MHC class II T cell modulating polypeptides and methods of use thereof for treating type 1 diabetes mellitus (T1D) - Patent Application 20070123333

Abstract

The present disclosure provides T cell modulating polypeptides (TMPs) comprising a type 1 diabetes (T1D) associated peptide epitope, an MHC class II polypeptide, one or more immune modulating polypeptides, a TGF-β polypeptide, and a masking polypeptide. The TMPs of the present disclosure are useful for modulating the activity of T cells. Thus, the present disclosure provides compositions and methods for modulating the activity of T cells, as well as compositions and methods for treating individuals with T1D.

Description

[Technical Field]

[0001] cross reference This application claims the benefit of U.S. Provisional Patent Application No. 63 / 177,641, filed April 21, 2021, which is incorporated herein by reference in its entirety.

[0002] Incorporation by reference of sequence listing provided as a text file A text file "CUEB-142WO_SEQ_LIST_ST25.txt", created on March 25, 2022, and having a size of 498 KB, is provided with this application as a Sequence Listing. The contents of the text file are incorporated herein by reference in their entirety. [Background technology]

[0003] preface 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 are responsible for capturing foreign or abnormal proteins (e.g., resulting from genetic mutations in cancer cells) and degrading them into small fragments suitable for signaling for inspection by the larger immune system, including T cells. Specifically, APCs degrade proteins into small peptide fragments, which then pair with proteins of the major histocompatibility complex ("MHC") and present them on the cell surface. Cell surface presentation of MHC containing peptide fragments, also known as T cell epitopes, provides a fundamental stepping stone for T cell surveillance, enabling specific recognition. Peptide fragments can be derived from pathogens, tumors, or natural host proteins (self-proteins). Additionally, APCs can recognize other foreign components, such as bacterial toxins, viral proteins, DNA, and RNA, whose presence signals an increased threat level. APCs relay this information to T cells via additional costimulatory signals to generate a more effective response.

[0004] T cells recognize peptide-major histocompatibility complex ("pMHC") complexes through 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 adequately address a world full of potential threats, the human body contains a large number of distinct T cells (approximately 10,000,000), each with distinct TCRs. Furthermore, any given T cell specific for a particular T cell peptide initially represents only a small portion of the total T cell population. While normally dormant and limited in number, T cells with a specific TCR can be easily activated and expanded by APCs to produce extremely potent T cell responses 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 exemplified below. Conversely, widespread non-specific activation of overactive T cell responses against self- or common antigens can result in T cells that inappropriately attack and destroy healthy tissues or cells. Summary of the Invention

[0005] overview The present disclosure provides T cell modulating polypeptides (TMPs) comprising a type 1 diabetes (T1D)-associated peptide epitope, an MHC class II polypeptide, one or more immunomodulatory polypeptides, a TGF-β polypeptide, and a masking polypeptide. The TMPs of the present disclosure are useful for modulating T cell activity. Accordingly, the present disclosure provides compositions and methods for modulating T cell activity, as well as compositions and methods for treating individuals with T1D. [Brief explanation of the drawings]

[0006] [Figure 1A] 1 shows a schematic diagram of a TMP of the present disclosure. [Figure 1B] 1 shows a schematic diagram of a TMP of the present disclosure. [Figure 1C] 1 shows a schematic diagram of a TMP of the present disclosure. [Figure 1D]1 shows a schematic diagram of a TMP of the present disclosure. [Figure 1E] 1 shows a schematic diagram of a TMP of the present disclosure. [Figure 1F] 1 shows a schematic diagram of a TMP of the present disclosure. [Figure 1G] 1 shows a schematic diagram of a TMP of the present disclosure. [Figure 1H] 1 shows a schematic diagram of a TMP of the present disclosure. [Figure 2] 1 shows a schematic diagram of a TMP of the disclosure, with an immunomodulatory polypeptide at position 2. [Figure 3] 1 shows a schematic diagram of a TMP of the disclosure, with an immunomodulatory polypeptide at position 3. [Figure 4] 1 shows the amino acid sequence of the HLA class II DRA (sometimes referred to as DRA1) alpha chain (SEQ ID NO: 1). [Figure 5-1] FIG. 5 shows the amino acid sequence of the HLA class II DRB1 β chain. [Figure 5-2] See description of Figure 5-1. [Figure 5-3] See description of Figure 5-1. [Figure 5-4] See description of Figure 5-1. [Figure 6] 1 shows the amino acid sequence of the HLA class II DRB3 β chain (from top to bottom: SEQ ID NOs: 37 to 40). [Figure 7-1] FIG. 7 shows the amino acid sequence of the HLA class II DRB4 β chain (SEQ ID NOs: 41 to 42). [Figure 7-2] See description of Figure 7-1. [Figure 8] 1 shows the amino acid sequence of the HLA class II DRB5 β chain (SEQ ID NO: 43). [Figure 9] The amino acid sequence of the HLA class II DMA α chain (SEQ ID NO: 44) is shown. [Figure 10] The amino acid sequence of the HLA class II DMB β chain (SEQ ID NO: 45) is shown. [Figure 11] The amino acid sequence of the HLA class II DOA α chain (SEQ ID NO: 46) is shown. [Figure 12]1 shows the amino acid sequence of the HLA class II DOB β chain (SEQ ID NO: 47). [Figure 13] The amino acid sequences of the HLA class II DPA1 α chain (SEQ ID NOs: 48 to 49) are shown. [Figure 14-1] FIG. 14 shows the amino acid sequence of the HLA class II DPB1 β chain (from top to bottom: SEQ ID NOs: 50 to 61). [Figure 14-2] See description of Figure 14-1. [Figure 15-1] FIG. 15 shows the amino acid sequence of the HLA class II DQA1 α chain (from top to bottom: SEQ ID NOs: 62 to 72). [Figure 15-2] See description of Figure 15-1. [Figure 16] The amino acid sequence of the HLA class II DQA2 α chain (SEQ ID NO: 73) is shown. [Figure 17-1] FIG. 17 shows the amino acid sequence of the HLA class II DQB1 β chain (from top to bottom: SEQ ID NOs: 74 to 85). [Figure 17-2] See description of Figure 17-1. [Figure 18] A to B show the amino acid sequences of the HLA class II DQB2 β chain (SEQ ID NO: 86 and SEQ ID NO: 87, respectively). [Figure 19A] Figures 19A-19O show the amino acid sequences of wild-type (Figure 19A) and variant (Figures 19B-9N) DRA*0101 α chains. [Figure 19B] See legend to Figure 19A. [Figure 19C] See legend to Figure 19A. [Figure 19D] See legend to Figure 19A. [Figure 19E] See legend to Figure 19A. [Figure 19F] See legend to Figure 19A. [Figure 19G] See legend to Figure 19A. [Figure 19H] See legend to Figure 19A. [Figure 19I] See legend to Figure 19A. [Figure 19J] See legend to Figure 19A. [Figure 19K] See legend to Figure 19A. [Figure 19L] See legend to Figure 19A. [Figure 19M] See legend to Figure 19A. [Figure 19N] See legend to Figure 19A. [Figure 19O] See legend to Figure 19A. [Figure 20A] The amino acid sequence of the wild-type DRB1*0401 β chain is shown. [Figure 20B] The amino acid sequence of the variant DRB1*0401 β chain is shown. [Figure 20C] The amino acid sequence of the variant DRB1*0401 β chain is shown. [Figure 20D] The amino acid sequence of the variant DRB1*0401 β chain is shown. [Figure 20E] The amino acid sequence of the variant DRB1*0401 β chain is shown. [Figure 20F] The amino acid sequence of the variant DRB1*0401 β chain is shown. [Figure 20G] The amino acid sequence of the variant DRB1*0401 β chain is shown. [Figure 20H] The amino acid sequence of the variant DRB1*0401 β chain is shown. [Figure 20I] The amino acid sequence of the variant DRB1*0401 β chain is shown. [Figure 20J] The amino acid sequence of the variant DRB1*0401 β chain is shown. [Figure 21A] 1 shows the amino acid sequence of an immunoglobulin Fc polypeptide. [Figure 21B] 1 shows the amino acid sequence of an immunoglobulin Fc polypeptide. [Figure 21C] 1 shows the amino acid sequence of an immunoglobulin Fc polypeptide. [Figure 21D] 1 shows the amino acid sequence of an immunoglobulin Fc polypeptide. [Figure 21E]1 shows the amino acid sequence of an immunoglobulin Fc polypeptide. [Figure 21F] 1 shows the amino acid sequence of an immunoglobulin Fc polypeptide. [Figure 21G] 1 shows the amino acid sequence of an immunoglobulin Fc polypeptide. [Figure 21H] 1 shows the amino acid sequence of an immunoglobulin Fc polypeptide. [Figure 21I] 1 shows the amino acid sequence of an immunoglobulin Fc polypeptide. [Figure 21J] 1 shows the amino acid sequence of an immunoglobulin Fc polypeptide. [Figure 21K] 1 shows the amino acid sequence of an immunoglobulin Fc polypeptide. [Figure 21L] 1 shows the amino acid sequence of an immunoglobulin Fc polypeptide. [Figure 21M] 1 shows the amino acid sequence of an immunoglobulin Fc polypeptide. [Figure 22A] 1 shows the amino acid sequence of the wild-type IL-2 polypeptide. [Figure 22B] The amino acid sequence of IL-2Rα is shown. [Figure 22C] The amino acid sequence of IL-2Rβ is shown. [Figure 22D] The amino acid sequence of IL-2Rγ is shown. [Figure 22E] 1 shows the amino acid sequence of the PD-L1 ectodomain polypeptide. [Figure 22F] The amino acid sequence of the 4-1BBL polypeptide is shown. [Figure 23A] The amino acid sequences of the three different isoforms of TGF-β preproprotein are shown. [Figure 23B] The amino acid sequence of the mature TGF-β protein is shown. [Figure 23C] The amino acid sequences of the three different isoforms of TGF-β preproprotein are shown. [Figure 23D] The amino acid sequence of the mature TGF-β protein is shown. [Figure 23E]The amino acid sequences of the three different isoforms of TGF-β preproprotein are shown. [Figure 23F] The amino acid sequence of the mature TGF-β protein is shown. [Figure 23G] The amino acid sequence of the mature form of TGF-β3 containing the C77S substitution is shown. [Figure 24-1] Figure 24 shows an alignment of the amino acid sequences of TGF-β isoforms 1 to 3, with residues corresponding to mature TGF-β2 shown in bold, except that amino acid residues Lys25, Cys77, Ile92, and Lys94 of TGF-β2 and the corresponding residues in the other forms of TGF-β isoforms 1 and 3 are underlined and italicized rather than bolded. [Figure 24-2] See description of Figure 24-1. [Figure 25A] The amino acid sequence of TGF-β receptor type 1 (TβRI) is shown. [Figure 25B] The amino acid sequence of the ectodomain of TGF-β receptor type 1 (TβRI) is shown. [Figure 25C] The amino acid sequence of TGF-β receptor type 2 (TβRII) is shown. [Figure 25D] The amino acid sequence of the ectodomain of TGF-β receptor type 2 (TβRII) is shown. [Figure 25E] The amino acid sequence of TGF-β receptor type 2 (TβRII) is shown. [Figure 25F] The amino acid sequence of the ectodomain of TGF-β receptor type 2 (TβRII) is shown. [Figure 25G] The amino acid sequence of a fragment of the ectodomain of TGF-β receptor type 2 (TβRII) is shown. [Figure 25H] The amino acid sequence of a fragment of the ectodomain of TGF-β receptor type 2 (TβRII) is shown. [Figure 25I] The amino acid sequence of a fragment of the ectodomain of TGF-β receptor type 2 (TβRII) is shown. [Figure 25J] The amino acid sequence of a fragment of the ectodomain of TGF-β receptor type 2 (TβRII) is shown. [Figure 25K]The amino acid sequence of TGF-β receptor type 3 (TβRIII) is shown. [Figure 25L] The amino acid sequence of the ectodomain of TGF-β receptor type 3 (TβRIII) is shown. [Figure 25M] The amino acid sequence of TGF-β receptor type 3 (TβRIII) is shown. [Figure 25N] The amino acid sequence of the ectodomain of TGF-β receptor type 3 (TβRIII) is shown. [Figure 26A] 1 shows the amino acid sequence of an exemplary TMP (construct 4415-4417). [Figure 26B] 1 shows the amino acid sequence of an exemplary TMP (construct 4415-4417). [Figure 27A] 1 shows the amino acid sequence of an exemplary TMP (construct 4418-4420). [Figure 27B] 1 shows the amino acid sequence of an exemplary TMP (construct 4418-4420). [Figure 28] 1 shows a schematic diagram of a TMP comprising a scaffold polypeptide with mutually specific binding sequences. [Figure 29A] The amino acid sequence of TMP lacking MOD (construct 4415-4416) is shown. [Figure 29B] The amino acid sequence of TMP lacking MOD (construct 4415-4416) is shown. [Figure 30A] The amino acid sequence of TMP lacking MOD (construct 4418-4419) is shown. [Figure 30B] The amino acid sequence of TMP lacking MOD (construct 4418-4419) is shown. [Figure 31A] The amino acid sequence of TMP lacking MOD (construct 3858-3859) is shown. [Figure 31B] The amino acid sequence of TMP lacking MOD (construct 3858-3859) is shown. [Figure 32A] 1 shows the amino acid sequence of an exemplary TMP (construct 3858-3869). [Figure 32B]1 shows the amino acid sequence of an exemplary TMP (construct 3858-3869). [Figure 33A] 1 shows the amino acid sequence of an exemplary TMP (construct 3870-3871). [Figure 33B] 1 shows the amino acid sequence of an exemplary TMP (construct 3870-3871). [Figure 34A] A schematic diagram of a particular TMP is shown. [Figure 34B] 1 shows an SDS-PAGE analysis of certain TMPs. [Figure 35] The effect of specific TMPs on the percentage of FoxP3-positive cells in the T cell population is shown. [Figure 36] 1 shows an SDS-PAGE analysis of certain TMPs. DETAILED DESCRIPTION OF THE INVENTION

[0007] definition The terms "polynucleotide" and "nucleic acid" are used interchangeably herein to refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, the terms include, but are not limited to, single-, double-, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers containing purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases.

[0008] The terms "peptide," "polypeptide," and "protein" are used interchangeably herein to refer to polymeric forms of amino acids of any length, including coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides with modified peptide backbones. Furthermore, as used herein, "polypeptide" refers to proteins containing modifications to the native sequence, such as deletions, additions, and substitutions (generally conservative substitutions in nature, as known to those of skill in the art), so long as the protein retains the desired activity. These modifications may be intentional, such as through site-directed mutagenesis, or accidental, such as mutations by the host producing the protein or errors during polymerase chain reaction (PCR) amplification or other recombinant DNA techniques. Reference herein to a particular residue or residue number of a known polypeptide, e.g., position 72 or 75 of the human DRA MHC class II polypeptide, is understood to refer to the amino acid at that position in the wild-type polypeptide (i.e., I72 or K75). To the extent that the sequence of a wild-type polypeptide is modified by either the addition or deletion of one or more amino acids, those skilled in the art will understand that a reference to a particular residue or residue number will be correspondingly modified to refer to the same particular amino acid in the modified polypeptide, which is understood to be at the position number after modification. For example, if a human DRA MHC class II polypeptide is modified by the addition of one amino acid at the N-terminus, a reference to position 72 or 75, or residue 172 or K75, will be understood to refer to the amino acid at position 73 or 76, or residues 173 and K76. Similarly, reference herein to a substitution of a particular amino acid at a particular position, e.g., I72C, will be understood to refer to the substitution of a cysteine ​​for the amino acid at position 72 of the wild-type polypeptide, i.e., isoleucine. For example, if a wild-type polypeptide is modified to change the amino acid at position 72 from isoleucine to an alternative amino acid, a reference to I72C will be understood to refer to a cysteine ​​substitution of the alternative amino acid.In such cases, if the polypeptide is further modified by the addition or deletion of one or more amino acids, a reference to I72C is understood to refer to the substitution of the alternative amino acid at the modified position number with a cysteine. Reference to a "non-native Cys residue" in a polypeptide, e.g., a DRA MHC class II polypeptide, means that the polypeptide contains a Cys residue at a position where no Cys is present in the corresponding wild-type polypeptide. This is an amino acid present in the wild-type sequence, e.g., DRA. * Positions 72 or 75 of the O101 polypeptide (see Figures 13G and 13H) were * This can be achieved by conventional protein engineering, substituting cysteine ​​for the isoleucine (I) or lysine (K) residues present in the O101 polypeptide (see Figure 13A).

[0009] A polynucleotide or polypeptide having a certain percentage of "sequence identity" to another polynucleotide or polypeptide means that, when aligned and compared, that percentage of bases or amino acids are the same and in the same relative positions. Sequence identity can be determined in a number of different ways. To determine sequence identity, 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 sites on the World Wide Web, including ncbi.nlm.nili.gov / BLAST, ebi.ac.uk / Tools / msa / tcoffee / , ebi.ac.uk / Tools / msa / muscle / , and mafft.cbrc.jp / alignment / software / . See, e.g., Altschul et al. (1990) J. Mol. Biol. 215:403-10. Unless otherwise specified, "sequence identity" referred to herein is determined by BLAST (Basic Local Alignment Search Tool) as described in Altschul et al. (1990) J. Mol. Biol. 215:403.

[0010] As used herein, "masked" means that a molecule (e.g., a masked polypeptide or masked protein) is bound or otherwise engaged by a masking molecule (e.g., a polypeptide, protein, or protein fragment), thereby limiting the availability of the masked molecule to other proteins (e.g., cell surface receptors) that also have affinity for the molecule.

[0011] A mutually specific binding sequence is a dimerization sequence that allows asymmetric pairing (heterodimerization) of polypeptides. A mutually specific binding sequence prefers heterodimer formation (as opposed to homodimer formation) with its cognate binding partner, the corresponding mutually specific binding sequence(s). A key-in-hole (or key-into-hole) Fc polypeptide pair is an example of a mutually specific binding sequence and its corresponding mutually specific binding sequence.

[0012] "Tandem," as used herein to describe the arrangement of MOD polypeptides, means that two or more MODs are arranged adjacently on a polypeptide, separated, if at all, by a linker.

[0013] As used herein, the term "in vivo" refers to any process or procedure that occurs within the body, for example, within the body of a T1D patient.

[0014] As used herein, "in vitro" refers to any process or procedure that occurs outside the body.

[0015] The term "conservative amino acid substitution" refers to the interchangeability of amino acid residues in proteins with similar side chains. For example, the group of amino acids with aliphatic side chains consists of glycine, alanine, valine, leucine, and isoleucine; the group of amino acids with aliphatic hydroxyl side chains consists of serine and threonine; the group of amino acids with amide-containing side chains consists of asparagine and glutamine; the group of amino acids with aromatic side chains consists of phenylalanine, tyrosine, and tryptophan; the group of amino acids with basic side chains consists of lysine, arginine, and histidine; the group of amino acids with acidic side chains consists of glutamic acid and aspartic acid; and the group of amino acids with sulfur-containing side chains consists of cysteine ​​and methionine. Exemplary conservative amino acid substitutions are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine-glycine, and asparagine-glutamine.

[0016] The term "binding," as used herein (e.g., with respect to binding of a T cell regulatory antigen-presenting polypeptide to a polypeptide on a T cell (e.g., a T cell receptor)), refers to a non-covalent interaction between two molecules. Non-covalent binding refers to a direct association between two molecules, for example, by electrostatic, hydrophobic, ionic, and / or hydrogen-bonding interactions (including interactions such as salt bridges and water bridges). "Covalent binding" or "covalent bond," as used herein, refers to the formation of one or more covalent chemical bonds between two different molecules.

[0017] As used herein, the term "immunological synapse" or "immune synapse" generally refers to the natural interface between two immune cells that interact in an adaptive immune response, including, for example, the interface between an antigen-presenting cell (APC) or target cell and an effector cell, e.g., a lymphocyte, an effector T cell, a natural killer cell, etc. The immunological synapse between an APC and a T cell is generally initiated by the interaction of a T cell antigen receptor with 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.

[0018] "T cells" are helper T cells (CD4 + cells), cytotoxic T cells (CD8 + These include all types of immune cells that express CD3, including immune cells (IL-1), regulatory T cells (Tregs), and NK-T cells.

[0019] The term "immunomodulating polypeptide" (also referred to as "MOD"), as used herein, includes a polypeptide, or a portion of a polypeptide on an antigen-presenting cell (APC) (e.g., dendritic cell, B cell, etc.), that specifically binds to a cognate immune co-regulatory polypeptide on a T cell, thereby providing a signal that mediates a T cell response (including, but not limited to, proliferation, activation, differentiation, etc.) in addition to the primary signal (e.g., that provided by binding of the TCR / CD3 complex to a peptide-loaded major histocompatibility complex (MHC) polypeptide).

[0020] "Heterologous," as used herein, means a nucleotide or polypeptide not found in the native nucleic acid or protein, respectively.

[0021] "Recombinant," as used herein, means that a particular nucleic acid (DNA or RNA) is the product of various combinations of cloning, restriction, polymerase chain reaction (PCR), and / or ligation steps that result in a construct having structural coding or non-coding sequences distinguishable from the endogenous nucleic acid found in a natural system. A DNA sequence encoding a polypeptide can be assembled from cDNA fragments or a series of synthetic oligonucleotides to provide a synthetic nucleic acid expressible from a recombinant transcription unit contained in a cell or a cell-free transcription-translation system.

[0022] The terms "recombinant expression vector" or "DNA construct" are used interchangeably herein to refer to a DNA molecule comprising a vector and at least one insert. Recombinant expression vectors are typically constructed for the expression and / or propagation of 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 a DNA regulatory sequence.

[0023] As used herein, the term "affinity" refers to the equilibrium constant for the reversible binding of two agents (e.g., an antibody and an antigen), and is also referred to as the dissociation constant (K D As used herein, the term "avidity" refers to the resistance of a complex of two or more agents to dissociation after dilution.

[0024] The terms "treatment," "treating," and the like are generally used herein to mean obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic, in that it completely or partially prevents a disease or its symptoms, and / or therapeutic, in that it partially or completely cures a disease and / or adverse effects that may result from the disease. As used herein, "treatment" encompasses any treatment of a disease or condition in a mammal, including (a) preventing the onset of a disease or condition in a subject who may be predisposed to, but has not yet been diagnosed with, the disease or condition; (b) inhibiting a disease or condition, i.e., preventing its development; and / or (c) alleviating the disease, i.e., inducing regression of the disease. Therapeutic agents may be administered before, during, or after the onset of disease or injury. Treatment of ongoing disease is of particular interest, where treatment stabilizes or reduces undesirable clinical symptoms in the patient. Such treatment is desirably administered before complete loss of function in the affected tissue. The subject treatments are desirably administered during, and in some cases after, the symptomatic stage of the disease.

[0025] The terms "individual," "subject," "host," and "patient" are used interchangeably herein to refer to any mammalian subject for whom 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.), etc.

[0026] Unless otherwise specified, the term "substantially" is intended to encompass both "entirely" and "largely but not entirely." For example, an Ig Fc that "does not substantially induce cell lysis" means an Ig Fc that does not induce cell lysis at all, or that largely, but not completely, does not induce cell lysis.

[0027] As used herein, the term "about" when used in relation to a quantity indicates that the quantity may vary by 10%. For example, "about 100" means an amount of 90 to 110. When "about" is used in the context of a range, "about" when used with respect to the lower limit of a range means that the lower limit includes an amount 10% lower than the lower limit of the range, and "about" when used with respect to the upper limit of a range means that the upper limit includes an amount 10% higher than the upper limit of the range. For example, about 100 to about 1000 means that the range spans 90 to 1100.

[0028] The terms "purify," "isolate," and the like refer to removing a desired substance, e.g., TMP, from a solution containing undesired substances, e.g., contaminants, or removing undesired substances from a solution containing the desired substance, leaving essentially only the desired substance. In some cases, a purified substance may be essentially free of other substances, e.g., contaminants.

[0029] It is to be understood that the present disclosure is not limited to particular embodiments described, as such may, of course, vary. 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, as the scope of the present disclosure, as set forth in the appended claims, will be limited only by the appended claims.

[0030] When a range of values ​​is described, it is understood that each value between the upper and lower limit of that range, to the tenth of the unit of the lower limit unless expressly stated otherwise, and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these narrower ranges may independently be included in the narrower ranges, and are also encompassed within the disclosure, provided that any specific limit in the stated range is excluded. When the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0031] 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 disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, the preferred methods and materials are described below. All publications mentioned herein are incorporated by reference to disclose and describe the methods and / or materials cited in connection with the publications.

[0032] 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 expressly stated otherwise. Thus, for example, a reference to an "immunomodulating polypeptide" includes a plurality of such immunomodulating polypeptides; a reference to "Treg" includes one or more Treg and equivalents thereof known to those skilled in the art, and so forth. It should also be noted that the claims may be drafted to exclude any optional element or any element included in a listing or other list of elements sharing a common general or specific characteristic, property, or activity. Accordingly, this statement is intended to serve as a premise for the use of exclusive terminology such as "only," "only," or the use of limitation "by negative" in connection with the recitation of claim elements.

[0033] For clarity, some features of the present disclosure are described in the context of separate embodiments, but it is recognized that they may also be provided in combination in a single embodiment. Conversely, to avoid clutter, various features of the present disclosure are described in the context of a single embodiment, but they may also be provided separately or in any suitable subcombination. All combinations of the embodiments of the present disclosure are expressly embraced by the present disclosure, and each and every combination is disclosed herein as if individually and expressly disclosed. In addition, all subcombinations of the various embodiments and elements thereof are also expressly embraced by the present disclosure, and each and every such subcombination is disclosed herein as if individually and expressly disclosed.

[0034] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein constitutes an admission that the present disclosure is not entitled to antedate such publication by virtue of prior disclosure. Further, the dates of publication provided may be different from the actual publication dates, which may need to be independently confirmed. The headings in this disclosure are for convenience of reference only and do not define, describe, or limit the scope of the disclosure or the appended claims.

[0035] Detailed Description The present disclosure provides T cell modulating polypeptides (TMPs) comprising a type 1 diabetes (T1D)-associated peptide epitope, an MHC class II polypeptide, one or more optional immunomodulatory polypeptides ("MODs"), a TGF-β polypeptide, and a masking polypeptide (the TGF-β polypeptide and masking polypeptide together comprise a "masked TGF-β MOD"). The TMPs of the present disclosure are useful for modulating T cell activity. Accordingly, the present disclosure provides compositions and methods for modulating T cell activity, as well as compositions and methods for treating individuals with T1D. In any of the embodiments described herein, in some cases, the TMP does not comprise a MOD, and such TMPs are useful for delivering TGFβ.

[0036] T-cell Modulatory Polypeptides ("TMPs") The present disclosure provides a TMP comprising: i) a peptide presenting a type 1 diabetes-associated epitope capable of being bound by a T cell receptor (a "T1D peptide"); ii) a TGF-β polypeptide; iii) a masking polypeptide; iv) an MHC class II polypeptide (i.e., an MHC class II α chain polypeptide and an MHC class II β chain polypeptide); and v) optionally, one or more MODs; wherein the TMP optionally comprises one or more scaffolding polypeptides (e.g., one or more immunoglobulin (Ig) Fc polypeptides); and the TMP optionally comprises one or more independently selected linker polypeptides (e.g., each of the one or more independently selected linker polypeptides is between any two of the foregoing polypeptides). The TMP can be in the form of a single-chain polypeptide, a heterodimeric polypeptide, and a dimer or multimer of such single-chain and heterodimeric polypeptides; exemplary configurations are described below.

[0037] Furthermore, although the TMPs of the present disclosure may include both one or more masked TGF-β MODs and one or more additional MODs, such as wild-type or variant IL-2, PD-L1 and / or 4-1BBL MOD (as described above), if desired, the TMPs of the present disclosure may include only one or more masked TGF-β MODs. That is, one or more additional MODs, such as wild-type or variant IL-2, PD-L1 and / or 4-1BBL MOD, need not be included in the TMPs of the present disclosure. Accordingly, in the following discussion, it will be understood that the presence of one or more MODs in addition to one or more masked TGF-β MODs is optional.

[0038] A brief overview of exemplary TMP structures A) In some instances, a TMP of the present disclosure comprises a heterodimer comprising a scaffold polypeptide (e.g., Ig Fc) having a mutually specific binding sequence, the heterodimer comprising: a) a first polypeptide comprising: i) a T1D peptide, ii) an MHC class II β polypeptide, iii) an MHC class II α polypeptide, and iv) a first scaffold polypeptide comprising a first mutually specific binding sequence; and b) a second polypeptide comprising: i) a T1D peptide, ii) an MHC class II β polypeptide, iii) an MHC class II α polypeptide, and iv) a second scaffold polypeptide comprising a mutually specific binding sequence corresponding to the mutually specific binding sequence of the first polypeptide, wherein the mutually specific binding sequences and the corresponding mutually specific binding sequences interact with each other in the heterodimer, wherein the first or second polypeptide comprises a TGF-β polypeptide, the first or second polypeptide comprises a masking polypeptide, and the first and / or second polypeptide optionally comprise one or more MODs, and the TMP optionally comprises one or more independently selected linker polypeptides. Examples of such TMPs are shown schematically in Figures 1A-1D. In some cases, the first and second polypeptides are linked by a disulfide bond, for example, between an MHC class II β polypeptide and an MHC class II α polypeptide, as shown schematically in Figures 1E-1H.

[0039] B) In some cases, a TMP of the present disclosure comprises one or more heterodimers, each heterodimer comprising a) a first polypeptide chain comprising i) a T1D peptide and a first MHC class II polypeptide, and b) a second polypeptide comprising a second MHC class II polypeptide, wherein the first and / or second polypeptides optionally comprise one or more MODs, the first or second polypeptide comprises a TGF-β polypeptide, the first or second polypeptide comprises a masking polypeptide, the TMP optionally comprises one or more independently selected linker polypeptides, and the first or second polypeptide chain comprises an Ig Fc polypeptide. A TMP of the present disclosure may be a homodimer comprising two such heterodimers, wherein the Ig Fc polypeptide of one heterodimer is disulfide-linked to the Ig Fc polypeptide of the other heterodimer. Examples of such TMPs are shown schematically in Figures 2A-2D.

[0040] C) In some cases, the TMPs of the present disclosure are single-chain polypeptides, i.e., a single polypeptide chain comprising: i) a T1D peptide; ii) an MHC class II polypeptide (e.g., an MHC class II β polypeptide and an MHC class II α polypeptide); iii) optionally, one or more MODs; iv) a TGF-β polypeptide; v) a masking polypeptide; optionally, one or more independently selected linker polypeptides; and optionally, an Ig Fc polypeptide. Examples of such TMPs are shown schematically in Figures 3A-3D. The TMPs of the present disclosure may be homodimers comprising two copies of such single-chain polypeptides, wherein the single-chain polypeptide comprises an Ig Fc polypeptide, and the Ig Fc polypeptide of one of the two single-chain polypeptides is disulfide-linked to the other Ig Fc polypeptide of the two single-chain polypeptides. Alternatively, the TMPs may comprise heterodimers of two different single-chain TMPs connected by polypeptides comprising mutually specific binding sequences, as described below.

[0041] The aforementioned components are described in more detail below. References herein to an MHC class II α polypeptide may include both the α1 and α2 domains of the class II MHC α chain, and references herein to an MHC class II β polypeptide may include both the β1 and β2 domains of the class II MHC β chain. These four domains represent all or most of the extracellular class II protein required for presentation of epitope peptides.

[0042] A) Heterodimeric TMP containing mutually specific binding sequences In some cases, the TMP of the present disclosure comprises a heterodimer comprising a scaffold polypeptide (e.g., an Ig Fc polypeptide) having a mutually specific binding sequence, the heterodimer comprising: a) a first polypeptide comprising: i) a T1D peptide, ii) an MHC class II β polypeptide, iii) an MHC class II α polypeptide, and iv) a first scaffold polypeptide comprising a first mutually specific binding sequence; and b) a second polypeptide comprising: i) a T1D peptide, ii) an MHC class II β polypeptide, iii) an MHC class II α polypeptide, and iv) a second scaffold polypeptide comprising a mutually specific binding sequence corresponding to the mutually specific binding sequence of the first polypeptide, wherein the mutually specific binding sequences and the corresponding mutually specific binding sequences interact with each other in the heterodimer, wherein the first or second polypeptide comprises a TGF-β polypeptide, the first or second polypeptide comprises a masking polypeptide, and the first and / or second polypeptide optionally comprise one or more MODs. The TMP optionally comprises one or more independently selected linker polypeptides. Non-limiting examples of such TMPs are shown schematically in Figures 1A-1D. While Figures 1A-1D illustrate the scaffold polypeptide as a knobs-in-holes ("KiH") Fc polypeptide, it should be understood that other scaffold polypeptides having mutually specific binding sequences may be used.

[0043] Arrangement of components The components of such a TMP can be arranged in various ways. In some cases, a TMP comprises at least one heterodimer, the heterodimer comprising: a) a first polypeptide comprising: i) a T1D peptide, ii) an MHC class II β polypeptide, iii) an MHC class II α polypeptide, iv) a first scaffold polypeptide comprising a first mutually specific binding sequence, and v) a TGF-β polypeptide; and b) a second polypeptide comprising: i) a T1D peptide, ii) an MHC class II β polypeptide, iii) an MHC class II α polypeptide, iv) a second scaffold polypeptide comprising a mutually specific binding sequence corresponding to the mutually specific binding sequence of the first polypeptide, wherein the mutually specific binding sequence and the corresponding mutually specific binding sequence interact with each other in the heterodimer; and v) a masking polypeptide, wherein the first and / or second polypeptide comprises one or more MODs. In some cases, the one or more MODs and the TGF-β polypeptide are on the same polypeptide. In some cases, the one or more MODs and the masking polypeptide are on the same polypeptide. In some cases, the mutually specific binding sequence and the corresponding mutually specific binding sequence comprise a KiH sequence.

[0044] In some cases, the TMP comprises at least one heterodimer, the heterodimer comprising: a) a first polypeptide comprising: i) a T1D peptide, ii) an MHC class II β polypeptide, iii) an MHC class II α polypeptide, iv) a first scaffold polypeptide comprising a first mutually specific binding sequence, v) a masking polypeptide, and vi) a TGF-β polypeptide; and b) a second polypeptide comprising: i) a T1D peptide, ii) an MHC class II β polypeptide, iii) an MHC class II α polypeptide, and iv) a second scaffold polypeptide comprising a mutually specific binding sequence corresponding to the mutually specific binding sequence of the first polypeptide, wherein the mutually specific binding sequences and the corresponding mutually specific binding sequences interact with each other in the heterodimer, and wherein the first and / or second polypeptide comprise one or more MODs. In some cases, the one or more MODs are on the first polypeptide. In some cases, the one or more MODs are on the second polypeptide. In some cases, the mutually specific binding sequences and the corresponding mutually specific binding sequences comprise KiH sequences.

[0045] Further examples of various arrangements of the components of a TMP are shown schematically in Figures 1A-1D, in which the scaffold polypeptide is a KiH Ig Fc polypeptide and the masking polypeptide is referred to as "mask." For example, as shown schematically in FIG. 1A, in some cases, the TMP is a heterodimer comprising: a) a first polypeptide comprising, from N-terminus to C-terminus: i) a T1D peptide; ii) an MHC class II β chain polypeptide; iii) an MHC class II α chain polypeptide; iv) a first Ig Fc comprising a reciprocal specific binding sequence (illustrated as a KiH sequence); and v) a TGF-β polypeptide; and b) a second polypeptide comprising, from N-terminus to C-terminus: i) a T1D peptide; ii) an MHC class II β chain polypeptide; iii) an MHC class II α chain polypeptide; iv) a second Ig Fc comprising a reciprocal specific binding sequence (illustrated as a KiH sequence); v) a masking polypeptide; and vi) one or more MODs; wherein the first Ig Fc polypeptide dimerizes with the second Ig Fc polypeptide, and the TMP comprises an optional peptide linker between any two of the TMP's components (any two adjacent components).

[0046] As another example, as shown schematically in FIG. 1B, in some cases, the TMP is a heterodimer comprising: a) a first polypeptide comprising, from N-terminus to C-terminus: i) a T1D peptide; ii) an MHC class II β chain polypeptide; iii) an MHC class II α chain polypeptide; iv) a first Ig Fc comprising a reciprocal specific binding sequence (illustrated as a KiH sequence); v) a masking polypeptide; and vi) a TGF-β polypeptide; and b) a second polypeptide comprising, from N-terminus to C-terminus: i) a T1D peptide; ii) an MHC class II β chain polypeptide; iii) an MHC class II α chain polypeptide; iv) a second Ig Fc comprising a reciprocal specific binding sequence (illustrated as a KiH sequence); and v) one or more MODs; wherein the first Ig Fc polypeptide dimerizes with the second Ig Fc polypeptide; and the TMP comprises an optional peptide linker between any two of the TMP components (any two adjacent components).

[0047] As another example, as shown schematically in FIG. 1C, in some cases, the TMP is a heterodimer comprising: a) a first polypeptide comprising, from N-terminus to C-terminus, i) a T1D peptide, ii) an MHC class II β chain polypeptide, iii) an MHC class II α chain polypeptide, iv) a first Ig Fc comprising a reciprocal specific binding sequence (illustrated as a KiH sequence), and v) a TGF-β polypeptide; and b) a second polypeptide comprising, from N-terminus to C-terminus, i) a T1D peptide, ii) an MHC class II β chain polypeptide, iii) an MHC class II α chain polypeptide, iv) one or more MODs, v) a second Ig Fc comprising a reciprocal specific binding sequence (illustrated as a KiH sequence), and vi) a masking polypeptide, wherein the first Ig Fc polypeptide dimerizes with the second Ig Fc polypeptide, and the TMP comprises an optional peptide linker between any two of the TMP's components (any two adjacent components).

[0048] As another example, as shown schematically in FIG. 1D , in some cases, the TMP is a heterodimer comprising: a) a first polypeptide comprising, in N-terminal to C-terminal order: i) a T1D peptide, ii) an MHC class II β chain polypeptide, iii) an MHC class II α chain polypeptide, iv) a first Ig Fc comprising a reciprocal specific binding sequence (illustrated as a KiH sequence), v) a masking polypeptide, and vi) a TGF-β polypeptide; and b) a second polypeptide comprising, in N-terminal to C-terminal order: i) a T1D peptide, ii) an MHC class II β chain polypeptide, iii) an MHC class II α chain polypeptide, iv) one or more MODs, and v) a second Ig Fc comprising a reciprocal specific binding sequence (illustrated as a KiH sequence), wherein the first Ig Fc polypeptide dimerizes with the second Ig Fc polypeptide, and the TMP comprises an optional peptide linker between any two of the TMP components (any two adjacent components).

[0049] In some cases, as shown schematically in Figures 1E-1H, the MHC class II α chain polypeptide and the MHC class II β chain polypeptide contain Cys residues not present in naturally occurring MHC class II α chain and MHC class II β chain polypeptides, forming disulfide bonds between the Cys residues, thereby disulfide linking the first and second polypeptides. Suitable MHC class II α chain and MHC class II β chain polypeptides containing introduced Cys residues are described below. Additionally, when the scaffold protein is an Ig Fc polypeptide, one or more disulfide bonds can form between the Ig Fc polypeptide of the first polypeptide and the Ig Fc polypeptide of the second polypeptide. Such disulfide bonds are shown schematically in Figure 28.

[0050] The amino acid sequences of exemplary TMPs, which are heterodimers comprising Ig Fc polypeptides with mutually specific binding sequences, are shown in 1) Figures 26A and 26B (the first polypeptide is "Construct 4415" (Figure 26A) and the second polypeptide is "Construct 4417" (Figure 26B)), 2) Figures 27A and 27B (the first polypeptide is "Construct 4418" (Figure 27A) and the second polypeptide is "Construct 4420" (Figure 27B)), 3) Figures 32A-32B (the first polypeptide is "Construct 3858" (Figure 32A) and the second polypeptide is "Construct 3869" (Figure 32B)), and 4) Figures 33A-33B (the first polypeptide is "Construct 3870" (Figure 33A) and the second polypeptide is "Construct 3871" (Figure 33B)).

[0051] TMP without additional mods As noted above, any of the above-described embodiments can be provided without including a MOD other than a masked TGF-β MOD. Non-limiting examples of such TMPs are shown below: 1) Figures 29A-29B (where the first polypeptide is "Construct 4415" (Figure 29A) and the second polypeptide is "Construct 4416" (Figure 29B)), 2) Figures 30A-30B (where the first polypeptide is "Construct 4418" (Figure 30A) and the second polypeptide is "Construct 4419" (Figure 30B)), and 3) Figures 31A-31B (where the first polypeptide is "Construct 3858" (Figure 31A) and the second polypeptide is "Construct 3859" (Figure 31B)). Such TMPs are useful, for example, when complexed with an MHC class II polypeptide present in the TMP, to selectively deliver TGFβ to T cells expressing on their surface a TCR specific for a peptide present in the TMP.

[0052] Scaffold polypeptides with mutually specific binding sequences The scaffold polypeptide functions, inter alia, as a structural element providing a framework for organizing the other components of the TMP. When the masking polypeptide and the TGF-β polypeptide are located in trans (on different polypeptides of the TMP), the scaffold polypeptide, forming mutually specific and non-mutually specific duplexes (or higher order structures), can maintain the association of the masking polypeptide and the TGF-β polypeptide. Depending on the nature of the scaffold, it may act as an organizing element providing higher order structure in terms of protein folding and dimerization or multimerization (e.g., homodimerization or heterodimerization). The scaffold may also contribute to serum stability, particularly if it is an Ig heavy chain constant region (e.g., Ig Fc). In some cases, a suitable scaffold polypeptide is a half-life extending polypeptide. In some cases, a suitable backbone polypeptide increases the in vivo half-life (e.g., serum half-life) of a TMP by at least about 10%, at least about 15%, at least about 25%, at least about 50%, at least about 2-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, compared to a control TMP having a backbone polypeptide comprising a different non-Ig sequence. As an example, in some cases, an Ig Fc polypeptide sequence (e.g., one comprising a mutually specific Ig sequence such as a KiH sequence pair) increases the stability and / or in vivo half-life (e.g., serum half-life) of a TMP compared to a control TMP in which the Ig Fc polypeptide sequence is replaced with a linker (e.g., repeats of GGGS amino acids of equal sequence length; SEQ ID NO: 234). The increase in in vivo half-life can be at least about 10%, at least about 15%, 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.When an Ig Fc polypeptide is employed in a TMP, the Ig Fc can contain a mutation that prevents spontaneous dimerization of the TMP (see, e.g., Tianlei Ying et al., J. Biol. Chem., 287(23), pp. 19399-19408 (June 1, 2012)), and may further contain a mutation (e.g., the LALA mutation described below) that reduces or substantially eliminates the ability of the Ig polypeptide to induce cell lysis via, for example, complement-dependent cytotoxicity (CDC) and / or antibody-dependent cellular cytotoxicity (ADCC).

[0053] If the scaffold polypeptide of a TMP contains one or more amino acid sequences that allow the scaffold polypeptide to interact (specifically bind) with another scaffold polypeptide, the TMP can form a homodimer. A TMP can also contain one or more amino acid sequences that allow the scaffold polypeptide to interact (specifically bind) with other scaffold polypeptides to form higher-order structures. Sequences that form higher-order multimeric structures allow the formation of higher-order TMPs (trimers, tetramers, pentamers, etc.). For example, a scaffold polypeptide containing an IgM Fc region allows the formation of pentamer (especially if a J chain sequence is also expressed) or hexamer TMP. Petrusic et al., Med Hypotheses. 77(6):959-61 (2011).

[0054] A variety of polypeptides that specifically bind to each other or to themselves with sufficient affinity can be utilized as dimerization sequences for TMP (see, e.g., U.S. Patent Publication No. 2003 / 0138440). The polypeptides can be relatively compact in size (e.g., less than about 300, 250, 225, 200, 175, 150, 125, 100, 75, or 50 amino acids, etc.). Dimerization / multimerization polypeptides include, but are not limited to, immunoglobulin heavy chain constant region (Ig Fc) polypeptides (polypeptides comprising the CH2-CH3 regions of Ig; see, for example, ); Fc KiH polypeptides; polypeptides of the collectin family containing a collagen domain consisting of collagen repeats Gly-Xaa-Yaa (e.g., ACRP30 or ACRP30-like proteins); coiled-coil domains; leucine-zipper domains; Fos / Jun binding pairs; Ig heavy chain region 1 (CH1) and light chain constant region CL sequences (CH1 / CL pairs, such as a CH1 sequence paired with a kappa or lambda Ig light chain constant region sequence).

[0055] In some cases, the scaffold polypeptide comprises an Ig heavy chain constant region (CH2-CH3) polypeptide sequence that functions as a dimerization or multimerization sequence (see, e.g., Figures 21A-21M). In some cases, the Ig polypeptide has a reduced ability to induce cell lysis, e.g., via activation of complement-dependent cytotoxicity (CDC) and / or antibody-dependent cellular cytotoxicity (ADCC), and thus may comprise mutations that reduce or substantially eliminate the ability of the Ig polypeptide to induce cell lysis. In some cases, the Ig Fc polypeptide comprises an amino acid sequence having at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to an Ig Fc polypeptide shown in any one of Figures 21A-21M. Such an Ig Fc polypeptide can covalently link polypeptides of TMP together, e.g., by forming one or two interchain disulfide bonds. As described below, additional disulfide bonds can be introduced to stabilize dimers, particularly when mutually specific Ig sequence pairs such as KiH polypeptide pairs are employed.

[0056] In one embodiment, the backbone polypeptide of TMP comprises an amino acid sequence having at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to at least 150 contiguous amino acids (at least 175, at least 200, at least 225, at least 250, at least 275, at least 300, at least 325, or at least 350 contiguous amino acids) or all amino acids of the IgA Fc sequence shown in Figure 2K. In one embodiment, the scaffold polypeptide comprises an amino acid sequence having at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity for at least 150 contiguous amino acids (at least 175, at least 200, at least 225, at least 250, at least 275, at least 300, at least 325, or at least 350 contiguous amino acids), or all amino acids, of the IgD Fc sequence shown in Figure 21. In one embodiment, the scaffold polypeptide comprises an amino acid sequence having at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity for at least 125 contiguous amino acids (at least 150, at least 175, or at least 200 contiguous amino acids), or all amino acids, of the IgE Fc sequence shown in Figure 21L. In one embodiment, the scaffold polypeptide comprises an amino acid sequence having at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to at least 125 contiguous amino acids (at least 150, at least 175, or at least 200 contiguous amino acids) or all amino acids of a wild-type IgG1 Fc polypeptide, such as the IgG1 Fc amino acid sequence shown in any one of Figures 21A-21F.In one embodiment, the scaffold polypeptide comprises an amino acid sequence having at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity for at least 125 contiguous amino acids (at least 150, at least 175, at least 200, at least 225, at least 250, at least 275, or at least 300), or all amino acids, to the IgG2 Fc polypeptide amino acid sequence shown in Figure 21G. In one embodiment, the scaffold polypeptide comprises a sequence having at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity for at least 125 contiguous amino acids (at least 150, at least 175, at least 200, or at least 225), or all amino acids, to the IgG3 Fc amino acid sequence shown in Figure 21H. In one embodiment, the scaffold polypeptide comprises an amino acid sequence having at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity for at least 125 contiguous amino acids (at least 150, at least 175, at least 200, at least 225, or at least 250), or all amino acids, to the IgG4 Fc amino acid sequence shown in Figure 21M. In one embodiment, the scaffold polypeptide comprises an amino acid sequence having at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity for at least 125 contiguous amino acids (at least 150, at least 175, at least 200, at least 225, or at least 250), or all amino acids, to the IgM Fc polypeptide sequence shown in Figure 21J. The scaffold polypeptides described above can be covalently linked together by the formation of one or two interchain disulfide bonds between cysteines flanking the hinge region.

[0057] In some cases, the dimerization sequence of the scaffold polypeptide present in the TMP has at least about 70% (e.g., at least about 80%, 90%, 95%, 98%, 99%, or 100%) amino acid sequence identity to the human IgG1 Fc polypeptide shown in Figure 21F and includes a substitution of N297 with alanine (N297A substitution, or N77 as numbered in Figure 21F). In some cases, the dimerization sequence of the scaffold polypeptide present in the TMP includes the amino acid sequence shown in Figure 21A (human IgG1 Fc), except for the substitution of N297 (N77 in the amino acid sequence shown in Figure 21A) with an amino acid other than asparagine. The substitution at N297 leads to the removal of carbohydrate modifications, such that the antibody sequence has reduced binding of complement component 1q ("C1q") compared to the wild-type protein, and therefore reduced complement-dependent cytotoxicity.

[0058] In some cases, the dimerization sequence of the scaffold polypeptide present in the TMP comprises the amino acid sequence shown in Figure 21A (human IgG1 Fc), except for substitution of L234 (L14 in the amino acid sequence shown in Figure 21A) with an amino acid other than leucine. L234 and other amino acids in the lower hinge region of IgG (e.g., amino acids 234-LLGGPS-239 (SEQ ID NO: 172), corresponding to amino acids 14-19 in Figure 21A) are involved in binding to the Fc lambda receptor (FcλR), and thus mutations at that position reduce binding to the receptor (compared to the wild-type protein). In some cases, the dimerization sequence of the scaffold polypeptide present in the TMP comprises the amino acid sequence shown in Figure 21A (human IgG1 Fc), except for substitution of L235 (L15 in the amino acid sequence shown in Figure 21A) with an amino acid other than leucine. In some cases, the dimerization sequence of the scaffold polypeptide present in the TMP comprises the amino acid sequence shown in Figure 21A (e.g., a wild-type human IgG1 sequence) including the L234A and L235A ("LALA") substitutions (positions corresponding to positions 14 and 15 of the wild-type amino acid sequence shown in Figure 21A; see, e.g., SEQ ID NO: 113). See Figure 21B. These two mutations reduce or substantially eliminate the ability of the IgG1 Fc to induce cell lysis via, for example, activation of complement-dependent cytotoxicity (CDC) and / or antibody-dependent cellular cytotoxicity (ADCC).

[0059] In some cases, the dimerization sequence of the scaffold polypeptide present in TMP comprises the amino acid sequence shown in FIG. 21A (human IgG1 Fc) with a substitution of P331 (P111 of the amino acid sequence shown in FIG. 21A) with an amino acid other than proline; in some cases, the substitution is a P331S substitution. Substitutions at P331, like substitutions at N297, reduce binding to C1q compared to the wild-type protein, thereby reducing complement-dependent cytotoxicity. Substitutions of D270, K322, and / or P329 (corresponding to D50, K122, and P119 in FIG. 21A), e.g., with alanine, can be used alone or in place of the P331 substitution to reduce binding to C1q. As noted above, in some cases, the dimerization sequence of the scaffold polypeptide present in TMP is an IgG1 Fc polypeptide comprising an L234A and / or an L235A substitution (a substitution of Ala for leucine at L14 and / or L15 of the amino acid sequence shown in Figure 21A). See Figure 21B. In some cases, the dimerization sequence of the scaffold polypeptide present in TMP comprises the amino acid sequence shown in Figure 21A (wild-type human IgG1 Fc), except for the substitution of an amino acid other than leucine at L234 and / or L235 (L14 and / or L15 of the amino acid sequence shown in Figure 21A) and the substitution of an amino acid other than proline at P331 (P111 of the amino acid sequence shown in Figure 2D). In some cases, the dimerization sequence of the scaffold polypeptide present in the TMP comprises the "triple mutant" amino acid sequence shown in Figure 21E (human IgG1 Fc), which contains the L234F, L235E, and P331S substitutions (corresponding to amino acid positions 14, 15, and 111 of the amino acid sequence shown in Figure 21E).

[0060] As described above, the Ig Fc polypeptide of each polypeptide chain of a heterodimeric TMP can contain a mutually specific dimerization sequence, e.g., a KiH sequence, that allows the two chains to selectively dimerize. The mutually specific binding sequence, particularly when based on an Ig Fc sequence variant, prefers heterodimer formation with its cognate polypeptide sequence (i.e., between the mutually specific sequence and its corresponding mutually specific sequence). Such mutually specific polypeptide sequences include KiH and a KiH sequence that facilitates the formation of one or more disulfide bonds. For example, one mutually specific binding pair includes the T366Y and Y407T mutant pair at the CH3 domain interface of IgG1 or corresponding residues in other immunoglobulins. See Ridgway et al., Protein Engineering 9:7, 617-621 (1996). A second mutually specific binding pair involves a knob formed by a T366W substitution and a hole formed by a triple substitution of T366S, L368A, and Y407V on the complementary Ig Fc sequence. See Xu et al. mAbs 7:1, 231-242 (2015). Another mutually specific binding pair has a first Fc polypeptide containing Y349C, T366S, L368A, and Y407V substitutions and a second Ig Fc polypeptide containing S354C and T366W substitutions (a disulfide bond can form between Y349C and S354C). See, e.g., Brinkmann and Konthermann, mAbs 9:2, 182-212 (2015). Ig Fc polypeptide sequences, with or without KiH modification, can be stabilized by the formation of disulfide bonds between the Ig Fc polypeptides (eg, disulfide bonds in the hinge region).

[0061] Some interspecific binding sequences based on immunoglobulin sequences are summarized in Table 1 below, with cross-references in brackets "{}" to the numbering of amino acid positions that appear in the wild-type IgG1 sequence shown in Figure 21A: i) hinge: amino acids 1-10, ii) CH2: amino acids 11-120, iii) CH3: amino acids 121-227, iv) Fc amino acids 11-227.

[0062] Table 1. Interspecific immunoglobulin sequences and their cognate corresponding interspecific sequences TIFF2024517658000002.tif160169Table 1 is modified from Ha et al., Frontiers in Immunol. 7:1-16 (2016). * The amino acids form stabilizing disulfide bonds.

[0063] In addition to the cross-specific pairs of sequences in Table 1, an Ig Fc polypeptide can include a cross-specific "SEED" sequence that has 45 residues derived from IgA in the IgG1 CH3 domain of the cross-specific sequence and 57 residues derived from IgG1 in the IgA CH3 domain in the corresponding cross-specific sequence. See Ha et al., Frontiers in Immunol. 7:1-16 (2016).

[0064] The Ig Fc polypeptide present in the TMP can comprise a reciprocal specific binding sequence selected from the group consisting of knob-in-hole (KiH), knob-in-hole with stabilizing disulfide (KiHs-s), HA-TF, ZW-1, 7.8.60, DD-KK, EW-RVT, EW-RVTs-s, A107, or SEED sequence, or its corresponding reciprocal specific binding sequence.

[0065] The TMP can comprise an Ig Fc polypeptide comprising a T146W KiH sequence substitution, and its corresponding mutually specific binding partner polypeptide comprises an Ig Fc polypeptide having T146W, L148A, and Y187V KiH sequence substitutions, wherein the Ig Fc polypeptide comprises a sequence having at least 80%, at least 90%, at least 95%, or at least 97% sequence identity over at least 100 (e.g., at least 125, 150, 170, 180, 190, 200, 210, 220, or all 227) contiguous amino acids of the wild-type IgG1 of Figure 21A. One or both of the Ig Fc polypeptides optionally include a substitution at one or more of L234 and L235 (e.g., L234A / L235A "LALA" or L234F / L235E), N297 (e.g., N297A), P331 (e.g., P331S), L351 (e.g., L351K), T366 (e.g., T366S), P395 (e.g., P395V), F405 (e.g., F405R), Y407 (e.g., Y407A), and K409 (e.g., K409Y). These substitutions appear at L14 and L15 (e.g., L14A / L15A "LALA" or L14F / L15E), N77 (e.g., N77A), P111 (e.g., P111S), L131 (e.g., L131K), T146 (e.g., T146S), P175 (e.g., P175V), F185 (e.g., F185R), Y187 (e.g., Y187A), and K189 (e.g., K189Y) in the wild-type IgG1 sequence in Figure 21A.

[0066] In some cases, the TMP comprises an Ig Fc polypeptide comprising a T146W KiH sequence substitution and its corresponding mutually specific binding partner Ig Fc polypeptide comprises a T146S, L148A, and Y187V KiH sequence substitution, the Ig Fc polypeptide comprises an amino acid sequence having at least 80%, at least 90%, at least 95%, or at least 97% sequence identity for at least 100 (e.g., at least 125, 150, 170, 180, 190, 200, 210, 220, or all 227) contiguous amino acids of a wild-type IgG1 in Figure 21A, and one or both of the Ig Fc polypeptides may comprise an L14 and / or L15 substitution (e.g., a "LALA" substitution of L234A and L235A), and / or an additional substitution such as N77 (N297, e.g., N297A or N297G).

[0067] In some cases, the TMP comprises an Ig Fc polypeptide comprising T146W and S134C KiHs-s substitutions, and its corresponding mutually specific binding partner Ig Fc polypeptide comprises T146S, L148A, Y187V and Y129C KiHs-s substitutions, and the Ig Fc polypeptide comprises an amino acid sequence having at least 80%, at least 90%, at least 95%, or at least 97% sequence identity for at least 100 (e.g., at least 125, 150, 170, 180, 190, 200, 210, 220, or all 227) consecutive amino acids of a wild-type IgG1 of Figure 21A; One or both of the Fc polypeptides may include an L14 and / or L15 substitution (e.g., a "LALA" substitution of L234A and L235A), and / or an additional substitution such as N77 (N297 e.g., N297A or N297G).

[0068] In some cases, the TMP comprises an Ig Fc polypeptide comprising S144H and F185A HA-TF substitutions, and its corresponding mutually specific binding partner Ig Fc polypeptide comprises Y129T and T174F HA-TF substitutions, wherein the Ig Fc polypeptide comprises an amino acid sequence having at least 80%, at least 90%, at least 95%, or at least 97% sequence identity over at least 100 (e.g., at least 125, 150, 170, 180, 190, 200, 210, 220, or all 227) contiguous amino acids of a wild-type IgG1 in Figure 21A, and one or both of the Ig Fc polypeptides may comprise an additional substitution, such as an L14 and / or L15 substitution (e.g., a "LALA" substitution at L234A and L235A), and / or N77 (N297, e.g., N297A or N297G).

[0069] In some cases, the TMP comprises an Ig Fc polypeptide comprising T130V, L131Y, F185A, and Y187V ZW1 substitutions, and its corresponding mutually specific binding partner Ig Fc polypeptide comprises T130V, T146L, K172L, and T174W ZW1 substitutions, and the Ig Fc polypeptide comprises an amino acid sequence having at least 80%, at least 90%, at least 95%, or at least 97% sequence identity for at least 100 (e.g., at least 125, 150, 170, 180, 190, 200, 210, 220, or all 227) consecutive amino acids of a wild-type IgG1 of Figure 21A; One or both of the Fc polypeptide sequences may include an L14 and / or L15 substitution (e.g., a "LALA" substitution of L234A and L235A), and / or an additional substitution such as N77 (N297 e.g., N297A or N297G).

[0070] In some cases, the TMP comprises an Ig Fc polypeptide comprising K140D, D179M, and Y187A 7.8.60 substitutions, and its corresponding mutually specific binding partner Ig Fc polypeptide comprises T130V E125R, Q127R, T146V, and K189V 7.8.60 substitutions, and the Ig Fc polypeptide comprises an amino acid sequence having at least 80%, at least 90%, at least 95%, or at least 97% sequence identity for at least 100 (e.g., at least 125, 150, 170, 180, 190, 200, 210, 220, or all 227) consecutive amino acids of a wild-type IgG1 of Figure 21A; One or both of the Fc polypeptide sequences may include an L14 and / or L15 substitution (e.g., a "LALA" substitution of L234A and L235A), and / or an additional substitution such as N77 (N297 e.g., N297A or N297G).

[0071] In some cases, the TMP comprises an Ig Fc polypeptide comprising K189D and K172D DD-KK substitutions, and its corresponding mutually specific binding partner Ig Fc polypeptide comprises an IgG1 sequence with T130V D179K and E136K DD-KK substitutions, wherein the Ig Fc polypeptide comprises an amino acid sequence having at least 80%, at least 90%, at least 95%, or at least 97% sequence identity over at least 100 (e.g., at least 125, 150, 170, 180, 190, 200, 210, 220, or all 227) contiguous amino acids of the wild-type IgG1 of Figure 21A, and one or both of the Ig Fc polypeptides may comprise an additional substitution such as an L14 and / or L15 substitution (e.g., a "LALA" substitution of L234A and L235A), and / or N77 (N297, e.g., N297A or N297G).

[0072] In some cases, the TMP comprises an Ig Fc polypeptide comprising K140E and K189W EW-RVT substitutions, and its corresponding mutually specific binding partner Ig Fc polypeptide comprises T130V Q127R, D179V, and F185T EW-RVT substitutions, and the Ig Fc polypeptide comprises an amino acid sequence having at least 80%, at least 90%, at least 95%, or at least 97% sequence identity for at least 100 (e.g., at least 125, 150, 170, 180, 190, 200, 210, 220, or all 227) consecutive amino acids of a wild-type IgG1 of Figure 21A; One or both of the Fc polypeptides may include an L14 and / or L15 substitution (e.g., a "LALA" substitution of L234A and L235A; corresponding to L14 and L15 in the amino acid sequence shown in Figure 21A), and / or an additional substitution such as N77 (N297, e.g., N297A or N297G).

[0073] In some cases, the TMP comprises an Ig Fc polypeptide comprising K140E, K189W, and Y129C EW-RVTs-s substitutions, and its corresponding mutually specific binding partner Ig Fc polypeptide comprises T130V Q127R, D179V, F185T, and S134C EW-RVTs-s substitutions, and the Ig Fc polypeptide comprises an amino acid sequence having at least 80%, at least 90%, at least 95%, or at least 97% sequence identity over at least 100 (e.g., at least 125, 150, 170, 180, 190, 200, 210, 220, or all 227) contiguous amino acids of the wild-type IgG1 of Figure 21A. One or both of the Ig Fc polypeptides may include an L14 and / or L15 substitution (e.g., a "LALA" substitution of L234A and L235A; corresponding to L14 and L15 in the amino acid sequence shown in Figure 21A), and / or an additional substitution such as N77 (N297, e.g., N297A or N297G).

[0074] In some cases, the TMP comprises an Ig Fc polypeptide comprising K150E and K189W A107 substitutions, and its corresponding mutually specific binding partner Ig Fc polypeptide comprises T130V E137N, D179V, and F185T A107 substitutions, wherein the Ig Fc polypeptide comprises an amino acid sequence having at least 80%, at least 90%, at least 95%, or at least 97% sequence identity over at least 100 (e.g., at least 125, 150, 170, 180, 190, 200, 210, 220, or all 227) contiguous amino acids of a wild-type IgG1 in Figure 21A, and one or both of the Ig Fc polypeptides may comprise an additional substitution, such as an L14 and / or L15 substitution (e.g., a "LALA" substitution of L234A and L235A), and / or N77 (N297, e.g., N297A or N297G).

[0075] As a non-limiting example, in some cases, a) the first polypeptide of TMP has the following amino acid sequence: 26A ) comprising an Ig Fc polypeptide (referred to as "IgG Fc(LALA)KiH chain A(S354C;T366W)" in Figure 26A ) comprising an amino acid sequence having at least 90%, at least 95%, or at least 98% identity with respect to TIFF2024517658000003.tif24164, wherein amino acid 14 is Leu, amino acid 15 is Leu, amino acid 134 is Cys, and amino acid 146 is Trp.

[0076] and b) the second polypeptide of TMP has the following amino acid sequence: 26B)), comprising an amino acid sequence having at least 90%, at least 95%, or at least 98% identity with respect to TIFF2024517658000004.tif24163, wherein amino acid 14 is Leu, amino acid 15 is Leu, amino acid 129 is Cys, amino acid 146 is Ser, amino acid 148 is Ala, and amino acid 187 is Val.

[0077] In some cases, a) the first polypeptide of the TMP has the following amino acid sequence: TIFF2024517658000005.tif24164 (referred to in Figure 26A as "IgG Fc(LALA)KiHchainA(S354C;T366W)"), and b) a second polypeptide of TMP having the following amino acid sequence: 26B).

[0078] B) Heterodimeric TMP containing disulfide linkages In some cases, the TMP of the present disclosure comprises one or more heterodimers, each heterodimer comprising a) a first polypeptide chain comprising i) a T1D peptide and 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 MODs, the first or second polypeptide comprises a TGF-β polypeptide, and the first or second polypeptide comprises a masking polypeptide, the TMP optionally comprises one or more independently selected linker polypeptides, and the first or second polypeptide chain comprises an Ig Fc polypeptide. In some cases, the first polypeptide comprises a TGF-β polypeptide and the second polypeptide comprises a masking polypeptide. In some cases, the first polypeptide comprises a TGF-β polypeptide and the second polypeptide comprises a TGF-β polypeptide. In some cases, the first polypeptide comprises a TGF-β polypeptide and a masking polypeptide, and the second polypeptide does not comprise a TGF-β polypeptide or a masking polypeptide. In some cases, the first polypeptide does not comprise a TGF-β polypeptide or a masking polypeptide, and the second polypeptide comprises a TGF-β polypeptide and a masking polypeptide. A TMP of the present disclosure may be a homodimer comprising two such heterodimers, where the Ig Fc polypeptide of one heterodimer is disulfide-linked to the Ig Fc polypeptide of the other heterodimer. Non-limiting examples of such TMPs are shown schematically in Figures 2A-2D. A TMP may comprise two disulfide-linked heterodimers. When both heterodimers comprise an Ig Fc polypeptide, disulfide bonds spontaneously form between the respective Ig Fc polypeptides, covalently linking the two heterodimers to each other. Alternatively, a TMP of the present disclosure may be a heterodimer comprising two such heterodimers, where the Ig Fc polypeptide of one heterodimer is disulfide-linked to the Ig Fc polypeptide of the other heterodimer using the mutually specific binding sequences described above.

[0079] Arrangement of components In some cases, the TMP comprises at least one heterodimer, each heterodimer comprising: a) a first polypeptide comprising i) a T1D peptide, and ii) a first MHC class II polypeptide, and iii) optionally a linker linking the T1D peptide to the 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 MODs, the first or second polypeptide comprises a TGF-β polypeptide, and the first or second polypeptide comprises a masking polypeptide, and optionally, the first and second polypeptides of the heterodimer are covalently linked to each other via at least one disulfide bond.

[0080] In some cases, the TMP comprises at least one heterodimer, each heterodimer comprising: a) a first polypeptide comprising i) a T1D peptide; ii) an MHC class II α chain polypeptide or an MHC class II β chain polypeptide; ii) optionally, a linker linking the T1D peptide to the first MHC class II polypeptide; and b) i) an MHC class II α chain polypeptide if the first polypeptide comprises an MHC class II β chain polypeptide; or ii) an MHC class II α chain polypeptide if the first polypeptide comprises an MHC class II β chain polypeptide. and a second polypeptide comprising a beta chain polypeptide, wherein i) the first and / or second polypeptide comprises one or more MODs, ii) the first or second polypeptide comprises a TGF-β polypeptide, and iii) the first or second polypeptide comprises a masking polypeptide, optionally wherein the first and second polypeptides of the heterodimer are covalently linked to each other via at least one disulfide bond, and optionally wherein the TMP optionally comprises one or more independently selected linker polypeptides (e.g., each of the one or more independently selected linker polypeptides is between any two of the foregoing polypeptides).

[0081] As a first non-limiting example, in some cases, a TMP comprises a1) a first polypeptide comprising i) a T1D peptide, ii) an MHC class II beta polypeptide, and b1) a second polypeptide comprising i) an MHC class II alpha polypeptide, ii) a TGF-beta polypeptide, iii) a masking polypeptide, iv) one or more MODs, and v) an Ig Fc polypeptide. In some cases, the first polypeptide components are connected by one or more independently selected linkers, and in some cases, the second polypeptide components are connected by one or more independently selected linkers.

[0082] As a second non-limiting example, in some cases, a TMP comprises: a2) a first polypeptide comprising: i) a T1D peptide, ii) an MHC class II β polypeptide, and iii) a TGF-β polypeptide or a masking polypeptide; and b2) a second polypeptide comprising: i) an MHC class II α polypeptide, ii) a masking polypeptide if the first polypeptide comprises a TGF-β polypeptide, or a TGF-β polypeptide if the first polypeptide comprises a masking polypeptide, iii) one or more MODs, and iv) an Ig Fc polypeptide. In some cases, the components of the first polypeptide are connected by one or more independently selected linkers, and in some cases, the components of the second polypeptide are connected by one or more independently selected linkers.

[0083] As a third non-limiting example, in some cases, the TMP comprises: a3) a first polypeptide comprising: i) a T1D peptide, ii) an MHC class II β polypeptide, iii) a TGF-β polypeptide or a masking polypeptide, and iv) an Ig Fc polypeptide; and b3) a second polypeptide comprising: i) an MHC class II α polypeptide, ii) a masking polypeptide if the first polypeptide comprises a TGF-β polypeptide, or a TGF-β polypeptide if the first polypeptide comprises a masking polypeptide, and iii) one or more MODs. In some cases, the components of the first polypeptide are connected by one or more independently selected linkers, and in some cases, the components of the second polypeptide are connected by one or more independently selected linkers.

[0084] As a fourth non-limiting example, in some cases, a TMP comprises: a4) a first polypeptide comprising: i) a T1D peptide; ii) an MHC class II β polypeptide; iii) a TGF-β polypeptide or a masking polypeptide; and iv) one or more MODs; and b4) a second polypeptide comprising: i) an MHC class II α polypeptide; ii) a masking polypeptide if the first polypeptide comprises a TGF-β polypeptide, or a TGF-β polypeptide if the first polypeptide comprises a masking polypeptide; and iii) an Ig Fc polypeptide. In some cases, the components of the first polypeptide are connected by one or more independently selected linkers, and in some cases, the components of the second polypeptide are connected by one or more independently selected linkers.

[0085] As a fifth non-limiting example, in some cases, a TMP comprises: a5) a first polypeptide comprising: i) a T1D peptide, ii) an MHC class II β polypeptide, iii) a TGF-β polypeptide or a masking polypeptide, and iv) one or more MODs, and v) an Ig Fc polypeptide; and b5) a second polypeptide comprising: i) an MHC class II α polypeptide, and ii) a masking polypeptide if the first polypeptide comprises a TGF-β polypeptide, or a TGF-β polypeptide if the first polypeptide comprises a masking polypeptide. In some cases, the components of the first polypeptide are connected by one or more independently selected linkers, and in some cases, the components of the second polypeptide are connected by one or more independently selected linkers.

[0086] As a sixth non-limiting example, in some cases, a TMP comprises a6) a first polypeptide comprising i) a T1D peptide, ii) an MHC class II beta polypeptide, iii) a TGF-β polypeptide, iv) a masking polypeptide, v) one or more MODs, and vi) an Ig Fc polypeptide; and b6) a second polypeptide comprising an MHC class II alpha polypeptide. In some cases, the components of the first polypeptide are connected by one or more independently selected linkers.

[0087] In some cases, as shown schematically in Figure 2A, a TMP comprises: a) a first polypeptide comprising, in N-terminal to C-terminal order: i) a T1D peptide, ii) an MHC class II β chain polypeptide, iii) a masking polypeptide, and iv) an Ig Fc polypeptide; and b) a second polypeptide comprising, in N-terminal to C-terminal order: i) an MHC class II α chain polypeptide, ii) a TGF-β polypeptide, and iii) one or more MODs; wherein the TMP optionally comprises one or more independently selected linker polypeptides between any two of the components. The first and second polypeptides are covalently linked to each other by a disulfide bond, e.g., a disulfide bond formed between Cys residues of the MHC class II α chain and β chain, as described below.

[0088] In some cases, a TMP comprises: a) a first polypeptide comprising, in N-terminal to C-terminal order, i) a T1D peptide, ii) an MHC class II β chain polypeptide, iii) a TGF-β polypeptide, and iv) an Ig Fc polypeptide; and b) a second polypeptide comprising, in N-terminal to C-terminal order, i) an MHC class II α chain polypeptide, ii) a masking polypeptide, and iii) one or more MODs; wherein the TMP optionally comprises one or more independently selected linker polypeptides between any two of the components. The first and second polypeptides are covalently linked to each other by a disulfide bond, e.g., a disulfide bond formed between Cys residues of the MHC class II α chain and β chain, as described below.

[0089] In some cases, as shown schematically in Figure 2B, a TMP comprises: a) a first polypeptide comprising, in N-terminal to C-terminal order: i) a T1D peptide, ii) an MHC class II β chain polypeptide, iii) a masking polypeptide, iv) an Ig Fc polypeptide, and v) one or more MODs; and b) a second polypeptide comprising, in N-terminal to C-terminal order: i) an MHC class II α chain polypeptide, ii) a TGF-β polypeptide; and optionally, one or more independently selected linker polypeptides between any two of the components. The first and second polypeptides are covalently linked to each other by a disulfide bond, e.g., a disulfide bond formed between Cys residues of the MHC class II α chain and β chain, as described below.

[0090] In some cases, a TMP comprises: a) a first polypeptide comprising, in N-terminal to C-terminal order: i) a T1D peptide, ii) an MHC class II β chain polypeptide, iii) a TGF-β polypeptide, iv) an Ig Fc polypeptide, and v) one or more MODs; and b) a second polypeptide comprising, in N-terminal to C-terminal order: i) an MHC class II α chain polypeptide, ii) a masking polypeptide; wherein the TMP optionally comprises one or more independently selected linker polypeptides between any two of the components. The first and second polypeptides are covalently linked to each other by a disulfide bond, e.g., a disulfide bond formed between Cys residues of the MHC class II α chain and β chain, as described below.

[0091] In some cases, as shown schematically in Figure 2C, a TMP comprises: a) a first polypeptide comprising, in N-terminal to C-terminal order: i) a T1D peptide, ii) an MHC class II β chain polypeptide, iii) a masking polypeptide, and iv) an Ig Fc polypeptide; and b) a second polypeptide comprising, in N-terminal to C-terminal order: i) one or more MODs, ii) an MHC class II α chain polypeptide, and iii) a TGF-β polypeptide; wherein the TMP optionally comprises one or more independently selected linker polypeptides between any two of the components. The first and second polypeptides are covalently linked to each other by a disulfide bond, e.g., a disulfide bond formed between Cys residues of the MHC class II α chain and β chain, as described below.

[0092] In some cases, a TMP comprises: a) a first polypeptide comprising, in N-terminal to C-terminal order, i) a T1D peptide, ii) an MHC class II β chain polypeptide, iii) a TGF-β polypeptide, and iv) an Ig Fc polypeptide; and b) a second polypeptide comprising, in N-terminal to C-terminal order, i) one or more MODs, ii) an MHC class II α chain polypeptide, and iii) a masking polypeptide, wherein the TMP optionally comprises one or more independently selected linker polypeptides between any two of the components. The first and second polypeptides are covalently linked to each other by a disulfide bond, e.g., a disulfide bond formed between Cys residues of the MHC class II α chain and β chain, as described below.

[0093] In some cases, as shown schematically in Figure 2D, a TMP comprises: a) a first polypeptide comprising, in N-terminal to C-terminal order: i) a T1D peptide, ii) an MHC class II β chain polypeptide, iii) a masking polypeptide, and iv) a TGF-β polypeptide; and b) a second polypeptide comprising, in N-terminal to C-terminal order: i) one or more MODs, ii) an MHC class II α chain polypeptide, and iii) an Ig Fc polypeptide; wherein the TMP optionally comprises one or more independently selected linker polypeptides between any two of the components. The first and second polypeptides are covalently linked to each other by a disulfide bond, e.g., a disulfide bond formed between Cys residues of the MHC class II α chain and β chain, as described below.

[0094] In some cases, a TMP comprises: a) a first polypeptide comprising, in N-terminal to C-terminal order, i) a T1D peptide, ii) an MHC class II β chain polypeptide, iii) a TGF-β polypeptide, and iv) a masking polypeptide; and b) a second polypeptide comprising, in N-terminal to C-terminal order, i) one or more MODs, ii) an MHC class II α chain polypeptide, and iii) an Ig Fc polypeptide, wherein the TMP optionally comprises one or more independently selected linker polypeptides between any two of the components. The first and second polypeptides are covalently linked to each other by a disulfide bond, e.g., a disulfide bond formed between Cys residues of the MHC class II α chain and β chain, as described below.

[0095] Disulfide Linkage As described above, the first and second polypeptides of the TMP heterodimer are covalently linked to each other via at least one disulfide bond. For example, at least one disulfide bond exists between i) a Cys present in the first MHC class II polypeptide and a Cys present in the second MHC class II polypeptide, or ii) a Cys present in the peptide linker of the first polypeptide and a Cys present in the MHC class II polypeptide present in the second polypeptide, or ii) a Cys present in the peptide linker of the second polypeptide and a Cys present in the MHC class II polypeptide present in the first polypeptide, or iv) a Cys present in the linker of the first polypeptide and a Cys present in the linker of the second polypeptide.

[0096] 1) Disulfide bonds between two MHC class II polypeptides As described above, in some cases, a first polypeptide of a TMP heterodimer comprises a first MHC class II polypeptide comprising an amino acid substitution resulting in a Cys (the "first Cys"), and a second polypeptide comprises a second MHC class II polypeptide comprising an amino acid substitution resulting in a Cys (the "second Cys"), and the heterodimer comprises a disulfide bond formed between the first Cys and the second Cys. For example, in some cases, a first polypeptide comprises an MHC class II β polypeptide comprising an amino acid substitution resulting in a Cys (the "first Cys"), and a second polypeptide comprises an MHC class II α polypeptide comprising an amino acid substitution resulting in a Cys (the "second Cys"), and the heterodimer comprises a disulfide bond formed between the first Cys and the second Cys.

[0097] Arrangement of components In some cases, for example, a TMP comprises: a) a first polypeptide comprising, from N-terminus to C-terminus: i) a T1D peptide; and ii) a first MHC class II polypeptide comprising a substitution of an amino acid (other than Cys) with a Cys (the "first Cys"), wherein the components of the first polypeptide are optionally connected by a linker that does not contain Cys; and b) a second MHC class II polypeptide comprising, from N-terminus to C-terminus: i) one or more MODs; ii) a second MHC class II polypeptide comprising a substitution of an amino acid (other than Cys) with a Cys (the "second Cys"); and iii) optionally an Ig and a second polypeptide comprising an Fc polypeptide, optionally connected by one or more linkers that do not contain Cys, wherein the first and second polypeptides are linked via a disulfide bond between a first Cys and a second Cys, and the first or second polypeptide comprises a TGF-β polypeptide, and the first or second polypeptide comprises a masking polypeptide. In any of the above embodiments, in some cases, one or more MODs are PD-L1 polypeptides or variants thereof, in some cases, one or more MODs are 4-1BBL polypeptides or variants thereof, in some cases, one or more MODs are IL-2 polypeptides or variants thereof, and in some cases, one or more MODs are FasL polypeptides or variants thereof. In some cases, the T1D peptide is a proinsulin peptide. In some cases, the T1D peptide is a GAD peptide. In any of the above embodiments, in some cases, the first MHC class II polypeptide is an MHC class II β chain polypeptide and the second MHC class II polypeptide is an MHC class II α chain polypeptide.

[0098] As another example, in some cases, a TMP comprises: a) a first polypeptide comprising, from N-terminus to C-terminus, i) a T1D peptide, and ii) a first MHC class II polypeptide comprising a substitution of an amino acid (other than Cys) with Cys (the "first Cys"), wherein the components of the first polypeptide are optionally connected by a linker that does not contain Cys; and b) a second MHC class II polypeptide comprising, from N-terminus to C-terminus, i) a substitution of an amino acid (other than Cys) with Cys (the "second Cys"), ii) one or more MODs, and iii) optionally an Ig and a second polypeptide comprising an Fc polypeptide, optionally connected by one or more linkers that do not contain Cys, wherein the first and second polypeptides are linked via a disulfide bond between a first Cys and a second Cys, and the first or second polypeptide comprises a TGF-β polypeptide, and the first or second polypeptide comprises a masking polypeptide. In any of the above embodiments, in some cases, one or more MODs are PD-L1 polypeptides or variants thereof, in some cases, one or more MODs are 4-1BBL polypeptides or variants thereof, in some cases, one or more MODs are IL-2 polypeptides or variants thereof, and in some cases, one or more MODs are FasL polypeptides or variants thereof. In some cases, the T1D peptide is a proinsulin peptide. In some cases, the T1D peptide is a GAD peptide. In any of the above embodiments, in some cases, the first MHC class II polypeptide is an MHC class II β chain polypeptide and the second MHC class II polypeptide is an MHC class II α chain polypeptide.

[0099] As another example, in some cases, a TMP comprises: a) a first polypeptide comprising, from N-terminus to C-terminus, i) a T1D peptide, and ii) a first MHC class II polypeptide comprising a substitution of an amino acid (other than Cys) with a Cys (the "first Cys"), wherein the components of the first polypeptide are optionally connected by a linker that does not contain Cys; and b) a second MHC class II polypeptide comprising, from N-terminus to C-terminus, i) a substitution of an amino acid (other than Cys) with a Cys (the "second Cys"), ii) an Ig and iii) a second polypeptide comprising one or more MODs, wherein the components of the second polypeptide are optionally connected by a linker that does not contain Cys, wherein the first and second polypeptides are linked via a disulfide bond between a first Cys and a second Cys, and wherein the first or second polypeptide comprises a TGF-β polypeptide, and the first or second polypeptide comprises a masking polypeptide. In any of the above embodiments, in some cases, one or more MODs are PD-L1 polypeptides or variants thereof, in some cases, one or more MODs are 4-1BBL polypeptides or variants thereof, in some cases, one or more MODs are IL-2 polypeptides or variants thereof, and in some cases, one or more MODs are FasL polypeptides or variants thereof. In some cases, the T1D peptide is a proinsulin peptide. In some cases, the T1D peptide is a GAD peptide. In any of the above embodiments, in some cases, the first MHC class II polypeptide is an MHC class II β chain polypeptide and the second MHC class II polypeptide is an MHC class II α chain polypeptide.

[0100] As another example, in some cases, a TMP comprises a) a first polypeptide comprising, from N-terminus to C-terminus, i) one or more MODs, ii) a T1D peptide, and iii) a first MHC class II polypeptide comprising a substitution of an amino acid (other than Cys) with a Cys (the "first Cys"), where the components of the first polypeptide are optionally connected by one or more linkers that do not contain Cys; and b) a second polypeptide comprising, from N-terminus to C-terminus, i) a second MHC class II polypeptide comprising a substitution of an amino acid (other than Cys) with a Cys (the "second Cys"), and ii) optionally an Ig Fc polypeptide, where the Ig When Fc is present, the second polypeptide component optionally comprises a second polypeptide connected by a Cys-free linker, wherein the first and second polypeptides are linked via a disulfide bond between the first Cys and the second Cys, and the first or second polypeptide comprises a TGF-β polypeptide, and the first or second polypeptide comprises a masking polypeptide. In any of the above embodiments, in some cases, one or more MODs are PD-L1 polypeptides or variants thereof, in some cases, one or more MODs are 4-1BBL polypeptides or variants thereof, in some cases, one or more MODs are IL-2 polypeptides or variants thereof, and in some cases, one or more MODs are FasL polypeptides or variants thereof. In some cases, the T1D peptide is a proinsulin peptide. In some cases, the T1D peptide is a GAD peptide. In any of the above embodiments, in some cases, the first MHC class II polypeptide is an MHC class II β chain polypeptide and the second MHC class II polypeptide is an MHC class II α chain polypeptide.

[0101] As another example, in some cases, a TMP comprises a) a first polypeptide comprising, from N-terminus to C-terminus, i) a T1D peptide, ii) a first MHC class II polypeptide comprising a substitution of an amino acid (other than Cys) with Cys (the "first Cys"), and iii) one or more MODs, wherein the components of the first polypeptide are optionally connected by one or more linkers that do not contain Cys; and b) a second polypeptide comprising, from N-terminus to C-terminus, i) a second MHC class II polypeptide comprising a substitution of an amino acid (other than Cys) with Cys (the "second Cys"), and ii) optionally an Ig Fc polypeptide, wherein the Ig Fc polypeptide is optionally an Ig Fc polypeptide. When Fc is present, the second polypeptide component optionally comprises a second polypeptide connected by a Cys-free linker, wherein the first and second polypeptides are linked via a disulfide bond between the first Cys and the second Cys, and the first or second polypeptide comprises a TGF-β polypeptide, and the first or second polypeptide comprises a masking polypeptide. In any of the above embodiments, in some cases, one or more MODs are PD-L1 polypeptides or variants thereof, in some cases, one or more MODs are 4-1BBL polypeptides or variants thereof, in some cases, one or more MODs are IL-2 polypeptides or variants thereof, and in some cases, one or more MODs are FasL polypeptides or variants thereof. In some cases, the T1D peptide is a proinsulin peptide. In some cases, the T1D peptide is a GAD peptide. In any of the above embodiments, in some cases, the first MHC class II polypeptide is an MHC class II β chain polypeptide and the second MHC class II polypeptide is an MHC class II α chain polypeptide.

[0102] Position of Cys residues As described above, in some cases, the TMP in some cases comprises a heterodimer comprising: i) a first polypeptide chain comprising a T1D peptide, 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 MODs, the first or second polypeptide comprises a masking polypeptide, the first or second polypeptide comprises a TGF-β polypeptide, and optionally the first and / or second polypeptide comprises an Ig Fc polypeptide, wherein the first MHC class II polypeptide comprises a substitution of an amino acid (other than Cys) with a Cys (the "first Cys"), and the second MHC class II polypeptide comprises a substitution of an amino acid (other than Cys) with a Cys (the "second" Cys), and the first polypeptide and the second polypeptide are connected by a disulfide bond formed between the first Cys and the second Cys.

[0103] The first MHC class II polypeptide can be a DRB MHC class II polypeptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 5 or FIG. 20A, and having an amino acid substitution selected from P5C, F7C, Q10C, N19C, G20C, H33C, G151C, D152C, and W153C. The second MHC class II polypeptide can be a DRA MHC class II polypeptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence shown in Figure 4 or Figure 19A, and with amino acid substitutions selected from E3C, E4C, F12C, G28C, D29C, I72C, K75C, T80C, P81C, I82C, T93C, N94C, and S95C.

[0104] As an example, in some cases, the first MHC class II polypeptide is a DRB MHC class II polypeptide that includes a substitution at a residue selected from the group consisting of P5C, H33C, G151C, and W153, and the second MHC class II polypeptide is a DRA MHC class II polypeptide that includes a substitution at a residue selected from the group consisting of P81C, I82C, and D29C. For example, an MHC class II β chain polypeptide can comprise an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence shown in Figure 5 or Figure 20A, wherein the polypeptide comprises a Cys at a residue position selected from the group consisting of 5, 33, 151, and 153; an MHC class II α chain polypeptide can comprise an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence shown in Figure 4 or Figure 19A, wherein the polypeptide comprises a Cys at a residue selected from the group consisting of 81, 82, and 29.

[0105] As another example, a disulfide can be formed between one of the specific pairs of Cys residues in Table 2 below.

[0106] (Table 2) TIFF2024517658000007.tif200110

[0107] For example, a DRB MHC class II polypeptide can comprise an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 5 or FIG. 20A, and a DRA MHC class II polypeptide can comprise an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 4 or FIG. 19A, wherein the β chain polypeptide and the α chain polypeptide are connected by a disulfide formed between pairs of Cys residues selected from the group consisting of β chain polypeptide residue 5 and α chain polypeptide residue 81; β chain polypeptide residue 33 and α chain polypeptide residue 81; β chain polypeptide residue 33 and α chain polypeptide residue 82; β chain polypeptide residue 151 and α chain polypeptide residue 29; and β chain polypeptide residue 153 and α chain polypeptide residue 29.

[0108] In some cases, the TMP comprises a DRB MHC class II polypeptide comprising a P5C substitution and a DRA MHC class II polypeptide comprising a P81C substitution, wherein the first and second polypeptides of the TMP are linked via a disulfide bond between a Cys at residue 5 in the DRB MHC class II polypeptide and a Cys at residue 81 in the DRA MHC class II polypeptide. In some cases, a linker that does not contain a Cys can be optionally used to connect the components of the first or second polypeptide. In some cases, one or more MODs present in the TMP are a PD-L1 polypeptide or a variant thereof, an IL-2 polypeptide or a variant thereof, or a FasL polypeptide or a variant thereof. In some cases, the TMP comprises an Ig Fc polypeptide, and in some cases, the IgFc polypeptide is a human IgG1 Fc polypeptide optionally comprising L234A and L235A (L14A and L15A of the amino acid sequence shown in Figure 21A) substitutions. In some cases, the DRB MHC class II polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Figure 20B and comprises a Cys at position 5. In some cases, the DRA MHC class II polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Figure 19J and comprises a Cys at position 81. In some cases, the T1D peptide is a proinsulin peptide. In some cases, the T1D peptide is a proinsulin peptide selected from SLQPLALEGSLQKRG (SEQ ID NO: 175; proIns 76-90), SLQPLALEGSLQSRG (SEQ ID NO: 159; proIns 76-90; K88S), and GAGSLQPLALEGSLQKRG (SEQ ID NO: 176; proIns 73-90).In some cases, the T1D peptide is a GAD peptide. In some cases, the T1D peptide is a GAD peptide selected from NFFRMVISNPAAT (SEQ ID NO: 177; GAD65 555-567) and NFIRMVISNPAAT (SEQ ID NO: 163; GAD65 555-567; F557I).

[0109] In some cases, the TMP comprises a DRB MHC class II polypeptide comprising an H33C substitution and a DRA MHC class II polypeptide comprising a P81C substitution, wherein the first and second polypeptides of the TMP are linked via a disulfide bond between a Cys at residue 33 in the DRB MHC class II polypeptide and a Cys at residue 81 in the DRA MHC class II polypeptide. In some cases, a linker that does not contain a Cys can be optionally used to connect the components of the first or second polypeptide. In some cases, one or more MODs present in the TMP are a PD-L1 polypeptide or a variant thereof, an IL-2 polypeptide or a variant thereof, or a FasL polypeptide or a variant thereof. In some cases, the TMP comprises an Ig Fc polypeptide, and in some cases, the IgFc polypeptide is a human IgG1 Fc polypeptide optionally comprising L234A and L235A (L14A and L15A of the amino acid sequence shown in Figure 21A) substitutions. In some cases, the DRB MHC class II polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence shown in Figure 20G, and comprises a Cys at position 33. In some cases, the DRA MHC class II polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence shown in Figure 19J, and comprises a Cys at position 81. In some cases, the T1D peptide is a proinsulin peptide. In some cases, the T1D peptide is a proinsulin peptide selected from SLQPLALEGSLQKRG (SEQ ID NO: 175; proIns 76-90), SLQPLALEGSLQSRG (SEQ ID NO: 159; proIns 76-90; K88S), and GAGSLQPLALEGSLQKRG (SEQ ID NO: 176; proIns 73-90).In some cases, the T1D peptide is a GAD peptide. In some cases, the T1D peptide is a GAD peptide selected from NFFRMVISNPAAT (SEQ ID NO: 177; GAD65 555-567) and NFIRMVISNPAAT (SEQ ID NO: 163; GAD65 555-567; F557I).

[0110] In some cases, the TMP comprises a DRB MHC class II polypeptide comprising an H33C substitution and a DRA MHC class II polypeptide comprising an I82C substitution, wherein the first and second polypeptides of the TMP are linked via a disulfide bond between a Cys at residue 33 in the DRB MHC class II polypeptide and a Cys at residue 81 in the DRA MHC class II polypeptide. In some cases, a linker that does not contain a Cys can be optionally used to connect the components of the first or second polypeptide. In some cases, one or more MODs present in the TMP are a PD-L1 polypeptide or a variant thereof, an IL-2 polypeptide or a variant thereof, or a FasL polypeptide or a variant thereof. In some cases, the TMP comprises an Ig Fc polypeptide, and in some cases, the IgFc polypeptide is a human IgG1 Fc polypeptide optionally comprising L234A and L235A (L14A and L15A of the amino acid sequence shown in Figure 21A) substitutions. In some cases, the DRB MHC class II polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence shown in Figure 20G and comprises a Cys at position 33. In some cases, the DRA MHC class II polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence shown in Figure 19K and comprises a Cys at position 82. In some cases, the T1D peptide is a proinsulin peptide. In some cases, the T1D peptide is a proinsulin peptide selected from SLQPLALEGSLQKRG (SEQ ID NO: 175; proIns 76-90), SLQPLALEGSLQSRG (SEQ ID NO: 159; proIns 76-90; K88S), and GAGSLQPLALEGSLQKRG (SEQ ID NO: 176; proIns 73-90).In some cases, the T1D peptide is a GAD peptide. In some cases, the T1D peptide is a GAD peptide selected from NFFRMVISNPAAT (SEQ ID NO: 177; GAD65 555-567) and NFIRMVISNPAAT (SEQ ID NO: 163; GAD65 555-567; F557I).

[0111] In some cases, the TMP comprises a DRB MHC class II polypeptide comprising a G151C substitution and a DRA MHC class II polypeptide comprising a D29C substitution, wherein the first and second polypeptides of the TMP are linked via a disulfide bond between a Cys at residue 33 in the DRB MHC class II polypeptide and a Cys at residue 81 in the DRA MHC class II polypeptide. In some cases, a linker that does not contain a Cys can be optionally used to connect the components of the first or second polypeptide. In some cases, one or more MODs present in the TMP are a PD-L1 polypeptide or a variant thereof, an IL-2 polypeptide or a variant thereof, or a FasL polypeptide or a variant thereof. In some cases, the TMP comprises an Ig Fc polypeptide, and in some cases, the IgFc polypeptide is a human IgG1 Fc polypeptide that optionally comprises L234A and L235A (L14A and L15A of the amino acid sequence shown in Figure 21A) substitutions. In some cases, the DRB MHC class II polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Figure 20H, and comprises a Cys at position 151. In some cases, the DRA MHC class II polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Figure 19F, and comprises a Cys at position 29. In some cases, the T1D peptide is a proinsulin peptide. In some cases, the T1D peptide is a proinsulin peptide selected from SLQPLALEGSLQKRG (SEQ ID NO: 175; proIns 76-90), SLQPLALEGSLQSRG (SEQ ID NO: 159; proIns 76-90; K88S), and GAGSLQPLALEGSLQKRG (SEQ ID NO: 176; proIns 73-90).In some cases, the T1D peptide is a GAD peptide. In some cases, the T1D peptide is a GAD peptide selected from NFFRMVISNPAAT (SEQ ID NO: 177; GAD65 555-567) and NFIRMVISNPAAT (SEQ ID NO: 163; GAD65 555-567; F557I).

[0112] In some cases, the TMP comprises a DRB MHC class II polypeptide comprising a W153C substitution and a DRA MHC class II polypeptide comprising a D29C substitution, wherein the first and second polypeptides of the TMP are linked via a disulfide bond between a Cys at residue 33 in the DRB MHC class II polypeptide and a Cys at residue 81 in the DRA MHC class II polypeptide. In some cases, a Cys-free linker can be optionally used to connect the components of the first or second polypeptide. In some cases, one or more MODs present in the TMP are a PD-L1 polypeptide or a variant thereof, an IL-2 polypeptide or a variant thereof, or a FasL polypeptide or a variant thereof. In some cases, the TMP comprises an Ig Fc polypeptide, and in some cases, the IgFc polypeptide is a human IgG1 Fc polypeptide optionally comprising L234A and L235A (L14A and L15A of the amino acid sequence shown in Figure 21A) substitutions. In some cases, the DRB MHC class II polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Figure 20J, and comprises a Cys at position 153. In some cases, the DRA MHC class II polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Figure 19F, and comprises a Cys at position 29. In some cases, the T1D peptide is a proinsulin peptide. In some cases, the T1D peptide is a proinsulin peptide selected from SLQPLALEGSLQKRG (SEQ ID NO: 175; proIns 76-90), SLQPLALEGSLQSRG (SEQ ID NO: 159; proIns 76-90; K88S), and GAGSLQPLALEGSLQKRG (SEQ ID NO: 176; proIns 73-90).In some cases, the T1D peptide is a GAD peptide. In some cases, the T1D peptide is a GAD peptide selected from NFFRMVISNPAAT (SEQ ID NO: 177; GAD65 555-567) and NFIRMVISNPAAT (SEQ ID NO: 163; GAD65 555-567; F557I).

[0113] 2) Disulfide bond between the peptide linker and the MHC class II polypeptide In some cases, the TMP includes a peptide linker between the T1D peptide and the MHC class II polypeptide. If desired, the linker can include a Cys that can be used to form a disulfide bond between the two polypeptides of the heterodimer.

[0114] Arrangement of components In some cases, the TMP comprises: a) a first polypeptide comprising, in order from N-terminus to C-terminus, i) a T1D peptide, ii) a peptide linker comprising Cys, and iii) an MHC class II beta polypeptide; and b) a second polypeptide comprising, in order from N-terminus to C-terminus (optionally comprising one or more linkers that do not contain Cys): i) one or more MODs, ii) an MHC class II alpha chain, and (optionally) iii) an Ig Fc polypeptide, wherein the first or second polypeptide comprises a TGF-beta polypeptide, and the first or second polypeptide comprises a masking polypeptide. The Cys-containing peptide linker can comprise an amino acid sequence selected from (CGGGS)(GGGGS)n (SEQ ID NO: 178), (GCGGS)(GGGGS)n (SEQ ID NO: 179), (GGCGS)(GGGGS)n (SEQ ID NO: 180), (GGGCS)(GGGGS)n (SEQ ID NO: 181), and (GGGGC)(GGGGS)n (SEQ ID NO: 182), where n is an integer between 1 and 10 (e.g., where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). In any of the above embodiments, in some cases, the MOD is a PD-L1 polypeptide or a variant thereof; in some cases, the MOD is an IL-2 polypeptide or a variant thereof; and in some cases, the MOD is a FasL polypeptide or a variant thereof. In some cases, the T1D peptide is a proinsulin peptide. In some cases, the T1D peptide is a GAD peptide. In any of the above embodiments, in some cases, the MHC class II α chain comprises an amino acid substitution of an amino acid (other than Cys) with Cys such that Cys in the MHC class II α chain forms a disulfide bond with Cys in the first polypeptide linker.

[0115] In some cases, the TMP comprises: a) a first polypeptide comprising, in order from N-terminus to C-terminus, i) a T1D peptide, ii) a peptide linker comprising Cys, and iii) an MHC class II beta polypeptide; and b) a second polypeptide comprising, in order from N-terminus to C-terminus (optionally comprising one or more linkers that do not contain Cys), i) an MHC class II alpha chain, ii) one or more MODs, and iii) an Ig Fc polypeptide, wherein the first or second polypeptide comprises a TGF-beta polypeptide, and the first or second polypeptide comprises a masking polypeptide. A peptide linker containing Cys can comprise an amino acid sequence selected from, for example, (CGGGS)(GGGGS)n (SEQ ID NO: 178), (GCGGS)(GGGGS)n (SEQ ID NO: 179), (GGCGS)(GGGGS)n (SEQ ID NO: 180), (GGGCs)(GGGGS)n (SEQ ID NO: 181), and (GGGGC)(GGGGS)n (SEQ ID NO: 182), where n is an integer between 1 and 10 (e.g., where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). Other peptide linkers containing Cys may also be used. In any of the above embodiments, in some cases, one or more MODs are PD-L1 polypeptides or variants thereof; in some cases, one or more MODs are 4-1BBL polypeptides or variants thereof; in some cases, one or more MODs are IL-2 polypeptides or variants thereof; and in some cases, one or more MODs are FasL polypeptides or variants thereof. In some cases, the T1D peptide is a proinsulin peptide. In some cases, the T1D peptide is a GAD peptide. In any of the above embodiments, in some cases, the MHC class II α chain comprises an amino acid substitution of an amino acid (other than Cys) with Cys, such that Cys in the MHC class II α chain forms a disulfide bond with Cys in the linker.

[0116] In some cases, the TMP comprises: a) a first polypeptide comprising, in order from N-terminus to C-terminus, i) a T1D peptide, ii) a peptide linker comprising Cys, and iii) an MHC class II beta polypeptide; and b) a second polypeptide comprising, in order from N-terminus to C-terminus (optionally comprising one or more linkers that do not contain Cys), i) an MHC class II alpha chain, and ii) one or more MODs, wherein the first or second polypeptide comprises an Ig Fc polypeptide, the first or second polypeptide comprises a TGF-beta polypeptide, and the first or second polypeptide comprises a masking polypeptide. A peptide linker containing Cys can comprise an amino acid sequence selected from, for example, (CGGGS)(GGGGS)n (SEQ ID NO: 178), (GCGGS)(GGGGS)n (SEQ ID NO: 179), (GGCGS)(GGGGS)n (SEQ ID NO: 180), (GGGCs)(GGGGS)n (SEQ ID NO: 181), and (GGGGC)(GGGGS)n (SEQ ID NO: 182), where n is an integer between 1 and 10 (e.g., where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). Other peptide linkers containing Cys may also be used. In any of the above embodiments, in some cases, one or more MODs are PD-L1 polypeptides or variants thereof; in some cases, one or more MODs are 4-1BBL polypeptides or variants thereof; in some cases, one or more MODs are IL-2 polypeptides or variants thereof; and in some cases, one or more MODs are FasL polypeptides or variants thereof. In some cases, the T1D peptide is a proinsulin peptide. In some cases, the T1D peptide is a GAD peptide. In any of the above embodiments, in some cases, the MHC class II α chain comprises an amino acid substitution of an amino acid (other than Cys) with Cys, such that Cys in the MHC class II α chain forms a disulfide bond with Cys in the linker.

[0117] In some cases, the TMP comprises: a) a first polypeptide comprising, in order from N-terminus to C-terminus, i) one or more MODs, ii) a T1D peptide, ii) a peptide linker comprising Cys, and iii) an MHC class II beta polypeptide; and b) a second polypeptide comprising, in order from N-terminus to C-terminus, i) an MHC class II alpha chain, and (optionally) ii) an Ig Fc polypeptide, optionally connected by a linker not comprising Cys, wherein the first or second polypeptide comprises a TGF-beta polypeptide, and the first or second polypeptide comprises a masking polypeptide. A peptide linker containing Cys can comprise an amino acid sequence selected from, for example, (CGGGS)(GGGGS)n (SEQ ID NO: 178), (GCGGS)(GGGGS)n (SEQ ID NO: 179), (GGCGS)(GGGGS)n (SEQ ID NO: 180), (GGGCs)(GGGGS)n (SEQ ID NO: 181), and (GGGGC)(GGGGS)n (SEQ ID NO: 182), where n is an integer between 1 and 10 (e.g., where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). Other peptide linkers containing Cys may also be used. In any of the above embodiments, in some cases, one or more MODs are PD-L1 polypeptides or variants thereof; in some cases, one or more MODs are 4-1BBL polypeptides or variants thereof; in some cases, one or more MODs are IL-2 polypeptides or variants thereof; and in some cases, one or more MODs are FasL polypeptides or variants thereof. In some cases, the T1D peptide is a proinsulin peptide. In some cases, the T1D peptide is a GAD peptide. In any of the above embodiments, in some cases, the MHC class II α chain comprises an amino acid substitution of an amino acid (other than Cys) with Cys, such that Cys in the MHC class II α chain forms a disulfide bond with Cys in the linker.

[0118] In some cases, the TMP comprises: a) a first polypeptide comprising, in order from N-terminus to C-terminus, i) a T1D peptide, ii) a peptide linker comprising Cys, iii) an MHC class II beta polypeptide, and iv) one or more MODs; and b) a second polypeptide comprising, in order from N-terminus to C-terminus, i) an MHC class II alpha chain, and (optionally) ii) an Ig Fc polypeptide, optionally connected by a linker not comprising Cys, wherein the first or second polypeptide comprises a TGF-beta polypeptide, and the first or second polypeptide comprises a masking polypeptide. A peptide linker containing Cys can comprise an amino acid sequence selected from, for example, (CGGGS)(GGGGS)n (SEQ ID NO: 178), (GCGGS)(GGGGS)n (SEQ ID NO: 179), (GGCGS)(GGGGS)n (SEQ ID NO: 180), (GGGCs)(GGGGS)n (SEQ ID NO: 181), and (GGGGC)(GGGGS)n (SEQ ID NO: 182), where n is an integer between 1 and 10 (e.g., where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). Other peptide linkers containing Cys may also be used. In any of the above embodiments, in some cases, one or more MODs are PD-L1 polypeptides or variants thereof; in some cases, one or more MODs are 4-1BBL polypeptides or variants thereof; in some cases, one or more MODs are IL-2 polypeptides or variants thereof; and in some cases, one or more MODs are FasL polypeptides or variants thereof. In some cases, the T1D peptide is a proinsulin peptide. In some cases, the T1D peptide is a GAD peptide. In any of the above embodiments, in some cases, the MHC class II α chain comprises an amino acid substitution of an amino acid (other than Cys) with Cys, such that Cys in the MHC class II α chain forms a disulfide bond with Cys in the linker.

[0119] Position of Cys residues As described above, in some cases, the TMP comprises a heterodimer comprising: i) a T1D peptide, ii) a peptide linker comprising Cys, and iii) 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 MODs, the first or second polypeptide comprises a TGF-β polypeptide, the first or second polypeptide comprises a masking polypeptide, and optionally the first or second polypeptide comprises an Ig Fc polypeptide, and the second MHC class II polypeptide comprises a substitution of an amino acid (other than Cys) with Cys, and the first and second polypeptides are linked via a disulfide bond between the Cys in the peptide linker and the Cys provided by the substitution in the second MHC class II polypeptide.

[0120] For example, in some cases, the peptide linker can comprise an amino acid sequence selected from (CGGGS)(GGGGS)n (SEQ ID NO: 178), (GCGGS)(GGGGS)n (SEQ ID NO: 179), (GGCGS)(GGGGS)n (SEQ ID NO: 180), (GGGCs)(GGGGS)n (SEQ ID NO: 181), and (GGGGC)(GGGGS)n (SEQ ID NO: 182), where n is an integer between 1 and 10 (e.g., where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; generally, n=2 or 3 is useful), and the first MHC class II polypeptide is a DRB MHC class II polypeptide and the second MHC class II polypeptide is a DRA MHC class II polypeptide comprising a substitution of an amino acid (other than Cys) with Cys.

[0121] Specific examples of Cys-containing linkers between the T1D peptide and the DRB MHC class II polypeptide and Cys residues in the DRA MHC class II polypeptide that can form disulfide bonds are listed in Table 3 below.

[0122] (Table 3) TIFF2024517658000008.tif159128

[0123] In some cases, when the first and second MHC class II polypeptides are DRB and DRA polypeptides, respectively, the DRB MHC class II polypeptide can comprise an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence shown in Figure 5 or Figure 20A. The DRA MHC class II polypeptide can have an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence shown in Figure 4 or Figure 19A, and can include, for example, a Cys at position 72 or position 75. In any of the above embodiments, n=0, 1, 2, 3, 4, or more, but typically 2 or 3 is used, creating a total linker length of 15 or 20 amino acids, although longer lengths may be possible. In any of the above embodiments, a Cys-free linker can optionally be used to connect other components of the first or second polypeptide. In some cases, the Ig Fc polypeptide is present and is a human IgG1 Fc polypeptide, optionally including L234A and L235A (L14A and L15A of the amino acid sequence shown in FIG. 21A) substitutions. In some cases, the T1D peptide is a proinsulin peptide. In some cases, the T1D peptide is a proinsulin peptide selected from SLQPLALEGSLQKRG (SEQ ID NO: 175; proIns 76-90), SLQPLALEGSLQSRG (SEQ ID NO: 159; proIns 76-90; K88S), and GAGSLQPLALEGSLQKRG (SEQ ID NO: 176; proIns 73-90). In some cases, the T1D peptide is a GAD peptide. In some cases, the T1D peptide is a GAD peptide selected from NFFRMVISNPAAT (SEQ ID NO: 177; GAD65 555-567) and NFIRMVISNPAAT (SEQ ID NO: 163; GAD65 555-567; F557I).In any of the above embodiments, in some cases, the one or more MODs is a PD-L1 polypeptide or a variant thereof, a 4-1BBL polypeptide or a variant thereof, an IL-2 polypeptide or a variant thereof, or a FasL polypeptide or a variant thereof.

[0124] 3) a disulfide bond between the peptide linkers in the first and second polypeptides; In some cases, the TMP comprises a first Cys-containing peptide linker between the T1D peptide and the MHC class II polypeptide in the first polypeptide and a second Cys-containing peptide linker between the two components in the second polypeptide, in which case the Cys residues of the first and second Cys-containing linkers can be used to form disulfide bonds between the two polypeptides of the heterodimer.

[0125] In some cases, the TMP comprises a) a first polypeptide comprising, in order from N-terminus to C-terminus: i) a T1D peptide; ii) a first peptide linker comprising Cys; and iii) an MHC class II β polypeptide (e.g., a DRB MHC class II polypeptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence depicted in FIG. 5 or FIG. 20A); and b) a first polypeptide comprising, in order from N-terminus to C-terminus: i) one or more MODs; ii) an MHC class II α chain (e.g., a DRA MHC class II polypeptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence depicted in FIG. 4 or FIG. 19A); and (optionally) iii) an Ig and a second polypeptide comprising an Fc polypeptide, wherein the first or second polypeptide comprises a TGF-β polypeptide, and the first or second polypeptide comprises a masking polypeptide. The second polypeptide comprises a second peptide linker comprising Cys, and the second peptide linker is located either between the MOD(s) and the MHC class II α chain or between the MHC class II α chain and the Ig Fc (if present). The second polypeptide may optionally comprise one or more linkers that do not contain Cys. A peptide linker containing Cys can comprise an amino acid sequence selected from (CGGGS)(GGGGS)n (SEQ ID NO: 178), (GCGGS)(GGGGS)n (SEQ ID NO: 179), (GGCGS)(GGGGS)n (SEQ ID NO: 180), (GGGCS)(GGGGS)n (SEQ ID NO: 181), and (GGGGC)(GGGGS)n (SEQ ID NO: 182), where n is an integer between 1 and 10 (e.g., where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10).In any of the above embodiments, in some cases, one or more MODs are PD-L1 polypeptides or variants thereof, in some cases, one or more MODs are 4-1BBL polypeptides or variants thereof, in some cases, one or more MODs are IL-2 polypeptides or variants thereof, and in some cases, one or more MODs are FasL polypeptides or variants thereof. In some cases, the T1D peptide is a proinsulin peptide. In some cases, the T1D peptide is a GAD peptide.

[0126] In some cases, the TMP comprises a) a first polypeptide comprising, from N-terminus to C-terminus, i) a T1D peptide, ii) a first peptide linker comprising Cys, and iii) an MHC class II β polypeptide (e.g., a DRB MHC class II polypeptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence depicted in FIG. 5 or FIG. 20A ); and b) a first polypeptide comprising, from N-terminus to C-terminus, i) an MHC class II α chain (e.g., a DRA MHC class II polypeptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence depicted in FIG. 4 or FIG. 19A ), ii) an Ig and iii) a second polypeptide comprising one or more MODs, wherein the first or second polypeptide comprises a TGF-β polypeptide, and the first or second polypeptide comprises a masking polypeptide. The second polypeptide comprises a second peptide linker comprising Cys, and the second peptide linker is located either between the MHC class II α chain and the Ig Fc or between the Ig Fc and one or more MODs. The second polypeptide may optionally comprise one or more linkers that do not contain Cys. A peptide linker containing Cys can comprise an amino acid sequence selected from (CGGGS)(GGGGS)n (SEQ ID NO: 178), (GCGGS)(GGGGS)n (SEQ ID NO: 179), (GGCGS)(GGGGS)n (SEQ ID NO: 180), (GGGCS)(GGGGS)n (SEQ ID NO: 181), and (GGGGC)(GGGGS)n (SEQ ID NO: 182), where n is an integer between 1 and 10 (e.g., where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10).In any of the above embodiments, in some cases, one or more MODs are PD-L1 polypeptides or variants thereof, in some cases, one or more MODs are 4-1BBL polypeptides or variants thereof, in some cases, one or more MODs are IL-2 polypeptides or variants thereof, and in some cases, one or more MODs are FasL polypeptides or variants thereof. In some cases, the T1D peptide is a proinsulin peptide. In some cases, the T1D peptide is a GAD peptide.

[0127] C) Single-stranded TMP As noted above, in some cases, the TMP of the present disclosure is a single-chain (single polypeptide chain) TMP. A single-chain TMP comprises: i) a T1D peptide; ii) an MHC class II β chain polypeptide; iii) an MHC class II α chain polypeptide; iv) one or more MODs; v) a TGF-β polypeptide; vi) a masking polypeptide; and, optionally, one or more independently selected linker polypeptides. A single-chain TMP of the present disclosure may also comprise an Ig Fc polypeptide. A single-chain TMP may comprise two or more MODs, where the two or more MODs may have the same or different amino acid sequences. Non-limiting examples of single-chain TMPs are shown schematically in Figures 3A-3D.

[0128] Arrangement of components The arrangement of the components of a single-chain TMP can vary. As a first, non-limiting example, in some cases, the single-chain TMP comprises, from N-terminus to C-terminus, i) a T1D peptide, ii) an MHC class II β polypeptide, iii) an MHC class II α polypeptide, iv) a TGF-β polypeptide or a masking polypeptide, v) a masking polypeptide or a TGF-β polypeptide, vi) an Ig Fc polypeptide, and vii) one or more MODs. The components of a single-chain TMP can be connected by one or more independently selected linkers.

[0129] As a second non-limiting example, in some cases, a single-chain TMP comprises, from N-terminus to C-terminus, i) a T1D peptide, ii) an MHC class II β polypeptide, iii) an MHC class II α polypeptide, iv) a TGF-β polypeptide or a masking polypeptide, v) a masking polypeptide or a TGF-β polypeptide, vi) one or more MODs, and vii) an Ig Fc polypeptide. The components of the first polypeptide may be connected by one or more independently selected linkers.

[0130] As a third non-limiting example, in some cases, a single-chain TMP comprises, in order from N-terminus to C-terminus: i) a T1D peptide, ii) an MHC class II beta polypeptide, iii) an MHC class II alpha polypeptide, iv) one or more MODs, v) a TGF-β polypeptide or a masking polypeptide, vi) a masking polypeptide or a TGF-β polypeptide, and vii) an Ig Fc polypeptide. The components of the single-chain TMP may be connected by one or more independently selected linkers.

[0131] As a fourth non-limiting example, in some cases, a single-chain TMP comprises, in order from N-terminus to C-terminus, i) a T1D peptide, ii) an MHC class II beta polypeptide, iii) an MHC class II alpha polypeptide, iv) one or more MODs, v) an Ig Fc polypeptide, vi) a TGF-β polypeptide or a masking polypeptide, and vii) a masking polypeptide or a TGF-β polypeptide. The components of the single-chain TMP may be connected by one or more independently selected linkers.

[0132] As a fifth non-limiting example, in some cases, a single-chain TMP comprises, from N-terminus to C-terminus, i) a T1D peptide, ii) an MHC class II β polypeptide, iii) an MHC class II α polypeptide, iv) an Ig Fc polypeptide, v) one or more MODs, vi) a TGF-β polypeptide or a masking polypeptide, and vii) a masking polypeptide or a TGF-β polypeptide. The components of the single-chain TMP may be connected by one or more independently selected linkers.

[0133] As a sixth non-limiting example, in some cases, a single-chain TMP comprises, in order from N-terminus to C-terminus: i) a T1D peptide, ii) an MHC class II beta polypeptide, iii) an MHC class II alpha polypeptide, iv) one or more MODs, v) a TGF-β polypeptide or a masking polypeptide, vi) a masking polypeptide or a TGF-β polypeptide, and vii) an Ig Fc polypeptide. The components of the single-chain TMP may be connected by one or more independently selected linkers.

[0134] As a seventh non-limiting example, in some cases, a single-chain TMP comprises, in order from N-terminus to C-terminus, i) a T1D peptide, ii) an MHC class II β polypeptide, iii) an MHC class II α polypeptide, iv) an Ig Fc polypeptide, v) a TGF-β polypeptide or a masking polypeptide, vi) a masking polypeptide or a TGF-β polypeptide, and vii) one or more MODs. The components of a single-chain TMP may be connected by one or more independently selected linkers.

[0135] In some cases, as shown schematically in Figure 3A, in some cases, the single-chain TMP comprises, from N-terminus to C-terminus, i) a T1D peptide, ii) an MHC class II β chain polypeptide, iii) an MHC class II α chain polypeptide, iv) a masking polypeptide, v) a TGF-β polypeptide, vi) an Ig Fc polypeptide, and vii) one or more MODs, and optionally one or more independently selected linker polypeptides. In some cases, the single-chain TMP comprises, from N-terminus to C-terminus, i) a T1D peptide, ii) an MHC class II β chain polypeptide, iii) an MHC class II α chain polypeptide, iv) a TGF-β polypeptide, v) a masking polypeptide, vi) an Ig Fc polypeptide, and vii) one or more MODs, and optionally one or more independently selected linker polypeptides. In some cases, as shown schematically in Figure 3B, in some cases, the single-chain TMP comprises, from N-terminus to C-terminus, i) a T1D peptide, ii) an MHC class II β chain polypeptide, iii) an MHC class II α chain polypeptide, iv) one or more MODs, v) an Ig Fc polypeptide, vi) a masking polypeptide, and vii) a TGF-β polypeptide, and optionally one or more independently selected linker polypeptides. In some cases, the single-chain TMP comprises, from N-terminus to C-terminus, i) a T1D peptide, ii) an MHC class II β chain polypeptide, iii) an MHC class II α chain polypeptide, iv) one or more MODs, v) an Ig Fc polypeptide, vi) a TGF-β polypeptide, and vii) a masking polypeptide, and optionally one or more independently selected linker polypeptides.In some cases, as shown schematically in Figure 3C, in some cases, the single-chain TMP comprises, from N-terminal to C-terminal order: i) a T1D peptide, ii) an MHC class II β chain polypeptide, iii) an MHC class II α chain polypeptide, iv) an Ig Fc polypeptide, v) a masking polypeptide, vi) a TGF-β polypeptide, vii) one or more MODs, and optionally one or more independently selected linker polypeptides. In some cases, the single-chain TMP comprises, from N-terminal to C-terminal order: i) a T1D peptide, ii) an MHC class II β chain polypeptide, iii) an MHC class II α chain polypeptide, iv) an Ig Fc polypeptide, v) a TGF-β polypeptide, vi) a masking polypeptide, vii) one or more MODs, and optionally one or more independently selected linker polypeptides. In some cases, as shown schematically in Figure 3D, in some cases, the single-chain TMP comprises, from N-terminus to C-terminus, i) a T1D peptide, ii) an MHC class II β chain polypeptide, iii) an MHC class II α chain polypeptide, iv) an Ig Fc polypeptide, v) one or more MODs, vi) a masking polypeptide, and vii) a TGF-β polypeptide, and optionally, one or more independently selected linker polypeptides. In some cases, the single-chain TMP comprises, from N-terminus to C-terminus, i) a T1D peptide, ii) an MHC class II β chain polypeptide, iii) an MHC class II α chain polypeptide, iv) an Ig Fc polypeptide, v) one or more MODs, vi) a TGF-β polypeptide, and vii) a masking polypeptide, and optionally, one or more independently selected linker polypeptides. In any one of the above embodiments, the TMP may comprise a single MOD. In any one of the above embodiments, the TMP may comprise two copies of MOD, which may be in tandem or separated by a linker. In any one of the above embodiments, the TMP may comprise three copies of MOD, which may be in tandem or separated by a linker.

[0136] In any one of the above embodiments, the TMP comprises a peptide linker between one or more of: i) the T1D peptide and the MHC class II β chain polypeptide, ii) the MHC class II β chain polypeptide and the MHC class II α chain polypeptide, iii) the MHC class II α chain polypeptide and the masking polypeptide, iv) the MHC class II α chain polypeptide and the MOD, v) the MHC class II α chain polypeptide and the Ig Fc polypeptide, vi) the masking polypeptide and the TGF-β polypeptide, vii) the TGF-β polypeptide and the Ig Fc polypeptide, viii) the Ig Fc polypeptide and the MOD, ix) the MOD and the Ig Fc polypeptide, or any other two components of the TMP. Exemplary suitable linkers include (GGGGS)n (SEQ ID NO: 183), where n is 1, 2, 3, 4, 5, 6, 7, or 8; AAAGG (SEQ ID NO: 184), and GGSAAAGG (SEQ ID NO: 162). In any of the above embodiments, in some cases, the Ig Fc is an IgG1 Fc polypeptide or variant thereof. In any of the above embodiments, in some cases, the Ig Fc is an IgG4 Fc polypeptide or variant thereof. In any of the above embodiments, in some cases, one or more MODs are PD-L1 polypeptides or variants thereof, in some cases, one or more MODs are 4-1BBL polypeptides or variants thereof, in some cases, one or more MODs are IL-2 polypeptides or variants thereof, and in some cases, one or more MODs are FasL polypeptides or variants thereof. In some cases, the T1D peptide is a proinsulin peptide. In some cases, the T1D peptide is a GAD peptide. In any of the above embodiments, in some cases, the TMP comprises one or more intrachain disulfide bonds.

[0137] In some cases, the single-chain TMP contains one or more intrachain disulfide bonds, which can be formed a) between a Cys present in an MHC class II α chain polypeptide and a Cys present in an MHC class II β chain polypeptide, or b) between a Cys present in the peptide linker and an MHC class II polypeptide.

[0138] The TMP may comprise a homodimer of two identical single-chain TMPs. When the single-chain TMP comprises an Ig Fc polypeptide, the homodimer may comprise one or more disulfide bonds formed between the Ig Fc polypeptides of the two single-chain TMPs present in the homodimer. Alternatively, the TMP may comprise a heterodimer of two different single-chain TMPs, each comprising a polypeptide having an Ig Fc polypeptide comprising the above-described mutually specific binding sequence, e.g., a KiH sequence.

[0139] Homodimers and heterodimers The present disclosure provides proteins comprising two of the TMPs of the present disclosure. In some cases, the protein is a homodimer comprising two TMP heterodimers. In some cases, the protein is a homodimer comprising two single-chain TMPs. In some cases, the protein is a heterodimer comprising two TMP heterodimers. In some cases, the protein is a heterodimer comprising two single-chain TMPs.

[0140] The present disclosure provides a protein that is a homodimer comprising two TMP heterodimers, e.g., a first TMP heterodimer and a second TMP heterodimer, where the first TMP heterodimer and the second TMP heterodimer are the same (i.e., the first polypeptides of the first TMP heterodimer and the second TMP heterodimer have the same amino acid sequence, and the second polypeptides of the first TMP heterodimer and the second TMP heterodimer have the same amino acid sequence). The first TMP heterodimer and the second TMP heterodimer can be covalently linked to each other, for example, via one or more disulfide bonds between the Ig Fc polypeptide present in the first TMP heterodimer and the Ig Fc polypeptide present in the second TMP heterodimer.

[0141] The present disclosure provides proteins that are heterodimers comprising two TMP heterodimers, e.g., a first TMP heterodimer and a second TMP heterodimer, where the first TMP heterodimer and the second TMP heterodimer are not the same (i.e., the first polypeptide of the first TMP heterodimer and the second TMP heterodimer have different amino acid sequences and / or the second polypeptide of the first TMP heterodimer and the second TMP heterodimer have different amino acid sequences). In some cases, the first and second polypeptides of the first and second TMP heterodimers are identical except for their different MODs. For example, in some cases, the first TMP heterodimer comprises one or more MODs, where the one or more MODs comprise an IL-2 polypeptide, and the second TMP heterodimer comprises one or more MODs, where the one or more MODs comprise one or more PD-L1 polypeptides. As another example, in some cases, a first TMP heterodimer comprises one or more MODs, wherein the one or more MODs comprise an IL-2 polypeptide, and a second TMP heterodimer comprises one or more MODs, wherein the one or more MODs comprise one or more 4-1BBL polypeptides.

[0142] The present disclosure provides a protein that is a homodimer comprising two single-chain TMPs, e.g., a first single-chain TMP and a second single-chain TMP, where the first single-chain TMP and the second single-chain TMP are the same (have the same amino acid sequence). The first single-chain TMP and the second single-chain TMP may be covalently linked to each other, for example, via one or more disulfide bonds between the Ig Fc polypeptide present in the first single-chain TMP and the Ig Fc polypeptide present in the second single-chain TMP.

[0143] The present disclosure provides proteins that are heterodimers comprising two single-chain TMPs, e.g., a first single-chain TMP and a second single-chain TMP, where the first single-chain TMP and the second single-chain TMP are not the same (have different amino acid sequences). In such cases, the two single-chain TMPs may comprise polypeptides having the mutually specific binding sequences described above, e.g., Ig Fc polypeptides comprising KiH sequences. In some cases, the first and second polypeptides of the first and second single-chain TMPs are identical except for their different MODs. For example, in some cases, the first single-chain TMP comprises one or more MODs, where the one or more MODs comprise an IL-2 polypeptide, and the second single-chain TMP comprises one or more MODs, where the one or more MODs comprise one or more PD-L1 polypeptides. As another example, in some cases, a first single-chain TMP comprises one or more MODs, wherein the one or more MODs comprise an IL-2 polypeptide, and a second single-chain TMP comprises one or more MODs, wherein the one or more MODs comprise one or more 4-1BBL polypeptides. The first single-chain TMP and the second single-chain TMP may be covalently linked to each other, for example, via one or more disulfide bonds between an Ig Fc polypeptide present in the first single-chain TMP and an Ig Fc polypeptide present in the second single-chain TMP.

[0144] Immunomodulatory Polypeptides ("MODs") Suitable MODs for inclusion in the TMPs 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. In some cases, the MOD is selected from an IL-2 polypeptide, a 4-1BBL polypeptide, an ICOS-L polypeptide, an OX-40L polypeptide, a CD80 polypeptide, a CD86 polypeptide, a PD-L1 polypeptide, a FasL polypeptide, and a PD-L2 polypeptide. In some cases, the MOD is selected from an IL-2 polypeptide, a PD-L1 polypeptide, a 4-1BBL polypeptide, and a FasL polypeptide.

[0145] As described above, a MOD can include a wild-type amino acid sequence or can include one or more amino acid substitutions relative to the wild-type amino acid sequence. A MOD can include only the extracellular portion of a full-length MOD. Thus, for example, a MOD can, in some cases, exclude one or more of the signal peptide, transmembrane domain, and intracellular domain typically found in a native MOD.

[0146] In some cases, a MOD suitable for inclusion in a TMP comprises all or a portion (e.g., the extracellular portion) of the amino acid sequence of a native MOD. In other cases, a MOD suitable for inclusion in a TMP is a variant MOD that comprises at least one amino acid substitution compared to the amino acid sequence of the native MOD. In some cases, a variant MOD exhibits a binding affinity for an immune co-modulatory polypeptide ("co-MOD") that is lower than the affinity of the corresponding native MOD (e.g., a MOD that does not include the amino acid substitution(s) present in the variant) for the immune co-modulatory polypeptide.

[0147] A suitable MOD that exhibits reduced affinity for a co-MOD may differ from the wild-type MOD by 1 amino acid (aa) to 20 amino acids. For example, in some cases, a variant MOD present in a TMP differs in amino acid sequence from the corresponding wild-type MOD by 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids. As another example, in some cases, a variant MOD present in a TMP differs in amino acid sequence from the corresponding wild-type MOD by 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids. By way of example, in some cases, a variant MOD present in a TMP contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions compared to the corresponding reference (e.g., wild-type) MOD.

[0148] A variant MOD suitable for inclusion in a TMP exhibits reduced affinity for the cognate co-MOD compared to the affinity of the corresponding wild-type MOD for the cognate co-MOD.

[0149] Exemplary pairs of MOD and cognate co-MOD include, but are not limited to, those listed in Table 4 below.

[0150] (Table 4) TIFF2024517658000009.tif145128

[0151] The binding affinity between a MOD and its cognate co-MOD can be determined by biolayer interferometry (BLI) using purified MOD and purified cognate co-MOD according to the procedures described in PCT Application Publication WO2020 / 132138A1.

[0152] MODs and variants, including reduced affinity variants, of PD-L1, CD80, CD86, 4-1BBL, and IL-2, are described in the published literature, e.g., PCT Publications WO2020132138A1 and WO2019 / 051091, the disclosures of which regarding MODs and specific variant MODs of PD-L1, CD80, CD86, 4-1BBL, and IL-2 are expressly incorporated herein by reference, particularly in paragraphs

[0260] to

[0455] of WO2020132138A1 and paragraphs

[0157] to

[0352] of WO2019 / 051091.

[0153] Of particular interest is MOD, a variant of the cytokine IL-2. Wild-type IL-2 binds to the IL-2 receptor (IL-2R) on the surface of T cells. Wild-type IL-2 has a strong affinity for IL-2R and binds to and activates most or virtually all CD8+ T cells. Therefore, synthetic forms of wild-type IL-2, such as the drug aldesleukin (trade name Proleukin®), are known to have serious side effects when administered to humans for the treatment of cancer, because IL-2 indiscriminately activates both target and non-target T cells.

[0154] The IL-2 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-2, human IL-2Rα, IL2Rβ, and IL-2Rγ are known. See, e.g., the above-mentioned PCT application publications WO2020132138A1 and WO2019 / 051091. For example, a wild-type IL-2 polypeptide can have the amino acid sequence shown in FIG. 22A. The amino acid sequences of human IL-2Rα, human IL-2Rβ, and human IL-2Rγ are shown in FIGS. 22B, 22C, and 22D, respectively.

[0155] In some cases, an IL-2 variant MOD of the present disclosure exhibits reduced binding to IL-2Rα, thereby minimizing or substantially reducing Treg activation by the IL-2 variant. Alternatively or additionally, in some cases, an IL-2 variant MOD of the present disclosure exhibits reduced binding to IL-2Rβ and / or IL-2Rγ, thereby causing the IL-2 variant MOD to exhibit an overall reduced affinity for IL-2R. In some cases, an IL-2 variant MOD of the present disclosure exhibits both properties, i.e., exhibits reduced or substantially no binding to IL-2Rα and also exhibits reduced binding to IL-2Rβ and / or IL-2Rγ, causing the IL-2 variant polypeptide to exhibit an overall reduced affinity for IL-2R. For example, IL-2 variants with substitutions at H16 and F42 exhibit reduced binding to IL-2Rα and IL-2Rβ. See Quayle et al., Clin Cancer Res; 26(8) April 15, 2020, which discloses that the binding affinity of IL-2 polypeptides containing H16A and F42A substitutions to human IL-2Rα and IL-2Rβ is reduced by 110-fold and 3-fold, respectively, compared to wild-type IL2 binding, primarily due to faster off-rates for each of these interactions. TMPs containing such variants, including variants that exhibit reduced binding to IL-2Rα and IL-2Rβ, have demonstrated the ability to preferentially bind to and activate IL-2 receptors on T cells containing target TCRs specific for the peptide epitope on the TMP, and are therefore less likely to deliver IL-2 to non-target T cells, i.e., T cells that do not contain a TCR that specifically binds to the peptide epitope on the TMP. That is, binding of IL-2 variant MOD to its costimulatory polypeptide on T cells is driven substantially by binding of the MHC epitope portion rather than by binding of IL-2.

[0156] Accordingly, suitable IL-2 variant MODs include polypeptides comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the wild-type IL-2 amino acid sequence shown in Figure 22 A, and having one or more amino acid differences from the wild-type IL-2 amino acid sequence shown in Figure 22 A. In some cases, such variant IL-2 polypeptides of the present disclosure exhibit reduced binding affinity to IL-2R compared to the binding affinity of an IL-2 polypeptide comprising the wild-type IL-2 amino acid sequence shown in Figure 22A. For example, in some cases, a variant IL-2 polypeptide binds to IL-2R with a binding affinity 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 the binding affinity of an IL-2 polypeptide comprising the wild-type IL-2 amino acid sequence shown in Figure 22A to IL-2R (e.g., an IL-2R comprising a polypeptide comprising an amino acid sequence shown in Figures 22B-22D), when assayed under the same conditions.

[0157] In some cases, a suitable variant IL-2 polypeptide has the amino acid sequence: TIFF2024517658000010.tif17154, i.e., the variant IL-2 polypeptide has the amino acid sequence of wild-type IL-2 but includes H16A and F42A substitutions (shown in bold). Alternatively, the foregoing sequence may be employed but with substitutions other than Ala at H16 and / or F42, e.g., H16T may be employed in place of H16A. In some cases, the variant IL-2 polypeptide present in the TMP has the amino acid sequence: In some cases, the variant IL-2 polypeptide present in the TMP has the amino acid sequence: TIFF2024517658000012.tif24157. In some cases, the TMP comprises two copies of such a variant IL-2 polypeptide.

[0158] In some cases, the MOD present in the TMP is a PD-L1 polypeptide. PD-L1 variants that may be suitable as MODs are disclosed in PCT Application Publications WO2019 / 051091 and WO2017 / 201131. In some cases, the PD-L1 polypeptide of the TMP comprises an amino acid sequence that has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following PD-L1 ectodomain amino acid sequence:FT VTVPKDLYVV EYGSNMTIEC KFPVEKQLDL AALIVYWEME DKNIIQFVHG EEDLKVQHSS YRQRARLLKD QLSLGNAALQ ITDVKLQDAG VYRCMISYGG ADYKRITVKV NAPYNKINQR ILVVDPVTSE HELTCQAEGY PKAEVIWTSS DHQVLSGKTT TTNSKREEKL FNVTSTLRIN TTTNEIFYCT FRRLDPEENH TAELVIPGNI LNVSIKI (SEQ ID NO: 130). See also Figure 22E.

[0159] In some cases, the MOD present in TMP is a 4-1BBL polypeptide. In some cases, the 4-1BBL polypeptide of TMP comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following 4-1BBL amino acid sequence: DPAGLLDLRQG MFAQLVAQNV LLIDGPLSWY SDPGLAGVSL TGGLSYKEDT KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPA (SEQ ID NO: 131). See also Figure 22F.

[0160] In some cases, the MOD present in the TMP is a FasL polypeptide, e.g., an extracellular domain of a FasL polypeptide. In some cases, the FasL polypeptide of the TMP comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following FasL extracellular domain amino acid sequence: QLFHLQKE LAELRESTSQ MHTASSLEKQ IGHPSPPPEK KELRKVAHLT GKSNSRSMPL EWEDTYGIVL LSGVKYKKGG LVINETGLYF VYSKVYFRGQ SCNNLPLSHK VYMRNSKYPQ DLVMMEGKMM SYCTTGQMWA RSSYLGAVFN LTSADHLYVN VSELSLVNFE ESQTFFGLYK L (SEQ ID NO: 187).

[0161] Class II MHC polypeptides As described above, the TMPs of the present disclosure comprise MHC class II polypeptides. The TMPs may comprise MHC class II polypeptides of various species, including human MHC polypeptides (HLA polypeptides), rodent (e.g., mouse, rat, etc.) MHC polypeptides, and MHC polypeptides of other mammalian species (e.g., lagomorphs, non-human primates, canines, felines, and ungulates (e.g., horses, cows, sheep, goats, etc.)). Examples of MHC class II polypeptides are shown in Figures 4-18, 19A-19N, and 20A-20J.

[0162] In humans, MHC proteins are called human leukocyte antigens (HLA). HLA class II loci include 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).

[0163] For purposes of this disclosure, the term "MHC polypeptide" is meant to include Class II MHC polypeptides, including α and β chains or portions thereof. More specifically, MHC Class II polypeptides include the α1 and α2 domains of the Class II MHC α chain and the β1 and β2 domains of the Class II MHC β chain, which represent all or most of the extracellular Class II protein required for presentation of epitope peptides. In some cases, both the α and β Class II MHC polypeptide sequences in the TMP are of human origin or are variants of such polypeptides.

[0164] Unless otherwise specified, the MHC class II polypeptides in the TMPs are not intended to include the membrane anchor domains (transmembrane regions) of the MHC class II α and β chains, or any portions thereof that are sufficient to anchor the resulting TMP or peptides thereof to the membrane of the cell in which the TMP is expressed (e.g., a eukaryotic cell, such as a mammalian cell, such as a Chinese hamster ovary or "CHO" cell). Similarly, unless otherwise specified, the MHC class II polypeptides in the TMPs described herein do not include leader portions and / or intracellular portions (e.g., cytoplasmic tails) that may be present in some naturally occurring MHC class II proteins.

[0165] The TMPs of the present disclosure comprise MHC class II polypeptides. Naturally occurring class II MHC polypeptides include α and β chains (e.g., HLA α and β chains). MHC class II polypeptides include MHC class II DP α and β polypeptides, DM α and β polypeptides, DO α and β polypeptides, DQ α and β polypeptides, and DR α and β polypeptides. As used herein, "class II MHC polypeptide" can include a class II MHC α chain polypeptide, a class II MHC β chain polypeptide, or only a portion of a class II MHC α chain and / or β chain polypeptide. For example, a "class II MHC polypeptide" can be a polypeptide that comprises i) only the α1 domain of a class II MHC α chain, ii) only the α2 domain of a class II MHC α chain, iii) only the α1 and α2 domains of a class II MHC α chain, iv) only the β1 domain of a class II MHC β chain, v) only the β2 domain of a class II MHC β chain, vi) only the β1 and β2 domains of a class II MHC β chain, vii) the α1 domain of a class II MHC α chain, the β1 domain of a class II MHC β chain, and the β2 domain of a class II MHC, etc. In many instances, an MHC class II α chain polypeptide comprises only the α1 and α2 domains of a class II MHC α chain polypeptide, and an MHC class II β chain polypeptide comprises only the β1 and β2 domains of a class II MHC β chain polypeptide.

[0166] The human MHC or HLA loci are highly polymorphic in nature, and the HLA nomenclature site operated by the Anthony Nolan Research Institute (available on the World Wide Web at hla.alleles.org / nomenclature / index.html) indicates that as of April 8, 2019, there are 7 DRA alleles, 2,479 DRB1 alleles, 1 DRB2 allele, 225 DRB3 alleles, 121 DRB4 alleles, 85 DRB5 alleles, 3 DRB6 alleles, 2 DRB7 alleles, 1 DRB8 allele, 6 DRB9 alleles, 149 DQA1 alleles, 1,561 DQB1 alleles, 106 DPA1 alleles, 1,360 DPB1 alleles, 7 DMA alleles, 13 DMB alleles, 12 DOA alleles, and 13 DOB alleles. As used herein, the term "class II MHC polypeptide" includes allelic forms of any known class II MHC polypeptide.

[0167] A TMP can comprise a class II MHC α chain without the leader portion, transmembrane portion, and intracellular portion (e.g., cytoplasmic tail) that may be present in a naturally occurring class II MHC α chain. Thus, a TMP can comprise only the α1 and α2 portions of a class II MHC α chain, without the leader portion, transmembrane portion, and intracellular portion (e.g., cytoplasmic tail) that may be present in a naturally occurring class II MHC α chain.

[0168] A TMP can comprise a class II MHC β chain, without the leader portion, transmembrane portion, and intracellular portion (e.g., cytoplasmic tail) that may be present in a naturally occurring class II MHC β chain. Thus, a TMP can comprise only the β1 and β2 domain portions of a class II MHC β chain, without the leader portion, transmembrane portion, and intracellular portion (e.g., cytoplasmic tail) that may be present in a naturally occurring class II MHC β chain.

[0169] a) MHC class II alpha chain The MHC class II alpha chain comprises an α1 domain and an α2 domain. In some cases, the α1 and α2 domains present in an antigen-presenting cell are derived from the same MHC class II α chain polypeptide. In some cases, the α1 and α2 domains present in an antigen-presenting cell are derived from two different MHC class II α chain polypeptides. As noted above, reference herein to an MHC class II α polypeptide can include both the α1 and α2 domains of the class II MHC α chain.

[0170] An MHC class II alpha chain suitable for inclusion in a TMP may lack a signal peptide. An MHC class II alpha chain suitable for inclusion in a TMP may have a length of about 60 amino acids (aa) to about 200 amino acids. For example, an MHC class II alpha chain suitable for inclusion in a TMP may 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 140 amino acids, about 140 amino acids to about 170 amino acids, or about 170 amino acids to about 200 amino acids. An MHC class II α1 domain suitable for inclusion in a TMP may have a length of about 30 amino acids to about 95 amino acids. For example, an MHC class II α1 domain suitable for inclusion in a TMP may have a length of about 30 amino acids to about 50 amino acids, about 50 amino acids to about 70 amino acids, or about 70 amino acids to about 95 amino acids. In one embodiment, the MHC class II α1 domain of a TMP is about 70 amino acids to about 95 amino acids. An MHC class II α2 domain suitable for inclusion in a TMP can have a length of about 30 to about 95 amino acids, for example, an MHC class II α2 domain suitable for inclusion in a TMP can have a length of about 30 to about 50 amino acids, about 50 to about 70 amino acids, or about 70 to about 95 amino acids. In one embodiment, the MHC class II α2 domain of a TMP is about 70 to about 95 amino acids.

[0171] DRA Polypeptides In some cases, a suitable MHC class II α chain polypeptide is a 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 to at least 150, at least 160, or at least 170 contiguous amino acids of the amino acid sequence from amino acid 26 to amino acid 203 of the DRA amino acid sequence shown in Figure 4, including naturally occurring allelic variants thereof. 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).

[0172] "DRA polypeptide" includes allelic variants, e.g., naturally occurring allelic variants. Thus, in some cases, a suitable DRA polypeptide may be a DRA polypeptide. * 01:02:01 (see Figure 4), or an allelic variant thereof. In some cases, the allelic variant has a valine instead of a leucine at position 242 (see Figure 4), thereby altering the DRA * DRA different from 01:02 * 01:01:01:01 allelic variant.

[0173] Suitable DRAs for inclusion in a TMP include the DRAs shown in FIG. * It may have 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 at least 160, at least 170, or at least 180 contiguous amino acids of the sequence from amino acid 26 to amino acid 216 of the 01:02 sequence. "DRA polypeptide" includes allelic variants, e.g., naturally occurring allelic variants.

[0174] Thus, in some cases, a suitable DRA polypeptide may have the following amino acid sequence: IKEEH VIIQAEFYLN PDQSGEFMFD FDGDEIFHVD MAKKETVWRL EEFGRFASFE AQGALANIAV DKANLEIMTK RSNYTPITNV PPEVTVLTNS PVELREPNVL ICFIDKFTPP VVNVTWLRNG KPVTTGVSET VFLPREDHLF RKFHYLPFLP STEDVYDCRV EHWGLDEPLL KHW (SEQ ID NO: 188, DRA * 01:02 (amino acids 26-203, see Figure 4), or an allelic variant thereof. In some cases, the allelic variant has a valine instead of a leucine at position 242 of the sequence in Figure 4, thereby mimicking the DRA * DRA different from 01:02:01 * 01:01:01:01 allelic variant. In some cases, a DRA polypeptide suitable for inclusion in a TMP contains an amino acid substitution relative to a wild-type DRA polypeptide, where the amino acid substitution replaces an amino acid (other than Cys) with Cys.

[0175] A TMP can include a variant DRA polypeptide containing a non-naturally occurring Cys residue, for example, a TMP can include a variant DRA polypeptide containing an amino acid substitution selected from E3C, E4C, F12C, G28C, D29C, I72C, K75C, T80C, P81C, I82C, T93C, N94C, and S95C.

[0176] A DRA α1 domain suitable for inclusion in a TMP may comprise an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: VIIQAEFYLN PDQSGEFMFD FDGDEIFHVD MAKKETVWRL EEFGRFASFE AQGALANIAV DKANLEIMTK RSNYTPITN (SEQ ID NO: 189), including naturally occurring allelic variants thereof, and may have a length of approximately 84 amino acids (e.g., 80, 81, 82, 83, 84, 85, or 86 amino acids).

[0177] A DRA α2 domain suitable for inclusion in a suitable TMP may comprise an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: V PPEVTVLTNSPVELREPNVL ICFIDKFTPP VVNVTWLRNG KPVTTGVSET VFLPREDHLF RKFHYLPFLP STEDVYDCRV EHWGLDEPLL KHW (SEQ ID NO: 190), including naturally occurring allelic variants thereof, and may have a length of about 94 amino acids (e.g., 90, 91, 92, 93, 94, 95, 96, 97, or 98 amino acids).

[0178] DMA Polypeptide In some cases, a suitable MHC class II α chain polypeptide is a DMA polypeptide, such as the DMA polypeptide shown in Figure 9, including naturally occurring allelic variants thereof. * 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 01:01 amino acid sequence. 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).

[0179] "DMA polypeptide" includes allelic variants, e.g., naturally occurring allelic variants. Thus, in some cases, a suitable DMA polypeptide is * 01:01:01 (see Figure 9), or an allelic variant thereof.

[0180] A suitable DMA α1 domain may comprise an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: VPEA PTPMWPDDLQ NHTFLHTVYC QDGSPSVGLS EAYDEDQLFF FDFSQNTRVP RLPEFADWAQ EQGDAPAILF DKEFCEWMIQ QIGPKLDGKI PVSR (SEQ ID NO: 191), including naturally occurring allelic variants thereof, and may have a length of approximately 98 amino acids (e.g., 94, 95, 96, 97, 98, 99, 100, or 101 amino acids).

[0181] A suitable DMA α2 domain may comprise an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: GFPIAE VFTLKPLEFG KPNTLVCFVS NLFPPMLTVN WQHHSVPVEG FGPTFVSAVD GLSFQAFSYL NFTPEPSDIF SCIVTHEIDR YTAIAYW (SEQ ID NO: 192), including naturally occurring allelic variants thereof, and may have a length of approximately 93 amino acids (e.g., 90, 91, 92, 93, 94, 95, 96, or 97 amino acids).

[0182] DOA Polypeptide In some cases, a suitable MHC class II α chain polypeptide is a DOA polypeptide. The DOA polypeptide is the DOA polypeptide shown in FIG. *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 with amino acids 26-204 of the O1:01 amino acid sequence. 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).

[0183] "DOA polypeptide" includes allelic variants, e.g., naturally occurring allelic variants. Thus, in some cases, a suitable DOA polypeptide is * 01:011 (see Figure 11), or an allelic variant thereof. In some cases, the allelic variant comprises amino acids 26-204 of DOA * DOA by having arginine instead of cysteine ​​at position 80 (R80C) compared to 01:01 * 01:02 or DOA by having valine instead of leucine at position 74 (L74V) * 01:03 Could be a variant.

[0184] A preferred DOA α1 domain has the following amino acid sequence, including naturally occurring allelic variants thereof: The α1 domain sequence may comprise an amino acid sequence having 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 TIFF2024517658000013.tif17156 and may have a length of about 85 amino acids (e.g., 83, 84, 85, 86, 87, or 88 amino acids). * 01:02 and DOA * The L74V and / or R80C substitutions found in 01:03 can be incorporated (amino acids corresponding to L74 and R80 are shown in italics and bold).

[0185] A suitable DOA α2 domain may comprise an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: VPPRVTVLPK SRVELGQPNI LICIVDNIFP PVINITWLRN GQTVTEGVAQ TSFYSQPDHL FRKFHYLPFV PSAEDVYDCQ VEHWGLDAPL LRHW (SEQ ID NO: 194), including naturally occurring allelic variants thereof, and may have a length of approximately 94 amino acids (e.g., 91, 92, 93, 94, 95, 96, or 97 amino acids).

[0186] DPA1 polypeptide In some cases, a suitable MHC class II α chain polypeptide is a DPA1 polypeptide. The DPA1 polypeptide can have at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to amino acids 29-209 of the DPA1 amino acid sequence shown in Figure 13. 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).

[0187] "DPA1 polypeptide" includes allelic variants, e.g., naturally occurring allelic variants. Thus, in some cases, a suitable DPA1 polypeptide is DPA1 * 01:03 (see Figure 13), or an allelic variant thereof.

[0188] A suitable DPA1 α1 domain may comprise an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: AIKADHVSTY AAFVQTHRPT GEFMFEFDED EMFYVDLDKK ETVWHLEEFG QAFSFEAQGG LANIAILNNN LNTLIQRSNH TQATN (SEQ ID NO: 195), including natural allelic variants thereof, and may have a length of approximately 87 amino acids (e.g., 84, 85, 86, 87, 88, or 89 amino acids).

[0189] A suitable DPA1 α2 domain may comprise an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: DPPEV TVFPKEPVEL GQPNTLICHI DKFFPPVLNV TWLCNGELVT EGVAESLFLP RTDYSFHKFH YLTFVPSAED FYDCRVEHWG LDQPLLKHW (SEQ ID NO: 196), including natural allelic variants thereof, and may have a length of approximately 97 amino acids (e.g., 91, 92, 93, 94, 95, 96, or 97 amino acids).

[0190] Another DPA1 polypeptide is DPA1 * 02:01 (see FIG. 13 ), or a variant thereof having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity. *02:01, and may have a length of about 87 amino acids (e.g., 84, 85, 86, 87, 88, or 89 amino acids). * It may comprise an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to amino acids 116 to 209 of 02:01:01:01, and may have a length of approximately 97 amino acids (e.g., 91, 92, 93, 94, 95, 96, or 97 amino acids).

[0191] DQA1 polypeptide In some cases, a suitable MHC class II α chain polypeptide is a DQA1 polypeptide. A suitable DQA1 polypeptide can 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 24-204 of any of the DQA1 amino acid sequences shown in FIG. 15, including naturally occurring allelic variants thereof. In some cases, the DQA1 polypeptide has a length of approximately 181 amino acids (e.g., 177, 178, 179, 180, 181, 182, or 183 amino acids). In one embodiment, the DQA1 α chain polypeptide is the DQA1 α chain polypeptide of ImMunoGeneTics ("IMGT") / HLA Acc. No.: HLA00601 in FIG. 15. * 15, IMGT / HLA Accession Number: HLA00603, GenBank NP_002113.* 15 , IMGT / HLA Acc No.: HLA00607, and the DQA1 α 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 24-204 of the DQA1 α chain amino acid sequence of 01:02. * 15. In one embodiment, the DQA1 α 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 24-204 of the DQA1 α chain amino acid sequence of IMGT / HLA Acc No. HLA00609 in FIG. * In one embodiment, the DQA1 α 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 24-204 of the 03:01 α chain amino acid sequence. ... * 15. In one embodiment, the DQA1 α 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 24-204 of the DQA1 α chain amino acid sequence of IMGT / HLA Acc No. HLA00613 in FIG. * 15 , IMGT / HLA Acc No.: HLA00620. *It 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 24 to 204 of the 06:01 alpha chain amino acid sequence.

[0192] "DQA1 polypeptide" includes allelic variants, e.g., naturally occurring allelic variants. Thus, in some cases, a suitable DQA1 polypeptide includes the following amino acid sequence: EDIVADH VASCGVNLYQ FYGPSGQYTH EFDGDEQFYV DLERKETAWR WPEFSKFGGF DPQGALRNMA VAKHNLNIMI KRYNSTAATN EVPEVTVFSK SPVTLGQPNT LICLVDNIFP PVVNITWLSN GQSVTEGVSE TSFLSKSDHS FFKISYLTFL PSADEIYDCK VEHWGLDQPL LKHW (SEQ ID NO: 197), or an allelic variant thereof.

[0193] A suitable DQA1 α1 domain may comprise an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: EDIVADH VASCGVNLYQ FYGPSGQYTH EFDGDEQFYV DLERKETAWR WPEFSKFGGF DPQGALRNMA VAKHNLNIMI KRYNSTAATN (SEQ ID NO: 198), including naturally occurring allelic variants thereof, and may have a length of approximately 87 amino acids (e.g., 84, 85, 86, 87, 88, or 89 amino acids).

[0194] A suitable DQA1α2 domain may comprise an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: EVPEVTVFSK SPVTLGQPNT LICLVDNIFP PVVNITWLSN GQSVTEGVSE TSFLSKSDHS FFKISYLTFL PSADEIYDCK VEHWGLDQPL LKHW (SEQ ID NO: 199), including natural allelic variants thereof, and may have a length of approximately 94 amino acids (e.g., 91, 92, 93, 94, 95, 96, or 97 amino acids).

[0195] DQA2 polypeptide In some cases, a suitable MHC class II α chain polypeptide is a DQA2 polypeptide. The DQA2 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 to amino acids 24-204 of the DQA2 amino acid sequence shown in Figure 16. 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).

[0196] "DQA2 polypeptide" includes allelic variants, e.g., naturally occurring allelic variants. Thus, in some cases, a suitable DQA2 polypeptide includes the following amino acid sequence: EDIVADH VASYGVNFYQ SHGPSGQYTH EFDGDEEFYV DLETKETVWQ LPMFSKFISF DPQSALRNMA VGKHTLEFMM RQSNSTAATN EVPEVTVFSK FPVTLGQPNT LICLVDNIFP PVVNITWLSN GHSVTEGVSE TSFLSKSDHS FFKISYLTFL PSADEIYDCK VEHWGLDEPL LKHW (SEQ ID NO: 200), or an allelic variant thereof.

[0197] A suitable DQA2 α1 domain may comprise an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: EDIVADH VASYGVNFYQ SHGPSGQYTH EFDGDEEFYV DLETKETVWQ LPMFSKFISF DPQSALRNMA VGKHTLEFMM RQSNSTAATN (SEQ ID NO: 201), including naturally occurring allelic variants thereof, and may have a length of approximately 87 amino acids (e.g., 84, 85, 86, 87, 88, or 89 amino acids).

[0198] A suitable DQA2 α2 domain may comprise an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: EVPEVTVFSK FPVTLGQPNT LICLVDNIFP PVVNITWLSN GHSVTEGVSE TSFLSKSDHS FFKISYLTFL PSADEIYDCK VEHWGLDEPL LKHW (SEQ ID NO: 202), including naturally occurring allelic variants thereof, and may have a length of about 94 amino acids (e.g., 91, 92, 93, 94, 95, 96, or 97 amino acids).

[0199] b) MHC class II beta chain An MHC class II beta chain comprises a β1 domain and a β2 domain. In some cases, the β1 and β2 domains present in a TMP are derived from the same MHC class II β chain polypeptide. In some cases, the β1 and β2 domains present in a TMP are derived from two different MHC class II β chain polypeptides. As noted above, reference herein to an MHC class II β polypeptide can include both the β1 and β2 domains of a class II MHC β chain.

[0200] The MHC class II beta chain sequence suitable for inclusion in a TMP lacks a signal peptide. The MHC class II beta chain suitable for inclusion in a TMP may have a length of about 60 to about 210 amino acids. For example, the MHC class II beta chain suitable for inclusion in a TMP may have a length of about 60 to about 90 amino acids, about 90 to about 120 amino acids, about 120 to about 150 amino acids, about 150 to about 180 amino acids, or about 180 to about 210 amino acids. The MHC class II β1 domain suitable for inclusion in a TMP may have a length of about 30 to about 105 amino acids. For example, the MHC class II β1 domain suitable for inclusion in a TMP may have a length of about 30 to about 50 amino acids, about 50 to about 70 amino acids, about 70 to about 90 amino acids, or about 90 to about 105 amino acids. An MHC class II β2 domain suitable for inclusion in a TMP may have a length of about 30 to about 105 amino acids, for example, an MHC class II β2 domain suitable for inclusion in a TMP may have a length of about 30 to about 50 amino acids, about 50 to about 70 amino acids, about 70 to about 90 amino acids, or about 90 to about 105 amino acids.

[0201] MHC class II β chain polypeptides suitable for inclusion in a TMP can contain an amino acid substitution relative to a wild-type MHC class II β chain polypeptide, where the amino acid substitution replaces an amino acid (other than Cys) with Cys. For example, in some cases, the MHC class II β chain polypeptide is a variant DRB1 MHC class II polypeptide containing an amino acid substitution selected from the group consisting of P5C, F7C, Q10C, N19C, G20C, H33C, G151C, D152C, and W153C. In some cases, the MHC class II The β-chain polypeptide has the following DRB1 amino acid sequence: GDTRPRFLEQVKHECHFFNGTERVRFLDRYFYHQEEYVRFDSDVGEYRAVTELGRPDAEYWNSQKDLLEQKRAAVDTYCRHNYGVGESFTVQRRVYPEVTVYPAKTQPLQHHNLLVCSVNGFYPASIEVRWFRNGQEEKTGVVSTGLIQNGDWTFQTLVMLETVPRSGEVYTCQVEHPSLTSP The variant DRB1 polypeptide comprises an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to LTVEWRARSESAQSKM (SEQ ID NO: 103), and comprises an amino acid substitution selected from the group consisting of P5C, F7C, Q10C, N19C, G20C, H33C, G151C, D152C, and W153C. In some cases, the MHC class II β chain polypeptide is a variant DRB3 polypeptide, a variant DRB4 polypeptide, or a variant DRB5 polypeptide comprising an amino acid substitution corresponding to any of the foregoing amino acid substitutions of the variant DRB1 polypeptide.For example, as shown in FIG. 7B, i) the amino acid corresponding to P5 in DRB1 is P5 in the mature DRB3 polypeptide (lacking the N-terminal signal peptide MVCLKLPGGSSLAALTVTLMVLSSRLAFA (SEQ ID NO: 203)), ii) the amino acid corresponding to F7 in DRB1 is F7 in the mature DRB3 polypeptide, iii) the amino acid corresponding to Q10 in DRB1 is L10 in the mature DRB3 polypeptide, and iv) the amino acid corresponding to N19 in DRB1 is L10 in the mature DRB3 polypeptide. v) the amino acid corresponding to G20 in DRB1 is G20 in the mature DRB3 polypeptide; vi) the amino acid corresponding to H33 in DRB1 is N33 in the mature DRB3 polypeptide; vii) the amino acid corresponding to G151 in DRB1 is G151 in the mature DRB3 polypeptide; viii) the amino acid corresponding to D152 in DRB1 is D152 in the mature DRB3 polypeptide; and ix) the amino acid corresponding to W153 in DRB1 is W153 in the mature DRB3 polypeptide. As another example, as shown in FIG. 7C, i) the amino acid corresponding to P5 in DRB1 is P15 in the mature DRB4 polypeptide (lacking the N-terminal signal peptide MVCLKLPGGSCMAALTVTL (SEQ ID NO: 204)), ii) the amino acid corresponding to F7 in DRB1 is F17 in the mature DRB4 polypeptide, iii) the amino acid corresponding to Q10 in DRB1 is Q20 in the mature DRB4 polypeptide, and iv) the amino acid corresponding to N19 in DRB1 is N20 in the mature DRB4 polypeptide. 29, v) the amino acid corresponding to G20 in DRB1 is G30 in the mature DRB4 polypeptide, vi) the amino acid corresponding to H33 in DRB1 is N43 in the mature DRB4 polypeptide, vii) the amino acid corresponding to G151 in DRB1 is G161 in the mature DRB4 polypeptide, viii) the amino acid corresponding to D152 in DRB1 is D162 in the mature DRB4 polypeptide, and ix) the amino acid corresponding to W153 in DRB1 is W153 in the mature DRB4 polypeptide.As another example, as shown in FIG. 7D, i) the amino acid corresponding to P5 in DRB1 is P15 in the mature DRB5 polypeptide (lacking the N-terminal signal peptide MVCLKLPGGSYMAKLTVTL (SEQ ID NO: 205)), ii) the amino acid corresponding to F7 in DRB1 is F17 in the mature DRB5 polypeptide, iii) the amino acid corresponding to Q10 in DRB1 is Q20 in the mature DRB5 polypeptide, and iv) the amino acid corresponding to N19 in DRB1 is N20 in the mature DRB5 polypeptide. 29, v) the amino acid corresponding to G20 in DRB1 is G30 in the mature DRB5 polypeptide, vi) the amino acid corresponding to H33 in DRB1 is N43 in the mature DRB5 polypeptide, vii) the amino acid corresponding to G151 in DRB1 is G161 in the mature DRB5 polypeptide, viii) the amino acid corresponding to D152 in DRB1 is D162 in the mature DRB5 polypeptide, and ix) the amino acid corresponding to W153 in DRB1 is W163 in the mature DRB5 polypeptide.

[0202] DRB1 polypeptide 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 to at least 170, at least 180, or at least 190 contiguous amino acids of the sequence from amino acid 30 to amino acid 227 of any of the DRB1 amino acid sequences shown in Figure 5, including naturally occurring allelic variants. Figure 5 shows the DRB1 precursor protein, in which amino acids 1-29 are the signal sequence (underlined), 30-124 form the β1 region (bold), 125-227 are the β2 region (bold and underlined), and 228-250 are the transmembrane region. In some cases, a DRB1 polypeptide suitable for inclusion in a TMP comprises an amino acid substitution relative to a wild-type DRB1 polypeptide, wherein the amino acid substitution replaces an amino acid (other than Cys) with Cys.

[0203] In one embodiment, the DRB1 β chain polypeptide of TMP is DRB1-1 (DRB1 * 01:01) beta chain amino acid sequence Swiss-Prot / UniProt reference ("sp") P04229.2. * 03:01) beta chain amino acid sequence sp. P01912.2. The DRB1 beta chain polypeptide of TMP 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 at least 170, at least 180, or at least 190 consecutive amino acids of the sequence from amino acid 30 to amino acid 227 of the DRB1 beta chain amino acid sequence sp. P01912.2. In one embodiment, the DRB1 beta chain polypeptide of TMP is DRB1-4 (DRB1 * 04:01) beta chain amino acid sequence sp. P13760.1 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 at least 170, at least 180, or at least 190 consecutive amino acids of the sequence from amino acid 30 to amino acid 227 of the beta chain amino acid sequence sp. P13760.1. In one embodiment, the DRB1 β chain polypeptide of TMP is DRB1-7 (DRB1 *07:01) beta chain amino acid sequence sp P13761.1 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 at least 170, at least 180, or at least 190 consecutive amino acids of the sequence from amino acid 30 to amino acid 227 of the beta chain amino acid sequence sp P13761.1. In one embodiment, the DRB1 beta chain polypeptide of TMP is DRB1-8 (DRB1 * 08:01) beta chain amino acid sequence sp Q30134.2. The DRB1 beta chain polypeptide of TMP 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 at least 170, at least 180, or at least 190 consecutive amino acids of the sequence from amino acid 30 to amino acid 227 of the DRB1 beta chain amino acid sequence sp Q30134.2. In one embodiment, the DRB1 beta chain polypeptide of TMP is DRB1-9 (DRB1) in FIG. * 09:01) beta chain amino acid sequence sp Q9TQE0.1 from amino acid 30 to amino acid 227. In one embodiment, the DRB1 β chain polypeptide of TMP 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 at least 170, at least 180, or at least 190 consecutive amino acids of the sequence from amino acid 30 to amino acid 227 of the DRB1 β chain amino acid sequence sp Q9TQE0.1. * 10:01) beta chain amino acid sequence sp Q30167.2 from amino acid 30 to amino acid 227. In one embodiment, the DRB1 β chain polypeptide of TMP 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 at least 170, at least 180, or at least 190 consecutive amino acids of the sequence from amino acid 30 to amino acid 227 of the DRB1 β chain amino acid sequence sp Q30167.2. *11:01) beta chain amino acid sequence sp. P20039.1 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 at least 170, at least 180, or at least 190 consecutive amino acids of the sequence from amino acid 30 to amino acid 227 of the beta chain amino acid sequence sp. P20039.1. In one embodiment, the DRB1 β chain polypeptide of TMP 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 at least 170, at least 180, or at least 190 consecutive amino acids of the sequence from amino acid 30 to amino acid 227 of the beta chain amino acid sequence sp. P20039.1. * 12:01) beta chain amino acid sequence sp Q95IE3.1. The DRB1 beta chain polypeptide of TMP 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 at least 170, at least 180, or at least 190 consecutive amino acids of the sequence from amino acid 30 to amino acid 227 of sp Q95IE3.1. In one embodiment, the DRB1 beta chain polypeptide of TMP is DRB1-13 (DRB1) in FIG. * 13:01) beta chain amino acid sequence sp Q5Y7A7.1 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 at least 170, at least 180, or at least 190 consecutive amino acids of the sequence from amino acid 30 to amino acid 227. In one embodiment, the DRB1 β chain polypeptide of TMP 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 at least 170, at least 180, or at least 190 consecutive amino acids of the sequence from amino acid 30 to amino acid 227 of the DRB1 β chain amino acid sequence sp Q5Y7A7.1. * 14:01) beta chain amino acid sequence sp Q9GIY3.1. The DRB1 beta chain polypeptide of TMP 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 at least 170, at least 180, or at least 190 consecutive amino acids of the sequence from amino acid 30 to amino acid 227 of sp Q9GIY3.1. In one embodiment, the DRB1 beta chain polypeptide of TMP is DRB1-15 (DRB1 *15:01) beta chain amino acid sequence sp. P01911 from amino acid 30 to amino acid 227. In one embodiment, the DRB1 β chain polypeptide of TMP 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 at least 170, at least 180, or at least 190 consecutive amino acids of the sequence from amino acid 30 to amino acid 227 of the DRB1 β chain amino acid sequence sp. P01911. * 16:01) beta chain amino acid sequence sp Q29974.1 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 at least 170, at least 180, or at least 190 contiguous amino acids of the sequence from amino acid 30 to amino acid 227. In some cases, the DRB1 beta chain polypeptide has a length of about 198 amino acids (e.g., 195, 196, 197, 198, 199, 200, 201, or 202 amino acids).

[0204] A "DRB1 polypeptide" suitable for inclusion in a TMP can include an allelic variant, e.g., a naturally occurring allelic variant. Thus, in some cases, a suitable DRB1 polypeptide may be the DRB1 polypeptide provided in FIG. * 04:01 (DRB1-4) or an allelic variant thereof.

[0205] Another suitable DRB1 polypeptide is the following DRB1 *The 04:01 amino acid sequence may comprise a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity for at least 170, at least 180, or at least 190 consecutive amino acids of the amino acid sequence: GDTRPRFLEQVKHECHFFNGTERVRFLDRYFYHQEEYVRFDSDVGEYRAVTELGRPDAEYWNSQKDLLEQKRAAVDTYCRHNYGVGESFTVQRRVYPEVTVYPAKTQPLQHHNLLVCSVNGFYPASIEVRWFRNGQEEKTGVVSTGLIQNGDWTFQTLVMLETVPRSGEVYTCQVEHPSLTSPLTVEWRARSESAQSKM (SEQ ID NO: 103), which may have one or more cysteine ​​substitutions. In one embodiment, the cysteine ​​substitution is a P5C substitution. In one embodiment, the cysteine ​​substitution is a G151C substitution. In one embodiment, the cysteine ​​substitution is a W153C substitution.

[0206] A suitable DRB1β1 domain may comprise an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: DTRPRFLEQVKHECHFFNGTERVRFLDRYFYHQEEYVRFDSDVGEYRAVTELGRPDAEYWNSQKDLLEQKRAAVDTYCRHNYGVGESFTVQRRV (SEQ ID NO: 206), including naturally occurring allelic variants thereof, and may have a length of about 95 amino acids (e.g., 92, 93, 94, 95, 96, 97, or 98 amino acids).

[0207] A suitable DRB1 β1 domain has the following amino acid sequence: TIFF2024517658000014.tif11164, in which P5 is substituted with Cys (shown in bold and italics).

[0208] A suitable DRB1β2 domain may comprise an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: YPEVTVYPAKTQPLQHHNLLVCSVNGFYPGSIEVRWFRNGQEEKTGVVSTGLIQNGDWTFQTLVMLETVPRSGEVYTCQVEHPSLTSPLTVEWRARSESAQSK (SEQ ID NO: 208), including natural allelic variants thereof, and may have a length of about 103 amino acids (e.g., 100, 101, 102, 103, 104, 105, or 106 amino acids).

[0209] A suitable DRB1 β2 domain has the following amino acid sequence: TIFF2024517658000015.tif11164, in which W153 is substituted with Cys (shown in bold and italics).

[0210] DRB3 polypeptide 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 to amino acids 30-227 of any of the DRB3 amino acid sequences shown in Figure 6, which represents a DRB3 precursor protein, in which amino acids 1-29 are a signal sequence (underlined), 30-124 form the β1 region (shown in bold), 125-227 form the β2 region, and 228-250 are the transmembrane region. In one embodiment, the DRB3 β chain polypeptide is DRB1-3 (DRB3) in Figure 6. *01:01) beta chain amino acid sequence of GenBank NP_072049.1. In one embodiment, the DRB3 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 to amino acids 30-227 of the DRB1-3 beta chain amino acid sequence of GenBank accession EAX03632.1 in Figure 6. In one embodiment, the DRB3 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 to amino acids 30-227 of the DRB1-3 beta chain amino acid sequence of GenBank accession CAA23781.1 in Figure 6. * 02:01) beta chain amino acid sequence. 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 30-227 of the DRB1-3 (DRB3) beta chain amino acid sequence of GenBank AAN15205.1 in FIG. 6. * 03:01) beta chain amino acid sequence. In some cases, a DRB3 polypeptide suitable for inclusion in a TMP contains an amino acid substitution relative to a wild-type DRB3 polypeptide, where the amino acid substitution replaces an amino acid (other than Cys) with Cys.

[0211] A "DRB3 polypeptide" suitable for incorporation into a TMP may include an allelic variant, e.g., a naturally occurring allelic variant. Thus, in some cases, a suitable DRB3 polypeptide may be the DRB3 polypeptide provided in FIG. *01:01, or an allelic variant thereof. Thus, in some cases, a suitable DRB3 polypeptide comprises a sequence having at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity for at least 170, at least 180, or at least 190 contiguous amino acids of the following sequence: DTRPRFLELR KSECHFFNGT ERVRYLDRYF HNQEEFLRFD SDVGEYRAVT ELGRPVAESW NSQKDLLEQK RGRVDNYCRH NYGVGESFTV QRRVHPQVTV YPAKTQPLQH HNLLVCSVSG FYPGSIEVRW FRNGQEEKAG VVSTGLIQNG DWTFQTLVML ETVPRSGEVY TCQVEHPSVT SALTVEWRAR SESAQSK (SEQ ID NO: 210), or an allelic variant thereof. In some cases, a DRB3 polypeptide suitable for inclusion in a TMP contains an amino acid substitution relative to a wild-type DRB3 polypeptide, where the amino acid substitution replaces an amino acid (other than Cys) with Cys. Thus, for example, in some cases, the MHC class II β chain polypeptide is a variant DRB3 MHC class II polypeptide containing a non-naturally occurring Cys at an amino acid selected from the group consisting of P5C, F7C, L10C, N19C, G20C, N33C, G151C, D152C, and W153C of the mature DRB3 polypeptide (which lacks the N-terminal signal peptide MVCLKLPGGSSLAALTVTLMVLSSRLAFA (SEQ ID NO: 203) shown in FIG. 6).

[0212] A suitable DRB3 β1 domain may comprise an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: DTRPRFLELR KSECHFFNGT ERVRYLDRYF HNQEEFLRFD SDVGEYRAVT ELGRPVAESW NSQKDLLEQK RGRVDNYCRH NYGVGESFTV (SEQ ID NO: 211), including naturally occurring allelic variants thereof, and may have a length of about 95 amino acids (e.g., 93, 94, 95, 96, 97, or 98 amino acids). A suitable DRB3 β1 domain may comprise the following amino acid sequence: DTRPRFLELR KSECHFFNGT ERVRYLDRYF HNQEEFLRFD SDVGEYRAVT ELGRPVAESW NSQKDLLEQK RGRVDNYCRH NYGVGESFTV QRRV (SEQ ID NO: 211), or a naturally occurring allelic variant. A suitable DRB3 β2 domain may comprise an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: HPQVTV YPAKTQPLQH HNLLVCSVSG FYPGSIEVRW FRNGQEEKAG VVSTGLIQNG DWTFQTLVML ETVPRSGEVY TCQVEHPSVT SALTVEWRAR SESAQSK (SEQ ID NO: 212), including naturally occurring allelic variants thereof, and may have a length of about 103 amino acids (e.g., 100, 101, 102, 103, 104, or 105 amino acids). A suitable DRB3 β2 domain may comprise the following amino acid sequence: HPQVTV YPAKTQPLQH HNLLVCSVSG FYPGSIEVRW FRNGQEEKAG VVSTGLIQNG DWTFQTLVML ETVPRSGEVY TCQVEHPSVT SALTVEWRAR SESAQSK (SEQ ID NO: 212), or a naturally occurring allelic variant thereof.

[0213] DRB4 polypeptide In some cases, a suitable MHC class II β chain polypeptide is a DRB4 polypeptide. The DRB4 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 DRB4 amino acid sequence shown in Figure 7. 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). In some cases, a DRB4 polypeptide suitable for inclusion in a TMP contains an amino acid substitution relative to a wild-type DRB4 polypeptide, where the amino acid substitution replaces an amino acid (other than Cys) with Cys.

[0214] A "DRB4 polypeptide" suitable for inclusion in a TMP may include an allelic variant, e.g., a naturally occurring allelic variant. Thus, in some cases, a suitable DRB4 polypeptide may be the DRB4 polypeptide provided in FIG. * 01:01 (SEQ ID NO: 41) or DRB4 * 01:03 (SEQ ID NO:42), or an allelic variant thereof. In some cases, a DRB4 polypeptide suitable for inclusion in a TMP comprises an amino acid substitution relative to a wild-type DRB4 polypeptide, where the amino acid substitution replaces an amino acid (other than Cys) with Cys. Thus, for example, in some cases, the MHC class II β chain polypeptide is a variant DRB4 MHC class II polypeptide comprising a non-naturally occurring Cys residue, e.g., the variant DRB4 MHC class II polypeptide comprises an amino acid substitution selected from the group consisting of P15C, F17C, Q20C, N29C, G30C, N43C, G161C, D162C, and W163C of the mature DRB4 polypeptide (which lacks the N-terminal signal peptide MVCLKLPGGSCMAALTVTL (SEQ ID NO:204) shown in FIG. 7).

[0215] A suitable DRB4 β1 domain may comprise an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: T VLSSPLALAG DTQPRFLEQA KCECHFLNGT ERVWNLIRYI YNQEEYARYN SDLGEYQAVT ELGRPDAEYW NSQKDLLERR RAEVDTYCRY NYGVVESFTV (SEQ ID NO: 213), including naturally occurring allelic variants thereof, and may have a length of about 95 amino acids (e.g., 93, 94, 95, 96, 97, or 98 amino acids).

[0216] A suitable DRB4 β2 domain may comprise an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: QPKVTV YPSKTQPLQH HNLLVCSVNG FYPGSIEVRW FRNGQEEKAG VVSTGLIQNG DWTFQTLVML ETVPRSGEVY TCQVEHPSMM SPLTVQWSAR SESAQSK (SEQ ID NO: 214), including naturally occurring allelic variants thereof, and may have a length of about 103 amino acids (e.g., 100, 101, 102, 103, 104, or 105 amino acids).

[0217] DRB5 polypeptide The TMP may comprise an MHC class II β chain polypeptide of a DRB5 allele. 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). In some cases, the DRB5 polypeptide suitable for inclusion in the TMP comprises an amino acid substitution relative to the wild-type DRB5 polypeptide, wherein the amino acid substitution is a substitution of an amino acid (other than Cys) with Cys.

[0218] DRB5 * 01:01 TMP is provided in Figure 8. * DRB5, comprising an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity for at least 170, at least 180, or at least 190 consecutive amino acids of the sequence from amino acid 30 to amino acid 227 of the 01:01 amino acid sequence. * 01:01 polypeptide. In some cases, a DRB5 polypeptide suitable for inclusion in a TMP comprises an amino acid substitution relative to a wild-type DRB5 polypeptide, where the amino acid substitution replaces an amino acid (other than Cys) with Cys. Thus, for example, in some cases, the MHC class II β chain polypeptide is a variant DRB5 MHC class II polypeptide comprising a non-naturally occurring Cys residue, e.g., the variant DRB5 MHC class II polypeptide comprises an amino acid substitution selected from the group consisting of P15C, F17C, Q20C, N29C, G30C, N43C, G161C, D162C, and W163C of the mature DRB5 polypeptide (shown in FIG. 8 (lacking the N-terminal signal peptide MVCLKLPGGSYMAKLTVTL (SEQ ID NO: 205)) or a naturally occurring allelic variant thereof).

[0219] TMP is provided in Figure 8. * DRB5, comprising an amino acid sequence having 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 to 124 of the 01:01 amino acid sequence. * The TMP may comprise a DRB5 β1 domain polypeptide, as shown in FIG. *01:01 DRB5 comprising an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity for at least 170, at least 180, or at least 190 consecutive amino acids of the sequence from amino acid 30 to amino acid 227 of the β2 domain amino acid sequence or a naturally occurring allelic variant thereof. * 01:01Contains polypeptides.

[0220] DMB Polypeptide In some cases, a suitable MHC class II β chain polypeptide is a DMB polypeptide. The DMB 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 to amino acids 19-207 of the DMB amino acid sequence shown in Figure 10. In some cases, the DMB polypeptide has a length of about 189 amino acids (e.g., 187, 188, 189, 190, or 191 amino acids).

[0221] "DMB polypeptide" includes allelic variants, e.g., naturally occurring allelic variants. Thus, in some cases, a suitable DMB polypeptide is the DMB polypeptide provided in Figure 10. * 01:03 (SEQ ID NO: 45), or an allelic variant thereof.

[0222] A suitable DMB β1 domain may comprise an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: GG FVAHVESTCL LDDAGTPKDF TYCISFNKDL LTCWDPEENK MAPCEFGVLN SLANVLSQHL NQKDTLMQRL RNGLQNCATH TQPFWGSLTN RT (SEQ ID NO: 215), including naturally occurring allelic variants thereof, and may have a length of approximately 94 amino acids (e.g., 92, 93, 94, 95, 96, or 97 amino acids).

[0223] A suitable DMB β2 domain may comprise an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: RPPSVQVA KTTPFNTREP VMLACYVWGF YPAEVTITWR KNGKLVMPHS SAHKTAQPNG DWTYQTLSHL ALTPSYGDTY TCVVEHTGAP EPILRDW (SEQ ID NO: 216), including naturally occurring allelic variants thereof, and may have a length of about 95 amino acids (e.g., 93, 94, 95, 96, 97, or 98 amino acids).

[0224] DOB Polypeptide In some cases, a suitable MHC class II β chain polypeptide is a 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 to amino acids 27-214 of the DOB amino acid sequence shown in Figure 12. In some cases, the DOB polypeptide has a length of about 188 amino acids (e.g., 186, 187, 188, 189, or 190 amino acids).

[0225] "DOB polypeptide" includes allelic variants, e.g., naturally occurring allelic variants. Thus, in some cases, a suitable DOB polypeptide is the DOB polypeptide provided in FIG. * 01:01 (SEQ ID NO: 47), or an allelic variant thereof.

[0226] A suitable DOB β1 domain may comprise an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: TDSP EDFVIQAKAD CYFTNGTEKV QFVVRFIFNL EEYVRFDSDV GMFVALTKLG QPDAEQWNSR LDLLERSRQA VDGVCRHNYR LGAPFTVGRK (SEQ ID NO: 217), including naturally occurring allelic variants thereof, and may have a length of approximately 94 amino acids (e.g., 92, 93, 94, 95, 96, or 97 amino acids).

[0227] A suitable DOB β2 domain may comprise an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: VQPEVTVYPE RTPLLHQHNL LHCSVTGFYP GDIKIKWFLN GQEERAGVMS TGPIRNGDWT FQTVVMLEMT PELGHVYTCL VDHSSLLSPV SVEW (SEQ ID NO: 218), including naturally occurring allelic variants thereof, and may have a length of approximately 94 amino acids (e.g., 92, 93, 94, 95, 96, or 97 amino acids).

[0228] DPB1 polypeptide 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 to amino acids 30-215 of any of the DPB1 amino acid sequences shown in Figure 14. 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 is the DPB1 of IMGT / HLA Acc number: HLA00514 in Figure 14. * In one embodiment, the DRB3 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 30-227 of the DRB3 β chain amino acid sequence of DPB1 in IMGT / HLA Acc number: HLA00517 in FIG. * In one embodiment, the DRB3 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 30-227 of the DRB3 β chain amino acid sequence of DPB1 in IMGT / HLA Acc No. HLA00520 in FIG. * In one embodiment, the DRB3 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 30-227 of the DRB3 β chain amino acid sequence of DPB1 in IMGT / HLA Accession Number: HLA00521 in Figure 14, GenBank NP_002112.3. *In one embodiment, the DRB3 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 30-227 of the DRB3 β chain amino acid sequence of DPB1 in IMGT / HLA Acc number: HLA00524 in FIG. * In one embodiment, the DRB3 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 30-227 of the DRB3 β chain amino acid sequence of DPB1 in IMGT / HLA Acc number: HLA00528 in FIG. * In one embodiment, the DRB3 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 30-227 of the DRB3 β chain amino acid sequence of DPB1 of IMGT / HLA Acc No. HLA00590 in FIG. * In one embodiment, the DRB3 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 30-227 of the DRB3 β chain amino acid sequence of DPB1 71:01 IMGT / HLA Acc number: HLA02046 in FIG. * In one embodiment, the DRB3 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 30-227 of the DRB3 β chain amino acid sequence of DPB1 104:01, IMGT / HLA Acc number: HLA10364 in FIG. *It 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 141:01 beta chain amino acid sequence.

[0229] "DPB1 polypeptide" includes allelic variants, e.g., naturally occurring allelic variants. Thus, in some cases, a suitable DPB1 polypeptide includes the following amino acid sequence: R ATPENYLFQG RQECYAFNGT QRFLERYIYN REEFARFDSD VGEFRAVTEL GRPAAEYWNS QKDILEEKRA VPDRMCRHNY ELGGPMTLQR RVQPRVNVSP SKKGPLQHHN LLVCHVTDFY PGSIQVRWFL NGQEETAGVV STNLIRNGDW TFQILVMLEM TPQQGDVYTC QVEHTSLDSP VTVEW (SEQ ID NO: 219), or an allelic variant thereof.

[0230] A suitable DPB1 β1 domain may comprise an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: R ATPENYLFQG RQECYAFNGT QRFLERYIYN REEFARFDSD VGEFRAVTEL GRPAAEYWNS QKDILEEKRA VPDRMCRHNY ELGGPMTLQR R (SEQ ID NO: 220), including natural allelic variants thereof, and may have a length of approximately 92 amino acids (e.g., 90, 91, 92, 93, or 94 amino acids).

[0231] A suitable DPB1 β2 domain may comprise an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: VQPRVNVSP SKKGPLQHHN LLVCHVTDFY PGSIQVRWFL NGQEETAGVV STNLIRNGDW TFQILVMLEM TPQQGDVYTC QVEHTSLDSP VTVEW (SEQ ID NO: 221), including natural allelic variants thereof, and may have a length of approximately 94 amino acids (e.g., 92, 93, 94, 95, 96, or 97 amino acids).

[0232] DQB1 polypeptide 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 to amino acids 33-220 of the DQB1 amino acid sequence shown in Figure 17. 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).

[0233] "DQB1 polypeptide" includes allelic variants, e.g., naturally occurring allelic variants. Thus, in some cases, a suitable DQB1 polypeptide is the DQB1 polypeptide provided in FIG. * 06:02 (SEQ ID NO: 85), or an allelic variant thereof.

[0234] A suitable DQB1 β1 domain may comprise an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: RDSPEDFV FQFKGMCYFT NGTERVRLVT RYIYNREEYA RFDSDVGVYR AVTPQGRPDA EYWNSQKEVL EGTRAELDTV CRHNYEVAFR GILQRR (SEQ ID NO: 222), including natural allelic variants thereof, and may have a length of approximately 94 amino acids (e.g., 92, 93, 94, 95, or 96 amino acids).

[0235] A suitable DQB1 β2 domain may comprise an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: VEPT VTISPSRTEA LNHHNLLVCS VTDFYPGQIK VRWFRNDQEE TAGVVSTPLI RNGDWTFQIL VMLEMTPQRG DVYTCHVEHP SLQSPITVEW (SEQ ID NO: 223), including naturally occurring allelic variants thereof, and may have a length of approximately 94 amino acids (e.g., 92, 93, 94, 95, or 96 amino acids).

[0236] DQB2 polypeptide In some cases, a suitable MHC class II β chain polypeptide is a DQB2 polypeptide. The DQB2 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 to amino acids 33-215 of the DQB2 amino acid sequence shown in Figure 18A or Figure 18B. 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).

[0237] "DQB2 polypeptide" includes allelic variants, e.g., naturally occurring allelic variants. Thus, in some cases, a suitable DQB2 polypeptide includes the following amino acid sequence: DFLVQFK GMCYFTNGTE RVRGVARYIY NREEYGRFDS DVGEFQAVTE LGRSIEDWNN YKDFLEQERA AVDKVCRHNY EAELRTTLQR QVEPTVTISP SRTEALNHHN LLVCSVTDFY PAQIKVRWFR NDQEETAGVV STSLIRNGDW TFQILVMLEI TPQRGDIYTC QVEHPSLQSP ITVEW (SEQ ID NO: 224), or an allelic variant thereof.

[0238] A suitable DQB2 β1 domain may comprise an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: DFLVQFK GMCYFTNGTE RVRGVARYIY NREEYGRFDS DVGEFQAVTE LGRSIEDWNN YKDFLEQERA AVDKVCRHNY EAELRTTLQR QVEPTV (SEQ ID NO: 225), including naturally occurring allelic variants thereof, and may have a length of approximately 94 amino acids (e.g., 92, 93, 94, 95, 96, or 97 amino acids).

[0239] A suitable DQB2 β2 domain may comprise an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: TISP SRTEALNHHN LLVCSVTDFY PAQIKVRWFR NDQEETAGVV STSLIRNGDW TFQILVMLEI TPQRGDIYTC QVEHPSLQSP ITVEW (SEQ ID NO: 226), including naturally occurring allelic variants thereof, and may have a length of about 94 amino acids (e.g., 92, 93, 94, 95, 96, or 97 amino acids).

[0240] MHC class II alleles and haplotypes associated with disease risk Certain MHC class II alleles and haplotypes are associated with disease, e.g., increasing the risk of developing certain diseases. See, e.g., 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 th ed.; Margaritte-Jeannin et al. (2004) Tissue Antigens 63:562; and Kurko et al. (2013) Clin. Rev. Allergy Immunol. 45:170.

[0241] MHC class II polypeptides in type 1 diabetes mellitus (T1D) Alleles / isoforms that have been shown to be highly associated with T1D are suitable sources of MHC II α1, α2, β1, and β2 polypeptide sequences for incorporation into TMPs intended for the treatment of T1D. T1D is associated with alleles belonging to HLA-DR3 and HLA-DR4 haplotypes / serotypes, with the highest risk being associated with HLA-DQ8 (e.g., HLA-DQB1 * 03:02) and HLA-DQ2 serotype alleles. Some high-risk and medium-risk haplotypes and their association with various DR serotypes are shown in Table 5 below, adapted from Kantarova and Buc, Physiol. Res. 56:255-266 (2007).

[0242] (Table 5) TIFF2024517658000016.tif117137

[0243] The stereotypically defined DR3 and DR4 protein isoforms / haplotypes of the DRB1 gene are associated with a higher risk of developing T1D in individuals expressing such alleles. * 03:01, * 03:02, * 03:03, and * Contains the allele encoding the 03:04 protein, HLA-DRB1 * The 0301 allele is often associated with a predisposition to T1D. * 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 protein-encoding alleles. Certain HLA-DR4 alleles (e.g., HLA-DRB1 * 0401, HLA-DRB1 * 0402, and HLA-DRB1 * 0405) predisposes individuals to T1D, but HLA-DRB1 * 04:03 Allele / Isoform May Confer Protection. DRB1 * 16:01 has also been shown to be more prevalent in children with diabetes compared to healthy controls (Deja, et al., Mediators of Inflammation 2006:1-7 (2006)).

[0244] Although T1D is associated with the DR3 and DR4 alleles, as discussed above, the risk factors most strongly associated with T1D include HLA-DQ8 serotype (e.g., HLA-DQB1 * 03:02 isoform) and HLA DQ2 serotype (e.g., HLA-DQB1 *The HLA-DQ8 T1D susceptibility serotype is the presence of an allele belonging to the HLA-DQ8.1 serotype (HLA-DQA1 * 03:01 / DQB1 * 03:02), including the HLA-DQ2 serotype associated with T1D (HLA-DQB1 * O2) is DQB1 * 02:01). Jones, et al., Nat. Rev. Immunol. 2006, 6:271-282. In contrast, HLADQB1 * Individuals carrying the 0602 allele appear to be protected from type 1 diabetes. Ibid. DQ2 is most common in Western Europe, North Africa, and East Africa, and is observed at high frequencies in parts of Spain and Ireland.

[0245] The DQB1 locus alone has been reported to be associated with T1D when position β57 is a neutral residue such as Ala or Ser. Both DQ2 and DQ8 serotypes associated with T1D do not have Asp at position β57, but instead have Ala at that position, which confers T1D susceptibility (e.g., HLA-DQB1 in Figure 19B). * 02:01 and HLA-DQB1 in Figure 19C * In contrast, DQB1 with Asp at position β57 (position 89 in Figures 11A-11B) * 06:02 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 a critical residue in peptide-binding pocket 9 (P9) of DQB1, which is involved in antigen presentation and interaction with the T cell receptor (TCR).

[0246] HLA haplotype DQA1 * 03:01-DRB1 * Individuals with 03:02 are particularly likely to have DQA1 * 05:01-DRB1 *When combined with 02:01, there is a significant susceptibility to T1D (10-20 fold increase). See Notkins, AL, J. Biol. Chem., 2002, 277(46):43545-48. The group with a typical defined susceptibility to T1D includes HLA-DR4.1 (HLA-DR1 * 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, e.g., Jones et al., Nat. Rev. Immunol. 2006, 6:271-282). * 04:05-DQβ1 * 04:01 / DRβ1 * 08:02-DQβ1 * The 03:02 genotype has been shown to be associated with acute-onset and slowly progressive T1D. Fulminant diabetes mellitus was associated with DRβ1 in a Japanese population study by Kawabata, et al., Diabetologia 2009, 52:2513-21. * 04:05-DQβ1 * 04:01 / DRβ1 * 04:05-DQβ1 * It was associated with the 04:01 genotype.

[0247] Although the association between T1D and HLA-DR is not as strong as that between HLA-DQ and HLA-DR, insulin-reactive T cells derived from lymph nodes that deplete the pancreas of T1D patients appear to be restricted to HLA-DR4.1, rather than HLA-DQ8 or HLA-DQ2 (Kent et al., Nature 2005 435:224-228).

[0248] 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 TMPs of the present disclosure. * α1 and α2 polypeptides derived from 0501 and DQB1 * In one embodiment, the TMP is DQ2.5-like, comprising β1 and β2 polypeptides adopted from DQA1. * α1 and α2 polypeptides derived from 0301 and DQB1 * 0302 and is DQ8.1-like, comprising β1 and β2 polypeptides adopted from 0302.

[0249] Backbone Polypeptide The TMPs of the present disclosure, whether heterodimeric or single-chain, can comprise an immunoglobulin or non-immunoglobulin backbone. The TMP polypeptides of the present disclosure, whether heterodimeric or single-chain, can comprise an Fc polypeptide or another suitable backbone polypeptide.

[0250] Suitable scaffold polypeptides include antibody-based scaffold polypeptides and non-antibody-based scaffolds. Non-antibody-based scaffolds include, for example, albumin, XTEN (extended recombinant) polypeptide, transferrin, Fc receptor polypeptide, elastin-like polypeptide (see, for example, Hassouneh et al. (2012) Methods Enzymol. 502:215; for example, a polypeptide comprising a pentapeptide repeat unit of (Val-Pro-Gly-X-Gly; SEQ ID NO: 227) (wherein X is any amino acid other than proline)), albumin-binding polypeptide, silk-like polypeptide (see, for example, Valluzzi et al. (2002) Philos Trans R Soc Lond B Biol Sci. 357:165), silk-elastin-like polypeptide (SELP; see, for example, 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.

[0251] In some cases, a suitable scaffold polypeptide is a half-life extending polypeptide. Thus, in some cases, a suitable scaffold polypeptide increases the in vivo half-life (e.g., serum half-life) of a polypeptide (e.g., TMP) compared to a control polypeptide lacking the scaffold polypeptide. For example, in some cases, the scaffold polypeptide increases the in vivo half-life (e.g., serum half-life) of a 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 compared to a control polypeptide lacking the scaffold polypeptide. By way of example, in some cases, the Fc polypeptide increases the in vivo half-life (e.g., serum half-life) of the 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, relative to a control polypeptide lacking the Fc polypeptide.

[0252] Fc polypeptide In some cases, the TMP comprises an Ig Fc polypeptide. The Ig Fc polypeptide is also referred to herein as an "Fc polypeptide." The Ig Fc polypeptide of the TMP can be human IgG1 Fc, human IgG2 Fc, human IgG3 Fc, human IgG4 Fc, etc., or a variant of a wild-type Ig Fc polypeptide. Variants include naturally occurring variants, non-naturally occurring variants, and combinations thereof. Exemplary Ig Fc polypeptides are described below, any of which may further comprise a mutually specific binding sequence, if desired.

[0253] In some cases, the Fc polypeptide present in the TMP comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to an Fc amino acid sequence shown in any one of Figures 21A-21M.

[0254] In some cases, the Fc polypeptide present in the TMP is an IgG1 Fc polypeptide or a variant of an IgG1 Fc polypeptide. For example, in some cases, the Fc polypeptide present in the TMP comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to the human IgG1 Fc polypeptide shown in Figure 21A. As another example, in some cases, the Fc polypeptide present in the TMP 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 Fc polypeptide shown in Figure 21B, wherein the Ig Fc polypeptide comprises an Ala at position 14 and an Ala at position 15. In any of the above embodiments, the Ig Fc polypeptide may have an N77 substitution, i.e., the Ig Fc polypeptide may have an amino acid other than Asn at position 77, and in some cases, the Ig Fc polypeptide has Ala at position 77. In some cases, the Fc polypeptide present in the TMP comprises the amino acid sequence shown in Figure 21A. In some cases, the Fc polypeptide present in the TMP comprises the amino acid sequence shown in Figure 21B.

[0255] In some cases, the Fc polypeptide present in the TMP is an IgG1 Fc polypeptide or a variant of an IgG1 Fc polypeptide, where the variant includes a naturally occurring variant, a non-naturally occurring variant, and combinations thereof. For example, in some cases, the Fc polypeptide present in the TMP comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to the human IgG1 Fc polypeptide shown in Figure 21C, wherein the Ig Fc polypeptide comprises a Glu at position 136 and a Met at position 138. As another example, in some cases, the Fc polypeptide present in the TMP comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to the human IgG1 Fc polypeptide shown in Figure 21D, wherein the Ig Fc polypeptide has Ala at positions 14 and 15, and the Fc polypeptide comprises a Glu at position 136 and a Met at position 138. In any of the above embodiments, the Ig Fc polypeptide may have an N77 substitution, i.e., the Ig Fc polypeptide may have an amino acid other than Asn at position 77, and in some cases, the Ig Fc polypeptide has Ala at position 77. In some cases, the Fc polypeptide present in the TMP comprises the amino acid sequence shown in Figure 21C. In some cases, the Fc polypeptide present in the TMP comprises the amino acid sequence shown in Figure 21D.

[0256] In some cases, the Fc polypeptide present in the TMP comprises the amino acid sequence shown in Figure 21E (human IgG1 Fc comprising an L234F substitution, an L235E substitution, and a P331S substitution; L234 corresponds to amino acid 14 of the amino acid sequence shown in Figure 21E, L235 corresponds to amino acid 15 of the amino acid sequence shown in Figure 21E, and P331 corresponds to amino acid 111 of the amino acid sequence shown in Figure 21E). In some cases, the Fc polypeptide present in the TMP comprises the amino acid sequence shown in Figure 21F, including an N279A (N77A of the amino acid sequence shown in Figure 21F) substitution.

[0257] 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 IgG2 Fc polypeptide shown in Figure 21G, e.g., the Fc polypeptide comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to amino acids 99-325 of the human IgG2 Fc polypeptide shown in Figure 21G (e.g., an Ig Fc polypeptide has a length of about 227 amino acids). 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 Figure 21H, e.g., the Fc polypeptide comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to amino acids 19-246 of the human IgG3 Fc polypeptide shown in Figure 21H (e.g., an Ig Fc polypeptide has a length of about 228 amino acids).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 Figure 21J, e.g., the Fc polypeptide comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to amino acids 1-276 of the human IgM Fc polypeptide shown in Figure 21J. 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 Figure 21K, e.g., the Fc polypeptide comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to amino acids 1-234 of the human IgA Fc polypeptide shown in Figure 21K.

[0258] 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 21 M. 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 21 M (e.g., an Ig Fc polypeptide has a length of about 228 amino acids).

[0259] In some cases, the IgG4 Fc polypeptide comprises the following amino acid sequence: PPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 228).

[0260] In some cases, the Ig Fc employed in the TMP contains one or more amino acid substitutions in the wild-type sequence such that the Ig Fc does not substantially induce cell lysis. For example, in some cases, the Fc polypeptide present in the TMP contains the amino acid sequence shown in Figure 21A (human IgG1 Fc), except for the substitution of L234 (L14 in the amino acid sequence shown in Figure 21A) with an amino acid other than leucine, or the substitution of L235 (L15 in the amino acid sequence shown in Figure 21A) with an amino acid other than leucine. Examples include the L234A (L14A) substitution and the L235A (L15A) substitution.

[0261] TGF-β polypeptides As described above, the TMP of the present disclosure includes at least one TGF-β polypeptide reversibly masked by a polypeptide that binds to the TGF-β polypeptide (a "masking polypeptide"), which together form a masked TGF-β MOD. The masking polypeptide can be, for example, a TGF-β receptor polypeptide or an antibody that functions to reversibly mask the TGF-β polypeptide present in the TMP, which TGF-β polypeptide is otherwise capable of acting as an agonist of the cellular TGF receptor. The masked TGF-β MOD provides an active TGF-β polypeptide (e.g., a TGF-β signaling pathway agonist). The TGF-β polypeptide and the masking polypeptide (e.g., a TGF-β receptor fragment) interact with each other to reversibly mask the TGF-β polypeptide, thereby allowing the TGF-β polypeptide to interact with its cellular receptor. Additionally, the masking sequence competes with cellular receptors that can scavenge TGF-β, such as non-signaling TβRIII, thereby enabling the TGF-β MOD (and thus the TMP) to efficiently deliver active TGF-β agonists to target cells. The TMP constructs discussed herein allow for the specific presentation of reversibly masked TGF-β epitopes to target T cells, but also provide sites for presenting one or more additional MODs. Thus, the ability of the TMP constructs to include one or more additional MODs allows for the combined presentation of TGF-β and additional MOD(s) to induce target T cell responses in a substantially epitope-specific / selective manner, resulting in modulation of the target T cells. The TMP can thereby deliver one or more masked TGF-β MODs in an epitope-selective (e.g., dependent / specific) manner, (i) forming an active immune synapse with target T cells, such as CD4+ cells, that are selective for the epitope, and (ii) modulating (e.g., controlling / regulating) the response of the target T cells to the epitope.When bound to the TCR of a T cell, the effect of a TMP containing a masked TGF-β MOD on a T cell depends on whether any additional MODs are present as part of the TMP, and if so, which additional MOD(s) are present.

[0262] Furthermore, although a TMP of the present disclosure can contain both one or more masked TGF-β MODs and one or more additional MODs, such as wild-type or variant IL-2, PD-L1, and / or 4-1BBL MOD (as described above), if desired, a TMP of the present disclosure can contain only one or more masked TGF-β MODs. That is, one or more additional MODs, such as wild-type or variant IL-2, PD-L1, and / or 4-1BBL MOD, need not be included in a TMP of the present disclosure. A TMP of the present disclosure containing a masked TGF-β MOD can function as a means of producing a TGF-β-driven T cell response. For example, TGF-β itself can inhibit the development of effector cell function of T cells, activate macrophages, and / or promote tissue repair after local immune and inflammatory effects have subsided.

[0263] Although a masked TGF-β MOD comprises a masked TGF-β polypeptide, this TGF-β polypeptide-masking complex is reversible, "breathing" between an open state in which the TGF-β polypeptide is available to the cellular receptor and a closed state in which the mask is bound to the TGF-β polypeptide, allowing the TGF-β polypeptide to still act as a TβR agonist. Thus, the masking polypeptide functions to bind to the TGF-β polypeptide and prevent it from forming a tight complex with, for example, ubiquitous non-signaling TβR3 molecules that would otherwise be able to scavenge free TGF-β. Furthermore, because the active form of TGF-β is a dimer with higher affinity for TβR3, substitutions that limit dimerization (e.g., a C77S substitution replacing the cysteine ​​at position 77 with a serine) can be incorporated into the TGF-β sequence to avoid scavenging by the receptor.

[0264] One effect of the masking sequence is to reduce the effective affinity of TGF-β1, TGF-β2, and TGF-β3 polypeptides for TβR. At the same time, the masking polypeptide can alter the affinity for TGF-β polypeptides, allowing them to dissociate more easily from the TGF-β polypeptides and increasing the availability of the TGF-β polypeptides to cellular TβR proteins. That is, when the affinity of the masking polypeptide for TGF-β polypeptides is reduced, the masked TGF-β MODs remain in an open state for a longer period of time. Although TGF-β polypeptides can bind to cellular receptors in an open state, because the TβRII protein is generally the first peptide in the heterodimeric TβR1 / TβR2 signaling complex that interacts with TGF-β, controlling the affinity of the TGF-β polypeptide for TβRII can effectively control the formation of TGF-β into the active signaling complex. For example, incorporating substitutions at one or more, two or more, or all three of Lys25, Ile92, and / or Lys94 of TGF-β2 (or the corresponding positions in TGF-β1 and TGF-β3) reduces affinity for the TβRII polypeptide, allowing interaction between the TCR of the target cell and the TMP MHC polypeptide and epitope, effectively controlling binding and allowing for target cell-specific interaction.

[0265] When a TβRII polypeptide is used as a masking polypeptide, the possibility of direct interaction with cellular TβRI receptors and off-target signaling can be addressed by appropriate modification of the masking sequence. If it is desired to block / limit TβRI-mediated signaling by the masked TGF-β polypeptide and / or to alter (e.g., reduce) the affinity of the masked TβRII polypeptide for TGF-β, it is possible to incorporate N-terminal deletions and / or amino acid substitutions into the masked TβRII polypeptide. Modifications that can be made include deletion of N-terminal amino acids (e.g., N-terminal Δ14 or Δ25 deletions) and / or substitutions at one or more of L27, F30, D32, S49, I50, T51, S52, I53, E55, V77, D118, and / or E119. Some specific TβRII modifications that result in reduced association of TβRI and TβRII and reduced affinity for TGF-β include any one or more of L27A, F30A, D32A, D32N, S49A, I50A, T51A, S52A, S52L, I53A, E55A, V77A, D118A, D118R, E119A, and / or E119Q.

[0266] The TGF-β polypeptide present in the TMP is, in some cases, a variant TGF-β polypeptide, e.g., a variant TGF-β polypeptide that has reduced affinity for at least one class of TGF-β receptor or is selective for at least one class of TGF-β receptor compared to a wild-type TGF-β polypeptide.

[0267] While TGF-β1, TGF-β2, or TGF-β3 polypeptides can be incorporated into TMPs as part of the masked TGF-β polypeptide, various factors can influence the selection of a particular TGF-β polypeptide and the specific sequence and amino acid substitutions employed. For example, TGF-β1 and TGF-β3 polypeptides undergo "clipping" of their amino acid sequences when expressed in certain mammalian cell lines (e.g., CHO cells). In addition, dimerized TGF-β (e.g., TGF-β2) has a higher affinity for TβR3 (betaglycan receptor) than for the TβR2 receptor, potentially resulting in off-target binding or loss of biologically active masked protein, leading to a large in vivo pool of non-signaling TβR3 molecules. To minimize high-affinity off-target binding to TβR3, it may be desirable to replace residues that result in dimeric TGF-β molecules connected by disulfide bonds. Thus, cysteine ​​77 (C77) may be substituted with an amino acid other than cysteine ​​(eg, serine, forming a C77S substitution).

[0268] The amino acid sequence of a TGF-β polypeptide is known in the art. In some cases, the TGF-β polypeptide present in the masked TGF-β polypeptide is a TGF-β1 polypeptide. In some cases, the TGF-β polypeptide present in the masked TGF-β polypeptide is a TGF-β2 polypeptide. In some cases, the TGF-β polypeptide present in the masked TGF-β polypeptide is a TGF-β3 polypeptide.

[0269] Suitable TGF-β polypeptides can have a length of about 70 to about 125 amino acids, for example, a length of about 70 to about 80 amino acids, about 80 to about 90 amino acids, about 90 to about 100 amino acids, about 100 to about 105 amino acids, about 105 to about 110 amino acids, about 110 to about 112 amino acids, about 113 to about 120 amino acids, or about 120 to about 125 amino acids. A suitable TGF-β polypeptide may comprise an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity for at least 80, at least 90, at least 100, or at least 110 consecutive amino acids of the mature form of a human TGF-β1 polypeptide, a human TGF-β2 polypeptide, or a human TGF-β3 polypeptide.

[0270] Figures 23A-23G provide the amino acid sequences of TGF-β polypeptides, including the TGF-β1 preproprotein (Figure 23A), TGF-β2 preprotein (Figure 23C), and TGF-β3 preprotein (Figure 23E), the mature form of TGF-β1 (Figure 23B), the mature form of TGF-β2 (Figure 23D), and the mature form of TGF-β3 (Figure 23F). The mature form of TGF-β3 containing the C77S substitution is provided in Figure 23G. An alignment of the amino acid sequences of Homo sapiens TGF-β1, TGF-β2, and TGF-β3 polypeptides is provided in Figure 24.

[0271] (i) TGF-β1 polypeptide A suitable TGF-β1 polypeptide has the following TGF-β1 amino acid sequence: The TGF-β1 polypeptide may comprise an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to at least 70, at least 80, at least 90, at least 100, at least 110, or 112 amino acids of TIFF2024517658000017.tif17157 (SEQ ID NO: 133; 112 amino acids in length), wherein the TGF-β1 polypeptide has a length of approximately 112 amino acids. The TGF-β1 preproprotein is shown in Figure 23A. In Figure 23A, amino acids R25, C77, V92, and R94 are shown in bold and italic.

[0272] In some cases, a suitable TGF-β1 polypeptide comprises a C77S substitution. Thus, in some cases, a suitable TGF-β1 polypeptide has the following TGF-β1 amino acid sequence: TIFF2024517658000018.tif17157, wherein amino acid 77 is Ser. Positions 25, 77, 92, and 94 are shown in bold and italics.

[0273] (ii) TGF-β2 polypeptide A suitable TGF-β2 polypeptide has the following TGF-β2 amino acid sequence: The TGF-β2 polypeptide may comprise an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to at least 70, at least 80, at least 90, at least 100, at least 110, or 112 amino acids of TIFF2024517658000019.tif17153, wherein the TGF-β2 polypeptide has a length of approximately 112 amino acids. The TGF-β2 preproprotein is shown in Figure 23C. In Figure 23C, residues Lys25, Cys77, Ile92, and Lys94 are shown in bold and italic.

[0274] In some cases, a suitable TGF-β2 polypeptide comprises a C77S substitution. Thus, in some cases, a suitable TGF-β2 polypeptide has the following TGF-β2 amino acid sequence: The amino acid sequence 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 to at least 70, at least 80, at least 90, at least 100, at least 110, or 112 amino acids of TIFF2024517658000020.tif17160, wherein amino acid 77 is substituted with Ser. Ser77 is shown in bold and italics.

[0275] (iii) TGF-β3 polypeptide A suitable TGF-β3 polypeptide has the following TGF-β3 amino acid sequence: The TGF-β3 polypeptide may comprise an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to at least 70, at least 80, at least 90, at least 100, at least 110, or 112 amino acids of TIFF2024517658000021.tif17155, wherein the TGF-β3 polypeptide has a length of approximately 112 amino acids. The TGF-β3 isoform 1 preproprotein is shown in Figure 23E. In Figure 23E, positions 25, 92, and 94 are shown in bold and italicized.

[0276] In some cases, a suitable TGF-β3 polypeptide comprises a C77S substitution. In some cases, a suitable TGF-β3 polypeptide has the following TGF-β3 amino acid sequence: The amino acid sequence may comprise an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to at least 70, at least 80, at least 90, at least 100, at least 110, or 112 amino acids of TIFF2024517658000022.tif17162, wherein amino acid 77 is Ser. Positions 25, 92, and 94 are shown in bold and italics.

[0277] (iv) additional TGF-β polypeptide sequence changes In addition to sequence changes that alter TGF-β dimerization (e.g., cysteine ​​77 substitutions such as C77S), TGF-β1, TGF-β2, and TGF-β3 polypeptides with sequence changes that affect affinity and other properties can be incorporated into TGF-β polypeptides. When a variant TGF-β with reduced affinity for a masking polypeptide (e.g., a TβR polypeptide such as a TβRII polypeptide) is present in a TGF-β polypeptide, these components readily dissociate, increasing the availability of the TGF-β polypeptide to cellular TβR proteins. Because the TβRII protein is generally the first peptide in the heterodimeric TβR signaling complex that interacts with TGF-β, interaction with TβRII effectively controls TGF-β's entry into the active signaling complex. Therefore, variants that control the affinity of TGF-β for TβRII effectively control the entry of the masked TGF-β MOD into the active signaling complex.

[0278] The present disclosure provides TMPs that include variant masked TβR (e.g., TβRII) polypeptide sequences and / or variant TGF-β polypeptides that have altered (e.g., decreased) affinity for each other (compared to otherwise identical TGF-β polypeptides that do not contain the sequence change(s)). The affinity between a TGF-β polypeptide and a TβR (e.g., TβRII) polypeptide can be determined using BLI (blood level intensity), as described above for a MOD and its co-MOD.

[0279] (a) Additional TGF-β2 sequence variants The present disclosure provides a TMP comprising either a wild-type (wt) or variant TGF-β2 polypeptide, where the variant polypeptide has reduced affinity for a masked TβR present in the TMP (compared to an otherwise identical wild-type TGF-β polypeptide that does not contain the sequence alteration).

[0280] In some cases, the TMP comprises a variant TGF-β2 polypeptide that has greater than 85% (e.g., greater than 90%, 95%, 98% or 99%) sequence identity for at least 100 consecutive amino acids of the amino acid sequence shown in Figure 23D and includes substitutions that reduce the affinity of the variant TGF-β2 polypeptide for the TβRII receptor sequence.

[0281] In some cases, the TMP comprises a variant TGF-β (e.g., TGF-β2) polypeptide comprising substitutions of one or more, two or more, or all three of Lys25, Ile92, and / or Lys94 (see Figure 23D for residue locations and Figure 24 for corresponding residues in TGF-β1 and TGF-β3). These amino acid residues have been shown to affect the affinity of TGF-β2 for TβRII polypeptides (see Crescenzo et al., J. Mol. Biol. 355:47-62 (2006)). As an example, the TMP comprises a TGF-β2 polypeptide having an amino acid other than Lys or Arg at position 25 (Figure 23D). The TMP can comprise a TGF-β2 polypeptide having an amino acid other than Ile or Val at position 92 of the amino acid sequence shown in Figure 23D (or an amino acid other than Ile, Val, or Leu at position 92). A TMP can include a TGF-β2 polypeptide having an amino acid other than Lys or Arg at position 94 of the amino acid sequence shown in Figure 23D. A TMP can include a TGF-β2 polypeptide containing substitutions at one or more, two or more, or all three of Lys25, Ile92, and / or Lys94 of the amino acid sequence shown in Figure 23D.

[0282] (b) Additional TGF-β1 and TGF-β3 sequence variants In some cases, the TMP comprises a variant TGF-β1 or TGF-β3 polypeptide containing substitutions at one or more, two or more, or all three of the amino acid positions corresponding to Lys25, Ile92, and / or Lys94 in TGF-β2. In TGF-β1 or TGF-β3, the corresponding amino acids are Lys25 to Arg25, Ile92 to Val92, and Lys94 to Arg94, each of which is a conservative substitution. See, e.g., Figure 23B for TGF-β1 (mature form) and Figure 23F for TGF-β3 (mature form).

[0283] As an example, in some cases, the TMP comprises a TGF-β1 or β3 polypeptide having an amino acid other than Arg or Lys at position 25. As another example, in some cases, the TMP comprises a TGF-β1 or β3 polypeptide having an amino acid other than Val or Ile at position 92 (or an amino acid other than Ile, Val, or Leu at position 92). As another example, in some cases, the TMP comprises a TGF-β2 polypeptide having an amino acid other than Arg or Lys. As another example, in some cases, the TMP comprises a TGF-β1 or β3 polypeptide having substitutions at one or more, two or more, or all three of Arg25, Val92, and / or Arg94. As another example, in some cases, the TMP comprises a TGF-β1 or β3 polypeptide having substitutions at one or more, two or more, or all three of Arg25, Val92, and / or Arg94.

[0284] Masking Polypeptides As noted above, the TMPs of the present disclosure include a TGF-β polypeptide and a masking polypeptide. The polypeptide or polypeptide complex that binds to and masks the TGF-β polypeptide ("masking polypeptide") can take a variety of forms, including, for example, TGF-βRI ("TβRI"), TGF-βII ("TβRII"), TGF-βRIII ("TβRIII"), and anti-TGF-β antibodies.

[0285] TGF-β receptor polypeptide Masking of TGF-β in a masked TGF-β polypeptide can be achieved by utilizing a TGF-β receptor fragment (e.g., the ectodomain sequence of TβRI, TβRII, or TβRIII) that includes a polypeptide sequence sufficient for binding to a TGF-β polypeptide (e.g., TGF-β1, TGF-β2, or TGF-β3). In one embodiment, the masking sequence comprises all or a portion of the TβRI, TβRII, or TβRIII ectodomain. Examples of amino acid sequences of TGF-β receptor polypeptides are provided in Figures 25A-25N. The amino acid sequence of a TβRI ectodomain polypeptide is shown in Figure 25B. The amino acid sequence of a TβRII ectodomain polypeptide is shown in Figure 25D and Figures 25F-J.

[0286] (1) TGF-β receptor I (TβRI) The masking polypeptide can be derived from TβRI (e.g., isoform 1; SEQ ID NO: 141) and can include all or a portion of the TβRI ectodomain (amino acids 34-126). A TβRI polypeptide suitable for masking TGF-β can 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 at least 70, at least 80, at least 90, at least 100, or 103 amino acids of the following TβRI ectodomain amino acid sequence: LQCFCHL CTKDNFTCVT DGLCFVSVTE TTDKVIHNSM CIAEIDLIPR DRPFVCAPSS KTGSVTTTYC CNQDHCNKIE LPTTVKSSPG LGPVEL (SEQ ID NO: 142).

[0287] (2) TGF-β receptor II (TβRII) The masking polypeptide can be derived from TβRII (e.g., isoform A) and can include all or part of the TβRII ectodomain sequence (amino acids 24-177). A TβRII isoform A polypeptide suitable for masking TGF-β has the following TβRII isoform A ectodomain amino acid sequence: The amino acid sequence may comprise an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, or at least 154 amino acids of TIFF2024517658000023.tif24157. The position of the aspartic acid residue corresponding to D118 in the B isoform is shown in bold and italics.

[0288] The masking polypeptide may be derived from TβRII isoform B (SEQ ID NO: 145) and may contain all or part of the TβRII ectodomain sequence (amino acids 24-166). A TβRII isoform B polypeptide suitable for masking TGF-β has the TβRII isoform B ectodomain amino acid sequence: The masking polypeptide 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 at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, or 143 amino acids of TIFF2024517658000024.tif24156. As described below, any one or more of F30, D32, S52, E55, or D118 (italic and bold) may be substituted with an amino acid other than the natural amino acid at that position (e.g., alanine). The masking polypeptide may include a D118A or D118R substitution. The masking polypeptide may comprise a D118A or D118R substitution and one or more of an F30A, D32N, S52L and / or E55A substitution.

[0289] While the ectodomain of TβRII can be used as a masking polypeptide, this region of the protein contains a charged hydrophobic patch, resulting in an unfavorable pI and potentially toxic to cells expressing the polypeptide. Additionally, combining the TβRII ectodomain with an active TGF-β polypeptide can form a complex that can bind to TβRI on the cell surface and activate signaling receptors (e.g., signaling via the Smad pathway). Modifying the TβRII ectodomain sequence used to mask TGF-β by removing or altering sequences involved in TβRI association can prevent unintended cell stimulation by masked TGF-β, except through its cell surface heterodimer, the TβRI / TβRII complex. Modification of TβRII can also alter (e.g., decrease) its affinity for TGF-β (e.g., TGF-β3), thereby enabling controlled unmasking of TGF-β and its availability as a signaling molecule. Polypeptides containing TβR (e.g., TβRII) peptides with the highest affinity for TGF-β (e.g., TGF-β3) mask TGF-β polypeptides most tightly, requiring higher doses to achieve the same effect. In contrast, amino acid substitutions in TβRII that result in lower affinity unmask TGF-β polypeptides and are biologically effective at lower doses.

[0290] Thus, where it is desirable to block / limit signaling by a TGF-β polypeptide through TβRI and / or to alter (e.g., decrease) the affinity of a masked TβRII polypeptide for TGF-β, a number of TβRII modifications can be incorporated into the TβRII polypeptide sequence. Modifications that can be made include those described above for the N-terminal amino acids, e.g., deletion of 14 or 25 N-terminal amino acids (1-14 amino acids or 1-25 amino acids; Δ14, Δ25 modifications), and / or substitution of one or more of L27, F30, D32, S49, 150, T51, S52, 153, E55, V77, D118, and / or E119. Some specific TβRII modifications that result in reduced association of TβRI and TβRII and reduced affinity for TGF-β include any one or more of L27A, F30A, D32A, D32N, S49A, I50A, T51A, S52A, S52L, I53A, E55A, V77A, D118A, D118R, E119A, and / or E119Q, based on the amino acid numbering of the amino acid sequence shown in Figure 25F. For the effects of these substitutions on TGF-β3-TβRII and TβRI-TβRII complexes, see, e.g., J. Groppe et al. Mol Cell 29, 157-168, (2008) and De Crescenzo et al. JMB 355, 47-62 (2006). Modifications of TβRII, including an N-terminal Δ25 deletion and / or a substitution at F24 (e.g., an F24A substitution), substantially or completely block signaling through the canonical SMAD signaling pathway. In one embodiment, the aspartic acid at position 118 (D118) of the mature TβRII B isoform (e.g., the amino acid sequence shown in Figure 25F) is replaced with an amino acid other than Asp or Glu, e.g., Ala, resulting in a "D118A" substitution, or Arg, resulting in a D118R substitution. The Asp residue corresponding to D118 is shown in bold and underlined in Figure 25F. N-terminal deletions of 1 to 25 amino acids in length (e.g., a Δ25 deletion) and / or substitutions at F24 (e.g., an F24A substitution) may be combined with a D118 substitution (e.g., D118A or D118R).N-terminal deletions of 1 to 25 amino acids in length (e.g., a Δ25 deletion) and / or substitutions at F24 (e.g., an F24A substitution) may also be combined with substitutions at any of L27, F30, D32, S49, 150, T51, S52, I53, E55, V77, D118, and / or E119 (e.g., a D118A) substitutions, particularly any of the specific substitutions listed for the affinity-altering amino acid sequence positions above shown in Figure 25F.

[0291] N-terminal deletion of a TβRII polypeptide also abolishes its interaction with TβRI, preventing masked TGF-β polypeptides containing the TβRII polypeptide from acting as a constitutively active complex and participating in and activating TβRI signaling. A 14-amino acid deletion (Δ14) of a TβRII polypeptide substantially reduces the protein's interaction with TβRI, and a Δ25-amino acid deletion of TβRII appears to completely abolish the interaction with TβRI. N-terminal deletions also substantially alter the protein's pI, with Δ14 TβRII ectodomain mutants exhibiting pIs of approximately 4.5 to 5.0 (e.g., approximately 4.74). Thus, masking polypeptides can include TβRII ectodomain polypeptides with N-terminal deletions of 14 to 25 amino acids (e.g., 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids). Modified ectodomain sequences that can be utilized to mask TGF-β polypeptides, including those that limit interaction with TβRI, are described in the following paragraphs.

[0292] In one embodiment, the masking polypeptide comprises the TβRII isoform B ectodomain sequence: The masking polypeptide comprises a 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 at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, or 142 amino acids of TIFF2024517658000025.tif17158. Any one or more of F30, D32, S52, E55, or D118 (italicized and bolded) can be substituted with an amino acid other than the natural amino acid at that position (e.g., alanine). In one embodiment, the masking polypeptide comprises a TβRII isoform B ectodomain with a D118A substitution. In one embodiment, the TGF-β masking sequence comprises a TβRII isoform B ectodomain sequence with a D118A substitution and one or more of an F30A, D32N, S52L and / or E55A substitution.

[0293] Combinations of N-terminal deletions of TβRII, such as 14 to 25 amino acids (e.g., 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids), that block unintended cell signaling due to interaction of the masked TGF-β / TβRII complex with TβRI, may be combined with other TβRII ectodomain substitutions, including those at any one or more of F30, D32, S52, E55, and / or D118. The combination of deletions and substitutions ensures that the masked TGF-β MOD does not trigger cell signaling other than that mediated by the cell membrane-bound TβRI and TβRII receptors.

[0294] In one embodiment, the masking polypeptide comprises the TβRII isoform B ectodomain sequence in which amino acids 1 to 14 (Δ14) have been deleted: The masking polypeptide comprises a 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 at least 70, at least 80, at least 90, at least 100, at least 110, or 114 amino acids of TIFF2024517658000026.tif17157. Any one or more of F30, D32, S52, E55, or D118 (italicized and bolded) may be substituted with an amino acid other than the natural amino acid at that position (e.g., alanine). In one embodiment, the masking polypeptide comprises a TβRII isoform B ectodomain having a D118A substitution. In one embodiment, the sequence-masked TGF-β comprises the amino acid sequence TIFF2024517658000027.tif18156, which has a D118A substitution and one or more of F30A, D32N, S52L and / or E55A substitutions.

[0295] In one embodiment, the masking polypeptide has the sequence: The masking polypeptide comprises an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to at least 70, at least 80, at least 90, at least 100, or 104 amino acids of TIFF2024517658000028.tif17156. Any one or more of F30, D32, S52, E55, or D118 (italicized and bolded) can be substituted with an amino acid other than the natural amino acid at that position (e.g., alanine). In one embodiment, the masking polypeptide has a D118A substitution. In one embodiment, the masking polypeptide has a D118A substitution and one or more of F30A, D32N, S52L, and / or E55A substitutions. In one embodiment, the masking polypeptide has a D118A and an F30A substitution. In one embodiment, the masking polypeptide has a D118A and a D32N substitution. In one embodiment, the masking polypeptide has a D118A and a S52L substitution. In one embodiment, the masking polypeptide has a D118A and an E55A substitution.

[0296] (3) TGF-β receptor III (TβRIII) In one embodiment, the masking polypeptide may be derived from TβRIII and may comprise all or a portion of the TβRIII ectodomain (amino acids 27-787 of the A isoform or 27-786 of the B isoform). In some cases, a suitable masking polypeptide comprises an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to at least 70, at least 80, at least 90, at least 100, or 120 amino acids of the TβRIII A or B isoform ectodomain sequence.

[0297] (c) Antibody Although a TGF-β receptor polypeptide (e.g., an ectodomain sequence) can function to bind to and mask a TGF-β polypeptide in a TMP, other polypeptides that bind to a TGF-β polypeptide can also be employed as masking polypeptides. Suitable polypeptides that can be used to mask TGF-β include antibodies with affinity for TGF-β (e.g., antibodies specific for one or more of TGF-β1, TGF-β2, or TGF-β3), including antibody fragments such as nanobodies with affinity for a TGF-β polypeptide and single-chain anti-TGF-β antibodies (e.g., either of which can be humanized). Several antibodies, including scFv antibodies, that bind to and neutralize TGF-β have been described. See, e.g., U.S. Pat. No. 9,090,685. Throughout the embodiments and / or aspects described in the present disclosure, the TβR (e.g., TβRII) polypeptide used to mask a TGF-β polypeptide can be replaced with a masking antibody (e.g., scFv or nanobody) with affinity for a TGF-β polypeptide.

[0298] In some cases, an antibody (e.g., a single-chain antibody) can be used as the masking polypeptide, and the antibody can be limited to the isoform of the TGF-β polypeptide(s) to be masked. For example, if a TGF-β isoform is present in the masked TGF-β MOD, it can be masked using a single-chain antibody based on methelimumab (CAT192) against TGF-β1 (e.g., Lord et al., mAbs 10(3):444-452(2018)). In another embodiment, a single-chain antibody specific for TGF-β2 is used to mask the TGF-β isoform. In another embodiment, a single-chain antibody specific for TGF-β3 is used to mask the TGF-β isoform. Single chain antibodies may also be specific for an ectodomain selected from the group consisting of a combination of TGF-β isoforms (e.g., TGF-β1 & TGF-β2, TGF-β1 & TGF-β3, and TGF-β2 & TGF-β3). Single chain antibodies may also be pan-specific for the TGF-β1, TGF-β2, and TGF-β3 ectodomain sequences present in a masked TGF-β MOD. See, e.g., WO 2014 / 164709. Antibodies and single chain antibodies with desired specificity and affinity for TGF-β isoforms can be prepared by a variety of methods, including screening hybridomas and / or modifying (e.g., combinatorially modifying) the variable region sequences of antibodies with affinity for the target TGF-β polypeptide sequence.

[0299] In one embodiment, the masking polypeptide comprises a single chain antibody that masks TGF-β (e.g., TGF-β3). In one such embodiment, the single chain amino acid sequence is specific for TGF-β3 containing a C77S substitution.

[0300] Linker As noted above, a TMP of the present disclosure may include one or more linker peptides between the first and second polypeptide components of the TMP, e.g., between a T1D peptide and an MHC polypeptide, between an MHC polypeptide and an Ig Fc polypeptide, between a first MHC polypeptide and a second MHC polypeptide, between a MOD and an MHC polypeptide, etc. As used herein, the phrase "an optional peptide linker between any two of the components of a TMP" refers to a peptide linker between any two adjacent polypeptides within a TMP. For example, as used herein, the phrase "an optional peptide linker between any two of the components of a TMP" refers to a peptide linker between one or more of: i) a peptide and an MHC class II polypeptide; ii) a first MHC class II polypeptide and a second MHC class II polypeptide; iii) an MHC class II polypeptide and an Ig Fc polypeptide; iv) an MHC class II polypeptide and a masking polypeptide; v) an MHC class II polypeptide and a TGF-β polypeptide; vi) an Ig Fc polypeptide and a MOD; vii) an Ig Fc polypeptide and a TGF-β polypeptide; viii) a first MOD and a second MOD; ix) a masking polypeptide and a TGF-β polypeptide; and x) a TGF-β polypeptide and a MOD.

[0301] As described above, in some cases, a TMP may contain a Cys-containing peptide linker between the T1D peptide and the MHC class II polypeptide, for example, between the T1D peptide and the MHC class II β chain polypeptide. Generally, a Cys-containing peptide linker is used in either the first or second polypeptide of a TMP to intentionally promote disulfide bond formation between the linker and a desired site on the other polypeptide. When a Cys-containing linker is inserted into one polypeptide of a TMP, the remaining linkers in the TMP do not contain Cys to prevent disulfide bond formation at unnecessary sites on the TMP. However, as an exception, a Cys-containing linker can be used in each of the first and second polypeptides when it is desired to link the first and second polypeptides via a disulfide bond formed between the linkers. Thus, the TMP may comprise a) a Cys-containing peptide linker between the T1D peptide and the MHC class II polypeptide, e.g., between the T1D peptide and the MHC class II β chain polypeptide, and b) at least one additional peptide linker that does not contain Cys.

[0302] Suitable linkers (also referred to as "spacers") can be readily 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 45-50 amino acids in length. Suitable linkers can be 45, 46, 47, 48, 49, or 50 amino acids in length.

[0303] Cys-containing linkers A peptide linker containing Cys can comprise an amino acid sequence selected from (CGGGS)(GGGGS)n (SEQ ID NO: 178), (GCGGS)(GGGGS)n (SEQ ID NO: 179), (GGCGS)(GGGGS)n (SEQ ID NO: 180), (GGGCS)(GGGGS)n (SEQ ID NO: 181), and (GGGGC)(GGGGS)n (SEQ ID NO: 182), where n is an integer between 1 and 10 (e.g., where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). In some cases, the TMPs of the present disclosure include heterodimers comprising first and second polypeptides, wherein the first polypeptide comprises a T1D peptide and an MHC class II polypeptide (e.g., an MHC class II β chain polypeptide), and wherein the first polypeptide comprises a linker between the T1D peptide and the MHC class II polypeptide (e.g., an MHC class II β chain polypeptide), and the linker comprises an amino acid sequence selected from (CGGGS)(GGGGS)n (SEQ ID NO: 178), (GCGGS)(GGGGS)n (SEQ ID NO: 179), (GGCGS)(GGGGS)n (SEQ ID NO: 180), (GGGCS)(GGGGS)n (SEQ ID NO: 181), and (GGGGC)(GGGGS)n (SEQ ID NO: 182), where n is an integer between 1 and 10 (e.g., where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10).

[0304] Non-Cys containing linkers Exemplary linkers include glycine polymers (G) n , glycine-serine polymers (e.g., (GS) n , (GSGGS) n (SEQ ID NO: 233) and (GGGS) n(SEQ ID NO:234) (where n is an integer of at least one), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers can be used; both Gly and Ser are relatively unstructured and can therefore function as intermediate tethers between components. Glycine polymers can also be used because glycine has significantly more Φ-Ψ space available than alanine and is less restrictive than residues with long 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:235), GGSGG (SEQ ID NO:236), GSGSG (SEQ ID NO:237), GSGGG (SEQ ID NO:238), GGGSG (SEQ ID NO:239), GSSSG (SEQ ID NO:240), and the like. Exemplary linkers can include, for example, Gly(Ser4)n (SEQ ID NO:241), where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some cases, the linker includes the amino acid sequence (GSSSS)n (SEQ ID NO:242), where n is 4. In some cases, the linker includes the amino acid sequence (GSSSS)n (SEQ ID NO:243), where n is 5. Exemplary linkers can include, for example, (GlyGlyGlyGlySer)n (SEQ ID NO:244) (also referred to as a "G4S" linker), 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:245), where n is 1. In some cases, the linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO:246), where n is 2. In some cases, the linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO:160), where n is 3. In some cases, the linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO:171), where n is 4. In some cases, the linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO:161), where n is 5.In some cases, the linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO:247), where n is 6. In some cases, the linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO:248), where n is 7. In some cases, the linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO:249), where n is 8. In some cases, the linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO:250), where n is 9. In some cases, the linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO:259), where n is 10. In some cases, the linker comprises the amino acid sequence AAAGG (SEQ ID NO:184). In some cases, the linker comprises the amino acid sequence GGSAAAGG (SEQ ID NO:162). AAAGG (SEQ ID NO: 184) and GGSAAAGG (SEQ ID NO: 162) linkers have been found to be useful, for example, for linking MHC class II alpha chain polypeptides (e.g., DRA class II polypeptides) to Ig Fc polypeptides.

[0305] Peptides presenting T1D-associated epitopes ("T1D peptides") As used herein, a "T1D peptide" refers to a peptide that, when present in a TMP of the present disclosure, presents a T1D-associated epitope capable of binding to a TCR on the surface of a T cell. A T1D peptide can be about 4 to about 25 amino acids in length. For example, a T1D peptide can be 4 to 10, 8 to 12, 10 to 15, 12 to 20, 15 to 20, 15 to 25, or 20 to 25 amino acids in length. For example, a T1D peptide present in a TMP can be 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. In some cases, the T1D peptide present in the TMP has a length of 8 to 20 amino acids, e.g., 8 amino acids, 9 amino acids, 10 amino acids, 11 amino acids, 12 amino acids, 13 amino acids, 14 amino acids, 15 amino acids, 16 amino acids, 17 amino acids, 18 amino acids, 19 amino acids, or 20 amino acids.

[0306] Antigens associated with type 1 diabetes (T1D) include, for example, preproinsulin, proinsulin, insulin, insulin B chain, insulin A chain, the 65 kDa isoform of glutamic acid decarboxylase (GAD65), the 67 kDa isoform of glutamic acid 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, e.g., Mallone et al. (2011) Clin. Dev. Immunol. 2011:513210 and U.S. Patent 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 individuals with that autoimmune disorder. In this case, such autoantibodies and / or autoreactive T cells mediate the pathological conditions associated with the autoimmune disorder. A T1D peptide suitable for inclusion in a TMP of the present disclosure can be an epitope-presenting T1D peptide of 4 amino acids to about 25 amino acids in length of any one of the T1D-associated antigens described above.

[0307] As one non-limiting example, the T1D peptide is proinsulin 73-90 (GAGSLQPLALEGSLQKR; SEQ ID NO: 251). As another non-limiting example, the T1D peptide is the following insulin (InsA(1-15) peptide: GIVDQCCTSICSLYQ (SEQ ID NO: 252). As another non-limiting example, the T1D peptide is the following insulin (InsA(1-15; D4E) peptide: GIVEQCCTSICSLYQ (SEQ ID NO: 253). As another non-limiting example, the T1D peptide is the following GAD65(555-567) peptide; NFFRMVISNPAAT (SEQ ID NO: 177). As another non-limiting example, the T1D peptide is the following GAD65( 555-567; F557I) peptide; NFIRMVISNPAAT (SEQ ID NO: 163). As another non-limiting example, a T1D peptide is the following islet antigen 2 (IA2) peptide: SFYLKNVQTQETRTLTQFHF (SEQ ID NO: 254). As another non-limiting example, a T1D peptide is the following proinsulin peptide: SLQPLALEGSLQSRG (SEQ ID NO: 159). As another non-limiting example, a T1D peptide is the following proinsulin peptide GSLQPLALEGSLQSRGIV (SEQ ID NO: 255; proIns 75-92(K88S)).

[0308] In some cases, a suitable T1D peptide has the following human preproinsulin amino acid sequence (amino acids 1-24 (underlined) are the signal peptide): The T1D peptide comprises 4 to 25 contiguous amino acids of an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to amino acids 25 to 110 of TIFF2024517658000029.tif17149, and the T1D peptide has a length of 4 amino acids (aa), 5 amino acids, 6 amino acids, 7 amino acids, 8 amino acids, 9 amino acids, 10 amino acids, 11 amino acids, 12 amino acids, 13 amino acids, 14 amino acids, 15 amino acids, 16 amino acids, 17 amino acids, 18 amino acids, 19 amino acids, 20 amino acids, 21 amino acids, 22 amino acids, 23 amino acids, 24 amino acids, or 25 amino acids. In some cases, the T1D peptide has the amino acid sequence: GAGSLQPLALEGSLQKRG (SEQ ID NO: 176) (proIns 73-90). In some cases, the T1D peptide has the amino acid sequence: SLQPLALEGSLQKRG (SEQ ID NO: 175) (proIns 76-90). In some cases, the T1D peptide has the amino acid sequence: SLQPLALEGSLQSRG (SEQ ID NO: 159) (proIns 76-90; K88S). In some cases, the T1D peptide has the amino acid sequence: QPLALEGSLQKRG (SEQ ID NO: 257). In some cases, the T1D peptide has the amino acid sequence: QPLALEGSLQSRG (SEQ ID NO: 258).

[0309] nucleic acid The present disclosure provides a nucleic acid comprising a nucleotide sequence encoding a TMP of the present disclosure. In some cases, when TMP comprises two different polypeptide chains, the present disclosure provides a single nucleic acid comprising a nucleotide sequence encoding both the first and second polypeptides of TMP. In other cases, when TMP comprises two different polypeptide chains, the present disclosure provides a) a first nucleic acid comprising a nucleotide sequence encoding the first polypeptide of TMP, and b) a second nucleic acid comprising a nucleotide sequence encoding the second polypeptide of TMP. In other cases, when TMP is a single-chain TMP, the present disclosure provides a nucleic acid comprising a nucleotide sequence encoding the single-chain TMP. In some cases, the nucleic acid is a recombinant expression vector, and thus the present disclosure provides a recombinant expression vector comprising a nucleotide sequence encoding TMP.

[0310] Separate nucleic acids encoding the individual polypeptide chains of TMP The present disclosure provides a nucleic acid comprising the nucleotide sequence encoding the TMP of the present disclosure.As mentioned above, in some cases, when TMP comprises two different polypeptide chains, each polypeptide chain of TMP is encoded by a separate nucleic acid.In some cases, the nucleotide sequence encoding each polypeptide chain of TMP is operably linked to a transcription control element, for example, a promoter (such as a promoter that functions in eukaryotic cells), and the promoter can be a constitutive promoter or an inducible promoter.

[0311] For example, the present disclosure provides a first nucleic acid and a second nucleic acid, wherein the first nucleic acid comprises a nucleotide sequence encoding a first polypeptide of TMP, and the second nucleic acid comprises a nucleotide sequence encoding a second polypeptide of TMP. In some cases, the nucleotide sequences encoding the first and second polypeptides are operably linked to a transcriptional control element. In some cases, the transcriptional control element is a promoter that functions in eukaryotic cells. In some cases, the nucleic acids are present in separate expression vectors.

[0312] A single nucleic acid encoding two or more polypeptides present in TMP The present disclosure provides nucleic acids comprising nucleotide sequences encoding a first polypeptide and a second polypeptide of TMP. In some cases, the nucleotide sequences encoding the first polypeptide and the second polypeptide of TMP comprise a proteolytically cleavable linker inserted between the nucleotide sequence encoding the first polypeptide and the nucleotide sequence encoding the second polypeptide. In some cases, the nucleotide sequences encoding the first polypeptide and the second polypeptide of TMP comprise an internal ribosome entry site (IRES) inserted between the nucleotide sequence encoding the first polypeptide and the nucleotide sequence encoding the second polypeptide. In some cases, the nucleotide sequences encoding the first polypeptide and the second polypeptide of TMP comprise a ribosome skipping signal (or cis-acting hydrolase element, CHYSEL) inserted between the nucleotide sequence encoding the first polypeptide and the nucleotide sequence encoding the second polypeptide. Examples of nucleic acids in which a proteolytically cleavable linker is provided between the nucleotide sequence encoding the first and second polypeptides of TMP are described below, and in any of these embodiments, an IRES or ribosomal skipping signal can be used in place of the nucleotide sequence encoding the proteolytically cleavable linker.

[0313] In some cases, a first nucleic acid (e.g., a recombinant expression vector, mRNA, viral RNA, etc.) comprises a nucleotide sequence encoding a first polypeptide chain of TMP, and a second nucleic acid (e.g., a recombinant expression vector, mRNA, viral RNA, etc.) comprises a nucleotide sequence encoding a second polypeptide chain of TMP. In some cases, the nucleotide sequence encoding the first polypeptide and the second nucleotide sequence encoding the second polypeptide are each operably linked to a transcriptional control element, e.g., a promoter (such as a promoter functional in eukaryotic cells), which can be a constitutive promoter or an inducible promoter.

[0314] Nucleic acid encoding single-chain TMP The present disclosure provides nucleic acid, comprising the nucleotide sequence encoding single-chain TMP described herein.In some cases, the nucleotide sequence encoding single-chain TMP is operably linked to one or more transcriptional control elements.In some cases, the transcriptional control element is a promoter that functions in eukaryotic cells.

[0315] Recombinant Expression Vectors The present disclosure provides a recombinant expression vector comprising the nucleic acid of the present disclosure.In some cases, the recombinant expression vector is a non-viral vector.In some cases, the recombinant expression vector is a viral construct, such as a recombinant adeno-associated virus construct (see, for example, U.S. Patent No. 7,078,387), a recombinant adenovirus construct, a recombinant lentivirus construct, a recombinant retrovirus construct, a non-integrating viral vector, etc.

[0316] Suitable expression vectors include viral vectors (e.g., viral vectors based on vaccinia virus, poliovirus, adenovirus (see, e.g., Li et al., Invest Opthalmol Vis Sci 35:2543 2549, 1994; Borras et al., Gene Ther 6:515 524, 1999; Li and Davidson, PNAS 92:7700 7704, 1995; Sakamoto et al., H Gene Ther 5:1088 1097, 1999; WO94 / 12649, WO93 / 03769, WO93 / 19191, WO94 / 28938, WO95 / 11984, and WO95 / 00655), adeno-associated virus (see, e.g., Ali et al., Hum Gene Ther 9:81 86,1998, Flannery et al.,PNAS 94:6916 6921,1997;Bennett et al.,Invest Opthalmol Vis Sci 38:2857 2863,1997;Jomary et al.,Gene Ther 4:683 690,1997,Rolling et al.,Hum Gene Ther 10:641 648,1999;Ali et al.,Hum Mol Genet 5:591 594,1996;Srivastava WO93 / 09239, Samulski et al.,J.Vir.(1989)63:3822-3828;Mendelson et al.,Virol.(1988)166:154-165;and Flotte et al. al., PNAS (1993) 90:10613-10617), SV40, herpes simplex virus, human immunodeficiency virus (e.g., Miyoshi et al., PNAS 94:10319 23, 1997; Takahashi et al., J Virol 73:7812 7816, 1999), retroviral vectors (e.g., vectors derived from murine leukemia virus, spleen necrosis virus, and retroviruses such as Rous sarcoma virus, Harvey sarcoma virus, avian leukosis virus, lentivirus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus), and the like. Numerous suitable expression vectors are known to those of skill in the art, and many are commercially available.

[0317] Depending on the host / vector system utilized, any of a number of suitable transcriptional and translational control elements, including constitutive and inducible promoters, transcriptional enhancer elements, transcriptional terminators, etc., can be used in the expression vector (see, e.g., Bitter et al. (1987) Methods in Enzymology, 153:516-544).

[0318] In some cases, the nucleotide sequence encoding TMP is operably linked to a regulatory element, e.g., a transcriptional regulatory element such as a promoter. The transcriptional regulatory element can be functional in either eukaryotic cells (e.g., mammalian cells) or prokaryotic cells (e.g., bacterial or archaeal cells). In some cases, the nucleotide sequence encoding TMP is operably linked to multiple regulatory elements capable of expressing the nucleotide sequence in both prokaryotic and eukaryotic cells.

[0319] Non-limiting examples of suitable eukaryotic promoters (promoters that function in eukaryotic cells) include those derived from cytomegalovirus (CMV), immediate early herpes simplex virus (HSV) thymidine kinase, early and late SV40, long terminal repeats (LTRs) from retrovirus, and mouse metallothionein-I. Selection of an appropriate vector and promoter is well within the level of one of ordinary skill in the art. Expression vectors also contain a ribosome binding site for translation initiation and a transcription terminator. Expression vectors may also include sequences appropriate for amplifying expression.

[0320] Genetically modified host cells The present disclosure provides genetically modified host cells, wherein the host cells are genetically modified with a nucleic acid(s) of the present disclosure.The present disclosure provides genetically modified host cells, wherein the host cells are genetically modified with a recombinant expression vector of the present disclosure.

[0321] Suitable host cells include eukaryotic cells such as yeast cells, insect cells, and mammalian cells. In some cases, the host cells are cells of a mammalian cell line. Suitable mammalian cell lines include human cell lines, non-human primate cell lines, rodent (e.g., mouse, rat) cell lines, and the like. Suitable mammalian cell lines include, but are not limited to, HeLa cells (e.g., American Type Culture Collection (ATCC) No. CCL-2), CHO cells (e.g., ATCC Nos. CRL9618, CCL61, CRL9096), 293 cells (e.g., ATCC No. CRL-1573), Vero cells, NIH 3T3 cells (e.g., ATCC No. CRL-1658), Huh-7 cells, BHK cells (e.g., ATCC No. CCL10), PC12 cells (ATCC No. CRL1721), COS cells, COS-7 cells (ATCC No. CRL1651), RAT1 cells, mouse L cells (ATCC No. CCLI.3), human embryonic kidney (HEK) cells (ATCC No. CRL1573), HLHepG2 cells, and the like.

[0322] Genetically modified host cells can be used to produce TMP. Expression vector(s) containing nucleotide sequence(s) encoding polypeptide(s) are introduced into the host cells to generate genetically modified host cells, which produce the polypeptide(s). For example, the genetically modified host cells are cultured in a suitable culture medium under conditions such that TMP is synthesized by the genetically modified host cells. TMP can then be obtained from the culture medium and / or lysate of the genetically modified host cells. TMP can be purified from the cell culture medium and / or cell lysate.

[0323] Methods for Producing TMP The present disclosure provides a method for producing the TMP of the present disclosure. The method generally includes culturing a genetically modified host cell (e.g., a genetically modified host cell of the present disclosure) in a culture medium with a recombinant expression vector(s) containing a nucleotide sequence(s) encoding TMP, and isolating TMP from the genetically modified host cell and / or the culture medium. As described above, in some cases, individual polypeptide chains of TMP are encoded by separate recombinant expression vectors. In some cases, all polypeptide chains of TMP are encoded by a single recombinant expression vector. When TMP is a single-chain TMP, one recombinant expression vector encodes the single-chain TMP.

[0324] Isolation of TMP from expression host cells (eg, lysates of genetically modified host cells) and / or the culture medium in which the host cells have been cultured can be carried out using standard protein purification methods.

[0325] For example, a lysate of the expression host can be prepared and the lysate purified using high performance liquid chromatography (HPLC), exclusion chromatography (e.g., size exclusion chromatography), gel electrophoresis, affinity chromatography, or other purification techniques. Alternatively, if TMP is secreted from the expression host cells into the culture medium, TMP can be purified from the culture medium using HPLC, exclusion chromatography, gel electrophoresis, affinity chromatography, or other purification techniques. In some cases, TMP is purified to produce a composition that contains, for example, at least 80%, at least about 85%, at least about 95%, or at least about 99.5% by weight of TMP, relative to contaminants associated with the method of product preparation and its purification. Percentages may be based on total protein.

[0326] In some cases, for example, when the TMP comprises an affinity tag, the TMP can be purified using an immobilized binding partner of the affinity tag. For example, when the TMP comprises an Ig Fc polypeptide, the TMP can be isolated from the genetically modified mammalian host cells and / or the culture medium in which the mammalian cells have been cultured, and isolation of the TMP or APP can be performed by affinity chromatography, for example, using a protein A column, a protein G column, or the like. One example of a suitable mammalian cell is a CHO cell, for example, an Expi-CHO-S™ cell (e.g., ThermoFisher Scientific, catalog number A29127).

[0327] In some cases, the first and second polypeptides of TMP self-assemble into heterodimers by spontaneously forming disulfide bonds between the aforementioned Cys residues in the first and second polypeptides. Also, as described above, when both heterodimers contain an Ig Fc polypeptide, a disulfide bond spontaneously forms between each Ig Fc polypeptide, covalently linking the two heterodimers to each other.

[0328] composition The present disclosure provides compositions (including pharmaceutical compositions) comprising the TMPs of this disclosure.The present disclosure provides compositions (including pharmaceutical compositions) comprising the nucleic acids or recombinant expression vectors of this disclosure.

[0329] Compositions containing TMP In addition to TMP, the compositions of the present disclosure may include one or more pharmaceutically acceptable components / excipients: salts, e.g., NaCl, MgCl, KCl, MgSO, etc.; buffers, e.g., Tris buffer, N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES), 2-(N-morpholino)ethanesulfonic acid (MES), 2-(N-morpholino)ethanesulfonic acid sodium salt (MES), 3-(N-morpholino)propanesulfonic acid (MOPS), N-tris[hydroxymethyl]methyl-3-aminopropanesulfonic acid (TAPS), etc.; solubilizers; surfactants, e.g., non-ionic surfactants such as Tween-20; protease inhibitors; glycerol, etc. Pharmaceutically acceptable excipients are described, for example, in "Remington: The Science and Practice of Pharmacy," 1999. th Ed. (1995) or latest edition, Mack Publishing Co; A. Gennaro (2000) “Remington: The Science and Practice of Pharmacy”, 20th edition, Lippincott, Williams, & Wilkins; Pharmaceutical Dosage Forms and Drug Delivery Systems (1999) H.C. Cansel et al., eds. 7 th ed., Lippincott, Williams, & Wilkins; and Handbook of Pharmaceutical Excipients (2000) AHKibbe et al., eds., 3 rd and is well described in various publications, including ed. Amer. Pharmaceutical Assoc.

[0330] A pharmaceutical composition can include i) a TMP of the present disclosure and ii) a pharmaceutically acceptable excipient. In some cases, the subject pharmaceutical compositions are suitable for administration to a subject, e.g., are sterile. For example, in some cases, the subject pharmaceutical compositions are suitable for administration to a human subject, e.g., the compositions are sterile and free of detectable pyrogens and / or other toxins or contain detectable pyrogens and / or other toxins below acceptable limits.

[0331] For example, the compositions may include aqueous solutions, powder forms, granules, tablets, pills, suppositories, capsules, suspensions, sprays, etc. The compositions may be formulated for various routes of administration, as described below.

[0332] When TMP is administered directly into tissues as an injection (e.g., subcutaneously, intraperitoneally, intramuscularly, intralymphaticly, and / or intravenously), the formulation can be provided as a ready-to-use dosage form or as a non-aqueous form (e.g., a reconstitutable, shelf-stable powder) or an aqueous form, such as a liquid comprising pharmaceutically acceptable carriers and excipients. Protein-containing formulations can also be provided to extend the serum half-life of the subject protein after administration. For example, proteins can be provided in liposomal formulations prepared as colloids or other conventional techniques for extending serum half-life. Various methods are available for preparing liposomes, such as those described in Szoka et al. 1980 Ann. Rev. Biophys. Bioeng. 9:467, U.S. Patent Nos. 4,235,871, 4,501,728, and 4,837,028. Preparations can also be provided in controlled-release or sustained-release forms.

[0333] In some cases, a composition of the present disclosure includes a) TMP and b) saline (e.g., 0.9% NaCl). In some cases, the composition is sterile. In some cases, the composition is suitable for administration to a human subject, e.g., the composition is sterile and free of detectable pyrogens and / or other toxins. Thus, the present disclosure provides a composition comprising a) TMP of the present disclosure and b) saline (e.g., 0.9% NaCl), wherein the composition is sterile and free of detectable pyrogens and / or other toxins or has detectable pyrogens and / or other toxins below acceptable limits.

[0334] The concentration of TMP in the formulation can vary widely (e.g., from less than about 0.1% (usually about or at least about 2%) to 20% to 50% or more by weight) and is typically selected based primarily on fluid volume, viscosity, and patient-dependent factors according to the particular mode of administration selected and patient needs.

[0335] The present disclosure provides a container containing a composition of the present disclosure, for example, a liquid composition. The container may be, for example, a syringe, an ampoule, or the like. In some cases, the container is sterile. In some cases, both the container and the composition are sterile.

[0336] Compositions Comprising Nucleic Acids or Recombinant Expression Vectors The present disclosure provides compositions, e.g., pharmaceutical compositions, comprising the nucleic acids or recombinant expression vectors of the present disclosure. A wide variety of pharmaceutically acceptable excipients are known in the art and need not be discussed in detail herein. Pharmaceutically acceptable excipients are described, for example, in A. Gennaro (2000) "Remington: The Science and Practice of Pharmacy", 20th edition, Lippincott, Williams, & Wilkins; Pharmaceutical Dosage Forms and Drug Delivery Systems (1999) H.C. Cansel et al., eds. 7 thed., Lippincott, Williams, & Wilkins; and Handbook of Pharmaceutical Excipients (2000) AHKibbe et al., eds., 3 rd and is well described in various publications, including ed. Amer. Pharmaceutical Assoc.

[0337] The pharmaceutical formulations of the present disclosure may contain nucleic acids or recombinant expression vectors in amounts of about 0.001% to about 90% (w / w). In the following description of the formulations, "subject nucleic acids or recombinant expression vectors" is understood to include nucleic acids or recombinant expression vectors. For example, in some cases, the subject formulations include nucleic acids or recombinant expression vectors.

[0338] The subject nucleic acids or recombinant expression vectors can be mixed, encapsulated, conjugated, or otherwise associated with other compounds or mixtures of compounds; such compounds can include, for example, liposomes or receptor-targeting molecules. The subject nucleic acids or recombinant expression vectors can be formulated in combination with one or more components that aid in uptake, distribution, and / or absorption.

[0339] method The TMPs of the present disclosure are useful for modulating the activity of T cells. Accordingly, the present disclosure provides methods of modulating the activity of T cells, generally involving contacting a target T cell with a TMP of the present disclosure.

[0340] The present disclosure provides methods for selectively modulating the activity of epitope-specific T cells, comprising contacting a T cell with a TMP, wherein contacting the T cell with the TMP selectively modulates the activity of the epitope-specific T cell. In some cases, the contacting occurs in vivo. In some cases, the contacting occurs in vitro.

[0341] Accordingly, the present disclosure also provides a method for selectively delivering a TGF-β polypeptide to epitope-specific target T cells, comprising contacting the T cells with a TMP comprising a masked TGF-β MOD, wherein contacting the epitope-specific T cells with the TMP comprising the masked TGF-β MOD selectively provides TGF-β modulation to the epitope-specific T cells. In some cases, the contacting occurs in vivo. In some cases, the contacting occurs in vitro. As used herein, the terms "selectively deliver" and "selectively provide" mean that a majority of the T cells to which the TMP provides TGF-β modulation contain a TCR that specifically or preferentially binds to the epitope of the TMP.

[0342] In some cases, TMPs reduce the activity of autoreactive T cells and / or autoreactive B cells. In some cases, TMPs increase the number and / or activity of regulatory T cells (Tregs), thereby resulting in a reduction in the activity of autoreactive T cells and / or autoreactive B cells.

[0343] In some cases, the T cells contacted with the TMP are regulatory T cells (Tregs). Tregs are CD4 + , FOXP3 + , and CD25 + Tregs can suppress autoreactive T cells. In some cases, the methods of the present disclosure activate Tregs, thereby reducing the activity of autoreactive T cells.

[0344] The present disclosure provides a method for increasing the proliferation of Tregs, the method comprising contacting Tregs with a TMP, wherein the contacting increases the proliferation of Tregs. The present disclosure provides a method for increasing the number of Tregs in an individual, the method comprising administering a TMP to the individual, wherein the administration results in an increase in the number of Tregs in the individual. For example, the number of Tregs can be increased by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 2-fold, at least 2.5-fold, at least 5-fold, at least 10-fold, or more than 10-fold.

[0345] Treatment method The present disclosure provides therapeutic methods comprising administering to an individual a TMP of the present disclosure, or one or more nucleic acids or expression vectors encoding TMP, in an amount effective to selectively regulate the activity of epitope-specific (e.g., T1D peptide-specific) T cells in the individual and treat the individual. In some cases, the therapeutic methods of the present disclosure comprise administering to an individual in need thereof one or more recombinant expression vectors comprising a nucleotide sequence encoding TMP. In some cases, the therapeutic methods comprise administering to an individual in need thereof one or more mRNA molecules comprising a nucleotide sequence encoding TMP. In some cases, the therapeutic methods of the present disclosure comprise administering to an individual in need thereof TMP e. TMP is useful for treating type 1 diabetes (T1D).

[0346] The present disclosure provides a method for selectively modulating the activity of epitope-specific T cells in an individual, comprising administering to the individual an effective amount of TMP or one or more nucleic acids (e.g., expression vectors, mRNA, etc.) comprising a nucleotide sequence encoding TMP, wherein the TMP selectively modulates the activity of epitope-specific T cells in the individual. Selectively modulating the activity of epitope-specific T cells can treat a disease or disorder in the individual. Accordingly, the present disclosure provides a therapeutic method comprising administering an effective amount of TMP to an individual in need thereof. The therapeutic method of the present disclosure comprising administering an effective amount of TMP is suitable for treating T1D.

[0347] The present disclosure provides a method of...

Claims

1. a) a peptide presenting a type 1 diabetes-related epitope capable of being bound by a T cell receptor ("T1D peptide"); b) a TGF-β3 polypeptide comprising an amino acid sequence having at least 95% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 137; c) a masking polypeptide comprising a TGF-β receptor type 2 (TβRII) polypeptide comprising an amino acid sequence having at least 95% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 144 or 146; d) an MHC class II α chain polypeptide; and e) an MHC class II β chain polypeptide; and f) one or more immunomodulatory polypeptides (MODs); 1. A T cell modulating polypeptide (TMP) comprising: at least one of the one or more MODs is a variant IL-2 polypeptide; the one or more variant IL-2 polypeptides exhibit a binding affinity to an IL-2 receptor, wherein the binding affinity of such one or more variant IL-2 polypeptides is lower than the binding affinity of a wild-type human IL-2 polypeptide to the same IL-2 receptor when assayed under the same conditions in a Bio-Layer Interferometry (BLI) assay; The TMP comprises an immunoglobulin (Ig) Fc scaffold polypeptide; The TMP may comprise one or more independently selected linker polypeptides. TMP.

2. The TMP of claim 1, wherein the T1D peptide has a length of about 8 amino acids to about 20 amino acids and comprises a proinsulin peptide or a glutamic acid decarboxylase (GAD) peptide.

3. The T1D peptide, a) a proinsulin peptide selected from SLQPLALEGSLQKRG (SEQ ID NO: 175; proIns 76-90), SLQPLALEGSLQSRG (SEQ ID NO: 159; proIns 76-90; K88S), and GAGSLQPLALEGSLQKRG (SEQ ID NO: 176; proIns 73-90); or b) a GAD peptide selected from NFFRMVISNPAAT (SEQ ID NO: 177; GAD65 555-567) and NFIRMVISNPAAT (SEQ ID NO: 163; GAD65 555-567; F557I); 3. The TMP of claim 2.

4. The TMP of claim 1, wherein the at least one variant IL-2 polypeptide exhibits reduced binding to both the IL-2 receptor alpha chain and the IL-2 receptor beta chain.

5. The TMP of claim 4, wherein the at least one variant IL-2 polypeptide comprises substitutions at H16 and F42 based on the numbering of SEQ ID NO:

185.

6. The TMP of claim 5, wherein the at least one variant IL-2 polypeptide comprises an H16A substitution and an F42A substitution, or an H16T substitution and an F42A substitution.

7. The TMP of claim 1, wherein the TβRII polypeptide comprises any one, any two, any three, any four, or a total of five substitutions selected from the group consisting of F30, D32, S52, E55, and D118.

8. The TMP of claim 7, wherein the TGF-β3 polypeptide comprises a C77S substitution and / or the TβRII polypeptide comprises a D118A or D118R substitution.

9. The TMP of claim 1, wherein the Ig Fc polypeptide is a variant Ig Fc polypeptide comprising one or more sequence alterations compared to a wild-type polypeptide, and wherein the ability of the Ig Fc polypeptide to induce cell lysis via complement-dependent cytotoxicity (CDC) and / or antibody-dependent cellular cytotoxicity (ADCC) is reduced or substantially eliminated.

10. The TMP described in claim 1, wherein the MHC class II alpha polypeptide comprises an amino acid sequence having at least 95% amino acid sequence identity to a DRA1*01:01 polypeptide, and the MHC class II beta polypeptide comprises an amino acid sequence having at least 95% amino acid sequence identity to a DRB1*04:01 polypeptide.

11. The T1D peptide, a) a proinsulin peptide selected from SLQPLALEGSLQKRG (SEQ ID NO: 175; proIns 76-90), SLQPLALEGSLQSRG (SEQ ID NO: 159; proIns 76-90; K88S), and GAGSLQPLALEGSLQKRG (SEQ ID NO: 176; proIns 73-90); or b) a GAD peptide selected from NFFRMVISNPAAT (SEQ ID NO: 177; GAD65 555-567) and NFIRMVISNPAAT (SEQ ID NO: 163; GAD65 555-567; F557I); the at least one variant IL-2 polypeptide exhibits reduced binding to both the α chain of the IL-2 receptor and the β chain of the IL-2 receptor; the TGF-β3 polypeptide comprises a C77S substitution, and / or the TβRII polypeptide comprises a D118A or D118R substitution; 5. The TMP of claim 4, wherein the Ig Fc polypeptide is a variant Ig Fc polypeptide comprising one or more sequence changes compared to a wild-type polypeptide, and the ability of the Ig Fc polypeptide to induce cell lysis via complement dependent cytotoxicity (CDC) and / or antibody dependent cellular cytotoxicity (ADCC) is reduced or substantially eliminated.

12. The TMP of claim 11, wherein the at least one variant IL-2 polypeptide comprises substitutions at H16 and F42 based on the numbering of SEQ ID NO:

185.

13. The TMP of claim 12, wherein the at least one variant IL-2 polypeptide comprises an H16A substitution and an F42A substitution, or an H16T substitution and an F42A substitution.

14. The TMP of claim 11, wherein the MHC class II alpha polypeptide comprises an amino acid sequence having at least 95% amino acid sequence identity to a DRA1*01:01 polypeptide, and the MHC class II beta polypeptide comprises an amino acid sequence having at least 95% amino acid sequence identity to a DRB1*04:01 polypeptide.

15. The TMP comprising at least one heterodimer, each heterodimer comprising: a) i) the T1D peptide, and ii) an MHC class II α chain polypeptide or an MHC class II β chain polypeptide, and iii) a linker linking the T1D peptide to the first MHC class II polypeptide. A first polypeptide comprising: b) a second polypeptide comprising: i) an MHC class II α chain polypeptide when said first polypeptide comprises an MHC class II β chain polypeptide; or ii) an MHC class II β chain polypeptide when said first polypeptide comprises an MHC class II α chain polypeptide; and Including, said first and / or said second polypeptide comprises said one or more MODs, the first or second polypeptide comprises the TGF-β3 polypeptide; the first or second polypeptide comprises the TβRII masking polypeptide, the first polypeptide and the second polypeptide of the heterodimer are covalently linked to each other via at least one disulfide bond; 2. The TMP of claim 1.

16. A method for treating a cancer cell line comprising the steps of: (a) administering to a patient a therapeutically effective amount of a TGF-β3 polypeptide; and (b) administering to a patient a therapeutically effective amount of a TGF-β3 polypeptide. the TMP comprises a Cys-containing linker between the T1D peptide and the MHC class II β chain polypeptide; a disulfide bond is formed between the Cys in the linker and the Cys in the MHC class II α chain polypeptide; the linker comprises an amino acid sequence selected from (CGGGS)(GGGGS)n (SEQ ID NO:178), (GCGGS)(GGGGS)n (SEQ ID NO:179), (GGCGS)(GGGGS)n (SEQ ID NO:180), (GGGCS)(GGGGS)n (SEQ ID NO:181), and (GGGGC)(GGGGS)n (SEQ ID NO:182), where n is an integer from 1 to 10; 16. The TMP of claim 15.

17. The MHC class II β chain polypeptide is a variant DRB MHC class II polypeptide comprising an amino acid substitution selected from the group consisting of P5C, F7C, Q10C, N19C, G20C, H33C, G151C, D152C, and W153C; and / or the MHC class II α chain polypeptide is a variant DRA MHC class II polypeptide comprising an amino acid substitution selected from E3C, E4C, F12C, G28C, D29C, I72C, K75C, T80C, P81C, I82C, T93C, N94C, and S95C; 17. The TMP of claim 16.

18. The method of claim 17, wherein: a) the amino acid substitution in the DRA MHC class II polypeptide is P81C and the amino acid substitution in the DRB MHC class II polypeptide is F7C; or b) the amino acid substitution in the DRA MHC class II polypeptide is P81C and the amino acid substitution in the DRB MHC class II polypeptide is P5C; c) the amino acid substitution in the DRA MHC class II polypeptide is P81C and the amino acid substitution in the DRB MHC class II polypeptide is H33C; d) the amino acid substitution in the DRA MHC class II polypeptide is E4C and the amino acid substitution in the DRB MHC class II polypeptide is N19C; e) the amino acid substitution in the DRA MHC class II polypeptide is E4C and the amino acid substitution in the DRB MHC class II polypeptide is G20C; f) the amino acid substitution in the DRA MHC class II polypeptide is T93C and the amino acid substitution in the DRB MHC class II polypeptide is Q156C; g) the amino acid substitution in the DRA MHC class II polypeptide is T93C and the amino acid substitution in the DRB MHC class II polypeptide is W153C; h) the amino acid substitution in the DRA MHC class II polypeptide is F12C and the amino acid substitution in the DRB MHC class II polypeptide is F7C; i) the amino acid substitution in the DRA MHC class II polypeptide is F12C and the amino acid substitution in the DRB MHC class II polypeptide is Q10C; j) the amino acid substitution in the DRA MHC class II polypeptide is T80C and the amino acid substitution in the DRB MHC class II polypeptide is F7C; k) the amino acid substitution in the DRA MHC class II polypeptide is T80C and the amino acid substitution in the DRB MHC class II polypeptide is P5C; l) the amino acid substitution in the DRA MHC class II polypeptide is T80C and the amino acid substitution in the DRB MHC class II polypeptide is I82C; m) the amino acid substitution in the DRA MHC class II polypeptide is I82C and the amino acid substitution in the DRB MHC class II polypeptide is F7C; n) the amino acid substitution in the DRA MHC class II polypeptide is I82C and the amino acid substitution in the DRB MHC class II polypeptide is P5C; o) the amino acid substitution in the DRA MHC class II polypeptide is I82C and the amino acid substitution in the DRB MHC class II polypeptide is H33C; p) the amino acid substitution in the DRA MHC class II polypeptide is G28C and the amino acid substitution in the DRB MHC class II polypeptide is G151C; q) the amino acid substitution in the DRA MHC class II polypeptide is G28C and the amino acid substitution in the DRB MHC class II polypeptide is D152C; r) the amino acid substitution in the DRA MHC class II polypeptide is G28C and the amino acid substitution in the DRB MHC class II polypeptide is W153C; s) the amino acid substitution in the DRA MHC class II polypeptide is D29C and the amino acid substitution in the DRB MHC class II polypeptide is G151C; t) the amino acid substitution in the DRA MHC class II polypeptide is D29C and the amino acid substitution in the DRB MHC class II polypeptide is D152C; u) the amino acid substitution in the DRA MHC class II polypeptide is D29C and the amino acid substitution in the DRB MHC class II polypeptide is W153C; v) the amino acid substitution in the DRA MHC class II polypeptide is N94C and the amino acid substitution in the DRB MHC class II polypeptide is Q156C; w) the amino acid substitution in the DRA MHC class II polypeptide is N94C and the amino acid substitution in the DRB MHC class II polypeptide is N120C; y) the amino acid substitution in the DRA MHC class II polypeptide is S95C and the amino acid substitution in the DRB MHC class II polypeptide is Q156C; z) the amino acid substitution in the DRA MHC class II polypeptide is S95C and the amino acid substitution in the DRB MHC class II polypeptide is N120C; aa) the amino acid substitution in the DRA MHC class II polypeptide is E3C and the amino acid substitution in the DRB MHC class II polypeptide is N19C; or bb) the amino acid substitution in the DRA MHC class II polypeptide is E3C and the amino acid substitution in the DRB MHC class II polypeptide is G20C; 18. The TMP of claim 17.

19. The T1D peptide, a) a proinsulin peptide selected from SLQPLALEGSLQKRG (SEQ ID NO: 175; proIns 76-90), SLQPLALEGSLQSRG (SEQ ID NO: 159; proIns 76-90; K88S), and GAGSLQPLALEGSLQKRG (SEQ ID NO: 176; proIns 73-90); or b) a GAD peptide selected from NFFRMVISNPAAT (SEQ ID NO: 177; GAD65 555-567) and NFIRMVISNPAAT (SEQ ID NO: 163; GAD65 555-567; F557I); the at least one variant IL-2 polypeptide exhibits reduced binding to both the α chain of the IL-2 receptor and the β chain of the IL-2 receptor; the TGF-β3 polypeptide comprises a C77S substitution, and / or the TβRII polypeptide comprises a D118A or D118R substitution; 16. The TMP of claim 15, wherein the Ig Fc polypeptide is a variant Ig Fc polypeptide comprising one or more sequence changes compared to a wild-type polypeptide, and the ability of the Ig Fc polypeptide to induce cell lysis via complement dependent cytotoxicity (CDC) and / or antibody dependent cellular cytotoxicity (ADCC) is reduced or substantially eliminated.

20. The T1D peptide, a) a proinsulin peptide selected from SLQPLALEGSLQKRG (SEQ ID NO: 175; proIns 76-90), SLQPLALEGSLQSRG (SEQ ID NO: 159; proIns 76-90; K88S), and GAGSLQPLALEGSLQKRG (SEQ ID NO: 176; proIns 73-90); or b) a GAD peptide selected from NFFRMVISNPAAT (SEQ ID NO: 177; GAD65 555-567) and NFIRMVISNPAAT (SEQ ID NO: 163; GAD65 555-567; F557I); the at least one variant IL-2 polypeptide exhibits reduced binding to both the α chain of the IL-2 receptor and the β chain of the IL-2 receptor; the TGF-β3 polypeptide comprises a C77S substitution, and / or the TβRII polypeptide comprises a D118A or D118R substitution; 19. The TMP of claim 18, wherein the Ig Fc polypeptide is a variant Ig Fc polypeptide comprising one or more sequence changes compared to a wild-type polypeptide, and the ability of the Ig Fc polypeptide to induce cell lysis via complement dependent cytotoxicity (CDC) and / or antibody dependent cellular cytotoxicity (ADCC) is reduced or substantially eliminated.

21. A TMP comprising a homodimer of two TMPs, each TMP being a TMP according to any one of claims 15 to 20.

22. One or more nucleic acids comprising one or more nucleotide sequences encoding the first and / or second polypeptide of TMP described in any one of claims 15 to 20.

23. An expression vector comprising one or more nucleic acids described in claim 22.

24. A genetically modified host cell genetically modified with one or more nucleic acids described in claim 22.

25. A method for producing TMP, comprising culturing in vitro a genetically modified host cell according to claim 24 in a culture medium under conditions such that the host cell synthesizes TMP.

26. A pharmaceutical composition comprising the TMP described in any one of claims 1 to 20.

27. A pharmaceutical composition comprising the TMP of claim 21.

28. The pharmaceutical composition of claim 26 for use in a method for increasing the number of regulatory T cells (Treg) in an individual, comprising administering an effective amount of the pharmaceutical composition to the individual.

29. The pharmaceutical composition of claim 27 for use in a method for increasing the number of regulatory T cells (Treg) in an individual, comprising administering an effective amount of the pharmaceutical composition to the individual.

30. The pharmaceutical composition of claim 26 for use in a method for treating type I diabetes in an individual comprising administering to the individual an effective amount of the pharmaceutical composition.

31. The pharmaceutical composition of claim 27 for use in a method for treating type I diabetes in an individual comprising administering to the individual an effective amount of the pharmaceutical composition.

32. The TMP of claim 1, comprising a single polypeptide chain.

33. The TMP of claim 32, wherein the single polypeptide chain comprises one or more intrachain disulfide bonds, the components of which may be connected by one or more independently selected linkers.

34. The TMP comprising at least one heterodimer, each heterodimer comprising: a) i) the T1D peptide; ii) an MHC class II β polypeptide; iii) an MHC class II α polypeptide, and iv) a first scaffold polypeptide comprising a first mutually specific binding sequence; A first polypeptide comprising: b) i) the T1D peptide; ii) an MHC class II β polypeptide; iii) an MHC class II α polypeptide, and iv) a second scaffold polypeptide comprising a mutually specific binding sequence corresponding to said mutually specific binding sequence of said first polypeptide. and a second polypeptide comprising: wherein the mutual specific binding sequence and the corresponding mutual specific binding sequence interact with each other in the heterodimer; said first and / or said second polypeptide comprises said one or more MODs, the first or second polypeptide comprises the TGF-β3 polypeptide; the first or second polypeptide comprises the TβRII masking polypeptide, The first polypeptide component and / or the second polypeptide component may be connected by one or more independently selected linkers; 34. The TMP of claim 33.

35. The T1D peptide, a) a proinsulin peptide selected from SLQPLALEGSLQKRG (SEQ ID NO: 175; proIns 76-90), SLQPLALEGSLQSRG (SEQ ID NO: 159; proIns 76-90; K88S), and GAGSLQPLALEGSLQKRG (SEQ ID NO: 176; proIns 73-90); or b) a GAD peptide selected from NFFRMVISNPAAT (SEQ ID NO: 177; GAD65 555-567) and NFIRMVISNPAAT (SEQ ID NO: 163; GAD65 555-567; F557I); the at least one variant IL-2 polypeptide exhibits reduced binding to both the α chain of the IL-2 receptor and the β chain of the IL-2 receptor; the TGF-β3 polypeptide comprises a C77S substitution, and / or the TβRII polypeptide comprises a D118A or D118R substitution; 35. The TMP of claim 34, wherein the Ig Fc polypeptide is a variant Ig Fc polypeptide comprising one or more sequence changes compared to a wild-type polypeptide, and the ability of the Ig Fc polypeptide to induce cell lysis via complement dependent cytotoxicity (CDC) and / or antibody dependent cellular cytotoxicity (ADCC) is reduced or substantially eliminated.

36. The single polypeptide chain, in order from N-terminus to C-terminus, a) the T1D peptide; MHC class II β polypeptide, MHC class II α polypeptide, the TGF-β polypeptide or the masking polypeptide, the masking polypeptide or the TGF-β polypeptide, Ig Fc polypeptides, and said one or more MODs; or b) the T1D peptide; MHC class II β polypeptide, MHC class II α polypeptide, the TGF-β polypeptide or the masking polypeptide, the masking polypeptide or the TGF-β polypeptide, the one or more MODs, and an Ig Fc polypeptide, or c) the T1D peptide; MHC class II β polypeptide, MHC class II α polypeptide, said one or more MODs, the TGF-β polypeptide or the masking polypeptide, the masking polypeptide or the TGF-β polypeptide, and an Ig Fc polypeptide, or d) the T1D peptide; MHC class II β polypeptide, MHC class II α polypeptide, said one or more MODs, Ig Fc polypeptides, the TGF-β polypeptide or the masking polypeptide, and the masking polypeptide or the TGF-β polypeptide, or e) the T1D peptide; MHC class II β polypeptide, MHC class II α polypeptide, Ig Fc polypeptides, said one or more MODs, the TGF-β polypeptide or the masking polypeptide, and the masking polypeptide or the TGF-β polypeptide, or f) the T1D peptide; MHC class II β polypeptide, MHC class II α polypeptide, said one or more MODs, the TGF-β polypeptide or the masking polypeptide, the masking polypeptide or the TGF-β polypeptide, and an Ig Fc polypeptide, or g) the T1D peptide; MHC class II β polypeptide, MHC class II α polypeptide, Ig Fc polypeptides, the TGF-β polypeptide or the masking polypeptide, the masking polypeptide or the TGF-β polypeptide, and The one or more MODs Including, the components may be connected by one or more independently selected linkers; 36. The TMP of claim 35.

37. A TMP comprising a homodimer of two single-chain TMPs described in any one of claims 32 to 36.

38. A nucleic acid comprising a nucleotide sequence encoding a single-chain TMP according to any one of claims 32 to 36.

39. An expression vector comprising the nucleic acid described in claim 38.

40. A genetically modified host cell genetically modified with the nucleic acid described in claim 38.

41. A method for producing TMP, comprising culturing in vitro a genetically modified host cell according to claim 40 in a culture medium under conditions such that the host cell synthesizes TMP.

42. A pharmaceutical composition comprising the TMP described in any one of claims 32 to 36.

43. A pharmaceutical composition comprising the TMP described in claim 37.

44. The pharmaceutical composition of claim 42 for use in a method for increasing the number of regulatory T cells (Treg) in an individual, comprising administering an effective amount of the pharmaceutical composition to the individual.

45. The pharmaceutical composition of claim 43 for use in a method for increasing the number of regulatory T cells (Treg) in an individual, comprising administering an effective amount of the pharmaceutical composition to the individual.

46. ​​The pharmaceutical composition of claim 42 for use in a method for treating type I diabetes in an individual comprising administering to the individual an effective amount of the pharmaceutical composition.

47. The pharmaceutical composition of claim 43 for use in a method for treating type I diabetes in an individual comprising administering to the individual an effective amount of the pharmaceutical composition.

Images