEQ ID NO: 78).

[0175] In some cases, the epitope-presenting peptide is the following human preproinsulin amino acid sequence (where amino acids 1-24 (underlined) are the signal peptide): Contains 4 to 25 consecutive amino acids of an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to amino acids 25-110 of TIFF0007691927000089.tif17136, where the epitope-presenting peptide has a length of 4 amino acids (aa), 5aa, 6aa, 7, aa, 8aa, 9aa, 10aa, 11aa, 12aa, 13aa, 14aa, 15aa, 16aa, 17aa, 18aa, 19aa, 20aa, 21aa, 22aa, 23aa, 24aa, or 25aa. In some cases, the epitope-presenting peptide has the amino acid sequence: GAGSLQPLALEGSLQKRG (SEQ ID NO: 434). In some cases, the epitope-presenting peptide has the amino acid sequence: SLQPLALEGSLQKRG (SEQ ID NO: 435). In some cases, the epitope-presenting peptide has the amino acid sequence: SLQPLALEGSLQSRG (SEQ ID NO: 78). In some cases, the epitope-presenting peptide has the amino acid sequence: QPLALEGSLQKRG (SEQ ID NO: 436). In some cases, the epitope-presenting peptide has the amino acid sequence: QPLALEGSLQSRG (SEQ ID NO: 437).

[0176] Antigens associated with celiac disease include, for example, tissue transglutaminase and gliadin. Suitable epitope-presenting peptides for inclusion in the TMAPP of the present disclosure can be epitope-presenting peptides that are 4 amino acids to about 25 amino acids in length of any one of the above-described celiac-related antigens. Other antigens associated with celiac disease include, for example, secalin, hordein, avenin, and glutenin. Examples of secalin include rye secalin. Examples of hordein include barley hordein. Examples of glutenin include wheat glutenin. See, for example, U.S. 2016 / 0279233.

[0177] For example, suitable celiac-related peptides, in some cases, are the following gamma-gliadin amino acid sequences: TIFF0007691927000090.tif44133 having 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 a polypeptide comprising an amino acid sequence of about 4 to about 25 contiguous amino acids of the peptide. In some cases, the epitope is the Glia-α9 epitope. Glia-α9 is the major (immunodominant) epitope recognized by most patients with celiac disease (CD). The Glia-α9 epitope includes, for example, QPFPQPQ (SEQ ID NO: 80), and is selectively deamidated by transglutaminase 2 to be presented by HLA-DQ2 as the amino acid sequence PFPQPELPY (SEQ ID NO: 82), and PFPQPQLPY (SEQ ID NO: 81) induces a strong T cell response.

[0178] In some cases, the epitope-presenting peptide includes a sequence selected from QLQPFPQPELPY (SEQ ID NO: 83; gliadin alpha 1a peptide) or a modified equivalent thereof LQPFPQPELPY (SEQ ID NO: 84), PQPELPYPQPE (SEQ ID NO: 85; gliadin alpha 2 peptide), and QPFPQPEQPFPW (SEQ ID NO: 86; gliadin omega peptide).

[0179] In some cases, the gliadin epitope presenting peptide is modified for increased expression and contains a sequence selected from TIFF0007691927000091.tif44148.

[0180] In some cases, the gliadin epitope presenting peptide is modified for increased expression and contains cysteine for immobilizing the peptide in the binding groove. In one embodiment, the peptide contains the alpha1a gliadin peptide sequence QLQPFPQPCLPY (SEQ ID NO: 101), and in another embodiment, the alpha2 gliadin peptide sequence PQPELCYPQPE (SEQ ID NO: 102).

[0181] Immunomodulatory polypeptide (「MOD」) Immunomodulatory polypeptides suitable for inclusion in the TMAPPs of the present disclosure include, but are not limited to, IL-2, CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L, Fas ligand (FasL), inducible costimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM), CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, HVEM, lymphotoxin beta receptor, 3 / TR6, ILT3, ILT4, and HVEM.

[0182] In some cases, the immunomodulatory polypeptide is selected from a 4-1BBL polypeptide, a B7-1 polypeptide, a B7-2 polypeptide, an ICOS-L polypeptide, an OX-40L polypeptide, a CD80 polypeptide, a CD86 polypeptide, a PD-L1 polypeptide, a FasL polypeptide, a TGFβ polypeptide, and a PD-L2 polypeptide. The immunomodulatory polypeptide may contain only the extracellular portion of the full-length immunomodulatory polypeptide. Thus, for example, the immunomodulatory polypeptide can, in some cases, exclude one or more of the signal peptide, transmembrane domain, and intracellular domain that are normally found in native immunomodulatory polypeptides.

[0183] In some cases, an immunomodulatory polypeptide suitable for inclusion in the TMAPP of the present disclosure comprises all or part (e.g., the extracellular portion) of the amino acid sequence of a native immunomodulatory polypeptide. In other cases, an immunomodulatory polypeptide suitable for inclusion in the TMAPP of the present disclosure is a variant immunomodulatory polypeptide that comprises at least one amino acid substitution as compared to the amino acid sequence of a native immunomodulatory polypeptide. In some cases, the variant immunomodulatory polypeptide exhibits a lower binding affinity for an immunocoregulatory polypeptide than the affinity of the corresponding native immunomodulatory polypeptide (e.g., an immunomodulatory polypeptide that does not include the amino acid substitution(s) present in the variant) for the immunocoregulatory polypeptide.

[0184] Low-affinity variant immunomodulatory polypeptide Suitable immune regulatory domains that exhibit a reduced affinity for the immune co-regulatory domain can have a difference of 1 amino acid (aa) to 20 aa from the wild-type immune regulatory domain. For example, in some cases, the variant immune regulatory polypeptides present in the TMAPP of the present disclosure have an amino acid sequence that differs from the corresponding wild-type immune regulatory polypeptide by 1 aa, 2 aa, 3 aa, 4 aa, 5 aa, 6 aa, 7 aa, 8 aa, 9 aa, or 10 aa. As another example, in some cases, the variant immune regulatory polypeptides present in the TMAPP of the present disclosure have an amino acid sequence that differs from the corresponding wild-type immune regulatory polypeptide by 11 aa, 12 aa, 13 aa, 14 aa, 15 aa, 16 aa, 17 aa, 18 aa, 19 aa, or 20 aa. As an example, in some cases, the variant immune regulatory polypeptides present in the TMAPP of the present disclosure contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions compared to the corresponding reference (e.g., wild-type) immune regulatory polypeptide. In some cases, the variant immune regulatory polypeptides present in the TMAPP of the present disclosure contain a single amino acid substitution compared to the corresponding reference (e.g., wild-type) immune regulatory polypeptide. In some cases, the variant immune regulatory polypeptides present in the TMAPP of the present disclosure contain two amino acid substitutions (e.g., amino acid substitutions of 2 or less) compared to the corresponding reference (e.g., wild-type) immune regulatory polypeptide. In some cases, the variant immune regulatory polypeptides present in the TMAPP of the present disclosure contain three amino acid substitutions (e.g., amino acid substitutions of 3 or less) compared to the corresponding reference (e.g., wild-type) immune regulatory polypeptide. In some cases, the variant immune regulatory polypeptides present in the TMAPP of the present disclosure contain four amino acid substitutions (e.g., amino acid substitutions of 4 or less) compared to the corresponding reference (e.g., wild-type) immune regulatory polypeptide. In some cases, the variant immune regulatory polypeptides present in the TMAPP of the present disclosure contain five amino acid substitutions (e.g., amino acid substitutions of 5 or less) compared to the corresponding reference (e.g., wild-type) immune regulatory polypeptide.In some cases, the variant immunomodulatory polypeptide present in the TMAPP of the present disclosure comprises six amino acid substitutions (e.g., amino acid substitutions of six or less) compared to the corresponding reference (e.g., wild-type) immunomodulatory polypeptide. In some cases, the variant immunomodulatory polypeptide present in the TMAPP of the present disclosure comprises seven amino acid substitutions (e.g., amino acid substitutions of seven or less) compared to the corresponding reference (e.g., wild-type) immunomodulatory polypeptide. In some cases, the variant immunomodulatory polypeptide present in the TMAPP of the present disclosure comprises eight amino acid substitutions (e.g., amino acid substitutions of eight or less) compared to the corresponding reference (e.g., wild-type) immunomodulatory polypeptide. In some cases, the variant immunomodulatory polypeptide present in the TMAPP of the present disclosure comprises nine amino acid substitutions (e.g., amino acid substitutions of nine or less) compared to the corresponding reference (e.g., wild-type) immunomodulatory polypeptide. In some cases, the variant immunomodulatory polypeptide present in the TMAPP of the present disclosure comprises ten amino acid substitutions (e.g., amino acid substitutions of ten or less) compared to the corresponding reference (e.g., wild-type) immunomodulatory polypeptide.

[0185] In some cases, the variant immunomodulatory polypeptides present in the TMAPPs of the present disclosure contain 11 amino acid substitutions (e.g., amino acid substitutions of 11 or less) compared to the corresponding reference (e.g., wild-type) immunomodulatory polypeptide. In some cases, the variant immunomodulatory polypeptides present in the TMAPPs of the present disclosure contain 12 amino acid substitutions (e.g., amino acid substitutions of 12 or less) compared to the corresponding reference (e.g., wild-type) immunomodulatory polypeptide. In some cases, the variant immunomodulatory polypeptides present in the TMAPPs of the present disclosure contain 13 amino acid substitutions (e.g., amino acid substitutions of 13 or less) compared to the corresponding reference (e.g., wild-type) immunomodulatory polypeptide. In some cases, the variant immunomodulatory polypeptides present in the TMAPPs of the present disclosure contain 14 amino acid substitutions (e.g., amino acid substitutions of 14 or less) compared to the corresponding reference (e.g., wild-type) immunomodulatory polypeptide. In some cases, the variant immunomodulatory polypeptides present in the TMAPPs of the present disclosure contain 15 amino acid substitutions (e.g., amino acid substitutions of 15 or less) compared to the corresponding reference (e.g., wild-type) immunomodulatory polypeptide. In some cases, the variant immunomodulatory polypeptides present in the TMAPPs of the present disclosure contain 16 amino acid substitutions (e.g., amino acid substitutions of 16 or less) compared to the corresponding reference (e.g., wild-type) immunomodulatory polypeptide. In some cases, the variant immunomodulatory polypeptides present in the TMAPPs of the present disclosure contain 17 amino acid substitutions (e.g., amino acid substitutions of 17 or less) compared to the corresponding reference (e.g., wild-type) immunomodulatory polypeptide. In some cases, the variant immunomodulatory polypeptides present in the TMAPPs of the present disclosure contain 18 amino acid substitutions (e.g., amino acid substitutions of 18 or less) compared to the corresponding reference (e.g., wild-type) immunomodulatory polypeptide. In some cases, the variant immunomodulatory polypeptides present in the TMAPPs of the present disclosure contain 19 amino acid substitutions (e.g., amino acid substitutions of 19 or less) compared to the corresponding reference (e.g., wild-type) immunomodulatory polypeptide.In some cases, the variant immunomodulatory polypeptides present in the TMAPPs of the present disclosure contain 20 amino acid substitutions (e.g., 20 or fewer amino acid substitutions) compared to the corresponding reference (e.g., wild-type) immunomodulatory polypeptides.

[0186] As described above, variant immunomodulatory polypeptides suitable for inclusion in the TMAPPs of the present disclosure exhibit a decrease in affinity compared to the affinity of the corresponding wild-type immunomodulatory polypeptide for the homologous immunocoregulatory polypeptide of the same species.

[0187] Exemplary pairs of immunomodulatory polypeptides and homologous immunocoregulatory polypeptides include, but are not limited to: a) 4-1BBL (immunomodulatory polypeptide) and 4-1BB (homologous immunocoregulatory polypeptide), b) PD-L1 (immunomodulatory polypeptide) and PD1 (homologous immunocoregulatory polypeptide), c) IL-2 (immunomodulatory polypeptide) and IL-2 receptor (homologous immunocoregulatory polypeptide), d) CD80 (immunomodulatory polypeptide) and CD28 (homologous immunocoregulatory polypeptide), e) CD86 (immunomodulatory polypeptide) and CD28 (homologous immunocoregulatory polypeptide), f) OX40L (CD252) (immunomodulatory polypeptide) and OX40 (CD134) (homologous immunocoregulatory polypeptide), g) Fas ligand (immunomodulatory polypeptide) and Fas (homologous immunocoregulatory polypeptide), h) ICOS-L (immunomodulatory polypeptide) and ICOS (homologous immunocoregulatory polypeptide), i) ICAM (immunomodulatory polypeptide) and LFA-1 (homologous immunocoregulatory polypeptide), j) CD30L (immunomodulatory polypeptide) and CD30 (homologous immunocoregulatory polypeptide), k) CD40 (immunomodulatory polypeptide) and CD40L (homologous immunocoregulatory polypeptide), l) CD83 (immunomodulatory polypeptide) and CD83L (homologous immunocoregulatory polypeptide), m) HVEM (CD270) (immunomodulatory polypeptide) and CD160 (homologous immunocoregulatory polypeptide), n) JAG1 (CD339) (immunomodulatory polypeptide) and Notch (homologous immunocoregulatory polypeptide), o) JAG1 (immunomodulatory polypeptide) and CD46 (homologous immunocoregulatory polypeptide), p) CD80 (immunomodulatory polypeptide) and CTLA4 (homologous immunocoregulatory polypeptide), q) CD86 (immunomodulatory polypeptide) and CTLA4 (homologous immunocoregulatory polypeptide), and r) CD70 (immunomodulatory polypeptide) and CD27 (homologous immunocoregulatory polypeptide).

[0188] In some cases, the variant immunomodulatory polypeptides present in the TMAPPs of the present disclosure have a binding affinity of 100 nM to 100 μM for the homologous immunocoregulatory polypeptides. For example, in some cases, the variant immunomodulatory polypeptides present in the TMAPPs of the present disclosure have a binding affinity of about 100 nM to 150 nM, about 150 nM to about 200 nM, about 200 nM to about 250 nM, about 250 nM to about 300 nM, about 300 nM to about 350 nM, about 350 nM to about 400 nM, about 400 nM to about 500 nM, about 500 nM to about 600 nM, about 600 nM to about 700 nM, about 700 nM to about 800 nM, about 800 nM to about 900 nM, about 900 nM to about 1 μM, about 1 μM to about 5 μM, about 5 μM to about 10 μM, about 10 μM to about 15 μM, about 15 μM to about 20 μM, about 20 μM to about 25 μM, about 25 μM to about 50 μM, about 50 μM to about 75 μM, or about 75 μM to about 100 μM for the homologous immunocoregulatory polypeptides.

[0189] Determination of Binding Affinity The binding affinity between an immunomodulatory polypeptide and its cognate immunocoregulatory polypeptide can be determined by biolayer interferometry (BLI) using the purified immunomodulatory polypeptide and the purified cognate immunocoregulatory polypeptide. The binding affinity between TMAPP and its cognate immunocoregulatory polypeptide can also be determined by BLI using the purified TMAPP and the cognate immunocoregulatory polypeptide. The BLI method is well known to those skilled in the art. See, for example, Lad et al. (2015) J. Biomol. Screen. 20(4):498-507; and Shah and Duncan (2014) J. Vis. Exp. 18:e51383. The specific and relative binding affinities between the immunomodulatory polypeptide and its cognate immunocoregulatory polypeptide, or between synTac and its cognate immunocoregulatory polypeptide, as described in the present disclosure, can be determined using the following procedure.

[0190] To determine the binding affinity between TMAPP and its homologous immunomodulatory polypeptides, a BLI assay can be performed as follows using an Octet RED 96 (Pal ForteBio) instrument or a similar instrument. Fix TMAPP (e.g., the TMAPP of the present disclosure; control TMAPP (control TMAPP contains a wild-type immunomodulatory polypeptide)) onto an insoluble support (“biosensor”). The immobilized TMAPP is the “target”. The immobilization can be carried out by immobilizing a capture antibody onto the insoluble support, and the capture antibody immobilizes the TMAPP. For example, the immobilization can be carried out by immobilizing an anti-Fc (e.g., anti-human IgG Fc) antibody onto the insoluble support, and the immobilized anti-Fc antibody binds to and immobilizes the TMAPP (TMAPP contains an Ig Fc polypeptide). Apply the immunomodulatory polypeptide to the immobilized TMAPP at several different concentrations and record the response of the instrument. The assay is performed in a liquid medium containing 25 mM HEPES (pH 6.8), 5% poly(ethylene glycol) 6000, 50 mM KCl, 0.1% bovine serum albumin, and 0.02% Tween 20 nonionic surfactant. The binding of the immunomodulatory polypeptide to the immobilized TMAPP is carried out at 30°C. As a positive control for binding affinity, an anti-MHC class II monoclonal antibody can be used. For example, an anti-HLA-DR3 monoclonal antibody such as the 16-23 antibody (Sigma; also referred to as “16.23”; see, for example, Pious et al. (1985) J. Exp. Med. 162:1193; Mellins et al. (1991) J. Exp. Med. 174:1607; ECACC Hybridoma Collection 16-23, ECACC 99043001) can be used as a positive control for binding affinity. As another example, 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 is created using serial dilutions of the anti-MHC class II monoclonal antibody.The immunomodulatory polypeptide or anti-MHC class II mAb is the "analyte". BLI analyzes the interference wave of white light reflected from two surfaces of i) the immobilized polypeptide ("target") and ii) the internal reference layer. A change in the number of molecules ("analyte"; e.g., immunomodulatory polypeptide, anti-HLA antibody) binding to the biosensor chip causes a shift in the interference wave, and this shift in the interference wave can be measured in real time. Two kinetic terms that describe the affinity of the target / analyte interaction are the association constant (k. a ) and the dissociation constant (k d ). The ratio of these two terms (k d / a ) gives the affinity constant K D .

[0191] As described above, the determination of the binding affinity between an immune modulating polypeptide (e.g., IL-2 or IL-2 variant) and its cognate immune co-modulating polypeptide (e.g., IL-2R) can also be determined by BLI. The assay is similar to that described above for TMAPP. Using an Octet RED 96 (Pal ForteBio) instrument or a similar instrument, the BLI assay can be performed as follows: An immune modulating polypeptide (e.g., a variant IL-2 polypeptide of the present disclosure) that is a component of TMAPP of the present disclosure and a control immune modulating polypeptide (wherein the control immune modulating polypeptide includes a wild-type immune modulating polypeptide, e.g., wild-type IL-2) are immobilized on an insoluble support ("biosensor"). The immune modulating polypeptide is the "target". The immobilization can be performed by immobilizing a capture antibody on the insoluble support, which immobilizes the immune modulating polypeptide. For example, if the target is fused to an immunoaffinity tag (e.g., FLAG, human IgG Fc), immobilization can be performed by immobilizing an appropriate antibody against the immunoaffinity tag (e.g., anti-human IgG Fc) on an insoluble support, and the immobilized antibody binds to and immobilizes the immune modulating polypeptide (immunomodulating polypeptides include Ig Fc polypeptides). An immune co-modulating polypeptide (or multiple polypeptides) is applied to the immobilized immune modulating polypeptide at several different concentrations, and the response of the instrument is recorded. Alternatively, an immune co-modulating polypeptide (or multiple polypeptides) is immobilized on a biosensor (e.g., for an IL-2 receptor heterotrimer, e.g., a monomeric subunit, a heterodimeric subcomplex, or a complete heterotrimer), and an immune modulating polypeptide is applied to the immobilized immune co-modulating polypeptide(s) at several different concentrations, and the response of the instrument is recorded. The assay is carried out in a liquid medium containing 25 mM HEPES (pH 6.8), 5% poly(ethylene glycol) 6000, 50 mM KCl, 0.1% bovine serum albumin, and 0.02% non-ionic surfactant Tween 20. Binding of immune co-regulatory polypeptides to immobilized immune modulatory polypeptides is carried out at 30° C.BLI analyzes the interference wave of white light reflected from two surfaces of i) an immobilized polypeptide ("target") and ii) an internal reference layer. A change in the number of molecules ("analyte"; e.g., immunomodulatory polypeptide) that bind to the biosensor chip causes a shift in the interference wave, and this shift in the interference wave can be measured in real time. Two kinetic terms that describe the affinity of the target / analyte interaction are the association constant (k a ), and the dissociation constant (k d ). The ratio of these two terms (k d / a ) gives the affinity constant K D . Thus, by determining both the binding affinity of a wild-type immunomodulatory polypeptide (e.g., IL-2) for its receptor (e.g., IL-2R) and the binding affinity of a variant immunomodulatory polypeptide (e.g., an IL-2 variant disclosed herein) for its cognate immunomodulatory polypeptide (e.g., its receptor) (e.g., IL-2R), it becomes possible to determine the relative binding affinity of the variant immunomodulatory polypeptide for the cognate immunomodulatory polypeptide compared to the wild-type immunomodulatory polypeptide. That is, it can be determined whether the binding affinity of the variant immunomodulatory polypeptide for its receptor (its cognate immunomodulatory polypeptide) is decreased compared to the binding affinity of the wild-type immunomodulatory polypeptide for the same cognate immunomodulatory polypeptide, and if so, by what percentage the binding affinity is less than that of the wild-type immunomodulatory polypeptide.

[0192] The BLI assay is performed in a multi-well plate. To perform the assay, determine the plate layout, determine the assay steps, and assign the biosensor to the Octet Data Acquisition software. Hydrate the biosensor assembly. Equilibrate the hydrated biosensor assembly and the assay plate on the Octet instrument for 10 minutes. Once data is acquired, load the acquired data into the Octet Data Analysis software. Perform data processing by specifying methods for subtraction of reference, alignment of the y-axis, correction between steps, and the Savitzky-Golay filter in the processing window. Perform data analysis by specifying the steps to analyze (association and dissociation), the curve fitting model (1:1), the fitting method (global), and the window of interest (in seconds) in the analysis window. Evaluate the quality of the fit. The K D values of the trace (analyte concentration) of each data can be averaged if they are within a three-fold range. K D error values must be within 10-fold of the affinity constant value, and the R 2 values should be greater than 0.95. See, for example, Abdiche et al. (2008) J. Anal. Biochem. 377:209.

[0193] In some cases, the ratio of i) the binding affinity of a control TMAPP (where the control TMAPP comprises a wild-type immunomodulatory polypeptide) for a cognate immunocoregulatory polypeptide to ii) the binding affinity of a TMAPP of the present disclosure comprising a variant of the wild-type immunomodulatory polypeptide for the cognate immunocoregulatory 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 102:1, at least 5×10 2 :1, at least 10 3 :1, at least 5×10 3:1. At least 10 4 :1. At least 10 5 :1. Or at least 10 6 :1. In some cases, i) the binding affinity of a control TMAPP (where the control TMAPP comprises a wild - type immunomodulatory polypeptide) for a homologous immunocoregulatory polypeptide and ii) the binding affinity of the TMAPP of the present disclosure comprising a variant of the wild - type immunomodulatory polypeptide for the homologous immunocoregulatory polypeptide, when measured by BLI, is from 1.5:1 to 10 6 :1. For example, 1.5:1 to 10:1, 10:1 to 50:1, 50:1 to 10 2 :1. 10 2 :1 to 10 3 :1. 10 3 :1 to 10 4 :1. 10 4 :1 to 10 5 :1. Or 10 5 :1 to 10 6 :1. Within the range of 1.

[0194] The epitopes present in the TMAPP of the present disclosure bind to the T - cell receptor (TCR) on T - cells with an affinity of at least 100 μM (e.g., at least 10 μM, at least 1 μM, at least 100 nM, at least 10 nM, or at least 1 nM). In some cases, the epitopes present in the TMAPP of the present disclosure are from about 10 -4 M to about 5 × 10 -4 M, about 5 × 10 -4 M to about 10 -5 M, about 10 -5 M to 5 × 10 -5 M, about 5 × 10 -5 M to 10 -6 M, about 10 -6 M to about 5 × 10 -6 M, about 5 × 10 -6 M to about 10 -7 M, about 10 -7 M to about 5 × 10 -7 M, about 5 × 10 -7 M to about 10 -8 M, or about 10 -8 M to about 10 -9It binds to the TCR on T cells with the affinity of M. In other words, in some cases, the epitopes present in the TMAPP of the present disclosure bind to the TCR on T cells with an affinity of about 1 nM to about 5 nM, about 5 nM to about 10 nM, about 10 nM to about 50 nM, about 50 nM to about 100 nM, about 0.1 μM to about 0.5 μM, about 0.5 μM to about 1 μM, about 1 μM to about 5 μM, about 5 μM to about 10 μM, about 10 μM to about 25 μM, about 25 μM to about 50 μM, about 50 μM to about 75 μM, about 75 μM to about 100 μM.

[0195] In some cases, the variant immunomodulatory polypeptides present in the TMAPP of the present disclosure have a binding affinity of 1 nM to 100 nM, or 100 nM to 100 μM for the homologous immunocoregulatory polypeptides. For example, in some cases, the variant immunomodulatory polypeptides present in the TMAPP of the present disclosure have a binding affinity of about 100 nM to 150 nM, about 150 nM to about 200 nM, about 200 nM to about 250 nM, about 250 nM to about 300 nM, about 300 nM to about 350 nM, about 350 nM to about 400 nM, about 400 nM to about 500 nM, about 500 nM to about 600 nM, about 600 nM to about 700 nM, about 700 nM to about 800 nM, about 800 nM to about 900 nM, about 900 nM to about 1 μM, about 1 μM to about 5 μM, about 5 μM to about 10 μM, about 10 μM to about 15 μM, about 15 μM to about 20 μM, about 20 μM to about 25 μM, about 25 μM to about 50 μM, about 50 μM to about 75 μM, or about 75 μM to about 100 μM for the homologous immunocoregulatory polypeptides. In some cases, the variant immunomodulatory polypeptides present in the TMAPP of the present disclosure have a binding affinity of about 1 nM to about 5 nM, about 5 nM to about 10 nM, about 10 nM to about 50 nM, about 50 nM to about 100 nM for the homologous immunocoregulatory polypeptides.

[0196] PD-L1 variant As one non-limiting example, in some cases, the variant immunomodulatory polypeptides present in the TMAPP of the present disclosure are variant PD-L1 polypeptides. Wild-type PD-L1 binds to PD1.

[0197] The wild-type human PD-L1 polypeptide has the following amino acid sequence: It may include TIFF0007691927000092.tif31140.

[0198] The wild-type human PD-L1 ectodomain has the following amino acid sequence: It may include TIFF0007691927000093.tif31132.

[0199] The wild-type PD-1 polypeptide has the following amino acid sequence: It may include TIFF0007691927000094.tif37133.

[0200] In some cases, the variant PD-L1 polypeptide exhibits a reduced binding affinity compared to PD-1 (e.g., a PD-1 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 105), a PD-L1 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 103 or SEQ ID NO: 104. For example, in some cases, the variant PD-L1 polypeptide of the present disclosure has a binding affinity that is at least 10% lower, at least 15% lower, at least 20% lower, at least 25% lower, at least 30% lower, at least 35% lower, at least 40% lower, at least 45% lower, at least 50% lower, at least 55% lower, at least 60% lower, at least 65% lower, at least 70% lower, at least 75% lower, at least 80% lower, at least 85% lower, at least 90% lower, at least 95% lower, or more than 95% lower than the binding affinity of a PD-L1 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 103 or SEQ ID NO: 104, and binds to PD-1 (e.g., a PD-1 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 105).

[0201] In some cases, the variant PD-L1 polypeptide has a binding affinity of 1 nM to 1 mM for PD-1. In some cases, the variant PD-L1 polypeptide of the present disclosure has a binding affinity of 100 nM to 100 μM for PD-1. As another example, in some cases, the variant PD-L1 polypeptide has a binding affinity of about 100 nM to 150 nM, about 150 nM to about 200 nM, about 200 nM to about 250 nM, about 250 nM to about 300 nM, about 300 nM to about 350 nM, about 350 nM to about 400 nM, about 400 nM to about 500 nM, about 500 nM to about 600 nM, about 600 nM to about 700 nM, about 700 nM to about 800 nM, about 800 nM to about 900 nM, about 900 nM to about 1 μM, about 1 μM to about 5 μM, about 5 μM to about 10 μM, about 10 μM to about 15 μM, about 15 μM to about 20 μM, about 20 μM to about 25 μM, about 25 μM to about 50 μM, about 50 μM to about 75 μM, or about 75 μM to about 100 μM for PD1 (e.g., a PD1 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 105).

[0202] In some cases, the variant PD-L1 polypeptide has a single amino acid substitution as compared to the PD-L1 amino acid sequence set forth in SEQ ID NO: 103 or SEQ ID NO: 104. In some cases, the variant PD-L1 polypeptide has 2 to 10 amino acid substitutions as compared to the PD-L1 amino acid sequence set forth in SEQ ID NO: 103 or SEQ ID NO: 104. In some cases, the variant PD-L1 polypeptide has two amino acid substitutions as compared to the PD-L1 amino acid sequence set forth in SEQ ID NO: 103 or SEQ ID NO: 104. In some cases, the variant PD-L1 polypeptide has three amino acid substitutions as compared to the PD-L1 amino acid sequence set forth in SEQ ID NO: 103 or SEQ ID NO: 104. In some cases, the variant PD-L1 polypeptide has four amino acid substitutions as compared to the PD-L1 amino acid sequence set forth in SEQ ID NO: 103 or SEQ ID NO: 104. In some cases, the variant PD-L1 polypeptide has five amino acid substitutions as compared to the PD-L1 amino acid sequence set forth in SEQ ID NO: 103 or SEQ ID NO: 104. In some cases, the variant PD-L1 polypeptide has six amino acid substitutions as compared to the PD-L1 amino acid sequence set forth in SEQ ID NO: 103 or SEQ ID NO: 104. In some cases, the variant PD-L1 polypeptide has seven amino acid substitutions as compared to the PD-L1 amino acid sequence set forth in SEQ ID NO: 103 or SEQ ID NO: 104. In some cases, the variant PD-L1 polypeptide has eight amino acid substitutions as compared to the PD-L1 amino acid sequence set forth in SEQ ID NO: 103 or SEQ ID NO: 104. In some cases, the variant PD-L1 polypeptide has nine amino acid substitutions as compared to the PD-L1 amino acid sequence set forth in SEQ ID NO: 103 or SEQ ID NO: 104. In some cases, the variant PD-L1 polypeptide has ten amino acid substitutions as compared to the PD-L1 amino acid sequence set forth in SEQ ID NO: 103 or SEQ ID NO: 104.

[0203] Preferred PD-L1 variants are the following amino acid sequences: A polypeptide comprising an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF0007691927000095.tif30133 (wherein X is any amino acid other than Asp). In some cases, X is Ala. In some cases, X is Arg.

[0204] Suitable PD-L1 variants have the following amino acid sequences: A polypeptide comprising an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF0007691927000096.tif30133 (wherein X is any amino acid other than Ile). In some cases, X is Asp.

[0205] Suitable PD-L1 variants have the following amino acid sequences: A polypeptide comprising an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF0007691927000097.tif30133 (wherein X is any amino acid other than Glu). In some cases, X is Arg.

[0206] CD80 variant In some cases, the variant immunomodulatory polypeptides present in the TMAPP of the present disclosure are variant CD80 polypeptides. Wild-type CD80 binds to CD28.

[0207] The wild-type amino acid sequence of the extracellular domain of human CD80 can be as follows: TIFF0007691927000098.tif31149.

[0208] The wild-type CD28 amino acid sequence can be as follows: TIFF0007691927000099.tif37148。

[0209] The wild-type CD28 amino acid sequence can be as follows: TIFF0007691927000100.tif24133。

[0210] The wild-type CD28 amino acid sequence can be as follows: TIFF0007691927000101.tif17140。

[0211] In some cases, the variant CD80 polypeptide exhibits a reduced binding affinity for CD28 as compared to the binding affinity of the CD80 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 109 for CD28. For example, in some cases, the variant CD80 polypeptide has a binding affinity for CD28 (e.g., a CD28 polypeptide comprising the amino acid sequence set forth in one of SEQ ID NOs: 110, 111, or 112) that is at least 10% lower, at least 15% lower, at least 20% lower, at least 25% lower, at least 30% lower, at least 35% lower, at least 40% lower, at least 45% lower, at least 50% lower, at least 55% lower, at least 60% lower, at least 65% lower, at least 70% lower, at least 75% lower, at least 80% lower, at least 85% lower, at least 90% lower, at least 95% lower, or more than 95% lower than the binding affinity of the CD80 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 109 for CD28.

[0212] In some cases, the variant CD80 polypeptide has a binding affinity for CD28 of 100 nM to 100 μM. As another example, in some cases, the 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: 110, SEQ ID NO: 111, or SEQ ID NO: 112) of about 100 nM to 150 nM, about 150 nM to about 200 nM, about 200 nM to about 250 nM, about 250 nM to about 300 nM, about 300 nM to about 350 nM, about 350 nM to about 400 nM, about 400 nM to about 500 nM, about 500 nM to about 600 nM, about 600 nM to about 700 nM, about 700 nM to about 800 nM, about 800 nM to about 900 nM, about 900 nM to about 1 μM, about 1 μM to about 5 μM, about 5 μM to about 10 μM, about 10 μM to about 15 μM, about 15 μM to about 20 μM, about 20 μM to about 25 μM, about 25 μM to about 50 μM, about 50 μM to about 75 μM, or about 75 μM to about 100 μM.

[0213] In some cases, the variant CD80 polypeptide has a single amino acid substitution compared to the CD80 amino acid sequence set forth in SEQ ID NO: 109. In some cases, the variant CD80 polypeptide has 2 to 10 amino acid substitutions compared to the CD80 amino acid sequence set forth in SEQ ID NO: 109. In some cases, the variant CD80 polypeptide has 2 amino acid substitutions compared to the CD80 amino acid sequence set forth in SEQ ID NO: 109. In some cases, the variant CD80 polypeptide has 3 amino acid substitutions compared to the CD80 amino acid sequence set forth in SEQ ID NO: 109. In some cases, the variant CD80 polypeptide has 4 amino acid substitutions compared to the CD80 amino acid sequence set forth in SEQ ID NO: 109. In some cases, the variant CD80 polypeptide has 5 amino acid substitutions compared to the CD80 amino acid sequence set forth in SEQ ID NO: 109. In some cases, the variant CD80 polypeptide has 6 amino acid substitutions compared to the CD80 amino acid sequence set forth in SEQ ID NO: 109. In some cases, the variant CD80 polypeptide has 7 amino acid substitutions compared to the CD80 amino acid sequence set forth in SEQ ID NO: 109. In some cases, the variant CD80 polypeptide has 8 amino acid substitutions compared to the CD80 amino acid sequence set forth in SEQ ID NO: 109. In some cases, the variant CD80 polypeptide has 9 amino acid substitutions compared to the CD80 amino acid sequence set forth in SEQ ID NO: 109. In some cases, the variant CD80 polypeptide has 10 amino acid substitutions compared to the CD80 amino acid sequence set forth in SEQ ID NO: 109.

[0214] Suitable CD80 variants include polypeptides comprising an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to any one of the following amino acid sequences: TIFF0007691927000102.tif31149(where X is any amino acid other than Asn). In some cases, X is Ala; TIFF0007691927000103.tif31149(where X is any amino acid other than Asn). In some cases, X is Ala; TIFF0007691927000104.tif31149(where X is any amino acid other than Ile). In some cases, X is Ala; TIFF0007691927000105.tif31149(where X is any amino acid other than Lys). In some cases, X is Ala; TIFF0007691927000106.tif31150(where X is any amino acid other than Gln). In some cases, X is Ala; TIFF0007691927000107.tif31149(where X is any amino acid other than Asp). In some cases, X is Ala; TIFF0007691927000108.tif31149(where X is any amino acid other than Leu). In some cases, X is Ala; TIFF0007691927000109.tif31149(where X is any amino acid other than Tyr). In some cases, X is Ala; TIFF0007691927000110.tif32150(where X is any amino acid other than Gln). In some cases, X is Ala; TIFF0007691927000111.tif31149(where X is any amino acid other than Met). In some cases, X is Ala; TIFF0007691927000112.tif31149(where X is any amino acid other than Val). In some cases, X is Ala; TIFF0007691927000113.tif31149 (where X is any amino acid other than Ile). In some cases, X is Ala; TIFF0007691927000114.tif31149 (where X is any amino acid other than Tyr). In some cases, X is Ala; TIFF0007691927000115.tif31149 (where X is any amino acid other than Asp). In some cases, X is Ala; TIFF0007691927000116.tif31149 (where X is any amino acid other than Phe). In some cases, X is Ala; TIFF0007691927000117.tif31149 (where X is any amino acid other than Ser). In some cases, X is Ala; TIFF0007691927000118.tif31150 (where X is any amino acid other than Pro). In some cases, X is Ala.

[0215] CD86 variant In some cases, the variant immunomodulatory polypeptide present in the TTMAPP of the present disclosure is a variant CD86 polypeptide. Wild-type CD86 binds to CD28.

[0216] The amino acid sequence of the complete ectodomain of wild-type human CD86 may be as follows: TIFF0007691927000119.tif23149.

[0217] The amino acid sequence of the IgV domain of wild-type human CD86 may be as follows: TIFF0007691927000120.tif11149.

[0218] In some cases, the variant CD86 polypeptide exhibits a reduced binding affinity for CD28 as compared to the binding affinity of the CD86 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 130 or SEQ ID NO: 131 for CD28. For example, in some cases, the variant CD86 polypeptide has a binding affinity for CD28 (e.g., a CD28 polypeptide comprising the amino acid sequence set forth in one of SEQ ID NO: 110, 111, or 112) that is at least 10% lower, at least 15% lower, at least 20% lower, at least 25% lower, at least 30% lower, at least 35% lower, at least 40% lower, at least 45% lower, at least 50% lower, at least 55% lower, at least 60% lower, at least 65% lower, at least 70% lower, at least 75% lower, at least 80% lower, at least 85% lower, at least 90% lower, at least 95% lower, or more than 95% lower than the binding affinity of the CD86 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 130 or SEQ ID NO: 131 for CD28.

[0219] In some cases, the variant CD86 polypeptide has a binding affinity for CD28 of 100 nM to 100 μM. As another example, in some cases, the variant CD86 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 NO: 110, 111, or 112) of about 100 nM to 150 nM, about 150 nM to about 200 nM, about 200 nM to about 250 nM, about 250 nM to about 300 nM, about 300 nM to about 350 nM, about 350 nM to about 400 nM, about 400 nM to about 500 nM, about 500 nM to about 600 nM, about 600 nM to about 700 nM, about 700 nM to about 800 nM, about 800 nM to about 900 nM, about 900 nM to about 1 μM, about 1 μM to about 5 μM, about 5 μM to about 10 μM, about 10 μM to about 15 μM, about 15 μM to about 20 μM, about 20 μM to about 25 μM, about 25 μM to about 50 μM, about 50 μM to about 75 μM, or about 75 μM to about 100 μM.

[0220] In some cases, the variant CD86 polypeptide has a single amino acid substitution as compared to the CD86 amino acid sequence set forth in SEQ ID NO: 130. In some cases, the variant CD86 polypeptide has 2 to 10 amino acid substitutions as compared to the CD86 amino acid sequence set forth in SEQ ID NO: 130. In some cases, the variant CD86 polypeptide has two amino acid substitutions as compared to the CD86 amino acid sequence set forth in SEQ ID NO: 130. In some cases, the variant CD86 polypeptide has three amino acid substitutions as compared to the CD86 amino acid sequence set forth in SEQ ID NO: 130. In some cases, the variant CD86 polypeptide has four amino acid substitutions as compared to the CD86 amino acid sequence set forth in SEQ ID NO: 130. In some cases, the variant CD86 polypeptide has five amino acid substitutions as compared to the CD86 amino acid sequence set forth in SEQ ID NO: 130. In some cases, the variant CD86 polypeptide has six amino acid substitutions as compared to the CD86 amino acid sequence set forth in SEQ ID NO: 130. In some cases, the variant CD86 polypeptide has seven amino acid substitutions as compared to the CD86 amino acid sequence set forth in SEQ ID NO: 130. In some cases, the variant CD86 polypeptide has eight amino acid substitutions as compared to the CD86 amino acid sequence set forth in SEQ ID NO: 130. In some cases, the variant CD86 polypeptide has nine amino acid substitutions as compared to the CD86 amino acid sequence set forth in SEQ ID NO: 130. In some cases, the variant CD86 polypeptide has ten amino acid substitutions as compared to the CD86 amino acid sequence set forth in SEQ ID NO: 130.

[0221] In some cases, the variant CD86 polypeptide has a single amino acid substitution compared to the CD86 amino acid sequence set forth in SEQ ID NO: 131. In some cases, the variant CD86 polypeptide has 2 to 10 amino acid substitutions compared to the CD86 amino acid sequence set forth in SEQ ID NO: 131. In some cases, the variant CD86 polypeptide has 2 amino acid substitutions compared to the CD86 amino acid sequence set forth in SEQ ID NO: 131. In some cases, the variant CD86 polypeptide has 3 amino acid substitutions compared to the CD86 amino acid sequence set forth in SEQ ID NO: 131. In some cases, the variant CD86 polypeptide has 4 amino acid substitutions compared to the CD86 amino acid sequence set forth in SEQ ID NO: 131. In some cases, the variant CD86 polypeptide has 5 amino acid substitutions compared to the CD86 amino acid sequence set forth in SEQ ID NO: 131. In some cases, the variant CD86 polypeptide has 6 amino acid substitutions compared to the CD86 amino acid sequence set forth in SEQ ID NO: 131. In some cases, the variant CD86 polypeptide has 7 amino acid substitutions compared to the CD86 amino acid sequence set forth in SEQ ID NO: 131. In some cases, the variant CD86 polypeptide has 8 amino acid substitutions compared to the CD86 amino acid sequence set forth in SEQ ID NO: 131. In some cases, the variant CD86 polypeptide has 9 amino acid substitutions compared to the CD86 amino acid sequence set forth in SEQ ID NO: 131. In some cases, the variant CD86 polypeptide has 10 amino acid substitutions compared to the CD86 amino acid sequence set forth in SEQ ID NO: 131.

[0222] Suitable CD86 variants include polypeptides comprising an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to any one of the following amino acid sequences: TIFF0007691927000121.tif24149(where X is any amino acid other than Asn). In some cases, X is Ala; TIFF0007691927000122.tif24149(where X is any amino acid other than Asp). In some cases, X is Ala; TIFF0007691927000123.tif24149(where X is any amino acid other than Trp). In some cases, X is Ala; TIFF0007691927000124.tif23149(where X is any amino acid other than His). In some cases, X is Ala; TIFF0007691927000125.tif11149(where X is any amino acid other than Asn). In some cases, X is Ala; TIFF0007691927000126.tif11149(where X is any amino acid other than Asp). In some cases, X is Ala; TIFF0007691927000127.tif11149(where X is any amino acid other than Trp). In some cases, X is Ala; TIFF0007691927000128.tif11149(where X is any amino acid other than His). In some cases, X is Ala; TIFF0007691927000129.tif24149(where X is any amino acid other than Val). In some cases, X is Ala; TIFF0007691927000130.tif11149(where X is any amino acid other than Val). In some cases, X is Ala; TIFF0007691927000131.tif24149(where X is any amino acid other than Gln). In some cases, X is Ala; TIFF0007691927000132.tif11150(where X is any amino acid other than Gln). In some cases, X is Ala; TIFF0007691927000133.tif23149(where X is any amino acid other than Phe). In some cases, X is Ala; TIFF0007691927000134.tif11149(where X is any amino acid other than Phe). In some cases, X is Ala; TIFF0007691927000135.tif24149(where X is any amino acid other than Leu). In some cases, X is Ala; TIFF0007691927000136.tif11149(where X is any amino acid other than Leu). In some cases, X is Ala; TIFF0007691927000137.tif24150(where X is any amino acid other than Tyr). In some cases, X is Ala; TIFF0007691927000138.tif11150(where X is any amino acid other than Tyr). In some cases, X is Ala; TIFF0007691927000139.tif24149(where the first X is any amino acid other than Asn and the second X is any amino acid other than His). In some cases, both the first and second X are Ala; TIFF0007691927000140.tif11149(where the first X is any amino acid other than Asn and the second X is any amino acid other than His). In some cases, both the first and second X are Ala; TIFF0007691927000141.tif23150(where X 1 is any amino acid other than Asp and X 2 is any amino acid other than His). In some cases, X1 is Ala, and X 2 is Ala; TIFF0007691927000142.tif17144 (where the first X is any amino acid other than Asn and the second X is any amino acid other than His). In some cases, both the first and second X are Ala; TIFF0007691927000143.tif24149 (where X 1 is any amino acid other than Asn, X 2 is any amino acid other than Asp, X 3 is any amino acid other than His). In some cases, X 1 is Ala, X 2 is Ala, X 3 is Ala; and TIFF0007691927000144.tif17146 (where X 1 is any amino acid other than Asn, X 2 is any amino acid other than Asp, X 3 is any amino acid other than His). In some cases, X 1 is Ala, X 2 is Ala, X 3 is Ala.

[0223] 4-1BBL variant In some cases, the variant immunomodulatory polypeptide present in the TMAPP of the present disclosure is a variant 4-1BBL polypeptide. Wild-type 4-1BBL binds to 4-1BB (CD137).

[0224] The wild-type 4-1BBL amino acid sequence can be as follows: TIFF0007691927000145.tif37146.

[0225] In some cases, the variant 4-1BBL polypeptide is a variant of the tumor necrosis factor (TNF) homology domain (THD) of human 4-1BBL.

[0226] The wild-type amino acid sequence of human 4-1BBL THD can be, for example, one of SEQ ID NOs: 157-159 below: TIFF0007691927000146.tif79137。

[0227] The wild-type 4-1BB amino acid sequence can be as follows: TIFF0007691927000147.tif37150。

[0228] In some cases, the variant 4-1BBL polypeptide exhibits a decreased binding affinity for 4-1BB as compared to the binding affinity of a 4-1BBL polypeptide comprising the amino acid sequence set forth in one of SEQ ID NOs: 156-159. For example, in some cases, the variant 4-1BBL polypeptides of the present disclosure, when assayed under the same conditions, have a binding affinity for a 4-1BB polypeptide (e.g., a 4-1BB polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 160) that is at least 10% lower, at least 15% lower, at least 20% lower, at least 25%, at least 30% lower, at least 35% lower, at least 40% lower, at least 45% lower, at least 50% lower, at least 55% lower, at least 60% lower, at least 65% lower, at least 70% lower, at least 75% lower, at least 80% lower, at least 85% lower, at least 90% lower, at least 95% lower, or more than 95% lower than that of a 4-1BBL polypeptide comprising the amino acid sequence set forth in one of SEQ ID NOs: 156-159.

[0229] In some cases, the variant 4-1BBL polypeptide has a binding affinity for 4-1BB of 100 nM to 100 μM. As another example, in some cases, the 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: 160) of about 100 nM to 150 nM, about 150 nM to about 200 nM, about 200 nM to about 250 nM, about 250 nM to about 300 nM, about 300 nM to about 350 nM, about 350 nM to about 400 nM, about 400 nM to about 500 nM, about 500 nM to about 600 nM, about 600 nM to about 700 nM, about 700 nM to about 800 nM, about 800 nM to about 900 nM, about 900 nM to about 1 μM, about 1 μM to about 5 μM, about 5 μM to about 10 μM, about 10 μM to about 15 μM, about 15 μM to about 20 μM, about 20 μM to about 25 μM, about 25 μM to about 50 μM, about 50 μM to about 75 μM, or about 75 μM to about 100 μM.

[0230] In some cases, the variant 4-1BBL polypeptide has a single amino acid substitution as compared to the 4-1BBL amino acid sequence set forth in one of SEQ ID NOs: 156-159. In some cases, the variant 4-1BBL polypeptide has 2 to 10 amino acid substitutions as compared to the 4-1BBL amino acid sequence set forth in one of SEQ ID NOs: 156-159. In some cases, the variant 4-1BBL polypeptide has two amino acid substitutions as compared to the 4-1BBL amino acid sequence set forth in one of SEQ ID NOs: 156-159. In some cases, the variant 4-1BBL polypeptide has three amino acid substitutions as compared to the 4-1BBL amino acid sequence set forth in one of SEQ ID NOs: 156-159. In some cases, the variant 4-1BBL polypeptide has four amino acid substitutions as compared to the 4-1BBL amino acid sequence set forth in one of SEQ ID NOs: 156-159. In some cases, the variant 4-1BBL polypeptide has five amino acid substitutions as compared to the 4-1BBL amino acid sequence set forth in one of SEQ ID NOs: 156-159. In some cases, the variant 4-1BBL polypeptide has six amino acid substitutions as compared to the 4-1BBL amino acid sequence set forth in one of SEQ ID NOs: 156-159. In some cases, the variant 4-1BBL polypeptide has seven amino acid substitutions as compared to the 4-1BBL amino acid sequence set forth in one of SEQ ID NOs: 156-159. In some cases, the variant 4-1BBL polypeptide has eight amino acid substitutions as compared to the 4-1BBL amino acid sequence set forth in one of SEQ ID NOs: 156-159. In some cases, the variant 4-1BBL polypeptide has nine amino acid substitutions as compared to the 4-1BBL amino acid sequence set forth in one of SEQ ID NOs: 156-159. In some cases, the variant 4-1BBL polypeptide has ten amino acid substitutions as compared to the 4-1BBL amino acid sequence set forth in one of SEQ ID NOs: 156-159.

[0231] Suitable 4-1BB variants include polypeptides comprising an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to any one of the following amino acid sequences: TIFF0007691927000148.tif24135 (where X is any amino acid other than Lys). In some cases, X is Ala; TIFF0007691927000149.tif24135 (where X is any amino acid other than Gln). In some cases, X is Ala; TIFF0007691927000150.tif24135 (where X is any amino acid other than Met). In some cases, X is Ala; TIFF0007691927000151.tif24135 (where X is any amino acid other than Phe). In some cases, X is Ala; TIFF0007691927000152.tif24135 (where X is any amino acid other than Gln). In some cases, X is Ala; TIFF0007691927000153.tif24135 (where X is any amino acid other than Leu). In some cases, X is Ala; TIFF0007691927000154.tif24135 (where X is any amino acid other than Val). In some cases, X is Ala; TIFF0007691927000155.tif24135 (where X is any amino acid other than Gln). In some cases, X is Ala; TIFF0007691927000156.tif24135 (where X is any amino acid other than Asn). In some cases, X is Ala; TIFF0007691927000157.tif24135(where X is any amino acid other than Val). In some cases, X is Ala; TIFF0007691927000158.tif24135(where X is any amino acid other than Leu). In some cases, X is Ala; TIFF0007691927000159.tif24135(where X is any amino acid other than Leu). In some cases, X is Ala; TIFF0007691927000160.tif24136(where X is any amino acid other than Ile). In some cases, X is Ala; TIFF0007691927000161.tif24135(where X is any amino acid other than Asp). In some cases, X is Ala; TIFF0007691927000162.tif24135(where X is any amino acid other than Gly). In some cases, X is Ala; TIFF0007691927000163.tif25135(where X is any amino acid other than Pro). In some cases, X is Ala; TIFF0007691927000164.tif24135(where X is any amino acid other than Leu). In some cases, X is Ala; TIFF0007691927000165.tif24135(where X is any amino acid other than Ser). In some cases, X is Ala; TIFF0007691927000166.tif24135(where X is any amino acid other than Trp). In some cases, X is Ala; TIFF0007691927000167.tif24135(where X is any amino acid other than Tyr). In some cases, X is Ala; TIFF0007691927000168.tif24135(where X is any amino acid other than Ser). In some cases, X is Ala; TIFF0007691927000169.tif24135(where X is any amino acid other than Asp). In some cases, X is Ala; TIFF0007691927000170.tif24135(where X is any amino acid other than Pro). In some cases, X is Ala; TIFF0007691927000171.tif24135(where X is any amino acid other than Gly). In some cases, X is Ala; TIFF0007691927000172.tif24135(where X is any amino acid other than Leu). In some cases, X is Ala; TIFF0007691927000173.tif24135(where X is any amino acid other than Gly). In some cases, X is Ala; TIFF0007691927000174.tif24135(where X is any amino acid other than Val). In some cases, X is Ala; TIFF0007691927000175.tif24135(where X is any amino acid other than Ser). In some cases, X is Ala; TIFF0007691927000176.tif23135(where X is any amino acid other than Leu). In some cases, X is Ala; TIFF0007691927000177.tif24135(where X is any amino acid other than Thr). In some cases, X is Ala; TIFF0007691927000178.tif24135(where X is any amino acid other than Gly). In some cases, X is Ala; TIFF0007691927000179.tif24135(where X is any amino acid other than Gly). In some cases, X is Ala; TIFF0007691927000180.tif24135(where X is any amino acid other than Leu). In some cases, X is Ala; TIFF0007691927000181.tif24135(where X is any amino acid other than Ser). In some cases, X is Ala; TIFF0007691927000182.tif24135(where X is any amino acid other than Tyr). In some cases, X is Ala; TIFF0007691927000183.tif25135(where X is any amino acid other than Glu). In some cases, X is Ala; TIFF0007691927000184.tif24135(where X is any amino acid other than Asp). In some cases, X is Ala; TIFF0007691927000185.tif24135(where X is any amino acid other than Thr). In some cases, X is Ala; TIFF0007691927000186.tif24135(where X is any amino acid other than Lys). In some cases, X is Ala; TIFF0007691927000187.tif24135(where X is any amino acid other than Glu). In some cases, X is Ala; TIFF0007691927000188.tif24135(where X is any amino acid other than Phe). In some cases, X is Ala; TIFF0007691927000189.tif24135(where X is any amino acid other than Phe). In some cases, X is Ala; TIFF0007691927000190.tif24135(where X is any amino acid other than Gln). In some cases, X is Ala; TIFF0007691927000191.tif24135(where X is any amino acid other than Leu). In some cases, X is Ala; TIFF0007691927000192.tif24135(where X is any amino acid other than Glu). In some cases, X is Ala; TIFF0007691927000193.tif24135(where X is any amino acid other than Leu). In some cases, X is Ala; TIFF0007691927000194.tif24135(where X is any amino acid other than Arg). In some cases, X is Ala; TIFF0007691927000195.tif24135(where X is any amino acid other than Arg). In some cases, X is Ala; TIFF0007691927000196.tif24135(where X is any amino acid other than Val). In some cases, X is Ala; TIFF0007691927000197.tif24135(where X is any amino acid other than Val). In some cases, X is Ala; TIFF0007691927000198.tif24135(where X is any amino acid other than Gly). In some cases, X is Ala; TIFF0007691927000199.tif24135(where X is any amino acid other than Glu). In some cases, X is Ala; TIFF0007691927000200.tif24135(where X is any amino acid other than Gly). In some cases, X is Ala; TIFF0007691927000201.tif24135(where X is any amino acid other than Ser). In some cases, X is Ala; TIFF0007691927000202.tif24135(where X is any amino acid other than Asp). In some cases, X is Ala; TIFF0007691927000203.tif24135(where X is any amino acid other than Leu). In some cases, X is Ala; TIFF0007691927000204.tif24135(where X is any amino acid other than Pro). In some cases, X is Ala; TIFF0007691927000205.tif24135(where X is any amino acid other than Ser). In some cases, X is Ala; TIFF0007691927000206.tif24135(where X is any amino acid other than Ser). In some cases, X is Ala; TIFF0007691927000207.tif24135(where X is any amino acid other than Glu). In some cases, X is Ala; TIFF0007691927000208.tif24135(where X is any amino acid other than Arg). In some cases, X is Ala; TIFF0007691927000209.tif24135(where X is any amino acid other than Asn). In some cases, X is Ala; TIFF0007691927000210.tif24135(where X is any amino acid other than Ser). In some cases, X is Ala; TIFF0007691927000211.tif24135(where X is any amino acid other than Phe). In some cases, X is Ala; TIFF0007691927000212.tif24135(where X is any amino acid other than Gln). In some cases, X is Ala; TIFF0007691927000213.tif24135(where X is any amino acid other than Arg). In some cases, X is Ala; TIFF0007691927000214.tif24135(where X is any amino acid other than Leu). In some cases, X is Ala; TIFF0007691927000215.tif24135(where X is any amino acid other than Gly). In some cases, X is Ala; TIFF0007691927000216.tif24135(where X is any amino acid other than Val). In some cases, X is Ala; TIFF0007691927000217.tif24135(where X is any amino acid other than His). In some cases, X is Ala; TIFF0007691927000218.tif24135(where X is any amino acid other than Leu). In some cases, X is Ala; TIFF0007691927000219.tif24135(where X is any amino acid other than His). In some cases, X is Ala; TIFF0007691927000220.tif24135(where X is any amino acid other than Thr). In some cases, X is Ala; TIFF0007691927000221.tif24135(where X is any amino acid other than Glu). In some cases, X is Ala; TIFF0007691927000222.tif24135(where X is any amino acid other than Arg). In some cases, X is Ala; TIFF0007691927000223.tif24135(where X is any amino acid other than Arg). In some cases, X is Ala; TIFF0007691927000224.tif24135(where X is any amino acid other than His). In some cases, X is Ala; TIFF0007691927000225.tif24135(where X is any amino acid other than Trp). In some cases, X is Ala; TIFF0007691927000226.tif24135(where X is any amino acid other than Leu). In some cases, X is Ala; TIFF0007691927000227.tif24135(where X is any amino acid other than Thr). In some cases, X is Ala; TIFF0007691927000228.tif24135(where X is any amino acid other than Gln). In some cases, X is Ala; TIFF0007691927000229.tif24135(where X is any amino acid other than Gly). In some cases, X is Ala; TIFF0007691927000230.tif24135(where X is any amino acid other than Thr). In some cases, X is Ala; TIFF0007691927000231.tif24135(where X is any amino acid other than Val). In some cases, X is Ala.

[0232] IL-2 variant In some cases, the variant immunomodulatory polypeptide present in the TMAPP of the present disclosure is a variant IL-2 polypeptide. Wild-type IL-2 binds to the IL-2 receptor (IL-2R).

[0233] The wild-type IL-2 amino acid sequence can be as follows: TIFF0007691927000232.tif23149。

[0234] Wild-type IL2 binds to the IL2 receptor (IL2R) on the surface of cells. The IL2 receptor is, in some cases, a heterotrimeric polypeptide comprising an alpha chain (IL-2Rα; also referred to as CD25), a beta chain (IL-2Rβ; also referred to as CD122), and a gamma chain (IL-2Rγ; also referred to as CD132). The amino acid sequences of human IL-2Rα, IL2Rβ, and IL-2Rγ can be as follows. TIFF0007691927000233.tif166140。

[0235] In some cases, when the TMAPP of the present disclosure comprises a variant IL-2 polypeptide, the "allogeneic immunomodulatory polypeptide" is an IL-2R comprising the polypeptides contained in the amino acid sequences of SEQ ID NOs: 246, 247, and 248.

[0236] In some cases, the variant IL-2 polypeptide exhibits a decreased binding affinity for the IL-2R as compared to the binding affinity of the IL-2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 245 for the IL-2R. For example, in some cases, the variant IL-2 polypeptide, when assayed under the same conditions, has a binding affinity for the IL-2R (e.g., the IL-2R comprising the polypeptide contained in the amino acid sequences set forth in SEQ ID NOs: 246-248) that is at least 10% lower, at least 15% lower, at least 20% lower, at least 25% lower, at least 30% lower, at least 35% lower, at least 40% lower, at least 45% lower, at least 50% lower, at least 55% lower, at least 60% lower, at least 65% lower, at least 70% lower, at least 75% lower, at least 80% lower, at least 85% lower, at least 90% lower, at least 95% lower, or more than 95% lower than the binding affinity of the IL-2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 245 for the IL-2R.

[0237] In some cases, the variant IL-2 polypeptide has a binding affinity for IL-2R of 100 nM to 100 μM. As another example, in some cases, the variant IL-2 polypeptide has a binding affinity for IL-2R (e.g., IL-2R including a polypeptide having the amino acid sequence set forth in SEQ ID NOs: 246-248) of about 100 nM to 150 nM, about 150 nM to about 200 nM, about 200 nM to about 250 nM, about 250 nM to about 300 nM, about 300 nM to about 350 nM, about 350 nM to about 400 nM, about 400 nM to about 500 nM, about 500 nM to about 600 nM, about 600 nM to about 700 nM, about 700 nM to about 800 nM, about 800 nM to about 900 nM, about 900 nM to about 1 μM, about 1 μM to about 5 μM, about 5 μM to about 10 μM, about 10 μM to about 15 μM, about 15 μM to about 20 μM, about 20 μM to about 25 μM, about 25 μM to about 50 μM, about 50 μM to about 75 μM, or about 75 μM to about 100 μM.

[0238] In some cases, the variant IL-2 polypeptide has a single amino acid substitution compared to the IL-2 amino acid sequence set forth in SEQ ID NO: 245. In some cases, the variant IL-2 polypeptide has 2 to 10 amino acid substitutions compared to the IL-2 amino acid sequence set forth in SEQ ID NO: 245. In some cases, the variant IL-2 polypeptide has 2 amino acid substitutions compared to the IL-2 amino acid sequence set forth in SEQ ID NO: 245. In some cases, the variant IL-2 polypeptide has 3 amino acid substitutions compared to the IL-2 amino acid sequence set forth in SEQ ID NO: 245. In some cases, the variant IL-2 polypeptide has 4 amino acid substitutions compared to the IL-2 amino acid sequence set forth in SEQ ID NO: 245. In some cases, the variant IL-2 polypeptide has 5 amino acid substitutions compared to the IL-2 amino acid sequence set forth in SEQ ID NO: 245. In some cases, the variant IL-2 polypeptide has 6 amino acid substitutions compared to the IL-2 amino acid sequence set forth in SEQ ID NO: 245. In some cases, the variant IL-2 polypeptide has 7 amino acid substitutions compared to the IL-2 amino acid sequence set forth in SEQ ID NO: 245. In some cases, the variant IL-2 polypeptide has 8 amino acid substitutions compared to the IL-2 amino acid sequence set forth in SEQ ID NO: 245. In some cases, the variant IL-2 polypeptide has 9 amino acid substitutions compared to the IL-2 amino acid sequence set forth in SEQ ID NO: 245. In some cases, the variant IL-2 polypeptide has 10 amino acid substitutions compared to the IL-2 amino acid sequence set forth in SEQ ID NO: 245.

[0239] Suitable IL-2 variants include polypeptides comprising an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to any one of the following amino acid sequences: TIFF0007691927000234.tif17128(where X is any amino acid other than Phe). In some cases, X is Ala; TIFF0007691927000235.tif17128(where X is any amino acid other than Asp). In some cases, X is Ala; TIFF0007691927000236.tif17150(where X is any amino acid other than Glu). In some cases, X is Ala; TIFF0007691927000237.tif17128(where X is any amino acid other than His). In some cases, X is Ala. In some cases, X is Arg. In some cases, X is Asn. In some cases, X is Asp. In some cases, X is Cys. In some cases, X is Glu. In some cases, X is Gln. In some cases, X is Gly. In some cases, X is Ile. In some cases, X is Lys. In some cases, X is Leu. In some cases, X is Met. In some cases, 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; TIFF0007691927000238.tif17128(where X is any amino acid other than Tyr). In some cases, X is Ala; TIFF0007691927000239.tif18128(where X is any amino acid other than Gln). In some cases, X is Ala; TIFF0007691927000240.tif17129(where X 1is any amino acid other than His, and X 2 is any amino acid other than Phe). In some cases, X 1 is Ala. In some cases, X 2 is Ala. In some cases, X 1 is Ala and X 2 is Ala. In some cases, X 1 is Thr and X 2 is Ala; TIFF0007691927000241.tif17129 (where X 1 is any amino acid other than Asp, and X 2 is any amino acid other than Phe). In some cases, X 1 is Ala. In some cases, X 2 is Ala. In some cases, X 1 is Ala and X 2 is Ala; TIFF0007691927000242.tif17131 (where X 1 is any amino acid other than Glu, and X 2 is any amino acid other than Asp, and X 3 is any amino acid other than Phe). In some cases, X 1 is Ala. In some cases, X 2 is Ala. In some cases, X 3 is Ala. In some cases, X 1 is Ala and X 2 is Ala and X 3 is Ala; TIFF0007691927000243.tif17131 (where X 1 is any amino acid other than His, and X 2 is any amino acid other than Asp, and X 3 is any amino acid other than Phe). In some cases, X 1 is Ala. In some cases, X 2is Ala. In some cases, X 3 is Ala. In some cases, X 1 is Ala, and X 2 is Ala, and X 3 is Ala; TIFF0007691927000244.tif18129(where X 1 is any amino acid other than Asp, and X 2 is any amino acid other than Phe, and X 3 is any amino acid other than Gln). In some cases, X 1 is Ala. In some cases, X 2 is Ala. In some cases, X 3 is Ala. In some cases, X 1 is Ala, and X 2 is Ala, and X 3 is Ala; TIFF0007691927000245.tif17131(where X 1 is any amino acid other than Asp, and X 2 is any amino acid other than Phe, and X 3 is any amino acid other than Tyr). In some cases, X 1 is Ala. In some cases, X 2 is Ala. In some cases, X 3 is Ala. In some cases, X 1 is Ala, and X 2 is Ala, and X 3 is Ala; TIFF0007691927000246.tif17132(where X 1 is any amino acid other than His, and X 2 is any amino acid other than Asp, and X 3 is any amino acid other than Phe, and X 4 is any amino acid other than Tyr). In some cases, X 1 is Ala. In some cases, X2 is Ala. In some cases, X 3 is Ala. In some cases, X 4 is Ala. In some cases, X 1 is Ala and X 2 is Ala and X 3 is Ala and X 4 is Ala; TIFF0007691927000247.tif18131 (where X 1 is any amino acid other than Asp, and X 2 is any amino acid other than Phe, and X 3 is any amino acid other than Tyr, and X 4 is any amino acid other than Gln). In some cases, X 1 is Ala. In some cases, X 2 is Ala. In some cases, X 3 is Ala. In some cases, X 4 is Ala. In some cases, X 1 is Ala and X 2 is Ala and X 3 is Ala and X 4 is Ala; TIFF0007691927000248.tif18132 (where X 1 is any amino acid other than His, and X 2 is any amino acid other than Asp, and X 3 is any amino acid other than Phe, and X 4 is any amino acid other than Tyr, and X 5 is any amino acid other than Gln). In some cases, X 1 is Ala. In some cases, X 2 is Ala. In some cases, X 3 is Ala. In some cases, X 4 is Ala. In some cases, X 5is Ala. In some cases, X 1 is Ala, and X 2 is Ala, and X 3 is Ala, and X 4 is Ala, and X 5 is Ala; TIFF0007691927000249.tif18129(where X 1 is any amino acid other than His, and X 2 is any amino acid other than Phe, and X 3 is any amino acid other than Gln). In some cases, X 1 is Ala. In some cases, X 2 is Ala. In some cases, X 3 is Ala. In some cases, X 1 is Ala, and X 2 is Ala, and X 3 is Ala.

[0240] TGF-β As described above, in some cases, the immunomodulatory polypeptide present in the TMAPP of the present disclosure is a TGF-β polypeptide. The amino acid sequence of the TGF-β polypeptide is known in the art. In some cases, the immunomodulatory polypeptide present in the TMAPP of the present disclosure is a TGF-β1 polypeptide. The immunomodulatory polypeptide present in the TMAPP of the present disclosure is a TGF-β2 polypeptide. The immunomodulatory polypeptide present in the TMAPP of the present disclosure is a TGF-β3 polypeptide. Suitable TGF-β polypeptides may include amino acid sequences having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the mature form of the human TGF-β1 polypeptide, the human TGF-β2 polypeptide, or the human TGF-β3 polypeptide. Suitable TGF-β polypeptides may have a length of about 100 amino acids to about 125 amino acids. For example, suitable TGF-β polypeptides may have a length of about 100 amino acids to about 105 amino acids, about 105 amino acids to about 110 amino acids, about 110 amino acids to about 115 amino acids, about 115 amino acids to about 120 amino acids, or about 120 amino acids to about 125 amino acids.

[0241] Suitable TGF-β1 polypeptides are the following TGF-β1 amino acid sequences: TIFF0007691927000250.tif24146 may include an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity thereto, wherein the TGF-β1 polypeptide has a length of about 112 amino acids.

[0242] In some cases, suitable TGF-β1 polypeptides include the C77S substitution. Thus, in some cases, suitable TGF-β1 polypeptides are the following TGF-β1 amino acid sequences: It includes an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF0007691927000251.tif24146 (where amino acid 77 is Ser).

[0243] A preferred TGF-β2 polypeptide has the following TGF-β2 amino acid sequence: It may include an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF0007691927000252.tif24150, where the TGF-β2 polypeptide has a length of about 112 amino acids.

[0244] In some cases, a preferred TGF-β2 polypeptide includes the C77S substitution. Thus, in some cases, a preferred TGF-β2 polypeptide has the following TGF-β2 amino acid sequence: It includes an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF0007691927000253.tif24150 (where amino acid 77 is Ser).

[0245] A preferred TGF-β3 polypeptide has the following TGF-β3 amino acid sequence: It may include an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF0007691927000254.tif24147, where the TGF-β3 polypeptide has a length of about 112 amino acids.

[0246] In some cases, a suitable TGF-β3 polypeptide comprises a C77S substitution. Thus, in some cases, a suitable TGF-β3 polypeptide has the following TGF-β3 amino acid sequence: TIFF0007691927000255.tif24151 (where amino acid 77 is Ser) and comprises an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity thereto.

[0247] Dimerization pair As noted above, in some cases, the antigen-presenting polypeptides of the disclosure (including the TMAPPs of the disclosure) comprise a polypeptide dimerization pair. For example, the antigen-presenting polypeptides of the disclosure (including the TMAPPs of the disclosure) are multimeric polypeptides comprising at least a first and a second polypeptide, and in some cases, the first polypeptide comprises a first member of the dimerization pair and the second polypeptide comprises a second member of the dimerization pair.

[0248] 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) that comprise a collagen domain consisting of collagen repeats Gly-Xaa-Xaa. Other dimerization peptides include coiled-coil domains and leucine zipper domains. The collagen domain can comprise (Gly-Xaa-Xaa) n where Xaa is any amino acid and n is an integer from 10 to 40. In some cases, the 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, for example, U.S. Patent Application Publication No. 2003 / 0138440.

[0249] In some cases, the dimerization pair comprises two leucine zipper polypeptides that bind to each other. Non-limiting examples of leucine zipper polypeptides include, for example, any one of the following peptide sequences: TIFF0007691927000256.tif44140。

[0250] In some cases, the leucine zipper polypeptide has the following amino acid sequence: TIFF0007691927000257.tif4150。

[0251] Additional leucine zipper polypeptides are known in the art, and any of them are suitable for use in the antigen-presenting polypeptides of the present disclosure.

[0252] The collagen oligomerization peptide may have the following amino acid sequence: TIFF0007691927000258.tif11151。

[0253] Coiled-coil dimerization peptides are known in the art. For example, the coiled-coil dimerization peptide may include any one of the following peptide sequences: TIFF0007691927000259.tif31128。

[0254] In some cases, the dimerization peptide contains at least one cysteine residue. Examples include, for example, TIFF0007691927000260.tif17128。

[0255] Additional polypeptides The polypeptide chain of the TMAPP of the present disclosure (including the TMAPP of the present disclosure) may include one or more polypeptides in addition to those described above. Suitable additional polypeptides include epitope tags and affinity domains. One or more additional polypeptides may be included at the N-terminus of the polypeptide chain of the TMAPP of the present disclosure, at the C-terminus of the polypeptide chain of the TMAPP of the present disclosure, or internally within the polypeptide chain of the TMAPP of the present disclosure.

[0256] Epitope tag Suitable epitope tags include, but are not limited to, hemagglutinin (HA; e.g., YPYDVPDYA (SEQ ID NO: 288)), FLAG (e.g., DYKDDDDK (SEQ ID NO: 289)), c-myc (e.g., EQKLISEEDL; SEQ ID NO: 290), etc.

[0257] Affinity domain An affinity domain includes a peptide sequence that can interact with a binding partner (e.g., one immobilized on a solid support useful for identification or purification). A DNA sequence encoding a plurality of consecutive single amino acids such as histidine fused to an expressed protein can be used for one-step purification of a recombinant protein by high-affinity binding to a resin column such as nickel sepharose. Exemplary affinity domains include His5 (HHHHH) (SEQ ID NO: 291), HisX6 (HHHHHH) (SEQ ID NO: 292), C-myc (EQKLISEEDL) (SEQ ID NO: 290), Flag (DYKDDDDK) (SEQ ID NO: 289), StrepTag (WSHPQFEK) (SEQ ID NO: 293), hemagglutinin, e.g., HA tag (YPYDVPDYA) (SEQ ID NO: 288), glutathione-S-transferase (GST), thioredoxin, cellulose binding domain, RYIRS (SEQ ID NO: 294), Phe-His-His-Thr (SEQ ID NO: 295), chitin binding domain, S-peptide, T7 peptide, SH2 domain, C-terminal RNA tag, TIFF0007691927000261.tif4128, metal-binding domains, such as zinc-binding domains or calcium-binding domains, such as calcium-binding proteins, such as calmodulin, troponin C, calcineurin B, myosin light chain, recoverin, S-modulin, visinin, VILIP, neurocalcin, hippocalcin, frequenin, caltractin, the large subunit of calpain, S100 proteins, parvalbumin, calbindin D9K, calbindin D28K, and calretinin-derived inteins, biotin, streptavidin, MyoD, Id, leucine zipper sequences, as well as maltose-binding protein.

[0258] Drug conjugate The polypeptide chain of the TMAPP of the present disclosure may include a small molecule drug linked (e.g., covalently associated) to the polypeptide chain. For example, if the TMAPP of the present disclosure includes an Fc polypeptide, the Fc polypeptide may include a covalently linked small molecule drug. The polypeptide chain of the TMAPP of the present disclosure may include a cytotoxic agent linked (e.g., covalently associated) to the polypeptide chain. For example, if the TMAPP of the present disclosure includes an Fc polypeptide, the Fc polypeptide may include a covalently linked cytotoxic agent. The cytotoxic agent includes prodrugs.

[0259] The drug may be directly or indirectly linked to the polypeptide chain of the TMAPP of the present disclosure. For example, if the TMAPP of the present disclosure includes an Fc polypeptide, the drug may be directly or indirectly linked to the Fc polypeptide. Direct linkage may involve direct linkage to an amino acid side chain. Indirect linkage may be linkage via a linker. The drug may be linked to the polypeptide chain (e.g., Fc polypeptide) of the TMAPP of the present disclosure via a thioether bond, an amide bond, a carbamate bond, a disulfide bond, or an ether bond.

[0260] Linkers include cleavable linkers and non-cleavable linkers. In some cases, the linker is a linker cleavable by a protease. Suitable linkers include, for example, peptides (e.g., 2 to 10 amino acids in length, e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids in length), alkyl chains, poly(ethylene glycol), disulfide groups, thioether groups, acid-labile groups, photo-labile groups, peptidase-labile groups, and esterase-labile groups.Non-limiting examples of suitable linkers are: i) N-succinimidyl-[(N-maleimidopropionamido)-tetraethyleneglycol] ester (NHS-PEG4-maleimide), ii) N-succinimidyl 4-(2-pyridyldithio)butanoate (SPDB), N-succinimidyl 4-(2-pyridyldithio)2-sulfobutanoate (sulfo-SPDB), N-succinimidyl 4-(2-pyridyldithio)pentaenoate (SPP), N-succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxy-(6-amidocaproate) (LC-SMCC), κ-maleimidoundecanoic acid N-succinimidyl ester (KMUA), γ-maleimidobutyric acid N-succinimidyl ester (GMBS), ε-maleimidocaproic acid N-hydroxysuccinimide ester (EMCS), m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), N-(α-maleimidoacetoxy)-succinimide ester (AMAS), succinimidyl-6-(β-maleimidopropionamido)hexanoate (SMPH), N-succinimidyl 4-(p-maleimidophenyl)butyrate (SMPB), N-(p-maleimidophenyl)isocyanate (PMPI), N-succinimidyl 4(2-pyridylthio)pentaenoate (SPP), N-succinimidyl(4-iodo-acetyl)aminobenzoate (SIAB), 6-maleimidocaproyl (MC), maleimidopropanoyl (MP), p-aminobenzyloxycarbonyl (PAB), N-succinimidyl 4-(maleimidomethyl)cyclohexanecarboxylate (SMCC), the "long-chain" analog of SMCC (LC-SMCC), N-succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxy-(6-amidocaproate), 3-maleimidopropanoic acid N-succinimidyl ester (BMPS), N-succinimidyl iodoacetate (SIA), N-succinimidyl bromoacetate (SBA), and N-succinimidyl 3-(bromoacetamido)propionate (SBAP).

[0261] A polypeptide (e.g., an Fc polypeptide) can be modified with a cross-linking agent such as N-succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC), sulfo-SMCC, maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), sulfo-MBS or succinimidyl iodoacetate as described in the literature to introduce 1 to 10 reactive groups. Then, the modified Fc polypeptide is reacted with a thiol-containing cytotoxic agent to generate a conjugate.

[0262] For example, when the TMAPP of the present disclosure contains an Fc polypeptide, the polypeptide chain containing the Fc polypeptide may be of the formula (A)-(L)-(C), where (A) is a polypeptide chain containing the Fc polypeptide, (L) is a linker if present, and (C) is a cytotoxic agent. (L), if present, links (A) to (C). In some cases, the polypeptide chain containing the Fc polypeptide may contain two or more cytotoxic agents (e.g., 2, 3, 4, or 5, or more than 5 cytotoxic agents).

[0263] Suitable drugs include, for example, rapamycin. Suitable drugs include, for example, retinoids such as all-trans retinoic acid (ATRA), vitamin D3, vitamin D3 analogs, and the like. As noted above, in some cases, the drug is a cytotoxic agent. Cytotoxic agents are known in the art. Suitable cytotoxic agents can be any compound that causes cell death, induces cell death, or decreases cell viability in some way, such as maytansinoids and maytansinoid analogs, benzodiazepines, taxoids, CC-1065 and CC-1065 analogs, duocarmycin and duocarmycin analogs, enediynes such as calicheamicin, dolastatin and dolastatin analogs containing auristatin, tomamycin derivatives, leptomycin derivatives, methotrexate, cisplatin, carboplatin, daunorubicin, doxorubicin, vincristine, vinblastine, melphalan, mitomycin C, chlorambucil, and morpholinodoxorubicin.

[0264] For example, in some cases, the cytotoxic agent is a compound that inhibits microtubule formation in eukaryotic cells. Such agents include, for example, maytansinoids, benzodiazepines, taxoids, CC-1065, duocarmycin, duocarmycin analogs, calicheamicin, dolastatin, dolastatin analogs, auristatin, tomamycin, and leptomycin, or any prodrug of any of the foregoing. Maytansinoid compounds include, for example, N(2’)-deacetyl-N(2’)-(3-mercapto-1-oxopropyl)-maytansine (DM1), N(2’)-deacetyl-N(2’)-(4-mercapto-1-oxopentyl)-maytansine (DM3), and N(2’)-deacetyl-N2-(4-mercapto-4-methyl-1-oxopentyl)-maytansine (DM4). Benzodiazepines include, for example, indolinobenzodiazepines and oxazolidinobenzodiazepines.

[0265] Cytotoxic agents are known in the art. Suitable cytotoxic agents can be any compound that causes cell death, induces cell death, or in some way reduces cell viability, for example, maytansinoids and maytansinoid analogs, benzodiazepines, taxoids, CC-1065 and CC-1065 analogs, duocarmycins and duocarmycin analogs, enediynes such as calicheamicin, dolastatin and dolastatin analogs including auristatin, tomamycin derivatives, leptomycin derivatives, methotrexate, cisplatin, carboplatin, daunorubicin, doxorubicin, vincristine, vinblastine, melphalan, mitomycin C, chlorambucil, and morpholino doxorubicin.

[0266] Cytotoxic agents include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracinedione, maytansine or its analogs or derivatives, auristatin or its functional peptide analogs or derivatives, dolastatin 10 or 15 or their analogs, irinotecan or its analogs, mitoxantrone, mitramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, calicheamicin or its analogs or derivatives, antimetabolites, 6-mercaptopurine, 6-thioguanine, cytarabine, fludarabine, 5-fluorouracil, dacarbazine, hydroxyurea, asparaginase, gemcitabine, cladribine, alkylating agents, platinum derivatives, duocarmycin A, duocarmycin SA, rachelmycin (CC-1065) or its analogs or derivatives, antibiotics, pyrrolo[2,1-c][1,4]-benzodiazepine (PDB), diphtheria toxin, ricin toxin, cholera toxin, Shiga-like toxin, LT toxin, C3 toxin, Shiga toxin, pertussis toxin, tetanus toxin, soybean Bowman-Birk protease inhibitor, Pseudomonas aeruginosa exotoxin, alloricin, saporin, modicine, geranin, abrin A chain, modicine A chain, alpha-sarcin, Aleurites fordii protein, dianthin protein, Phytolacca americana protein, momordica charantia inhibitor, curcin, crocin, saponaria officinalis inhibitor, gelonin, mitogelin, restrictocin, phenomycin, enomycin toxin, ribonuclease (RNase), DNase I, Staphylococcal enterotoxin A, Phytolacca americana antiviral protein, diphtheria bacterium toxin, and Pseudomonas aeruginosa endotoxin.

[0267] In some cases, the cytotoxin is a compound that inhibits microtubule formation in eukaryotic cells. Such agents include, for example, maytansinoids, benzodiazepines, taxoids, CC-1065, duocarmycin, duocarmycin analogs, calicheamicin, dolastatin, dolastatin analogs, auristatin, tomatimycin, and leptomycin, or prodrugs of any one of the foregoing. Maytansinoid compounds include, for example, N(2’)-deacetyl-N(2’)-(3-mercapto-1-oxopropyl)-maytansin (DM1), N(2’)-deacetyl-N(2’)-(4-mercapto-1-oxopentyl)-maytansin (DM3), and N(2’)-deacetyl-N2-(4-mercapto-4-methyl-1-oxopentyl)-maytansin (DM4). Benzodiazepines include, for example, indolinobenzodiazepines and oxazolidinobenzodiazepines.

[0268] Exemplary T cell regulatory antigen-presenting polypeptides The following are non-limiting examples of the multimeric TMAPPs of the present disclosure that employ the following polypeptide pairs: 1) the 3003 polypeptide shown in FIG. 40C and the 2639 polypeptide shown in FIG. 40B, 2) the 3004 polypeptide shown in FIG. 40D and the 2639 polypeptide shown in FIG. 40B, 3) the 3005 polypeptide shown in FIG. 40E and the 2639 polypeptide shown in FIG. 40B, 4) the 2932 polypeptide shown in FIG. 40A and the 3213 polypeptide shown in FIG. 40F, and 5) the 2932 polypeptide shown in FIG. 40A and the 3214 polypeptide shown in FIG. 40G.

[0269] 1) 3003 + 2639. In some cases, the multimeric TMAPP of the present disclosure comprises: a) a first polypeptide comprising, in order from the N-terminus to the C-terminus, i) an immunomodulatory polypeptide, ii) a linker, iii) an HLA α1 polypeptide, iv) an HLA α2 polypeptide, and v) an Ig Fc polypeptide; and b) a second polypeptide comprising, in order from the N-terminus to the C-terminus, i) a peptide presenting an epitope capable of binding to a TCR, ii) a linker, iii) an HLA β1 polypeptide, and iv) an HLA β2 polypeptide. In some cases, the immunomodulatory polypeptide is a PD-L1 polypeptide. In some cases, the PD-L1 polypeptide has the following amino acid sequence: TIFF0007691927000262.tif24151 and has a length of 220 amino acids. In some cases, both the HLA α1 and HLA α2 polypeptides have the following amino acid sequence: TIFF0007691927000263.tif24151 and have a length of 189 amino acids. In some cases, the Ig Fc polypeptide is an IgG1 Fc polypeptide comprising the L14A and L15A substitutions. For example, the Ig Fc polypeptide has the following amino acid sequence: TIFF0007691927000264.tif31151 and has a length of 226 amino acids. In some cases, the peptide epitope is a self-peptide. For example, in some cases, the peptide epitope is SLQPLALEGSLQSRG (SEQ ID NO: 78). In some cases, both the HLA β1 and β2 polypeptides have the following amino acid sequence: TIFF0007691927000265.tif24150 and have a length of 199 amino acids. A suitable linker can be TIFF0007691927000266.tif11128.

[0270] 2) 3004 + 2639. In some cases, the multimeric TMAPP of the present disclosure comprises: a) a first polypeptide comprising, in order from the N-terminus to the C-terminus, i) an HLA α1 polypeptide, ii) an HLA α2 polypeptide, iii) a linker, iv) an immunomodulatory polypeptide, v) a linker, and vi) an Ig Fc polypeptide; and b) a second polypeptide comprising, in order from the N-terminus to the C-terminus, i) a peptide presenting an epitope capable of binding to a TCR, ii) a linker, iii) an HLA β1 polypeptide, and iv) an HLA β2 polypeptide. In some cases, the immunomodulatory polypeptide is a PD-L1 polypeptide. In some cases, the PD-L1 polypeptide has the following amino acid sequence: TIFF0007691927000267.tif24151 and has a length of 220 amino acids. In some cases, both the HLA α1 and HLA α2 polypeptides have the following amino acid sequence: TIFF0007691927000268.tif24151 and has a length of 189 amino acids. In some cases, the Ig Fc polypeptide is an IgG1 Fc polypeptide comprising the substitutions L14A and L15A. For example, the Ig Fc polypeptide has the following amino acid sequence: TIFF0007691927000269.tif31151 and has a length of 226 amino acids. In some cases, the peptide epitope is a self-peptide. For example, in some cases, the peptide epitope is TIFF0007691927000270.tif4128. In some cases, both the HLA β1 and β2 polypeptides have the following amino acid sequence: TIFF0007691927000271.tif24150 and has a length of 199 amino acids. A suitable linker can be TIFF0007691927000272.tif11128.

[0271] 3) 3005 + 2639. In some cases, the multimeric TMAPP of the present disclosure comprises: a) a first polypeptide comprising, in order from the N-terminus to the C-terminus, i) an HLA α1 polypeptide, ii) an HLA α2 polypeptide, iii) a linker, iv) an Ig Fc polypeptide, and v) an immunomodulatory polypeptide; and b) a second polypeptide comprising, in order from the N-terminus to the C-terminus, i) a peptide presenting an epitope capable of binding to a TCR, ii) a linker, iii) an HLA β1 polypeptide, and iv) an HLA β2 polypeptide. In some cases, the immunomodulatory polypeptide is a PD-L1 polypeptide. In some cases, the PD-L1 polypeptide has the following amino acid sequence: TIFF0007691927000273.tif24151 and has a length of 220 amino acids. In some cases, both the HLA α1 and HLA α2 polypeptides have the following amino acid sequence: TIFF0007691927000274.tif24151 and has a length of 189 amino acids. In some cases, the Ig Fc polypeptide is an IgG1 Fc polypeptide comprising the substitutions L14A and L15A. For example, the Ig Fc polypeptide has the following amino acid sequence: TIFF0007691927000275.tif31151 and has a length of 226 amino acids. In some cases, the peptide epitope is a self-peptide. For example, in some cases, the peptide epitope is TIFF0007691927000276.tif4128. In some cases, both the HLA β1 and β2 polypeptides have the following amino acid sequence: TIFF0007691927000277.tif24150 and has a length of 199 amino acids. A suitable linker can be TIFF0007691927000278.tif11128.

[0272] 4) 2932 + 3213. In some cases, the multimeric TMAPP of the present disclosure comprises a first polypeptide comprising, in order from the N-terminus to the C-terminus, i) an HLA α1 polypeptide, ii) an HLA α2 polypeptide, iii) a linker, and iv) an Ig Fc polypeptide, and a second polypeptide comprising, in order from the N-terminus to the C-terminus, i) an immunomodulatory polypeptide, ii) a linker, iii) a peptide presenting an epitope capable of binding to a TCR, iv) a linker, v) an HLA β1 polypeptide, and vi) an HLA β2 polypeptide. In some cases, the immunomodulatory polypeptide is a PD-L1 polypeptide. In some cases, the PD-L1 polypeptide has the following amino acid sequence: TIFF0007691927000279.tif24151 and has a length of 220 amino acids. In some cases, both the HLA α1 and HLA α2 polypeptides have the following amino acid sequence: TIFF0007691927000280.tif24151 and has a length of 189 amino acids. In some cases, the Ig Fc polypeptide is an IgG1 Fc polypeptide comprising the substitutions L14A and L15A. For example, the Ig Fc polypeptide has the following amino acid sequence: TIFF0007691927000281.tif31151 and has a length of 226 amino acids. In some cases, the peptide epitope is a self-peptide. For example, in some cases, the peptide epitope is TIFF0007691927000282.tif4128. In some cases, both the HLA β1 and β2 polypeptides have the following amino acid sequence: TIFF0007691927000283.tif24150 and has a length of 199 amino acids. A suitable linker can be TIFF0007691927000284.tif11128.

[0273] 5) 2932 + 3214. In some cases, the multimeric TMAPP of the present disclosure comprises: a) a first polypeptide comprising, in order from the N-terminus to the C-terminus, i) an HLA α1 polypeptide, ii) an HLA α2 polypeptide, iii) a linker, and iv) an Ig Fc polypeptide; and b) a second polypeptide comprising, in order from the N-terminus to the C-terminus, i) a peptide presenting an epitope capable of binding to a TCR, ii) a linker, iii) an HLA β1 polypeptide, iv) an HLA β2 polypeptide, and v) an immunomodulatory polypeptide. In some cases, the immunomodulatory polypeptide is a PD-L1 polypeptide. In some cases, the PD-L1 polypeptide has the following amino acid sequence: TIFF0007691927000285.tif24151 and has a length of 220 amino acids. In some cases, both the HLA α1 and HLA α2 polypeptides have the following amino acid sequence: TIFF0007691927000286.tif24151 and has a length of 189 amino acids. In some cases, the Ig Fc polypeptide is an IgG1 Fc polypeptide comprising the substitutions L14A and L15A. For example, the Ig Fc polypeptide has the following amino acid sequence: TIFF0007691927000287.tif31151 and has a length of 226 amino acids. In some cases, the peptide epitope is a self-peptide. For example, in some cases, the peptide epitope is TIFF0007691927000288.tif4128. In some cases, both the HLA β1 and β2 polypeptides have the following amino acid sequence: TIFF0007691927000289.tif24150 and has a length of 199 amino acids. A suitable linker can be TIFF0007691927000290.tif11128.

[0274] Additional exemplary multimeric TMAPP The following are non-limiting examples of the multimeric TMAPPs of the present disclosure that employ the following polypeptide pairs: 1) the 1452 polypeptide shown in FIG. 26A and the 1661 polypeptide shown in FIG. 34A, 2) the 1659 polypeptide shown in FIG. 33A and the 1664 polypeptide shown in FIG. 35A, 3) the 1637 polypeptide shown in the figure and the 1408 polypeptide shown in FIG. 25A, the 1639 polypeptide shown in FIG. 31A and the 1640 polypeptide shown in FIG. 32A, and 5) the 1711 polypeptide shown in FIG. 37A and the 1705 polypeptide shown in FIG. 38A. The TMAPPs administered to an individual in need thereof generally do not contain a leader sequence or a histidine tag as shown in the figures above.

[0275] 1) 1452 + 1661. In some cases, the multimeric TMAPP of the present disclosure includes, in order from the N-terminus to the C-terminus: a) a first polypeptide comprising, i) an epitope, ii) a linker, iii) an HLA β1 polypeptide, iv) an HLA α1 polypeptide, v) an HLA α2 polypeptide, vi) a dimerization polypeptide, and vii) an Ig Fc polypeptide; and b) a second polypeptide comprising, in order from the N-terminus to the C-terminus, i) a first immunomodulatory polypeptide (e.g., a variant immunomodulatory polypeptide having low affinity for its homologous immunocoregulatory polypeptide), ii) a second immunomodulatory polypeptide (e.g., a variant immunomodulatory polypeptide having low affinity for its homologous immunocoregulatory polypeptide), iii) an HLA β2 polypeptide, and iv) a dimerization polypeptide. As one non-limiting example, the multimeric TMAPP of the present disclosure may include, in order from the N-terminus to the C-terminus: a) a first polypeptide comprising, i) an epitope, ii) a linker, iii) an HLA DRB1 β1 polypeptide, iv) an HLA DRA α1 polypeptide, v) an HLA DRA α2 polypeptide, vi) a leucine zipper dimerization polypeptide, and vii) an IgG1 Fc polypeptide; and b) a second polypeptide comprising, in order from the N-terminus to the C-terminus, i) a first immunomodulatory polypeptide (e.g., a variant IL-2 polypeptide comprising the substitutions H16A and F42A), ii) a second immunomodulatory polypeptide (e.g., a variant IL-2 polypeptide comprising the substitutions H16A and F42A), iii) an HLA DRB β2 polypeptide, and iv) a leucine zipper dimerization polypeptide. In some cases, the epitope is a hemagglutinin epitope, e.g., PKYVKQNTLKLAT (SEQ ID NO: 303). In some cases, the variant IL-2 polypeptide has the following amino acid sequence: TIFF0007691927000291.tif17151 (the substitutions H16A and F42A are underlined). In some cases, the HLA-DRB1 β1 polypeptide has the following amino acid sequence: It includes TIFF0007691927000292.tif11151. In some cases, the HLA DRA α1 polypeptide has the following amino acid sequence It includes TIFF0007691927000293.tif11151. In some cases, the HLA DRA α2 polypeptide has the following amino acid sequence It includes TIFF0007691927000294.tif11149. In some cases, the leucine zipper dimerization polypeptide has the following amino acid sequence: It includes TIFF0007691927000295.tif4150. In some cases, the IgG1 Fc polypeptide has the following amino acid sequence: It includes TIFF0007691927000296.tif31151. In some cases, the first polypeptide includes the 1452 - amino acid sequence shown in Figure 26A without a leader sequence, and without a C - terminal linker and histidine tag. For example, in some cases, the first polypeptide includes amino acids 21 - 628 of the 1452 - amino acid sequence shown in Figure 26A. In some cases, the second polypeptide includes the 1661 - amino acid sequence shown in Figure 34A without a leader sequence. For example, in some cases, the second polypeptide includes amino acids 21 - 491 of the amino acid sequence shown in Figure 34A. In some cases, the epitope of the first polypeptide is not PKYVKQNTLKLAT (SEQ ID NO: 303), but instead is replaced by a different epitope.

[0276] 2) 1659 + 1664. In some cases, the multimeric TMAPP of the present disclosure comprises: a) a first polypeptide comprising, in order from the N-terminus to the C-terminus, i) an epitope, ii) an HLA β1 polypeptide, iii) an HLA α1 polypeptide, iv) an HLA α2 polypeptide, and v) an Ig Fc polypeptide; and b) a second polypeptide comprising, in order from the N-terminus to the C-terminus, i) a first immunomodulatory polypeptide (e.g., a variant immunomodulatory polypeptide having low affinity for its cognate co-immunomodulatory polypeptide), ii) a second immunomodulatory polypeptide (e.g., a variant immunomodulatory polypeptide having low affinity for its cognate co-immunomodulatory polypeptide), and iii) an HLA β2 polypeptide. As one non-limiting example, the multimeric TMAPP of the present disclosure may comprise: a) a first polypeptide comprising, in order from the N-terminus to the C-terminus, i) an epitope, ii) an HLA DRB1 β1 polypeptide, iii) an HLA DRA α1 polypeptide, iv) an HLA DRA α2 polypeptide, and v) an IgG1 Fc polypeptide; and b) a second polypeptide comprising, in order from...

Claims

1. A multimeric T cell regulatory antigen-presenting polypeptide, a) a first polypeptide comprising, in order from the N-terminus to the C-terminus, i) a proinsulin polypeptide epitope capable of binding to a T cell receptor (TCR), said peptide epitope having a length of 10 to 15 amino acids or 15 to 20 amino acids, ii) an MHC class II β1 polypeptide, and iii) an MHC class II β2 polypeptide said first polypeptide, and b) a second polypeptide comprising, in order from the N-terminus to the C-terminus, i) a PD-L1 immunomodulatory polypeptide, ii) an MHC class II α1 polypeptide, iii) an MHC class II α2 polypeptide, and iv) an immunoglobulin (Ig) Fc polypeptide said second polypeptide, and comprising, wherein the MHC class II α1 polypeptide and the MHC class II α2 polypeptide comprise an MHC class II α-chain polypeptide selected from the group consisting of DRA*0101 polypeptide, DQA1*05:01 polypeptide, and DQA1*03:01 polypeptide, and the MHC class II β1 polypeptide and the MHC class II β2 polypeptide comprise an MHC class II β-chain polypeptide selected from the group consisting of DRB1*04:01 polypeptide, DRB1*03:01 polypeptide, DRB1*04:02 polypeptide, DRB1*04:05 polypeptide, DQB1*02:01 polypeptide, and DQB1*03:02 polypeptide, said multimeric T cell regulatory antigen-presenting polypeptide.

2. a) the MHC class II α1 polypeptide comprises the DRA1*01:01 polypeptide and the MHC class II β1 polypeptide comprises the DRB1*04:01 polypeptide, or b) the MHC class II α1 polypeptide comprises the DQA1*0501 polypeptide and the MHC class II β1 polypeptide comprises the DQB1*0201 polypeptide, or c) the MHC class II α1 polypeptide comprises the DQA1*0301 polypeptide and the MHC class II β1 polypeptide comprises the DQB1*0302 polypeptide, The multimeric T cell regulatory antigen-presenting polypeptide according to Claim 1.

3. The immunomodulatory polypeptide is a) comprising the amino acid sequence of a natural immunomodulatory polypeptide, or b) A variant immunomodulatory polypeptide comprising an amino acid sequence having 1 to 10 amino acid substitutions as compared to the amino acid sequence of a natural immunomodulatory polypeptide, wherein said variant immunomodulatory polypeptide has a reduced affinity for said immunocoregulatory polypeptide as compared to the affinity of said natural immunomodulatory polypeptide for the immunocoregulatory polypeptide. The T cell regulatory antigen-presenting polypeptide according to claim 1 or 2.

4. The T cell regulatory antigen-presenting polypeptide according to any one of claims 1 to 3, wherein said proinsulin peptide epitope comprises the amino acid sequence SLQP LALE GSLQ SRG (SEQ ID NO: 78).

5. a) Said first polypeptide, in order from the N-terminus to the C-terminus, i) a proinsulin peptide epitope comprising the amino acid sequence SLQP LALE GSLQ SRG (SEQ ID NO: 78); ii) a linker; and iii) an MHC class II beta chain polypeptide comprising, b) Said second polypeptide, in order from the N-terminus to the C-terminus, i) a PD-L1 immunomodulatory polypeptide; ii) a linker; iii) an MHC class II alpha chain polypeptide; iv) a linker; and v) an Ig Fc polypeptide comprising an amino acid sequence having at least 90% amino acid sequence identity to the IgG1 Fc amino acid sequence set forth in SEQ ID NO: 378 comprising, The T cell regulatory antigen-presenting polypeptide according to claim 1.

6. The T cell regulatory antigen-presenting polypeptide according to claim 5, wherein said Ig Fc polypeptide comprises Ala at position 14 and Ala at position 15 based on the amino acid numbers set forth in SEQ ID NO:

378.

7. Said MHC class II beta chain polypeptide comprises the DRB1*0401 amino acid sequence set forth in SEQ ID NO: 58, Said MHC class II alpha chain polypeptide comprises the DRA1*0101 amino acid sequence set forth in SEQ ID NO: 57, The T cell regulatory antigen-presenting polypeptide according to claim 5 or 6.

8. A pharmaceutical composition comprising the T cell regulatory antigen-presenting polypeptide according to any one of claims 1 to 7.

9. One or more nucleic acids comprising a nucleotide sequence encoding the T cell regulatory antigen-presenting polypeptide according to any one of claims 1 to 7.

10. One or more expression vectors comprising the one or more nucleic acids according to claim 9.

11. A host cell genetically modified by one or more nucleic acids according to claim 9 or one or more expression vectors according to claim 10.

12. CD4 specific for type 1 diabetes-related epitopes of an individual ＋ and / or CD8 ＋ The pharmaceutical composition according to claim 8 for use in a method of reducing the number and / or activity of autoreactive T cells, wherein the method comprises contacting said CD4 ＋ T cells with a T cell regulatory antigen-presenting polypeptide according to any one of claims 1 to 7, and said contacting reduces the number and / or activity of said CD4 ＋ and / or CD8 ＋ T cells, said pharmaceutical composition.

13. A pharmaceutical composition according to claim 8 for use in a method of treating type 1 diabetes in an individual, the method comprising administering to an individual in need thereof an effective amount of a T cell regulatory antigen-presenting polypeptide according to any one of claims 1 to 7.

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