T cell modulating polypeptides and methods of use thereof

Abstract

The present disclosure provides T cell modulating polypeptides (TMPs), which include a MOD, a class I HLA polypeptide (a class I HLA heavy chain polypeptide and a β2-microglobulin polypeptide), and a KRAS peptide that presents an epitope to a T cell receptor (e.g., a KRAS peptide that includes a cancer-associated mutation). The TMPs are useful for modulating the activity of T cells and 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 / 163,270, filed March 19, 2021, the entire contents of which are incorporated herein by reference.

[0002] Incorporation by reference of sequence listings provided as text files A sequence listing is attached hereto as a text file "CUEB-140WO SeqListing ST25.txt" of size 462KB, created on March 15, 2022. 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 to 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. This specific regulation is dependent 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 typically 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 include an immune modulating polypeptide (MOD), a class I HLA polypeptide (a class I HLA heavy chain polypeptide and a β2 microglobulin polypeptide), and a KRAS peptide that presents an epitope to a T cell receptor (e.g., a KRAS peptide having a cancer-associated mutation). 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. 3 shows the amino acid sequence of wild-type human β2M polypeptide (SEQ ID NO:3). [Figure 1B] FIG. 4 shows the amino acid sequence of a β2M polypeptide having an R12C substitution (SEQ ID NO:4). [Figure 2A] FIG. 1 shows the amino acid sequence of an immunoglobulin Fc polypeptide (SEQ ID NO:5). [Figure 2B] FIG. 1 shows the amino acid sequence of an immunoglobulin Fc polypeptide (SEQ ID NO:6). [Figure 2C] FIG. 1 shows the amino acid sequence of an immunoglobulin Fc polypeptide (SEQ ID NO:7). [Figure 2D] FIG. 1 shows the amino acid sequence of an immunoglobulin Fc polypeptide (SEQ ID NO:8). [Figure 2E] FIG. 1 shows the amino acid sequence of an immunoglobulin Fc polypeptide (SEQ ID NO:9). [Figure 2F] FIG. 1 shows the amino acid sequence of an immunoglobulin Fc polypeptide (SEQ ID NO:10). [Figure 2G] FIG. 1 shows the amino acid sequence of an immunoglobulin Fc polypeptide (SEQ ID NO:11). [Figure 2H] FIG. 1 shows the amino acid sequence of an immunoglobulin Fc polypeptide (SEQ ID NO:12). [Figure 2I] FIG. 1 shows the amino acid sequence of an immunoglobulin Fc polypeptide (SEQ ID NO:13). [Figure 2J] FIG. 1 shows the amino acid sequence of an immunoglobulin Fc polypeptide (SEQ ID NO:14). [Figure 2K] FIG. 1 shows the amino acid sequence of an immunoglobulin Fc polypeptide (SEQ ID NO:15). [Figure 2L] FIG. 1 shows the amino acid sequence of an immunoglobulin Fc polypeptide (SEQ ID NO: 16). [Figure 2M] FIG. 1 shows the amino acid sequence of an immunoglobulin Fc polypeptide (SEQ ID NO:17). [Figure 3A] FIG. 1 shows the amino acid sequence of wild-type HLA-A*0201 (SEQ ID NO: 18). [Figure 3B] FIG. 1 shows the amino acid sequence of the mutant (SEQ ID NO: 19). [Figure 3C] FIG. 2 shows the amino acid sequence of the mutant (SEQ ID NO: 20). [Figure 3D] FIG. 2 shows the amino acid sequence of the mutant (SEQ ID NO:21). [Figure 3E] FIG. 2 shows the amino acid sequence of the mutant (SEQ ID NO: 22). [Figure 4A] FIG. 2 shows the amino acid sequence of wild-type HLA-A*1101 (SEQ ID NO: 23). [Figure 4B] FIG. 2 shows the amino acid sequence of the mutant (SEQ ID NO:24). [Figure 4C] FIG. 2 shows the amino acid sequence of the mutant (SEQ ID NO:25). [Figure 4D] FIG. 2 shows the amino acid sequence of the mutant (SEQ ID NO:26). [Figure 4E] FIG. 2 shows the amino acid sequence of the mutant (SEQ ID NO:27). [Figure 5A] FIG. 2 shows wild type HLA-A*2402 (SEQ ID NO: 28). [Figure 5B] FIG. 2 shows the amino acid sequence of the mutant (SEQ ID NO:29). [Figure 5C] FIG. 3 shows the amino acid sequence of the mutant (SEQ ID NO:30). [Figure 5D] FIG. 3 shows the amino acid sequence of the mutant (SEQ ID NO:31). [Figure 5E] FIG. 3 shows the amino acid sequence of the mutant (SEQ ID NO:32). [Figure 6A] FIG. 1 shows the amino acid sequence of wild-type HLA-A*3303 (SEQ ID NO: 33). [Figure 6B] FIG. 3 shows the amino acid sequence of the mutant (SEQ ID NO:34). [Figure 6C] FIG. 3 shows the amino acid sequence of the mutant (SEQ ID NO:35). [Figure 6D] FIG. 3 shows the amino acid sequence of the mutant (SEQ ID NO:36). [Figure 6E] FIG. 3 shows the amino acid sequence of the mutant (SEQ ID NO:37). [Figure 7A-1] FIG. 1 shows a comparison of the amino acid sequences of the HLA-A heavy chain (SEQ ID NOs: 38 to 46, respectively). [Figure 7A-2] FIG. 1 shows a comparison of the amino acid sequences of the HLA-A heavy chain (SEQ ID NOs: 38 to 46, respectively). [Figure 7B] FIG. 47 shows the consensus sequence. [Figure 8A-1] FIG. 1 shows a comparison of amino acid sequences of HLA-B heavy chains (SEQ ID NOs: 48 to 54, respectively). [Figure 8A-2] FIG. 1 shows a comparison of amino acid sequences of HLA-B heavy chains (SEQ ID NOs: 48 to 54, respectively). [Figure 8B] FIG. 5 shows the consensus sequence (SEQ ID NO:55). [Figure 9A-1] FIG. 1 shows a comparison of the amino acid sequences of the HLA-C heavy chain (SEQ ID NOs: 56 to 64, respectively). [Figure 9A-2] FIG. 1 shows a comparison of the amino acid sequences of the HLA-C heavy chain (SEQ ID NOs: 56 to 64, respectively). [Figure 9B] FIG. 6 shows the consensus sequence (SEQ ID NO:65). [Figure 10] FIG. 1 shows the consensus amino acid sequences of HLA-E, HLA-F, and HLA-G heavy chains (SEQ ID NOs: 66-68, 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 47), HLA-B (sequence number 55), HLA-C (sequence number 65), HLA-E (sequence number 66), HLA-F (sequence number 67), and HLA-G (sequence number 68). [Figure 12] FIG. 1 is a schematic diagram showing a specific example of the arrangement of immunomodulatory polypeptides in a "single-chain" TMP of the present disclosure. [Figure 13]FIG. 1 is a schematic depicting a disulfide-bonded single-chain TMP of the present disclosure. [Figure 14A] FIG. 1 shows the amino acid sequence (SEQ ID NO:69) of an illustrative example of a single-chain TMP of the present disclosure. [Figure 14B] FIG. 7 shows the amino acid sequence of an illustrative single-chain TMP of the present disclosure (SEQ ID NO:70). [Figure 14C] FIG. 7 shows the amino acid sequence of an illustrative example of a single-chain TMP of the present disclosure (SEQ ID NO:71). [Figure 14D] FIG. 1 shows the amino acid sequence (SEQ ID NO:72) of an illustrative example of a single-chain TMP of the present disclosure. [Figure 14E] FIG. 1 shows the amino acid sequence (SEQ ID NO:73) of an illustrative example of a single-chain TMP of the present disclosure. [Figure 14F] FIG. 1 shows the amino acid sequence (SEQ ID NO:74) of an illustrative example of a single-chain TMP of the present disclosure. [Figure 14G] FIG. 1 shows the amino acid sequence (SEQ ID NO:75) of an illustrative example of a single-chain TMP of the present disclosure. [Figure 14H] FIG. 1 shows the amino acid sequence (SEQ ID NO:76) of an illustrative example of a single-chain TMP of the present disclosure. [Figure 14I] FIG. 7 shows the amino acid sequence of an illustrative example of a single-chain TMP of the present disclosure (SEQ ID NO:77). [Figure 14J] FIG. 1 shows the amino acid sequence (SEQ ID NO:78) of an illustrative example of a single-chain TMP of the present disclosure. [Figure 14K] FIG. 1 shows the amino acid sequence (SEQ ID NO:79) of an illustrative example of a single-chain TMP of the present disclosure. [Figure 14L] FIG. 1 shows the amino acid sequence (SEQ ID NO:80) of an illustrative example of a single-chain TMP of the present disclosure. [Figure 14M] FIG. 1 shows the amino acid sequence (SEQ ID NO:81) of an illustrative example of a single-chain TMP of the present disclosure. [Figure 14N] FIG. 1 shows the amino acid sequence (SEQ ID NO:82) of an illustrative example of a single-chain TMP of the present disclosure. [Figure 14O] FIG. 1 shows the amino acid sequence (SEQ ID NO:83) of an illustrative example of a single-chain TMP of the present disclosure. [Figure 14P] FIG. 1 shows the amino acid sequence of an illustrative example of a single-chain TMP of the present disclosure (SEQ ID NO:84). [Figure 14Q] FIG. 1 shows the amino acid sequence of an illustrative example of a single-chain TMP of the present disclosure (SEQ ID NO:85). [Figure 14R] FIG. 1 shows the amino acid sequence of an illustrative example of a single-chain TMP of the present disclosure (SEQ ID NO:86). [Figure 14S] FIG. 1 shows the amino acid sequence (SEQ ID NO:87) of an illustrative example of a single-chain TMP of the present disclosure. [Figure 14T] FIG. 1 shows the amino acid sequence (SEQ ID NO:88) of an illustrative example of a single-chain TMP of the present disclosure. [Figure 14U] FIG. 1 shows the amino acid sequence (SEQ ID NO:89) of an illustrative example of a single-chain TMP of the present disclosure. [Figure 14V] FIG. 1 shows the amino acid sequence (SEQ ID NO:90) of an illustrative example of a single-chain TMP of the present disclosure. [Figure 15A] FIG. 1 shows the in vitro activity of specific examples of single-chain TMPs of the present disclosure. [Figure 15B] FIG. 1 shows the in vitro activity of specific examples of single-chain TMPs of the present disclosure. [Figure 15C] FIG. 1 shows the in vitro activity of specific examples of single-chain TMPs of the present disclosure. [Figure 15D] FIG. 1 shows the in vitro activity of specific examples of single-chain TMPs of the present disclosure. [Figure 15E] FIG. 1 shows the in vitro activity of specific examples of single-chain TMPs of the present disclosure. [Figure 16A] FIG. 1 shows the amino acid sequence (SEQ ID NO:91) of an illustrative example of a single-chain TMP of the present disclosure. [Figure 16B] FIG. 1 shows the amino acid sequence (SEQ ID NO:92) of an illustrative example of a single-chain TMP of the present disclosure. [Figure 16C] FIG. 1 shows the amino acid sequence (SEQ ID NO:93) of an illustrative example of a single-chain TMP of the present disclosure. [Figure 16D] FIG. 1 shows the amino acid sequence (SEQ ID NO:94) of an illustrative example of a single-chain TMP of the present disclosure. [Figure 17A]FIG. 9 depicts the amino acid sequence of wild-type IL-2 polypeptide (SEQ ID NO:95). [Figure 17B] FIG. 1 depicts the amino acid sequence of the IL-2Rα polypeptide (SEQ ID NO:96). [Figure 17C] FIG. 97 depicts the amino acid sequence of the IL-2Rβ polypeptide. [Figure 17D] FIG. 1 depicts the amino acid sequence of the IL-2Rγ polypeptide (SEQ ID NO:98). [Figure 18A] FIG. 1 shows the amino acid sequence of the HLA-E heavy chain (SEQ ID NO: 99). [Figure 18B] FIG. 1 shows the amino acid sequence of the HLA-E heavy chain (SEQ ID NO: 100). [Figure 18C] FIG. 1 shows the amino acid sequence of the HLA-E heavy chain (SEQ ID NO: 101). [Figure 18D] FIG. 1 shows the amino acid sequence of the HLA-E heavy chain (SEQ ID NO: 102). [Figure 19A] FIG. 1 shows the amino acid sequence of the HLA-G heavy chain (SEQ ID NO: 103). [Figure 19B] FIG. 1 shows the amino acid sequence of the HLA-G heavy chain (SEQ ID NO: 104). [Figure 19C] FIG. 1 shows the amino acid sequence of the HLA-G heavy chain (SEQ ID NO: 105). [Figure 19D] FIG. 1 shows the amino acid sequence of the HLA-G heavy chain (SEQ ID NO: 106). 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 to the native sequence of the protein, such as deletions, additions, and substitutions (which are generally 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 that produces 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 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 subsequent amino acid substitution at position 84 will refer to the substitution of the alternative amino acid.In such cases, if the polypeptide is also modified by the addition or deletion of one or more amino acids, then reference to the 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, e.g., Altschul et al. (1990), J. Mol. Bioi. 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" encompasses 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 co-stimulatory polypeptide 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 MOD of the TMP can bind to a cognate costimulatory polypeptide (i.e., a co-MOD) 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 specific binding site or receptor. "Non-specific binding" generally refers to the binding of a ligand to a site other than its specific 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, completely or partially, the disease or its symptoms, and / or therapeutic, in the sense of resulting in a partial or complete cure of the disease and / or the deleterious effects resulting from the disease. As used herein, the term "treatment" encompasses any treatment of a disease or condition in a mammal. "Treatment" includes: (a) preventing the 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 the disease or one or more symptoms associated therewith, e.g., arresting its development; and / or (c) relieving the disease, i.e., causing regression of the disease. Therapeutic agents can be administered before, during, and / or after the onset of the disease or injury. Treatment of ongoing disease is of particular interest when the treatment stabilizes or reduces the undesirable clinical symptoms of the patient. 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, as shown in Figures 17 and 18, 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 present disclosure is not limited to particular embodiments described, as such, can, of course, vary. It should also be made clear that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be 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 nearest tenth of a unit, 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 indicates otherwise. Thus, for example, a reference to a "T cell modulating polypeptide" includes a plurality of such polypeptides, and a reference to "the immunomodulating polypeptide" refers to one or more immunomodulating polypeptides and equivalents thereof known to those skilled in the art, and so forth. It should also be noted that the claims may be drafted to exclude any non-essential element. This is the premise for using limiting terms such as "only" and "only" or making "negative" limitations when 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 if each and every combination were 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 if each and every subcombination were 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), where the TMP is a single chain polypeptide comprising: i) a KRAS 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, optionally comprising an immunoglobulin (Ig) Fc polypeptide or a non-Ig 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 spontaneously forms between the IgFc polypeptides in the two single chain TMPs, linking the two single chain TMPs to form a homodimer.

[0033] The TMPs of the present disclosure contain at least one rigid peptide linker (described below) between components of the TMP to reduce the possibility of unfavorable interactions between different polypeptide domains within the TMP. For example, in some cases, a single-chain TMP contains a rigid peptide linker between an IgFc polypeptide and a MOD. A rigid peptide linker may also be disposed between a first MOD and a second MOD. As described below, a TMP containing a rigid peptide linker may have higher thermal stability than a control TMP that does not contain a rigid peptide linker.

[0034] Alternatively, or in addition, the TMP of the present disclosure includes at least one short flexible peptide linker (described below) between components of the TMP to reduce the possibility of unfavorable interactions between different polypeptide domains within the TMP. For example, in some cases, the single-chain TMP includes a short flexible peptide linker between the IgFc polypeptide and the MOD. A short flexible peptide linker may also be disposed between the first MOD and the second MOD. As described below, the TMP that includes a short flexible peptide linker may have higher thermal stability compared to a control TMP that does not include a rigid peptide linker.

[0035] In some cases, the TMP comprises, in order from N-terminus to C-terminus, i) a KRAS peptide, 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, in order from N-terminus to C-terminus, i) a KRAS peptide, 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 disposed between two or more of these components. Such an arrangement of components is referred to as MOD arrangement 2 in FIG. 12.

[0036] In some cases, the TMP comprises, in order from N-terminus to C-terminus, i) a KRAS peptide, 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, in order from N-terminus to C-terminus, i) a KRAS peptide, 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 disposed between two or more of these components. Such an arrangement of components is referred to as MOD arrangement 3 in FIG. 12.

[0037] In some cases, the TMP comprises, in order from N-terminus to C-terminus, i) one or more MODs, ii) a KRAS peptide, iii) a first MHC polypeptide, iv) a second MHC polypeptide, and v) an IgFc polypeptide. In some cases, the TMP comprises, in order from N-terminus to C-terminus, i) one or more MODs, ii) a KRAS peptide, iii) a first class I MHC polypeptide, iv) a second class I MHC polypeptide, and v) an IgFc polypeptide. In some cases, the TMP comprises, in order from N-terminus to C-terminus, i) one or more MODs, ii) a KRAS peptide, 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 disposed between two or more of these 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) immunomodulatory polypeptide or a mutant of a wt immunomodulatory polypeptide. If the MOD comprises a mutant, the MOD may have reduced binding to its co-MOD (e.g., reduced binding to one or more chains or domains of the co-MOD). In such cases, the reduced affinity of the MOD for the co-MOD, combined with the affinity of the KRAS peptide for the TCR, may increase the selectivity of the TMP.

[0039] The binding affinity of MOD and its co-MOD can be measured by biolayer interferometry (BLI) using purified MOD and purified co-MOD. The binding affinity of MOD present in TMP and its co-MOD can be measured by BLI using purified TMP and co-MOD. BLI methods are 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.

[0040] 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.

[0041] KRAS peptides The TMP of the present disclosure includes a KRAS peptide. The KRAS peptide, when present in an MHC / peptide complex (e.g., an HLA / peptide complex), presents a KRAS epitope to a T cell (i.e., a TCR present on the surface of a T cell). As used herein, the term "KRAS peptide" refers to a peptide that presents a KRAS epitope to a TCR when it binds to an MHC complex. As used herein, the term "KRAS epitope" refers to an epitope present on a KRAS protein. As used herein, the terms "KRAS" and "KRAS protein" are synonymous and refer to a protein having an amino acid sequence present in any one of the following (i) to (iii): (i) a KRAS4A polypeptide, (ii) KRAS4B, and (iii) variants (e.g., including mutant forms) of (i) and (ii) that occur in human cancers. As used herein, the term "KRAS polypeptide" refers to a polypeptide having a sequence of amino acids present in all or a portion of a KRAS protein, or, where specified, a polypeptide having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to all or a portion of a KRAS protein or to all or a portion of a variant (e.g., including mutated form) thereof that occurs in human cancers.

[0042] KRAS (also known as "KRAS proto-oncogene GTPase," "Kirsten rat sarcoma viral oncogene homolog," and "K-Ras P21 protein") is a GTPase that regulates cell proliferation. When KRAS is mutated, it leads to uncontrolled cell proliferation and cancer.

[0043] A wild-type (normal, non-cancer associated) KRAS polypeptide may have the following amino acid sequence: TIFF2024512470000002.tif35165

[0044] A wild-type (normal, non-cancer associated) KRAS polypeptide may have the following amino acid sequence: TIFF2024512470000003.tif36165

[0045] Mutant forms of KRAS are associated with certain cancers, and at least some of the mutant forms of KRAS are present on the surface of certain cancer cells. See, e.g., Prior et al. (2012) Cancer Res. 72:2457, and Warren and Holt (2010) Human Immunology 71:245. In SEQ ID NO: 1 and SEQ ID NO: 2, amino acids G12, G13, T35, I36, E49, Q61, K127, and A156 are shown in bold and underlined. Substitutions of one or more of these residues may be present in cancer-associated forms of KRAS polypeptides. Cancer-associated KRAS polypeptides may include one or more of the following i) to vii): i) a substitution at G12 (e.g., G12C, G12V, G125, G12A, G12R, G12F, or G12D), ii) a substitution at G13 (e.g., G13C, G13D, G13R, G13V, G13S, or G13A), iii) a substitution at T35 (e.g., T35I), iv) a substitution at I36 (e.g., I36L or I36M), v) a substitution at E49 (e.g., E49K), vi) a substitution at Q61 (e.g., Q61H, Q61R, Q61P, Q61E, Q61K, Q61L, or Q61K), vii) a substitution at K117 (e.g., K117N), and viii) a substitution at A146 (e.g., A146T or A146V). Here, the amino acid numbers are as shown in SEQ ID NO: 1 and SEQ ID NO: 2. See, for example, US2019 / 0194192.

[0046] For example, a cancer-associated mutant form of a KRAS polypeptide may have one or more amino acid substitutions relative to the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2. In some cases, a cancer-associated mutant form of a KRAS polypeptide has only a single amino acid substitution relative to the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2. In some cases, a cancer-associated mutant form of a KRAS polypeptide has only two amino acid substitutions relative to the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2. In some cases, a cancer-associated mutant form of a KRAS polypeptide has only three amino acid substitutions relative to the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2. In some cases, a cancer-associated mutant form of a KRAS polypeptide has only four amino acid substitutions relative to the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2. In some cases, a cancer-associated mutant form of a KRAS polypeptide has only five amino acid substitutions relative to the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2.

[0047] For example, KRAS(G12D) (KRAS polypeptide having a G to D substitution at amino acid position 12 based on the amino acid numbering set forth in SEQ ID NO:1) is associated with pancreatic ductal adenocarcinoma (PDAC). KRAS(G12V) (KRAS polypeptide having a G to V substitution at amino acid position 12 based on the amino acid numbering set forth in SEQ ID NO:1 or SEQ ID NO:2) is also associated with pancreatic cancer. KRAS(G12R) (KRAS polypeptide having a G to R substitution at amino acid position 12 based on the amino acid numbering set forth in SEQ ID NO:1 or SEQ ID NO:2) is also associated with pancreatic cancer. See, e.g., Waters and Der (2018) Cold Spring Harb. Perspect. Med. 8:(9).pii:a031435.doi:10.1101 / cshperspect.a031435. As another specific example, KRAS(G12C) (KRAS polypeptide having a G to C substitution at amino acid position 12 based on the amino acid numbering set forth in SEQ ID NO:1 or SEQ ID NO:2) is associated with lung cancer, e.g., non-small cell lung cancer. See, e.g., Roman et al. (2018) Mol. Cancer 17:33. Other mutant forms of KRAS (e.g., G12A, G12C, G12D, G12R, G12S, G12V, G13A, G13C, G13D, G13R, G13S, G13V) are associated with various cancers, including, e.g., cholangiocarcinoma, gallbladder cancer, adenocarcinoma, rectal adenocarcinoma, endometrial cancer, hematopoietic tumors, and lung cancer. See, e.g., Prior et al. (2012) Cancer Res. 72:2457.

[0048] As another specific example, a cancer-associated mutant form of a KRAS polypeptide can have an amino acid substitution at amino acid 61 of a KRAS polypeptide (e.g., a KRAS polypeptide having the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:2). For example, a cancer-associated mutant form of a KRAS polypeptide can have an amino acid substitution such as Q61H, Q61L, Q61E, Q61R, or Q61K.

[0049] Generally, a KRAS peptide / MHC complex present in a TMP of the present disclosure binds to a T cell receptor (TCR) on a T cell with an affinity of at least 100 μM (e.g., at least 10 μM, at least 1 μM, at least 100 nM, at least 10 nM, or at least 1 nM). Moreover, generally speaking, a TMP will bind to a T cell having a TCR and a co-MOD that binds to the KRAS peptide / MHC complex with greater affinity (e.g., 25% greater) than the TMP will bind to another T cell having the same co-MOD and a TCR that does not substantially bind to the KRAS peptide / MHC complex. For example, a KRAS peptide / MHC complex will bind to a TCR with an affinity of at least 10 μM (e.g., at least 10 μM, at least 1 μM, at least 100 nM, at least 10 nM, or at least 1 nM). -7 It binds with an affinity less than M.

[0050] The KRAS epitope present in TMP is a peptide that is specifically bound by T cells. That is, this epitope is specifically bound by epitope-specific T cells, i.e., T cells having a TCR specific for the KRAS epitope. Epitope-specific T cells bind to an epitope having a certain standard amino acid sequence, but do not substantially bind to an epitope different from this standard amino acid sequence. For example, epitope-specific T cells bind to an epitope having a certain standard amino acid sequence, and do not substantially bind to an epitope different from this standard amino acid sequence, even if they bind to it. -6 Less than M, 10 -5 Less than M or 10 -4 Epitope-specific T cells bind with an affinity of less than 10 -7 M, at least 10 -8 M, at least 10 -9 M, or at least 10 -10 It is capable of binding to an epitope for which it is specific with an affinity of M.

[0051] In some cases, a suitable KRAS peptide is a peptide at least 4 amino acids in length, e.g., a peptide from 4 amino acids to about 20 amino acids in length (e.g., 4 amino acids (aa) in length, 5 amino acids in length, 6 amino acids in length, 7 amino acids in length, 8 amino acids in length, 9 amino acids in length, 10 amino acids in length, 11 amino acids in length, 12 amino acids in length, 13 amino acids in length, 14 amino acids in length, 15 amino acids in length, 16 amino acids in length, 17 amino acids in length, 18 amino acids in length, 19 amino acids in length, and 20 amino acids in length (including ranges of 9-11 amino acids in length, 6-15 amino acids in length, 8-12 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 KRAS peptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to one of the following KRAS amino acid sequences (A)-(C): (A) MTEYKLVVVG AGGVGKSALT IQLIQNHFVD EYDPTIEDSY RKQVVIDGET CLWDILDTAG QEEYSAMRDQ YMRTGEGFLC VFAINNTKSF EDIHHYREQI KRVKDSEDVP MVLVGNKCDL PSRTVDTKQA QDLARSYGIP FIETSAKTRQ GVDDAFYTLV REIRKHKEKM SKDGKKKKKK SKTKCVIM (SEQ ID NO:1), where the KRAS polypeptide comprises one or more (e.g., 1, 2, 3, 4, or 5) amino acid substitutions relative to the amino acid sequence set forth in SEQ ID NO:1, and the one or more amino acid substitutions can include a substitution associated with cancer (e.g., a substitution present in a KRAS polypeptide in a cancer cell); (B) MTEYKLVVVG AGGVGKSALT IQLIQNHFVD EYDPTIEDSY RKQVVIDGET CLLDILDTAG QEEYSAMRDQ YMRTGEGFLC VFAINNTKSF EDIHHYREQI KRVKDSEDVP MVLVGNKCDL PSRTVDTKQA QDLARSYGIP FIETSAKTRQ RVEDAFYTLV REIRQYRLKK ISKEEKTPGC VKIKKCIIM (SEQ ID NO:2), where the KRAS polypeptide includes one or more (e.g., 1, 2, 3, 4, or 5) amino acid substitutions relative to the amino acid sequence set forth in SEQ ID NO:1, and the one or more amino acid substitutions can include a substitution associated with cancer (e.g., a substitution present in a KRAS polypeptide in a cancer cell); and (C)MTEY (X1) L ( GVDDAFYTLV REIRKHKEKM SKDGKKKKKK SKTKCVIM (sequence number 107) (wherein X1 is Lys, Phe, or Leu, X2 is Val or Leu, X3 is Val or Thr, X4 is Val or Thr, X5 is Gly, Asp, Cys, Val, or Ser, and X6 is Gly, Cys, or Asp, wherein one or both of X5 and X6 are not Cys).

[0052] Non-limiting examples of suitable KRAS peptides include peptides comprising a sequence selected from the group consisting of VVGADGVGK (SEQ ID NO:108), VVGACGVGK (SEQ ID NO:109), VVGAVGVGK (SEQ ID NO:110), VVVGADGVGK (SEQ ID NO:111), VVVGAVGVGK (SEQ ID NO:112), VVVGACGVGK (SEQ ID NO:113), VTGADGVGK (SEQ ID NO:114), VTGAVGVGK (SEQ ID NO:115), VTGACGVGK (SEQ ID NO:116), VTVGADGVGK (SEQ ID NO:117), VTVGAVGVGK (SEQ ID NO:118), and VTVGACGVGK (SEQ ID NO:119), wherein the KRAS peptide is 9 or 10 amino acids in length, or at least 9 or 10 amino acids in length.

[0053] Further non-limiting examples of suitable KRAS peptides include peptides comprising a sequence selected from the group consisting of VVVGAGDVGK (SEQ ID NO: 120), VVGAGDVGK (SEQ ID NO: 121), VVVGARGVGK (SEQ ID NO: 122), and VVGARGVGK (SEQ ID NO: 123), wherein the KRAS peptide is 9 or 10 amino acids in length, or at least 9 or 10 amino acids in length.

[0054] Non-limiting examples of suitable KRAS peptides include peptides comprising a sequence selected from the group consisting of LVVVGADGV (SEQ ID NO:124), LVVVGAVGV (SEQ ID NO:125), LVVVGACGV (SEQ ID NO:126), KLVVVGADGV (SEQ ID NO:127), KLVVVGAVGV (SEQ ID NO:128), KLVVVGACGV (SEQ ID NO:129), LLVVGADGV (SEQ ID NO:130), LLVVGAVGV (SEQ ID NO:131), LLVVGACGV (SEQ ID NO:132), FLVVVGADGV (SEQ ID NO:133), FLVVVGAVGV (SEQ ID NO:135), and FLVVVGACGV (SEQ ID NO:135), wherein the KRAS peptide is 9 or 10 amino acids in length, or at least 9 or 10 amino acids in length.

[0055] Further non-limiting examples of suitable KRAS peptides include peptides comprising a sequence selected from the group consisting of KLVVVGAGDV (SEQ ID NO: 136) and KLVVVGARGV (SEQ ID NO: 137), wherein the KRAS peptide is 9 or 10 amino acids in length, or at least 9 or 10 amino acids in length.

[0056] Further non-limiting examples of suitable KRAS peptides include peptides comprising a sequence selected from the group consisting of GAGDVGKSAL (SEQ ID NO:138), AGDVGKSAL (SEQ ID NO:139), DVGKSALTI (SEQ ID NO:140), GAVGVGKSAL (SEQ ID NO:141), AVGVGKSAL (SEQ ID NO:142), YKLVVVGAV (SEQ ID NO:143), ARGVGKSAL (SEQ ID NO:144), GARGVGKSAL (SEQ ID NO:145), EYKLVVVGAR (SEQ ID NO:146), RGVGKSALTI (SEQ ID NO:147), LVVGARGV (SEQ ID NO:148), GADGVGKSAL (SEQ ID NO:149), ACGVGKSAL (SEQ ID NO:150), and GACGVGKSAL (SEQ ID NO:151), wherein the KRAS peptide is 9 or 10 amino acids in length, or at least 9 or 10 amino acids in length.

[0057] Further non-limiting examples of suitable KRAS peptides include peptides comprising a sequence selected from the group consisting of VVVGAGGVGK (SEQ ID NO:229), VVVGACGVGK (SEQ ID NO:113), VVVGADGVGK (SEQ ID NO:111), VVVGARGVGK (SEQ ID NO:122), VVVGAVGVGK (SEQ ID NO:112), and VVVGAVGVGK (SEQ ID NO:110), wherein the KRAS peptide is 9 or 10 amino acids in length, or at least 9 or 10 amino acids in length. Such peptides can be used in combination with β2M polypeptide and A * 03:01 HLA-A heavy chain. Such peptides may present epitopes upon binding to an HLA complex containing the β2M polypeptide and HLA A heavy chain. *When bound to an HLA complex containing the 11:01 heavy chain, the epitope may be presented. In some cases, a 9 or 10 amino acid long peptide such as VVVGARGVGK (SEQ ID NO: 122), VVGARGVGK (SEQ ID NO: 123), VVVGAVGVGK (SEQ ID NO: 112), or VVGAVGVGK (SEQ ID NO: 110) may be associated with a β2M polypeptide and an A * When bound to an HLA complex containing the 30:01 HLA-A heavy chain, it presents an epitope. In some cases, a 9 or 10 amino acid long peptide such as VVVGACGVGK (SEQ ID NO: 113), VVVGADGVGK (SEQ ID NO: 111), VVVGARGVGK (SEQ ID NO: 122), VVVGAVGVGK (SEQ ID NO: 112), or VVGAVGVGK (SEQ ID NO: 110) binds to the β2M polypeptide and A * Upon binding to an HLA complex containing the 68:01 HLA heavy chain, it presents an epitope.

[0058] Further specific, non-limiting examples of suitable KRAS peptides include peptides comprising the amino acid sequence GADGVGKSAL (SEQ ID NO: 149) or GARGVGKSAL (SEQ ID NO: 145), where the KRAS peptide is 9 or 10 amino acids in length, or at least 9 or 10 amino acids in length. Such peptides are useful in the synthesis of β2M polypeptides and B * Upon binding to an HLA complex containing the 07:02 HLA-B heavy chain, it may present an epitope.

[0059] Further specific, non-limiting examples of suitable KRAS peptides include peptides comprising a sequence selected from the group consisting of AVGVGKSAL (SEQ ID NO:142), GAVGVGKSAL (SEQ ID NO:141), GAVGVGKSA (SEQ ID NO:155), GACGVGKSA (SEQ ID NO:230), GADGVGKSAL (SEQ ID NO:149), and GADGVGKSA (SEQ ID NO:231), where the KRAS peptide is 9 or 10 amino acids in length, or at least 9 or 10 amino acids in length. In some cases, a 9 amino acid long peptide, such as AVGVGKSAL (SEQ ID NO:142), is used in combination with a β2M polypeptide and a C *01:02 When bound to an HLA complex containing the HLA-C heavy chain, it presents an epitope. In some cases, a 10 amino acid long peptide such as GAVGVGKSAL (SEQ ID NO: 141) or a 9 amino acid long peptide such as GAVGVGKSA (SEQ ID NO: 155) binds to a β2M polypeptide and a C * 03:03 When bound to an HLA complex containing the HLA-C heavy chain, it presents an epitope. In some cases, 9- or 10-amino acid peptides such as GACGVGKSA (SEQ ID NO: 230), GADGVGKSAL (SEQ ID NO: 149), GAVGVGKSAL (SEQ ID NO: 141), or GAVGVGKSA (SEQ ID NO: 155) are associated with the β2M polypeptide and C * 03:04 When bound to an HLA complex containing the HLA-C heavy chain, it presents an epitope. In some cases, a 10 amino acid long peptide such as GADGVGKSAL (SEQ ID NO: 149) or a 9 amino acid long peptide such as GADGVGKSA (SEQ ID NO: 231) binds to a β2M polypeptide and C * 08:02 When bound to an HLA complex containing the HLA-C heavy chain, it presents an epitope.

[0060] In some cases, the TMP regulates the activity of T cells with TCR specific for the G12V form of the KRAS polypeptide, as described above. In such cases, the KRAS peptide present in the TMP can, for example, comprise one of the following amino acid sequences: VVGAVGVGK (SEQ ID NO: 110), VVVGAVGVGK (SEQ ID NO: 112), VGAVGVGKS (SEQ ID NO: 152), VGAVGVGKSA (SEQ ID NO: 153), AVGVGKSAL (SEQ ID NO: 142), AVGVGKSALT (SEQ ID NO: 154), GAVGVGKSAL (SEQ ID NO: 141), GAVGVGKSA (SEQ ID NO: 155), LVVVGAVGVG (SEQ ID NO: 156), LVVVGAVGV (SEQ ID NO: 125), KLVVVGAVGV (SEQ ID NO: 128), and KLVVVGAVG (SEQ ID NO: 157). Wherein, the KRAS peptide is 9 or 10 amino acids long, or at least 9 or 10 amino acids long.

[0061] In some cases, the KRAS 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 * 0203, A * 0301, A * 1101, A * 2301, A * 2402, A * 2407, A * 3101, A * 3303, A * 3401 and / or A * In one embodiment, the KRAS epitope peptide present in the TMP is restricted to HLA-B 6801. * 0702, B * 0801, B * 1502, B * 2705, B * 3802, B * 3802, B * 3901, B * 3902, B * 4001, B * 4601, B * 5101 and / or B * In one embodiment, the KRAS epitope peptide present in the TMP is C5301-restricted. * 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.

[0062] As specific, non-limiting examples, KRAS peptides include VVGADGVGK (SEQ ID NO: 108), VVGACGVGK (SEQ ID NO: 109), VVGAVGVGK (SEQ ID NO: 110), VVVGADGVGK (SEQ ID NO: 111), VVVGAVGVGK (SEQ ID NO: 112), VVVGACGVGK (SEQ ID NO: 113), VTGADGVGK (SEQ ID NO: 114), VTGAVGVGK (SEQ ID NO: 115), VTGACGVGK (SEQ ID NO: 116), and VTVGADGVGK (SEQ ID NO: 117). , VTVGAVGVGK (SEQ ID NO: 118), VTVGACGVGK (SEQ ID NO: 119), VVVGAGDVGK (SEQ ID NO: 120), VVGAGDVGK (SEQ ID NO: 121), VVVGARGVGK (SEQ ID NO: 122), and VVGARGVGK (SEQ ID NO: 123), wherein the KRAS peptide is 9 or 10 amino acids in length, or at least 9 or 10 amino acids in length, and * When bound to an HLA complex containing the 1101HLA-A heavy chain, the peptide presents an epitope. * It can be presented in complex with an HLA complex containing the 6801HLA-A heavy chain.

[0063] As specific, non-limiting examples, the KRAS peptide comprises a peptide selected from the group consisting of LVVVGADGV (SEQ ID NO: 124), LVVVGAVGV (SEQ ID NO: 125), LVVVGACGV (SEQ ID NO: 126), KLVVVGADGV (SEQ ID NO: 127), KLVVVGAVGV (SEQ ID NO: 128), KLVVVGACGV (SEQ ID NO: 129), LLVVGADGV (SEQ ID NO: 130), LLVVGAVGV (SEQ ID NO: 131), LLVVGACGV (SEQ ID NO: 132), FLVVVGADGV (SEQ ID NO: 133), FLVVVGAVGV (SEQ ID NO: 134), and FLVVVGACGV (SEQ ID NO: 135), the KRAS peptide being 9 or 10 amino acids in length, or at least 9 or 10 amino acids in length, and a β2M polypeptide and an A polypeptide. * Upon binding to an HLA complex containing the 0201HLA-A heavy chain, it presents an epitope.

[0064] As a further illustrative example, the following KRAS peptides can present an epitope upon binding to an HLA complex containing a β2M polypeptide and an HLA-A heavy chain: GAGDVGKSAL (SEQ ID NO: 138), which binds to a β2M polypeptide and an HLA-A heavy chain. * 3801HLA-A heavy chain), AGDVGKSAL (SEQ ID NO: 139) (which can present an epitope when bound to an HLA complex containing the β2M polypeptide and B0702, B * 3801, or B * 3901HLA-A heavy chain), DVGKSALTI (SEQ ID NO: 140) (which is capable of presenting an epitope when bound to an HLA complex containing the β2M polypeptide and B * 5101HLA-A heavy chain), GAVGVGKSAL (SEQ ID NO: 141) (which is capable of presenting an epitope when bound to an HLA complex containing the β2M polypeptide and B * 0702 or B * 3801HLA-A heavy chain), AVGVGKSAL (SEQ ID NO: 142) (which is capable of presenting an epitope when bound to an HLA complex containing the β2M polypeptide and B * 0702HLA-A heavy chain), YKLVVVGAV (SEQ ID NO: 143), which is capable of presenting an epitope upon binding to an HLA complex containing the β2M polypeptide and A * 0203 or B * 3902, which may present an epitope when bound to an HLA complex containing the HLA-A heavy chain), ARGVGKSAL (SEQ ID NO: 144), which may present an epitope when bound to an HLA complex containing the β2M polypeptide and B * 0702, B * 2705, or B * 3901HLA-A heavy chain), GARGVGKSAL (SEQ ID NO: 145) (which can present an epitope when bound to an HLA complex containing the β2M polypeptide and B * 0702HLA-A heavy chain), EYKLVVVGAR (SEQ ID NO: 146) (which is capable of presenting an epitope upon binding to an HLA complex containing a β2M polypeptide and an A *3101HLA-A heavy chain), RGVGKSALTI (SEQ ID NO: 147) (which can present an epitope when bound to an HLA complex containing the β2M polypeptide and B * 0702HLA-A heavy chain), LVVVGARGV (SEQ ID NO: 148) (which is capable of presenting an epitope upon binding to an HLA complex containing a β2M polypeptide and A * 0203HLA-A heavy chain), GADGVGKSAL (SEQ ID NO: 149) (which can present an epitope when bound to an HLA complex containing the β2M polypeptide and B * 3801HLA-A heavy chain), ACGVGKSAL (SEQ ID NO: 150) (which can present an epitope when bound to an HLA complex containing the β2M polypeptide and B * 0702, which may present an epitope when bound to an HLA complex containing the HLA-A heavy chain, and GACGVGKSAL (SEQ ID NO: 151), which may present an epitope when bound to an HLA complex containing the β2M polypeptide and B * 3801 may present an epitope upon binding to an HLA complex containing the HLA-A heavy chain).

[0065] Whether a peptide (e.g., a KRAS peptide containing a KRAS 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 specific binding assays in this publication is incorporated herein by reference, particularly including paragraphs

[0217] to

[0225] .

[0066] 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.

[0067] 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, including human MHC (also known as human leukocyte antigen (HLA)) polypeptides, rodent (e.g., mouse, rat, etc.) MHC polypeptides, and MHC polypeptides of other mammalian species (e.g., lagomorphs, non-human primates, canines, felines, ungulates (e.g., equines, bovidae, ovines, caprines, etc.)). The term "MHC polypeptide" is meant to encompass class I MHC polypeptides (e.g., beta-2 microglobulin and MHC class I heavy chains).

[0068] In some cases, the first MHC polypeptide is an MHC class I beta-2 microglobulin (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 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 is 270 amino acids long, 271 amino acids long, 272 amino acids long, 273 amino acids long, 274 amino acids long, 275 amino acids long, 276 amino acids long, 277 amino acids long, 278 amino acids long, 279 amino acids long, 280 amino acids long, 281 amino acids long, 282 amino acids long, 283 amino acids long, 284 amino acids long, 285 amino acids long, 286 amino acids long, 287 amino acids long, 288 amino acids long, 289 amino acids long, or 290 amino acids long.

[0069] 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 is 270 amino acids long, 271 amino acids long, 272 amino acids long, 273 amino acids long, 274 amino acids long, 275 amino acids long, 276 amino acids long, 277 amino acids long, 278 amino acids long, 279 amino acids long, 280 amino acids long, 281 amino acids long, 282 amino acids long, 283 amino acids long, 284 amino acids long, 285 amino acids long, 286 amino acids long, 287 amino acids long, 288 amino acids long, 289 amino acids long, or 290 amino acids long. 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 contain transmembrane or cytoplasmic domains. In one embodiment, the MHC class I heavy chain polypeptide of the TMP may comprise an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to 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.

[0070] 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.

[0071] 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 these alleles 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.

[0072] 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 comprises 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.

[0073] 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 some cases, Cys-84 forms an intrachain disulfide bond with Cys-236); 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).

[0074] 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).

[0075] 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 comprises 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).

[0076] 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 comprises 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).

[0077] 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 comprises 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).

[0078] 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.

[0079] 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.

[0080] 7A , in some cases, i) aac1 (amino acid cluster 1) may be the amino acid sequence GTLRG (SEQ ID NO: 158) 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: 159) 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: 160) 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: 159) 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). aac3) may be the amino acid sequence AQTTK (SEQ ID NO: 161) 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., 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) aac5 (amino acid cluster 5) may be the amino acid sequence VETRP (SEQ ID NO: 162) 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., V is replaced with I or L, E is replaced with D, T is replaced with S, and / or R is replaced with K); and / or vi) aac6 (amino acid cluster 6) may be the amino acid sequence GDGTF (SEQ ID NO: 163) 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., D is replaced with E, T is replaced with S, or F is replaced with L, W, or Y).

[0081] With reference to FIG. 8A , in some cases, i) aac1 (amino acid cluster 1) may be the amino acid sequence RNLRG (SEQ ID NO: 164) 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 T or I, and / or L is replaced with A, and / or the second R is replaced with L, and / or G is replaced with R), and ii) aac2 (amino acid cluster 2) may be the amino acid sequence YNQSE (SEQ ID NO: 159) 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 T or I, and / or L is replaced with A, and / or the second R is replaced with L, and / or G is replaced with R). aac3 (amino acid cluster 3) may be the amino acid sequence TAADT (SEQ ID NO: 165) or 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 is replaced with S, and / or the A is replaced with G, and / or the D is replaced with E, and / or the second T is replaced with S); iii) aac4 (amino acid cluster 4) may be the amino acid sequence TAADT (SEQ ID NO: 165) or 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 is replaced with S, and / or the A is replaced with G, and / or the D is replaced with E, and / or the second T is replaced with S); iv) aac5 (amino acid cluster 6) may be the amino acid sequence TAADT (SEQ ID NO: 165) or 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 is replaced with S, and / or the A is replaced with G, and / or the D is replaced with E, and / or the second T is replaced with S); Cluster 4) may be the amino acid sequence AQITQ (SEQ ID NO: 166) 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: 162) 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: 167) 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).

[0082] With reference to FIG. 9A , in some cases, i) aac1 (amino acid cluster 1) may be the amino acid sequence RNLRG (SEQ ID NO: 164) 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: 159) 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: 165) or 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 is replaced with S, and / or the A is replaced with G, and / or the D is replaced with E, and / or the second T is replaced with S). iv) aac4 (amino acid cluster 4) may be the amino acid sequence AQITQ (SEQ ID NO: 166) 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: 162) 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: 163) 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).

[0083] 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 (as well as HLA-A, HLA-B, and HLA-C alleles) can be found on the World Wide Web at: hla.alleles.org / nomenclature / index.html, the European Bioinformatics Institute (www(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).

[0084] Specific, non-limiting examples of suitable HLA-E alleles include HLA-E * 0101(HLA-E * 01:01:01:01), HLA-E * 01:03(HLA-E * 01:03:01:01), HLA-E * 01:04, HLA-E * 01:05, HLA-E * 01:06, HLA-E * 01:07, HLA-E * 01:09, and HLA-E * 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 18A-18D. * FIG. 18B shows the amino acid sequence of HLA-E 01:01 (wild type) with the Y84C and A2346C substitutions. * FIG. 18C shows the amino acid sequence of HLA-E 01:01. * FIG. 18D shows the amino acid sequence of HLA-E 01:03 (wild type) with the Y84C and A2346C substitutions. * The amino acid sequence of 01:03 is shown.

[0085] Specific, non-limiting examples of suitable HLA-F alleles include HLA-F * 0101(HLA-F * 01:01:01:01), HLA-F * 01:02, HLA-F * 01:03 (HLA-F * 01:03:01:01), HLA-F * 01:04, HLA-F * 01:05, and HLA-F * 01:06. Non-limiting examples of suitable HLA-G alleles include HLA-G * 0101(HLA-G * 01:01:01:01), HLA-G * 01:02, HLA-G * 01:03(HLA-G * 01:03:01:01), HLA-G * 01:04 (HLA-G * 01:04:01:01), HLA-G * 01:06, HLA-G * 01:07, HLA-G * 01:08, HLA-G * 01:09, HLA-G * 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 * 01:01:01:01) 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 19A-19D. Figure 19A shows the amino acid sequence of HLA-G * FIG. 19B shows the amino acid sequence of HLA-G 01:01 (wild type) with the Y84C and A2346C substitutions. * FIG. 19C shows the amino acid sequence of HLA-G * FIG. 19D shows the amino acid sequence of HLA-G 01:04 (wild type) with the Y84C and A2346C substitutions. * The amino acid sequence of 01:04 is shown.

[0086] The consensus sequences for those HLA-E, HLA-F and HLA-G alleles that lack 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.

[0087] 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.

[0088] 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.

[0089] 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).

[0090] 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).

[0091] 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.

[0092] 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.

[0093] Intrachain and interchain 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 comprises an IgFc polypeptide, the TMP can be dimerized such that one or more interchain disulfide bonds link the IgFc polypeptides in two TMPs 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.

[0094] When the KRAS peptide of the 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 the TMP. In some cases, the TMP has a Cys in the β2M polypeptide and a Cys in the MHC class I heavy chain, and the 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 KRAS peptide and the β2M polypeptide in the TMP and 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 and ii) a second Cys present in the MHC class I heavy chain. Linkers containing Cys are described in more detail below.

[0095] Generally, potential disulfide bond positions in TMP are those where the residues of TMP are 5 angstroms or less apart. Such positions refer to positions where a Cys residue can be substituted for a residue present in the polypeptide (if not naturally occurring). For example, Cys residues in TMP can 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 such 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 are representative of amino acids that can form disulfide bonds in TMP when substituted with Cys. Similarly, disulfide bonds can 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.

[0096] TMP is, for example, i) a KRAS peptide (e.g., a KRAS peptide that is between 4 amino acids and about 25 amino acids in length, which KRAS peptide is bound by the TCR when complexed with an MHC polypeptide); ii) a first MHC polypeptide; and iii) a peptide linker between the KRAS peptide and the first MHC polypeptide, wherein the peptide linker has a Cys residue and the first MHC polypeptide is a β2M polypeptide having an amino acid substitution introducing a Cys residue; iv) a second MHC polypeptide, wherein the second MHC polypeptide is a class I heavy chain having 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 having a first intrachain disulfide bond between a Cys residue in the peptide linker (the peptide linker between the KRAS 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 having 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.

[0097] 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. 4C , FIG. 5C , and FIG. 6C .

[0098] 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).

[0099] 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: 20) * 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: 25) * 1101(Y84C, A236C) amino acid sequence (wherein amino acid 84 is Cys and amino acid 236 is Cys), (iii) HLA-A shown in FIG. 5C (SEQ ID NO: 30) * 2402 (Y84C, A236C) amino acid sequence (wherein amino acid 84 is Cys and amino acid 236 is Cys), (iv) HLA-A shown in FIG. 6C (SEQ ID NO: 35) * 3303 (Y84C, A236C) amino acid sequence (wherein amino acid 84 is Cys and amino acid 236 is Cys).

[0100] 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: 38) *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: 40) * 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: 42) * 2301 amino acid sequence (wherein amino acid 84 is Cys and amino acid 236 is Cys), (iv) HLA-A shown in Figure 7A (SEQ ID NO: 44) * 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: 46) * 3401 amino acid sequence (wherein amino acid 84 is Cys and amino acid 236 is Cys).

[0101] 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%, at least 99%, or 100% amino acid sequence identity to any one of the amino acid sequences shown in Figures 18A-18D.

[0102] 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 one of the following sequences: (i) and (ii): (i) HLA-E as shown in FIG. 18B (SEQ ID NO: 100) * 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. 18D (SEQ ID NO: 102) * 01:03 (Y84C, A236C) amino acid sequence (wherein amino acid 84 is Cys and amino acid 236 is Cys).

[0103] 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%, at least 99%, or 100% amino acid sequence identity to any one of the amino acid sequences shown in Figures 19A-19D.

[0104] 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 one of the following sequences: (i) and (ii): (i) HLA-G as shown in FIG. 19B (SEQ ID NO: 104) * 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. 19D (SEQ ID NO: 106) * 01:04 (Y84C, A236C) amino acid sequence (wherein amino acid 84 is Cys and amino acid 236 is Cys).

[0105] HLA / peptide binding assay Whether a peptide (e.g., a KRAS peptide containing a KRAS 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 specific binding assays in this publication is incorporated herein by reference, particularly including paragraphs

[0217] to

[0225] .

[0106] 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.

[0107] Immunomodulating Polypeptides 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. Suitable MODs that exhibit 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.

[0108] As discussed above, a MOD may include a mutant of a wild-type immunomodulatory polypeptide that has reduced binding to its co-MOD (e.g., reduced binding to one or more chains or domains of the co-MOD). For example, a mutant MOD present in a TMP can bind to its co-MOD with an affinity that is at least 10% lower, at least 15% lower, at least 20% lower, at least 25% lower, at least 30% lower, at least 35% lower, at least 40% lower, at least 45% lower, at least 50% lower, at least 55% lower, at least 60% lower, at least 65% lower, at least 70% lower, at least 75% lower, at least 80% lower, at least 85% lower, at least 90% lower, at least 95% lower, or more than 95% lower than the affinity of the corresponding wild-type MOD for the co-MOD.

[0109] Specific pairs of a MOD and its co-MOD include, but are not limited to, those shown in Table 1 below.

[0110] [Table 1]

[0111] One or more MODs can be present in any of a variety of locations in the TMP, as shown diagrammatically in Figure 12. Although Figure 12 shows the location of two copies of the mutant IL-2 polypeptide, the MODs can be any number and variety of MODs, as described herein. As shown in Figure 12, the MODs can be 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).

[0112] Immune modulating polypeptides and variants, including affinity-reduced variants, of PD-L1, CD80, CD86, 4-1BBL, and IL-2 have been described in the published literature (e.g., published PCT applications 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.

[0113] 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.

[0114] 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.

[0115] In some cases, an IL-2 variant MOD of the disclosure has reduced binding to IL-2Rα, such that activation of regulatory T cells (Tregs) by the IL-2 variant is minimized or substantially reduced. Alternatively, or additionally, in some cases, an IL-2 variant MOD of the disclosure has 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, an IL-2 variant MOD of the disclosure exhibits 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, an IL-2 variant having substitutions at H16 and F42 exhibits reduced binding to IL-2Rα and IL-2Rβ. See Quayle et al., Clin Cancer Res; 26(8)April 15,2020, which shows that the binding affinity of IL-2 polypeptides with H16A and F42A substitutions to human IL-2Rα and IL-2Rβ was reduced by 110-fold and 3-fold, respectively, compared to that of wild-type IL2, 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, binding of the IL-2 mutant MOD to its costimulatory polypeptide on T cells is driven substantially by binding of the MHC-epitope portion rather than by binding of IL-2.

[0116] Thus, suitable IL-2 mutant MODs include polypeptides that comprise 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 that 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 mutant IL-2 polypeptides of the disclosure have a lower binding affinity to 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.

[0117] 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. TIFF2024512470000005.tif23165 i.e., the mutant IL-2 polypeptide has the amino acid sequence of wild type IL-2 but with an H16A substitution and an F42A substitution (shown in bold). Alternatively, the above sequence can be employed but with a substitution 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 following amino acid sequence: TIFF2024512470000006.tif24165In one case, the mutant IL-2 polypeptide present in the TMP has the following amino acid sequence: TIFF2024512470000007.tif25165In some cases, the TMP contains two copies of such a mutant IL-2 polypeptide.

[0118] In some cases, the 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: 170).

[0119] 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: 171).

[0120] 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: 172).

[0121] 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: 173).

[0122] 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: 174).

[0123] 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: 175).

[0124] 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: 176).

[0125] 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: 177).

[0126] Scaffold Polypeptides The TMP may comprise an Fc polypeptide, or may comprise another suitable scaffold polypeptide.

[0127] 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: 193), 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.

[0128] 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., blood 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., blood 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.

[0129] 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.

[0130] 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.

[0131] 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 Ala at position 77. In some cases, the Fc polypeptide present in the TMP comprises the amino acid sequence shown in Figure 2A. In some cases, the Fc polypeptide present in the TMP comprises the amino acid sequence shown in Figure 2B.

[0132] 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 a 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 may have an N77 substitution. That is, the IgFc polypeptide may have an amino acid other than Asn at position 77. In some cases, the IgFc polypeptide has Ala at position 77. In some cases, the Fc polypeptide present in the TMP comprises the amino acid sequence shown in FIG. 2C. In some cases, the Fc polypeptide present in the TMP comprises the amino acid sequence shown in FIG. 2D.

[0133] In some cases, the Fc polypeptide present in the TMP comprises the amino acid sequence shown in Figure 2E (human IgG1Fc having 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 comprises the amino acid sequence shown in Figure 2F, which has an N279A substitution (N77A of the amino acid sequence shown in Figure 2F).

[0134] 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.

[0135] 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).

[0136] In some cases, the IgG4 Fc polypeptide comprises the amino acid sequence: PPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 178).

[0137] 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 comprises 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.

[0138] 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 and the β2M polypeptide.

[0139] As used herein, the phrase "an optional peptide linker between any two of the 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 of the components of the TMP" refers to a peptide linker between any one or more of: i) a peptide 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.

[0140] Suitable linkers (also called "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.

[0141] Flexible Peptide Linker Specific flexible peptide linkers include glycine polymers (G), glycine-serine polymers (e.g., (GS) n , (GSGGS) n (SEQ ID NO: 179), (GGGGS)n (SEQ ID NO: 180), and (GGGS) n(SEQ ID NO:181) (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 significantly more access to phi-psi space than alanine and is much less restricted than residues with longer side chains (see Scheraga, Rev. Computational Chem. 11173-142 (1992)). Particular linkers can have amino acid sequences such as, but are not limited to, GGSG (SEQ ID NO:182), GGSGG (SEQ ID NO:183), GSGSG (SEQ ID NO:184), GSGGG (SEQ ID NO:185), GGGSG (SEQ ID NO:186), GSSSG (SEQ ID NO:187).

[0142] Specific flexible peptide linkers include, for example, (GGGGS)n (SEQ ID NO:235) (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:235), 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:188), where n is 2. In some cases, the linker has the amino acid sequence (GGGGS)n (SEQ ID NO:189), where n is 3. In some cases, the linker has the amino acid sequence (GGGGS)n (SEQ ID NO:190), where n is 4. In some cases, the linker has the amino acid sequence (GGGGS)n (SEQ ID NO:191), where n is 7. In some cases, the linker has the amino acid sequence AAAGG (SEQ ID NO:192). Also suitable is a linker having the amino acid sequence AAAGG (SEQ ID NO: 192). In the TMPs of the disclosure, the β2M polypeptide can be linked to the MHC heavy chain polypeptide by a (GGGGS)n (SEQ ID NO: 235) linker, where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, e.g., n=3, n=4, or n=7.

[0143] The term "short flexible peptide linker" as used in this disclosure refers to a flexible peptide linker composed of less than 15 amino acids, i.e., 2-14 amino acids. For example, a short flexible peptide linker can be composed of 2-4, 2-5, or 3-6 amino acids (e.g., the GGS linker discussed in Example 1), or 4-8, 5-10, or 10-14 amino acids. Included within this range are flexible peptide linkers composed of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 amino acids.

[0144] 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 may have a specific secondary and / or tertiary structure in solution, and typically have a length sufficient to form a secondary or tertiary structure. Rigid peptide linkers include proline-rich peptide linkers and peptide linkers with 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.

[0145] Specific examples of rigid peptide linkers include (EAAAK)n (SEQ ID NO: 236), A(EAAAK)nA (SEQ ID NO: 245), A(EAAAK)nALEA(EAAAK)nA (SEQ ID NO: 238), (Lys-Pro)n (SEQ ID NO: 242), (Glu-Pro)n (SEQ ID NO: 239), (Thr-Pro-Arg)n (SEQ ID NO: 240), and (Ala-Pro)n (SEQ ID NO: 241), where n is an integer of 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: 194) include EAAAK (SEQ ID NO: 194), (EAAAK)2 (SEQ ID NO: 197), (EAAAK)3 (SEQ ID NO: 198), A(EAAAK)4ALEA(EAAAK)4A (SEQ ID NO: 199), and AEAAAKEAAAKA (SEQ ID NO: 200). Non-limiting examples of suitable rigid peptide linkers having (AP)n include PAPAP (SEQ ID NO: 201) (also referred to herein as "(AP)2"), APAPAPAP (SEQ ID NO: 202) (also referred to herein as "(AP)4"), APAPAPAPAPAP (SEQ ID NO: 203) (also referred to herein as "(AP)6"), APAPAPAPAPAPAPAP (SEQ ID NO: 204) (also referred to herein as "(AP)8"), and APAPAPAPAPAPAPAPAPAPAP (SEQ ID NO: 205) (also referred to herein as "(AP)10"). Non-limiting illustrative examples of suitable rigid peptide linkers having (KP)n include KPKP (SEQ ID NO:206) (also referred to herein as "(KP)2"), KPKPKPKP (SEQ ID NO:207) (also referred to herein as "(KP)4"), KPKPKPKPKPKP (SEQ ID NO:208) (also referred to herein as "(KP)6"), KPKPKPKPKPKPKPKP (SEQ ID NO:209) (also referred to herein as "(KP)8"), and KPKPKPKPKPKPKPKPKPKP (SEQ ID NO:210) (also referred to herein as "(KP)10").Non-limiting illustrative examples of suitable rigid peptide linkers having (EP)n include EPEP (SEQ ID NO:211) (also referred to herein as "(EP)2"), EPEPEPEPEP (SEQ ID NO:212) (also referred to herein as "(EP)4"), EPEPEPEPEPEPEP (SEQ ID NO:213) (also referred to herein as "(EP)6"), EPEPEPEPEPEPEPEP (SEQ ID NO:214) (also referred to herein as "(EP)8"), and EPEPEPEPEPEPEPEPEPEPEP (SEQ ID NO:215) (also referred to herein as "(EP)10").

[0146] In general, a TMP may contain a rigid peptide linker and / or a short flexible peptide linker between any two of the 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 comprising one or more MODs of configuration 2, it is used 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. (ii) In a TMP containing one or more MODs of configuration 3, they are used between any 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 comprising one or more MODs of configuration 4, used between any one or more of: i) the KRAS 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.

[0147] Thus, the present disclosure provides a method for increasing the thermal stability of a TMP comprising one or more MODs of Configuration 2, Configuration 3, or Configuration 4, the method comprising placing a rigid peptide linker or a short flexible linker between two or more of the components of the TMP, as described herein.

[0148] As shown in Example 1 below, it has been determined that the use of a rigid or short flexible peptide linker, instead of a flexible peptide linker, between an IgFc polypeptide and a MOD in a TMP having one or more MODs of configuration 3 can result in increased thermal stability of the resulting TMP compared to a similar TMP having a longer flexible peptide linker, such as a (G4S)3 linker (i.e., (GGGGS)3 linker) (SEQ ID NO: 189). 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, resulting in increased thermal stability as measured by accelerated stability testing as described in Example 1.

[0149] 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 in a TMP having one or more MODs of configuration 3, improves thermal stability as measured by the 37°C accelerated thermal stability test described in Example 1 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, compared to the thermal stability of a control TMP containing a flexible peptide linker that is a (GGGGS)3 linker (SEQ ID NO: 189) instead of the rigid peptide linker or short flexible peptide linker.

[0150] 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 in a TMP having one or more MODs of configuration 3, improves thermal stability as measured by the 42°C accelerated thermal stability test described in Example 1 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: 189) instead of the rigid peptide linker or short flexible peptide linker.

[0151] 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.

[0152] In some cases, the peptide linker between the KRAS peptide and the β2M polypeptide has the amino acid sequence GCGGS (SEQ ID NO: 216). In some cases, the peptide linker between the KRAS peptide and the β2M polypeptide has the amino acid sequence GCGGS(GGGGS)n (SEQ ID NO: 217), 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: 218), where n is 2.

[0153] In some cases, the peptide linker between the KRAS peptide and the β2M polypeptide has the amino acid sequence CGGGS (SEQ ID NO: 219). In some cases, the peptide linker has the amino acid sequence CGGGS(GGGGS)n (SEQ ID NO: 220), where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, e.g., 1, 2, or 3.

[0154] In some cases, the peptide linker between the KRAS peptide and the β2M polypeptide has the amino acid sequence GGCGS (SEQ ID NO: 221). In some cases, the peptide linker has the amino acid sequence GGCGS(GGGGS)n (SEQ ID NO: 222), where n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, e.g., 1, 2 or 3.

[0155] In some cases, the peptide linker between the KRAS peptide and the β2M polypeptide has the amino acid sequence GGGCS (SEQ ID NO: 223). In some cases, the peptide linker has the amino acid sequence GGGCS(GGGGS)n (SEQ ID NO: 224), where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, e.g., 1, 2, or 3.

[0156] In some cases, the peptide linker between the KRAS peptide and the β2M polypeptide has the amino acid sequence GGGGC (SEQ ID NO: 225). In some cases, the peptide linker has the amino acid sequence GGGGC(GGGGS)n (SEQ ID NO: 226), where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, e.g., 1, 2, or 3.

[0157] 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 human IgG1Fc 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 IgG1Fc 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.

[0158] Thus, the disclosure provides a protein, the protein comprising a) a first TMP and b) a second TMP, which may be identical to the first TMP, where the first and second TMP are covalently bonded to one another, the covalent bond may be a disulfide bond between an IgFc polypeptide in the first TMP and an IgFc polypeptide in the second TMP.

[0159] 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 T366Y and Y407T mutation pair 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.

[0160] Interspecies dimerization sequences may also be used to allow binding of TMP to non-TMP molecules, which can confer additional functionality to TMP. For example, TMP can bind 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 TMP to localize to tissues that contain the cancer-associated antigen.

[0161] 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.

[0162] 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).

[0163] In some cases, the TMP comprises i) a KRAS peptide, ii) a first MHC class I polypeptide (wherein the first MHC class I polypeptide is a β2M polypeptide), iii) a second MHC class I polypeptide (wherein 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.

[0164] In the case of a TMP comprising one or more MODs of configuration 2, one or more peptide linkers may be disposed between i) the KRAS 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) between the MODs if the TMP comprises two or more MODs in tandem. As noted above, in such a TMP, a rigid peptide linker or a short flexible peptide linker may be disposed 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) between further MODs in tandem, such as between the first MOD and the second MOD, if there are multiple MODs in tandem. In some cases, the rigid peptide linker has an amino acid sequence selected from EAAAK (SEQ ID NO: 194), A(EAAAK)n (SEQ ID NO: 244), A(EAAAK)nA (SEQ ID NO: 245), (AP)n (SEQ ID NO: 246), (EP)n (SEQ ID NO: 247), and (KP)n (SEQ ID NO: 248), 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 to 4, 2 to 5, 3 to 6, 4 to 8, 5 to 10, or 10 to 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 KRAS peptide and the β2M polypeptide may further comprise a Cys-containing linker as described above.

[0165] In the case of a TMP comprising one or more MODs of configuration 3, one or more peptide linkers may be disposed between i) the KRAS 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 a TMP, a rigid peptide linker or a short flexible peptide linker may be disposed between one or more of i) the IgFc polypeptide and the MOD, and ii) if multiple MODs are present in tandem, between 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: 194), A(EAAAK)n (SEQ ID NO: 244), A(EAAAK)nA (SEQ ID NO: 245), (AP)n (SEQ ID NO: 246), (EP)n (SEQ ID NO: 247), and (KP)n (SEQ ID NO: 248), 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 to 4, 2 to 5, 3 to 6, 4 to 8, 5 to 10, or 10 to 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 KRAS peptide and the β2M polypeptide may further comprise a Cys-containing linker as described above.

[0166] 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.

[0167] In any of the above TMPs, the KRAS peptide has an amino acid sequence selected from any of the KRAS peptide sequences set forth below. For example, the KRAS peptide may be selected from the following KRAS peptide sequences: VVGADGVGK (SEQ ID NO: 108), VVGACGVGK (SEQ ID NO: 109), VVGAVGVGK (SEQ ID NO: 110), VVVGADGVGK (SEQ ID NO: 111), VVVGAVGVGK (SEQ ID NO: 112), VVVGACGVGK (SEQ ID NO: 113), VTGADGVGK (SEQ ID NO: 114), VTGAVGVGK (SEQ ID NO: 115), VTGACGVGK (SEQ ID NO: 116), VTVGADGVGK (SEQ ID NO: 117), VTVGAVGVGK (SEQ ID NO: 118), VTVGACGVGK (SEQ ID NO: 119), LVVGA The polypeptide may have an amino acid sequence selected from the group consisting of DGV (SEQ ID NO: 124), LVVVGAVGV (SEQ ID NO: 125), LVVVGACGV (SEQ ID NO: 126), KLVVVGADGV (SEQ ID NO: 127), KLVVVGAVGV (SEQ ID NO: 128), KLVVVGACGV (SEQ ID NO: 129), LLVVGADGV (SEQ ID NO: 130), LLVVGAVGV (SEQ ID NO: 131), LLVVGACGV (SEQ ID NO: 132), FLVVVGADGV (SEQ ID NO: 133), FLVVVGAVGV (SEQ ID NO: 134), and FLVVVGACGV (SEQ ID NO: 135).

[0168] 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 having a Y84C substitution and / or an A236C substitution. * In some cases, the HLA heavy chain polypeptide is an HLA-A 0201 polypeptide having a Y84C substitution and / or an A236C substitution. * In some cases, the HLA heavy chain polypeptide is an HLA-E 1101 polypeptide having 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 having a Y84C substitution and / or an A236C substitution. * 0101 and / or HLA-G * 01:04A * 1101 polypeptide.

[0169] In some cases, the TMP comprises 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 having an H16A substitution and an F42A substitution, or a mutant IL-2 polypeptide having an H16T substitution and an F42A substitution).

[0170] In some cases, the IgFc polypeptide is a human IgG1Fc polypeptide variant that does not substantially induce cell lysis, for example an IgG1Fc polypeptide having a L234A substitution and a L235A substitution as shown in Figure 2B or Figure 2D.

[0171] 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 human IgG1Fc polypeptide mutant that does not substantially cause cell lysis, such as a human IgG1Fc polypeptide having an L234A substitution and an L235A substitution 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 having an H16A substitution and an F42A substitution, or a mutant IL-2 polypeptide having an H16T substitution and an F42A substitution. In some cases, the KRAS peptide has an amino acid sequence selected from any of the KRAS peptide sequences described above. For example, the KRAS peptides are VVGADGVGK (SEQ ID NO: 108), VVGACGVGK (SEQ ID NO: 109), VVGAVGVGK (SEQ ID NO: 110), VVVGADGVGK (SEQ ID NO: 111), VVVGAVGVGK (SEQ ID NO: 112), VVVGACGVGK (SEQ ID NO: 113), VTGADGVGK (SEQ ID NO: 114), VTGAVGVGK (SEQ ID NO: 115), VTGACGVGK (SEQ ID NO: 116), VTVGADGVGK (SEQ ID NO: 117), VTVGAVGVGK (SEQ ID NO: 118), VTVGACGVGK (SEQ ID NO: 119), LVVGA The polypeptide may have an amino acid sequence selected from the group consisting of DGV (SEQ ID NO: 124), LVVVGAVGV (SEQ ID NO: 125), LVVVGACGV (SEQ ID NO: 126), KLVVVGADGV (SEQ ID NO: 127), KLVVVGAVGV (SEQ ID NO: 128), KLVVVGACGV (SEQ ID NO: 129), LLVVGADGV (SEQ ID NO: 130), LLVVGAVGV (SEQ ID NO: 131), LLVVGACGV (SEQ ID NO: 132), FLVVVGADGV (SEQ ID NO: 133), FLVVVGAVGV (SEQ ID NO: 134), and FLVVVGACGV (SEQ ID NO: 135).In such 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: 194), A(EAAAK)n (SEQ ID NO: 244), A(EAAAK)nA (SEQ ID NO: 245), (AP)n (SEQ ID NO: 246), (EP)n (SEQ ID NO: 247), and (KP)n (SEQ ID NO: 248), where n is an integer between 1 and 10 (e.g., n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). Meanwhile, in some cases, the TMP comprises a short flexible peptide linker between the mutant IL-2MOD and the IgFc polypeptide present in the TMP, where 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. In some cases, the TMP comprises two copies of a mutant IL-2MOD. In some cases, the TMP comprises two copies of a mutant IL-2MOD, and the TMP comprises rigid peptide linkers a) 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, the rigid peptide linkers having an amino acid sequence selected from EAAAK (SEQ ID NO: 194), A(EAAAK)n (SEQ ID NO: 244), A(EAAAK)nA (SEQ ID NO: 245), (AP)n (SEQ ID NO: 246), (EP)n (SEQ ID NO: 247), and (KP)n (SEQ ID NO: 248), where n is an integer from 1 to 10 (e.g., n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10).

[0172] 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. *1101 polypeptide, optionally comprising a Y84 substitution and / or an A236C substitution, *1101 polypeptide). In some cases, the IgFc polypeptide is a human IgG1Fc polypeptide variant that does not substantially cause cell lysis, such as a human IgG1Fc polypeptide having an L234A substitution and an L235A substitution 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 having an H16A substitution and an F42A substitution, or a mutant IL-2 polypeptide having an H16T substitution and an F42A substitution. In some cases, the KRAS peptide has an amino acid sequence selected from any of the KRAS peptide sequences described above. For example, the KRAS peptides are VVGADGVGK (SEQ ID NO: 108), VVGACGVGK (SEQ ID NO: 109), VVGAVGVGK (SEQ ID NO: 110), VVVGADGVGK (SEQ ID NO: 111), VVVGAVGVGK (SEQ ID NO: 112), VVVGACGVGK (SEQ ID NO: 113), VTGADGVGK (SEQ ID NO: 114), VTGAVGVGK (SEQ ID NO: 115), VTGACGVGK (SEQ ID NO: 116), VTVGADGVGK (SEQ ID NO: 117), VTVGAVGVGK (SEQ ID NO: 118), VTVGACGVGK (SEQ ID NO: 119), LVVGA The polypeptide may have an amino acid sequence selected from the group consisting of DGV (SEQ ID NO: 124), LVVVGAVGV (SEQ ID NO: 125), LVVVGACGV (SEQ ID NO: 126), KLVVVGADGV (SEQ ID NO: 127), KLVVVGAVGV (SEQ ID NO: 128), KLVVVGACGV (SEQ ID NO: 129), LLVVGADGV (SEQ ID NO: 130), LLVVGAVGV (SEQ ID NO: 131), LLVVGACGV (SEQ ID NO: 132), FLVVVGADGV (SEQ ID NO: 133), FLVVVGAVGV (SEQ ID NO: 134), and FLVVVGACGV (SEQ ID NO: 135).In such 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: 194), A(EAAAK)n (SEQ ID NO: 244), A(EAAAK)nA (SEQ ID NO: 245), (AP)n (SEQ ID NO: 246), (EP)n (SEQ ID NO: 247), and (KP)n (SEQ ID NO: 248), 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 two copies of mutant IL-2MOD. In some cases, the TMP comprises two copies of a mutant IL-2MOD, and the TMP comprises rigid peptide linkers a) 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, the rigid peptide linkers having an amino acid sequence selected from EAAAK (SEQ ID NO: 194), A(EAAAK)n (SEQ ID NO: 244), A(EAAAK)nA (SEQ ID NO: 245), (AP)n (SEQ ID NO: 246), (EP)n (SEQ ID NO: 247), and (KP)n (SEQ ID NO: 248), where n is an integer between 1 and 10 (e.g., n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). Alternatively, in some cases, the TMP comprises a short flexible peptide linker between the mutant IL-2MOD and the IgFc polypeptide present in the TMP, where 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.

[0173] 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 human IgG1Fc polypeptide variant that does not substantially cause cell lysis, such as a human IgG1Fc polypeptide having an L234A substitution and an L235A substitution 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 having an H16A substitution and an F42A substitution, or a mutant IL-2 polypeptide having an H16T substitution and an F42A substitution. In some cases, the KRAS peptide has an amino acid sequence selected from any of the KRAS peptide sequences above. For example, the KRAS peptides are VVGADGVGK (SEQ ID NO: 108), VVGACGVGK (SEQ ID NO: 109), VVGAVGVGK (SEQ ID NO: 110), VVVGADGVGK (SEQ ID NO: 111), VVVGAVGVGK (SEQ ID NO: 112), VVVGACGVGK (SEQ ID NO: 113), VTGADGVGK (SEQ ID NO: 114), VTGAVGVGK (SEQ ID NO: 115), VTGACGVGK (SEQ ID NO: 116), VTVGADGVGK (SEQ ID NO: 117), VTVGAVGVGK (SEQ ID NO: 118), VTVGACGVGK (SEQ ID NO: 119), LVVGA The polypeptide may have an amino acid sequence selected from the group consisting of DGV (SEQ ID NO: 124), LVVVGAVGV (SEQ ID NO: 125), LVVVGACGV (SEQ ID NO: 126), KLVVVGADGV (SEQ ID NO: 127), KLVVVGAVGV (SEQ ID NO: 128), KLVVVGACGV (SEQ ID NO: 129), LLVVGADGV (SEQ ID NO: 130), LLVVGAVGV (SEQ ID NO: 131), LLVVGACGV (SEQ ID NO: 132), FLVVVGADGV (SEQ ID NO: 133), FLVVVGAVGV (SEQ ID NO: 134), and FLVVVGACGV (SEQ ID NO: 135).In such 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: 194), A(EAAAK)n (SEQ ID NO: 244), A(EAAAK)nA (SEQ ID NO: 245), (AP)n (SEQ ID NO: 246), (EP)n (SEQ ID NO: 247), and (KP)n (SEQ ID NO: 248), 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 two copies of mutant IL-2MOD. In some cases, the TMP comprises two copies of a mutant IL-2MOD, and the TMP comprises rigid peptide linkers a) 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, the rigid peptide linkers having an amino acid sequence selected from EAAAK (SEQ ID NO: 194), A(EAAAK)n (SEQ ID NO: 244), A(EAAAK)nA (SEQ ID NO: 245), (AP)n (SEQ ID NO: 246), (EP)n (SEQ ID NO: 247), and (KP)n (SEQ ID NO: 248), where n is an integer between 1 and 10 (e.g., n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). Alternatively, in some cases, the TMP comprises a short flexible peptide linker between the mutant IL-2MOD and the IgFc polypeptide present in the TMP, where 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.

[0174] As discussed above, the TMP can include one or more intrachain disulfide bonds. For example, the TMP can include a β2M polypeptide having an R12C substitution and a class I MHC heavy chain polypeptide having an A236C substitution, whereby a 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. As another example, the TMP can include any of the following: i) GCGGS (GGGGS) n(SEQ ID NO:249) sequence, where n is 1, 2, or 3, and ii) a class I MHC heavy chain polypeptide having a Y84C substitution, whereby a disulfide bond is formed between a Cys in the peptide linker and a Cys at position 84 of the class I MHC heavy chain polypeptide. n (SEQ ID NO:249) sequence, where n is 1, 2, or 3; and ii) a class I MHC heavy chain polypeptide having a Y84C substitution and a 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." The TMP can a) contain a G2C / Y84C disulfide bond and no R12C / A236C disulfide bond, b) contain an R12C / A236C disulfide bond and no G2C / Y84C disulfide bond, or c) contain a G2C / Y84C disulfide bond and an R12C / A236C disulfide bond. In some cases, the 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.

[0175] The TMP can be one that a) comprises a G2C / Y84C disulfide bond, does not comprise a R12C / A236C disulfide bond, and b) comprises at least one MOD that is configuration 2 or configuration 3. The TMP can be one that a) comprises a R12C / A236C disulfide bond, does not comprise a G2C / Y84C disulfide bond, and b) comprises at least one MOD that is configuration 2 or configuration 3. The TMP can be one that comprises a) a G2C / Y84C disulfide bond and a R12C / A236C disulfide bond, and b) at least one MOD that is configuration 2 or configuration 3.

[0176] 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 position 84 and alanine at position 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.

[0177] 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 position 84 and position 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.

[0178] 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 a Cys at position 84 and alanine at position 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.

[0179] As a non-limiting example, TMP may include the amino acid sequence (TMP4074) shown in FIG. 14A. As a non-limiting example, TMP may include the amino acid sequence (TMP4390) shown in FIG. 14B. As a non-limiting example, TMP may include the amino acid sequence (TMP4391) shown in FIG. 14C. As a non-limiting example, TMP may include the amino acid sequence (TMP4467) shown in FIG. 14D. As a non-limiting example, TMP may include the amino acid sequence (TMP4468) shown in FIG. 14E. As a non-limiting example, TMP may include the amino acid sequence (TMP4469) shown in FIG. 14F. As a non-limiting example, TMP may include the amino acid sequence (TMP4470) shown in FIG. 14G. As a non-limiting example, TMP may include the amino acid sequence (TMP4471) shown in FIG. 14H. As a non-limiting example, TMP may include the amino acid sequence (TMP4472) shown in FIG. 14I. As a non-limiting example, TMP may include the amino acid sequence (TMP4473) shown in FIG. 14J. As a non-limiting example, TMP may include the amino acid sequence (TMP4478) shown in FIG. 14K. As a non-limiting example, TMP may include the amino acid sequence (TMP4514) shown in FIG. 14L. As a non-limiting example, TMP may include the amino acid sequence (TMP4515) shown in FIG. 14M. As a non-limiting example, TMP may include the amino acid sequence (TMP4545) shown in FIG. 14N. As a non-limiting example, TMP may include the amino acid sequence (TMP4546) shown in FIG. 14O. As a non-limiting example, TMP may include the amino acid sequence (TMP4547) shown in FIG. 14P. As a specific, non-limiting example, TMP can include the amino acid sequence shown in Figure 14Q (TMP4548). As a specific, non-limiting example, TMP can include the amino acid sequence shown in Figure 14R (TMP4549). As a specific, non-limiting example, TMP can include the amino acid sequence shown in Figure 14S (TMP4550). As a specific, non-limiting example, TMP can include the amino acid sequence shown in Figure 14T (TMP4551).As a specific, non-limiting example, the TMP can include the amino acid sequence shown in Figure 14U (TMP4552). As a specific, non-limiting example, the TMP can include the amino acid sequence shown in Figure 14V (TMP4553).

[0180] Further non-limiting examples of TMPs are shown in Figures 16A-16D, where "(X)" is a rigid peptide linker. For example, (X) is a rigid peptide linker having an amino acid sequence selected from EAAAK (SEQ ID NO: 194), A(EAAAK)n (SEQ ID NO: 244), A(EAAAK)nA (SEQ ID NO: 245), (AP)n (SEQ ID NO: 246), (EP)n (SEQ ID NO: 247), and (KP)n (SEQ ID NO: 248), where n is an integer from 1 to 10 (e.g., n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). As a specific example, TMP can have the amino acid sequence shown in Figure 16A, where (X) is (AP)n (n is 2 to 8). As another specific example, TMP can have the amino acid sequence shown in Figure 16A, where (X) is (EP)n (n is 2 to 8). As another specific example, TMP can have the amino acid sequence shown in FIG. 16A, where (X) is (KP)n (n is 2 to 8). As another specific example, TMP can have the amino acid sequence shown in FIG. 16A, where (X) is a peptide containing EAAAK (SEQ ID NO: 194), for example, (X) is A(EAAAK)nA (SEQ ID NO: 245) (where n is 1 or 2). As another specific example, TMP can have the amino acid sequence shown in FIG. 16B, where (X) is (EP)n (n is 2 to 8). As another specific example, TMP can have the amino acid sequence shown in FIG. 16B, where (X) is (KP)n (n is 2 to 8). As another embodiment, the TMP can have the amino acid sequence shown in FIG. 16B, where (X) is a peptide comprising EAAAK (SEQ ID NO: 194), e.g., (X) is A(EAAAK)nA (SEQ ID NO: 245), where n is 1 or 2. As another embodiment, the TMP can have the amino acid sequence shown in FIG. 16C, where (X) is (EP)n, where n is 2 to 8. As another embodiment, the TMP can have the amino acid sequence shown in FIG. 16C, where (X) is (KP)n, where n is 2 to 8.As another specific example, the TMP can have the amino acid sequence shown in FIG. 16C, where (X) is a peptide comprising EAAAK (SEQ ID NO: 194), for example, (X) is A(EAAAK)nA (SEQ ID NO: 245) (where n is 1 or 2). As another specific example, the TMP can have the amino acid sequence shown in FIG. 16D, where (X) is (EP)n (where n is 2 to 8). As another specific example, the TMP can have the amino acid sequence shown in FIG. 16D, where (X) is (KP)n (where n is 2 to 8). As another specific example, the TMP can have the amino acid sequence shown in FIG. 16D, where (X) is a peptide comprising EAAAK (SEQ ID NO: 194), for example, (X) is A(EAAAK)nA (SEQ ID NO: 245) (where n is 1 or 2). In any of the specific TMPs shown in FIGS. 16A to 16D, the KRAS peptide can be replaced with a different KRAS peptide. Suitable KRAS peptides are described herein. In any of the exemplary TMPs shown in Figures 16A-16D, the peptide linker between the two copies of the mutant IL-2 polypeptide can be replaced with a rigid peptide linker.

[0181] 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.

[0182] 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.

[0183] 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.

[0184] The present disclosure further provides a genetically modified host cell, which is genetically modified with a nucleic acid or expression vector as described above.

[0185] 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.

[0186] In some cases, the host cell is a mammalian cell that has been genetically modified so that it does not synthesize endogenous MHC β2M.

[0187] 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.

[0188] 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.

[0189] 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.

[0190] 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.

[0191] 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.

[0192] 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.

[0193] 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.

[0194] Compositions Comprising Nucleic Acids or Recombinant Expression Vectors The present disclosure provides pharmaceutical compositions comprising a nucleic acid (e.g., DNA, RNA, or mRNA) or a recombinant expression vector encoding TMP. Published PCT applications WO2020132138A1 and WO2019 / 051091 disclose methods for preparing such compositions. See, e.g., paragraphs

[0537] to

[0546] of WO2020132138A1 and paragraphs

[0423] to

[0432] of WO2019 / 051091. These disclosures are incorporated herein by reference as if expressly set forth herein.

[0195] Methods for modulating T cell activity The disclosure provides methods of selectively modulating activity of epitope-specific T cells (e.g., T cells specific for a KRAS epitope, such as a KRAS peptide that includes a cancer-associated mutation), the methods comprising contacting the T cells with a TMP or a dimerized TMP, where contacting the T cells with the TMP or the dimerized TMP selectively modulates 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.

[0196] When the TMP or dimerized TMP contains an activating polypeptide, MOD, contacting a T cell with the TMP or dimerized TMP activates the epitope-specific T cell, increases the cytotoxic activity of the T cell against cancer cells expressing the KRAS epitope, and / or increases the number of epitope-specific T cells.

[0197] 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 KRAS 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 the KRAS 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 the target KRAS epitope versus the increase in the number of T cells not specific for the 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) cytotoxicity (e.g., CD8 + ) activating T cells; ii) cytotoxicity (e.g., CD8 + iii) inducing cytotoxic activity of T cells; and iv) cytotoxicity (e.g., CD8 + ) inducing the production and release of cytotoxins (e.g., perforin, granzymes, granulysin) by T cells; iv) cytotoxicity (e.g., CD8 + ) Inducing T cell proliferation.

[0198] As described above, in some cases, TMP or dimerized TMP induces the proliferation of epitope-specific T cells when administered to an individual in need thereof. The increase in the proportion of epitope-specific T cells can be measured by conventional flow cytometry (see, for example, Example 2 below). Thus, for example, the proportion of total CD8+ T cells specific for KRAS epitopes 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.

[0199] The disclosure provides a method of selectively delivering a MOD to a target T cell, the method comprising contacting a mixed population of T cells with a TMP or dimerized TMP, the mixed population of T cells comprising target T cells and non-target T cells, the target T cells being specific for a KRAS epitope present in the TMP or dimerized TMP (e.g., the target T cells being specific for a KRAS 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, and the contacting results in activation and / or expansion 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 the individual.

[0200] The present disclosure provides a method for detecting the presence of target T cells that bind to an epitope of interest (e.g., a cancer epitope, a KRAS peptide containing a cancer-associated mutation) in a mixed population of T cells obtained from an individual, the method comprising the steps of a) contacting the mixed population of T cells in vitro with a TMP or a dimerized TMP, where the TMP or dimerized TMP comprises a KRAS 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.

[0201] Treatment method The present disclosure provides a method of treating an individual, comprising administering to the individual TMP or dimerized TMP or one or more nucleic acids encoding TMP in an amount 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 comprises administering to an individual in need of treatment one or more recombinant expression vectors comprising a nucleotide sequence encoding TMP. In some cases, the method of treatment comprises administering to an individual in need of treatment one or more nucleic acid molecules (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, for example, cancer expressing a KRAS peptide (e.g., a mutant KRAS peptide as described above). Thus, the present disclosure provides a method of treatment comprising administering to an individual in need of treatment an effective amount of TMP or dimerized TMP.

[0202] TMP or dimerized TMP can be administered to an individual in need of treatment to treat cancer in the individual, where the cancer expresses a KRAS peptide present in TMP or dimerized TMP. For example, the cancer can be one whose cells express or overexpress KRAS (e.g., a mutant form of KRAS as described above). The present disclosure provides a method of treating cancer in an individual, comprising administering to the individual an effective amount of TMP or dimerized TMP, or an effective amount of one or more nucleic acids (e.g., expression vectors, mRNA, etc.) comprising a nucleotide sequence encoding TMP or dimerized TMP, where the TMP or dimerized TMP comprises a T cell epitope that is a KRAS epitope, and the TMP or dimerized TMP comprises a stimulatory 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 (i.e., the amount of cancer in the body) in the individual or maintains the total tumor burden in the patient relatively stable for a sufficient period of time such that the patient has "stable disease" as measured by standard Response Evaluation Criteria in Solid Tumors (RECIST). See, e.g., Aykan and Ozatli (2020) World J. Clin. Oncol. 11:53.

[0203] 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 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.

[0204] 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.

[0205] 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 the TMP or dimerized TMP or when the TMP or dimerized TMP is not administered.

[0206] 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.

[0207] In some cases, an effective amount of TMP or dimerized TMP is an amount that, when administered once or multiple times to an individual (individual with a tumor) in need of treatment, 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 (individual with a tumor) in need of treatment, as a monotherapy or 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 is 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. 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 overall survival 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 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 of the individual in the absence of administration of TMP or dimerized TMP.

[0208] 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, and the same method can be used with a series of samples to determine whether and by how much it has been reduced.

[0209] Cancers treatable by the method of the present disclosure include cancers in which cancer cells express mutant forms of KRAS.Specific examples of such cancers include adenocarcinomas and hematopoietic tumors.Specific examples of cancers treatable by the method of the present disclosure include 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.

[0210] As mentioned above, in some cases, in carrying out the method for treating a subject, TMP or dimerized TMP is administered to the individual in need of treatment.In other cases, in carrying out the method for treating a subject, one or more nucleic acids that contain the nucleotide sequence (e.g., DNA or mRNA) that codes for TMP or dimerized TMP are administered to the individual in need of treatment.For example, in other cases, one or more nucleic acids (e.g., one or more recombinant expression vectors) are administered to the individual in need of treatment.

[0211] 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, 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.

[0212] 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.

[0213] 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 can be readily determined by those of skill in the art by a variety of means.

[0214] 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.

[0215] 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.

[0216] 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.

[0217] 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.

[0218] 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. The suitable dosage of TMP or dimerized TMP can be the same as the dosage of TMP monotherapy (as described above), or it can be less than or more than the dosage of monotherapy. Suitable additional therapeutic agents include, for example, i) immune checkpoint inhibitors, ii) cancer chemotherapeutic agents, iii) agents that inhibit cancer-associated mutant forms of KRAS, and iv) one or more other TMPs or dimerized TMPs. Suitable additional therapies include, for example, radiation therapy, surgery (e.g., surgical removal of tumors), and the like.

[0219] In some cases, the method includes 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 method includes administering to an individual in need thereof a) a first composition comprising TMP or dimerized TMP, and b) a second composition comprising an agent that inhibits a cancer-associated mutant form of KRAS (e.g., KRAS(G12C or G12V)). In some cases, the method includes 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.

[0220] TMP or dimerized TMP can be administered to an individual in need thereof at the same time or at a different time than one or more additional therapeutic agents are administered.

[0221] 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.

[0222] As another specific example, the method of treatment can include administering TMP or dimerized TMP and an immune checkpoint inhibitor (e.g., an antibody specific for an immune checkpoint) together. "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. On the other hand, TMP or dimerized TMP and an immune checkpoint inhibitor can be administered on different schedules (including different days and different weeks) and at different frequencies. 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.

[0223] 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.

[0224] 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-870,893 (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.

[0225] 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 disclosure may 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.

[0226] In some cases, the at least one additional therapeutic agent is an agent that selectively inhibits a mutant form of KRAS (e.g., KRAS(G12C), KRAS(K117A), etc.). Specific examples of agents that selectively inhibit a mutant form of KRAS include ARS-1620, AMG510, KRA-533, and MRTX849.

[0227] 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 of treatment: a) a first composition comprising a first TMP or dimerized TMP, where the first TMP or dimerized TMP is a TMP or dimerization, and b) a second composition comprising a second TMP or dimerized TMP, where the second TMP or dimerized TMP is a different TMP or dimerized TMP from the first TMP or dimerized TMP, e.g., comprises a different KRAS epitope and / or one or more different MODs. Additionally or alternatively, the one or more additional TMPs or dimerized TMPs can comprise an epitope that is a cancer-associated peptide other than a KRAS peptide epitope.

[0228] Suitable subjects for treatment Subjects suitable for treatment with the methods of the present disclosure include individuals with cancer, such as 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 but have initially been effective but have subsequently become resistant to the treatment. Subjects suitable for treatment include individuals with cancer whose cancer cells express a mutant form of KRAS, where the mutant form of KRAS is a cancer-associated mutant form. Subjects suitable for treatment methods include individuals with cancer such as multiple myeloma, B-cell lymphoma, breast cancer, lung cancer, ovarian cancer, pancreatic cancer, colorectal cancer, prostate cancer, kidney cancer, acute myeloid leukemia, mesothelioma, thyroid cancer, head and neck cancer, gastric cancer, urothelial cancer, cervical cancer, and ovarian endometrial cancer.

[0229] 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.

[0230] Detection Method The TMP or dimerized TMP of the present disclosure is useful for diagnostic and therapeutic applications. As described below, when used for diagnostic applications, the TMP or dimerized TMP of the present disclosure can further include a detectable label, and the binding of the TMP or dimerized TMP to target T cells is detected by detecting the detectable label. When used for such diagnostic applications, the 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 the KRAS epitope. Like TMP, APP can also dimerize. Thus, the following discussion of detection methods using TMP and dimerized TMP is believed to apply to the use of APP and dimerized APP as well. The construct shown as "4515" in Figure 14M is a specific example of such an APP.

[0231] Thus, 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 KRAS epitope present in the TMP / APP or dimerized TMP / APP, i.e., that the T cell has a T cell receptor specific for a KRAS epitope present in the TMP / APP or dimerized TMP / APP.

[0232] 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.

[0233] 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, a detectable label 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.

[0234] 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, Se, Lurian, 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 proteins and kindling proteins, 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, 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.

[0235] 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.

[0236] 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.

[0237] 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 Pieces or 10 8 ~10 9 1 or 10 9 The antibody may be present in a sample containing more than one T cell.

[0238] HLA / peptide binding assay Whether a particular peptide (e.g., a KRAS peptide containing a KRAS 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] .

[0239] 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.

[0240] Specific Non-Limiting Aspects of the Disclosure Mode Set A The above-mentioned aspects, including the embodiments of the subject matter, 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 employed, 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 intended to be limited to the combinations of aspects explicitly stated below.

[0241] Aspect 1 A single chain T cell modulating polypeptide (TMP) comprising: i) a KRAS peptide comprising a KRAS epitope expressed on a cancer cell, the KRAS peptide being about 4 to about 25 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; v) immunoglobulin (Ig) Fc polypeptides; Including, The TMP is (a) a 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) a 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). Including, Optionally, the TMP comprises one or more additional peptide linkers.

[0242] Aspect 2 The TMP according to embodiment 1, 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-2M.

[0243] Aspect 3 3. The TMP according to embodiment 1 or embodiment 2, wherein said IgFc polypeptide does not substantially induce cell lysis, and optionally said IgFc is an IgG1Fc polypeptide comprising one or more amino acid substitutions selected from the group consisting of N297A, L234A, L235A, L234F, L235E, and P331S.

[0244] Aspect 4 The TMP according to any one of embodiments 1 to 3, comprising one or more intrachain disulfide bonds.

[0245] Aspect 5 The TMP according to embodiment 4, wherein the β2M polypeptide and the MHC heavy chain polypeptide are linked by a disulfide bond, said disulfide bond linking a Cys residue in the β2M polypeptide to a Cys residue in 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.

[0246] Aspect 6 6. The TMP according to embodiment 4 or embodiment 5, wherein the β2-microglobulin polypeptide is linked to the KRAS peptide by a first linker comprising a Cys, and a disulfide bond is formed between the Cys present in the first linker and the Cys present in the MHC heavy chain polypeptide, and optionally the first linker comprises the sequence CGGGS(GGGGS)n (SEQ ID NO: 220) or the sequence GCGGS(GGGGS)n (SEQ ID NO: 217), where n is an integer from 1 to 10, e.g., 2 or 3, and a disulfide bond is formed between the Cys in the linker and the Cys replacing Tyr-84 in the MHC heavy chain polypeptide.

[0247] Aspect 7 The MHC heavy chain polypeptide is HLA-A * 0201 Polypeptide, HLA-A * 1101 Polypeptide, HLA-A * 3303 polypeptide, and HLA-A * 7. The TMP according to any one of embodiments 1 to 6, comprising an amino acid sequence having at least 95% amino acid sequence identity to an HLA-A polypeptide selected from the group consisting of: 2401 polypeptides.

[0248] Aspect 8 The TMP according to any one of embodiments 1 to 6, wherein said MHC heavy chain polypeptide comprises an amino acid sequence having at least 95% amino acid sequence identity to an HLA-E polypeptide or an HLA-G polypeptide.

[0249] Aspect 9 9. The TMP according to any one of aspects 1-8, wherein the 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 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.

[0250] Aspect 10 The TMP according to embodiment 9, wherein said at least one immune modulating polypeptide is a mutant IL-2 having 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.

[0251] Aspect 11 The TMP according to embodiment 9 or 10, comprising two copies of said immunomodulatory polypeptide.

[0252] Aspect 12 The TMP according to embodiment 11, wherein two copies of said immunomodulatory polypeptide are arranged in tandem, and optionally a peptide linker is arranged between said two copies.

[0253] Aspect 13 The TMP according to any one of aspects 1 to 12, wherein the KRAS peptide is between 8 amino acids and 12 amino acids in length.

[0254] Aspect 14 The KRAS peptide is A) VVGADGVGK (SEQ ID NO:108), VVGACGVGK (SEQ ID NO:109), VVGAVGVGK (SEQ ID NO:110), VVVGADGVGK (SEQ ID NO:111), VVVGAVGVGK (SEQ ID NO:112), VVVGACGVGK (SEQ ID NO:113), VTGADGVGK (SEQ ID NO:114), VTGAVGVGK (SEQ ID NO:115), VTGACGVGK (SEQ ID NO:116), VTVGADGVGK (SEQ ID NO:117), VTVGAVGVGK (SEQ ID NO:118), and VTVGACGVGK (SEQ ID NO:119), wherein the KRAS peptide is 9 or 10 amino acids in length, or at least 9 or 10 amino acids in length; B) VVVGAGDVGK (SEQ ID NO: 120), VVGAGDVGK (SEQ ID NO: 121), VVVGARGVGK (SEQ ID NO: 122), and VVGARGVGK (SEQ ID NO: 123), wherein the KRAS peptide is 9 or 10 amino acids in length, or 9 or 10 amino acids in length; C) LVVVGADGV (SEQ ID NO:124), LVVVGAVGV (SEQ ID NO:125), LVVVGACGV (SEQ ID NO:126), KLVVVGADGV (SEQ ID NO:127), KLVVVGAVGV (SEQ ID NO:128), KLVVVGACGV (SEQ ID NO:129), LLVVGADGV (SEQ ID NO:130), LLVVGAVGV (SEQ ID NO:131), LLVVGACGV (SEQ ID NO:132), FLVVVGADGV (SEQ ID NO:133), FLVVVGAVGV (SEQ ID NO:134), and FLVVVGACGV (SEQ ID NO:135), wherein the KRAS peptide is 9 or 10 amino acids in length, or 9 or 10 amino acids in length; D) KLVVVGAGDV (SEQ ID NO: 136) and KLVVVGARGV (SEQ ID NO: 137), wherein the KRAS peptide is 9 or 10 amino acids in length; E) GAGDVGKSAL (SEQ ID NO: 138), AGDVGKSAL (SEQ ID NO: 139), DVGKSALTI (SEQ ID NO: 140), GAVGVGKSAL (SEQ ID NO: 141), AVGVGKSAL (SEQ ID NO: 142), YKLVVVGAV (SEQ ID NO: 143), ARGVGKSAL (SEQ ID NO: 144), GARGVGKSAL (SEQ ID NO: 145), EYKLVVVGAR (SEQ ID NO: 146), RGVGKSALTI (SEQ ID NO: 147), LVVGARGV (SEQ ID NO: 148), GADGVGKSAL (SEQ ID NO: 149), ACGVGKSAL (SEQ ID NO: 150), and GACGVGKSAL (SEQ ID NO: 151), wherein the KRAS peptide is 9 or 10 amino acids in length; and F) VVGAVGVGK (SEQ ID NO: 110), VVVGAVGVGK (SEQ ID NO: 112), VGAVGVGKS (SEQ ID NO: 152), VGAVGVGKSA (SEQ ID NO: 153), AVGVGKSAL (SEQ ID NO: 142), AVGVGKSALT (SEQ ID NO: 154), GAVGVGKSAL (SEQ ID NO: 141), GAVGVGKSA (SEQ ID NO: 155), LVVVGAVGVG (SEQ ID NO: 156), LVVVGAVGV (SEQ ID NO: 125), KLVVVGAVGV (SEQ ID NO: 128), and KLVVVGAVG (SEQ ID NO: 157), wherein the KRAS peptide is 9 or 10 amino acids in length. 14. The TMP according to any one of embodiments 1 to 13, comprising a sequence selected from the group consisting of:

[0255] Aspect 15 A) the KRAS peptide is KLVVVGADGV (SEQ ID NO: 127) and the MHC heavy chain polypeptide is HLA-A * comprises an amino acid sequence having at least 95% amino acid sequence identity to a 0201 polypeptide; or B) the KRAS peptide is VVVGADGVGK (SEQ ID NO: 111) or VVGAVGVGKV (SEQ ID NO: 110) and the MHC heavy chain polypeptide is HLA-A11 * 01 polypeptide, comprising an amino acid sequence having at least 95% amino acid sequence identity to A T cell modulating polypeptide according to any one of embodiments 1 to 13.

[0256] Aspect 16 From the N-terminus to the C-terminus, i) the KRAS 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) a rigid peptide linker or a short flexible peptide linker, and ix) the one or more immunomodulatory polypeptides. Including, 16. The TMP according to any one of aspects 1 to 15, optionally wherein when two or more immunomodulatory polypeptides are present, a peptide linker is disposed between each of said two or more immunomodulatory polypeptides.

[0257] Aspect 17 From the N-terminus to the C-terminus, i) the KRAS peptide, ii) a flexible peptide linker; iii) the β2M polypeptide; iv) a flexible peptide linker; v) the MHC class I heavy chain polypeptide, vi) a rigid peptide linker or a short flexible peptide linker; vii) a first copy of the at least one immunomodulatory polypeptide; viii) a flexible peptide linker, and ix) a second copy of said at least one immunomodulatory polypeptide. Including, 17. The TMP according to embodiment 16, optionally wherein said peptide linker between said KRAS peptide and said β2M polypeptide comprises a cysteine.

[0258] Aspect 18 From the N-terminus to the C-terminus, i) the KRAS peptide, ii) a flexible peptide linker; iii) the β2M polypeptide; iv) a flexible peptide linker; v) the MHC class I heavy chain polypeptide, vi) a rigid peptide linker or a short flexible peptide linker; vii) a first copy of said at least one immunomodulatory polypeptide; viii) a rigid peptide linker or a short flexible peptide linker, and ix) a second copy of said at least one immunomodulatory polypeptide. Including, 17. The TMP according to embodiment 16, optionally wherein said peptide linker between said KRAS peptide and said β2M polypeptide comprises a cysteine.

[0259] Aspect 19 The TMP according to embodiment 17 or embodiment 18, wherein the first and second copies of the at least one immune modulating polypeptide are mutant IL-2 with 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.

[0260] Aspect 20 From the N-terminus to the C-terminus, i) the KRAS 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) said rigid peptide linker or a short flexible peptide linker, and ix) IgFc polypeptide Including, 16. The TMP according to any one of aspects 1-15, optionally wherein said peptide linker between said KRAS peptide and said β2M polypeptide comprises a cysteine.

[0261] Aspect 21 Each of the optional one or more additional peptide linkers, when present, is selected from the following: i) CGGGS(GGGGS)n (SEQ ID NO: 220), where n is an integer from 1 to 10; ii) GCGGS(GGGGS)n (SEQ ID NO: 217), where n is an integer from 1 to 10; iii) (GGGGS)n(GCGGS) (SEQ ID NO: 228), where n is an integer from 1 to 10; iv) (GGGGS)n (SEQ ID NO: 235), where n is an integer from 1 to 10; and v) AAAGG (SEQ ID NO: 192) The TMP according to any one of embodiments 1 to 20, independently selected from the group consisting of:

[0262] Aspect 22 Each of the optional one or more additional peptide linkers, when present, is selected from the following: i) CGGGS(GGGGS)n (SEQ ID NO: 220), where n is 2, 3, or 4; ii) GCGGS(GGGGS)n (SEQ ID NO:217), where n is 2, 3, or 4; iii) (GGGGS)n(GCGGS) (SEQ ID NO: 228) (wherein n is 2, 3, or 4); iv) (GGGGS)n (SEQ ID NO: 180), where n is 2, 3, 4, or 7; and v) AAAGG (SEQ ID NO: 192) The TMP according to any one of embodiments 1 to 20, independently selected from the group consisting of:

[0263] Aspect 23 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:246) (wherein n is an integer from 1 to 10); ii) (EP)n (SEQ ID NO:247), where n is an integer from 1 to 10; iii) (KP)n (SEQ ID NO:248), where n is an integer from 1 to 10; and iv) Peptide containing EAAAK (SEQ ID NO: 194) 23. The TMP according to any one of embodiments 1 to 22, independently selected from the group consisting of:

[0264] Aspect 24 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:203) (wherein n is 2 to 6); ii) (EP)n (SEQ ID NO:213) (wherein n is 2 to 6); iii) (KP)n (SEQ ID NO:208) (wherein n is 2 to 6); and iv) A(EAAAK)nA (SEQ ID NO: 200) (wherein n is 1 or 2) 23. The TMP according to any one of embodiments 1 to 22, independently selected from the group consisting of:

[0265] Aspect 25 a) the β2M polypeptide is linked to the KRAS peptide by a first linker comprising the sequence CGGGS(GGGGS)n (SEQ ID NO:220) or the sequence GCGGS(GGGGS)n (SEQ ID NO:217), where n is an integer from 1 to 10, and optionally n is 2 or 3; b) the MHC heavy chain polypeptide comprises a Cys at residue 84 and a Cys at residue 236; c) the β2M polypeptide comprises a Cys at residue 12; d) the 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; e) the Cys of the linker between the KRAS peptide and the β2M polypeptide is linked by a disulfide bond to the Cys that replaces Tyr84 of the MHC heavy chain polypeptide; f) the β2M polypeptide is linked to the MHC heavy chain polypeptide by a (GGGGS)n linker (SEQ ID NO:235), where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, and optionally n is 3 or 7; g) the MHC heavy chain polypeptide is HLA-A * 0201 Polypeptide, HLA-A * 1101 Polypeptide, HLA-A * 3303 polypeptide, HLA-A * 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-2401 polypeptide, an HLA-E polypeptide, and an HLA-G polypeptide; h) the TMP comprises two immunomodulatory polypeptides, the two immunomodulatory polypeptides being identical and arranged in tandem, and the TMP comprises a mutant IL-2, the mutant IL-2 comprising i) an H16A substitution and an F42A substitution, or ii) an H16T substitution and an F42A substitution; and i) the TMP comprises at least one rigid peptide linker, the at least one rigid peptide linker being selected from the group consisting of a peptide comprising: i) (AP)n (SEQ ID NO:246), where n is an integer from 1 to 10; ii) (EP)n (SEQ ID NO:247), where n is an integer from 1 to 10, optionally n is 2 to 6; iii) (KP)n (SEQ ID NO:248), where n is an integer from 1 to 10; and iv) EAAAK (SEQ ID NO:194); A TMP according to embodiment 1.

[0266] Aspect 26 a) the β2M polypeptide is linked to the KRAS peptide by a first linker comprising the sequence GCGGS(GGGGS)n (SEQ ID NO:218), where n is 2; f) the β2M polypeptide is linked to the MHC heavy chain polypeptide by a (GGGGS)n (SEQ ID NO:253) linker, where n is 3 or 7; g) the MHC heavy chain polypeptide is HLA-A * comprising an amino acid sequence having at least 95% amino acid sequence identity to a 1101 polypeptide; h) the TMP comprises two immunomodulatory polypeptides, the two immunomodulatory polypeptides being identical and arranged in tandem, and comprising a mutant IL-2 comprising an H16A substitution and an F42A substitution, and the two immunomodulatory polypeptides are linked by a peptide linker; and i) the at least one rigid peptide linker comprises an amino acid sequence (AP)n, where n is an integer from 1 to 10, optionally n is from 2 to 6; A TMP according to embodiment 25.

[0267] Aspect 27 A TMP according to embodiment 1, comprising an amino acid sequence shown in any one of Figures 14A-14L and Figures 14N-14V.

[0268] Aspect 28 The TMP comprises an amino acid sequence shown in any one of Figures 16A to 16D; (X) is: i) (AP)n (SEQ ID NO:246) (wherein n is an integer from 1 to 10); ii) (EP)n (SEQ ID NO:247), where n is an integer from 1 to 10; iii) (KP)n (SEQ ID NO:248), where n is an integer from 1 to 10; and iv) Peptide containing EAAAK (SEQ ID NO: 194) The TMP according to embodiment 1, selected from the group consisting of:

[0269] Aspect 29 (X) is: i) (AP)n (where n is 2 to 6) (SEQ ID NO: 203); ii) (EP)n, where n is 2 to 6 (SEQ ID NO:213); iii) (KP)n, where n is 2 to 6 (SEQ ID NO: 208); and iv) A(EAAAK)nA (SEQ ID NO: 200) (wherein n is 1 or 2) 29. The TMP according to embodiment 28, selected from the group consisting of:

[0270] Aspect 30 the TMP comprises at least one short, flexible peptide linker; 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; A TMP according to any one of embodiments 1 to 22.

[0271] Aspect 31 The TMP according to embodiment 30, wherein the short flexible peptide linker comprises a number of amino acids selected from the group consisting of 2-4, 2-5, 3-6, and 4-8, and optionally the short flexible peptide linker is GGS.

[0272] Aspect 32 a) the β2M polypeptide is linked to the KRAS peptide by a first linker comprising the sequence CGGGS(GGGGS)n (SEQ ID NO:220) or the sequence GCGGS(GGGGS)n (SEQ ID NO:217), where n is an integer from 1 to 10, and optionally n is 2 or 3; b) the MHC heavy chain polypeptide comprises a Cys at residue 84 and a Cys at residue 236; c) the β2M polypeptide comprises a Cys at residue 12; d) the 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; e) the Cys of the linker between the KRAS peptide and the β2M polypeptide is linked by a disulfide bond to the Cys that replaces Tyr84 of the MHC heavy chain polypeptide; f) the β2M polypeptide is linked to the MHC heavy chain polypeptide by a (GGGGS)n (SEQ ID NO:235), where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, and optionally n is 3 or 7, linker; g) the MHC heavy chain polypeptide is HLA-A * 0201 Polypeptide, HLA-A * 1101 Polypeptide, HLA-A * 3303 Polypeptide, HLA-A * 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-2401 polypeptide, an HLA-E polypeptide, and an HLA-G polypeptide; h) the TMP comprises two immunomodulatory polypeptides, the two immunomodulatory polypeptides being identical and arranged in tandem, and the TMP comprises a mutant IL-2, the mutant IL-2 comprising i) an H16A substitution and an F42A substitution, or ii) an H16T substitution and an F42A substitution; The TMP according to embodiment 30 or 31.

[0273] Aspect 33 a) the β2M polypeptide is linked to the KRAS peptide by a first linker comprising the sequence GCGGS(GGGGS)n (SEQ ID NO:218), where n is 2; f) the β2M polypeptide is linked to the MHC heavy chain polypeptide by a (GGGGS)n (SEQ ID NO:253) linker, where n is 3 or 7; g) the MHC heavy chain polypeptide is HLA-A * comprising an amino acid sequence having at least 95% amino acid sequence identity to a 1101 polypeptide; h) the TMP comprises two immunomodulatory polypeptides, the two immunomodulatory polypeptides being identical and arranged in tandem, and comprising a mutant IL-2 having an H16A substitution and an F42A substitution, and the two immunomodulatory polypeptides being linked by a peptide linker; A TMP according to embodiment 32.

[0274] Aspect 34 A homodimer comprising a first and a second TMP according to any one of aspects 1 to 33, the first and second TMPs are identical and covalently linked by one or more disulfide bonds between the IgFc polypeptides of the first and second TMPs; The homodimer.

[0275] Aspect 35 A heterodimer comprising a first and a second TMP according to any one of aspects 1 to 33, the first and second TMPs are not identical and are linked by one or more covalent bonds; The heterodimer.

[0276] Aspect 36 A nucleic acid comprising a nucleotide sequence encoding a TMP according to any one of embodiments 1 to 33.

[0277] Aspect 37 A recombinant expression vector comprising a nucleic acid according to embodiment 36.

[0278] Aspect 38 A cell genetically modified with a nucleic acid according to embodiment 36 or a recombinant expression vector according to embodiment 37.

[0279] Aspect 39 a) a TMP according to any one of embodiments 1 to 33, a homodimer according to embodiment 34, or a heterodimer according to embodiment 35; b) a pharma- ceutically acceptable excipient; 13. A pharmaceutical composition comprising:

[0280] Aspect 40 A method for producing a TMP according to any one of aspects 1 to 33, a homodimer according to aspect 34, or a heterodimer according to aspect 35, comprising the steps of: Culturing in vitro a cell according to embodiment 38 in a cell culture medium. The method comprising:

[0281] Aspect 41 1. A method for selectively modulating the activity of T cells specific for a KRAS peptide epitope, comprising: contacting said T cells with a T cell modulating polypeptide according to any one of aspects 1 to 33, a homodimer according to aspect 34, or a heterodimer according to aspect 35. Including, The method, wherein the contacting selectively modulates activity of the epitope-specific T cells.

[0282] Aspect 42 1. A method of treating a KRAS associated cancer in a patient having said cancer, comprising: administering to said patient an effective amount of a pharmaceutical composition comprising a TMP according to any one of embodiments 1 to 33, a homodimer according to embodiment 34, or a heterodimer according to embodiment 35. The method comprising:

[0283] Aspect 43 The method according to embodiment 42, further comprising the step of co-administering to said patient one or more immune checkpoint inhibitors.

[0284] Aspect 44 The method according to aspect 43, wherein said immune checkpoint inhibitor is an antibody specific for PD-L1, PD-1, or CTLA4, and optionally said immune checkpoint inhibitor is selected from the group consisting of nivolumab, tremelimumab, pembrolizumab, ipilimumab, cemiplimab, atezolizumab, avelumab, tislelizumab, durvalumab, and combinations thereof.

[0285] Aspect 45 1. A method for improving the thermal stability of a T cell modulating polypeptide (TMP), comprising: The TMP may be selected from the group consisting of: i) a KRAS peptide containing a KRAS epitope expressed on a cancer cell, the KRAS being about 4 to about 25 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 polypeptides; Including, when the TMP comprises one or more MODs of configuration 2, the method comprises placing 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 MOD, and (ii) the IgFc polypeptide and the MOD, and optionally, when two or more MODs are present, placing a rigid peptide linker or a short flexible linker between at least two of the two or more MODs; and When the TMP contains one or more MODs of configuration 3, the method comprises placing 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, placing a rigid peptide linker or a short flexible linker between at least two of the two or more MODs.

[0286] Aspect 46 Each rigid peptide linker is selected from the group consisting of: i) (AP)n (SEQ ID NO:246) (wherein n is an integer from 1 to 10); ii) (EP)n (SEQ ID NO:247), where n is an integer from 1 to 10; iii) (KP)n (SEQ ID NO:248), where n is an integer from 1 to 10; and iv) Peptide containing EAAAK (SEQ ID NO: 194) The method according to embodiment 45, wherein the compound is independently selected from the group consisting of:

[0287] Aspect 47 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:203) (wherein n is 2 to 6); ii) (EP)n (SEQ ID NO:213) (wherein n is 2 to 6); iii) (KP)n (SEQ ID NO:208) (wherein n is 2 to 6); and iv) A(EAAAK)nA (SEQ ID NO: 200) (wherein n is 1 or 2) The method according to embodiment 45, wherein the compound is independently selected from the group consisting of:

[0288] Aspect 48 The method according to embodiment 45, 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.

[0289] Aspect 49 The method according to embodiment 45, 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.

[0290] Mode Set B The above-mentioned aspects, including the embodiments of the subject matter, 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 employed, 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 intended to be limited to the combinations of aspects explicitly stated below.

[0291] Aspect 1 A single chain T cell modulating polypeptide (TMP) comprising: i) a KRAS peptide comprising a KRAS epitope expressed on a cancer cell, the KRAS peptide being about 7 amino acids to about 16 amino acids in length, and optionally, being 8 amino acids to 12 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; v) immunoglobulin (Ig) Fc polypeptides; Including, The TMP is (a) at least one rigid peptide linker, optionally wherein 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 wherein the TMP comprises a short flexible peptide linker between the IgFc polypeptide and one of the one or more immune modulating polypeptides; Optionally, the TMP comprises one or more additional peptide linkers.

[0292] Aspect 2 The TMP according to embodiment 1, wherein said IgFc polypeptide comprises an amino acid sequence having at least about 95% amino acid sequence identity to an amino acid sequence shown in any one of Figures 2A-2M.

[0293] Aspect 3 The TMP according to embodiment 2, wherein said IgFc polypeptide does not substantially induce cell lysis, and optionally said IgFc is an IgG1Fc polypeptide comprising one or more amino acid substitutions selected from the group consisting of N297A, L234A, L235A, L234F, L235E, and P331S.

[0294] Aspect 4 The TMP according to embodiment 1, comprising one or more intrachain disulfide bonds.

[0295] Aspect 5 (a) the β2M polypeptide and the MHC heavy chain polypeptide are linked by a disulfide bond, the disulfide bond linking a Cys residue in the β2M polypeptide to a Cys residue in 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 Cys present in the first linker is linked to a Cys present in the MHC heavy chain polypeptide by a disulfide bond, optionally the first linker comprises the sequence CGGGS(GGGGS)n (SEQ ID NO: 220) or the sequence GCGGS(GGGGS)n (SEQ ID NO: 217), where n is an integer between 1 and 10, e.g., 2 or 3, and the Cys in the linker is linked to a Cys that replaces Tyr84 in the MHC heavy chain polypeptide by a disulfide bond; or (c) the β2M polypeptide and the MHC heavy chain polypeptide are linked by a disulfide bond, the disulfide bond linking a Cys residue in the β2M polypeptide to a Cys residue in the MHC heavy chain polypeptide, and optionally, a Cys at amino acid residue 12 of the β2M polypeptide is disulfide-linked 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 wherein a Cys present in said first linker is linked by a disulfide bond to a Cys present in said MHC heavy chain polypeptide, and optionally said first linker comprises the sequence CGGGS(GGGGS)n (SEQ ID NO: 220) or the sequence GCGGS(GGGGS)n (SEQ ID NO: 217), where n is an integer between 1 and 10, e.g. 2 or 3, and wherein said Cys in said linker is linked by a disulfide bond to a Cys that replaces Tyr84 in said MHC heavy chain polypeptide. A TMP according to embodiment 4.

[0296] Aspect 6 The MHC heavy chain polypeptide is HLA-A * 0201 Polypeptide, HLA-A * 1101 Polypeptide, HLA-A * 3303 polypeptide, and HLA-A * 2401 polypeptides.

[0297] Aspect 7 The TMP according to embodiment 1, wherein said MHC heavy chain polypeptide comprises an amino acid sequence having at least 95% amino acid sequence identity to an HLA-E polypeptide or an HLA-G polypeptide.

[0298] Aspect 8 2. The TMP according to embodiment 1, wherein the at least one immunomodulatory polypeptide is a wild-type or mutant form of an activated immunomodulatory polypeptide selected from the group consisting of an IL-2 polypeptide, a 4-1BBL polypeptide, a CD80 polypeptide, a CD86 polypeptide, or a combination thereof, and optionally at least one of the at least one immunomodulatory polypeptide 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.

[0299] Aspect 9 The TMP according to embodiment 8, wherein said at least one immune modulating polypeptide is a mutant IL-2 having 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.

[0300] Aspect 10 The KRAS peptide is A) VVGADGVGK (SEQ ID NO:108), VVGACGVGK (SEQ ID NO:109), VVGAVGVGK (SEQ ID NO:110), VVVGADGVGK (SEQ ID NO:111), VVVGAVGVGK (SEQ ID NO:112), VVVGACGVGK (SEQ ID NO:113), VTGADGVGK (SEQ ID NO:114), VTGAVGVGK (SEQ ID NO:115), VTGACGVGK (SEQ ID NO:116), VTVGADGVGK (SEQ ID NO:117), VTVGAVGVGK (SEQ ID NO:118), and VTVGACGVGK (SEQ ID NO:119), wherein the KRAS peptide is 9 or 10 amino acids in length, or at least 9 or 10 amino acids in length; B) VVVGAGDVGK (SEQ ID NO: 120), VVGAGDVGK (SEQ ID NO: 121), VVVGARGVGK (SEQ ID NO: 122), and VVGARGVGK (SEQ ID NO: 123), wherein the KRAS peptide is 9 or 10 amino acids in length, or 9 or 10 amino acids in length; C) LVVVGADGV (SEQ ID NO:124), LVVVGAVGV (SEQ ID NO:125), LVVVGACGV (SEQ ID NO:126), KLVVVGADGV (SEQ ID NO:127), KLVVVGAVGV (SEQ ID NO:128), KLVVVGACGV (SEQ ID NO:129), LLVVGADGV (SEQ ID NO:130), LLVVGAVGV (SEQ ID NO:131), LLVVGACGV (SEQ ID NO:132), FLVVVGADGV (SEQ ID NO:133), FLVVVGAVGV (SEQ ID NO:134), and FLVVVGACGV (SEQ ID NO:135), wherein the KRAS peptide is 9 or 10 amino acids in length, or 9 or 10 amino acids in length; D) KLVVVGAGDV (SEQ ID NO: 136) and KLVVVGARGV (SEQ ID NO: 137), wherein the KRAS peptide is 9 or 10 amino acids in length; E) GAGDVGKSAL (SEQ ID NO: 138), AGDVGKSAL (SEQ ID NO: 139), DVGKSALTI (SEQ ID NO: 140), GAVGVGKSAL (SEQ ID NO: 141), AVGVGKSAL (SEQ ID NO: 142), YKLVVVGAV (SEQ ID NO: 143), ARGVGKSAL (SEQ ID NO: 144), GARGVGKSAL (SEQ ID NO: 145), EYKLVVVGAR (SEQ ID NO: 146), RGVGKSALTI (SEQ ID NO: 147), LVVGARGV (SEQ ID NO: 148), GADGVGKSAL (SEQ ID NO: 149), ACGVGKSAL (SEQ ID NO: 150), and GACGVGKSAL (SEQ ID NO: 151), wherein the KRAS peptide is 9 or 10 amino acids in length; and F) VVGAVGVGK (SEQ ID NO: 110), VVVGAVGVGK (SEQ ID NO: 112), VGAVGVGKS (SEQ ID NO: 152), VGAVGVGKSA (SEQ ID NO: 153), AVGVGKSAL (SEQ ID NO: 142), AVGVGKSALT (SEQ ID NO: 154), GAVGVGKSAL (SEQ ID NO: 141), GAVGVGKSA (SEQ ID NO: 155), LVVVGAVGVG (SEQ ID NO: 156), LVVVGAVGV (SEQ ID NO: 125), KLVVVGAVGV (SEQ ID NO: 128), and KLVVVGAVG (SEQ ID NO: 157), wherein the KRAS peptide is 9 or 10 amino acids in length. The TMP according to embodiment 1, comprising a sequence selected from the group consisting of:

[0301] Aspect 11 A) the KRAS peptide is KLVVVGADGV (SEQ ID NO: 127) and the MHC heavy chain polypeptide is HLA-A * comprises an amino acid sequence having at least 95% amino acid sequence identity to a 0201 polypeptide; or B) the KRAS peptide is VVVGADGVGK (SEQ ID NO: 111) or VVGAVGVGK (SEQ ID NO: 110) and the MHC heavy chain polypeptide is HLA-A11 * 01 polypeptide, comprising an amino acid sequence having at least 95% amino acid sequence identity to A T cell modulating polypeptide according to embodiment 1.

[0302] Aspect 12 From the N-terminus to the C-terminus, i) the KRAS 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) a rigid peptide linker or a short flexible peptide linker, and ix) the one or more immunomodulatory polypeptides. Including, Optionally, when two or more immunomodulatory polypeptides are present, a peptide linker is disposed between each of the two or more immunomodulatory polypeptides; or From the N-terminus to the C-terminus, i) the KRAS peptide, ii) a flexible peptide linker; iii) the β2M polypeptide; iv) a flexible peptide linker; v) the MHC class I heavy chain polypeptide, vi) a flexible peptide linker; vii) the IgFc polypeptide, viii) a rigid peptide linker or a short flexible peptide linker; ix) a first copy of the at least one immunomodulatory polypeptide; x) a peptide linker, and xi) a second copy of said at least one immunomodulatory polypeptide. Including, Optionally, the peptide linker between the KRAS peptide and the β2M polypeptide comprises a cysteine; or From the N-terminus to the C-terminus, i) the KRAS peptide, ii) a flexible peptide linker; iii) the β2M polypeptide; iv) a flexible peptide linker; v) the MHC class I heavy chain polypeptide, vi) a rigid peptide linker or a short flexible peptide linker; vii) a first copy of said at least one immunomodulatory polypeptide; viii) a rigid peptide linker or a short flexible peptide linker; ix) a second copy of the at least one immunomodulatory polypeptide; x) a peptide linker, and xi) the IgFc polypeptide Including, Optionally, the peptide linker between the KRAS peptide and the β2M polypeptide comprises a cysteine; or From the N-terminus to the C-terminus, i) the KRAS 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) said rigid peptide linker or a short flexible peptide linker, and ix) IgFc polypeptide Including, Optionally, the peptide linker between the KRAS peptide and the β2M polypeptide comprises a cysteine, and optionally, when two or more immune modulating polypeptides are present, a peptide linker is disposed between each of the two or more immune modulating polypeptides. A TMP according to embodiment 1.

[0303] Aspect 13 Each of the optional one or more additional peptide linkers, when present, is selected from the following: i) CGGGS(GGGGS)n (SEQ ID NO:220), where n is an integer from 1 to 10, and optionally n is 2, 3, or 4; ii) GCGGS(GGGGS)n (SEQ ID NO:217), 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; and v)AAAGG The TMP according to embodiment 1, independently selected from the group consisting of:

[0304] Aspect 14 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, and optionally n is from 2 to 6; ii) (EP)n, where n is an integer from 1 to 10, and optionally n is from 2 to 6; iii) (KP)n, where n is an integer from 1 to 10, and optionally n is from 2 to 6; and iv) a peptide comprising EAAAK (optionally, n is 1 or 2) The TMP according to embodiment 1, independently selected from the group consisting of:

[0305] Aspect 15 a) the β2M polypeptide is linked to the KRAS peptide by a first linker comprising the sequence CGGGS(GGGGS)n (SEQ ID NO:220) or the sequence GCGGS(GGGGS)n (SEQ ID NO:217), where n is an integer from 1 to 10, and optionally n is 2 or 3; b) the MHC heavy chain polypeptide comprises a Cys at residue 84 and a Cys at residue 236; c) the β2M polypeptide comprises a Cys at residue 12; d) the 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; e) the Cys of the linker between the KRAS peptide and the β2M polypeptide is linked by a disulfide bond to the Cys that replaces Tyr84 of the MHC heavy chain polypeptide; f) the β2M polypeptide is linked to the MHC heavy chain polypeptide by a (GGGGS)n linker, where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, and optionally n is 3 or 7; g) the MHC heavy chain polypeptide is HLA-A * 0201 Polypeptide, HLA-A * 1101 Polypeptide, HLA-A * 3303 polypeptide, HLA-A * 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-2401 polypeptide, an HLA-E polypeptide, and an HLA-G polypeptide; h) the TMP comprises two immunomodulatory polypeptides, the two immunomodulatory polypeptides being identical and arranged in tandem, and the TMP comprises a mutant IL-2, the mutant IL-2 comprising i) an H16A substitution and an F42A substitution, or ii) an H16T substitution and an F42A substitution; and i) the TMP comprises at least one rigid peptide linker, the at least one rigid peptide linker being selected from the group consisting of: i) (AP)n, where n is an integer between 1 and 10; ii) (EP)n, where n is an integer between 1 and 10, optionally n is between 2 and 6; iii) (KP)n, where n is an integer between 1 and 10; and iv) a peptide comprising EAAAK; A TMP according to embodiment 1.

[0306] Aspect 16 a) the β2M polypeptide is linked to the KRAS peptide by a first linker comprising the sequence GCGGS(GGGGS)n (SEQ ID NO:217), where n is 2; f) the β2M polypeptide is linked to the MHC heavy chain polypeptide by a (GGGGS)n linker, where n is 3 or 7; g) the MHC heavy chain polypeptide is HLA-A * comprising an amino acid sequence having at least 95% amino acid sequence identity to a 1101 polypeptide; h) the TMP comprises two immunomodulatory polypeptides, the two immunomodulatory polypeptides being identical and arranged in tandem, and comprising a mutant IL-2 comprising an H16A substitution and an F42A substitution, and the two immunomodulatory polypeptides are linked by a peptide linker; and i) the at least one rigid peptide linker comprises an amino acid sequence (AP)n, where n is an integer from 1 to 10, optionally n is from 2 to 6; A TMP according to embodiment 15.

[0307] Aspect 17 A TMP according to embodiment 1, comprising an amino acid sequence shown in any one of Figures 14A-14L and Figures 14N-14V.

[0308] Aspect 18 the TMP comprises at least one short, flexible peptide linker; 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; A TMP according to embodiment 1.

[0309] Aspect 19 A homodimer comprising a first and a second TMP according to any one of aspects 1 to 18, the first and second TMPs are identical and covalently linked by one or more disulfide bonds between the IgFc polypeptides of the first and second TMPs; The homodimer.

[0310] Aspect 20 A heterodimer comprising a first and a second TMP according to any one of aspects 1 to 18, the first and second TMPs are not identical and are linked by one or more covalent bonds; The heterodimer.

[0311] Aspect 21 A nucleic acid comprising a nucleotide sequence encoding a TMP according to any one of embodiments 1 to 18.

[0312] Aspect 22 22. A recombinant expression vector comprising a nucleic acid according to embodiment 21.

[0313] Aspect 23 A pharmaceutical composition comprising a TMP according to any one of embodiments 1 to 18, a homodimer according to embodiment 19, or a heterodimer according to embodiment 20.

[0314] Aspect 24 1. A method for selectively modulating the activity of T cells specific for a KRAS peptide epitope, comprising: contacting said T cells with a T cell modulating polypeptide according to any one of aspects 1 to 18, a homodimer according to aspect 19, or a heterodimer according to aspect 20. Including, The method, wherein the contacting selectively modulates activity of the epitope-specific T cells.

[0315] Aspect 25 1. A method of treating a KRAS associated cancer in a patient having said cancer, comprising: administering to said patient an effective amount of a pharmaceutical composition comprising a TMP according to any one of embodiments 1 to 18, a homodimer according to embodiment 19, or a heterodimer according to embodiment 20. The method comprising:

[0316] Aspect 26 further comprising co-administering to the patient one or more immune checkpoint inhibitors; Optionally, the immune checkpoint inhibitor is an antibody specific for PD-L1, PD-1, TIGIT, or CTLA4. A method according to embodiment 25. EXAMPLES

[0317] The following examples are provided to provide one of ordinary skill in the art with a complete disclosure and description of how to make and use the present disclosure, and are not intended to limit the scope of the disclosure, nor are they intended to represent that the following experiments are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.), but experimental error and bias should be accounted for. Unless otherwise noted, "parts" are parts by weight, "temperature" is degrees Celsius, and "pressure" is at or near atmospheric pressure. Standard abbreviations may be used, e.g., bp is base pair, kb is kilobase, pl is picoliter, s or sec is seconds, min is minutes, h or hr is hours, aa is amino acid, kb is kilobase, bp is base pair, nt is nucleotide, im is intramuscular, ip is intraperitoneal, and sc is subcutaneous.

[0318] Example 1: Thermal stability of dimerized TMP The thermal stability of dimerized TMP was evaluated by accelerated stability studies conducted at 4° C., 37° C., and 42° C. The dimerized TMP compositions were maintained in solution (phosphate buffered saline (PBS) containing 500 mM NaCl, pH 7.4) at the above temperatures at a concentration of 10 mg dimerized TMP / mL solution for 14 days. The percentage of monomer remaining in solution was measured by size exclusion chromatography on days 1, 7, and 14. The PBS solution was as follows: 10.14 mM disodium hydrogen phosphate, 1.76 mM potassium dihydrogen phosphate, 2.7 mM KCl, and 0.5 M NaCl, pH 7.4.

[0319] TMP, which spontaneously forms homodimers, was tested for its stability at 4°C, 37°C, and 42°C. The amino acid sequences of the single-chain TMP tested are shown in Figure 19C and Figures 14A-14V. The amino acid sequence of TMP4074 is shown in Figure 19C. TMP4074 is an HLA-A peptide that contains: i) G12V(7-16) (VVVGAVGVGK (SEQ ID NO: 112)) as a peptide epitope, (ii) the linker GCGGS(GGGGS)2 (SEQ ID NO: 218), iii) β2M with amino acid 12 being Cys(R12C), iv) the (GGGGS)3 (SEQ ID NO: 189) linker, v) a Cys at amino acid 84 (Y84C) and a Cys at amino acid 236 (A236C). * The TMPs comprise a 11:01 allele heavy chain polypeptide, vi) an AAAGG (SEQ ID NO: 192) linker, vii) an IgFc polypeptide with Ala residues at positions 14 and 15 (L234A and L235A), viii) a (GGGGS)3 (SEQ ID NO: 189) linker, ix) a first copy of a mutant IL-2 polypeptide with Ala residues at amino acids 16 and 42 (H16A and F42A), x) a (GGGGS)4 (SEQ ID NO: 190) linker, and xi) a second copy of the mutant IL-2 polypeptide. The TMPs spontaneously formed homodimers with disulfide bonds. The disulfide bonds were between the IgFc polypeptides of each TMP. The dimerized TMPs tested differ from dimerized TMP4074 as shown in Table 2, "Construct Characteristics," below.

[0320] The data are summarized in Table 2 below, showing the % monomer remaining after 14 days at the indicated temperatures.

[0321] [Table 2]

[0322] It is apparent from the above data, particularly the thermal stability data for dimerized TMP4470 and 4471, that replacement of the flexible (G4S)3 (SEQ ID NO: 189) linker between the IgFc polypeptide and the mutant IL-2 polypeptide in dimerized TMP4470 with a rigid peptide linker ((AP)4 (SEQ ID NO: 202) or A(EAAAK)2A (SEQ ID NO: 220)) placed between the IgFc polypeptide and the mutant IL-2 polypeptide improves the stability properties of the dimerized TMP at both 37° C. and 42° C. compared to TMP4074. As also shown by the stability data for dimerized TMP4467, shortening the flexible peptide linker between the IgFc polypeptides from a (GGGGS)3 (SEQ ID NO: 189) linker to a GGS linker also improved the thermal stability properties at both 37° C. and 42° C. compared to TMP4074.

[0323] Example 2: In vitro activity of dimerized TMP The ability of dimerized TMP to activate mutant KRAS G12V-reactive T cells was examined by immunohistochemistry using human CD8 + The antibody was developed using human CD8 T cells. + The T cells were transduced with a T cell receptor (TCR1) containing the constant region of the mouse TCR (mTCR) reactive to KRAS G12V (amino acids 7-16) presented by HLA-A11. + T cells were labeled as "TCR-transduced CD8 + T cells”. + T cells were mixed with autologous peripheral blood mononuclear cells (PBMCs) to prepare a cell mixture. The cell mixture was contacted with various concentrations of single-chain TMP presenting KRAS G12V 7-16 and incubated at 37°C for 9 days. Total cells were harvested, and total CD8 + The percentage of KRAS-reactive T cells among T cells was measured by flow cytometry to examine the presence of mTCR+ cells, and the data are shown in Figures 15A-15E.

[0324] As can be seen from the data, dimerized TMPs containing rigid peptide linkers, i.e., 4470 (with a rigid (AP)4 linker between the IgFc polypeptide and the mutant IL-2 polypeptide) and 4471 (with a rigid peptide linker A(EAAAK)2A (SEQ ID NO: 200) between the IgFc polypeptide and the mutant IL-2 polypeptide), showed in vitro activity. Similarly, the dimerized construct 4467, which has a shorter GGS linker between the IgFc polypeptide and the mutant IL-2 polypeptide, also showed in vitro activity.

[0325] 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 can be substituted without departing from the essential spirit and scope of the present disclosure. In addition, many modifications can be made to adapt a particular situation, material, composition of matter, 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 following claims.

Claims

1. A single chain T cell modulating polypeptide (TMP) comprising: i) a KRAS peptide comprising a KRAS epitope expressed on a cancer cell, the KRAS peptide being about 7 to about 16 amino acids in length, and optionally, being 8 to 12 amino acids in length; ii) a first major histocompatibility complex (MHC) polypeptide that 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; v) immunoglobulin (Ig) Fc polypeptide; Including, The TMP is (a) at least one rigid peptide linker, wherein 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, wherein the TMP comprises a short, flexible peptide linker between the IgFc polypeptide and one of the one or more immunomodulatory polypeptides; The TMP, wherein the TMP comprises one or more additional peptide linkers.

2. 2. The TMP of claim 1, wherein said IgFc polypeptide comprises an amino acid sequence having at least about 95% amino acid sequence identity to an amino acid sequence shown in any one of Figures 2A-2M.

3. The TMP of claim 1 or 2, wherein the IgFc polypeptide does not substantially induce cell lysis.

4. The TMP of claim 3, wherein the IgFc is an IgG1Fc polypeptide comprising one or more amino acid substitutions selected from the group consisting of N297A, L234A, L235A, L234F, L235E, and P331S.

5. 2. The TMP of claim 1, comprising one or more intrachain disulfide bonds.

6. (a) the β2M polypeptide and the MHC heavy chain polypeptide are bound by a disulfide bond, and the disulfide bond bonds a Cys residue in the β2M polypeptide and a Cys residue in 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 Cys present in the first linker is linked to a Cys present in the MHC heavy chain polypeptide by a disulfide bond; or (c) the β2M polypeptide and the MHC heavy chain polypeptide are linked by a disulfide bond, the disulfide bond linking a Cys residue in the β2M polypeptide to a Cys residue in 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, the β2-microglobulin polypeptide is linked to the peptide by a first linker comprising a Cys, and a Cys present in the first linker is linked to a Cys present in the MHC heavy chain polypeptide by a disulfide bond; The TMP according to claim 4. (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:220) or the sequence GCGGS(GGGGS)n (SEQ ID NO:217), where n is an integer from 1 to 10, e.g., 2 or 3, and the Cys in the linker is linked to the Cys that replaces Tyr84 of the MHC heavy chain polypeptide by a disulfide bond; or (c) the first linker comprises the sequence CGGGS(GGGGS)n (SEQ ID NO:220) or the sequence GCGGS(GGGGS)n (SEQ ID NO:217), where n is an integer from 1 to 10, e.g., 2 or 3, and the Cys in the linker is linked to the Cys that replaces Tyr84 of the MHC heavy chain polypeptide by a disulfide bond; The TMP according to claim 6.

8. The MHC heavy chain polypeptide is HLA-A ＊ 0201 polypeptide, HLA-A ＊ 1101 polypeptide, HLA-A ＊ 3303 polypeptide, and HLA-A ＊ 2. The TMP of claim 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: 2401 polypeptides.

9. 2. The TMP of claim 1, wherein the MHC heavy chain polypeptide comprises an amino acid sequence having at least 95% amino acid sequence identity to an HLA-E polypeptide or an HLA-G polypeptide.

10. 2. The TMP of claim 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 an IL-2 polypeptide, a 4-1BBL polypeptide, a CD80 polypeptide, a CD86 polypeptide, or a combination thereof, and optionally, at least one of the at least one immunomodulatory polypeptide is a mutant immunomodulatory polypeptide, which 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.

11. 11. The TMP of claim 10, wherein the at least one immunomodulatory polypeptide is a mutant IL-2 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 substitutions at H16 and F42.

13. The TMP of claim 12, wherein the 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 10 to 13, comprising two copies of the immunomodulatory polypeptide.

15. The KRAS peptide is A) VVGADGVGK (SEQ ID NO:108), VVGACGVGK (SEQ ID NO:109), VVGAVGVGK (SEQ ID NO:110), VVVGADGVGK (SEQ ID NO:111), VVVGAVGVGK (SEQ ID NO:112), VVVGACGVGK (SEQ ID NO:113), VTGADGVGK (SEQ ID NO:114), VTGAVGVGK (SEQ ID NO:115), VTGACGVGK (SEQ ID NO:116), VTVGADGVGK (SEQ ID NO:117), VTVGAVGVGK (SEQ ID NO:118), and VTGACGVGK (SEQ ID NO:119), wherein the KRAS peptide is 9 or 10 amino acids in length, or at least 9 or 10 amino acids in length; B) VVVGAGDVGK (SEQ ID NO: 120), VVGAGDVGK (SEQ ID NO: 121), VVVGARGVGK (SEQ ID NO: 122), and VVGARGVGK (SEQ ID NO: 123), wherein the KRAS peptide is 9 or 10 amino acids in length, or 9 or 10 amino acids in length; C) LVVVGADGV (SEQ ID NO: 124), LVVVGAVGV (SEQ ID NO: 125), LVVVGACGV (SEQ ID NO: 126), KLVVVGADGV (SEQ ID NO: 127), KLVVVGAVGV (SEQ ID NO: 128), KLVVVGACGV (SEQ ID NO: 129), LLVVGADGV (SEQ ID NO: 130), LLVVGAVGV (SEQ ID NO: 131), LLVVGACGV (SEQ ID NO: 132), FLVVVGADGV (SEQ ID NO: 133), FLVVVGAVGV (SEQ ID NO: 134), and FLVVVGACGV (SEQ ID NO: 135), wherein the KRAS peptide is 9 or 10 amino acids in length, or 9 or 10 amino acids in length; D) KLVVVGAGDV (SEQ ID NO: 136) and KLVVVGARGV (SEQ ID NO: 137), wherein the KRAS peptide is 9 or 10 amino acids in length; E) GAGDVGKSAL (SEQ ID NO: 138), AGDVGKSAL (SEQ ID NO: 139), DVGKSALTI (SEQ ID NO: 140), GAVGVGKSAL (SEQ ID NO: 141), AVGVGKSAL (SEQ ID NO: 142), YKLVVVGAV (SEQ ID NO: 143), ARGVGKSAL (SEQ ID NO: 144), GARGVGKSAL (SEQ ID NO: 145), EYKLVVVGAR (SEQ ID NO: 146), RGVGKSALTI (SEQ ID NO: 147), LVVVGARGV (SEQ ID NO: 148), GADGVGKSAL (SEQ ID NO: 149), ACGVGKSAL (SEQ ID NO: 150), and GACGVGKSAL (SEQ ID NO: 151), wherein the KRAS peptide is 9 or 10 amino acids in length; and F) VVGAVGVGK (SEQ ID NO: 110), VVVGAVGVGK (SEQ ID NO: 112), VGAVGVGKS (SEQ ID NO: 152), VGAVGVGKSA (SEQ ID NO: 153), AVGVGKSAL (SEQ ID NO: 142), AVGVGKSALT (SEQ ID NO: 154), GAVGVGKSAL (SEQ ID NO: 141), GAVGVGKSA (SEQ ID NO: 155), LVVVGAVGVG (SEQ ID NO: 156), LVVVGAVGV (SEQ ID NO: 125), KLVVVGAVGV (SEQ ID NO: 128), and KLVVVGAVG (SEQ ID NO: 157), wherein the KRAS peptide is 9 or 10 amino acids in length. The TMP of any one of claims 1 to 14, comprising a sequence selected from the group consisting of:

16. A) the KRAS peptide is KLVVVGADGV (SEQ ID NO: 127) and the MHC heavy chain polypeptide is HLA-A ＊ comprises an amino acid sequence having at least 95% amino acid sequence identity to a 0201 polypeptide; or B) the KRAS peptide is VVVGADGVGK (SEQ ID NO: 111) or VVGAVGVGK (SEQ ID NO: 110) and the MHC heavy chain polypeptide is HLA-A11 ＊ 01 polypeptide, comprising an amino acid sequence having at least 95% amino acid sequence identity to The TMP according to any one of claims 1 to 15.

17. From the N-terminus to the C-terminus, i) the KRAS 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) a rigid peptide linker or a short flexible peptide linker, and ix) the one or more immunomodulatory polypeptides. Including, Optionally, when two or more immunomodulatory polypeptides are present, a peptide linker is disposed between each of said two or more immunomodulatory polypeptides; or From the N-terminus to the C-terminus, i) the KRAS peptide; ii) a flexible peptide linker; iii) the β2M polypeptide; iv) a flexible peptide linker; v) the MHC class I heavy chain polypeptide, vi) a flexible peptide linker; vii) the IgFc polypeptide; viii) a rigid peptide linker or a short flexible peptide linker; ix) a first copy of the at least one immunomodulatory polypeptide; x) a peptide linker, and xi) a second copy of said at least one immunomodulatory polypeptide. Including, Optionally, the peptide linker between the KRAS peptide and the β2M polypeptide comprises a cysteine; or From the N-terminus to the C-terminus, i) the KRAS peptide; ii) a flexible peptide linker; iii) the β2M polypeptide; iv) a flexible peptide linker; v) the MHC class I heavy chain polypeptide, vi) a rigid peptide linker or a short flexible peptide linker; vii) a first copy of said at least one immunomodulatory polypeptide; viii) a rigid peptide linker or a short flexible peptide linker; ix) a second copy of the at least one immunomodulatory polypeptide; x) a peptide linker, and xi) the IgFc polypeptide Including, Optionally, the peptide linker between the KRAS peptide and the β2M polypeptide comprises a cysteine; or From the N-terminus to the C-terminus, i) the KRAS 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) said rigid peptide linker or a short flexible peptide linker, and ix) IgFc polypeptide Including, Optionally, the peptide linker between the KRAS peptide and the β2M polypeptide comprises a cysteine, and optionally, when two or more immunomodulatory polypeptides are present, a peptide linker is disposed between each of the two or more immunomodulatory polypeptides. A TMP according to any one of claims 1 to 16.

18. Each of said optional one or more additional peptide linkers, when present, is selected from the following: i) CGGGS(GGGGS)n (SEQ ID NO:220), where n is an integer from 1 to 10, and optionally n is 2, 3, or 4; ii) GCGGS(GGGGS)n (SEQ ID NO:217), where n is an integer from 1 to 10, and optionally n is 2, 3, or 4; iii) (GGGGS)n(GCGGS) (SEQ ID NO:228), where n is an integer from 1 to 10, and optionally n is 2, 3, or 4; iv) (GGGGS)n (SEQ ID NO:180), where n is an integer from 1 to 10, and optionally n is 2, 3, 4, or 7; and v) AAAGG (SEQ ID NO: 192) The TMP of any one of claims 1 to 17, independently selected from the group consisting of:

19. the TMP comprises at least one rigid peptide linker; Each rigid peptide linker is i) (AP)n (SEQ ID NO:246), where n is an integer from 1 to 10, and optionally n is from 2 to 6; ii) (EP)n (SEQ ID NO:247), where n is an integer from 1 to 10, and optionally n is from 2 to 6; iii) (KP)n (SEQ ID NO:248), where n is an integer from 1 to 10, and optionally n is from 2 to 6; and iv) a peptide comprising EAAAK (SEQ ID NO: 194), where n is 1 or 2. The TMP of any one of claims 1 to 18, independently selected from the group consisting of:

20. a) the β2M polypeptide is linked to the KRAS peptide by a first linker comprising the sequence CGGGS(GGGGS)n (SEQ ID NO:220) or the sequence GCGGS(GGGGS)n (SEQ ID NO:217), where n is an integer from 1 to 10, and optionally n is 2 or 3; b) the MHC heavy chain polypeptide comprises a Cys at residue 84 and a Cys at residue 236; c) the β2M polypeptide comprises a Cys at residue 12; d) 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; e) the Cys of the linker between the KRAS peptide and the β2M polypeptide is linked by a disulfide bond to the Cys that replaces Tyr84 of the MHC heavy chain polypeptide; f) the β2M polypeptide is linked to the MHC heavy chain polypeptide by a (GGGGS)n linker, where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, and optionally n is 3 or 7; g) the MHC heavy chain polypeptide is HLA-A ＊ 0201 polypeptide, HLA-A ＊ 1101 polypeptide, HLA-A ＊ 3303 polypeptide, HLA-A ＊ 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-2401 polypeptide, an HLA-E polypeptide, and an HLA-G polypeptide; h) the TMP comprises two immunomodulatory polypeptides, the two immunomodulatory polypeptides being identical and arranged in tandem, and the TMP comprises a mutant IL-2, the mutant IL-2 comprising i) an H16A substitution and an F42A substitution, or ii) an H16T substitution and an F42A substitution; and i) the TMP comprises at least one rigid peptide linker, the at least one rigid peptide linker being selected from the group consisting of a peptide 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, optionally n is 2 to 6; iii) (KP)n, where n is an integer from 1 to 10; and iv) EAAAK (SEQ ID NO: 194); The TMP according to any one of claims 1 to 19.

21. a) the β2M polypeptide is linked to the KRAS peptide by a first linker comprising the sequence GCGGS(GGGGS)n (SEQ ID NO:217), where n is 2; f) the β2M polypeptide is linked to the MHC heavy chain polypeptide by a (GGGGS)n linker, where n is 3 or 7; g) the MHC heavy chain polypeptide is HLA-A ＊ 1101 polypeptide; h) the TMP comprises two immunomodulatory polypeptides, the two immunomodulatory polypeptides being identical and arranged in tandem, and comprising a mutant IL-2 comprising an H16A substitution and an F42A substitution, and the two immunomodulatory polypeptides being linked by a peptide linker; and i) said at least one rigid peptide linker comprises an amino acid sequence (AP)n, where n is an integer from 1 to 10, optionally n is from 2 to 6; 21. The TMP of claim 20.

22. 2. The TMP of claim 1, comprising an amino acid sequence as shown in any one of Figures 14A-14L and Figures 14N-14V.

23. the TMP comprises at least one short, flexible peptide linker; each short flexible peptide linker is independently selected from the group consisting of flexible peptide linkers comprising a number of amino acids selected from 2-4, 2-5, 3-6, 4-8, 5-10, or 10-14; The TMP according to any one of claims 1 to 18.

24. The TMP of claim 23, comprising at least one short flexible peptide linker comprising a number of amino acids selected from 2 to 4, 2 to 5, 3 to 6, or 4 to 8.

25. The TMP of claim 23, wherein the short flexible peptide linker is GGS.

26. A homodimer comprising a first and a second TMP according to any one of claims 1 to 22, the first and second TMPs are identical and covalently linked by one or more disulfide bonds between the IgFc polypeptides of the first and second TMPs; The homodimer.

27. A heterodimer comprising a first and a second TMP according to any one of claims 1 to 22, the first and second TMPs are not identical and are linked by one or more covalent bonds; The heterodimer.

28. A nucleic acid comprising a nucleotide sequence encoding the TMP according to any one of claims 1 to 25.

29. A cell genetically modified with the nucleic acid described in claim 28.

30. A pharmaceutical composition comprising the TMP of any one of claims 1 to 25, the homodimer of claim 26, or the heterodimer of claim 27, and a pharma- ceutically acceptable excipient.

31. A method for producing the TMP according to any one of claims 1 to 25, the homodimer according to claim 26, or the heterodimer according to claim 27, comprising the steps of: Culturing in vitro the cell of claim 29 in a cell culture medium. The method comprising:

32. A pharmaceutical composition for selectively regulating the activity of T cells specific for a KRAS peptide epitope, comprising a TMP described in any one of claims 1 to 25, a homodimer described in claim 26, or a heterodimer described in claim 27.

33. A pharmaceutical composition for treating a cancer associated with KRAS in a patient having said cancer, comprising a TMP according to any one of claims 1 to 25, a homodimer according to claim 26, or a heterodimer according to claim 27.

34. The pharmaceutical composition of claim 33, administered to the patient in combination with an immune checkpoint inhibitor, wherein the immune checkpoint inhibitor is an antibody specific for PD-L1, PD-1, TIGIT, or CTLA4.

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