T cell modulating polypeptides and methods of use thereof

Abstract

The present disclosure provides T cell modulating polypeptides (TMPs) that include an immunomodulating polypeptide, a class I HLA polypeptide (a class I HLA heavy chain polypeptide and a β2 microglobulin polypeptide), and a peptide that presents an epitope to a T cell receptor. The TMPs of the present disclosure are useful for modulating the activity of T cells and are useful for modulating an immune response in an individual.

Description

[Technical field]

[0001] cross reference This application claims the benefit of U.S. Provisional Patent Application No. 63 / 163,265, filed March 19, 2021, the entire contents of which are incorporated herein by reference.

[0002] Incorporation by reference of sequence listing provided as a text file A sequence listing is attached hereto as a text file "CUEB-139WO_SeqListing_ST25.txt" created on March 15, 2022 and having a size of 672 KB. The entire contents of this text file are incorporated herein by reference. [Background technology]

[0003] Introduction The adaptive immune response requires the binding of small peptide antigens to the T cell receptor (TCR) present on the surface of T cells. These small peptide antigens are non-covalently presented on the surface of antigen-presenting cells (APCs) by the major histocompatibility complex (MHC, also called human leukocyte antigen (HLA) complex in humans). This binding represents the targeting mechanism of the immune system and is a molecular interaction required for the regulation (activation or inhibition) and effector function of T cells. After epitope-specific cell targeting, the targeted T cell is activated by the binding of a costimulatory protein present on the APC with a corresponding costimulatory protein on the T cell. Both signals (i.e., epitope / TCR binding and binding of APC costimulatory proteins to T cell costimulatory proteins) are necessary to provide the specificity and activation or inhibition of the T cell. Costimulatory proteins on APCs are also called "immunomodulatory" proteins because they regulate the activity of T cells when they bind to costimulatory proteins on T cells. The specific regulation depends on which immunomodulatory protein on the APC binds to which immunomodulatory protein on the T cell. TCRs are specific for a particular epitope, whereas T cell costimulatory proteins are not epitope specific, but rather are generally expressed on all T cells or on large subsets of T cells. Summary of the Invention

[0004] overview The present disclosure provides single-chain T cell modulating polypeptides (TMPs) and dimers thereof. The single-chain T cell modulating polypeptides (TMPs) and dimers thereof comprise an immunomodulating polypeptide (MOD), a class I HLA polypeptide (a class I HLA heavy chain polypeptide and a β2 microglobulin polypeptide), and a peptide epitope (e.g., a cancer-associated peptide or a viral peptide) that presents the epitope to a T cell receptor. The TMPs are useful for modulating the activity of T cells and for modulating immune responses in individuals. [Brief description of the drawings]

[0005] [Figure 1A]FIG. 4 shows the amino acid sequence of wild-type human β2M polypeptide (SEQ ID NO:448). [Figure 1B] FIG. 4 shows the amino acid sequence of a β2M polypeptide containing an R12C substitution (SEQ ID NO:449). [Figure 2A] FIG. 4 shows the amino acid sequence of an immunoglobulin Fc polypeptide (SEQ ID NO:450). [Figure 2B] FIG. 4 shows the amino acid sequence of an immunoglobulin Fc polypeptide (SEQ ID NO:451). [Figure 2C] FIG. 4 shows the amino acid sequence of an immunoglobulin Fc polypeptide (SEQ ID NO:452). [Figure 2D] FIG. 4 shows the amino acid sequence of an immunoglobulin Fc polypeptide (SEQ ID NO:453). [Figure 2E] FIG. 4 shows the amino acid sequence of an immunoglobulin Fc polypeptide (SEQ ID NO:454). [Figure 2F] FIG. 4 shows the amino acid sequence of an immunoglobulin Fc polypeptide (SEQ ID NO:455). [Figure 2G] FIG. 4 shows the amino acid sequence of an immunoglobulin Fc polypeptide (SEQ ID NO:456). [Figure 2H] FIG. 4 shows the amino acid sequence of an immunoglobulin Fc polypeptide (SEQ ID NO:457). [Figure 2I] FIG. 4 shows the amino acid sequence of an immunoglobulin Fc polypeptide (SEQ ID NO:458). [Figure 2J] FIG. 4 shows the amino acid sequence of an immunoglobulin Fc polypeptide (SEQ ID NO:459). [Figure 2K] FIG. 4 shows the amino acid sequence of an immunoglobulin Fc polypeptide (SEQ ID NO:460). [Figure 2L] FIG. 4 shows the amino acid sequence of an immunoglobulin Fc polypeptide (SEQ ID NO:461). [Figure 2M] FIG. 4 shows the amino acid sequence of an immunoglobulin Fc polypeptide (SEQ ID NO:462). [Figure 3A]FIG. 1 shows the amino acid sequence of wild-type HLA-A*0201 (SEQ ID NO: 463). [Figure 3B] FIG. 4 shows the amino acid sequence of the mutant (SEQ ID NO: 464). [Figure 3C] FIG. 3 shows the amino acid sequence of the mutant (SEQ ID NO:373). [Figure 3D] FIG. 4 shows the amino acid sequence of the mutant (SEQ ID NO: 465). [Figure 3E] FIG. 4 shows the amino acid sequence of the mutant (SEQ ID NO: 464). [Figure 4A] FIG. 1 shows the amino acid sequence of wild-type HLA-A*1101 (SEQ ID NO: 467). [Figure 4B] FIG. 4 shows the amino acid sequence of the mutant (SEQ ID NO: 468). [Figure 4C] FIG. 3 shows the amino acid sequence of the mutant (SEQ ID NO:374). [Figure 4D] FIG. 4 shows the amino acid sequence of the mutant (SEQ ID NO: 469). [Figure 4E] FIG. 4 shows the amino acid sequence of the mutant (SEQ ID NO: 470). [Figure 5A] FIG. 1 shows wild type HLA-A*2402 (SEQ ID NO: 471). [Figure 5B] FIG. 4 shows the amino acid sequence of the mutant (SEQ ID NO: 472). [Figure 5C] FIG. 3 shows the amino acid sequence of the mutant (SEQ ID NO:375). [Figure 5D] FIG. 473 shows the amino acid sequence of the mutant. [Figure 5E] FIG. 4 shows the amino acid sequence of the mutant (SEQ ID NO: 474). [Figure 6A] FIG. 4 shows the amino acid sequence of wild-type HLA-A*3303 (SEQ ID NO: 475). [Figure 6B] FIG. 4 shows the amino acid sequence of the mutant (SEQ ID NO: 476). [Figure 6C] FIG. 3 shows the amino acid sequence of the mutant (SEQ ID NO:376). [Figure 6D] FIG. 477 shows the amino acid sequence of the mutant. [Figure 6E] FIG. 478 shows the amino acid sequence of the mutant. [Figure 7A-1] FIG. 1 shows a comparison of the amino acid sequences of the HLA-A heavy chains (SEQ ID NOs: 377, 599, 378, 600, 379, 601, 380, 602, and 381, respectively). [Figure 7A-2] See description of Figure 7A-1. [Figure 7B] FIG. 4 shows the consensus sequence (SEQ ID NO: 479). [Figure 8A-1] FIG. 1 shows a comparison of amino acid sequences of HLA-B heavy chains (SEQ ID NOs: 480 to 486, respectively). [Figure 8A-2] See description of Figure 8A-1. [Figure 8B] FIG. 4 shows the consensus sequence (SEQ ID NO: 487). [Figure 9A-1] FIG. 1 shows a comparison of amino acid sequences of HLA-C heavy chains (SEQ ID NOs: 488 to 496, respectively). [Figure 9A-2] See description of Figure 9A-1. [Figure 9B] FIG. 4 shows the consensus sequence (SEQ ID NO:497). [Figure 10] FIG. 1 shows the consensus amino acid sequences of HLA-E, HLA-F, and HLA-G heavy chains (SEQ ID NOs: 498, 499, 500, respectively). (Variable amino acid (aa) positions are shown as sequentially numbered "X" residues, with amino acid positions 84, 139, and 236 double underlined.) [Figure 11] FIG. 1 shows a comparison of consensus amino acid sequences for HLA-A (sequence number 479), HLA-B (sequence number 487), HLA-C (sequence number 497), HLA-E (sequence number 498), HLA-F (sequence number 499), and HLA-G (sequence number 500). [Figure 12] FIG. 1 is a schematic diagram showing a specific example of the arrangement of immunomodulatory polypeptides in a TMP. [Figure 13] FIG. 1 is a schematic representation of disulfide-bonded TMP. [Figure 14] FIG. 2 shows the amino acid sequence of alpha-fetoprotein (SEQ ID NO:501). [Figure 15A] FIG. 5 shows the amino acid sequence of the WT-1 polypeptide (SEQ ID NO:502). [Figure 15B] FIG. 5 depicts the amino acid sequence of the WT-1 polypeptide (SEQ ID NO:503). [Figure 15C] FIG. 5 shows the amino acid sequence of the WT-1 polypeptide (SEQ ID NO:504). [Figure 15D] FIG. 5 depicts the amino acid sequence of the WT-1 polypeptide (SEQ ID NO:505). [Figure 15E] FIG. 5 depicts the amino acid sequence of the WT-1 polypeptide (SEQ ID NO:506). [Figure 16A] FIG. 5 shows the amino acid sequence of the HPV E6 polypeptide (SEQ ID NO:507). [Figure 16B] FIG. 5 depicts the amino acid sequence of the HPV E7 polypeptide (SEQ ID NO:508). [Figure 17A] FIG. 5 depicts the amino acid sequence of wild-type IL-2 polypeptide (SEQ ID NO:509). [Figure 17B] FIG. 5 shows the amino acid sequence of the IL-2Rα polypeptide (SEQ ID NO:510). [Figure 17C] FIG. 5 shows the amino acid sequence of the IL-2Rβ polypeptide (SEQ ID NO:511). [Figure 17D] FIG. 5 depicts the amino acid sequence of the IL-2Rγ polypeptide (SEQ ID NO:512). [Figure 18A] FIG. 1 shows the amino acid sequence of an exemplary embodiment of TMP (SEQ ID NO:513). [Figure 18B] FIG. 1 shows the amino acid sequence of an illustrative example of TMP (SEQ ID NO:514). [Figure 18C] FIG. 1 shows the amino acid sequence of an exemplary embodiment of TMP (SEQ ID NO:515). [Figure 18D] FIG. 1 shows the amino acid sequence of an exemplary embodiment of TMP (SEQ ID NO:516). [Figure 18E] FIG. 1 shows the amino acid sequence of an exemplary embodiment of TMP (SEQ ID NO:517). [Figure 18F] FIG. 1 shows the amino acid sequence of an exemplary embodiment of TMP (SEQ ID NO:518). [Figure 18G] FIG. 1 shows the amino acid sequence of an exemplary embodiment of TMP (SEQ ID NO:519). [Figure 18H] FIG. 1 shows the amino acid sequence of an exemplary embodiment of TMP (SEQ ID NO:520). [Figure 18I] FIG. 1 shows the amino acid sequence of an exemplary embodiment of TMP (SEQ ID NO:521). [Figure 18J] FIG. 1 shows the amino acid sequence of an exemplary embodiment of TMP (SEQ ID NO:522). [Figure 18K] FIG. 1 shows the amino acid sequence of an exemplary embodiment of TMP (SEQ ID NO:523). [Figure 18L] FIG. 1 shows the amino acid sequence of an exemplary embodiment of TMP (SEQ ID NO:524). [Figure 18M] FIG. 1 shows the amino acid sequence of an exemplary embodiment of TMP (SEQ ID NO:525). [Figure 18N] FIG. 1 shows the amino acid sequence of an exemplary embodiment of TMP (SEQ ID NO:526). [Figure 18O] FIG. 1 shows the amino acid sequence of an exemplary embodiment of TMP (SEQ ID NO:527). [Figure 18P] FIG. 1 shows the amino acid sequence of an exemplary embodiment of TMP (SEQ ID NO:528). [Figure 18Q] FIG. 1 shows the amino acid sequence of an exemplary embodiment of TMP (SEQ ID NO:529). [Figure 18R] FIG. 1 shows the amino acid sequence of an exemplary embodiment of TMP (SEQ ID NO:530). [Figure 18S] FIG. 1 shows the amino acid sequence of an exemplary embodiment of TMP (SEQ ID NO:531). [Figure 18T] FIG. 1 shows the amino acid sequence of an exemplary embodiment of TMP (SEQ ID NO:532). [Figure 19A] FIG. 1 shows the amino acid sequence of an exemplary embodiment of TMP (SEQ ID NO:533). [Figure 19B]FIG. 1 shows the amino acid sequence of an exemplary embodiment of TMP (SEQ ID NO:534). [Figure 19C] FIG. 1 shows the amino acid sequence of an exemplary embodiment of TMP (SEQ ID NO:535). [Figure 19D] FIG. 1 shows the amino acid sequence of an exemplary embodiment of TMP (SEQ ID NO:536). [Figure 20A] FIG. 1 shows the amino acid sequence of an exemplary embodiment of TMP (SEQ ID NO:537). [Figure 20B] FIG. 1 shows the amino acid sequence of an exemplary embodiment of TMP (SEQ ID NO:538). [Figure 20C] FIG. 1 shows the amino acid sequence of an exemplary embodiment of TMP (SEQ ID NO:539). [Figure 20D] FIG. 1 shows the amino acid sequence of an exemplary embodiment of TMP (SEQ ID NO:540). [Figure 20E] FIG. 1 shows the amino acid sequence of an exemplary embodiment of TMP (SEQ ID NO:541). [Figure 20F] FIG. 1 shows the amino acid sequence of an exemplary embodiment of TMP (SEQ ID NO:542). [Figure 20G] FIG. 1 shows the amino acid sequence of an exemplary embodiment of TMP (SEQ ID NO:543). [Figure 21A] FIG. 5 shows the amino acid sequence of an illustrative example of TMP having a rigid peptide linker (SEQ ID NO:544). [Figure 21B] FIG. 5 shows the amino acid sequence of an illustrative example of TMP having a rigid peptide linker (SEQ ID NO:545). [Figure 21C] FIG. 5 shows the amino acid sequence of an illustrative example of TMP having a rigid peptide linker (SEQ ID NO:546). [Figure 21D] FIG. 5 shows the amino acid sequence of an illustrative example of TMP having a rigid peptide linker (SEQ ID NO:547). [Figure 22A] FIG. 5 depicts the amino acid sequence of the HLA-E heavy chain (SEQ ID NO:548). [Figure 22B] FIG. 3 shows the amino acid sequence of the HLA-E heavy chain (SEQ ID NO:382). [Figure 22C] FIG. 1 shows the amino acid sequence of the HLA-E heavy chain (SEQ ID NO:549). [Figure 22D] FIG. 3 shows the amino acid sequence of the HLA-E heavy chain (SEQ ID NO:383). [Figure 23A] FIG. 5 shows the amino acid sequence of the HLA-G heavy chain (SEQ ID NO:550). [Figure 23B] FIG. 3 shows the amino acid sequence of the HLA-G heavy chain (SEQ ID NO:384). [Figure 23C] FIG. 5 shows the amino acid sequence of the HLA-G heavy chain (SEQ ID NO:551). [Figure 23D] FIG. 3 shows the amino acid sequence of the HLA-G heavy chain (SEQ ID NO:385). [Figure 24A] FIG. 5 depicts the amino acid sequence of the MUC-1 polypeptide (SEQ ID NO:552). [Figure 24B] FIG. 5 depicts the amino acid sequence of the MUC-1 polypeptide (SEQ ID NO:553). [Figure 24C] FIG. 5 depicts the amino acid sequence of the MUC-1 polypeptide (SEQ ID NO:554). [Figure 24D] FIG. 5 depicts the amino acid sequence of the MUC-1 polypeptide (SEQ ID NO:555). [Figure 24E] FIG. 5 depicts the amino acid sequence of the MUC-1 polypeptide (SEQ ID NO:556). [Figure 24F] FIG. 5 depicts the amino acid sequence of the MUC-1 polypeptide (SEQ ID NO:557). [Figure 24G] FIG. 5 depicts the amino acid sequence of the MUC-1 polypeptide (SEQ ID NO:558). [Fig. 24H] FIG. 5 depicts the amino acid sequence of the MUC-1 polypeptide (SEQ ID NO:559). [Figure 24I] FIG. 5 depicts the amino acid sequence of the MUC-1 polypeptide (SEQ ID NO:560). [Figure 24J] FIG. 5 depicts the amino acid sequence of the MUC-1 polypeptide (SEQ ID NO:561). [Figure 24K] FIG. 5 depicts the amino acid sequence of the MUC-1 polypeptide (SEQ ID NO:562). [Figure 24L]FIG. 5 depicts the amino acid sequence of the MUC-1 polypeptide (SEQ ID NO:563). [Figure 24M] FIG. 5 depicts the amino acid sequence of the MUC-1 polypeptide (SEQ ID NO:564). [Figure 25A] FIG. 5 provides the amino acid sequence of the survivin polypeptide (SEQ ID NO:565). [Figure 25B] FIG. 5 provides the amino acid sequence of the survivin polypeptide (SEQ ID NO:566). [Figure 25C] FIG. 5 provides the amino acid sequence of the survivin polypeptide (SEQ ID NO:567). [Figure 26A] FIG. 1 depicts the cytomegalovirus polypeptide amino acid sequence (SEQ ID NO:568). [Figure 26B] FIG. 1 depicts the cytomegalovirus polypeptide amino acid sequence (SEQ ID NO:569). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0006] definition The terms "polynucleotide" and "nucleic acid" are used interchangeably herein to refer to a polymer of any length of nucleotides, either ribonucleotides or deoxyribonucleotides. Thus, the term encompasses, but is not limited to, single-, double-, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers composed of purine and pyrimidine bases, or other naturally occurring, chemically or biochemically modified, non-natural, or derivatized nucleotide bases.

[0007] The terms "peptide," "polypeptide," and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. The polymers may include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides with modified peptide backbones. Additionally, the term "polypeptide" as used herein also refers to modifications made to the native sequence of the protein, such as deletions, additions, and substitutions (usually benign, as known to those of skill in the art), so long as the protein maintains a desired activity. These modifications may be deliberate, such as by site-directed mutagenesis, or accidental, such as mutations in the host producing the protein, or errors in polymerase chain reaction (PCR) amplification or other recombinant DNA techniques. Reference herein to a particular residue or residue number in a known polypeptide is understood to refer to the amino acid at that position in the wild-type polypeptide. When the sequence of a wild-type polypeptide is modified by either the addition or deletion of one or more amino acids, it will be clear to one skilled in the art that reference to a particular residue or residue number will be modified accordingly and will refer to that same particular amino acid in the modified polypeptide (i.e., it will be present at the modified position number). For example, if an MHC class I polypeptide is modified by adding one amino acid to the N-terminus, reference to position 84 or a particular residue at position 84 will refer to the amino acid at position 85 on the modified polypeptide. Similarly, when reference is made herein to the substitution of a particular amino acid at a particular position (e.g., Y84), it will refer to the substitution of the amino acid at position 84 in the wild-type polypeptide. Thus, a Y84C substitution refers to the substitution of a Cys residue for the Tyr residue present in the wild-type sequence. For example, if a wild-type polypeptide is modified to change the amino acid at position 84 from the wild-type amino acid to an alternative amino acid, the amino acid substitution at position 84 refers to the substitution of the alternative amino acid.In such cases, if the polypeptide is also altered by the addition or deletion of one or more amino acids, reference to a substitution will refer to the substitution of an alternative amino acid at the altered position number. When referring to a "non-naturally occurring Cys residue" in a polypeptide (e.g., an MHC class I polypeptide), it means that the polypeptide has a Cys residue at a position where no Cys is present in the corresponding wild-type polypeptide. This can be achieved by conventional protein engineering to substitute a cysteine ​​for an amino acid present in the wild-type sequence.

[0008] A polynucleotide or polypeptide has a certain percentage of "sequence identity" to another polynucleotide or polypeptide. This means that the percentage of bases or amino acids are the same and in the same relative positions when the two sequences are compared side by side. Sequence identity can be determined in many different ways. To determine sequence identity, a variety of convenient methods and computer programs (e.g., BLAST, T-COFFEE, MUSCLE, MAFFT, etc.) can be used to match sequences. Computer programs are available on the World Wide Web at sites such as ncbi.nlm.nili.gov / BLAST, ebi.ac.uk / Tools / msa / tcoffee / , ebi.ac.uk / Tools / msa / muscle / , mafft.cbrc.jp / alignment / software / , etc. See, for example, Altschul et al. (1990), J. Mol. Biol. 215:403-10. Unless otherwise specified, "sequence identity" referred to herein is determined by BLAST (Basic Local Alignment Search Tool) using default parameters as described in Altschul et al. ((1990) J. Mol. Biol. 215:403).

[0009] 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. The group of amino acids with sulfur-containing side chains consists of cysteine ​​and methionine. Specific examples of conservative amino acid substitution groups include valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine-glycine, and asparagine-glutamine.

[0010] The term "immunological synapse" or "immune synapse" as used herein generally refers to the natural contact area (interface) between two interacting immune cells involved in adaptive immune responses. This contact area includes, for example, the contact area between an antigen-presenting cell (APC) or target cell and an effector cell (e.g., lymphocyte, effector T cell, natural killer cell, etc.). The immunological synapse between an APC and a T cell is typically induced by the interaction of the T cell antigen receptor with a major histocompatibility complex molecule (e.g., as described in Bromley et al., Annu Rev Immunol. 2001;19:375-96, the disclosure of which is incorporated herein by reference in its entirety).

[0011] The term "T cells" includes all types of immune cells that express CD3. "T cells" include T helper cells (CD4 + cells), cytotoxic T cells (CD8 + These include T-cells, T regulatory cells (Tregs), and NK-T cells.

[0012] As used herein, the term "immunomodulatory polypeptide" (also referred to herein as "MOD") refers to a polypeptide that specifically binds to a cognate costimulatory polypeptide (also referred to herein as "co-MOD") on a T cell to generate a signal. This signal, along with the primary signal provided by, for example, the binding of a peptide-loaded major histocompatibility complex (MHC) polypeptide to the TCR / CD3 complex, mediates a T cell response. T cell responses include, but are not limited to, proliferation, activation, and differentiation. As described herein, MODs include, but are not limited to, the following: A wild-type polypeptide or a variant of a wild-type polypeptide, such as a cytokine (e.g., IL-2), CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L, Fas ligand (FasL), inducible costimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM), CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, HVEM, lymphotoxin beta receptor, 3 / TR6, ILT3, ILT4, HVEM, an agonist or antibody that binds to a Toll ligand receptor, and a ligand that specifically binds to B7-H3. The immunomodulatory domain or "MOD" of the TMP of the present disclosure is capable of binding to a cognate costimulatory polypeptide present on a target T cell.

[0013] As used herein, the term "in vivo" refers to any action or process that occurs within a living organism.

[0014] As used herein, the term "in vitro" refers to any action or process that occurs outside of a living organism.

[0015] The term "heterologous" as used herein refers to a nucleotide or polypeptide that is not present in the native nucleic acid or protein, respectively.

[0016] As used herein, the term "recombinant" means that a particular nucleic acid (DNA or RNA) is the product of various combinations of cloning, restriction enzyme, polymerase chain reaction (PCR) and / or ligation steps, resulting in a construct having structural coding or non-coding sequences that are distinct from the endogenous nucleic acid present in a natural system. A DNA sequence encoding a polypeptide can be assembled from cDNA fragments or a series of synthetic oligonucleotides, resulting in a synthetic nucleic acid expressible from a recombinant transcription unit contained in a cellular or cell-free transcription and translation system.

[0017] The terms "recombinant expression vector" and "DNA construct" are used interchangeably herein and refer to a DNA molecule that includes a vector and at least one insert. Recombinant expression vectors are typically generated for the purpose of expressing and / or propagating an insert(s) or for the purpose of constructing other recombinant nucleotide sequences. The insert(s) may or may not be operably linked to a promoter sequence and may or may not be operably linked to a DNA regulatory sequence.

[0018] 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 The term "avidity" as used herein refers to the resistance of a complex of two or more agents to dissociation after dilution. The terms "immunoreactive" and "preferentially bind" are used interchangeably herein with respect to antibodies and / or antigen-binding fragments.

[0019] As used herein, the term "binding" (e.g., with respect to binding of TMP 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, e.g., through electrostatic, hydrophobic, ionic, and / or hydrogen bonding interactions (including interactions such as salt bridges and water bridges). "Affinity" refers to the strength of the non-covalent bond, with higher binding affinity and lower K D "Specific binding" generally refers to the binding of a ligand to its designated binding site or receptor. "Non-specific binding" generally refers to the binding of a ligand to a site other than its designated binding site or receptor. As used herein, the term "covalent" or "covalent bond" refers to the formation of one or more covalent chemical bonds between two different molecules.

[0020] As used herein, the terms "treatment", "treating" and the like generally refer to obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic, in the sense of preventing a disease or its symptoms completely or partially, and / or may be therapeutic, in the sense of curing a disease and / or adverse effects resulting from the disease partially or completely. As used herein, the term "treatment" encompasses any treatment of a disease or condition in a mammal. "Treatment" includes: (a) preventing a disease or condition from occurring in a subject who may be susceptible to the disease or condition but has not yet been diagnosed as having the disease or condition; (b) inhibiting a disease or condition, i.e., arresting its development; and / or (c) relieving the disease, i.e., causing regression of the disease. Therapeutic agents can be administered before, during, or after the onset of the disease or injury. Treatment of ongoing disease is of particular interest when the treatment stabilizes or reduces the patient's undesirable clinical symptoms. It is desirable for such treatment to occur before complete loss of function of the affected tissue. Desirably, the subject is treated during the symptomatic period, and optionally after the symptomatic period.

[0021] 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. Unless otherwise specified, the terms "individual," "subject," "host," and "patient" refer to humans.

[0022] Unless otherwise specified, the term "substantially" is intended to encompass both the meanings of "completely" and "mostly but not completely." For example, an IgFc that "does not substantially induce cytolysis" means an IgFc that does not induce any cytolysis or that induces very little cytolysis.

[0023] The term "about" used herein for an amount indicates that the amount may vary by 10% of the stated amount. For example, "about 100" means an amount of 90 to 110. When "about" is used in the context of a range, "about" used for the lower limit of the range means that the lower limit includes a value 10% lower than the lower limit, and "about" used for the upper limit of the range means that the upper limit includes a value 10% higher than the upper limit. For example, about 100 to about 1000 means that the range extends to 90 to 1100.

[0024] As used herein, the term "MHC heavy chain polypeptide" refers collectively to the domains of an MHC heavy chain polypeptide present in a TMP. For example, an MHC heavy chain polypeptide can include an α1 domain, an α2 domain, and an α3 domain.

[0025] Before further describing the present disclosure, it should be made clear that the disclosure is not limited to particular embodiments described, as such, can, of course, vary. It should be further made clear that the terminology used herein is for the purpose of describing particular embodiments only, since the scope of the present disclosure is limited only by the appended claims.

[0026] Where a range of numerical values ​​is given, unless the context clearly indicates otherwise, each numerical value between the upper and lower limits of that range, and any other numerical values ​​within the stated or stated range, to the tenth of the unit of the lower limit, is to be construed as being encompassed in the disclosure. Each of the upper and lower limits of such smaller ranges may be independently included in the smaller ranges, which are also encompassed in the disclosure (subject to any specifically excluded limit in a stated range). Where a stated range includes one or both of the upper and lower limits, ranges excluding one or both of those limits are also within the scope of the disclosure.

[0027] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of this disclosure, the preferred methods and materials are described herein. All publications mentioned herein are incorporated by reference. Thus, the methods and / or materials are disclosed and described by citing the publications.

[0028] 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, a reference to a "T cell modulating polypeptide" includes a plurality of such polypeptides, and a reference to "the MOD" refers to one or more MODs and equivalents thereof known to those of skill in the art, and so forth. It should be further noted that the claims may be drafted to exclude any non-essential element. This presupposes the use of limiting terms such as "only" and "only" or "negative" limitations in describing the elements of the claims.

[0029] As will be apparent, features of the present disclosure that are described for clarity in the context of separate embodiments may also be combined into a single embodiment. Conversely, various features of the present disclosure that are described for brevity in the context of a single embodiment may be taken separately or in any suitable subcombination. All combinations of the embodiments according to the present disclosure are specifically included in the present disclosure, and each and every combination can be considered as being expressly disclosed herein. In addition, all subcombinations of the various embodiments and elements thereof are also specifically included in the present disclosure, and each and every subcombination can be considered as being expressly disclosed herein.

[0030] 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 disclosure is not entitled to antedate such publications. Further, the publication dates provided may be different from the actual publication dates, which may need to be confirmed individually.

[0031] Detailed Description The present disclosure provides T cell modulating polypeptides (TMPs) comprising a MOD and an epitope-presenting peptide. The TMPs are useful for modulating the activity of T cells and for modulating an immune response in an individual.

[0032] T-cell Modulating Polypeptides The present disclosure provides a T cell modulating polypeptide (TMP), wherein the TMP is a single chain polypeptide comprising: i) a peptide that presents an epitope to a T cell receptor (TCR) upon binding to a major histocompatibility complex (MHC) polypeptide; ii) a first MHC polypeptide; iii) a second MHC polypeptide; and iv) one or more MODs, and optionally, an immunoglobulin (Ig) Fc polypeptide or a non-Ig molecular scaffold. As described below, the single chain TMP can self-assemble into a dimer, for example, when the TMP comprises an IgFc (e.g., IgG1Fc). A disulfide bond between the IgFc polypeptides in two single chain TMPs spontaneously forms, linking the two single chain TMPs to form a homodimer.

[0033] As used herein, the term "peptide epitope" (sometimes referred to herein as "peptide" or "epitope") refers to a peptide that presents an epitope to the TCR upon binding to an MHC complex.

[0034] Generally, a TMP binds to a T cell having a co-MOD and a TCR that binds to the peptide / MHC complex of the TMP with greater affinity (e.g., 25% greater) than the TMP binds to another T cell having the same co-MOD and a TCR that does not substantially bind to the peptide / MHC complex.

[0035] In some cases, the TMP comprises the following components, in order from N-terminus to C-terminus: i) a peptide epitope, ii) a first MHC polypeptide, iii) a second MHC polypeptide, iv) one or more MODs, and v) an IgFc polypeptide. In some cases, the TMP comprises the following components, in order from N-terminus to C-terminus: i) a peptide epitope, ii) a β2M polypeptide, iii) a class I MHC heavy chain polypeptide, iv) one or more MODs, and v) an IgFc polypeptide. As described below, a peptide linker may be present between two or more components. Such an arrangement of components is referred to as MOD arrangement 2 in FIG. 12.

[0036] In some cases, the TMP comprises the following components, in order from N-terminus to C-terminus: i) a peptide epitope, ii) a first MHC polypeptide, iii) a second MHC polypeptide, iv) an IgFc polypeptide, and v) one or more MODs. In some cases, the TMP comprises the following components, in order from N-terminus to C-terminus: i) a peptide epitope, ii) a β2M polypeptide, iii) a class I MHC heavy chain polypeptide, iv) an IgFc polypeptide, and v) one or more MODs. As described below, a peptide linker may be present between two or more components. Such an arrangement of components is referred to as MOD arrangement 3 in FIG. 12.

[0037] In some cases, the TMP comprises the following components, in order from N-terminus to C-terminus: i) one or more MODs, ii) a peptide epitope, iii) a first MHC polypeptide, iv) a second MHC polypeptide, and v) an IgFc polypeptide. In some cases, the TMP comprises the following components, in order from N-terminus to C-terminus: i) one or more MODs, ii) a peptide epitope, iii) a β2M polypeptide, iv) a class I MHC heavy chain polypeptide, and v) an IgFc polypeptide. As described below, a peptide linker may be present between two or more components. Such an arrangement of components is referred to as MOD arrangement 4 in FIG. 12.

[0038] The MOD may comprise either a wild-type (wt) MOD or a mutant of the wtMOD. When the MOD comprises a mutant, it may exhibit reduced binding to the co-MOD (e.g., reduced binding to one or more chains or domains of the co-MOD). In such a case, the reduced affinity of the MOD for the co-MOD in combination with the affinity of the peptide for the TCR may enhance the selectivity of the TMP. The binding affinity of the MOD to its co-MOD can be measured by biolayer interferometry (BLI) using purified MOD and purified co-MOD. The binding affinity of the MOD present in the TMP to its co-MOD can be measured by BLI using purified TMP and co-MOD. 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. Unless otherwise specified herein, the affinity of a MOD for a co-MOD, or the affinity of a MOD on a TMP for a co-MOD, is measured using BLI as described in PCT application WO2020 / 132138, published on June 25, 2020. See, e.g., paragraphs

[0056] to

[0057] therein.

[0039] Peptide epitopes As described above, a TMP typically comprises a peptide epitope having a length of at least about four amino acids, and presents the epitope to a T cell (i.e., to a TCR present on the surface of the T cell) when the peptide is present in an MHC / peptide complex (e.g., an HLA / peptide complex).

[0040] Peptide epitopes present in the TMPs of the disclosure are at least 4 amino acids in length, e.g., 4-20 amino acids (aa) (e.g., 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids), including amino acid lengths ranging from 6-15 amino acids, 8-12 amino acids, 8-10 amino acids, 9-11 amino acids, 5-10 amino acids, 10-15 amino acids, and 15-20 amino acids. In some cases, the peptides are 8, 9, 10, or 11 amino acids in length.

[0041] A peptide epitope present in a TMP is a peptide that is specifically bound by a T cell, i.e., an epitope that is specifically bound by an epitope-specific T cell (i.e., a T cell having a TCR specific for the epitope).

[0042] In some cases, the epitope peptides present in the TMP present epitopes specific for alleles of HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, or HLA-G. In one embodiment, the epitope peptides present in the TMP present epitopes specific for alleles of HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, or HLA-G. * 0101, A * 0201, A * 0301, A * 1101, A * 2301, A * 2402, A * 2407, A * 3303 and / or A * In one embodiment, the epitope peptide present in the TMP is restricted to HLA-B 3401. * 0702, B * 0801, B * 1502, B * 3802, B * 4001, B * 4601 and / or B *In one embodiment, the epitope peptide present in the TMP is C * 0102, C * 0303, C * 0304, C * 0401, C * 0602, C * 0701, C * 702, C * 0801 and / or C * This presents an epitope restricted to 1502.

[0043] The TMP of the present disclosure includes peptide epitopes other than KRAS peptides associated with cancer. That is, the peptides present in the TMP are not KRAS peptides associated with cancer. For example, the peptide epitopes present in the TMP are not peptides of about 4 amino acids to about 20 amino acids in a KRAS polypeptide, where the KRAS polypeptide may have the following amino acid sequence: MTEYKLVVVG AGGVGKSALT IQLIQNHFVD EYDPTIEDSY RKQVVIDGET CLWDILDTAG QEEYSAMRDQ YMRTGEGFLC VFAINNTKSF EDIHHYREQI KRVKDSEDVP MVLVGNKCDL PSRTVDTKQA QDLARSYGIP FIETSAKTRQ GVDDAFYTLV REIRKHKEKM SKDGKKKKKK SKTKCVIM (SEQ ID NO: 1). For example, the peptide epitopes present in the TMP of the present disclosure are not any of the following peptides:VVVGAGDVGK (SEQ ID NO: 2), VVGAGDVGK (SEQ ID NO: 3), VVVGARGVGK (SEQ ID NO: 4), VVGARGVGK (SEQ ID NO: 5), LVVVGADGV (SEQ ID NO: 6), LVVGAVGV (SEQ ID NO: 7), LVVVGACGV (SEQ ID NO: 8), KLVVVGADGV (SEQ ID NO: 9), KLVVVGAVGV (SEQ ID NO: 10), KLVVVGACGV (SEQ ID NO: 11), LLVVGADGV (SEQ ID NO: 12), LLVVGAVGV (SEQ ID NO: 13), LLVVGACGV (SEQ ID NO: 14), FLVVVGADGV (SEQ ID NO: 15), FLVVVGAVGV (SEQ ID NO: 16), FLVVVGACGV (SEQ ID NO: 17), KLVVVGAGDV (SEQ ID NO: 18), KLVVVGARGV (SEQ ID NO: 19), GAGDVGKSAL (SEQ ID NO: 20), AGDVGKSAL (SEQ ID NO: 21), DVGK SALTI (SEQ ID NO:22), GAVGVGKSAL (SEQ ID NO:23), AVGVGKSAL (SEQ ID NO:24), YKLVVVGAV (SEQ ID NO:25), ARGVGKSAL (SEQ ID NO:26), GARGVGKSAL (SEQ ID NO:27), EYKLVVVGAR (SEQ ID NO:28), RGVGKSALTI (SEQ ID NO:29), LVVVGARGV (SEQ ID NO:30), GADGVGKSAL (SEQ ID NO:31), ACGVGKSAL (SEQ ID NO:32), GACGVGKSAL (SEQ ID NO:33), VVGAVGVGK (SEQ ID NO:34), VVVGAVGVGK (SEQ ID NO:35), VGAVGVGKS (SEQ ID NO:36), VGAVGVGKSA (SEQ ID NO:37), AVGVGKSALT (SEQ ID NO:38), GAVGVGKSA (SEQ ID NO:39), LVVVGAVGVG (SEQ ID NO:40), and KLVVVGAVG (SEQ ID NO:41).

[0044] Suitable peptide epitopes include, but are not limited to, epitopes present in cancer-associated antigens. Cancer-associated antigens are known in the art. For example, see Cheever et al. (2009) Clin.Cancer Res.15:5323. Cancer-associated antigens include, but are not limited to: α-Folate receptor, Carbonic anhydrase IX (CAIX), CD19, CD20, CD22, CD30, CD33, CD44v7 / 8, Carcinoembryonic antigen (CEA), Epithelial glycoprotein-2 (EGP-2), Epithelial glycoprotein-40 (EGP-40), Folate binding protein (FBP), Fetal acetylcholine receptor, Ganglioside antigen GD2, Her2 / neu, IL-13R-a2, Kappa light chain, LeY, L1 cell adhesion molecule, Melanoma-associated antigen (MAGE), MAGE-A1, Mesothelin, MUC1, NKG2D ligand, Carcinoembryonic antigen (h5T4), Prostate stem cell antigen (PSCA), Prostate specific membrane antigen (PSMA), Tumor associated glycoprotein-72 (TAG-72), Vascular endothelial growth factor receptor-2 (VEGF-R2) (see, e.g., Vigneron et al. (2013) Cancer Immunity 13:15 and Vigneron (2015) BioMed Res. Int'l Article ID 948501), and epidermal growth factor receptor (EGFR) vIII polypeptides (see, e.g., Wong et al. (1992) Proc. Natl. Acad. Sci. USA 89:2965 and Miao et al. (2014) PLoSOne 9:e94281).

[0045] In some cases, suitable peptide epitopes are peptide fragments of the following polypeptides that are 4 to 20 amino acids in length, e.g., 6 to 15 amino acids in length, 8 to 12 amino acids in length, 8 to 10 amino acids in length, 9 to 11 amino acids in length, 5 to 10 amino acids in length, 10 to 15 amino acids in length, and 15 to 20 amino acids in length. MUC1 polypeptide, LMP2 polypeptide, epidermal growth factor receptor (EGFR) vIII polypeptide, HER-2 / neu polypeptide, melanoma antigen family A, 3 (MAGE A3) polypeptide, p53 polypeptide, mutant p53 polypeptide, NY-ESO-1 polypeptide, folate hydrolase (prostate specific membrane antigen (PSMA)) polypeptide, carcinoembryonic antigen (CEA) polypeptide, melanoma antigen recognized by T cells (melanA / MART1) polypeptide, Ras polypeptide, gp100 polypeptide, proteinase 3 (PR1) polypeptide, bcr-abl polypeptide, tyrosinase polypeptide, survivin polypeptide, prostate specific antigen (PSA) polypeptide, hTERT polypeptide, sarcoma translocation breakpoint polypeptide, synovial sarcoma X (SSX) breakpoint polypeptide, EphA2 polypeptide, acid phosphatase prostate (PAP) polypeptide, melanoma inhibitor of apoptosis (ML-IAP) polypeptide, epithelial cell adhesion molecule (EpCAM) polypeptide, ETS fusion) polypeptide, NA17 polypeptide, paired box-3 (PAX3) polypeptide, anaplastic lymphoma kinase (ALK) polypeptide, androgen receptor polypeptide, cyclin B1 polypeptide, N-myc proto-oncogene (MYCN) polypeptide, Ras homolog gene family member C (RhoC) polypeptide, tyrosinase-related protein-2 (TRP-2) polypeptide, mesothelin polypeptide, prostate stem cell antigen (PSCA) polypeptide, melanoma associated antigen-1 (MAGE A1) polypeptide, cytochrome P4501B1 (CYP1B1) polypeptide, placenta-specific protein 1 (PLAC1) polypeptide, BORIS polypeptide (also known as CCCTC-binding factor or CTCF), ETV6-AML polypeptide, breast cancer antigen NY-BR-1 polypeptide (also known as ankyrin repeat domain-containing protein 30A), regulator of G protein signaling (RGS5) polypeptide, squamous cell carcinoma antigen recognized by T cells (SART3) polypeptide, carbonic anhydrase IX polypeptide, paired box-5 (PAX5) polypeptide, OY-TES1 (testis antigen (also known as acrosin-binding protein)) polypeptide, sperm protein 17 polypeptide, lymphocyte-specific protein tyrosine kinase (LCK) polypeptide, high molecular weight melanoma-associated antigen (HMW-MAA), A-kinase anchor protein a polypeptide, such as an AKAP-4 polypeptide, a synovial sarcoma X breakpoint 2 (SSX2) polypeptide, an X antigen family member 1 (XAGE1) polypeptide, a B7 homolog 3 (B7H3 (also known as CD276)) polypeptide, a legumain polypeptide (LGMN1 (also known as asparaginyl endopeptidase)), a tyrosine kinase-2 with Ig and EGF homology domains 2 (Tie-2 (also known as angiopoietin-1 receptor)) polypeptide, a P antigen family member 4 (PAGE4) polypeptide, a vascular endothelial growth factor receptor 2 (VEGF2) polypeptide, a MAD-CT-1 polypeptide, a fibroblast activation protein (FAP) polypeptide, a platelet-derived growth factor receptor beta (PDGFβ) polypeptide, a MAD-CT-2 polypeptide, or a Fos-related antigen-1 (FOSL) polypeptide.

[0046] The amino acid sequences of cancer associated antigens are known in the art, see, e.g., MUC1 (GenBank CAA56734), LMP2 (GenBank CAA47024), EGFRvIII (GenBank NP_001333870), HER-2 / neu (GenBank AAI67147), MAGE-A3 (GenBank AAH11744), p53 (GenBank BAC16799), NY-ESO-1 (GenBank CAA05908), PSMA (GenBank AAH25672), CEA (GenBank AAA51967), melan / MART1 (GenBank NP_005502), Ras (GenBank NP_001123914), gp100 (GenBank AAC60634), bcr-abl (GenBank AAB60388), tyrosinase (GenBank AAB60319), survivin (GenBank AAC51660), PSA (GenBank CAD54617), hTERT (GenBank BAC11010), SSX (GenBank NP_001265620), Eph2A (GenBank NP_004422), PAP (GenBank AAH16344), ML-IAP (GenBank AAH14475), EpCAM (GenBank NP_002345), ERG (TMPRSS2 ETS fusion) (GenBank ACA81385), PAX3 (GenBank AAI01301), ALK (GenBank NP_004295), androgen receptor (GenBank NP_000035), cyclin B1 (GenBank CAO99273), MYAN (GenBank NP_001280157), RhoC (GenBank AAH52808), TRP-2 (GenBank AAC60627), mesothelin (GenBank AAH09272), PSCA (GenBank AAH65183), MAGE A1 (GenBank NP_004979), CYP1B1 (GenBank AAM50512), PLAC1 (GenBank AAG22596), BORIS (GenBank NP_001255969), ETV6 (GenBank NP_001978), NY-BR1 (GenBankNP_443723), SART3 (GenBank NP_055521), carbonic anhydrase IX (GenBank EAW58359), PAX5 (GenBank NP_057953), OY-TES1 (GenBank NP_115878), sperm protein 17 (GenBank AAK20878), LCK (GenBank NP_001036236), HMW-MAA (GenBank NP_001888), AKAP-4 (GenBank NP_003877), SSX2 (GenBank CAA60111), XAGE1 (GenBank NP_001091073, XP_001125834, XP_001125856, and XP_001125872), B7H3 (GenBank NP_001019907, XP_947368, XP_950958, XP_950960, XP_950962, XP_950963, XP_950965, and NP_000450), PAGE4 (GenBank NP_001305806), VEGFR2 (GenBank NP_002244), MAD-CT-1 (GenBank NP_005893, NP_056215), FAP (GenBank NP_004451), PDGFβ (GenBank NP_002600), MAD-CT-2(GenBank NP_001138574), and FOSL (GenBank NP_005429). These polypeptides are also described, for example, in Cheever et al. (2009) Clin. Cancer Res. 15:5323 and references cited therein, Wagner et al. (2003) J. Cell. Sci. 116:1653, Matsui et al. (1990) Oncogene 5:249, Zhang et al. (1996) Nature 383:168.

[0047] AFP peptide In some cases, the TMP comprises an alpha-fetoprotein (AFP) peptide as a peptide epitope. In some cases, the AFP peptide epitope present in the TMP can be a peptide 4-20 amino acids in length, e.g., 6-15 amino acids in length, 8-12 amino acids in length, 8-10 amino acids in length, 9-11 amino acids in length, 5-10 amino acids in length, 10-15 amino acids in length, and 15-20 amino acids in length, 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 AFP amino acid sequence shown in Figure 14.

[0048] Specific examples of AFP peptides suitable for inclusion in a TMP include, but are not limited to, the following: AITRKMAAT (449-457, SEQ ID NO: 42), AYTKKAPQL (434-442, SEQ ID NO: 43), LLNQHACAV (218-226, SEQ ID NO: 44), KLVLDVAHV (257-265, SEQ ID NO: 45), FMNKFIYEI (158-166, SEQ ID NO: 46), SIPLFQVPE (135-143, SEQ ID NO: 47), LLNFTESRT (12-20, SEQ ID NO: 48), FVQEATYKF (54-62, SEQ ID NO: 49), ATYKEVSKM (58-66, SEQ ID NO: 50), KEVS KMVKD (61-69, SEQ ID NO: 51), RHNCFLAHK (121-129, SEQ ID NO: 52), ATAATCCQL (456-464, SEQ ID NO: 53), YIQESQALA (404-412, SEQ ID NO: 54), QLTSSELMAI (441-450, SEQ ID NO: 55), KLSQKFTKV (242-250, SEQ ID NO: 56), KELRESSLL (211-219, SEQ ID NO: 57), SLVVDETYV (514-522, SEQ ID NO: 58), ILLWAARYD (178-186, SEQ ID NO: 59), KIIP SCCKA (187 to 195, SEQ ID NO: 60), CRGDVLDCL (270 to 278, SEQ ID NO: 61), QQDTLSNKI (291 to 299, SEQ ID NO: 62), TMKQEFLINL (547 to 556, SEQ ID NO: 63), NLVKQKPQI (555 to 563, SEQ ID NO: 64), AVIADFSGL (570 to 578, SEQ ID NO: 65), LLACGEGAA (469 to 477, SEQ ID NO: 66), LACGEGAAD (470 to 478, SEQ ID NO: 67), KAPQLTSSEL (438 to 447, SEQ ID NO: 68), Y ICSQQDTL (287 to 295, SEQ ID NO: 69), TECCKLTTL (300 to 308, SEQ ID NO: 70), CTAEISLADL (37 to 46, SEQ ID NO: 71), VTKELRESSL (209 to 218, SEQ ID NO: 72), IMSYICSQQD (284 to 293, SEQ ID NO: 73), TRTFQAITV (232 to 240, SEQ ID NO: 74), FQKLGEYYL (419 to 427, SEQ ID NO: 75), RVAKGYQEL (372 to 380, SEQ ID NO: 76), SYQCTAEISL (34 to 43, SEQ ID NO: 77),KQEFLINLV (549-557, SEQ ID NO: 78), MKWVESIFL (1-9, SEQ ID NO: 79), PNVPGVGQC (492-500, SEQ ID NO: 80), AADIIIGHL (476-484, SEQ ID NO: 81), QVPEPVTSC (140-148, SEQ ID NO: 82), TTLERGQCII (306-315, SEQ ID NO: 83), KMAATAATC (453-461, SEQ ID NO: 84), QAQGVALQTM (539-548, SEQ ID NO: 85), FQAITVTKL (235 to 243, SEQ ID NO: 86), LLEKCFQTE (380 to 388, SEQ ID NO: 87), VAYTKKAPQ (433 to 441, SEQ ID NO: 88), KYIQESQAL (403 to 411, SEQ ID NO: 89), GVALQTMKQ (542 to 550, SEQ ID NO: 90), GQEQEVCFA (585 to 593, SEQ ID NO: 91), SEEGRHNCFL (117 to 126, SEQ ID NO: 92), RHPFLYAP TI (169 to 178, SEQ ID NO: 93), TEIQKLVLDV (253 to 262, SEQ ID NO: 94), RRHPQLAVSV (360 to 369, SEQ ID NO: 95), GEYYLQNAFL (423 to 432, SEQ ID NO: 96), NRRPCFSSLV (507 to 516, SEQ ID NO: 97), LQTMKQEFLI (545 to 554, SEQ ID NO: 98), IADFSGLLEK (572 to 581, SEQ ID NO: 99), GLLEKCCQGQ (577 to 58 6, SEQ ID NO: 100), TLSNKITEC (294-302, SEQ ID NO: 101), LQDGEKIMSY (278-287, SEQ ID NO: 102), GLFQKLGBY (417-425, SEQ ID NO: 103), NEYGIASILD (24-33, SEQ ID NO: 104), KMVKDALTAI (65-74, SEQ ID NO: 105), FLASFVHEY (350-358, SEQ ID NO: 106), and AQFVQEATY (52-60, SEQ ID NO: 107). The position of the peptide in the amino acid sequence shown in FIG. 14 is shown in brackets followed by the sequence identifier.

[0049] In some cases, AFP peptides suitable for inclusion in a TMP are selected from the group consisting of: FMNKFIYEI (158-166, SEQ ID NO: 46), LLNFTESRT (12-20, SEQ ID NO: 48), YIQESQALA (404-412, SEQ ID NO: 54), QLTSSELMAI (441-450, SEQ ID NO: 55), ILLWAARYD (178-186, SEQ ID NO: 59), TMKQEFLINL (547-556, SEQ ID NO: 63), NLVKQKPQI (SEQ ID NO: 64), YICSQQDTL (287-295, SEQ ID NO: 69), MKWVESIFL (1-9, SEQ ID NO: 79), PVGVGQC (492-500, SEQ ID NO: 80), FQAITVTKL (235-243, SEQ ID NO: 86), and GVALQTMKQ (542-550, SEQ ID NO: 90).

[0050] In some cases, AFP peptides suitable for inclusion in a TMP are selected from the group consisting of: KYIQESQAL (SEQ ID NO:89), EYYLQNAFL (SEQ ID NO:108), AYTKKAPQL (SEQ ID NO:43), EYSRRHPQL (SEQ ID NO:109), AYEEDRETF (SEQ ID NO:110), SYANRRPCF (SEQ ID NO:111), CFAEEGQKL (SEQ ID NO:112), RSCGLFQKL (SEQ ID NO:113), IFLIFLLNF (SEQ ID NO:114), KPEGLSPNL (SEQ ID NO:115), FMNKFIYEI (SEQ ID NO:46), and GLSPNLNRFL (SEQ ID NO:116).

[0051] In some cases, the AFP peptides present in the TMP are related to HLA-A * Presents the 2402-restricted epitope. HLA-A * Non-limiting examples of AFP peptides displaying 2402-restricted epitopes include: KYIQESQAL (SEQ ID NO:89), EYYLQNAFL (SEQ ID NO:108), AYTKKAPQL (SEQ ID NO:43), EYSRRHPQL (SEQ ID NO:109), RSCGLFQKL (SEQ ID NO:113), and AYEEDRETF (SEQ ID NO:110).

[0052] In some cases, the AFP peptide present in the TMP is KYIQESQAL (SEQ ID NO: 89). In some cases, the AFP peptide present in the TMP is EYYLQNAFL (SEQ ID NO: 108). In some cases, the AFP peptide present in the TMP is AYTKKAPQL (SEQ ID NO: 43). In some cases, the AFP peptide present in the TMP is EYSRRHPQL (SEQ ID NO: 109). In some cases, the AFP peptide present in the TMP is RSCGLFQKL (SEQ ID NO: 113).

[0053] In some cases, the AFP peptides present in the TMP are related to HLA-A * Presents 0201-restricted epitope. HLA-A * Specific, non-limiting examples of AFP peptides displaying a 0201-restricted epitope include: FMNKFIYEI (SEQ ID NO: 46) and GLSPNLNRFL (SEQ ID NO: 116).

[0054] WT1 peptide In some cases, the TMP comprises a Wilms Tumor-1 (WT-1) peptide as a peptide epitope. The amino acid sequences of WT-1 isoforms are shown in Figures 3A-3E. WT-1 peptides that display one or more epitopes are referred to herein as "WT-1 peptides" or "WT-1 epitopes." In some cases, the WT-1 epitopes present in the TMP can be peptides 4-20 amino acids in length, e.g., 6-15 amino acids in length, 8-12 amino acids in length, 8-10 amino acids in length, 9-11 amino acids in length, 5-10 amino acids in length, 10-15 amino acids in length, and 15-20 amino acids in length, 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 shown in any one of Figures 15A-15E.

[0055] Specific examples of WT-1 peptides suitable for inclusion in a TMP include, but are not limited to, those shown below.CMTWNQMNLGATLKG (SEQ ID NO: 117), WNQMNLGATLKGVAA (SEQ ID NO: 118), CMTWNYMNLGATLKG (SEQ ID NO: 119), WNYMNLGATLKGVAA (SEQ ID NO: 120), MTWNQMNLGATLKGV (SEQ ID NO: 121), TWNQMNLGATLKGVA (SEQ ID NO: 122), CMTWNLMNLGATLKG (SEQ ID NO: 123), MTWNLMNLGATLKGV (SEQ ID NO: 124), TWNLMNLGATLKGVA (SEQ ID NO: 125), WNLMNLGATLKGVAA (SEQ ID NO: 126) 6), MNLGATLK (SEQ ID NO: 127), MTWNYMNLGATLKGV (SEQ ID NO: 128), TWNYMNLGATLKGVA (SEQ ID NO: 129), CMTWNQMNLGATLKGVA (SEQ ID NO: 130), CMTWNLMNLGATLKGVA (SEQ ID NO: 131), CMTWNYMNLGATLKGVA (SEQ ID NO: 132), GYLRNPTAC (SEQ ID NO: 133), GALRNPTAL (SEQ ID NO: 134), YALRNPTAC (SEQ ID NO: 135), GLLRNPTAC (SEQ ID NO: 136), RYRPHPGAL (SEQ ID NO: 137), Y QRPHPGAL (SEQ ID NO: 138), RLRPHPGAL (SEQ ID NO: 139), RIRPHPGAL (SEQ ID NO: 140), QFPNHSFKHEDPMGQ (SEQ ID NO: 141), HSFKHEDPY (SEQ ID NO: 142), QFPNHSFKHEDPM (SEQ ID NO: 143), QFPNHSFKHEDPY (SEQ ID NO: 144), KRPFMCAYPGCNK (SEQ ID NO: 145), KRPFMCAYPGCYK (SEQ ID NO: 146), FMCAYPGCY (SEQ ID NO: 147), FMCAYPGCK (SEQ ID NO: 148), KRPFMCAYPGCNKRY (SEQ ID NO: 149), No. 149), SEKRPFMCAYPGCNK (SEQ ID NO: 150), KRPFMCAYPGCYKRY (SEQ ID NO: 151), NLMNLGATL (SEQ ID NO: 152), VLDFAPPGA (SEQ ID NO: 153), RMFPNAPYL (SEQ ID NO: 154), CMTWNQMN (SEQ ID NO: 155), CYTWNQMNL (SEQ ID NO: 156), NYMNLGATL (SEQ ID NO: 157), YMFPNAPYL (SEQ ID NO: 158), SLGEQQYSV (SEQ ID NO: 159), CMTWNQMNL (SEQ ID NO: 160), and NQMNLGATL (SEQ ID NO: 161).In some cases, the WT-1 peptide present in the TMP is CMTWNQMN (SEQ ID NO: 155). In some cases, the WT-1 peptide present in the TMP is CYTWNQMNL (SEQ ID NO: 156).

[0056] In some cases, the WT-1 peptide present in the TMP is associated with HLA-A * Presents the 2402-restricted epitope. HLA-A * Examples of WT-1 peptides presenting the 2402-restricted epitope include the following: CMTWNQMN (SEQ ID NO: 155), NYMNLGATL (SEQ ID NO: 157) (WT-1 239 to 247, Q240Y), CYTWNQMNL (SEQ ID NO: 156) (WT-1 235 to 243), CMTWNQMNL (SEQ ID NO: 160) (WT-1 235 to 243), NQMNLGATL (SEQ ID NO: 161) (WT-1 239 to 247), and NLMNLGATL (SEQ ID NO: 152) (WT-1 239 to 247, Q240L).

[0057] In some cases, the WT-1 peptide present in the TMP is associated with HLA-A * Presents 0201-restricted epitope. HLA-A * Examples of WT-1 peptides presenting a 0201-restricted epitope include the following: VLDFAPPGA (SEQ ID NO: 153) (WT-1 37-45), RMFPNAPYL (SEQ ID NO: 154) (WT-1 126-134), YMFPNAPYL (SEQ ID NO: 158) (WT-1 126-134, R126Y), SLGEQQYSV (SEQ ID NO: 159) (WT-1 187-195), and NLMNLGATL (SEQ ID NO: 152) (WT-1 239-247, Q240L).

[0058] HPV peptides In some cases, the TMP includes a human papillomavirus (HPV) peptide as a peptide epitope. The HPV peptide suitable for inclusion in the TMP can be a peptide of HPV E6 polypeptide or HPV E7 polypeptide. The HPV epitope can be an epitope of any of the various genotypes of HPV, including, for example, HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59, HPV68, HPV73, or HPV82. In some cases, the epitope is an HPV E6 epitope. In some cases, the epitope is an HPV E7 epitope. The amino acid sequence of the HPV E6 polypeptide is shown in Figure 3A. The amino acid sequence of the HPV E7 polypeptide is shown in Figure 3B. In this specification, an HPV peptide that presents one or more epitopes is referred to as an "HPV peptide" or an "HPV epitope." In some cases, the HPV epitope present in the TMP is a peptide 4-20 amino acids in length, e.g., 6-15 amino acids in length, 8-12 amino acids in length, 8-10 amino acids in length, 9-11 amino acids in length, 5-10 amino acids in length, 10-15 amino acids in length, and 15-20 amino acids in length, 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 HPV E6 amino acid sequence shown in Figure 16A or the HPV E7 amino acid sequence shown in Figure 16B.

[0059] Specific examples of HPV E6 peptides suitable for inclusion in a TMP include, but are not limited to, E6 18-26 (KLPQLCTEL, SEQ ID NO: 162), E6 26-34 (LQTTIHDII, SEQ ID NO: 163), E6 49-57 (VYDFAFRDL, SEQ ID NO: 164), E6 52-60 (FAFRDLCIV, SEQ ID NO: 165), E6 75-83 (KFYSKISEY, SEQ ID NO: 166), and E6 80-88 (ISEYRHYCY, SEQ ID NO: 167).

[0060] Specific examples of HPV E7 peptides suitable for inclusion in a TMP include, but are not limited to, E7 7-15 (TLHEYMLDL, SEQ ID NO: 168), E7 11-19 (YMLDLQPET, SEQ ID NO: 169), E7 44-52 (QAEPDRAHY, SEQ ID NO: 170), E7 49-57 (RAHYNIVTF (SEQ ID NO: 171), E7 61-69 (CDSTLRLCV, SEQ ID NO: 172), and E7 67-76 (LCVQSTHVDI, SEQ ID NO: 173), E7 82-90 (LLMGTLGIV, SEQ ID NO: 174), E7 86-93 (TLGIVCPI, SEQ ID NO: 175), and E7 92-93 (LLMGTLGIVCPI, SEQ ID NO: 176).

[0061] In some cases, a suitable HPV peptide for inclusion in a TMP is an HPV E6 peptide that binds to HLA-A24 (e.g., is an HLA-A2401-restricted epitope), non-limiting examples of which include: VYDFAFRDL (SEQ ID NO: 164), CYSLYGTTL (SEQ ID NO: 177), EYRHYCYSL (SEQ ID NO: 178), KLPQLCTEL (SEQ ID NO: 162), DPQERPRKL (SEQ ID NO: 179), HYCYSLYGT (SEQ ID NO: 180), DFAFRDLCI (SEQ ID NO: 181), LYGTTLEQQY (SEQ ID NO: 182), HYCYSLYGTT (SEQ ID NO: 183), EVYDFAFRDL (SEQ ID NO: 184), EYRHYCYSLY (SEQ ID NO: 185), VYDFAFRDLC (SEQ ID NO: 186), YCYSIYGTTL (SEQ ID NO: 187), VYCKTVLEL (SEQ ID NO: 188), VYGDTLEKL (SEQ ID NO: 189), and LTNTGLYNLL (SEQ ID NO: 190).

[0062] In some cases, HPV peptides suitable for inclusion in a TMP are selected from the group consisting of: DLQPETTDL (SEQ ID NO: 191), TLHEYMLDL (SEQ ID NO: 168), TPTLHEYML (SEQ ID NO: 168), RAHYNIVTF (SEQ ID NO: 171), GTLGIVCCPI (SEQ ID NO: 192), EPDRAHYNI (SEQ ID NO: 193), QLFLNTLSF (SEQ ID NO: 194), FQQLFLNTL (SEQ ID NO: 195), and AFQQLFLNTL (SEQ ID NO: 196).

[0063] In some cases, the preferred HPV peptide is HLA-A * Presents the 2401-restricted epitope. HLA-A * Non-limiting examples of HPV peptides displaying 2401 restricted epitopes include VYDFAFRDL (SEQ ID NO: 164), RAHYNIVTF (SEQ ID NO: 171), CDSTLRLCV (SEQ ID NO: 172), and LCVQSTHVDI (SEQ ID NO: 173).

[0064] MUC1 peptide In some cases, the TMP comprises a mucin-1 (MUC-1) peptide as a peptide epitope. In some cases, suitable MUC1 peptides are peptides at least 4-20 amino acids in length, e.g., 6-15 amino acids in length, 8-12 amino acids in length, 8-10 amino acids in length, 9-11 amino acids in length, 5-10 amino acids in length, 10-15 amino acids in length, and 15-20 amino acids in length, of a MUC1 polypeptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the MUC1 amino acid sequence shown in any one of Figures 24A-24M.

[0065] Non-limiting examples of suitable MUC1 peptides include: i) STAPPAHGV (SEQ ID NO: 197), ii) STAPPVHNV (SEQ ID NO: 198), iii) SLAPPVHNV (SEQ ID NO: 199), iv) SLAPPAHGV (SEQ ID NO: 200), v) SAPDTRPAP (SEQ ID NO: 201), vi) VTSAPDTRPAPGSTAPPAHG (SEQ ID NO: 202), vii) PDTRPAPGSTAPPAHGVTSA (SEQ ID NO: 203), and viii) LLLLTVLTV (SEQ ID NO: 204).

[0066] In some cases, the MUC1 peptide present in the TMP presents an epitope specific for an allele of HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, or HLA-G. * 0101, A * 0201, A * 0301, A * 1101, A * 2301, A * 2402, A * 2407, A * 3303 and / or A * In one embodiment, the epitope peptide present in the TMP is restricted to HLA-B 3401. * 0702, B * 0801, B * 1502, B * 3802, B * 4001, B * 4601 and / or B * In one embodiment, the epitope peptide present in the TMP is C * 0102, C * 0303, C * 0304, C * 0401, C * 0602, C * 0701, C * 702, C * 0801 and / or C * This presents an epitope restricted to 1502.

[0067] In one embodiment, the MUC1 peptide STAPPAHGV (SEQ ID NO: 197) is a β2M polypeptide and * When bound to an HLA complex containing the 1101HLA-A heavy chain, the epitope is presented. As another example, the MUC1 peptide STAPPAHGV (SEQ ID NO: 197) binds to the β2M polypeptide and A * When bound to an HLA complex containing the 0201HLA-A heavy chain, the epitope is presented. As another example, the MUC1 peptide STAPPVHNV (SEQ ID NO: 198) binds to a β2M polypeptide and an A * When bound to an HLA complex containing the 0201HLA-A heavy chain, the epitope is presented. As another example, the MUC1 peptide SLAPPVHNV (SEQ ID NO: 199) binds to a β2M polypeptide and an A * When bound to an HLA complex containing the β2M polypeptide and the A heavy chain, the epitope is presented. * Upon binding to an HLA complex containing the 0201HLA-A heavy chain, it presents an epitope.

[0068] MAGE-A4 peptides In some cases, the TMP comprises a melanoma associated antigen 4 (MAGE-A4) peptide as a peptide epitope. In some cases, suitable MAGE-A4 peptides are peptide fragments of 4-20 amino acids in length, e.g., 6-15 amino acids in length, 8-12 amino acids in length, 8-10 amino acids in length, 9-11 amino acids in length, 5-10 amino acids in length, 10-15 amino acids in length, and 15-20 amino acids in length, of a MAGE-A4 polypeptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the Homo sapiens MAGE-A4 amino acid sequence:

[0069] MSSEQKSQHC KPEEGVEAQE EALGLVGAQA PTTEEQEAAV SSSSPLVPGT LEEVPAAESA GPPQSPQGAS ALPTTISFTC WRQPNEGSSS QEEEGPSTSP DAESLFREAL SNKVDELAHF LLRKYRAKEL VTKAEMLERV IKNYKRCFPV IFGKASESLK MIFGIDVKEV DPASNTYTLV TCLGLSYDGL LGNNQIFPKT GLLIIVLGTI AMEGDSASEE EIWEELGVMG VYDGREHTVY GEPRKLLTQD WVQENYLEYR QVPGSNPARY EFLWGPRALA ETSYVKVLEH VVRVNARVRI AYPSLREAAL LEEEEGV (SEQ ID NO: 205).

[0070] In one embodiment, a suitable MAGE-A4 peptide has the amino acid sequence GVYDGREHTV (SEQ ID NO: 206, p230-239) and is 10 amino acids long. In another embodiment, a suitable MAGE-A4 peptide has the amino acid sequence NYKRCFPVI (SEQ ID NO: 207, p143-151) and is 9 amino acids long. In another embodiment, a suitable MAGE-A4 peptide has the amino acid sequence EVDPASNTY (SEQ ID NO: 208) and is 9 amino acids long. In another embodiment, a suitable MAGE-A4 peptide has the amino acid sequence SESLKMIF (SEQ ID NO: 209) and is 8 amino acids long. In another embodiment, a suitable MAGE-A4 peptide has the amino acid sequence SESLICMIF (SEQ ID NO: 210) and is 9 amino acids long.

[0071] In some cases, the MAGE-A4 peptide is HLA-restricted. For example, the peptide GVYDGREHTV (SEQ ID NO: 206) is HLA-A2 restricted.

[0072] NY-ESO-1 peptide In some cases, the TMP comprises a cancer / testis antigen-1 (CTAG1B) peptide as a peptide epitope. CTAG1B is also known as LAGE2, LAGE3, or NY-ESO-1 (New York Esophageal Squamous Cell Carcinoma 1). Thus, in some cases, the TMP comprises a NY-ESO-1 peptide as a peptide epitope. In some cases, suitable NY-ESO-1 peptides are peptide fragments of 4-20 amino acids in length, e.g., 6-15 amino acids in length, 8-12 amino acids in length, 8-10 amino acids in length, 9-11 amino acids in length, 5-10 amino acids in length, 10-15 amino acids in length, and 15-20 amino acids in length, of a NY-ESO-1 polypeptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following NY-ESO-1 amino acid sequence:

[0073] MQAEGRGTGG STGDADGPGG PGIPDGPGGN AGGPGEAGAT GGRGPRGAGA ARASGPGGGA PRGPHGGAAS GLNGCCRCGA RGPESRLLEF YLAMPFATPM EAELARRSLA QDAPPLPVPG VLLKEFTVSG NILTIRLTAA DHRQLQLSIS SCLQQLSLLM WITQCFLPVF LAQPPSGQRR (sequence number 211).

[0074] In one embodiment, a suitable NY-ESO-1 peptide has the amino acid sequence SLLMWITQCFL (SEQ ID NO:212) and is 11 amino acids in length. In another embodiment, a suitable NY-ESO-1 peptide has the amino acid sequence SLLMWITQC (SEQ ID NO:213) and is 9 amino acids in length. In another embodiment, a suitable NY-ESO-1 peptide has the amino acid sequence QLSLLMWIT (SEQ ID NO:214) and is 9 amino acids in length. In another embodiment, a suitable NY-ESO-1 peptide has the amino acid sequence SLLMWITQCFLPVF (SEQ ID NO:215) and is 14 amino acids in length (NY-ESO-1 157-170Other suitable NY-ESO-1 peptides include, for example, MLMAQEALAFL (SEQ ID NO: 216), YLAMPFATPME (SEQ ID NO: 217), ASGPGGGAPR (SEQ ID NO: 218), LAAQERRVPR (SEQ ID NO: 219), TVSGNILTIR (SEQ ID NO: 220), APRGPHGGAASGL (SEQ ID NO: 221), MPFATPMEAEL (SEQ ID NO: 222), KEFTVSGNLLTI (SEQ ID NO: 223), MPFATPMEA (SEQ ID NO: 224), FATPMEAELAR (SEQ ID NO: 225), LAMPFATPM (SEQ ID NO: 226), and ARGPESRLL (SEQ ID NO: 227).

[0075] Survivin peptides In some cases, the TMP comprises a survivin peptide as a peptide epitope. Survivin is also known in the art as Baculoviral IAP Repeat Containing 5 (BIRC5) and Inhibitor of Apoptosis 4 (IAP4). In some cases, suitable survivin peptides are peptide fragments of 4-20 amino acids in length, e.g., 6-15 amino acids in length, 8-12 amino acids in length, 8-10 amino acids in length, 9-11 amino acids in length, 5-10 amino acids in length, 10-15 amino acids in length, and 15-20 amino acids in length, of a survivin polypeptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence shown in any one of Figures 25A-25C.

[0076] In one embodiment, a suitable survivin peptide has the amino acid sequence ELTLGEFLKL (SEQ ID NO: 228, survivin 95-104) and is 10 amino acids long. In another embodiment, a suitable survivin peptide has the amino acid sequence TLGEFLKLDRERAKN (SEQ ID NO: 229) and is 15 amino acids long. In another embodiment, a suitable survivin peptide has the amino acid sequence QMFFCF (SEQ ID NO: 230) and is 6-10 amino acids long. In another embodiment, a suitable survivin peptide has the amino acid sequence DLAQMFFCFKELEGW (SEQ ID NO: 231) and is 15 amino acids long. In another embodiment, a suitable survivin peptide has the amino acid sequence AQMFFCFKEL (SEQ ID NO: 232) and is 10 amino acids long. In another embodiment, a suitable survivin peptide has the amino acid sequence QMFFCFKEL (SEQ ID NO: 233) and is 9 amino acids long.

[0077] Mesothelin Peptides In some cases, the TMP comprises a mesothelin peptide as a peptide epitope. In some cases, a suitable mesothelin peptide is a peptide fragment 4-20 amino acids in length, e.g., 6-15 amino acids in length, 8-12 amino acids in length, 8-10 amino acids in length, 9-11 amino acids in length, 5-10 amino acids in length, 10-15 amino acids in length, 15-20 amino acids in length, of a mesothelin polypeptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following mesothelin amino acid sequence:

[0078] MALPTARPLL GSCGTPALGS LLFLLFSLGW VQPSRTLAGE TGQEAAPLDG VLANPPNISS LSPRQLLGFP CAEVSGLSTE RVRELAVALA QKNVKLSTEQ LRCLAHRLSE PPEDLDALPL DLLLFLNPDA FSGPQACTRF FSRITKANVD LLPRGAPERQ RLLPAALACW GVRGSLLSEA DVRALGGLAC DLPGRFVAES AEVLLPRLVS CPGPLDQDQQ EAARAALQGG GPPYGPPSTW SVSTMDALRG LLPVLGQPII RSIPQGIVAA WRQRSSRDPS WRQPERTILR PRFRREVEKT ACPSGKKARE IDESLIFYKK WELEACVDAA LLATQMDRVN AIPFTYEQLD VLKHKLDELY PQGYPESVIQ HLGYLFLKMS PEDIRKWNVT SLETLKALLE VNKGHEMSPQ VATLIDRFVK GRGQLDKDTL DTLTAFYPGY LCSLSPEELS SVPPSSIWAV RPQDLDTCDP RQLDVLYPKA RLAFQNMNGS EYFVKIQSFL GGAPTEDLKA LSQQNVSMDL ATFMKLRTDA VLPLTVAEVQ KLLGPHVEGL KAEERHRPVR DWILRQRQDD LDTLGLGLQG GIPNGYLVLD LSMQEALSGT PCLLGPGPVL TVLALLLAST LA(SEQ ID NO: 234).

[0079] Non-limiting examples of suitable mesothelin peptides include mesothelin A2 (20-28) peptide SLLFLLFSL (SEQ ID NO: 235), mesothelin A2 (530-538) peptide VLPLTVAEV (SEQ ID NO: 236), mesothelin A3 (83-91) peptide ELAVALAQK (SEQ ID NO: 237), mesothelin A3 (225-233) peptide ALQGGGPPY (SEQ ID NO: 238), mesothelin A24 (435-443) peptide FYPGYLCSL (SEQ ID NO: 239), and mesothelin A24 (475-483) peptide LYPKARLAF (SEQ ID NO: 240).

[0080] MART-1 peptide In some cases, the TMP comprises a melanoma antigen-1 (MART-1) peptide recognized by T cells as a peptide epitope. In some cases, suitable MART-1 peptides are peptide fragments of 4-20 amino acids in length, e.g., 6-15 amino acids in length, 8-12 amino acids in length, 8-10 amino acids in length, 9-11 amino acids in length, 5-10 amino acids in length, 10-15 amino acids in length, and 15-20 amino acids in length, of a MART-1 polypeptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following MART-1 amino acid sequence: MPREDAHFIY GYPKKGHGHS YTTAEEAAGI GILTVILGVL LLIGCWYCRR RNGYRALMDK SLHVGTQCAL TRRCPQEGFD HRDSKVSLQE KNCEPVVPNA PPAYEKLSAE QSPPPYSP (SEQ ID NO: 241).As one specific, non-limiting example, a suitable MART-1 peptide has the amino acid sequence ELAGIGILTV (SEQ ID NO: 242) and is 10 amino acids in length.

[0081] Non-classical HLA peptides In some cases, the TMP comprises a non-classical HLA peptide as a peptide epitope. For example, in some cases, suitable non-classical HLA peptides are peptide fragments of HLA-G polypeptides that are 4-20 amino acids long, e.g., 6-15 amino acids long, 8-12 amino acids long, 8-10 amino acids long, 9-11 amino acids long, 5-10 amino acids long, 10-15 amino acids long, and 15-20 amino acids long. In one non-limiting example, a suitable HLA-G peptide has the amino acid sequence VMARPTLFL (SEQ ID NO: 243) and is 9 amino acids long.

[0082] Viral peptides In some cases, the TMP comprises as a peptide epitope a viral peptide epitope. Suitable peptide epitopes include, but are not limited to, epitopes present in infectious disease pathogens, such as epitopes presented by virus-encoded polypeptides. Specific examples of viral infectious disease pathogens include: Adenoviruses, Adeno-associated viruses, Alphaviruses (Togaviruses), Eastern Equine Encephalitis Virus, Eastern Equine Encephalomyelitis Virus, Venezuelan Equine Encephalomyelitis Vaccine Strain TC-83, Western Equine Encephalomyelitis Virus, Arenaviruses, Lymphocytic Choriomeningitis Virus (non-neurotropic strains), Tacaribe Virus Complex, Bunyaviruses, Bunyamwera Virus, Rift Valley Fever Virus Vaccine Strain MP-12, Chikungunya Virus, Caliciviruses, Coronaviruses, Cowpox Virus, Flaviviruses (Togaviruses) Group B Arboviruses, Dengue Virus Serotypes 1, 2, 3, and 4, Yellow Fever Virus Vaccine Strain 17D, Hepatitis A, B, C, D, and E Viruses, Cytomegalovirus, Epstein-Barr Virus, Eastern Equine Encephalitis Virus, Herpes Simplex Virus Types 1 and 2, Herpes Zoster, Human Herpes Virus Types 6 and 7, Hepatitis C Virus Hepatitis B virus (HBV), influenza viruses types A, B, and C, papovavirus, Newcastle disease virus, measles virus, mumps virus, parainfluenza viruses types 1, 2, 3, and 4, polyomaviruses (JC virus, BK virus), respiratory syncytial virus, human parvovirus (B19), coxsackieviruses A and B, echovirus, poliovirus, rhinovirus, alastrim (variola minor virus), smallpox virus (variola major virus), whitepox reovirus, coltivirus, human rotavirus, orbivirus (Colorado tick fever virus), rabies virus, vesicular stomatitis virus, rubivirus (rubella), Semliki Forest virus, Saint Louis encephalitis virus, Venezuelan equine encephalitis virus, Venezuelan equine encephalomyelitis virus, arenaviruses (akaSouth American hemorrhagic fever viruses), flexaviruses, lymphocytic choriomeningitis virus (LCM) (neurotropic strains), Hantaviruses (including Hantaan virus), Rift Valley fever virus, Japanese encephalitis virus, Yellow fever virus, Monkeypox virus, Human immunodeficiency virus (HIV) types 1 and 2, Human T-cell lymphotropic virus (HTLV) types 1 and 2, Simian immunodeficiency virus (SIV), Vesicular stomatitis virus, Guanarito virus, Lassa fever virus, Junin virus, Machupo virus, Sabia virus, Crimean-Congo hemorrhagic fever virus, Ebola virus, Marburg virus, Tick-borne encephalitis virus complex (flavivirus) (Central European tick-borne encephalitis, Polar Eastern tick-borne encephalitis), Hansalova virus, Hypr virus, Kumringe virus, Kyasanur Forest disease virus, Omsk hemorrhagic fever virus, Russian spring-summer encephalitis virus, Herpesvirus simie (Herpes B or monkey B virus), Cercopithecus herpesvirus 1 (Herpes B virus), Equine morbillivirus (Hendra and Hendra-like viruses), Nipah virus, Variola major virus (Smallpox virus), Variola minor virus (Alastrim), African swine fever virus, African horsesickness virus, Akabane virus, Avian influenza virus (highly pathogenic), Bluetongue virus, Camelpox virus, Classical swine fever virus, Rickettsia (Cowdria ruminantium (heartwater disease) virus, foot and mouth disease virus, goat pox virus, Japanese encephalitis virus, lumpy skin disease virus, malignant catarrhal fever virus, Menangle virus, Newcastle disease virus (VVND), vesicular stomatitis virus (foreign), and Zika virus. Antigens encoded by such viruses are known in the art. Peptide epitopes suitable for use in TMP can include peptides from any known viral antigen. In some cases, HPV antigens are specifically excluded. In some cases, HBV antigens are specifically excluded. In some cases, the viral epitope is an epitope present in a viral antigen encoded by a virus that infects most humans, such as, for example, cytomegalovirus (CMV), Epstein-Barr virus (EBV), human papillomavirus, influenza virus, adenovirus, and the like.

[0083] A non-limiting example of an influenza virus peptide is an influenza virus matrix protein (M1) peptide, e.g., M1(58-66), having the amino acid sequence GILGFVFTL (SEQ ID NO: 244) and being 9 amino acids in length. A non-limiting example of an EBV peptide is an EBV nuclease antigen 3B (EBNA3B) peptide having the amino acid sequence IVTDFSVIK (SEQ ID NO: 245) and being 9 amino acids in length. Another non-limiting example of an EBV peptide is an EBNA3B peptide having the amino acid sequence AVFDRKSDAK (SEQ ID NO: 246) and being 9 amino acids in length.

[0084] In some cases, the TMP comprises a CMV peptide as a peptide epitope. In some cases, the CMV peptide epitope present in the TMP is a peptide derived from CMV pp65. In some cases, the CMV peptide epitope present in the TMP is a peptide derived from CMV gB (glycoprotein B).

[0085] For example, in some cases, a CMV peptide epitope present in a TMP is a peptide of a CMV polypeptide having a length of 4-20 amino acids, e.g., 6-15 amino acids, 8-12 amino acids, 8-10 amino acids, 9-11 amino acids, 5-10 amino acids, 10-15 amino acids, and 15-20 amino acids, and includes an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the CMV pp65 polypeptide amino acid sequence shown in Figure 26A or to the CMV gB polypeptide amino acid sequence shown in Figure 26B.

[0086] As one specific, non-limiting example, the CMV peptide epitope present in TMP has the amino acid sequence NLVPMVATV (SEQ ID NO:247) and is 9 amino acids in length.

[0087] In some cases, the CMV epitopes present in the TMP present epitopes specific for HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, or HLA-G alleles. In some cases, the epitope peptides present in the TMP present epitopes specific for HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, or HLA-G alleles. * 0101, A * 0201, A * 0301, A * 1101, A * 2301, A * 2402, A * 2407, A * 3303 and / or A * In some cases, the CMV epitopes present in the TMP are restricted to HLA-B 3401. * 0702, B * 0801, B * 1502, B * 3802, B * 4001, B * 4601 and / or B * In some cases, the CMV epitopes present in the TMP are restricted to CMV 5301. * 0102, C * 0303, C * 0304, C * 0401, C * 0602, C * 0701, C * 702, C * 0801 and / or C * In one embodiment, the TMP presents an epitope restricted to HLA-A 1502. In one embodiment, the TMP presents an epitope restricted to HLA-A 1502. * 0201 class I heavy chain polypeptide, and c) a β2M polypeptide.

[0088] In some cases, the CMV epitope present in the TMP comprises a CMV peptide having the amino acid sequence ATVQGQNLK (SEQ ID NO: 248) and is 9 amino acids in length. In some cases, the CMV epitope present in the TMP comprises a CMV peptide having the amino acid sequence VMAPRTLIL (SEQ ID NO: 249) and is 9 amino acids in length.

[0089] In some cases, the TMP comprises a SARS-CoV-2 peptide. In some cases, the SARS-CoV-2 peptide present in the TMP is a SARS-CoV-2 peptide derived from a surface glycoprotein encoded by SARS-CoV-2 and is selected from the group consisting of: NLTTRTQL (SEQ ID NO: 250), LPPAYTNSF (SEQ ID NO: 251), KVFRSSVLH (SEQ ID NO: 252), LPFFSNVTW (SEQ ID NO: 253), PFFSNVTWF (SEQ ID NO: 254), RFDNPVLPF (SEQ ID NO: 255), LPFNDGVYF (SEQ ID NO: 256), GVYFASTEK (SEQ ID NO: 257), TEKSNIIRGW (SEQ ID NO: 258), TLDSKTQSL (SEQ ID NO: 259), GVYYHKNNK (SEQ ID NO: 260), YYHKNNKSW (SEQ ID NO: 261), or a combination thereof. Column number 261), VYSSANNCTF (SEQ ID NO: 262), FEYVSQPFL (SEQ ID NO: 263), EYVSQPFLM (SEQ ID NO: 264), FVFKNIDGY (SEQ ID NO: 265), TPINLVRDL (SEQ ID NO: 266), LPQGFSAL (SEQ ID NO: 267), LPIGINITRF (SEQ ID NO: 268), INITRFQTL (SEQ ID NO: 269), LLALHRSYL (SEQ ID NO: 270), WTAGAAAYY (SEQ ID NO: 271), YYVGYLQPRTF (SEQ ID NO: 272), Y LQPRTFLL (SEQ ID NO: 273), YLQPRTFL (SEQ ID NO: 274), SETKCTLKSF (SEQ ID NO: 275), TLKSFTVEK (SEQ ID NO: 276), QPTESIVRF (SEQ ID NO: 277), RFPNITNLCPF (SEQ ID NO: 278), GEVFNATRF (SEQ ID NO: 279), NATRFASVY (SEQ ID NO: 280), LYNSASFSTF (SEQ ID NO: 281), NSASFSTFK (SEQ ID NO: 282), RQIAPGQTGK (SEQ ID NO: 283), KIADYNYKL (SEQ ID NO: 284), NYNYLYRLF (SEQ ID NO: 285), RLFRKSNLK (SEQ ID NO: 286), KPFERDISTEI (SEQ ID NO: 287), YFPLQSYGF (SEQ ID NO: 288), QPYRVVVL (SEQ ID NO: 289), PYRVVVLSF (SEQ ID NO: 290), GPKKSTNLV (SEQ ID NO: 291), TSNQVAVLY (SEQ ID NO: 292), VYSTGSNVF (SEQ ID NO: 293), AEHVNNSY (SEQ ID NO: 294), IPIGAGICASY (SEQ ID NO: 295),SPRRARSVA (SEQ ID NO: 296), VASQSIIAY (SEQ ID NO: 297), SIIAYTMSL (SEQ ID NO: 298), LGAENSVAY (SEQ ID NO: 299), AYSNNSIAIPTNF (SEQ ID NO: 300), IPTNFTISV (SEQ ID NO: 301), TEILPVSMTK (SEQ ID NO: 302), QEVFAQVKQIY (SEQ ID NO: 303), KQIYKTPPIK (SEQ ID NO: 304), IYKTPPIKDF (SEQ ID NO: No. 305), LLFNKVTLA (SEQ ID NO: 306), TLADAGFIK (SEQ ID NO: 307), LADAGFIKQY (SEQ ID NO: 308), ADAGFIKQY (SEQ ID NO: 309), VLPPLLTDEMIAQY (SEQ ID NO: 310), IPFAMQMAY (SEQ ID NO: 311), SSTASALGK (SEQ ID NO: 312), VLNDILSRL (SEQ ID NO: 313), RLDKVEAEV (SEQ ID NO: 314), VEAEVQIDRL (SEQ ID NO: 315), AEVQIDRLI (SEQ ID NO: 316), LITGRLQSL (SEQ ID NO: 317), RLQSLQTYV (SEQ ID NO: 318), AEIRASANL (SEQ ID NO: 319), ASANLAATK (SEQ ID NO: 320), HLMSFPQSA (SEQ ID NO: 321), FPQSAPHGVVF (SEQ ID NO: 322), APHGVVFL (SEQ ID NO: 323), VTYVPAQEK (SEQ ID NO: 324), TYVPAQEKNF ( SEQ ID NO: 325), REGVFVSNGTHW (SEQ ID NO: 326), GTHWFVTQR (SEQ ID NO: 327), TVYDPLQPELDSFK (SEQ ID NO: 328), KEIDRLNEV (SEQ ID NO: 329), QELGKYEQYIKW (SEQ ID NO: 330), YEQYIKWPW (SEQ ID NO: 331), QYIKWPWYI (SEQ ID NO: 332), FIAGLIAIV (SEQ ID NO: 333), and SEPVLKGVKL (SEQ ID NO: 334).

[0090] In some cases, the SARS-CoV-2 peptide epitope has the amino acid sequence RLQSLQTYV (SEQ ID NO: 318) and is 9 amino acids in length. In some cases, the SARS-CoV-2 peptide epitope has the amino acid sequence YLQPRTFLL (SEQ ID NO: 273) and is 9 amino acids in length.

[0091] In some cases, the peptide epitope is a SARS-CoV-2 peptide derived from a membrane glycoprotein encoded by SARS-CoV-2. In some cases, the SARS-CoV-2 peptide present in the TMP is a SARS-CoV-2 peptide derived from a membrane glycoprotein encoded by SARS-CoV-2 and is selected from the group consisting of: GTITVEELK (SEQ ID NO: 335), EELKKLLEQW (SEQ ID NO: 336), KLLEQWNLV (SEQ ID NO: 337), FAYANRNRF (SEQ ID NO: 338), YANRNRFLY (SEQ ID NO: 339), SYFIASFRLF (SEQ ID NO: 340), RLFARTRSM (SEQ ID NO: 341), VPLHGTIL (SEQ ID NO: 342), SELVIGAVIL (SEQ ID NO: 343), HLRIAGHHL (SEQ ID NO: 344), RIAGHHLGR (SEQ ID NO: 345), KEITVATSRTL (SEQ ID NO: 346), ATSRTLSYYK (SEQ ID NO: 347), ASQRVAGDSGFAAY (SEQ ID NO: 348), and VAGDSGFAAY (SEQ ID NO: 349).

[0092] In some cases, the peptide epitope is a SARS-CoV-2 peptide derived from the nucleocapsid phosphoprotein encoded by SARS-CoV-2. In some cases, the peptide epitope is a SARS-CoV-2 peptide derived from the nucleocapsid phosphoprotein encoded by SARS-CoV-2 and is selected from the group consisting of: LPNNTASWF (SEQ ID NO: 350), KFPGQGVPI (SEQ ID NO: 351), NTNSSPDDQIGYY (SEQ ID NO: 352), SPLWYFYL (SEQ ID NO: 353), LLDRLNQL (SEQ ID NO: 354), KAYNVTQAF (SEQ ID NO: 355), QELIRQGTDYKW (SEQ ID NO: 356), ASAFFGMSR (SEQ ID NO: 357), SRIGMEVTPSGTW (SEQ ID NO: 358), GMEVTPSGTWL (SEQ ID NO: 359), TPSGTWLTY (SEQ ID NO: 360), AYKTFPPTEPK (SEQ ID NO: 361), and LPAADLDDF (SEQ ID NO: 362).

[0093] MHC Polypeptides As discussed above, TMPs include MHC polypeptides. For purposes of this disclosure, the term "major histocompatibility complex (MHC) polypeptide" is meant to encompass MHC polypeptides of various species. MHC polypeptides of various species include 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., equines, bovidae, ovines, caprines, etc.)). The term "MHC polypeptide" is meant to encompass class I MHC polypeptides (e.g., β-2 microglobulin (β2M) and MHC class I heavy chains).

[0094] In some cases, the first MHC polypeptide is an MHC class I β2M polypeptide and the second MHC polypeptide is an MHC class I heavy chain (H chain) (MHC-H). In other cases, the first MHC polypeptide is an MHC class I heavy chain polypeptide and the second MHC polypeptide is a β2M polypeptide. In some cases, both the β2M chain and the MHC-H chain are of human origin. That is, the MHC-H chain is an HLA heavy chain or a variant thereof. Unless otherwise specified, the TMP does not include the membrane anchor domain (transmembrane region) of the MHC class I heavy chain or a portion of the MHC class I heavy chain sufficient to anchor the resulting TMP to the cell in which it is expressed (e.g., a eukaryotic cell such as a mammalian cell). In some cases, the MHC class I heavy chain present in the TMP does not include a signal peptide, a transmembrane domain, or an intracellular domain (cytoplasmic tail) associated with the native MHC class I heavy chain. Thus, for example, in some cases, the MHC class I heavy chain present in the TMP comprises only the α1, α2, and α3 domains of the MHC class I heavy chain. In some cases, the MHC class I heavy chain present in the TMP is about 270 amino acids long to about 290 amino acids long. In some cases, the MHC class I heavy chain present in the TMP has a length of 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, or 290 amino acids.

[0095] MHC class I heavy chain In some cases, the MHC class I heavy chain polypeptide present in the TMP comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to all or a portion (e.g., 50, 75, 100, 150, 200, or 250 contiguous amino acids) of the amino acid sequence of any of the human HLA heavy chain polypeptides shown in Figures 3-11. In some cases, the MHC class I heavy chain has a length of 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, or 290 amino acids. In some cases, the MHC class I heavy chain polypeptide present in the TMP has between 1 and 30, 1 and 5, 5 and 10, 10 and 15, 15 and 20, 20 and 25, or 25 and 30 amino acid insertions, deletions, and / or substitutions (in addition to the positions indicated as variable in the heavy chain consensus sequence) of any one of the amino acid sequences shown in Figures 3-11. As discussed above, MHC class I heavy chains typically do not include transmembrane or cytoplasmic domains. In one embodiment, the MHC class I heavy chain polypeptide of the TMP comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to amino acids 25-300 (lacking all or substantially all of the leader, transmembrane, and cytoplasmic sequences) or amino acids 25-365 (lacking the leader) of a human HLA-A heavy chain polypeptide.

[0096] In some cases, the MHC polypeptide of the TMP is a human MHC polypeptide (human MHC polypeptides are also called "human leukocyte antigen" (HLA) polypeptides). In some cases, the MHC polypeptide of the TMP is a class I HLA polypeptide, such as 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.

[0097] In some cases, the TMP comprises an HLA-A heavy chain polypeptide. The HLA-A heavy chain peptide sequences or portions thereof that may be incorporated into the TMP include, but are not limited to, the following alleles: * 0101, A * 0201, A * 0301, A * 1101, A * 2301, A * 2402, A * 2407, A * 3303 and A *3401, any of which may have a mutation at one or more of positions 84, 139, and 236 (a mutation selected from 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) as shown in Figures 3-6). Furthermore, HLA-A 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 the whole or part (e.g., 50, 75, 100, 150, 200, or 250 consecutive amino acids) of the sequences of these HLA-A alleles may be used (e.g., which may have 1 to 25, 1 to 5, 5 to 10, 10 to 15, 15 to 20, 20 to 25, or 25 to 30 amino acid insertions, deletions, and / or substitutions). Certain specific examples are shown below.

[0098] HLA-A02 In some cases, the MHC class I heavy chain polypeptide of the TMP 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 amino acid sequence shown in Figure 3A. In some cases, the HLA-A heavy chain polypeptide suitable for inclusion in the TMP has the amino acid sequence shown in Figure 3A. This HLA-A heavy chain polypeptide is referred to as "HLA-A *As described in WO2018 / 119114 and WO2020 / 132138, the TMP may include one or more mutations from wild-type HLA-A02. Such mutations include the provision of Cys residues capable of forming disulfide bonds. The disulfide bonds may be, for example, (i) between the β2M and the MHC class I heavy chain, and / or (ii) between the MHC heavy chain and the linker that attaches the peptide epitope to the β2M polypeptide, and / or (iii) an intrachain disulfide bond within the MHC class I heavy chain polypeptide.

[0099] To facilitate the formation of such disulfide bonds, the heavy chain polypeptide can be provided with one or more non-naturally occurring Cys residues. For example, the MHC class I 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 A), B), or C): A) the HLA-A02(Y84C, A236C) amino acid sequence shown in FIG. 3C, in which amino acid 84 is Cys and amino acid 236 is Cys (in one case, Cys-136 forms a disulfide bond with Cys-12 of a mutant β2M polypeptide containing an R12C substitution); or B) the HLA-A02(Y84A, A236C) amino acid sequence shown in FIG. 3D, in which 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 a mutant β2M polypeptide containing an R12C substitution); or C) HLA-A02 (Y84C, A139C) amino acid sequence shown in FIG. 3E, where amino acid 84 is Cys and amino acid 139 is Cys (in some cases, Cys-84 forms an intrachain disulfide bond with Cys-139).

[0100] As described above, in some cases, the MHC class I 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 HLA-A02 (Y84A, A236 wild type) amino acid sequence shown in FIG. 3B (wherein amino acid 84 is Tyr and amino acid 236 is Ala).

[0101] HLA-A11(HLA-A * 1101) In some cases, the MHC class I heavy chain polypeptide of the TMP 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 amino acid sequence shown in Figure 4A. In some cases, the HLA-A heavy chain polypeptide suitable for inclusion in the TMP has the amino acid sequence shown in Figure 4A. This HLA-A heavy chain polypeptide is referred to as "HLA-A * The variant is also referred to as "HLA-A1101" or simply "HLA-A11." Examples of the variant include an MHC class I heavy chain polypeptide comprising 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 A) to D). A) HLA-A11 (Y84A, A236 wild type) amino acid sequence shown in FIG. 4B (wherein amino acid 84 is Tyr and amino acid 236 is Ala); B) the HLA-A11 (Y84C, A236C) amino acid sequence shown in FIG. 4C (wherein amino acid 84 is Cys and amino acid 236 is Cys); C) the HLA-A11 (Y84A, A236C) amino acid sequence shown in FIG. 4D, in which 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 a mutant β2M polypeptide containing an R12C substitution); and D) HLA-A11 (Y84C, A139C) amino acid sequence shown in Figure 4E, where amino acid 84 is Cys and amino acid 139 is Cys (in some cases, Cys-84 forms an intrachain disulfide bond with Cys-139).

[0102] HLA-A24(HLA-A * 2402) In some cases, the MHC class I heavy chain polypeptide of the TMP 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 amino acid sequence shown in Figure 5A. In some cases, the HLA-A heavy chain polypeptide suitable for inclusion in the TMP has the amino acid sequence shown in Figure 5A. This HLA-A heavy chain polypeptide is referred to as "HLA-A * The variant is also referred to as "HLA-A24" or simply "HLA-A24." Examples of the variant include an MHC class I heavy chain polypeptide comprising 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 A) to D). A) The amino acid sequence of HLA-A24 (Y84A, A236 wild type) shown in FIG. 5B (wherein amino acid 84 is Tyr and amino acid 236 is Ala); B) the HLA-A24(Y84C, A236C) amino acid sequence shown in FIG. 5C, where amino acid 84 is Cys and amino acid 236 is Cys; C) the HLA-A24(Y84A, A236C) amino acid sequence shown in FIG. 5D, in which 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 a mutant β2M polypeptide containing an R12C substitution); and D) HLA-A24(Y84C, A139C) amino acid sequence shown in FIG. 5E, where amino acid 84 is Cys and amino acid 139 is Cys (in some cases, Cys-84 forms an intrachain disulfide bond with Cys-139).

[0103] HLA-A33(HLA-A * 3303) In some cases, the MHC class I heavy chain polypeptide of the TMP 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 amino acid sequence shown in Figure 6A. In some cases, the HLA-A heavy chain polypeptide suitable for inclusion in the TMP has the amino acid sequence shown in Figure 6A. This HLA-A heavy chain polypeptide is referred to as "HLA-A * The variant is also referred to as "HLA-A33" or simply "HLA-A33." Examples of the variant include an MHC class I heavy chain polypeptide comprising 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 A) to D). A) The amino acid sequence of HLA-A24 (Y84A, A236 wild type) shown in FIG. 6B (wherein amino acid 84 is Tyr and amino acid 236 is Ala); B) the HLA-A24(Y84C, A236C) amino acid sequence shown in FIG. 6C, where amino acid 84 is Cys and amino acid 236 is Cys; C) the HLA-A24(Y84A, A236C) amino acid sequence shown in FIG. 6D, in which 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 a mutant β2M polypeptide containing an R12C substitution); and D) HLA-A24(Y84C, A139C) amino acid sequence shown in FIG. 6E, where amino acid 84 is Cys and amino acid 139 is Cys (in some cases, Cys-84 forms an intrachain disulfide bond with Cys-139).

[0104] 7-9 show sequence comparisons of mature HLA class I heavy chain amino acid sequences (not including leader sequences or transmembrane or intracellular domains). The amino acid sequences aligned in FIG. 7A are the HLA-A class I heavy chains of the following alleles: * 0101, A * 0201, A * 0301, A * 1101, A * 2301, A * 2402, A * 2407, A * 3303 and A * 3401. The amino acid sequences aligned in FIG. 8A are the HLA-B class I heavy chains of the following alleles: * 0702, B * 0801, B * 1502, B * 3802, B * 4001, B * 4601 and B * 5301. The amino acid sequences aligned in FIG. 9A are the HLA-C class I heavy chains of the following alleles: * 0102, C * 0303, C * 0304, C * 0401, C * 0602, C * 0701, C * 0801 and C *1502. The alignment shows positions (positions 84 and 139 of the mature protein) where a cysteine ​​residue may be introduced (e.g., by substitution) to form a disulfide bond that stabilizes the HLA H chain-β2M complex. The alignment also shows position 236 (of the mature polypeptide) which may be substituted with a cysteine ​​residue that can form an interchain disulfide bond with β2M (e.g., at amino acid 12). The boxes adjacent to residues 84, 139, and 236 show five amino acid groups on either side of six sets of five residues designated aac1 (amino acid cluster 1), aac2 (amino acid cluster 2), aac3 (amino acid cluster 3), aac4 (amino acid cluster 4), aac5 (amino acid cluster 5), and aac6 (amino acid cluster 6). The five residues may be substituted with from 1 to 5 amino acids independently selected from (i) any naturally occurring amino acid, or (ii) any naturally occurring amino acid except proline or glycine.

[0105] Figures 7A, 8A and 9A show alignments of the amino acid sequences of mature HLA-A, HLA-B and HLA-C class I heavy chains, respectively. These sequences are shown for the extracellular portion of the mature protein (not including the leader sequence, transmembrane domain or intracellular domain). The positions of amino acid residues 84, 139 and 236, as well as their adjacent residues (aac1-aac6), are also shown. The adjacent residues may be substituted with (i) any naturally occurring amino acid, or (ii) one to five amino acids independently selected from any naturally occurring amino acid except proline or glycine. Figures 7B, 8B and 9B show consensus amino acid sequences for the HLA-A, HLA-B and HLA-C sequences shown in Figures 7A, 8A and 9A, respectively. The consensus sequences sequentially number the positions of the variable amino acids as "X" residues, and double underline the positions of amino acids 84, 139 and 236.

[0106] 7A , in some cases, i) aac1 (amino acid cluster 1) may be the amino acid sequence GTLRG (SEQ ID NO: 363) or may be a sequence in which one or two amino acids have been deleted or substituted with other naturally occurring amino acids (e.g., L is replaced with I, V, A, or F); ii) aac2 (amino acid cluster 2) may be the amino acid sequence YNQSE (SEQ ID NO: 364) or may be a sequence in which one or two amino acids have been deleted or substituted with other naturally occurring amino acids (e.g., N is replaced with Q, Q is replaced with N, and / or E is replaced with D); iii) aac3 (amino acid cluster 3) may be the amino acid sequence TAADM (SEQ ID NO: 365) or may be a sequence in which one or two amino acids have been deleted or substituted with other naturally occurring amino acids (e.g., T is replaced with S, A is replaced with G, D is replaced with E, and / or M is replaced with L, V, or I); and iv) aac4 (amino acid cluster 4) may be the amino acid sequence YNQSE (SEQ ID NO: 366) or may be a sequence in which one or two amino acids have been deleted or substituted with other naturally occurring amino acids (e.g., T is replaced with S, A is replaced with G, D is replaced with E, and / or M is replaced with L, V, or I). aac5 (amino acid cluster 5) may be the amino acid sequence VETRP (SEQ ID NO: 367) or may be the amino acid sequence in which one or two amino acids have been deleted or substituted with other naturally occurring amino acids (e.g., A is replaced with G, Q is replaced with N, or T is replaced with S, and / or K is replaced with R or Q); v) aac6 (amino acid cluster 6) may be the amino acid sequence GDGTF (SEQ ID NO: 368) or may be the amino acid sequence in which one or two amino acids have been deleted or substituted with other naturally occurring amino acids (e.g., D is replaced with E, T is replaced with S, or F is replaced with L, W, or Y).

[0107] With reference to FIG. 8A , in some cases, i) aac1 (amino acid cluster 1) may be the amino acid sequence RNLRG (SEQ ID NO: 369) or a sequence in which one or two amino acids have been deleted or substituted with other naturally occurring amino acids (e.g., N replaced with T or I, and / or L replaced with A, and / or the second R replaced with L, and / or G replaced with R), and ii) aac2 (amino acid cluster 2) may be the amino acid sequence YNQSE (SEQ ID NO: 364) or a sequence in which one or two amino acids have been deleted or substituted with other naturally occurring amino acids (e.g., N replaced with T or I, and / or L replaced with A, and / or the second R replaced with L, and / or G replaced with R). aac3 (amino acid cluster 3) may be the amino acid sequence TAADT (SEQ ID NO: 370) or a sequence in which one or two amino acids have been deleted or substituted with other naturally occurring amino acids (e.g., replacing N with Q, replacing Q with N, and / or replacing E with D); iii) aac3 (amino acid cluster 3) may be the amino acid sequence TAADT (SEQ ID NO: 370) or a sequence in which one or two amino acids have been deleted or substituted with other naturally occurring amino acids (e.g., replacing the first T with S, and / or replacing A with G, and / or replacing D with E, and / or replacing the second T with S); iv) aac4 (amino acid cluster 4) may be the amino acid sequence TAADT (SEQ ID NO: 370) or a sequence in which one or two amino acids have been deleted or substituted with other naturally occurring amino acids (e.g., replacing the first T with S, and / or replacing A with G, and / or replacing D with E, and / or replacing the second T with S); Cluster 4) may be the amino acid sequence AQITQ (SEQ ID NO: 371) or a sequence in which one or two amino acids have been deleted or substituted with other naturally occurring amino acids (e.g., A is replaced with G, and / or the first Q is replaced with N, and / or I is replaced with L or V, and / or T is replaced with S, and / or the second Q is replaced with N); v) aac5 (amino acid cluster 5) may be the amino acid sequence VETRP (SEQ ID NO: 367) or a sequence in which one or two amino acids have been deleted or substituted with other naturally occurring amino acids (e.g., A is replaced with G, and / or the first Q is replaced with N, and / or I is replaced with L or V, and / or T is replaced with S, and / or the second Q is replaced with N); and / or vi) aac6 (amino acid cluster 6) may be the amino acid sequence GDRTF (SEQ ID NO: 372) or a sequence in which one or two amino acids have been 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).

[0108] 9A , in some cases, i) aac1 (amino acid cluster 1) may be the amino acid sequence RNLRG (SEQ ID NO: 369) or a sequence in which one or two amino acids have been deleted or substituted with other naturally occurring amino acids (e.g., N is replaced with K, and / or L is replaced with A or I, and / or the second R is replaced with H, and / or G is replaced with T or S), and ii) aac2 (amino acid cluster 2) may be the amino acid sequence YNQSE (SEQ ID NO: 364) or a sequence in which one or two amino acids have been deleted or substituted with other naturally occurring amino acids (e.g., N is replaced with K, and / or L is replaced with A or I, and / or the second R is replaced with H, and / or G is replaced with T or S), aac3 (amino acid cluster 3) may be the amino acid sequence TAADT (SEQ ID NO: 370) or may be a sequence in which one or two amino acids have been deleted or substituted with other naturally occurring amino acids (e.g., N with Q, Q with N, and / or E with D); and iii) aac3 (amino acid cluster 3) may be the amino acid sequence TAADT (SEQ ID NO: 370) or may be a sequence in which one or two amino acids have been deleted or substituted with other naturally occurring amino acids (e.g., the first T with S, and / or the A with G, and / or the D with E, and / or the second T with S). iv) aac4 (amino acid cluster 4) may be the amino acid sequence AQITQ (SEQ ID NO: 371) or a sequence in which one or two amino acids have been deleted or substituted with other naturally occurring amino acids (e.g., A is replaced with G, and / or the first Q is replaced with N, and / or I is replaced with L, and / or the second Q is replaced with N or K); v) aac5 (amino acid cluster 5) may be the amino acid sequence VETRP (SEQ ID NO: 367) or a sequence in which one or two amino acids have been deleted or substituted with other naturally occurring amino acids (e.g., A is replaced with G, and / or the first Q is replaced with N, and / or I is replaced with L, and / or the second Q is replaced with N or K); and / or vi) aac6 (amino acid cluster 6) may be the amino acid sequence GDGTF (SEQ ID NO: 368) or a sequence in which one or two amino acids have been deleted or substituted with other naturally occurring amino acids (e.g., D is replaced with E and / or T is replaced with S and / or F is replaced with L, W or Y).

[0109] Non-classical HLA-E, HLA-F, and HLA-G MHC class I heavy chains In some cases, the TMP comprises a non-classical MHC class I heavy chain polypeptide. Non-classical HLA heavy chain polypeptides or portions thereof that can be incorporated into the TMP include, but are not limited to, HLA-E, HLA-F, and HLA-G alleles. Amino acid sequences for HLA-E, HLA-F, and HLA-G heavy chain polypeptides (and HLA-A, B, and C alleles) can be found on the World Wide Web at: hla.alleles.org / nomenclature / index.html, the European Bioinformatics Institute (www(dot)ebi(dot)ac(dot)uk), which is a division of the European Molecular Biology Laboratory (EMBL), and the National Center for Biotechnology Information (www(dot)ncbi(dot)nlm(dot)nih(dot)gov).

[0110] Specific, non-limiting examples of suitable HLA-E alleles include HLA-E * 0101(HLA-E * 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 * Among these, the isoform HLA-E * 0101 and HLA-E *01:03 is of particular interest because it is a common allele and differs by only one amino acid (Arg or Gly at position 107). For example, the amino acid sequences of suitable HLA-E heavy chain polypeptides are shown in Figures 22A-22D. * FIG. 22B shows the amino acid sequence of HLA-E 01:01 (wild type), and FIG. 22C shows the amino acid sequence of HLA-E 01:01 (wild type) containing the Y84C and A2346C substitutions. * FIG. 22C shows the amino acid sequence of HLA-E 01:01. * FIG. 22D shows the amino acid sequence of HLA-E 01:03 (wild type) with Y84C and A2346C substitutions. * The amino acid sequence of 01:03 is shown.

[0111] Specific, non-limiting examples of suitable HLA-F alleles include HLA-F * 0101(HLA-F * 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 HLA-G * 0101(HLA-G * 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 * 01:10, HLA-G * 01:10, HLA-G * 01:11, HLA-G *01:12, HLA-G * 01:14, HLA-G * 01:15, HLA-G * 01:16, HLA-G * 01:17, HLA-G * 01:18, HLA-G * 01:19, HLA-G * 01:20, and HLA-G * Among these, the isoform HLA-G * 0101(HLA-G * 01:01:01) and HLA-G * 01:04 (HLA-G * Of particular interest is the allele 01:04:01:01) which is a common allele. For example, the amino acid sequences of suitable HLA-G heavy chain polypeptides are shown in Figures 23A-23D. Figure 23A shows the amino acid sequence of HLA-G * FIG. 23B shows the amino acid sequence of HLA-G 01:01 (wild type) with Y84C and A2346C substitutions. * FIG. 23C shows the amino acid sequence of HLA-G * FIG. 23D shows the amino acid sequence of HLA-G 01:04 (wild type) with Y84C and A2346C substitutions. * The amino acid sequence of 01:04 is shown.

[0112] The consensus sequences for those HLA-E, HLA-F and HLA-G alleles lacking all or substantially all of the leader, transmembrane and cytoplasmic sequences are shown in Figure 10 and compared in Figure 11 with the consensus sequences for the HLA-A, HLA-B and HLA-C alleles described above.

[0113] FIG. 10 shows the consensus sequences for each of HLA-E, HLA-F, and HLA-G, with variable amino acid positions numbered consecutively as "X" residues, and amino acid positions 84, 139, and 236 double underlined.

[0114] FIG. 11 shows a comparison of the consensus amino acid sequences of HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, and HLA-G shown in FIGS. 7-11. Variable residues in each sequence are listed as "X" with the consecutive numbers removed. The positions of amino acids 84, 139, and 236 are shown along with the adjacent 5 amino acid cluster. The 5 amino acid cluster may be substituted with 1 to 5 amino acids independently selected from (i) any naturally occurring amino acid, or (ii) any naturally occurring amino acid except proline or glycine.

[0115] Any of the above HLA-E, HLA-F, and / or HLA-G alleles may have a substitution at one or more of positions 84, 139, and / or 236, as shown in Figure 11, with respect to the consensus sequence. In some cases, the substitution may be selected from a tyrosine to alanine substitution at position 84 (Y84A) or a cysteine ​​substitution (Y84C), or in the case of HLA-F, an R84A or R84C substitution, an alanine to cysteine ​​substitution at position 139 (A139C), or in the case of HLA-F, V139C, and an alanine to cysteine ​​substitution at position 236 (A236C). Additionally, 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 those consensus sequences shown in FIG. 11 may be used (e.g., the sequences may have 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 changes in the variable residues described therein).

[0116] Beta-2 microglobulin The β2-microglobulin (β2M) polypeptide of the TMP can be a human β2M polypeptide, a non-human primate β2M polypeptide, a murine β2M polypeptide, etc. In certain embodiments, 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 amino acid sequence depicted in Figure 1A (wild-type human β2M). In some cases, the β2M polypeptide present in the TMP comprises the amino acid sequence depicted in Figure 1A (wild-type human β2M).

[0117] In some cases, the MHC polypeptide present in the TMP has a single amino acid substitution with respect to a reference MHC polypeptide (wherein the reference MHC polypeptide can be a wild-type MHC polypeptide), where the single amino acid substitution is an amino acid substitution with a cysteine ​​(Cys) residue. Such a cysteine ​​residue can form a disulfide bond with a natural or non-natural cysteine ​​residue present in the MHC heavy chain of the TMP. As used herein, the expression "non-natural Cys residue" in an MHC class I polypeptide means that the polypeptide has a Cys residue at a position where no Cys is present in the corresponding wild-type polypeptide. This can be achieved by general protein engineering to substitute a cysteine ​​for an amino acid present in the wild-type sequence.

[0118] In some cases, the β2M polypeptide present in TMP comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence shown in Figure 1B, where amino acid 12 is Cys. That is, the β2M has a non-naturally occurring Cys at position 12 as a result of an R12C substitution. In some cases, the β2M polypeptide present in TMP comprises the amino acid sequence shown in Figure 1B.

[0119] Intra- and inter-chain disulfide bonds In some cases, the TMP has one or more intrachain disulfide bonds. Figure 13 shows a schematic diagram of such a disulfide-bonded TMP. Furthermore, when the TMP has an IgFc polypeptide, the TMP can be dimerized such that the IgFc polypeptides in the two TMPs are linked by one or more interchain disulfide bonds to form a homodimer. In some cases, the dimerized TMP has i) an interchain disulfide bond (e.g., one or two disulfide bonds between the IgFc polypeptides present in the two TMPs), and ii) one or more intrachain disulfide bonds.

[0120] When the peptide epitope of TMP is linked to the β2M polypeptide by a linker having a Cys, at least one of the one or more intrachain disulfide bonds links a Cys in the linker to a Cys in the MHC class I heavy chain in TMP. In some cases, TMP has a Cys in the β2M polypeptide and a Cys in the MHC class I heavy chain, and TMP has an intrachain disulfide bond linking a Cys in the β2M polypeptide to a Cys in the MHC class I heavy chain. In some cases, the TMP has a) a first intrachain disulfide bond linking i) a Cys present in the linker between the peptide epitope and the β2M polypeptide in the TMP to ii) a first Cys present in the MHC class I heavy chain in the TMP, and b) a second intrachain disulfide bond linking i) a Cys present in the β2M polypeptide in the TMP to ii) a second Cys present in the MHC class I heavy chain. Linkers containing Cys are described in more detail below.

[0121] Generally, potential positions for disulfide bonds in TMP are those where the residues of TMP are 5 angstroms or less apart. Such positions refer to positions where a Cys residue may be substituted for a residue present in the polypeptide (if not naturally occurring). For example, Cys residues in TMP may be linked via a disulfide bond between two Cys residues that are generally not more than about 5 angstroms apart from each other in TMP. In some cases, one or both of the Cys residues are not naturally occurring. Amino acids in the β2M polypeptide and MHC class I heavy chain of TMP that are not more than 5 angstroms apart from each other may form disulfide bonds in TMP when substituted with Cys. Similarly, disulfide bonds may be formed between a Cys residue in the linker and a natural or non-natural Cys residue in the MHC heavy chain, where the two Cys residues are not more than about 5 angstroms apart from each other. However, not all residue pairs separated by about 5 angstroms or less are suitable for disulfide bond formation or result in disulfide bonds that stabilize the resulting TMP or result in enhanced expression.

[0122] TMP is, for example, i) a peptide epitope (e.g., a peptide between 4 amino acids and about 25 amino acids in length that is bound by the TCR when complexed with an MHC polypeptide); ii) a first MHC polypeptide; and iii) a peptide linker between the peptide and the first MHC polypeptide, wherein the peptide linker has a Cys residue and the first MHC polypeptide is a β2M polypeptide comprising an amino acid substitution introducing a Cys residue; iv) a second MHC polypeptide, wherein the second MHC polypeptide is a class I heavy chain comprising a Y84C substitution and an A236C substitution (HLA-A *0201 (shown in FIG. 3A ) or at the corresponding positions in other class I heavy chain alleles), the TMP has a first intrachain disulfide bond between a Cys residue in the peptide linker (the peptide linker between the peptide and the first MHC polypeptide) and a Cys residue at amino acid position 84 of the MHC class I heavy chain polypeptide, and the TMP has a second intrachain disulfide bond between an introduced Cys residue in the β2M polypeptide and a Cys residue at amino acid position 236 of the MHC class I heavy chain polypeptide; v) at least one MOD; and, optionally, vi) An IgFc polypeptide. Such an intrachain disulfide bond is shown diagrammatically in Figure 13.

[0123] Y84C and A236C substitutions (HLA-A * Non-limiting examples of MHC class I heavy chains having the amino acid numbering of 0201 (as shown in FIG. 3A ) or based on the amino acid numbering at the corresponding positions in other class I heavy chain alleles) are shown in FIG. 3C , FIG. 4C , FIG. 5C , and FIG. 6C .

[0124] In some cases, the TMP comprises an HLA-A class I heavy chain polypeptide. In some cases, the HLA-A heavy chain polypeptide present in the TMP (e.g., a TMP having one or more intrachain disulfide bonds) is an HLA-A class I heavy chain polypeptide shown in FIG. * 0101, HLA-A * 0201, HLA-A * 0301, HLA-A * 1101, HLA-A * 2301, HLA-A * 2402, HLA-A * 2407, HLA-A * 3303, or HLA-A *3401, wherein the HLA-A heavy chain polypeptide has i) a Y84C substitution (wherein amino acid 236 is Ala) (e.g., as shown in Figures 3B, 4B, 5B, and 6B); ii) a Y84C substitution and a A236C substitution (e.g., as shown in Figures 3C, 4C, 5C, and 6C); or iii) a Y84A substitution and a A236C substitution (e.g., as shown in Figures 3D, 4D, 5D, and 6D).

[0125] In some cases, the HLA-A heavy chain polypeptide present in the TMP comprises an amino acid sequence having at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to one of the following sequences: (i)-(iv): (i) HLA-A as shown in FIG. 3C (SEQ ID NO: 373) * 0201 (Y84C, A236C) amino acid sequence, where amino acid 84 is Cys and amino acid 236 is Cys, (ii) HLA-A as shown in FIG. 4C (SEQ ID NO: 374) * 1101(Y84C, A236C) amino acid sequence (wherein amino acid 84 is Cys and amino acid 236 is Cys), (iii) HLA-A shown in Figure 5C (SEQ ID NO: 375) * 2402 (Y84C, A236C) amino acid sequence (wherein amino acid 84 is Cys and amino acid 236 is Cys), (iv) HLA-A shown in Figure 6C (SEQ ID NO: 376) * 3303 (Y84C, A236C) amino acid sequence (wherein amino acid 84 is Cys and amino acid 236 is Cys).

[0126] In some cases, the HLA-A heavy chain polypeptide present in the TMP comprises an amino acid sequence having at least 95%, at least 98%, or at least 99% amino acid sequence identity to any one of the following sequences: (i)-(v): (i) HLA-A as shown in FIG. 7A (SEQ ID NO: 377)* 0101 amino acid sequence, where amino acid 84 is Cys and amino acid 236 is Cys, (ii) HLA-A as shown in FIG. 7A (SEQ ID NO: 378) * 0301 amino acid sequence, where amino acid 84 is Cys and amino acid 236 is Cys, (iii) HLA-A as shown in FIG. 7A (SEQ ID NO: 379) * 2301 amino acid sequence (wherein amino acid 84 is Cys and amino acid 236 is Cys), (iv) HLA-A as shown in FIG. 7A (SEQ ID NO: 380) * 2407 amino acid sequence (wherein amino acid 84 is Cys and amino acid 236 is Cys), (v) HLA-A as shown in FIG. 7A (SEQ ID NO: 381) * 3401 amino acid sequence (wherein amino acid 84 is Cys and amino acid 236 is Cys).

[0127] In some cases, the TMP comprises an HLA-E class I heavy chain polypeptide. In some cases, the HLA-E heavy chain polypeptide present in a TMP (e.g., a TMP having one or more intrachain disulfide bonds) comprises an amino acid sequence having at least 95%, at least 98%, or at least 99%, or 100% amino acid sequence identity to any one of the amino acid sequences shown in Figures 22A-22D.

[0128] In some cases, the HLA-E heavy chain polypeptide present in the TMP comprises an amino acid sequence having at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to any one of the following sequences: (i) and (ii): (i) HLA-E as shown in FIG. 22B (SEQ ID NO: 382) * 01:01 (Y84C, A236C) amino acid sequence (wherein amino acid 84 is Cys and amino acid 236 is Cys), (ii) HLA-E as shown in FIG. 22D (SEQ ID NO: 383) *01:03 (Y84C, A236C) amino acid sequence (wherein amino acid 84 is Cys and amino acid 236 is Cys).

[0129] In some cases, the TMP comprises an HLA-G class I heavy chain polypeptide. In some cases, the HLA-G heavy chain polypeptide present in the TMP (e.g., a TMP having one or more intrachain disulfide bonds) comprises an amino acid sequence having at least 95%, at least 98%, or at least 99%, or 100% amino acid sequence identity to any one of the amino acid sequences shown in Figures 23A-23D.

[0130] In some cases, the HLA-G heavy chain polypeptide present in the TMP comprises an amino acid sequence having at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to any one of the following sequences: (i) and (ii): (i) HLA-G as shown in FIG. 23B (SEQ ID NO: 384) * 01:01 (Y84C, A236C) amino acid sequence (wherein amino acid 84 is Cys and amino acid 236 is Cys), (ii) HLA-G as shown in FIG. 23D (SEQ ID NO: 385) * 01:04 (Y84C, A236C) amino acid sequence (wherein amino acid 84 is Cys and amino acid 236 is Cys).

[0131] HLA / peptide binding assay Whether a peptide binds to class I HLA (including HLA heavy chain and β2M polypeptide) and can effectively present an epitope to a TCR when bound to an HLA complex can be determined using any of a number of well-known methods. Assays include binding assays and T cell activation assays, including cell-based binding assays, biochemical binding assays, T cell activation assays, ELISPOT assays, cytotoxicity assays, and detection of antigen-specific T cells using peptide-HLA tetramers. Such assays are described in the published scientific literature as well as in published PCT application WO2020132138A1. The disclosure of this publication regarding specific binding assays is incorporated herein by reference, particularly including paragraphs

[0217] to

[0225] .

[0132] In another embodiment, multimers (e.g., tetramers) of peptide-HLA complexes are produced with fluorescent or heavy metal tags. The multimers can be used to identify and quantify specific T cells by flow cytometry (FACS) or mass cytometry (CyTOF). Detection of epitope-specific T cells provides direct evidence that peptide-bound HLA molecules can bind to specific TCRs on a subset of antigen-specific T cells. See, e.g., Klenerman et al. (2002) Nature Reviews Immunol. 2:263.

[0133] Immunomodulatory Polypeptides (MODs) In some cases, the MOD present in the TMP is a wild-type (wt) MOD. As noted above, in other cases, the MOD present in the TMP is a mutant of wtMOD that has a reduced affinity for the co-MOD compared to the affinity of the corresponding wild-type MOD for the co-MOD. A suitable MOD that exhibits reduced affinity for the co-MOD may differ from the wild-type MOD by 1 to 20 amino acids. For example, in some cases, the mutant MOD present in the TMP differs in amino acid sequence from the corresponding wild-type MOD by 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids. As another specific example, in some cases, the mutant MOD present in the TMP differs in amino acid sequence from the corresponding wild-type MOD by 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids.

[0134] As discussed above, the MOD may include a mutant of the wtMOD, which has reduced binding to the co-MOD (e.g., reduced binding to one or more chains or domains of the co-MOD). For example, the affinity with which a mutant MOD present in a TMP binds to a co-MOD is at least 10% lower, at least 15% lower, at least 20% lower, at least 25% lower, at least 30% lower, at least 35% lower, at least 40% lower, at least 45% lower, at least 50% lower, at least 55% lower, at least 60% lower, at least 65% lower, at least 70% lower, at least 75% lower, at least 80% lower, at least 85% lower, at least 90% lower, at least 95% lower, or greater than 95% lower than the affinity of the corresponding wild-type MOD for the co-MOD.

[0135] Specific pairs of a MOD and its co-MOD include, but are not limited to, those shown in Table 1 below.

[0136] [Table 1]

[0137] As shown diagrammatically in Figure 12, one or more MODs can be present in any of a variety of locations in the TMP. Although Figure 12 shows the location of two copies of the mutant IL-2 polypeptide, the MOD can be any of the number and variety of MODs as described herein. As shown in Figure 12, the MOD can be 1), 2), or 3): 1) C-terminal to the MHC class I heavy chain and N-terminal to the IgFc polypeptide (i.e., between the MHC class I heavy chain polypeptide and the IgFc polypeptide, which is designated "Configuration 2" in Figure 12), 2) C-terminal to the IgFc polypeptide (which is designated "Configuration 3" in Figure 12), or 3) N-terminal to the peptide epitope (which is designated "Configuration 4" in Figure 12).

[0138] Wild-type immune modulating polypeptides and variants, including variants with reduced affinity, of PD-L1, CD80, CD86, 4-1BBL, and IL-2 have been described in the published literature (e.g., published WO2020132138A1 and WO2019 / 051091). The disclosures of these publications relating to MODs and specific mutant MODs of PD-L1, CD80, CD86, 4-1BBL, and IL-2 (including, in particular, paragraphs

[0260] to

[0455] of WO2020132138A1 and paragraphs

[0157] to

[0352] of WO2019 / 051091) are hereby incorporated by reference as if expressly set forth herein.

[0139] Of particular interest is MOD, a mutant of the cytokine IL-2. Wild-type IL-2 binds to the IL-2 receptor (IL-2R) on the surface of T cells. Wild-type IL-2 has a strong affinity for IL-2R and binds to activate most or virtually all CD8+ T cells. For this reason, synthetic wild-type IL-2, such as the drug aldesleukin (trade name Proleukin®), is known to have severe side effects when administered to humans for cancer treatment. This is because IL-2 indiscriminately activates both target and non-target T cells.

[0140] IL-2 receptors, in some cases, are heterotrimeric polypeptides having an alpha chain (IL-2Rα (also referred to as CD25)), a beta chain (IL-2Rβ (also referred to as CD122)), and a gamma chain (IL-2Rγ (also referred to as CD132)). The amino acid sequences of human IL-2, human IL-2Rα, IL2Rβ, and IL-2Rγ are known. See, for example, the above-mentioned published PCT applications WO2020132138A1 and WO2019 / 051091. For example, a wild-type IL-2 polypeptide can have the amino acid sequence shown in FIG. 17A. The amino acid sequences of human IL-2Rα, human IL-2Rβ, and human IL-2Rγ are shown in FIGS. 17B, 17C, and 17D, respectively.

[0141] In some cases, the IL-2 variant MODs of the disclosure exhibit reduced binding to IL-2Rα, thereby minimizing or substantially reducing activation of Tregs by the IL-2 variants. Alternatively, or additionally, in some cases, the IL-2 variant MODs of the disclosure exhibit reduced binding to IL-2Rβ and / or IL-2Rγ, such that the IL-2 variant MOD exhibits an overall reduced affinity for IL-2R. In some cases, the IL-2 variant MODs of the disclosure exhibit both properties, i.e., reduced or substantially no binding to IL-2Rα, and also reduced binding to IL-2Rβ and / or IL-2Rγ, such that the IL-2 variant polypeptide exhibits an overall reduced affinity for IL-2R. For example, IL-2 variants having substitutions at H16 and F42 exhibit reduced binding to IL-2Rα and IL-2Rβ. See Quayle et al., Clin Cancer Res; 26(8) April 15, 2020. This document shows that the binding affinity of IL-2 polypeptides containing H16A and F42A substitutions to human IL-2Rα and IL-2Rβ was reduced by 110-fold and 3-fold, respectively, compared to wild-type IL2 binding, which is primarily due to faster dissociation rates for each of these interactions. TMPs containing such mutants (including mutants that exhibit reduced binding to IL-2Rα and IL-2Rβ) demonstrated the ability to preferentially bind and activate IL-2 receptors on T cells that contain a target TCR specific for a peptide epitope on the TMP. Thus, TMPs containing such mutants (including mutants that exhibit reduced binding to IL-2Rα and IL-2Rβ) are less likely to deliver IL-2 to non-target T cells (i.e., T cells that do not contain a TCR that specifically binds to a peptide epitope on the TMP). That is, the binding of the IL-2 mutant MOD to its costimulatory polypeptide on T cells is substantially driven by the binding of the MHC-epitope moiety rather than the binding of IL-2.

[0142] Accordingly, suitable IL-2 variant MODs include polypeptides comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the wild-type IL-2 amino acid sequence shown in Figure 17 A, and which differ by one or more amino acids from the wild-type IL-2 amino acid sequence shown in Figure 17 A. In some cases, such variant IL-2 polypeptides of the disclosure exhibit a lower binding affinity for IL-2R compared to the binding affinity of an IL-2 polypeptide having the wild-type IL-2 amino acid sequence shown in Figure 17A. For example, in some cases, the binding affinity of a mutant IL-2 polypeptide to IL-2R (e.g., an IL-2R comprising a polypeptide having an amino acid sequence as depicted in Figures 17B-17D) is at least 10% lower, at least 15% lower, at least 20% lower, at least 25% lower, at least 30% lower, at least 35% lower, at least 40% lower, at least 45% lower, at least 50% lower, at least 55% lower, at least 60% lower, at least 65% lower, at least 70% lower, at least 75% lower, at least 80% lower, at least 85% lower, at least 90% lower, at least 95% lower, or more than 95% lower) than the binding affinity of an IL-2 polypeptide having the wild-type IL-2 amino acid sequence depicted in Figure 17A to IL-2R, when measured under the same conditions.

[0143] In certain cases, suitable variant IL-2 polypeptides 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 amino acid sequence below. TIFF2024518680000003.tif17154 (SEQ ID NO: 386). That is, the mutant IL-2 polypeptide has the amino acid sequence of wild type IL-2 but with substitutions of H16A and F42A (shown in bold). Alternatively, the above sequence can be employed but with substitutions other than Ala at H16 and / or F42, e.g., H16T can be employed in place of H16A. In some cases, the mutant IL-2 polypeptide present in the TMP has the amino acid sequence: APTSSSTKKT QLQLEALLLD LQMILNGINN YKNPKLTRML TAKFYMPKKA TELKHLQCLEEELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNRWITFCQSIIS TLT (SEQ ID NO: 387). In some cases, the mutant IL-2 polypeptide present in the TMP has the amino acid sequence: APTSSSTKKT QLQLETLLLD LQMILNGINN YKNPKLTRML TAKFYMPKKA TELKHLQCLEEELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNRWITFCQSIIS TLT (SEQ ID NO: 388). In some cases, the TMP contains two copies of such a mutant IL-2 polypeptide.

[0144] In some cases, a MOD present in the TMP is a PD-L1 polypeptide. In some cases, the PD-L1 polypeptide of the TMP comprises an amino acid sequence that has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following PD-L1 extracellular domain amino acid sequence: FT VTVPKDLYVV EYGSNMTIEC KFPVEKQLDL AALIVYWEME DKNIIQFVHG EEDLKVQHSS YRQRARLLKD QLSLGNAALQ ITDVKLQDAG VYRCMISYGG ADYKRITVKV NAPYNKINQR ILVVDPVTSE HELTCQAEGY PKAEVIWTSS DHQVLSGKTT TTNSKREEKL FNVTSTLRIN TTTNEIFYCT FRRLDPEENH TAELVIPGNI LNVSIKI (SEQ ID NO: 389).

[0145] In some cases, the MOD present in the TMP is a 4-1BBL polypeptide. In some cases, the 4-1BBL polypeptide of the TMP comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following 4-1BBL amino acid sequence: DPAGLLDLRQG MFAQLVAQNV LLIDGPLSWY SDPGLAGVSL TGGLSYKEDT KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPA (SEQ ID NO: 390).

[0146] In some cases, the MOD present in the TMP is an ICOS-L polypeptide. In some cases, the ICOS-L polypeptide of the TMP comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following ICOS-L amino acid sequence: QEKEVRAMVG SDVELSCACP EGSRFDLNDV YVYWQTSESK TVVTYHIPQN SSLENVDSRY RNRALMSPAG MLRGDFSLRL FNVTPQDEQK FHCLVLSQSL GFQEVLSVEV TLHVAANFSV PVVSAPHSPS QDELTFTCTS INGYPRPNVY WINKTDNSLL DQALQNDTVF LNMRGLYDVV SVLRIARTPS VNIGCCIENV LLQQNLTVGS QTGNDIGERD KITENPVSTG EKNAATWSIL (SEQ ID NO: 391).

[0147] In some cases, the T cell modulating polypeptide of the multimeric polypeptide is an OX40L polypeptide. In some cases, the OX40L polypeptide of the TMP comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following OX40L amino acid sequence: L QVSHRYPRIQ SIKVQFTEYK KEKGFILTSQ KEDEIMKVQN NSVIINCDGF YLISLKGYFS QEVNISLHYQ KDEEPLFQLK KVRSVNSLMV ASLTYKDKVY LNVTTDNTSL DDFHVNGGEL ILIHQNPGEF CVL (SEQ ID NO: 392).

[0148] In some cases, the T cell regulatory polypeptide of the multimeric polypeptide is a PD-L2 polypeptide. In some cases, the PD-L2 polypeptide of the multimeric 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 amino acids 20 to 273 of the PD-L2 amino acid sequence: L FTVTVPKELY IIEHGSNVTL ECNFDTGSHV NLGAITASLQ KVENDTSPHR ERATLLEEQL PLGKASFHIP QVQVRDEGQY QCIIIYGVAW DYKYLTLKVK ASYRKINTHI LKVPETDEVE LTCQATGYPL AEVSWPNVSV PANTSHSRTP EGLYQVTSVL RLKPPPGRNF SCVFWNTHVR ELTLASIDLQ SQMEPRTHPT (SEQ ID NO: 393).

[0149] In some cases, the MOD present in the TMP is a CD80 polypeptide. In some cases, the CD80 polypeptide of the TMP comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following CD80 amino acid sequence: VIHVTK EVKEVATLSC GHNVSVEELA QTRIYWQKEK KMVLTMMSGD MNIWPEYKNR TIFDITNNLS IVILALRPSD EGTYECVVLK YEKDAFKREH LAEVTLSVKA DFPTPSISDF EIPTSNIRRI ICSTSGGFPE PHLSWLENGE ELNAINTTVS QDPETELYAV SSKLDFNMTT NHSFMCLIKY GHLRVNQTFN WNTTKQEHFP DN (SEQ ID NO: 394).

[0150] In some cases, the MOD present in the TMP is a CD86 polypeptide. In some cases, the CD86 polypeptide of the TMP comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the CD86 amino acid sequence: APLKIQAYFNETADLPCQFANSQNQSLSELVVFWQDQENLVLNEVYLGKEKFDSVHSKYMNRTSFDSDSWTLRLHNLQIKDKGLYQCIIHHKKPTGMIRIHQMNSELSVLANFSQPEIVPISNITENVYINLTCSSIHGYPEPKKMSVLLRTKNSTIEYDGIMQKSQDNVTELYDVSISLSVSFPDVTSNMTIFCILETDKTRLLSSPFSIELEDPQPPPDHIP (SEQ ID NO: 395).

[0151] In some cases, the MOD present in the TMP is a FasL polypeptide, e.g., an extracellular domain of a FasL polypeptide. In some cases, the FasL polypeptide of the TMP comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following FasL extracellular domain amino acid sequence: QLFHLQKE LAELRESTSQ MHTASSLEKQ IGHPSPPPEK KELRKVAHLT GKSNSRSMPL EWEDTYGIVL LSGVKYKKGG LVINETGLYF VYSKVYFRGQ SCNNLPLSHK VYMRNSKYPQ DLVMMEGKMM SYCTTGQMWA RSSYLGAVFN LTSADHLYVN VSELSLVNFE ESQTFFGLYK L (SEQ ID NO: 396).

[0152] Scaffold Polypeptides The TMP may comprise an Fc polypeptide, or may comprise another suitable scaffold polypeptide.

[0153] 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, for example, Hassouneh et al. (2012) Methods Enzymol. 502:215, e.g., polypeptides having pentapeptide repeat units of Val-Pro-Gly-X-Gly (SEQ ID NO: 446), where X is any amino acid except proline), albumin-binding polypeptides, silk-like polypeptides (see, for example, Valluzzi et al. (2002) Philos Trans R Soc Lond B Biol Sci. 357:165), silk-elastin-like polypeptides (SELPs, see, for example, Megeed et al. (2002) Adv Drug Deliv Rev. 54:1075), and the like. Suitable XTEN polypeptides include, for example, those disclosed in WO2009 / 023270, WO2010 / 091122, WO2007 / 103515, US2010 / 0189682, and US2009 / 0092582. See also, Schellenberger et al. (2009) Nat Biotechnol. 27:1186. Suitable albumin polypeptides include, for example, human serum albumin.

[0154] In some cases, a suitable scaffold polypeptide is a half-life extending polypeptide. Thus, in some cases, a suitable scaffold polypeptide increases the in vivo half-life (e.g., serum half-life) of TMP compared to a control TMP without the scaffold polypeptide. For example, in some cases, a scaffold polypeptide increases the in vivo half-life (e.g., serum half-life) of TMP by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 50%, at least about 2-fold, at least about 2.5-fold, at least about 5-fold, at least about 10-fold, at least about 25-fold, at least about 50-fold, at least about 100-fold, or more than 100-fold compared to a control TMP without the scaffold polypeptide. As a specific example, in some cases, the Fc polypeptide increases the in vivo half-life (e.g., serum half-life) of the TMP by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 50%, at least about 2-fold, at least about 2.5-fold, at least about 5-fold, at least about 10-fold, at least about 25-fold, at least about 50-fold, at least about 100-fold, or more than 100-fold, relative to a control TMP lacking the Fc polypeptide.

[0155] Fc Polypeptides In some cases, the TMP comprises an IgFc polypeptide. The IgFc polypeptide is also referred to herein as an "Fc polypeptide." The IgFc polypeptide of the TMP can be human IgG1Fc, human IgG2Fc, human IgG3Fc, human IgG4Fc, etc., or a variant of a wild-type IgFc polypeptide. Variants include naturally occurring variants, non-naturally occurring variants, and combinations thereof.

[0156] In some cases, the Fc polypeptide present in the TMP comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to an Fc amino acid sequence shown in any one of Figures 2A-2M.

[0157] In some cases, the Fc polypeptide present in the TMP is an IgG1 Fc polypeptide, or a variant of an IgG1 Fc polypeptide. For example, in some cases, the Fc polypeptide present in the TMP comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to the human IgG1 Fc polypeptide shown in Figure 2A. As another specific example, in some cases, the Fc polypeptide present in the TMP comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to the Fc polypeptide shown in Figure 2B, where the IgFc polypeptide has an Ala at position 14 and an Ala at position 15. In any of the above embodiments, the IgFc polypeptide may have an N77 substitution. That is, the IgFc polypeptide can have an amino acid other than Asn at position 77. Here, in some cases, the IgFc polypeptide has an Ala at position 77. In some cases, the Fc polypeptide present in the TMP has the amino acid sequence shown in Figure 2A. In some cases, the Fc polypeptide present in the TMP has the amino acid sequence shown in Figure 2B.

[0158] In some cases, the Fc polypeptide present in the TMP is an IgG1 Fc polypeptide or a variant of an IgG1 Fc polypeptide. Variants include naturally occurring variants, non-naturally occurring variants, and combinations thereof. For example, in some cases, the Fc polypeptide present in the TMP comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to the human IgG1 Fc polypeptide shown in Figure 2C, where the IgFc polypeptide has a Glu at position 136 and a Met at position 138. As another specific example, in some cases, the Fc polypeptide present in the TMP comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to the human IgG1 Fc polypeptide shown in FIG. 2D, where the IgFc polypeptide has Ala at positions 14 and 15, and the Fc polypeptide has Glu at position 136 and Met at position 138. In any of the above embodiments, the IgFc polypeptide can have an N77 substitution. That is, the IgFc polypeptide can have an amino acid other than Asn at position 77, and in some cases, the IgFc polypeptide has Ala at position 77. In some cases, the Fc polypeptide present in the TMP has the amino acid sequence shown in FIG. 2C. In some cases, the Fc polypeptide present in the TMP has the amino acid sequence shown in FIG. 2D.

[0159] In some cases, the Fc polypeptide present in the TMP has the amino acid sequence shown in Figure 2E (human IgG1Fc comprising an L234F substitution, an L235E substitution, and a P331S substitution, where L234 corresponds to amino acid 14 of the amino acid sequence shown in Figure 3I, L235 corresponds to amino acid 15 of the amino acid sequence shown in Figure 2E, and P331 corresponds to amino acid 111 of the amino acid sequence shown in Figure 2E). In some cases, the Fc polypeptide present in the TMP has the amino acid sequence shown in Figure 2F, which has an N279A substitution (N77A of the amino acid sequence shown in Figure 2F).

[0160] In some cases, the Fc polypeptide comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to the human IgG2 Fc polypeptide shown in Figure 2G. For example, the Fc polypeptide comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to amino acids 99-325 of the human IgG2 Fc polypeptide shown in Figure 2G (e.g., when the IgFc polypeptide is about 227 amino acids in length). In some cases, the Fc polypeptide comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to the human IgG3Fc polypeptide shown in Figure 2H. For example, the Fc polypeptide comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to amino acids 19-246 of the human IgG3Fc polypeptide shown in Figure 2H (e.g., when the IgFc polypeptide is about 228 amino acids in length). In some cases, the Fc polypeptide comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to the human IgM Fc polypeptide shown in Figure 2J. For example, the Fc polypeptide comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to amino acids 1-276 of the human IgM Fc polypeptide shown in Figure 2J.In some cases, the Fc polypeptide comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to the human IgA Fc polypeptide shown in Figure 2K. For example, the Fc polypeptide comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to amino acids 1-234 of the human IgA Fc polypeptide shown in Figure 2K.

[0161] In some cases, the Fc polypeptide comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to the human IgG4 Fc polypeptide shown in Figure 2M. In some cases, the Fc polypeptide comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to amino acids 100-327 of the human IgG4 Fc polypeptide shown in Figure 2M (e.g., when the IgFc polypeptide is about 228 amino acids in length).

[0162] In some cases, the IgG4 Fc polypeptide has the amino acid sequence: PPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 397).

[0163] In some cases, the IgFc employed in the TMP has one or more amino acid substitutions in the wild-type sequence such that the IgFc does not substantially induce cell lysis. For example, in some cases, the Fc polypeptide present in the TMP has the amino acid sequence shown in Figure 2A (human IgG1 Fc), except that L234 (L14 in the amino acid sequence shown in Figure 2A) is substituted with an amino acid other than leucine, or L235 (L15 in the amino acid sequence shown in Figure 2A) is substituted with an amino acid other than leucine. Specific examples include the L234A (L14A) substitution and the L235A (L15A) substitution.

[0164] Linker The TMP may include one or more peptide linkers, i.e., the TMP may include a linker stretch of two or more consecutive amino acids, where the one or more linkers are present between any one or more of: i) the MHC class I heavy chain polypeptide and the IgFc polypeptide (wherein such a linker is referred to herein as "L1"), ii) the MOD and the MHC class I polypeptide (wherein such a linker is referred to herein as "L2"), iii) the first MOD and the second MOD (wherein such a linker is referred to herein as "L3"), iv) the peptide and the MHC class I polypeptide, and v) the peptide epitope and the β2M polypeptide.

[0165] As used herein, the phrase "an optional peptide linker between any two components of the TMP" refers to a peptide linker between any two adjacent polypeptides in the TMP. For example, as used herein, the phrase "an optional peptide linker between any two components of the TMP" refers to a peptide linker between any one or more of: i) a peptide epitope and a β2M polypeptide; ii) a β2M polypeptide and an MHC class I heavy chain polypeptide; iii) an MHC class I heavy chain polypeptide and an IgFc polypeptide; iv) an MHC class I heavy chain polypeptide and a MOD; v) an IgFc polypeptide and a MOD; and vi) a first MOD and a second MOD. As described below, the linker may be a flexible peptide linker (including a short flexible peptide linker) or a rigid peptide linker.

[0166] Suitable linkers (also referred to as "spacers") can be readily selected and can be of any of a number of suitable lengths, for example, 1 to 25 amino acids, 3 to 20 amino acids, 2 to 15 amino acids, 3 to 12 amino acids, 4 to 10 amino acids, 5 to 9 amino acids, 6 to 8 amino acids, or 7 to 8 amino acids. Suitable linkers can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids in length. In some cases, the linker is 25 to 50 amino acids in length, for example, 25 to 30, 30 to 35, 35 to 40, 40 to 45, or 45 to 50 amino acids in length.

[0167] Flexible Peptide Linker Exemplary flexible peptide linkers include glycine polymers (G)n, glycine-serine polymers (including, for example, (GS)n, (GSGGS)n (SEQ ID NO: 398), (GGGS)n (SEQ ID NO: 399), and (GGGS)n (SEQ ID NO: 400), where n is an integer of at least 1 and can be an integer between 1 and 10), glycine-alanine polymers, alanine-serine polymers, and other flexible peptide linkers known in the art. Glycine and glycine-serine polymers can be used. Both Gly and Ser are relatively amorphous and therefore can function as neutral tethers between components. Glycine polymers can be used. Glycine has access to significantly more phi-phi space than alanine and is much less restricted than residues with long side chains (see Scheraga, Rev. Computational Chem. 11173-142 (1992)). Exemplary flexible peptide linkers can have amino acid sequences such as, but not limited to, GGSG (SEQ ID NO: 401), GGSGG (SEQ ID NO: 402), GSGSG (SEQ ID NO: 403), GSGGG (SEQ ID NO: 404), GGGSG (SEQ ID NO: 405), GSSSG (SEQ ID NO: 406).

[0168] Specific flexible peptide linkers include, for example, (GGGGS)n (SEQ ID NO:572) (also referred to as a "G4S" linker), where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some cases, the linker has the amino acid sequence (GGGGS)n (SEQ ID NO:572), where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some cases, the linker has the amino acid sequence (GGGGS)n (SEQ ID NO:407), where n is 2. In some cases, the linker has the amino acid sequence (GGGGS)n (SEQ ID NO:408), where n is 3. In some cases, the linker has the amino acid sequence (GGGGS)n (SEQ ID NO:409), where n is 4. In some cases, the linker has the amino acid sequence (GGGGS)n (SEQ ID NO:410), where n is 7. In some cases, the linker has the amino acid sequence AAAGG (SEQ ID NO:411). Also suitable is a linker having the amino acid sequence AAAGG (SEQ ID NO: 411). In the TMP of the disclosure, the β2M polypeptide can be linked to the MHC heavy chain polypeptide by a (GGGGS)n (SEQ ID NO: 572) linker, where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, e.g., n=3, n=4, or n=7.

[0169] The term "short flexible peptide linker" as used in this disclosure refers to a flexible peptide linker consisting of less than 15 amino acids, i.e., 2-14 amino acids. For example, a short flexible peptide linker can consist of 2-4, 2-5, or 3-6 amino acids (e.g., a GGS linker), or 4-8, 5-10, or 10-14 amino acids. Included within this range are flexible peptide linkers consisting of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 amino acids.

[0170] Rigid Peptide Linkers In some cases, the peptide linker is a rigid peptide linker. The term "rigid peptide linker" as used herein refers to a linker of two or more consecutive amino acids that effectively separates protein domains by maintaining a substantially constant distance / spatial separation between the domains, thereby reducing or substantially eliminating unfavorable interactions between the domains. Rigid peptide linkers are known in the art and generally adopt a relatively well-defined conformation in solution. Rigid peptide linkers include those that have a specific secondary and / or tertiary structure in solution, and typically have a length sufficient to impart a secondary or tertiary structure to the linker. Rigid peptide linkers include proline-rich peptide linkers and peptide linkers that have an inflexible helical structure, such as an alpha helix structure. Rigid peptide linkers are described, for example, in Chen et al. (2013) Adv. Drug Deliv. Rev. 65:1357 and Klein et al. (2014) Protein Engineering, Design & Selection 27:325.

[0171] Specific examples of rigid peptide linkers include (EAAAK)n (SEQ ID NO: 573), A(EAAAK)n (SEQ ID NO: 574), A(EAAAK)nA (SEQ ID NO: 575), A(EAAAK)nALEA(EAAAK)nA (SEQ ID NO: 576), (Lys-Pro)n (SEQ ID NO: 577), (Glu-Pro)n (SEQ ID NO: 578), (Thr-Pro-Arg)n (SEQ ID NO: 579), and (Ala-Pro)n (SEQ ID NO: 580), where n is an integer from 1 to 20 (e.g., n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20). Non-limiting examples of suitable rigid peptide linkers having EAAAK (SEQ ID NO: 412) include EAAAK (SEQ ID NO: 412), (EAAAK)2 (SEQ ID NO: 416), (EAAAK)3 (SEQ ID NO: 417), A(EAAAK)4ALEA(EAAAK)4A (SEQ ID NO: 418), and AEAAAKEAAAKA (SEQ ID NO: 419). Non-limiting examples of suitable rigid peptide linkers having (AP)n include PAPAP (SEQ ID NO: 420) (also referred to herein as "(AP)2"), APAPAPAP (SEQ ID NO: 421) (also referred to herein as "(AP)4"), APAPAPAPAPAPAP (SEQ ID NO: 422) (also referred to herein as "(AP)6"), APAPAPAPAPAPAPAP (SEQ ID NO: 423) (also referred to herein as "(AP)8"), and APAPAPAPAPAPAPAPAPAPAP (SEQ ID NO: 424) (also referred to herein as "(AP)10"). Non-limiting illustrative examples of suitable rigid peptide linkers having (KP)n include KPKP (SEQ ID NO:425) (also referred to herein as "(KP)2"), KPKPKPKP (SEQ ID NO:426) (also referred to herein as "(KP)4"), KPKPKPKPKPKP (SEQ ID NO:427) (also referred to herein as "(KP)6"), KPKPKPKPKPKPKPKP (SEQ ID NO:428) (also referred to herein as "(KP)8"), and KPKPKPKPKPKPKPKPKPKP (SEQ ID NO:429) (also referred to herein as "(KP)10").Non-limiting illustrative examples of suitable rigid peptide linkers having (EP)n include EPEP (SEQ ID NO:430) (also referred to herein as "(EP)2"), EPEPEPEPEP (SEQ ID NO:431) (also referred to herein as "(EP)4"), EPEPEPEPEPEPEP (SEQ ID NO:432) (also referred to herein as "(EP)6"), EPEPEPEPEPEPEPEP (SEQ ID NO:433) (also referred to herein as "(EP)8"), and EPEPEPEPEPEPEPEPEPEPEP (SEQ ID NO:434) (also referred to herein as "(EP)10").

[0172] Generally, when a TMP comprises a rigid peptide linker and / or a short flexible peptide linker, the TMP may comprise a rigid peptide linker and / or a short flexible peptide linker between any two components of the TMP, but typically one or more rigid peptide linkers and / or short flexible peptide linkers are used, as follows: (i) In a TMP having one or more MODs of configuration 2, it is used between one or more of: i) the MHC class I heavy chain polypeptide and the MOD, ii) the MOD and the IgFc polypeptide, and iii) if multiple MODs are present in tandem, between the first MOD and the second MOD. (ii) In a TMP having one or more MODs of configuration 3, it is used between one or more of: i) the IgFc polypeptide and the MOD, and ii) if multiple MODs are present in tandem, between the first MOD and the second MOD. (iii) in a TMP having one or more MODs of configuration 4, used between one or more of: i) the epitope and the MOD, ii) the MHC class I heavy chain polypeptide and the IgFc polypeptide, and iii) between the first MOD and the second MOD when multiple MODs are present in tandem.

[0173] Thus, the present disclosure provides methods of increasing the thermal stability of a TMP comprising one or more MODs of Configuration 2, Configuration 3, or Configuration 4.

[0174] In TMPs having one or more MODs of configuration 3, it has been found that the use of a rigid or short flexible peptide linker, instead of a flexible peptide linker, between the IgFc polypeptide and the MOD results in increased thermal stability of the resulting TMP compared to a similar TMP having a long flexible peptide linker, such as a (G4S)3 (i.e., (GGGGS)3) linker (SEQ ID NO: 408). Without wishing to be bound by any particular theory, it is believed that the rigid or short flexible peptide linker reduces or prevents interactions between the MOD and other polypeptides in the TMP that can occur with flexible peptide linkers having 15 or more amino acids, thereby enhancing thermal stability as measured using an accelerated stability test as described below.

[0175] In some cases, the use of a rigid peptide linker or a short flexible peptide linker, when present between an IgFc polypeptide and a MOD of a TMP having one or more MODs of configuration 3, increases the thermal stability as measured by the 37° C. accelerated thermal stability test described below by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, or at least about 10-fold, as compared to the thermal stability of a control TMP containing a flexible peptide linker that is a (GGGGS)3 linker (SEQ ID NO:408) instead of a rigid peptide linker or a short flexible peptide linker.

[0176] In some cases, the use of a rigid peptide linker or a short flexible peptide linker, when present between an IgFc polypeptide and a MOD of a TMP having one or more MODs of configuration 3, increases the thermal stability as measured by the 42°C accelerated thermal stability test described below by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, or at least about 10-fold, as compared to the thermal stability of a control TMP containing a flexible peptide linker that is a (GGGGS)3 linker (SEQ ID NO:408) instead of a rigid peptide linker or a short flexible peptide linker.

[0177] Accelerated thermal stability testing can be performed as follows: Thermal stability of dimerized TMP can be evaluated by accelerated stability testing performed at 4°C, 37°C, and 42°C. A composition of dimerized TMP is maintained in solution (phosphate buffered saline (PBS), pH 7.4, containing 500 mM NaCl) at the above temperatures at a concentration of 10 mg dimerized TMP / mL solution for 14 days. After 1 day, 7 days, and 14 days, the percentage of monomer present in the solution is measured by size exclusion chromatography. The PBS solution is as follows: 10.14 mM sodium phosphate (dibasic), 1.76 mM potassium phosphate (monobasic), 2.7 mM KCl, and 0.5 M NaCl, pH 7.4.

[0178] Cysteine-Containing Linkers As described above, in some cases, the linker peptide has a cysteine ​​residue, which can form an intrachain disulfide bond with a cysteine ​​residue present elsewhere in the TMP polypeptide chain. For example, as described above, in some cases, the TMP, or dimerized TMP, such as a homodimer, contains a linker between the peptide epitope and a β2M polypeptide having a cysteine ​​residue, which forms an intrachain disulfide bond with a cysteine ​​residue in the MHC class I heavy chain polypeptide present in the TMP. For example, when the TMP, or dimerized TMP, such as a homodimer, has a cysteine-containing linker between the peptide epitope and a β2M polypeptide, the cysteine ​​residue in the linker forms an intrachain disulfide bond with the cysteine ​​residue at amino acid 236 (e.g., formed by A236C substitution) in the MHC class I heavy chain polypeptide present in the TMP.

[0179] In some cases, the peptide linker between the peptide and the β2M polypeptide has the amino acid sequence GCGGS (SEQ ID NO: 435). In some cases, the peptide linker between the peptide and the β2M polypeptide has the amino acid sequence GCGGS(GGGGS)n (SEQ ID NO: 436), where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, e.g., 1, 2, or 3. In some cases, the peptide linker between the peptide and the β2M polypeptide has the amino acid sequence GCGGS(GGGGS)n (SEQ ID NO: 437), where n is 2.

[0180] In some cases, the peptide linker between the peptide and the β2M polypeptide has the amino acid sequence CGGGS (SEQ ID NO: 438). In some cases, the peptide linker has the amino acid sequence CGGGS(GGGGS)n (SEQ ID NO: 439), where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, e.g., 1, 2, or 3.

[0181] In some cases, the peptide linker between the peptide and the β2M polypeptide has the amino acid sequence GGCGS (SEQ ID NO: 440). In some cases, the peptide linker has the amino acid sequence GGCGS(GGGGS)n (SEQ ID NO: 441), where n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, e.g., 1, 2 or 3.

[0182] In some cases, the peptide linker between the peptide and the β2M polypeptide has the amino acid sequence GGGCS (SEQ ID NO: 442). In some cases, the peptide linker has the amino acid sequence GGGCS(GGGGS)n (SEQ ID NO: 443), where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, e.g., 1, 2, or 3.

[0183] In some cases, the peptide linker between the peptide and the β2M polypeptide has the amino acid sequence GGGGC (SEQ ID NO: 444). In some cases, the peptide linker has the amino acid sequence GGGGC(GGGGS)n (SEQ ID NO: 445), where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, e.g., 1, 2, or 3.

[0184] Dimerized TMP In some cases, the TMP may form a dimer. That is, the present disclosure provides a polypeptide comprising a dimer of two TMPs. Thus, the present disclosure provides a protein that is a dimerized TMP having two TMPs covalently linked to each other. The covalent bond of the dimer may be one or more disulfide bonds between the IgFc polypeptide in the first TMP and the IgFc polypeptide in the second TMP. As an example, the IgFc may be a variant of a human IgG1Fc polypeptide, which variant has a substantially reduced ability to cause complement-dependent cytotoxicity (CDC) or antibody-dependent cellular cytotoxicity (ADCC) (e.g., the IgFc polypeptide of FIG. 2B or FIG. 2D). When the TMP comprises an IgFc polypeptide, the TMP typically self-assembles into a dimer by spontaneously forming a disulfide bond with the IgFc polypeptide of another TMP. Thus, for example, the IgFc polypeptides of the first TMP and the second TMP may be linked to each other by one or more disulfide bonds. In many cases, the two TMPs contain IgFc polypeptides that are identical in amino acid sequence to one another and spontaneously form one or more disulfide bonds, thereby forming a dimerized TMP that is a homodimer.

[0185] Thus, the present disclosure provides a protein, the protein comprising a) a first TMP and b) a second TMP, which may optionally be identical to the first TMP, where the first and second TMP are covalently bonded to each other, the covalent bond may be a disulfide bond between an IgFc polypeptide in the first TMP and an IgFc polypeptide in the second TMP.

[0186] Optionally, the IgFc polypeptide of each TMP can include an interspecies dimerization sequence, e.g., a knob-in-hole sequence, that allows for selective dimerization of two different TMPs. An interspecies binding sequence is advantageous for forming heterodimers with its cognate polypeptide sequence (i.e., the interspecies sequence and its corresponding interspecies sequence), and is particularly advantageous for forming heterodimers based on IgFc sequence variants. Such interspecies polypeptide sequences include knob-in-hole and knob-in-hole sequences that promote the formation of one or more disulfide bonds. For example, one interspecies binding pair includes the mutant pair T366Y and Y407T at the CH3 domain interface of IgG1, or the corresponding residues in other immunoglobulins. See Ridgway et al., Protein Engineering 9:7, 617-621 (1996). A second interspecies binding pair requires the formation of a knob by the T366W substitution and a hole by three substitutions T366S, L368A, and Y407V on the complementary IgFc sequence. See Xu et al. mAbs 7:1, 231-242 (2015). Another interspecies binding pair has a first Fc polypeptide with Y349C, T366S, L368A, and Y407V substitutions and a second IgFc polypeptide with S354C and T366W substitutions (a disulfide bond can form between Y349C and S354C). See, e.g., Brinkmann and Konthermann, mAbs 9:2, 182-212 (2015). With or without knobs-in-holes modifications, the IgFc polypeptide sequence may be stabilized by the formation of disulfide bonds between the IgFc polypeptides (e.g., disulfide bonds in the hinge region). Thus, in some cases, the dimerized TMP may be a heterodimer, with the two TMP chains not having the same amino acid sequence.

[0187] Interspecies dimerization sequences may also be used to allow for linkage of TMP to non-TMP molecules that can confer additional functionality to the TMP. For example, TMP can be linked to a molecule that contains a polypeptide (e.g., an antibody or binding fragment thereof, e.g., an scFv) that binds to a cancer-associated antigen, thereby allowing the TMP to localize to tissues that contain the cancer-associated antigen.

[0188] Other Polypeptides The polypeptide chain of TMP may include one or more polypeptides and conjugate drugs in addition to those described above. Suitable other polypeptides (including epitope tags and affinity domains) and drug conjugates are described in the above-mentioned published PCT applications WO2020132138A1 and WO2019 / 051091, the disclosures of which regarding epitope tags, affinity domains, and drug conjugates are incorporated herein by reference, in particular in paragraphs

[0498] to

[0508] of WO2020132138A1 and paragraphs

[0353] to

[0363] of WO2019 / 051091. One or more other polypeptides may be included at the N-terminus of the TMP polypeptide chain, at the C-terminus of the TMP polypeptide chain, or within the polypeptide chain of TMP. As described above, other polypeptides may also be attached to TMP using interspecies sequences.

[0189] Specific TMP and dimerized TMP In the following description, the description of a specific TMP is intended to include both TMP and dimerized TMP. Dimerized TMP has two TMPs that are linked by one or more covalent bonds, such as one or more disulfide bonds that are formed spontaneously between the IgFc polypeptides therein. Such dimerized TMPs can be either: i) a homodimer having two TMPs (where both TMPs have the same amino acid sequence), or ii) a heterodimer having two TMPs (where the two TMPs differ from each other in amino acid sequence).

[0190] In some cases, a TMP comprises the following components i)-v): i) a peptide epitope, ii) a first MHC class I polypeptide, where the first MHC class I polypeptide is a β2M polypeptide, iii) a second MHC class I polypeptide, where the second MHC class I polypeptide is an MHC class I heavy chain polypeptide, iv) at least one MOD, and v) an IgFc polypeptide. The TMP may include one or more peptide linkers between one or more of these components.

[0191] In the case of a TMP comprising one or more MODs of configuration 2, one or more peptide linkers may be present between: i) the peptide and the β2M polypeptide, ii) the β2M polypeptide and the MHC class I heavy chain polypeptide, iii) the MHC class I heavy chain polypeptide and the MOD, iv) the MOD, the IgFc polypeptide and the MOD, and vi) if the TMP comprises more than one MOD in tandem, between the MODs. As noted above, in such TMPs, a rigid peptide linker or a short flexible peptide linker may be present between any one or more of: i) the MHC class I heavy chain polypeptide and the MOD, ii) the MOD and the IgFc polypeptide, and iii) if multiple MODs are present in tandem, between the first MOD and the second MOD. In some cases, the rigid peptide linker has an amino acid sequence selected from EAAAK (SEQ ID NO: 412), A(EAAAK)n (SEQ ID NO: 582), A(EAAAK)nA (SEQ ID NO: 583), (AP)n (SEQ ID NO: 584), (EP)n (SEQ ID NO: 585), and (KP)n (SEQ ID NO: 586), where n is an integer from 1 to 10 (e.g., n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). In some cases, the short flexible peptide linker is composed of 2-4, 2-5, 3-6, 4-8, 5-10, or 10-14 amino acids. In some cases, the short flexible peptide linker is GGS. Generally, a flexible peptide linker will be provided between components that are not linked by a rigid peptide linker or a short flexible peptide linker, where the linker between the peptide and the β2M polypeptide may further comprise a Cys-containing linker as described above.

[0192] In the case of a TMP comprising one or more MODs of configuration 3, one or more peptide linkers may be present between: i) the peptide and the β2M polypeptide, ii) the β2M polypeptide and the MHC class I heavy chain polypeptide, iii) the MHC class I heavy chain polypeptide and the IgFc polypeptide, iv) the IgFc polypeptide and the MOD, and v) if the TMP comprises two or more MODs in tandem, between the MODs. As noted above, in such TMPs, a rigid peptide linker or a short flexible peptide linker may be present between any one or more of: i) the IgFc polypeptide and the MOD, and ii) if more than one MOD is present in tandem, one or more of the MODs (e.g., between the first MOD and the second MOD if two MODs are present in tandem). In some cases, the rigid peptide linker has an amino acid sequence selected from EAAAK (SEQ ID NO: 412), A(EAAAK)n (SEQ ID NO: 582), A(EAAAK)nA (SEQ ID NO: 583), (AP)n (SEQ ID NO: 584), (EP)n (SEQ ID NO: 585), and (KP)n (SEQ ID NO: 586), where n is an integer from 1 to 10 (e.g., n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). In some cases, the short flexible peptide linker is composed of 2-4, 2-5, 3-6, 4-8, 5-10, or 10-14 amino acids. In some cases, the short flexible peptide linker is GGS. Generally, a flexible peptide linker will be provided between components that are not linked by a rigid peptide linker or a short flexible peptide linker, where the linker between the peptide and the β2M polypeptide may further comprise a Cys-containing linker as described above.

[0193] As noted above, in some cases, at least one MOD present in the TMP is a wild-type MOD, and in other cases, at least one MOD present in the TMP is a mutant MOD that has a lower affinity for a co-MOD than the affinity of the corresponding wild-type MOD for the co-MOD.

[0194] In some cases, the peptide epitope is an AFP peptide, a WT1 peptide, an HPV peptide, a MUC1 peptide, a MAGE A4 peptide, a NY-ESO-1 peptide, a survivin peptide, a mesothelin peptide, or a viral peptide (e.g., a CMV peptide), such peptides being as described above.

[0195] In the above TMP, in some cases, the second MHC polypeptide is an HLA heavy chain, and the HLA heavy chain is HLA-A * 0201 Polypeptide, HLA-A * 1101 polypeptide, HLA-A24 polypeptide, HLA-E polypeptide (e.g., HLA-E * 0101 or HLA-E * 01.03), or HLA-G polypeptides (e.g., HLA-G * 0101 and / or HLA-G * 01:04). In some cases, the HLA 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 HLA-A * In some cases, the HLA heavy chain polypeptide is an HLA-A 0201 polypeptide that includes a Y84C substitution and / or an A236C substitution. * In some cases, the HLA heavy chain polypeptide is an HLA-A 0201 polypeptide that includes a Y84C substitution and / or an A236C substitution. * In some cases, the HLA heavy chain polypeptide is an HLA-E 1101 polypeptide that includes a Y84C substitution and / or an A236C substitution. * 0101 or HLA-E * 01.03 * In some cases, the HLA heavy chain polypeptide is an HLA-G polypeptide that contains a Y84C substitution and / or an A236C substitution. * 0101 or HLA-G * 01:04A * 1101 polypeptide.

[0196] In some cases, the TMP has two MODs, where the two MODs have the same amino acid sequence, e.g., the MOD is a mutant IL-2 polypeptide having a lower binding affinity to both the α and β chains of IL2R compared to wtIL-2 having the sequence of Figure 17A (e.g., a mutant IL-2 polypeptide comprising H16A and F42A substitutions, or a mutant IL-2 polypeptide comprising H16T and F42A substitutions).

[0197] In some cases, the IgFc polypeptide is a human IgG1 Fc polypeptide variant that does not substantially induce cell lysis, for example an IgG1 Fc polypeptide having L234A and L235A substitutions as shown in Figure 2B or Figure 2D.

[0198] In some cases, the TMP comprises a MOD of configuration 3, wherein the HLA heavy chain polypeptide is a wild-type or mutant HLA-A * 0201 polypeptide (e.g., any one of HLA-A of Figure 3A, 3B, 3C, 3D, or 3E) * HLA-A polypeptides having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% amino acid sequence identity to the 0201 polypeptide. * HLA-A 0201 polypeptide, optionally comprising a Y84 substitution and / or an A236C substitution. *17A , 17B , and 17C ). In some cases, the IgFc polypeptide is a variant of a human IgG1Fc polypeptide that does not substantially cause cell lysis, such as a human IgG1Fc polypeptide comprising the L234A and L235A substitutions as shown in FIG. 2B or FIG. 2D . In some cases, the MOD is a mutant IL-2 polypeptide that has a lower binding affinity to both the α and β chains of IL2R compared to wtIL-2 having the sequence of FIG. 17A , such as a mutant IL-2 polypeptide comprising the H16A and F42A substitutions, or a mutant IL-2 polypeptide comprising the H16T and F42A substitutions. In some cases, the peptide is an AFP peptide, a WT1 peptide, an HPV peptide, a MUC1 peptide, a MAGE A4 peptide, a NY-ESO-1 peptide, a survivin peptide, a mesothelin peptide, or a viral peptide (e.g., a CMV peptide), such peptides being as described above. In some cases, the TMP comprises a rigid peptide linker between the mutant IL-2MOD and the IgFc polypeptide present in the TMP, where the rigid peptide linker has an amino acid sequence selected from EAAAK (SEQ ID NO:412), A(EAAAK)n (SEQ ID NO:582), A(EAAAK)nA (SEQ ID NO:583), (AP)n (SEQ ID NO:584), (EP)n (SEQ ID NO:585), and (KP)n (SEQ ID NO:586), where n is an integer between 1 and 10 (e.g., n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). In some cases, the TMP has two copies of mutant IL-2MOD. In some cases, the TMP has two copies of a mutant IL-2MOD, and the TMP comprises a) a rigid peptide linker between a first mutant IL-2MOD and an IgFc polypeptide present in the TMP, and b) between the first mutant IL-2MOD and a second mutant IL-2MOD, said rigid peptide linker having an amino acid sequence selected from EAAAK (SEQ ID NO:412), A(EAAAK)n (SEQ ID NO:582), A(EAAAK)nA (SEQ ID NO:583), (AP)n (SEQ ID NO:584), (EP)n (SEQ ID NO:585), and (KP)n (SEQ ID NO:586), where n is an integer between 1 and 10 (e.g., n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10).In some cases, the TMP comprises a short flexible peptide linker between the mutant IL-2MOD and the IgFc polypeptide present in the TMP, wherein the short flexible peptide linker is comprised of 2-4, 2-5, 3-6, 4-8, 5-10, or 10-14 amino acids. In some cases, the short flexible peptide linker is GGS.

[0199] In some cases, the TMP comprises a MOD of configuration 3, wherein the HLA heavy chain polypeptide is a wild-type or mutant HLA-A * 1101 polypeptide (e.g., any one of HLA-A of FIG. 4A, 4B, 4C, 4D, or 4E) * HLA-A having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% amino acid sequence identity to the 1101 polypeptide. * HLA-A 1101 polypeptide, optionally comprising a Y84 substitution and / or an A236C substitution. *1101 polypeptide). In some cases, the IgFc polypeptide is a variant of a human IgG1Fc polypeptide that does not substantially cause cell lysis, such as a human IgG1Fc polypeptide comprising the L234A and L235A substitutions as shown in FIG. 2B or FIG. 2D. In some cases, the MOD is a mutant IL-2 polypeptide that has a lower binding affinity to both the α and β chains of IL2R compared to wtIL-2 having the sequence of FIG. 17A, such as a mutant IL-2 polypeptide comprising the H16A and F42A substitutions, or a mutant IL-2 polypeptide comprising the H16T and F42A substitutions. In some cases, the peptide is an AFP peptide, a WT1 peptide, an HPV peptide, a MUC1 peptide, a MAGE A4 peptide, a NY-ESO-1 peptide, a survivin peptide, a mesothelin peptide, or a viral peptide (e.g., a CMV peptide), such peptides being as described above. In some cases, the TMP comprises a rigid peptide linker between the mutant IL-2MOD and the IgFc polypeptide present in the TMP, where the rigid peptide linker has an amino acid sequence selected from EAAAK (SEQ ID NO:412), A(EAAAK)n (SEQ ID NO:582), A(EAAAK)nA (SEQ ID NO:583), (AP)n (SEQ ID NO:584), (EP)n (SEQ ID NO:585), and (KP)n (SEQ ID NO:586), where n is an integer between 1 and 10 (e.g., n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). In some cases, the TMP has two copies of mutant IL-2MOD. In some cases, the TMP has two copies of a mutant IL-2MOD, and the TMP comprises a) a rigid peptide linker between a first mutant IL-2MOD and an IgFc polypeptide present in the TMP, and b) between the first mutant IL-2MOD and a second mutant IL-2MOD, said rigid peptide linker having an amino acid sequence selected from EAAAK (SEQ ID NO:412), A(EAAAK)n (SEQ ID NO:582), A(EAAAK)nA (SEQ ID NO:583), (AP)n (SEQ ID NO:584), (EP)n (SEQ ID NO:585), and (KP)n (SEQ ID NO:586), where n is an integer between 1 and 10 (e.g., n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10).In some cases, the TMP comprises a short flexible peptide linker between the mutant IL-2MOD and the IgFc polypeptide present in the TMP, wherein the short flexible peptide linker is comprised of 2-4, 2-5, 3-6, 4-8, 5-10, or 10-14 amino acids. In some cases, the short flexible peptide linker is GGS.

[0200] In some cases, the TMP comprises a MOD of configuration 3, wherein the HLA heavy chain polypeptide is a wild-type or mutant HLA-A * 2402 polypeptide (e.g., any one of HLA-A of FIG. 5A, 5B, 5C, 5D, or 5E) * HLA-A having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% amino acid sequence identity to the 2402 polypeptide. * HLA-A 2402 polypeptide, optionally comprising a Y84 substitution and / or an A236C substitution. *2402 polypeptide). In some cases, the IgFc polypeptide is a variant of a human IgG1Fc polypeptide that does not substantially cause cell lysis, such as a human IgG1Fc polypeptide comprising the L234A and L235A substitutions as shown in FIG. 2B or FIG. 2D. In some cases, the MOD is a mutant IL-2 polypeptide that has a lower binding affinity to both the α and β chains of IL2R compared to wtIL-2 having the sequence of FIG. 17A, such as a mutant IL-2 polypeptide comprising the H16A and F42A substitutions, or a mutant IL-2 polypeptide comprising the H16T and F42A substitutions. In some cases, the peptide is an AFP peptide, a WT1 peptide, an HPV peptide, a MUC1 peptide, a MAGE A4 peptide, a NY-ESO-1 peptide, a survivin peptide, a mesothelin peptide, or a viral peptide (e.g., a CMV peptide), such peptides being as described above. In some cases, the TMP comprises a rigid peptide linker between the mutant IL-2MOD and the IgFc polypeptide present in the TMP, where the rigid peptide linker has an amino acid sequence selected from EAAAK (SEQ ID NO:412), A(EAAAK)n (SEQ ID NO:582), A(EAAAK)nA (SEQ ID NO:583), (AP)n (SEQ ID NO:584), (EP)n (SEQ ID NO:585), and (KP)n (SEQ ID NO:586), where n is an integer between 1 and 10 (e.g., n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). In some cases, the TMP has two copies of mutant IL-2MOD. In some cases, the TMP has two copies of a mutant IL-2MOD, and the TMP comprises a) a rigid peptide linker between a first mutant IL-2MOD and an IgFc polypeptide present in the TMP, and b) between the first mutant IL-2MOD and a second mutant IL-2MOD, said rigid peptide linker having an amino acid sequence selected from EAAAK (SEQ ID NO:412), A(EAAAK)n (SEQ ID NO:582), A(EAAAK)nA (SEQ ID NO:583), (AP)n (SEQ ID NO:584), (EP)n (SEQ ID NO:585), and (KP)n (SEQ ID NO:586), where n is an integer between 1 and 10 (e.g., n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10).In some cases, the TMP comprises a short flexible peptide linker between the mutant IL-2MOD and the IgFc polypeptide present in the TMP, wherein the short flexible peptide linker is comprised of 2-4, 2-5, 3-6, 4-8, 5-10, or 10-14 amino acids. In some cases, the short flexible peptide linker is GGS.

[0201] As described above, the TMP can include one or more intrachain disulfide bonds. For example, the TMP can include a β2M polypeptide that includes an R12C substitution and a class I MHC heavy chain polypeptide that includes an A236C substitution, whereby a disulfide bond is formed between Cys at position 12 of the β2M polypeptide and Cys at position 236 of the class I MHC heavy chain polypeptide. In another embodiment, the TMP includes i) a peptide epitope and a β2M polypeptide linked by a peptide linker having the sequence GCGGS(GGGGS)n (SEQ ID NO:587), where n is 1, 2, or 3, and ii) a class I MHC heavy chain polypeptide that includes a Y84C substitution, such that a disulfide bond is formed between Cys in the peptide linker and Cys at position 84 of the class I MHC heavy chain polypeptide. In another embodiment, a TMP comprises i) a peptide epitope and a β2M polypeptide linked by a peptide linker having the sequence GCGGS(GGGGS)n (SEQ ID NO:587), where n is 1, 2, or 3, wherein the β2M polypeptide comprises an R12C substitution, and ii) a class I MHC heavy chain polypeptide comprising a Y84C substitution and an A236C substitution, whereby a) a first disulfide bond is formed between a Cys of the peptide linker and a Cys at position 84 of the class I MHC heavy chain polypeptide, and b) a second disulfide bond is formed between a Cys at position 12 of the β2M polypeptide and a Cys at position 236 of the class I MHC heavy chain polypeptide. For simplicity, the first disulfide bond is referred to as "G2C / Y84C" and the second disulfide bond is referred to as "R12C / A236C." TMP can contain a) a G2C / Y84C disulfide bond (but not a R12C / A236C disulfide bond), b) a R12C / A236C disulfide bond (but not a G2C / Y84C disulfide bond), or c) a G2C / Y84C disulfide bond and a R12C / A236C disulfide bond.In some cases, an MHC class I heavy chain has a non-native Cys at position 84 and a non-native residue at position 139, thereby forming an intrachain disulfide bond between Cys-84 and Cys-139.

[0202] The TMP can comprise a) a G2C / Y84C disulfide bond (but not an R12C / A236C disulfide bond) and b) at least one MOD of configuration 2 or 3. The TMP can comprise a) an R12C / A236C disulfide bond (but not a G2C / Y84C disulfide bond) and b) at least one MOD of configuration 2 or 3. The TMP can comprise a) a G2C / Y84C disulfide bond and an R12C / A236C disulfide bond and b) at least one MOD of configuration 2 or 3.

[0203] In some cases, the TMP comprises an MHC class I heavy chain polypeptide, the MHC class I heavy chain polypeptide comprising (i) an HLA-A0201(Y84A, A236C) polypeptide having an Ala at position 84 and a Cys at position 236, or (ii) an HLA-A0201(Y84C, A139C) polypeptide having a Cys at positions 84 and 139, or (iii) an HLA-A0201(Y84C, A236) polypeptide having a Cys at position 84 and an alanine at position 236. Examples of these are shown in Figures 3B-3E.

[0204] In some cases, the TMP comprises an MHC class I heavy chain polypeptide, the MHC class I heavy chain polypeptide comprising (i) an HLA-A polypeptide having an Ala at position 84 and a Cys at position 236; * 1101(Y84A, A236C) polypeptide, or (ii) HLA-A with Cys at positions 84 and 236 * 1101(Y84C, A236C) polypeptide, or (iii) HLA-A having a Cys at position 84 and an alanine at position 236 * 1101(Y84C, A236) polypeptide. Specific examples of these are shown in Figures 4B-4E.

[0205] In some cases, the TMP comprises an MHC class I heavy chain polypeptide, the MHC class I heavy chain polypeptide comprising (i) an HLA-A24(Y84A, A236C) polypeptide having an Ala at position 84 and a Cys at position 236, or (ii) an HLA-A24(Y84C, A236C) polypeptide having Cys at positions 84 and 236, or (iii) an HLA-A24(Y84C, A236) polypeptide having a Cys at position 84 and an alanine at position 236. Examples of these are shown in Figures 5B-5E.

[0206] The peptide contained in the TMP may be any one of the above peptides. In Figures 19A to 19D, "(X)" represents a peptide epitope as described above, for example, an AFP peptide, a WT1 peptide, an HPV peptide, a MUC1 peptide, a MAGE A4 peptide, a NY-ESO-1 peptide, a survivin peptide, a mesothelin peptide, or a viral peptide (for example, a CMV peptide). Similarly, in Figures 18A to 18T, "(X)" represents a peptide epitope as described above, for example, an AFP peptide, a WT1 peptide, an HPV peptide, a MUC1 peptide, a MAGE A4 peptide, a NY-ESO-1 peptide, a survivin peptide, a mesothelin peptide, or a viral peptide (for example, a CMV peptide).

[0207] As discussed above, in some cases, the TMP comprises one or more rigid peptide linkers. As non-limiting examples, as shown in Figures 21A-21D, "(XX)" represents a rigid peptide linker and "(X)" represents a peptide epitope as described above (e.g., an AFP peptide, a WT1 peptide, an HPV peptide, a MUC1 peptide, a MAGE A4 peptide, a NY-ESO-1 peptide, a survivin peptide, a mesothelin peptide, or a viral peptide (e.g., a CMV peptide)). In some cases, the rigid peptide linker has an amino acid sequence selected from EAAAK (SEQ ID NO: 412), A(EAAAK)n (SEQ ID NO: 582), A(EAAAK)nA (SEQ ID NO: 583), (AP)n (SEQ ID NO: 584), (EP)n (SEQ ID NO: 585), and (KP)n (SEQ ID NO: 586), where n is an integer from 1 to 10 (e.g., n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10).

[0208] In some cases, the TMP comprises a MOD of configuration 2 or configuration 3 as shown in FIG. 12, wherein the HLA heavy chain polypeptide is a wild-type or mutant HLA-A * 0201 polypeptide (e.g., HLA-A * 0201 polypeptide, or an HLA-A polypeptide having a sequence as shown in any one of Figures 3A-3E. * In some cases, the IgFc polypeptide is a human IgG1Fc polypeptide that does not substantially cause cell lysis (e.g., a human IgG1Fc polypeptide comprising L234A and L235A substitutions as shown in Figures 2B and 2D). In some cases, the TMP has an intrachain disulfide bond that is between (i) two Cys residues in the MHC class I heavy chain polypeptide, (ii) between a Cys in the linker between the β2M polypeptide and the heavy chain polypeptide and / or between the peptide epitope and the β2M polypeptide and a Cys in the MHC class I heavy chain. In some cases, the MOD is a mutant IL-2 polypeptide comprising H16A and F42A substitutions or H16T and F42A substitutions.

[0209] In some cases, the TMP comprises a MOD of configuration 2 or configuration 3 as shown in FIG. 12, wherein the HLA heavy chain polypeptide is a wild-type or mutant HLA-A24 polypeptide (HLA-A * 2402) (e.g., HLA-A containing the A236C substitution * 2402 polypeptide, or an HLA-A polypeptide having an amino acid sequence as shown in any one of Figures 5A-5E. * In some cases, the IgFc polypeptide is a human IgG1Fc polypeptide that does not substantially cause cell lysis (e.g., a human IgG1Fc polypeptide comprising L234A and L235A substitutions as shown in Figures 2B and 2D). In some cases, the TMP has an intrachain disulfide bond that is between (i) two Cys residues in the MHC class I heavy chain polypeptide, (ii) between a Cys in the linker between the β2M polypeptide and the heavy chain polypeptide and / or between the peptide epitope and the β2M polypeptide and a Cys in the MHC class I heavy chain. In some cases, the MOD is a mutant IL-2 polypeptide comprising H16A and F42A substitutions or H16T and F42A substitutions.

[0210] In some cases, the TMP comprises a MOD of configuration 2 or configuration 3 as shown in FIG. 12, wherein the HLA heavy chain polypeptide is a wild-type or mutant HLA-A * 1101 polypeptides (disclosed herein) (e.g., HLA-A * 1101 polypeptide, or an HLA-A polypeptide having an amino acid sequence as shown in any one of Figures 4A-4E. *In some cases, the IgFc polypeptide is a human IgG1Fc polypeptide that does not substantially cause cell lysis (e.g., a human IgG1Fc polypeptide comprising L234A and L235A substitutions as shown in FIG. 2B or FIG. 2D). In some cases, the TMP has an intrachain disulfide bond that is (i) between two Cys residues in the MHC class I heavy chain polypeptide, (ii) between a Cys in the linker between the β2M polypeptide and the heavy chain polypeptide and / or between the peptide epitope and the β2M polypeptide and a Cys in the MHC class I heavy chain. In some cases, the MOD is a mutant IL-2 polypeptide comprising H16A and F42A substitutions or H16T and F42A substitutions.

[0211] The TMP can comprise a) a G2C / Y84C disulfide bond (but not a R12C / A236C disulfide bond) and b) at least one MOD of configuration 2 or 3. The TMP can comprise a) a R12C / A236C disulfide bond (but not a G2C / Y84C disulfide bond) and at least one MOD of configuration 2 or 3. The TMP can comprise a) a G2C / Y84C disulfide bond and a R12C / A236C disulfide bond and at least one MOD of configuration 2 or 3.

[0212] In some cases, the TMP comprises an MHC class I heavy chain polypeptide, the MHC class I heavy chain polypeptide comprising (i) an HLA-A0201(Y84A, A236C) polypeptide having an Ala at position 84 and a Cys at position 236, or (ii) an HLA-A0201(Y84C, A139C) polypeptide having Cys at positions 84 and 139, or (iii) an HLA-A0201(Y84C, A236) polypeptide having a Cys at position 84 and an alanine at position 236, e.g., as shown in Figures 3A-3E.

[0213] In some cases, the TMP comprises an MHC class I heavy chain polypeptide, the MHC class I heavy chain polypeptide comprising (i) an HLA-A polypeptide having an Ala at position 84 and a Cys at position 236; * 1101(Y84A, A236C) polypeptide, or (ii) HLA-A with Cys at positions 84 and 236 * 1101(Y84C, A236C) polypeptide, or (iii) HLA-A having a Cys at position 84 and an alanine at position 236 * 1101(Y84C, A236) polypeptide, for example, as shown in Figures 4A-4E.

[0214] In some cases, the TMP comprises a second polypeptide, the second polypeptide comprising (i) an HLA-A24(Y84A, A236C) polypeptide having an Ala at position 84 and a Cys at position 236, or (ii) an HLA-A24(Y84C, A236C) polypeptide having Cys at positions 84 and 236, or (iii) an HLA-A24(Y84C, A236) polypeptide having a Cys at position 84 and an alanine at position 236, for example, as shown in Figures 5A-5E.

[0215] As specific, non-limiting examples, the TMP can have an amino acid sequence as shown in any one of Figures 19A-19D, where "(X)" is an AFP peptide, a WT1 peptide, an HPV peptide, a MUC1 peptide, a MAGE A4 peptide, a NY-ESO-1 peptide, a survivin peptide, a mesothelin peptide, or a viral peptide (e.g., a CMV peptide).

[0216] As further non-limiting specific examples, the TMP can have an amino acid sequence as shown in any one of Figures 18A-18T, where "(X)" is an AFP peptide, a WT1 peptide, an HPV peptide, a MUC1 peptide, a MAGE A4 peptide, a NY-ESO-1 peptide, a survivin peptide, a mesothelin peptide, or a viral peptide (e.g., a CMV peptide).

[0217] As a further non-limiting example, the TMP can have an amino acid sequence as shown in any one of Figures 20A-20G. The construct numbers, MHC heavy chain alleles, and peptide epitopes present in the TMPs shown in Figures 20A-20G are summarized in Table 2 below.

[0218] [Table 2]

[0219] The peptide present in any of the constructs shown in Figures 20A-20G may be replaced with a different peptide. For example, the peptide present in any of the constructs shown in Figures 20A-20G may be replaced with an AFP peptide, a WT1 peptide, an HPV peptide, a MUC1 peptide, a MAGE A4 peptide, a NY-ESO-1 peptide, a survivin peptide, or a mesothelin peptide, as described above.

[0220] Nucleic Acids, Recombinant Expression Vectors, and Modified Host Cells The present disclosure provides a nucleic acid comprising the nucleotide sequence encoding the TMP of the present disclosure.In some cases, the nucleotide sequence encoding the TMP is operably linked to one or more transcriptional regulators.In some cases, the transcriptional regulator is a promoter that functions in eukaryotic cells.In some cases, the nucleic acid is present in a recombinant expression vector.

[0221] Thus, the present disclosure provides a recombinant expression vector that comprises the nucleic acid encoding TMP.In some cases, recombinant expression vector is a non-viral vector.In some cases, recombinant expression vector is a viral construct, such as recombinant adeno-associated virus construct (see, for example, U.S. Patent No. 7,078,387), recombinant adenovirus construct, recombinant lentivirus construct, recombinant retrovirus construct, non-integrated viral vector, etc.

[0222] Suitable expression vectors are well known to those of skill in the art. See, for example, published PCT applications WO2020132138A1 and WO2019 / 051091, the disclosures of which regarding such expression vectors are hereby incorporated by reference as if expressly set forth herein, in particular paragraphs

[0515] to

[0520] of WO2020132138A1 and paragraphs

[0401] to

[0406] of WO2019 / 051091.

[0223] The present disclosure further provides a genetically modified host cell, wherein said host cell is genetically modified with a nucleic acid or expression vector as described above.

[0224] Suitable host cells include eukaryotic cells such as yeast cells, insect cells, mammalian cells, etc. In some cases, the host cells are cells of a mammalian cell line. Suitable mammalian cell lines include human cell lines, non-human primate cell lines, rodent (e.g., mouse, rat) cell lines, etc. Suitable mammalian cell lines include, but are not limited to, HeLa cells (e.g., American Type Culture Collection (ATCC) No. CCL-2), CHO cells (e.g., ATCC Nos. CRL9618, CCL61, CRL9096), 293 cells (e.g., ATCC No. CRL-1573), Vero cells, NIH3T3 cells (e.g., ATCC No. CRL-1658), Huh-7 cells, BHK cells (e.g., ATCC No. CCL10), PC12 cells (ATCC No. CRL1721), COS cells, COS-7 cells (ATCC No. CRL1651), RAT1 cells, mouse L cells (ATCC No. CCLI.3), human embryonic kidney (HEK) cells (ATCC No. CRL1573), HLHepG2 cells, and the like.

[0225] In some cases, the host cell is a mammalian cell that has been genetically modified so that it does not synthesize endogenous MHC β2M.

[0226] In some cases, the host cell is a mammalian cell that has been genetically modified not to synthesize endogenous MHC class I heavy chains. In some cases, the host cell is a mammalian cell that has been genetically modified not to synthesize endogenous MHC β2M and not to synthesize endogenous MHC class I heavy chains.

[0227] Methods for Producing T Cell Modulating Polypeptides The TMP of the present disclosure can be produced by in vitro culturing of the genetically modified host cells of the present disclosure in a suitable culture medium, such culturing resulting in the production of the TMP. For example, a mammalian host cell (e.g., a CHO cell) can be genetically modified with a recombinant expression vector comprising a nucleotide sequence encoding the TMP of the present disclosure, and the genetically modified mammalian host cell can be cultured in vitro in a suitable culture medium, whereby the genetically modified mammalian host cell produces the TMP. The TMP can be isolated, for example, from the culture medium in which the genetically modified mammalian host cell is cultured, and / or from a cell lysate of the genetically modified mammalian host cell. The TMP can be isolated using any of a variety of established methods. If the TMP comprises an IgFc polypeptide at its C-terminus, the C-terminal Lys residue can be removed from the C-terminus of the IgFc polypeptide by intracellular processing. See, e.g., van den Bremer et al. (2015) mAbs 7:4 and Sissolak et al. (2019) J. Industrial Microbiol. & Biotechnol. 46: 1167. Also, as described above, two TMPs, each of which contains an IgFc polypeptide (e.g., IgG1Fc), can spontaneously form a homodimer of the two TMPs, where the individual TMPs are linked by one or more disulfide bonds between their respective IgFc portions.

[0228] composition The present disclosure provides compositions (e.g., pharmaceutical compositions) that include the TMP or dimerized TMP disclosed herein. The present disclosure provides compositions (e.g., pharmaceutical compositions) that include a nucleic acid or recombinant expression vector.

[0229] Compositions containing TMP or dimerized TMP The compositions can include one or more pharma- ceutically acceptable excipients in addition to TMP or dimerized TMP. Such excipients can be, for example, carriers, diluents, buffers, salts, solubilizers, surfactants, stabilizers, or other additives that, for example, aid in the manufacturing process and can protect, maintain, or enhance stability, bioavailability, and / or patient acceptability. Pharmaceutically acceptable excipients are well known to those of skill in the art.

[0230] When TMP or dimerized TMP is administered directly to tissues as an injection (e.g., subcutaneously, intraperitoneally, intramuscularly, and / or intravenously), the formulation can be provided in a ready-to-use form (which can be directly injected or infused into a patient or mixed with saline for infusion), or optionally in a non-aqueous form (e.g., a shelf-stable powder that can be reconstituted by adding water), or in an aqueous form (e.g., a liquid comprised of pharma- ceutically acceptable carriers and excipients). Formulations can also be provided to enhance the blood half-life of TMP following administration. For example, TMP or dimerized TMP can be provided in a liposomal formulation prepared as a colloid, or in other conventional techniques to enhance blood half-life. Formulations can also be provided in controlled or sustained release forms.

[0231] The concentration of TMP or dimerized TMP in the liquid composition formulation can vary widely (e.g., from less than about 0.1% by weight, usually at least about 2% by weight, up to 20% to 50% by weight or more). Concentrations falling within this range are about 5 to about 15 mg / mL, about 8 to about 12 mg / mL, about 9 to about 11 mg / mL, e.g., about 5 mg / mL, about 6 mg / mL, about 7 mg / mL, about 8 mg / mL, about 9 mg / mL, about 10 mg / mL, about 11 mg / mL, about 12 mg / mL, about 13 mg / mL, about 14 mg / mL, and about 15 mg / mL. The concentration can depend on a number of factors, including the stability of the TMP in the liquid composition.

[0232] In some cases, the TMP or dimerized TMP is present in a liquid composition. In some cases, the composition includes a) TMP or dimerized TMP, and b) saline (e.g., 0.9% NaCl). In some cases, the composition is sterile and suitable for administration to a human subject.

[0233] Methods for modulating T cell activity The present disclosure provides a method of selectively modulating the activity of epitope-specific T cells (e.g., T cells specific for a peptide epitope present in a TMP, such as a cancer-associated peptide or a viral peptide or a viral-associated peptide), the method comprising contacting the T cells with a TMP or a dimerized TMP, wherein contacting the T cells with the TMP or the dimerized TMP selectively modulates the activity of the epitope-specific T cells. In some cases, the contacting is performed in vitro. In some cases, the contacting is performed in vivo.

[0234] When the TMP or dimerized TMP comprises a MOD that is an activating polypeptide and the peptide is a cancer associated peptide, contacting a T cell with the TMP or dimerized TMP activates epitope-specific T cells, increases the cytotoxic activity of the T cell against cancer cells expressing the cancer associated peptide epitope, and / or increases the number of epitope-specific T cells. When the TMP or dimerized TMP comprises a MOD that is an activating polypeptide and the peptide is a viral peptide or a virus associated peptide, contacting a T cell with the TMP or dimerized TMP activates epitope-specific T cells, increases the cytotoxic activity of the T cell against virus-infected cells expressing the viral peptide or a virus associated peptide, and / or increases the number of epitope-specific T cells.

[0235] In some cases, the TMP or dimerized TMP, when administered to an individual in need thereof, induces both epitope-specific and epitope-nonspecific T cell responses. That is, in some cases, the TMP, when administered to an individual in need thereof, induces (i) an epitope-specific T cell response by modulating the activity of a first T cell that presents both a TCR specific for a peptide epitope present in the TMP and a co-MOD that binds to a MOD present in the TMP, and (ii) an epitope-nonspecific T cell response by modulating the activity of a second T cell that presents a TCR specific for an epitope other than a peptide epitope present in the TMP and a co-MOD that binds to a MOD present in the TMP. The ratio of epitope-specific 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 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, about 50:1 to about 100:1, or more than 100:1. This ratio is determined by measuring the increase in the number of T cells specific for a target peptide epitope (e.g., a cancer-associated peptide, a viral peptide, or a viral-associated peptide) versus the increase in the number of T cells not specific for that target epitope. For example, conventional flow cytometry methods can be used. "Modulating the activity" of T cells can include one or more of the following in the presence of an activating MOD such as mutant IL-2: i) activating cytotoxic (e.g., CD8+) T cells, ii) activating cytotoxic (e.g., CD8 + ) inducing the cytotoxic activity of T cells; iii) inducing the production and release of cytotoxins (e.g., perforin, granzymes, granulysin) by cytotoxic (e.g., CD8+) T cells; iv) inducing the production and release of cytotoxins (e.g., perforin, granzymes, granulysin) by cytotoxic (e.g., CD8 + ) Inducing T cell proliferation.

[0236] As described above, in some cases, TMP or dimerized TMP induces proliferation of epitope-specific T cells when administered to an individual in need thereof. The increase in the percentage of epitope-specific T cells can be measured by conventional flow cytometry. Thus, for example, the percentage of total CD8+ T cells specific for a peptide epitope can increase by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 50%, at least about 75%, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 4-fold, or more than 4-fold after contact with TMP.

[0237] The disclosure provides a method of selectively delivering a MOD to a target T cell, the method comprising contacting a TMP or dimerized TMP with a mixed population of T cells, the mixed population of T cells comprising target T cells and non-target T cells, the target T cells being specific for a peptide epitope present in the TMP or dimerized TMP, whereby one or more MODs present in the TMP or dimerized TMP are delivered to the target T cells. In some cases, the population of T cells is in vitro. In some cases, the population of T cells is in vivo within an individual. In some cases, the method comprises administering the TMP or dimerized TMP to an individual. In some cases, the target T cells are cytotoxic T cells. In some cases, the mixed population of T cells is an in vitro population of mixed T cells obtained from an individual, whereby the contacting results in activation and / or proliferation of the target T cells to generate a population of activated and / or expanded target T cells. In some cases, the method further comprises administering the population of activated and / or expanded target T cells to an individual.

[0238] The present disclosure provides a method for detecting the presence of target T cells that bind an epitope of interest (e.g., a cancer epitope, a viral epitope) in a mixed population of T cells obtained from an individual, the method comprising: a) contacting a TMP or a dimerized TMP with the mixed population of T cells in vitro, where the TMP or dimerized TMP comprises a peptide epitope of interest, and b) detecting activation and / or proliferation of the T cells in response to the contact, where activated and / or proliferated T cells indicate the presence of the target T cells.

[0239] Treatment method The present disclosure provides a method of treating an individual, comprising administering to the individual an amount of TMP or dimerized TMP, or one or more nucleic acids encoding TMP, effective to treat the individual. Also provided is TMP or dimerized TMP for use in a method of treating a human or non-human animal. In some cases, the method of treatment of the present disclosure comprises administering to an individual in need of treatment one or more recombinant expression vectors comprising a nucleotide sequence encoding TMP or dimerized TMP. In some cases, the method of treatment of the present disclosure comprises administering to an individual in need of treatment one or more nucleic acids (e.g., mRNA molecules) comprising a nucleotide sequence encoding TMP or dimerized TMP. In some cases, the method of treatment comprises administering to an individual in need of treatment TMP or dimerized TMP. Treatable diseases include, for example, cancer, such as cancers expressing AFP peptide, WT1 peptide, HPV peptide, MUC1 peptide, MAGE A4 peptide, NY-ESO-1 peptide, survivin peptide, mesothelin peptide, or viral peptide (e.g., CMV peptide). Treatable diseases include diseases caused by infectious diseases, such as viral infections.

[0240] Cancer Treatment A TMP of the present disclosure can be administered to an individual in need of treatment to treat a cancer in the individual, where the cancer expresses or overexpresses a peptide present in the TMP (e.g., an AFP peptide, a WT1 peptide, an HPV peptide, a MUC1 peptide, a MAGE A4 peptide, a NY-ESO-1 peptide, a survivin peptide, a mesothelin peptide, or a viral peptide (e.g., a CMV peptide)). For example, the cancer can be one whose cancer cells express or overexpress an AFP peptide, a WT1 peptide, an HPV peptide, a MUC1 peptide, a MAGE A4 peptide, a NY-ESO-1 peptide, a survivin peptide, or a mesothelin peptide. The present disclosure provides methods of treating cancer in an individual, the methods comprising administering to the individual an effective amount of TMP or dimerized TMP, or one or more nucleic acids (e.g., expression vectors, mRNA, etc.) comprising a nucleotide sequence encoding TMP, where the TMP or dimerized TMP comprises a peptide that presents a T cell epitope, and the TMP or dimerized TMP comprises an activating MOD. In some cases, an effective amount of TMP or dimerized TMP is an amount that, when administered once or multiple times to an individual in need of treatment, either as a monotherapy or as part of a combination therapy (e.g., as part of a combination therapy with an immune checkpoint inhibitor, as described below), reduces the total tumor burden in the individual (i.e., the amount of cancer in the body) or maintains the total tumor burden in the patient relatively stable for a sufficient period of time to result in "stable disease" in the patient, as assessed by standard Response Evaluation Criteria in Solid Tumors (RECIST).

[0241] In some cases, an effective amount of TMP or dimerized TMP is an amount that, when administered once or multiple times to an individual in need of treatment, either as monotherapy or as part of a combination therapy (e.g., as part of a combination therapy with an immune checkpoint inhibitor), as described below, reduces tumor size by a sufficient amount and for a sufficient duration to result in a "partial response" in the patient, as measured by standard solid tumor response criteria.

[0242] In some cases, an effective amount of TMP or dimerized TMP is an amount that, when administered once or multiple times to an individual in need of treatment, either as a monotherapy or as part of a combination therapy (e.g., as part of a combination therapy with an immune checkpoint inhibitor), reduces tumor size by a sufficient amount and for a sufficient duration to result in a "complete response" in the patient, as measured by standard solid tumor response criteria.

[0243] In some cases, an effective amount of TMP or dimerized TMP is an amount that, when administered one or more times to an individual in need of treatment, either as a monotherapy or as part of a combination therapy (e.g., as part of a combination therapy with an immune checkpoint inhibitor), reduces the number of cancer cells in an individual by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%, compared to the number of cancer cells in the individual before administration of TMP or dimerized TMP or when TMP or dimerized TMP is not administered.

[0244] In some cases, an effective amount of TMP or dimerized TMP is an amount that, when administered once or multiple times to an individual in need of treatment, either as a monotherapy or as part of a combination therapy (e.g., as part of a combination therapy with an immune checkpoint inhibitor), reduces the number of cancer cells in the individual, including to substantially undetectable levels.

[0245] In some cases, an effective amount of TMP or dimerized TMP is an amount that, when administered once or multiple times to an individual in need of treatment (an individual having a tumor), either as a monotherapy or as part of a combination therapy (e.g., as part of a combination therapy with an immune checkpoint inhibitor), reduces tumor volume in the individual. For example, in some cases, an effective amount of TMP or dimerized TMP is an amount that, when administered once or multiple times to an individual in need of treatment (an individual having a tumor), either as a monotherapy or as part of a combination therapy (e.g., as part of a combination therapy with an immune checkpoint inhibitor), reduces tumor volume in the individual by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%, compared to the tumor volume in the individual before administration of TMP or dimerized TMP or when TMP or dimerized TMP is not administered. Tumor volume was determined using the formula (length x width x width) / 2, where length represents the maximum tumor diameter and width represents the tumor diameter perpendicular thereto.

[0246] In some cases, an effective amount of TMP or dimerized TMP is an amount that, when administered one or more times to an individual in need of treatment, increases the survival time of the individual. For example, in some cases, an effective amount of TMP or dimerized TMP is an amount that, when administered one or more times to an individual in need of treatment, either as a monotherapy or as part of a combination therapy (e.g., as part of a combination therapy with an immune checkpoint inhibitor), increases the survival time of the individual by at least 1 month, at least 2 months, at least 3 months, between 3 months and 6 months, between 6 months and 1 year, between 1 year and 2 years, between 2 years and 5 years, between 5 years and 10 years, or more than 10 years, compared to the expected survival time of the individual in the absence of administration of TMP or dimerized TMP.

[0247] In some cases, an effective amount of TMP or dimerized TMP is an amount that, when administered once or multiple times to an individual in need of treatment, either as a monotherapy or as part of a combination therapy (e.g., as part of a combination therapy with an immune checkpoint inhibitor), reduces the level of circulating tumor DNA (ctDNA) in the patient by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% compared to the ctDNA level in the individual before administration of the TMP or dimerized TMP or when the TMP or dimerized TMP is not administered. The level of ctDNA can be determined using any known method, see, for example, Cescon et al. (2020) Nature Cancer 1:276.

[0248] Cancers treatable by the methods of the present disclosure include cancers in which the cancer cells express an AFP peptide, a WT1 peptide, an HPV peptide, a MUC1 peptide, a MAGE A4 peptide, a NY-ESO-1 peptide, a survivin peptide, a mesothelin peptide, a MART1 peptide, or a viral peptide (e.g., a CMV peptide, an influenza virus peptide, an EBV peptide, etc.).

[0249] For example, TMPs containing the AFP peptide or dimerized TMPs can be used to treat cancers such as hepatocellular carcinoma, pancreatic cancer, gastric cancer, colorectal cancer, hepatoblastoma, or ovarian yolk sac tumors.

[0250] In another embodiment, TMPs or dimerized TMPs containing WT-1 peptides can be used to treat cancers, such as WT-1-expressing cancers, including leukemia, desmoplastic small round cell tumor, gastric cancer, colon cancer, lung cancer, breast cancer, germ cell tumor, ovarian cancer, uterine cancer, thyroid cancer, liver cancer, kidney cancer, Kaposi's sarcoma, sarcoma, hepatocellular carcinoma, Wilms' tumor, acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), non-small cell lung cancer (NSCLC), myeloma, pancreatic cancer, colorectal cancer, mesothelioma, soft tissue sarcoma, neuroblastoma, and nephroblastoma.

[0251] In another embodiment, TMPs containing HPV peptides or dimerized TMPs can be used to treat cancers, such as HPV-expressing (HPV+) cancers, including head and neck cancer, cervical cancer, prostate cancer, ovarian cancer, reproductive cancer, and the like.

[0252] In another embodiment, TMPs or dimerized TMPs containing MUC-1 peptides can be used to treat cancers that express or overexpress MUC-1. Specific examples include adenocarcinomas and hematological malignancies. Specific examples include, for example, multiple myeloma, B-cell lymphoma, breast cancer, lung cancer, ovarian cancer, pancreatic cancer, colorectal cancer, prostate cancer, renal cancer, acute myeloid leukemia, mesothelioma, thyroid cancer, head and neck cancer, gastric cancer, urothelial cancer, cervical cancer, and ovarian endometrial cancer.

[0253] In another embodiment, TMPs containing MAGE A4 peptides or dimerized TMPs can be used to treat MAGE-A4 positive cancers, including, for example, melanoma, bladder cancer, head and neck cancer, lung cancer, esophageal cancer, breast cancer, colon cancer, and ovarian cancer.

[0254] As another embodiment, TMP or dimerized TMP containing the NY-ESO-1 peptide can be used to treat lung cancer, esophageal cancer, breast cancer, pancreatic cancer, colorectal cancer, and ovarian cancer.

[0255] As another example, TMPs containing survivin peptides or dimerized TMPs can be used to treat esophageal cancer (eg, esophageal squamous cell carcinoma), melanoma, breast cancer, and leukemia.

[0256] As another embodiment, TMPs containing mesothelin peptides or dimerized TMPs can be used to treat mesothelin-expressing cancers, such as mesothelioma, pancreatic cancer, ovarian cancer, and lung adenocarcinoma.

[0257] Treating infection The present disclosure provides a method for treating an infectious disease in an individual, for example, an infectious disease caused by a pathogenic virus. The method of the present disclosure can increase the number and / or activity of epitope-specific T cells in an individual. In some cases, the epitope-specific T cells are T cells specific to an epitope present on a virus-infected cell, and contacting the epitope-specific T cells with a TMP increases the cytotoxic activity of the T cells against the virus-infected cell. In some cases, the epitope-specific T cells are T cells specific to an epitope present on a virus-infected cell, and contacting the epitope-specific T cells with a TMP increases the number of epitope-specific T cells.

[0258] Thus, the present disclosure provides a method of treating a viral infection in an individual, the method comprising administering to the individual an effective amount of TMP or dimerized TMP or one or more nucleic acids comprising a nucleotide sequence encoding a TMP, where the TMP comprises a T cell epitope that is a viral epitope, and the TMP comprises a stimulatory (activating) MOD. In some cases, an effective amount of TMP or dimerized TMP is an amount that, when administered one or more times to an individual in need thereof, reduces the number of virally infected cells in the individual. For example, in some cases, an effective amount of TMP or dimerized TMP is an amount that, when administered one or more times to an individual in need thereof, reduces the number of virally infected cells in the individual by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%, compared to the number of virally infected cells in the individual before administration of the TMP or dimerized TMP or when the TMP or dimerized TMP is not administered. In some cases, an effective amount of TMP or dimerized TMP is an amount that, when administered one or more times to an individual in need thereof, reduces the number of virally infected cells in the individual to undetectable levels.

[0259] In some cases, the effective amount of TMP or dimerized TMP is an amount that, when administered once or multiple times to an individual in need thereof, reduces the number of viruses (e.g., virus particles) in the individual. For example, in some cases, the effective amount of TMP or dimerized TMP is an amount that, when administered once or multiple times to an individual in need thereof, reduces the number of viruses in the individual by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%, compared to the number of viruses in the individual before administering TMP or dimerized TMP or when not administering TMP or dimerized TMP. In some cases, the effective amount of TMP or dimerized TMP is an amount that, when administered once or multiple times to an individual in need thereof, reduces the number of viruses in the individual to undetectable levels. The number of viruses in an individual can be determined using any of a number of well-known methods for determining viral load, including, for example, measuring viral genomic copies using the polymerase chain reaction.

[0260] Dosage The appropriate dosage of TMP or dimerized TMP can be determined by the attending physician or other qualified medical personnel based on various clinical factors. As is well known in the medical field, the dosage for any one patient depends on many factors. Such factors include the patient's size, body surface area, age, the particular polypeptide or nucleic acid being administered, the patient's sex, the time and route of administration, general health, and other drugs administered at the same time. The dosage of TMP or dimerized TMP per administration can be between 0.1 mg / kg body weight and 20 mg / kg body weight, for example, between 0.1 mg / kg body weight and 10 mg / kg body weight, for example, between 0.5 mg / kg body weight and 5 mg / kg body weight, between 1 mg / kg body weight and 5 mg / kg body weight, between 5 mg / kg body weight and 10 mg / kg body weight, between 10 mg / kg body weight and 15 mg / kg body weight, between 15 mg / kg body weight and 20 mg / kg body weight, although dosages beyond this specific range are also contemplated, especially considering the aforementioned factors. When the formulation is a continuous infusion, the dosage can be in the range of 1 μg to 10 mg per kg of body weight per minute. The dosage of TMP or dimerized TMP can be about 1 mg / kg to 50 mg / kg of body weight, for example, as follows: about 1 mg / kg to about 5 mg / kg of body weight, about 5 mg / kg to about 10 mg / kg of body weight, about 10 mg / kg to about 15 mg / kg of body weight, about 15 mg / kg to about 20 mg / kg of body weight, about 20 mg / kg to about 25 mg / kg of body weight, about 25 mg / kg to about 30 mg / kg of body weight, about 30 mg / kg to about 35 mg / kg of body weight, about 35 mg / kg to about 40 mg / kg of body weight, or about 40 mg / kg to about 50 mg / kg of body weight. Specific amounts of TMP or dimerized TMP include 1 mg / kg body weight to 5 mg / kg body weight, 5 mg / kg body weight to 10 mg / kg body weight, about 1 mg / kg body weight to about 5 mg / kg body weight, and about 5 mg / kg body weight to about 10 mg / kg body weight.

[0261] Those skilled in the art can easily estimate how many times administration should be repeated based on measurements of residence time and concentration of the administered drug in body fluids or tissues.After successful treatment, it may be desirable to subject the patient to maintenance therapy to prevent recurrence of the disease, in which case the maintenance dose of TMP or dimerized TMP administered is about 1 mg / kg body weight to about 5 mg / kg body weight, about 5 mg / kg body weight to about 10 mg / kg body weight, about 10 mg / kg body weight to about 15 mg / kg body weight, about 15 mg / kg body weight to about 20 mg / kg body weight, or more than 20 mg / kg body weight.

[0262] Those of skill in the art will readily appreciate that dosages can vary as a function of the particular TMP or dimerized TMP, the severity of the symptoms, and the susceptibility of the subject to side effects. Preferred dosages for a given compound are readily determinable by those of skill in the art by a variety of means.

[0263] In some cases, TMP or dimerized TMP, nucleic acid, or recombinant expression vector is administered multiple times. The frequency of administration of TMP or dimerized TMP, nucleic acid, or recombinant expression vector can vary depending on any of a variety of factors, such as the severity of symptoms. For example, in some cases, TMP or dimerized TMP, nucleic acid, or recombinant expression vector is administered once a month, twice a month, three times a month, every other week (qow), once a week (qw), once every two weeks, once every three weeks, once every four weeks, twice a week (biw), three times a week (tiw), four times a week, five times a week, six times a week, every other day (qod), every day (qd), twice a day (qid), or three times a day (tid). When TMP or dimerized TMP is administered intravenously, dosing once a week, once every two weeks, once every three weeks, once every four weeks, or once a month can generally be employed at the initiation of treatment.

[0264] The administration period of TMP or dimerized TMP, nucleic acid, or recombinant expression vector, e.g., the period during which TMP or dimerized TMP, nucleic acid, or recombinant expression vector is administered, can vary depending on any of a variety of factors, e.g., patient response, etc. For example, TMP or dimerized TMP, nucleic acid, or recombinant expression vector can be administered for a period of about 1 day to about 1 week, about 2 weeks to about 4 weeks, about 1 month to about 2 months, about 2 months to about 4 months, about 4 months to about 6 months, about 6 months to about 8 months, about 8 months to about 1 year, about 1 year to about 2 years, or about 2 years to about 4 years, or longer.

[0265] Route of administration The active agent (TMP or dimerized TMP, nucleic acid, or recombinant expression vector) is administered to an individual using any available method and route suitable for drug delivery, including in vivo and in vitro methods, and systemic and local administration routes.

[0266] The TMP or dimerized TMP of the present disclosure is typically delivered by intravenous administration, although other conventional pharma- ceutically acceptable routes of administration can also be used, such as intratumoral, peritumoral, intramuscular, intralymphatic, intratracheal, intracranial, subcutaneous, intradermal, topical, intraarterial, rectal, nasal, oral, and other enteral and parenteral routes of administration. If necessary, multiple routes of administration can be combined or adjusted depending on the TMP or dimerized TMP and / or the desired effect. The TMP or dimerized TMP, or the nucleic acid or recombinant expression vector, can be administered in a single dose or multiple doses.

[0267] Combination therapy TMP or dimerized TMP can be administered to an individual in need thereof in combination with one or more additional therapeutic agents or therapies. A suitable dosage of TMP or dimerized TMP can be the same as the dosage of TMP or dimerized TMP monotherapy (described above), or it can be less than or greater than the dosage of the monotherapy.

[0268] TMP or dimerized TMP can be administered to an individual in need thereof at the same time that one or more additional therapeutic agents are administered, or at a different time.

[0269] Thus, for example, a method of treatment can include administering TMP or dimerized TMP and at least one additional therapeutic agent together. By "administering together" is meant administering both TMP or dimerized TMP and at least one additional therapeutic agent to an individual (not necessarily simultaneously) to achieve a certain therapeutic effect. The administration of TMP or dimerized TMP and at least one additional therapeutic agent can be substantially simultaneous, for example, TMP or dimerized TMP can be administered to an individual within about 1 minute to about 24 hours (e.g., within about 1 minute, within about 5 minutes, within about 15 minutes, within about 30 minutes, within about 1 hour, within about 4 hours, within about 8 hours, within about 12 hours, or within about 24 hours) of the administration of the at least one additional therapeutic agent. In some cases, TMP or dimerized TMP is administered to an individual who is undergoing or has undergone treatment with at least one additional therapeutic agent. The administration of TMP or dimerized TMP and at least one additional therapeutic agent can be performed at different times and / or at different frequencies.

[0270] Combination Therapy in Cancer Treatment TMP or dimerized TMP can be administered to an individual in need thereof in combination with one or more additional therapeutic agents or therapies.The suitable dosage of TMP or dimerized TMP is typically the same as the dosage of TMP monotherapy (as described above), or may be less than or greater than the dosage of monotherapy.Suitable additional therapeutic agents include, for example, i) immune checkpoint inhibitors, ii) cancer chemotherapy drugs, and iii) one or more other TMPs or dimerized TMPs.Suitable additional therapies include, for example, radiation therapy, surgery (e.g., surgical removal of tumor), etc.

[0271] In some cases, the methods include administering to an individual in need thereof a) a first composition comprising TMP or dimerized TMP, and b) a second composition comprising an immune checkpoint inhibitor.In some cases, the methods include administering to an individual in need thereof a) a first composition comprising TMP or dimerized TMP, and b) a second composition comprising another TMP or dimerized TMP.

[0272] TMP or dimerized TMP can be administered to an individual in need thereof at the same time that one or more additional therapeutic agents are administered, or at different times.

[0273] Thus, for example, a method of treatment can include administering TMP or dimerized TMP and at least one additional therapeutic agent together. By "administering together" is meant administering both TMP or dimerized TMP and at least one additional therapeutic agent to an individual (not necessarily simultaneously) to achieve a certain therapeutic effect. The administration of TMP or dimerized TMP and at least one additional therapeutic agent can be substantially simultaneous, for example, TMP or dimerized TMP can be administered to an individual within about 1 minute to about 24 hours (e.g., within about 1 minute, within about 5 minutes, within about 15 minutes, within about 30 minutes, within about 1 hour, within about 4 hours, within about 8 hours, within about 12 hours, or within about 24 hours) of the administration of the at least one additional therapeutic agent. In some cases, TMP or dimerized TMP is administered to an individual who is undergoing or has undergone treatment with at least one additional therapeutic agent. The administration of TMP or dimerized TMP and at least one additional therapeutic agent can be performed at different times and / or at different frequencies.

[0274] As another specific example, the method of treatment can include administering TMP or dimerized TMP together with an immune checkpoint inhibitor (e.g., an antibody specific for an immune checkpoint). "Administering together" means administering both TMP or dimerized TMP and an antibody specific for an immune checkpoint to an individual (not necessarily simultaneously) to achieve a certain therapeutic effect. The administration of TMP or dimerized TMP and an antibody specific for an immune checkpoint can be performed substantially simultaneously, for example, TMP or dimerized TMP can be administered to an individual within about 1 minute to about 24 hours (e.g., within about 1 minute, within about 5 minutes, within about 15 minutes, within about 30 minutes, within about 1 hour, within about 2 hours, within about 4 hours, within about 8 hours, within about 12 hours, or within about 24 hours) of the administration of the antibody specific for an immune checkpoint. Alternatively, TMP or dimerized TMP and an immune checkpoint inhibitor can be administered on different schedules, for example, on different days, different weeks, different frequencies, etc. In some cases, the TMP or dimerized TMP is administered to an individual who is undergoing or has undergone treatment with an antibody specific for an immune checkpoint.

[0275] Specific immune checkpoint inhibitors include inhibitors that target immune checkpoint polypeptides such as CD27, CD28, CD40, CD122, CD96, CD73, CD47, OX40, GITR, CSF1R, JAK, PI3K delta, PI3K gamma, TAM, arginase, CD137 (also known as 4-1BB), ICOS, A2AR, B7-H3, B7-H4, BTLA, CTLA-4, LAG3, TIM3, VISTA, CD96, TIGIT, CD122, PD-1, PD-L1, and PD-L2. In some cases, the immune checkpoint polypeptide is a stimulatory checkpoint molecule selected from CD27, CD28, CD40, ICOS, OX40, GITR, CD122, and CD137. In some cases, the immune checkpoint polypeptide is an inhibitory checkpoint molecule selected from A2AR, B7-H3, B7-H4, BTLA, CTLA-4, IDO, KIR, LAG3, PD-1, TIM3, CD96, TIGIT, and VISTA.

[0276] In some cases, the immune checkpoint inhibitor is an antibody specific for an immune checkpoint. Suitable anti-immune checkpoint antibodies include, but are not limited to, the following: Nivolumab (Bristol-Myers Squibb), pembrolizumab (Merck), pidilizumab (Curetech), AMP-224 (GlaxoSmithKline / Amplimmune), MPDL3280A (Roche), MDX-1105 (Medarex,Inc. / Bristol Myer Squibb), MEDI-4736 (Medimmune / AstraZeneca), allerumab (Merck Serono), ipilimumab (YERVOY, (Bristol-Myers Squibb)), tremelimumab (Pfizer), pidilizumab (CureTech,Ltd.), IMP321 (Immutep SA), MGA271 (Macrogenics), BMS-986016 (Bristol-Myers Squibb), lirilumab (Bristol-Myers Squibb), urelumab (Bristol-Meyers Squibb), PF-05082566 (Pfizer), IPH2101 (Innate Pharma / Bristol-Myers Squibb), MEDI-6469 (MedImmune / AZ), CP-870893 (Genentech), mogamulizumab (Kyowa Hakko Kirin), varlilumab (CelIDex Therapeutics), avelumab (EMD Serono), galiximab (Biogen Idec), AMP-514 (Amplimmune / AZ), AUNP 12 (Aurigene and Pierre Fabre), indoximod (NewLink Genetics), NLG-919 (NewLink Genetics), INCB024360 (Incyte), KN035, and combinations thereof. For example, in some cases, the immune checkpoint inhibitor is an anti-PD-1 antibody. Suitable anti-PD-1 antibodies include, for example, nivolumab, pembrolizumab (also known as MK-3475), pidilizumab, SHR-1210, PDR001, and AMP-224.In some cases, the anti-PD-1 monoclonal antibody is nivolumab, pembrolizumab or PDR001. Suitable anti-PD1 antibodies are described in US Patent Publication No. 2017 / 0044259. For pidilizumab, see, for example, Rosenblatt et al. (2011) J. Immunother. 34: 409-18. In some cases, the immune checkpoint inhibitor is an anti-CTLA-4 antibody. In some cases, the anti-CTLA-4 antibody is ipilimumab or tremelimumab. For tremelimumab, see, for example, Ribas et al. (2013) J. Clin. Oncol. 31: 616-22. In some cases, the immune checkpoint inhibitor is an anti-PD-L1 antibody. In some cases, the anti-PD-L1 monoclonal antibody is BMS-935559, MEDI4736, MPDL3280A (also known as RG7446), KN035, or MSB0010718C. In some cases, the anti-PD-L1 monoclonal antibody is MPDL3280A (atezolizumab) or MEDI4736 (durvalumab). For durvalumab, see, e.g., WO2011 / 066389. For atezolizumab, see, e.g., U.S. Patent No. 8,217,149.

[0277] Of these checkpoint inhibitors, antibodies against PD-1, PD-L1, and CTLA-4 are the most common, with at least nivolumab, tremelimumab, pembrolizumab, ipilimumab, cemiplimab, atezolizumab, avelumab, tislelizumab, and durvalumab approved by the FDA and / or regulatory agencies outside the U.S. The TMPs and dimerized TMPs of the present disclosure can also be administered with a combination of multiple checkpoint inhibitors, for example, a combination of i) an antibody against PD-1 or PD-L1 and (ii) an antibody against CTLA-4.

[0278] In some cases, the at least one additional therapeutic agent comprises one or more additional TMPs or dimerized TMPs. In some cases, the method comprises administering to an individual in need thereof a) a first composition comprising a first TMP, and b) a second composition comprising a second TMP, wherein the second TMP is a different TMP than the first TMP, e.g., comprises a different peptide epitope and / or one or more different MODs.

[0279] Combination Therapy for Treating Viral Infections In some cases, the method includes administering to an individual in need of treatment a) a first composition comprising TMP or dimerized TMP, and b) a second composition comprising a second antiviral therapeutic agent. Antiviral agents are known in the art, and any known antiviral agent can be used as the second therapeutic agent.

[0280] Suitable subjects for treatment Subjects suitable for treatment with the methods of the present disclosure include individuals with cancer, including, for example, individuals who have been diagnosed with cancer, individuals who have been treated for cancer but have not been effective, and individuals who have been treated for cancer that was initially effective but has subsequently become resistant to the treatment. Subjects suitable for treatment include individuals with cancer whose cancer cells express or overexpress a cancer-associated peptide, such as an AFP peptide, a WT1 peptide, an HPV peptide, a MUC1 peptide, a MAGE A4 peptide, a NY-ESO-1 peptide, a survivin peptide, a mesothelin peptide, or a viral peptide (e.g., a CMV peptide).

[0281] For example, when a TMP or dimerized TMP comprises an AFP peptide, subjects suitable for treatment with such a TMP or dimerized TMP include individuals with hepatocellular carcinoma, pancreatic cancer, gastric cancer, colorectal cancer, hepatoblastoma, or ovarian yolk sac tumor.

[0282] As another specific example, when a TMP or dimerized TMP comprises a WT-1 peptide, subjects suitable for treatment with such a TMP or dimerized TMP include individuals with leukemia, desmoplastic small round cell tumor, gastric cancer, colon cancer, lung cancer, breast cancer, germ cell tumor, ovarian cancer, uterine cancer, thyroid cancer, liver cancer, kidney cancer, Kaposi's sarcoma, sarcoma, hepatocellular carcinoma, Wilms' tumor, acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), non-small cell lung cancer (NSCLC), myeloma, pancreatic cancer, colorectal cancer, mesothelioma, soft tissue sarcoma, neuroblastoma, or nephroblastoma.

[0283] In another embodiment, when TMP or dimerized TMP comprises HPV peptide, the subjects suitable for treatment with such TMP or dimerized TMP include individuals with head and neck cancer, cervical cancer, prostate cancer, ovarian cancer, or reproductive cancer.When TMP or dimerized TMP comprises HPV peptide epitope, TMP or dimerized TMP can be administered to an individual in need of treatment to treat cervical cancer in the individual.When TMP or dimerized TMP comprises HPV peptide epitope, TMP or dimerized TMP can be administered to an individual in need of treatment to treat prostate cancer in the individual.When TMP or dimerized TMP comprises HPV peptide epitope, TMP or dimerized TMP can be administered to an individual in need of treatment to treat ovarian cancer in the individual.In some cases, TMP or dimerized TMP is administered to an individual who is infected with HPV and has atypical cells of unknown significance (ACUS). In some cases, the TMP or dimerized TMP is administered to an individual who is infected with HPV and has had an abnormal Pap smear. In some cases, the TMP or dimerized TMP is administered to an individual who is infected with HPV and has been diagnosed with a precursor to cervical cancer, such as a squamous intraepithelial lesion.

[0284] As another example, when a TMP or dimerized TMP comprises a MUC-1 peptide, subjects suitable for treatment with such a TMP or dimerized TMP include individuals with multiple myeloma, B cell lymphoma, breast cancer, lung cancer, ovarian cancer, pancreatic cancer, colorectal cancer, prostate cancer, renal cancer, acute myeloid leukemia, mesothelioma, thyroid cancer, head and neck cancer, gastric cancer, urothelial cancer, cervical cancer, or ovarian endometrial cancer.

[0285] As another specific example, when the TMP or dimerized TMP comprises a MAGE A4 peptide, subjects suitable for treatment with such a TMP or dimerized TMP include individuals with melanoma, bladder cancer, head and neck cancer, lung cancer, esophageal cancer, breast cancer, colon cancer, or ovarian cancer.

[0286] As another specific example, when the TMP or dimerized TMP comprises the NY-ESO-1 peptide, subjects suitable for treatment with such a TMP or dimerized TMP include individuals with lung cancer, esophageal cancer, breast cancer, pancreatic cancer, colorectal cancer, or ovarian cancer.

[0287] As another specific example, when a TMP or dimerized TMP comprises a survivin peptide, subjects suitable for treatment with such a TMP or dimerized TMP include individuals with esophageal cancer (e.g., esophageal squamous cell carcinoma), melanoma, breast cancer, or leukemia.

[0288] As another specific example, when a TMP or dimerized TMP comprises a mesothelin peptide, subjects suitable for treatment with such a TMP or dimerized TMP include individuals with mesothelioma, pancreatic cancer, ovarian cancer, or lung adenocarcinoma.

[0289] In some cases, the subject is an individual undergoing treatment with an immune checkpoint inhibitor. In some cases, the subject is an individual who has undergone treatment with an immune checkpoint inhibitor, but whose disease has progressed despite such treatment. In some cases, the subject is an individual undergoing or has undergone treatment with a cancer chemotherapy drug. In some cases, the subject is an individual preparing to undergo treatment with an immune checkpoint inhibitor, or an individual undergoing or has undergone treatment with an immune checkpoint inhibitor. In some cases, the subject is an individual preparing to undergo, undergoing, or having undergone treatment with a cancer chemotherapy drug, radiation therapy, surgery, and / or another therapeutic agent.

[0290] Detection Method TMP or dimerized TMP is useful for diagnostic and therapeutic applications. As described below, when used for diagnostic applications, TMP or dimerized TMP can also contain a detectable label, and the binding of TMP or dimerized TMP to target T cells is detected by detecting this detectable label. When used for such diagnostic applications, TMP or dimerized TMP can be free of one or more MODs, in which case the TMP or dimerized TMP without MODs is called an antigen-presenting polypeptide (APP), which in this case presents a peptide epitope. Like TMP, APP can also dimerize. Therefore, the following discussion of detection methods using TMP and dimerized TMP is considered to be equally applicable to the use of APP and dimerized APP.

[0291] The present disclosure provides a method of detecting the presence and / or activation of an antigen-specific T cell, the method comprising contacting a T cell with TMP / APP or dimerized TMP / APP of the present disclosure and detecting binding of the TMP / APP or dimerized TMP / APP to the T cell and / or activation of the T cell. The present disclosure provides a method of detecting an antigen-specific T cell, the method comprising contacting a T cell with TMP / APP or dimerized TMP / APP of the present disclosure, wherein binding of the TMP / APP or dimerized TMP / APP to the T cell indicates that the T cell is specific for a peptide epitope present in TMP / APP or dimerized TMP / APP, i.e., the T cell has a T cell receptor specific for a peptide epitope present in TMP / APP or dimerized TMP / APP.

[0292] In some cases, TMP / APP or dimerized TMP / APP comprises a detectable label. Suitable detectable labels include, but are not limited to, radioisotopes, fluorescent polypeptides, or enzymes that produce fluorescent products and enzymes that produce colored products. When TMP / APP or dimerized TMP / APP comprises a detectable label, binding of TMP / APP or dimerized TMP / APP to T cells is detected by detecting the detectable label.

[0293] In some cases, TMP / APP or dimerized TMP / APP comprises a detectable label suitable for use in in vivo imaging. Such labels include, for example, detectable labels suitable for use in positron emission tomography (PET), single photon emission computed tomography (SPECT), near infrared (NIR) optical imaging, X-ray imaging, computer-assisted tomography (CAT), or magnetic resonance imaging (MRI), or other in vivo imaging methods. Specific examples of labels suitable for in vivo imaging include gadolinium chelates (e.g., gadolinium chelates with DTPA (diethylenetriaminepentaacetic acid), DTPA-bismethylamide (BMA), DOTA (dodecanetetraacetic acid), or HP-DO3A (1,4,7-tris(carboxymethyl)-10-(2'-hydroxypropyl)-1,4,7,10-tetraazacyclododecane)), iron chelates, magnesium chelates, manganese chelates, copper chelates, chromium chelates, iodine-based materials, and radionuclides. Suitable radionuclides include: 123 I, 125 I, 130 I, 131 I, 133 I, 135 I, 47 Sc, 72 As, 72 Se, 90 Y, 88 Y, 97 Ru, 100 Pd, 101 mRh, 119 Sb, 128 Ba, 197 Hg, 211 At, 212 Bi, 212 Pb, 109 Pd, 111 In, 67 Ga, 68 Ga, 64 Cu, 67 Cu, 75 Br, 77 Br, 99 mTc, 14 C. 13 N, 15 O. 32 P, 33 P, and18 In some cases, the detectable label includes, but is not limited to, 11 C. 13 N, 15 O. 18 F, 64 Cu, 68 Ga, 78 Br, 82 Rb, 86 Y, 90 Y, 22 Na, 26 Al, 40 K, 83 Sr, 89 Zr, or 124 In some cases, the detectable label is a positron-emitting isotope such as I. 64 Cu. For example, see Woodham, Andrew et al., In vivo detection of antigen-specific CD8+ T cells by immuno-positron emission tomography, Nature Methods Articles (2020) https: / / doi.org / 10.1038 / s41592-020-0934-5.

[0294] Suitable fluorescent proteins include, but are not limited to, green fluorescent protein (GFP) or variants thereof, blue fluorescent variants of GFP (BFP), cyan fluorescent variants of GFP (CFP), yellow fluorescent variants of GFP (YFP), enhanced GFP (EGFP), enhanced CFP (ECFP), enhanced YFP (EYFP), GFPS65T, emerald, topaz (TYFP), Venus, citrine, mCitrine, GFPuv, destabilized EGFP (dEGFP), destabilized ECFP (dECFP), destabilized EYFP (dEYFP), mCFPm , Cerulean, T-Sapphire, CyPet, YPet, mKO, HcRed, t-HcRed, DsRed, DsRed2, DsRed-monomer, J-Red, dimer2, t-dimer2 (12), mRFP1, pocilloporin, Renilla GFP, Monster GFP, paGFP, Kaede protein and kindling protein, phycobiliproteins, and phycobiliprotein conjugates including B-phycoerythrin, R-phycoerythrin, and allophycocyanin. Other examples of fluorescent proteins include mHoneydew, mBanana, mOrange, dTomato, tdTomato, mTangerine, mStrawberry, mCherry, mGrapel, mRaspberry, mGrape2, and mPlum (Shaner et al. (2005) Nat. Methods 2:905-909). Any of a variety of fluorescent and colored proteins from Anthozoan species are suitable for use, for example, as described in Matz et al. (1999) Nature Biotechnol. 17:969-973.

[0295] Suitable enzymes include, but are not limited to, horseradish peroxidase (HRP), alkaline phosphatase (AP), β-galactosidase (GAL), glucose-6-phosphate dehydrogenase, β-N-acetylglucosaminidase, β-glucuronidase, invertase, xanthine oxidase, firefly luciferase, glucose oxidase (GO), and the like.

[0296] In some cases, binding of TMP / APP or dimerized TMP / APP to T cells is detected using a detectably labeled antibody specific for TMP / APP or dimerized TMP / APP. The antibody specific for TMP / APP or dimerized TMP / APP can contain a detectable label, such as a radioisotope, a fluorescent polypeptide, an enzyme that produces a fluorescent product, or an enzyme that produces a colored product.

[0297] In some cases, the detected T cells are present in a sample that contains a plurality of T cells. For example, the detected T cells may be present in a sample that contains 10 to 10 9 T cells, e.g., 10–10 2 pieces, 10 2 ~10 4 pieces, 10 4 ~10 6 pieces, 10 6 ~10 7 pieces, 10 7 ~10 8 pcs or 10 8 ~10 9 1 or 10 9 The antibody may be present in a sample containing more than one T cell.

[0298] HLA / peptide binding assay Whether a particular peptide (e.g., a peptide containing a cancer-associated epitope) binds to class I HLA (including an HLA heavy chain and a β2M polypeptide) and can effectively present the epitope to a TCR when bound to an HLA complex can be determined using any of a number of well-known methods. Assays include binding assays and T cell activation assays, including cell-based binding assays, biochemical binding assays, T cell activation assays, ELISPOT assays, cytotoxicity assays, and detection of antigen-specific T cells using peptide-HLA tetramers. Such assays are described in the published scientific literature as well as in published PCT application WO2020132138A1. The disclosure of this publication regarding specific binding assays is incorporated herein by reference, particularly including paragraphs

[0217] to

[0225] .

[0299] In another embodiment, multimers (e.g., tetramers) of peptide-HLA complexes are produced with fluorescent or heavy metal tags. The multimers can be used to identify and quantify specific T cells by flow cytometry (FACS) or mass cytometry (CyTOF). Detection of epitope-specific T cells provides direct evidence that peptide-bound HLA molecules can bind to specific TCRs on a subset of antigen-specific T cells. See, e.g., Klenerman et al. (2002) Nature Reviews Immunol. 2:263.

[0300] Specific Non-Limiting Aspects of the Disclosure Mode Set A The aspects including the embodiments of the subject matter described above may be useful alone or in combination with one or more other aspects or embodiments. Without limiting the above description, certain non-limiting aspects of the present disclosure are given below. As will be clear to those skilled in the art upon reading this disclosure, each of the individually numbered aspects may be used, or they may be combined with any of the preceding or succeeding individually numbered aspects. This supports all combinations of such aspects, and is not limited to the combinations of aspects set forth below.

[0301] Aspect 1 A single chain T cell modulating polypeptide (TMP) comprising: i) a peptide comprising an epitope expressed on a cancer cell or a virus, the peptide being about 4 to about 25 amino acids in length and being other than a KRAS peptide; ii) a first major histocompatibility complex (MHC) polypeptide; iii) a second MHC polypeptide; iv) at least one immunomodulatory polypeptide; Including, the first major histocompatibility complex (MHC) polypeptide is a β2-microglobulin polypeptide and the second MHC polypeptide is an MHC class I heavy chain polypeptide; Optionally, the TMP comprises an immunoglobulin (Ig) Fc polypeptide, and optionally comprises one or more peptide linkers linking one or more components of the TMP.

[0302] Aspect 2 The TMP according to embodiment 1, wherein said peptide is an alpha-fetoprotein (AFP) peptide.

[0303] Aspect 3 The AFP peptides are selected from the group consisting of AITRKMAAT (SEQ ID NO: 42), AYTKKAPQL (SEQ ID NO: 43), LLNQHACAV (SEQ ID NO: 44), KLVLDVAHV (SEQ ID NO: 45), FMNKFIYEI (SEQ ID NO: 46), SIPLFQVPE (SEQ ID NO: 47), LLNFTESRT (SEQ ID NO: 48), FVQEATYKF (SEQ ID NO: 49), ATYKEVSKM (SEQ ID NO: 50), KEVSKMVKD (SEQ ID NO: 51), RHNCFLAHK (SEQ ID NO: 52), ATAATCCQL (SEQ ID NO: 53), YIQESQALA (SEQ ID NO: 54), QLTS SELMAI (SEQ ID NO: 55), KLSQKFTKV (SEQ ID NO: 56), KELRESSLL (SEQ ID NO: 57), SLVVDETYV (SEQ ID NO: 58), ILLWAARYD (SEQ ID NO: 59), KIIPSCCKA (SEQ ID NO: 60), CRGDVLDCL (SEQ ID NO: 61), QQDTLSNKI (SEQ ID NO: 62), TMKQEFLINL (SEQ ID NO: 63), NLVKQKPQI (SEQ ID NO: 64), AVIADFSGL (SEQ ID NO: 65), LLACGEGAA (SEQ ID NO: 66), LACGEGAAD (SEQ ID NO: 67), KAPQLTSSEL (SEQ ID NO: 68) ), YICSQQDTL (SEQ ID NO: 69), TECCKLTTL (SEQ ID NO: 70), CTAEISLADL (SEQ ID NO: 71), VTKELRESSL (SEQ ID NO: 72), IMSYICSQQD (SEQ ID NO: 73), TRTFQAITV (SEQ ID NO: 74), FQKLGEYYL (SEQ ID NO: 75), RVAKGYQEL (SEQ ID NO: 76), SYQCTAEISL (SEQ ID NO: 77), KQEFLINLV (SEQ ID NO: 78), MKWVESIFL (SEQ ID NO: 79), PVGNPGVGQC (SEQ ID NO: 80), AADIIIGHL (SEQ ID NO: 81), QVPEPVTSC (SEQ ID NO: 82), TTLERGQCII (SEQ ID NO: 83), KMAATAATC (SEQ ID NO: 84), QAQGVALQTM (SEQ ID NO: 85), FQAITVTKL (SEQ ID NO: 86), LLEKCFQTE (SEQ ID NO: 87), VAYTKKAPQ (SEQ ID NO: 88), KYIQESQAL (SEQ ID NO: 89), GVALQTMKQ (SEQ ID NO: 90), GQEQEVCFA (SEQ ID NO: 91), SEEGRHNCFL (SEQ ID NO: 92), RHPFLYAPTI (SEQ ID NO: 93), TEIQKLVLDV (SEQ ID NO: 94), RRHPQLAVSV (SEQ ID NO: 95),A TMP according to embodiment 2 comprising an amino acid sequence selected from the group consisting of GEYYLQNAFL (SEQ ID NO: 96), NRRPCFSSLV (SEQ ID NO: 97), LQTMKQEFLI (SEQ ID NO: 98), IADFSGLLEK (SEQ ID NO: 99), GLLEKCCQGQ (SEQ ID NO: 100), TLSNKITEC (SEQ ID NO: 101), LQDGEKIMSY (SEQ ID NO: 102), GLFQKLGBY (SEQ ID NO: 103), NEYGIASILD (SEQ ID NO: 104), KMVKDALTAI (SEQ ID NO: 105), FLASFVHEY (SEQ ID NO: 106), and AQFVQEATY (SEQ ID NO: 107).

[0304] Aspect 4 The TMP according to embodiment 1, wherein said peptide is a Wilms Tumor-1 (WT-1) peptide.

[0305] Aspect 5 The WT-1 peptide epitope is selected from the group consisting of NLMNLGATL (SEQ ID NO: 152), NYMNLGATL (SEQ ID NO: 157), CMTWNQMNLGATLKG (SEQ ID NO: 117), WNQMNLGATLKGVAA (SEQ ID NO: 118), CMTWNYMNLGATLKG (SEQ ID NO: 119), WNYMNLGATLKGVAA (SEQ ID NO: 120), MTWNQMNLGATLKGV (SEQ ID NO: 121), TWNQMNLGATLKGVA (SEQ ID NO: 122), CMTWNLMNLGATLKG (SEQ ID NO: 123), MTWNLMNLGATLKGV ( SEQ ID NO: 124), TWNLMNLGATLKGVA (SEQ ID NO: 125), WNLMNLGATLKGVAA (SEQ ID NO: 126), MNLGATLK (SEQ ID NO: 127), MTWNYMNLGATLKGV (SEQ ID NO: 128), TWNYMNLGATLKGVA (SEQ ID NO: 129), CMTWNQMNLGATLKGVA (SEQ ID NO: 130), CMTWNLMNLGATLKGVA (SEQ ID NO: 131), CMTWNYMNLGATLKGVA (SEQ ID NO: 132), GYLRNPTAC (SEQ ID NO: 133), GALRNPTAL (SEQ ID NO: 134), Y ALRNPTAC (SEQ ID NO: 135), GLLRNPTAC (SEQ ID NO: 136), RYRPHPGAL (SEQ ID NO: 137), YQRPHPGAL (SEQ ID NO: 138), RLRPHPGAL (SEQ ID NO: 139), RIRPHPGAL (SEQ ID NO: 140), QFPNHSFKHEDPMGQ (SEQ ID NO: 141), HSFKHEDPY (SEQ ID NO: 142), QFPNHSFKHEDPM (SEQ ID NO: 143), QFPNHSFKHEDPY (SEQ ID NO: 144), KRPFMCAYPGCNK (SEQ ID NO: 145), KRPFMCAYPGCYK (SEQ ID NO: 146) ), FMCAYPGCY (SEQ ID NO: 147), FMCAYPGCK (SEQ ID NO: 148), KRPFMCAYPGCNKRY (SEQ ID NO: 149), SEKRPFMCAYPGCNK (SEQ ID NO: 150), KRPFMCAYPGCYKRY (SEQ ID NO: 151), NLMNLGATL (SEQ ID NO: 152), VLDFAPPGA (SEQ ID NO: 153), RMFPNAPYL (SEQ ID NO: 154), CMTWNQMN (SEQ ID NO: 155), CYTWNQMNL (SEQ ID NO: 156), NYMNLGATL (SEQ ID NO: 157), YMFPNAPYL (SEQ ID NO: 158),A TMP according to embodiment 4, comprising an amino acid sequence selected from the group consisting of SLGEQQYSV (SEQ ID NO: 159), CMTWNQMNL (SEQ ID NO: 160), and NQMNLGATL (SEQ ID NO: 161).

[0306] Aspect 6 The TMP according to embodiment 4, wherein the WT-1 peptide comprises the amino acid sequence CMTWNQMNL (SEQ ID NO: 160), CYTWNQMNL (SEQ ID NO: 156), NYMNLGATL (SEQ ID NO: 157), VLDFAPPGA (SEQ ID NO: 153), YMFPNAPYL (SEQ ID NO: 158), SLGEQQYSV (SEQ ID NO: 159), RMFPNAPYL (SEQ ID NO: 154), and NLMNLGATL (SEQ ID NO: 152).

[0307] Aspect 7 The first MHC polypeptide is a β2M polypeptide and the second MHC polypeptide is HLA-A * The TMP according to embodiment 4, comprising an amino acid sequence having at least 95% amino acid sequence identity to a 2402 polypeptide, and said epitope is selected from the group consisting of RMFPNAPYL (SEQ ID NO: 154), CYTWNQMNL (SEQ ID NO: 156), and NYMNLGATL (SEQ ID NO: 157).

[0308] Aspect 8 The first MHC polypeptide is a β2M polypeptide and the second MHC polypeptide is HLA-A * The TMP according to embodiment 4, comprising an amino acid sequence having at least 95% amino acid sequence identity to a 0201 polypeptide, and said epitope is selected from the group consisting of VLDFAPPGA (SEQ ID NO: 153), RMFPNAPYL (SEQ ID NO: 154), and YMFPNAPYL (SEQ ID NO: 158).

[0309] Aspect 9 The TMP according to embodiment 1, wherein said peptide is a human papillomavirus (HPV) peptide epitope.

[0310] Aspect 10 The HPV peptide epitopes are selected from the group consisting of E6 18 to 26 (KLPQLCTEL (SEQ ID NO: 162)), E6 26 to 34 (LQTTIHDII (SEQ ID NO: 163)), E6 49 to 57 (VYDFAFRDL (SEQ ID NO: 164)), E6 52 to 60 (FAFRDLCIV (SEQ ID NO: 165)), E6 75 to 83 (KFYSKISEY (SEQ ID NO: 166)), E6 80 to 88 (ISEYRHYCY (SEQ ID NO: 167)), E7 7 to 15 (TLHEYMLDL (SEQ ID NO: 168)), E7 11 to 19 (YMLDLQPET (SEQ ID NO: 169)), E7 44 to 52 (QAEPDRAHY (SEQ ID NO: 170)), E7 49 to 57 (RAHYNIVTF (SEQ ID NO: 171)), E7 The TMP according to embodiment 9, wherein E7 is 61 to 69 (CDSTLRLCV (SEQ ID NO: 172)), and E7 is 67 to 76 (LCVQSTHVDI (SEQ ID NO: 173)), E7 82 to 90 (LLMGTLGIV (SEQ ID NO: 174)), E7 86 to 93 (TLGIVCPI (SEQ ID NO: 175)), or E7 92 to 93 (LLMGTLGIVCPI (SEQ ID NO: 176)).

[0311] Aspect 11 The HPV peptide epitope is a) the amino acid sequence of HPV E6 VYDFAFRDL (SEQ ID NO: 164), or b) A TMP according to embodiment 9, comprising an HPV E7 amino acid sequence selected from the group consisting of RAHYNIVTF (SEQ ID NO: 171), CDSTLRLCV (SEQ ID NO: 172), and LCVQSTHVDI (SEQ ID NO: 173).

[0312] Aspect 12 The TMP according to embodiment 1, wherein said peptide is a MUC-1 peptide.

[0313] Aspect 13 The TMP according to embodiment 12, wherein said MUC-1 peptide comprises an amino acid sequence selected from the group consisting of STAPPAHGV (SEQ ID NO: 197), STAPPVHNV (SEQ ID NO: 198), SLAPPVHNV (SEQ ID NO: 199), SLAPPAHGV (SEQ ID NO: 200), SAPDTRPAP (SEQ ID NO: 201), VTSAPDTRPAPGSTAPPAHG (SEQ ID NO: 202), PDTRPAPGSTAPPAHGVTSA (SEQ ID NO: 203), and LLLLTVLTV (SEQ ID NO: 204).

[0314] Aspect 14 The TMP according to embodiment 1, wherein said peptide is a melanoma associated antigen-4 (MAGE-A4) peptide.

[0315] Aspect 15 The TMP according to embodiment 14, wherein said MAGE-A4 peptide comprises an amino acid sequence selected from GVYDGREHTV (SEQ ID NO: 206), NYKRCFPVI (SEQ ID NO: 207), EVDPASNTY (SEQ ID NO: 208), SESLKMIF (SEQ ID NO: 209), and SESLICMIF (SEQ ID NO: 210).

[0316] Aspect 16 The TMP according to embodiment 1, wherein said peptide is the NY-ESO-1 peptide.

[0317] Aspect 17 The TMP according to embodiment 16, wherein the NY-ESO-1 peptide comprises an amino acid sequence selected from SLLMWITQCFL (SEQ ID NO: 212), SLLMWITQC (SEQ ID NO: 213), QLSLLMWIT (SEQ ID NO: 214), and SLLMWITQCFLPVF (SEQ ID NO: 215).

[0318] Aspect 18 The TMP according to embodiment 1, wherein said peptide is a survivin peptide.

[0319] Aspect 19 The TMP according to embodiment 18, wherein said survivin peptide comprises an amino acid sequence selected from TLGEFLKLDRERAKN (SEQ ID NO: 229), QMFFCF (SEQ ID NO: 230), DLAQMFFCFKELEGW (SEQ ID NO: 231), AQMFFCFKEL (SEQ ID NO: 232), and QMFFCFKEL (SEQ ID NO: 233).

[0320] Aspect 20 The TMP according to embodiment 1, wherein said peptide is a mesothelin peptide.

[0321] Aspect 21 The TMP according to embodiment 20, wherein the mesothelin peptide comprises an amino acid sequence selected from SLLFLLFSL (SEQ ID NO: 235), VLPLTVAEV (SEQ ID NO: 236), ELAVALAQK (SEQ ID NO: 237), ALQGGGPPY (SEQ ID NO: 238), FYPGYLCSL (SEQ ID NO: 239), and LYPKARLAF (SEQ ID NO: 240).

[0322] Aspect 22 The TMP according to embodiment 1, wherein said peptide is a peptide of a viral antigen.

[0323] Aspect 23 The TMP according to embodiment 22, wherein said viral antigen is a cytomegalovirus (CMV) polypeptide.

[0324] Aspect 24 The TMP according to embodiment 23, wherein said CMV polypeptide is a CMVpp65 polypeptide.

[0325] Aspect 25 The TMP according to embodiment 24, wherein said peptide has the amino acid sequence NLVPMVATV (SEQ ID NO: 247) and is 9 amino acids in length.

[0326] Aspect 26 26. The TMP according to any one of aspects 1 to 25, wherein the β2M polypeptide and the MHC heavy chain polypeptide are linked by a disulfide bond, the disulfide bond linking a Cys residue of the β2M polypeptide to a Cys residue of the MHC heavy chain polypeptide, and optionally, a Cys at amino acid residue 12 of the β2M polypeptide is disulfide bonded to a Cys at amino acid residue 236 of the MHC heavy chain polypeptide.

[0327] Aspect 27 27. The TMP according to any one of aspects 1 to 26, wherein the β2-microglobulin polypeptide is attached to the peptide by a first linker comprising a Cys, and a disulfide bond links a Cys present in the first linker to a Cys present in the MHC heavy chain polypeptide, and optionally, the first linker comprises the sequence CGGGS(GGGGS)n (SEQ ID NO: 439) or GCGGS(GGGGS)n (SEQ ID NO: 436), where n is an integer from 1 to 10, e.g. 2 or 3, and a disulfide bond links a Cys in the linker to a Cys that replaces Tyr84 in the MHC heavy chain polypeptide.

[0328] Aspect 28 The MHC heavy chain polypeptide is HLA-A * 0201 Polypeptide, HLA-A * 1101 Polypeptide, HLA-A * 3303 Polypeptide, HLA-A * 28. The TMP according to any one of embodiments 1 to 27, comprising an amino acid sequence having at least 95% amino acid sequence identity to an HLA-A polypeptide selected from the group consisting of a 2401 polypeptide, an HLA-E polypeptide, and an HLA-G polypeptide.

[0329] Aspect 29 29. The TMP according to any one of aspects 1-28, wherein said at least one immunomodulatory polypeptide is a wild-type or mutant form of an activated immunomodulatory polypeptide selected from the group consisting of IL-2, 4-1BBL, CD80, CD86, or a combination thereof, and optionally wherein at least one of said at least one immunomodulatory polypeptide is a mutant immunomodulatory polypeptide, wherein said mutant immunomodulatory polypeptide exhibits a reduced affinity for a cognate costimulatory polypeptide compared to the affinity of a corresponding wild-type immunomodulatory polypeptide for said cognate costimulatory polypeptide.

[0330] Aspect 30 The TMP according to embodiment 29, wherein said at least one immune modulating polypeptide is an IL-2 mutant exhibiting reduced binding affinity to IL-2Rα and IL-2Rβ, and optionally said mutant IL-2 polypeptide comprises i) an H16A substitution and an F42A substitution, or ii) an H16T substitution and an F42A substitution.

[0331] Aspect 31 The TMP according to embodiment 29 or 30, comprising two copies of said immunomodulatory polypeptide.

[0332] Aspect 32 The TMP according to embodiment 31, wherein the two copies of said immunomodulatory polypeptide are in tandem, and optionally there is a peptide linker between said two copies.

[0333] Aspect 33 33. A TMP according to any one of embodiments 1 to 32, comprising an IgFc polypeptide.

[0334] Aspect 34 34. The TMP according to embodiment 33, wherein said IgFc polypeptide does not substantially induce cell lysis, and optionally said IgFc polypeptide comprises one or more amino acid substitutions selected from N297A, L234A, L235A, L234F, L235E, and P331S.

[0335] Aspect 35 The TMP according to embodiment 33 or embodiment 34, wherein said IgFc polypeptide comprises an amino acid sequence having at least 95% amino acid sequence identity to an amino acid sequence shown in any one of Figures 2A to 2M.

[0336] Aspect 36 From the N-terminus to the C-terminus, i) the peptide, ii) an optional peptide linker; iii) the β2M polypeptide; iv) an optional peptide linker; v) the MHC class I heavy chain polypeptide, vi) an optional peptide linker; vii) an IgFc polypeptide; viii) an optional peptide linker, and ix) A TMP according to any one of embodiments 1 to 35, comprising said one or more immunomodulatory polypeptides.

[0337] Aspect 37 From the N-terminus to the C-terminus, i) the peptide, ii) an optional peptide linker; iii) the β2M polypeptide; iv) an optional peptide linker; v) the MHC class I heavy chain polypeptide, vi) an optional peptide linker; vii) the one or more immunomodulatory polypeptides, viii) an optional peptide linker, and ix) A TMP according to any one of embodiments 1 to 35, comprising an IgFc polypeptide.

[0338] Aspect 38 Each of the optional peptide linkers, if present, is selected from the following: i) CGGGS(GGGGS)n (SEQ ID NO:439), where n is an integer from 1 to 10; ii) GCGGS(GGGGS)n (SEQ ID NO: 436), where n is an integer from 1 to 10; iii) (GGGGS)n(GCGGS) (SEQ ID NO: 447), where n is an integer from 1 to 10; iv) (GGGGS)n (SEQ ID NO:572), where n is an integer from 1 to 10; v) AAAGG (SEQ ID NO:411); vi) (AP)n (SEQ ID NO:584) (wherein n is an integer from 1 to 10); vii) (EP)n (SEQ ID NO:585) (wherein n is an integer from 1 to 10); viii) (KP)n (SEQ ID NO:586), where n is an integer from 1 to 10; and ix) Peptide containing EAAAK (SEQ ID NO: 412) The TMP according to any one of embodiments 1 to 37, independently selected from the group consisting of:

[0339] Aspect 39 Each of the optional peptide linkers, if present, is selected from the following: i) CGGGS(GGGGS)n (SEQ ID NO:588), where n is 2, 3, or 4; ii) GCGGS(GGGGS)n (SEQ ID NO:589), where n is 2, 3, or 4; iii) (GGGGS)n(GCGGS) (SEQ ID NO:590), where n is 2, 3, or 4; iv) (GGGGS)n (SEQ ID NO:591), where n is 2, 3, or 4; v) AAAGG (SEQ ID NO:411); vi) (AP)n (SEQ ID NO:592) (wherein n is 2 to 10); vii) (EP)n (SEQ ID NO:593) (wherein n is 2 to 10); viii) (KP)n (SEQ ID NO:594) (wherein n is 2 to 10); and ix) AEAAAKEAAAKA (SEQ ID NO: 419) The TMP according to any one of embodiments 1 to 37, independently selected from the group consisting of:

[0340] Aspect 40 the β2M polypeptide is linked to the peptide by a first linker comprising the sequence CGGGS(GGGGS)n (SEQ ID NO:439) or GCGGS(GGGGS)n (SEQ ID NO:436), where n is an integer between 1 and 10, e.g., 2 or 3; the MHC heavy chain polypeptide has a Cys at residue 84 and a Cys at residue 236; the β2M polypeptide has a Cys at residue 12, Cys at amino acid residue 12 of the β2M polypeptide is disulfide bonded to Cys at amino acid residue 236 of the MHC heavy chain polypeptide; a disulfide bond links the Cys of the linker to the Cys that replaces Tyr84 of the MHC heavy chain polypeptide; the β2M polypeptide is linked to the MHC heavy chain polypeptide by a (GGGGS)n linker (SEQ ID NO:572), where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, e.g., n is 3 or 7; The MHC heavy chain polypeptide is HLA-A * 0201 Polypeptide, HLA-A * 1101 Polypeptide, HLA-A * 3303 polypeptide, and HLA-A * 2401 polypeptides, the at least one immunomodulatory polypeptide is an IL-2 variant comprising i) an H16A substitution and an F42A substitution, or ii) an H16T substitution and an F42A substitution; and 38. The TMP according to embodiment 36 or embodiment 37, wherein said polypeptide comprises two identical immunomodulatory polypeptides arranged in tandem, said two immunomodulatory polypeptides comprising an IL-2 variant comprising i) an H16A substitution and an F42A substitution or ii) an H16T substitution and an F42A substitution.

[0341] Aspect 41 39. The TMP according to any one of aspects 1 to 39, wherein the TMP comprises an amino acid sequence as shown in any one of Figures 13A to 13D and Figures 18A to 18T, (X) is a peptide, and (X) is a peptide according to any one of aspects 2 to 25.

[0342] Aspect 42 42. The TMP according to any one of aspects 1-41, wherein the TMP comprises: (a) at least one rigid peptide linker (optionally, the TMP comprises a rigid peptide linker between the IgFc polypeptide and one of the one or more immunomodulatory polypeptides); and / or (b) at least one short flexible peptide linker consisting of 2 to 14 amino acids (optionally, the TMP comprises a short flexible peptide linker between the IgFc polypeptide and one of the one or more immunomodulatory polypeptides).

[0343] Aspect 43 the TMP comprises at least one rigid peptide linker; and Each rigid peptide linker is selected from the group consisting of: i) (AP)n (SEQ ID NO:584) (wherein n is an integer from 1 to 10); ii) (EP)n (SEQ ID NO:585), where n is an integer from 1 to 10; iii) (KP)n (SEQ ID NO:586), where n is an integer from 1 to 10; and iv) Peptide containing EAAAK (SEQ ID NO: 412) The TMP according to embodiment 42, independently selected from the group consisting of:

[0344] Aspect 44 the TMP comprises at least one rigid peptide linker; and Each rigid peptide linker is selected from the group consisting of: i) (AP)n (SEQ ID NO:595) (wherein n is 2 to 6); ii) (EP)n (SEQ ID NO:596) (wherein n is 2 to 6); iii) (KP)n (SEQ ID NO:597) (wherein n is 2 to 6); and iv) A(EAAAK)nA (SEQ ID NO: 598) (wherein n is 1 or 2) The TMP according to embodiment 42, independently selected from the group consisting of:

[0345] Aspect 45 i) a homodimer comprising two identical TMPs according to any one of aspects 1 to 44, optionally wherein the two TMPs are linked to each other by one or more disulfide bonds linking an immunoglobulin (Ig) Fc polypeptide of one TMP to an IgFc polypeptide of the other TMP; or (ii) A heterodimer comprising two different TMPs according to any one of embodiments 1 to 44, optionally wherein the two TMPs are linked to each other by an interspecies linker sequence. A protein comprising any one of the following:

[0346] Aspect 46 A pharmaceutical composition comprising a TMP according to any one of aspects 1 to 44.

[0347] Aspect 47 A pharmaceutical composition comprising a protein according to embodiment 45.

[0348] Aspect 48 A nucleic acid comprising a nucleotide sequence encoding a TMP according to any one of embodiments 1 to 44.

[0349] Aspect 49 1. A method for selectively modulating the activity of T cells specific for a cancer-associated peptide epitope, comprising: contacting said T cells with a TMP according to any one of aspects 1 to 44 or a protein according to aspect 45 Including, The method, wherein the contacting selectively modulates activity of the epitope-specific T cells.

[0350] Aspect 50 1. A method of treating cancer in a patient having cancer, comprising: administering to said patient an effective amount of a pharmaceutical composition comprising a TMP according to any one of embodiments 1 to 44, a protein according to embodiment 45, or a pharmaceutical composition according to embodiment 46 or embodiment 47. The method comprising:

[0351] Aspect 51 The method according to embodiment 50, further comprising the step of co-administering to said patient an immune checkpoint inhibitor, optionally wherein said immune checkpoint inhibitor is an antibody specific for PD-L1, PD-1, or CTLA4.

[0352] Aspect 52 1. A method of treating an infectious disease in a patient having said infectious disease, comprising: administering to said patient an effective amount of a pharmaceutical composition comprising a TMP according to any one of aspects 1, 9 to 11 and 22 to 44, a protein according to aspect 45, or a pharmaceutical composition according to aspect 46 or aspect 47. A method comprising:

[0353] Aspect 52 1. A method of treating an infectious disease in a patient having said infectious disease, comprising: administering to said patient an effective amount of a pharmaceutical composition comprising a TMP according to any one of aspects 1, 9 to 11 and 22 to 44, a protein according to aspect 45, or a pharmaceutical composition according to aspect 46 or aspect 47. The method comprising:

[0354] Aspect 53 1. A method for increasing the thermal stability of a T cell modulating polypeptide (TMP), comprising: The TMP may be selected from the group consisting of: i) a peptide epitope expressed on a cancer cell or a virus, the peptide epitope being about 4 to about 20 amino acids in length; ii) a first major histocompatibility complex (MHC) polypeptide which is a β2-microglobulin (β2M) polypeptide; iii) a second MHC polypeptide which is an MHC class I heavy chain polypeptide; iv) one or more immunomodulatory polypeptides (MODs); v) immunoglobulin (Ig) Fc polypeptide; Including, When the TMP comprises one or more MODs of configuration 2, the method comprises providing a rigid peptide linker or a short flexible linker between any one or more of: (i) the MHC class I heavy chain polypeptide and the MODs, and (ii) the IgFc polypeptide and the MODs, and optionally, when two or more MODs are present, between at least two of the two or more MODs; When the TMP contains one or more MODs of configuration 3, the method includes the step of providing a rigid peptide linker or a short flexible linker between the IgFc polypeptide and the MOD, and optionally, when two or more MODs are present, between at least two of the two or more MODs.

[0355] Aspect 54 Each rigid peptide linker is selected from the group consisting of: i) (AP)n (SEQ ID NO:584) (wherein n is an integer from 1 to 10); ii) (EP)n (SEQ ID NO:585), where n is an integer from 1 to 10; iii) (KP)n (SEQ ID NO:586), where n is an integer from 1 to 10; and iv) Peptide containing EAAAK (SEQ ID NO: 412) The method according to embodiment 53, wherein the compound is independently selected from the group consisting of:

[0356] Aspect 55 the TMP comprises at least one rigid peptide linker; Each rigid peptide linker is selected from the group consisting of: i) (AP)n (SEQ ID NO:595) (wherein n is 2 to 6); ii) (EP)n (SEQ ID NO:596) (wherein n is 2 to 6); iii) (KP)n (SEQ ID NO:597) (wherein n is 2 to 6); and iv) A(EAAAK)nA (SEQ ID NO: 598) (wherein n is 1 or 2) The method according to embodiment 53, wherein the compound is independently selected from the group consisting of:

[0357] Aspect 56 The method according to embodiment 53, wherein each short flexible peptide linker is independently selected from the group consisting of flexible peptide linkers comprising a number of amino acids selected from the group consisting of 2-4, 2-5, 3-6, 4-8, 5-10, and 10-14.

[0358] Aspect 57 54. The method according to embodiment 53, wherein each short flexible peptide linker is independently selected from the group consisting of 2 to 4 amino acids, 2 to 5 amino acids, 3 to 6 amino acids, and 4 to 8 amino acids, and optionally said short flexible peptide linker is GGS.

[0359] Aspect 58 The method according to embodiment 53, wherein said peptide epitope is an AFP peptide, a WT1 peptide, an HPV peptide, a MUC1 peptide, a MAGE A4 peptide, a NY-ESO-1 peptide, a survivin peptide, a mesothelin peptide, or a viral peptide.

[0360] Aspect 59 The method according to any one of aspects 49 to 58, wherein the protein is a homodimer comprising two identical TMPs according to any one of aspects 1 to 44, and optionally the two TMPs are linked to each other by one or more disulfide bonds linking the immunoglobulin (Ig) Fc polypeptide of one TMP to the IgFc polypeptide of the other TMP.

[0361] Mode Set B The aspects including the embodiments of the subject matter described above may be useful alone or in combination with one or more other aspects or embodiments. Without limiting the above description, certain non-limiting aspects of the present disclosure are given below. As will be clear to those skilled in the art upon reading this disclosure, each of the individually numbered aspects may be used, or they may be combined with any of the preceding or succeeding individually numbered aspects. This supports all combinations of such aspects, and is not limited to the combinations of aspects set forth below.

[0362] Aspect 1 A single chain T cell modulating polypeptide (TMP) comprising: i) a peptide comprising an epitope expressed on a cancer cell, the peptide being about 7 to about 16 amino acids in length, optionally 8 to 12 amino acids in length, and other than a KRAS peptide; ii) a first major histocompatibility complex (MHC) polypeptide; iii) a second MHC polypeptide; iv) at least one immunomodulatory polypeptide; Including, the first major histocompatibility complex (MHC) polypeptide is a β2-microglobulin polypeptide and the second MHC polypeptide is an MHC class I heavy chain polypeptide; Optionally, the TMP comprises an immunoglobulin (Ig) Fc polypeptide, and optionally comprises one or more peptide linkers linking one or more components of the TMP.

[0363] Aspect 2 The TMP according to embodiment 1, wherein said peptide is an alpha-fetoprotein (AFP) peptide, a Wilms' tumor-1 (WT-1) peptide, a human papillomavirus (HPV) peptide epitope, a MUC-1 peptide, a melanoma associated antigen-4 (MAGE-A4) peptide, a NY-ESO-1 peptide, a survivin peptide, a mesothelin peptide, or a peptide of a viral antigen.

[0364] Aspect 3 (a) the β2M polypeptide and the MHC heavy chain polypeptide are linked by a disulfide bond, the disulfide bond linking a Cys residue of the β2M polypeptide to a Cys residue of the MHC heavy chain polypeptide, and optionally, a Cys at amino acid residue 12 of the β2M polypeptide is disulfide bonded to a Cys at amino acid residue 236 of the MHC heavy chain polypeptide; (b) the β2-microglobulin polypeptide is linked to the peptide by a first linker comprising a Cys, and a disulfide bond links a Cys present in the first linker to a Cys present in the MHC heavy chain polypeptide, optionally wherein the first linker comprises the sequence CGGGS(GGGGS)n (SEQ ID NO: 439) or GCGGS(GGGGS)n (SEQ ID NO: 436), where n is an integer between 1 and 10, e.g., 2 or 3, and a disulfide bond links a Cys in the linker to a Cys that replaces Tyr84 in the MHC heavy chain polypeptide; or (c) the β2M polypeptide and the MHC heavy chain polypeptide are linked by a disulfide bond, the disulfide bond linking a Cys residue of the β2M polypeptide to a Cys residue of the MHC heavy chain polypeptide, and optionally, a Cys at amino acid residue 12 of the β2M polypeptide is disulfide bonded to a Cys at amino acid residue 236 of the MHC heavy chain polypeptide, and the β2-microglobulin polypeptide is connected to the peptide by a first linker comprising a Cys. 2. The TMP according to embodiment 1, wherein said first linker is linked to a Cys present in said MHC heavy chain polypeptide and a disulfide bond links a Cys present in said first linker to a Cys present in said MHC heavy chain polypeptide, optionally wherein said first linker comprises the sequence CGGGS(GGGGS)n (SEQ ID NO: 439) or GCGGS(GGGGS)n (SEQ ID NO: 436), where n is an integer from 1 to 10, e.g. 2 or 3, and a disulfide bond links a Cys in said linker to a Cys that replaces Tyr84 in said MHC heavy chain polypeptide.

[0365] Aspect 4 The MHC heavy chain polypeptide is HLA-A * 0201 Polypeptide, HLA-A * 1101 Polypeptide, HLA-A * 3303 Polypeptide, HLA-A * 2. The TMP according to embodiment 1, comprising an amino acid sequence having at least 95% amino acid sequence identity to an HLA-A polypeptide selected from the group consisting of a 2401 polypeptide, an HLA-E polypeptide, and an HLA-G polypeptide.

[0366] Aspect 5 2. The TMP according to embodiment 1, wherein the at least one immunomodulatory polypeptide is a wild-type or mutant form of an activating immunomodulatory polypeptide selected from the group consisting of IL-2, 4-1BBL, CD80, CD86, or a combination thereof, and optionally at least one of the at least one immunomodulatory polypeptides is a mutant immunomodulatory polypeptide, wherein the mutant immunomodulatory polypeptide exhibits a reduced affinity for a cognate costimulatory polypeptide compared to the affinity of a corresponding wild-type immunomodulatory polypeptide for the cognate costimulatory polypeptide.

[0367] Aspect 6 The TMP according to embodiment 5, wherein said at least one immune modulating polypeptide is an IL-2 mutant exhibiting reduced binding affinity to IL-2Rα and IL-2Rβ, and optionally said mutant IL-2 polypeptide comprises i) an H16A substitution and an F42A substitution, or ii) an H16T substitution and an F42A substitution.

[0368] Aspect 7 2A-2M。 The TMP according to embodiment 1, comprising an IgFc polypeptide, optionally wherein said IgFc polypeptide comprises an amino acid sequence having at least 95% amino acid sequence identity to any one of the amino acid sequences shown in Figures 2A-2M.

[0369] Aspect 8 The TMP according to embodiment 7, wherein said IgFc polypeptide does not substantially induce cell lysis, and optionally said IgFc polypeptide comprises one or more amino acid substitutions selected from N297A, L234A, L235A, L234F, L235E, and P331S.

[0370] Aspect 9 From the N-terminus to the C-terminus, i) the peptide, ii) an optional peptide linker; iii) the β2M polypeptide; iv) an optional peptide linker; v) the MHC class I heavy chain polypeptide, vi) an optional peptide linker; vii) an IgFc polypeptide; viii) an optional peptide linker, and ix) the one or more immunomodulatory polypeptides. Contains; or i) the peptide, ii) an optional peptide linker; iii) the β2M polypeptide; iv) an optional peptide linker; v) the MHC class I heavy chain polypeptide, vi) an optional peptide linker; vii) the one or more immunomodulatory polypeptides, viii) an optional peptide linker, and ix) IgFc polypeptide The TMP according to embodiment 1, comprising:

[0371] Aspect 10 Each of the optional peptide linkers, if present, is selected from the following: i) CGGGS(GGGGS)n, where n is an integer from 1 to 10, and optionally n is 2, 3, or 4; ii) GCGGS(GGGGS)n, where n is an integer from 1 to 10, and optionally n is 2, 3, or 4; iii) (GGGGS)n(GCGGS), where n is an integer from 1 to 10, and optionally n is 2, 3, or 4; iv) (GGGGS)n, where n is an integer from 1 to 10, and optionally n is 2, 3, 4, or 7; v)AAAGG; vi) (AP)n, where n is an integer from 1 to 10, and optionally n is from 2 to 10; vii) (EP)n, where n is an integer from 1 to 10, and optionally n is from 2 to 10; viii) (KP)n, where n is an integer from 1 to 10, and optionally n is from 2 to 10; and ix) Peptides containing EAAAK, and optionally AEAAAKEAAAKA The TMP according to embodiment 1, independently selected from the group consisting of:

[0372] Aspect 11 The TMP according to embodiment 1, wherein said TMP comprises an amino acid sequence as shown in any one of FIGS. 13A-13D and 18A-18T, and (X) is a peptide according to embodiment 2.

[0373] Aspect 12 (a) at least one rigid peptide linker (optionally, the TMP comprises a rigid peptide linker between the IgFc polypeptide and one of the one or more immunomodulatory polypeptides); and / or (b) at least one short flexible peptide linker comprising 2 to 14 amino acids (optionally, the TMP comprises a short flexible peptide linker between the IgFc polypeptide and one of the one or more immune modulating polypeptides). The TMP according to embodiment 1, comprising:

[0374] Aspect 13 the TMP comprises at least one rigid peptide linker; Each rigid peptide linker is selected from the group consisting of: i) (AP)n, where n is an integer from 1 to 10; ii) (EP)n, where n is an integer from 1 to 10; iii) (KP)n, where n is an integer from 1 to 10; and iv) Peptide containing EAAAK 13. The TMP according to embodiment 12, independently selected from the group consisting of:

[0375] Aspect 14 below: (i) a homodimer comprising two identical TMPs according to any one of embodiments 1 to 13, optionally wherein the two TMPs are linked to each other by one or more disulfide bonds linking an immunoglobulin (Ig) Fc polypeptide of one TMP to an IgFc polypeptide of the other TMP; or (ii) A heterodimer comprising two different TMPs according to any one of embodiments 1 to 13, optionally wherein the two TMPs are linked to each other by an interspecies linkage sequence. TMP, including any of the following:

[0376] Aspect 15 A pharmaceutical composition comprising a TMP according to any one of aspects 1 to 13.

[0377] Aspect 16 A nucleic acid comprising a nucleotide sequence encoding a TMP according to any one of embodiments 1 to 13.

[0378] Aspect 17 1. A method for selectively modulating the activity of T cells specific for a cancer-associated peptide epitope, comprising: contacting said T cells with a TMP according to any one of embodiments 1 to 13. Including, The method, wherein the contacting selectively modulates activity of the epitope-specific T cells.

[0379] Aspect 18 1. A method of treating cancer in a patient having cancer, comprising: administering to said patient an effective amount of a pharmaceutical composition according to embodiment 15. The method comprising:

[0380] Aspect 19 The method according to embodiment 18, further comprising the step of co-administering to said patient one or more immune checkpoint inhibitors, optionally wherein said one or more immune checkpoint inhibitors comprise an antibody specific for PD-L1, PD-1, CTLA4, or TIGIT.

[0381] Although the present disclosure has been described with reference to specific embodiments thereof, it will be apparent to those skilled in the art that various modifications can be made and equivalents substituted without departing from the essential spirit and scope of the disclosure. In addition, many modifications can be made to adapt a particular situation, material, composition, method, method step or steps to the objective, spirit and scope of the present disclosure. All such modifications are intended to be within the scope of the claims appended hereto.

Claims

1. A single chain T cell modulating polypeptide (TMP) comprising: i) a peptide comprising an epitope expressed on a cancer cell, the peptide being between 7 and 16 amino acids in length and being other than a KRAS peptide; ii) a first major histocompatibility complex (MHC) polypeptide; iii) a second MHC polypeptide; iv) one or more immunomodulatory polypeptides; Including, the first major histocompatibility complex (MHC) polypeptide is a β2-microglobulin (β2M) polypeptide and the second MHC polypeptide is an MHC class I heavy chain polypeptide; the β2M polypeptide comprises an amino acid sequence having at least 95% amino acid sequence identity to the amino acid sequence shown in FIG. 1A (SEQ ID NO:448); said first and second MHC polypeptides, together with said peptide, form a peptide-MHC complex that presents said peptide to a T cell receptor; the TMP comprises an immunoglobulin (Ig) Fc polypeptide; the TMP comprises one or more peptide linkers linking one or more components of the TMP; The TMP is (a) a rigid peptide linker between said IgFc polypeptide and said one or more immunomodulatory polypeptides, comprising: i) (AP)n, where n is an integer from 1 to 10; ii) (EP)n, where n is an integer from 1 to 10; iii) (KP)n, where n is an integer from 1 to 10; and iv) Peptides containing EAAAK or (b) a short, flexible peptide linker comprising 2 to 14 amino acids positioned between the IgFc polypeptide and the one or more immunomodulatory polypeptides. Including, The TMP.

2. In the order from the N-terminus to the C-terminus, i) the peptide, ii) a peptide linker; iii) the β2M polypeptide; iv) an optional peptide linker; v) the MHC class I heavy chain polypeptide, vi) an optional peptide linker; vii) an IgFc polypeptide; viii) said rigid peptide linker or a short flexible peptide linker, and ix) the one or more immunomodulatory polypeptides; or From the N-terminus to the C-terminus, i) the peptide, ii) a peptide linker; iii) the β2M polypeptide; iv) an optional peptide linker; v) the MHC class I heavy chain polypeptide, vi) an optional peptide linker; vii) the one or more immunomodulatory polypeptides; viii) said rigid peptide linker or a short flexible peptide linker, and ix) IgFc polypeptide 2. The TMP of claim 1 , comprising:

3. The rigid peptide linker is: i) (AP)n, where n is 2 to 6; ii) (EP)n, where n is 2 to 6; iii) (KP)n, where n is 2 to 6; or iv) peptides containing A(EAAAK)nA, where n is 1 or 2 is selected from the short flexible peptide linker has a length selected from 2 to 4 amino acids, 2 to 5 amino acids, 3 to 6 amino acids, or 4 to 8 amino acids; 3. The TMP according to claim 1 or 2.

4. A TMP described in any one of claims 1 to 3, wherein the IgFc comprises an amino acid sequence having at least 95% amino acid sequence identity to any one of the amino acid sequences set forth in any one of SEQ ID NOs: 450 to 462, and the IgFc polypeptide does not substantially induce cell lysis.

5. The TMP of any one of claims 1 to 4, wherein the IgFc polypeptide comprises one or more amino acid substitutions selected from N297A, L234A, L235A, L234F, L235E, or P331S.

6. 6. The TMP of any one of claims 1 to 5, wherein the peptide is an alpha-fetoprotein (AFP) peptide, a Wilms' tumor-1 (WT-1) peptide, a human papillomavirus (HPV) peptide epitope, a MUC-1 peptide, a melanoma associated antigen-4 (MAGE-A4) peptide, a NY-ESO-1 peptide, a survivin peptide, a mesothelin peptide, or a peptide of a viral antigen.

7. (a) the β2M polypeptide and the MHC heavy chain polypeptide are linked by a disulfide bond, the disulfide bond linking a Cys residue of the β2M polypeptide to a Cys residue of the MHC heavy chain polypeptide; (b) the β2-microglobulin polypeptide is linked to the peptide by a first linker that includes a Cys, and a disulfide bond links a Cys present in the first linker and a Cys present in the MHC heavy chain polypeptide; or (c) the β2M polypeptide and the MHC heavy chain polypeptide are linked by a disulfide bond, the disulfide bond linking a Cys residue of the β2M polypeptide to a Cys residue of the MHC heavy chain polypeptide, and optionally a Cys at amino acid residue 12 of the β2M polypeptide is disulfide bonded to a Cys at amino acid residue 236 of the MHC heavy chain polypeptide, and the β2-microglobulin polypeptide is linked to the peptide by a first linker comprising a Cys, and a disulfide bond links a Cys present in the first linker to a Cys present in the MHC heavy chain polypeptide. The TMP according to any one of claims 1 to 6. (a) a Cys at amino acid residue 12 of the β2M polypeptide is disulfide bonded to a Cys at amino acid residue 236 of the MHC heavy chain polypeptide; (b) the first linker comprises the sequence CGGGS(GGGGS)n (SEQ ID NO:439) or GCGGS(GGGGS)n (SEQ ID NO:436), where n is an integer from 1 to 10, and a disulfide bond connects a Cys in the linker to the Cys that replaces Tyr84 of the MHC heavy chain polypeptide; or (c) the Cys at amino acid residue 12 of the β2M polypeptide is disulfide bonded to the Cys at amino acid residue 236 of the MHC heavy chain polypeptide, and the β2-microglobulin polypeptide is linked to the peptide by a first linker comprising a Cys, and a disulfide bond connects the Cys in the linker to the Cys that replaces Tyr84 of the MHC heavy chain polypeptide; The TMP according to claim 7.

9. The MHC heavy chain polypeptide is HLA-A ＊ 0201 polypeptide, HLA-A ＊ 1101 polypeptide, HLA-A ＊ 3303 polypeptide, HLA-A ＊ 9. The TMP of any one of claims 1 to 8, comprising an amino acid sequence having at least 95% amino acid sequence identity to an HLA-A polypeptide selected from the group consisting of an HLA-E polypeptide, an HLA-2401 polypeptide, and an HLA-E polypeptide.

10. 10. The TMP of any one of claims 1 to 9, wherein the at least one immunomodulatory polypeptide is a wild-type or mutant form of an activating immunomodulatory polypeptide selected from the group consisting of IL-2, 4-1BBL, CD80, CD86, and combinations thereof, and optionally at least one of the at least one immunomodulatory polypeptides is a mutant immunomodulatory polypeptide, which exhibits reduced affinity for a cognate costimulatory polypeptide compared to the affinity of a corresponding wild-type immunomodulatory polypeptide for the cognate costimulatory polypeptide.

11. 11. The TMP of claim 10, wherein the at least one immunomodulatory polypeptide is an IL-2 variant that exhibits reduced binding affinity to IL-2Rα and IL-2Rβ.

12. The TMP of claim 11, wherein the at least one mutant IL-2 polypeptide comprises an amino acid other than histidine at position 16 and an amino acid other than phenylalanine at position 42 based on the numbering of SEQ ID NO:

509.

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

14. A TMP according to any one of claims 1 to 13, comprising a rigid peptide linker between the IgFc polypeptide and the one or more immune modulating polypeptides.

15. The TMP of claim 14, wherein the rigid peptide linker is (AP)n, where n is an integer from 2 to 6.

16. A TMP according to any one of claims 1 to 13, comprising a short flexible peptide linker positioned between the IgFc polypeptide and the one or more immunomodulatory polypeptides.

17. The TMP of claim 16, wherein the short flexible peptide linker comprises 2 to 4 amino acids, 2 to 5 amino acids, 3 to 6 amino acids, or 4 to 8 amino acids.

18. The TMP of claim 16, wherein the short flexible peptide linker is GGS. (i) a homodimer comprising two identical TMPs according to any one of claims 1 to 18; or (ii) A heterodimer comprising two different TMPs according to any one of claims 1 to 18, wherein the two TMPs of the heterodimer are linked to each other by an interspecies linkage sequence. TMP comprising any one of the following:

20. The TMP of claim 19, comprising a homodimer comprising two identical TMPs described in any one of claims 1 to 18, wherein the two TMPs are linked to each other by one or more disulfide bonds formed between the immunoglobulin (Ig) Fc polypeptide of one TMP and the IgFc polypeptide of the other TMP.

21. A nucleic acid comprising a nucleotide sequence encoding the TMP described in any one of claims 1 to 18.

22. A pharmaceutical composition comprising a TMP described in any one of claims 1 to 20 or a nucleic acid described in claim 21.

23. The pharmaceutical composition of claim 22 for use in treating cancer in a patient.

24. The pharmaceutical composition of claim 23, administered in combination with an immune checkpoint inhibitor that is an antibody specific for PD-L1, PD-1, TIGIT, or CTLA4.

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