T cell modulating polypeptides and methods of use thereof

Abstract

The present disclosure provides T cell modulatory polypeptides (TMPs) that include an immunomodulatory polypeptide, 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 containing a cancer-associated mutation). The TMPs are useful for modulating the activity of T cells and modulating an individual's immune response. TIFF2022548472000020.tif41165

Description

[Technical Field]

[0001] cross reference This application claims the benefit of U.S. Provisional Patent Application No. 62 / 903,441, filed September 20, 2019, U.S. Provisional Patent Application No. 62 / 990,693, filed March 17, 2020, and U.S. Provisional Patent Application No. 63 / 048,561, filed July 6, 2020, each of which is incorporated by reference in its entirety. [Background technology]

[0002] preface The adaptive immune response involves the engagement of T cell receptors (TCRs) on the surface of T cells with small peptide antigens noncovalently presented on the surface of antigen-presenting cells (APCs) by the major histocompatibility complex (MHC, also known in humans as the human leukocyte antigen (HLA) complex). This engagement represents the immune system's targeting mechanism and is a molecular interaction required for T cell regulation (activation or inhibition) and effector function. Following epitope-specific cell targeting, targeted T cells are activated through the engagement of costimulatory proteins found on APCs with corresponding costimulatory proteins on T cells. Both signals—epitope / TCR binding and the engagement of APC costimulatory proteins with T cell costimulatory proteins—are required to promote T cell specificity and activation or inhibition. Costimulatory proteins on APCs are also called "immunoregulatory" proteins because they regulate T cell activity upon binding to them. Specific regulation is a function of which immunooregulatory proteins on APCs bind to which costimulatory proteins on T cells. TCRs are specific for a given epitope, however T cell costimulatory proteins are not epitope specific and are generally expressed on all T cells or on large T cell subsets. Summary of the Invention

[0003] The present disclosure provides heterodimeric and single-chain T cell modulatory polypeptides (TMPs), as well as dimers thereof, that include an immunomodulatory polypeptide, 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 containing a cancer-associated mutation). The TMPs regulate T cell activity and help regulate an individual's immune response. [The present invention 1001] T cell modulatory polypeptide (TMP), i) a KRAS peptide comprising a KRAS epitope expressed on cancer cells and having a length of at least 4 amino acids; ii) a first major histocompatibility complex (MHC) polypeptide; iii) a second MHC polypeptide, and iv) at least one immunomodulatory polypeptide Including, The T cell modulatory polypeptide, wherein the first major histocompatibility complex (MHC) polypeptide is a β2-microglobulin polypeptide and the second MHC polypeptide is an MHC class I heavy chain polypeptide. [The present invention 1002] 1001. The T cell modulating polypeptide of the present invention, wherein the T cell modulating polypeptide further comprises an Ig Fc polypeptide that does not substantially induce cell lysis, and optionally, the IgG1 Fc polypeptide comprises one or more amino acid substitutions selected from N297A, L234A, L235A, L234F, L235E, and P331S. [The present invention 1003] 1001 or 1002, wherein the β2M polypeptide and the MHC heavy chain polypeptide are connected by a disulfide bond connecting a Cys residue of the β2M polypeptide and a Cys residue of the MHC heavy chain polypeptide, and optionally, a Cys at amino acid residue 12 of the β2M polypeptide is disulfide bonded to a Cys at amino acid residue 236 of the MHC heavy chain polypeptide. [The present invention 1004] 10. The T cell modulatory polypeptide of any one of claims 1001 to 1003, wherein the β2-microglobulin polypeptide is connected to the KRAS peptide by a first linker comprising a Cys, wherein a disulfide bond links a Cys present in the first linker to a Cys present in the MHC heavy chain polypeptide, and optionally, the first linker comprises the sequence CGGGS(GGGGS)n (SEQ ID NO: 142) or GCGGS(GGGGS)n (SEQ ID NO: 140), wherein n is an integer between 1 and 10, e.g., 2 or 3, and a disulfide bond links a Cys of the linker to a Cys substituted for Tyr84 of the MHC heavy chain polypeptide. [The present invention 1005] The MHC heavy chain polypeptide is HLA-A * 0201 polypeptide, HLA-A * 1101 polypeptide, HLA-A * 3303 polypeptide, and HLA-A * 2401 polypeptides. 1004. A T cell modulatory polypeptide of the invention comprising an amino acid sequence having at least 95% amino acid sequence identity to an HLA-A polypeptide selected from the group consisting of: 1004 T cell modulatory polypeptides. [The present invention 1006] The T cell regulatory polypeptide of any of claims 1001 to 1005, wherein the at least one immunoregulatory polypeptide is a wild-type or variant form of an activating immunoregulatory polypeptide selected from the group consisting of IL-2, 4-1BBL, CD80, CD86, or a combination thereof, and optionally at least one of the at least one immunoregulatory polypeptides is a variant immunoregulatory polypeptide that exhibits lower affinity for a cognate costimulatory polypeptide compared to the affinity of the corresponding wild-type immunoregulatory polypeptide for the cognate costimulatory polypeptide. [The present invention 1007] 1006. The T cell modulatory polypeptide of the present invention, wherein said at least one immune modulatory polypeptide is a variant IL-2 that does not substantially bind to IL-2Rα and has reduced affinity for IL-2Rβ, and optionally said variant IL-2 polypeptide comprises i) an H16A substitution and an F42A substitution, or ii) an H16T substitution and an F42A substitution. [The present invention 1008] 1007. The T cell regulatory polypeptide of any one of claims 1001 to 1007, wherein the KRAS peptide comprises a sequence selected from the group consisting of: A) VVGADGVGK (SEQ ID NO: 176), VVGACGVGK (SEQ ID NO: 177), VVGAVGVGK (SEQ ID NO: 178), VVVGADGVGK (SEQ ID NO: 179), VVVGAVGVGK (SEQ ID NO: 180), VVVGACGVGK (SEQ ID NO: 181), VTGADGVGK (SEQ ID NO: 182), VTGAVGVGK (SEQ ID NO: 183), VTGACGVGK (SEQ ID NO: 184), VTVGADGVGK (SEQ ID NO: 185), VTVGAVGVGK (SEQ ID NO: 186), and VTVGACGVGK (SEQ ID NO: 187); wherein the KRAS peptides are 9 or 10 amino acids in length, or at least 9 or 10 amino acids in length; B) VVVGAGDVGK (SEQ ID NO: 188), VVGAGDVGK (SEQ ID NO: 189), VVVGARGVGK (SEQ ID NO: 190), and VVGARGVGK (SEQ ID NO: 191); wherein the KRAS peptide is 9 or 10 amino acids in length, or at least 9 or 10 amino acids in length; C) LVVVGADGV (SEQ ID NO: 192), LVVVGAVGV (SEQ ID NO: 193), LVVVGACGV (SEQ ID NO: 194), KLVVVGADGV (SEQ ID NO: 195), KLVVVGAVGV (SEQ ID NO: 196), KLVVVGACGV (SEQ ID NO: 197), LLVVGADGV (SEQ ID NO: 198), LLVVGAVGV (SEQ ID NO: 199), LLVVGACGV (SEQ ID NO: 200), FLVVVGADGV (SEQ ID NO: 201), FLVVVGAVGV (SEQ ID NO: 202), and FLVVVGACGV (SEQ ID NO: 203); wherein the KRAS peptide is 9 or 10 amino acids in length, or at least 9 or 10 amino acids in length; D) KLVVVGAGDV (SEQ ID NO: 204), and KLVVVGARGV (SEQ ID NO: 205); wherein the KRAS peptide is 9 or 10 amino acids in length, or at least 9 or 10 amino acids in length; E) GAGDVGKSAL (SEQ ID NO: 206), AGDVGKSAL (SEQ ID NO: 207), DVGKSALTI (SEQ ID NO: 208), GAVGVGKSAL (SEQ ID NO: 209), AVGVGKSAL (SEQ ID NO: 210), YKLVVVGAV (SEQ ID NO: 211), ARGVGKSAL (SEQ ID NO: 212), GARGVGKSAL (SEQ ID NO: 213), EYKLVVVGAR (SEQ ID NO: 214), RGVGKSALTI (SEQ ID NO: 215), LVVVGARGV (SEQ ID NO: 216), GADGVGKSAL (SEQ ID NO: 217), ACGVGKSAL (SEQ ID NO: 218), and GACGVGKSAL (SEQ ID NO: 219); wherein the KRAS peptides are 9 or 10 amino acids in length, or at least 9 or 10 amino acids in length; and F) VVGAVGVGK (SEQ ID NO: 178), VVVGAVGVGK (SEQ ID NO: 180), VGAVGVGKS (SEQ ID NO: 222), VGAVGVGKSA (SEQ ID NO: 223), AVGVGKSAL (SEQ ID NO: 210), AVGVGKSALT (SEQ ID NO: 225), GAVGVGKSAL (SEQ ID NO: 209), GAVGVGKSA (SEQ ID NO: 227), LVVVGAVGVG (SEQ ID NO: 228), LVVVGAVGV (SEQ ID NO: 193), KLVVVGAVGV (SEQ ID NO: 196), and KLVVVGAVG (SEQ ID NO: 231); wherein the KRAS peptide is 9 or 10 amino acids in length, or at least 9 or 10 amino acids in length. [The present invention 1009] A) the KRAS peptide is KLVVVGADGV (SEQ ID NO: 195) 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: 179) or VVGAVGVGK (SEQ ID NO: 178), 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 A T cell regulatory polypeptide according to any one of 1001 to 1008 of the present invention. [The present invention 1010] 1009. The T cell modulatory polypeptide of any of claims 1001 to 1009, wherein the TMP comprises a heterodimer, the heterodimer comprising: a1) the first polypeptide comprises, in order from the N-terminus to the C-terminus: i) the KRAS peptide, and ii) β2-microglobulin polypeptide and b1) the second polypeptide comprises, in order from the N-terminus to the C-terminus: i) at least one immunomodulatory polypeptide; ii) an MHC class I heavy chain polypeptide, and iii) Ig Fc polypeptide Contains, or a3) the first polypeptide comprises, in order from the N-terminus to the C-terminus: i) the KRAS peptide, and ii) β2-microglobulin polypeptide and b3) the second polypeptide comprises, in order from the N-terminus to the C-terminus: i) an MHC class I heavy chain polypeptide, and ii) an Ig Fc polypeptide, and iii) at least one immunomodulatory polypeptide Including, wherein said Ig Fc polypeptide is a human IgG1 Fc polypeptide that does not substantially induce cell lysis, and optionally comprises the amino acid sequence of Figure 3G. [The present invention 1011] the β2-microglobulin polypeptide is connected to the KRAS peptide by a first linker comprising the sequence CGGGS(GGGGS)n (SEQ ID NO: 142) or GCGGS(GGGGS)n (SEQ ID NO: 140), wherein n is an integer between 1 and 10, e.g., 2 or 3; the MHC heavy chain polypeptide comprises a Cys at residue 84 and a Cys at residue 236; the β2M polypeptide comprises a Cys at residue 12; Cys at amino acid residue 12 of the β2M polypeptide is disulfide bonded to Cys at amino acid residue 236 of the MHC heavy chain polypeptide; a disulfide bond links the Cys of the linker to the Cys substituted for Tyr84 of the MHC heavy chain polypeptide; The MHC heavy chain polypeptide is HLA-A * 0201 polypeptide, HLA-A * 1101 polypeptide, HLA-A * 3303 polypeptide, and HLA-A* 2401 polypeptides, the at least one immunomodulatory polypeptide is a variant IL-2 comprising i) an H16A substitution and an F42A substitution or ii) an H16T substitution and an F42A substitution; and the polypeptide comprises two immunomodulatory polypeptides that are identical, in tandem, and comprise a variant IL-2 comprising i) an H16A substitution and an F42A substitution, or ii) an H16T substitution and an F42A substitution; A heterodimeric T cell modulating polypeptide of the present invention. [The present invention 1012] A) the KRAS peptide is KLVVVGADGV (SEQ ID NO: 195) 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: 179) or VVGAVGVGK (SEQ ID NO: 178), 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 A heterodimeric T cell modulating polypeptide of the present invention 1010 or 1011. [The present invention 1013] The TMP i) a KRAS peptide comprising a KRAS epitope expressed on cancer cells and having a length of at least 4 amino acids; ii) β2-microglobulin polypeptide; iii) an MHC class I heavy chain polypeptide, and iv) at least one immunomodulatory polypeptide The T cell regulatory polypeptide of any one of 1001 to 1009 of the present invention, which is a single polypeptide chain comprising: [The present invention 1014] The single-chain T cell modulating polypeptides are, 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 Ig Fc polypeptide, and v) one or more immunomodulatory polypeptides; the Ig Fc polypeptide is a human IgG1 Fc polypeptide that does not substantially induce cell lysis, and optionally comprises the amino acid sequence of Figure 3G; 1013 single-chain T cell modulating polypeptides of the present invention. [The present invention 1015] the β2 microglobulin polypeptide is connected to the KRAS peptide by a first linker comprising the sequence CGGGS(GGGGS)n (SEQ ID NO: 142) or GCGGS(GGGGS)n (SEQ ID NO: 140), wherein n is an integer between 1 and 10, e.g., 2 or 3; the MHC heavy chain polypeptide comprises a Cys at residue 84 and a Cys at residue 236; the β2M polypeptide comprises a Cys at residue 12; Cys at amino acid residue 12 of the β2M polypeptide is disulfide bonded to Cys at amino acid residue 236 of the MHC heavy chain polypeptide; a disulfide bond links the Cys of the linker to the Cys substituted for Tyr84 of the MHC heavy chain polypeptide; the β2M polypeptide is connected 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, e.g., n=3 or 7; The MHC heavy chain polypeptide is HLA-A * 0201 polypeptide, HLA-A * 1101 polypeptide, HLA-A * 3303 polypeptide, and HLA-A * 2401 polypeptides, the at least one immunomodulatory polypeptide is a variant IL-2 comprising i) an H16A substitution and an F42A substitution or ii) an H16T substitution and an F42A substitution; the polypeptide comprises two immunomodulatory polypeptides that are identical, in tandem, and comprise a variant IL-2 comprising i) an H16A substitution and an F42A substitution, or ii) an H16T substitution and an F42A substitution; 1014 single-chain T cell modulating polypeptides of the present invention. [The present invention 1016] A) the KRAS peptide is KLVVVGADGV (SEQ ID NO: 195) 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: 179) or VVGAVGVGK (SEQ ID NO: 178), 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 1015 single-chain T cell modulating polypeptides of the present invention. [The present invention 1017] A T cell modulating polypeptide, wherein the TMP is a homodimer comprising a first and a second heterodimeric TMP of any one of claims 1010 to 1012 of the present invention or a first and a second single-chain TMP of any one of claims 1013 to 1016 of the present invention, wherein the first and second heterodimers are identical and covalently linked by one or more disulfide bonds between the Ig Fc polypeptides of the first and second heterodimers. [The present invention 1018] A nucleic acid comprising a nucleotide sequence encoding the first or second polypeptide of the heterodimeric TMP of any one of 1010 to 1012 of the present invention or the single-chain TMP of any one of 1013 to 1016 of the present invention. [The present invention 1019] 18. A method of selectively modulating the activity of T cells specific for a KRAS peptide epitope, the method comprising contacting the T cells with a T cell modulating polypeptide according to any one of aspects 1 to 17, wherein the contacting selectively modulates the activity of the epitope-specific T cells. [The present invention 1020] A method for treating a KRAS-associated cancer in a patient having the cancer, the method comprising administering to the patient an effective amount of a pharmaceutical composition comprising any of the T cell regulatory polypeptides of the present inventions 1001 to 1017, and optionally further comprising co-administering to the patient an immune checkpoint inhibitor, optionally wherein the immune checkpoint inhibitor is an antibody specific for PD-L1, PD-1, or CTLA4. [Brief explanation of the drawings]

[0004] [Figure 1A] 1A-1F are schematic diagrams of various exemplary TMPs of the present disclosure. [Figure 1B] See legend to Figure 1A. [Figure 1C] See legend to Figure 1A. [Figure 1D] See legend to Figure 1A. [Figure 1E] See legend to Figure 1A. [Figure 1F] See legend to Figure 1A. [Figure 2A] 2A-2F are schematic diagrams of various disulfide-bonded TMPs of the present disclosure. [Figure 2B] See legend to Figure 2A. [Figure 2C] See legend to Figure 2A. [Figure 2D] See legend to Figure 2A. [Figure 2E] See legend to Figure 2A. [Figure 2F] See legend to Figure 2A. [Figure 3A] 3A-3G provide the amino acid sequences of immunoglobulin Fc polypeptides, which are set forth in SEQ ID NOs: 19-30. [Figure 3B] See legend to Figure 3A. [Figure 3C] See legend to Figure 3A. [Figure 3D] See legend to Figure 3A. [Figure 3E] See legend to Figure 3A. [Figure 3F] See legend to Figure 3A. [Figure 3G] See legend to Figure 3A. [Figure 4]

[0023] Figure 1 provides a multiple amino acid sequence alignment of the β2-microglobulin (β2M) precursor (i.e., including the leader sequence) from humans (Homo sapiens) (NP_004039.1, SEQ ID NO: 31), chimpanzees (Pan troglodytes) (NP_001009066.1, SEQ ID NO: 31), rhesus monkeys (Macaca mulatta) (NP_001040602.1, SEQ ID NO: 32), cattle (Bos taurus) (NP_776318.1, SEQ ID NO: 33), and mice (Mus musculus) (NP_033865.2, SEQ ID NO: 34). Amino acids 1-20 are the signal peptide. [Figure 5A] 1 provides the amino acid sequences of the full-length human HLA heavy chains of alleles A*0101 (SEQ ID NO: 35), A*1101 (SEQ ID NO: 36), A*2402 (SEQ ID NO: 37), and A*3303 (SEQ ID NO: 38). [Figure 5B] 1 provides the amino acid sequence of the full-length human HLA heavy chain of allele B*0702 (SEQ ID NO: 39). [Figure 5C] 1 provides the amino acid sequence of the full-length human HLA-C heavy chain (SEQ ID NO: 40). [Figure 6-1] An alignment of 11 mature MHC class I heavy chain amino acid sequences, excluding the leader sequence, transmembrane domain, and intracellular domain, is provided. The sequences are set forth in SEQ ID NOs: 41 to 51, from top to bottom. [Figure 6-2] This is a continuation of Figure 6-1. [Figure 7A-1] An alignment of HLA-A heavy chain amino acid sequences (SEQ ID NOs: 52 to 60, respectively) is provided. [Figure 7A-2] This shows a continuation of Figure 7A-1. [Figure 7B] The HLA-A heavy chain consensus sequence (SEQ ID NO: 61) is provided. [Figure 8A-1] An alignment of HLA-B heavy chain amino acid sequences (SEQ ID NOs: 62 to 68, respectively) is provided. [Figure 8A-2] This shows a continuation of Figure 8A-1. [Figure 8B] The HLA-B heavy chain consensus sequence (SEQ ID NO: 69) is provided. [Figure 9A-1] An alignment of HLA-C heavy chain amino acid sequences (SEQ ID NOs: 70 to 78, respectively) is provided. [Figure 9A-2] This shows a continuation of Figure 9A-1. [Figure 9B] The HLA-C heavy chain consensus sequence (SEQ ID NO: 79) is provided. [Figure 10] Consensus amino acid sequences for each of the HLA-E, -F, and -G heavy chains are provided (SEQ ID NOS: 80-82, respectively). Variable amino acid (aa) positions are shown as consecutively numbered "X" residues. Amino acid positions 84, 139, and 236 are double underlined. [Figure 11] An alignment of the consensus amino acid sequences of HLA-A (SEQ ID NO: 83), -B (SEQ ID NO: 84), -C (SEQ ID NO: 85), -E (SEQ ID NO: 86), -F (SEQ ID NO: 87), and -G (SEQ ID NO: 88) is provided. [Figure 12A] 12A-12D provide schematic diagrams of the multiple disulfide bonded TMPs of the present disclosure. [Figure 12B] See legend to Figure 12A. [Figure 12C] See legend to Figure 12A. [Figure 12D] See legend to Figure 12A. [Figure 13A] Figures 13A-13L provide amino acid sequences of examples of polypeptides suitable for inclusion in the TMPs of the present disclosure. The sequences in Figures 13A-13F are set forth in SEQ ID NOs: 255-260. The sequences in Figures 13G-13L are set forth in SEQ ID NOs: 339-344. [Figure 13B] See legend to Figure 13A. [Figure 13C] See legend to Figure 13A. [Figure 13D] See legend to Figure 13A. [Figure 13E] See legend to Figure 13A. [Figure 13F] See legend to Figure 13A. [Figure 13G] See legend to Figure 13A. [Figure 13H] See legend to Figure 13A. [Figure 13I] See legend to Figure 13A. [Figure 13J] See legend to Figure 13A. [Figure 13K] See legend to Figure 13A. [Figure 13L] See legend to Figure 13A. [Figure 14A] 14A-14JJ provide the amino acid sequences of examples of polypeptides suitable for inclusion in the TMPs of the present disclosure. The sequences are set forth in SEQ ID NOs:261-296. [Figure 14B] See legend to Figure 14A. [Figure 14C] See legend to Figure 14A. [Figure 14D] See legend to Figure 14A. [Figure 14E] See legend to Figure 14A. [Figure 14F] See legend to Figure 14A. [Figure 14G] See legend to Figure 14A. [Figure 14H] See legend to Figure 14A. [Figure 14I] See legend to Figure 14A. [Figure 14J] See legend to Figure 14A. [Figure 14K] See legend to Figure 14A. [Figure 14L] See legend to Figure 14A. [Figure 14M] See legend to Figure 14A. [Figure 14N] See legend to Figure 14A. [Figure 14O] See legend to Figure 14A. [Figure 14P] See legend to Figure 14A. [Figure 14Q] See legend to Figure 14A. [Figure 14R] See legend to Figure 14A. [Figure 14S] See legend to Figure 14A. [Figure 14T] See legend to Figure 14A. [Figure 14U] See legend to Figure 14A. [Figure 14V] See legend to Figure 14A. [Figure 14W] See legend to Figure 14A. [Figure 14X] See legend to Figure 14A. [Figure 14Y] See legend to Figure 14A. [Figure 14Z] See legend to Figure 14A. [Figure 14AA] See legend to Figure 14A. [Figure 14BB] See legend to Figure 14A. [Figure 14CC] See legend to Figure 14A. [Figure 14DD] See legend to Figure 14A. [Figure 14EE] See legend to Figure 14A. [Figure 14FF] See legend to Figure 14A. [Figure 14GG] See legend to Figure 14A. [Figure 14HH] See legend to Figure 14A. [Figure 14II] See legend to Figure 14A. [Figure 14JJ] See legend to Figure 14A. [Figure 15A] 15A-15JJ provide the amino acid sequences of examples of polypeptides suitable for inclusion in the TMPs of the present disclosure. The sequences are set forth in SEQ ID NOs: 297-332. [Figure 15B] See legend to Figure 15A. [Figure 15C] See legend to Figure 15A. [Figure 15D] See legend to Figure 15A. [Figure 15E]See legend to Figure 15A. [Figure 15F] See legend to Figure 15A. [Figure 15G] See legend to Figure 15A. [Figure 15H] See legend to Figure 15A. [Figure 15I] See legend to Figure 15A. [Figure 15J] See legend to Figure 15A. [Figure 15K] See legend to Figure 15A. [Figure 15L] See legend to Figure 15A. [Figure 15M] See legend to Figure 15A. [Figure 15N] See legend to Figure 15A. [Figure 15O] See legend to Figure 15A. [Figure 15P] See legend to Figure 15A. [Figure 15Q] See legend to Figure 15A. [Figure 15R] See legend to Figure 15A. [Figure 15S] See legend to Figure 15A. [Figure 15T] See legend to Figure 15A. [Figure 15U] See legend to Figure 15A. [Figure 15V] See legend to Figure 15A. [Figure 15W] See legend to Figure 15A. [Figure 15X] See legend to Figure 15A. [Figure 15Y] See legend to Figure 15A. [Figure 15Z] See legend to Figure 15A. [Figure 15AA] See legend to Figure 15A. [Figure 15BB] See legend to Figure 15A. [Figure 15CC] See legend to Figure 15A. [Figure 15DD] See legend to Figure 15A. [Figure 15EE] See legend to Figure 15A. [Figure 15FF]See legend to Figure 15A. [Figure 15GG] See legend to Figure 15A. [Figure 15HH] See legend to Figure 15A. [Figure 15II] See legend to Figure 15A. [Figure 15JJ] See legend to Figure 15A. [Figure 16A] 16A-16C provide schematic diagrams of example configurations of disulfide-bonded TMPs of the present disclosure. [Figure 16B] See legend to Figure 16A. [Figure 16C] See legend to Figure 16A. [Figure 17] 1 provides a schematic diagram of an example of the location of an immunomodulatory polypeptide in a "split-chain" TMP of the present disclosure. [Figure 18] 1 provides a schematic diagram of an example of the location of an immunomodulatory polypeptide in a "single-chain" TMP of the present disclosure. [Figure 19A] 19A-19P provide the amino acid sequences of several examples of single-chain TMPs of the present disclosure. The sequences are set forth in SEQ ID NOs: 333-338 and 593-602. [Figure 19B] See legend to Figure 19A. [Figure 19C] See legend to Figure 19A. [Figure 19D] See legend to Figure 19A. [Figure 19E] See legend to Figure 19A. [Figure 19F] See legend to Figure 19A. [Figure 19G] See legend to Figure 19A. [Figure 19H] See legend to Figure 19A. [Figure 19I] See legend to Figure 19A. [Figure 19J] See legend to Figure 19A. [Figure 19K] See legend to Figure 19A. [Figure 19L] See legend to Figure 19A. [Figure 19M] See legend to Figure 19A. [Figure 19N]See legend to Figure 19A. [Figure 19O] See legend to Figure 19A. [Figure 19P] See legend to Figure 19A. [Figure 20] 1 shows the effect of KRAS G12V(7-16)HLA-A11 TMP on the proliferation of KRAS G12V(7-16)-specific CD8+ T cells in vitro. [Figure 21A] 21A-21JJ provide the amino acid sequences for the HLA-A heavy chain amino acid sequences and the β2M amino acid sequences of the TMPs of the disclosure. [Figure 21B] See legend to Figure 21A. [Figure 21C] See legend to Figure 21A. [Figure 21D] See legend to Figure 21A. [Figure 21E] See legend to Figure 21A. [Figure 21F] See legend to Figure 21A. [Figure 21G] See legend to Figure 21A. [Figure 21H] See legend to Figure 21A. [Figure 21I] See legend to Figure 21A. [Figure 21J] See legend to Figure 21A. [Figure 21K] See legend to Figure 21A. [Figure 21L] See legend to Figure 21A. [Figure 21M] See legend to Figure 21A. [Figure 21N] See legend to Figure 21A. [Figure 21O] See legend to Figure 21A. [Figure 21P] See legend to Figure 21A. [Figure 21Q] See legend to Figure 21A. [Figure 21R] See legend to Figure 21A. [Figure 21S] See legend to Figure 21A. [Figure 21T] See legend to Figure 21A. [Figure 21U]See legend to Figure 21A. [Figure 21V] See legend to Figure 21A. [Figure 21W] See legend to Figure 21A. [Figure 21X] See legend to Figure 21A. [Figure 21Y] See legend to Figure 21A. [Figure 21Z] See legend to Figure 21A. [Figure 21AA] See legend to Figure 21A. [Figure 21BB] See legend to Figure 21A. [Figure 21CC] See legend to Figure 21A. [Figure 21DD] See legend to Figure 21A. [Figure 21EE] See legend to Figure 21A. [Figure 21FF] See legend to Figure 21A. [Figure 21GG] See legend to Figure 21A. [Figure 21HH] See legend to Figure 21A. [Figure 21II] See legend to Figure 21A. [Figure 21JJ] See legend to Figure 21A. [Figure 22A] 22A-22BB provide the amino acid sequences of the HLA-A heavy chain amino acid sequences of the TMPs of the present disclosure. [Figure 22B] See legend to Figure 22A. [Figure 22C] See legend to Figure 22A. [Figure 22D] See legend to Figure 22A. [Figure 22E] See legend to Figure 22A. [Figure 22F] See legend to Figure 22A. [Figure 22G] See legend to Figure 22A. [Figure 22H] See legend to Figure 22A. [Figure 22I] See legend to Figure 22A. [Figure 22J] See legend to Figure 22A. [Figure 22K]See legend to Figure 22A. [Figure 22L] See legend to Figure 22A. [Figure 22M] See legend to Figure 22A. [Figure 22N] See legend to Figure 22A. [Figure 22O] See legend to Figure 22A. [Figure 22P] See legend to Figure 22A. [Figure 22Q] See legend to Figure 22A. [Figure 22R] See legend to Figure 22A. [Figure 22S] See legend to Figure 22A. [Figure 22T] See legend to Figure 22A. [Figure 22U] See legend to Figure 22A. [Figure 22V] See legend to Figure 22A. [Figure 22W] See legend to Figure 22A. [Figure 22X] See legend to Figure 22A. [Figure 22Y] See legend to Figure 22A. [Figure 22Z] See legend to Figure 22A. [Figure 22AA] See legend to Figure 22A. [Figure 22BB] See legend to Figure 22A. DETAILED DESCRIPTION OF THE INVENTION

[0005] definition The terms "polynucleotide" and "nucleic acid" are used interchangeably herein to refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, the term includes, but is not limited to, single-, double-, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers that contain purine and pyrimidine bases or other naturally occurring, chemically or biochemically modified, non-naturally occurring, or derivatized nucleotide bases.

[0006] The terms "peptide," "polypeptide," and "protein" are used interchangeably herein to refer to polymeric forms of amino acids of any length, and may include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides with modified peptide backbones. Furthermore, as used herein, "polypeptide" refers to proteins containing modifications to the native sequence, such as deletions, additions, and substitutions (generally essentially conservative, as would be known to one of skill in the art), so long as the protein maintains a desired activity. These modifications may be intentional, such as through site-directed mutagenesis, or may be accidental, such as through mutations of hosts producing the protein or errors in polymerase chain reaction (PCR) amplification or other recombinant DNA methods. Reference herein to a specific residue or residue number of a known polypeptide is understood to refer to the amino acid at that position in the wild-type polypeptide. To the extent that the sequence of the wild-type polypeptide is modified, either by the addition or deletion of one or more amino acids, those of skill in the art will understand that a reference to a specific residue or residue number, to refer to the same specific amino acid in the modified polypeptide, is accordingly modified and understood to be the residue at the modified position number. For example, if an MHC class I polypeptide is modified by adding one amino acid to the N-terminus, a reference to position 84 or a specific residue at position 84 is understood to refer to the amino acid at position 85 of the modified polypeptide. Similarly, a reference herein to a specific amino acid substitution at a specific position, e.g., Y84, is understood to refer to the amino acid substitution at amino acid position 84 of the wild-type polypeptide. Thus, a Y84C substitution is understood to be a substitution of a Tyr residue present in the wild-type sequence with a Cys residue. For example, if a wild-type polypeptide is modified to change the amino acid at position 84 from its wild-type amino acid to an alternative amino acid, the substitution of the amino acid at position 84 will be understood to refer to a substitution with an alternative amino acid. In such cases, if the polypeptide is also modified by the addition or deletion of one or more amino acids, a reference to a substitution will be understood to refer to a substitution with an alternative amino acid at the modified position number.Reference to a "non-native Cys residue" in a polypeptide, e.g., an MHC class I polypeptide, means that the polypeptide contains 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, substituting cysteine ​​for the amino acid present in the wild-type sequence.

[0007] A polynucleotide or polypeptide has a certain percent "sequence identity" to another polynucleotide or polypeptide, meaning that the percentage of bases or amino acids are the same and in the same relative positions when the two sequences are aligned and compared. Sequence identity can be measured using a number of different methods. To measure sequence identity, sequences may be aligned using a variety of convenient methods and computer programs (e.g., BLAST, T-COFFEE, MUSCLE, MAFFT, etc.) available on the world wide web at sites including ncbi.nlm.nili.gov / BLAST, ebi.ac.uk / Tools / msa / tcoffee / , ebi.ac.uk / Tools / msa / muscle / , and mafft.cbrc.jp / alignment / software / . See, e.g., Altschul et al. (1990), J. Mol. Biol. 215:403-10. Unless otherwise specified, "sequence identity" referred to herein is determined by BLAST (basic local alignment search tool) as described in Altschul et al. (1990) J. Mol. Biol. 215:403.

[0008] The term "conservative amino acid substitution" refers to the interchangeability of amino acid residues in proteins that have similar side chains. For example, the group of amino acids with aliphatic side chains consists of glycine, alanine, valine, leucine, and isoleucine; the group of amino acids with aliphatic hydroxyl side chains consists of serine and threonine; the group of amino acids with amide-containing side chains consists of asparagine and glutamine; the group of amino acids with aromatic side chains consists of phenylalanine, tyrosine, and tryptophan; the group of amino acids with basic side chains consists of lysine, arginine, and histidine; the group of amino acids with acidic side chains consists of glutamic acid and aspartic acid; and the group of amino acids with sulfur-containing side chains consists of cysteine ​​and methionine. Exemplary conservative amino acid substitution groups are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine-glycine, and asparagine-glutamine.

[0009] As used herein, the term "immunological synapse" or "immune synapse" generally refers to the natural interface between two interacting immune cells of the adaptive immune response (e.g., including the interface 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 initiated by the interaction of a T cell antigen receptor with a major histocompatibility complex molecule, as described, for example, in Bromley et al., Annu Rev Immunol. 2001, 19:375-96 (the entire disclosure of which is incorporated herein by reference).

[0010] "T cells" include all types of immune cells that express CD3, such as helper T cells (CD4 + cells), cytotoxic T cells (CD8 + These include T cells, T suppressor cells (Tregs), and NK-T cells.

[0011] As used herein, the term "immunomodulatory polypeptide" (also referred to herein as "MOD") refers to a polypeptide that specifically binds to a cognate costimulatory polypeptide on a T cell, thereby producing signals that mediate T cell responses, including, but not limited to, proliferation, activation, differentiation, etc., in addition to the primary signal produced, for example, by binding of the TCR / CD3 complex to a peptide-binding major histocompatibility complex (MHC) polypeptide. As described herein, immunomodulatory polypeptides can include, but are not limited to, wild-type or variants of wild-type polypeptides, such as cytokines (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, agonists or antibodies that bind to Toll ligand receptors, and ligands that specifically bind to B7-H3. The immunomodulatory domain or "MOD" of the TMPs of the present disclosure can bind to cognate costimulatory polypeptides present on target T cells.

[0012] As used herein, the term "in vivo" refers to any process or procedure that occurs inside the body.

[0013] As used herein, "in vitro" refers to any process or procedure that occurs outside the body.

[0014] As used herein, "heterologous" refers to a nucleotide or polypeptide that is not found in the native nucleic acid or protein, respectively.

[0015] As used herein, "recombinant" means that a particular nucleic acid (DNA or RNA) is the product of various combinations of cloning, restriction, polymerase chain reaction (PCR), and / or ligation steps that result in a construct having structural coding or non-coding sequences that are distinguishable from endogenous nucleic acids present in natural systems. A DNA sequence encoding a polypeptide may be assembled from cDNA fragments or a series of synthetic oligonucleotides to obtain a synthetic nucleic acid capable of expression by a recombinant transcription unit contained within a cellular or cell-free transcription and translation system.

[0016] The terms "recombinant expression vector" or "DNA construct" are used interchangeably herein to refer to a DNA molecule comprising a vector and at least one insert. Recombinant expression vectors are typically constructed for the purpose of expressing and / or propagating an insert(s) or for 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.

[0017] As used herein, the term "affinity" refers to the equilibrium constant for the reversible binding of two substances (e.g., an antibody and an antigen), and also refers to the dissociation constant (K D) The affinity may be at least 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, at least 100-fold, or at least 1,000-fold, or more, greater than the antibody affinity for an unrelated amino acid sequence. The affinity of an antibody for a target protein may be, for example, from about 100 nanomolar (nM) to about 0.1 nM, from about 100 nM to about 1 picomolar (pM), or from about 100 nM to about 1 femtomolar (fM), or more. As used herein, the term "avidity" refers to the resistance of a complex of two or more substances to dissociation after dilution. The terms "immunoreactive" and "preferentially bind" are used interchangeably herein with respect to antibodies and / or antigen-binding fragments.

[0018] As used herein (e.g., with respect to binding of TMP to a polypeptide on a T cell (e.g., a T cell receptor)), the term "binding" refers to a non-covalent interaction between two molecules. Non-covalent binding refers to a direct association between two molecules, e.g., by electrostatic, hydrophobic, ionic, and / or hydrogen-bonding interactions, including interactions such as salt bridges and water bridges. Non-covalent interactions typically occur over a 10 -6 Under M, 10 -7 Under M, 10 -8 Under M, 10 -9 Under M, 10 -10 Under M, 10 -11 Under M, 10 -12 Under M, 10 -13 Under M, 10 -14 Less than M or 10 -15 A dissociation constant (K D "Affinity" refers to the strength of non-covalent binding, with high binding affinity being characterized by a low K D "Specific binding" generally correlates with at least about 10 -7M or more, e.g., 5 x 10 -7 M, 10 -8 M, 5 x 10 -8 M, 10 -9 M and higher affinity binding. "Non-specific binding" generally refers to binding with an affinity of about 10 -7 Binding with an affinity of less than M (e.g., 10 -6 M, 10 -5 M, 10 -4 "Specific binding" refers to binding with an affinity of 1 μM to 100 μM (e.g., binding of a ligand to a moiety other than its designated binding site or receptor). However, in some contexts, e.g., binding between a TCR and a peptide / MHC complex, "specific binding" may be in the range of 1 μM to 100 μM, or 100 μM to 1 mM. As used herein, "covalent binding" or "covalent bond" refers to the formation of one or more covalent chemical bonds between two different molecules.

[0019] As used herein, "phosphate buffered saline" or "PBS" refers to an aqueous buffer solution typically available as a concentrated solution. Unless otherwise specified, the PBS solution used in this disclosure contains sodium chloride (500 mM), sodium dihydrogen phosphate (10 mM), potassium dihydrogen phosphate (2 mM), potassium chloride (2.7 mM), and the balance water. The pH of PBS is 7.5±0.15. The buffer is prepared in 18.2 megaohm DNase- and RNase-free water and filtered through a 0.22 micron filter.

[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 terms of completely or partially preventing a disease or its symptoms, and / or therapeutic, in terms of partially or completely curing a disease and / or side effects resulting from the disease. As used herein, "treatment" encompasses any treatment of a disease or condition in a mammal, including (a) preventing the onset of the disease or condition in a subject susceptible to, but not yet diagnosed with, the disease or condition; (b) suppressing the disease or condition, i.e., arresting its development; and / or (c) alleviating the disease, i.e., causing regression of the disease. Therapeutic agents may be administered before, during, or after the onset of a disease or disorder. Treatment of an ongoing disease (wherein treatment stabilizes or suppresses undesirable clinical symptoms in a patient) is particularly advantageous. Such treatment is desirably performed before complete loss of function of the affected tissue. The therapeutic agent is desirably administered during, and in some cases after, the symptomatic stage of the disease.

[0021] The terms "individual," "subject," "host," and "patient" are used interchangeably herein to refer to any mammalian subject for whom diagnosis, treatment, or therapy is desired. Mammals include, for example, humans, non-human primates, rodents (e.g., rats, mice), lagomorphs (e.g., rabbits), and ungulates (e.g., cows, sheep, pigs, horses, goats, etc.).

[0022] Unless otherwise specified, the term "substantially" is intended to encompass both "completely" and "largely but not completely." For example, an Ig Fc that "does not substantially induce cell lysis" means an Ig Fc that induces no cell lysis or that induces very little cell lysis.

[0023] As used herein, the term "about" when used in connection with a quantity indicates that the quantity may vary by 10% of the stated amount. For example, "about 100" means an amount of 90 to 110. When "about" is used in connection with a range, "about" when used in connection with the lower limit of the range means that the lower limit includes an amount 10% lower than the lower limit of the range, and "about" when used in connection with the upper limit of the range means that the upper limit includes an amount 10% higher than the upper limit of the range. For example, about 100 to about 1000 means that the range spans 90 to 1100.

[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 the α1, α2, and α3 domains.

[0025] Before the present disclosure is further described, it is to be understood that this disclosure is not limited to particular embodiments described, as such may, of course, vary. It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting, as the scope of the present disclosure will be limited only by the appended claims.

[0026] When a range of numerical values ​​is provided, it is understood that each intervening value (to one-tenth of the unit of the lower limit, unless the context clearly dictates otherwise) between the upper and lower limits of that range, and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these narrower ranges may independently be included in the narrower range and are also encompassed within the disclosure, subject to any specifically excluded value in the stated range. When an stated range includes one or both of those upper and lower limits, ranges excluding either or both of those included limits are also encompassed within the disclosure.

[0027] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of this disclosure, the preferred methods and materials are described below. All publications mentioned herein are incorporated by reference to disclose and describe the methods and / or materials in connection with the content in which the publications are cited.

[0028] It should be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, a reference to a "T cell modulating polypeptide" includes a plurality of such polypeptides; a reference to an "immunomodulating polypeptide" includes a reference to one or more immunomodulating polypeptides and equivalents thereof known to those skilled in the art; and so forth. It should be further noted that the claims may be drafted to exclude any element. Accordingly, this statement intends that the use of exclusive terminology, such as "solely," "only," or the use of a "negative" limitation in connection with the recitation of claim elements, serves as antecedent.

[0029] It is to be understood that certain features of the present disclosure that are, for clarity, described in the context of individual embodiments, may also be provided in combination within a single embodiment. Conversely, various features of the present disclosure that are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination. All combinations of the embodiments belonging to the present disclosure are expressly embraced by the present disclosure and are disclosed herein as if all combinations were individually and specifically disclosed. In addition, all subcombinations of the various embodiments and elements thereof are also expressly embraced by the present invention and are disclosed herein as if all such subcombinations were individually and specifically 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 publication. Further, the dates of publication provided may be different from the actual publication dates, which may need to be independently confirmed.

[0031] Detailed Description The present disclosure provides T cell modulating polypeptides, including immunomodulating polypeptides and including epitope-presenting peptides. TMPs are useful for modulating T cell activation and for modulating immune responses in individuals.

[0032] T-cell modulating polypeptide The present disclosure provides a T cell modulatory polypeptide (TMP) comprising a) a first polypeptide and b) a second polypeptide, wherein the TMP comprises: 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 immunomodulatory polypeptides, and optionally, an immunoglobulin (Ig) Fc polypeptide or a non-Ig scaffold. A TMP comprising a first polypeptide and a second polypeptide may also be referred to herein as a "split-chain TMP" or "heterodimeric TMP." As described herein, the first and second polypeptides of such a TMP are typically covalently linked to each other by one or more disulfide bonds, which may confer high stability and / or high expression to the TMP.

[0033] However, in some instances, the TMP of the present disclosure may comprise multiple different polypeptides linked together to form a single polypeptide chain. Such a single-chain TMP may comprise, for example, i) a KRAS peptide that presents an epitope to a T cell receptor (TCR) upon binding to a major histocompatibility complex (MHC) polypeptide, ii) first and second MHC polypeptides, and iii) one or more immunomodulatory polypeptides, and optionally, an immunoglobulin (Ig) Fc polypeptide or a non-Ig scaffold. As described below, heterodimeric TMPs and single-chain TMPs can self-assemble into dimers, for example, when the TMP comprises an Ig Fc, e.g., an IgG1 Fc. In such cases, disulfide bonds spontaneously form to link the two TMPs.

[0034] As used herein, the term "KRAS peptide" refers to a peptide having a length of at least 4 amino acids, e.g., 4 amino acids to about 25 amino acids (e.g., a length of 4 amino acids (aa), 5 aa, 6 aa, 7 aa, 8 aa, 9 aa, 10 aa, 11 aa, 12 aa, 13 aa, 14 aa, 15 aa, 16 aa, 17 aa, 18 aa, 19 aa, 20 aa, 21 aa, 22 aa, 23 aa, 24 aa, or 25 aa, including within the ranges of 4 to 20 amino acids, 6 to 18 amino acids, 8 to 15 amino acids, 8 to 12 amino acids, 5 to 10 amino acids, 10 to 20 amino acids, and 15 to 25 amino acids), which presents a KRAS epitope to a TCR when the KRAS peptide binds to an MHC complex. As used herein, the term "KRAS epitope" refers to an epitope found 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 found in any of the following: (i) a KRAS4A polypeptide, (ii) a KRAS4B polypeptide, and (iii) variants of (i) and (ii) that occur in human cancers, including, for example, mutant forms. As used herein, the term "KRAS polypeptide" refers to a polypeptide having an amino acid sequence found in all or a portion of a KRAS protein, or, if specified, a polypeptide having, for example, 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 found in all or a portion of a KRAS protein or a variant, including, for example, a mutant form, that occurs in human cancers.

[0035] 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 mutated, KRAS fails to regulate cell proliferation, which can lead to cancer.

[0036] A wild-type (normal, non-cancer associated) KRAS polypeptide has the following amino acid sequence: TIFF0007756072000001.tif23160 (SEQ ID NO: 1).

[0037] A wild-type (normal, non-cancer associated) KRAS polypeptide has the following amino acid sequence: TIFF0007756072000002.tif24159 (SEQ ID NO: 2).

[0038] 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, for example, 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, the amino acids G12, G13, T35, I36, E49, Q61, K127, and A156 are bold and underlined, and one or more substitutions of these residues may be present in cancer-associated KRAS polypeptides. Cancer-associated KRAS polypeptides can include one or more of the following: 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 or I36M), v) substitution of E49 (e.g., E49K), vi) substitution of Q61 (e.g., Q61H, Q61R, Q61P, Q61E, Q61K, Q61L, or Q61K), vii) substitution of K117 (e.g., K117N), and viii) substitution of A146 (e.g., A146T or A146V), where the amino acid numbers are as set forth in SEQ ID NO: 1 and SEQ ID NO: 2. See, e.g., US2019 / 0194192.

[0039] For example, a cancer-associated mutant KRAS polypeptide may have one or more amino acid substitutions compared to the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:2. In some examples, a cancer-associated mutant KRAS polypeptide has only a single amino acid substitution compared to the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:2. In some examples, a cancer-associated mutant KRAS polypeptide has only two amino acid substitutions compared to the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:2. In some examples, a cancer-associated mutant KRAS polypeptide has only three amino acid substitutions compared to the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:2. In some examples, a cancer-associated mutant KRAS polypeptide has only four amino acid substitutions compared to the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:2. In some examples, a cancer-associated mutant KRAS polypeptide has only five amino acid substitutions compared to the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:2.

[0040] For example, KRAS(G12D) (a 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) (a 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) (a 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:0.1101 / cshperspect.a031435. As another example, KRAS(G12C) (a 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, for example, cholangiocarcinoma, gallbladder cancer, adenocarcinoma, rectal adenocarcinoma, endometrial cancer, hematopoietic tumors, and lung cancer. See, e.g., Prior et al. (20120 Cancer Res. 72:2457).

[0041] As another example, a cancer-associated mutant 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 KRAS polypeptide can have an amino acid substitution such as Q61H, Q61L, Q61E, Q61R, or Q61K.

[0042] As described above, in some examples, the present disclosure provides a TMP comprising a heterodimer, the heterodimer comprising a) a first polypeptide comprising a first MHC polypeptide, and b) a second polypeptide comprising a second MHC polypeptide, wherein the first polypeptide or the second polypeptide comprises a KRAS peptide (e.g., a KRAS peptide comprising a cancer-associated mutation, where the KRAS peptide has a length of at least 4 amino acids (e.g., from 4 amino acids to about 25 amino acids), and the KRAS peptide presents an epitope to a T cell receptor upon binding to an MHC complex), and the first polypeptide and / or the second polypeptide comprise one or more immunomodulatory polypeptides, which may be the same as or different from each other, and optionally an Ig Fc polypeptide or a non-Ig scaffold. In some examples, at least one of the one or more immunomodulatory polypeptides is a variant immunomodulatory polypeptide that exhibits reduced affinity for a cognate costimulatory polypeptide compared to the affinity of a corresponding wild-type immunomodulatory polypeptide for the cognate costimulatory polypeptide.

[0043] The KRAS peptide / MHC complex present in the TMPs 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). Generally speaking, the TMPs of the present disclosure bind to a T cell having a cognate costimulatory polypeptide and a TCR that binds to the KRAS peptide / MHC complex of the TMP with a higher affinity, e.g., 25% higher, than the affinity with which the same TMP binds to a second T cell having the same cognate costimulatory polypeptide but a TCR that does not substantially bind to the KRAS peptide / MHC complex; e.g., the KRAS peptide / MHC complex binds to a T cell with an affinity of at least 1 ... -7 Binds to TCR with an affinity less than M

[0044] The present disclosure provides a TMP, which is a heterodimer comprising a) a first polypeptide comprising a first MHC polypeptide, and b) a second polypeptide comprising a second MHC polypeptide, wherein the first polypeptide or the second polypeptide comprises a KRAS peptide, wherein the first polypeptide and / or the second polypeptide comprise one or more immunomodulatory polypeptides which may be the same or different, wherein at least one of the one or more immunomodulatory polypeptides may be a wild-type immunomodulatory polypeptide or a variant of a wild-type immunomodulatory polypeptide, wherein the variant immunomodulatory polypeptide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid substitutions compared to the amino acid sequence of the corresponding wild-type immunomodulatory polypeptide, and wherein the first polypeptide or the second polypeptide optionally comprises an Ig Fc polypeptide or a non-Ig scaffold. In some examples, at least one of the one or more immunomodulatory domains is a variant immunomodulatory polypeptide that exhibits reduced affinity for a cognate costimulatory polypeptide compared to the affinity of the corresponding wild-type immunomodulatory polypeptide for its cognate costimulatory polypeptide, e.g., a ratio of (i) the binding affinity of the wild-type immunomodulatory polypeptide for its cognate costimulatory polypeptide to (ii) the binding affinity of the variant of the wild-type immunomodulatory polypeptide for its cognate costimulatory polypeptide of 1.5:1-10, as measured by biolayer interferometry. 6 :1, wherein the variant immunomodulatory polypeptide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid substitutions compared to the amino acid sequence of the corresponding wild-type immunomodulatory polypeptide, and the first polypeptide or the second polypeptide optionally comprises an Ig Fc polypeptide or a non-Ig scaffold.

[0045] In some examples, a heterodimeric TMP of the present disclosure may comprise: a) a first polypeptide comprising, in N-terminal to C-terminal order: i) a KRAS peptide, ii) a first MHC polypeptide, and b) a second polypeptide comprising, in N-terminal to C-terminal order: i) a second MHC polypeptide, and ii) optionally an immunoglobulin (Ig) Fc polypeptide or a non-Ig scaffold, wherein the TMP comprises one or more immunomodulatory domains, which may be the same or different, and at least one of the one or more immunomodulatory domains is located at the C-terminus of the first polypeptide: A) the first polypeptide; B) the second polypeptide; C) the second polypeptide; at the N-terminus of the second polypeptide, C) at the C-terminus of the second polypeptide, or D) at the C-terminus of the first polypeptide and the N-terminus of the second polypeptide, and at least one of the one or more immunomodulatory domains can be a wild-type immunomodulatory polypeptide or a variant of a wild-type immunomodulatory polypeptide, wherein the variant immunomodulatory polypeptide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid substitutions compared to the amino acid sequence of the corresponding wild-type immunomodulatory polypeptide.

[0046] The present disclosure provides a TMP comprising as a basic scaffold: a) a first polypeptide comprising, in N-terminal to C-terminal order: i) a KRAS peptide, ii) a first MHC polypeptide, and b) a second polypeptide comprising, in N-terminal to C-terminal order: i) a second MHC polypeptide, and ii) optionally an Ig Fc polypeptide or a non-Ig scaffold. The TMP of the present disclosure further comprises one or more immunomodulatory polypeptides, at least one of which is A) at the C-terminus of the first polypeptide, B) at the N-terminus of the second polypeptide, C) at the C-terminus of the second polypeptide, or D) at the C-terminus of the first polypeptide and the N-terminus of the second polypeptide. In some examples, at least one of the one or more immunomodulatory polypeptides is a variant immunomodulatory polypeptide that exhibits reduced affinity for a cognate costimulatory polypeptide compared to the affinity of a corresponding wild-type immunomodulatory polypeptide for the cognate costimulatory polypeptide.

[0047] The KRAS peptide / MHC complex present in the TMP of the present disclosure binds to the 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).

[0048] As noted above, in some examples, the TMPs of the present disclosure comprise a single polypeptide chain. Such single-chain TMPs of the present disclosure comprise: i) a first MHC polypeptide, ii) a second MHC polypeptide, iii) a KRAS peptide that presents an epitope to a TCR upon binding to the MHC polypeptide, and iv) one or more immunomodulatory polypeptides, and optionally, an Ig Fc polypeptide or a non-Ig scaffold.

[0049] In some examples, a single-chain TMP of the disclosure 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 immunomodulatory polypeptides, and v) an Ig Fc polypeptide. In some examples, a single-chain TMP of the disclosure 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 immunomodulatory polypeptides, and v) an Ig Fc polypeptide. This arrangement of components is referred to as MOD position 2 in Figure 18.

[0050] In some examples, a single-chain TMP of the disclosure 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 Ig Fc polypeptide, and v) one or more immunomodulatory polypeptides. In some examples, a single-chain TMP of the disclosure 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 Ig Fc polypeptide, and v) one or more immunomodulatory polypeptides. This arrangement of components is referred to as MOD position 3 in Figure 18.

[0051] In some examples, a single-chain TMP of the present disclosure comprises, in N-terminal to C-terminal order: i) one or more immunomodulatory polypeptides, ii) a KRAS peptide, iii) a first MHC polypeptide, iv) a second MHC polypeptide, and v) an Ig Fc polypeptide. In some examples, a single-chain TMP of the present disclosure comprises, in N-terminal to C-terminal order: i) one or more immunomodulatory polypeptides, ii) a KRAS peptide, iii) a first Class I MHC polypeptide, iv) a second Class I MHC polypeptide, and v) an Ig Fc polypeptide. In some examples, a single-chain TMP of the present disclosure comprises, in N-terminal to C-terminal order: i) one or more immunomodulatory polypeptides, ii) a KRAS peptide, iii) a β2M polypeptide, iv) a Class I MHC heavy chain polypeptide, and v) an Ig Fc polypeptide. This arrangement of components is referred to as MOD position 4 in Figure 18.

[0052] In some instances, the KRAS peptide / MHC complex present in the TMP of the present disclosure is about 10 -4 M ~ approx. 5×10 -4 M, about 5 x 10 -4 M ~ about 10 -5 M, about 10 -5 M~5×10 -5 M, about 5 x 10 -5 M~10 -6 M, about 10 -6 M ~ approx. 5×10 -6 M, about 5 x 10 -6 M ~ about 10 -7 M, about 10 -7 M ~ approx. 5×10 -7 M, about 5 x 10 -7 M ~ about 10 -8 M, or about 10 -8 M ~ about 10 -9Expressed another way, in some examples, the KRAS peptide / MHC complex present in the TMP of the present disclosure binds to the TCR on a T cell with an affinity of about 1 nM to about 5 nM, about 5 nM to about 10 nM, about 10 nM to about 50 nM, about 50 nM to about 100 nM, about 0.1 μM to about 0.5 μM, about 0.5 μM to about 1 μM, about 1 μM to about 5 μM, about 5 μM to about 10 μM, about 10 μM to about 25 μM, about 25 μM to about 50 μM, about 50 μM to about 75 μM, or about 75 μM to about 100 μM.

[0053] In some examples, an immunomodulatory polypeptide present in a TMP of the present disclosure binds to its cognate costimulatory polypeptide 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 immunomodulatory polypeptide for its cognate costimulatory polypeptide.

[0054] In some examples, variant immunomodulatory polypeptides present in TMPs of the disclosure have a binding affinity for their cognate costimulatory polypeptides of 1 nM to 100 nM, or 100 nM to 100 μM. For example, in some examples, variant immunomodulatory polypeptides present in TMPs of the disclosure have a binding affinity for their cognate costimulatory polypeptides of about 100 nM to 150 nM, about 150 nM to about 200 nM, about 200 nM to about 250 nM, about 250 nM to about 300 nM, about 300 nM to about 350 nM, about 350 nM to about 400 nM, about 400 nM to about 500 nM, about 500 nM to about 60 In some instances, a variant immunomodulatory polypeptide present in a TMP of the present disclosure has a binding affinity of about 1 nM to about 5 nM, about 5 nM to about 10 nM, about 10 nM to about 50 nM, about 50 nM to about 75 nM, or about 75 nM to about 100 nM to its cognate costimulatory polypeptide.

[0055] The combination of the reduced affinity of an immunomodulatory polypeptide for its cognate costimulatory polypeptide and the affinity of a KRAS peptide for a TCR results in improved selectivity of the TMPs of the present disclosure, e.g., a TMP of the present disclosure selectively binds to a first T cell that presents both i) a TCR specific for a KRAS peptide present in the TMP and ii) a costimulatory polypeptide that binds to the immunomodulatory polypeptide present in the TMP, compared to binding to a second T cell that presents i) a TCR specific for an epitope other than the epitope present in the TMP and ii) a costimulatory polypeptide that binds to the immunomodulatory polypeptide present in the TMP. For example, a TMP of the disclosure binds to a first T cell with an affinity that is at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 2-fold, at least 2.5-fold, at least 5-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 50-fold, at least 100-fold, or more than 100-fold higher than the affinity with which a TMP of the disclosure binds to a second T cell.

[0056] In some examples, when administered to an individual in need thereof, the TMP of the present disclosure induces both epitope-specific and non-epitope-specific T cell responses. In other words, in some examples, when administered to an individual in need thereof, the TMP of the present disclosure induces an epitope-specific T cell response by modulating the activation of a first T cell that presents both i) a TCR specific for a KRAS epitope present in the TMP and ii) a costimulatory polypeptide that binds to an immunomodulatory polypeptide present in the TMP, and induces an epitope-non-specific T cell response by modulating the activation of a second T cell that presents i) a TCR specific for an epitope other than the KRAS epitope present in the TMP and ii) a costimulatory polypeptide that binds to an immunomodulatory polypeptide present in the TMP. The ratio of epitope-specific T cell responses to epitope-nonspecific T cell responses is at least 2:1, at least 5:1, at least 10:1, at least 15:1, at least 20:1, at least 25:1, at least 50:1, or at least 100:1. The ratio of epitope-specific T cell responses to epitope-nonspecific T cell responses is about 2:1 to about 5:1, about 5:1 to about 10:1, about 10:1 to about 15:1, about 15:1 to about 20:1, about 20:1 to about 25:1, about 25:1 to about 50:1, or about 50:1 to about 100:1, or greater than 100:1. Examples of "modulating activation" of T cells include: i) cytotoxicity (e.g., CD8 + ) activating T cells; ii) cytotoxic (e.g., CD8 + ) inducing T cell cytotoxic activity; iii) 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; and v) suppressing activation of autoreactive T cells.

[0057] The combination of the reduced affinity of an immunomodulatory polypeptide for its cognate costimulatory polypeptide and the affinity of the KRAS epitope for the TCR results in improved selectivity for the TMPs of the present disclosure. Thus, for example, a TMP of the present disclosure binds to a first T cell that presents both i) a TCR specific for a KRAS epitope present in the TMP and ii) a costimulatory polypeptide that binds to the immunomodulatory polypeptide present in the TMP with a higher avidity than the TMP of the present disclosure binds to a second T cell that presents i) a TCR specific for an epitope other than the epitope present in the TMP and ii) a costimulatory polypeptide that binds to the immunomodulatory polypeptide present in the TMP.

[0058] The binding affinity between an immunomodulatory polypeptide and its cognate costimulatory polypeptide can be measured by biolayer interferometry (BLI) using a purified immunomodulatory polypeptide and a purified cognate costimulatory polypeptide. The binding affinity between TMP and its cognate costimulatory polypeptide can be measured by BLI using purified TMP and a cognate costimulatory polypeptide. 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.

[0059] BLI assays can be performed using an Octet RED 96 (Pal ForteBio) instrument or similar instrument as follows: TMP (e.g., a TMP of the present disclosure, a control TMP (the control TMP contains a wild-type immunomodulatory polypeptide)) is immobilized on an insoluble support ("biosensor"). The immobilized TMP is the "target." Immobilization can be performed by immobilizing a capture antibody on the insoluble support, which immobilizes the TMP. For example, immobilization can be performed by immobilizing an anti-Fc (e.g., anti-human IgG Fc) antibody on the insoluble support, which binds to and immobilizes the TMP (the TMP contains an Ig Fc polypeptide). Several different concentrations of costimulatory polypeptide are added to the immobilized TMP, and the instrument's response is recorded. The assay is performed in a liquid medium containing 25 mM HEPES pH 6.8, 5% poly(ethylene glycol) 6000, 50 mM KCl, 0.1% bovine serum albumin, and 0.02% Tween 20 non-ionic surfactant. Binding of costimulatory polypeptides to immobilized TMP is performed at 30°C. As a positive control for binding affinity, an anti-MHC class I monoclonal antibody can be used. For example, a K of 7 nM D Anti-HLA class I monoclonal antibody W6 / 32 (American Type Culture Collection No. HB-95, Parham et al. (1979) J. Immunol. 123:342) with α-heptane-1-phosphate dehydrogenase (α-heptane) can be used. A standard curve can be generated using serial dilutions of the anti-MHC class I monoclonal antibody. The costimulatory polypeptide, i.e., anti-MHC class I mAb, is the "analyte." BLI analyzes the interference pattern of white light reflected from two surfaces: i) the immobilized polypeptide ("target") and ii) an internal reference layer. Changes in the number of molecules ("analytes," e.g., costimulatory polypeptides, anti-HLA antibodies) bound to the biosensor chip result in a shift in the interference pattern, which can be measured in real time. Two kinetic terms describing the affinity of the target / analyte interaction are the binding rate constant (k a ) and dissociation constant (k d ) The ratio of these two terms (k d / a ) to determine the affinity constant (K D ) is required.

[0060] BLI assays are performed using multiwell plates. To run the assay, use Octet Data Acquisition software to define the plate layout, define the assay steps, and assign biosensors. Hydrate the biosensor assembly. Equilibrate the hydrated biosensor assembly and assay plate on the Octet instrument for 10 minutes. Once data is acquired, load the acquired data into Octet Data Analysis software. Process the data in the Processing window by specifying the methods for reference subtraction, y-axis alignment, inter-step correction, and Savitzky-Golay filtering. Analyze the data in the Analysis window by specifying the analysis steps (association and dissociation) and selecting the curve fitting model (1:1), fitting method (global), and window of interest (seconds). Assess the quality of the fit. If the K of each data record is within a 3-fold range, D The values ​​(analyte concentrations) can be averaged. D The error value must be within one order of magnitude of the affinity constant value, R 2 The value should be greater than 0.95. See, e.g., Abdiche et al. (2008) J. Anal. Biochem. 377:209.

[0061] Unless otherwise specified herein, the affinity of a TMP of the present disclosure for a cognate costimulatory polypeptide, or the affinity of a control TMP (wherein the control TMP comprises a wild-type immunomodulatory polypeptide) for a cognate costimulatory polypeptide, is measured using BLI as described above.

[0062] In some examples, the ratio of i) the binding affinity of a control TMP (wherein the control comprises a wild-type immunomodulatory polypeptide) to its cognate costimulatory polypeptide and ii) the binding affinity of a TMP of the disclosure comprising a variant form of a wild-type immunomodulatory polypeptide to its cognate costimulatory polypeptide, as measured by BLI (described above), is at least 1.5:1, at least 2:1, at least 5:1, at least 10:1, at least 15:1, at least 20:1, at least 25:1, at least 50:1, at least 100:1, at least 500:1, at least 10 2 :1, at least 5 × 10 2 :1, at least 10 3 :1, at least 5 × 10 3 :1, at least 10 4 :1, at least 10 5 :1, or at least 10 6 In some examples, the ratio of i) the binding affinity of a control TMP (wherein the control comprises a wild-type immunomodulatory polypeptide) to its cognate costimulatory polypeptide to ii) the binding affinity of a TMP of the disclosure comprising a variant of a wild-type immunomodulatory polypeptide to its cognate costimulatory polypeptide is 1.5:1 to 10, as measured by BLI. 6 :1, for example, 1.5:1 to 10:1, 10:1 to 50:1, 50:1 to 10 2 :1, 10 2 :1~10 3 :1, 10 3 :1~10 4 :1, 10 4 :1~10 5 :1, or 10 5 :1~10 6 :1 range.

[0063] In one example, the immunomodulatory polypeptides include a control TMP that comprises a wild-type IL-2 polypeptide and a TMP of the disclosure that comprises a variant IL-2 polypeptide (comprising 1 to 10 amino acid substitutions compared to the amino acid sequence of the wild-type IL-2 polypeptide), e.g., an IL-2 variant comprising H16A and F42A substitutions as described herein, and the ratio of i) the binding affinity of the control TMP to the IL-2 receptor (i.e., cognate costimulatory polypeptide) to ii) the binding affinity of the TMP of the disclosure to the IL-2 receptor is at least 1.5:1, at least 2:1, at least 5:1, at least 10:1, at least 15:1, at least 20:1, at least 25:1, at least 50:1, at least 100:1, at least 500:1, at least 10 2 :1, at least 5 × 10 2 :1, at least 10 3 :1, at least 5 × 10 3 :1, at least 10 4 :1, at least 10 5 :1, or at least 10 6 In some examples, the control TMP comprises a wild-type IL-2 polypeptide and the TMP of the disclosure comprises a variant IL-2 polypeptide (comprising 1 to 10 amino acid substitutions compared to the amino acid sequence of the wild-type IL-2 polypeptide) as the immunomodulatory polypeptide, and the ratio of i) the binding affinity of the control TMP to the IL-2 receptor (i.e., cognate costimulatory polypeptide) to ii) the binding affinity of the TMP of the disclosure to the IL-2 receptor is 1.5:1 to 10, as measured by BLI. 6 :1, for example, 1.5:1 to 10:1, 10:1 to 50:1, 50:1 to 10 2 :1, 10 2 :1~10 3 :1, 10 3 :1~10 4 :1, 10 4 :1~10 5 :1 or 10 5 :1~10 6 :1 range.

[0064] In another example, the control TMP comprises a wild-type CD80 polypeptide and the TMP of the disclosure comprises a variant CD80 polypeptide (comprising 1 to 10 amino acid substitutions compared to the amino acid sequence of the wild-type CD80 polypeptide) as the immunomodulatory polypeptide, and the ratio of i) the binding affinity of the control TMP to a CTLA4 polypeptide (i.e., a cognate costimulatory polypeptide) to ii) the binding affinity of the TMP of the disclosure to a CTLA4 polypeptide is at least 1.5:1, at least 2:1, at least 5:1, at least 10:1, at least 15:1, at least 20:1, at least 25:1, at least 50:1, at least 100:1, at least 500:1, at least 10 2 :1, at least 5 × 10 2 :1, at least 10 3 :1, at least 5 × 10 3 :1, at least 10 4 :1, at least 10 5 :1, or at least 10 6 :1.

[0065] In another example, the control TMP comprises a wild-type CD80 polypeptide and the TMP of the disclosure comprises a variant CD80 polypeptide (comprising 1 to 10 amino acid substitutions compared to the amino acid sequence of the wild-type CD80 polypeptide) as the immunomodulatory polypeptide, and the ratio of i) the binding affinity of the control TMP to a CD28 polypeptide (i.e., a cognate costimulatory polypeptide) to ii) the binding affinity of the TMP of the disclosure to a CD28 polypeptide is at least 1.5:1, at least 2:1, at least 5:1, at least 10:1, at least 15:1, at least 20:1, at least 25:1, at least 50:1, at least 100:1, at least 500:1, at least 10 2 :1, at least 5 × 10 2 :1, at least 10 3 :1, at least 5 × 10 3 :1, at least 10 4 :1, at least 10 5 :1, or at least 10 6 :1.

[0066] In another example, the immunomodulatory polypeptides include a control TMP that comprises a wild-type 4-1BBL polypeptide and a TMP of the disclosure that comprises a variant 4-1BBL polypeptide (comprising 1 to 10 amino acid substitutions compared to the amino acid sequence of the wild-type 4-1BBL polypeptide), and the ratio of i) the binding affinity of the control TMP to a 4-1BB polypeptide (i.e., a cognate costimulatory polypeptide) to ii) the binding affinity of the TMP of the disclosure to a 4-1BB polypeptide is at least 1.5:1, at least 2:1, at least 5:1, at least 10:1, at least 15:1, at least 20:1, at least 25:1, at least 50:1, at least 100:1, at least 500:1, at least 10 2 :1, at least 5 × 10 2 :1, at least 10 3 :1, at least 5 × 10 3 :1, at least 10 4 :1, at least 10 5 :1, or at least 10 6 :1.

[0067] In another example, the control TMP comprises a wild-type CD86 polypeptide and the TMP of the disclosure comprises a variant CD86 polypeptide (comprising 1 to 10 amino acid substitutions compared to the amino acid sequence of the wild-type CD86 polypeptide) as the immunomodulatory polypeptide, and the ratio of i) the binding affinity of the control TMP to a CD28 polypeptide (i.e., a cognate costimulatory polypeptide) to ii) the binding affinity of the TMP of the disclosure to a CD28 polypeptide is at least 1.5:1, at least 2:1, at least 5:1, at least 10:1, at least 15:1, at least 20:1, at least 25:1, at least 50:1, at least 100:1, at least 500:1, at least 10 2 :1, at least 5 × 10 2 :1, at least 10 3 :1, at least 5 × 10 3 :1, at least 10 4 :1, at least 105 :1, or at least 10 6 :1.

[0068] The binding affinity of a TMP of the present disclosure for a target T cell can be measured by the following method: A) contacting a TMP of the present disclosure with a target T cell that expresses on its surface i) a cognate costimulatory polypeptide that binds to the parent wild-type immunomodulatory polypeptide and ii) a T cell receptor that substantially binds to the epitope (the TMP comprises an epitope tag, such that the TMP binds to the target T cell); B) contacting the target T cell-bound TMP with a fluorescently labeled binding agent (e.g., a fluorescently labeled antibody) that binds to the epitope tag to generate a TMP / target T cell / binding agent complex; and C) measuring the mean fluorescence intensity (MFI) of the TMP / target T cell / binding agent complex using flow cytometry. The epitope tag can be, for example, a FLAG tag, a hemagglutinin tag, a c-myc tag, a poly(histidine) tag, etc. The MFI measured over a range of TMP concentrations provides an indication of affinity. The MFI measured over a range of TMP concentrations provides the half maximal effective concentration (EC) of the TMP. 50 In some examples, the EC of the TMP of the present disclosure on the target T cells is 50 is in the nM range and is the EC of TMP on control T cells (control T cells express on their surface i) a cognate costimulatory polypeptide that binds to the parent wild-type immunomodulatory polypeptide and ii) a T cell receptor that does not bind to an epitope present in the TMP). 50 In some examples, the EC 50 and EC of TMP on target T cells. 50 The ratio of is at least 1.5:1, at least 2:1, at least 5:1, at least 10:1, at least 15:1, at least 20:1, at least 25:1, at least 50:1, at least 100:1, at least 500:1, at least 10 2 :1, at least 5 × 10 2 :1, at least 10 3 :1, at least 5 × 10 3 :1, at least 10 4:1, at least 10 5 :1, or at least 10 6 :1. EC of the TMP of the present disclosure relative to control T cells 50 and EC of TMP on target T cells. 50 The ratio is a manifestation of the selectivity of TMP.

[0069] In some examples, when measured as described in the immediately preceding paragraph, a TMP of the present disclosure comprising a low-affinity MOD exhibits selective binding to a target T cell compared to binding of the TMP to a control T cell comprising i) a cognate costimulatory polypeptide that binds to the parent wild-type MOD and ii) a T cell receptor that substantially binds to an epitope other than the epitope present in the TMP.

[0070] KRAS peptides As noted above, the TMPs of the present disclosure typically comprise a KRAS peptide that is at least about four amino acids in length and that presents a KRAS epitope to a T cell when in an MHC / peptide complex (eg, an HLA / peptide complex).

[0071] The KRAS peptides present in the TMPs of the present disclosure can be at least 4 amino acids in length, e.g., 4 amino acids to about 25 amino acids in length (e.g., 4 amino acids (aa), 5 aa, 6 aa, 7 aa, 8 aa, 9 aa, 10 aa, 11 aa, 12 aa, 13 aa, 14 aa, 15 aa, 16 aa, 17 aa, 18 aa, 19 aa, 20 aa, 21 aa, 22 aa, 23 aa, 24 aa, or 25 aa, including within the ranges of 9-10 amino acids, 4-20 amino acids, 6-18 amino acids, 8-15 amino acids, 8-12 amino acids, 5-10 amino acids, 10-20 amino acids, and 15-25 amino acids in length). In some examples, the KRAS peptide is 9 or 10 amino acids in length.

[0072] The KRAS epitope present in the TMP of the present disclosure is a peptide that is specifically bound by T cells, i.e., the epitope is specifically bound by epitope-specific T cells, i.e., T cells with a TCR specific for the KRAS epitope. Epitope-specific T cells bind to an epitope with a reference amino acid sequence, but do not substantially bind to an epitope different from the reference amino acid sequence. For example, epitope-specific T cells bind to an epitope with a reference amino acid sequence, but do not substantially bind to an epitope different from the reference amino acid sequence, even if they do bind to an epitope different from the reference amino acid sequence. -6 Under M, 10 -5 Less than M or 10 -4 Epitope-specific T cells bind to a specific epitope with an affinity of at least 10 -7 M, at least 10 -8 M, at least 10 -9 M, or at least 10 -10 It can bind with an affinity of M.

[0073] In some examples, a suitable KRAS peptide is at least 4 amino acids in length, e.g., 4 amino acids to about 25 amino acids (e.g., 9-10 amino acids, 4-20 amino acids, 6-18 amino acids, 9-10 amino acids, 4-20 amino acids, 6-18 amino acids, 9-10 amino acids, 9-20 amino acids, 9-20 amino acids, 9-20 amino acids, 9-30 amino acids, 9-30 amino acids, 9-40 amino acids, 9-40 amino acids, 9-50 amino acids, 9-50 amino acids, 9-60 amino acids, 9-60 amino acids, 9-70 amino acids, 9-70 amino acids, 9-80 amino acids, 9-80 amino acids, 9-90 amino acids, 9-10 amino acids, 9-10 amino acids, 9-20 amino acids, 9-10 amino acids, 9-20 amino acids, 9-10 amino acids, 9-20 amino acids, 9-30 amino acids, 9-10 amino acids, 9-20 amino acids, 9-30 amino acids, 9-40 amino acids, 9-5 ... and KRAS polypeptides of 4 amino acids (aa), 5 aa, 6 aa, 7 aa, 8 aa, 9 aa, 10 aa, 11 aa, 12 aa, 13 aa, 14 aa, 15 aa, 16 aa, 17 aa, 18 aa, 19 aa, 20 aa, 21 aa, 22 aa, 23 aa, 24 aa, or 25 aa, including within the lengths of 8 to 15 amino acids, 8 to 12 amino acids, 5 to 10 amino acids, 10 to 20 amino acids, and 15 to 25 amino acids: (A) MTEYKLVVVG AGGVGKSALT IQLIQNHFVD EYDPTIEDSY RKQVVIDGET CLWDILDTAG QEEYSAMRDQ YMRTGEGFLC VFAINNTKSF EDIHHYREQI KRVKDSEDVP MVLVGNKCDL PSRTVDTKQA QDLARSYGIP FIETSAKTRQ GVDDAFYTLV REIRKHKEKM SKDGKKKKKK SKTKCVIM (SEQ ID NO: 1), wherein the KRAS polypeptide comprises one or more (e.g., 1, 2, 3, 4, or 5) amino acid substitutions compared to the amino acid sequence set forth in SEQ ID NO: 1, and the one or more amino acid substitutions may include substitutions associated with cancer, e.g., substitutions found in KRAS polypeptides of cancer cells; (B) MTEYKLVVVG AGGVGKSALT IQLIQNHFVD EYDPTIEDSY RKQVVIDGET CLLDILDTAG QEEYSAMRDQ YMRTGEGFLC VFAINNTKSF EDIHHYREQI KRVKDSEDVP MVLVGNKCDL PSRTVDTKQA QDLARSYGIP FIETSAKTRQ RVEDAFYTLV REIRQYRLKK ISKEEKTPGC VKIKKCIIM (SEQ ID NO: 2), wherein the KRAS polypeptide comprises one or more (e.g., 1, 2, 3, 4, or 5) amino acid substitutions compared to the amino acid sequence set forth in SEQ ID NO: 1, and the one or more amino acid substitutions may include substitutions associated with cancer, e.g., substitutions found in KRAS polypeptides of cancer cells; and (C)MTEY (X1) L ( GVDDAFYTLV REIRKHKEKM SKDGKKKKKK SKTKCVIM (SEQ ID NO: 89), in which 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, and one or both of X5 and X6 are not Cys.

[0074] Non-limiting examples of suitable KRAS peptides include peptides comprising a sequence selected from the group consisting of VVGADGVGK (SEQ ID NO: 176), VVGACGVGK (SEQ ID NO: 177), VVGAVGVGK (SEQ ID NO: 178), VVVGADGVGK (SEQ ID NO: 179), VVVGAVGVGK (SEQ ID NO: 180), VVVGACGVGK (SEQ ID NO: 181), VTGADGVGK (SEQ ID NO: 182), VTGAVGVGK (SEQ ID NO: 183), VTGACGVGK (SEQ ID NO: 184), VTVGADGVGK (SEQ ID NO: 185), VTVGAVGVGK (SEQ ID NO: 186), and VTVGACGVGK (SEQ ID NO: 187), wherein the KRAS peptide is 9 or 10 amino acids in length, or at least 9 or 10 amino acids in length.

[0075] Additional non-limiting examples of suitable KRAS peptides include peptides comprising a sequence selected from the group consisting of VVVGAGDVGK (SEQ ID NO: 188), VVGAGDVGK (SEQ ID NO: 189), VVVGARGVGK (SEQ ID NO: 190), and VVGARGVGK (SEQ ID NO: 191), wherein the KRAS peptide is 9 or 10 amino acids in length, or at least 9 or 10 amino acids in length.

[0076] Non-limiting examples of suitable KRAS peptides include peptides comprising a sequence selected from the group consisting of LVVVGADGV (SEQ ID NO: 192), LVVVGAVGV (SEQ ID NO: 193), LVVVGACGV (SEQ ID NO: 194), KLVVVGADGV (SEQ ID NO: 195), KLVVVGAVGV (SEQ ID NO: 196), KLVVVGACGV (SEQ ID NO: 197), LLVVGADGV (SEQ ID NO: 198), LLVVGAVGV (SEQ ID NO: 199), LLVVGACGV (SEQ ID NO: 200), FLVVVGADGV (SEQ ID NO: 201), FLVVVGAVGV (SEQ ID NO: 202), and FLVVVGACGV (SEQ ID NO: 203), wherein the KRAS peptide is 9 or 10 amino acids in length, or at least 9 or 10 amino acids in length.

[0077] Additional non-limiting examples of suitable KRAS peptides include peptides comprising a sequence selected from the group consisting of KLVVVGAGDV (SEQ ID NO: 204) and KLVVVGARGV (SEQ ID NO: 205), wherein the KRAS peptide is 9 or 10 amino acids in length, or at least 9 or 10 amino acids in length.

[0078] Additional non-limiting examples of suitable KRAS peptides include peptides comprising a sequence selected from the group consisting of GAGDVGKSAL (SEQ ID NO: 206), AGDVGKSAL (SEQ ID NO: 207), DVGKSALTI (SEQ ID NO: 208), GAVGVGKSAL (SEQ ID NO: 209), AVGVGKSAL (SEQ ID NO: 210), YKLVVVGAV (SEQ ID NO: 211), ARGVGKSAL (SEQ ID NO: 212), GARGVGKSAL (SEQ ID NO: 213), EYKLVVVGAR (SEQ ID NO: 214), RGVGKSALTI (SEQ ID NO: 215), LVVVGARGV (SEQ ID NO: 216), GADGVGKSAL (SEQ ID NO: 217), ACGVGKSAL (SEQ ID NO: 218), and GACGVGKSAL (SEQ ID NO: 219), wherein the KRAS peptide is 9 or 10 amino acids in length, or at least 9 or 10 amino acids in length.

[0079] In some examples, the TMPs of the present disclosure modulate the activity of T cells comprising a TCR specific for the G12V form of a KRAS polypeptide, as described above. In such cases, the KRAS peptide present in the TMP of the present disclosure can include, for example, one of the following amino acid sequences: VVGAVGVGK (SEQ ID NO: 178), VVVGAVGVGK (SEQ ID NO: 180), VGAVGVGKS (SEQ ID NO: 222), VGAVGVGKSA (SEQ ID NO: 223), AVGVGKSAL (SEQ ID NO: 210), AVGVGKSALT (SEQ ID NO: 225), GAVGVGKSAL (SEQ ID NO: 209), GAVGVGKSA (SEQ ID NO: 227), LVVVGAVGVG (SEQ ID NO: 228), LVVVGAVGV (SEQ ID NO: 193), KLVVVGAVGV (SEQ ID NO: 196), and KLVVVGAVG (SEQ ID NO: 231), and the KRAS peptide is 9 or 10 amino acids in length, or at least 9 or 10 amino acids in length.

[0080] In some examples, the KRAS peptide present in the TMP of the present disclosure presents an epitope specific for HLA-A, -B, -C, -E, -F, or -G alleles. In one embodiment, the KRAS peptide present in the TMP presents an epitope specific for HLA-A, -B, -C, -E, -F, or -G alleles. * 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 C * 0102, C * 0303, C * 0304, C * 0401, C * 0602, C * 0701, C * 702, C * 0801, and / or C * It presents an epitope restricted to 1502.

[0081] Non-limiting examples include VVGADGVGK (SEQ ID NO: 176), VVGACGVGK (SEQ ID NO: 177), VVGAVGVGK (SEQ ID NO: 178), VVVGADGVGK (SEQ ID NO: 179), VVVGAVGVGK (SEQ ID NO: 180), VVVGACGVGK (SEQ ID NO: 181), VTGADGVGK (SEQ ID NO: 182), VTGAVGVGK (SEQ ID NO: 183), VTGACGVGK (SEQ ID NO: 184), VTVGADGVGK (SEQ ID NO: 185), VTVGAVGVG KRAS peptides, including peptides selected from the group consisting of K (SEQ ID NO: 186), VTVGACGVGK (SEQ ID NO: 187), VVVGAGDVGK (SEQ ID NO: 188), VVGAGDVGK (SEQ ID NO: 189), VVVGARGVGK (SEQ ID NO: 190), and VVGARGVGK (SEQ ID NO: 191), wherein the KRAS peptides are 9 or 10 amino acids in length, or at least 9 or 10 amino acids in length, are conjugated to a β2M polypeptide and an A. * Such peptides also present epitopes when bound to an HLA complex containing a β2M polypeptide and an A heavy chain. * 6801 and HLA-A heavy chains.

[0082] As a non-limiting example, a KRAS peptide comprising a peptide selected from the group consisting of LVVVGADGV (SEQ ID NO: 192), LVVVGAVGV (SEQ ID NO: 193), LVVVGACGV (SEQ ID NO: 194), KLVVVGADGV (SEQ ID NO: 195), KLVVVGAVGV (SEQ ID NO: 196), KLVVVGACGV (SEQ ID NO: 197), LLVVGADGV (SEQ ID NO: 198), LLVVGAVGV (SEQ ID NO: 199), LLVVGACGV (SEQ ID NO: 200), FLVVVGADGV (SEQ ID NO: 201), FLVVVGAVGV (SEQ ID NO: 202), and FLVVVGACGV (SEQ ID NO: 203), wherein the KRAS peptide is 9 or 10 amino acids in length, or at least 9 or 10 amino acids in length, is a β2M polypeptide and an A polypeptide. * 0201 HLA-A heavy chain and presents the epitope when bound to an HLA complex containing the heavy chain.

[0083] As a further example, the following KRAS peptide can present an epitope when bound to an HLA complex containing a β2M polypeptide and an HLA-A heavy chain as follows: GAGDVGKSAL (SEQ ID NO: 206) (β2M polypeptide and B * 3801 HLA-A heavy chain), AGDVGKSAL (SEQ ID NO: 207) (β2M polypeptide and B0702, B * 3801 or B * 3901 HLA-A heavy chain), DVGKSALTI (SEQ ID NO: 208) (β2M polypeptide and B * 5101 HLA-A heavy chain), GAVGVGKSAL (SEQ ID NO: 209) (β2M polypeptide and B * 0702 or B * 3801 HLA-A heavy chain), AVGVGKSAL (SEQ ID NO: 210) (β2M polypeptide and B *0702 HLA-A heavy chain), YKLVVVGAV (SEQ ID NO: 211) (β2M polypeptide and A * 0203 or B * 3902 HLA-A heavy chain), ARGVGKSAL (SEQ ID NO: 212) (β2M polypeptide and B * 0702, B * 2705, or B * 3901 HLA-A heavy chain), GARGVGKSAL (SEQ ID NO: 213) (β2M polypeptide and B * 0702 HLA-A heavy chain), EYKLVVVGAR (SEQ ID NO: 214) (β2M polypeptide and A * 3101 HLA-A heavy chain), RGVGKSALTI (SEQ ID NO: 215) (β2M polypeptide and B * 0702 HLA-A heavy chain), LVVVGARGV (SEQ ID NO: 216) (β2M polypeptide and A * β2M polypeptide and B * 3801 HLA-A heavy chain), ACGVGKSAL (SEQ ID NO: 218) (β2M polypeptide and B * 0702 HLA-A heavy chain), and GACGVGKSAL (SEQ ID NO: 219) (β2M polypeptide and B * 3801 HLA-A heavy chain and can present epitopes when bound to an HLA complex containing the HLA-A heavy chain).

[0084] MHC polypeptides As noted above, the TMPs of the present disclosure include MHC polypeptides. For purposes of this disclosure, the term "major histocompatibility complex (MHC) polypeptide" is intended to include various types of MHC polypeptides, 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, and ungulates (e.g., horses, cattle, sheep, goats, etc.)). The term "MHC polypeptide" is intended to include class I MHC polypeptides (e.g., beta-2 microglobulin and MHC class I heavy chains).

[0085] In some examples, the first MHC polypeptide is an MHC class I β2M (β2M) polypeptide and the second MHC polypeptide is an MHC class I heavy chain (H chain) ("MHC-H"). In other examples, the first MHC polypeptide is an MHC class I heavy chain polypeptide and the second MHC polypeptide is a β2M polypeptide. In some examples, both the β2M chain and the MHC-H chain are human, i.e., the MHC-H chain is an HLA heavy chain or a variant thereof. Unless otherwise specified, the TMPs of the present disclosure do not include the membrane-anchoring domain (transmembrane region) of an MHC class I heavy chain or a portion of an MHC class I heavy chain sufficient to anchor the resulting TMP in a cell that expresses it (e.g., a eukaryotic cell such as a mammalian cell). In some examples, the MHC class I heavy chain present in the TMPs of the present disclosure does not include the signal peptide, transmembrane domain, or intracellular domain (cytoplasmic tail) associated with a naturally occurring MHC class I heavy chain. Thus, for example, in some examples, the MHC class I heavy chain present in a TMP of the present disclosure comprises only the α1, α2, and α3 domains of the MHC class I heavy chain. In some examples, the MHC class I heavy chain present in a TMP of the present disclosure has a length of about 270 amino acids (aa) to about 290 aa. In some examples, the MHC class I heavy chain present in a TMP of the present disclosure has a length of 270 aa, 271 aa, 272 aa, 273 aa, 274 aa, 275 aa, 276 aa, 277 aa, 278 aa, 279 aa, 280 aa, 281 aa, 282 aa, 283 aa, 284 aa, 285 aa, 286 aa, 287 aa, 288 aa, 289 aa, or 290 aa.

[0086] In some examples, the MHC polypeptide of the TMP is a human MHC polypeptide, which is also referred to as a "human leukocyte antigen" ("HLA") polypeptide. In some examples, 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.

[0087] MHC class I heavy chain In some examples, the MHC class I heavy chain polypeptide present in a TMP of the present disclosure comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to all or a portion (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 7-13. In some examples, the MHC class I heavy chain has a length of 270 aa, 271 aa, 272 aa, 273 aa, 274 aa, 275 aa, 276 aa, 277 aa, 278 aa, 279 aa, 280 aa, 281 aa, 282 aa, 283 aa, 284 aa, 285 aa, 286 aa, 287 aa, 288 aa, 289 aa, or 290 aa. In some examples, the MHC class I heavy chain polypeptide present in a TMP of the disclosure comprises 1-30, 1-5, 5-10, 10-15, 15-20, 20-25, or 25-30 amino acid insertions, deletions, and / or substitutions of any one of the amino acid sequences shown in Figures 7-13 (in addition to those positions indicated as variable in the heavy chain consensus sequence). In some examples, the MHC class I heavy chain does not include a transmembrane or cytoplasmic domain. By way of example, the MHC class I heavy chain polypeptide of a TMP of the present disclosure can include 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 sequence, transmembrane sequence, and cytoplasmic sequence) or amino acids 25-365 (lacking the leader) of the human HLA-A heavy chain polypeptide shown in any one of Figures 5A, 5B, and 5C.

[0088] Figures 5A, 5B, and 5C provide the amino acid sequence of the human leukocyte antigen (HLA) class I heavy chain polypeptide. Amino acids 1-24, the signal sequence, are shown in bold and underlined. Entry 3A.1 in Figure 5A is the amino acid sequence of the HLA-A heavy chain (HLA-A * 01:01:01:01 or A* 0101) (NCBI accession number NP_001229687.1), and SEQ ID NO: 35, entry 3A.2, is HLA-A * 1101 is derived from SEQ ID NO: 36, and entry 3A.3 is derived from HLA-A * 2402 is derived from SEQ ID NO: 37, and entry 3A.4 is derived from HLA-A * 3303 is derived from SEQ ID NO: 38. FIG. 5B shows the sequence HLA-B * 07:02:01(HLA-B * 0702) NCBI GenBank accession number NP_005505.2 (see also GenBank accession number AUV50118.1). Figure 5C shows the sequence HLA-C * 0701 (GenBank accession number NP_001229971.1) (HLA-C * 07:01:01:01 or HLA-Cw * 070101, HLA-Cw * 07, see GenBank accession number CAO78194.1).

[0089] As described above, the first and second polypeptides of a heterodimeric TMP typically contain one or more disulfide bonds to confer increased stability and / or increased expression to the TMP. One or more disulfide bonds may be formed between Cys residues present within the same polypeptide, i.e., intrachain disulfide bonds. Alternatively, or in addition to such intrachain disulfide bonds, one or more interchain disulfide bonds can be formed between Cys residues provided in the first and second polypeptides, e.g., (i) a non-naturally occurring Cys residue can be provided in both MHC class I polypeptides, i.e., the β2M polypeptide and the MHC class I heavy chain polypeptide, and / or (ii) a linker comprising a Cys residue can be provided in both the first and second polypeptides, and / or (iii) a linker comprising a Cys residue can be provided in one of the first and second polypeptides (e.g., between the epitope of the first polypeptide and β2M) and a non-naturally occurring Cys residue can be provided in the other MHC class I polypeptide (e.g., in the MHC class I heavy chain polypeptide). Exemplary configurations are described below.

[0090] Figure 6 shows an alignment of 11 mature MHC class I heavy chain amino acid sequences (not including their leader sequences or transmembrane or intracellular domains). The aligned sequences include human HLA-A, HLA-B, and HLA-C, mouse H2K protein sequence, three variants of HLA-A (variant 1, variant 2C, and variant 2CP), and three human HLA-A variants (HLA-A * 1101, HLA-A * 2402, and HLA-A * 3303). Shown in the alignment are positions (84 and 139 of the mature protein) where cysteine ​​residues can be introduced (e.g., by substitution) to form disulfide bonds that stabilize the MHC chain-β2M complex. Also shown in the alignment is position 236 (of the mature polypeptide) which can be substituted with a cysteine ​​residue (e.g., at aa 12) that can form an interchain disulfide bond with β2M. Arrows are shown above each of these positions, and the residues are in bold. The seventh HLA-A sequence shown in the alignment (variant 2c) shows the sequence of variant 2 with C residue substitutions at positions 84, 139, and 236. The boxes flanking residues 84, 139, and 236 indicate groups of five amino acids on either side of six sets of five residues that 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, denoted 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").

[0091] With reference to FIG. 6 , in some examples, i) aac1 (amino acid cluster 1) may be the amino acid sequence GTLRG (SEQ ID NO: 98) or a sequence in which one or two amino acids have been deleted or substituted with other naturally occurring amino acids (e.g., L has been substituted with I, V, A, or F); ii) aac2 (amino acid cluster 2) may be the amino acid sequence YNQSE (SEQ ID NO: 99) or a sequence in which one or two amino acids have been deleted or substituted with other naturally occurring amino acids (e.g., N has been substituted with Q, Q has been substituted with N, and / or E has been substituted with D); iii) aac3 (amino acid cluster 3) may be the amino acid sequence TAADM (SEQ ID NO: 100) or a sequence in which one or two amino acids have been deleted or substituted with other naturally occurring amino acids (e.g., T has been substituted with S, A has been substituted with G, D has been substituted with E, and / or M has been substituted with L, V, or I); and iv) aac4 (amino acid cluster 4) may be the amino acid sequence YNQSE (SEQ ID NO: 99) or a sequence in which one or two amino acids have been deleted or substituted with other naturally occurring amino acids (e.g., T has been substituted with S, A has been substituted with G, D has been substituted with E, and / or M has been substituted with L, V, or I). 4) may be the amino acid sequence AQTTK (SEQ ID NO: 101) 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 substituted with G, Q is substituted with N, T is substituted with S, and / or K is substituted with R or Q); v) aac5 (amino acid cluster 5) may be the amino acid sequence VETRP (SEQ ID NO: 102) or a sequence in which one or two amino acids have been deleted or substituted with other naturally occurring amino acids (e.g., V is substituted with I or L, E is substituted with D, T is substituted with S, and / or R is substituted with K); and / or vi) aac6 (amino acid cluster 6) may be the amino acid sequence GDGTF (SEQ ID NO: 103) 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 substituted with E, T is substituted with S, or F is substituted with L, W, or Y).

[0092] Figures 7-9 show alignments of mature HLA class I heavy chain amino acid sequences (not including leader sequences or transmembrane or intracellular domains). The aligned amino acid sequences in Figure 7A are HLA-A class I heavy chains of the following alleles: A * 0101, A * 0201, A * 0301, A * 1101, A * 2301, A * 2402, A * 2407, A * 3303 and A * 3401. The aligned amino acid sequences in Figure 8A are the HLA-B class I heavy chains of the following alleles: B * 0702, B * 0801, B * 1502, B * 3802, B * 4001, B * 4601 and B * 5301. The aligned amino acid sequences in Figure 9A are the HLA-C class I heavy chains of the following alleles: C * 0102, C * 0303, C * 0304, C * 0401, C * 0602, C * 0701, C * 0801 and C *1502. The alignment shows positions (84 and 139 of the mature protein) where cysteine ​​residues can be introduced (e.g., by substitution) to form disulfide bonds to stabilize the HLA H chain-β2M complex. The alignment also shows position 236 (of the mature polypeptide), which can be substituted (e.g., at aa 12) by a cysteine ​​residue capable of forming an interchain disulfide bond with β2M. The boxes adjacent to residues 84, 139, and 236 indicate five amino acid groups on either side of six sets of five residues, designated aac1 (for "amino acid cluster 1"), aac2 (for "amino acid cluster 2"), aac3 (for "amino acid cluster 3"), aac4 (for "amino acid cluster 4"), aac5 (for "amino acid cluster 5"), and aac6 (for "amino acid cluster 6"), which may be substituted by one to five amino acids independently selected from (i) natural amino acids or (ii) natural amino acids excluding proline or glycine.

[0093] 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. The sequences represent the extracellular portions of the mature proteins (excluding leader sequences, transmembrane domains, or intracellular domains). Also shown are aa residues 84, 139, and 236, as well as their adjacent residues (aac1 through aac6), which may be substituted with (i) any naturally occurring amino acid or (ii) one to five amino acids independently selected from naturally occurring amino acids excluding proline or glycine, as described in Figure 6. 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. In the consensus sequences, variable amino acid positions are indicated as consecutively numbered "X" residues, with amino acid positions 84, 139, and 236 double-underlined.

[0094] With reference to FIG. 7A , in some examples, i) aac1 (amino acid cluster 1) may be the amino acid sequence GTLRG (SEQ ID NO: 98) or a sequence from which one or two amino acids have been deleted or substituted with other naturally occurring amino acids (e.g., L has been substituted with I, V, A, or F); ii) aac2 (amino acid cluster 2) may be the amino acid sequence YNQSE (SEQ ID NO: 99) or a sequence from which one or two amino acids have been deleted or substituted with other naturally occurring amino acids (e.g., N has been substituted with Q, Q has been substituted with N, and / or E has been substituted with D); iii) aac3 (amino acid cluster 3) may be the amino acid sequence TAADM (SEQ ID NO: 100) or a sequence from which one or two amino acids have been deleted or substituted with other naturally occurring amino acids (e.g., T has been substituted with S, A has been substituted with G, D has been substituted with E, and / or M has been substituted with L, V, or I); and iv) aac4 ( Amino acid cluster 4) may be the amino acid sequence AQTTK (SEQ ID NO: 101) or a sequence from 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, 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: 102) or a sequence from 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: 103) or a sequence from 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).

[0095] With reference to FIG. 8A , in some examples, i) aac1 (amino acid cluster 1) may be the amino acid sequence RNLRG (SEQ ID NO: 104) or a sequence from which one or two amino acids have been deleted or substituted with other naturally occurring amino acids (e.g., N replaced with T or I, and / or L replaced with A, and / or a second R replaced with L, and / or G replaced with R), and ii) aac2 (amino acid cluster 2) may be the amino acid sequence YNQSE (SEQ ID NO: 99) or a sequence from which one or two amino acids have been deleted. aac3 (amino acid cluster 3) may be the amino acid sequence TAADT (SEQ ID NO: 105) or a sequence in which one or two amino acids have been deleted or substituted with other natural amino acids (e.g., replacing N with Q, replacing Q with N, and / or replacing E with D); iii) aac3 (amino acid cluster 3) may be the amino acid sequence TAADT (SEQ ID NO: 105) or a sequence in which one or two amino acids have been deleted or substituted with other natural amino acids (e.g., replacing the first T with S, and / or replacing A with G, and / or replacing D with E, and / or replacing the second T with S); iv) aac4 (amino acid cluster 4) may be the amino acid sequence TAADT (SEQ ID NO: 105) or a sequence in which one or two amino acids have been deleted or substituted with other natural amino acids (e.g., replacing the first T with S, and / or replacing A with G, and / or replacing D with E, and / or replacing the second T with S); v) aac5 (amino acid cluster 5) may be the amino acid sequence VETRP (SEQ ID NO: 102) or a sequence obtained by deleting or substituting one or two amino acids with other natural amino acids (e.g., A is substituted with G, and / or the first Q is substituted with N, and / or I is substituted with L or V, and / or T is substituted with S, and / or the second Q is substituted with N); may be a sequence in which other naturally occurring amino acids are substituted (e.g., V is substituted with I or L, E is substituted with D, T is substituted with S, and / or R is substituted with K); and / or vi) aac6 (amino acid cluster 6) may be the amino acid sequence GDRTF (SEQ ID NO: 107) or a sequence in which one or two amino acids have been deleted from that sequence or substituted with other naturally occurring amino acids (e.g., D is substituted with E, and / or T is substituted with S, and / or R is substituted with K or H, and / or F is substituted with L, W, or Y).

[0096] With reference to FIG. 9A , in some examples, i) aac1 (amino acid cluster 1) may be the amino acid sequence RNLRG (SEQ ID NO: 104) or a sequence from which one or two amino acids have been deleted or substituted with other natural amino acids (e.g., N is replaced with K, and / or L is replaced with A or I, and / or 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: 99) or a sequence from which one or two amino acids have been deleted or substituted with other natural amino acids. iii) aac3 (amino acid cluster 3) may be the amino acid sequence TAADT (SEQ ID NO: 105) or a sequence in which one or two amino acids have been deleted or substituted with other natural amino acids (e.g., a first T has been replaced with S, and / or an A has been replaced with G, and / or a D has been replaced with E, and / or a second T has been replaced with S). iv) aac4 (amino acid cluster 4) may be the amino acid sequence AQITQ (SEQ ID NO: 106) or a sequence obtained by deleting one or two amino acids from that sequence or substituting them with other natural amino acids (e.g., substituting A with G, and / or the first Q with N, and / or I with L, and / or the second Q with N or K), and v) aac5 (amino acid cluster 5) may be the amino acid sequence VETRP (SEQ ID NO: 102) or a sequence obtained by deleting one or two amino acids from that sequence. and / or vi) aac6 (amino acid cluster 6) may be the amino acid sequence GDGTF (SEQ ID NO: 103) or a sequence in which one or two amino acids have been deleted or substituted with other naturally occurring amino acids (e.g., D has been substituted with E, and / or T has been substituted with S, and / or F has been substituted with L, W, or Y).

[0097] HLA-A In some examples, the TMP of the present disclosure comprises an HLA-A heavy chain polypeptide. HLA-A heavy chain peptide sequences or portions thereof that can be incorporated into the TMP of the present disclosure include, but are not limited to, allele A * 0101, A * 0201, A * 0301, A * 1101, A * 2301, A * 2402, A * 2407, A * 3303 and A * 3401, which are aligned in Figure 7A without all or substantially all of the leader, transmembrane, and cytoplasmic sequences. Any of these alleles may contain a mutation at one or more of positions 84, 139, and / or 236 (shown in Figure 7A) selected from the following: tyrosine to alanine at position 84 (Y84A), tyrosine to cysteine ​​at position 84 (Y84C), alanine to cysteine ​​at position 139 (A139C), and alanine to cysteine ​​substitution at position 236 (A236C). Additionally, 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 all or a portion (e.g., 50, 75, 100, 150, 200, or 250 consecutive amino acids) of the sequence of an HLA-A allele may also be used (e.g., which may include insertions, deletions, and / or substitutions of 1-25, 1-5, 5-10, 10-15, 15-20, 20-25, or 25-30 amino acids). A TMP of the present disclosure may comprise an MHC class I heavy chain comprising an amino acid sequence shown in any one of Figures 21A-21AA, 21DD-21FF, 21HH, and 22A-22BB.

[0098] In some examples, the TMP of the present disclosure has the HLA-A consensus amino acid sequence shown in Figure 21 II (SEQ ID NO: 61), where X1 is F, Y, S, or T; X2 is K or R; X3 is Q, G, E, or R; X4 is N or E; X5 is R or G; X6 is N or K; X7 is M or V; X8 is H or Q; X9 is T or I; and X10 is H or Q. is D or H, X11 is A, V, or E, X12 is N or D, X13 is G or R, X14 is T or I, X15 is L or A, X16 is R or L, X17 is G or R, X18 is A or D, X19 is I, L, or V, X20 is I, R, or M, X21 is F or Y, and X22 is S or P, X23 is W or G, X24 is R, H, or Q, X25 is D or Y, X26 is N or K, X27 is T or I, X28 is K or Q, X29 is R or H, X30 is A or T, X31 is A or V, X32 is H or R, X33 is R, L, Q, or W, and X34 is V or A X35 is D or E, X36 is R or T, X37 is D or E, X38 is W or G, X39 is P or A, X40 is P or A, X41 is V or I, X42 is S or G, X43 is A or S, X44 is Q or E, and X45 is P or L.

[0099] By way of example, the MHC class I heavy chain polypeptide of 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 human HLA-A heavy chain amino acid sequence shown in Figure 21A (SEQ ID NO: 108) or 21B (SEQ ID NO: 109). The HLA-A heavy chain polypeptide of Figure 21B is an "HLA-A" heavy chain polypeptide. *In some instances, the C-terminal Pro is not included in the TMPs of the present disclosure. For example, in some instances, an HLA-A02 polypeptide suitable for inclusion in a TMP of the present disclosure 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 amino acid sequence shown in Figure 21C (SEQ ID NO: 110).

[0100] HLA-A (Y84A, A236C) In some examples, the MHC class I heavy chain polypeptide comprises a Y84A substitution and an A236C substitution. For example, in some examples, 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 human HLA-A heavy chain (Y84A, A236C) amino acid sequence shown in Figure 21D (SEQ ID NO: 111) (amino acid 84 is Ala and amino acid 236 is Cys). In some examples, Cys-236 forms an interchain disulfide bond with Cys-12 of a variant β2M polypeptide comprising an R12C substitution.

[0101] In some examples, an HLA-A heavy chain polypeptide suitable for inclusion in a TMP of the present disclosure is an HLA-A02(Y84A, A236C) polypeptide comprising the amino acid sequence of Figure 21E (SEQ ID NO: 112). In some examples, an HLA-A heavy chain polypeptide suitable for inclusion in a TMP of the present disclosure is an HLA-A02(Y84A, A236C) polypeptide comprising the amino acid sequence shown in Figure 21F (SEQ ID NO: 113).

[0102] HLA-A(Y84C, A139C) In some examples, the MHC class I heavy chain polypeptide comprises a Y84C substitution and an A139C substitution. For example, in some examples, 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 human HLA-A heavy chain (Y84C, A139C) amino acid sequence shown in Figure 21G (SEQ ID NO: 114) (amino acid 84 is Cys and amino acid 139 is Cys). In some examples, Cys-84 forms an intrachain disulfide bond with Cys-139.

[0103] HLA-A (Y84C, A139C, A236C) In some examples, an MHC class I heavy chain polypeptide suitable for inclusion in a TMP of the present disclosure comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the human HLA-A heavy chain (Y84C, A139C, A236C) amino acid sequence shown in Figure 21H (SEQ ID NO: 120), where amino acid 84 is a Cys, amino acid 139 is a Cys, and amino acid 236 is a Cys. In some examples, Cys-84 forms an intrachain disulfide bond with Cys-139. The Cys at amino acid 236 can form a disulfide bond with a Cys residue of a second polypeptide chain. For example, the Cys at amino acid 236 can form a disulfide bond with the Cys-12 residue of a β2M polypeptide comprising an R12C substitution.

[0104] In some examples, an HLA-A heavy chain polypeptide suitable for inclusion in a TMP of the present disclosure is an HLA-A02 (Y84C, A139C, A236C) polypeptide comprising the amino acid sequence shown in Figure 21I (SEQ ID NO: 232).

[0105] HLA-A11 (HLA-A * 1101) As one non-limiting example, 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 human HLA-A11 heavy chain amino acid sequence shown in Figure 21J (SEQ ID NO: 115). Such MHC class I heavy chains can be prominent in Asian populations, including populations of individuals of Asian descent.

[0106] HLA-A11(Y84A, A236C) As one non-limiting example, in some instances, the MHC class I heavy chain polypeptide is an HLA-A11 allele comprising a Y84A substitution and an A236C substitution. For example, in some instances, 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 human HLA-A A11 heavy chain (Y84A, A236C) amino acid sequence shown in Figure 21K (SEQ ID NO: 116) (amino acid 84 is Ala and amino acid 236 is Cys). In some instances, Cys-236 forms an interchain disulfide bond with Cys-12 of a variant β2M polypeptide comprising an R12C substitution (amino acid 84 is Cys and amino acid 236 is Cys), as shown in Figure 21L (SEQ ID NO: 233).

[0107] HLA-A24 (HLA-A * 2402) As one non-limiting example, the MHC class I heavy chain polypeptide of a TMP of the present disclosure can include 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 human HLA-A24 heavy chain amino acid sequence shown in Figure 21M (SEQ ID NO: 117). Such an MHC class I heavy chain can be prominent in Asian populations, including populations of individuals of Asian descent. In some examples, amino acid 84 is Ala. In some examples, amino acid 84 is Cys. In some examples, amino acid 236 is Cys. In some examples, amino acid 84 is Ala and amino acid 236 is Cys. In some examples, amino acid 84 is Cys and amino acid 236 is Cys.

[0108] As one non-limiting example, the MHC class I heavy chain polypeptide of the TMP of the present disclosure is selected from the group consisting of human HLA-A24 (HLA-A24), shown in Figure 21N (SEQ ID NO: 586), * The heavy chain amino acid sequence of the MHC class I heavy chain may comprise an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the heavy chain amino acid sequence of MHC class I heavy chain (also referred to as MHC class I heavy chain 2402). Such MHC class I heavy chains may be prominent in Asian populations, including populations of individuals of Asian descent. In some examples, amino acid X1 at position 84 is Ala. In some examples, amino acid 84 is Cys. In some examples, amino acid X2 at position 236 is Cys. In some examples, amino acid 84 is Ala and amino acid 236 is Cys. In some examples, amino acid 84 is Cys and amino acid 236 is Cys.

[0109] In some examples, the MHC class I heavy chain polypeptide of the TMP of the present disclosure is selected from the group consisting of human HLA-A24 (HLA-A24) and human HLA-A14 (HLA-A14) as shown in one of the following figures: *2402) heavy chain amino acid sequence: (i) Figure 21O (SEQ ID NO: 235) (amino acid 84 is Tyr and amino acid 236 is Ala) (amino acids 84 and 236 are bold and underlined) (MHC class I heavy chain length is approximately 275 amino acids) (ii) TMP Figure 21P (SEQ ID NO: 236) (amino acid 84 is Ala and amino acid 236 is Ala (amino acids 84 and 236 are bold and underlined) (MHC class I heavy chain length is approximately 275 amino acids) (iii) TMP Figure 21Q (SEQ ID NO: 237) (amino acid 84 is Tyr and amino acid 236 is Cys) (amino acids 84 and 236 are bold and underlined) (MHC class I heavy chain length is approximately 275 amino acids) (iv) TMP Figure 21R (SEQ ID NO: 238) (amino acid 84 is Ala and amino acid 236 is Cys) (amino acids 84 and 236 are bold and underlined) (MHC class I heavy chain length is approximately 275 amino acids) (v) TMP Figure 21S (SEQ ID NO: 239) (amino acid 84 is Cys and amino acid 236 is Ala) (amino acids 84 and 236 are bold and underlined) (MHC class I heavy chain length is approximately 275 amino acids) (vi) TMP Figure 21T (SEQ ID NO: 240) (amino acid 84 is Cys and amino acid 236 is Cys) (amino acids 84 and 236 are bold and underlined) (MHC class I heavy chain length is approximately 275 amino acids)

[0110] HLA-A33 (HLA-A * 3303) As one non-limiting example, an MHC class I heavy chain polypeptide of a TMP of the present disclosure can include 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 human HLA-A33 heavy chain amino acid sequence shown in Figure 21U (SEQ ID NO: 118). Such an MHC class I heavy chain can be prominent in Asian populations, including populations of individuals of Asian descent. In some examples, amino acid 84 is Ala. In some examples, amino acid 84 is Cys. In some examples, amino acid 236 is Cys. In some examples, amino acid 84 is Ala and amino acid 236 is Cys. In some examples, amino acid 84 is Cys and amino acid 236 is Cys.

[0111] HLA-B In some examples, the TMPs of the present disclosure comprise an HLA-B heavy chain polypeptide. HLA-B heavy chain peptide sequences or portions thereof that may be incorporated into the TMPs of the present disclosure include, but are not limited to, alleles: B * 0702, B * 0801, B * 1502, B * 3802, B * 4001, B * 4601 and B *5301, which are aligned in Figure 8A without all or substantially all of the leader, transmembrane, and cytoplasmic sequences. Any of these alleles may contain a mutation at one or more of positions 84, 139, and / or 236 (shown in Figure 8A) selected from the following: tyrosine to alanine at position 84 (Y84A), tyrosine to cysteine ​​at position 84 (Y84C), alanine to cysteine ​​at position 139 (A139C), and alanine to cysteine ​​substitution at position 236 (A236C). Additionally, HLA-B polypeptides comprising an amino acid sequence having at least 75% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%) or 100% amino acid sequence identity to all or a portion (e.g., 50, 75, 100, 150, 200, or 250 contiguous amino acids) of the sequence of an HLA-B allele may also be used (e.g., which may include insertions, deletions, and / or substitutions of 1 to 25, 1 to 5, 5 to 10, 10 to 15, 15 to 20, 20 to 25, or 25 to 30 amino acids).

[0112] In some examples, the TMP of the disclosure has the HLA-B consensus amino acid sequence shown in Figure 21JJ (SEQ ID NO: 69) (X1 is H, Y, or D, X2 is A or S, X3 is M or V, X4 is A, S, or T, X5 is Q or L, X6 is A or T, X7 is E, M, K, or T, X8 is A or T, X9 is E or N, X10 is I or K, X11 is Y, F, S, or C, X12 is N or Q, X13 is A or T, X14 is D or Y, X15 is E or V, X16 is S or N, and X X17 is T, N, or I, X18 is A or L, X19 is L or R, X20 is R or G, X21 is T or I, X22 is L or I, X23 is R or S, X24 is R or S, X25 is S or T, X26 is L or W, X27 is E or V, X28 is R, D, L, or W, X29 is A or T, X30 is L, E, or T, X31 is E or D, X32 is K or T, X33 is E or Q, and X34 is I or V.

[0113] As an example, the MHC class I heavy chain polypeptide of a TMP of the present disclosure can comprise an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the human HLA-B heavy chain amino acid sequence shown in Figure 21V (SEQ ID NO: 119).

[0114] HLA-B (Y84A, A236C) As one non-limiting example, in some instances, the MHC class I heavy chain polypeptide is an HLA-B polypeptide comprising a Y84A substitution and an A236C substitution. For example, in some instances, 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 human HLA-B heavy chain (Y84A, A236C) amino acid sequence shown in Figure 21W (SEQ ID NO: 121) (amino acid 84 is Ala and amino acid 236 is Cys). In some instances, Cys-236 forms an interchain disulfide bond with Cys-12 of a variant β2M polypeptide comprising an R12C substitution.

[0115] HLA-B (Y84C, A139C) In some examples, the MHC class I heavy chain polypeptide comprises a Y84C substitution and an A139C substitution. For example, in some examples, 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 human HLA-B heavy chain (Y84C, A139C) amino acid sequence shown in Figure 21X (SEQ ID NO: 122) (amino acid 84 is Cys and amino acid 139 is Cys). In some examples, Cys-84 forms an intrachain disulfide bond with Cys-139.

[0116] HLA-B * 0702 As an example, in some instances, the MHC class I heavy chain polypeptide present in the TMP of the present disclosure may be HLA-B *0702 (SEQ ID NO: 62), or a sequence having at least 75% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%) or 100% amino acid sequence identity to all or a portion (e.g., 50, 75, 100, 150, 200, or 250 contiguous amino acids) of that sequence (e.g., which may include 1-25, 1-5, 5-10, 10-15, 15-20, 20-25, or 25-30 amino acid insertions, deletions, and / or substitutions). In some examples, the HLA-B heavy chain polypeptide of a TMP of the present disclosure comprises the sequence labeled HLA-B in FIG. 6 or "B" in FIG. 8A. * If a sequence has less than 100% identity to the sequence labeled "0702," it may contain a mutation at one or more of positions 84, 139, and / or 236 selected from the following: a tyrosine to alanine substitution at position 84 (Y84A), a tyrosine to cysteine ​​substitution at position 84 (Y84C), an alanine to cysteine ​​at position 139 (A139C), and an alanine to cysteine ​​substitution at position 236 (A236C). In some examples, the HLA-B heavy chain polypeptide of the TMP of the present disclosure contains the Y84A and A236C substitutions. In some examples, the HLA-B heavy chain polypeptide of the TMP of the present disclosure contains the Y84A and A236C substitutions. * The 0702 heavy chain polypeptide comprises Y84C and A139C substitutions. In some examples, the HLA-B heavy chain polypeptide of a TMP of the present disclosure comprises Y84C, A139C, and A236C substitutions.

[0117] HLA-C In some examples, the TMP of the present disclosure comprises an HLA-C heavy chain polypeptide. HLA-C heavy chain polypeptides or portions thereof that can be incorporated into the TMP of the present disclosure include, but are not limited to, alleles: C * 0102, C * 0303, C * 0304, C * 0401, C * 0602, C * 0701, C * 0801 and C *1502, which align without including all or substantially all of the leader, transmembrane, and cytoplasmic sequences in Figure 9A. Any of these alleles may contain a mutation at one or more of positions 84, 139, and / or 236 (shown in Figure 9A) selected from the following: a tyrosine to alanine substitution at position 84 (Y84A), a tyrosine to cysteine ​​substitution at position 84 (Y84C), an alanine to cysteine ​​substitution at position 139 (A139C), or an alanine to cysteine ​​substitution at position 236 (A236C). Additionally, HLA-C polypeptides comprising an amino acid sequence having at least 75% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%) or 100% amino acid sequence identity to all or a portion (e.g., 50, 75, 100, 150, 200, or 250 consecutive amino acids) of the sequence of an HLA-C allele may also be used (e.g., which may include insertions, deletions, and / or substitutions of 1 to 25, 1 to 5, 5 to 10, 10 to 15, 15 to 20, 20 to 25, or 25 to 30 amino acids).

[0118] In some examples, the TMP of the present disclosure has the following HLA-C consensus amino acid sequence: TIFF0007756072000003.tif38163 (SEQ ID NO: 79), wherein X1 is C or G, X2 is R or K, X3 is F, Y, S, or D, X4 is R or W, X5 is H or R, X6 is A or S, X7 is Q or R, X8 is A or E, X9 is N or K, X10 is T or A, X11 is S or N, X12 is N or K, X13 is A or D, X14 is G or R, X15 is T or I, X16 is L or I, and X17 is W or R; X18 is C, Y, F, or S, X19 is L or V, X20 is Y or H, X21 is D or N, X22 is Y, F, S, or L, X23 is L or W, X24 is E, A, or T, X25 is R, L, or W, X26 is L or T, X27 is E or K, X28 is E or K, X29 is H or P, X30 is R or V, X31 is W or R, X32 is V or M, X33 is E or Q, X34 is M or V, X35 is P or Q, X36 is R or S, and X37 is P or G.

[0119] As an example, the MHC class I heavy chain polypeptide of the TMP of the present disclosure can comprise an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the human HLA-C heavy chain amino acid sequence shown in Figure 21Y (SEQ ID NO: 123).

[0120] HLA-C (Y84A, A236C) As one non-limiting example, in some instances, the MHC class I heavy chain polypeptide is an HLA-C polypeptide comprising a Y84A substitution and an A236C substitution. For example, in some instances, 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 human HLA-C heavy chain (Y84A, A236C) amino acid sequence shown in Figure 21Z (SEQ ID NO: 124) (amino acid 84 is Ala and amino acid 236 is Cys). In some instances, Cys-236 forms an interchain disulfide bond with Cys-12 of a variant β2M polypeptide comprising an R12C substitution.

[0121] HLA-C (Y84C, A139C) In some examples, the MHC class I heavy chain polypeptide comprises a Y84C substitution and an A139C substitution. For example, in some examples, 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 human HLA-C heavy chain (Y84C, A139C) amino acid sequence shown in Figure 21AA (SEQ ID NO: 125) (amino acid 84 is Cys and amino acid 139 is Cys). In some examples, Cys-84 forms an intrachain disulfide bond with Cys-139.

[0122] HLA-C * 0701 In some examples, the MHC class I heavy chain polypeptide of the TMP of the present disclosure is selected from the group consisting of HLA-C *0701 (labeled HLA-C in FIG. 6), or an amino acid sequence having at least 75% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%) or 100% amino acid sequence identity to all or a portion (e.g., 50, 75, 100, 150, 200, or 250 contiguous amino acids) of that sequence (e.g., which may include insertions, deletions, and / or substitutions of 1-25, 1-5, 5-10, 10-15, 15-20, 20-25, or 25-30 amino acids). In some examples, the HLA-C heavy chain polypeptide of a TMP of the present disclosure is the HLA-C heavy chain polypeptide of FIG. 9A. * If it has less than 100% identity to the sequence labeled 0701, it may contain a mutation at one or more of positions 84, 139, and / or 236 selected from the following: a tyrosine to alanine substitution at position 84 (Y84A), a tyrosine to cysteine ​​substitution at position 84 (Y84C), an alanine to cysteine ​​substitution at position 139 (A139C), an alanine to cysteine ​​substitution at position 236 (A236C). In some examples, the HLA-C heavy chain polypeptide of a T-cell MMP of the present disclosure contains the Y84A and A236C substitutions. In some examples, the HLA-C heavy chain polypeptide of a T-cell MMP of the present disclosure contains the Y84A and A236C substitutions. * The 0701 heavy chain polypeptide or epitope complex thereof comprises Y84C and A139C substitutions. In some examples, the HLA-C heavy chain polypeptide of a TMP of the present disclosure comprises Y84C, A139C, and A236C substitutions.

[0123] Non-classical HLA-E, HLA-F, and HLA-G MHC class I heavy chains In some examples, the TMPs of the present disclosure include non-classical MHC class I heavy chain polypeptides. Non-classical HLA heavy chain polypeptides, or portions thereof, that can be incorporated into the TMPs of the present disclosure include, but are not limited to, those of HLA-E, HLA-F, and HLA-G alleles. The amino acid sequences of HLA-E, HLA-F, and HLA-G heavy chain polypeptides (and HLA-A, HLA-B, and HLA-C alleles) can be found on the World Wide Web at hla.alleles.org / nomenclature / index.html, at the European Bioinformatics Institute (www.ebi.ac.uk), which is part of the European Molecular Biology Laboratory (EMBL), and at the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov).

[0124] 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 * Non-limiting examples of suitable HLA-F alleles include, but are not limited to, HLA-F 01:10. * 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 *Non-limiting examples of suitable HLA-G alleles include, but are not limited to, HLA-G 01:06. * 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 * 01:22. 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 aligned with the consensus sequences for the HLA-A, HLA-B, and HLA-C alleles described above in Figure 11.

[0125] Figure 1 shows the consensus sequences for each of HLA-E, HLA-F, and HLA-G, with variable aa positions shown as consecutively numbered "X" residues, with aas 84, 139, and 236 double underlined.

[0126] Figure 11 shows an alignment of the consensus amino acid sequences of HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, and HLA-G shown in Figures 7-11. Variable residues in each sequence are indicated as "X" with the consecutive numbers omitted. Also shown are positions aas 84, 139, and 236, along with their adjacent five amino acid clusters, which 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, as shown in Figure 6.

[0127] Any of the above HLA-E, HLA-F, and / or HLA-G alleles can include substitutions at one or more of positions 84, 139, and / or 236, as shown for the consensus sequence in Figure 11. In some examples, the substitutions can be selected from the following: a tyrosine to alanine (Y84A) or cysteine ​​(Y84C), or in the case of HLA-F, an R84A or R84C substitution at position 84, an alanine to cysteine ​​(A139C), or in the case of HLA-F, V139C, at position 139, 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 any of the consensus sequences set forth in FIG. 11 may also be used (e.g., the sequences may include insertions, deletions, and / or substitutions of 1-25, 1-5, 5-10, 10-15, 15-20, 20-25, or 25-30 amino acids in addition to changes at the variable residues set forth in the figure).

[0128] Mouse H2K In some examples, the MHC class I heavy chain polypeptide present in a TMP of the disclosure comprises the amino acid sequence of mouse H2K (SEQ ID NO: 45) (mouse H2K in FIG. 6), or a 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 that sequence (e.g., which may include insertions, deletions, and / or substitutions of 1-25, 1-5, 5-10, 10-15, 15-20, 20-25, or 25-30 amino acids). In some examples, when a mouse H2K heavy chain polypeptide of a TMP of the present disclosure has less than 100% identity to the sequence labeled Mouse H2K in Figure 6, it may contain a mutation selected from the following at one or more of positions 84, 139, and / or 236: tyrosine to alanine at position 84 (Y84A), tyrosine to cysteine ​​at position 84 (Y84C), alanine to cysteine ​​at position 139 (A139C), and alanine to cysteine ​​at position 236 (A236C). In some examples, a mouse H2K heavy chain polypeptide of a TMP of the present disclosure contains a Y84A and A236C substitution. In some examples, a mouse H2K heavy chain polypeptide of a TMP of the present disclosure contains a Y84C and A139C substitution. In some examples, the murine H2K heavy chain polypeptide of a TMP of the present disclosure comprises Y84C, A139C, and A236C substitutions.

[0129] Combination examples Table 1 below shows various combinations of MHC class I heavy chain sequence modifications that can be incorporated into the TMPs of the present disclosure.

[0130] (Table 1) TIFF0007756072000004.tif148141TIFF0007756072000005.tif146141The sequence identity range is the acceptable range of sequence identity of the MHC-H polypeptide sequence to be incorporated into the TMP compared to the corresponding portion of the sequence listed in Figures 6-11, not counting variable residues in the consensus sequence.

[0131] Beta-2 microglobulin The β2-microglobulin (β2M) polypeptide of the TMP of the present disclosure can be a human β2M polypeptide, a non-human primate β2M polypeptide, a mouse β2M polypeptide, etc. In some examples, the β2M polypeptide comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the β2M amino acid sequence set forth in Figure 4. In some examples, 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 amino acids 21-119 of the β2M amino acid sequence set forth in Figure 4.

[0132] In some examples, a suitable β2M polypeptide comprises the amino acid sequence shown in Figure 21CC (SEQ ID NO: 126), and the HLA class I heavy chain polypeptide comprises the amino acid sequence shown in Figure 21HH (SEQ ID NO: 127) (the cysteine ​​residue indicated by {C} forms a disulfide bond between the α1 helix and the α2-1 helix, and this residue forms a disulfide bond with the β2M polypeptide cysteine ​​at position 12). In the above sequences, "aa1" is "amino acid cluster 1," "aa2" is "amino acid cluster 2," "aa3" is "amino acid cluster 3," "aa4" is "amino acid cluster 4," "aa5" is "amino acid cluster 5," and "aa6" is "amino acid cluster 6," see, e.g., Figure 8. Each occurrence of aa1, aa2, aa3, aa4, aa5, and aa6 is independently selected to be 1 to 5 amino acid residues, which are either i) independently selected from any naturally occurring (e.g., encoded) amino acid, or ii) proline is any naturally occurring amino acid except glycine.

[0133] In some examples, the MHC polypeptide comprises a single amino acid substitution compared to a reference MHC polypeptide (which may be a wild-type MHC polypeptide), where the single amino acid substitution replaces the amino acid with a cysteine ​​(Cys) residue. Such a non-naturally occurring cysteine ​​residue, when present in an MHC polypeptide of a first polypeptide of a heterodimeric TMP of the present disclosure or in an MHC polypeptide of a single-chain TMP, can form a disulfide bond with a cysteine ​​residue present in a second polypeptide chain of the TMP of the present disclosure or in another MHC polypeptide of the single-chain TMP.

[0134] In some examples, a first MHC polypeptide within a first polypeptide of a heterodimeric TMP of the present disclosure and / or a second MHC polypeptide within a second polypeptide of a heterodimeric TMP of the present disclosure comprises an amino acid substitution to replace an amino acid with a cysteine, such that the non-native cysteine ​​in the first MHC polypeptide forms a disulfide bond with a cysteine ​​in the second MHC polypeptide, or the cysteine ​​in the first MHC polypeptide forms a disulfide bond with a non-native cysteine ​​in the second MHC polypeptide, or the non-native cysteine ​​in the first MHC polypeptide forms a disulfide bond with a non-native cysteine ​​in the second MHC polypeptide. Similarly, one of the MHC polypeptides within a single-chain TMP of the present disclosure comprises an amino acid substitution to replace an amino acid with a cysteine, such that the non-native cysteine ​​in the MHC polypeptide forms a disulfide bond with a native or non-native cysteine ​​in a different MHC polypeptide within the TMP.

[0135] For example, in some instances, the following pairs of residues in HLA β2-microglobulin and HLA class I heavy chains are present: 1) β2M residue 12, HLA class I heavy chain residue 236; 2) β2M residue 12, HLA class I heavy chain residue 237; 3) β2M residue 8, HLA class I heavy chain residue 234; 4) β2M residue 10, HLA class I heavy chain residue 235; 5) β2M residue 24, HLA class I heavy chain residue 236; 6) β2M residue 28, HLA class I heavy chain residue 232; 7) β2M residue 98, HLA class I heavy chain residue residue 192, 8) β2M residue 99, HLA class I heavy chain residue 234, 9) β2M residue 3, HLA class I heavy chain residue 120, 10) β2M residue 31, HLA class I heavy chain residue 96, 11) β2M residue 53, HLA class I heavy chain residue 35, 12) β2M residue 60, HLA class I heavy chain residue 96, 13) β2M residue 60, HLA class I heavy chain residue 122, 14) β2M residue 63, HLA class I heavy chain residue 27, 15) β2M residue Arg3, HLA class I heavy chain residue Gly120, 16) β2M residue Hi s31, HLA class I heavy chain residue Gln96, 17) β2M residue Asp53, HLA class I heavy chain residue Arg35, 18) β2M residue Trp60, HLA class I heavy chain residue Gln96, 19) β2M residue Trp60, HLA class I heavy chain residue Asp122, 20) β2M residue Tyr63, HLA class I heavy chain residue Tyr27, 21) β2M residue Lys6, HLA class I heavy chain residue Glu232, 22) β2M residue Gln8, HLA class I heavy chain residue Arg234, 23) β2M residue Tyr1 One of the following residues is substituted with cysteine: 1) HLA class I heavy chain residue Pro235; 2) β2M residue Ser11; 2) HLA class I heavy chain residue Gln242; 25) β2M residue Asn24; 26) β2M residue Ser28; 27) β2M residue Asp98; 28) β2M residue Met99; and 30) HLA class I heavy chain residue Arg234 (residue numbers refer to the mature polypeptide). The amino acid numbering of the MHC / HLA class I heavy chain is based on the mature MHC / HLA class I heavy chain without the signal peptide. For example, in some cases, residue 236 of the mature HLA-A amino acid sequence is substituted with Cys.In some instances, residue 236 of the mature HLA-B amino acid sequence is substituted with Cys. In some instances, residue 236 of the mature HLA-C amino acid sequence is substituted with Cys. In some instances, residue 32 of the amino acid sequence set forth in Figure 4 (corresponding to Arg-12 of mature β2M) is substituted with Cys.

[0136] In some examples, the β2M polypeptide comprises the amino acid sequence shown in Figure 21BB (SEQ ID NO: 128) or Figure 21CC (SEQ ID NO: 129).

[0137] In some examples, the HLA class I heavy chain polypeptide comprises the amino acid sequence shown in Figure 21DD (SEQ ID NO: 130), 21EE (SEQ ID NO: 131) or 21FF (SEQ ID NO: 132).

[0138] In some examples, the β2M polypeptide of a TMP of the present disclosure comprises the amino acid sequence of Figure 21CC (sequence number 133), and the HLA class I heavy chain polypeptide of the TMP comprises the amino acid sequence of Figure 21EE (sequence number 134) (the Cys residues underlined and shown in bold form disulfide bonds with each other within the TMP).

[0139] In some examples, the β2M polypeptide comprises the amino acid sequence of Figure 21GG (SEQ ID NO: 135).

[0140] In some examples, the MHC polypeptides in the heterodimer or single-chain TMP of the present disclosure are disulfide bonded to each other via i) a Cys residue present in the linker connecting the peptide epitope and the β2M polypeptide, and ii) a Cys residue present in the MHC class I heavy chain. In some examples, the Cys residue present in the MHC class I heavy chain is a Cys introduced as a Y84C substitution. In some examples, the linker connecting the peptide epitope and the β2M polypeptide is GCGGS(GGGGS)n (SEQ ID NO: 583), where n is 1, 2, 3, 4, 5, 6, 7, 8, or 9. For example, in some examples, the linker comprises the amino acid sequence GCGGSGGGGSGGGGSGGGGS (SEQ ID NO: 137). In another example, the linker comprises the amino acid sequence GCGGSGGGGSGGGGGS (SEQ ID NO: 138). Examples of disulfide-linked first and second polypeptides of heterodimeric TMPs of the present disclosure are shown schematically in Figures 2A-2F.

[0141] Multi-disulfide bond TMP In some examples, the first and second polypeptides of the heterodimeric TMP of the present disclosure are linked to each other by at least two disulfide bonds (i.e., two interchain disulfide bonds). Examples of such multiply disulfide-linked TMPs are shown schematically in Figures 12A and 12B and Figure 16C. Furthermore, when a heterodimeric TMP or single-chain TMP of the present disclosure includes an Ig Fc polypeptide, the heterodimeric TMP can be dimerized, with a disulfide bond linking the Ig Fc polypeptides in the two heterodimeric TMPs. Such an arrangement is shown schematically in Figures 12C and 12D, where the disulfide bonds are represented by dashed lines. Unless otherwise specified, the reference to at least two disulfide bonds in the description of a multiply disulfide-linked TMPP in this section does not refer to the disulfide bond linking the Ig Fc polypeptides of the dimerized TMP.

[0142] For example, in some instances, the first and second polypeptides of the heterodimeric TMP of the present disclosure are linked to each other by two interchain disulfide bonds. As another example, in some instances, the first and second polypeptides of the TMP of the present disclosure are linked to each other by three interchain disulfide bonds. As another example, in some instances, the first and second polypeptides of the TMP of the present disclosure are linked to each other by four interchain disulfide bonds.

[0143] In some examples, when a peptide epitope of a heterodimeric or single-chain TMP of the present disclosure is linked to a β2M polypeptide by a linker containing Cys, at least one of the at least two disulfide bonds links a Cys in the linker to a Cys in the MHC class I heavy chain in the TMP. In some examples, when a peptide epitope of a TMP of the present disclosure is linked to an MHC class I heavy chain polypeptide by a linker, at least one of the at least two disulfide bonds links a Cys in the linker to a Cys in the β2M polypeptide present in the TMP.

[0144] In some examples, a multiply disulfide bonded TMP (e.g., a double disulfide bonded TMP) of the present disclosure exhibits increased stability and / or expression compared to a control TMP containing only one of at least two disulfide bonds. In some examples, a multiply disulfide bonded TMP (e.g., a double disulfide bonded TMP) of the present disclosure exhibits increased in vitro stability compared to a control TMP containing only one of at least two disulfide bonds. For example, in some examples, a multiply disulfide bonded TMP (e.g., a double disulfide bonded TMP) of the present disclosure exhibits at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 50%, at least 2-fold, at least 5-fold, or at least 10-fold increased in vitro stability compared to a control TMP containing only one of at least two disulfide bonds.

[0145] Stability is determined by measuring the percentage of TMP remaining in solution after a specified time in solution at a specified temperature. Stability can be measured in vitro in PBS buffer containing 500 mM NaCl (described above) for a specified period of time at a specified temperature (e.g., in a solution at a temperature between 37°C and 42°C for a period of 1 hour to 28 days, e.g., 1 hour at 37°C, 1 day at 37°C, 5 days at 37°C, 1 hour at 42°C, 1 day at 42°C, 5 days at 42°C, 5 days at 37°C, 10 days at 37°C, 14 days at 37°C, 28 days at 37°C, etc.) compared to a control TMMP lacking at least one disulfide bond between the first and second polypeptides of the heterodimer. TMMP can be present in the PBS buffer at a concentration of 0.1 mg / mL to 10 mg / mL, for example, about 1 mL, about 2 mL, about 3 mL, about 4 mL, about 5 mL, about 6 mL, about 7 mL, about 8 mL, about 9 mL, or about 10 mL, and the buffer can be kept at 37°C or 42°C for 1 hour, 5 days, 10 days, 14 days, 21 days, or 28 days.

[0146] Whether a multiple disulfide bond TMP of the present disclosure exhibits increased in vitro stability compared to a control TMP containing only one of the at least two disulfide bonds can be determined by measuring the amount of each TMP present in samples held at, for example, 37°C and / or 42°C for 1 hour, 5 days, 10 days, 14 days, 21 days, or 28 days, as described above.

[0147] For example, in some instances, a multiply disulfide-bonded TMP (e.g., a double-disulfide-bonded TMP) of the present disclosure exhibits at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 50%, at least 2-fold, at least 5-fold, or at least 10-fold greater in vitro stability than a control TMP containing only one of the at least two disulfides when the TMP is stored at 37°C for a period of time (e.g., from about 1 week to about 2 weeks, from about 2 weeks to about 4 weeks, or from about 4 weeks to about 2 months). For example, in some instances, the amount of disulfide-linked heterodimeric TMP remaining after in vitro storage of a multiply disulfide-linked TMP of the present disclosure (e.g., a double-disulfide-linked TMP) at 37°C for 28 days is at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 50%, at least 2-fold, at least 5-fold, or at least 10-fold greater than the amount of disulfide-linked heterodimeric TMP remaining after in vitro storage of a control TMP (a TMP containing only one of the at least two disulfide bonds present in the multiply disulfide-linked TMP) at 37°C for 28 days.

[0148] In some instances, the multiple disulfide bond TMPs of the present disclosure exhibit greater in vivo stability than a control TMP comprising only one of the at least two disulfide bonds, e.g., in some instances, the multiple disulfide bond TMPs of the present disclosure exhibit at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 50%, at least 2-fold, at least 5-fold, or at least 10-fold greater in vivo stability than a control TMP comprising only one of the at least two disulfide bonds.

[0149] In some examples, the presence of two disulfide bonds in a multiply disulfide-bonded TMP of the present disclosure (e.g., a double-disulfide-bonded TMP) increases the amount of disulfide-bonded heterodimer or single-chain TMP produced compared to the amount of disulfide-bonded heterodimer TMP produced when the TMP is a control TMP containing only one of the at least two disulfide bonds. For example, a multiply disulfide-bonded TMP of the present disclosure (e.g., a double-disulfide-bonded TMP) can be produced in mammalian cells in an in vitro cell culture in which the mammalian cells are cultured in a liquid cell culture medium. The TMP can be secreted into the cell culture medium. The cells can be lysed to produce a cell lysate, and the TMP can be present in the cell lysate. The TMP can be purified from the cell culture medium and / or the cell lysate. For example, if the TMP comprises an IgG1 Fc polypeptide, the cell culture medium and / or cell lysate can be contacted with immobilized Protein A (e.g., the cell culture medium and / or cell lysate can be applied to a Protein A column in which Protein A has been immobilized on beads). TMP present in the cell culture medium and / or cell lysate binds to the immobilized Protein A. After washing the column to remove unbound material, the bound TMP is eluted to produce a Protein A eluate. The amount of disulfide-linked heterodimer or single-chain TMP present in the Protein A eluate is at least 0.5%, at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, or at least 10% higher than the amount of disulfide-linked heterodimer or single-chain TMP present in the Protein A eluate when the TMP is a control TMP that contains only one of the at least two disulfide bonds present in a multi-disulfide-linked TMP (e.g., a double-disulfide-linked TMP). In some examples, the percentage of total TMP protein in the eluate that is unaggregated disulfide-linked heterodimer or single-chain TMP is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%.The Protein A eluate may be subjected to size exclusion chromatography (SEC) and / or one or more other additional purification steps.

[0150] In some examples, the T cell modulatory polypeptides of the present disclosure comprise at least one heterodimer comprising: a) i) a KRAS peptide (KRAS peptides can be at least 4 amino acids in length, e.g., 4 amino acids to about 25 amino acids (e.g., 4 amino acids (aa), 5 aa, 6 aa, 7 aa, 8 aa, 9 aa, 10 aa, 11 aa, 12 aa, 13 aa, 14 aa, 15 aa, 16 aa, 17 aa, 18 aa, 19 aa, 20 aa, 21 aa, 22 aa, 23 aa, 24 aa, or 25 aa, and can be 4 to 20 amino acids, 6 to 18 amino acids, 8 to 15 amino acids, 8 to 12 amino acids, 5 to 10 amino acids, 10 to 20 amino acids, and 15 to 25 amino acids in length). a) a peptide within the range (b) and ii) a first polypeptide comprising a first MHC polypeptide, b) a second polypeptide comprising a second MHC polypeptide, and c) at least one immunomodulatory polypeptide (wherein the first and / or second polypeptide comprises an immunomodulatory polypeptide, and the heterodimer comprises at least two disulfide bonds (e.g., two disulfide bonds) between the first and second polypeptides (e.g., the heterodimer comprises i) a first disulfide bond linking the first and second polypeptides, and ii) a second disulfide bond linking the first and second polypeptides)). In other words, the first polypeptide contains a first Cys residue that forms a disulfide bond (first disulfide bond) with a first Cys residue of the second polypeptide, and the first polypeptide contains a second Cys residue that forms a disulfide bond (second disulfide bond) with a second Cys residue of the second polypeptide.

[0151] In some examples, a TMP of the present disclosure comprises a) a first polypeptide comprising, in order from N-terminus to C-terminus, i) a KRAS peptide, ii) a peptide linker, and iii) a β2M polypeptide; and b) a second polypeptide comprising an MHC class I heavy chain polypeptide, wherein one or both of the first and second polypeptides comprise at least one immunomodulatory polypeptide, wherein the TMP comprises a) a first disulfide bond between i) a Cys present in the linker between the KRAS peptide and the β2M polypeptide and ii) a first Cys introduced into the MHC class I heavy chain polypeptide, and b) at least a second disulfide bond between the first and second polypeptides, wherein the at least second disulfide bond is between i) a Cys in the first polypeptide that is C-terminal to the Cys present in the linker and ii) a Cys in the second polypeptide that is C-terminal to the first Cys introduced into the MHC class I heavy chain polypeptide.

[0152] Generally speaking, potential positions for disulfide bonds in heterodimers or single-chain TMP are those where residues of different polypeptides of TMP are separated by a distance of 5 angstroms or less. Such positions represent potential positions where a Cys residue, if not naturally occurring, can substitute for a residue present in the polypeptide. For example, the first and second polypeptides of a heterodimeric TMP can potentially be linked via a disulfide bond between two Cys residues that are generally within about 5 angstroms of each other in the heterodimer. In some cases, one or both of the Cys residues is non-naturally occurring. Amino acids within 5 angstroms of each other in the β2M and MHC heavy chains of a heterodimer or single-chain TMP represent amino acids that, when substituted with Cys, can form disulfide bonds in the TMPs of the present disclosure. Similarly, disulfide bonds can form between Cys residues in a linker and natural or non-natural Cys residues in an MHC heavy chain, where the two Cys residues are separated by no more than about 5 angstroms from each other. However, not all pairs of residues, particularly those separated by no more than about 5 angstroms, are suitable for disulfide bond formation or provide disulfide bonds that stabilize the resulting TMP or enhance expression.

[0153] The multiply disulfide-linked heterodimeric TMP (e.g., a double disulfide-linked TMP) of the present disclosure can include, for example, a) a first polypeptide comprising i) a KRAS peptide (e.g., a KRAS peptide having a length of 4 amino acids to about 25 amino acids, which, when complexed with an MHC polypeptide, is bound by a TCR); and ii) a first MHC polypeptide (the first polypeptide comprises a peptide linker between the KRAS peptide and the first MHC polypeptide, the peptide linker comprising a Cys residue, the first MHC polypeptide comprising a β2M polypeptide comprising an amino acid substitution introducing the Cys residue); and b) a second polypeptide comprising a second MHC polypeptide (the second MHC polypeptide comprises an HLA-A polypeptide, *and c) at least one immunomodulatory polypeptide, wherein the first and / or second polypeptide comprises at least one immunomodulatory polypeptide. Examples are shown schematically in Figures 12A and 12B.

[0154] In some examples, the peptide linker comprises the amino acid sequence GCGGS (SEQ ID NO: 139). In some examples, the peptide linker comprises the amino acid sequence GCGGS(GGGGS)n (SEQ ID NO: 140), where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, e.g., 1, 2, or 3.

[0155] In some examples, the peptide linker comprises the amino acid sequence CGGGS (SEQ ID NO: 141). In some examples, the peptide linker comprises the amino acid sequence CGGGS(GGGGS)n (SEQ ID NO: 142), where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, e.g., 1, 2, or 3.

[0156] In some examples, the peptide linker comprises the amino acid sequence GGCGS (SEQ ID NO: 587). In some examples, the peptide linker comprises the amino acid sequence GGCGS(GGGGS)n (SEQ ID NO: 592), where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, e.g., 1, 2, or 3.

[0157] In some examples, the peptide linker comprises the amino acid sequence GGGCS (SEQ ID NO: 588). In some examples, the peptide linker comprises the amino acid sequence GGGCS(GGGGS)n (SEQ ID NO: 589), where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, e.g., 1, 2, or 3.

[0158] In some examples, the peptide linker comprises the amino acid sequence GGGGC (SEQ ID NO: 590). In some examples, the peptide linker comprises the amino acid sequence GGGGC(GGGGS)n (SEQ ID NO: 591), where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, e.g., 1, 2, or 3.

[0159] The following are HLA-A * 7A-7C are non-limiting examples of MHC class I heavy chains containing the Y84C and A236C substitutions based on the amino acid numbering of 0201 (shown in FIG. 7A), or substitutions at the corresponding positions in another class I heavy chain allele.

[0160] HLA-A In some examples, the multiple disulfide-linked heterodimer or single-chain TMP (e.g., a double disulfide-linked TMP) of the present disclosure comprises: i) a KRAS peptide (e.g., a KRAS peptide between 4 amino acids and 25 amino acids in length, which TCR binds when the peptide is complexed with an MHC polypeptide, e.g., the KRAS peptide comprises a cancer-associated mutation); ii) a first MHC polypeptide; iii) a peptide linker between the peptide and the first MHC polypeptide (the peptide linker comprises a Cys residue, and the first MHC polypeptide is a β2M polypeptide comprising an amino acid substitution introducing a Cys residue); and iv) an HLA-A polypeptide comprising an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence shown in Figure 22A (SEQ ID NO: 143). and a second polypeptide comprising an MHC class I heavy chain (amino acid 84 is Cys and amino acid 236 is Cys), and the TMP comprises at least one immunomodulatory polypeptide. In some examples, the peptide linker comprises the amino acid sequence GCGGS (SEQ ID NO: 139). In some examples, the peptide linker comprises the amino acid sequence GCGGS(GGGGS)n (SEQ ID NO: 140), where n is an integer between 1 and 10, e.g., 1, 2, or 3. In some examples, the β2M polypeptide comprises an R12C substitution. For example, the β2M polypeptide can comprise an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence shown in Figure 21CC (SEQ ID NO: 144), where amino acid 12 is Cys. The at least one immunomodulatory polypeptide can be a wild-type or variant of an immunomodulatory polypeptide, e.g., selected from the group consisting of a cytokine (e.g., IL-2), a 4-1BBL polypeptide, a CD80 polypeptide, a CD86 polypeptide, or a combination thereof. In some examples, the at least one immunomodulatory polypeptide is a reduced affinity variant, e.g., a reduced affinity variant of IL-2, as described elsewhere herein.In some examples, the TMP comprises an Ig Fc polypeptide, for example, a human IgG1 Fc, which does not substantially induce cell lysis as shown in Figure 3G.

[0161] In some examples, the multiply disulfide-bonded heterodimer or single-chain TMP (e.g., double-disulfide-bonded TMP) of the present disclosure comprises an HLA-A class I heavy chain polypeptide. In some examples, the HLA-A heavy chain polypeptide present in the multiply disulfide-bonded TMP (e.g., double-disulfide-bonded TMP) of the present disclosure comprises an HLA-A class I heavy chain polypeptide shown in FIG. 7A. * 0101, HLA-A * 0201, HLA-A * 0202, HLA-A * 1101, HLA-A * 2301, HLA-A * 2402, HLA-A * 2407, HLA-A * 3303, or HLA-A * 3401, and the HLA-A heavy chain polypeptide comprises a Y84C and an A236C substitution.

[0162] In some examples, the HLA-A heavy chain polypeptide present in a multiple disulfide-linked heterodimer or single-chain TMP (e.g., a double-disulfide-linked TMP) of the present disclosure comprises an amino acid sequence having at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to one of the following sequences: (i) HLA-A shown in Figure 22B (SEQ ID NO: 145) * 0101 (Y84C, A236C) amino acid sequence (amino acid 84 is Cys and amino acid 236 is Cys), (ii) HLA-A shown in Figure 22C (SEQ ID NO: 146) * 0201 (Y84C, A236C) amino acid sequence (amino acid 84 is Cys and amino acid 236 is Cys), (iii) HLA-A shown in Figure 22D (SEQ ID NO: 147)* 0202 (Y84C, A236C) amino acid sequence (amino acid 84 is Cys and amino acid 236 is Cys), (iv) HLA-A shown in Figure 22E (SEQ ID NO: 148) * 1101(Y84C, A236C) amino acid sequence (amino acid 84 is Cys and amino acid 236 is Cys), (v) HLA-A shown in Figure 22F (SEQ ID NO: 149) * 2301 (Y84C, A236C) amino acid sequence (amino acid 84 is Cys and amino acid 236 is Cys), (vi) HLA-A shown in Figure 22G (SEQ ID NO: 150) * 2402 (Y84C, A236C) amino acid sequence (amino acid 84 is Cys and amino acid 236 is Cys), (vii) HLA-A shown in Figure 22H (SEQ ID NO: 151) * 2407 (Y84C, A236C) amino acid sequence (amino acid 84 is Cys and amino acid 236 is Cys), (viii) HLA-A shown in Figure 22I (SEQ ID NO: 152) * 3303 (Y84C, A236C) amino acid sequence (amino acid 84 is Cys and amino acid 236 is Cys), (ix) HLA-A shown in Figure 22J (SEQ ID NO: 153) * 3401 (Y84C, A236C) amino acid sequence (amino acid 84 is Cys and amino acid 236 is Cys).

[0163] HLA-B In some examples, a multiple disulfide-linked heterodimer or single-chain TMP (e.g., a double-disulfide-linked TMP) of the present disclosure includes: i) a KRAS peptide (to which a TCR binds when the peptide is complexed with an MHC polypeptide of the TMP); ii) a β2M polypeptide including a non-naturally occurring Cys residue; iii) a peptide linker between the KRAS peptide and the β2M polypeptide; and iv) an HLA-B MHC class I heavy chain polypeptide including an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence shown in Figure 22K (SEQ ID NO: 154), where amino acid 84 is Cys and amino acid 236 is Cys. In some examples, the peptide linker includes the amino acid sequence GCGGS (SEQ ID NO: 139). In some examples, the peptide linker comprises the amino acid sequence GCGGS(GGGGS)n (SEQ ID NO: 140), where n is an integer between 1 and 10, e.g., 1, 2, or 3. In some examples, the β2M polypeptide comprises an R12C substitution. For example, the β2M polypeptide can comprise an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence of Figure 21CC (SEQ ID NO: 155) (amino acid 12 is Cys). The at least one immunomodulatory polypeptide of the TMP can be, for example, a wild-type or variant immunomodulatory polypeptide selected from the group consisting of a cytokine (e.g., IL-2), 4-1BBL, CD80, CD86, and combinations thereof. In some examples, the at least one immunomodulatory polypeptide is a low-affinity variant, such as an IL-2 variant, as described elsewhere herein. In some examples, the TMP comprises an Ig Fc polypeptide, for example, a variant human IgG1 Fc polypeptide that does not substantially induce cell lysis.

[0164] In some examples, the multiply disulfide-bonded heterodimer or single-chain TMP of the present disclosure comprises an HLA-B class I heavy chain polypeptide. In some examples, the HLA-B heavy chain polypeptide present in the multiply disulfide-bonded TMP (e.g., double disulfide-bonded TMP) of the present disclosure comprises an HLA-B class I heavy chain polypeptide shown in FIG. 8A. * 0702, HLA-B * 0801, HLA-B * 1502, HLA-B * 3802, HLA-B * 4001, HLA-B * 4601, or HLA-B * The HLA-B heavy chain polypeptide comprises an amino acid sequence having at least 95%, at least 98%, or at least 99% amino acid sequence identity to the 5301 amino acid sequence, and the HLA-B heavy chain polypeptide comprises a Y84C and an A236C substitution.

[0165] HLA-B * 0702(Y84C, A236C) In some examples, the HLA-B heavy chain polypeptide present in a multiple disulfide-bonded heterodimer or single-chain TMP (e.g., a double-disulfide-bonded TMP) of the present disclosure comprises an amino acid sequence having at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following sequence (amino acid 84 is Cys and amino acid 236 is Cys): (i) HLA-B as shown in Figure 22L (SEQ ID NO: 156) * 0702 (Y84C, A236C) amino acid sequence (amino acid 84 is Cys and amino acid 236 is Cys), (ii) HLA-B as shown in Figure 22M (SEQ ID NO: 157) * 0801 (Y84C, A236C) amino acid sequence (amino acid 84 is Cys and amino acid 236 is Cys), (iii) HLA-B as shown in Figure 22N (SEQ ID NO: 158) * 1502 (Y84C, A236C) amino acid sequence (amino acid 84 is Cys and amino acid 236 is Cys), (iv) HLA-B as shown in Figure 22O (SEQ ID NO: 159)* 3802 (Y84C, A236C) amino acid sequence (amino acid 84 is Cys and amino acid 236 is Cys), (v) HLA-B as shown in Figure 22P (SEQ ID NO: 160) * 4001 (Y84C, A236C) amino acid sequence (amino acid 84 is Cys and amino acid 236 is Cys), (vi) HLA-B as shown in Figure 22Q (SEQ ID NO: 161) * 4601 (Y84C, A236C) amino acid sequence (amino acid 84 is Cys and amino acid 236 is Cys), (vii) HLA-B as shown in Figure 22R (SEQ ID NO: 162) * 5301 (Y84C, A236C) amino acid sequence (amino acid 84 is Cys and amino acid 236 is Cys).

[0166] HLA-C In some examples, a multiple disulfide-linked heterodimer or single-chain TMP (e.g., a double-disulfide-linked TMP) of the present disclosure includes: i) a KRAS peptide (to which a TCR binds when the peptide is complexed with an MHC polypeptide of the TMP); ii) a β2M polypeptide including a non-naturally occurring Cys residue; iii) a peptide linker between the KRAS peptide and the β2M polypeptide; and iv) an HLA-CMHC class I heavy chain amino acid sequence including an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence shown in Figure 22S (SEQ ID NO: 163) (amino acid 84 is Cys and amino acid 236 is Cys); and c) at least one immunomodulatory polypeptide (the first and / or second polypeptides include at least one immunomodulatory polypeptide). In some examples, the peptide linker includes the amino acid sequence GCGGS (SEQ ID NO: 139). In some examples, the peptide linker comprises the amino acid sequence GCGGS(GGGGS)n (SEQ ID NO: 140), where n is an integer between 1 and 10, e.g., 1, 2, or 3. In some examples, the β2M polypeptide comprises an R12C substitution. For example, the β2M polypeptide can comprise an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence shown in Figure 21CC (SEQ ID NO: 164) (amino acid 12 is Cys). The at least one immunomodulatory polypeptide comprises, for example, a wild-type or variant immunomodulatory polypeptide selected from the group consisting of a cytokine (e.g., IL-2), a 4-1BBL polypeptide, a CD80 polypeptide, a CD86 polypeptide, or a combination thereof. In some examples, the at least one immunomodulatory polypeptide is a reduced affinity variant, e.g., a reduced affinity variant of IL-2, as described elsewhere herein. In some examples, the TMP comprises an Ig Fc polypeptide, for example, a variant human IgG1 Fc polypeptide that does not substantially induce cell lysis.

[0167] In some examples, the multiply disulfide-bonded TMP (e.g., double disulfide-bonded TMP) of the present disclosure comprises an HLA-C class I heavy chain polypeptide. In some examples, the HLA-C heavy chain polypeptide present in the multiply disulfide-bonded TMP (e.g., double disulfide-bonded TMP) of the present disclosure comprises an HLA-C class I heavy chain polypeptide shown in FIG. 9A. * 0102, HLA-C * 0303, HLA-C * 0304, HLA-C * 0401, HLA-C * 0602, HLA-C * 0701, HLA-C * 0702, HLA-C * 0801, or HLA-C * The HLA-C heavy chain polypeptide comprises an amino acid sequence having at least 95%, at least 98%, or at least 99% amino acid sequence identity to the 1502 amino acid sequence, and the HLA-C heavy chain polypeptide comprises a Y84C and an A236C substitution.

[0168] In some examples, the HLA-C heavy chain polypeptide present in a multiply disulfide-bonded TMP (e.g., a double-disulfide-bonded TMP) of the present disclosure comprises an amino acid sequence having at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to one of the following sequences: (i) HLA-C shown in Figure 22T (SEQ ID NO: 165) * 01:02 (Y84C, A236C) amino acid sequence (amino acid 84 is Cys and amino acid 236 is Cys) (ii) HLA-C shown in Figure 22U (SEQ ID NO: 166) * 03:03 (Y84C, A236C) amino acid sequence (amino acid 84 is Cys and amino acid 236 is Cys) (iii) HLA-C shown in Figure 22V (SEQ ID NO: 167) * 03:04 (Y84C, A236C) amino acid sequence (amino acid 84 is Cys and amino acid 236 is Cys) (iv) HLA-C shown in Figure 22W (SEQ ID NO: 168) *04:01 (Y84C, A236C) amino acid sequence (amino acid 84 is Cys and amino acid 236 is Cys) (v) HLA-C as shown in Figure 22X (SEQ ID NO: 169) * 06:02 (Y84C, A236C) amino acid sequence (amino acid 84 is Cys and amino acid 236 is Cys) (vi) HLA-C shown in Figure 22Y (SEQ ID NO: 170) * 07:01 (Y84C, A236C) amino acid sequence (amino acid 84 is Cys and amino acid 236 is Cys) (vii) HLA-C shown in Figure 22Z (SEQ ID NO: 171) * 07:02 (Y84C, A236C) amino acid sequence (amino acid 84 is Cys and amino acid 236 is Cys) (viii) HLA-C shown in Figure 22AA (SEQ ID NO: 172) * 08:01 (Y84C, A236C) amino acid sequence (amino acid 84 is Cys and amino acid 236 is Cys), and (ix) HLA-C shown in Figure 22BB (SEQ ID NO: 173) * 15:02 (Y84C, A236C) amino acid sequence (amino acid 84 is Cys and amino acid 236 is Cys).

[0169] Disulfide-linked TMP presenting antigen (i.e., TMP lacking MOD) The present disclosure provides an antigen-presenting polypeptide (APP) comprising a heterodimer or single-chain polypeptide (or a homodimer of two such polypeptides), the APP comprising: i) a KRAS peptide (which peptide is bound by a TCR when complexed with an MHC polypeptide of TMP), ii) a β2M polypeptide, optionally comprising a non-naturally occurring Cys residue, iii) a peptide linker between the KRAS peptide and the β2M polypeptide, which linker optionally comprises a Cys residue, and iv) an HLA-C MHC class I heavy chain comprising an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to any of the amino acid sequences shown in Figures 21A-21AA, 21DD-21FF, 21HH-21JJ, or 22A-22BB. The heterodimer or single-chain APP has the same configuration as either the multiply disulfide-bonded heterodimer or single-chain TMP described above, and can be single- or multiply disulfide-bonded in the same manner as either the multiply disulfide-bonded heterodimer or single-chain TMP described above, the only difference being that, unlike the TMP described above, APP does not contain a MOD. As noted above, the disulfide bonds referred to herein are not intended to refer to disulfide bonds between Ig Fc polypeptides of APP, such as the human IgG1 Fc polypeptide of Figure 3G, which do not substantially induce cell lysis.

[0170] Examples of heterodimeric APP include a) an APP comprising i) the "4027" polypeptide shown in Figure 19O, and ii) the "4030" polypeptide shown in Figure 14Q, and b) an APP comprising i) the "4027" polypeptide shown in Figure 19O, and ii) the "4029" polypeptide shown in Figure 14K. Examples of single-chain APP include the polypeptides designated "4238" (Figure 19G), "4241" (Figure 19H), and "4334" (Figure 19I).

[0171] The APPs of the present disclosure are useful for diagnostic and therapeutic applications. As described below, when used for diagnostic applications, the APPs can also contain a detectable label, whereby binding of the APP to target T cells is detected by detecting the detectable label.

[0172] Therefore, the present disclosure provides a method for detecting antigen-specific T cells. The method includes contacting T cells with the APP of the present disclosure and detecting the binding of the APP to the T cells. The present disclosure provides a method for detecting antigen-specific T cells, the method includes contacting T cells with the APP of the present disclosure, and the binding of the APP to the T cells indicates that the T cells are specific for an epitope present in the APP.

[0173] In some examples, APP comprises detectable label.Suitable detectable label includes but is not limited to radioisotope, fluorescent polypeptide, or the enzyme that produces fluorescent product and the enzyme that produces colored product.When APP comprises detectable label, the binding of APP to T cell is detected by detecting detectable label.

[0174] In some examples, the APPs of the present disclosure comprise a detectable label suitable for use in in vivo imaging, such as positron emission tomography (PET), single photon emission computed tomography (SPECT), near-infrared (NIR) optical imaging, X-ray imaging, computed tomography (CAT), magnetic resonance imaging (MRI), or other in vivo imaging methods. Examples of labels suitable for in vivo imaging include gadolinium chelators (e.g., gadolinium chelators including DTPA (diethylenetriaminepentaacetic acid), DTPA-bismethylamide (BMA), DOTA (dodecanetetraacetic acid), or HP-DO3A (1,4,7-tris(carboxymethyl)-10-(2'-hydroxypropyl)-1,4,7,10-tetraazacyclidodecane)), iron chelators, magnesium chelators, manganese chelators, copper chelators, chromium chelators, 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, and 18 In some examples, 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 I. In some instances, the detectable label is 64Cu. See, for example, Woodham, Andrew et al., In vivo detection of antigen-specific CD8+ T cells by immunopositron emission tomography, Nature Methods Articles (2020) https: / / doi.org / 10.1038 / s41592-020-0934-5.

[0175] Suitable fluorescent proteins include green fluorescent protein (GFP) or variants thereof, blue fluorescent variants of GFP (BFP), cyan fluorescent variants of GFP (CFP), yellow fluorescent variants of GFP (YFP), enhanced GFP (EGFP), enhanced CFP (ECFP), enhanced YFP (EYFP), GFPS65T, Emerald, Topaz (TYFP), Venus, Citrine, mCitrine, GFPuv, destabilized EGFP (dEGFP), destabilized ECFP (dECFP), destabilized EYFP (dEYFP), mCFPm, Cerulean, T-Sapphire, CyPet, YPet, mKO, HcRed, t-HcRed, DsRed, DsRed2, DsRed monomer, J-Red, dimer2, t-dimer2(12), mRFP1, pocilloporin, Renilla GFP, and Monster. Phycobiliproteins and phycobiliprotein complexes, including, but not limited to, GFP, paGFP, Kaede protein and kindling protein, B-phycoerythrin, R-phycoerythrin, and allophycocyanin, are also suitable. Other examples of fluorescent proteins include mHoneydew, mBanana, mOrange, dTomato, tdTomato, mTangerine, mStrawberry, mCherry, mGrape1, mRaspberry, mGrape2, and mPlum (Shaner et al. (2005) Nat. Methods 2:905-909). Suitable for use are any of the various fluorescent and colored proteins from anthozoans, as described, for example, in Matz et al. (1999) Nature Biotechnol. 17:969-973.

[0176] Suitable enzymes include 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.

[0177] In some examples, the binding of APP to T cell is detected by using detectably labeled APP-specific antibody.APP-specific antibody can comprise detectable label, such as radioisotope, fluorescent polypeptide, or the enzyme that produces fluorescent product or the enzyme that produces colored product.

[0178] In some examples, the T cells to be detected are present in a sample containing a plurality of T cells. For example, the T cells to be detected are present in a sample containing 10 to 10 9 T cells, e.g., 10–10 2 pieces, 10 2 ~10 pieces 4 pieces, 10 4 ~10 pieces 6 pieces, 10 6 ~10 pieces 7 pieces, 10 7 ~10 pieces 8 pieces or 10 8 ~10 pieces 9 pieces or 10 9 The antibody may be present in a sample containing more than one T cell.

[0179] HLA / peptide binding assay Whether a given 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 and in published PCT application WO2020132138A1, the disclosures of which are expressly incorporated herein by reference, particularly as they relate to specific binding assays, including paragraphs

[0217] -

[0225] .

[0180] In another example, multimers (e.g., tetramers) of peptide-HLA complexes are generated with fluorescent or heavy metal tags. The multimers can then be used to identify and quantify specific T cells via flow cytometry (FACS) or mass cytometry (CyTOF). Detection of epitope-specific T cells provides direct evidence that peptide-bound HLA molecules can bind to specific TCRs on a subset of antigen-specific T cells. See, for example, Klenerman et al. (2002) Nature Reviews Immunol. 2:263.

[0181] Immunomodulatory Polypeptides In some examples, an immunomodulatory polypeptide, or "MOD," present in a TMP of the present disclosure is a wild-type immunomodulatory polypeptide. In other examples, an immunomodulatory polypeptide present in a TMP of the present disclosure is a variant immunomodulatory polypeptide that has reduced affinity for a costimulatory polypeptide compared to the affinity of a corresponding wild-type immunomodulatory polypeptide for that costimulatory polypeptide. Suitable immunomodulatory domains that exhibit reduced affinity for costimulatory domains can have a difference of 1 amino acid (aa) to 20 aa from the wild-type immunomodulatory domain. For example, in some examples, a variant immunomodulatory polypeptide present in a TMP of the present disclosure differs in amino acid sequence from the corresponding wild-type immunomodulatory polypeptide by 1 aa, 2 aa, 3 aa, 4 aa, 5 aa, 6 aa, 7 aa, 8 aa, 9 aa, or 10 aa. As another example, in some instances, a variant immunomodulatory polypeptide present in a TMP of the present disclosure differs in amino acid sequence from the corresponding wild-type immunomodulatory polypeptide by 11 aa, 12 aa, 13 aa, 14 aa, 15 aa, 16 aa, 17 aa, 18 aa, 19 aa, or 20 aa.

[0182] Exemplary pairs of immunomodulatory polypeptides and their cognate costimulatory polypeptides include, but are not limited to, those pairs set forth in Table 1 below.

[0183] (Table 1) TIFF0007756072000006.tif145128

[0184] In some examples, variant immunomodulatory polypeptides present in TMPs of the disclosure have a binding affinity for their cognate costimulatory polypeptides of 100 nM to 100 μM. For example, in some examples, variant immunomodulatory polypeptides present in TMPs of the disclosure have a binding affinity for their cognate costimulatory polypeptides of about 100 nM to 150 nM, about 150 nM to about 200 nM, about 200 nM to about 250 nM, about 250 nM to about 300 nM, about 300 nM to about 350 nM, about 350 nM to about 400 nM, about 400 nM to about 500 nM, about 500 nM to about 60 The binding affinity is about 0 nM, about 600 nM to about 700 nM, about 700 nM to about 800 nM, about 800 nM to about 900 nM, about 900 nM to about 1 μM, about 1 μM to about 5 μM, about 5 μM to about 10 μM, about 10 μM to about 15 μM, about 15 μM to about 20 μM, about 20 μM to about 25 μM, about 25 μM to about 50 μM, about 50 μM to about 75 μM, or about 75 μM to about 100 μM.

[0185] Variant immunomodulatory polypeptides present in TMPs of the present disclosure exhibit reduced affinity for cognate costimulatory polypeptides. Similarly, TMPs of the present disclosure comprising variant immunomodulatory polypeptides exhibit reduced affinity for cognate costimulatory polypeptides. Thus, for example, TMPs of the present disclosure comprising variant immunomodulatory polypeptides have binding affinities of 100 nM to 100 μM for cognate costimulatory polypeptides. For example, in some examples, TMPs of the present disclosure comprising variant immunomodulatory polypeptides have binding affinities of about 100 nM to 150 nM, about 150 nM to about 200 nM, about 200 nM to about 250 nM, about 250 nM to about 300 nM, about 300 nM to about 350 nM, about 350 nM to about 400 nM, about 400 nM to about 500 nM, or about 500 nM to about 600 nM for cognate costimulatory polypeptides. M, about 600 nM to about 700 nM, about 700 nM to about 800 nM, about 800 nM to about 900 nM, about 900 nM to about 1 μM, about 1 μM to about 5 μM, about 5 μM to about 10 μM, about 10 μM to about 15 μM, about 15 μM to about 20 μM, about 20 μM to about 25 μM, about 25 μM to about 50 μM, about 50 μM to about 75 μM, or about 75 μM to about 100 μM.

[0186] As shown generally in Figure 17, the immunomodulatory polypeptide (i.e., one or more immunomodulatory polypeptides) can be present at any of a variety of positions in the heterodimeric TMP of the present disclosure. While Figure 17 shows the location of two copies of the variant IL-2 polypeptide, the immunomodulatory polypeptide can be any number of any variety of immunomodulatory polypeptides as described herein. As shown in Figure 17, the immunomodulatory polypeptide can be 1) N-terminal to the MHC class I heavy chain, 2) C-terminal to the MHC class I heavy chain and N-terminal to the Ig Fc polypeptide, i.e., between the MHC class I heavy chain and the Ig Fc polypeptide, 3) C-terminal to the Ig Fc polypeptide, 4) N-terminal to the peptide epitope, or 5) C-terminal to the β2M polypeptide.

[0187] As shown generally in Figure 18, the immunomodulatory polypeptide (i.e., one or more immunomodulatory polypeptides) can be present in any of a variety of positions in the single-chain TMP of the present disclosure. While Figure 18 shows the location of two copies of a variant IL-2 polypeptide, the immunomodulatory polypeptide can be any number of any variety of immunomodulatory polypeptides as described herein. As shown in Figure 18, the immunomodulatory polypeptide can be 1) C-terminal to the MHC class I heavy chain and N-terminal to the Ig Fc polypeptide, 2) C-terminal to the Ig Fc polypeptide, or 3) N-terminal to the peptide epitope.

[0188] Immunomodulatory polypeptides and variants (including low affinity variants), such as PD-L1, CD80, CD86, 4-1BBL, and IL-2, are described in the published literature, for example, published PCT applications WO2020132138A1 and WO2019 / 051091, the disclosures of which are expressly incorporated by reference herein, including paragraphs

[0260] to

[0455] of WO2020132138A1 and paragraphs

[0157] to

[0352] of WO2019 / 051091, as they relate to immunomodulatory polypeptides of PD-L1, CD80, CD86, 4-1BBL, IL-2 and certain variant immunomodulatory polypeptides.

[0189] MOD, a variant of the cytokine IL-2, is of particular interest. 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, binding and activating most or virtually all CD8+ T cells. For this reason, synthetic forms of wild-type IL-2, such as the drug aldesleukin (trade name Proleukin®), are known to have severe side effects when administered to humans for the treatment of cancer, due to their indiscriminate activation of both target and non-target T cells.

[0190] The IL-2 receptor is, in some instances, a heterotrimeric polypeptide comprising an α chain (IL-2Rα, also known as CD25), a β chain (IL-2Rβ, also known as CD122), and a γ chain (IL-2Rγ, also known as CD132). The amino acid sequences of human IL-2 (SEQ ID NO: 15), human IL-2Rα (SEQ ID NO: 16), IL2Rβ (SEQ ID NO: 17), and IL-2Rγ (SEQ ID NO: 18) are known. See, e.g., the above-mentioned published PCT applications WO2020132138A1 and WO2019 / 051091.

[0191] In some examples, an IL-2 variant MOD of the disclosure exhibits substantially reduced or no binding to IL-2Rα, thereby minimizing or substantially reducing Treg activation by the IL-2 variant. In some examples, an IL-2 variant MOD of the disclosure exhibits reduced affinity for IL-2Rβ and / or IL-2Rγ, thereby causing the IL-2 variant MOD to exhibit lower overall affinity for IL-2R. In some examples, an IL-2 variant MOD of the disclosure exhibits both properties, i.e., substantially reduced or no binding to IL-2Rα and reduced affinity for IL-2Rβ and / or IL-2Rγ, causing the IL-2 variant polypeptide to exhibit lower overall affinity for IL-2R. TMPs containing such variants, including variants that do not substantially bind to IL-2Rα and have reduced affinity for IL-2Rβ, have demonstrated the ability to preferentially bind to and activate IL-2 receptors on T cells having a target TCR specific for a peptide epitope on the TMP, but are therefore unlikely to deliver IL-2 to non-target T cells, i.e., T cells that do not have a TCR that specifically binds to a peptide epitope on the TMP. That is, binding of IL-2 variant MODs to costimulatory polypeptides on T cells is substantially driven by binding of the MHC-epitope portion, rather than by binding of IL-2.

[0192] Thus, suitable IL-2 variant MODs include polypeptides comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity for IL-2R to the amino acid sequence set forth in SEQ ID NO: 15. In some examples, such variant IL-2 polypeptides of the disclosure exhibit reduced binding affinity for IL-2R compared to the binding affinity of an IL-2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 15. For example, in some examples, the variant IL-2 polypeptide binds to IL-2R with a binding affinity that is at least 10% lower, at least 15% lower, at least 20% lower, at least 25% lower, at least 30% lower, at least 35% lower, at least 40% lower, at least 45% lower, at least 50% lower, at least 55% lower, at least 60% lower, at least 65% lower, at least 70% lower, at least 75% lower, at least 80% lower, at least 85% lower, at least 90% lower, at least 95% lower, or greater than 95% lower than the binding affinity of an IL-2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 15 to IL-2R (e.g., an IL-2R comprising a polypeptide comprising the amino acid sequence set forth in SEQ ID NOs: 16-18), when assayed under the same conditions. In some examples, such variant IL-2 polypeptides have a binding affinity for IL-2R of 100 nM to 100 μM. As another example, in some cases, the variant IL-2 polypeptide has an activity of about 100 nM to 150 nM, about 150 nM to about 200 nM, about 200 nM to about 250 nM, about 250 nM to about 300 nM, about 300 nM to about 350 nM, about 350 nM to about 400 nM, or about 400 nM to about 500 nM relative to IL-2R (e.g., IL-2R comprising a polypeptide comprising the amino acid sequence set forth in SEQ ID NOs: 16 to 18). nM, about 500 nM to about 600 nM, about 600 nM to about 700 nM, about 700 nM to about 800 nM, about 800 nM to about 900 nM, about 900 nM to about 1 μM, about 1 μM to about 5 μM, about 5 μM to about 10 μM, about 10 μM to about 15 μM, about 15 μM to about 20 μM, about 20 μM to about 25 μM, about 25 μM to about 50 μM, about 50 μM to about 75 μM, or about 75 μM to about 100 μM.

[0193] In some instances, a suitable variant IL-2 polypeptide has the amino acid sequence: The 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 TIFF0007756072000007.tif17154 (SEQ ID NO: 241), i.e., the variant IL-2 polypeptide has the amino acid sequence of wild-type IL-2 but with H16A and F42A substitutions (shown in bold). Alternatively, the foregoing sequences may be used but with substitutions other than Ala at H16 and / or F42, e.g., H16T may be used in place of H16A.

[0194] Scaffold Polypeptide The TMPs of the present disclosure may comprise an Fc polypeptide or another suitable scaffold polypeptide.

[0195] 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 comprising pentapeptide repeat units of (Val-Pro-Gly-X-Gly, SEQ ID NO: 561) (wherein 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.

[0196] In some cases, a suitable scaffold polypeptide is a polypeptide that extends half-life. Thus, in some cases, a suitable scaffold polypeptide extends the in vivo half-life (e.g., serum half-life) of TMP compared to a control TMP lacking the scaffold polypeptide. For example, in some cases, the scaffold polypeptide extends the in vivo half-life (e.g., serum half-life) of TMP by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 50%, at least about 2-fold, at least about 2.5-fold, at least about 5-fold, at least about 10-fold, at least about 25-fold, at least about 50-fold, at least about 100-fold, or more than 100-fold compared to a control TMP lacking the scaffold polypeptide. As an example, in some instances, the Fc polypeptide extends 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, compared to a control TMP lacking the Fc polypeptide.

[0197] Fc polypeptide In some examples, the first and / or second polypeptide chains of a TMP of the present disclosure comprise an Fc polypeptide. The Fc polypeptide of a TMP of the present disclosure may be human IgG1 Fc, human IgG2 Fc, human IgG3 Fc, human IgG4 Fc, etc. In some examples, 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 amino acid sequence of the Fc region set forth in Figures 3A-3G. In some examples, the Fc region 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 depicted in FIG. 3A , and optionally includes a substitution of N77, e.g., the Fc polypeptide includes an N77A substitution. In some examples, 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 depicted in FIG. 3A, 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 to 325 of the human IgG2 Fc polypeptide depicted in FIG. 3A.In some examples, the Fc polypeptide comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to the human IgG3 Fc polypeptide depicted in FIG. 3A , 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 to 246 of the human IgG3 Fc polypeptide depicted in FIG. 3A . In some examples, 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 depicted in Figure 3B, 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 depicted in Figure 3B. In some examples, 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 depicted in Figure 3C, 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 depicted in Figure 3C.

[0198] In some examples, 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 3 C. In some examples, 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 to 327 of the human IgG4 Fc polypeptide shown in Figure 3 C.

[0199] In some examples, the IgG4 Fc polypeptide has the following amino acid sequence: PPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEK TISKAKGQPREPQVYTLPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 175).

[0200] Typically, the Ig Fc used in a TMP contains one or more amino acid substitutions in the wild-type sequence, such that the Ig Fc "does not substantially induce cell lysis." For example, in some instances, the Fc polypeptide present in a TMP contains the amino acid sequence shown in Figure 3A (human IgG1 Fc), except for the substitution of L234 (L14 in the amino acid sequence shown in Figure 3A) with an amino acid other than leucine, or the substitution of L235 (L15 in the amino acid sequence shown in Figure 3A) with an amino acid other than leucine.

[0201] In some examples, the Fc polypeptide present in TMP comprises the amino acid sequence shown in Figure 3G (human IgG1 Fc including an L234A substitution and an L235A substitution corresponding to positions 14 and 15 of the amino acid sequence shown in Figure 3G). In some examples, the Fc polypeptide present in TMP comprises the amino acid sequence shown in Figure 3A (human IgG1 Fc), except for the substitutions of amino acids other than leucine at L234 and L235 (L14 and L15 of the amino acid sequence shown in Figure 3A). In some examples, the Fc polypeptide present in TMP comprises the amino acid sequence shown in Figure 3A (human IgG1 Fc), except for the substitutions of amino acids other than leucine at L234 and L235 (L14 and L15 of the amino acid sequence shown in Figure 3A) and the substitution of amino acid P331 (P111 of the amino acid sequence shown in Figure 3A) with an amino acid other than proline. In some examples, the Fc polypeptide present in the TMP comprises the amino acid sequence shown in Figure 3E (human IgG1 Fc containing L234F, L235E, and P331S substitutions (corresponding to amino acids 14, 15, and 111 of the amino acid sequence shown in Figure 3E). In some examples, the Fc polypeptide present in the TMP is an IgG1 Fc polypeptide containing L234A and L235A substitutions (substitution of L14 and L15 of the amino acid sequence shown in Figure 3A with Ala), as shown in Figure 3G.

[0202] Linker The TMPs of the present disclosure can include one or more linkers, the one or more linkers being located at one or more of: i) between an MHC class I polypeptide and an Ig Fc polypeptide (such a linker is referred to herein as "L1"); ii) between an immunomodulatory polypeptide and an MHC class I polypeptide (such a linker is referred to herein as "L2"); iii) between a first immunomodulatory polypeptide and a second immunomodulatory polypeptide (such a linker is referred to herein as "L3"); iv) between a peptide antigen ("epitope") and an MHC class I polypeptide; v) between an MHC class I polypeptide and a dimerization polypeptide (e.g., a first or second member of a dimerization pair); and vi) between a dimerization polypeptide (e.g., a first or second member of a dimerization pair) and an IgFc polypeptide.

[0203] Suitable linkers (also called "spacers") can be readily selected and can be any of a number of suitable lengths, such as 1 to 25 amino acids, 3 to 20 amino acids, 2 to 15 amino acids, 3 to 12 amino acids, etc., including 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 examples, the linker is 25 to 50 amino acids in length, e.g., 25 to 30, 30 to 35, 35 to 40, 40 to 45, or 45 to 50 amino acids in length.

[0204] Exemplary linkers include glycine polymers (G) n , glycine-serine polymers (e.g., (GS) n , (GSGGS) n (SEQ ID NO: 366), and (GGGS) n(SEQ ID NO:367), where n is an integer of at least 1), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers may also be used; both Gly and Ser are relatively free and can therefore serve as intermediate tethers between components. Glycine polymers may also be used; glycine has considerably more access to the φ-ψ space than alanine and is much less restricted than residues with longer side chains (see Scheraga, Rev. Computational Chem. 11173-142 (1992)). Exemplary linkers may include amino acid sequences including, but not limited to, GGSG (SEQ ID NO:368), GGSGG (SEQ ID NO:369), GSGSG (SEQ ID NO:370), GSGGG (SEQ ID NO:371), GGGSG (SEQ ID NO:372), GSSSG (SEQ ID NO:373), and the like. Exemplary linkers may include, for example, Gly(Ser4)n (SEQ ID NO: 374) (n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). In some examples, the linker comprises the amino acid sequence (GSSSS)n (SEQ ID NO: 375) (n is 4). In some examples, the linker comprises the amino acid sequence (GSSSS)n (SEQ ID NO: 376) (n is 5).

[0205] Exemplary linkers include (GGGGS)n (SEQ ID NO: 377), also referred to as a "G4S" linker, where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some examples, the linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO: 377), where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some examples, the linker comprises the amino acid sequence AAAGG (SEQ ID NO: 387). A linker having the amino acid sequence AAAGG (SEQ ID NO: 387) is also suitable. In the single-chain TMPs of the present disclosure, the β2M polypeptide can be connected to the MHC heavy chain polypeptide by a (GGGGS)n (SEQ ID NO: 377) linker, where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, e.g., n=3 or 7.

[0206] In some instances, a linker polypeptide present in a first polypeptide of a TMP of the present disclosure comprises a cysteine ​​residue capable of forming a disulfide bond with a cysteine ​​residue present in a second polypeptide of a TMP of the present disclosure. In some instances, for example, a suitable linker may comprise the amino acid sequence TIFF0007756072000008.tif4128 (SEQ ID NO: 388). In another example, a suitable linker may include the amino acid sequence GCGGS(GGGGS)n (SEQ ID NO: 389), where n is 1, 2, 3, 4, 5, 6, 7, 8, or 9. For example, in some examples, the linker includes the amino acid sequence GCGGSGGGGSGGGGSGGGGS (SEQ ID NO: 390). In another example, the linker includes the amino acid sequence GCGGSGGGGSGGGGS (SEQ ID NO: 391).

[0207] Dimerized TMP In some examples, the single-chain and heterodimeric TMPs of the present disclosure can form dimers, i.e., the present disclosure provides polypeptides comprising dimers of the TMPs of the present disclosure. The present disclosure provides, for example, proteins (dimerized TMPs of the present disclosure) comprising: A) a first heterodimer comprising a) i) a KRAS peptide, and ii) a first polypeptide comprising a first MHC polypeptide, and b) i) a second polypeptide comprising a second MHC polypeptide, wherein the first heterodimer comprises one or more immunomodulatory polypeptides; and B) a) i) a KRAS peptide, and ii) a first polypeptide comprising a first MHC polypeptide, and b) i) a second polypeptide comprising a second MHC polypeptide, wherein the second heterodimer comprises one or more immunomodulatory polypeptides, wherein the first heterodimer and the second heterodimer are covalently linked to each other. Alternatively, the dimerized TMP may comprise two single-chain TMPs covalently linked to each other. The covalent bond of the dimer can be a disulfide bond between the Ig Fc polypeptide of the first single-chain or heterodimeric TMP and the Ig Fc polypeptide of the second single-chain or heterodimeric TMP. When the TMP contains an Ig Fc polypeptide, such as a human IgG1 Fc polypeptide that does not substantially induce cell lysis (e.g., the polypeptide of Figure 3G), the TMP typically self-assembles into a dimer by spontaneously forming a disulfide bond with the IgG1 Fc polypeptide of another TMP. Thus, for example, the Ig Fc polypeptides within the first single-chain or heterodimeric TMP and the second single-chain or heterodimer can be linked to each other by one or more disulfide bonds. In some examples, the two TMPs have identical amino acid sequences. In some examples, the first heterodimer and the second heterodimer are covalently linked to each other via the C-terminal region of the second polypeptide of the first heterodimer and the C-terminal region of the second polypeptide of the second heterodimer. In some examples, the first heterodimer and the second heterodimer are covalently linked to each other via the C-terminal amino acid of the second polypeptide of the first heterodimer and the C-terminal region of the second polypeptide of the second heterodimer.For example, in some cases, the C-terminal amino acid of the second polypeptide of the first heterodimer and the C-terminal region of the second polypeptide of the second heterodimer are linked to each other directly or via a linker. The linker can be a peptide linker. The peptide linker can have a length of 1 to 200 amino acids (e.g., 1 to 5 aa, 5 to 10 aa, 10 to 25 aa, 25 to 50 aa, 50 to 100 aa, 100 to 150 aa, or 150 to 200 aa). In some cases, the peptide epitope of the first heterodimer and the peptide epitope of the second heterodimer comprise the same amino acid sequence. In some cases, the first MHC polypeptide of the first and second heterodimers is MHC class I β2-microglobulin, and the second MHC polypeptide of the first and second heterodimers is an MHC class I heavy chain. In some examples, the first heterodimeric immunomodulatory polypeptide and the second heterodimeric immunomodulatory polypeptide comprise the same amino acid sequence. In some examples, the first heterodimeric immunomodulatory polypeptide and the second heterodimeric immunomodulatory polypeptide comprise 1 to 10 amino acid substitutions compared to the corresponding parent wild-type immunomodulatory polypeptide, wherein the 1 to 10 amino acid substitutions result in reduced affinity binding of the variant immunomodulatory polypeptide to a cognate costimulatory polypeptide. In some examples, the first heterodimeric immunomodulatory polypeptide and the second heterodimeric immunomodulatory polypeptide are each independently selected from the group consisting of wild-type and variant polypeptides of IL-2, 4-1BBL, PD-L1, CD80, CD86, ICOS-L, OX-40L, FasL, JAG1 (CD339), TGFβ, CD70, and ICAM. Examples of suitable MHC polypeptides, immunomodulatory polypeptides, and peptide epitopes are described herein.

[0208] In some examples, the single-chain TMP of the present disclosure is dimerized. Thus, the present disclosure provides a protein comprising a) a first single-chain TMP of the present disclosure and b) a second single-chain TMP of the present disclosure, wherein the first and second single-chain TMPs are covalently bonded to each other. The covalent bond can be a disulfide bond between the Ig Fc polypeptide of the first single-chain TMP and the Ig Fc polypeptide of the second single-chain TMP.

[0209] Additional Polypeptides The polypeptide chain of the TMP of the present disclosure may include one or more polypeptides and conjugated drugs in addition to those described above. Suitable additional polypeptides containing epitope tags and affinity domains, as well as drug conjugates, are described in the above-mentioned published PCT applications WO2020132138A1 and WO2019 / 051091, the disclosures of which are expressly incorporated herein by reference as they relate to epitope tags, affinity domains, and drug conjugates, specifically including paragraphs

[0498] to

[0508] of WO2020132138A1 and paragraphs

[0353] to

[0363] of WO2019 / 051091. The one or more additional polypeptides may be included at the N-terminus of the polypeptide chain of TMP, at the C-terminus of the polypeptide chain of TMP, or within the polypeptide chain of TMP.

[0210] Exemplary TMP In some examples, a TMP of the present disclosure comprises at least one heterodimer comprising: a) a first polypeptide comprising i) a KRAS peptide, and ii) a first MHC polypeptide, b) a second polypeptide comprising a second MHC polypeptide, and c) at least one immunomodulatory polypeptide, wherein the first and / or second polypeptide comprises an immunomodulatory polypeptide and optionally comprises Ig Fc. Thus, in some examples, a TMP of the present disclosure comprises at least one heterodimer comprising: a) i) a KRAS peptide, ii) a first MHC polypeptide, and iii) a first polypeptide comprising at least one immunomodulatory polypeptide, and b) a second polypeptide comprising a second MHC polypeptide, and optionally comprises Ig Fc. In other examples, the TMP of the present disclosure comprises at least one heterodimer comprising a) a first polypeptide comprising i) a KRAS peptide and ii) a first MHC polypeptide, and b) a second polypeptide comprising i) a second MHC polypeptide and ii) at least one immunomodulatory polypeptide, optionally comprising Ig Fc. In some examples, the TMP of the present disclosure comprises at least one heterodimer comprising a) a first polypeptide comprising i) a KRAS peptide, ii) a first MHC polypeptide, and iii) at least one immunomodulatory polypeptide, and b) a second polypeptide comprising i) a second MHC polypeptide and ii) at least one immunomodulatory polypeptide, optionally comprising Ig Fc. In some examples, the at least one immunomodulatory polypeptide is a wild-type immunomodulatory polypeptide. In other cases, the at least one immunomodulatory polypeptide is a variant immunomodulatory polypeptide that exhibits reduced affinity for a costimulatory polypeptide compared to the affinity of the corresponding wild-type immunomodulatory polypeptide for the costimulatory polypeptide.

[0211] As described above and shown generally in Figure 17, an immunomodulatory polypeptide (i.e., one or more immunomodulatory polypeptides) can be present in a TMP of the present disclosure at any of a variety of positions. While Figure 17 shows the location of two copies of a variant IL-2 polypeptide, the immunomodulatory polypeptide can be any number and variety of immunomodulatory polypeptides, as described herein. As shown in Figure 17, the immunomodulatory polypeptide can be 1) the N-terminus of the MHC class I heavy chain (position 1), 2) the C-terminus of the MHC class I heavy chain and the N-terminus of the Ig Fc polypeptide, i.e., between the MHC class I heavy chain and the Ig Fc polypeptide (position 2), 3) the C-terminus of the Ig Fc polypeptide (position 3), 4) the N-terminus of the peptide epitope (position 4), or 5) the C-terminus of the β2M polypeptide (position 5). Thus, in some examples, a TMP of the present disclosure comprises one of the following scaffolds having a defined arrangement of components, wherein the first MHC polypeptide is a β2M polypeptide and the second MHC polypeptide is an HLA heavy chain polypeptide: a) a first polypeptide comprising, in N-terminal to C-terminal order, i) a KRAS peptide, and ii) a first MHC polypeptide, and b) a second polypeptide comprising, in N-terminal to C-terminal order, i) at least one immunomodulatory polypeptide, ii) a second MHC polypeptide, and iii) an Ig Fc polypeptide (this arrangement is referred to as MOD position 1); a) a first polypeptide comprising, in N-terminal to C-terminal order, i) a KRAS peptide, and ii) a first MHC polypeptide; and b) a second polypeptide comprising, in N-terminal to C-terminal order, i) a second MHC polypeptide, ii) at least one immunomodulatory polypeptide, and iii) an Ig Fc polypeptide (this arrangement is referred to as MOD position 2), TMP a) a first polypeptide comprising, from N-terminus to C-terminus, i) a KRAS peptide, and ii) a first MHC polypeptide, and b) a second polypeptide comprising, from N-terminus to C-terminus, i) a second MHC polypeptide, ii) an Ig Fc polypeptide, and iii) at least one immunomodulatory polypeptide (this arrangement is referred to as MOD position 3). a) a first polypeptide comprising, in N-terminal to C-terminal order, i) at least one immunomodulatory polypeptide, ii) a KRAS peptide, and iii) a first MHC polypeptide; and b) a second polypeptide comprising, in N-terminal to C-terminal order, i) a second MHC polypeptide, and ii) an Ig Fc polypeptide (this arrangement is referred to as MOD position 4); a) a first polypeptide comprising, in N-terminal to C-terminal order, i) a KRAS peptide, ii) a first MHC polypeptide, and iii) at least one immunomodulatory polypeptide; and b) a second polypeptide comprising, in N-terminal to C-terminal order, i) a second MHC polypeptide, and ii) an Ig Fc polypeptide (this arrangement is referred to as MOD position 5);

[0212] In the above scaffolds, either of the first and second polypeptide components may optionally be connected to a subsequent polypeptide component by a linker. In some examples, the peptide linker is located between: i) the second MHC polypeptide and an Ig Fc polypeptide; ii) the epitope and the first MHC polypeptide; iii) the first MHC polypeptide and an immunomodulatory polypeptide; and (if the TMP comprises two immunomodulatory polypeptides on the first polypeptide chain) iv) between the two immunomodulatory polypeptides; v) the second MHC polypeptide and an Ig Fc polypeptide; or vi) between one or more of the first MHC polypeptide and the immunomodulatory polypeptide(s). In some examples, the peptide linker comprises the amino acid sequence AAAGG (SEQ ID NO: 387). In some examples, the peptide linker comprises the amino acid sequence (GGGGS)n, where n is an integer between 1 and 10 (e.g., n is 2, 3, or 4).

[0213] In any of the above scaffolds, the KRAS peptide has an amino acid sequence selected from the group consisting of VVGADGVGK (SEQ ID NO: 176), VVGACGVGK (SEQ ID NO: 177), VVGAVGVGK (SEQ ID NO: 178), VVVGADGVGK (SEQ ID NO: 179), VVVGAVGVGK (SEQ ID NO: 180), VVVGACGVGK (SEQ ID NO: 181), VTGADGVGK (SEQ ID NO: 182), VTGAVGVGK (SEQ ID NO: 183), VTGACGVGK (SEQ ID NO: 184), VTVGADGVGK (SEQ ID NO: 185), VTVGAVGVGK (SEQ ID NO: 186). SEQ ID NO: 186), VTVGACGVGK (SEQ ID NO: 187), LVVVGADGV (SEQ ID NO: 192), LVVVGAVGV (SEQ ID NO: 193), LVVVGACGV (SEQ ID NO: 194), KLVVVGADGV (SEQ ID NO: 195), KLVVVGAVGV (SEQ ID NO: 196), KLVVVGACGV (SEQ ID NO: 197), LLVVGADGV (SEQ ID NO: 198), LLVVGAVGV (SEQ ID NO: 199), LLVVGACGV (SEQ ID NO: 200), FLVVVGADGV (SEQ ID NO: 201), FLVVVGAVGV (SEQ ID NO: 202), FLVVVGACGV (SEQ ID NO: 203).

[0214] In the above scaffold, in some instances, the second MHC polypeptide is HLA-A * 0201 polypeptide, HLA-A * In some embodiments, the HLA heavy chain polypeptide is an HLA heavy chain comprising an amino acid sequence having at least 95% amino acid sequence identity to an HLA-A1101 polypeptide or an HLA-A24 polypeptide. * In some examples, the HLA heavy chain polypeptide is an HLA-A 0201 polypeptide. * In some examples, the HLA heavy chain polypeptide is an HLA-A 0201 polypeptide. * 1101 polypeptide.

[0215] In some examples, the scaffold comprises two immunomodulatory polypeptides, wherein the two immunomodulatory polypeptides have the same amino acid sequence, for example, the immunomodulatory polypeptides are variant IL-2 polypeptides comprising H16A and F42A substitutions, or variant IL-2 polypeptides comprising H16T and F42A substitutions.

[0216] In some examples, the Ig Fc polypeptide is a variant of a human IgG1 Fc polypeptide that does not substantially induce cell lysis, for example, an IgG1 Fc polypeptide comprising L234A and L235A substitutions as shown in Figure 3G.

[0217] In some instances, the first and second polypeptides are disulfide bonded to each other.

[0218] In some examples, the TMP of the present disclosure comprises a scaffold having a MOD position 1 or position 3 configuration, and the HLA heavy chain polypeptide is a wild-type or variant HLA-A * 0201 polypeptide, e.g., HLA-A comprising an A236C substitution or a sequence as shown in Figure 22C *0201 polypeptide, or a variant thereof. In some examples, the Ig Fc polypeptide is a human IgG1 Fc polypeptide that does not substantially cause cell lysis, for example, a human IgG1 Fc polypeptide comprising L234A and L235A substitutions as shown in FIG. 3G. In some examples, the first and second polypeptides are disulfide bonded to each other. In some examples, the immunomodulatory polypeptide is a variant IL-2 polypeptide comprising H16A and F42A substitutions or H16T and F42A substitutions. In some examples, the KRAS peptide has an amino acid sequence selected from the group consisting of VVGADGVGK (SEQ ID NO: 176), VVGACGVGK (SEQ ID NO: 177), VVGAVGVGK (SEQ ID NO: 178), VVVGADGVGK (SEQ ID NO: 179), VVVGAVGVGK (SEQ ID NO: 180), VVVGACGVGK (SEQ ID NO: 181), VTGADGVGK (SEQ ID NO: 182), VTGAVGVGK (SEQ ID NO: 183), VTGACGVGK (SEQ ID NO: 184), VTVGADGVGK (SEQ ID NO: 185), VTVGAVGVGK (SEQ ID NO: 186), VTVGACGVGK (SEQ ID NO: 187), LVVVGADGV (SEQ ID NO: 192), LVVVGAVGV (SEQ ID NO: 193), LVVVGACGV (SEQ ID NO: 194), KLVVVGADGV (SEQ ID NO: 195), KLVVVGAVGV (SEQ ID NO: 196), KLVVVGACGV (SEQ ID NO: 197), LLVVGADGV (SEQ ID NO: 198), LLVVGAVGV (SEQ ID NO: 199), LLVVGACGV (SEQ ID NO: 200), FLVVVGADGV (SEQ ID NO: 201), FLVVVGAVGV (SEQ ID NO: 202), FLVVVGACGV (SEQ ID NO: 203).

[0219] In some examples, the TMPs of the present disclosure comprise a scaffold having a MOD position 1 or position 3 configuration, and the HLA heavy chain polypeptide is a wild-type or variant HLA-A24 polypeptide (HLA-A * 2402), e.g., an HLA-A comprising an A236C substitution or an amino acid sequence shown in any one of Figures 21O, 21P, 21Q, 21R, 21S, or 21T. *0201 polypeptide, or a variant thereof. In some examples, the Ig Fc polypeptide is a human IgG1 Fc polypeptide that does not substantially cause cell lysis, for example, a human IgG1 Fc polypeptide comprising L234A and L235A substitutions as shown in FIG. 3G. In some examples, the first and second polypeptides are disulfide bonded to each other. In some examples, the immunomodulatory polypeptide is a variant IL-2 polypeptide comprising H16A and F42A substitutions or H16T and F42A substitutions. In some examples, the KRAS peptide has an amino acid sequence selected from the group consisting of VVGADGVGK (SEQ ID NO: 176), VVGACGVGK (SEQ ID NO: 177), VVGAVGVGK (SEQ ID NO: 178), VVVGADGVGK (SEQ ID NO: 179), VVVGAVGVGK (SEQ ID NO: 180), VVVGACGVGK (SEQ ID NO: 181), VTGADGVGK (SEQ ID NO: 182), VTGAVGVGK (SEQ ID NO: 183), VTGACGVGK (SEQ ID NO: 184), VTVGADGVGK (SEQ ID NO: 185), VTVGAVGVGK (SEQ ID NO: 186), VTVGACGVGK (SEQ ID NO: 187), LVVVGADGV (SEQ ID NO: 192), LVVVGAVGV (SEQ ID NO: 193), LVVVGACGV (SEQ ID NO: 194), KLVVVGADGV (SEQ ID NO: 195), KLVVVGAVGV (SEQ ID NO: 196), KLVVVGACGV (SEQ ID NO: 197), LLVVGADGV (SEQ ID NO: 198), LLVVGAVGV (SEQ ID NO: 199), LLVVGACGV (SEQ ID NO: 200), FLVVVGADGV (SEQ ID NO: 201), FLVVVGAVGV (SEQ ID NO: 202), FLVVVGACGV (SEQ ID NO: 203).

[0220] In some examples, the TMP of the present disclosure comprises a scaffold having a MOD position 1 or position 3 configuration, and the HLA heavy chain polypeptide is a wild-type or variant HLA-A polypeptide as disclosed herein. * 1101 polypeptide, e.g., an HLA-A polypeptide containing an A236C substitution or having an amino acid sequence as shown in one of Figures 21J or 21K. *1101 polypeptide, or a variant thereof. In some examples, the Ig Fc polypeptide is a human IgG1 Fc polypeptide that does not substantially cause cell lysis, for example, a human IgG1 Fc polypeptide comprising L234A and L235A substitutions as shown in FIG. 3G. In some examples, the first and second polypeptides are disulfide bonded to each other. In some examples, the immunomodulatory polypeptide is a variant IL-2 polypeptide comprising H16A and F42A substitutions or H16T and F42A substitutions. In some examples, the KRAS peptide has an amino acid sequence selected from the group consisting of VVGADGVGK (SEQ ID NO: 176), VVGACGVGK (SEQ ID NO: 177), VVGAVGVGK (SEQ ID NO: 178), VVVGADGVGK (SEQ ID NO: 179), VVVGAVGVGK (SEQ ID NO: 180), VVVGACGVGK (SEQ ID NO: 181), VTGADGVGK (SEQ ID NO: 182), VTGAVGVGK (SEQ ID NO: 183), VTGACGVGK (SEQ ID NO: 184), VTVGADGVGK (SEQ ID NO: 185), VTVGAVGVGK (SEQ ID NO: 186), VTVGACGVGK (SEQ ID NO: 187), LVVVGADGV (SEQ ID NO: 192), LVVVGAVGV (SEQ ID NO: 193), LVVVGACGV (SEQ ID NO: 194), KLVVVGADGV (SEQ ID NO: 195), KLVVVGAVGV (SEQ ID NO: 196), KLVVVGACGV (SEQ ID NO: 197), LLVVGADGV (SEQ ID NO: 198), LLVVGAVGV (SEQ ID NO: 199), LLVVGACGV (SEQ ID NO: 200), FLVVVGADGV (SEQ ID NO: 201), FLVVVGAVGV (SEQ ID NO: 202), FLVVVGACGV (SEQ ID NO: 203).

[0221] In some examples, the TMP of the present disclosure comprises a scaffold having a MOD position 1 or position 3 configuration, and the HLA heavy chain polypeptide is a wild-type or variant HLA-A polypeptide as disclosed herein. * 1101 polypeptide, e.g., an HLA-A polypeptide containing an A236C substitution or having an amino acid sequence as shown in one of Figures 21J or 21K. *1101 polypeptide, or a variant thereof. In some examples, the Ig Fc polypeptide is a human IgG1 Fc polypeptide that does not substantially cause cell lysis, for example, a human IgG1 Fc polypeptide comprising L234A and L235A substitutions as shown in FIG. 3G. In some examples, the first and second polypeptides are disulfide bonded to each other. In some examples, the immunomodulatory polypeptide is a variant IL-2 polypeptide comprising H16A and F42A substitutions or H16T and F42A substitutions. In some examples, the KRAS peptide has an amino acid sequence selected from the group consisting of VVGADGVGK (SEQ ID NO: 176), VVGACGVGK (SEQ ID NO: 177), VVGAVGVGK (SEQ ID NO: 178), VVVGADGVGK (SEQ ID NO: 179), VVVGAVGVGK (SEQ ID NO: 180), VVVGACGVGK (SEQ ID NO: 181), VTGADGVGK (SEQ ID NO: 182), VTGAVGVGK (SEQ ID NO: 183), VTGACGVGK (SEQ ID NO: 184), VTVGADGVGK (SEQ ID NO: 185), VTVGAVGVGK (SEQ ID NO: 186), VTVGACGVGK (SEQ ID NO: 187), LVVVGADGV (SEQ ID NO: 192), LVVVGAVGV (SEQ ID NO: 193), LVVVGACGV (SEQ ID NO: 194), KLVVVGADGV (SEQ ID NO: 195), KLVVVGAVGV (SEQ ID NO: 196), KLVVVGACGV (SEQ ID NO: 197), LLVVGADGV (SEQ ID NO: 198), LLVVGAVGV (SEQ ID NO: 199), LLVVGACGV (SEQ ID NO: 200), FLVVVGADGV (SEQ ID NO: 201), FLVVVGAVGV (SEQ ID NO: 202), FLVVVGACGV (SEQ ID NO: 203). In the MOD Position 1 configuration, in some instances, one or more independently selected peptide linkers are positioned between one or more of: i) the KRAS peptide and the first MHC polypeptide; ii) the immunomodulatory polypeptide and the second MHC polypeptide (wherein the TMP comprises two immunomodulatory polypeptides on the second polypeptide chain between the two immunomodulatory polypeptides); and / or iii) the second MHC polypeptide and the Ig Fc polypeptide.In the MOD position 3 configuration, in some cases, one or more independently selected peptide linkers are positioned between one or more of: i) the KRAS peptide and a first MHC polypeptide; ii) the second MHC polypeptide and an Ig Fc polypeptide; and iii) the Ig Fc and an immunomodulatory polypeptide (wherein the TMP comprises two immunomodulatory polypeptides on the second polypeptide chain between the two immunomodulatory polypeptides). In some cases, the peptide linker comprises the amino acid sequence AAAGG (SEQ ID NO: 387). In some cases, the peptide linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO: 377), where n is an integer between 1 and 10 (e.g., n is 2, 3, or 4).

[0222] Furthermore, as described above and shown schematically in Figures 16A-16C, the first and second polypeptide chains of a TMP of the present disclosure can be linked by one or more disulfide bonds. For example, a TMMP of the present disclosure can include a) a first polypeptide chain comprising a β2M polypeptide having an R12C substitution, and b) a second polypeptide chain comprising a Class I MHC heavy chain polypeptide having an A236C substitution, whereby a disulfide bond is formed between Cys at position 12 of the β2M polypeptide of the first polypeptide chain and Cys at position 236 of the Class I MHC heavy chain polypeptide of the second polypeptide chain. As another example, a TMMP of the present disclosure can include, a) from N- to C-terminus, i) a peptide epitope, ii) a GCGGS (GGGGS) peptide, n (SEQ ID NO:582), where n is 1, 2, or 3, and iii) a first polypeptide comprising a β2M polypeptide, and b) a second polypeptide comprising a Class I MHC heavy chain polypeptide having a Y84C substitution, whereby a disulfide bond is formed between Cys of the peptide linker of the first polypeptide chain and Cys at position 84 of the Class I MHC heavy chain polypeptide of the second polypeptide chain. In other examples, the TMP of the present disclosure comprises, a) from N-terminus to C-terminus, i) a peptide epitope, ii) GCGGS (GGGGS) n(SEQ ID NO:582), where n is 1, 2, or 3; iii) a first polypeptide comprising a β2M polypeptide having an R12C substitution; and b) a second polypeptide comprising a Class I MHC heavy chain polypeptide having a Y84C substitution and an A236C substitution, whereby i) a first disulfide bond is formed between Cys in the peptide linker of the first polypeptide chain and Cys at position 84 of the Class I MHC heavy chain polypeptide of the second polypeptide chain, and ii) a second disulfide bond is formed between Cys at position 12 of the β2M polypeptide of the first polypeptide chain and Cys at position 236 of the Class I MHC heavy chain polypeptide of the second polypeptide chain. For simplicity, the first disulfide bond is referred to as "G2C / Y84C" and the second disulfide bond is referred to as "R12C / A236C." The TMPs of the present disclosure a) contain a G2C / Y84C disulfide bond and no R12C / A236C disulfide bond, b) contain no G2C / Y84C disulfide bond and a R12C / A236C disulfide bond, or c) contain a G2C / Y84C disulfide bond and a R12C / A236C disulfide bond.

[0223] The TMPs of the disclosure may a) comprise a G2C / Y84C disulfide bond and not comprise an R12C / A236C disulfide bond, and b) comprise at least one immunomodulatory polypeptide at position 1 or 3. The TMPs of the disclosure may a) comprise a G2C / Y84C disulfide bond and not comprise an R12C / A236C disulfide bond, and b) comprise at least one immunomodulatory polypeptide at position 2, 4, or 5.

[0224] The TMPs of the disclosure may a) contain an R12C / A236C disulfide bond and no G2C / Y84C disulfide bond and comprise at least one immunomodulatory polypeptide at position 1 or 3. The TMPs of the disclosure may a) contain an R12C / A236C disulfide bond and no G2C / Y84C disulfide bond and comprise at least one immunomodulatory polypeptide at position 2, 4, or 5.

[0225] The TMPs of the disclosure may comprise a) a G2C / Y84C disulfide bond and a R12C / A236C disulfide bond, and b) at least one immunomodulatory polypeptide at position 1 or 3. The TMPs of the disclosure may comprise a) a G2C / Y84C disulfide bond and a R12C / A236C disulfide bond, and b) at least one immunomodulatory polypeptide at position 2, 4, or 5.

[0226] In some examples, a TMP of the present disclosure comprises a second polypeptide comprising: (i) an HLA-A0201(Y84A, A236C) polypeptide comprising an Ala at position 84 and a Cys at position 236, or (ii) an HLA-A0201(Y84C, A139C) polypeptide comprising Cys at positions 84 and 139, or (iii) an HLA-A0201(Y84C, A236) polypeptide comprising a Cys at position 84 and an alanine at position 236, e.g., as shown in Figures 13A, 13B, or 13C, respectively.

[0227] In some examples, the single-chain TMP of the disclosure is capable of targeting (i) an HLA-A polypeptide containing Ala at position 84 and Cys at position 236, e.g., as shown in Figure 13D, Figure 13E, or Figure 13F, respectively. * 1101(Y84A, A236C) polypeptide, or (ii) HLA-A containing Cys at positions 84 and 236 * 1101(Y84C, A236C) polypeptide, or (iii) HLA-A containing Cys at position 84 and alanine at position 236 * The MHC class I heavy chain polypeptide comprises the 1101(Y84C, A236) polypeptide.

[0228] In some examples, the TMP of the present disclosure comprises a second polypeptide comprising: (i) an HLA-A24(Y84A, A236C) polypeptide comprising Ala at position 84 and Cys at position 236, or (ii) an HLA-A24(Y84C, A236C) polypeptide comprising Cys at positions 84 and 236, or (iii) an HLA-A24(Y84C, A236) polypeptide comprising Cys at position 84 and alanine at position 236, as shown in Figure 13G, Figure 13H, or Figure 13I, respectively.

[0229] As a non-limiting example, a TMP of the present disclosure can include one of the combinations of first and second polypeptides set forth in Table 2 below.

[0230] (Table 2) TIFF0007756072000009.tif237102TIFF0007756072000010.tif111128

[0231] As a non-limiting example, a TMP of the present disclosure can include one of the combinations of first and second polypeptides set forth in Table 3 below.

[0232] (Table 3) TIFF0007756072000011.tif93128TIFF0007756072000012.tif22894TIFF0007756072000013.tif39128

[0233] In some examples, the heterodimeric TMP of the present disclosure comprises a class I MHC heavy chain comprising an intrachain disulfide bond, for example, in some examples, the heterodimeric TMP of the present disclosure comprises a class I MHC heavy chain comprising an intrachain disulfide bond formed between Cys residues resulting from Y84C and A139C substitutions. In some examples, such heterodimeric TMP also includes a class I MHC heavy chain comprising an A236C substitution, wherein Cys-236 is capable of forming a disulfide bond with a second polypeptide chain comprising: i) a peptide epitope; ii) a β2M polypeptide comprising an R12C substitution (whereby Cys-12 forms a disulfide bond with Cys-236 of the class I MHC heavy chain); and iii) a peptide linker between the peptide epitope and the β2M polypeptide (the linker comprises the amino acid sequence (GGGGS)n, where n is an integer between 1 and 9 (e.g., n is 1, 2, or 3).

[0234] By way of example, a TMP of the present disclosure may include one of the combinations of first and second polypeptides set forth in Table 4 below to provide a heterodimeric TMP comprising a Class I MHC A02 heavy chain allele having Y84C, A139C, and A236C substitutions, where the immunomodulatory polypeptide is at position 1 or position 3, as shown in FIG. 17.

[0235] (Table 4) TIFF0007756072000014.tif122155

[0236] As another example, a heterodimeric TMP of the disclosure can include a) a first polypeptide comprising the amino acid sequence shown in Figure 13L and a second polypeptide comprising the amino acid sequence shown in Figure 14K. As another example, a heterodimeric TMP of the disclosure can include a) a first polypeptide comprising the amino acid sequence shown in Figure 13L and a second polypeptide comprising the amino acid sequence shown in Figure 14Q.

[0237] As described above, in some examples, the TMP of the present disclosure is a heterodimeric TMP comprising a first polypeptide and a second polypeptide, wherein the first polypeptide and the second polypeptide are linked by one or more disulfide bonds, e.g., a single disulfide bond or two disulfide bonds. For example, as described above, the TMP of the present disclosure may a) comprise a G2C / Y84C disulfide bond and no R12C / A236C disulfide bond, b) comprise a R12C / A236C disulfide bond and no G2C / Y84C disulfide bond, or c) comprise a G2C / Y84C disulfide bond and an R12C / A236C disulfide bond. In some examples, for example, if a TMP of the present disclosure contains a G2C / Y84C disulfide bond but does not contain an R12C / A236C disulfide bond, the β2M polypeptide will not contain an R12C substitution (with Arg at position 12 instead) and the Class I MHC heavy chain polypeptide will not contain an A236C substitution (with Ala at position 236 instead). In other words, the β2M polypeptide and the Class I MHC heavy chain polypeptide will not contain a "free" (unpaired) Cys residue at position 12 of the β2M and position 236 of the Class I MHC heavy chain polypeptide. Similarly, in some instances, for example, when a TMP of the present disclosure contains an R12C / A236C disulfide bond but not a G2C / Y84C disulfide bond, the linker between the peptide epitope and the β2M polypeptide does not contain a Cys substitution (and instead the linker is a (GGGGS)n linker, where n is an integer from 1 to 5), and the Class I MHC polypeptide does not contain a Y84C substitution (instead has a Tyr at position 84). In other words, neither the linker between the peptide epitope and the β2M polypeptide nor the Class I MHC polypeptide contains a "free" (unpaired) Cys residue within the linker or at position 84 of the Class I MHC polypeptide.

[0238] Single strand TMP As described above and shown generally in Figure 18, the immunomodulatory polypeptide (i.e., one or more immunomodulatory polypeptides) can be present in a single-chain TMP of the present disclosure at any of a variety of positions. While Figure 18 shows the location of two copies of the variant IL-2 polypeptide, the immunomodulatory polypeptide can be any number and any variety of immunomodulatory polypeptides as described herein. As shown in Figure 18, the immunomodulatory polypeptide can be 1) the C-terminus of the MHC class I heavy chain and the N-terminus of the Ig Fc polypeptide, 2) the C-terminus of the Ig Fc polypeptide, or 3) the N-terminus of the peptide epitope.

[0239] The MHC class I polypeptides (i.e., β2M and heavy chain polypeptides), immunomodulatory polypeptides, Ig Fc components, and linkers of the single-chain TMPs of the present disclosure are the same as those described above for the heterodimeric TMPs. Furthermore, the single-chain TMPs may contain the same disulfide bonds as the heterodimeric TMPs, i.e., intrachain disulfides between the MHC class I heavy chain polypeptides, e.g., between the two Cys residues mentioned above (e.g., Cys-84 and Cys-139 of the heavy chain), interchain disulfides between the β2M and heavy chain polypeptides (e.g., between R12C of β2M and Cys at residue 236 of the heavy chain), and / or disulfides connecting the Cys of the MHC class I heavy chain to the Cys residue of the linker between the epitope and the β2M polypeptide. In some examples, the MHC class I heavy chain is an A02 allele MHC class I heavy chain. In some examples, the MHC class I heavy chain comprises Y84C and A139C substitutions, whereby an intrachain disulfide bond is formed between Cys-84 and Cys-139. In some examples, the MHC class I heavy chain is an A02 allele MHC class I heavy chain.

[0240] In some examples, the single-chain TMP of the present disclosure comprises a scaffold having a MOD position 2 or position 3 configuration, as shown in FIG. 18, and the HLA heavy chain polypeptide is a wild-type or variant HLA-A * 0201 polypeptide, e.g., an HLA-A polypeptide comprising an A236C substitution, or the sequence shown in Figure 22C *0201 polypeptide, or a variant thereof. In some examples, the Ig Fc polypeptide is a human IgG1 Fc polypeptide that does not substantially cause cell lysis, for example, a human IgG1 Fc polypeptide comprising L234A and L235A substitutions as shown in FIG. 3G. In some examples, the single-chain TMP comprises an intrachain disulfide bond (i) between two Cys residues of the MHC class I heavy chain polypeptide, (ii) between the β2M and heavy chain polypeptide, and / or between a Cys of the MHC class I heavy chain and a Cys of the linker between the KRAS epitope and the β2M polypeptide. In some examples, the immunomodulatory polypeptide is a variant IL-2 polypeptide comprising H16A and F42A substitutions or H16T and F42A substitutions. In some examples, the KRAS peptide has an amino acid sequence selected from the group consisting of VVGADGVGK (SEQ ID NO: 176), VVGACGVGK (SEQ ID NO: 177), VVGAVGVGK (SEQ ID NO: 178), VVVGADGVGK (SEQ ID NO: 179), VVVGAVGVGK (SEQ ID NO: 180), VVVGACGVGK (SEQ ID NO: 181), VTGADGVGK (SEQ ID NO: 182), VTGAVGVGK (SEQ ID NO: 183), VTGACGVGK (SEQ ID NO: 184), VTVGADGVGK (SEQ ID NO: 185), VTVGAVGVGK (SEQ ID NO: 186), VTVGACGVGK (SEQ ID NO: 187), LVVVGADGV (SEQ ID NO: 192), LVVVGAVGV (SEQ ID NO: 193), LVVVGACGV (SEQ ID NO: 194), KLVVVGADGV (SEQ ID NO: 195), KLVVVGAVGV (SEQ ID NO: 196), KLVVVGACGV (SEQ ID NO: 197), LLVVGADGV (SEQ ID NO: 198), LLVVGAVGV (SEQ ID NO: 199), LLVVGACGV (SEQ ID NO: 200), FLVVVGADGV (SEQ ID NO: 201), FLVVVGAVGV (SEQ ID NO: 202), FLVVVGACGV (SEQ ID NO: 203).

[0241] In some examples, the single-chain TMP of the present disclosure comprises a scaffold having a MOD position 2 or position 3 configuration as shown in FIG. 18, and the HLA heavy chain polypeptide is a wild-type or variant HLA-A24 polypeptide (also referred to as HLA-A*2402), e.g., an HLA-A* polypeptide comprising an A236C substitution or any one of the amino acid sequences shown in FIGS. 21O, 21P, 21Q, 21R, 21S, or 21T. *0201 polypeptide, or a variant thereof. In some examples, the Ig Fc polypeptide is a human IgG1 Fc polypeptide that does not substantially cause cell lysis, for example, a human IgG1 Fc polypeptide comprising L234A and L235A substitutions as shown in FIG. 3G. In some examples, the single-chain TMP comprises an intrachain disulfide bond (i) between two Cys residues of the MHC class I heavy chain polypeptide, (ii) between the β2M and heavy chain polypeptide, and / or between a Cys of the MHC class I heavy chain and a Cys of the linker between the KRAS epitope and the β2M polypeptide. In some examples, the immunomodulatory polypeptide is a variant IL-2 polypeptide comprising H16A and F42A substitutions or H16T and F42A substitutions. In some examples, the KRAS peptide has an amino acid sequence selected from the group consisting of VVGADGVGK (SEQ ID NO: 176), VVGACGVGK (SEQ ID NO: 177), VVGAVGVGK (SEQ ID NO: 178), VVVGADGVGK (SEQ ID NO: 179), VVVGAVGVGK (SEQ ID NO: 180), VVVGACGVGK (SEQ ID NO: 181), VTGADGVGK (SEQ ID NO: 182), VTGAVGVGK (SEQ ID NO: 183), VTGACGVGK (SEQ ID NO: 184), VTVGADGVGK (SEQ ID NO: 185), VTVGAVGVGK (SEQ ID NO: 186), VTVGACGVGK (SEQ ID NO: 187), LVVVGADGV (SEQ ID NO: 192), LVVVGAVGV (SEQ ID NO: 193), LVVVGACGV (SEQ ID NO: 194), KLVVVGADGV (SEQ ID NO: 195), KLVVVGAVGV (SEQ ID NO: 196), KLVVVGACGV (SEQ ID NO: 197), LLVVGADGV (SEQ ID NO: 198), LLVVGAVGV (SEQ ID NO: 199), LLVVGACGV (SEQ ID NO: 200), FLVVVGADGV (SEQ ID NO: 201), FLVVVGAVGV (SEQ ID NO: 202), FLVVVGACGV (SEQ ID NO: 203).

[0242] In some examples, the TMP of the present disclosure comprises a scaffold having a MOD position 2 or position 3 configuration as shown in FIG. 18, and the HLA heavy chain polypeptide is a wild-type or variant HLA-A polypeptide as disclosed herein. *1101 polypeptide, e.g., an HLA-A polypeptide containing an A236C substitution or having an amino acid sequence as shown in one of Figures 21J or 21K. * 1101 polypeptide, or a variant thereof. In some examples, the Ig Fc polypeptide is a human IgG1 Fc polypeptide that does not substantially cause cell lysis, for example, a human IgG1 Fc polypeptide comprising L234A and L235A substitutions as shown in FIG. 3G. In some examples, the single-chain TMP comprises an intrachain disulfide bond (i) between two Cys residues of the MHC class I heavy chain polypeptide, (ii) between the β2M and heavy chain polypeptide, and / or between a Cys of the MHC class I heavy chain and a Cys of the linker between the KRAS epitope and the β2M polypeptide. In some examples, the immunomodulatory polypeptide is a variant IL-2 polypeptide comprising H16A and F42A substitutions or H16T and F42A substitutions. In some examples, the KRAS peptide has an amino acid sequence selected from the group consisting of VVGADGVGK (SEQ ID NO: 176), VVGACGVGK (SEQ ID NO: 177), VVGAVGVGK (SEQ ID NO: 178), VVVGADGVGK (SEQ ID NO: 179), VVVGAVGVGK (SEQ ID NO: 180), VVVGACGVGK (SEQ ID NO: 181), VTGADGVGK (SEQ ID NO: 182), VTGAVGVGK (SEQ ID NO: 183), VTGACGVGK (SEQ ID NO: 184), VTVGADGVGK (SEQ ID NO: 185), VTVGAVGVGK (SEQ ID NO: 186), VTVGACGVGK (SEQ ID NO: 187), LVVVGADGV (SEQ ID NO: 192), LVVVGAVGV (SEQ ID NO: 193), LVVVGACGV (SEQ ID NO: 194), KLVVVGADGV (SEQ ID NO: 195), KLVVVGAVGV (SEQ ID NO: 196), KLVVVGACGV (SEQ ID NO: 197), LLVVGADGV (SEQ ID NO: 198), LLVVGAVGV (SEQ ID NO: 199), LLVVGACGV (SEQ ID NO: 200), FLVVVGADGV (SEQ ID NO: 201), FLVVVGAVGV (SEQ ID NO: 202), FLVVVGACGV (SEQ ID NO: 203).

[0243] As described above, the polypeptides of a single-chain TMP can be linked by one or more disulfide bonds. For example, a TMP of the present disclosure can include a β2M polypeptide with an R12C substitution and a Class I MHC heavy chain polypeptide with an A236C substitution, thereby forming a disulfide bond between Cys at position 12 of the β2M polypeptide and Cys at position 236 of the Class I MHC heavy chain polypeptide. As another example, a single-chain TMP of the present disclosure can include: i) GCGGS (GGGGS) n (SEQ ID NO: 582) 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 Cys of the peptide linker and Cys at position 84 of the class I MHC heavy chain polypeptide. In another example, a single-chain TMP of the present disclosure can include: i) a KRAS epitope and a β2M polypeptide connected by a peptide linker comprising the sequence (SEQ ID NO: 582), 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 Cys of the peptide linker and Cys at position 84 of the class I MHC heavy chain polypeptide. n(SEQ ID NO:582) sequence, where n is 1, 2, or 3; and ii) a Class I MHC heavy chain polypeptide having a Y84C substitution and an A236C substitution, whereby a) a first disulfide bond is formed between Cys of the peptide linker and Cys at position 84 of the Class I MHC heavy chain polypeptide, and b) a second disulfide bond is formed between Cys at position 12 of the β2M polypeptide and Cys at position 236 of the Class I MHC heavy chain polypeptide. For simplicity, the first disulfide bond is referred to as "G2C / Y84C" and the second disulfide bond is referred to as "R12C / A236C." A single-chain TMP of the disclosure 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 examples, the MHC class I heavy chain contains a non-naturally occurring Cys at position 84 and a non-naturally occurring residue at position 139, thereby forming an intrachain disulfide bond between Cys-84 and Cys-139.

[0244] A single-chain TMP of the disclosure may a) comprise a G2C / Y84C disulfide bond and no R12C / A236C disulfide bond, and b) comprise at least one immunomodulatory polypeptide at position 2 or 3. A single-chain TMP of the disclosure may a) comprise an R12C / A236C disulfide bond and no G2C / Y84C disulfide bond, and b) comprise at least one immunomodulatory polypeptide at position 2 or 3. A single-chain TMP of the disclosure may a) comprise a G2C / Y84C disulfide bond and an R12C / A236C disulfide bond, and b) comprise at least one immunomodulatory polypeptide at position 2 or 3.

[0245] In some examples, a single-chain TMP of the disclosure comprises an MHC class I heavy chain polypeptide comprising: (i) an HLA-A0201(Y84A, A236C) polypeptide comprising an Ala at position 84 and a Cys at position 236; or (ii) an HLA-A0201(Y84C, A139C) polypeptide comprising Cys at positions 84 and 139; or (iii) an HLA-A0201(Y84C, A236) polypeptide comprising a Cys at position 84 and an alanine at position 236, e.g., as shown in Figures 13A, 13B, or 13C, respectively.

[0246] In some examples, the single-chain TMP of the disclosure is capable of targeting (i) an HLA-A polypeptide containing Ala at position 84 and Cys at position 236, e.g., as shown in Figure 13D, Figure 13E, or Figure 13F, respectively. * 1101(Y84A, A236C) polypeptide, or (ii) HLA-A containing Cys at positions 84 and 236 * 1101(Y84C, A236C) polypeptide, or (iii) HLA-A containing Cys at position 84 and alanine at position 236 * The MHC class I heavy chain polypeptide comprises the 1101(Y84C, A236) polypeptide.

[0247] In some examples, the TMP of the present disclosure comprises a second polypeptide comprising: (i) an HLA-A24(Y84A, A236C) polypeptide comprising Ala at position 84 and Cys at position 236, or (ii) an HLA-A24(Y84C, A236C) polypeptide comprising Cys at positions 84 and 236, or (iii) an HLA-A24(Y84C, A236) polypeptide comprising Cys at position 84 and alanine at position 236, as shown in Figure 13G, Figure 13H, or Figure 13I, respectively.

[0248] As one non-limiting example, a single-chain TMP of the present disclosure can comprise the amino acid sequence shown in Figure 19A (TMP "4095"). As another non-limiting example, a single-chain TMP of the present disclosure can comprise the amino acid sequence shown in Figure 19B (TMP "4073"). As another non-limiting example, a single-chain TMP of the present disclosure can comprise the amino acid sequence shown in Figure 19C (TMP "4074"). As another non-limiting example, a single-chain TMP of the present disclosure can comprise the amino acid sequence shown in Figure 19D (TMP "4333"). As another non-limiting example, a single-chain TMP of the present disclosure can comprise the amino acid sequence shown in Figure 19E (TMP "4335"). As another non-limiting example, a single-chain TMP of the present disclosure can comprise the amino acid sequence shown in Figure 19F. As another non-limiting example, a single-chain TMP of the present disclosure can comprise the amino acid sequence shown in Figure 19G (TMP "4238"). As another non-limiting example, a single-chain TMP of the present disclosure can comprise the amino acid sequence shown in Figure 19H (TMP "4241"). As another non-limiting example, a single-chain TMP of the present disclosure can comprise the amino acid sequence shown in Figure 19I (TMP "4334"). As another non-limiting example, a single-chain TMP of the present disclosure can comprise the amino acid sequence shown in Figure 19J (TMP "4144"). As another non-limiting example, a single-chain TMP of the present disclosure can comprise the amino acid sequence shown in Figure 19K (TMP "4145"). As another non-limiting example, a single-chain TMP of the present disclosure can comprise the amino acid sequence shown in Figure 19L (TMP "4146").

[0249] Methods for producing multimeric T cell modulating polypeptides Methods for obtaining TMPs comprising one or more variant immunomodulatory polypeptides that exhibit lower affinity for a cognate costimulatory polypeptide compared to the affinity of the corresponding parent wild-type immunomodulatory polypeptide for the costimulatory polypeptide are disclosed in published PCT application WO2020132138A1 and the aforementioned WO2019 / 051091, the disclosures of which are expressly incorporated by reference herein as they relate to methods of producing TMPs, including specifically paragraphs

[0560] to

[0583] of WO2020132138A1 and paragraphs

[0364] to

[0387] of WO2019 / 051091.

[0250] nucleic acid The present disclosure provides a nucleic acid comprising a nucleotide sequence encoding a TMP of the present disclosure.The present disclosure provides a nucleic acid comprising a nucleotide sequence encoding a TMP of the present disclosure.

[0251] The present disclosure provides a nucleic acid comprising a nucleotide sequence encoding a TMP of the present disclosure. In some examples, each polypeptide chain of the heterodimeric TMP of the present disclosure is encoded within an individual nucleic acid. In some examples, all polypeptide chains of the heterodimeric or single-chain TMP of the present disclosure are encoded within a single nucleic acid. In some examples, a first nucleic acid comprises a nucleotide sequence encoding a first polypeptide of the heterodimeric TMP of the present disclosure, and a second nucleic acid comprises a nucleotide sequence encoding a second polypeptide of the heterodimeric TMP of the present disclosure. In some examples, a single nucleic acid comprises a nucleotide sequence encoding a first polypeptide of the TMP of the present disclosure and a second polypeptide of the heterodimeric TMP of the present disclosure.

[0252] Individual nucleic acids encoding the individual polypeptide chains of the polypeptide In some cases, the individual polypeptide chains of the heterodimeric TMP of the present disclosure are encoded within individual nucleic acids. In some cases, the nucleotide sequences encoding the individual polypeptide chains of the TMP of the present disclosure are operably linked to a transcriptional control element, such as a promoter, for example, a promoter that is functional in eukaryotic cells, and the promoter may be a constitutive promoter or an inducible promoter.

[0253] Thus, for example, the present disclosure provides a first nucleic acid and a second nucleic acid, wherein the first nucleic acid comprises a distinct nucleotide sequence encoding a first polypeptide of a heterodimeric TMP, and the second nucleic acid comprises a distinct nucleotide sequence encoding a second polypeptide of the heterodimeric TMP of the present disclosure. For example, in MOD position 1 described above for heterodimeric TMP (see FIG. 17), where the first MHC polypeptide comprises a β2M polypeptide and the second polypeptide comprises an HLA heavy chain polypeptide, the first nucleic acid comprises, from N-terminus to C-terminus, i) a KRAS peptide and ii) a β2M polypeptide, and the second nucleic acid encodes a second polypeptide comprising, from N-terminus to C-terminus, i) at least one immunomodulatory polypeptide, ii) an HLA heavy chain polypeptide, and iii) an Ig Fc polypeptide. As noted above, linkers may optionally be included between the individual components of both the first and second polypeptides. Similarly, for example, at MOD position 3, the first nucleic acid encodes a first polypeptide comprising, from N- to C-terminus, i) a KRAS peptide, and ii) a β2M polypeptide, and the second nucleic acid encodes a second polypeptide comprising, from N- to C-terminus, i) an HLA heavy chain polypeptide, ii) an Ig Fc polypeptide, and iii) at least one immunomodulatory polypeptide. Again, linkers can optionally be included between the individual components of both the first and second polypeptides.

[0254] In some cases, the nucleotide sequences encoding the first and second polypeptides are operably linked to a transcriptional control element. In some cases, the transcriptional control element is a promoter that functions in eukaryotic cells. In some cases, the nucleic acids are present on separate expression vectors.

[0255] Nucleic acids encoding two or more polypeptides present in a polypeptide The present disclosure also provides a single nucleic acid comprising a nucleotide sequence encoding at least a first polypeptide and a second polypeptide of a heterodimeric or single-chain TMP of the present disclosure. TMP methods for preparing heterodimeric TMP using a single nucleic acid are disclosed in published PCT applications WO2020132138A1 and WO2019 / 051091, the disclosures of which are expressly incorporated herein by reference as they relate to nucleic acids encoding TMP, specifically including paragraphs

[0507] to

[0514] of WO2020132138A1 and paragraphs

[0393] to

[0400] of WO2019 / 051091.

[0256] Recombinant Expression Vectors The present disclosure provides a recombinant expression vector that comprises the nucleic acid of the present disclosure.In some examples, the recombinant expression vector is a non-viral vector.In some examples, the recombinant expression vector is a viral construct, for example, 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-integrating viral vector, etc.

[0257] Suitable expression vectors are disclosed in published PCT applications WO2020132138A1 and WO2019 / 051091, the disclosures of which are expressly incorporated herein by reference as they relate to such expression vectors, including specifically paragraphs

[0515] to

[0520] of WO2020132138A1 and paragraphs

[0401] to

[0406] of WO2019 / 051091.

[0258] Genetically modified host cells The present disclosure provides genetically modified host cells, which host cells are genetically modified with a nucleic acid of the present disclosure.

[0259] Suitable host cells include eukaryotic cells such as yeast, insect cells, and mammalian cells. In some cases, the host cells are cells of a mammalian cell line. Suitable mammalian cell lines include human cell lines, non-human primate cell lines, rodent (e.g., mouse, rat) cell lines, and the like. Suitable mammalian cell lines include, but are not limited to, HeLa cells (e.g., American Type Culture Collection (ATCC) No. CCL-2), CHO cells (e.g., ATCC Nos. CRL9618, CCL61, CRL9096), 293 cells (e.g., ATCC No. CRL-1573), Vero cells, NIH 3T3 cells (e.g., ATCC No. CRL-1658), Huh-7 cells, BHK cells (e.g., ATCC No. CCL10), PC12 cells (ATCC No. CRL1721), COS cells, COS-7 cells (ATCC No. CRL1651), RAT1 cells, mouse L cells (ATCC No. CCLI.3), human embryonic kidney (HEK) cells (ATCC No. CRL1573), HLHepG2 cells, and the like.

[0260] In some instances, the host cell is a mammalian cell that has been genetically engineered not to synthesize endogenous MHC β2-M.

[0261] In some examples, the host cell is a mammalian cell that has been genetically modified not to synthesize endogenous MHC class I heavy chains. In some examples, the host cell is a mammalian cell that has been genetically modified not to synthesize endogenous MHC β2-M and not to synthesize endogenous MHC class I heavy chains.

[0262] As described above, TMP is, in some cases, a single-chain polypeptide (e.g., consisting of a single polypeptide chain or a homodimer of a single polypeptide chain). It has been observed that single-chain TMP is produced intact and full-length, i.e., without cleavage of the polypeptide chain. Single-chain TMP can, in some cases, be produced in larger amounts than heterodimeric TMP.

[0263] composition The present disclosure provides compositions (including pharmaceutical compositions) comprising the TMP (synTac) of the present disclosure.The present disclosure provides compositions (including pharmaceutical compositions) comprising the TMP of the present disclosure.The present disclosure provides compositions (including pharmaceutical compositions) comprising the nucleic acid or recombinant expression vector of the present disclosure.

[0264] Compositions containing TMP In addition to the TMP of the present disclosure, the compositions of the present disclosure may include one or more of the following: salts, e.g., NaCl, MgCl, KCl, MgSO, etc., buffers, solubilizers, surfactants, e.g., non-ionic surfactants such as Tween-20, protease inhibitors, glycerol, etc. The compositions may also include pharmaceutically acceptable excipients, a variety of which are known in the art and need not be described in detail herein. Examples of pharmaceutically acceptable salts, buffers, excipients, formulations, dosage forms, and the like are disclosed in published PCT applications WO2020132138A1 and WO2019 / 051091, the disclosures of which are expressly incorporated herein by reference as they relate to compositions comprising the TMP of the present disclosure, specifically including paragraphs

[0526] to

[0536] of WO2020132138A1 and paragraphs

[0412] to

[0422] of WO2019 / 051091.

[0265] When the TMP of the present disclosure is administered directly to a tissue as an injection (e.g., subcutaneously, intraperitoneally, intramuscularly, and / or intravenously), the formulation may be directly injected or infused into a patient, mixed with saline for infusion, or provided as a ready-to-use dosage form that can be in a non-aqueous form (e.g., a shelf-stable powder that can be reconstituted) or in an aqueous form (e.g., a liquid comprised of a pharmaceutically acceptable carrier and pharmaceutically acceptable excipients). Formulations may also be provided to extend the serum half-life of the TMP after administration. For example, TMP may be provided as a liposomal formulation prepared as a colloid, or using other conventional techniques to extend serum half-life. Various methods for preparing liposomes are available, as described, for example, in Szoka et al. 1980 Ann. Rev. Biophys. Bioeng. 9:467, U.S. Patent Nos. 4,235,871, 4,501,728, and 4,837,028. The formulations may also be provided in controlled or sustained release forms.

[0266] The concentration of TMP of the present disclosure in liquid composition formulations can vary over a wide range (e.g., from less than about 0.1%, usually (or at least) about 2%, up to 20%-50% or more by weight). Concentrations within this range include concentrations of about 5 to about 15 mg / mL, including 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, and the concentration can depend on many factors, including the stability of the TMP in the liquid composition.

[0267] In some examples, the TMP of the present disclosure is present in a liquid composition. In some examples, the composition of the present disclosure includes a) the TMP of the present disclosure, and b) saline (e.g., 0.9% NaCl). In some examples, the composition is sterile and suitable for administration to a human subject.

[0268] Compositions Comprising Nucleic Acids or Recombinant Expression Vectors The present disclosure provides compositions, e.g., pharmaceutical compositions, comprising the nucleic acids or recombinant expression vectors of the present disclosure. Published PCT applications WO2020132138A1 and WO2019 / 051091 disclose methods for preparing such compositions. See paragraphs

[0537] to

[0546] of WO2020132138A1 and paragraphs

[0423] to

[0432] of WO2019 / 051091, the disclosures of which are expressly incorporated herein by reference.

[0269] Methods for modulating T cell activity The present disclosure provides methods for selectively modulating the activity of epitope-specific T cells (e.g., T cells specific for a KRAS epitope, such as a KRAS peptide comprising a cancer-associated mutation), the methods comprising contacting the T cells with a TMP of the present disclosure, wherein contacting the T cells with the TMP of the present disclosure selectively modulates the activity of the epitope-specific T cells. In some examples, the contacting is performed in vitro. In some examples, the contacting is performed in vivo.

[0270] When a TMP of the present disclosure comprises an immunomodulatory polypeptide that is an activating polypeptide, contacting T cells with the TMP activates epitope-specific T cells. In some examples, the epitope-specific T cells are T cells specific for an epitope present on a cancer cell, and contacting the epitope-specific T cells with the TMP increases the cytotoxic activity of the T cells against the cancer cell and / or increases the number of epitope-specific T cells.

[0271] The present disclosure provides a method for modulating an immune response in an individual, the method comprising administering an effective amount of a TMP of the present disclosure to the individual. Administration of the TMP induces an epitope-specific T cell response (e.g., a cancer epitope-specific T cell response) and an epitope-nonspecific T cell response, wherein the ratio of the epitope-specific T cell response to the epitope-nonspecific T cell response is at least 2:1. In some examples, the ratio of the epitope-specific T cell response to the epitope-nonspecific T cell response is at least 5:1. In some examples, the ratio of the epitope-specific T cell response to the epitope-nonspecific T cell response is at least 10:1. In some examples, the ratio of the epitope-specific T cell response to the epitope-nonspecific T cell response is at least 25:1. In some examples, the ratio of the epitope-specific T cell response to the epitope-nonspecific T cell response is at least 50:1. In some cases, the ratio of epitope-specific T cell responses to epitope-nonspecific T cell responses is at least 100:1. In some cases, the individual is human. In some cases, the modulation increases cytotoxic T cell responses against cancer cells, for example, cancer cells expressing an antigen that presents the same epitope presented by the KRAS peptide present in the TMP, and / or increases the number of T cells specific for the KRAS epitope. In some cases, administration is intravenous, subcutaneous, intramuscular, systemic, intralymphatic, distal to the treatment site, local, or at or near the treatment site.

[0272] The present disclosure provides methods for selectively delivering immunomodulatory polypeptides to target T cells, the methods comprising contacting a mixed population of T cells with a TMP of the present disclosure, the mixed population of T cells comprising target T cells and non-target T cells, where the target T cells are specific for an epitope present in the TMP (e.g., the target T cells are specific for an epitope present in the TMP), and the contacting step delivers one or more immunomodulatory polypeptides present in the TMP to the target T cells. In some examples, the population of T cells is in vitro. In some examples, the population of T cells is in vivo within an individual. In some examples, the method comprises administering the TMP to the individual. In some examples, the T cells are cytotoxic T cells. In some examples, the mixed population of T cells is an in vitro population of mixed T cells obtained from an individual, and the contacting step results in activation and / or proliferation of the target T cells to generate a population of activated and / or expanded target T cells, and in some of these examples, the method further comprises administering the population of activated and / or expanded target T cells to the individual.

[0273] The present disclosure provides methods for detecting the presence of target T cells that bind to an epitope of interest (e.g., a cancer epitope, a KRAS peptide comprising a cancer-associated mutation) in a mixed population of T cells obtained from an individual, the method comprising: a) contacting the mixed population of T cells in vitro with a TMP of the present disclosure, wherein the TMP comprises the KRAS epitope of interest; and b) detecting activation and / or proliferation of the T cells in response to said contact, wherein activated and / or proliferated T cells indicate the presence of the target T cells.

[0274] Treatment method The present disclosure provides a method for treating an individual, the method comprising administering to the individual an amount of the TMP of the present disclosure or one or more nucleic acids encoding the TMP to the individual that is effective for treating the individual.The TMP of the present disclosure is also provided for use in a method for treating a human or non-human animal body.In some examples, the treatment method of the present disclosure comprises administering to an individual in need thereof one or more recombinant expression vectors comprising a nucleotide sequence encoding the TMP of the present disclosure.In some examples, the treatment method of the present disclosure comprises administering to an individual in need thereof one or more mRNA molecules comprising a nucleotide sequence encoding the TMP of the present disclosure.In some examples, the treatment method of the present disclosure comprises administering to an individual in need thereof the TMP of the present disclosure.Treatable conditions include, for example, cancers such as those described above, for example, cancers that express KRAS polypeptides, for example, mutant KRAS polypeptides.

[0275] In some examples, when administered to an individual in need thereof, the TMP of the present disclosure induces both epitope-specific and epitope-nonspecific T cell responses. In other words, in some examples, when administered to an individual in need thereof, the TMP of the present disclosure induces an epitope-specific T cell response by modulating the activation of a first T cell that presents both i) a TCR specific for an epitope present in the TMP and ii) a costimulatory polypeptide that binds to an immunomodulatory polypeptide present in the TMP, and induces an epitope-nonspecific T cell response by modulating the activation of a second T cell that presents i) a TCR specific for an epitope other than the epitope present in the TMP and ii) a costimulatory polypeptide that binds to an immunomodulatory polypeptide 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 T cell responses to epitope-nonspecific T cell responses is about 2:1 to about 5:1, about 5:1 to about 10:1, about 10:1 to about 15:1, about 15:1 to about 20:1, about 20:1 to about 25:1, about 25:1 to about 50:1, or about 50:1 to about 100:1, or more than 100:1. Examples of "modulating activation" of T cells include: i) cytotoxicity (e.g., CD8 + ) activating T cells; ii) cytotoxic (e.g., CD8 + ) inducing T cell cytotoxic activity; iii) cytotoxicity (e.g., CD8 + ) inducing the production and release of cytotoxins (e.g., perforin, granzymes, granulysin) by T cells, and iv) cytotoxicity (e.g., CD8 + ) increasing the number of T cells, etc.

[0276] When a MOD is a low-affinity variant of a wild-type MOD, the combination of the MOD's lower affinity for its cognate costimulatory polypeptide and the affinity of the epitope for the TCR results in improved selectivity for the TMPs of the present disclosure. Thus, for example, a TMP of the present disclosure binds to a first T cell that presents both i) a TCR specific for an epitope other than the epitope present in the TMP and ii) a costimulatory polypeptide that binds to the immunomodulatory polypeptide present in the TMP with a higher avidity than the avidity with which a TMP of the present disclosure binds to a second T cell that presents i) a TCR specific for an epitope other than the epitope present in the TMP and ii) a costimulatory polypeptide that binds to the immunomodulatory polypeptide present in the TMP.

[0277] Thus, the present disclosure provides a method for selectively modulating the activation of epitope-specific T cells in an individual, the method comprising administering to the individual an effective amount of a TMP of the present disclosure or one or more nucleic acids (e.g., expression vectors, mRNA, etc.) comprising a nucleotide sequence encoding the TMP, wherein the TMP selectively modulates the activation of epitope-specific T cells in the individual. Selective modulation of the activation of epitope-specific T cells makes it possible to treat a disease or disorder in the individual. Thus, the present disclosure provides a method of treatment comprising administering an effective amount of a TMP of the present disclosure to an individual in need thereof.

[0278] In some examples, the immunomodulatory polypeptide ("MOD") is an activating polypeptide, and the TMP activates epitope-specific T cells. In some examples, the TMP promotes activation of T cells specific for a KRAS epitope. In some examples, the MOD is an activating polypeptide, and the TMP activates epitope-specific T cells (e.g., T cells specific for a KRAS epitope). In some examples, the T cells are activated by T helper cells (CD4 + cells), cytotoxic T cells (CD8 + In some instances, the epitope is a cancer epitope, and the TMP is a cancer cell (e.g., a T helper cell (CD4 +cells), cytotoxic T cells (CD8 + Increases the activity of CD4 + CD4 is required for T cell activation + Increased proliferation of T cells and / or CD4 + This may include the induction or enhancement of cytokines released by T cells. NK-T cells and / or CD8 + Cell activation involves NK-T cells and / or CD8 + Increased proliferation of cells and / or NK-T cells and / or CD8 + This may include inducing the release of cytokines such as interferon gamma by cells.

[0279] The TMP of the present disclosure can be administered to an individual in need thereof to treat cancer in the individual, where the cancer expresses a KRAS peptide present in the TMP.For example, the cancer can be a cancer in which cancer cells express or overexpress KRAS, for example, a mutant form of KRAS, as described above.The present disclosure provides a method for treating cancer in an individual, the method comprising administering to the individual an effective amount of the TMP of the present disclosure or one or more nucleic acids (e.g., expression vectors, mRNA, etc.) comprising a nucleotide sequence encoding the TMP, wherein the TMP comprises a T cell epitope that is a KRAS epitope, and the TMP comprises a stimulatory immunomodulatory polypeptide.In some examples, an "effective amount" of the TMP of the present disclosure refers to an amount that, when administered in one or more doses to an individual in need thereof, reduces the number of cancer cells in the individual. For example, in some instances, an "effective amount" of a TMP of the present disclosure is an amount that, when administered in one or more doses to an individual in need thereof, reduces the number of cancer cells in the individual by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% compared to the number of cancer cells in the individual before or in the absence of TMP administration. In some instances, an "effective amount" of a TMP of the present disclosure is an amount that, when administered in one or more doses to an individual in need thereof, reduces the number of cancer cells in the individual (including reducing them to substantially undetectable levels).

[0280] In some instances, an "effective amount" of a TMP of the present disclosure refers to an amount that, when administered in one or more doses to an individual in need thereof, reduces tumor weight in the individual. For example, in some instances, an "effective amount" of a TMP of the present disclosure refers to an amount that, when administered in one or more doses to an individual in need thereof (an individual having a tumor), reduces tumor weight 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 weight in the individual before or in the absence of TMP administration. In some instances, an "effective amount" of a TMP of the present disclosure refers to an amount that, when administered in one or more doses to an individual in need thereof (an individual having a tumor), reduces tumor volume in the individual. For example, in some instances, an "effective amount" of a TMP of the present disclosure is an amount that, when administered in one or more doses to an individual in need thereof (an individual having a tumor), reduces the 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 or in the absence of TMP administration. In some instances, an "effective amount" of a TMP of the present disclosure is an amount that, when administered in one or more doses to an individual in need thereof, extends the survival of the individual. For example, in some instances, an "effective amount" of a TMP of the present disclosure is an amount that, when administered in one or more doses to an individual in need thereof, extends 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 TMP administration.

[0281] Cancers that can be treated with the method of the present disclosure include cancers in which cancer cells express mutant KRAS. Examples include adenocarcinoma and hematological tumors. Examples of cancers that can be treated with 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 carcinoma, cervical cancer, and ovarian endometrial cancer.

[0282] As described above, in some cases, in carrying out the present treatment method, the TMP of the present disclosure is administered to an individual in need thereof as TMP itself. In other cases, in carrying out the present treatment method, one or more nucleic acids comprising a nucleotide sequence encoding the TMP of the present disclosure are administered to an individual in need thereof. Thus, in other cases, one or more nucleic acids of the present disclosure, such as one or more recombinant expression vectors of the present disclosure, are administered to an individual in need thereof.

[0283] As described above, the APP of the present disclosure can also be administered to patients for therapeutic purposes in cases where it is desired to engage the TCR of T cells specific for the KRAS peptide of APP. In such cases, the presence of natural immunomodulatory polypeptides in the patient can affect the regulation of T cells when APP engages the TCR.

[0284] formulation Suitable formulations are as described above, and include a pharmaceutically acceptable excipient. In some examples, suitable formulations include a) a TMP of the present disclosure and b) a pharmaceutically acceptable excipient. In some examples, suitable formulations include a) a nucleic acid comprising a nucleotide sequence encoding a TMP of the present disclosure, and b) a pharmaceutically acceptable excipient, and in some examples, the nucleic acid is mRNA. In some examples, suitable formulations include a) a first nucleic acid comprising a nucleotide sequence encoding a first polypeptide of a TMP of the present disclosure, b) a second nucleic acid comprising a nucleotide sequence encoding a second polypeptide of a TMP of the present disclosure, and c) a pharmaceutically acceptable excipient. In some examples, suitable formulations include a) a recombinant expression vector comprising a nucleotide sequence encoding a TMP of the present disclosure, and b) a pharmaceutically acceptable excipient. In some examples, a suitable formulation comprises: a) a first recombinant expression vector comprising a nucleotide sequence encoding a first polypeptide of a TMP of the present disclosure, b) a second recombinant expression vector comprising a nucleotide sequence encoding a second polypeptide of a TMP of the present disclosure, and c) a pharmaceutically acceptable excipient. Suitable pharmaceutically acceptable excipients are described above.

[0285] dose Appropriate dosages can be determined by the attending physician or other qualified medical personnel based on various clinical factors. As is well known in the medical arts, dosages for any single patient depend on many factors, including the patient's size, body surface area, age, the particular polypeptide or nucleic acid being administered, the patient's sex, the duration and route of administration, general health, and other medications being administered concomitantly. The TMP of the present disclosure may be administered in an amount of 0.1 mg / kg body weight to 20 mg / kg body weight per dose, e.g., 0.1 mg / kg body weight to 10 mg / kg body weight, e.g., 0.5 mg / kg body weight to 5 mg / kg body weight, 1 mg / kg body weight to 5 mg / kg body weight, 5 mg / kg body weight to 10 mg / kg body weight, 10 mg / kg body weight to 15 mg / kg body weight, or 15 mg / kg body weight to 20 mg / kg body weight, although dosages outside this exemplary range are contemplated, particularly considering the above factors. If the regimen is a continuous infusion, it may be in the range of 1 μg to 10 mg per kilogram of body weight per minute. The TMP of the present disclosure may be administered in an amount of about 1 mg / kg to 50 mg / kg body weight, e.g., about 1 mg / kg to about 5 mg / kg body weight, about 5 mg / kg to about 10 mg / kg body weight, about 10 mg / kg to about 15 mg / kg body weight, about 15 mg / kg to about 20 mg / kg body weight, about 20 mg / kg to about 25 mg / kg body weight, about 25 mg / kg to about 30 mg / kg body weight, about 30 mg / kg to about 35 mg / kg body weight, about 35 mg / kg to about 40 mg / kg body weight, or about 40 mg / kg to about 50 mg / kg body weight.

[0286] Those skilled in the art can readily estimate repetition rates for administration based on measured residence times and concentrations of the administered agent in bodily fluids or tissues. Following successful treatment, it may be desirable to administer a patient maintenance therapy to prevent recurrence of the disease state, wherein the TMP of the present disclosure is administered at a maintenance dose ranging from 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 greater than 20 mg / kg body weight.

[0287] Those of skill in the art will readily appreciate that dosage levels may vary depending on the particular TMP, the severity of the symptoms, and the subject's susceptibility to side effects. Preferred dosages for any compound can be readily determined by those of skill in the art using a variety of methods.

[0288] In some examples, multiple doses of the TMP, nucleic acid, or recombinant expression vector of the present disclosure are administered. The frequency of administration of the TMP, nucleic acid, or recombinant expression vector of the present disclosure can vary depending on various factors, such as the severity of symptoms. For example, in some examples, the TMP, nucleic acid, or recombinant expression vector of the present disclosure 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), daily (qd), twice a day (qid), or three times a day (tid).

[0289] The duration of administration of the TMP, nucleic acid, or recombinant expression vector of the present disclosure, for example, the period during which the TMP, nucleic acid, or recombinant expression vector of the present disclosure is administered, can vary depending on various factors, for example, the patient's response, etc. For example, the TMP, nucleic acid, or recombinant expression vector of the present disclosure may be administered for a period ranging from about 1 day to about 1 week, from about 2 weeks to about 4 weeks, from about 1 month to about 2 months, from about 2 months to about 4 months, from about 4 months to about 6 months, from about 6 months to about 8 months, from about 8 months to about 1 year, from about 1 year to about 2 years, or from about 2 years to about 4 years, or longer.

[0290] Route of administration The active agent (a TMP of the present disclosure, a nucleic acid of the present disclosure, or a recombinant expression vector of the present disclosure) is administered to an individual using any available method and route suitable for drug delivery, including in vivo and in vitro methods, as well as systemic and local administration routes.

[0291] The TMPs of the present disclosure are typically delivered via intravenous administration, although other conventional pharmaceutically acceptable routes of administration may also be used, including intratumoral, peritumoral, intramuscular, intralymphatic, intratracheal, intracranial, subcutaneous, intradermal, topical, intraarterial, rectal, nasal, oral, and other enteral and parenteral routes of administration. Routes of administration may be combined as needed, or tailored depending on the TMP and / or the desired effect. The TMPs of the present disclosure, or the nucleic acids or recombinant expression vectors of the present disclosure, may be administered in a single dose or multiple doses.

[0292] A TMP of the present disclosure, a nucleic acid of the present disclosure, or a recombinant expression vector of the present disclosure may be administered to a host using any available conventional method and route suitable for general drug delivery, including systemic or local routes. Generally, routes of administration contemplated for use in the methods of the present disclosure include, but are not necessarily limited to, enteral, parenteral, and inhalation routes.

[0293] Combination therapy The TMP of the present disclosure can be administered to an individual in need thereof in combination with one or more additional therapeutic agents or treatments. A suitable dose of TMP can be the same as the dose (above) of monotherapy with APP, or it can be less or more than the dose 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 additional TMPs. Suitable additional therapeutic treatments include, for example, radiation, surgery (e.g., surgical removal of tumors), etc.

[0294] In some examples, the method includes administering to an individual in need thereof a) a first composition comprising a TMP of the present disclosure, and b) a second composition comprising an immune checkpoint inhibitor. In some examples, the method includes administering to an individual in need thereof a) a first composition comprising a TMP of the present disclosure, and b) a second composition comprising an agent that inhibits a cancer-associated mutant form of KRAS, such as KRAS(G12C). In some examples, the method includes administering to an individual in need thereof a) a first composition comprising a TMP of the present disclosure, and b) a second composition comprising a second TMP.

[0295] The TMP of the present disclosure can be administered to an individual in need thereof at the same time as, or at different times than, the administration of one or more additional therapeutic agents.

[0296] Thus, for example, a therapeutic method of the present disclosure can include co-administration of a TMP of the present disclosure with at least one additional therapeutic agent. "Co-administration" refers to administering both a TMP of the present disclosure and at least one additional therapeutic agent to an individual, although not necessarily simultaneously, to achieve a therapeutic effect resulting from the administration of both the TMP and the at least one additional therapeutic agent. The administration of the TMP and the at least one additional therapeutic agent can be substantially simultaneous; for example, the 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 examples, the TMP of the present disclosure is administered to an individual who is undergoing or has been treated with at least one additional therapeutic agent. The administration of the TMP and the at least one additional therapeutic agent can occur at different times and / or at different frequencies.

[0297] As another example, a therapeutic method of the present disclosure may include co-administration of a TMP of the present disclosure with an immune checkpoint inhibitor, such as an antibody specific for an immune checkpoint. "Co-administration" refers to administering both a TMP of the present disclosure and an antibody specific for an immune checkpoint to an individual, although not necessarily simultaneously, to achieve a therapeutic effect resulting from administering both the TMP and the immune checkpoint inhibitor. The administration of the TMP and the antibody specific for an immune checkpoint polypeptide can be substantially simultaneous; for example, the 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 the immune checkpoint. In some examples, a TMP of the present disclosure is administered to an individual who is undergoing or has been treated with an antibody specific for an immune checkpoint. The administration of the TMP and immune checkpoint-specific antibody can be at different times and / or at different frequencies.

[0298] Exemplary immune checkpoint inhibitors include inhibitors that target immune checkpoint polypeptides such as CD27, CD28, CD40, CD122, CD96, CD73, CD47, OX40, GITR, CSF1R, JAK, PI3Kδ, PI3Kγ, 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 examples, the immune checkpoint polypeptide is a stimulatory checkpoint molecule selected from CD27, CD28, CD40, ICOS, OX40, GITR, CD122, and CD137. In some examples, 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.

[0299] In some instances, the immune checkpoint inhibitor is an antibody specific for an immune checkpoint. Suitable anti-immune checkpoint antibodies include 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), arelumab (Merck Serono), ipilimumab (YERVOY, (Bristol-Myers Squibb), tremelimumab (Pfizer), pidilizumab (CureTech, Ltd.), IMP321 (Immutep SA), MGA271 (Macrogenics), BMS-986016 (Bristol-Myers Squibb), and pembrolizumab (CureTech, Ltd.). Squibb), lirilumab (Bristol-Myers Squibb), urelumab (Bristol-Myers 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), AUNP12 (Aurigene and Pierre Fabre), indoximod (NewLink Genetics), NLG-919 (NewLink Examples of suitable anti-PD-1 antibodies include, but are not limited to, nivolumab, pembrolizumab (also known as MK-3475), pidilizumab, SHR-1210, PDR001, and AMP-224.In some examples, the anti-PD-1 monoclonal antibody is nivolumab, pembrolizumab, or PDR001. Suitable anti-PD1 antibodies are described in U.S. Patent Publication No. 2017 / 0044259. For pidilizumab, see, e.g., Rosenblatt et al. (2011) J. Immunother. 34:409-18. In some examples, the immune checkpoint inhibitor is an anti-CTLA-4 antibody. In some examples, the anti-CTLA-4 antibody is ipilimumab or tremelimumab. For tremelimumab, see, e.g., Ribas et al. (2013) J. Clin. Oncol. 31:616-22. In some examples, the immune checkpoint inhibitor is an anti-PD-L1 antibody. In some examples, the anti-PD-L1 monoclonal antibody is BMS-935559, MEDI4736, MPDL3280A (also known as RG7446), KN035, or MSB0010718C. In some embodiments, 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.

[0300] In some examples, the at least one additional therapeutic agent is an agent that selectively inhibits mutant KRAS, such as KRAS(G12C), KRAS(K117A), etc. Examples of agents that selectively inhibit mutant KRAS include ARS-1620, AMG510, KRA-533, and MRTX849.

[0301] The AMG150 has the following structure: I have TIFF0007756072000015.tif51128.

[0302] ARS-1620 has the following structure: I have TIFF0007756072000016.tif62128.

[0303] In some examples, the at least one additional therapeutic agent comprises one or more additional TMPs. In some examples, the method includes administering to an individual in need thereof: a) a first composition comprising a first TMP (where the first TMP is a TMP of the present disclosure), and b) a second composition comprising a second TMP (where the second TMP is a TMP of the present disclosure that is different from the first TMP of the present disclosure, e.g., comprises a different KRAS epitope and / or one or more different MODs). Additionally or alternatively, the one or more additional TMPs may comprise an epitope that is a cancer-associated peptide other than a KRAS peptide epitope.

[0304] Suitable subjects for treatment Suitable subjects for treatment using the methods of the present disclosure include individuals with cancer, including individuals who have been diagnosed with cancer, individuals who have been treated for cancer but have not responded to the treatment, and individuals who have been treated for cancer and initially responded but have subsequently stopped responding to the treatment. Suitable subjects for treatment using the methods of the present disclosure include individuals with cancer whose cancer cells express mutant KRAS, where the mutant KRAS is a cancer-associated mutant. Suitable subjects for treatment using the methods of the present disclosure include individuals with cancers such as 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.

[0305] In some examples, the subject is an individual undergoing treatment with an immune checkpoint inhibitor. In some examples, the subject is an individual who has been treated with an...

Claims

1. T cell modulatory polypeptides (TMPs), The TMP is i) a KRAS peptide comprising a KRAS epitope expressed on cancer cells and having a length of 8 to 12 amino acids; ii) a β2-microglobulin (β2M) polypeptide containing a Cys residue; iii) an MHC class I heavy chain polypeptide comprising at least two Cys residues; iv) immunoglobulin (Ig) Fc polypeptides, and v) two variant IL-2 polypeptides that do not substantially bind to IL-2Rα and have reduced affinity for IL-2Rβ, each of which contains an amino acid other than histidine at position 16 and an amino acid other than phenylalanine at position 42 compared to the amino acid sequence set forth in SEQ ID NO:

15. is a single polypeptide chain comprising the β2M polypeptide is connected to the KRAS peptide by a first linker comprising a Cys residue; the TMP comprises one or more independently selected linkers; the TMP comprises a first intrachain disulfide bond linking a Cys residue of the β2M polypeptide to a Cys amino acid residue of the MHC class I heavy chain polypeptide; and the TMP comprises a second intrachain disulfide bond connecting a Cys of the linker between the β2M and the KRAS to a Cys of the MHC class I heavy chain polypeptide; The TMP.

2. From the N-terminus to the C-terminus, i) a KRAS peptide, ii) a β2M polypeptide, iii) an MHC class I heavy chain polypeptide, iv) an Ig Fc polypeptide, and v) two variant IL-2 polypeptides; the Ig Fc polypeptide is a human IgG1 Fc polypeptide that does not substantially induce cell lysis; the TMP comprises one or more independently selected linkers; 2. The TMP of claim 1.

3. 3. The TMP of claim 2, wherein the human IgG1 Fc polypeptide that does not substantially induce cell lysis has at least 95% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO:

30.

4. the β2M polypeptide is connected to the KRAS peptide by a first linker comprising the sequence CGGGS(GGGGS)n (SEQ ID NO: 142) or GCGGS(GGGGS)n (SEQ ID NO: 140), where n is an integer from 1 to 10, e.g., 2 or 3; the MHC class I heavy chain polypeptide comprises a Cys at residue 84 and a Cys at residue 236 compared to the amino acid sequence set forth in SEQ ID NO: 108; the β2M polypeptide comprises a Cys at residue 12 compared to the amino acid sequence set forth in SEQ ID NO: 128; Cys at amino acid residue 12 of the β2M polypeptide is disulfide bonded to Cys at amino acid residue 236 of the MHC class I heavy chain polypeptide; a disulfide bond connects the Cys of the linker to the Cys substituted for Tyr84 of the MHC class I heavy chain polypeptide; the β2M polypeptide is connected to the MHC class I heavy chain polypeptide by a (GGGGS)n linker, where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, e.g., n=3 or 7; The TMP comprises two variant IL-2 polypeptides in tandem having identical amino acid sequences.

4. The TMP according to claim 2 or 3.

5. 5. The TMP of claim 4, wherein the two variant IL-2 polypeptides comprise i) an H16A substitution and an F42A substitution or ii) an H16T substitution and an F42A substitution compared to the amino acid sequence set forth in SEQ ID NO:

15.

6. The TMP of any one of claims 1 to 5, wherein the KRAS peptide comprises an amino acid sequence selected from the group consisting of: A) VVGADGVGK (SEQ ID NO: 176), VVGACGVGK (SEQ ID NO: 177), VVGAVGVGK (SEQ ID NO: 178), VVVGADGVGK (SEQ ID NO: 179), VVVGAVGVGK (SEQ ID NO: 180), VVVGACGVGK (SEQ ID NO: 181), VTGADGVGK (SEQ ID NO: 182), VTGAVGVGK (SEQ ID NO: 183), VTGACGVGK (SEQ ID NO: 184), VTVGADGVGK (SEQ ID NO: 185), VTGAVGVGK (SEQ ID NO: 186), and VTVGACCGVGK (SEQ ID NO: 187); 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: 188), VVGAGDVGK (SEQ ID NO: 189), VVVGARGVGK (SEQ ID NO: 190), and VVGARGVGK (SEQ ID NO: 191); wherein the KRAS peptide is 9 or 10 amino acids in length, or at least 9 or 10 amino acids in length; C) LVVVGADGV (SEQ ID NO:192), LVVVGAVGV (SEQ ID NO:193), LVVVGACGV (SEQ ID NO:194), KLVVVGADGV (SEQ ID NO:195), KLVVVGAVGV (SEQ ID NO:196), KLVVVGACGV (SEQ ID NO:197), LLVVGADGV (SEQ ID NO:198), LLVVGAVGV (SEQ ID NO:199), LLVVGACGV (SEQ ID NO:200), FLVVVGADGV (SEQ ID NO:201), FLVVVGAVGV (SEQ ID NO:202), and FLVVVGACGV (SEQ ID NO:203); wherein the KRAS peptide is 9 or 10 amino acids in length, or at least 9 or 10 amino acids in length; D) KLVVVGAGDV (SEQ ID NO: 204), and KLVVVGARGV (SEQ ID NO: 205); wherein the KRAS peptide is 9 or 10 amino acids in length, or at least 9 or 10 amino acids in length; E) GAGDVGKSAL (SEQ ID NO: 206), AGDVGKSAL (SEQ ID NO: 207), DVGKSALTI (SEQ ID NO: 208), GAVGVGKSAL (SEQ ID NO: 209), AVGVGKSAL (SEQ ID NO: 210), YKLVVVGAV (SEQ ID NO: 211), ARGVGKSAL (SEQ ID NO: 212), GARGVGKSAL (SEQ ID NO: 213), EYKLVVVGAR (SEQ ID NO: 2 14), RGVGKSALTI (SEQ ID NO:215), LVVVGARGV (SEQ ID NO:216), GADGVGKSAL (SEQ ID NO:217), ACGVGKSAL (SEQ ID NO:218), and GACGVGKSAL (SEQ ID NO:219); wherein the KRAS peptide is 9 or 10 amino acids in length, or at least 9 or 10 amino acids in length; and F) VVGAVGVGK (SEQ ID NO: 178), VVVGAVGVGK (SEQ ID NO: 180), VGAVGVGKS (SEQ ID NO: 222), VGAVGVGKSA (SEQ ID NO: 223), AVGVGKSAL (SEQ ID NO: 210), AVGVGKSALT (SEQ ID NO: 225), GAVGVGKSAL (SEQ ID NO: 209), GAVGVGKSA (SEQ ID NO: 227), LVVVGAVGVG (SEQ ID NO: 228), LVVVGAVGV (SEQ ID NO: 193), KLVVVGAVGV (SEQ ID NO: 196), and KLVVVGAVG (SEQ ID NO: 231); wherein the KRAS peptide is 9 or 10 amino acids in length, or at least 9 or 10 amino acids in length.

7. T cell modulatory polypeptides (TMPs), The TMP is i) a KRAS peptide comprising a KRAS epitope expressed on cancer cells and having a length of 8 to 12 amino acids; ii) a β2-microglobulin (β2M) polypeptide containing a Cys residue; iii) an MHC class I heavy chain polypeptide comprising at least two Cys residues; iv) immunoglobulin (Ig) Fc polypeptides, and v) A variant IL-2 polypeptide, which does not substantially bind to IL-2Rα and has reduced affinity for IL-2Rβ, and which comprises an amino acid other than histidine at position 16 and an amino acid other than phenylalanine at position 42 compared to the amino acid sequence set forth in SEQ ID NO:

15. is a single polypeptide chain comprising the β2M polypeptide is connected to the KRAS peptide by a first linker comprising a Cys residue; the TMP comprises one or more independently selected linkers; the TMP comprises a first intrachain disulfide bond linking a Cys residue of the β2M polypeptide to a Cys amino acid residue of the MHC class I heavy chain polypeptide; and the TMP comprises a second intrachain disulfide bond connecting a Cys of the linker between the β2M and the KRAS to a Cys of the MHC class I heavy chain polypeptide; The TMP, wherein the KRAS peptide comprises an amino acid sequence selected from the following: A) VVGADGVGK (SEQ ID NO: 176), VVGACGVGK (SEQ ID NO: 177), VVGAVGVGK (SEQ ID NO: 178), VVVGADGVGK (SEQ ID NO: 179), VVVGAVGVGK (SEQ ID NO: 180), VVVGACGVGK (SEQ ID NO: 181), VTGADGVGK (SEQ ID NO: 182), VTGAVGVGK (SEQ ID NO: 183), VTGACGVGK (SEQ ID NO: 184), VTVGADGVGK (SEQ ID NO: 185), VTGAVGVGK (SEQ ID NO: 186), and VTVGACCGVGK (SEQ ID NO: 187); 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: 188), VVGAGDVGK (SEQ ID NO: 189), VVVGARGVGK (SEQ ID NO: 190), and VVGARGVGK (SEQ ID NO: 191); wherein the KRAS peptide is 9 or 10 amino acids in length, or at least 9 or 10 amino acids in length; C) LVVVGADGV (SEQ ID NO:192), LVVVGAVGV (SEQ ID NO:193), LVVVGACGV (SEQ ID NO:194), KLVVVGADGV (SEQ ID NO:195), KLVVVGAVGV (SEQ ID NO:196), KLVVVGACGV (SEQ ID NO:197), LLVVGADGV (SEQ ID NO:198), LLVVGAVGV (SEQ ID NO:199), LLVVGACGV (SEQ ID NO:200), FLVVVGADGV (SEQ ID NO:201), FLVVVGAVGV (SEQ ID NO:202), and FLVVVGACGV (SEQ ID NO:203); wherein the KRAS peptide is 9 or 10 amino acids in length, or at least 9 or 10 amino acids in length; D) KLVVVGAGDV (SEQ ID NO: 204), and KLVVVGARGV (SEQ ID NO: 205); wherein the KRAS peptide is 9 or 10 amino acids in length, or at least 9 or 10 amino acids in length; E) GAGDVGKSAL (SEQ ID NO: 206), AGDVGKSAL (SEQ ID NO: 207), DVGKSALTI (SEQ ID NO: 208), GAVGVGKSAL (SEQ ID NO: 209), AVGVGKSAL (SEQ ID NO: 210), YKLVVVGAV (SEQ ID NO: 211), ARGVGKSAL (SEQ ID NO: 212), GARGVGKSAL (SEQ ID NO: 213), EYKLVVVGAR (SEQ ID NO: 2 14), RGVGKSALTI (SEQ ID NO:215), LVVVGARGV (SEQ ID NO:216), GADGVGKSAL (SEQ ID NO:217), ACGVGKSAL (SEQ ID NO:218), and GACGVGKSAL (SEQ ID NO:219); wherein the KRAS peptide is 9 or 10 amino acids in length, or at least 9 or 10 amino acids in length; and F) VVGAVGVGK (SEQ ID NO: 178), VVVGAVGVGK (SEQ ID NO: 180), VGAVGVGKS (SEQ ID NO: 222), VGAVGVGKSA (SEQ ID NO: 223), AVGVGKSAL (SEQ ID NO: 210), AVGVGKSALT (SEQ ID NO: 225), GAVGVGKSAL (SEQ ID NO: 209), GAVGVGKSA (SEQ ID NO: 227), LVVVGAVGVG (SEQ ID NO: 228), LVVVGAVGV (SEQ ID NO: 193), KLVVVGAVGV (SEQ ID NO: 196), and KLVVVGAVG (SEQ ID NO: 231); wherein the KRAS peptide is 9 or 10 amino acids in length, or at least 9 or 10 amino acids in length.

8. The MHC class I heavy chain polypeptide comprises the amino acid sequence set forth in SEQ ID NO:

44. ＊ 0201 polypeptide, HLA-A comprising the amino acid sequence set forth in SEQ ID NO:49 ＊ 1101 polypeptide, HLA-A comprising the amino acid sequence set forth in SEQ ID NO:51 ＊ 3303 polypeptide, and HLA-A comprising the amino acid sequence set forth in SEQ ID NO: 50 ＊ 8. The TMP of any one of claims 1 to 7, 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. A) the KRAS peptide is KLVVVGADGV (SEQ ID NO: 195) and the MHC class I 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: 179), VVVGAVGVGK (SEQ ID NO: 180), VVGADGVGK (SEQ ID NO: 176), or VVGAVGVGK (SEQ ID NO: 178), and the MHC class I heavy chain polypeptide is HLA-A11 ＊ 01 polypeptide, comprising an amino acid sequence having at least 95% amino acid sequence identity to the The TMP according to any one of claims 1 to 8.

10. the Ig Fc polypeptide comprises one or more amino acid substitutions selected from N77A, L14A, L15A, L14F, L15E, and P111S, and has at least 95% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 30; The TMP according to any one of claims 1 to 9.

11. 11. The TMP of claim 10, wherein the Ig Fc polypeptide comprises L14A and L15A amino acid substitutions and has at least 98% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO:

30.

12. 1. A T cell modulatory polypeptide (TMP) comprising a heterodimer, said heterodimer comprising: a) a first polypeptide, i) a KRAS peptide comprising a KRAS epitope expressed on cancer cells and having a length of 8 to 12 amino acids; ii) a peptide linker comprising a Cys residue, and iii) β2 microglobulin (β2M) polypeptides containing Cys residues the first polypeptide comprising: b) a second polypeptide, i) one or more immunomodulatory polypeptides; ii) a major histocompatibility complex (MHC) class I heavy chain polypeptide comprising at least two Cys residues; and iii) immunoglobulin (Ig) Fc polypeptides the second polypeptide comprising Including, the first polypeptide and the second polypeptide are covalently linked to each other by at least first and second disulfide bonds, the first disulfide bond being between (i) a Cys residue of a Cys-containing linker between the KRAS peptide and the β2M polypeptide and (ii) a Cys residue of the MHC class I heavy chain polypeptide; and the second disulfide bond being between a Cys residue of the β2M polypeptide and a Cys residue of the MHC class I heavy chain polypeptide; and the TMP comprises one or more additional, independently selected peptide linkers between any two of the above components, and when the TMP comprises two or more immunomodulatory polypeptides, the immunomodulatory polypeptides are connected by one or more independently selected peptide linkers; The TMP wherein the KRAS peptide comprises an amino acid sequence selected from the following: A) VVGADGVGK (SEQ ID NO: 176), VVGACGVGK (SEQ ID NO: 177), VVGAVGVGK (SEQ ID NO: 178), VVVGADGVGK (SEQ ID NO: 179), VVVGAVGVGK (SEQ ID NO: 180), VVVGACGVGK (SEQ ID NO: 181), VTGADGVGK (SEQ ID NO: 182), VTGAVGVGK (SEQ ID NO: 183), VTGACGVGK (SEQ ID NO: 184), VTVGADGVGK (SEQ ID NO: 185), VTGAVGVGK (SEQ ID NO: 186), and VTVGACCGVGK (SEQ ID NO: 187); 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: 188), VVGAGDVGK (SEQ ID NO: 189), VVVGARGVGK (SEQ ID NO: 190), and VVGARGVGK (SEQ ID NO: 191); wherein the KRAS peptide is 9 or 10 amino acids in length, or at least 9 or 10 amino acids in length; C) LVVVGADGV (SEQ ID NO:192), LVVVGAVGV (SEQ ID NO:193), LVVVGACGV (SEQ ID NO:194), KLVVVGADGV (SEQ ID NO:195), KLVVVGAVGV (SEQ ID NO:196), KLVVVGACGV (SEQ ID NO:197), LLVVGADGV (SEQ ID NO:198), LLVVGAVGV (SEQ ID NO:199), LLVVGACGV (SEQ ID NO:200), FLVVVGADGV (SEQ ID NO:201), FLVVVGAVGV (SEQ ID NO:202), and FLVVVGACGV (SEQ ID NO:203); wherein the KRAS peptide is 9 or 10 amino acids in length, or at least 9 or 10 amino acids in length; D) KLVVVGAGDV (SEQ ID NO: 204), and KLVVVGARGV (SEQ ID NO: 205); wherein the KRAS peptide is 9 or 10 amino acids in length, or at least 9 or 10 amino acids in length; E) GAGDVGKSAL (SEQ ID NO: 206), AGDVGKSAL (SEQ ID NO: 207), DVGKSALTI (SEQ ID NO: 208), GAVGVGKSAL (SEQ ID NO: 209), AVGVGKSAL (SEQ ID NO: 210), YKLVVVGAV (SEQ ID NO: 211), ARGVGKSAL (SEQ ID NO: 212), GARGVGKSAL (SEQ ID NO: 213), EYKLVVVGAR (SEQ ID NO: 2 14), RGVGKSALTI (SEQ ID NO:215), LVVVGARGV (SEQ ID NO:216), GADGVGKSAL (SEQ ID NO:217), ACGVGKSAL (SEQ ID NO:218), and GACGVGKSAL (SEQ ID NO:219); wherein the KRAS peptide is 9 or 10 amino acids in length, or at least 9 or 10 amino acids in length; and F) VVGAVGVGK (SEQ ID NO: 178), VVVGAVGVGK (SEQ ID NO: 180), VGAVGVGKS (SEQ ID NO: 222), VGAVGVGKSA (SEQ ID NO: 223), AVGVGKSAL (SEQ ID NO: 210), AVGVGKSALT (SEQ ID NO: 225), GAVGVGKSAL (SEQ ID NO: 209), GAVGVGKSA (SEQ ID NO: 227), LVVVGAVGVG (SEQ ID NO: 228), LVVVGAVGV (SEQ ID NO: 193), KLVVVGAVGV (SEQ ID NO: 196), and KLVVVGAVG (SEQ ID NO: 231); wherein the KRAS peptide is 9 or 10 amino acids in length, or at least 9 or 10 amino acids in length.

13. a1) the first polypeptide comprises, in order from N-terminus to C-terminus: i) the KRAS peptide; ii) a Cys-containing peptide linker, and iii) β2M polypeptide and b1) the second polypeptide comprises, in order from the N-terminus to the C-terminus: i) one or more immunomodulatory polypeptides; ii) an MHC class I heavy chain polypeptide, and iii) Ig Fc polypeptide Including, the Ig Fc polypeptide is a human IgG1 Fc polypeptide that does not substantially induce cell lysis; the TMP comprises one or more independently selected linkers; 13. The T cell modulatory polypeptide of claim 12.

14. The T cell modulatory polypeptide of claim 13, wherein the human IgG1 Fc polypeptide that does not substantially induce cell lysis has at least 95% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO:

30.

15. a3) the first polypeptide comprises, in order from the N-terminus to the C-terminus: i) the KRAS peptide; ii) a Cys-containing peptide linker, and iii) β2M polypeptide and b3) the second polypeptide comprises, in order from the N-terminus to the C-terminus: i) an MHC class I heavy chain polypeptide, and ii) an Ig Fc polypeptide, and iii) one or more immunomodulatory polypeptides Including, the Ig Fc polypeptide is a human IgG1 Fc polypeptide that does not substantially induce cell lysis; the TMP comprises one or more independently selected linkers; 13. The T cell modulatory polypeptide of claim 12.

16. The T cell modulatory polypeptide of claim 15, wherein the human IgG1 Fc polypeptide that does not substantially induce cell lysis has at least 95% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO:

30.

17. 17. The TMP of any one of claims 12 to 16, wherein the at least one immunomodulatory polypeptide is a wild-type or variant form of an activating immunomodulatory polypeptide selected from the group consisting of IL-2, 4-1BBL, CD80, CD86, or a combination thereof.

18. 18. The T cell modulatory polypeptide of claim 17, wherein at least one of the at least one immunomodulatory polypeptides is a variant immunomodulatory polypeptide that exhibits reduced affinity for a cognate costimulatory polypeptide compared to the affinity of a corresponding wild-type immunomodulatory polypeptide for the cognate costimulatory polypeptide.

19. 19. The TMP of claim 17 or 18, wherein the at least one immunomodulatory polypeptide is a variant IL-2 that does not substantially bind to IL-2Rα and has reduced affinity for IL-2Rβ.

20. 20. The TMP of claim 19, wherein the at least one immunomodulatory polypeptide is a variant IL-2 polypeptide comprising an amino acid other than histidine at position 16 and an amino acid other than phenylalanine at position 42 compared to the amino acid sequence set forth in SEQ ID NO:

15.

21. 21. The TMP of claim 20, wherein the variant IL-2 polypeptide comprises i) an H16A substitution and an F42A substitution, or ii) an H16T substitution and an F42A substitution compared to the amino acid sequence set forth in SEQ ID NO:

15.

22. comprising two variant IL-2 polypeptides in tandem, each of the two variant IL-2 polypeptides comprises an amino acid other than histidine at position 16 and an amino acid other than phenylalanine at position 42 compared to the amino acid sequence set forth in SEQ ID NO: 15; A TMP according to any one of claims 12 to 21.

23. 23. The TMP of claim 22, wherein the two variant IL-2 polypeptides are separated by a linker.

24. the linker between the β2M polypeptide and the KRAS peptide comprises the sequence CGGGS(GGGGS)n (SEQ ID NO: 142) or GCGGS(GGGGS)n (SEQ ID NO: 140), where n is an integer from 1 to 10, e.g., 2 or 3; the MHC class I heavy chain polypeptide comprises a Cys at residue 84 and a Cys at residue 236 compared to the amino acid sequence set forth in SEQ ID NO: 108; the β2M polypeptide comprises a Cys at residue 12 compared to the amino acid sequence set forth in SEQ ID NO: 128; a first disulfide bond links a Cys of the linker between the β2M polypeptide and the KRAS peptide to a Cys at residue 84 of the MHC class I heavy chain polypeptide; a second disulfide bond links a Cys at amino acid residue 12 of the β2M polypeptide to a Cys at amino acid residue 236 of the MHC class I heavy chain polypeptide; each of the variant IL-2 polypeptides comprises an H16A substitution and an F42A substitution compared to the amino acid sequence set forth in SEQ ID NO: 15, and the variant IL-2 polypeptides are separated by a linker.

24. The TMP of claim 22 or 23.

25. The MHC class I heavy chain polypeptide comprises the amino acid sequence set forth in SEQ ID NO:

44. ＊ 0201 polypeptide, HLA-A comprising the amino acid sequence set forth in SEQ ID NO:49 ＊ 1101 polypeptide, HLA-A comprising the amino acid sequence set forth in SEQ ID NO:51 ＊ 3303 polypeptide, and HLA-A comprising the amino acid sequence set forth in SEQ ID NO: 50 ＊ 25. The TMP of any one of claims 12 to 24, 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.

26. A) the KRAS peptide is KLVVVGADGV (SEQ ID NO: 195) and the MHC class I 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: 179), VVVGAVGVGK (SEQ ID NO: 180), VVGADGVGK (SEQ ID NO: 176), or VVGAVGVGK (SEQ ID NO: 178), and the MHC class I heavy chain polypeptide is HLA-A11 ＊ 01 polypeptide, comprising an amino acid sequence having at least 95% amino acid sequence identity to the A TMP according to any one of claims 12 to 24.

27. 27. The T cell modulatory polypeptide of any one of claims 12 to 26, wherein the Ig Fc polypeptide comprises one or more amino acid substitutions selected from N77A, L14A, L15A, L14F, L15E, and P111S, and has at least 95% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO:

30.

28. 28. The T cell modulatory polypeptide of claim 27, wherein the Ig Fc polypeptide comprises amino acid substitutions L14A and L15A and has at least 98% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO:

30.

29. a) a first and a second single-chain TMP according to any one of claims 1 to 11, having identical amino acid sequences and covalently linked by one or more disulfide bonds between the Ig Fc polypeptides of the first and second single-chain TMPs; or b) a first and a second heterodimeric TMP according to any one of claims 12 to 28, having identical amino acid sequences and covalently linked by one or more disulfide bonds between the Ig Fc polypeptides of the first and second heterodimers. T cell modulatory polypeptide (TMP), a homodimer comprising:

30. A nucleic acid comprising a nucleotide sequence encoding the single-chain TMP according to any one of claims 1 to 11, or A nucleic acid comprising a nucleotide sequence encoding the first and second polypeptides of the heterodimeric TMP of any one of claims 12 to 29.

31. 30. An in vitro method of selectively modulating the activity of T cells specific for a KRAS peptide epitope, said method comprising contacting said T cells with a TMP according to any one of claims 1 to 29, wherein said contacting selectively modulates the activity of said epitope-specific T cells.

32. A pharmaceutical composition for treating a KRAS-related cancer in a patient having said cancer, comprising an effective amount of the TMP of any one of claims 1 to 29.

33. 33. The pharmaceutical composition of claim 32, wherein TMP is administered to the patient in an amount of 1 mg / kg to 5 mg / kg of body weight.

34. further comprising an immune checkpoint inhibitor, The TMP and the immune checkpoint inhibitor are administered simultaneously or at different times.

34. The pharmaceutical composition of claim 32 or 33.

35. The pharmaceutical composition of claim 34, wherein the immune checkpoint inhibitor is an antibody specific for PD-L1, PD-1, TIGIT, or CTLA4.

36. The pharmaceutical composition of claim 34, wherein the immune checkpoint inhibitor is an antibody specific for PD-1 or PD-L1.

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