Multimeric T cell modulating polypeptides and methods of use thereof

Abstract

The present disclosure provides a T cell regulatory multimeric polypeptide, which comprises an immunomodulatory polypeptide and an epitope-presenting Wilms tumor peptide. The T cell regulatory multimeric polypeptide is suitable for regulating the activity of T cells and for regulating the immune response in an individual.

Description

[0001] Cross-references

[0002] This application claims priority to U.S. Provisional Patent Application No. 62 / 782,167, filed December 19, 2018, and U.S. Provisional Patent Application No. 62 / 814,684, filed March 6, 2019, the disclosures of which are incorporated herein by reference in their entireties. Background Art

[0003] The adaptive immune response involves the engagement of the T cell receptor (TCR) present on the surface of the T cell with the small peptide antigen presented non-covalently on the surface of the antigen presenting cell (APC) by the major histocompatibility complex (MHC; also referred to as the human leukocyte antigen (HLA) complex in humans). This engagement represents the targeting mechanism of the immune system and is an essential molecular interaction for T cell regulation (activation or inhibition) and effector function. After epitope-specific cell targeting, the targeted T cell is activated by engaging the costimulatory proteins seen on the APC with the counterpart costimulatory proteins on the T cell. Driving T cell specificity and activation or inhibition requires two signals, i.e., epitope / TCR binding and the engagement of APC costimulatory proteins with T cell costimulatory proteins. TCR is specific for a given epitope; however, costimulatory proteins are not epitope-specific and are typically expressed on all T cells or on a large T cell subset. Summary of the Invention

[0004] The present disclosure provides T cell modulatory multimeric polypeptides (TMMPs) comprising an immunomodulatory polypeptide and an epitope-presenting Wilms tumor peptide. The T cell modulatory multimeric polypeptides are useful for modulating the activity of T cells and for modulating an immune response in an individual. BRIEF DESCRIPTION OF THE DRAWINGS

[0005] Figures 1A-1F is a schematic depiction of various TMMPs of the present disclosure.

[0006] Figures 2A-2F Schematic depictions of various disulfide-linked TMMPs disclosed herein.

[0007] Figures 3A-3E The amino acid sequence of the WT-1 polypeptide is provided. Figures 3A-3E The sequences are listed in SEQ ID NO: 399-403, respectively.

[0008] Figures 4A-4E Amino acid sequences of exemplary polypeptide chains of the TMMPs of the present disclosure are provided. Figures 4A-4E The sequences of exemplary polypeptide chains of are listed in SEQ ID NOs: 405-409, respectively. The epitope sequences are listed as follows: Figure 4D:CMTWNQMNL (SEQ ID NO: 266); Figure 4E : CYTWNQMNL (SEQ ID NO: 267).

[0009] Figures 5A-5G The amino acid sequence of an immunoglobulin Fc polypeptide is provided. Figures 5A-5G The sequences are listed in SEQ ID NOs: 410-421, respectively.

[0010] Figure 6 A multiple amino acid sequence alignment of the beta-2 microglobulin (β2M) precursor (i.e., including the leader sequence) from Homo sapiens (NP_004039.1; SEQ ID NO: 19), Pantroglodytes (NP_001009066.1; SEQ ID NO: 19), Macaca mulatta (NP_001040602.1; SEQ ID NO: 20), Bos taurus (NP_776318.1; SEQ ID NO: 21), and Mus musculus (NP_033865.2; SEQ ID NO: 22) is provided. Amino acids 1-20 are signal peptides.

[0011] Figures 7A-7C Provided are alleles A*0101 (SEQ ID NO: 23), A*1101 (SEQ ID NO: 24), A*2402 (SEQ ID NO: 25) and A*3303 (SEQ ID NO: 26) ( Figure 7A )'s amino acid sequence of the full-length human HLA heavy chain; allele B*0702 (SEQ ID NO: 27) ( Figure 7B )'s amino acid sequence of the full-length human HLA heavy chain; and the full-length human HLA-C heavy chain (SEQ ID NO: 28) ( Figure 7C )'s amino acid sequence.

[0012] Figure 8 Provided is an alignment of eleven mature MHC class I heavy chain amino acid sequences without leader sequence, transmembrane domain, and intracellular domain. Top to bottom: SEQ ID NOs: 41-51.

[0013] Figures 9A-9B Provides HLA-A heavy chain amino acid sequence ( Figure 9A ; SEQ ID NO: 198-206, respectively) and consensus sequence ( Figure 9B ; SEQ ID NO: 29).

[0014] Figures 10A-10B Provides HLA-B heavy chain amino acid sequence ( Figure 10A ; SEQ ID NO: 207-213, respectively) and the consensus sequence ( Figure 10B ; SEQ ID NO: 30).

[0015] Figures 11A-11B Provides HLA-C heavy chain amino acid sequence ( Figure 11A ; SEQ ID NO: 214-222, respectively) and consensus sequence ( Figure 11B ; SEQ ID NO: 31).

[0016] Figure 12 The consensus amino acid sequences for each of the HLA-E, HLA-F, and HLA-G heavy chains (SEQ ID NOs: 32-34, respectively) are provided. The various amino acid (aa) positions are indicated by sequentially numbered "X" residues; the positions of amino acids 84, 139, and 236 are double underlined.

[0017] Figure 13 An alignment of the consensus amino acid sequences of HLA-A (SEQ ID NO: 35), HLA-B (SEQ ID NO: 36), HLA-C (SEQ ID NO: 37), HLA-E (SEQ ID NO: 38), HLA-F (SEQ ID NO: 39), and HLA-G (SEQ ID NO: 40) is provided.

[0018] Figures 14A-14I Provided are the amino acid sequences of the polypeptide chains of the double-disulfide-linked TMMPs of the present disclosure. Figures 14A-14I The sequences of the polypeptide chains are listed in SEQ ID NOs: 422-430, respectively. The epitope sequences are listed as follows: Figure 14B :VLDFAPPGA(SEQ ID NO:259); Figure 14C :RMFPNAPYL(SEQ ID NO:260); Figure 14F :VLDFAPPGA(SEQ ID NO:259); Figure 14G :RMFPNAPYL(SEQ ID NO:260); Figure 14H :YMFPNAPYL(SEQ ID NO:264); Figure 14I : YMFPNAPYL (SEQ ID NO: 264).

[0019] Figure 15 Depicted are expression and stability data for the TMMP of the present disclosure containing the WT1 (37-45) epitope.

[0020] Figure 16Depicted are expression and stability data for the TMMP of the present disclosure containing the WT1 (126-134) epitope.

[0021] Figures 17A-17D A schematic depiction of a double disulfide-linked TMMP of the present disclosure is provided.

[0022] Figures 18A-18C Schematic depictions of examples of configurations of disulfide-linked TMMPs of the present disclosure are provided.

[0023] Figure 19 Schematic depictions of examples of the location of immunomodulatory polypeptides in the TMMPs of the present disclosure are provided.

[0024] Figures 20A-20R Amino acid sequences of exemplary polypeptide chains of the TMMPs of the present disclosure are provided. Figures 20A-20R The sequences of exemplary polypeptide chains of are listed in SEQ ID NOs: 431-448, respectively. The epitope sequences are listed below: Figure 20H :CYTWNQMNL (SEQ ID NO: 262); Figure 20I :CYTWNQMNL (SEQ ID NO: 262); Figure 20J :CYTWNQMNL (SEQ ID NO: 262); Figure 20K :CYTWNQMNL(SEQ ID NO:262); Figure 20L :CYTWNQMNL(SEQ ID NO:262); Figure 20M :NYMNLGATL(SEQ ID NO:263); Figure 20N :NYMNLGATL(SEQ ID NO:263); Figure 20O :NYMNLGATL(SEQ ID NO:263); Figure 20P :NYMNLGATL(SEQ ID NO:263); Figure 20Q :NYMNLGATL(SEQ ID NO:263); Figure 20R :NYMNLGATL (SEQ ID NO:263).

[0025] Figure 21 Depicts the effect of TMMP containing a WT1 peptide epitope and HLA-A*02 heavy chain on the expansion of antigen-specific CD8+ T cells.

[0026] Figure 22 Depicted are the effects of TMMP containing a WT1 peptide epitope on the expansion of WT1-specific CD8+ T cells from total PBMCs during 8-day restimulation culture following a 10-day prime culture.

[0027] Figure 23 Depicted are TNF-§ and IFN-γ production by WT1-specific CD8+ T cells expanded with WT1 37-45-containing TMMPs with G2C or R12C / G2C frameworks.

[0028] Figure 24 Depicts TNF-§ and IFN-γ production by WT1-specific CD8+ T cells expanded with TMMP containing WT1 126-134 with the R12C / G2C framework.

[0029] Figure 25 Delineating the role of disulfide bonds in IL-2-driven immune cell activation.

[0030] Figure 26 Depicted are the effects of TMMP containing variant IL-2 as immunomodulatory polypeptide on CGLL-2 proliferation compared to proleukine.

[0031] Figure 27 Binding of "1715+2380" TMPP to various Fc receptors is depicted.

[0032] definition

[0033] As used interchangeably herein, the terms "polynucleotide" and "nucleic acid" refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, the term includes, but is not limited to, single-stranded, double-stranded, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers containing purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases.

[0034] The terms "peptide," "polypeptide," and "protein" are used interchangeably herein and refer to a polymeric form of amino acids of any length, which may include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides with modified peptide backbones.

[0035] A polynucleotide or polypeptide having a certain percentage of "sequence identity" with another polynucleotide or polypeptide means that the percentage of bases or amino acids is the same and in the same relative position when comparing the two sequences when aligned. Sequence identity can be determined in many different ways. To determine sequence identity, sequences can be aligned using various convenient methods and computer programs available on the World Wide Web (e.g., BLAST, T-COFFEE, MUSCLE, MAFFT, etc.), including ncbi.nlm.nili.gov / BLAST, ebi.ac.uk / Tools / msa / tcoffee / , ebi.ac.uk / Tools / msa / muscle / , mafft.cbrc.jp / alignment / software / . See, for example, Altschul et al. (1990), J. Mol. Biol. 215:403-10.

[0036] The term "conservative amino acid substitution" refers to the interchangeability of amino acid residues with similar side chains in proteins. 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.

[0037] As used herein, the term "immunological synapse" or "immunological synapse" generally refers to the natural interface between two interacting immune cells of the adaptive immune response, including, for example, the interface between an antigen presenting cell (APC) or target cell and an effector cell, such as a lymphocyte, effector T cell, natural killer cell, etc. The immunological synapse between an APC and a T cell is typically initiated by the interaction between the T cell antigen receptor and a major histocompatibility complex molecule, as described, for example, in Bromley et al., Annu Rev Immunol. 2001; 19: 375-96, the disclosure of which is incorporated herein by reference in its entirety.

[0038] "T cells" include all types of immune cells expressing CD3, including T-helper cells (CD4 + cells), cytotoxic T-cells (CD8 +cells), T-regulatory cells (Treg) and NK-T cells.

[0039] As used herein, the term "immunomodulatory polypeptide" (also referred to as "co-stimulatory polypeptide") includes polypeptides on antigen presenting cells (APCs) (e.g., dendritic cells, B cells, etc.) that specifically bind to a cognate co-immunomodulatory polypeptide on a T cell, thereby providing a signal that mediates T cell responses, including but not limited to proliferation, activation, differentiation, etc., in addition to the primary signal provided by, for example, the binding of the TCR / CD3 complex to a peptide-loaded major histocompatibility complex (MHC) polypeptide. Immunomodulatory polypeptides may include, but are not limited to, CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L, Fas ligand (FasL), inducible co-stimulatory ligand (ICOS-L), intracellular adhesion molecule (ICAM), CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, HVEM, lymphotoxin beta receptor, 3 / TR6, ILT3, ILT4, HVEM, agonists or antibodies that bind to Toll ligand receptors, and ligands that specifically bind to B7-H3.

[0040] As described above, an "immunomodulatory polypeptide" (also referred to herein as "MOD") specifically binds to a cognate co-immunomodulatory polypeptide on a T cell.

[0041] The "immunomodulatory domain" ("MOD") of the TMMPs of the present disclosure binds to a cognate co-immunomodulatory polypeptide that may be present on a target T cell.

[0042] As used herein, "heterologous" means that the nucleotide or polypeptide is not found in naturally occurring nucleic acids or proteins, respectively.

[0043] As used herein, "recombinant" means that a particular nucleic acid (DNA or RNA) is the product of various combinations of cloning, restriction, polymerase chain reaction (PCR), and / or ligation steps that result in a construct having structural coding or non-coding sequences distinguishable from endogenous nucleic acids found in natural systems. A DNA sequence encoding a polypeptide can be assembled from cDNA fragments or a series of synthetic oligonucleotides to provide a synthetic nucleic acid capable of being expressed from a recombinant transcription unit contained in a cell or a cell-free transcription and translation system.

[0044] 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 generated for the purpose of expressing and / or propagating the insert or for the purpose of constructing other recombinant nucleotide sequences. The insert may or may not be operably linked to a promoter sequence and may or may not be operably linked to DNA regulatory sequences.

[0045] 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 expressed as the dissociation constant (K D ). Affinity can be at least 1 times greater, at least 2 times greater, at least 3 times greater, at least 4 times greater, at least 5 times greater, at least 6 times greater, at least 7 times greater, at least 8 times greater, at least 9 times greater, at least 10 times greater, at least 20 times greater, at least 30 times greater, at least 40 times greater, at least 50 times greater, at least 60 times greater, at least 70 times greater, at least 80 times greater, at least 90 times greater, at least 100 times greater, or at least 1,000 times greater or more times greater than the affinity of the antibody for an unrelated amino acid sequence. The affinity of the antibody for the target protein can be, for example, from about 100 nanomolar (nM) to about 0.1 nM, from about 100 nM to about 1 picomolar (pM), from about 100 nM to about 1 femtomolar (fM) or greater. As used herein, the term "avidity" refers to the resistance of a complex of two or more agents to dissociation upon dilution. The terms "immunoreactivity" and "preferential binding" are used interchangeably herein with respect to antibodies and / or antigen-binding fragments.

[0046] As used herein, the term "binding" (e.g., with respect to the binding of a TMMP 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 due to, for example, electrostatic, hydrophobic, ionic, and / or hydrogen bonding interactions (including interactions such as salt bridges and water bridges). Non-covalent binding interactions are typically characterized by a dissociation constant (K D ) is less than 10 -6 M, less than 10 -7 M, less than 10 -8 M, less than 10 -9 M, less than 10 -10 M, less than 10 -11 M, less than 10 -12 M, less than 10 -13 M, less than 10 -14 M or less than 10 -15 M. “Affinity” refers to the strength of non-covalent binding, and an increase in binding affinity is associated with a lower K D "Specific binding" generally refers to binding with a specificity of at least about 10 -7 M or larger, such as 5x10-7 M, 10 -8 M, 5x10 -8 M, 10 -9 M, and greater affinity binding. "Non-specific binding" generally refers to binding with an affinity of less than about 10 -7 M affinity binding (e.g., binding of a ligand to a portion other than its designated binding site or receptor) (e.g., at 10 -6 M, 10 -5 M, 10 -4 "covalent binding" or "covalent bond" as used herein refers to the formation of one or more covalent chemical bonds between two different molecules.

[0047] The terms "treatment", "treating", and the like are used herein to generally mean obtaining a desired pharmacological and / or physiological effect. The effect may be preventive in terms of completely or partially preventing a disease or its symptoms, and / or may be therapeutic in terms of partial or complete cure of the disease and / or adverse effects caused by the disease. As used herein, "treatment" encompasses any treatment of a disease or symptom in a mammal and includes: (a) preventing the disease or symptom from occurring in a subject who may be predisposed to acquiring the disease or symptom but has not yet been diagnosed with the disease; (b) inhibiting the disease or symptom, i.e., arresting its development; and / or (c) alleviating the disease, i.e., causing the disease to regress. The therapeutic agent may be administered before, during, or after the onset of the disease or injury. Of particular interest is the treatment of an ongoing disease, wherein the treatment stabilizes or alleviates the patient's undesirable clinical symptoms. Such treatment is ideally performed before complete loss of function in the diseased tissue. The target therapy will ideally be administered during the symptomatic stage of the disease and in some cases after the symptomatic stage of the disease.

[0048] The terms "individual," "subject," "host," and "patient" are used interchangeably herein and refer to any mammalian subject in need of diagnosis, treatment, or therapy. 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.), and the like.

[0049] Before further describing the present invention, it should be understood that the present invention is not limited to the specific embodiments described, and therefore the embodiments may of course vary. It should also be understood that the terminology used herein is only for the purpose of describing specific embodiments and is not intended to be limiting, as the scope of the present invention is only limited by the scope of the appended claims.

[0050] When a numerical range is provided, it is understood that each intervening value between the upper and lower limits of that range, to the tenth of the lower limit (unless the context clearly dictates otherwise), as well as any other stated or intervening value within that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the invention, subject to any specifically excluded limits in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also encompassed within the invention.

[0051] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one skilled in the art to which this invention pertains. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials to which the publications are referred.

[0052] It must be noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a T cell modulating multimeric polypeptide" includes a plurality of such polypeptides and reference to an "immunomodulatory polypeptide" includes reference to one or more immunomodulatory polypeptides and their equivalents known to those skilled in the art, and so forth. It should be further noted that the scope of the claims may be drafted to exclude any optional elements. Therefore, this statement is intended to serve as a precondition for the use of exclusive terminology such as "solely," "only," and the like in connection with reciting claim elements or the use of a "negative" limitation.

[0053] It should be understood that certain features of the present invention that are described for clarity in the context of separate embodiments may also be provided in combination in a single embodiment. Conversely, various features of the present invention that are described for brevity in the context of a single embodiment may also be provided separately or in any suitable subcombination. All combinations of embodiments to which the present invention relates are expressly included herein and are disclosed herein as if each and every combination were individually and expressly disclosed herein. Furthermore, all subcombinations of the various embodiments and elements thereof are also expressly included herein and are disclosed herein as if each and every such subcombination were individually and expressly disclosed herein.

[0054] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the publication dates provided may be different from the actual publication dates, which may need to be independently confirmed. DETAILED DESCRIPTION

[0055] The present disclosure provides T cell modulating multimeric polypeptides comprising an immunomodulatory polypeptide and an epitope-presenting Wilms' Tumor-1 (WT-1) peptide. TMMP is useful for modulating the activity of T cells and for modulating an immune response in an individual.

[0056] T cell regulatory multimeric polypeptide

[0057] The present disclosure provides a T cell modulatory multimeric polypeptide (TMMP) comprising: a) a first polypeptide; and b) a second polypeptide, wherein the TMMP comprises an epitope; a first major histocompatibility complex (MHC) polypeptide; a second MHC polypeptide; one or more immunomodulatory polypeptides; and optionally an immunoglobulin (Ig) Fc polypeptide or a non-Ig scaffold. The present disclosure provides a TMMP, wherein the TMMP is a heterodimer comprising: a) a first polypeptide comprising a first MHC polypeptide; and b) a second polypeptide comprising a second MHC polypeptide, wherein the first polypeptide or the second polypeptide comprises an epitope (e.g., a peptide presenting the epitope); wherein the first polypeptide and / or the second polypeptide comprises one or more immunomodulatory polypeptides, which may be the same or different; and optionally an Ig Fc polypeptide or a non-Ig scaffold. The TMMP of the present disclosure is also referred to herein as a "multimeric polypeptide of the present disclosure" or "synTac." In some cases, the peptide epitope present in the TMMP of the present disclosure is a WT-1 peptide.

[0058] The present disclosure provides a TMMP comprising a heterodimeric polypeptide comprising: a) a first polypeptide comprising: i) a peptide epitope; and ii) a first MHC polypeptide; b) a second polypeptide comprising a second MHC polypeptide; and c) at least one immunomodulatory polypeptide, wherein the first polypeptide and / or the second polypeptide comprises at least one (i.e., one or more) immunomodulatory polypeptide. Optionally, the first polypeptide or the second polypeptide comprises an Ig Fc polypeptide or a non-Ig scaffold. At least one of the one or more immunomodulatory polypeptides is a variant immunomodulatory polypeptide that exhibits a reduced affinity for a cognate co-immunomodulatory polypeptide compared to the affinity of the corresponding wild-type immunomodulatory polypeptide for the cognate co-immunomodulatory polypeptide. The epitope present in the TMMP of the present disclosure binds to a T cell receptor (TCR) on a T cell with an affinity of at least 100 μM (e.g., at least 10 μM, at least 1 μM, at least 100 nM, at least 10 nM, or at least 1 nM). The TMMP of the present disclosure binds to a first T cell with an affinity that is at least 25% greater than the affinity with which the TMMP binds to a second T cell, wherein the first T cell expresses on its surface a cognate co-immunomodulatory polypeptide and a TCR that binds to the epitope with an affinity of at least 100 μM, and wherein the second T cell expresses on its surface a cognate co-immunomodulatory polypeptide but does not express on its surface a TCR that binds to the epitope with an affinity of at least 100 μM (e.g., at least 10 μM, at least 1 μM, at least 100 nM, at least 10 nM, or at least 1 nM). In some cases, the peptide epitope present in the TMMP of the present disclosure is a WT-1 peptide.

[0059] The present disclosure provides a TMMP, wherein the TMMP is:

[0060] A) a heterodimer comprising: a) a first polypeptide comprising a first MHC polypeptide; and b) a second polypeptide comprising a second MHC polypeptide, wherein the first polypeptide or the second polypeptide comprises an epitope (e.g., a peptide that presents the epitope to a T cell); wherein the first polypeptide and / or the second polypeptide comprises one or more immunomodulatory polypeptides, which may be the same or different, and wherein at least one of the one or more immunomodulatory polypeptides may be a wild-type immunomodulatory polypeptide or a variant of a wild-type immunomodulatory polypeptide, wherein the variant immunomodulatory polypeptide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid substitutions compared to the amino acid sequence of the corresponding wild-type immunomodulatory polypeptide; and wherein the first polypeptide or the second polypeptide optionally comprises an Ig Fc polypeptide or a non-Ig scaffold; or

[0061] B) a heterodimer comprising: a) a first polypeptide comprising a first MHC polypeptide; and b) a second polypeptide comprising a second MHC polypeptide, wherein either the first polypeptide or the second polypeptide comprises an epitope; wherein the first polypeptide and / or the second polypeptide comprises one or more immunomodulatory polypeptides which may be the same or different,

[0062] wherein at least one of the one or more immunomodulatory polypeptides is a variant of a wild-type immunomodulatory polypeptide, wherein the variant immunomodulatory polypeptide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acid substitutions compared to the amino acid sequence of the corresponding wild-type immunomodulatory polypeptide,

[0063] wherein at least one of the one or more immunomodulatory domains is a variant immunomodulatory polypeptide that exhibits reduced affinity for a cognate co-immunomodulatory polypeptide compared to the affinity of a corresponding wild-type immunomodulatory polypeptide for a cognate co-immunomodulatory polypeptide, and wherein the epitope is at least 10 -7 The invention relates to a method for binding a TMMP polypeptide to a TCR on a T cell with an affinity of at least 10 μg / mL, such that: i) the TMMP polypeptide binds to the first T cell with an affinity that is at least 25% greater than the affinity with which TMMP binds to the second T cell, wherein the first T cell expresses a cognate co-immunomodulatory polypeptide on its surface and binds to the TCR on the T cell with an affinity of at least 10 μg / mL; -7 M, and wherein the second T cell expresses the cognate co-immunomodulatory polypeptide on its surface but does not express the cognate co-immunomodulatory polypeptide on its surface with an affinity of at least 10 -7 and / or ii) the ratio of the binding affinity of the control TMMP to the cognate co-immunomodulatory polypeptide to the binding affinity of the TMMP comprising the wild-type immunomodulatory polypeptide variant to the cognate co-immunomodulatory polypeptide is in the range of 1.5:1 to 10 when measured by biolayer interferometry. 6 : 1, wherein the control comprises a wild-type immunomodulatory polypeptide; and wherein the variant immunomodulatory polypeptide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acid substitutions compared to the amino acid sequence of the corresponding wild-type immunomodulatory polypeptide; and

[0064] wherein the first polypeptide or the second polypeptide optionally comprises an Ig Fc polypeptide or a non-Ig scaffold; or

[0065] C) a heterodimer comprising: a) a first polypeptide comprising, in order from N-terminus to C-terminus: i) an epitope; ii) a first MHC polypeptide; and b) a second polypeptide comprising, in order from N-terminus to C-terminus: i) a second MHC polypeptide; and ii) optionally an immunoglobulin (Ig) Fc polypeptide or a non-Ig scaffold, wherein the TMMP comprises one or more immunomodulatory domains, which may be the same or different, wherein at least one of the one or more immunomodulatory domains is: A) at the C-terminus of the first polypeptide; B) at the N-terminus of the second polypeptide; C) at the C-terminus of the second polypeptide; or D) at the C-terminus of the first polypeptide and at the N-terminus of the second polypeptide, and at least one of the one or more immunomodulatory domains may be a wild-type immunomodulatory polypeptide or a variant of a wild-type immunomodulatory polypeptide, wherein the variant immunomodulatory polypeptide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acid substitutions compared to the amino acid sequence of the corresponding wild-type immunomodulatory polypeptide; and

[0066] Optionally, at least one of the one or more immunomodulatory domains is a variant immunomodulatory polypeptide that exhibits reduced affinity for a cognate co-immunomodulatory polypeptide compared to the affinity of a corresponding wild-type immunomodulatory polypeptide for a cognate co-immunomodulatory polypeptide, and wherein the epitope is at least 10 -7 The invention relates to a method for binding TMMP to a TCR on a T cell with an affinity of at least 10 μg / mL, such that: i) the affinity of TMMP binding to a first T cell is at least 25% greater than the affinity of TMMP binding to a second T cell, wherein the first T cell expresses a cognate co-immunomodulatory polypeptide on its surface and binds to a TCR on a T cell with an affinity of at least 10 μg / mL; -7 M, and wherein the second T cell expresses the cognate co-immunomodulatory polypeptide on its surface but does not express the cognate co-immunomodulatory polypeptide on its surface with an affinity of at least 10 -7 and / or ii) the ratio of the binding affinity of the control TMMP to the cognate co-immunomodulatory polypeptide to the binding affinity of the TMMP comprising the wild-type immunomodulatory polypeptide variant to the cognate co-immunomodulatory polypeptide is in the range of 1.5:1 to 10 when measured by biolayer interferometry. 6 : 1, wherein the control comprises a wild-type immunomodulatory polypeptide; and wherein the variant immunomodulatory polypeptide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acid substitutions compared to the amino acid sequence of the corresponding wild-type immunomodulatory polypeptide. In some cases, the peptide epitope present in the TMMP of the present disclosure is the WT-1 peptide.

[0067] The present disclosure provides a TMMP comprising: a) a first polypeptide comprising, in order from N-terminus to C-terminus: i) an epitope; ii) a first MHC polypeptide; and b) a second polypeptide comprising, in order from N-terminus to C-terminus: i) a second MHC polypeptide; and ii) an optional Ig Fc polypeptide or a non-Ig scaffold. The TMMP of the present disclosure comprises one or more immunomodulatory polypeptides, wherein at least one of the one or more immunomodulatory polypeptides is: A) at the C-terminus of the first polypeptide; B) at the N-terminus of the second polypeptide; C) at the C-terminus of the second polypeptide; or D) at the C-terminus of the first polypeptide and at the N-terminus of the second polypeptide. At least one of the one or more immunomodulatory polypeptides is a variant immunomodulatory polypeptide that exhibits reduced affinity for a cognate co-immunomodulatory polypeptide compared to the affinity of a corresponding wild-type immunomodulatory polypeptide for the cognate co-immunomodulatory polypeptide. The epitope present in the TMMPs of the present disclosure binds to a T cell receptor (TCR) on a T cell with an affinity of at least 100 μM (e.g., at least 10 μM, at least 1 μM, at least 100 nM, at least 10 nM, or at least 1 nM). The TMMPs of the present disclosure bind to a first T cell with an affinity that is at least 25% greater than the affinity with which the TMMP binds to a second T cell, wherein the first T cell expresses a cognate co-immunomodulatory polypeptide on its surface and a TCR that binds to the epitope with an affinity of at least 100 μM, and wherein the second T cell expresses a cognate co-immunomodulatory polypeptide on its surface but does not express a TCR on its surface that binds to the epitope with an affinity of at least 100 μM (e.g., at least 10 μM, at least 1 μM, at least 100 nM, at least 10 nM, or at least 1 nM).

[0068] In some cases, an epitope present in a TMMP of the present disclosure binds to a TCR on a T cell with an affinity of about 10 -4 M is about 5x10 -4 M, about 5x10 -4 M to about 10 -5 M, about 10 -5 M to 5x10 -5 M, about 5x10 -5 M to 10 -6 M, about 10 -6 M is about 5x10 -6 M, about 5x10 -6 M to about 10 -7 M, about 10 -7 M is about 5x10 -7 M, about 5x10 -7 M to about 10 -8 M, or about 10 -8 M to about 10 - 9In other words, in some cases, the epitope present in the TMMP of the present disclosure binds to the TCR on a T cell with an affinity of about 1 nM to about 5 nM, about 5 nM to about 10 nM, about 10 nM to about 50 nM, about 50 nM to about 100 nM, about 0.1 μM to about 0.5 μM, about 0.5 μM to about 1 μM, about 1 μM to about 5 μM, about 5 μM to about 10 μM, about 10 μM to about 25 μM, about 25 μM to about 50 μM, about 50 μM to about 75 μM, about 75 μM to about 100 μM.

[0069] The immunomodulatory polypeptides present in the TMMPs of the present disclosure bind to their cognate co-immunomodulatory polypeptides with an affinity that is at least 10% less, at least 15% less, at least 20% less, at least 25% less, at least 30% less, at least 35% less, at least 40% less, at least 45% less, at least 50% less, at least 55% less, at least 60% less, at least 65% less, at least 70% less, at least 75% less, at least 80% less, at least 85% less, at least 90% less, at least 95% less, or greater than 95% less than the affinity of the corresponding wild-type immunomodulatory polypeptide for the cognate co-immunomodulatory polypeptide.

[0070] In some cases, the variant immunomodulatory polypeptides present in the TMMPs of the present disclosure have a binding affinity for a cognate co-immunomodulatory polypeptide of 1 nM to 100 nM or 100 nM to 100 μM. For example, in some cases, the variant immunomodulatory polypeptides present in the TMMPs of the present disclosure have a binding affinity for a cognate co-immunomodulatory polypeptide of about 100 nM to 150 nM, about 150 nM to about 200 nM, about 200 nM to about 250 nM, about 250 nM to about 300 nM, about 300 nM to about 350 nM, about 350 nM to about 400 nM, about 400 nM to about 500 nM, about 500 nM to about 100 μM. In some cases, the variant immunomodulatory polypeptides present in the TMMPs of the present invention have a binding affinity for a cognate co-immunomodulatory polypeptide of about 1 nM to about 5 nM, about 5 nM to about 10 nM, about 10 nM to about 50 nM, about 15 nM to about 20 nM, about 20 nM to about 25 nM, about 25 nM to about 50 nM, about 50 nM to about 75 nM, or about 75 nM to about 100 nM.

[0071] The combination of the reduced affinity of the immunomodulatory polypeptide for its cognate co-immunomodulatory polypeptide and the affinity of the epitope for the TCR provides for improved selectivity of the TMMPs of the present disclosure. For example, the TMMPs of the present disclosure selectively bind to a first T cell that exhibits: i) a TCR specific for an epitope present in the TMMP; and ii) a co-immunomodulatory polypeptide that binds to an immunomodulatory polypeptide present in the TMMP, compared to binding to a second T cell that exhibits: i) a TCR specific for an epitope other than an epitope present in the TMMP; and ii) a co-immunomodulatory polypeptide that binds to an immunomodulatory polypeptide present in the TMMP. For example, a TMMP of the present disclosure binds to a first T cell with an affinity that is 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, at least 15-fold, at least 20-fold, at least 25-fold, at least 50-fold, at least 100-fold, or greater than 100-fold greater than its affinity for binding to a second T cell.

[0072] In some cases, the TMMPs of the present disclosure induce epitope-specific T cell responses and epitope-nonspecific T cell responses when administered to an individual in need thereof. In other words, in some cases, the TMMPs of the present disclosure induce epitope-specific T cell responses when administered to an individual in need thereof by modulating the activity of a first T cell, the first T cell presenting: i) a TCR specific for an epitope present in the TMMP; ii) a co-immunomodulatory polypeptide that binds to an immunomodulatory polypeptide present in the TMMP; and inducing an epitope-nonspecific T cell response by modulating the activity of a second T cell, the second T cell presenting: i) a TCR specific for an epitope other than an epitope present in the TMMP; and ii) a co-immunomodulatory polypeptide that binds to an immunomodulatory polypeptide present in the TMMP. The ratio of epitope-specific T cell responses to epitope-nonspecific T cell responses is at least 2: 1, at least 5: 1, at least 10: 1, at least 15: 1, at least 20: 1, at least 25: 1, at least 50: 1, or at least 100: 1. The ratio of epitope-specific T cell responses to epitope-nonspecific T cell responses is about 2:1 to about 5:1, about 5:1 to about 10:1, about 10:1 to about 15:1, about 15:1 to about 20:1, about 20:1 to about 25:1, about 25:1 to about 50:1, or about 50:1 to about 100:1, or greater than 100: 1. "Regulating the activity of T cells" may include one or more of the following: i) activating cytotoxic (e.g., CD8 + ) T cells; ii) induce cytotoxicity (e.g. CD8 +) T cell cytotoxic activity; iii) induction of cytotoxicity (e.g. CD8 + ) the production and release of cytotoxins (e.g., perforin; granzyme; granulolytic protein) by T cells; iv) the inhibition of the activity of autoreactive T cells; etc.

[0073] The combination of the reduced affinity of an immunomodulatory polypeptide for its cognate co-immunomodulatory polypeptide and the affinity of the epitope for the TCR provides for improved selectivity of the TMMPs of the present disclosure. Thus, for example, a TMMP of the present disclosure binds to a first T cell with higher affinity than it binds to a second T cell, the first T cell exhibiting: i) a TCR specific for an epitope present in a TMMP; and ii) a co-immunomodulatory polypeptide that binds to an immunomodulatory polypeptide present in a TMMP, the second T cell exhibiting: i) a TCR specific for an epitope other than an epitope present in a TMMP; and ii) a co-immunomodulatory polypeptide that binds to an immunomodulatory polypeptide present in a TMMP.

[0074] The binding affinity between an immunomodulatory polypeptide and its cognate co-immunomodulatory polypeptide can be determined by biolayer interferometry (BLI) using purified immunomodulatory polypeptides and purified cognate co-immunomodulatory polypeptides. The binding affinity between TMMP and its cognate co-immunomodulatory polypeptide can be determined by BLI using purified TMMP and cognate co-immunomodulatory polypeptides. The BLI method is well known to those skilled in the art. See, for example, Lad et al. (2015) J. Biomol. Screen. 20 (4): 498-507; and Shah and Duncan (2014) J. Vis. Exp. 18: e51383.

[0075] BLI assays can be performed using an Octet RED 96 (Pal Forte Bio) instrument or a similar instrument as follows. A TMMP (e.g., a TMMP of the present disclosure; a control TMMP (wherein the control TMMP comprises a wild-type immunomodulatory polypeptide)) is immobilized on an insoluble support ("biosensor"). The immobilized TMMP is a "target". Immobilization can be achieved by immobilizing a capture antibody to an insoluble support, wherein the capture antibody immobilizes the TMMP. For example, immobilization can be achieved by immobilizing an anti-Fc (e.g., anti-human IgG Fc) antibody to an insoluble support, wherein the immobilized anti-Fc antibody binds to and immobilizes the TMMP (wherein the TMMP comprises an IgFc polypeptide). The co-immunomodulatory polypeptide is applied to the immobilized TMMP at several different concentrations, and the response of the instrument is recorded. The assay is performed in a liquid medium comprising 25 mM HEPES pH 6.8, 5% poly (ethylene glycol) 6000, 50 mM KCl, 0.1% bovine serum albumin, and 0.02% Tween 20 non-ionic detergent. Binding of the co-immunomodulatory polypeptide to the immobilized TMMP was performed at 30°C. As a positive control for binding affinity, an anti-MHC class I monoclonal antibody can be used. For example, the anti-HLA class I monoclonal antibody W6 / 32 (American Type Culture Collection No. HB-95; Parham et al. (1979) J. Immunol. 123:342) can be used, which has a K of 7 nM. D . A standard curve can be generated using serial dilutions of anti-MHC class I monoclonal antibodies. The co-immunomodulatory polypeptide or anti-MHC class I mAb is the "analyte". BLI analyzes the interference pattern of white light reflected from two surfaces: i) an immobilized polypeptide ("target"); and ii) an internal reference layer. Changes in the number of molecules ("analytes"; e.g., co-immunomodulatory polypeptide; anti-HLA antibodies) bound to the biosensor tip cause a shift in the interference pattern; this shift in the interference pattern can be measured in real time. Two kinetic terms that describe the affinity of the target / analyte interaction are the association constant (k a ) and dissociation constant (k d The ratio of these two terms (k d / a ) produces an affinity constant K D .

[0076] The BLI assay is performed in a multiwell plate. To operate the assay, define the plate layout, define the assay steps, and assign the biosensor in the Octet Data Acquisition software. Hydrate the biosensor assembly. Equilibrate the hydrated biosensor assembly and assay plate on the Octet instrument for 10 minutes. Once the data is collected, the collected data is loaded into the Octet Data Analysis software. In the processing window, the data is processed by specifying the reference subtraction, y-axis alignment, inter-step correction, and Savitzky-Golay filtering methods. In the analysis window, the data is analyzed by specifying the analysis (association and dissociation), selecting the curve fitting model (1:1), the fitting method (global), and the target window (in seconds). Evaluate the quality of the fit. If within a 3-fold range, the K of each data trace (analyte concentration) is calculated. D The values ​​are averaged. D The error should be within about one order of magnitude of the affinity constant value; R 2 The value should be above 0.95. See, for example, Abdiche et al. (2008) J. Anal. Biochem. 377:209.

[0077] Unless otherwise indicated herein, affinity of a TMMP of the present disclosure for a cognate co-immunomodulatory polypeptide or affinity of a control TMMP (wherein the control TMMP comprises a wild-type immunomodulatory polypeptide) for a cognate co-immunomodulatory polypeptide is determined using BLI as described above.

[0078] In some cases, the ratio of the binding affinity of i) a control TMMP (wherein the control comprises a wild-type immunomodulatory polypeptide) to the cognate co-immunomodulatory polypeptide to ii) the binding affinity of a TMMP comprising a wild-type immunomodulatory polypeptide variant of the present disclosure to the cognate co-immunomodulatory polypeptide when measured by BLI (as described above) is at least 1.5:1, at least 2:1, at least 5:1, at least 10:1, at least 15:1, at least 20:1, at least 25:1, at least 50:1, at least 100:1, at least 500:1, at least 100:1, 2 :1. At least 5x10 2 :1. At least 10 3 :1. At least 5x10 3 :1. At least 10 4 :1. At least 10 5 :1, or at least 10 6 In some cases, the ratio of the binding affinity of i) a control TMMP (wherein the control comprises a wild-type immunomodulatory polypeptide) to the cognate co-immunomodulatory polypeptide to the binding affinity of ii) a TMMP comprising a wild-type immunomodulatory polypeptide variant of the present disclosure to the cognate co-immunomodulatory polypeptide, when measured by BLI, ranges from 1.5:1 to 10.6 :1, for example, 1.5:1 to 10:1, 10:1 to 50:1, 50:1 to 10 2 :1, 10 2 :1 to 10 3 :1, 10 3 :1 to 10 4 :1, 10 4 :1 to 10 5 :1, or 10 5 :1 to 10 6 :1.

[0079] As an example, when a control TMMP comprises a wild-type IL-2 polypeptide, and a TMMP of the present disclosure comprises a variant IL-2 polypeptide (comprising 1 to 10 amino acid substitutions relative to the amino acid sequence of the wild-type IL-2 polypeptide) as an immunomodulatory polypeptide, the ratio of i) the binding affinity of the control TMMP to the IL-2 receptor (i.e., a cognate co-immunomodulatory polypeptide) to ii) the binding affinity of the TMMP of the present disclosure to the IL-2 receptor, when measured by BLI, is at least 1.5:1, at least 2:1, at least 5:1, at least 10:1, at least 15:1, at least 20:1, at least 25:1, at least 50:1, at least 100:1, at least 500:1, at least 10 2 :1. At least 5x10 2 :1. At least 10 3 :1. At least 5x10 3 :1. At least 10 4 :1. At least 10 5 :1, or at least 10 6 In some cases, when the control TMMP comprises a wild-type IL-2 polypeptide and the TMMP of the present disclosure comprises a variant IL-2 polypeptide (comprising 1 to 10 amino acid substitutions relative to the amino acid sequence of the wild-type IL-2 polypeptide) as the immunomodulatory polypeptide, the ratio of i) the binding affinity of the control TMMP to the IL-2 receptor (i.e., a cognate co-immunomodulatory polypeptide) to ii) the binding affinity of the TMMP of the present disclosure to the IL-2 receptor, when measured by BLI, ranges from 1.5:1 to 10:1. 6 :1, for example, 1.5:1 to 10:1, 10:1 to 50:1, 50:1 to 10 2 :1, 10 2 :1 to 10 3 :1, 10 3 :1 to 10 4 :1, 10 4 :1 to 10 5 :1, or 10 5 :1 to 10 6 :1.

[0080] As another example, when the control TMMP comprises a wild-type PD-L1 polypeptide, and the TMMP of the present disclosure comprises a variant PD-L1 polypeptide (comprising 1 to 10 amino acid substitutions relative to the amino acid sequence of the wild-type PD-L1 polypeptide) as the immunomodulatory polypeptide, the ratio of i) the binding affinity of the control TMMP to the PD-1 polypeptide (i.e., a cognate co-immunomodulatory polypeptide) to ii) the binding affinity of the TMMP of the present disclosure to the PD-1 polypeptide, when measured by BLI, is at least 1.5:1, at least 2:1, at least 5:1, at least 10:1, at least 15:1, at least 20:1, at least 25:1, at least 50:1, at least 100:1, at least 500:1, at least 100:1, 2 :1. At least 5x10 2 :1. At least 10 3 :1. At least 5x10 3 :1. At least 10 4 :1. At least 10 5 :1, or at least 10 6 :1.

[0081] As another example, when the control TMMP comprises a wild-type CD80 polypeptide, and when the TMMP of the present disclosure comprises a variant CD80 polypeptide (comprising 1 to 10 amino acid substitutions relative to the amino acid sequence of the wild-type CD80 polypeptide) as the immunomodulatory polypeptide, the ratio of i) the binding affinity of the control TMMP to the CTLA4 polypeptide (i.e., a cognate co-immunomodulatory polypeptide) to ii) the binding affinity of the TMMP of the present disclosure to the CTLA4 polypeptide, when measured by BLI, is at least 1.5:1, at least 2:1, at least 5:1, at least 10:1, at least 15:1, at least 20:1, at least 25:1, at least 50:1, at least 100:1, at least 500:1, at least 10 2 :1. At least 5x10 2 :1. At least 10 3 :1. At least 5x10 3 :1. At least 10 4 :1. At least 10 5 :1, or at least 10 6 :1.

[0082] As another example, when the control TMMP comprises a wild-type CD80 polypeptide, and when the TMMP of the present disclosure comprises a variant CD80 polypeptide (comprising 1 to 10 amino acid substitutions relative to the amino acid sequence of the wild-type CD80 polypeptide) as the immunomodulatory polypeptide, the ratio of i) the binding affinity of the control TMMP to the CD28 polypeptide (i.e., a cognate co-immunomodulatory polypeptide) to ii) the binding affinity of the TMMP of the present disclosure to the CD28 polypeptide, when measured by BLI, is at least 1.5:1, at least 2:1, at least 5:1, at least 10:1, at least 15:1, at least 20:1, at least 25:1, at least 50:1, at least 100:1, at least 500:1, at least 10 2 :1. At least 5x10 2 :1. At least 10 3 :1. At least 5x10 3 :1. At least 10 4 :1. At least 10 5 :1, or at least 10 6 :1.

[0083] As another example, when the control TMMP comprises a wild-type 4-1BBL polypeptide, and the TMMP of the present disclosure comprises a variant 4-1BBL polypeptide (comprising 1 to 10 amino acid substitutions relative to the amino acid sequence of the wild-type 4-1BBL polypeptide) as an immunomodulatory polypeptide, the ratio of i) the binding affinity of the control TMMP to the 4-1BB polypeptide (i.e., a cognate co-immunomodulatory polypeptide) to ii) the binding affinity of the TMMP of the present disclosure to the 4-1BB polypeptide, when measured by BLI, is at least 1.5:1, at least 2:1, at least 5:1, at least 10:1, at least 15:1, at least 20:1, at least 25:1, at least 50:1, at least 100:1, at least 500:1, at least 10 2 :1. At least 5x10 2 :1. At least 10 3 :1. At least 5x10 3 :1. At least 10 4 :1. At least 10 5 :1, or at least 10 6 :1.

[0084] As another example, when the control TMMP comprises a wild-type CD86 polypeptide, and the TMMP of the present disclosure comprises a variant CD86 polypeptide (comprising 1 to 10 amino acid substitutions relative to the amino acid sequence of the wild-type CD86 polypeptide) as the immunomodulatory polypeptide, the ratio of i) the binding affinity of the control TMMP to the CD28 polypeptide (i.e., a cognate co-immunomodulatory polypeptide) to ii) the binding affinity of the TMMP of the present disclosure to the CD28 polypeptide, when measured by BLI, is at least 1.5:1, at least 2:1, at least 5:1, at least 10:1, at least 15:1, at least 20:1, at least 25:1, at least 50:1, at least 100:1, at least 500:1, at least 10 2 :1. At least 5x10 2 :1. At least 10 3 :1. At least 5x10 3 :1. At least 10 4 :1. At least 10 5 :1 or at least 10 6 :1.

[0085] The binding affinity of the TMMPs of the present invention to target T cells can be measured in the following manner: A) contacting the TMMPs of the present invention with target T cells expressing on their surface: i) a cognate co-immunomodulatory polypeptide that binds to a parent wild-type immunomodulatory polypeptide; and ii) a T cell receptor that binds to an epitope, wherein the TMMP comprises an epitope tag such that the TMMP binds to the target T cell; B) contacting the target T cell-bound TMMP with a fluorescently labeled binding agent (e.g., a fluorescently labeled antibody) that binds to the epitope tag, thereby generating a TMMP / target T cell / binding agent complex; C) measuring the mean fluorescence intensity (MFI) of the TMMP / target T cell / binding agent complex using flow cytometry. The epitope tag can be, for example, a FLAG tag, a lectin tag, a c-myc tag, a poly(histidine) tag, or the like. The MFI measured over a concentration range of the TMMP library members provides a measure of affinity. The MFI measured over a concentration range of the TMMP library members provides a half-maximal effective concentration (EC) of the TMMP. 50 ). In some cases, the TMMPs disclosed herein are effective for the EC of target T cells. 50 in the nM range; and the EC values ​​of TMMP for control T cells (wherein the control T cells express on their surface: i) a cognate co-immunomodulatory polypeptide that binds the parent wild-type immunomodulatory polypeptide; and ii) a T cell receptor that does not bind to an epitope present in TMMP) 50 In some cases, the TMMPs disclosed herein have an EC 50 Same as TMMP for target T cell EC 50The ratio is at least 1.5:1, at least 2:1, at least 5:1, at least 10:1, at least 15:1, at least 20:1, at least 25:1, at least 50:1, at least 100:1, at least 500:1, at least 10 2 :1. At least 5x10 2 :1. At least 10 3 :1. At least 5x10 3 :1. At least 10 4 :1. At least 10 5 :1, or at least 10 6 : 1. The TMMP disclosed herein has an effect on the EC of control T cells 50 Same as TMMP for target T cell EC 50 The ratio is an expression of the selectivity for TMMP.

[0086] In some cases, a TMMP of the present disclosure exhibits selective binding to target T cells, when measured as described in the preceding paragraph, compared to binding of the TMMP library member to control T cells comprising: i) a cognate co-immunomodulatory polypeptide that binds to the parent wild-type immunomodulatory polypeptide; and ii) a T cell receptor that binds to an epitope other than an epitope present in the TMMP library member.

[0087] Dimerized TMMP

[0088] The TMMPs of the present disclosure can be dimerized; that is, the present disclosure provides multimeric polypeptides comprising dimers of the TMMPs of the present disclosure. Therefore, the present disclosure provides a TMMP comprising: A) a first heterodimer comprising: a) a first polypeptide comprising: i) a peptide epitope; and ii) a first major histocompatibility complex (MHC) polypeptide; and b) a second polypeptide comprising: i) a second MHC polypeptide, wherein the first heterodimer comprises one or more immunomodulatory polypeptides; and B) a second heterodimer comprising: a) a first polypeptide comprising: i) a peptide epitope; and ii) a first MHC polypeptide; and b) a second polypeptide comprising: i) a second MHC polypeptide, wherein the second heterodimer comprises one or more immunomodulatory polypeptides, and wherein the first heterodimer and the second heterodimer are covalently linked to each other. In some cases, the amino acid sequences of the two TMMPs are identical. In some cases, the first heterodimer and the second heterodimer are covalently linked to each other via the C-terminal region of the second polypeptide of the first heterodimer and the C-terminal region of the second polypeptide of the second heterodimer. In some cases, the first heterodimer and the second heterodimer are covalently linked to each other via the C-terminal amino acid of the second polypeptide of the first heterodimer and the C-terminal region of the second polypeptide of the second heterodimer; For example, in some cases, the C-terminal amino acid of the second polypeptide of the first heterodimer and the C-terminal region of the second polypeptide of the second heterodimer are directly or via a joint. The joint can be a peptide linker. The length that a peptide linker can have is 1 amino acid to 200 amino acids (for example, 1 amino acid (aa) to 5aa, 5aa to 10aa, 10aa to 25aa, 25aa to 50aa, 50aa to 100aa, 100aa to 150aa or 150aa to 200aa). In some cases, the peptide epitope of the first heterodimer and the peptide epitope of the second heterodimer comprise the same amino acid sequence. In some cases, the first MHC polypeptide of the first heterodimer and the second heterodimer is a class I MHC β2-microglobulin, and wherein the second MHC polypeptide of the first heterodimer and the second heterodimer is a class I MHC heavy chain. In some cases, the immunomodulatory polypeptide of the first heterodimer and the immunomodulatory polypeptide of the second heterodimer comprise the same amino acid sequence. In some cases, the immunomodulatory polypeptide of the first heterodimer and the immunomodulatory polypeptide of the second heterodimer are variant immunomodulatory polypeptides comprising 1 to 10 amino acid substitutions compared to the corresponding parent wild-type immunomodulatory polypeptide, and wherein these 1 to 10 amino acid substitutions result in a reduced affinity binding of the variant immunomodulatory polypeptide to the cognate co-immunomodulatory polypeptide.In some cases, the immunomodulatory polypeptide of the first heterodimer and the immunomodulatory polypeptide of the second heterodimer are each independently selected from the group consisting of: IL-2, 4-1BBL, PD-L1, CD80, CD86, ICOS-L, OX-40L, FasL, JAG1 (CD339), TGFβ, CD70 and ICAM. Examples of suitable MHC polypeptides, immunomodulatory polypeptides and peptide epitopes are described below.

[0089] MHC peptides

[0090] As described above, the TMMPs of the present disclosure include MHC polypeptides. For the purposes of the present disclosure, the term "major histocompatibility complex (MHC) polypeptide" is intended to include MHC polypeptides of different species, including human MHC (also known as human leukocyte antigen (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, ungulates (e.g., horses, cattle, sheep, goats, etc.), etc. The term "MHC polypeptide" is intended to include class I MHC polypeptides (e.g., beta-2 microglobulin and class I MHC heavy chains).

[0091] In some cases, the first MHC polypeptide is a class I MHC β2M (β2M) polypeptide, and the second MHC polypeptide is a class I MHC heavy chain (H chain) ("MHC-H"). In other cases, the first MHC polypeptide is a class I MHC heavy chain polypeptide; and the second MHC polypeptide is a β2M polypeptide. In some cases, both the β2M and MHC-H chains are of human origin; that is, the MHC-H chain is an HLA heavy chain or a variant thereof. Unless otherwise expressly stated, the TMMPs of the present disclosure do not include a membrane anchoring domain (transmembrane domain) of a class I MHC heavy chain or a portion of a class I MHC heavy chain sufficient to anchor the resulting TMMP to a cell expressing it (e.g., a eukaryotic cell, such as a mammalian cell). In some cases, the class I MHC heavy chain present in the TMMPs of the present disclosure does not include a signal peptide, a transmembrane domain, or an intracellular domain (cytoplasmic tail) associated with a native class I MHC heavy chain. Thus, for example, in some cases, the class I MHC heavy chain present in a TMMP of the present disclosure includes only the α1, α2, and α3 domains of the class I MHC heavy chain. In some cases, the class I MHC heavy chain present in a TMMP of the present disclosure has a length of about 270 amino acids (aa) to about 290 aa. In some cases, the class I MHC heavy chain present in a TMMP of the present disclosure has a length of 270 aa, 271 aa, 272 aa, 273 aa, 274 aa, 275 aa, 276 aa, 277 aa, 278 aa, 279 aa, 280 aa, 281 aa, 282 aa, 283 aa, 284 aa, 285 aa, 286 aa, 287 aa, 288 aa, 289 aa, or 290 aa.

[0092] In some cases, the MHC polypeptide of the TMMP is a human MHC polypeptide, wherein human MHC polypeptides are also referred to as "human leukocyte antigen" ("HLA") polypeptides. In some cases, the MHC polypeptide of the TMMP is a class I HLA polypeptide, for example, a β2-microglobulin polypeptide or a class I HLA heavy chain polypeptide. Class I HLA heavy chain polypeptides include HLA-A heavy chain polypeptides, HLA-B heavy chain polypeptides, HLA-C heavy chain polypeptides, HLA-E heavy chain polypeptides, HLA-F heavy chain polypeptides, and HLA-G heavy chain polypeptides.

[0093] MHC class I heavy chain

[0094] In some cases, the class I MHC heavy chain polypeptide present in the TMMP of the present disclosure 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 all or a portion of the amino acid sequence (e.g., 50, 75, 100, 150, 200, or 250 contiguous amino acids) of any of the human HLA heavy chain polypeptides depicted in Figures 7-13. In some cases, the class I MHC heavy chain has a length of 270aa, 271aa, 272aa, 273aa, 274aa, 275aa, 276aa, 277aa, 278aa, 279aa, 280aa, 281aa, 282aa, 283aa, 284aa, 285aa, 286aa, 287aa, 288aa, 289aa, or 290aa. In some cases, the class I MHC heavy chain polypeptide present in the TMMP of the present disclosure comprises 1-30, 1-5, 5-10, 10-15, 15-20, 20-25, or 25-30 amino acid insertions, deletions, and / or substitutions of any one of the amino acid sequences depicted in Figures 7-13 (except for those positions indicated as variable in the heavy chain consensus sequence). In some cases, the class I MHC heavy chain does not include a transmembrane domain or a cytoplasmic domain. As an example, the class I MHC heavy chain polypeptide of the TMMP of the present disclosure may comprise Figure 7A 、 7B and amino acids 25-300 (lacking all or substantially all of the leader sequence, transmembrane sequence, and cytoplasmic sequence) or amino acids 25-365 (lacking the leader sequence) of a human HLA-A heavy chain polypeptide depicted in any of 7C and 7D.

[0095] Figure 7A 、 7B The amino acid sequence of the human leukocyte antigen (HLA) class I heavy chain polypeptide is provided in Figures 1 and 7C. Signal sequence amino acids 1-24 are bolded and underlined. Figure 7A Entry: 3A.1 is HLA-A heavy chain (HLA-A*01:01:01:01 or A*0101) (NCBI accession NP_001229687.1) SEQ ID NO: 23; entry 3A.2 is from HLA-A*1101 SEQ ID NO: 24; entry 3A.3 is from HLA-A*2402 SEQ ID NO: 25 and entry 3A.4 is from HLA-A*3303 SEQ ID NO: 26. Figure 7BThe sequence HLA-B*07:02:01 (HLA-B*0702) is provided as NCBI GenBank accession NP_005505.2 (see also GenBank accession AUV50118.1.). Figure 7C The sequence HLA-C*0701 (GenBank accession NP_001229971.1) (HLA-C*07:01:01:01 or HLA-Cw*070101, HLA-Cw*07 see GenBank accession CAO78194.1) is provided.

[0096] Figure 8 An alignment of eleven mature class I MHC heavy chain amino acid sequences without a leader sequence or transmembrane or intracellular domain is provided. The aligned sequences are human HLA-A, HLA-B, and HLA-C, mouse H2K protein sequences, three HLA-A variants (var.1, var.2C, and var.2CP), and three human HLA-A variants (HLA-A*1101; HLA-A*2402; and HLA-A*3303). Positions (84 and 139 of the mature protein) where cysteine ​​residues can be introduced (e.g., by substitution) to form disulfide bonds to stabilize the MHC H chain-β2M complex are indicated in the alignment. Position 236 (the position of the mature polypeptide) is also shown, which can be substituted with a cysteine ​​residue to form an interchain disulfide bond with β2M (e.g., at aa12). Arrows appear above each of those positions and the residues are bolded. The seventh HLA-A sequence shown in the alignment (var. 2c) shows the sequence of variant 2 with C residues substituted at positions 84, 139, and 236. The framed flanking residues 84, 139, and 236 show groups of five amino acids on either side of those six groups of five residues, designated aac1 (for "amino acid cluster 1"), aac2 (for "amino acid cluster 2"), aac3 (for "amino acid cluster 3"), aac4 (for "amino acid cluster 4"), aac5 (for "amino acid cluster 5"), and aac6 (for "amino acid cluster 6"), which can be substituted with 1 to 5 amino acids independently selected from: (i) any naturally occurring amino acid or (ii) any naturally occurring amino acid other than proline or glycine.

[0097] about Figure 8In some cases: i) aac1 (amino acid cluster 1) may be the amino acid sequence of GTLRG (SEQ ID NO: 287), or a sequence in which one or two amino acids are deleted or substituted with other naturally occurring amino acids (e.g., L is replaced by I, V, A, or F); ii) aac2 (amino acid cluster 2) may be the amino acid sequence of YNQSE (SEQ ID NO: 288), or a sequence in which one or two amino acids are deleted or substituted with other naturally occurring amino acids (e.g., N is replaced by Q, Q is replaced by N, and / or E is replaced by D); iii) aac3 (amino acid cluster 3) may be the amino acid sequence of TAADM (SEQ ID NO: 289), or a sequence in which one or two amino acids are deleted or substituted with other naturally occurring amino acids (e.g., T is replaced by S, A is replaced by G, D is replaced by E, and / or M is replaced by L, V, or I); iv) aac4 (amino acid cluster 4) may be the amino acid sequence of AQTTK (SEQ ID NO: 290). NO: 290) or a sequence in which one or two amino acids are deleted or substituted with other naturally occurring amino acids (e.g., A is replaced by G, Q is replaced by N, or T is replaced by S, and / or K is replaced by R or Q); v) aac5 (amino acid cluster 5) may be the amino acid sequence VETRP (SEQ ID NO: 291) or a sequence in which one or two amino acids are deleted or substituted with other naturally occurring amino acids (e.g., V is replaced by I or L, E is replaced by D, T is replaced by S, and / or R is replaced by K); and / or vi) aac6 (amino acid cluster 6) may be the amino acid sequence GDGTF (SEQ ID NO: 292) or a sequence in which one or two amino acids are deleted or substituted with other naturally occurring amino acids (e.g., D is replaced by E, T is replaced by S, or F is replaced by L, W or Y).

[0098] Figures 9-11 provide an alignment of mature HLA class I heavy chain amino acid sequences (without leader sequence or transmembrane or intracellular domains). Figure 9A The amino acid sequences aligned in the table are for the following HLA-A class I heavy chains: A*0101, A*0201, A*0301, A*1101, A*2301, A*2402, A*2407, A*3303, and A*3401. Figure 10A The amino acid sequences aligned in the table are for the following HLA-B class I heavy chains: B*0702, B*0801, B*1502, B*3802, B*4001, B*4601, and B*5301. Figure 11AThe amino acid sequences aligned in the table are for the following HLA-C class I heavy chains: C*0102, C*0303, C*0304, C*0401, C*0602, C*0701, C*0801, and C*1502. Positions (84 and 139 of the mature protein) where cysteine ​​residues can be introduced (e.g., by substitution) to form disulfide bonds to stabilize the HLA H chain-β2M complex are indicated in the alignment. Position 236 (the position of the mature polypeptide) where a cysteine ​​residue can be substituted to form an interchain disulfide bond with β2M (e.g., at aa 12) is also shown. Box flanking residues 84, 139, and 236 show groups of five amino acids on either side of those six groups of five residues, designated aac1 (for "amino acid cluster 1"), aac2 (for "amino acid cluster 2"), aac3 (for "amino acid cluster 3"), aac4 (for "amino acid cluster 4"), aac5 (for "amino acid cluster 5"), and aac6 (for "amino acid cluster 6"), which residues can be independently selected from 1 to 5 amino acid substitutions of: (i) any naturally occurring amino acid or (ii) any naturally occurring amino acid other than proline or glycine.

[0099] Figure 9A 、 10A 11A and 11B provide alignments of the amino acid sequences of mature HLA-A, HLA-B, and HLA-C class I heavy chains, respectively. 11B provides the sequences of the extracellular portion of the mature protein (without the leader sequence or transmembrane domain or intracellular domain). Figure 8 Also shown are the positions of aa residues 84, 139, and 236 and their flanking residues (aac1 to aac6), which can be substituted with 1 to 5 amino acids independently selected from the following: (i) any naturally occurring amino acid or (ii) any naturally occurring amino acid other than proline or glycine. Figure 9B 、 10B and 11B respectively Figure 9A 、 10A The consensus amino acid sequence for the HLA-A, HLA-B, and HLA-C sequences provided in Figure 11A is shown. The consensus sequence shows variable amino acid positions as sequentially numbered "X" residues and the positions of amino acids 84, 139, and 236 are double underlined.

[0100] about Figure 9AIn some cases: i) aac1 (amino acid cluster 1) may be the amino acid sequence of GTLRG (SEQ ID NO: 287), or a sequence in which one or two amino acids are deleted or substituted with other naturally occurring amino acids (e.g., L is replaced by I, V, A, or F); ii) aac2 (amino acid cluster 2) may be the amino acid sequence of YNQSE (SEQ ID NO: 288), or a sequence in which one or two amino acids are deleted or substituted with other naturally occurring amino acids (e.g., N is replaced by Q, Q is replaced by N, and / or E is replaced by D); iii) aac3 (amino acid cluster 3) may be the amino acid sequence of TAADM (SEQ ID NO: 289), or a sequence in which one or two amino acids are deleted or substituted with other naturally occurring amino acids (e.g., T is replaced by S, A is replaced by G, D is replaced by E, and / or M is replaced by L, V, or I); iv) aac4 (amino acid cluster 4) may be the amino acid sequence of AQTTK (SEQ ID NO: 290). NO: 290) or a sequence in which one or two amino acids are deleted or substituted with other naturally occurring amino acids (e.g., A is replaced by G, Q is replaced by N, or T is replaced by S, and or K is replaced by R or Q); v) aac5 (amino acid cluster 5) may be the amino acid sequence VETRP (SEQ ID NO: 291) or a sequence in which one or two amino acids are deleted or substituted with other naturally occurring amino acids (e.g., V is replaced by I or L, E is replaced by D, T is replaced by S, and / or R is replaced by K); and / or vi) aac6 (amino acid cluster 6) may be the amino acid sequence GDGTF (SEQ ID NO: 292) or a sequence in which one or two amino acids are deleted or substituted with other naturally occurring amino acids (e.g., D is replaced by E, T is replaced by S, or F is replaced by L, W or Y).

[0101] about Figure 10AIn some cases: i) aac1 (amino acid cluster 1) may be the amino acid sequence of RNLRG (SEQ ID NO: 293), or a sequence in which one or two amino acids are deleted or substituted with other naturally occurring amino acids (e.g., N is replaced by T or I; and / or L is replaced by A; and / or the second R is replaced by L; and / or G is replaced by R); ii) aac2 (amino acid cluster 2) may be the amino acid sequence of YNQSE (SEQ ID NO: 288), or a sequence in which one or two amino acids are deleted or substituted with other naturally occurring amino acids (e.g., N is replaced by Q, Q is replaced by N, and / or E is replaced by D); iii) aac3 (amino acid cluster 3) may be the amino acid sequence of TAADT (SEQ ID NO: 294), or a sequence in which one or two amino acids are deleted or substituted with other naturally occurring amino acids (e.g., the first T is replaced by S; and / or A is replaced by G; and / or D is replaced by E; and / or the second T is replaced by S); iv) aac4 (amino acid cluster 4) may be the amino acid sequence of AQITQ (SEQ ID NO: 289). NO: 295) or a sequence in which one or two amino acids are deleted or substituted with other naturally occurring amino acids (e.g., A is replaced by G; and / or the first Q is replaced by N; and / or I is replaced by L or V; and / or T is replaced by S; and / or the second Q is replaced by N); v) aac5 (amino acid cluster 5) may be the amino acid sequence VETRP (SEQ ID NO: 291) or a sequence in which one or two amino acids are deleted or substituted with other naturally occurring amino acids (e.g., V is replaced by I or L, E is replaced by D, T is replaced by S, and / or R is replaced by K); and / or vi) aac6 (amino acid cluster 6) may be the amino acid sequence GDRTF (SEQ ID NO: 296) or a sequence in which one or two amino acids are deleted or substituted with other naturally occurring amino acids (e.g., D is replaced by E; and / or T is replaced by S; and / or R is replaced by K or H; and / or F is replaced by L, W or Y).

[0102] about Figure 11AIn some cases: i) aac1 (amino acid cluster 1) may be the amino acid sequence of RNLRG (SEQ ID NO: 293), or a sequence in which one or two amino acids are deleted or substituted with other naturally occurring amino acids (e.g., N is replaced by K; and / or L is replaced by A or I; and / or the second R is replaced by H; and / or G is replaced by T or S); ii) aac2 (amino acid cluster 2) may be the amino acid sequence of YNQSE (SEQ ID NO: 288), or a sequence in which one or two amino acids are deleted or substituted with other naturally occurring amino acids (e.g., N is replaced by Q, Q is replaced by N, and / or E is replaced by D); iii) aac3 (amino acid cluster 3) may be the amino acid sequence of TAADT (SEQ ID NO: 294), or a sequence in which one or two amino acids are deleted or substituted with other naturally occurring amino acids (e.g., the first T is replaced by S; and / or A is replaced by G; and / or D is replaced by E; and / or the second T is replaced by S); iv) aac4 (amino acid cluster 4) may be the amino acid sequence of AQITQ (SEQ ID NO: 289). NO: 295) or a sequence in which one or two amino acids are deleted or substituted with other naturally occurring amino acids (e.g., A is replaced by G; and / or the first Q is replaced by N; and / or I is replaced by L; and / or the second Q is replaced by N or K); v) aac5 (amino acid cluster 5) may be the amino acid sequence VETRP (SEQ ID NO: 291) or a sequence in which one or two amino acids are deleted or substituted with other naturally occurring amino acids (e.g., V is replaced by I or L, E is replaced by D, T is replaced by S, and / or R is replaced by K or H); and / or vi) aac6 (amino acid cluster 6) may be the amino acid sequence GDGTF (SEQ ID NO: 292) or a sequence in which one or two amino acids are deleted or substituted with other naturally occurring amino acids (e.g., D is replaced by E; and / or T is replaced by S; and / or F is replaced by L, W or Y).

[0103] HLA-A

[0104] In some cases, the TMMPs of the present disclosure comprise an HLA-A heavy chain polypeptide. HLA-A heavy chain peptide sequences or portions thereof that can be incorporated into the TMMPs of the present disclosure include, but are not limited to, the following alleles: A*0101, A*0201, A*0301, A*1101, A*2301, A*2402, A*2407, A*3303, and A*3401, which are not all or substantially all Figure 9A Any of those alleles may be included at positions 84, 139 and / or 236 (e.g. Figure 9A(as shown in the table above) selected from the group consisting of: tyrosine to alanine (Y84A) at position 84; tyrosine to cysteine ​​(Y84C) at position 84; alanine to cysteine ​​(A139C) at position 139; and alanine to cysteine ​​(A236C) at position 236. Alternatively, an HLA-A sequence (e.g., which may comprise 1-25, 1-5, 5-10, 10-15, 15-20, 20-25, or 25-30 amino acid insertions, deletions, and / or substitutions) having at least 75% (e.g., 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 all or a portion (e.g., 50, 75, 100, 150, 200, or 250 contiguous amino acids) of the sequence of those HLA-A alleles may also be used.

[0105] In some instances, a TMMP of the present disclosure comprises an HLA-A heavy chain polypeptide comprising the following HLA-A consensus amino acid sequence:

[0106] (SEQ ID NO:29), wherein X1 is F, Y, S or T; X2 is K or R; X3 is Q, G, E or R; X4 is N or E; X5 is R or G; X6 is N or K; X7 is M or V; X8 is H or Q; X9 is T or I; X10 is D or H; X11 is A, V or E; X12 is N or D; X13 is G or R; X14 is T or I; X15 is L or A; X16 is R or L; X17 is G or R; X18 is A or D; X19 is I, L or V; X20 is I, R or M; X21 is F or Y; X22 is S or P; X23 is W or G; X24 is R, H, or Q; X25 is D or Y; X26 is N or K; X27 is T or I; X28 is K or Q; X29 is R or H; X30 is A or T; X31 is A or V; X32 is H or R; X33 is R, L, Q, or W; X34 is V or A; X35 is D or E; X36 is R or T; X37 is D or E; X38 is W or G; X39 is P or A; X40 is P or A; X41 is V or I; X42 is S or G; X43 is A or S; X44 is Q or E; and X45 is P or L.

[0107] As an example, the class I MHC heavy chain polypeptide of a TMMP may comprise 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 human HLA-A heavy chain amino acid sequence: GSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDGETRKVKAHSQTHRVDLGTLRGYYNQSE AGSHTVQRMYGCDVGSDWRFLRGYHQYAYDGKDYIALKEDLRSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGTCVEWLRRYLENGKETLQRTDAPKTHMTHHAVSDHEATLRCWALSFYPAEITLTWQRDGEDQTQDTELVETRPAGDGTFQKWAAVVVPSGQEQRYTCHVQHEGLPKPLTLRWEP (SEQ ID NO: 297).

[0108] In some instances, an HLA-A heavy chain polypeptide suitable for inclusion in a TMMP of the present disclosure comprises the following amino acid sequence: GSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDGETRKVKAHSQTHRVDLGTLRGYYNQSEAGSHTVQRMYGCDVGSDWRFLRGYHQYAYDGKDYIALKEDLRSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGTCVEWLRRYLENGKETLQRTDAPKTHMTHHAVSDHEATLRCWALSFYPAEITLTWQRDGEDQTQDTELVETRPAGDGTFQKWAAVVVPSGQEQRYTCHVQHEGLPKPLTLRWEP (SEQ ID NO: 297). This HLA-A heavy chain polypeptide is also referred to as "HLA-A*0201" or simply "HLA-A02." In some cases, a C-terminal Pro is not included in the TMMPs of the present disclosure. For example, in some cases, an HLA-A02 polypeptide suitable for inclusion in a TMMP of the present disclosure comprises the following amino acid sequence: GSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDGETRKVKAHSQTHRVDLGTLRGYYNQSEAGSHTVQRMYGCDVGSDWRFLRGYHQYAYDGKDYIALKEDLRSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGTCVEWLRRYLENGKETLQRTDAPKTHMTHHAVSDHEATLRCWALSFYPAEITLTWQRDGEDQTQDTELVETRPAGDGTFQKWAAVVVPSGQEQRYTCHVQHEGLPKPLTLRWE (SEQ ID NO: 449).

[0109] HLA-A (Y84A; A236C)

[0110] In some cases, the class I MHC heavy chain polypeptide comprises a Y84A and A236C substitution. For example, in some cases, the class I MHC heavy chain polypeptide 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 human HLA-A heavy chain (Y84A; A236C) amino acid sequence: GSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDGETRKVKAHSQTHRVDLGTLRG A YNQSEAGSHTVQRMYGCDVGSDWRFLRGYHQYAYDGKDYIALKEDLRSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGTCVEWLRRYLENGKETLQRTDAPKTHMTHHAVSDHEATLRCWALSFYPAEITLTWQRDGEDQTQDTELVETRP C GDGTFQKWAAVVVPSGQEQRYTCHVQHEGLPKPLTLRWEP (SEQ ID NO: 298), wherein amino acid 84 is Ala and amino acid 236 is Cys. In some cases, Cys-236 forms an interchain disulfide bond with Cys-12 of the variant β2M polypeptide comprising an R12C substitution.

[0111] In some cases, an HLA-A heavy chain polypeptide suitable for inclusion in a TMMP of the present disclosure is an HLA-A02 (Y84A; A236C) polypeptide comprising the following amino acid sequence: GSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDGETRKVKAHSQTHRVDLGTLRG A YNQSEAGSHTVQRMYGCDVGSDWRFLRGYHQYAYDGKDYIALKEDLRSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGTCVEWLRRYLENGKETLQRTDAPKTHMTHHAVSDHEATLRCWALSFYPAEITLTWQRDGEDQTQDTELVETRP C GDGTFQKWAAVVVPSGQEQRYTCHVQHEGLPKPLTLRWEP (SEQ ID NO: 298).

[0112] In some cases, an HLA-A heavy chain polypeptide suitable for inclusion in a TMMP of the present disclosure is an HLA-A02 (Y84A; A236C) polypeptide comprising the following amino acid sequence: GSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDGETRKVKAHSQTHRVDLGTLRG A YNQSEAGSHTVQRMYGCDVGSDWRFLRGYHQYAYDGKDYIALKEDLRSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGTCVEWLRRYLENGKETLQRTDAPKTHMTHHAVSDHEATLRCWALSFYPAEITLTWQRDGEDQTQDTELVETRP C GDGTFQKWAAVVVPSGQEQRYTCHVQHEGLPKPLTLRWE (SEQ ID NO: 450).

[0113] HLA-A(Y84C;A139C)

[0114] In some cases, the class I MHC heavy chain polypeptide comprises a Y84C and an A139C substitution. For example, in some cases, the class I MHC heavy chain polypeptide 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 human HLA-A heavy chain (Y84C; A139C) amino acid sequence: GSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDGETRKVKAHSQTHRVDLGTLRG C YNQSEAGSHTVQRMYGCDVGSDWRFLRGYHQYAYDGKDYIALKEDLRSWTAADM C AQTTKHKWEAAHVAEQLRAYLEGTCVEWLRRYLENGKETLQRTDAPKTHMTHHAVSDHEATLRCWALSFYPAEITLTWQRDGEDQTQDTELVETRPAGDGTFQKWAAVVVPSGQEQRYTCHVQHEGLPKPLTLRWEP (SEQ ID NO: 299), wherein amino acid 84 is Cys and amino acid 139 is Cys. In some cases, Cys-84 forms an intrachain disulfide bond with Cys-139.

[0115] HLA-A11 (HLA-A*1101)

[0116] As a non-limiting example, the class I MHC heavy chain polypeptide of TMMP may comprise 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 human HLA-A11 heavy chain amino acid sequence: GSHSMRYFYTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDQETRNVKAQSQTDRVDLGTLRGYY NQSEDGSHTIQIMYGCDVGPDGRFLRGYRQDAYDGKDYIALNEDLRSWTAADMAAQITKRKWEAAHAAEQQRAYLEGTCVEWLRRYLENGKETLQRTDPPKTHMTHHPISDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLRWE (SEQ ID NO: 300). Such class I MHC heavy chains may be prominent in Asian populations, including populations of individuals of Asian descent.

[0117] HLA-A11(Y84A;A236C)

[0118] As a non-limiting example, in some cases, the class I MHC heavy chain polypeptide is an HLA-A11 allele comprising Y84A and A236C substitutions. For example, in some cases, the class I MHC heavy chain polypeptide 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 human HLA-A A11 heavy chain (Y84A; A236C) amino acid sequence: GSHSMRYFYTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDQETRNVKAQSQTDRVDLGTLRG A YNQSEDGSHTIQIMYGCDVGPDGRFLRGYRQDAYDGKDYIALNEDLRSWTAADMAAQITKRKWEAAHAAEQQRAYLEGTCVEWLRRYLENGKETLQRTDPPKTHMTHHPISDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRP CGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLRWE (SEQ ID NO: 301), wherein amino acid 84 is Ala and amino acid 236 is Cys. In some cases, Cys-236 forms an interchain disulfide bond with Cys-12 of the variant β2M polypeptide comprising an R12C substitution.

[0119] HLA-A24 (HLA-A*2402)

[0120] As a non-limiting example, a class I MHC heavy chain polypeptide of a TMMP of the present disclosure may comprise 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 human HLA-A24 heavy chain amino acid sequence: GSHSMRYFSTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDEETGKVKAHSQTDRENLRIALRYYNQSEAGSHTLQMMFGCDVGSDGRFLRGYHQY AYDGKDYIALKEDLRSWTAADMAAQITKRKWEAAHVAEQQRAYLEGTCVDGLRRYLENGKETLQRTDPPKTHMTHHPISDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLRWEPSSQPTVPIVGIIAGLVLLGAVITGAVVAAVMWRRNSSDRKGGSYSQAASSDSAQGSDVSLTACKV (SEQ ID NO: 302). This type of MHC class I heavy chain may be prominent in Asian populations, including populations of individuals of Asian descent. In some cases, amino acid 84 is Ala. In some cases, amino acid 84 is Cys. In some cases, amino acid 236 is Cys. In some cases, amino acid 84 is Ala and amino acid 236 is Cys. In some instances, amino acid 84 is Cys and amino acid 236 is Cys.

[0121] HLA-A33 (HLA-A*3303)

[0122] As a non-limiting example, a class I MHC heavy chain polypeptide of a TMMP of the present disclosure may comprise 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 human HLA-A33 heavy chain amino acid sequence: GSHSMRYFTTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDRNTRNVKAHSQIDRVDLGTLRGYYNQSEAGSHTIQMMYGCDVGSDGRFLRGYQQD AYDGKDYIALNEDLRSWTAADMAAQITQRKWEAARVAEQLRAYLEGTCVEWLRRYLENGKETLQRTDPPKTHMTHHAVSDHEATLRCWALSFYPAEITLTWQRDGEDQTQDTELVETRPAGDGTFQKWASVVVPSGQEQRYTCHVQHEGLPKPLTLRWEPSSQPTIPIVGIIAGLVLFGAVFAGAVVAAVRWRRKSSDRKGGSYSQAASSDSAQGSDMSLTACKV (SEQ ID NO: 303). This class I MHC heavy chain may be prominent in Asian populations, including populations of individuals of Asian descent. In some cases, amino acid 84 is Ala. In some cases, amino acid 84 is Cys. In some cases, amino acid 236 is Cys. In some cases, amino acid 84 is Ala and amino acid 236 is Cys. In some instances, amino acid 84 is Cys and amino acid 236 is Cys.

[0123] HLA-B

[0124] In some cases, the TMMPs of the present disclosure comprise an HLA-B heavy chain polypeptide. HLA-B heavy chain peptide sequences or portions thereof that can be incorporated into the TMMPs of the present disclosure include, but are not limited to, the following alleles: B*0702, B*0801, B*1502, B*3802, B*4001, B*4601, and B*5301, which are not all or substantially all Figure 10A Any of those alleles may be included at positions 84, 139 and / or 236 (e.g. Figure 10A84C); an alanine to cysteine ​​substitution at position 139 (A139C); and an alanine to cysteine ​​substitution at position 236 (A236C). Alternatively, an HLA-B polypeptide comprising an amino acid sequence having at least 75% (e.g., 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 all or a portion of the sequence (e.g., 50, 75, 100, 150, 200, or 250 contiguous amino acids) of those HLA-B alleles can also be used (e.g., which can comprise 1-25, 1-5, 5-10, 10-15, 15-20, 20-25, or 25-30 amino acid insertions, deletions, and / or substitutions).

[0125] In some instances, a TMMP of the present disclosure comprises an HLA-B heavy chain polypeptide comprising the following HLA-B consensus amino acid sequence:

[0126] GSHSMRYF X1 T X2X3 SRPGRGEPRFI X4 VGYVDDT X5 FVRFDSDA X6 SPR X7X8 PRAPWIEQEGPEYWDR X9 TQ X10X11 KT X12X13 TQ X14 Y X15X16 NL X17X18X19X20 YYNQSEAGSH X21X22QX23 MYGCDLGPDGRLLRGHDQSAYDGKDYIALNEDL X24 SWTAADTAAQI X25 QRK X26 EAAR X27 AEQ X28 R X29 YLEG X30 CVEWLRRYLENGK X31X32 L X33 RADPPKTHVTHHP X34SDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRPAGDRTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLRWEP (SEQ ID NO: 30), wherein X1 is H, Y or D; X2 is A or S; X3 is M or V; X4 is A, S or T; X5 is Q or L; X6 is A or T; X7 is E, MK or T; X8 is A or T; X9 is E or N; X10 is I or K; X11 is Y, F, S or C; X12 is N or Q; X13 is A or T; X14 is D or Y; X15 is E or V; X16 is S or N; X17 is T, N or I; X18 is A or L; X19 is L or R; X20 is R or G; X21 is T or I; X22 is L or I; X23 is R or S; X24 is R or S; X25 is S or T; X26 is L or W; X27 is E or V; X28 is R, D, L or W; X29 is A or T; X30 is L, E or T; X31 is E or D; X32 is K or T; X33 is E or Q; and X34 is I or V.

[0127] As an example, a class I MHC heavy chain polypeptide of a TMMP of the present disclosure can comprise 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 human HLA-B heavy chain amino acid sequence: GSHSMRYFYTSVSRPGRGEPRFISVGYVDDTQFVRFDSDAASPREEPRAPWIEQEGPEYWDRNTQIYKAQAQTDRESLRNLRGYYNQSEAGSHTLQSMYGCDVGPDGRLLRGHDQYAYDGKDYIALNEDLRSWTAADTAAQITQRKWEAAREAEQRRAYLEGECVEWLRRYLENGKDKLERADPPKTHVTHHPISDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRPAGDRTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLRWEP (SEQ ID NO:304).

[0128] HLA-B (Y84A; A236C)

[0129] As a non-limiting example, in some cases, the class I MHC heavy chain polypeptide is an HLA-B polypeptide comprising Y84A and A236C substitutions. For example, in some cases, the class I MHC heavy chain polypeptide 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 human HLA-B heavy chain (Y84A; A236C) amino acid sequence: GSHSMRYFYTSVSRPGRGEPRFISVGYVDDTQFVRFDSDAASPREEPRAPWIEQEGPEYWDRNTQIYKAQAQTDRESLRNLRG A YNQSEAGSHTLQSMYGCDVGPDGRLLRGHDQYAYDGKDYIALNEDLRSWTAADTAAQITQRKWEAAREAEQRRAYLEGECVEWLRRYLENGKDKLERADPPKTHVTHHPISDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRP C GDRTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLRWEP (SEQ ID NO: 305), wherein amino acid 84 is Ala and amino acid 236 is Cys. In some cases, Cys-236 forms an interchain disulfide bond with Cys-12 of the variant β2M polypeptide comprising an R12C substitution.

[0130] HLA-B (Y84C; A139C)

[0131] In some cases, the class I MHC heavy chain polypeptide comprises a Y84C and an A139C substitution. For example, in some cases, the class I MHC heavy chain polypeptide 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 human HLA-B heavy chain (Y84C; A139C) amino acid sequence: GSHSMRYFYTSVSRPGRGEPRFISVGYVDDTQFVRFDSDAASPREEPRAPWIEQEGPEYWDRNTQIYKAQAQTDRESLRNLRG C YNQSEAGSHTLQSMYGCDVGPDGRLLRGHDQYAYDGKDYIALNEDLRSWTAADT CAQITQRKWEAAREAEQRRAYLEGECVEWLRRYLENGKDKLERADPPKTHVTHHPISDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRPAGDRTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLRWEP (SEQ ID NO: 306), wherein amino acid 84 is Cys and amino acid 139 is Cys. In some cases, Cys-84 forms an intrachain disulfide bond with Cys-139.

[0132] HLA-B*0702

[0133] As an example, in some cases, the class I MHC heavy chain polypeptide present in the TMMP of the present disclosure comprises Figure 10A The amino acid sequence of HLA-B*0702 (SEQ ID NO: 207) in the present disclosure, or a sequence having at least 75% (e.g., 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 all or a portion (e.g., 50, 75, 100, 150, 200, or 250 consecutive amino acids) of that sequence (e.g., which may comprise 1-25, 1-5, 5-10, 10-15, 15-20, 20-25, or 25-30 amino acid insertions, deletions, and / or substitutions). In some cases, the HLA-B heavy chain polypeptide of the TMMP of the present disclosure is at least 100% identical to the HLA-B heavy chain polypeptide of the present disclosure. Figure 8 Sequence tagged HLA-B or Figure 10A In some cases, the HLA-B heavy chain polypeptide of a TMMP of the present disclosure comprises Y84A and A236C substitutions. In some cases, the HLA-B*0702 heavy chain polypeptide of a TMMP of the present disclosure comprises Y84C and A139C substitutions. In some cases, the HLA-B heavy chain polypeptide of a TMMP of the present disclosure comprises Y84C and A139C substitutions. In some cases, the HLA-B heavy chain polypeptide of a TMMP of the present disclosure comprises Y84C, A139C, and A236C substitutions.

[0134] HLA-C

[0135] In some cases, the TMMPs of the present disclosure comprise an HLA-C heavy chain polypeptide. HLA-C heavy chain polypeptides or portions thereof that can be incorporated into the TMMPs of the present disclosure include, but are not limited to, the following alleles: C*0102, C*0303, C*0304, C*0401, C*0602, C*0701, C*0801, and C*1502, in the absence of all or substantially all of the Figure 11A Any of those alleles may be included at positions 84, 139 and / or 236 (e.g. Figure 11A ) at one or more of the following mutations: a tyrosine to alanine substitution at position 84 (Y84A); a tyrosine to cysteine ​​substitution at position 84 (Y84C); an alanine to cysteine ​​substitution at position 139 (A139C); and an alanine to cysteine ​​substitution at position 236 (A236C). Additionally, HLA-C polypeptides comprising an amino acid sequence having at least 75% (e.g., 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 all or a portion of the sequence (e.g., 50, 75, 100, 150, 200, or 250 contiguous amino acids) of those HLA-C alleles can also be used (e.g., which may comprise 1-25, 1-5, 5-10, 10-15, 15-20, 20-25, or 25-30 amino acid insertions, deletions, and / or substitutions).

[0136] In some instances, a TMMP of the present disclosure comprises an HLA-C heavy chain polypeptide comprising the following HLA-C consensus amino acid sequence:

[0137] (SEQ ID NO:31), wherein X1 is C or G; X2 is R or K; X3 is F, Y, S or D; X4 is R or W; X5 is H or R; X6 is A or S; X7 is Q or R; X8 is A or E; X9 is N or K; X10 is T or A; X11 is S or N; X12 is N or K; X13 is A or D; X14 is G or R; X15 is T or I; X16 is L or I; X17 is W or R; X18 is C, Y, F or S; X19 is L or V; X20 is Y or H; X21 is D or N; X22 is Y, F, S or L; X23 is L or W; X24 is E, A or T; X25 is R, L or W; X26 is L or T; X27 is E or K; X28 is E or K; X29 is H or P; X30 is R or V; X31 is W or R; X32 is V or M; X33 is E or Q; X34 is M or V; X35 is P or Q; X36 is R or S; and X37 is P or G.

[0138] As an example, the class I MHC heavy chain polypeptide of the TMMP of the present disclosure may comprise 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 human HLA-C heavy chain amino acid sequence: CSHSMRYFDTAVSRPGRGEPRFISVGYVDDTQFVRFDSDAASPRGEPRAPWVEQEGPEYWDRETQNYKRQAQADRVSLRNLRGYYN QSEDGSHTLQRMYGCDLGPDGRLLRGYDQSAYDGKDYIALNEDLRSWTAADTAAQITQRKLEAARAAEQLRAYLEGTCVEWLRRYLENGKETLQRAEPPKTHVTHHPLSDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRPAGDGTFQKWAAVVVPSGQEQRYTCHMQHEGLQEPLTLSWEP (SEQ ID NO: 307).

[0139] HLA-C (Y84A; A236C)

[0140] As a non-limiting example, in some instances, the class I MHC heavy chain polypeptide is an HLA-C polypeptide comprising Y84A and A236C substitutions. For example, in some instances, the class I MHC heavy chain polypeptide 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 human HLA-C heavy chain (Y84A; A236C) amino acid sequence: CSHSMRYFDTAVSRPGRGEPRFISVGYVDDTQFVRFDSDAASPRGEPRAPWVEQEGPEYWDRETQNYKRQAQADRVSLRNLRG A YNQSEDGSHTLQRMYGCDLGPDGRLLRGYDQSAYDGKDYIALNEDLRSWTAADTAAQITQRKLEAARAAEQLRAYLEGTCVEWLRRYLENGKETLQRAEPPKTHVTHHPLSDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRP CGDGTFQKWAAVVVPSGQEQRYTCHMQHEGLQEPLTLSWEP (SEQ ID NO: 308), wherein amino acid 84 is Ala and amino acid 236 is Cys. In some cases, Cys-236 forms an interchain disulfide bond with Cys-12 of the variant β2M polypeptide comprising an R12C substitution.

[0141] HLA-C (Y84C; A139C)

[0142] In some instances, the class I MHC heavy chain polypeptide comprises a Y84C and an A139C substitution. For example, in some instances, the class I MHC heavy chain polypeptide 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 human HLA-C heavy chain (Y84C; A139C) amino acid sequence: CSHSMRYFDTAVSRPGRGEPRFISVGYVDDTQFVRFDSDAASPRGEPRAPWVEQEGPEYWDRETQNYKRQAQADRVSLRNLRG C YNQSEDGSHTLQRMYGCDLGPDGRLLRGYDQSAYDGKDYIALNEDLRSWTAADT C AQITQRKLEAARAAEQLRAYLEGTCVEWLRRYLENGKETLQRAEPPKTHVTHHPLSDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRPAGDGTFQKWAAVVVPSGQEQRYTCHMQHEGLQEPLTLSWEP (SEQ ID NO: 397), wherein amino acid 84 is Cys and amino acid 139 is Cys. In some cases, Cys-84 forms an intrachain disulfide bond with Cys-139.

[0143] HLA-C*0701

[0144] In some instances, the class I MHC heavy chain polypeptide of a TMMP of the present disclosure comprises Figure 11A The amino acid sequence of HLA-C*0701 in ( Figure 8or an amino acid sequence that has at least 75% (e.g., 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 all or a portion (e.g., 50, 75, 100, 150, 200, or 250 consecutive amino acids) of that sequence (e.g., which may include 1-25, 1-5, 5-10, 10-15, 15-20, 20-25, or 25-30 amino acid insertions, deletions, and / or substitutions). In some cases, the HLA-C heavy chain polypeptide of the TMMP of the present disclosure is Figure 11A When the sequence-tagged HLA-C*0701 in the present disclosure has less than 100% identity, it may comprise a mutation at one or more of positions 84, 139, and / or 236 selected from the following: a tyrosine to alanine substitution at position 84 (Y84A); a tyrosine to cysteine ​​substitution at position 84 (Y84C); an alanine to cysteine ​​substitution at position 139 (A139C); and an alanine to cysteine ​​substitution at position 236 (A236C). In some cases, the HLA-C heavy chain polypeptide of the TMMP of the present disclosure comprises Y84A and A236C substitutions. In some cases, the HLA-C*0701 heavy chain polypeptide of the T-cell-MMP or epitope conjugate thereof comprises Y84C and A139C substitutions. In some cases, the HLA-C heavy chain polypeptide of the TMMP of the present disclosure comprises Y84C, A139C, and A236C substitutions.

[0145] Nonclassical HLA-E, HLA-F, and HLA-G class I MHC heavy chains

[0146] In some cases, the TMMPs of the present disclosure comprise non-classical class I MHC heavy chain polypeptides. Non-classical HLA heavy chain polypeptides or portions thereof that can be incorporated into the TMMPs of the present disclosure include, but are not limited to, those of the HLA-E, HLA-F, and HLA-G alleles. The amino acid sequences of HLA-E, HLA-F, and HLA-G heavy chain polypeptides (as well as HLA-A, HLA-B, and HLA-C alleles) can be found on the World Wide Web at hla.alleles.org / nomenclature / index.html, the European Bioinformatics Institute (www.ebi.ac.uk), part of the European Molecular Biology Laboratory (EMBL), and the U.S. National Center for Biotechnology Information (www.ncbi.nlm.nih.gov).

[0147] Non-limiting examples of suitable HLA-E alleles include, but are not limited to, HLA-E*0101 (HLA-E*01:01:01:01), HLA-E*01:03 (HLA-E*01:03:01:01), HLA-E*01:04, HLA-E*01:05, HLA-E*01:06, HLA-E*01:07, HLA-E*01:09, and HLA-E*01:10. Non-limiting examples of suitable HLA-F alleles include, but are not limited to, HLA-F*0101 (HLA-F*01:01:01:01), HLA-F*01:02, HLA-F*01:03 (HLA-F*01:03:01:01), HLA-F*01:04, HLA-F*01:05, and HLA-F*01:06. Non-limiting examples of suitable HLA-G alleles include, but are not limited to, HLA-G*0101 (HLA-G*01:01:01:01), HLA-G*01:02, HLA-G*01:03 (HLA-G*01:03:01:01), HLA-G*01:04 (HLA-G*01:04:01:01), HLA-G*01:06, HLA-G*01:07, HLA-G*01:08, HLA-G*01:09, HLA-G*02:010, HLA-G*02:021, HLA-G*02:031, HLA-G*02:042, HLA-G*02:053, HLA-G*02:064, HLA-G*02:075, HLA-G*02:086, HLA-G*02:097, HLA-G*03:031 Consensus sequences for all or substantially all of the HLA E, HLA-F, and HLA-G alleles without the leader sequence, transmembrane sequence, and cytoplasmic sequence are provided in Figure 12 and compare it with Figure 13 The consensus sequences of the above-mentioned HLA-A, HLA-B and HLA-C alleles were aligned.

[0148] Figure 12 The consensus sequence for each of HLA-E, HLA-F, and HLA-G is provided, with variable aa positions indicated as sequentially numbered "X" residues and the positions of aa 84, 139, and 236 double underlined.

[0149] Figure 13 An alignment of the consensus amino acid sequences of HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, and HLA-G is provided in Figures 9-13. Variable residues in each sequence are listed as "X" where the sequence numbering has been removed. Figure 8, indicating the positions of aa 84, 139, and 236 and the five amino acid clusters flanking them, which can be substituted with 1 to 5 amino acids independently selected from: (i) any naturally occurring amino acid or (ii) any naturally occurring amino acid other than proline or glycine.

[0150] Any of the above-mentioned HLA-E, HLA-F and / or HLA-G alleles may comprise Figure 13 The consensus sequence shown in FIG. 1 is substituted at one or more of positions 84, 139, and / or 236. In some cases, these substitutions can be selected from: a tyrosine to alanine (Y84A) or cysteine ​​(Y84C) substitution at position 84, or in the case of HLA-F, an R84A or R84C substitution; a tyrosine to cysteine ​​(A139C) substitution at position 139, or V139C substitution at position 139; and a cysteine ​​to cysteine ​​substitution at position 236 (A236C). Figure 13 HLA-E, HLA-F, and / or HLA-G sequences having at least 75% (e.g., 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 all or a portion (e.g., 50, 75, 100, 150, 200, or 250 contiguous amino acids) of any of the listed consensus sequences (e.g., such sequences may contain 1-25, 1-5, 5-10, 10-15, 15-20, 20-25, or 25-30 amino acid insertions, deletions, and / or substitutions in addition to the listed variable residues).

[0151] Mouse H2K

[0152] In some cases, the class I MHC heavy chain polypeptide present in the TMMP of the present disclosure comprises mouse H2K (SEQ ID NO: 45) ( Figure 8 The present invention also provides a method for preparing a mouse H2K heavy chain polypeptide of the present invention and a method for preparing a mouse H2K heavy chain polypeptide of the present invention. The method also provides a method for preparing a mouse H2K heavy chain polypeptide of the present invention and a method for preparing a mouse H2K heavy chain polypeptide of the present invention. The method also provides a method for preparing a mouse H2K heavy chain polypeptide of the present invention and a method for preparing a mouse H2K heavy chain polypeptide of the present invention. Figure 8When the mouse H2K of the sequence tag in the present invention has less than 100% identity, it may include a mutation selected from the following at one or more of positions 84, 139 and / or 236: tyrosine to alanine (Y84A) at position 84; tyrosine to cysteine ​​(Y84C) at position 84; alanine to cysteine ​​(A139C) at position 139; and alanine to cysteine ​​substitution (A236C) at position 236. In some cases, the mouse H2K heavy chain polypeptide of the TMMP of the present invention comprises Y84A and A236C substitutions. In some cases, the mouse H2K heavy chain polypeptide of the TMMP of the present invention comprises Y84C and A139C substitutions. In some cases, the mouse H2K heavy chain polypeptide of the TMMP of the present invention comprises Y84C, A139C and A236C substitutions.

[0153] Exemplary combinations

[0154] Table 1 below presents various combinations of MHC class I heavy chain sequence modifications that may be incorporated into the TMMPs of the present disclosure.

[0155] Table 1

[0156]

[0157]

[0158]

[0159] Sequence identity ranges from the MHC-H polypeptide sequence incorporated into TMMP to Figure 8-13 The permissible ranges of sequence identity for corresponding portions of the sequences listed in do not count variable residues in the consensus sequence.

[0160] beta-2 microglobulin

[0161] The β2-microglobulin (β2M) polypeptide of the TMMP of the present disclosure can be a human β2M polypeptide, a non-human primate β2M polypeptide, a murine β2M polypeptide, etc. In some cases, the β2M polypeptide comprises Figure 6 In some cases, the β2M polypeptide 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 β2M polypeptide amino acid sequence depicted in Figure 6 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 amino acids 21 to 119 of the β2M amino acid sequence depicted in .

[0162] In some cases, a suitable β2M polypeptide comprises the following amino acid sequence:

[0163] IQRTPKIQVY SCHPAENGKS NFLNCYVSGF HPSDIEVDLLK NGERIEKVE HSDLSFSKDWSFYLLYYTEF TPTEKDEYAC RVN HVTLSQP KIVKWDRDM (SEQ ID NO: 311); and the HLA class I heavy chain polypeptide comprises the following amino acid sequence:

[0164] (aa5) (C) (aa6) QKWAAVVVPSGQEQRYTCHVQHEGLPKPLTLRWEP (SEQ ID NO: 309), wherein the cysteine ​​residue indicated as {C} forms a disulfide bond between the α1 and α2-1 helices and the (C) residue forms a disulfide bond with the β2M polypeptide cysteine ​​at position 12. In the above sequences, "aa1" is "amino acid cluster 1"; "aa2" is "amino acid cluster 2"; "aa3" is "amino acid cluster 3"; "aa4" is "amino acid cluster 4"; "aa5" is "amino acid cluster 5"; and "aa6" is "amino acid cluster 6"; see, e.g., Figure 10. aa1, aa2, aa3, aa4, aa5, and aa6 are selected at each occurrence and are independently selected to be 1-5 amino acid residues, wherein the amino acid residues are i) independently selected from any naturally occurring (e.g., encoded) amino acids or ii) any naturally occurring amino acid except proline or glycine.

[0165] In some cases, the MHC polypeptide comprises a single amino acid substitution relative to a reference MHC polypeptide (wherein the reference MHC polypeptide can be a wild-type MHC polypeptide), wherein the single amino acid substitution replaces an amino acid with a cysteine ​​(Cys) residue. Such a cysteine ​​residue, when present in the MHC polypeptide of the first polypeptide of the TMMP of the present disclosure, can form a disulfide bond with a cysteine ​​residue present in the second polypeptide chain of the TMMP of the present disclosure.

[0166] In some cases, the first MHC polypeptide in the first polypeptide of the TMMP of the present disclosure and / or the second MHC polypeptide in the second polypeptide of the TMMP of the present disclosure comprises an amino acid substitution to replace an amino acid with a cysteine, wherein the substituted cysteine ​​in the first MHC polypeptide forms a disulfide bond with a cysteine ​​in the second MHC polypeptide, wherein the cysteine ​​in the first MHC polypeptide forms a disulfide bond with the substituted cysteine ​​in the second MHC polypeptide, or wherein the substituted cysteine ​​in the first MHC polypeptide forms a disulfide bond with the substituted cysteine ​​in the second MHC polypeptide.

[0167] For example, in some cases, one of the following residue pairs in HLA β2-microglobulin and the HLA class I heavy chain is substituted with cysteine ​​(where the residue numbering is that of the mature polypeptide): 1) β2M residue 12, HLA class I heavy chain residue 236; 2) β2M residue 12, HLA class I heavy chain residue 237; 3) β2M residue 8, HLA class I heavy chain residue 234; 4) β2M residue 10, HLA class I heavy chain residue 235; 5) β2M residue 24, HLA class I heavy chain residue 236; 6) β2M residue 28, HLA class I heavy chain residue 232; 7) β2M residue 98, HLA class I heavy chain residue 192; 8) β2M residue 99, HLA class I heavy chain residue 234; 9) β2M residue 3, HLA class I heavy chain residue 120; 10) β2M residue 31, HLA class I heavy chain residue class I heavy chain residue 96; 11) β2M residue 53, HLA class I heavy chain residue 35; 12) β2M residue 60, HLA class I heavy chain residue 96; 13) β2M residue 60, HLA class I heavy chain residue 122; 14) β2M residue 63, HLA class I heavy chain residue 27; 15) β2M residue Arg3, HLA class I heavy chain residue Gly120; 16) β2M residue His31, HLA class I heavy chain residue Gln96; 17) β2M residue Asp53, HLA class I heavy chain residue Arg35; 18) β2M residue Trp60, HLA class I heavy chain residue Gln96; 19) β2M residue Trp60, HLA class I heavy chain residue Asp122; 20) β2M residue Tyr63, HLA Class I heavy chain residue Tyr27; 21) β2M residue Lys6, HLA class I heavy chain residue Glu232; 22) β2M residue Gln8, HLA class I heavy chain residue Arg234; 23) β2M residue Tyr10, HLA class I heavy chain residue Pro235; 24) β2M residue Ser11, HLA class I heavy chain residue Gln242; 25) β2M residue Asn24, HLA class I heavy chain residue Ala236; 26) β2M residue Ser28, HLA class I heavy chain residue Glu232; 27) β2M residue Asp98, HLA class I heavy chain residue His192; and 28) β2M residue Met99, HLA class I heavy chain residue Arg234. The amino acid numbering of the MHC / HLA class I heavy chain is based on the mature MHC / HLA class I heavy chain, without the signal peptide. For example, in some cases, residue 236 of the mature HLA-A amino acid sequence is substituted with Cys. In some cases, residue 236 of the mature HLA-B amino acid sequence is substituted with Cys. In some cases, residue 236 of the mature HLA-C amino acid sequence is substituted with Cys. In some cases, Figure 6Residue 32 of the amino acid sequence depicted in (corresponding to Arg-12 of mature β2M) is substituted with Cys.

[0168] In some cases, the β2M polypeptide comprises the following amino acid sequence: IQRTPKIQVY S R HPAENGKSNFLNCYVSGF HPSDIEVDLLKNGERIEKVE HSD LSFSKDW SFYLLYYTEF TPTEKDEYAC RVNHVTLSQPKIVKW DRDM (SEQ ID NO: 310). In some cases, the β2M polypeptide comprises the following amino acid sequence: IQRTPKIQVYS C HPAENGKS NFLNCYVSGF HPSDIEVD LLKNGERIEKVE HSDLSFSKDW SFYLLYYTEF TPTEKDEYACRVNHVTLSQP KIVKWDRDM (SEQ ID NO: 311).

[0169] In some instances, the HLA class I heavy chain polypeptide comprises the following amino acid sequence: GSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDGETRKVKAHSQTHRVDLGTLRGYYNQSEAGSHTVQRMYGCDVGSDWRFLRGYHQYAYDGKDYIALKEDLRSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGTCVEWLRRYLENGKETLQRTDAPKTHMTHHAVSDHEATLRCWALSFYPAEITLTWQRDGEDQTQDTELVETRP A GDGTFQKWAAVVVPSGQEQRYTCHVQHEGLPKPLTLRWEP (SEQ ID NO: 297).

[0170] In some instances, the HLA class I heavy chain polypeptide comprises the following amino acid sequence:

[0171] GSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDGETRKVKAHSQTHRVDLGTLRGYYNQSEAGSHTVQRMYGCDVGSDWRFLRGYHQYA YDGKDYIALKEDLRSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGTCVEWLRRYLENGKETLQRTDAPKTHMTHHAVSDHEATLRCWALSFYPAEITLTWQRDGEDQTQDTELVETRP C GDGTFQKWAAVVVPSGQEQRYTCHVQHEGLPKPLTLRWEP (SEQ ID NO: 312).

[0172] In some instances, the HLA class I heavy chain polypeptide comprises the following amino acid sequence: GSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDGETRKVKAHSQTHRVDLGTLRG A YNQSEAGSHTVQRMYGCDVGSDWRFLRGYHQYAYDGKDYIALKEDLRSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGTCVEWLRRYLENGKETLQRTDAPKTHMTHHAVSDHEATLRCWALSFYPAEITLTWQRDGEDQTQDTELVETRP C GDGTFQKWAAVVVPSGQEQRYTCHVQHEGLPKPLTLRWE (SEQ ID NO: 313).

[0173] In some cases, the β2M polypeptide comprises the following amino acid sequence:

[0174] IQRTPKIQVY S C HPAENGKS NFLNCYVSGF HPSDIEVDLLK NGERIEKVE HSDLSFSKDWSFYLLYYTEF TPTEKDEYAC RVN HVTLSQP KIVKWDRDM (SEQ ID NO: 311); and the HLA class I heavy chain polypeptide of the TMMP of the present disclosure comprises the following amino acid sequence:

[0175] (SEQ ID NO: 312), wherein the underlined and bold Cys residues form disulfide bonds with each other in TMMP.

[0176] In some cases, the β2M polypeptide comprises the following amino acid sequence: IQRTPKIQVYS C HPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDM (SEQ ID NO: 314).

[0177] In some cases, the first polypeptide and the second polypeptide of the TMMP of the present disclosure are linked to each other via a disulfide bond through: i) a Cys residue present in a linker linking the peptide epitope in the first polypeptide chain to the β2M polypeptide; and ii) a Cys residue present in the class I MHC heavy chain in the second polypeptide chain. In some cases, the Cys residue present in the class I MHC heavy chain is a Cys introduced as a Y84C substitution. In some cases, the linker linking the peptide epitope in the first polypeptide chain to the β2M polypeptide is GCGGS(G4S)n (SEQ ID No: 315), wherein n is 1, 2, 3, 4, 5, 6, 7, 8, or 9. For example, in some cases, the linker comprises the amino acid sequence GCGGSGGGGSGGGGSGGGGS (SEQ ID NO: 316). As another example, the linker comprises the amino acid sequence GCGGSGGGGSGGGGS (SEQ ID NO: 317). An example of a disulfide-linked first polypeptide and second polypeptide of the TMMP of the present disclosure is schematically depicted in Figures 2A-2F middle.

[0178] Multi-disulfide-bonded TMMP

[0179] In some cases, the first polypeptide and the second polypeptide of the TMMP of the present disclosure are linked to each other by at least two disulfide bonds (i.e., two interchain disulfide bonds). Examples of such multi-disulfide-linked TMMPs are schematically depicted in Figure 17A and 17B and Figures 18A-18C In addition, when the TMMP of the present disclosure comprises an IgFc polypeptide, the heterodimeric TMMP can be dimerized so that disulfide bonds link the IgFc polypeptides in the two heterodimeric TMMPs. This arrangement is schematically depicted in Figure 17C and 17D Unless otherwise indicated, the at least two disulfide bonds described in a multi-disulfide-linked TMMPP in this section do not refer to the disulfide bonds linking the IgFc polypeptides in the dimerized TMMP.

[0180] As described above, in some cases, the first polypeptide and the second polypeptide of the TMMP of the present disclosure are linked to each other by at least two disulfide bonds (i.e., two interchain disulfide bonds). For example, in some cases, the first polypeptide and the second polypeptide of the TMMP of the present disclosure are linked to each other by two interchain disulfide bonds. As another example, in some cases, the first polypeptide and the second polypeptide of the TMMP of the present disclosure are linked to each other by three interchain disulfide bonds. As another example, in some cases, the first polypeptide and the second polypeptide of the TMMP of the present disclosure are linked to each other by four interchain disulfide bonds.

[0181] In some cases, when the peptide epitope in the first polypeptide of the TMMP of the present disclosure is linked to the β2M polypeptide via a linker comprising Cys, at least one of the at least two disulfide bonds links the Cys in the linker to the Cys in the class I MHC heavy chain of the second polypeptide. In some cases, when the peptide epitope in the first polypeptide of the TMMP of the present disclosure is linked to the class I MHC heavy chain polypeptide via a linker, at least one of the at least two disulfide bonds links the Cys in the linker to the Cys present in the β2M polypeptide of the second polypeptide.

[0182] In some cases, a multi-disulfide-linked TMMP (e.g., a disulfide-linked TMMP) of the present disclosure exhibits increased stability compared to a control TMMP comprising only one of the at least two disulfide bonds. In some cases, a multi-disulfide-linked TMMP (e.g., a disulfide-linked TMMP) of the present disclosure exhibits increased in vitro stability compared to a control TMMP comprising only one of the at least two disulfide bonds. For example, in some cases, a multi-disulfide-linked TMMP (e.g., a disulfide-linked TMMP) of the present disclosure exhibits at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 50%, at least 2-fold, at least 5-fold, or at least 10-fold greater in vitro stability compared to a control TMMP comprising only one of the at least two disulfide bonds.

[0183] Whether a multi-disulfide-linked TMMP (e.g., a double-disulfide-linked TMMP) of the present disclosure exhibits increased in vitro stability compared to a control TMMP comprising only one of the at least two disulfide bonds can be determined by measuring the amount of disulfide-linked heterodimeric TMMP present in a sample over time and / or under specified conditions and / or during purification of the TMMP.

[0184] For example, in some cases, a multi-disulfide-linked TMMP (e.g., a double-disulfide-linked TMMP) of the present disclosure exhibits at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 50%, at least 2-fold, at least 5-fold, or at least 10-fold greater in vitro stability when the TMMP is stored at 37°C for a period of time (e.g., a period of about 1 week to about 2 weeks, about 2 weeks to about 4 weeks, or about 4 weeks to about 2 months) compared to a control TMMP comprising only one of the at least two disulfide bonds. For example, in some cases, the amount of disulfide-linked heterodimeric TMMP remaining after storage of a multi-disulfide-linked TMMP (e.g., a double disulfide-linked TMMP) of the present disclosure at 37°C for 28 days in vitro is at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 50%, at least 2-fold, at least 5-fold, or at least 10-fold greater than the amount of disulfide-linked heterodimeric TMMP remaining after storage of a control TMMP (including a TMMP having only one of the at least two disulfide bonds present in the multi-disulfide-linked TMMP) at 37°C for 28 days in vitro.

[0185] As an example, including Figure 14A and 14B The double disulfide bonded TMMP comprising polypeptides 1715 and 2380 depicted in FIG exhibits greater in vitro stability than the TMMP comprising polypeptides 2405 and 2380, wherein polypeptide 2405 is depicted in FIG Figure 14D wherein such TMMP comprises only one disulfide linkage, wherein a single disulfide bond is formed between: i) the Cys of the G2C linker between the epitope and β2M; and ii) the Cys provided by the Y84C substitution in the class I MHC heavy chain. As another example, a TMMP comprising Figure 14A and 14B The double disulfide bonded TMMP comprising polypeptides 1715 and 2380 depicted in FIG exhibits greater in vitro stability than the TMMP comprising polypeptides 1380 and 2380, wherein polypeptide 1380 is depicted in FIG Figure 14E wherein such TMMP comprises only one disulfide linkage, wherein the single disulfide bond is formed between: i) a Cys provided by an R12C substitution in the β2M polypeptide; and ii) a Cys provided by an A236C substitution in the class I MHC heavy chain.

[0186] In some cases, the multi-disulfide-linked TMMPs of the present disclosure exhibit increased in vivo stability compared to a control TMMP comprising only one of the at least two disulfide bonds. For example, in some cases, the multi-disulfide-linked TMMPs of the present disclosure exhibit at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 50%, at least 2-fold, at least 5-fold, or at least 10-fold greater in vivo stability compared to a control TMMP comprising only one of the at least two disulfide bonds.

[0187] In some cases, the presence of two disulfide bonds in a multi-disulfide-linked TMMP (e.g., a double-disulfide-linked TMMP) of the present disclosure increases the amount of disulfide-linked heterodimeric TMMP produced compared to a control TMMP comprising only one of the at least two disulfide bonds. For example, a multi-disulfide-linked TMMP (e.g., a double-disulfide-linked TMMP) of the present disclosure can be produced in mammalian cells cultured in vitro, wherein the mammalian cells are cultured in liquid cell culture medium. TMMP can be secreted into the cell culture medium. The cells can be lysed, thereby generating a cell lysate, and TMMP can be present in the cell lysate. TMMP can be purified from the cell culture medium and / or cell lysate. For example, when the TMMP comprises an IgG1 Fc polypeptide, the cell culture medium and / or cell lysate can be contacted with immobilized protein A (e.g., the cell culture medium and / or cell lysate can be applied to a protein A column, wherein protein A is immobilized on beads). The TMMP present in the cell culture medium and / or cell lysate is bound to immobilized protein A. After washing the column to remove unbound material, the bound TMMP is eluted to generate a protein A eluate. The amount of the disulfide-linked heterodimeric TMMP present in the protein A eluate is at least 0.5%, at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9% or at least 10% greater than the amount of the disulfide-linked heterodimeric TMMP present in the protein A eluate when the TMMP is a control TMMP comprising only one of at least two disulfide bonds present in the TMMP (e.g., a TMMP with two disulfide bonds) . In some cases, the percentage of the total TMMP protein that is non-aggregated disulfide-linked heterodimeric TMMP in the eluate is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99%. The Protein A eluate may be subjected to size exclusion chromatography (SEC) and / or one or more other additional purification steps.

[0188] In some cases, the T cell regulatory multimeric polypeptide of the present disclosure comprises at least one heterodimer comprising: a) a first polypeptide comprising: i) a WT1 peptide epitope, wherein the WT1 peptide is at least 4 amino acids in length (e.g., 4 amino acids to 25 amino acids; e.g., the WT1 peptide has a length of 4, 5, 6, 7, 8, 9, 10-15, 15-20, or 20-25 amino acids); and ii) a first MHC polypeptide; b) a second polypeptide comprising a second MHC polypeptide, and c) at least one immunomodulatory polypeptide, wherein the first polypeptide and / or the second polypeptide comprises an immunomodulatory polypeptide, and wherein the heterodimer comprises two disulfide bonds between the first polypeptide and the second polypeptide (i.e., the heterodimer comprises: i) a first disulfide bond linking the first polypeptide to the second polypeptide; and ii) a second disulfide bond linking the first polypeptide to the second polypeptide). In other words, the first Cys residue contained in the first polypeptide forms a disulfide bond with the first Cys residue in the second polypeptide (first disulfide bond); and the second Cys residue contained in the first polypeptide forms a disulfide bond with the second Cys residue in the second polypeptide (second disulfide bond).

[0189] In some cases, a TMMP of the present disclosure comprises: a) a first polypeptide comprising, in order from N-terminus to C-terminus: i) a peptide epitope; ii) a peptide linker; and iii) a β2M polypeptide; and b) a second polypeptide comprising a class I MHC heavy chain polypeptide, wherein one or both of the first and second polypeptides comprise at least one immunomodulatory polypeptide, wherein the TMMP comprises: a) a first disulfide bond between: i) a Cys present in the linker between the peptide epitope and the β2M polypeptide; and ii) a first Cys introduced into the class I MHC heavy chain polypeptide; and b) at least one second disulfide bond between the first polypeptide and the second polypeptide, wherein the at least one second disulfide bond is between: i) a Cys in the first polypeptide that is C-terminal to the Cys present in the linker; and ii) a Cys in the second polypeptide that is C-terminal to the first Cys introduced into the class I MHC heavy chain polypeptide.

[0190] In some cases, the first disulfide bond-forming Cys residue and the second disulfide bond-forming Cys residue in the first or second polypeptide of the TMMP of the present disclosure are separated from each other by about 10 amino acids to about 200 amino acids. For example, in some cases, the first disulfide bond-forming Cys residue and the second disulfide bond-forming Cys residue in the first or second polypeptide of the TMMP are separated by about 10 amino acids (aa) to about 15 aa, about 15 aa to about 20 aa, about 20 aa to about 25 aa, about 25 aa to about 30 aa, about 30 aa to about 40 aa, about 40 aa to about 50 aa, about 50 aa to about 60 aa, about 60 aa to about 70 aa, about 70 aa to about 80 aa. , about 80aa to about 90aa, about 90aa to about 100aa, about 100aa to about 110aa, about 110aa to about 120aa, about 120aa to about 130aa, about 130aa to about 140aa, about 140aa to about 150aa, about 150aa to about 160aa, about 160aa to about 170aa, about 170aa to about 180aa, about 180aa to about 190aa, or about 190aa to about 200aa.

[0191] As an example, in some cases, the first disulfide bond-forming Cys residue and the second disulfide bond-forming Cys residue in the first polypeptide of the TMMP of the present disclosure are separated from each other by about 10 amino acids to about 80 amino acid residues. For example, in some cases, the second disulfide bond-forming Cys residue in the first polypeptide is about 10 amino acids to about 80 amino acids C-terminal to the first disulfide bond-forming Cys residue in the first polypeptide (e.g., about 10 amino acids (aa) to about 15 aa, about 15 aa to about 20 aa, about 20 aa to about 25 aa, about 25 aa to about 30 aa, about 30 aa to about 40 aa, about 40 aa to about 50 aa, about 50 aa to about 60 aa, about 60 aa to about 70 aa, or about 70 aa to about 80 aa). In some cases, the second disulfide bond-forming Cys residue in the first polypeptide is 10 aa, 11 aa, 12 aa, 13 aa, 14 aa, 15 aa, 16 aa, 17 aa, 18 aa, 19 aa, 20 aa, 21 aa, 22 aa, 23 aa, 24 aa, or 25 aa from the C-terminus of the first disulfide bond-forming Cys residue in the first polypeptide. In some cases, the second disulfide bond-forming Cys residue in the first polypeptide is 15 aa from the C-terminus of the first disulfide bond-forming Cys residue in the first polypeptide. In some cases, the second disulfide bond-forming Cys residue in the first polypeptide is 20 aa from the C-terminus of the first disulfide bond-forming Cys residue in the first polypeptide. In some cases, the second disulfide bond-forming Cys residue in the first polypeptide is 25 aa from the C-terminus of the first disulfide bond-forming Cys residue in the first polypeptide.

[0192] In some cases, the first disulfide bond-forming Cys residue and the second disulfide bond-forming Cys residue in the second polypeptide of the TMMP of the present disclosure are separated from each other by about 140 amino acids to about 160 amino acids. For example, in some cases, the second disulfide bond-forming Cys residue in the second polypeptide is about 140 amino acids to about 160 amino acids C-terminal to the first disulfide bond-forming Cys residue in the second polypeptide. In some cases, the second disulfide bond forming Cys residue in the second polypeptide is at 140 amino acids (aa), 141 aa, 142 aa, 143 aa, 144 aa, 145 aa, 146 aa, 147 aa, 148 aa, 149 aa, 150 aa, 151 aa, 152 aa, 153 aa, 154 aa, 155 aa, 156 aa, 157 aa, 158 aa, 159 aa, or 160 aa C-terminal to the first disulfide bond forming Cys residue in the second polypeptide.

[0193] The multi-disulfide-linked TMMP (e.g., a double-disulfide-linked TMMP) of the present disclosure may comprise: a) a first polypeptide comprising: i) a WT1 peptide (e.g., a WT1 peptide having 4 to about 25 amino acids); and ii) a first MHC polypeptide, wherein the first polypeptide comprises a peptide linker between the WT1 peptide and the first MHC polypeptide, wherein the peptide linker comprises a Cys residue, and wherein the first MHC polypeptide is a β2M polypeptide comprising an amino acid substitution introducing a Cys residue; b) and a second polypeptide comprising a second MHC polypeptide, wherein the second MHC polypeptide is a class I heavy chain comprising a β2M polypeptide based on HLA-A*0201( Figure 9A ), or a Y84C substitution and an A236C substitution at the corresponding position in another class I heavy chain allele, wherein the TMMP comprises a disulfide bond between a Cys residue in the peptide linker and the Cys residue at amino acid position 84 of the class I heavy chain or the corresponding position in another class I heavy chain allele, and wherein the TMMP comprises a disulfide bond between a Cys residue introduced in the β2M polypeptide and a Cys at amino acid position 236 of the class I heavy chain or the corresponding position in another class I heavy chain allele; and c) at least one immunomodulatory polypeptide, wherein the first polypeptide and / or the second polypeptide comprises the at least one immunomodulatory polypeptide. Examples are schematically depicted in Figure 17A and Figure 17B middle.

[0194] In some cases, the peptide linker comprises the amino acid sequence GCGGS (SEQ ID NO: 318). In some cases, the peptide linker comprises the amino acid sequence GCGGS (GGGGS) n (SEQ ID NO: 319), wherein n is an integer from 1 to 10. In some cases, the peptide linker comprises the amino acid sequence GCGGS (GGGGS) n (SEQ ID NO: 398), wherein n is 1. In some cases, the peptide linker comprises the amino acid sequence GCGGS (GGGGS) n (SEQ ID NO: 320), wherein n is 2. In some cases, the peptide linker comprises the amino acid sequence GCGGS (GGGGS) n (SEQ ID NO: 321), wherein n is 3. In some cases, the peptide linker comprises the amino acid sequence GCGGS (GGGGS) n (SEQ ID NO: 322), wherein n is 4. In some cases, the peptide linker comprises the amino acid sequence GCGGS (GGGGS) n (SEQ ID NO: 323), wherein n is 5. In some cases, the peptide linker comprises the amino acid sequence GCGGS(GGGGS)n (SEQ ID NO: 324), wherein n is 6. In some cases, the peptide linker comprises the amino acid sequence GCGGS(GGGGS)n (SEQ ID NO: 325), wherein n is 7. In some cases, the peptide linker comprises the amino acid sequence GCGGS(GGGGS)n (SEQ ID NO: 326), wherein n is 8. In some cases, the peptide linker comprises the amino acid sequence GCGGS(GGGGS)n (SEQ ID NO: 327), wherein n is 9. In some cases, the peptide linker comprises the amino acid sequence GCGGS(GGGGS)n (SEQ ID NO: 328), wherein n is 10.

[0195] In some cases, the peptide linker comprises the amino acid sequence CGGGS (SEQ ID NO: 329). In some cases, the peptide linker comprises the amino acid sequence CGGGS (GGGGS) n (SEQ ID NO: 330), wherein n is an integer from 1 to 10. In some cases, the peptide linker comprises the amino acid sequence CGGGS (GGGGS) n (SEQ ID NO: 331), wherein n is 1. In some cases, the peptide linker comprises the amino acid sequence CGGGS (GGGGS) n (SEQ ID NO: 332), wherein n is 2. In some cases, the peptide linker comprises the amino acid sequence CGGGS (GGGGS) n (SEQ ID NO: 333), wherein n is 3. In some cases, the peptide linker comprises the amino acid sequence CGGGS (GGGGS) n (SEQ ID NO: 334), wherein n is 4. In some cases, the peptide linker comprises the amino acid sequence CGGGS (GGGGS) n (SEQ ID NO: 335), wherein n is 5. In some cases, the peptide linker comprises the amino acid sequence CGGGS(GGGGS)n (SEQ ID NO: 336), wherein n is 6. In some cases, the peptide linker comprises the amino acid sequence CGGGS(GGGGS)n (SEQ ID NO: 337), wherein n is 7. In some cases, the peptide linker comprises the amino acid sequence CGGGS(GGGGS)n (SEQ ID NO: 338), wherein n is 8. In some cases, the peptide linker comprises the amino acid sequence CGGGS(GGGGS)n (SEQ ID NO: 339), wherein n is 9. In some cases, the peptide linker comprises the amino acid sequence CGGGS(GGGGS)n (SEQ ID NO: 340), wherein n is 10.

[0196] The following are based on HLA-A*0201 ( Figure 9A Non-limiting examples of a class I MHC heavy chain having a Y84C substitution and an A236C substitution with amino acid numbering as depicted in ) or at the corresponding position in another class I heavy chain allele.

[0197] HLA-A

[0198] In some cases, a multi-disulfide-linked TMMP (e.g., a double-disulfide-linked TMMP) of the present disclosure comprises: a) a first polypeptide comprising: i) a WT1 peptide (e.g., a WT1 peptide having 4 to about 25 amino acids); and ii) a first MHC polypeptide, wherein the first polypeptide comprises a peptide linker between the WT1 peptide and the first MHC polypeptide, wherein the peptide linker comprises a Cys residue, and wherein the first MHC polypeptide is a β2M polypeptide comprising an amino acid substitution introducing a Cys residue; and b) a second polypeptide comprising an HLA-A The class I MHC heavy chain comprises an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99% or 100% amino acid sequence identity to the following amino acid sequence: GSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDGETRKVKAHSQTHRVDLGTLRGCYNQSEAGSHTVQRMYGCDVGSDWRFLRGYHQYAYDGKDYIALKEDLRSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGTCVEWLRRYLENGKETLQRTDAPKTHMTHHAVSDHEATLRCWALSFYPAEITLTWQRDGEDQTQDTELVETRPCGDGTFQKWAAVVVPSGQEQRYTCHVQHEGLPKPLTLRWEP (SEQ ID NO: 341), wherein amino acid 84 is Cys and amino acid 236 is Cys; and c) at least one immunomodulatory polypeptide, wherein the first polypeptide and / or the second polypeptide comprises the at least one immunomodulatory polypeptide. In some cases, the peptide linker comprises the amino acid sequence GCGGS (SEQ ID NO: 318). In some cases, the peptide linker comprises the amino acid sequence GCGGS(GGGGS)n (SEQ ID NO: 319), wherein n is an integer from 1 to 10. In some cases, the β2M polypeptide comprises an R12C substitution. For example, the β2M polypeptide can comprise an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: IQRTPKIQVYSCHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDM (SEQ ID NO: 314), wherein amino acid 12 is Cys.The at least one immunomodulatory polypeptide may be a polypeptide that activates / stimulates target T cells or suppresses / inhibits target T cells. For example, the at least one immunomodulatory polypeptide may be a cytokine (e.g., IL2 polypeptide, IL7 polypeptide, IL12 polypeptide, IL15 polypeptide, IL17 polypeptide, IL21 polypeptide, IL27 polypeptide, IL-23 polypeptide, TGFβ polypeptide, etc.; and including all family members, such as IL17A, IL-17B, IL-17C, IL-17D, IL-17E, IL-17F, IL-17E), 4-1BBL polypeptide, ICOS-L polypeptide, OX-40L polypeptide, CD80 polypeptide, CD86 polypeptide (CD80 and CD86 are also referred to as B7-1 and B7-2, respectively) , CD40 polypeptide, CD70 polypeptide, JAG1 (CD339) polypeptide, ICAM (CD540 polypeptide, PD-L1 polypeptide, FasL polypeptide, PD-L2 polypeptide, PD-1H (VISTA) polypeptide, ICOS-L (CD275) polypeptide, GITRL polypeptide, HVEM polypeptide, CXCL10 polypeptide, CXCL9 polypeptide, CXCL11 polypeptide, CXCL13 polypeptide, and CX3CL1 polypeptide, Galectin-9 polypeptide, CD83 polypeptide, CD30L polypeptide, HLA-G polypeptide, MICA polypeptide, MICB polypeptide, HVEM (CD270) polypeptide , lymphotoxin beta receptor polypeptide, 3 / TR6 polypeptide, ILT3 polypeptide, ILT4 polypeptide, CXCL10 polypeptide, CXCL9 polypeptide, CXCL11 polypeptide, CXCL13 polypeptide or CX3CL1 polypeptide. These immunomodulatory polypeptides may be wild-type polypeptides or variants of wild-type polypeptides. In some cases, the immunomodulatory polypeptide is an activating ("stimulating") immunomodulatory polypeptide; for example, the immunomodulatory polypeptide can produce an activating / stimulating effect on T cells. Examples of activating immunomodulatory polypeptides include, for example, CD80, CD86, 4-1BBL, OX40L, CD70, ICOS-L, CD40, ICAM (C D54), IL2, IL7, IL12, IL15, IL17, IL21, IL27, IL23, GITRL, TGFβ, and lymphotoxin beta receptor. In some cases, the immunomodulatory polypeptide is an inhibitory ("inhibitory") immunomodulatory polypeptide; for example, the immunomodulatory polypeptide can produce an inhibitory / suppressive effect on T cells. Examples of inhibitory immunomodulatory polypeptides include, for example, PD-1H, PD-L1, PD-L2, TGFβ, FasL, HVEM, Galectin-9, ILT3, and ILT4. Depending on the circumstances, the TGFβ polypeptide can produce an activating / stimulatory effect or an inhibitory / suppressive effect.

[0199] In some cases, the at least one immunomodulatory polypeptide is a reduced affinity variant, as described elsewhere herein. In some cases, the first polypeptide or the second polypeptide comprises an Ig Fc polypeptide.

[0200] In some instances, a multi-disulfide-linked TMMP (e.g., a double-disulfide-linked TMMP) of the present disclosure comprises an HLA-A class I heavy chain polypeptide. In some instances, an HLA-A heavy chain polypeptide present in a multi-disulfide-linked TMMP (e.g., a double-disulfide-linked TMMP) of the present disclosure comprises Figure 9A An amino acid sequence having at least 95%, at least 98%, or at least 99% amino acid sequence identity to the HLA-A*0101, HLA-A*0201, HLA-A*0202, HLA-A*1101, HLA-A*2301, HLA-A*2402, HLA-A*2407, HLA-A*3303, or HLA-A*3401 amino acid sequence depicted in, wherein the HLA-A heavy chain polypeptide comprises Y84C and A236C substitutions.

[0201] HLA-A*0101(Y84C;A236C)

[0202] In some cases, the HLA-A heavy chain polypeptide present in a multiply disulfide-linked TMMP (e.g., a double disulfide-linked TMMP) of the present disclosure comprises an amino acid sequence having at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following HLA-A*0101 (Y84C; A236C) amino acid sequence:

[0203] GSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQKMEPRAPWIEQEGPEYWDQETRNMKAHSQTDRANLGTLRGCYNQSEDGSHTIQIMYGCDVGPDGRFLRGYRQDAYDGKDYIALNEDLRSWTAADMA AQITKRKWEAVHAAEQRRVYLEGRCVDGLRRYLENGKETLQRTDPPKTHMTHHPISDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRPCGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLRWE(SEQ ID NO:343), wherein amino acid 84 is Cys and amino acid 236 is Cys.

[0204] HLA-A*0201(Y84C;A236C)

[0205] In some instances, an HLA-A heavy chain polypeptide present in a multiply disulfide-linked TMMP (e.g., a double disulfide-linked TMMP) of the present disclosure comprises an amino acid sequence having at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following HLA-A*0201 (Y84C; A236C) amino acid sequence: GSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDGETRKVKAHSQTHRVDL GTLRGCYNQSEAGSHTVQRMYGCDVGSDWRFLRGYHQYAYDGKDYIALKEDLRSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGTCVEWLRRYLENGKETLQRTDAPKTHMTHHAVSDHEATLRCWALSFYPAEITLTWQRDGEDQTQDTELVETRPCGDGTFQKWAAVVVPSGQEQRYTCHVQHEGLPKPLTLRWEP (SEQ ID NO: 341), wherein amino acid 84 is Cys and amino acid 236 is Cys.

[0206] HLA-A*0202(Y84C;A236C)

[0207] In some instances, an HLA-A heavy chain polypeptide present in a multiply disulfide-linked TMMP (e.g., a double disulfide-linked TMMP) of the present disclosure comprises an amino acid sequence having at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following HLA-A*0202 (Y84C; A236C) amino acid sequence: GSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDGETRKVKAHSQTHRVDL GTLRGCYNQSEAGSHTVQRMYGCDVGSDWRFLRGYHQYAYDGKDYIALKEDLRSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGTCVEWLRRYLENGKETLQRTDAPKTHMTHHAVSDHEATLRCWALSFYPAEITLTWQRDGEDQTQDTELVETRPCGDGTFQKWAAVVVPSGQEQRYTCHVQHEGLPKPLTLRWE (SEQ ID NO: 341), wherein amino acid 84 is Cys and amino acid 236 is Cys.

[0208] HLA-A*1101(Y84C;A236C)

[0209] In some instances, an HLA-A heavy chain polypeptide present in a multiply disulfide-linked TMMP (e.g., a double disulfide-linked TMMP) of the present disclosure comprises an amino acid sequence having at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following HLA-A*1101 (Y84C; A236C) amino acid sequence: GSHSMRYFYTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDQETRNVKAQSQTDRVDL GTLRGCYNQSEDGSHTIQIMYGCDVGPDGRFLRGYRQDAYDGKDYIALNEDLRSWTAADMAAQITKRKWEAAHAAEQQRAYLEGRCVEWLRRYLENGKETLQRTDPPKTHMTHHPISDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRPCGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLRWE (SEQ ID NO: 344), wherein amino acid 84 is Cys and amino acid 236 is Cys.

[0210] HLA-A*2301(Y84C;A236C)

[0211] In some cases, the HLA-A heavy chain polypeptide present in a multiply disulfide-linked TMMP (e.g., a double disulfide-linked TMMP) of the present disclosure comprises an amino acid sequence having at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following HLA-A*2301 (Y84C; A236C) amino acid sequence: GSHSMRYFSTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDEETGKVKAHSQTDRENL RIALRCYNQSEAGSHTLQMMFGCDVGSDGRFLRGYHQYAYDGKDYIALKEDLRSWTAADMAAQITQRKWEAARVAEQLRAYLEGTCVDGLRRYLENGKETLQRTDPPKTHMTHHPISDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRPCGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLRWE (SEQ ID NO: 345), wherein amino acid 84 is Cys and amino acid 236 is Cys.

[0212] HLA-A*2402(Y84C;A236C)

[0213] In some instances, an HLA-A heavy chain polypeptide present in a multiply disulfide-linked TMMP (e.g., a double disulfide-linked TMMP) of the present disclosure comprises an amino acid sequence having at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following HLA-A*2402 (Y84C; A236C) amino acid sequence: GSHSMRYFSTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDEETGKVKAHSQTDRENL RIALRCYNQSEAGSHTLQMMFGCDVGSDGRFLRGYHQYAYDGKDYIALKEDLRSWTAADMAAQITKRKWEAAHVAEQQRAYLEGTCVDGLRRYLENGKETLQRTDPPKTHMTHHPISDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRPCGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLRWE (SEQ ID NO: 346), wherein amino acid 84 is Cys and amino acid 236 is Cys.

[0214] HLA-A*2407(Y84C;A236C)

[0215] In some instances, an HLA-A heavy chain polypeptide present in a multiply disulfide-linked TMMP (e.g., a double disulfide-linked TMMP) of the present disclosure comprises an amino acid sequence having at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following HLA-A*2407 (Y84C; A236C) amino acid sequence: GSHSMRYFSTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDEETGKVKAQSQTDRENL RIALRCYNQSEAGSHTLQMMFGCDVGSDGRFLRGYHQYAYDGKDYIALKEDLRSWTAADMAAQITKRKWEAAHVAEQQRAYLEGTCVDGLRRYLENGKETLQRTDPPKTHMTHHPISDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRPCGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLRWE (SEQ ID NO: 347), wherein amino acid 84 is Cys and amino acid 236 is Cys.

[0216] HLA-A*3303(Y84C;A236C)

[0217] In some cases, the HLA-A heavy chain polypeptide present in a multiply-disulfide-linked TMMP (e.g., a double-disulfide-linked TMMP) of the present disclosure comprises an amino acid sequence having at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following HLA-A*3303 (Y84C; A236C) amino acid sequence:

[0218] GSHSMRYFTTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDRNTRNVKAHSQIDRVDLGTLRGCYNQSEAGSHTIQMMYGCDVGSDGRFLRGYQQDAYDGKDYIALNEDLRSWTAADMA AQITQRKWEAARVAEQLRAYLEGTCVEWLRRYLENGKETLQRTDPPKTHMTHHAVSDHEATLRCWALSFYPAEITLTWQRDGEDQTQDTELVETRPCGDGTFQKWASVVVPSGQEQRYTCHVQHEGLPKPLTLRWE(SEQ ID NO:348), wherein amino acid 84 is Cys and amino acid 236 is Cys.

[0219] HLA-A*3401(Y84C;A236C)

[0220] In some instances, an HLA-A heavy chain polypeptide present in a multiply disulfide-linked TMMP (e.g., a double disulfide-linked TMMP) of the present disclosure comprises an amino acid sequence having at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following HLA-A*3401 (Y84C; A236C) amino acid sequence: GSHSMRYFYTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDRNTRKVKAQSQTDRVDL GTLRGCYNQSEDGSHTIQRMYGCDVGPDGRFLRGYQQDAYDGKDYIALNEDLRSWTAADMAAQITQRKWETAHEAEQWRAYLEGTCVEWLRRYLENGKETLQRTDAPKTHMTHHAVSDHEATLRCWALSFYPAEITLTWQRDGEDQTQDTELVETRPCGDGTFQKWASVVVPSGQEQRYTCHVQHEGLPKPLTLRWE (SEQ ID NO: 349), wherein amino acid 84 is Cys and amino acid 236 is Cys.

[0221] HLA-B

[0222] In some cases, a multi-disulfide-linked TMMP (e.g., a double-disulfide-linked TMMP) of the present disclosure comprises: a) a first polypeptide comprising: i) a WT1 peptide (e.g., a WT1 peptide having 4 to about 25 amino acids); and ii) a first MHC polypeptide, wherein the first polypeptide comprises a peptide linker between the WT1 peptide and the first MHC polypeptide, wherein the peptide linker comprises a Cys residue, and wherein the first MHC polypeptide is a β2M polypeptide comprising an amino acid substitution introducing a Cys residue; and b) a second polypeptide comprising an HLA-B The class I MHC heavy chain comprises an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99% or 100% amino acid sequence identity to the following amino acid sequence: GSHSMRYFYTSVSRPGRGEPRFISVGYVDDTQFVRFDSDAASPREEPRAPWIEQEGPEYWDRNTQIYKAQAQTDRESLRNLRGCYNQSEAGSHTLQSMYGCDVGPDGRLLRGHDQYAYDGKDYIALNEDLRSWTAADTAAQITQRKWEAAREAEQRRAYLEGECVEWLRRYLENGKDKLERADPPKTHVTHHPISDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRPCGDRTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLRWEP (SEQ ID NO: 350), wherein amino acid 84 is Cys and amino acid 236 is Cys; and c) at least one immunomodulatory polypeptide, wherein the first polypeptide and / or the second polypeptide comprises the at least one immunomodulatory polypeptide. In some cases, the peptide linker comprises the amino acid sequence GCGGS (SEQ ID NO: 318). In some cases, the peptide linker comprises the amino acid sequence GCGGS(GGGGS)n (SEQ ID NO: 342), wherein n is an integer from 1 to 10. In some cases, the β2M polypeptide comprises an R12C substitution. For example, the β2M polypeptide can comprise an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: IQRTPKIQVYSCHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDM (SEQ ID NO: 314), wherein amino acid 12 is Cys.The at least one immunomodulatory polypeptide may be a polypeptide that activates / stimulates target T cells or suppresses / inhibits target T cells. For example, the at least one immunomodulatory polypeptide may be a cytokine (e.g., IL2 polypeptide, IL7 polypeptide, IL12 polypeptide, IL15 polypeptide, IL17 polypeptide, IL21 polypeptide, IL27 polypeptide, IL-23 polypeptide, TGFβ polypeptide, etc.; and including all family members, such as IL17A, IL-17B, IL-17C, IL-17D, IL-17E, IL-17F, IL-17E), 4-1BBL polypeptide, ICOS-L polypeptide, OX-40L polypeptide, CD80 polypeptide, CD86 polypeptide (CD80 and CD86 are also referred to as B7-1 and B7-2, respectively) , CD40 polypeptide, CD70 polypeptide, JAG1 (CD339) polypeptide, ICAM (CD540 polypeptide, PD-L1 polypeptide, FasL polypeptide, PD-L2 polypeptide, PD-1H (VISTA) polypeptide, ICOS-L (CD275) polypeptide, GITRL polypeptide, HVEM polypeptide, CXCL10 polypeptide, CXCL9 polypeptide, CXCL11 polypeptide, CXCL13 polypeptide, and CX3CL1 polypeptide, Galectin-9 polypeptide, CD83 polypeptide, CD30L polypeptide, HLA-G polypeptide, MICA polypeptide, MICB polypeptide, HVEM (CD270) polypeptide , lymphotoxin beta receptor polypeptide, 3 / TR6 polypeptide, ILT3 polypeptide, ILT4 polypeptide, CXCL10 polypeptide, CXCL9 polypeptide, CXCL11 polypeptide, CXCL13 polypeptide or CX3CL1 polypeptide. These immunomodulatory polypeptides may be wild-type polypeptides or variants of wild-type polypeptides. In some cases, the immunomodulatory polypeptide is an activating ("stimulating") immunomodulatory polypeptide; for example, the immunomodulatory polypeptide can produce an activating / stimulating effect on T cells. Examples of activating immunomodulatory polypeptides include, for example, CD80, CD86, 4-1BBL, OX40L, CD70, ICOS-L, CD40, ICAM (C D54), IL2, IL7, IL12, IL15, IL17, IL21, IL27, IL23, GITRL, TGFβ, and lymphotoxin beta receptor. In some cases, the immunomodulatory polypeptide is an inhibitory ("inhibitory") immunomodulatory polypeptide; for example, the immunomodulatory polypeptide can produce an inhibitory / suppressive effect on T cells. Examples of inhibitory immunomodulatory polypeptides include, for example, PD-1H, PD-L1, PD-L2, TGFβ, FasL, HVEM, Galectin-9, ILT3, and ILT4. Depending on the circumstances, the TGFβ polypeptide can produce an activating / stimulatory effect or an inhibitory / suppressive effect.

[0223] In some cases, the at least one immunomodulatory polypeptide is a reduced affinity variant, as described elsewhere herein. In some cases, the first polypeptide or the second polypeptide comprises an Ig Fc polypeptide.

[0224] In some cases, a multi-disulfide-linked TMMP (e.g., a double-disulfide-linked TMMP) of the present disclosure comprises an HLA-B class I heavy chain polypeptide. In some cases, an HLA-B heavy chain polypeptide present in a multi-disulfide-linked TMMP (e.g., a double-disulfide-linked TMMP) of the present disclosure comprises Figure 10A An amino acid sequence having at least 95%, at least 98%, or at least 99% amino acid sequence identity to the HLA-B*0702, HLA-B*0801, HLA-B*1502, HLA-B*3802, HLA-B*4001, HLA-B*4601, or HLA-B*5301 amino acid sequence depicted in, wherein the HLA-B heavy chain polypeptide comprises Y84C and A236C substitutions.

[0225] HLA-B*0702(Y84C;A236C)

[0226] In some cases, the HLA-B heavy chain polypeptide present in a multiply disulfide-linked TMMP (e.g., a double disulfide-linked TMMP) of the present disclosure comprises an amino acid sequence having at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following HLA-B*0702 (Y84C; A236C) amino acid sequence:

[0227] GSHSMRYFYTSVSRPGRGEPRFISVGYVDDTQFVRFDSDAASPREEPRAPWIEQEGPEYWDRNTQIYKAQAQTDRESLRNLRGCYNQSEAGSHTLQSMYGCDVGPDGRLLRGHDQYAYDGKDYIALNEDLRSWTAADTA AQITQRKWEAAREAEQRRAYLEGECVEWLRRYLENGKDKLERADPPKTHVTHHPISDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRPCGDRTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLRWE(SEQ ID NO:350), wherein amino acid 84 is Cys and amino acid 236 is Cys.

[0228] HLA-B*0801(Y84C;A236C)

[0229] In some cases, the HLA-B heavy chain polypeptide present in a multiply disulfide-linked TMMP (e.g., a double disulfide-linked TMMP) of the present disclosure comprises an amino acid sequence having at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following HLA-B*0801 (Y84C; A236C) amino acid sequence:

[0230] GSHSMRYFDTAMSRPGRGEPRISVGYVDDTQFVRFDSDAASPREEPRAPWIEQEGPEYWDRNTQIFKTNTQTDRESLRNLRGCYNQSEAGSHTLQSMYGCDVGPDGRLLRGHNQYAYDGKDYIALNEDLRSWTAADTA AQITQRKWEAARVAEQDRAYLEGTCVEWLRRYLENGKDTLERADPPKTHVTHHPISDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRPCGDRTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLRWE(SEQ ID NO:351), wherein amino acid 84 is Cys and amino acid 236 is Cys.

[0231] HLA-B*1502(Y84C;A236C)

[0232] In some cases, the HLA-B heavy chain polypeptide present in a multiply disulfide-linked TMMP (e.g., a double disulfide-linked TMMP) of the present disclosure comprises an amino acid sequence having at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following HLA-B*1502 (Y84C; A236C) amino acid sequence:

[0233] GSHSMRYFYTAMSRPGRGEPFIAVGYVDDTQFVRFDSDAASPRMAPRAPWIEQEGPEYWDRNTQISKTNTQTYRESLRNLRGCYNQSEAGSHIIQRMYGCDVGPDGRLLRGYDQSAYDGKDYIALNEDLSSWTAADTA AQITQRKWEAAREAEQLRAYLEGLCVEWLRRYLENGKETLQRADPPKTHVTHHPISDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRPCGDRTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLRWE(SEQ ID NO:352), wherein amino acid 84 is Cys and amino acid 236 is Cys.

[0234] HLA-B*3802(Y84C;A236C)

[0235] In some cases, the HLA-B heavy chain polypeptide present in a multiply disulfide-linked TMMP (e.g., a double disulfide-linked TMMP) of the present disclosure comprises an amino acid sequence having at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following HLA-B*3802 (Y84C; A236C) amino acid sequence:

[0236] GSHSMRYFYTSVSRPGRGEPRFISVGYVDDTQFVRFDSDAASPREEPRAPWIEQEGPEYWDRNTQICKTNTQTYRENLRTALRCYNQSEAGSHTLQRMYGCDVGPDGRLLRGHNQFAYDGKDYIALNEDLSSWTAADTA AQITQRKWEAARVAEQLRTYLEGTCVEWLRRYLENGKETLQRADPPKTHVTHHPISDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRPCGDRTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLRWE(SEQ ID NO:353), wherein amino acid 84 is Cys and amino acid 236 is Cys.

[0237] HLA-B*4001(Y84C;A2346C)

[0238] In some cases, the HLA-B heavy chain polypeptide present in a multiply disulfide-linked TMMP (e.g., a double disulfide-linked TMMP) of the present disclosure comprises an amino acid sequence having at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following HLA-B*4001 (Y84C; A236C) amino acid sequence:

[0239] GSHSMRYFHTAMSRPGRGEPRFITVGYVDDTLFVRFDSDATSPRKEPRAPWIEQEGPEYWDRETQISKTNTQTYRESLRNLRGCYNQSEAGSHTLQRMYGCDVGPDGRLLRGHNQYAYDGKDYIALNEDLRSWTAADTA AQISQRKLEAARVAEQLRAYLEGECVEWLRRYLENGKDKLERADPPKTHVTHHPISDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRPCGDRTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLRWE(SEQ ID NO:354), wherein amino acid 84 is Cys and amino acid 236 is Cys.

[0240] HLA-B*4601(Y84C;A236C)

[0241] In some cases, the HLA-B heavy chain polypeptide present in a multiply disulfide-linked TMMP (e.g., a double disulfide-linked TMMP) of the present disclosure comprises an amino acid sequence having at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following HLA-B*4601 (Y84C; A236C) amino acid sequence:

[0242] GSHSMRYFYTAMSRPGRGEPRFIAVGYVDDTQFVRFDSDAASPRMAPRAPWIEQEGPEYWDRETQKYKRQAQTDRVSLRNLRGCYNQSEAGSHTLQRMYGCDVGPDGRLLRGHDQSAYDGKDYIALNEDLSSWTAADTA AQITQRKWEAAREAEQWRAYLEGLCVEWLRRYLENGKETLQRADPPKTHVTHHPISDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRPCGDRTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLRWE(SEQ ID NO:355), wherein amino acid 84 is Cys and amino acid 236 is Cys.

[0243] HLA-B*5301(Y84C;A236C)

[0244] In some cases, the HLA-B heavy chain polypeptide present in a multiply disulfide-linked TMMP (e.g., a double disulfide-linked TMMP) of the present disclosure comprises an amino acid sequence having at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following HLA-B*5301 (Y84C; A236C) amino acid sequence:

[0245] GSHSMRYFYTAMSRPGRGEPRFIAVGYVDDTQFVRFDSDAASPRTEPRAPWIEQEGPEYWDRNTQIFKTNTQTYRENLRIALRCYNQSEAGSHIIQRMYGCDLGPDGRLLRGHDQSAYDGKDYIALNEDLSSWTAADTA AQITQRKWEAARVAEQLRAYLEGLCVEWLRRYLENGKETLQRADPPKTHVTHHPVSDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRPCGDRTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLRWE(SEQ ID NO:356), wherein amino acid 84 is Cys and amino acid 236 is Cys.

[0246] HLA-C

[0247] In some cases, a multi-disulfide-linked TMMP (e.g., a double-disulfide-linked TMMP) of the present disclosure comprises: a) a first polypeptide comprising: i) a WT-1 peptide (e.g., a WT-1 peptide having 4 to about 25 amino acids); and ii) a first MHC polypeptide, wherein the first polypeptide comprises a peptide linker between the WT-1 peptide and the first MHC polypeptide, wherein the peptide linker comprises a Cys residue, and wherein the first MHC polypeptide is a β2M polypeptide comprising an amino acid substitution introducing a Cys residue; and b) a second polypeptide comprising an HLA-C The class I MHC heavy chain comprises an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99% or 100% amino acid sequence identity to the following amino acid sequence: CSHSMRYFDTAVSRPGRGEPRFISVGYVDDTQFVRFDSDAASPRGEPRAPWVEQEGPEYWDRETQNYKRQAQADRVSLRNLRGCYNQSEDGSHTLQRMYGCDLGPDGRLLRGYDQSAYDGKDYIALNEDLRSWTAADTAAQITQRKLEAARAAEQLRAYLEGTCVEWLRRYLENGKETLQRAEPPKTHVTHHPLSDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRPCGDGTFQKWAAVVVPSGQEQRYTCHMQHEGLQEPLTLSWEP (SEQ ID NO: 357), wherein amino acid 84 is Cys and amino acid 236 is Cys; and c) at least one immunomodulatory polypeptide, wherein the first polypeptide and / or the second polypeptide comprises the at least one immunomodulatory polypeptide. In some cases, the peptide linker comprises the amino acid sequence GCGGS (SEQ ID NO: 318). In some cases, the peptide linker comprises the amino acid sequence GCGGS(GGGGS)n (SEQ ID NO: 342), wherein n is an integer from 1 to 10. In some cases, the β2M polypeptide comprises an R12C substitution. For example, the β2M polypeptide can comprise an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: IQRTPKIQVYSCHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDM (SEQ ID NO: 314), wherein amino acid 12 is Cys.The at least one immunomodulatory polypeptide may be a polypeptide that activates / stimulates target T cells or suppresses / inhibits target T cells. For example, the at least one immunomodulatory polypeptide may be a cytokine (e.g., IL2 polypeptide, IL7 polypeptide, IL12 polypeptide, IL15 polypeptide, IL17 polypeptide, IL21 polypeptide, IL27 polypeptide, IL-23 polypeptide, TGFβ polypeptide, etc.; and including all family members, such as IL17A, IL-17B, IL-17C, IL-17D, IL-17E, IL-17F, IL-17E), 4-1BBL polypeptide, ICOS-L polypeptide, OX-40L polypeptide, CD80 polypeptide, CD86 polypeptide (CD80 and CD86 are also referred to as B7-1 and B7-2, respectively) , CD40 polypeptide, CD70 polypeptide, JAG1 (CD339) polypeptide, ICAM (CD540 polypeptide, PD-L1 polypeptide, FasL polypeptide, PD-L2 polypeptide, PD-1H (VISTA) polypeptide, ICOS-L (CD275) polypeptide, GITRL polypeptide, HVEM polypeptide, CXCL10 polypeptide, CXCL9 polypeptide, CXCL11 polypeptide, CXCL13 polypeptide, and CX3CL1 polypeptide, Galectin-9 polypeptide, CD83 polypeptide, CD30L polypeptide, HLA-G polypeptide, MICA polypeptide, MICB polypeptide, HVEM (CD270) polypeptide , lymphotoxin beta receptor polypeptide, 3 / TR6 polypeptide, ILT3 polypeptide, ILT4 polypeptide, CXCL10 polypeptide, CXCL9 polypeptide, CXCL11 polypeptide, CXCL13 polypeptide or CX3CL1 polypeptide. These immunomodulatory polypeptides may be wild-type polypeptides or variants of wild-type polypeptides. In some cases, the immunomodulatory polypeptide is an activating ("stimulating") immunomodulatory polypeptide; for example, the immunomodulatory polypeptide can produce an activating / stimulating effect on T cells. Examples of activating immunomodulatory polypeptides include, for example, CD80, CD86, 4-1BBL, OX40L, CD70, ICOS-L, CD40, ICAM (C D54), IL2, IL7, IL12, IL15, IL17, IL21, IL27, IL23, GITRL, TGFβ, and lymphotoxin beta receptor. In some cases, the immunomodulatory polypeptide is an inhibitory ("inhibitory") immunomodulatory polypeptide; for example, the immunomodulatory polypeptide can produce an inhibitory / suppressive effect on T cells. Examples of inhibitory immunomodulatory polypeptides include, for example, PD-1H, PD-L1, PD-L2, TGFβ, FasL, HVEM, Galectin-9, ILT3, and ILT4. Depending on the circumstances, the TGFβ polypeptide can produce an activating / stimulatory effect or an inhibitory / suppressive effect.

[0248] In some cases, the at least one immunomodulatory polypeptide is a reduced affinity variant, as described elsewhere herein. In some cases, the first polypeptide or the second polypeptide comprises an Ig Fc polypeptide.

[0249] In some instances, a multi-disulfide-linked TMMP (e.g., a double-disulfide-linked TMMP) of the present disclosure comprises an HLA-C class I heavy chain polypeptide. In some instances, an HLA-C heavy chain polypeptide present in a multi-disulfide-linked TMMP (e.g., a double-disulfide-linked TMMP) of the present disclosure comprises Figure 11A An amino acid sequence having at least 95%, at least 98%, or at least 99% amino acid sequence identity to the HLA-C*0102, HLA-C*0303, HLA-C*0304, HLA-C*0401, HLA-C*0602, HLA-C*0701, HLA-C*0702, HLA-C*0801, or HLA-C*1502 amino acid sequence depicted in, wherein the HLA-C heavy chain polypeptide comprises Y84C and A236C substitutions.

[0250] HLA-C*01:02(Y84C;A236C)

[0251] In some instances, the HLA-C heavy chain polypeptide present in a multiply-disulfide-linked TMMP (e.g., a double-disulfide-linked TMMP) of the present disclosure comprises an amino acid sequence having at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following HLA-C*01:02 (Y84C; A236C) amino acid sequence:

[0252] CSHSMKYFFTSVSRPGRGEPRISVGYVDDTQFVRFDSDAASPRGEPRAPWVEQEGPEYWDRETQKYKRQAQTDRVSLRNLRGCYNQSEAGSHTLQWMCGCDLGPDGRLLRGYDQYAYDGKDYIALNEDLRSWTAADTAA QITQRKWEAAREAEQRRAYLEGTCVEWLRRYLENGKETLQRAEHPKTHVTHHPVSDHEATLRCWALGFYPAEITLTWQWDGEDQTQDTELVETRPCGDGTFQKWAAVMVPSGEEQRYTCHVQHEGLPEPLTLRWEP(SEQ ID NO:358), wherein amino acid 84 is Cys and amino acid 236 is Cys.

[0253] HLA-C*0303(Y84C;A236C)

[0254] In some cases, the HLA-C heavy chain polypeptide present in a multiply-disulfide-linked TMMP (e.g., a double-disulfide-linked TMMP) of the present disclosure comprises an amino acid sequence having at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following HLA-C*03:03 (Y84C; A236C) amino acid sequence:

[0255] GSHSMRYFYTAVSRPGRGEPHFIAVGYVDDTQFVRFDSDAASPRGEPRAPWVEQEGPEYWDRETQKYKRQAQTDRVSLRNLRGCYNQSEARSHIIQRMYGCDVGPDGRLLRGYDQYAYDGKDYIALNEDLRSWTAADTAA QITQRKWEAAREAEQLRAYLEGLCVEWLRRYLKNGKETLQRAEHPKTHVTHHPVSDHEATLRCWALGFYPAEITLTWQWDGEDQTQDTELVETRPCGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLTLRWEP(SEQ ID NO:359), wherein amino acid 84 is Cys and amino acid 236 is Cys.

[0256] HLA-C*0304(Y84C;A236C)

[0257] In some instances, the HLA-C heavy chain polypeptide present in a multiply-disulfide-linked TMMP (e.g., a double-disulfide-linked TMMP) of the present disclosure comprises an amino acid sequence having at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following HLA-C*03:04 (Y84C; A236C) amino acid sequence:

[0258] GSHSMRYFYTAVSRPGRGEPHFIAVGYVDDTQFVRFDSDAASPRGEPRAPWVEQEGPEYWDRETQKYKRQAQTDRVSLRNLRGCYNQSEAGSHIIQRMYGCDVGPDGRLLRGYDQYAYDGKDYIALNEDLRSWTAADTAA QITQRKWEAAREAEQLRAYLEGLCVEWLRRYLKNGKETLQRAEHPKTHVTHHPVSDHEATLRCWALGFYPAEITLTWQWDGEDQTQDTELVETRPCGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLTLRWEP(SEQ ID NO:360), wherein amino acid 84 is Cys and amino acid 236 is Cys.

[0259] HLA-C*0401(Y84C;A236C)

[0260] In some instances, the HLA-C heavy chain polypeptide present in a multiply-disulfide-linked TMMP (e.g., a double-disulfide-linked TMMP) of the present disclosure comprises an amino acid sequence having at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following HLA-C*04:01 (Y84C; A236C) amino acid sequence:

[0261] GSHSMRYFSTSVSWPGRGEPRFIAVGYVDDTQFVRFDSDAASPRGEPREPWVEQEGPEYWDRETQKYKRQAQADRVNLRKLRGCYNQSEDGSHTLQRMFGCDLGPDGRLLRGYNQFAYDGKDYIALNEDLRSWTAADTAA QITQRKWEAAREAEQRRAYLEGTCVEWLRRYLENGKETLQRAEHPKTHVTHHPVSDHEATLRCWALGFYPAEITLTWQWDGEDQTQDTELVETRPCGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLTLRWKP(SEQ ID NO:361), wherein amino acid 84 is Cys and amino acid 236 is Cys.

[0262] HLA-C*0602(Y84C;A236C)

[0263] In some instances, the HLA-C heavy chain polypeptide present in a multiply-disulfide-linked TMMP (e.g., a double-disulfide-linked TMMP) of the present disclosure comprises an amino acid sequence having at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following HLA-C*06:02 (Y84C; A236C) amino acid sequence:

[0264] CSHSMRYFDTAVSRPGRGEPFISVGYVDDTQFVRFDSDAASPRGEPRAPWVEQEGPEYWDRETQKYKRQAQADRVNLRKLRGCYNQSEDGSHTLQWMYGCDLGPDGRLLRGYDQSAYDGKDYIALNEDLRSWTAADTAA QITQRKWEAAREAEQWRAYLEGTCVEWLRRYLENGKETLQRAEHPKTHVTHHPVSDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRPCGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLTLRWEP(SEQ ID NO:362), wherein amino acid 84 is Cys and amino acid 236 is Cys.

[0265] HLA-C*0701(Y84C;A236C)

[0266] In some instances, the HLA-C heavy chain polypeptide present in a multiply-disulfide-linked TMMP (e.g., a double-disulfide-linked TMMP) of the present disclosure comprises an amino acid sequence having at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following HLA-C*07:01 (Y84C; A236C) amino acid sequence:

[0267] CSHSMRYFDTAVSRPGRGEPRISVGYVDDTQFVRFDSDAASPRGEPRAPWVEQEGPEYWDRETQNYKRQAQADRVSLRNLRGCYNQSEDGSHTLQRMYGCDLGPDGRLLRGYDQSAYDGKDYIALNEDLRSWTAADTAA QITQRKLEAARAAEQLRAYLEGTCVEWLRRYLENGQRAEPPKTHVTHHPLSDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRPCGDGTFQKWAAVVVPSGQEQRYTCHMQHEGLQEPLTLSWEP(SEQ ID NO:357), wherein amino acid 84 is Cys and amino acid 236 is Cys.

[0268] HLA-C*0702(Y84C;A236C)

[0269] In some instances, the HLA-C heavy chain polypeptide present in a multiply-disulfide-linked TMMP (e.g., a double-disulfide-linked TMMP) of the present disclosure comprises an amino acid sequence having at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following HLA-C*07:02 (Y84C; A236C) amino acid sequence:

[0270] CSHSMRYFDTAVSRPGRGEPRISVGYVDDTQFVRFDSDAASPRGEPRAPWVEQEGPEYWDRETQKYKRQAQADRVSLRNLRGCYNQSEDGSHTLQRMSGCDLGPDGRLLRGYDQSAYDGKDYIALNEDLRSWTAADTAA QITQRKLEAARAAEQLRAYLEGTCVEWLRRYLENGQRAEPPKTHVTHHPLSDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRPCGDGTFQKWAAVVVPSGQEQRYTCHMQHEGLQEPLTLSWEP(SEQ ID NO:404), wherein amino acid 84 is Cys and amino acid 236 is Cys.

[0271] HLA-C*0801(Y84C;A236C)

[0272] In some instances, the HLA-C heavy chain polypeptide present in a multiply-disulfide-linked TMMP (e.g., a double-disulfide-linked TMMP) of the present disclosure comprises an amino acid sequence having at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following HLA-C*08:01 (Y84C; A236C) amino acid sequence:

[0273] CSHSMRYFYTAVSRPGRGEPFIAVGYVDDTQFVQFDSDAASPRGEPRAPWVEQEGPEYWDRETQKYKRQAQTDRVSLRNLRGCYNQSEAGSHTLQRMYGCDLGPDGRLLRGYNQFAYDGKDYIALNEDLRSWTAADTAA QITQRKWEAARTAEQLRAYLEGTCVEWLRRYLENGKKTLQRAEHPKTHVTHHPVSDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRPCGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLTLRWGP(SEQ ID NO:363), wherein amino acid 84 is Cys and amino acid 236 is Cys.

[0274] HLA-C*1502(Y84C;A236C)

[0275] In some cases, the HLA-C heavy chain polypeptide present in a multiply-disulfide-linked TMMP (e.g., a double-disulfide-linked TMMP) of the present disclosure comprises an amino acid sequence having at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following HLA-C*15:02 (Y84C; A236C) amino acid sequence:

[0276] CSHSMRYFYTAVSRPGRGEPHFIAVGYVDDTQFVRFDSDAASPRGEPRAPWVEQEGPEYWDRETQNYKRQAQTDRVNLRKLRGCYNQSEAGSHIIQRMYGCDLGPDGRLLRGHDQLAYDGKDYIALNEDLRSWTAADTAA QITQRKWEAAREAEQLRAYLEGTCVEWLRRYLENGKETLQRAEHPKTHVTHHPVSDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRPCGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLTLRWEP(SEQ ID NO:364), wherein amino acid 84 is Cys and amino acid 236 is Cys.

[0277] Scaffold peptides

[0278] The TMMP may comprise an Fc polypeptide or may comprise another suitable scaffold polypeptide.

[0279] Suitable scaffold polypeptides include antibody-based scaffold polypeptides and non-antibody-based scaffolds. Non-antibody-based scaffolds include, for example, albumin, XTEN (extended recombinant) polypeptides, transferrin, Fc receptor polypeptides, elastin-like polypeptides (see, e.g., Hassouneh et al. (2012) Methods Enzymol. 502:215; e.g., a polypeptide comprising a pentapeptide repeat unit (Val-Pro-Gly-X-Gly; SEQ ID NO:59), wherein X is any amino acid other than proline), albumin-binding polypeptides, silk-like polypeptides (see, e.g., Valluzzi et al. (2002) Philos Trans R Soc Lond B Biol Sci. 357:165), silk-elastin-like polypeptides (SELP; see, e.g., Megeed et al. (2002) Adv Drug Deliv Rev. 54:1075), and the like. Suitable XTEN polypeptides include, for example, those disclosed in WO 2009 / 023270, WO 2010 / 091122, WO 2007 / 103515, US 2010 / 0189682, and US 2009 / 0092582; see also Schellenberger et al. (2009) Nat Biotechnol. 27: 1186). Suitable albumin polypeptides include, for example, human serum albumin.

[0280] Suitable scaffold polypeptides will in some cases be polypeptides that extend half-life. Thus, in some cases, suitable scaffold polypeptides increase the in vivo half-life (e.g., serum half-life) of TMMP compared to a control TMMP lacking the scaffold polypeptide. For example, in some cases, the scaffold polypeptide increases the in vivo half-life (e.g., serum half-life) of TMMP 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 greater than 100-fold compared to a control TMMP lacking the scaffold polypeptide. As an example, in some cases, the Fc polypeptide increases the in vivo half-life (e.g., serum half-life) of the TMMP 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 greater than 100-fold compared to a control TMMP lacking the Fc polypeptide.

[0281] Fc peptide

[0282] In some cases, the first polypeptide chain and / or the second polypeptide chain of the TMMP of the present disclosure comprises an Fc polypeptide. The Fc polypeptide of the TMMP of the present disclosure can be human IgG1 Fc, human IgG2 Fc, human IgG3 Fc, human IgG4 Fc, etc. In some cases, the Fc polypeptide comprises Figures 5A-5G In some instances, the Fc region comprises an amino acid sequence that is 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% identical to the amino acid sequence of the Fc region depicted in FIG. Figure 5A In some instances, the Fc region comprises an amino acid sequence that is 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% identical to the human IgG1 Fc polypeptide depicted in FIG. Figure 5A The human IgG1 Fc polypeptide depicted in has an amino acid sequence that is 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; and comprises an N77 substitution; e.g., the Fc polypeptide comprises an N77A substitution. In some cases, the Fc polypeptide comprises an N77A substitution. Figure 5AThe human IgG2 Fc polypeptide depicted in has an amino acid sequence that is 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; for example, an Fc polypeptide comprising Figure 5A In some cases, the Fc polypeptide comprises an amino acid sequence that is 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% identical to amino acids 99-325 of the human IgG2 Fc polypeptide depicted in FIG. Figure 5A The human IgG3 Fc polypeptide depicted in has an amino acid sequence that is 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; for example, an Fc polypeptide comprising Figure 5A In some cases, the Fc polypeptide comprises an amino acid sequence that has at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to amino acids 19-246 of the human IgG3 Fc polypeptide depicted in . Figure 5B The human IgM Fc polypeptides depicted in the foregoing have an amino acid sequence that is 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; for example, an Fc polypeptide comprising an amino acid sequence that is identical to Figure 5B , 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 depicted in . In some cases, the Fc polypeptide comprises an amino acid sequence that is identical to Figure 5C The human IgA Fc polypeptide depicted in has an amino acid sequence that is 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 identical; for example, an Fc polypeptide comprising Figure 5C amino acids 1-234 of the human IgA Fc polypeptide depicted in have an amino acid sequence of 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.

[0283] In some cases, the Fc polypeptide comprises Figure 5CIn some instances, the Fc polypeptide comprises an amino acid sequence that is 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% identical to the human IgG4 Fc polypeptide depicted in FIG. Figure 5C amino acids 100 to 327 of the human IgG4 Fc polypeptide depicted in have an amino acid sequence of 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.

[0284] In some instances, the IgG4 Fc polypeptide comprises the following amino acid sequence: PPCSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 365).

[0285] In some cases, the Fc polypeptide present in the TMMP comprises Figure 5A In some cases, the Fc polypeptide present in the TMMP comprises the amino acid sequence depicted in (human IgG1 Fc). Figure 5A The amino acid sequence depicted in (human IgG1 Fc), except that N297 is substituted with an amino acid other than asparagine ( Figure 5A N77 of the amino acid sequence depicted in ). In some cases, the Fc polypeptide present in the TMMP comprises Figure 5C The amino acid sequence depicted in (human IgG1 Fc comprising N297A substitution, the substitution being Figure 5A N77 of the amino acid sequence depicted in ). In some cases, the Fc polypeptide present in the TMMP comprises Figure 5A The amino acid sequence depicted in (human IgG1 Fc), except that L234 is substituted with an amino acid other than leucine ( Figure 5A L14 of the amino acid sequence depicted in ). In some cases, the Fc polypeptide present in the TMMP comprises Figure 5A The amino acid sequence depicted in (human IgG1 Fc), except that L235 is substituted with an amino acid other than leucine ( Figure 5AL15 of the amino acid sequence depicted in ).

[0286] In some cases, the Fc polypeptide present in the TMMP comprises Figure 5E In some cases, the Fc polypeptide present in the TMMP comprises Figure 5F In some cases, the Fc polypeptide present in the TMMP comprises Figure 5G The amino acid sequence depicted in (human IgG1 Fc containing L234A substitution and L235A substitution, which correspond to Figure 5G 14 and 15 of the amino acid sequence depicted in FIG). In some cases, the Fc polypeptide present in the TMMP comprises Figure 5A The amino acid sequence depicted in (human IgG1 Fc) with the exception of substitution of P331 with an amino acid other than proline ( Figure 5A In some cases, the Fc polypeptide present in the TMMP comprises Figure 5A The amino acid sequence depicted in (human IgG1 Fc) with the exception of substitutions of L234 and L235 with amino acids other than leucine ( Figure 5A L14 and L15 of the amino acid sequence depicted in ). In some cases, the Fc polypeptide present in the TMMP comprises Figure 5A The amino acid sequence depicted in (human IgG1 Fc) with the exception of substitutions of L234 and L235 with amino acids other than leucine ( Figure 5A L14 and L15 of the amino acid sequence depicted in ) and substitution of P331 with an amino acid other than proline ( Figure 5A In some cases, the Fc polypeptide present in the TMMP comprises Figure 5E The amino acid sequence depicted in (comprising L234F, L235E and P331S substitutions (corresponding to Figure 5E In some cases, the Fc polypeptide present in the TMMP is a human IgG1 Fc comprising L234A and L235A substitutions (with Ala to Figure 5A ) an IgG1 Fc polypeptide comprising a substitution of L14 and L15 of the amino acid sequence depicted in Figure 5G Depicted.

[0287] connector

[0288] The TMMPs of the present disclosure may comprise one or more linkers, wherein the one or more linkers are interposed between one or more of: i) a class I MHC polypeptide and an Ig Fc polypeptide, wherein this linker is referred to herein as "L1"; ii) an immunomodulatory polypeptide and a class I MHC polypeptide, wherein this linker is referred to herein as "L2"; iii) a first immunomodulatory polypeptide and a second immunomodulatory polypeptide, wherein this linker is referred to herein as "L3"; iv) a peptide antigen ("epitope") and a class I MHC polypeptide; v) a class I MHC polypeptide and a dimerizing polypeptide (e.g., a first or second member of a dimerizing pair); and vi) a dimerizing polypeptide (e.g., a first or second member of a dimerizing pair) and an Ig Fc polypeptide.

[0289] Suitable linkers (also referred to as "spacers") can be readily selected and can have any of a number of suitable lengths, such as 1 amino acid to 25 amino acids, 3 amino acids to 20 amino acids, 2 amino acids to 15 amino acids, 3 amino acids to 12 amino acids, including 4 amino acids to 10 amino acids, 5 amino acids to 9 amino acids, 6 amino acids to 8 amino acids, or 7 amino acids to 8 amino acids. 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. In some cases, the linker has a length of 25 to 50 amino acids, for example, a length of 25 to 30, 30 to 35, 35 to 40, 40 to 45, or 45 to 50 amino acids.

[0290] Exemplary linkers include glycine polymers (G) n , glycine-serine polymers (including, for example, (GS) n 、(GSGGS) n (SEQ ID NO: 366) and (GGGS) n(SEQ ID NO: 367), wherein n is an integer of at least 1), 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 serve as neutral tethers between components. Glycine polymers can be used; glycine enters the phi-psi space significantly more easily than even alanine and is much less constrained than residues with longer side chains (see Scheraga, Rev. Computational Chem. 11173-142 (1992)). Exemplary linkers can comprise amino acid sequences including, but not limited to, GGSG (SEQ ID NO: 368), GGSGG (SEQ ID NO: 369), GSGSG (SEQ ID NO: 370), GSGGG (SEQ ID NO: 371), GGGSG (SEQ ID NO: 372), GSSSG (SEQ ID NO: 373), and the like. Exemplary linkers can include, for example, Gly(Ser4)n (SEQ ID NO: 374), wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some cases, the linker comprises the amino acid sequence (GSSSS)n (SEQ ID NO: 375), wherein n is 4. In some cases, the linker comprises the amino acid sequence (GSSSS)n (SEQ ID NO: 376), wherein n is 5. In some cases, the linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO: 377), wherein n is 1. In some cases, the linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO: 378), wherein n is 2. In some cases, the linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO: 379), wherein n is 3. In some cases, the linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO: 380), wherein n is 4. In some cases, the linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO: 381), wherein n is 5. In some cases, the linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO: 382), wherein n is 6. In some cases, the linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO: 383), wherein n is 7, in some cases, the linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO: 384), wherein n is 8, in some cases, the linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO: 385), wherein n is 9, and in some cases, the linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO: 386), wherein n is 10.In some cases, the linker comprises the amino acid sequence AAAGG (SEQ ID NO: 387).

[0291] In some cases, the linker polypeptide present in the first polypeptide of the TMMP of the present disclosure includes a cysteine ​​residue that can form a disulfide bond with a cysteine ​​residue present in the second polypeptide of the TMMP of the present disclosure. In some cases, for example, a suitable linker comprises the amino acid sequence G C GGSGGGGSGGGGS (SEQ ID NO: 317). As another example, a suitable linker can comprise the amino acid sequence GCGGS(G4S)n (SEQ ID NO: 315), wherein n is 1, 2, 3, 4, 5, 6, 7, 8, or 9. For example, in some cases, the linker comprises the amino acid sequence GCGGSGGGGSGGGGSGGGGS (SEQ ID NO: 316). As another example, the linker comprises the amino acid sequence GCGGSGGGGSGGGGS (SEQ ID NO: 317).

[0292] Epitope

[0293] In some cases, the epitope (peptide presenting one or more epitopes) present in the TMMP of the present disclosure is a WT-1 peptide, for example, a WT-1 peptide that presents the epitope to the TCR in conjunction with MHC. The amino acid sequence of the WT-1 subtype is presented in Figures 3A-3E A WT-1 peptide presenting one or more epitopes is referred to herein as a "WT-1 peptide" or "WT-1 epitope." In some cases, the WT-1 epitope present in a TMMP of the present disclosure may be Figures 3A-3E A peptide of 4 to 25 consecutive amino acids (e.g., 4 amino acids (aa), 5 aa, 6 aa, 7 aa, 8 aa, 9 aa, 10-15 aa, 15-20 aa, or 20-25 aa) having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to any of the WT-1 amino acid sequences depicted in . In some cases, the WT-1 epitope present in the TMMP of the present disclosure may be a peptide of 4 to 25 consecutive amino acids (e.g., 4 amino acids (aa), 5 aa, 6 aa, 7 aa, 8 aa, 9 aa, 10-15 aa, 15-20 aa, or 20-25 aa) having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to any of the WT-1 amino acid sequences depicted in . Figure 3A A peptide of 4 to 25 consecutive amino acids (e.g., 4 amino acids (aa), 5 aa, 6 aa, 7 aa, 8 aa, 9 aa, 10-15 aa, 15-20 aa, or 20-25 aa) having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the WT-1 amino acid sequence depicted in . In some cases, the WT-1 epitope present in the TMMP of the present disclosure may be a peptide of 4 to 25 consecutive amino acids (e.g., 4 amino acids (aa), 5 aa, 6 aa, 7 aa, 8 aa, 9 aa, 10-15 aa, 15-20 aa, or 20-25 aa) having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the WT-1 amino acid sequence depicted in . Figure 3BA peptide of 4 to 25 consecutive amino acids (e.g., 4 amino acids (aa), 5 aa, 6 aa, 7 aa, 8 aa, 9 aa, 10-15 aa, 15-20 aa, or 20-25 aa) having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the WT-1 amino acid sequence depicted in . In some cases, the WT-1 epitope present in the TMMP of the present disclosure may be a peptide of 4 to 25 consecutive amino acids (e.g., 4 amino acids (aa), 5 aa, 6 aa, 7 aa, 8 aa, 9 aa, 10-15 aa, 15-20 aa, or 20-25 aa) having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the WT-1 amino acid sequence depicted in . Figure 3C A peptide of 4 to 25 consecutive amino acids (e.g., 4 amino acids (aa), 5 aa, 6 aa, 7 aa, 8 aa, 9 aa, 10-15 aa, 15-20 aa, or 20-25 aa) having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the WT-1 amino acid sequence depicted in . In some cases, the WT-1 epitope present in the TMMP of the present disclosure may be a peptide of 4 to 25 consecutive amino acids (e.g., 4 amino acids (aa), 5 aa, 6 aa, 7 aa, 8 aa, 9 aa, 10-15 aa, 15-20 aa, or 20-25 aa) having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the WT-1 amino acid sequence depicted in . Figure 3D A peptide of 4 to 25 consecutive amino acids (e.g., 4 amino acids (aa), 5 aa, 6 aa, 7 aa, 8 aa, 9 aa, 10-15 aa, 15-20 aa, or 20-25 aa) having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the WT-1 amino acid sequence depicted in . In some cases, the WT-1 epitope present in the TMMP of the present disclosure may be a peptide of 4 to 25 consecutive amino acids (e.g., 4 amino acids (aa), 5 aa, 6 aa, 7 aa, 8 aa, 9 aa, 10-15 aa, 15-20 aa, or 20-25 aa) having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the WT-1 amino acid sequence depicted in . Figure 3EIn some cases, the WT-1 epitope present in the TMMP of the present invention is a peptide of 4 to 25 consecutive amino acids (e.g., 4 amino acids (aa), 5 aa, 6 aa, 7 aa, 8 aa, 9 aa, 10-15 aa, 15-20 aa, or 20-25 aa) 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 the WT-1 amino acid sequence depicted in the disclosure. In some cases, the WT-1 epitope present in the TMMP of the present invention is 6 amino acids in length. In some cases, the WT-1 epitope present in the TMMP of the present invention is 7 amino acids in length. In some cases, the WT-1 epitope present in the TMMP of the present invention is 8 amino acids in length. In some cases, the WT-1 epitope present in the TMMP of the present invention is 9 amino acids in length. In some cases, the WT-1 epitope present in the TMMP of the present invention is 10 amino acids in length. In some cases, the WT-1 epitope present in the TMMP of the present invention is 11 amino acids in length. In some cases, the WT-1 epitope present in a TMMP of the present disclosure is 6 amino acids to 25 amino acids in length. In some cases, the WT-1 epitope present in a TMMP of the present disclosure is 6 amino acids to 20 amino acids in length. In some cases, the WT-1 epitope present in a TMMP of the present disclosure is 7 amino acids to 25 amino acids in length. In some cases, the WT-1 epitope present in a TMMP of the present disclosure is 7 amino acids to 20 amino acids in length. In some cases, the WT-1 epitope present in a TMMP of the present disclosure is at least 4 amino acids in length, at least 6 amino acids in length, or at least 7 amino acids in length.

[0294] The epitope present in the TMMP of the present disclosure may have a length of about 4 amino acids to about 25 amino acids, for example, the epitope may have a length of 4 amino acids (aa) to 10 aa, 10 aa to 15 aa, 15 aa to 20 aa, or 20 aa to 25 aa. For example, the epitope present in the TMMP of the present disclosure may have a length of 4 amino acids (aa), 5 aa, 6 aa, 7 aa, 8 aa, 9 aa, 10 aa, 11 aa, 12 aa, 13 aa, 14 aa, 15 aa, 16 aa, 17 aa, 18 aa, 19 aa, 20 aa, 21 aa, 22 aa, 23 aa, 24 aa, or 25 aa. In some cases, the epitope present in a TMMP has a length of 5 amino acids to 10 amino acids, eg, 5 aa, 6 aa, 7 aa, 8 aa, 9 aa, or 10 aa.

[0295] The WT-1 epitope present in the TMMP of the present disclosure is a peptide that is specifically bound by a T cell, i.e., the epitope is specifically bound by a WT-1 epitope-specific T cell. The epitope-specific T cell binds to the epitope having the reference amino acid sequence, but does not substantially bind to an epitope that differs from the reference amino acid sequence. For example, the epitope-specific T cell binds to the epitope having the reference amino acid sequence, and binds to an epitope that differs from the reference amino acid sequence, if any, with an affinity of less than 10 -6 M, less than 10 -5 M or less than 10 -4 M. Epitope-specific T cells can bind to the epitope for which they are specific with an affinity of at least 10 -7 M, at least 10 -8 M, at least 10 -9 M or at least 10 -10 M.

[0296] Examples of WT-1 peptides suitable for inclusion in the TMMPs of the present disclosure include, but are not limited to, CMTWNQMNLGATLKG (SEQ ID NO: 223), WNQMNLGATLKGVAA (SEQ ID NO: 224), CMTWNYMNLGATLKG (SEQ ID NO: 225), WNYMNLGATLKGVAA (SEQ ID NO: 226), MTWNQMNLGATLKGV (SEQ ID NO: 227), TWNQMNLGATLKGVA (SEQ ID NO: 228), CMTWNLMNLGATLKG (SEQ ID NO: 229), MTWNLMNLGATLKGV (SEQ ID NO: 230), TWNLMNLGATLKGVA (SEQ ID NO: 231), WNLMNLGATLKGVAA (SEQ ID NO: 232), MNLGATLK (SEQ ID NO: 233), MTWNYMNLGATLKGV (SEQ ID NO: 234), TWNYMNLGATLKGVA (SEQ ID NO: 235), CMTWNQMNLGATLKGVA (SEQ ID NO: 236), CMTWNLMNLGATLKGVA (SEQ ID NO: 237), CMTWNYMNLGATLKGVA (SEQ ID NO: 238), GYLRNPTAC (SEQ ID NO: 239), GALRNPTAL (SEQ ID NO: 240), YALRNPTAC (SEQ ID NO: 241), GLLRNPTAC (SEQ ID NO: 242), RYRPHPGAL (SEQ ID NO: 243), YQRPHPGAL (SEQ ID NO: 244), RLRPHPGAL (SEQ ID NO: 245), RIRPHPGAL (SEQ ID NO: 246), QFPNHSFKHEDPMGQ (SEQ ID NO: 247), HSFKHEDPY (SEQ ID NO: 248), QFPNHSFKHEDPM (SEQ ID NO: 249), QFPNHSFKHEDPY (SEQ ID NO: 250), KRPFMCAYPGCNK (SEQ ID NO: 251), KRPFMCAYPGCYK (SEQ ID NO: 252), FMCAYPGCY (SEQ ID NO: 253), FMCAYPGCK (SEQ ID NO: 254), KRPFMCAYPGCNKRY (SEQ ID NO: 255), SEKRPFMCAYPGCNK (SEQ IDNO: 256), KRPFMCAYPGCYKRY (SEQ ID NO: 257), NLMNLGATL (SEQ ID NO: 258), VLDFAPPGA (SEQ ID NO: 259); RMFPNAPYL (SEQ ID NO: 260); CMTWNQMN (SEQ ID NO: 261); CYTWNQMNL (SEQ ID NO: 269); NYMNLGATL (SEQ ID NO: 263); YMFPNAPYL (SEQ ID NO: 264); SLGEQQYSV (SEQ ID NO: 265); CMTWNQMNL (SEQ ID NO: 266); and NQMNLGATL (SEQ ID NO: 267). In some cases, the WT-1 peptide present in the TMMPs of the present disclosure is CMTWNQMN (SEQ ID NO: 261). In some cases, the WT-1 peptide present in the TMMPs of the present disclosure is CYTWNQMNL (SEQ ID NO: 269).

[0297] In some cases, the WT-1 peptide present in the TMMPs of the present disclosure presents an HLA-A*2402 restricted epitope. WT-1 peptides presenting an HLA-A*2402 restricted epitope include, for example, CMTWNQMN (SEQ ID NO: 261); NYMNLGATL (SEQ ID NO: 263) (WT-1 239-247; Q240Y); CYTWNQMNL (SEQ ID NO: 269) (WT-1 235-243); CMTWNQMNL (SEQ ID NO: 266) (WT-1 235-243); NQMNLGATL (SEQ ID NO: 267) (WT-1 239-247); and NLMNLGATL (SEQ ID NO: 258) (WT-1 239-247; Q240L).

[0298] In some cases, the WT-1 peptide present in the TMMP of the present disclosure presents an HLA-A*0201 restricted epitope. WT-1 peptides presenting an HLA-A*0201 restricted epitope include, for example, VLDFAPPGA (SEQ ID NO: 259) (WT-1 37-45); RMFPNAPYL (SEQ ID NO: 260) (WT-1 126-134); YMFPNAPYL (SEQ ID NO: 264) (WT-1 126-134; R126Y); SLGEQQYSV (SEQ ID NO: 265) (WT-1 187-195); and NLMNLGATL (SEQ ID NO: 258) (WT-1 239-247; Q240L).

[0299] HLA / peptide binding assay

[0300] Whether a given peptide (e.g., a WT-1 peptide) binds to class I HLA (comprising an HLA heavy chain and a β2M polypeptide) and can effectively present epitopes to the TCR when bound to the HLA complex can be determined using any of a number of well-known methods. Assays include binding assays and T cell activation assays.

[0301] Cell-based binding assays

[0302] As an example, a cell-based peptide-induced stability assay can be used to determine peptide-HLA class I binding. In this assay, the target peptide is allowed to bind to TAP-deficient cells, i.e., cells that have a defective transporter associated with the antigen processing (TAP) machinery and therefore have fewer surface class I molecules. Such cells include, for example, the human T2 cell line (T2(174xCEM.T2; American Type Culture Collection (ATCC) No. CRL-1992). Henderson et al. (1992) Science 255:1264. Without cytoplasmic peptide-deficient TAP-mediated transport into the endoplasmic reticulum, the assembled class I complex is structurally unstable and is only transiently retained on the cell surface. However, when T2 cells are incubated with exogenous peptides capable of binding to class I, surface peptide-HLA class I complexes are stable and can be detected by flow cytometry using, for example, a pan-anti-class I monoclonal antibody. The stability and consequently prolonged lifespan of the peptide-HLA complex on the cell surface conferred by the addition of peptide confirms its identity. Analysis can be performed using flow cytometry, for example, where the pan-HLA class I antibody contains a fluorescent label. Binding of the peptide to various allelic forms of the HLA H chain can be tested by genetically modifying T2 cells to express the allele HLA H chain of interest.

[0303] The following is a non-limiting example of the use of the T2 assay to assess peptide binding to HLA A*0201. T2 cells were washed in cell culture medium and concentrated to 10 6 cells / ml. The peptide of interest was prepared in cell culture medium and serially diluted to provide concentrations of 200 μM, 100 μM, 20 μM, and 2 μM. Cells were mixed 1:1 with each peptide dilution to yield a final volume of 200 μL and final peptide concentrations of 100 μM, 50 μM, 10 μM, and 1 μM. The HLA A*0201-binding peptide, GILGFVFTL (SEQ ID NO: 395), and the non-HLA A*0201-restricted peptide, HPVGEADYF (SEQ ID NO: 396) (HLA-B*3501), were included as positive and negative controls, respectively. The cell / peptide mixture was maintained at 37°C under 5% CO2 for ten minutes; then incubated overnight at room temperature. The cells were then incubated at 37°C for 2 hours and stained with a fluorescently labeled anti-human HLA antibody. The cells were then washed twice with phosphate-buffered saline and analyzed using flow cytometry. The binding intensity was measured using the mean fluorescence intensity (MFI) of anti-HLA antibody staining.

[0304] Biochemical binding assay

[0305] Binding of an HLA polypeptide (a complex of an HLA heavy chain polypeptide and a β2M polypeptide) to a target peptide can be tested in a cell-free in vitro assay system. For example, a labeled reference peptide (e.g., a fluorescent label) is bound to an HLA polypeptide (a complex of an HLA heavy chain polypeptide and a β2M polypeptide) to form an HLA-reference peptide complex. The ability of the target test peptide to displace the labeled reference peptide from the HLA-reference peptide complex is tested. The relative binding affinity is calculated as the amount of test peptide required to displace the bound reference peptide. See, for example, van der Burg et al. (1995) Human Immunol. 44:189.

[0306] As another example, the target peptide can be incubated with an HLA molecule (a complex of an HLA heavy chain and a β2M polypeptide), and the stability of the HLA / peptide complex can be measured in an immunoassay format. The ability of the target peptide to stabilize the HLA molecule is compared to the ability of a control peptide that presents a known T cell epitope. Stability is detected based on the presence or absence of the native conformation of the HLA / peptide complex as detected using an anti-HLA antibody. See, for example, Westrop et al. (2009) J. Immunol. Methods 341:76; Steinitz et al. (2012) Blood 119:4073; and U.S. Patent No. 9,205,144.

[0307] T cell activation assay

[0308] Whether a given peptide binds to HLA class I (comprising an HLA heavy chain and a β2M polypeptide) and whether it can effectively present the epitope to the TCR when bound to the HLA complex can be determined by assessing the T cell response to the peptide-HLA complex. Measurable T cell responses include, for example, interferon-γ (IFNγ) production, cytotoxic activity, etc.

[0309] ELISPOT assay

[0310] Suitable assays include, for example, the enzyme-linked immunospot (ELISPOT) assay. In this assay, CD8 T cells are measured after presentation of a target peptide in complex with HLA class I antigen-presenting cells (APCs). + IFNγ production by T cells. Antibodies against IFNγ are fixed to the wells of a multi-well plate. APCs are added to the wells and incubated with the target peptide for a period of time to allow the peptide to bind to HLA class I on the surface of the APCs. CD8 T cells specific for the peptide are then injected into the wells. + T cells are added to the wells and the plate is incubated for about 24 hours. The wells are then washed and any IFNγ bound to the fixed anti-IFNγ antibody is detected using a detectably labeled anti-IFNγ antibody. A colorimetric assay can be used. For example, the detectably labeled anti-IFNγ antibody can be a biotinylated anti-IFNγ antibody, which can be detected using, for example, a streptavidin conjugated to alkaline phosphatase. A BCIP / NBT (5-bromo-4-chloro-3-indolyl phosphate / nitro blue tetrazolium) solution is added to develop the assay. The presence of IFNγ-secreting T cells is identified by staining. A negative control includes APCs that have not been contacted with the peptide. APCs expressing various HLA H chain alleles can be used to determine whether the target peptide effectively binds to an HLA class I molecule comprising a specific HLA H chain.

[0311] Cytotoxicity assay

[0312] Whether a given peptide binds to a specific HLA class I H chain and can effectively present the epitope to the TCR when bound to an HLA class I complex containing the H chain can also be determined using cytotoxicity assays. Cytotoxicity assays involve incubating target cells with cytotoxic CD8 + The target cells are cultured with HLA class I T cells. The target cells display on their surface a peptide / HLA class I complex containing the peptide of interest and an HLA class I molecule containing the HLA H chain to be tested. The target cells can be radiolabeled, for example 51 Cr radiolabeled. Whether the target cells effectively present epitopes to cytotoxic CD8 + TCR on T cells, thereby CD8 + T cell-induced cytotoxic activity against target cells is measured by51 Cr is determined by release from lysed target cells. Specific cytotoxicity can be calculated as the amount of cytotoxic activity in the presence of the peptide minus the amount of cytotoxic activity in the absence of the peptide.

[0313] Detection of antigen-specific T cells using peptide-HLA tetramers

[0314] As another example, fluorescent or heavy metal tags are used to generate polymers (e.g., tetramers) of peptide-HLA complexes. The polymers can then be used to identify and quantify specific T cells via flow cytometry (FACS) or mass cytometry (CyTOF). Detection of epitope-specific T cells provides direct evidence that peptide-bound HLA molecules are able to bind to specific TCRs on antigen-specific T cell subsets. See, for example, Klenerman et al. (2002) Nature Reviews Immunol. 2: 263.

[0315] Immunomodulatory peptides

[0316] In some cases, the immunomodulatory polypeptide present in the TMMP of the present invention is a wild-type immunomodulatory polypeptide. In other cases, the immunomodulatory polypeptide present in the TMMP of the present invention is a variant immunomodulatory polypeptide whose affinity for a co-immunomodulatory polypeptide is reduced compared to the affinity of a corresponding wild-type immunomodulatory polypeptide for a co-immunomodulatory polypeptide. A suitable immunomodulatory domain that exhibits a reduced affinity for a co-immunomodulatory domain may have 1 amino acid (aa) to 20aa differences compared to a wild-type immunomodulatory domain. For example, in some cases, the amino acid sequence of the variant immunomodulatory polypeptide present in the TMMP of the present invention differs from the corresponding wild-type immunomodulatory polypeptide by 1aa, 2aa, 3aa, 4aa, 5aa, 6aa, 7aa, 8aa, 9aa or 10aa. As another example, in some cases, the amino acid sequence of the variant immunomodulatory polypeptide present in the TMMP of the present invention differs from the corresponding wild-type immunomodulatory polypeptide by 11 aa, 12 aa, 13 aa, 14 aa, 15 aa, 16 aa, 17 aa, 18 aa, 19 aa or 20 aa. As an example, in some cases, the variant immunomodulatory polypeptide present in the TMMP of the present invention comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions compared to the corresponding reference (e.g., wild-type) immunomodulatory polypeptide. In some cases, the variant immunomodulatory polypeptide present in the TMMP of the present invention comprises a single amino acid substitution compared to the corresponding reference (e.g., wild-type) immunomodulatory polypeptide. In some cases, the variant immunomodulatory polypeptide present in the TMMP of the present invention comprises 2 amino acid substitutions (e.g., no more than 2 amino acid substitutions) compared to the corresponding reference (e.g., wild-type) immunomodulatory polypeptide. In some cases, the variant immunomodulatory polypeptides present in the TMMPs of the present invention include 3 amino acid substitutions (e.g., no more than 3 amino acid substitutions) compared to the corresponding reference (e.g., wild-type) immunomodulatory polypeptide. In some cases, the variant immunomodulatory polypeptides present in the TMMPs of the present invention include 4 amino acid substitutions (e.g., no more than 4 amino acid substitutions) compared to the corresponding reference (e.g., wild-type) immunomodulatory polypeptide. In some cases, the variant immunomodulatory polypeptides present in the TMMPs of the present invention include 5 amino acid substitutions (e.g., no more than 5 amino acid substitutions) compared to the corresponding reference (e.g., wild-type) immunomodulatory polypeptide. In some cases, the variant immunomodulatory polypeptides present in the TMMPs of the present invention include 6 amino acid substitutions (e.g., no more than 6 amino acid substitutions) compared to the corresponding reference (e.g., wild-type) immunomodulatory polypeptide. In some cases, the variant immunomodulatory polypeptides present in the TMMPs of the present invention include 7 amino acid substitutions (e.g., no more than 7 amino acid substitutions) compared to the corresponding reference (e.g., wild-type) immunomodulatory polypeptide.In some cases, the variant immunomodulatory polypeptides present in the TMMPs of the present disclosure comprise 8 amino acid substitutions (e.g., no more than 8 amino acid substitutions) compared to the corresponding reference (e.g., wild-type) immunomodulatory polypeptide. In some cases, the variant immunomodulatory polypeptides present in the TMMPs of the present disclosure comprise 9 amino acid substitutions (e.g., no more than 9 amino acid substitutions) compared to the corresponding reference (e.g., wild-type) immunomodulatory polypeptide. In some cases, the variant immunomodulatory polypeptides present in the TMMPs of the present disclosure comprise 10 amino acid substitutions (e.g., no more than 10 amino acid substitutions) compared to the corresponding reference (e.g., wild-type) immunomodulatory polypeptide.

[0317] In some cases, a variant immunomodulatory polypeptide present in a TMMP of the present disclosure comprises 11 amino acid substitutions (eg, no more than 11 amino acid substitutions) compared to a corresponding reference (eg, wild-type) immunomodulatory polypeptide.

[0318] In some cases, a variant immunomodulatory polypeptide present in a TMMP of the present disclosure comprises 12 amino acid substitutions (eg, no more than 12 amino acid substitutions) compared to a corresponding reference (eg, wild-type) immunomodulatory polypeptide.

[0319] In some cases, a variant immunomodulatory polypeptide present in a TMMP of the present disclosure comprises 13 amino acid substitutions (eg, no more than 13 amino acid substitutions) compared to a corresponding reference (eg, wild-type) immunomodulatory polypeptide.

[0320] In some cases, a variant immunomodulatory polypeptide present in a TMMP of the present disclosure comprises 14 amino acid substitutions (eg, no more than 14 amino acid substitutions) compared to a corresponding reference (eg, wild-type) immunomodulatory polypeptide.

[0321] In some cases, a variant immunomodulatory polypeptide present in a TMMP of the present disclosure comprises 15 amino acid substitutions (eg, no more than 15 amino acid substitutions) compared to a corresponding reference (eg, wild-type) immunomodulatory polypeptide.

[0322] In some cases, a variant immunomodulatory polypeptide present in a TMMP of the present disclosure comprises 16 amino acid substitutions (eg, no more than 16 amino acid substitutions) compared to a corresponding reference (eg, wild-type) immunomodulatory polypeptide.

[0323] In some cases, a variant immunomodulatory polypeptide present in a TMMP of the present disclosure comprises 17 amino acid substitutions (eg, no more than 17 amino acid substitutions) compared to a corresponding reference (eg, wild-type) immunomodulatory polypeptide.

[0324] In some cases, a variant immunomodulatory polypeptide present in a TMMP of the present disclosure comprises 18 amino acid substitutions (eg, no more than 18 amino acid substitutions) compared to a corresponding reference (eg, wild-type) immunomodulatory polypeptide.

[0325] In some cases, a variant immunomodulatory polypeptide present in a TMMP of the present disclosure comprises 19 amino acid substitutions (eg, no more than 19 amino acid substitutions) compared to a corresponding reference (eg, wild-type) immunomodulatory polypeptide.

[0326] In some cases, a variant immunomodulatory polypeptide present in a TMMP of the present disclosure comprises 20 amino acid substitutions (eg, no more than 20 amino acid substitutions) compared to a corresponding reference (eg, wild-type) immunomodulatory polypeptide.

[0327] As discussed above, variant immunomodulatory polypeptides suitable for inclusion in the TMMPs of the present disclosure exhibit a reduced affinity for a cognate co-immunomodulatory polypeptide compared to the affinity of the corresponding wild-type immunomodulatory polypeptide for the cognate co-immunomodulatory polypeptide.

[0328] Exemplary pairs of immunomodulatory polypeptides and cognate co-immunomodulatory polypeptides include, but are not limited to:

[0329] a) 4-1BBL (immunomodulatory polypeptide) and 4-1BB (homologous co-immunomodulatory polypeptide);

[0330] b) PD-L1 (immunomodulatory polypeptide) and PD1 (homologous co-immunomodulatory polypeptide);

[0331] c) IL-2 (immunomodulatory polypeptide) and IL-2 receptor (homologous co-immunomodulatory polypeptide);

[0332] d) CD80 (immunomodulatory polypeptide) and CD86 (homologous co-immunomodulatory polypeptide);

[0333] e) CD86 (immunomodulatory polypeptide) and CD28 (homologous co-immunomodulatory polypeptide);

[0334] f) OX40L (CD252) (immunomodulatory polypeptide) and OX40 (CD134) (homologous co-immunomodulatory polypeptide);

[0335] g) Fas ligand (immunomodulatory polypeptide) and Fas (homologous co-immunomodulatory polypeptide);

[0336] h) ICOS-L (immunomodulatory polypeptide) and ICOS (homologous co-immunomodulatory polypeptide);

[0337] i) ICAM (immunomodulatory polypeptide) and LFA-1 (homologous co-immunomodulatory polypeptide);

[0338] j) CD30L (immunomodulatory polypeptide) and CD30 (homologous co-immunomodulatory polypeptide);

[0339] k) CD40 (immunomodulatory polypeptide) and CD40L (homologous co-immunomodulatory polypeptide);

[0340] l) CD83 (immunomodulatory polypeptide) and CD83L (homologous co-immunomodulatory polypeptide);

[0341] m) HVEM (CD270) (immunomodulatory polypeptide) and CD160 (homologous co-immunomodulatory polypeptide);

[0342] n) JAG1 (CD339) (immunomodulatory polypeptide) and Notch (homologous co-immunomodulatory polypeptide);

[0343] o) JAG1 (immunomodulatory polypeptide) and CD46 (homologous co-immunomodulatory polypeptide);

[0344] p) CD80 (immunomodulatory polypeptide) and CTLA4 (homologous co-immunomodulatory polypeptide);

[0345] q) CD86 (immunomodulatory polypeptide) and CTLA4 (cognate co-immunomodulatory polypeptide); and

[0346] r) CD70 (immunomodulatory polypeptide) and CD27 (homologous co-immunomodulatory polypeptide).

[0347] In some cases, the variant immunomodulatory polypeptides present in the TMMPs of the present disclosure have a binding affinity for a cognate co-immunomodulatory polypeptide of 100 nM to 100 μM. For example, in some cases, the variant immunomodulatory polypeptides present in the TMMPs of the present disclosure have a binding affinity for a cognate co-immunomodulatory polypeptide of about 100 nM to 150 nM, about 150 nM to about 200 nM, about 200 nM to about 250 nM, about 250 nM to about 300 nM, about 300 nM to about 350 nM, about 350 nM to about 400 nM, about 400 nM to about 500 nM, about 500 nM to about 1000 nM, about 1000 nM to about 1500 nM, about 1500 nM to about 2000 nM, about 2000 nM to about 2500 nM, about 2500 nM to about 3000 nM, about 3000 nM to about 3500 nM, about 3500 nM to about 4000 nM, about 4000 nM to about 5000 nM, about 5000 nM to about 10000 nM, about About 600 nM, about 600 nM to about 700 nM, about 700 nM to about 800 nM, about 800 nM to about 900 nM, about 900 nM to about 1 μM to about 1 μM to about 5 μM, about 5 μM to about 10 μM, about 10 μM to about 15 μM, about 15 μM to about 20 μM, about 20 μM to about 25 μM, about 25 μM to about 50 μM, about 50 μM to about 75 μM, or about 75 μM to about 100 μM.

[0348] The variant immunomodulatory polypeptides present in the TMMPs of the present disclosure exhibit a reduced affinity for the homologous co-immunomodulatory polypeptides. Similarly, the TMMPs of the present disclosure comprising variant immunomodulatory polypeptides exhibit a reduced affinity for the homologous co-immunomodulatory polypeptides. Thus, for example, the TMMPs of the present disclosure comprising variant immunomodulatory polypeptides have a binding affinity of 100 nM to 100 μM for the homologous co-immunomodulatory polypeptides. For example, in some cases, the TMMPs of the present disclosure comprising variant immunomodulatory polypeptides have the following binding affinities for the homologous co-immunomodulatory polypeptides: about 100 nM to 150 nM, about 150 nM to about 200 nM, about 200 nM to about 250 nM, about 250 nM to about 300 nM, about 300 nM to about 350 nM, about 350 nM to about 400 nM, about 400 nM to about 500 nM, about 500 nM to about 600 nM, about 600 nM to about 700 nM, about 700 nM to about 800 nM, about 800 nM to about 900 nM, about 900 nM to about 1000 nM, about 1000 nM to about 15 ... About 600 nM, about 600 nM to about 700 nM, about 700 nM to about 800 nM, about 800 nM to about 900 nM, about 900 nM to about 1 μM to about 1 μM to about 5 μM, about 5 μM to about 10 μM, about 10 μM to about 15 μM, about 15 μM to about 20 μM, about 20 μM to about 25 μM, about 25 μM to about 50 μM, about 50 μM to about 75 μM, or about 75 μM to about 100 μM.

[0349] like Figure 19 As schematically shown, an immunomodulatory polypeptide (ie, one or more immunomodulatory polypeptides) may be present at any of a variety of locations on a TMMP of the present disclosure. Figure 19 The positions of the two copies of the variant IL-2 polypeptide are depicted; however, the immunomodulatory polypeptide can be any of a variety of immunomodulatory polypeptides as described herein. Figure 19 As depicted, the immunomodulatory polypeptide can be: 1) at the N-terminus of the class I MHC heavy chain; 2) at the C-terminus of the class I MHC heavy chain and the N-terminus of the Ig Fc polypeptide; in other words, between the class I MHC heavy chain and the Ig Fc polypeptide; 3) at the C-terminus of the Ig Fc polypeptide; 4) at the N-terminus of a peptide epitope; or 5) at the C-terminus of a β2M polypeptide.

[0350] PD-L1 variants

[0351] As a non-limiting example, in some cases, the variant immunomodulatory polypeptide present in the TMMP of the present disclosure is a variant PD-L1 polypeptide. Wild-type PD-L1 binds to PD1.

[0352] The wild-type human PD-L1 polypeptide may comprise the following amino acid sequence: MRIFAVFIFM TYWHLLNAFTVTVPKDLYVV EYGSNMTIEC KFPVEKQLDL AALIVYWEME DKNIIQFVHG EEDLKVQHSS YRQRARLLKDQLSLGNAALQ ITDVKLQDAG VYRCMISYGG ADYKRITVKV NAPYNKINQR ILVVDPVTSE HELTCQAEGYPKAEVIWTSS DHQVLSGKTT TTNSKREEKL FNVTSTLRIN TTTNEIFYCT FRRLDPEENH TAELVIPGNILNVSIKICLT LSPST (SEQ ID NO: 1).

[0353] The wild-type human PD-L1 extracellular domain may comprise the following amino acid sequence: FT VTVPKDLYVV EYGSNMTIECKFPVEKQLDL AALIVYWEME DKNIIQFVHG EEDLKVQHSS YRQRARLLKD QLSLGNAALQ ITDVKLQDAGVYRCMISYGG ADYKRITVKV NAPYNKINQR ILVVDPVTSE HELTCQAEGY PKAEVIWTSS DHQVLSGKTTTTNSKREEKL FNVTSTLRIN TTTNEIFYCT FRRLDPEENH TAELVIPGNI LNVSIKI (SEQ ID NO: 2).

[0354] The wild-type PD-1 polypeptide may comprise the following amino acid sequence: PGWFLDSPDR PWNPPTFSPA LLVVTEGDNATFTCSFSNTS ESFVLNWYRM SPSNQTDKLA AFPEDRSQPG QDCRFRVTQL PNGRDFHMSV VRARRNDSGTYLCGAISLAP KAQIKESLRA ELRVTERRAE VPTAHPSPSP RPAGQFQTLV VGVVGGLLGS LVLLVWVLAVICSRAARGTI GARRTGQPLK EDPSAVPVFS VDYGELDFQW REKTPEPPVP CVPEQTEYAT IVFPSGMGTSSPARRGSADG PRSAQPLRPE DGHCSWPL (SEQ ID NO: 3). In some cases, when the TMMP of the present disclosure comprises a variant PD-L1 polypeptide, the "cognate co-immunomodulatory polypeptide" is a PD-1 polypeptide comprising the amino acid sequence of SEQ ID NO: 3.

[0355] In some instances, the variant PD-L1 polypeptide exhibits reduced binding affinity to PD-1 (e.g., a PD-1 polypeptide comprising the amino acid sequence set forth in SEQ ID NO:3) as compared to the binding affinity of a PD-L1 polypeptide comprising the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:2. For example, in some cases, the variant PD-L1 polypeptides of the present disclosure bind to PD-1 (e.g., a PD-1 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 3) with a binding affinity that is at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or greater than 95% less than the binding affinity of a PD-L1 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2.

[0356] In some cases, the variant PD-L1 polypeptide has a binding affinity for PD-1 of 1 nM to 1 mM. In some cases, the variant PD-L1 polypeptides of the present disclosure have a binding affinity for PD-1 of 100 nM to 100 μM. As another example, in some cases, the variant PD-L1 polypeptide has a binding affinity for PD1 (e.g., a PD1 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 3) of about 100 nM to 150 nM, about 150 nM to about 200 nM, about 200 nM to about 250 nM, about 250 nM to about 300 nM, about 300 nM to about 350 nM, about 350 nM to about 400 nM, about 400 nM to about 500 nM, about 500 nM to about 600 nM, about 600 nM to about 700 nM, about 700 nM to about 800 nM, about 800 nM to about 900 nM, about 900 nM to about 1000 nM, about 1000 nM to about 1500 nM, about 1500 nM to about 200 nM, about 200 nM to about 250 nM, about 250 nM to about 300 nM, about 300 nM to about 350 nM, about 350 nM to about 400 nM, about 400 nM to about 500 nM, about 500 nM to about 600 nM, about 600 nM to about 700 nM, about 700 nM to about 800 nM, about 800 nM to about 900 nM, about 900 nM to about 1 μM to about 1 μM to about 5 μM, about 5 μM to about 10 μM, about 10 μM to about 15 μM, about 15 μM to about 20 μM, about 20 μM to about 25 μM, about 25 μM to about 50 μM, about 50 μM to about 75 μM, or about 75 μM to about 100 μM.

[0357] In some cases, the variant PD-L1 polypeptide has a single amino acid substitution compared to the PD-L1 amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2. In some cases, the variant PD-L1 polypeptide has 2 to 10 amino acid substitutions compared to the PD-L1 amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2. In some cases, the variant PD-L1 polypeptide has 2 amino acid substitutions compared to the PD-L1 amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2. In some cases, the variant PD-L1 polypeptide has 3 amino acid substitutions compared to the PD-L1 amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2. In some cases, the variant PD-L1 polypeptide has 4 amino acid substitutions compared to the PD-L1 amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2. In some cases, the variant PD-L1 polypeptide has 5 amino acid substitutions compared to the PD-L1 amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2. In some cases, the variant PD-L1 polypeptide has 6 amino acid substitutions compared to the PD-L1 amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2. In some cases, the variant PD-L1 polypeptide has 7 amino acid substitutions compared to the PD-L1 amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2. In some cases, the variant PD-L1 polypeptide has 8 amino acid substitutions compared to the PD-L1 amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2. In some cases, the variant PD-L1 polypeptide has 9 amino acid substitutions compared to the PD-L1 amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2. In some cases, the variant PD-L1 polypeptide has 10 amino acid substitutions compared to the PD-L1 amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2.

[0358] Suitable PD-L1 variants include a polypeptide comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence:

[0359] FT VTVPK XLYVV EYGSNMTIEC KFPVEKQLDL AALIVY WEME DKNIIQFVHG EEDLKVQHSSYRQRARLLKD QLSLGNA ALQ ITDVKLQDAG VYRCMISYGG ADYKRITVKV NAPYNKIN QRILVVDPVTSE HELTCQAEGY PKAEVIWTSS DHQVLSGKTT TTNSKREEKL FNVTSTLRIN TTTNEIFYCTFRRLDPEENH TAE LVIPGNI LNVSIKI (SEQ ID NO: 52), wherein X is any amino acid except Asp. In some cases, X is Ala. In some cases, X is Arg.

[0360] Suitable PD-L1 variants include a polypeptide comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence:

[0361] FT VTVPKDLYVV EYGSNMTIEC KFPVEKQLDL AAL X VY WEME DKNIIQFVHG EEDLKVQHSSYRQRARLLKD QLSLGNA ALQ ITDVKLQDAG VYRCMISYGG ADYKRITVKV NAPYNKIN QRILVVDPVTSE HELTCQAEGY PKAEVIWTSS DHQVLSGKTT TTNSKREEKL FNVTSTLRIN TTTNEIFYCTFRRLDPEENH TAE LVIPGNI LNVSIKI (SEQ ID NO: 53), wherein X is any amino acid except He. In some cases, X is Asp.

[0362] Suitable PD-L1 variants include a polypeptide comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence:

[0363] FT VTVPKDLYVV EYGSNMTIEC KFPVEKQLDL AALIVY WEME DKNIIQFVHG E XDLKVQHSSYRQRARLLKD QLSLGNA ALQ ITDVKLQDAG VYRCMISYGG ADYKRITVKV NAPYNKIN QRILVVDPVTSE HELTCQAEGY PKAEVIWTSS DHQVLSGKTT TTNSKREEKL FNVTSTLRIN TTTNEIFYCTFRRLDPEENH TAE LVIPGNI LNVSIKI (SEQ ID NO: 54), wherein X is any amino acid except Glu. In some cases, X is Arg.

[0364] CD80 variants

[0365] In some cases, the variant immunomodulatory polypeptide present in the TMMP of the present disclosure is a variant CD80 polypeptide. Wild-type CD80 binds to CD28. Wild-type CD80 also binds to CD86.

[0366] The wild-type amino acid sequence of the extracellular domain of human CD80 may be as follows:

[0367] VIHVTK EVKEVATLSC GHNVSVEELA QTRIYWQKEK KM VLTMMSGD MNIWPEYKNRTIFDITNNLS IVILALRPSD EGTYE CVVLK YEKDAFKREH LAEVTLSVKA DFPTPSISDF EIPTSNIRRI ICSTSGGFPE PHLSWLENGE ELNAINTTVS QDPETELYAV SS KLDFNMTT NHSFMCLIKYGHLRVNQTFN WNTTKQEHFP DN (SEQ ID NO: 4).

[0368] The wild-type CD28 amino acid sequence may be as follows: MLRLLLALNL FPSIQVTG NK ILVKQSPMLVAYDNAVNLSC KYSYNLFSRE FRASLHKGL D SAVEVCVVYG NYSQQLQVYS KTGFNCDGKL GNESVTFYLQ NLYVNQTDIY FCKIEVMYPP PYLDNEKSNG TIIHVKGKHL CPSPLFPGPS KPFWVLVVVGGVLACYSLLV TVAFIIFWVR SKR SRLLHSD YMNMTPRRPG PTRKHYQPYA PPRDFAAYRS (SEQ ID NO: 5). In some cases, when the TMMP of the present disclosure comprises a variant CD80 polypeptide, the "cognate co-immunomodulatory polypeptide" is a CD28 polypeptide comprising the amino acid sequence of SEQ ID NO: 5.

[0369] The wild-type CD28 amino acid sequence may be as follows: MLRLLLALNL FPSIQVTG NK ILVKQSPMLVAYDNAVNLSW KHLCPSPLFP GPSKPFWVL V VVGGVLACYS LLVTVAFIIF WVRSKRSRLL HSDYMNMTPRRPGPTRKHYQ PYAPPRDFAA YRS (SEQ ID NO: 6)

[0370] The wild-type CD28 amino acid sequence may be as follows: MLRLLLALNL FPSIQVTG KH LCPSPLFPGPSKPFWVLVVV GGVLACYSLL VTVAFIIFWV RSKRSRLLHS DYMNMTPRRP GPTRKHYQPY APPRDFAAYRS (SEQ ID NO: 7).

[0371] In some cases, the variant CD80 polypeptide exhibits reduced binding affinity for CD28 compared to the binding affinity of the CD80 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 4. For example, in some cases, the variant CD80 polypeptide binds CD28 with an affinity that is at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or greater than 95% less than the binding affinity of a CD80 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 4 for CD28 (e.g., a CD28 polypeptide comprising the amino acid sequence set forth in one of SEQ ID NOs: 5, 6, or 7).

[0372] In some cases, the variant CD80 polypeptides have a binding affinity for CD28 of 100 nM to 100 μM. As another example, in some cases, the variant CD80 polypeptides of the present disclosure have a binding affinity for CD28 (e.g., a CD28 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7) of about 100 nM to 150 nM, about 150 nM to about 200 nM, about 200 nM to about 250 nM, about 250 nM to about 300 nM, about 300 nM to about 350 nM, about 350 nM to about 400 nM, about 400 nM to about 500 nM, about 500 nM to about 600 nM, about 600 nM to about 700 nM, about 800 nM to about 900 nM, about 900 nM to about 1000 nM, about 1000 nM to about 1500 nM, about 1500 nM to about 200 nM, about 200 nM to about 250 nM, about 250 nM to about 300 nM, about 300 nM to about 350 nM, about 350 nM to about 400 nM, about 400 nM to about 500 nM, about 500 nM to about 600 nM, about 600 nM to about 700 nM, about 700 nM to about 800 nM, about 800 nM to about 900 nM, about 900 nM to about 1 μM to about 1 μM to about 5 μM, about 5 μM to about 10 μM, about 10 μM to about 15 μM, about 15 μM to about 20 μM, about 20 μM to about 25 μM, about 25 μM to about 50 μM, about 50 μM to about 75 μM, or about 75 μM to about 100 μM.

[0373] In some cases, the variant CD80 polypeptide has a single amino acid substitution compared to the CD80 amino acid sequence set forth in SEQ ID NO: 4. In some cases, the variant CD80 polypeptide has 2 to 10 amino acid substitutions compared to the CD80 amino acid sequence set forth in SEQ ID NO: 4. In some cases, the variant CD80 polypeptide has 2 amino acid substitutions compared to the CD80 amino acid sequence set forth in SEQ ID NO: 4. In some cases, the variant CD80 polypeptide has 3 amino acid substitutions compared to the CD80 amino acid sequence set forth in SEQ ID NO: 4. In some cases, the variant CD80 polypeptide has 4 amino acid substitutions compared to the CD80 amino acid sequence set forth in SEQ ID NO: 4. In some cases, the variant CD80 polypeptide has 5 amino acid substitutions compared to the CD80 amino acid sequence set forth in SEQ ID NO: 4. In some cases, the variant CD80 polypeptide has 6 amino acid substitutions compared to the CD80 amino acid sequence set forth in SEQ ID NO: 4. In some cases, the variant CD80 polypeptide has 7 amino acid substitutions compared to the CD80 amino acid sequence set forth in SEQ ID NO: 4. In some cases, the variant CD80 polypeptide has 8 amino acid substitutions compared to the CD80 amino acid sequence set forth in SEQ ID NO: 4. In some cases, the variant CD80 polypeptide has 9 amino acid substitutions compared to the CD80 amino acid sequence set forth in SEQ ID NO: 4. In some cases, the variant CD80 polypeptide has 10 amino acid substitutions compared to the CD80 amino acid sequence set forth in SEQ ID NO: 4.

[0374] Suitable CD80 variants include a polypeptide comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to any of the following amino acid sequences:

[0375] VIHVTK EVKEVATLSC GH X VSVEELA QTRIYWQKEK KM VLTMMSGD MNIWPEYKNRTIFDITNNLS IVILALRPSD EGTYE CVVLK YEKDAFKREH LAEVTLSVKA DFPTPSISDF EIPTSNIRRI ICSTSGGFPE PHLSWLENGE ELNAINTTVS QDPETELYAV SS KLDFNMTT NHSFMCLIKYGHLRVNQTFN WNTTKQEHFP DN (SEQ ID NO: 55), wherein X is any amino acid except Asn. In some cases, X is Ala;

[0376] VIHVTK EVKEVATLSC GHNVSVEELA QTRIYWQKEK KM VLTMMSGD MNIWPEYKNRTIFDIT X NLS IVILALRPSD EGTYE CVVLK YEKDAFKREH LAEVTLSVKA DFPTPSISDF EIPTSNIRRI ICSTSGGFPE PHLSWLENGE ELNAINTTVS QDPETELYAV SS KLDFNMTT NHSFMCLIKYGHLRVNQTFN WNTTKQEHFP DN (SEQ ID NO: 56), wherein X is any amino acid except Asn. In some cases, X is Ala;

[0377] VIHVTK EVKEVATLSC GHNVSVEELA QTRIYWQKEK KM VLTMMSGD MNIWPEYKNRTIFDITNNLS X VILALRPSD EGTY ECVVLK YEKDAFKREH LAEVTLSVKA DFPTPSISDF EIPTSNIRRI ICSTSGGFPE PHLSWLENGE ELNAINTTVS QDPETELYAV S SKLDFNMTT NHSFMCLIKYGHLRVNQTFN WNTTKQEHFP DN (SEQ ID NO: 57), wherein X is any amino acid except Ile. In some cases, X is Ala;

[0378] VIHVTK EVKEVATLSC GHNVSVEELA QTRIYWQKEK KM VLTMMSGD MNIWPEYKNRTIFDITNNLS IVILALRPSD EGTYE CVVL X YEKDAFKREH LAEVTLSVKA DFPTPSISDF EIPTSNIRRI ICSTSGGFPE PHLSWLENGE ELNAINTTVS QDPETELYAV SS KLDFNMTT NHSFMCLIKYGHLRVNQTFN WNTTKQEHFP DN (SEQ ID NO: 58), wherein X is any amino acid except Lys. In some cases, X is Ala;

[0379] VIHVTK EVKEVATLSC GHNVSVEELA QTRIYWQKEK KM VLTMMSGD MNIWPEYKNRTIFDITNNLS IVILALRPSD EGTYE CVVLK YEKDAFKREH LAEVTLSVKA DFPTPSISDF EIPTSNIRRI ICSTSGGFPE PHLSWLENGE ELNAINTTVS X DPETELYAV SS KLDFNMTT NHSFMCLIKYGHLRVNQTFN WNTTKQEHFP DN (SEQ ID NO: 60), wherein X is any amino acid except Gln. In some cases, X is Ala;

[0380] VIHVTK EVKEVATLSC GHNVSVEELA QTRIYWQKEK KM VLTMMSGD MNIWPEYKNRTIFDITNNLS IVILALRPSD EGTYE CVVLK YEKDAFKREH LAEVTLSVKA DFPTPSISDF EIPTSNIRRI ICSTSGGFPE PHLSWLENGE ELNAINTTVS Q X PETELYAV SS KLDFNMTT NHSFMCLIKYGHLRVNQTFN WNTTKQEHFP DN (SEQ ID NO: 61), wherein X is any amino acid except Asp. In some cases, X is Ala;

[0381] VIHVTK EVKEVATLSC GHNVSVEE X A QTRIYWQKEK KM VLTMMSGD MNIWPEYKNRTIFDITNNLS IVILALRPSD EGTYE CVVLK YEKDAFKREH LAEVTLSVKA DFPTPSISDF EIPTSNIRRI ICSTSGGFPE PHLSWLENGE ELNAINTTVS QDPETELYAV SS KLDFNMTT NHSFMCLIKYGHLRVNQTFN WNTTKQEHFP DN (SEQ ID NO: 62), wherein X is any amino acid except Leu. In some cases, X is Ala;

[0382] VIHVTK EVKEVATLSC GHNVSVEELA QTRI XWQKEK KM VLTMMSGD MNIWPEYKNRTIFDITNNLS IVILALRPSD EGTYE CVVLK YEKDAFKREH LAEVTLSVKA DFPTPSISDF EIPTSNIRRI ICSTSGGFPE PHLSWLENGE ELNAINTTVS QDPETELYAV SS KLDFNMTT NHSFMCLIKYGHLRVNQTFN WNTTKQEHFP DN (SEQ ID NO: 63), wherein X is any amino acid except Tyr. In some cases, X is Ala;

[0383] VIHVTK EVKEVATLSC GHNVSVEELA QTRIYW X KEK KM VLTMMSGD MNIWPEYKNRTIFDITNNLS IVILALRPSD EGTYE CVVLK YEKDAFKREH LAEVTLSVKA DFPTPSISDF EIPTSNIRRI ICSTSGGFPE PHLSWLENGE ELNAINTTVS QDPETELYAV SS KLDFNMTT NHSFMCLIKYGHLRVNQTFN WNTTKQEHFP DN (SEQ ID NO: 64), wherein X is any amino acid except Gln. In some cases, X is Ala;

[0384] VIHVTK EVKEVATLSC GHNVSVEELA QTRIYWQKEK K X VLTMMSGD MNIWPEYKNRTIFDITNNLS IVILALRPSD EGTYE CVVLK YEKDAFKREH LAEVTLSVKA DFPTPSISDF EIPTSNIRRI ICSTSGGFPE PHLSWLENGE ELNAINTTVS QDPETELYAV SS KLDFNMTT NHSFMCLIKYGHLRVNQTFN WNTTKQEHFP DN (SEQ ID NO: 65), wherein X is any amino acid except Met. In some cases, X is Ala;

[0385] VIHVTK EVKEVATLSC GHNVSVEELA QTRIYWQKEK KM XLTMMSGD MNIWPEYKNRTIFDITNNLS IVILALRPSD EGTYE CVVLK YEKDAFKREH LAEVTLSVKA DFPTPSISDF EIPTSNIRRI ICSTSGGFPE PHLSWLENGE ELNAINTTVS QDPETELYAV SS KLDFNMTT NHSFMCLIKYGHLRVNQTFN WNTTKQEHFP DN (SEQ ID NO: 66), wherein X is any amino acid except Val. In some cases, X is Ala;

[0386] VIHVTK EVKEVATLSC GHNVSVEELA QTRIYWQKEK KM VLTMMSGD MN X WPEYKNRTIFDITNNLS IVILALRPSD EGTY ECVVLK YEKDAFKREH LAEVTLSVKA DFPTPSISDF EIPTSNIRRI ICSTSGGFPE PHLSWLENGE ELNAINTTVS QDPETELYAV S SKLDFNMTT NHSFMCLIKYGHLRVNQTFN WNTTKQEHFP DN (SEQ ID NO: 67), wherein X is any amino acid except Ile. In some cases, X is Ala;

[0387] VIHVTK EVKEVATLSC GHNVSVEELA QTRIYWQKEK KM VLTMMSGD MNIWPE X KNRTIFDITNNLS IVILALRPSD EGTYE CVVLK YEKDAFKREH LAEVTLSVKA DFPTPSISDF EIPTSNIRRI ICSTSGGFPE PHLSWLENGE ELNAINTTVS QDPETELYAV SS KLDFNMTT NHSFMCLIKYGHLRVNQTFN WNTTKQEHFP DN (SEQ ID NO: 68), wherein X is any amino acid except Tyr. In some cases, X is Ala;

[0388] VIHVTK EVKEVATLSC GHNVSVEELA QTRIYWQKEK KM VLTMMSGD MNIWPEYKNRTIF XITNNLS IVILALRPSD EGTYE CVVLK YEKDAFKREH LAEVTLSVKA DFPTPSISDF EIPTSNIRRI ICSTSGGFPE PHLSWLENGE ELNAINTTVS QDPETELYAV SS KLDFNMTT NHSFMCLIKYGHLRVNQTFN WNTTKQEHFP DN (SEQ ID NO: 69), wherein X is any amino acid except Asp. In some cases, X is Ala;

[0389] VIHVTK EVKEVATLSC GHNVSVEELA QTRIYWQKEK KM VLTMMSGD MNIWPEYKNRTIFDITNNLS IVILALRPSD EGTYE CVVLK YEKDAFKREH LAEVTLSVKA D X PTPSISDF EIPTSNIRRI ICSTSGGFPE PHLSWLENGE ELNAINTTVS QDPETELYAV S SKLDFNMTT NHSFMCLIKYGHLRVNQTFN WNTTKQEHFP DN (SEQ ID NO: 70), wherein X is any amino acid except Phe. In some cases, X is Ala;

[0390] VIHVTK EVKEVATLSC GHNVSVEELA QTRIYWQKEK KM VLTMMSGD MNIWPEYKNRTIFDITNNLS IVILALRPSD EGTYE CVVLK YEKDAFKREH LAEVTLSVKA DFPTPSISDF EIPTSNIRRI ICSTSGGFPE PHLSWLENGE ELNAINTTV X QDPETELYAV SS KLDFNMTT NHSFMCLIKYGHLRVNQTFN WNTTKQEHFP DN (SEQ ID NO: 71), wherein X is any amino acid except Ser. In some cases, X is Ala; and

[0391] VIHVTK EVKEVATLSC GHNVSVEELA QTRIYWQKEK KM VLTMMSGD MNIWPEYKNRTIFDITNNLS IVILALRPSD EGTYE CVVLK YEKDAFKREH LAEVTLSVKA DFPT XSISDF EIPTSNIRRI ICSTSGGFPE PHLSWLENGE ELNAINTTVS QDPETELYAV S SKLDFNMTT NHSFMCLIKYGHLRVNQTFN WNTTKQEHFP DN (SEQ ID NO: 72), wherein X is any amino acid except Pro. In some cases, X is Ala.

[0392] CD86 variants

[0393] In some cases, the variant immunomodulatory polypeptide present in the TMMP of the present disclosure is a variant CD86 polypeptide. Wild-type CD86 binds to CD28. In some cases, when the TMMP of the present disclosure comprises a variant CD86 polypeptide, the "cognate co-immunomodulatory polypeptide" is a CD28 polypeptide comprising the amino acid sequence of SEQ ID NO: 5.

[0394] The amino acid sequence of the entire extracellular domain of wild-type human CD86 may be as follows: APLKIQAYFNETADLPCQFANSQNQSLSELVVFWQDQENLVLNEVYLGKEKFDSVHSKYM N RTSF D SDS W TLRLHNLQIKDKGLYQCIIH H KKPTGMIRIHQMNSELSVLANFSQPEIVPISNITENVYINLTCSSIHGYPEPKKMSVLLRTKNSTIEYDGIMQKSQDNVTELYDVSISLSSVSFPDVTSNMTIFCILETDKTRLLSSPFSIELEDPQPPPDHIP (SEQ ID NO: 8).

[0395] The amino acid sequence of the IgV domain of wild-type human CD86 may be as follows: APLKIQAYFNETADLPCQFANSQNQSLSELVVFWQDQENLVLNE VYLGKEKFDSVHSKYM N RTSF D SDS W TLRLHNLQIKDKGLYQCIIH H KKPTGMIRIHQMNSELSVL (SEQ ID NO:9).

[0396] In some cases, the variant CD86 polypeptide exhibits reduced binding affinity for CD28 compared to the binding affinity of a CD86 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 8 or SEQ ID NO: 9. For example, in some cases, the variant CD86 polypeptide binds CD28 with an affinity that is at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or greater than 95% less than the binding affinity of a CD86 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 8 or SEQ ID NO: 9 (e.g., a CD28 polypeptide comprising the amino acid sequence set forth in one of SEQ ID NOs: 5, 6, or 7).

[0397] In some cases, the variant CD86 polypeptide has a binding affinity for CD28 of 100 nM to 100 μM. As another example, in some cases, the variant CD86 polypeptides of the present disclosure have a binding affinity for CD28 (e.g., a CD28 polypeptide comprising an amino acid sequence set forth in one of SEQ ID NOs: 5, 6, or 7) of about 100 nM to 150 nM, about 150 nM to about 200 nM, about 200 nM to about 250 nM, about 250 nM to about 300 nM, about 300 nM to about 350 nM, about 350 nM to about 400 nM, about 400 nM to about 500 nM, about 500 nM to about 600 nM, or about 700 nM to about 800 nM. About 600 nM to about 700 nM, about 700 nM to about 800 nM, about 800 nM to about 900 nM, about 900 nM to about 1 μM to about 1 μM to about 5 μM, about 5 μM to about 10 μM, about 10 μM to about 15 μM, about 15 μM to about 20 μM, about 20 μM to about 25 μM, about 25 μM to about 50 μM, about 50 μM to about 75 μM, or about 75 μM to about 100 μM.

[0398] In some cases, the variant CD86 polypeptide has a single amino acid substitution compared to the CD86 amino acid sequence set forth in SEQ ID NO: 8. In some cases, the variant CD86 polypeptide has 2 to 10 amino acid substitutions compared to the CD86 amino acid sequence set forth in SEQ ID NO: 8. In some cases, the variant CD86 polypeptide has 2 amino acid substitutions compared to the CD86 amino acid sequence set forth in SEQ ID NO: 8. In some cases, the variant CD86 polypeptide has 3 amino acid substitutions compared to the CD86 amino acid sequence set forth in SEQ ID NO: 8. In some cases, the variant CD86 polypeptide has 4 amino acid substitutions compared to the CD86 amino acid sequence set forth in SEQ ID NO: 8. In some cases, the variant CD86 polypeptide has 5 amino acid substitutions compared to the CD86 amino acid sequence set forth in SEQ ID NO: 8. In some cases, the variant CD86 polypeptide has 6 amino acid substitutions compared to the CD86 amino acid sequence set forth in SEQ ID NO: 8. In some cases, the variant CD86 polypeptide has 7 amino acid substitutions compared to the CD86 amino acid sequence set forth in SEQ ID NO: 8. In some cases, the variant CD86 polypeptide has 8 amino acid substitutions compared to the CD86 amino acid sequence set forth in SEQ ID NO: 8. In some cases, the variant CD86 polypeptide has 9 amino acid substitutions compared to the CD86 amino acid sequence set forth in SEQ ID NO: 8. In some cases, the variant CD86 polypeptide has 10 amino acid substitutions compared to the CD86 amino acid sequence set forth in SEQ ID NO: 8.

[0399] In some cases, the variant CD86 polypeptide has a single amino acid substitution compared to the CD86 amino acid sequence set forth in SEQ ID NO: 9. In some cases, the variant CD86 polypeptide has 2 to 10 amino acid substitutions compared to the CD86 amino acid sequence set forth in SEQ ID NO: 9. In some cases, the variant CD86 polypeptide has 2 amino acid substitutions compared to the CD86 amino acid sequence set forth in SEQ ID NO: 9. In some cases, the variant CD86 polypeptide has 3 amino acid substitutions compared to the CD86 amino acid sequence set forth in SEQ ID NO: 9. In some cases, the variant CD86 polypeptide has 4 amino acid substitutions compared to the CD86 amino acid sequence set forth in SEQ ID NO: 9. In some cases, the variant CD86 polypeptide has 5 amino acid substitutions compared to the CD86 amino acid sequence set forth in SEQ ID NO: 9. In some cases, the variant CD86 polypeptide has 6 amino acid substitutions compared to the CD86 amino acid sequence set forth in SEQ ID NO: 9. In some cases, the variant CD86 polypeptide has 7 amino acid substitutions compared to the CD86 amino acid sequence set forth in SEQ ID NO: 9. In some cases, the variant CD86 polypeptide has 8 amino acid substitutions compared to the CD86 amino acid sequence set forth in SEQ ID NO: 9. In some cases, the variant CD86 polypeptide has 9 amino acid substitutions compared to the CD86 amino acid sequence set forth in SEQ ID NO: 9. In some cases, the variant CD86 polypeptide has 10 amino acid substitutions compared to the CD86 amino acid sequence set forth in SEQ ID NO: 9.

[0400] Suitable CD86 variants include a polypeptide comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to any of the following amino acid sequences:

[0401] APLKIQAYFNETADLPCQFANSQNQSLSELVVFWQDQENLVLNEVYLGKEKFDSVHSKYM X RTSFDSDSWTLRLHNLQIKDKGLYQCIIHHKKPTGMIRIHQMNSELSVLANFSQPEIVPISNITENVYINLTCSSIHGYPEPKKMSVLLRTKNSTIEYDGIMQKSQDNVTELYDVSISLSVSFPDVTSNMTIFCILETDKTRLLSSPFSIELEDPQPPPDHIP (SEQ ID NO: 73), wherein X is any amino acid except Asn. In some cases, X is Ala;

[0402] APLKIQAYFNETADLPCQFANSQNQSLSELVVFWQDQENLVLNEVYLGKEKFDSVHSKYMNRTSF X SDSWTLRLHNLQIKDKGLYQCIIHHKKPTGMIRIHQMNSELSVLANFSQPEIVPISNITENVYINLTCSSIHGYPEPKKMSVLLRTKNSTIEYDGIMQKSQDNVTELYDVSISLSVSFPDVTSNMTIFCILETDKTRLLSSPFSIELEDPQPPPDHIP (SEQ ID NO: 74), wherein X is any amino acid except Asp. In some cases, X is Ala;

[0403] APLKIQAYFNETADLPCQFANSQNQSLSELVVFWQDQENLVLNEVYLGKEKFDSVHSKYMNRTSFDSDS X TLRLHNLQIKDKGLYQCIIHHKKPTGMIRIHQMNSELSVLANFSQPEIVPISNITENVYINLTCSSIHGYPEPKKMSVLLRTKNSTIEYDGIMQKSQDNVTELYDVSISLSVSFPDVTSNMTIFCILETDKTRLLSSPFSIELEDPQPPPDHIP (SEQ ID NO: 75), wherein X is any amino acid except Trp. In some cases, X is Ala;

[0404] APLKIQAYFNETADLPCQFANSQNQSLSELVVFWQDQENLVLNEVYLGKEKFDSVHSKYMNRTSFDSDSWTLRLHNLQIKDKGLYQCIIH X KKPTGMIRIHQMNSELSVLANFSQPEIVPISNITENVYINLTCSSIHGYPEPKKMSVLLRTKNSTIEYDGIMQKSQDNVTELYDVSISLSVSFPDVTSNMTIFCILETDKTRLLSSPFSIELEDPQPPPDHIP (SEQ ID NO: 76), wherein X is any amino acid except His. In some cases, X is Ala;

[0405] APLKIQAYFNETADLPCQFANSQNQSLSELVVFWQDQENLVLNEVYLGKEKFDSVHSKYMX RTSFDSDSWTLRLHNLQIKDKGLYQCIIHHKKPTGMIRIHQMNSELSVL (SEQ ID NO: 77), wherein X is any amino acid except Asn. In some cases, X is Ala;

[0406] APLKIQAYFNETADLPCQFANSQNQSLSELVVFWQDQENLVLNEVYLGKEKFDSVHSKYMNRTSF X SDSWTLRLHNLQIKDKGLYQCIIHHKKPTGMIRIHQMNSELSVL (SEQ ID NO: 78), wherein X is any amino acid except Asp. In some cases, X is Ala;

[0407] APLKIQAYFNETADLPCQFANSQNQSLSELVVFWQDQENLVLNEVYLGKEKFDSVHSKYMNRTSFDSDS X TLRLHNLQIKDKGLYQCIIHHKKPTGMIRIHQMNSELSVL (SEQ ID NO: 79), wherein X is any amino acid except Trp. In some cases, X is Ala;

[0408] APLKIQAYFNETADLPCQFANSQNQSLSELVVFWQDQENLVLNEVYLGKEKFDSVHSKYMNRTSFDSDSWTLRLHNLQIKDKGLYQCIIH X KKPTGMIRIHQMNSELSVL (SEQ ID NO: 80), wherein X is any amino acid except His. In some cases, X is Ala;

[0409] APLKIQAYFNETADLPCQFANSQNQSLSELVVFWQDQENL X LNEVYLGKEKFDSVHSKYMNRTSFDSDSWTLRLHNLQIKDKGLYQCIIHHKKPTGMIRIHQMNSELSVLANFSQPEIVPISNITENVYINLTCSSIHGYPEPKKMSVLLRTKNSTIEYDGIMQKSQDNVTELYDVSISLSVSFPDVTSNMTIFCILETDKTRLLSSPFSIELEDPQPPPDHIP (SEQ ID NO: 81), wherein X is any amino acid except Val. In some cases, X is Ala;

[0410] APLKIQAYFNETADLPCQFANSQNQSLSELVVFWQDQENL X LNEVYLGKEKFDSVHSKYMNRTSFDSDSWTLRLHNLQIKDKGLYQCIIHHKKPTGMIRIHQMNSELSVL (SEQ ID NO: 82), wherein X is any amino acid except Val. In some cases, X is Ala;

[0411] APLKIQAYFNETADLPCQFANSQNQSLSELVVFW X DQENLVLNEVYLGKEKFDSVHSKYMNRTSFDSDSWTLRLHNLQIKDKGLYQCIIHHKKPTGMIRIHQMNSELSVLANFSQPEIVPISNITENVYINLTCSSIHGYPEPKKMSVLLRTKNSTIEYDGIMQKSQDNVTELYDVSISLSVSFPDVTSNMTIFCILETDKTRLLSSPFSIELEDPQPPPDHIP (SEQ ID NO: 83), wherein X is any amino acid except Gln. In some cases, X is Ala;

[0412] APLKIQAYFNETADLPCQFANSQNQSLSELVVFW X DQENLVLNEVYLGKEKFDSVHSKYMNRTSFDSDSWTLRLHNLQIKDKGLYQCIIHHKKPTGMIRIHQMNSELSVL (SEQ ID NO: 84), wherein X is any amino acid except Gln. In some cases, X is Ala;

[0413] APLKIQAYFNETADLPCQFANSQNQSLSELVV XWQDQENLVLNEVYLGKEKFDSVHSKYMNRTSFDSDSWTLRLHNLQIKDKGLYQCIIHHKKPTGMIRIHQMNSELSVLANFSQPEIVPISNITENVYINLTCSSIHGYPEPKKMSVLLRTKNSTIEYDGIMQKSQDNVTELYDVSISLSVSFPDVTSNMTIFCILETDKTRLLSSPFSIELEDPQPPPDHIP (SEQ ID NO: 85), wherein X is any amino acid except Phe. In some cases, X is Ala;

[0414] APLKIQAYFNETADLPCQFANSQNQSLSELVV X WQDQENLVLNEVYLGKEKFDSVHSKYMNRTSFDSDSWTLRLHNLQIKDKGLYQCIIHHKKPTGMIRIHQMNSELSVL (SEQ ID NO: 86), wherein X is any amino acid except Phe. In some cases, X is Ala;

[0415] APLKIQAYFNETADLPCQFANSQNQSLSELVVFWQDQENLVLNEVYLGKEKFDSVHSKYMNRTSFDSDSWT X RLHNLQIKDKGLYQCIIHHKKPTGMIRIHQMNSELSVLANFSQPEIVPISNITENVYINLTCSSIHGYPEPKKMSVLLRTKNSTIEYDGIMQKSQDNVTELYDVSISLSVSFPDVTSNMTIFCILETDKTRLLSSPFSIELEDPQPPPDHIP (SEQ ID NO: 87), wherein X is any amino acid except Leu. In some cases, X is Ala;

[0416] APLKIQAYFNETADLPCQFANSQNQSLSELVVFWQDQENLVLNEVYLGKEKFDSVHSKYMNRTSFDSDSWT X RLHNLQIKDKGLYQCIIHHKKPTGMIRIHQMNSELSVL (SEQ ID NO: 88), wherein X is any amino acid except Leu. In some cases, X is Ala;

[0417] APLKIQAYFNETADLPCQFANSQNQSLSELVVFWQDQENLVLNEVYLGKEKFDSVHSK X MNRTSFDSDSWTLRLHNLQIKDKGLYQCIIHHKKPTGMIRIHQMNSELSVLANFSQPEIVPISNITENVYINLTCSSIHGYPEPKKMSVLLRTKNSTIEYDGIMQKSQDNVTELYDVSISLSVSFPDVTSNMTIFCILETDKTRLLSSPFSIELEDPQPPPDHIP (SEQ ID NO: 89), wherein X is any amino acid except Tyr. In some cases, X is Ala;

[0418] APLKIQAYFNETADLPCQFANSQNQSLSELVVFWQDQENLVLNEVYLGKEKFDSVHSK X MNRTSFDSDSWTLRLHNLQIKDKGLYQCIIHHKKPTGMIRIHQMNSELSVL (SEQ ID NO: 90), wherein X is any amino acid except Tyr. In some cases, X is Ala;

[0419] APLKIQAYFNETADLPCQFANSQNQSLSELVVFWQDQENLVLNEVYLGKEKFDSVHSKYM X RTSFDSDSWTLRLHNLQIKDKGLYQCIIH X KKPTGMIRIHQMNSELSVLANFSQPEIVPISNITENVYINLTCSSIHGYPEPKKMSVLLRTKNSTIEYDGIMQKSQDNVTELYDVSISLSVSFPDVTSNMTIFCILETDKTRLLSSPFSIELEDPQPPPDHIP (SEQ ID NO: 91), wherein the first X is any amino acid except Asn and the second X is any amino acid except His. In some cases, both the first X and the second X are Ala;

[0420] APLKIQAYFNETADLPCQFANSQNQSLSELVVFWQDQENLVLNEVYLGKEKFDSVHSKYM X RTSFDSDSWTLRLHNLQIKDKGLYQCIIH XKKPTGMIRIHQMNSELSVL (SEQ ID NO: 92), wherein the first X is any amino acid except Asn and the second X is any amino acid except His. In some cases, both the first X and the second X are Ala;

[0421] APLKIQAYFNETADLPCQFANSQNQSLSELVVFWQDQENLVLNEVYLGKEKFDSVHSKYMNRTSF X1 SDSWTLRLHNLQIKDKGLYQCIIH X2 KKPTGMIRIHQMNSELSVLANFSQPEIVPISNITENVYINLTCSSIHGYPEPKKMSVLLRTKNSTIEYDGIMQKSQDNVTELYDVSISLSVSFPDVTSNMTIFCILETDKTRLLSSPFSIELEDPQPPPDHIP (SEQ ID NO: 93), wherein X1 is any amino acid except Asp and X2 is any amino acid except His. In some cases, X1 is Ala and X2 is Ala;

[0422] APLKIQAYFNETADLPCQFANSQNQSLSELVVFWQDQENLVLNEVYLGKEKFDSVHSKYMNRTSF X1 SDSWTLRLHNLQIKDKGLYQCIIH X2 KKPTGMIRIHQMNSELSVL (SEQ ID NO: 94), wherein the first X is any amino acid except Asn and the second X is any amino acid except His. In some cases, the first X and the second X are both Ala;

[0423] APLKIQAYFNETADLPCQFANSQNQSLSELVVFWQDQENLVLNEVYLGKEKFDSVHSKYM X1 RTSF X2 SDSWTLRLHNLQIKDKGLYQCIIH X3KKPTGMIRIHQMNSELSVLANFSQPEIVPISNITENVYINLTCSSIHGYPEPKKMSVLLRTKNSTIEYDGIMQKSQDNVTELYDVSISLSVSFPDVTSNMTIFCILETDKTRLLSSPFSIELEDPQPPPDHIP (SEQ ID NO: 95), wherein X1 is any amino acid except Asn, X2 is any amino acid except Asp, and X3 is any amino acid except His. In some cases, X1 is Ala, X2 is Ala, and X3 is Ala; and

[0424] APLKIQAYFNETADLPCQFANSQNQSLSELVVFWQDQENLVLNEVYLGKEKFDSVHSKYM X1 RTSF X2 SDSWTLRLHNLQIKDKGLYQCIIH X3 KKPTGMIRIHQMNSELSVL (SEQ ID NO: 96), wherein X1 is any amino acid except Asn, X2 is any amino acid except Asp, and X3 is any amino acid except His. In some cases, X1 is Ala, X2 is Ala, and X3 is Ala.

[0425] 4-1BBL variant

[0426] In some cases, the variant immunomodulatory polypeptide present in the TMMP of the present disclosure is a variant 4-1BBL polypeptide.Wild-type 4-1BBL binds to 4-1BB (CD137).

[0427] The wild-type 4-1BBL amino acid sequence may be as follows: MEYASDASLD PEAPWP PAPR ARACRVLP WA LVAGLLLLLL LAAACAVFL A CPWAVSG ARA SPGSAASPRL REGPELSPDD PAGLLDLRQG MFAQLVAQNV LLIDGPLSWY SDPGLAGVSL TGGLSYKEDT KELVVAKAG V YYVFFQLELR RVVAGEGSGSVSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ GRLLHLSAGQ RLGVHLHTEA R ARHAWQLTQGATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 10).

[0428] In some instances, the variant 4-1BBL polypeptide is a variant of the tumor necrosis factor (TNF) homology domain (THD) of human 4-1BBL.

[0429] The wild-type amino acid sequence of THD of human 4-1BBL can be, for example, one of the following SEQ ID NOs: 11-13:

[0430] PAGLLDLRQG MFAQLVAQNV LLIDGPLSWY SDPGLAGVS L TGGLSYKEDT KELVVAKAGVYYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ G RLLHLSAGQRLGVHLHTEA RARHAWQLTQ GATVLGLFRV TP EIPAGLPS PRSE (SEQ ID NO: 11).

[0431] D PAGLLDLRQG MFAQLVAQNV LLIDGPLSWY SDPGLAG VSL TGGLSYKEDT KELVVAKAGVYYVFFQLELR RVVAGEGS GS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ GRLLHLSAGQRLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 12).

[0432] D PAGLLDLRQG MFAQLVAQNV LLIDGPLSWY SDPGLAG VSL TGGLSYKEDT KELVVAKAGVYYVFFQLELR RVVAGEGS GS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ GRLLHLSAGQRLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPA (SEQ ID NO: 13).

[0433] The wild-type 4-1BB amino acid sequence may be as follows: MGNSCYNIVA TLLLVLN FER TRSLQDPCSNCPAGTFCDNN RNQICSPCPP NSFSSAGGQR TCDICRQCKG VFRTRKECSS TSNAECDCTP GFHCLGAGCSM CEQDCKQGQ ELTKKGCKDC CFGTFNDQKR GICRPWTNCS LD GKSVLVNG TKERDVVCGPSPADLSPGAS SVTPPAPARE PGHS PQIISF FLALTSTALL FLLFFLTLRF SVVKRGRKKL LYIFKQPFMR PVQTTQEEDG CSCRFPEEEE GGCEL (SEQ ID NO: 14). In some cases, when the TMMP of the present disclosure comprises a variant 4-1BBL polypeptide, the "homologous co-immunomodulatory polypeptide" is a 4-1BB polypeptide comprising the amino acid sequence SEQ ID NO: 14.

[0434] In some cases, the variant 4-1BBL polypeptide exhibits reduced binding affinity for 4-1BB compared to the binding affinity of a 4-1BBL polypeptide comprising the amino acid sequence set forth in one of SEQ ID NOs: 10-13. For example, in some cases, a variant 4-1BBL polypeptide of the present disclosure binds to 4-1BB with an affinity that is at least 10% less, at least 15% less, at least 20% less, at least 25% less, at least 30% less, at least 35% less, at least 40% less, at least 45% less, at least 50% less, at least 55% less, at least 60% less, at least 65% less, at least 70% less, at least 75% less, at least 80% less, at least 85% less, at least 90% less, at least 95% less, or greater than 95% less than the binding affinity of a 4-1BBL polypeptide comprising the amino acid sequence set forth in one of SEQ ID NOs: 10-13 for a 4-1BB polypeptide (e.g., a 4-1BB polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 14), when measured under the same conditions.

[0435] In some cases, the variant 4-1BBL polypeptide has a binding affinity for 4-1BB of 100 nM to 100 μM. As another example, in some cases, the variant 4-1BBL polypeptide has a binding affinity for 4-1BB (e.g., a 4-1BB polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 14) of about 100 nM to 150 nM, about 150 nM to about 200 nM, about 200 nM to about 250 nM, about 250 nM to about 300 nM, about 300 nM to about 350 nM, about 350 nM to about 400 nM, about 400 nM to about 500 nM, about 500 nM to about 600 nM, about 600 nM to about 700 nM, about 700 nM to about 800 nM, about 800 nM to about 900 nM, about 900 nM to about 1000 nM, about 1000 nM to about 15 ... 0 nM to about 700 nM, about 700 nM to about 800 nM, about 800 nM to about 900 nM, about 900 nM to about 1 μM, about 1 μM to about 5 μM, about 5 μM to about 10 μM, about 10 μM to about 15 μM, about 15 μM to about 20 μM, about 20 μM to about 25 μM, about 25 μM to about 50 μM, about 50 μM to about 75 μM, or about 75 μM to about 100 μM.

[0436] In some cases, the variant 4-1BBL polypeptide has a single amino acid substitution compared to a 4-1BBL amino acid sequence listed in one of SEQ ID NOs: 10-13. In some cases, the variant 4-1BBL polypeptide has 2 to 10 amino acid substitutions compared to a 4-1BBL amino acid sequence listed in one of SEQ ID NOs: 10-13. In some cases, the variant 4-1BBL polypeptide has 2 amino acid substitutions compared to a 4-1BBL amino acid sequence listed in one of SEQ ID NOs: 10-13. In some cases, the variant 4-1BBL polypeptide has 3 amino acid substitutions compared to a 4-1BBL amino acid sequence listed in one of SEQ ID NOs: 10-13. In some cases, the variant 4-1BBL polypeptide has 4 amino acid substitutions compared to a 4-1BBL amino acid sequence listed in one of SEQ ID NOs: 10-13. In some cases, the variant 4-1BBL polypeptide has 5 amino acid substitutions compared to a 4-1BBL amino acid sequence listed in one of SEQ ID NOs: 10-13. In some cases, the variant 4-1BBL polypeptide has 6 amino acid substitutions compared to a 4-1BBL amino acid sequence listed in one of SEQ ID NOs: 10-13. In some cases, the variant 4-1BBL polypeptide has 7 amino acid substitutions compared to a 4-1BBL amino acid sequence listed in one of SEQ ID NOs: 10-13. In some cases, the variant 4-1BBL polypeptide has 8 amino acid substitutions compared to a 4-1BBL amino acid sequence listed in one of SEQ ID NOs: 10-13. In some cases, the variant 4-1BBL polypeptide has 9 amino acid substitutions compared to a 4-1BBL amino acid sequence listed in one of SEQ ID NOs: 10-13. In some cases, the variant 4-1BBL polypeptide has 10 amino acid substitutions compared to a 4-1BBL amino acid sequence listed in one of SEQ ID NOs: 10-13.

[0437] Suitable 4-1BBL variants include a polypeptide comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to any of the following amino acid sequences:

[0438] PAGLLDLRQG MFAQLVAQNV LLIDGPLSWY SDPGLAGVS L TGGLSY XEDT KELVVAKAGVYYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ G RLLHLSAGQRLGVHLHTEA RARHAWQLTQ GATVLGLFRV TP EIPAGLPS PRSE (SEQ ID NO: 97), wherein X is any amino acid except Lys. In some cases, X is Ala;

[0439] PAGLLDLRQG MFAQLVAQNV LLIDGPLSWY SDPGLAGVS L TGGLSYKEDT KELVVAKAGVYYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ G RLLHLSAGQRLGVHLHTEA RARHAW X LTQ GATVLGLFRV TP EIPAGLPS PRSE (SEQ ID NO: 98), wherein X is any amino acid except Gln. In some cases, X is Ala;

[0440] PAGLLDLRQG X FAQLVAQNV LLIDGPLSWY SDPGLAGVS L TGGLSYKEDT KELVVAKAGVYYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ G RLLHLSAGQRLGVHLHTEA RARHAWQLTQ GATVLGLFRV TP EIPAGLPS PRSE (SEQ ID NO: 99), wherein X is any amino acid except Met. In some cases, X is Ala;

[0441] PAGLLDLRQG M XAQLVAQNV LLIDGPLSWY SDPGLAGV SL TGGLSYKEDT KELVVAKAGVYYVFFQLELR RVVAGEGSG S VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ GRLLHLSAGQRLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 100), wherein X is any amino acid except Phe. In some cases, X is Ala;

[0442] PAGLLDLRQG MFA X LVAQNV LLIDGPLSWY SDPGLAGVS L TGGLSYKEDT KELVVAKAGVYYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ G RLLHLSAGQRLGVHLHTEA RARHAWQLTQ GATVLGLFRV TP EIPAGLPS PRSE (SEQ ID NO: 101), wherein X is any amino acid except Gln. In some cases, X is Ala;

[0443] PAGLLDLRQG MFAQ X VAQNV LLIDGPLSWY SDPGLAGVS L TGGLSYKEDT KELVVAKAGVYYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ G RLLHLSAGQRLGVHLHTEA RARHAWQLTQ GATVLGLFRV TP EIPAGLPS PRSE (SEQ ID NO: 102), wherein X is any amino acid except Leu. In some cases, X is Ala;

[0444] PAGLLDLRQG MFAQL XAQNV LLIDGPLSWY SDPGLAGVS L TGGLSYKEDT KELVVAKAGVYYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ G RLLHLSAGQRLGVHLHTEA RARHAWQLTQ GATVLGLFRV TP EIPAGLPS PRSE (SEQ ID NO: 103), wherein X is any amino acid except Val. In some cases, X is Ala;

[0445] PAGLLDLRQG MFAQLVA X NV LLIDGPLSWY SDPGLAGVS L TGGLSYKEDT KELVVAKAGVYYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ G RLLHLSAGQRLGVHLHTEA RARHAWQLTQ GATVLGLFRV TP EIPAGLPS PRSE (SEQ ID NO: 104), wherein X is any amino acid except Gln. In some cases, X is Ala;

[0446] PAGLLDLRQG MFAQLVAQ X V LLIDGPLSWY SDPGLAGVS L TGGLSYKEDT KELVVAKAGVYYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ G RLLHLSAGQRLGVHLHTEA RARHAWQLTQ GATVLGLFRV TP EIPAGLPS PRSE (SEQ ID NO: 105), wherein X is any amino acid except Asn. In some cases, X is Ala;

[0447] PAGLLDLRQG MFAQLVAQN XLLIDGPLSWY SDPGLAGVS L TGGLSYKEDT KELVVAKAGVYYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ G RLLHLSAGQRLGVHLHTEA RARHAWQLTQ GATVLGLFRV TP EIPAGLPS PRSE (SEQ ID NO: 106), wherein X is any amino acid except Val. In some cases, X is Ala;

[0448] PAGLLDLRQG MFAQLVAQNV X LIDGPLSWY SDPGLAGVS L TGGLSYKEDT KELVVAKAGVYYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ G RLLHLSAGQRLGVHLHTEA RARHAWQLTQ GATVLGLFRV TP EIPAGLPS PRSE (SEQ ID NO: 107), wherein X is any amino acid except Leu. In some cases, X is Ala;

[0449] PAGLLDLRQG MFAQLVAQNV L X IDGPLSWY SDPGLAGVS L TGGLSYKEDT KELVVAKAGVYYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ G RLLHLSAGQRLGVHLHTEA RARHAWQLTQ GATVLGLFRV TP EIPAGLPS PRSE (SEQ ID NO: 108), wherein X is any amino acid except Leu. In some cases, X is Ala;

[0450] PAGLLDLRQG MFAQLVAQNV LL XDGPLSWY SDPGLAGV SL TGGLSYKEDT KELVVAKAGVYYVFFQLELR RVVAGEGSG S VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ GRLLHLSAGQRLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 109), wherein X is any amino acid except Ile. In some cases, X is Ala;

[0451] PAGLLDLRQG MFAQLVAQNV LLI X GPLSWY SDPGLAGVS L TGGLSYKEDT KELVVAKAGVYYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ G RLLHLSAGQRLGVHLHTEA RARHAWQLTQ GATVLGLFRV TP EIPAGLPS PRSE (SEQ ID NO: 110), wherein X is any amino acid except Asp. In some cases, X is Ala;

[0452] PAGLLDLRQG MFAQLVAQNV LLID X PLSWY SDPGLAGVS L TGGLSYKEDT KELVVAKAGVYYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ G RLLHLSAGQRLGVHLHTEA RARHAWQLTQ GATVLGLFRV TP EIPAGLPS PRSE (SEQ ID NO: 111), wherein X is any amino acid except Gly. In some cases, X is Ala;

[0453] PAGLLDLRQG MFAQLVAQNV LLIGG XLSWY SDPGLAGV SL TGGLSYKEDT KELVVAKAGVYYVFFQLELR RVVAGEGSG S VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ GRLLHLSAGQRLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 112), wherein X is any amino acid except Pro. In some cases, X is Ala;

[0454] PAGLLDLRQG MFAQLVAQNV LLIGGP X SWY SDPGLAGVS L TGGLSYKEDT KELVVAKAGVYYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ G RLLHLSAGQRLGVHLHTEA RARHAWQLTQ GATVLGLFRV TP EIPAGLPS PRSE (SEQ ID NO: 113), wherein X is any amino acid except Leu. In some cases, X is Ala;

[0455] PAGLLDLRQG MFAQLVAQNV LLIGGPL X WY SDPGLAGV SL TGGLSYKEDT KELVVAKAGVYYVFFQLELR RVVAGEGSG S VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ GRLLHLSAGQRLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 114), wherein X is any amino acid except Ser. In some cases, X is Ala;

[0456] PAGLLDLRQG MFAQLVAQNV LLIGGPLS XY SDPGLAGVS L TGGLSYKEDT KELVVAKAGVYYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ G RLLHLSAGQRLGVHLHTEA RARHAWQLTQ GATVLGLFRV TP EIPAGLPS PRSE (SEQ ID NO: 115), wherein X is any amino acid except Trp. In some cases, X is Ala;

[0457] PAGLLDLRQG MFAQLVAQNV LLIGGPLSW X SDPGLAGVS L TGGLSYKEDT KELVVAKAGVYYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ G RLLHLSAGQRLGVHLHTEA RARHAWQLTQ GATVLGLFRV TP EIPAGLPS PRSE (SEQ ID NO: 116), wherein X is any amino acid except Tyr. In some cases, X is Ala;

[0458] PAGLLDLRQG MFAQLVAQNV LLIGGPLSWY X DPGLAGV SL TGGLSYKEDT KELVVAKAGVYYVFFQLELR RVVAGEGSG S VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ GRLLHLSAGQRLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 117), wherein X is any amino acid except Ser. In some cases, X is Ala;

[0459] PAGLLDLRQG MFAQLVAQNV LLIGGPLSWY S XPGLAGVS L TGGLSYKEDT KELVVAKAGVYYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ G RLLHLSAGQRLGVHLHTEA RARHAWQLTQ GATVLGLFRV TP EIPAGLPS PRSE (SEQ ID NO: 118), wherein X is any amino acid except Asp. In some cases, X is Ala;

[0460] PAGLLDLRQG MFAQLVAQNV LLIGGPLSWY SD X GLAGV SL TGGLSYKEDT KELVVAKAGVYYVFFQLELR RVVAGEGSG S VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ GRLLHLSAGQRLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 119), wherein X is any amino acid except Pro. In some cases, X is Ala;

[0461] PAGLLDLRQG MFAQLVAQNV LLIGGPLSWY SDP X LAGVS L TGGLSYKEDT KELVVAKAGVYYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ G RLLHLSAGQRLGVHLHTEA RARHAWQLTQ GATVLGLFRV TP EIPAGLPS PRSE (SEQ ID NO: 120), wherein X is any amino acid except Gly. In some cases, X is Ala;

[0462] PAGLLDLRQG MFAQLVAQNV LLIGGPLSWY SDPG XAGVS L TGGLSYKEDT KELVVAKAGVYYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ G RLLHLSAGQRLGVHLHTEA RARHAWQLTQ GATVLGLFRV TP EIPAGLPS PRSE (SEQ ID NO: 121), wherein X is any amino acid except Leu. In some cases, X is Ala;

[0463] PAGLLDLRQG MFAQLVAQNV LLIGGPLSWY SDPGLA X VS L TGGLSYKEDT KELVVAKAGVYYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ G RLLHLSAGQRLGVHLHTEA RARHAWQLTQ GATVLGLFRV TP EIPAGLPS PRSE (SEQ ID NO: 122), wherein X is any amino acid except Gly. In some cases, X is Ala;

[0464] PAGLLDLRQG MFAQLVAQNV LLIGGPLSWY SDPGLAG X SL TGGLSYKEDT KELVVAKAGVYYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ G RLLHLSAGQRLGVHLHTEA RARHAWQLTQ GATVLGLFRV TP EIPAGLPS PRSE (SEQ ID NO: 123), wherein X is any amino acid except Val. In some cases, X is Ala;

[0465] PAGLLDLRQG MFAQLVAQNV LLIGGPLSWY SDPGLAGV XL TGGLSYKEDT KELVVAKAGVYYVFFQLELR RVVAGEGSG S VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ GRLLHLSAGQRLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 124), wherein X is any amino acid except Ser. In some cases, X is Ala;

[0466] PAGLLDLRQG MFAQLVAQNV LLIGGPLSWY SDPGLAGVS X TGGLSYKEDT KELVVAKAGVYYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ G RLLHLSAGQRLGVHLHTEA RARHAWQLTQ GATVLGLFRV TP EIPAGLPS PRSE (SEQ ID NO: 125), wherein X is any amino acid except Leu. In some cases, X is Ala;

[0467] PAGLLDLRQG MFAQLVAQNV LLIGGPLSWY SDPGLAGVS L X GGLSYKEDT KELVVAKAGVYYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ G RLLHLSAGQRLGVHLHTEA RARHAWQLTQ GATVLGLFRV TP EIPAGLPS PRSE (SEQ ID NO: 126), wherein X is any amino acid except Thr. In some cases, X is Ala;

[0468] PAGLLDLRQG MFAQLVAQNV LLIGGPLSWY SDPGLAGVS LT XGLSYKEDT KELVVAKAGVYYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ G RLLHLSAGQRLGVHLHTEA RARHAWQLTQ GATVLGLFRV TP EIPAGLPS PRSE (SEQ ID NO: 127), wherein X is any amino acid except Gly. In some cases, X is Ala;

[0469] PAGLLDLRQG MFAQLVAQNV LLIGGPLSWY SDPGLAGVS L TG X LSYKEDT KELVVAKAGVYYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ G RLLHLSAGQRLGVHLHTEA RARHAWQLTQ GATVLGLFRV TP EIPAGLPS PRSE (SEQ ID NO: 128), wherein X is any amino acid except Gly. In some cases, X is Ala;

[0470] PAGLLDLRQG MFAQLVAQNV LLIGGPLSWY SDPGLAGVS L TGG X SYKEDT KELVVAKAGVYYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ G RLLHLSAGQRLGVHLHTEA RARHAWQLTQ GATVLGLFRV TP EIPAGLPS PRSE (SEQ ID NO: 129), wherein X is any amino acid except Leu. In some cases, X is Ala;

[0471] PAGLLDLRQG MFAQLVAQNV LLIGGPLSWY SDPGLAGVS L TGGL XYKEDT KELVVAKAGVYYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ G RLLHLSAGQRLGVHLHTEA RARHAWQLTQ GATVLGLFRV TP EIPAGLPS PRSE (SEQ ID NO: 130), wherein X is any amino acid except Ser. In some cases, X is Ala;

[0472] PAGLLDLRQG MFAQLVAQNV LLIGGPLSWY SDPGLAGVS L TGGLS X KEDT KELVVAKAGVYYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ G RLLHLSAGQRLGVHLHTEA RARHAWQLTQ GATVLGLFRV TP EIPAGLPS PRSE (SEQ ID NO: 131), wherein X is any amino acid except Tyr. In some cases, X is Ala;

[0473] PAGLLDLRQG MFAQLVAQNV LLIGGPLSWY SDPGLAGVS L TGGLSYK X DT KELVVAKAGVYYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ G RLLHLSAGQRLGVHLHTEA RARHAWQLTQ GATVLGLFRV TP EIPAGLPS PRSE (SEQ ID NO: 132), wherein X is any amino acid except Glu. In some cases, X is Ala;

[0474] PAGLLDLRQG MFAQLVAQNV LLIGGPLSWY SDPGLAGVS L TGGLSYKE XT KELVVAKAGVYYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ G RLLHLSAGQRLGVHLHTEA RARHAWQLTQ GATVLGLFRV TP EIPAGLPS PRSE (SEQ ID NO: 133), wherein X is any amino acid except Asp. In some cases, X is Ala;

[0475] PAGLLDLRQG MFAQLVAQNV LLIGGPLSWY SDPGLAGVS L TGGLSYKED X KELVVAKAGVYYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ G RLLHLSAGQRLGVHLHTEA RARHAWQLTQ GATVLGLFRV TP EIPAGLPS PRSE (SEQ ID NO: 134), wherein X is any amino acid except Thr. In some cases, X is Ala;

[0476] PAGLLDLRQG MFAQLVAQNV LLIGGPLSWY SDPGLAGVS L TGGLSYKEDT X ELVVAKAGVYYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ G RLLHLSAGQRLGVHLHTEA RARHAWQLTQ GATVLGLFRV TP EIPAGLPS PRSE (SEQ ID NO: 135), wherein X is any amino acid except Lys. In some cases, X is Ala;

[0477] PAGLLDLRQG MFAQLVAQNV LLIGGPLSWY SDPGLAGVS L TGGLSYKEDT K XLVVAKAGVYYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ G RLLHLSAGQRLGVHLHTEA RARHAWQLTQ GATVLGLFRV TP EIPAGLPS PRSE (SEQ ID NO: 136), wherein X is any amino acid except Glu. In some cases, X is Ala;

[0478] PAGLLDLRQG MFAQLVAQNV LLIGGPLSWY SDPGLAGVS L TGGLSYKEDT KELVVAKAGVYYV X FQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ G RLLHLSAGQRLGVHLHTEA RARHAWQLTQ GATVLGLFRV TP EIPAGLPS PRSE (SEQ ID NO: 137), wherein X is any amino acid except Phe. In some cases, X is Ala;

[0479] PAGLLDLRQG MFAQLVAQNV LLIGGPLSWY SDPGLAGVS L TGGLSYKEDT KELVVAKAGVYYVF X QLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ G RLLHLSAGQRLGVHLHTEA RARHAWQLTQ GATVLGLFRV TP EIPAGLPS PRSE (SEQ ID NO: 138), wherein X is any amino acid except Phe. In some cases, X is Ala;

[0480] PAGLLDLRQG MFAQLVAQNV LLIGGPLSWY SDPGLAGVS L TGGLSYKEDT KELVVAKAGVYYVFF XLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ G RLLHLSAGQRLGVHLHTEA RARHAWQLTQ GATVLGLFRV TP EIPAGLPS PRSE (SEQ ID NO: 139), wherein X is any amino acid except Gln. In some cases, X is Ala;

[0481] PAGLLDLRQG MFAQLVAQNV LLIGGPLSWY SDPGLAGVS L TGGLSYKEDT KELVVAKAGVYYVFFQ X ELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ G RLLHLSAGQRLGVHLHTEA RARHAWQLTQ GATVLGLFRV TP EIPAGLPS PRSE (SEQ ID NO: 140), wherein X is any amino acid except Leu. In some cases, X is Ala;

[0482] PAGLLDLRQG MFAQLVAQNV LLIGGPLSWY SDPGLAGVS L TGGLSYKEDT KELVVAKAGVYYVFFQL X LR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ G RLLHLSAGQRLGVHLHTEA RARHAWQLTQ GATVLGLFRV TP EIPAGLPS PRSE (SEQ ID NO: 141), wherein X is any amino acid except Glu. In some cases, X is Ala;

[0483] PAGLLDLRQG MFAQLVAQNV LLIGGPLSWY SDPGLAGVS L TGGLSYKEDT KELVVAKAGVYYVFFQLE X R RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ G RLLHLSAGQRLGVHLHTEA RARHAWQLTQ GATVLGLFRV TP EIPAGLPS PRSE (SEQ ID NO: 142), wherein X is any amino acid except Leu. In some cases, X is Ala;

[0484] PAGLLDLRQG MFAQLVAQNV LLIGGPLSWY SDPGLAGVS L TGGLSYKEDT KELVVAKAGVYYVFFQLEL X RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ G RLLHLSAGQRLGVHLHTEA RARHAWQLTQ GATVLGLFRV TP EIPAGLPS PRSE (SEQ ID NO: 143), wherein X is any amino acid except Arg. In some cases, X is Ala;

[0485] PAGLLDLRQG MFAQLVAQNV LLIGGPLSWY SDPGLAGVS L TGGLSYKEDT KELVVAKAGVYYVFFQLELR X VVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ G RLLHLSAGQRLGVHLHTEA RARHAWQLTQ GATVLGLFRV TP EIPAGLPS PRSE (SEQ ID NO: 144), wherein X is any amino acid except Arg. In some cases, X is Ala;

[0486] PAGLLDLRQG MFAQLVAQNV LLIGGPLSWY SDPGLAGVS L TGGLSYKEDT KELVVAKAGVYYVFFQLELR R X VAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ G RLLHLSAGQRLGVHLHTEA RARHAWQLTQ GATVLGLFRV TP EIPAGLPS PRSE (SEQ ID NO: 145), wherein X is any amino acid except Val. In some cases, X is Ala;

[0487] PAGLLDLRQG MFAQLVAQNV LLIGGPLSWY SDPGLAGVS L TGGLSYKEDT KELVVAKAGVYYVFFQLELR RV XAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ G RLLHLSAGQRLGVHLHTEA RARHAWQLTQ GATVLGLFRV TP EIPAGLPS PRSE (SEQ ID NO: 146), wherein X is any amino acid except Val. In some cases, X is Ala;

[0488] PAGLLDLRQG MFAQLVAQNV LLIGGPLSWY SDPGLAGVS L TGGLSYKEDT KELVVAKAGVYYVFFQLELR RVVA X EGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ G RLLHLSAGQRLGVHLHTEA RARHAWQLTQ GATVLGLFRV TP EIPAGLPS PRSE (SEQ ID NO: 147), wherein X is any amino acid except Gly. In some cases, X is Ala;

[0489] PAGLLDLRQG MFAQLVAQNV LLIGGPLSWY SDPGLAGVS L TGGLSYKEDT KELVVAKAGVYYVFFQLELR RVVAG X GSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ G RLLHLSAGQRLGVHLHTEA RARHAWQLTQ GATVLGLFRV TP EIPAGLPS PRSE (SEQ ID NO: 148), wherein X is any amino acid except Glu. In some cases, X is Ala;

[0490] PAGLLDLRQG MFAQLVAQNV LLIGGPLSWY SDPGLAGVS L TGGLSYKEDT KELVVAKAGVYYVFFQLELR RVVAGE X SGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ G RLLHLSAGQRLGVHLHTEA RARHAWQLTQ GATVLGLFRV TP EIPAGLPS PRSE (SEQ ID NO: 149), wherein X is any amino acid except Gly. In some cases, X is Ala;

[0491] PAGLLDLRQG MFAQLVAQNV LLIGGPLSWY SDPGLAGVS L TGGLSYKEDT KELVVAKAGVYYVFFQLELR RVVAGEG X GS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ G RLLHLSAGQRLGVHLHTEA RARHAWQLTQ GATVLGLFRV TP EIPAGLPS PRSE (SEQ ID NO: 150), wherein X is any amino acid except Ser. In some cases, X is Ala;

[0492] PAGLLDLRQG MFAQLVAQNV LLIGGPLSWY SDPGLAGVS L TGGLSYKEDT KELVVAKAGVYYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTV X LPPASS EARNSAFGFQ G RLLHLSAGQRLGVHLHTEA RARHAWQLTQ GATVLGLFRV TP EIPAGLPS PRSE (SEQ ID NO: 151), wherein X is any amino acid except Asp. In some cases, X is Ala;

[0493] PAGLLDLRQG MFAQLVAQNV LLIGGPLSWY SDPGLAGVS L TGGLSYKEDT KELVVAKAGVYYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVD X PPASS EARNSAFGFQ G RLLHLSAGQRLGVHLHTEA RARHAWQLTQ GATVLGLFRV TP EIPAGLPS PRSE (SEQ ID NO: 152), wherein X is any amino acid except Leu. In some cases, X is Ala;

[0494] PAGLLDLRQG MFAQLVAQNV LLIGGPLSWY SDPGLAGVS L TGGLSYKEDT KELVVAKAGVYYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDL XPASS EARNSAFGFQ G RLLHLSAGQRLGVHLHTEA RARHAWQLTQ GATVLGLFRV TP EIPAGLPS PRSE (SEQ ID NO: 153), wherein X is any amino acid except Pro. In some cases, X is Ala;

[0495] PAGLLDLRQG MFAQLVAQNV LLIGGPLSWY SDPGLAGVS L TGGLSYKEDT KELVVAKAGVYYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPA X S EARNSAFGFQ G RLLHLSAGQRLGVHLHTEA RARHAWQLTQ GATVLGLFRV TP EIPAGLPS PRSE (SEQ ID NO: 154), wherein X is any amino acid except Ser. In some cases, X is Ala;

[0496] PAGLLDLRQG MFAQLVAQNV LLIGGPLSWY SDPGLAGVS L TGGLSYKEDT KELVVAKAGVYYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPAS X EARNSAFGFQ G RLLHLSAGQRLGVHLHTEA RARHAWQLTQ GATVLGLFRV TP EIPAGLPS PRSE (SEQ ID NO: 155), wherein X is any amino acid except Ser. In some cases, X is Ala;

[0497] PAGLLDLRQG MFAQLVAQNV LLIGGPLSWY SDPGLAGVS L TGGLSYKEDT KELVVAKAGVYYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS X ARNSAFGFQ G RLLHLSAGQRLGVHLHTEA RARHAWQLTQ GATVLGLFRV TP EIPAGLPS PRSE (SEQ ID NO: 156), wherein X is any amino acid except Glu. In some cases, X is Ala;

[0498] PAGLLDLRQG MFAQLVAQNV LLIGGPLSWY SDPGLAGVS L TGGLSYKEDT KELVVAKAGVYYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EA X NSAFGFQ G RLLHLSAGQRLGVHLHTEA RARHAWQLTQ GATVLGLFRV TP EIPAGLPS PRSE (SEQ ID NO: 157), wherein X is any amino acid except Arg. In some cases, X is Ala;

[0499] PAGLLDLRQG MFAQLVAQNV LLIGGPLSWY SDPGLAGVS L TGGLSYKEDT KELVVAKAGVYYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EAR X SAFGFQ G RLLHLSAGQRLGVHLHTEA RARHAWQLTQ GATVLGLFRV TP EIPAGLPS PRSE (SEQ ID NO: 158), wherein X is any amino acid except Asn. In some cases, X is Ala;

[0500] PAGLLDLRQG MFAQLVAQNV LLIGGPLSWY SDPGLAGVS L TGGLSYKEDT KELVVAKAGVYYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARN X AFGFQ G RLLHLSAGQRLGVHLHTEA RARHAWQLTQ GATVLGLFRV TP EIPAGLPS PRSE (SEQ ID NO: 159), wherein X is any amino acid except Ser. In some cases, X is Ala;

[0501] PAGLLDLRQG MFAQLVAQNV LLIGGPLSWY SDPGLAGVS L TGGLSYKEDT KELVVAKAGVYYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSA XGFQ G RLLHLSAGQRLGVHLHTEA RARHAWQLTQ GATVLGLFRV TP EIPAGLPS PRSE (SEQ ID NO: 160), wherein X is any amino acid except Phe. In some cases, X is Ala;

[0502] PAGLLDLRQG MFAQLVAQNV LLIGGPLSWY SDPGLAGVS L TGGLSYKEDT KELVVAKAGVYYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ G RLLHLSAG X RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TP EIPAGLPS PRSE (SEQ ID NO: 161), wherein X is any amino acid except Gln. In some cases, X is Ala;

[0503] PAGLLDLRQG MFAQLVAQNV LLIGGPLSWY SDPGLAGVS L TGGLSYKEDT KELVVAKAGVYYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ G RLLHLSAGQ X LGVHLHTEA RARHAWQLTQ GATVLGLFRV TP EIPAGLPS PRSE (SEQ ID NO: 162), wherein X is any amino acid except Arg. In some cases, X is Ala;

[0504] PAGLLDLRQG MFAQLVAQNV LLIGGPLSWY SDPGLAGVS L TGGLSYKEDT KELVVAKAGVYYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ G RLLHLSAGQR X GVHLHTEA RARHAWQLTQ GATVLGLFRV T PEIPAGLPS PRSE (SEQ ID NO: 163), wherein X is any amino acid except Leu. In some cases, X is Ala;

[0505] PAGLLDLRQG MFAQLVAQNV LLIGGPLSWY SDPGLAGVS L TGGLSYKEDT KELVVAKAGVYYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ G RLLHLSAGQRL X VHLHTEA RARHAWQLTQ GATVLGLFRV TP EIPAGLPS PRSE (SEQ ID NO: 164), wherein X is any amino acid except Gly. In some cases, X is Ala;

[0506] PAGLLDLRQG MFAQLVAQNV LLIGGPLSWY SDPGLAGVS L TGGLSYKEDT KELVVAKAGVYYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ G RLLHLSAGQRLG X HLHTEA RARHAWQLTQ GATVLGLFRV TP EIPAGLPS PRSE (SEQ ID NO: 165), wherein X is any amino acid except Val. In some cases, X is Ala;

[0507] PAGLLDLRQG MFAQLVAQNV LLIGGPLSWY SDPGLAGVS L TGGLSYKEDT KELVVAKAGVYYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ G RLLHLSAGQRLGV X LHTEA RARHAWQLTQ GATVLGLFRV TP EIPAGLPS PRSE (SEQ ID NO: 166), wherein X is any amino acid except His. In some cases, X is Ala;

[0508] PAGLLDLRQG MFAQLVAQNV LLIGGPLSWY SDPGLAGVS L TGGLSYKEDT KELVVAKAGVYYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ G RLLHLSAGQRLGVH XHTEA RARHAWQLTQ GATVLGLFRV T PEIPAGLPS PRSE (SEQ ID NO: 167), wherein X is any amino acid except Leu. In some cases, X is Ala;

[0509] PAGLLDLRQG MFAQLVAQNV LLIGGPLSWY SDPGLAGVS L TGGLSYKEDT KELVVAKAGVYYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ G RLLHLSAGQRLGVHL X TEA RARHAWQLTQ GATVLGLFRV TP EIPAGLPS PRSE (SEQ ID NO: 168), wherein X is any amino acid except His. In some cases, X is Ala;

[0510] PAGLLDLRQG MFAQLVAQNV LLIGGPLSWY SDPGLAGVS L TGGLSYKEDT KELVVAKAGVYYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ G RLLHLSAGQRLGVHLH X EA RARHAWQLTQ GATVLGLFRV T PEIPAGLPS PRSE (SEQ ID NO: 169), wherein X is any amino acid except Thr. In some cases, X is Ala;

[0511] PAGLLDLRQG MFAQLVAQNV LLIGGPLSWY SDPGLAGVS L TGGLSYKEDT KELVVAKAGVYYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ G RLLHLSAGQRLGVHLHT X A RARHAWQLTQ GATVLGLFRV T PEIPAGLPS PRSE (SEQ ID NO: 170), wherein X is any amino acid except Glu. In some cases, X is Ala;

[0512] PAGLLDLRQG MFAQLVAQNV LLIGGPLSWY SDPGLAGVS L TGGLSYKEDT KELVVAKAGVYYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ G RLLHLSAGQRLGVHLHTEA X ARHAWQLTQ GATVLGLFRV TP EIPAGLPS PRSE (SEQ ID NO: 171), wherein X is any amino acid except Arg. In some cases, X is Ala;

[0513] PAGLLDLRQG MFAQLVAQNV LLIGGPLSWY SDPGLAGVS L TGGLSYKEDT KELVVAKAGVYYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ G RLLHLSAGQRLGVHLHTEA RA X HAWQLTQ GATVLGLFRV TP EIPAGLPS PRSE (SEQ ID NO: 172), wherein X is any amino acid except Arg. In some cases, X is Ala;

[0514] PAGLLDLRQG MFAQLVAQNV LLIGGPLSWY SDPGLAGVS L TGGLSYKEDT KELVVAKAGVYYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ G RLLHLSAGQRLGVHLHTEA RAR X AWQLTQ GATVLGLFRV TP EIPAGLPS PRSE (SEQ ID NO: 173), wherein X is any amino acid except His. In some cases, X is Ala;

[0515] PAGLLDLRQG MFAQLVAQNV LLIGGPLSWY SDPGLAGVS L TGGLSYKEDT KELVVAKAGVYYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ G RLLHLSAGQRLGVHLHTEA RARHA XQLTQ GATVLGLFRV TP EIPAGLPS PRSE (SEQ ID NO: 174), wherein X is any amino acid except Trp. In some cases, X is Ala;

[0516] PAGLLDLRQG MFAQLVAQNV LLIGGPLSWY SDPGLAGVS L TGGLSYKEDT KELVVAKAGVYYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ G RLLHLSAGQRLGVHLHTEA RARHAWQ X TQ GATVLGLFRV T PEIPAGLPS PRSE (SEQ ID NO: 175), wherein X is any amino acid except Leu. In some cases, X is Ala;

[0517] PAGLLDLRQG MFAQLVAQNV LLIGGPLSWY SDPGLAGVS L TGGLSYKEDT KELVVAKAGVYYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ G RLLHLSAGQRLGVHLHTEA RARHAWQL X Q GATVLGLFRV T PEIPAGLPS PRSE (SEQ ID NO: 176), wherein X is any amino acid except Thr. In some cases, X is Ala;

[0518] PAGLLDLRQG MFAQLVAQNV LLIGGPLSWY SDPGLAGVS L TGGLSYKEDT KELVVAKAGVYYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ G RLLHLSAGQRLGVHLHTEA RARHAWQLT X GATVLGLFRV TP EIPAGLPS PRSE (SEQ ID NO: 177), wherein X is any amino acid except Gln. In some cases, X is Ala;

[0519] PAGLLDLRQG MFAQLVAQNV LLIGGPLSWY SDPGLAGVS L TGGLSYKEDT KELVVAKAGVYYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ G RLLHLSAGQRLGVHLHTEA RARHAWQLTQ X ATVLGLFRV TP EIPAGLPS PRSE (SEQ ID NO: 178), wherein X is any amino acid except Gly. In some cases, X is Ala;

[0520] PAGLLDLRQG MFAQLVAQNV LLIGGPLSWY SDPGLAGVS L TGGLSYKEDT KELVVAKAGVYYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ G RLLHLSAGQRLGVHLHTEA RARHAWQLTQ GA X VLGLFRV T PEIPAGLPS PRSE (SEQ ID NO: 179), wherein X is any amino acid except Thr. In some cases, X is Ala; and

[0521] PAGLLDLRQG MFAQLVAQNV LLIGGPLSWY SDPGLAGVS L TGGLSYKEDT KELVVAKAGVYYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ G RLLHLSAGQRLGVHLHTEA RARHAWQLTQ GAT X LGLFRV TP EIPAGLPS PRSE (SEQ ID NO: 180), wherein X is any amino acid except Val. In some cases, X is Ala.

[0522] IL-2 variants

[0523] In some cases, the variant immunomodulatory polypeptide present in the TMMP of the present disclosure is a variant IL-2 polypeptide.Wild-type IL-2 binds to the IL-2 receptor (IL-2R), a heterotrimeric polypeptide comprising IL-2Rα, IL-2Rβ, and IL-2Rγ.

[0524] The wild-type IL-2 amino acid sequence may be as follows: APTSSSTKKT QLQL EH LLL D LQMILNGINNYKNPKLTRML T F KF Y MPKKA TELKHLQCLE EELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSETTF MCEYADE TATIVEFLNRWITFC Q SIIS TLT (SEQ ID NO: 15).

[0525] Wild-type IL2 binds to the IL2 receptor (IL2R) on the cell surface. In some cases, the IL2 receptor is a heterotrimeric polypeptide comprising an α chain (IL-2Rα; also known as CD25), a β chain (IL-2Rβ; also known as CD122: and a γ chain (IL-2Rγ; also known as CD132). The amino acid sequences of human IL-2Rα, IL2Rβ, and IL-2Rγ can be as follows.

[0526] Human IL-2Rα: ELCDDDPPE IPHATFKAMA YKEGTMLNCE CKR GFRRIKS GSLYMLCTGNSSHSSWDNQC QCTSSATRNT TKQVT PQPEE QKERKTTEMQ SPMQPVDQAS LPGHCREPPP WENEATERIY HFVVGQMVYY QCVQGYRALH RGPAESVCKM THGKTR WTQP QLICTGEMET SQFPGEEEKPQASPEGRPESE TSCLVTTTD F QIQTEMAATM ETSIFTTEYQ VAVAGCVFLL ISVLLLSGLT WQRRQRKSRR TI (SEQ ID NO: 16).

[0527] Human IL-2Rβ: VNG TSQFTCFYNS RANISCVWSQ DGALQDTSC Q VHAWPDRRRW NQTCELLPVSQASWACNLIL GAPDSQKLTT VDIVTLRVLC REGVRWRVMA IQDFKPFENL RLMAPISLQV V HVETHRCNISWEISQASHY FERHLEFEAR TLSPGHTWEE APLL TLKQKQ EWICLETLTP DTQYEFQVRV KPLQGEFTTWSPWSQP LAFR TKPAALGKDT IPWLGHLLVG LSGAFGFIIL VYLLINCRN T GPWLKKVLKCNTPDPSKFFS QLSSEHGGDV QKWLSSPFPS SSFSPGGLAP EISPLEVLER DKVTQLLLQQ DKVPEPASLSSNHS LTSCFT NQGYFFFHLP DALEIEACQV YFTYDPYSEE DPDEGVA GAP TGSSPQPLQPLSGEDDAYCT FPSRDDLLLF SPSLLGGPSP PSTAPGGSGA GEERMPPSLQ ERVPRDWDPQ PLGPPTPGVPDL VDFQPPPE LVLREAGEEV PDAGPREGVS FPWSRPPGQG EFRA LNARLP LNTDAYLSLQELQGQDPTHL V (SEQ ID NO:17).

[0528] Human IL-2Rγ: LNTTILTP NGNEDTTADF FLTTMPTDSL SVSTLP LPEV QCFVFNVEYMNCTWNSSSEP QPTNLTLHYW YKNSDND KVQ KCSHYLFSEE ITSGCQLQKK EIHLYQTFVV QLQDPREPRR QATQMLKLQN LVIPWAPENL TLHKLSESQL ELNWNNRFLN HCLEHLVQYR TDWDHSWTEQSVDYRHKFSL PSVDGQKRYT FRVRSRFNPL CGSAQHWSEW SHPIHWGSNT SKENPFLFAL E AVVISVGSMGLIISLLCVY FWLERTMPRI PTLKNLEDLV TEYH GNFSAW SGVSKGLAES LQPDYSERLC LVSEIPPKGGALGEGP ​​GASP CNQHSPYWAP PCYTLKPET (SEQ ID NO:18).

[0529] In some cases, when the TMMP of the present disclosure comprises a variant IL-2 polypeptide, the "cognate co-immunomodulatory polypeptide" is an IL-2R comprising a polypeptide comprising the amino acid sequences of SEQ ID NOs: 16, 17, and 18.

[0530] In some cases, the variant IL-2 polypeptide exhibits reduced binding affinity for IL-2R compared to the binding affinity of the IL-2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO:15. For example, in some cases, the variant IL-2 polypeptide binds to IL-2R with an affinity that is at least 10% less, at least 15% less, at least 20% less, at least 25% less, at least 30% less, at least 35% less, at least 40% less, at least 45% less, at least 50% less, at least 55% less, at least 60% less, at least 65% less, at least 70% less, at least 75% less, at least 80% less, at least 85% less, at least 90% less, at least 95% less, or greater than 95% less than the binding affinity of an IL-2 polypeptide comprising the sequence of amino acids set forth in SEQ ID NO: 15 for an IL-2R polypeptide (e.g., an IL-2R comprising a polypeptide comprising the sequence of amino acids set forth in SEQ ID NOs: 16-18), when measured under the same conditions.

[0531] In some cases, the variant IL-2 polypeptide has a binding affinity for IL-2R of 100 nM to 100 μM. As another example, in some cases, the variant IL-2 polypeptide has a binding affinity for IL-2R (e.g., an IL-2R comprising a polypeptide comprising the amino acid sequence set forth in SEQ ID NOs: 16-18) of about 100 nM to 150 nM, about 150 nM to about 200 nM, about 200 nM to about 250 nM, about 250 nM to about 300 nM, about 300 nM to about 350 nM, about 350 nM to about 400 nM, about 400 nM to about 500 nM, about 500 nM to about 600 nM, about 600 nM to about 700 nM, about 700 nM to about 800 nM, about 800 nM to about 900 nM, about 900 nM to about 1 μM to about 1 μM to about 5 μM, about 5 μM to about 10 μM, about 10 μM to about 15 μM, about 15 μM to about 20 μM, about 20 μM to about 25 μM, about 25 μM to about 50 μM, about 50 μM to about 75 μM, or about 75 μM to about 100 μM.

[0532] In some cases, the variant IL-2 polypeptide has a single amino acid substitution compared to the IL-2 amino acid sequence set forth in SEQ ID NO: 15. In some cases, the variant IL-2 polypeptide has 2 to 10 amino acid substitutions compared to the IL-2 amino acid sequence set forth in SEQ ID NO: 15. In some cases, the variant IL-2 polypeptide has 2 amino acid substitutions compared to the IL-2 amino acid sequence set forth in SEQ ID NO: 15. In some cases, the variant IL-2 polypeptide has 3 amino acid substitutions compared to the IL-2 amino acid sequence set forth in SEQ ID NO: 15. In some cases, the variant IL-2 polypeptide has 4 amino acid substitutions compared to the IL-2 amino acid sequence set forth in SEQ ID NO: 15. In some cases, the variant IL-2 polypeptide has 5 amino acid substitutions compared to the IL-2 amino acid sequence set forth in SEQ ID NO: 15. In some cases, the variant IL-2 polypeptide has 6 amino acid substitutions compared to the IL-2 amino acid sequence set forth in SEQ ID NO: 15. In some cases, the variant IL-2 polypeptide has 7 amino acid substitutions compared to the IL-2 amino acid sequence set forth in SEQ ID NO: 15. In some cases, the variant IL-2 polypeptide has 8 amino acid substitutions compared to the IL-2 amino acid sequence set forth in SEQ ID NO: 15. In some cases, the variant IL-2 polypeptide has 9 amino acid substitutions compared to the IL-2 amino acid sequence set forth in SEQ ID NO: 15. In some cases, the variant IL-2 polypeptide has 10 amino acid substitutions compared to the IL-2 amino acid sequence set forth in SEQ ID NO: 15.

[0533] Suitable IL-2 variants include a polypeptide comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to any of the following amino acid sequences:

[0534] APTSSSTKKT QLQLEHLLLD LQMILNGINN YKNPKLTRML T X KFYMPKKA TELKHLQCLEEELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNR WITFCQSIISTLT (SEQ ID NO: 181), wherein X is any amino acid except Phe. In some cases, X is Ala. In some cases, X is Met. In some cases, X is Pro. In some cases, X is Ser. In some cases, X is Thr. In some cases, X is Trp. In some cases, X is Tyr. In some cases, X is Val. In some cases, X is His;

[0535] APTSSSTKKT QLQLEHLLL X LQMILNGINN YKNPKLTRML TFKFYMPKKA TELKHLQCLEEELKPLEEVL NLAQSKNFHL R PRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNR WITFCQSIIS TLT (SEQ ID NO: 182), wherein X is any amino acid except Asp. In some cases, X is Ala;

[0536] APTSSSTKKT QLQL X HLLLD LQMILNGINN YKNPKLTRML TFKFYMPKKA TELKHLQCLEEELKPLEEVL NLAQSKNFHL R PRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNR WITFCQSIIS TLT (SEQ ID NO: 183), wherein X is any amino acid except Glu. In some cases, X is Ala.

[0537] APTSSSTKKT QLQLE XLLLD LQMILNGINN YKNPKLTRML TFKFYMPKKA TELKHLQCLEEELKPLEEVL NLAQSKNFHL R PRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNR WITFCQSIIS TLT (SEQ ID NO: 184), wherein X is any amino acid except His. In some cases, X is Ala. In some cases, X is Thr. In some cases, X is Asn. In some cases, X is Cys. In some cases, X is Gln. In some cases, X is Met. In some cases, X is Val. In some cases, X is Trp;

[0538] APTSSSTKKT QLQLE X LLLD LQMILNGINN YKNPKLTRML TFKFYMPKKA TELKHLQCLEEELKPLEEVL NLAQSKNFHL R PRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNR WITFCQSIIS TLT (SEQ ID NO: 185), wherein X is any amino acid except His. In some cases, X is Ala. In some cases, X is Arg. In some cases, X is Asn. In some cases, X is Asp. In some cases, X is Cys. In some cases, X is Glu. In some cases, X is Gln. In some cases, X is Gly. In some cases, X is Ile. In some cases, X is Lys. In some cases, X is Leu. In some cases, X is Met. In some cases, X is Phe. In some cases, X is Pro. In some cases, X is Ser. In some cases, X is Thr. In some cases, X is Tyr. In some cases, X is Trp. In some cases, X is Val;

[0539] APTSSSTKKT QLQLEHLLLLD LQMILNGINN YKNPKLTRML TFKF X MPKKA TELKHLQCLEEELKPLEEVL NLAQSKNFHL R PRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNR WITFCQSIIS TLT (SEQ ID NO: 186), wherein X is any amino acid except Tyr. In some cases, X is Ala;

[0540] APTSSSTKKT QLQLEHLLLD LQMILNGINN YKNPKLTRML TFKFYMPKKA TELKHLQCLEEELKPLEEVL NLAQSKNFHL R PRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNR WITFC X SIIS TLT (SEQ ID NO: 187), wherein X is any amino acid except Gln. In some cases, X is Ala;

[0541] APTSSSTKKT QLQLE X1 LLLD LQMILNGINN YKNPKLTRM LT X2 KFYMPKKA TELKHLQCLEEELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNR WIT FCQSIISTLT (SEQ ID NO: 188), wherein X1 is any amino acid except His, and wherein X2 is any amino acid except Phe. In some cases, X1 is Ala. In some cases, X2 is Ala. In some cases, X1 is Ala; and X2 is Ala. In some cases, X1 is Thr; and X2 is Ala;

[0542] APTSSSTKKT QLQLEHLLL X1 LQMILNGINN YKNPKLTRM LT X2 KFYMPKKA TELKHLQCLEEELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNR WIT FCQSIISTLT (SEQ ID NO: 189), wherein X1 is any amino acid except Asp; and wherein X2 is any amino acid except Phe. In some cases, X1 is Ala. In some cases, X2 is Ala. In some cases, X1 is Ala; and X2 is Ala;

[0543] APTSSSTKKT QLQL X1 HLLL X2 LQMILNGINN YKNPKLTRM LT X3KFYMPKKA TELKHLQCLEEELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNR WIT FCQSIISTLT (SEQ ID NO: 190), wherein X1 is any amino acid except Glu; wherein X2 is any amino acid except Asp; and wherein X3 is any amino acid except Phe. In some cases, X1 is Ala. In some cases, X2 is Ala. In some cases, X3 is Ala. In some cases, X1 is Ala; X2 is Ala; and X3 is Ala;

[0544] APTSSSTKKT QLQLE X1 LLL X2 LQMILNGINN YKNPKLTRM LT X3 KFYMPKKA TELKHLQCLEEELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNR WIT FCQSIISTLT (SEQ ID NO: 191), wherein X1 is any amino acid except His; wherein X2 is any amino acid except Asp; and wherein X3 is any amino acid except Phe. In some cases, X1 is Ala. In some cases, X2 is Ala. In some cases, X3 is Ala. In some cases, X1 is Ala; X2 is Ala; and X3 is Ala;

[0545] APTSSSTKKT QLQLEHLLL X1 LQMILNGINN YKNPKLTRM LT X2 KFYMPKKA TELKHLQCLEEELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNR WITFC X3 SIIS TLT (SEQ ID NO: 192), wherein X1 is any amino acid except Asp; wherein X2 is any amino acid except Phe; and wherein X3 is any amino acid except Gln. In some cases, X1 is Ala. In some cases, X2 is Ala. In some cases, X3 is Ala. In some cases, X1 is Ala; X2 is Ala; and X3 is Ala;

[0546] APTSSSTKKT QLQLEHLLLX1 LQMILNGINN YKNPKLTRM LT X2 KF X3 MPKKA TELKHLQCLEEELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNR WIT FCQSIISTLT (SEQ ID NO: 193), wherein X1 is any amino acid except Asp; wherein X2 is any amino acid except Phe; and wherein X3 is any amino acid except Tyr. In some cases, X1 is Ala. In some cases, X2 is Ala. In some cases, X3 is Ala. In some cases, X1 is Ala; X2 is Ala; and X3 is Ala;

[0547] APTSSSTKKT QLQLE X1 LLL X2 LQMILNGINN YKNPKLTRM LT X3 KF X4 MPKKA TELKHLQCLEEELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNR WIT FCQSIISTLT (SEQ ID NO: 194), wherein X1 is any amino acid except His; wherein X2 is any amino acid except Asp; wherein X3 is any amino acid except Phe; and wherein X4 is any amino acid except Tyr. In some cases, X1 is Ala. In some cases, X2 is Ala. In some cases, X3 is Ala. In some cases, X4 is Ala. In some cases, X1 is Ala; X2 is Ala; X3 is Ala; and X4 is Ala;

[0548] APTSSSTKKT QLQLEHLLL X1 LQMILNGINN YKNPKLTRM LT X2 KF X3 MPKKA TELKHLQCLEEELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNR WITFC X4SIIS TLT (SEQ ID NO: 195), wherein X1 is any amino acid except Asp; wherein X2 is any amino acid except Phe; wherein X3 is any amino acid except Tyr; and wherein X4 is any amino acid except Gln. In some cases, X1 is Ala. In some cases, X2 is Ala. In some cases, X3 is Ala. In some cases, X4 is Ala. In some cases, X1 is Ala; X2 is Ala; X3 is Ala; and X4 is Ala;

[0549] APTSSSTKKT QLQLE X1 LLL X2 LQMILNGINN YKNPKLTRM LT X3 KF X4 MPKKA TELKHLQCLEEELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNR WITFC X5 SIIS TLT (SEQ ID NO: 196), wherein X1 is any amino acid except His; wherein X2 is any amino acid except Asp; wherein X3 is any amino acid except Phe; wherein X4 is any amino acid except Tyr; and wherein X5 is any amino acid except Gln. In some cases, X1 is Ala. In some cases, X2 is Ala. In some cases, X3 is Ala. In some cases, X4 is Ala. In some cases, X5 is Ala. In some cases, X1 is Ala; X2 is Ala; X3 is Ala; X4 is Ala; X5 is Ala; and

[0550] APTSSSTKKT QLQLE X1 LLLD LQMILNGINN YKNPKLTRM LT X2 KFYMPKKA TELKHLQCLEEELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNR WITFCX 3SIIS TLT (SEQ ID NO: 197), wherein X1 is any amino acid except His; wherein X2 is any amino acid except Phe; and wherein X3 is any amino acid except Gln. In some cases, X1 is Ala. In some cases, X2 is Ala. In some cases, X3 is Ala. In some cases, X1 is Ala; X2 is Ala; and X3 is Ala.

[0551] Additional peptides

[0552] In addition to those polypeptides described above, the polypeptides of the TMMP of the present disclosure may also include one or more polypeptides. Suitable additional polypeptides include epitope tags and affinity domains. The one or more additional polypeptides may be included at the N-terminus of the TMMP polypeptide chain, at the C-terminus of the TMMP polypeptide chain, or within the TMMP polypeptide chain.

[0553] Epitope tag

[0554] Suitable epitope tags include, but are not limited to, hemagglutinin (HA; e.g., YPYDVPDYA (SEQ ID NO: 271); FLAG (e.g., DYKDDDDK (SEQ ID NO: 272); c-myc (e.g., EQKLISEEDL; SEQ ID NO: 273); and the like.

[0555] Affinity domain

[0556] Affinity domains include peptide sequences that interact with binding partners, such as those immobilized on a solid support for identification or purification. DNA sequences encoding multiple consecutive single amino acids, such as histidine, when fused to expressed proteins can be used to purify recombinant proteins in one step by high affinity binding to resin columns, such as nickel agarose. Exemplary affinity domains include His5 (HHHHH) (SEQ ID NO: 271), HisX6 (HHHHHH) (SEQ ID NO: 275), C-myc (EQKLISEEDL) (SEQ ID NO: 276), Flag (DYKDDDDK) (SEQ ID NO: 277), StrepTag (WSHPQFEK) (SEQ ID NO: 278), hemagglutinins such as HA Tag (YPYDVPDYA) (SEQ ID NO: 279), glutathione S-transferase (GST), thioredoxin, cellulose binding domain, RYIRS (SEQ ID NO: 280), Phe-His-His-Thr (SEQ ID NO: 281), chitin binding domain, S-peptide, T7 peptide, SH2 domain, C-terminal RNA tag, WEAAAREACCRECCARA (SEQ ID NO: 282), SEQ ID NO: 283, SEQ ID NO: 284, SEQ ID NO: 285, SEQ ID NO: 286, SEQ ID NO: 287, SEQ ID NO: 288, SEQ ID NO: 289, SEQ ID NO: 300, SEQ ID NO: 301, SEQ ID NO: 302, SEQ ID NO: 303, SEQ ID NO: 304, SEQ ID NO: 305, SEQ ID NO: 306, SEQ ID NO: 307, SEQ ID NO: 308, SEQ ID NO: 309, SEQ ID NO: 310, SEQ ID NO: 311, SEQ ID NO: 312, SEQ ID NO: 313, SEQ ID NO: 314, SEQ ID NO: 315 NO: 282), a metal binding domain, e.g., a zinc binding domain or a calcium binding domain, such as those from a calcium binding protein, e.g., calmodulin, troponin C, calcineurin B, myosin light chain, recoverin, S-modulin, cone opsin, VILIP, calcineurin, hippocampal calcium binding protein, neuronal calcium sensor protein (frequenin), caltractin, calpain subunit, S100 protein, parvalbumin, calbindin D9K, calbindin D28K, and calreticulin, intein, biotin, streptavidin, MyoD, Id, leucine zipper sequence, and maltose binding protein.

[0557] Drug conjugates

[0558] The polypeptide chain of the TMMP of the present disclosure may include a small molecule drug linked (e.g., covalently attached) to the polypeptide chain. For example, when the TMMP of the present disclosure includes an Fc polypeptide, the Fc polypeptide may include a covalently linked small molecule drug. In some cases, the small molecule drug is a cancer chemotherapeutic agent, such as a cytotoxic agent. The polypeptide chain of the TMMP of the present disclosure may include a cytotoxic agent linked (e.g., covalently attached) to the polypeptide chain. For example, when the TMMP of the present disclosure includes an Fc polypeptide, the Fc polypeptide may include a covalently linked cytotoxic agent. Cytotoxic agents include prodrugs.

[0559] The drug (e.g., a cancer chemotherapeutic agent) can be directly or indirectly linked to the polypeptide chain of the TMMP of the present disclosure. For example, when the TMMP of the present disclosure comprises an Fc polypeptide, the drug (e.g., a cancer chemotherapeutic agent) can be directly or indirectly linked to the Fc polypeptide. Direct linkage can involve direct linkage to an amino acid side chain. Indirect linkage can be linkage via a linker. The drug (e.g., a cancer chemotherapeutic agent) can be linked to the polypeptide chain (e.g., Fc polypeptide) of the TMMP of the present disclosure via a thioether bond, an amide bond, a carbamate bond, a disulfide bond, or an ether bond.

[0560] The linker includes a cleavable linker and a non-cleavable linker. In some cases, the linker is a protease-cleavable linker. Suitable linkers include, for example, peptides (e.g., 2 to 10 amino acids in length; e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids in length), alkyl chains, poly (ethylene glycol), disulfide groups, thioether groups, acid-labile groups, photolabile groups, peptidase-labile groups, and esterase-labile groups. Non-limiting examples of suitable linkers are: i) N-succinimidyl-[(N-maleimidopropionamido)-tetraethylene glycol] ester (NHS-PEG4-maleimide); ii) N-succinimidyl 4-(2-pyridyldithio)butyrate (SPDB); N-succinimidyl 4-(2-pyridyldithio)2-sulfobutyrate (sulfo-SPDB); N-succinimidyl 4-(2-pyridyldithio)pentanoate (SPP); N-succinimidyl-4-(N-maleimido)butyrate (SPDB); methyl)-cyclohexane-1-carboxyl-6-amidohexanoate (LC-SMCC); kappa-maleimidoceanoic acid N-succinimidyl ester (KMUA); gamma-maleimidobutyric acid N-succinimidyl ester (GMBS); epsilon-maleimidocaproic acid N-hydroxysuccinimide ester (EMCS); m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS); N-(α-maleimidoacetoxy)-succinimide ester (AMAS); succinimidyl-6-(β- maleimidopropionamide) hexanoate (SMPH); 4-(p-maleimidophenyl) butyric acid N-succinimidyl ester (SMPB); N-(p-maleimidophenyl) isocyanate (PMPI); 4-(2-pyridylthio) pentanoic acid N-succinimidyl ester (SPP); N-succinimidyl (4-iodoacetyl) aminobenzoate (SIAB); 6-maleimidocaproyl (MC); maleimidopropionyl (MP); p-aminobenzyloxycarbonyl (PAB); 4-( N-succinimidyl maleimidomethyl)cyclohexanecarboxylate (SMCC); N-succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxy-(6-amidohexanoate), a "long-chain" analog of SMCC (LC-SMCC); N-succinimidyl 3-maleimidopropionate (BMPS); N-succinimidyl iodoacetate (SIA); N-succinimidyl bromoacetate (SBA); and N-succinimidyl 3-(bromoacetamido)propionate (SBAP).

[0561] Polypeptides (e.g., Fc polypeptides) can be modified with cross-linking reagents such as 4-(N-maleimidomethyl)-cyclohexane-1-carboxylic acid succinimidyl ester (SMCC), sulfo-SMCC, maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), sulfo-MBS, or succinimidyl-iodoacetate to introduce 1-10 reactive groups. The modified Fc polypeptide is then reacted with a thiol-containing cytotoxic agent to produce a conjugate.

[0562] For example, when a TMMP of the present disclosure comprises an Fc polypeptide, the polypeptide chain comprising the Fc polypeptide may have the formula (A)-(L)-(C), wherein (A) is a polypeptide chain comprising an Fc polypeptide; wherein (L), if present, is a linker; and wherein (C) is a cytotoxic agent. (L), if present, links (A) to (C). In some cases, the polypeptide chain comprising the Fc polypeptide may comprise more than one cytotoxic agent (e.g., 2, 3, 4, or 5 or more cytotoxic agents).

[0563] Suitable drugs include, for example, rapamycin. Suitable drugs include, for example, retinoids, such as all-trans retinoic acid (ATRA); vitamin D3; vitamin D3 analogs; etc. As described above, in some cases, the drug is a cytotoxic agent. Cytotoxic agents are known in the art. Suitable cytotoxic agents can be any compound that causes cell death, or induces cell death, or reduces cell viability in some way, and include, for example, maytansinoids and maytansinoid analogs, benzodiazepines, taxoids, CC-1065 and CC-1065 analogs, duocarmycins and duocarmycin analogs, enediynes such as calicheamicin, dolestatin and dolestatin analogs (including auristatins), tomaymycin derivatives, leptomycin derivatives, methotrexate, cisplatin, carboplatin, daunomycin, doxorubicin, vincristine, vinblastine, melphalan, mitomycin C, nitrogen mustard phenylbutyric acid, and morpholinodoxorubicin.

[0564] For example, in some cases, cytotoxic agents are compounds that inhibit the formation of microtubules in eukaryotic cells. Such agents include, for example, maytansine, benzodiazepines, taxoids, CC-1065, duocarmycins, duocarmycin analogs, calicheamicins, Aplysia, Aplysia analogs, Auristatin, tomaymycins and leptomycins or a prodrug of any of the foregoing. Maytansine compounds include, for example, N (2 ')-deacetyl-N (2 ')-(3-mercapto-1-oxopropyl)-maytansine (DM1); N (2 ')-deacetyl-N (2 ')-(4-mercapto-1-oxopentyl)-maytansine (DM3); and N (2 ')-deacetyl-N2-(4-mercapto-4-methyl-1-oxopentyl)-maytansine (DM4). Benzodiazepines include, for example, indoline benzodiazepines and oxazolidinone benzodiazepines.

[0565] Cytotoxic agents include paclitaxel; cytokine B; gramicidin D; ethidium bromide; emetine; mitomycin; etoposide; teniposide; vincristine; vinblastine; colchicine; doxorubicin; daunomycin; dihydroxyanthraquinone; maytansine or its analogs or derivatives; auristatin or its functional peptide analogs or derivatives; urostomycin 10 or 15 or its analogs; irinotecan or its analogs; mitoxantrone; mithramycin; actinomycin D; 1-dehydrotestosterone; glucocorticoids; procaine; tetracaine; lidocaine; propranolol; puromycin; calicheamicin or its analogs or derivatives; antimetabolites; 6-mercaptopurine; 6-thioguanine; cytarabine; fludarabine; 5-fluorouracil; dacarbazine; hydroxyurea; asparaginase; gemcitabine; cladribine; alkylating agents; platinum. derivatives; duocarmycin A; duocarmycin SA; rachelmycin (CC-1065) or its analogs or derivatives; antibiotics; pyrrolo[2,1-c][1,4]-benzodiazepine (PDB); diphtheria toxin; ricin toxin; cholera toxin; Shiga-like toxin; LT toxin; C3 toxin; Shiga toxin; pertussis toxin; tetanus toxin; soybean Bowman-Birk protease inhibitor; Pseudomonas exotoxin; alorin; saponin; modeccin; gelanin; abrin A chain; modeccin A chain; α-sarcin; Aleurites fordii protein; dianthin protein; Phytolacca americana americana protein; momordica charantia inhibitor; curcin; crotonin; sapaonaria officinalis inhibitor; gelonin; mitogellin; restrictocin; phenomycin; enomycin toxin; ribonuclease (RNase); DNase I; Staphylococcal enterotoxin A; pokeweed antiviral protein; diphtheria toxin; and Pseudomonas exotoxin.

[0566] Exemplary TMMPs

[0567] The TMMPs of the present disclosure comprise at least one heterodimer comprising: a) a first polypeptide comprising: i) a WT-1 peptide epitope; and ii) a first MHC polypeptide; b) a second polypeptide comprising a second MHC polypeptide; and c) at least one immunomodulatory polypeptide, wherein the first polypeptide and / or the second polypeptide comprise an immunomodulatory polypeptide. Thus, in some cases, the TMMPs of the present disclosure comprise at least one heterodimer comprising: a) a first polypeptide comprising: i) a WT-1 peptide epitope; ii) a first MHC polypeptide; and iii) at least one immunomodulatory polypeptide; and b) a second polypeptide comprising a second MHC polypeptide. In other cases, the TMMPs of the present disclosure comprise at least one heterodimer comprising: a) a first polypeptide comprising: i) a WT-1 peptide epitope; and ii) a first MHC polypeptide; and b) a second polypeptide comprising: i) a second MHC polypeptide; and ii) at least one immunomodulatory polypeptide. In some cases, the TMMP of the present disclosure comprises at least one heterodimer comprising: a) a first polypeptide comprising: i) a WT-1 peptide epitope; ii) a first MHC polypeptide; and iii) at least one immunomodulatory polypeptide; and b) a second polypeptide comprising: i) a second MHC polypeptide; and ii) at least one immunomodulatory polypeptide. In some cases, the at least one immunomodulatory polypeptide is a wild-type immunomodulatory polypeptide. In other cases, the at least one immunomodulatory polypeptide is a variant immunomodulatory polypeptide that exhibits reduced affinity for a co-immunomodulatory polypeptide compared to the affinity of the corresponding wild-type immunomodulatory polypeptide for the co-immunomodulatory polypeptide. In some cases, the TMMP of the present disclosure comprises two immunomodulatory polypeptides, wherein the two immunomodulatory polypeptides have the same amino acid sequence.

[0568] In some cases, a TMMP of the present disclosure comprises: a) a first polypeptide comprising, in order from N-terminus to C-terminus: i) a WT-1 peptide epitope; ii) a first MHC polypeptide; and iii) at least one immunomodulatory polypeptide; and b) a second polypeptide comprising, in order from N-terminus ...

Claims

1. A T cell regulatory multimeric polypeptide (TMMP), comprising: at least one heterodimer, the at least one heterodimer comprising: a) a first polypeptide, the first polypeptide comprising: i) a Wilms tumor-1 (WT-1) peptide epitope, wherein the WT-1 peptide is 4 to 25 amino acids in length; and ii) class I major histocompatibility complex (MHC) polypeptides; wherein the first class I MHC polypeptide is a β2 microglobulin (β2M) polypeptide; b) a second polypeptide, the second polypeptide comprising: i) a second class I MHC polypeptide, wherein the second class I MHC polypeptide is a class I MHC heavy chain polypeptide; ii) one or more immunomodulatory polypeptides, wherein the one or more immunomodulatory polypeptides are one or more variant IL-2 polypeptides that exhibit reduced affinity for the IL-2 receptor and comprise a substitution of H16, or a substitution of H16 and a substitution of F42 based on the amino acid numbering of SEQ ID NO: 15; and iii) immunoglobulin (Ig) Fc polypeptide, wherein the first polypeptide and the second polypeptide are covalently linked to each other via at least a first disulfide bond and a second disulfide bond, wherein the first disulfide bond is formed between: (i) a Cys residue in a Cys-containing linker present between the WT-1 peptide epitope and the β2M polypeptide, and (ii) a Cys residue present in the class I MHC heavy chain polypeptide; and the second disulfide bond is formed between: a Cys residue in the β2M polypeptide and a Cys residue in the class I MHC heavy chain polypeptide.

2. The TMMP of claim 1, wherein: a) the first polypeptide comprises, in order from N-terminus to C-terminus: i) the WT-1 peptide epitope; ii) said Cys-containing linker; and iii) said β2M polypeptide; and b) the second polypeptide comprises, in order from N-terminus to C-terminus: i) one or more variant IL-2 polypeptides; ii) connectors; iii) said class I MHC heavy chain polypeptide; iv) connectors; and v) the Ig Fc polypeptide; The TMMP comprises more than one variant IL-2 polypeptide, and a linker is inserted between the variant IL-2 polypeptides.

3. The TMMP of claim 1, wherein: a) the first polypeptide comprises, in order from N-terminus to C-terminus: i) the WT-1 peptide epitope; ii) said Cys-containing linker; and iii) said β2M polypeptide; and b) the second polypeptide comprises, in order from N-terminus to C-terminus: i) the class I MHC heavy chain polypeptide; ii) connectors; iii) the Ig Fc polypeptide; iv) connectors; and v) one or more variant IL-2 polypeptides; The TMMP comprises more than one variant IL-2 polypeptide, and a linker is inserted between the variant IL-2 polypeptides.

4. The TMMP of any one of claims 1 to 3, wherein the variant IL-2 polypeptide comprises: i) an H16A substitution and a F42A substitution; or ii) an H16T substitution and a F42A substitution.

5. The TMMP according to any one of claims 1 to 3, wherein the amino acid sequence of the class I MHC heavy chain polypeptide is the amino acid sequence shown in SEQ ID NO: 199, SEQ ID NO: 201 or SEQ ID NO:

203.

6. The TMMP of claim 2, wherein the TMMP comprises two variant IL-2 polypeptides in series and connected via a linker, and wherein the amino acid sequence of each variant IL-2 is the amino acid sequence shown in SEQ ID NO: 188, wherein X1 is Ala and X2 is Ala, or wherein X1 is Thr and X2 is Ala, The amino acid sequence of the β2M polypeptide is the amino acid sequence shown in SEQ ID NO: 314, and wherein, a first disulfide bond is formed between (i) a Cys residue in a linker between the WT-1 peptide epitope and the β2M polypeptide and (ii) a Cys residue at position 84 of the class I MHC heavy chain polypeptide, and wherein a second disulfide bond is formed between a Cys residue at position 12 of the β2M polypeptide and a Cys residue at position 236 of the class I MHC heavy chain polypeptide, The amino acid sequence of the class I MHC heavy chain polypeptide is an amino acid sequence as shown in any one of SEQ ID NOs: 41-43 and 198-206, in which amino acid 84 is Cys, amino acid 236 is Cys, and Wherein, the amino acid sequence of the Ig Fc polypeptide is the amino acid sequence shown in any one of SEQ ID NOs: 418-421.

7. The TMMP of claim 6, wherein the variant IL-2 polypeptides are linked via a linker, and wherein the amino acid sequence of each variant IL-2 polypeptide is the amino acid sequence shown in SEQ ID NO: 188, wherein X1 is Ala, and X2 is Ala.

8. The TMMP according to claim 6 or 7, wherein the amino acid sequence of the class I MHC heavy chain polypeptide is the amino acid sequence shown in SEQ ID NO: 199, and wherein the epitope is VLDFAPPGA (SEQ ID NO: 259).

9. The TMMP of claim 3, wherein the TMMP comprises two variant IL-2 polypeptides connected in series and via a linker, and wherein the amino acid sequence of each variant IL-2 is the amino acid sequence shown in SEQ ID NO: 188, wherein X1 is Ala and X2 is Ala, or wherein X1 is Thr and X2 is Ala, wherein the amino acid sequence of the β2M polypeptide is the amino acid sequence shown in SEQ ID NO: 314, and wherein the first disulfide bond is formed between (i) the Cys residue in the linker between the WT-1 peptide epitope and the β2M polypeptide and (ii) the Cys residue at position 84 in the class I MHC heavy chain polypeptide, and wherein, The second disulfide bond is formed between the Cys residue at position 12 of the β2M polypeptide and the Cys residue at position 236 of the class I MHC heavy chain polypeptide, The amino acid sequence of the class I MHC heavy chain polypeptide is an amino acid sequence as shown in any one of SEQ ID NOs: 41-43 and 198-206, in which amino acid 84 is Cys, amino acid 236 is Cys, and Wherein, the amino acid sequence of the Ig Fc polypeptide is the amino acid sequence shown in any one of SEQ ID NOs: 418-421.

10. The TMMP of claim 9, wherein the variant IL-2 polypeptides are connected via a linker, and the amino acid sequence of each variant IL-2 polypeptide is the amino acid sequence shown in SEQ ID NO: 188, wherein X1 is Ala, and X2 is Ala.

11. The TMMP according to any one of claims 1 to 3, wherein the amino acid sequence of the linker between the WT-1 peptide epitope and the β2M polypeptide is the amino acid sequence shown in SEQ ID NO: 316 or SEQ ID NO:

317.

12. The TMMP according to claim 7, wherein the amino acid sequence of the first polypeptide is the amino acid sequence shown in SEQ ID NO: 423, The sequence of the class I MHC heavy chain polypeptide is the following amino acid sequence: GSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDGETRKVKAHSQTHRVDLGTLRGCYNQSEAGSHTVQRMYGCDVGSDWRFLRGYHQYAYDGKDYIALKEDLRSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGTCVEWLRRYLENGKETLQRTDAPKTHMTHHAVSDHEATLRCWALSFYPAEITLTWQRDGEDQTQDTELVETRPCGDGTFQKWAAVVVPSGQEQRYTCHVQHEGLPKPLTLRWE, and The amino acid sequence of the Ig Fc polypeptide is the amino acid sequence shown in SEQ ID NO:

421.

13. A TMMP as described in any one of claims 1 to 3, 6 or 9, comprising a first heterodimer and a second heterodimer, wherein the first heterodimer is identical to the second heterodimer in amino acid sequence and is covalently bound by one or more disulfide bonds between the Ig Fc polypeptides of the first heterodimer and the second heterodimer.

14. The TMMP of claim 12, comprising a first heterodimer and a second heterodimer, wherein the first heterodimer is identical to the second heterodimer in amino acid sequence and is covalently bound via one or more disulfide bonds between the Ig Fc polypeptides of the first heterodimer and the second heterodimer.

15. The TMMP of claim 14, wherein two disulfide bonds link the Ig Fc polypeptide of one heterodimer to the Ig Fc polypeptide of another heterodimer.

16. A pharmaceutical composition comprising the TMMP according to any one of claims 1 to 14.

17. A pharmaceutical composition comprising the TMMP according to claim 15.

18. A composition comprising one or more nucleic acids comprising nucleotide sequences encoding the first and second polypeptides of the TMMP of any one of claims 1-15.

19. A composition comprising one or more expression vectors, wherein the one or more expression vectors comprise nucleotide sequences encoding the first and second polypeptides of the TMMP of any one of claims 1-15.

20. A method for producing the TMMP of any one of claims 1 to 15, comprising culturing a host cell genetically modified with one or more expression vectors comprising nucleotide sequences encoding the first and second polypeptides of the multimeric polypeptide, and wherein the culturing is carried out under conditions such that the transgenic host cell produces TMMP.

21. A composition of a genetically modified host cell, wherein the host cell comprises one or more nucleic acids comprising a nucleotide sequence encoding a first and a second polypeptide of the TMMP of any one of claims 1-15.

22. A pharmaceutical composition as claimed in claim 16 or 17 for use in a method of treating cancer in a subject.

23. The pharmaceutical composition of claim 16 or 17, for use in a method of treating cancer in an individual, wherein the individual is also receiving treatment with a therapeutically effective amount of an immune checkpoint inhibitor.

24. A pharmaceutical composition as described in claim 16 or 17, for use in a method of treating cancer in an individual, wherein the individual is also treated with a therapeutically effective amount of an immune checkpoint inhibitor, and wherein the checkpoint inhibitor is an antibody against PD-1, PD-L1, CTLA4 or TIGIT.

25. A method for detecting the presence of a target T cell that binds a WT-1 epitope in a mixed T cell population obtained from an individual, the method comprising: a) contacting the mixed T cell population with the TMMP according to any one of claims 1 to 15 in vitro; and b) Detecting the activation and / or proliferation of WT-1 epitope-specific T cells.

26. The pharmaceutical composition of claim 16 or 17, for use in a method of treating cancer in an individual, wherein the individual is also treated with a therapeutically effective amount of an immune checkpoint inhibitor, and wherein the checkpoint inhibitor is an antibody against PD-1.

27. The pharmaceutical composition of claim 16 or 17, for use in a method of treating cancer in an individual, wherein the individual is also being treated with a therapeutically effective amount of an immune checkpoint inhibitor, and wherein the checkpoint inhibitor is pembrolizumab.

28. The pharmaceutical composition of claim 16 or 17, for use in a method of treating cancer in an individual, wherein the individual is also being treated with a therapeutically effective amount of an immune checkpoint inhibitor, and wherein the checkpoint inhibitor is nivolumab.

29. The pharmaceutical composition of claim 16 or 17, for use in a method of treating cancer in an individual, wherein the individual is also treated with a therapeutically effective amount of an immune checkpoint inhibitor, and wherein the checkpoint inhibitor is an antibody against PD-L1.

30. The pharmaceutical composition of claim 16 or 17, for use in a method of treating cancer in an individual, wherein the individual is also being treated with a therapeutically effective amount of an immune checkpoint inhibitor, and wherein the checkpoint inhibitor is atezolizumab.

31. The pharmaceutical composition of claim 16 or 17, for use in a method of treating cancer in an individual, wherein the individual is also being treated with a therapeutically effective amount of an immune checkpoint inhibitor, and wherein the checkpoint inhibitor is durvalumab.

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