Multimeric T cell regulatory polypeptides and methods of use thereof

Abstract

The present disclosure provides T cell regulatory multimeric polypeptides comprising an immunomodulatory polypeptide, an epitope-presenting peptide, and a Class I MHC polypeptide. The T cell regulatory multimeric polypeptides are useful for modulating the activity of T cells and modulating an immune response in an individual. TIFF2022534846000227.tif27146

Description

[Technical Field]

[0001] cross reference This application claims the benefit of U.S. Provisional Patent Application No. 62 / 854,200, filed May 29, 2019, U.S. Provisional Patent Application No. 62 / 872,048, filed July 9, 2019, and U.S. Provisional Patent Application No. 62 / 901,538, filed September 17, 2019, which applications are incorporated herein by reference in their entireties. [Background technology]

[0002] preface The adaptive immune response is driven by the binding of T cell receptors (TCRs) present on the surface of T cells to small peptide antigens noncovalently presented on the surface of antigen-presenting cells (APCs) via the major histocompatibility complex (MHC, also known in humans as the human leukocyte antigen (HLA) complex). This binding represents the targeting mechanism of the immune system and is a molecular interaction essential for T cell regulation (activation or suppression) and effector function. Following epitope-specific cell targeting, the targeted T cell is activated by the binding of costimulatory proteins present on the APC with corresponding costimulatory proteins on the T cell. Both signals—epitope / TCR binding and the binding of APC costimulatory proteins with T cell costimulatory proteins—are required to drive T cell specificity and activation or suppression. While TCRs are specific for a given epitope, costimulatory proteins are not epitope-specific; instead, they are generally expressed on all T cells or many T cell subsets. Summary of the Invention

[0003] overview The present disclosure provides T cell regulatory multimeric polypeptides (TMMPs) comprising an immunomodulatory polypeptide, a class I HLA polypeptide (a class I HLA heavy chain polypeptide, and a β2 microglobulin polypeptide), and a peptide that presents an epitope to a T cell receptor. The TMMPs are useful for modulating T cell activation and for modulating immune responses in individuals. [The present invention 1001] (a)(i) a peptide epitope, which is a peptide having a length of at least four amino acids; and (ii) a first major histocompatibility complex (MHC) polypeptide a first polypeptide comprising (b) a second polypeptide comprising a second MHC polypeptide; (c) at least one immunomodulatory polypeptide, wherein the first and / or second polypeptide comprises the at least one immunomodulatory polypeptide; (d) an immunoglobulin (Ig) Fc polypeptide or a non-Ig scaffold, wherein the first and / or second polypeptide comprises the Ig Fc polypeptide or the non-Ig scaffold; and (e) a tumor targeting polypeptide, wherein the first and / or second polypeptide comprises the tumor targeting polypeptide. 10. A T cell regulatory multimeric polypeptide comprising at least one heterodimer comprising: [The present invention 1002] 1001. A T cell regulatory multimeric polypeptide of the present invention, wherein at least one of the one or more immunomodulatory polypeptides is a variant immunomodulatory polypeptide that exhibits reduced affinity for the associated co-immunomodulatory polypeptide compared to the affinity of the corresponding wild-type immunomodulatory polypeptide for the associated co-immunomodulatory polypeptide. [The present invention 1003] (i) the T cell regulatory multimeric polypeptide binds to a first T cell with an affinity that is at least 25% greater than the affinity with which the T cell regulatory multimeric polypeptide binds to a second T cell; the first T cell has on its surface the relevant co-immunomodulatory polypeptide and at least 10 -7 expressing a TCR that binds to the epitope with an affinity of M; The second T cell expresses the relevant co-immunomodulatory polypeptide on its surface, but not more than 10 -7 does not express a TCR that binds the epitope with an affinity of M and / or (ii) the ratio of the binding affinity of a control T cell regulatory multimeric polypeptide comprising a wild-type immunomodulatory polypeptide to a related co-immunomodulatory polypeptide to the binding affinity of the T cell regulatory multimeric polypeptide comprising a variant form of the wild-type immunomodulatory polypeptide to the related co-immunomodulatory polypeptide is 1.5:1 to 10, as measured by biolayer interferometry. 6 in the range :1 like, The epitope is located on a T cell receptor (TCR) on a T cell at least 10 -7 A T cell regulatory multimeric polypeptide of the invention 1002 that binds with an affinity of M. [The present invention 1004] (a) the T cell regulatory multimeric polypeptide binds to the first T cell with an affinity that is at least 50%, at least 2-fold, at least 5-fold, or at least 10-fold greater than the affinity with which it binds to the second T cell; and / or (b) the variant immunomodulatory polypeptide is about 10 -4 M~about 10 -7 M, about 10 -4 M~about 10 -6 M, about 10 -4 M~about 10 -5 binds to said co-immunomodulating polypeptide with an affinity of M; and / or (c) the ratio of the binding affinity of a control T cell regulatory multimeric polypeptide comprising a wild-type immunomodulatory polypeptide to a related co-immunomodulatory polypeptide to the binding affinity of the T cell regulatory multimeric polypeptide comprising a variant form of the wild-type immunomodulatory polypeptide to the related co-immunomodulatory polypeptide is at least 10:1, at least 50:1, at least 10 2 :1, or at least 10 3 1003. A T cell regulatory multimeric polypeptide of the present invention, wherein the polypeptide is: [The present invention 1005] The T cell regulatory multimeric polypeptide of any of claims 1001 to 1004, wherein the second polypeptide comprises the Ig Fc polypeptide, and optionally the Ig Fc polypeptide is an IgG1 Fc polypeptide. [The present invention 1006] 1005. The T cell regulatory multimeric polypeptide of the invention, wherein the IgG1 Fc polypeptide comprises one or more amino acid substitutions selected from N297A, L234A, L235A, L234F, L235E, and P331S. [The present invention 1007] (a5) the first polypeptide comprises, in order from the N-terminus to the C-terminus: (i) the peptide epitope; (ii) the first MHC polypeptide, and (iii) comprising said at least one immunomodulatory polypeptide; and (b5) the second polypeptide comprises, in order from the N-terminus to the C-terminus: (i) the second MHC polypeptide; (ii) the Ig Fc polypeptide, and (iii) comprises said tumor targeting polypeptide; or (a1) the first polypeptide comprises, in order from the N-terminus to the C-terminus: (i) the peptide epitope, and (ii) comprising the first MHC polypeptide; and (b1) the second polypeptide comprises, in order from the N-terminus to the C-terminus: (i) said at least one immunomodulatory polypeptide; (ii) the second MHC polypeptide; (iii) the Ig Fc polypeptide, and (iv) comprising said tumor targeting polypeptide; or (a6) the first polypeptide comprises, in order from the N-terminus to the C-terminus: (i) the peptide epitope, and (ii) the first MHC polypeptide; (iii) comprising said at least one immunomodulatory polypeptide; and (b6) the second polypeptide comprises, in order from the N-terminus to the C-terminus: (i) the second MHC polypeptide; (ii) said at least one immunomodulatory polypeptide; (iii) the Ig Fc polypeptide, and (iv) comprising said tumor targeting polypeptide; or (a7) the first polypeptide comprises, in order from the N-terminus to the C-terminus: (i) the peptide epitope; (ii) the first MHC polypeptide; (iii) comprising said at least one immunomodulatory polypeptide; and (b7) the second polypeptide comprises, in order from the N-terminus to the C-terminus: (i) said at least one immunomodulatory polypeptide; (ii) the second MHC polypeptide; (iii) the Ig Fc polypeptide, and (iv) comprising said tumor targeting polypeptide; or (a4) the first polypeptide comprises, in order from the N-terminus to the C-terminus: (i) said at least one immunomodulatory polypeptide; (ii) the peptide epitope, and (ii) comprising the first MHC polypeptide; and (b4) the second polypeptide comprises, in order from the N-terminus to the C-terminus: (i) the second MHC polypeptide; (ii) the Ig Fc polypeptide, and (iii) comprises said tumor targeting polypeptide; or (a2) the first polypeptide comprises, in order from the N-terminus to the C-terminus: (i) the peptide epitope, and (ii) comprising the first MHC polypeptide; and (b2) the second polypeptide comprises, in order from the N-terminus to the C-terminus: (i) the second MHC polypeptide; (ii) said at least one immunomodulatory polypeptide; (iii) the Ig Fc polypeptide, and (iv) comprising said tumor targeting polypeptide; or (a8) the first polypeptide comprises, in order from the N-terminus to the C-terminus: (i) the peptide epitope; (ii) the first MHC polypeptide, and (iii) comprising said at least one immunomodulatory polypeptide; and (b8) the second polypeptide comprises, in order from the N-terminus to the C-terminus: (i) said at least one immunomodulatory polypeptide; (ii) the second MHC polypeptide; (iii) the Ig Fc polypeptide, and (iv) comprising said tumor targeting polypeptide; or (a9) the first polypeptide comprises, in order from the N-terminus to the C-terminus: (i) the peptide epitope; (ii) the first MHC polypeptide, and (iii) comprising said at least one immunomodulatory polypeptide; and (b9) the second polypeptide comprises, in order from the N-terminus to the C-terminus: (i) the second MHC polypeptide; (ii) said at least one immunomodulatory polypeptide; (iii) the Ig Fc polypeptide, and (iv) comprising said tumor targeting polypeptide; or (a3) the first polypeptide comprises, in order from the N-terminus to the C-terminus: (i) the peptide epitope, and (ii) comprising the first MHC polypeptide; and (b3) the second polypeptide comprises, in order from the N-terminus to the C-terminus: (i) the tumor-targeting polypeptide; (ii) the second MHC polypeptide; (iii) the Ig Fc polypeptide, and (iv) comprising said at least one immunomodulatory polypeptide; or (a10) the first polypeptide comprises, in order from the N-terminus to the C-terminus: (i) the peptide epitope, and (ii) comprising the first MHC polypeptide; and (b10) the second polypeptide comprises, in order from the N-terminus to the C-terminus: (i) the tumor-targeting polypeptide; (ii) the second MHC polypeptide; (iii) the at least one immunomodulatory polypeptide, and (iv) comprising said Ig Fc polypeptide; or (a11) the first polypeptide comprises, in order from the N-terminus to the C-terminus: (i) the peptide epitope; (ii) the first MHC polypeptide, and (iii) comprising the tumor targeting polypeptide; and (b11) the second polypeptide comprises, in order from the N-terminus to the C-terminus: (i) said at least one immunomodulatory polypeptide; (ii) the second MHC polypeptide, and (iii) comprising said Ig Fc polypeptide; or (a12) the first polypeptide comprises, in order from the N-terminus to the C-terminus: (i) the peptide epitope; (ii) the first MHC polypeptide, and (iii) comprising the tumor targeting polypeptide; and (b12) the second polypeptide comprises, in order from the N-terminus to the C-terminus: (i) the second MHC polypeptide; (ii) the at least one immunomodulatory polypeptide, and (iii) comprising the Ig Fc polypeptide; A T cell regulatory multimeric polypeptide according to any one of 1001 to 1006 of the present invention. [The present invention 1008] A T cell regulatory multimeric polypeptide of any of claims 1001 to 1007, wherein the first polypeptide comprises a peptide linker between the epitope and the first MHC polypeptide, and / or the second polypeptide comprises a peptide linker between the immunoregulatory polypeptide and the second MHC polypeptide. [The present invention 1009] 1008. The T cell regulatory multimeric polypeptide of the present invention, wherein the peptide linker comprises an amino acid sequence (GGGGS)n, where n is an integer of 1 to 10. [The present invention 1010] The T cell regulatory multimeric polypeptide of any of claims 1001 to 1009, wherein said first MHC polypeptide is a β2-microglobulin polypeptide and said second MHC polypeptide is an MHC class I heavy chain polypeptide. [The present invention 1011] The T cell regulatory multimeric polypeptide of any of claims 1001 to 1010, wherein the at least one immunomodulatory polypeptide is selected from the group consisting of a cytokine, a 4-1BBL polypeptide, an ICOS-L polypeptide, an OX-40L polypeptide, a CD80 polypeptide, a CD86 polypeptide, a PD-L1 polypeptide, a FasL polypeptide, a PD-L2 polypeptide, and combinations thereof. [The present invention 1012] 1012. The T cell regulatory multimeric polypeptide of any of claims 1001 to 1011, wherein said at least one immunomodulatory polypeptide is an IL-2 polypeptide. [The present invention 1013] 13. The T cell regulatory multimeric polypeptide of any of claims 1001 to 1012, wherein said multimeric polypeptide comprises at least two immunoregulatory polypeptides, and at least two of said immunoregulatory polypeptides are the same. [The present invention 1014] The T cell regulatory multimeric polypeptide of the present invention, wherein said two or more immunomodulatory polypeptides are present in tandem. [The present invention 1015] The T cell regulatory multimeric polypeptide of any of claims 1001 to 1014, wherein the tumor targeting polypeptide is an antibody specific to a cancer-associated antigen on the surface of a cancer cell. [The present invention 1016] The T cell regulatory multimeric polypeptide of the present invention 1015, wherein the antibody is specific to a Her2, CD19, WT1, MUC1, BCMA, mesothelin, or claudin polypeptide. [The present invention 1017] The T cell regulatory multimeric polypeptide of the present invention 1015 or 1016, wherein the antibody is an scFv. [The present invention 1018] The T cell regulatory multimeric polypeptide of the present invention 1015 or 1016, wherein the antibody is a nanobody. [The present invention 1019] The T cell regulatory multimeric polypeptide of any of claims 1001 to 1014, wherein the tumor-targeting polypeptide is an antibody specific to a cancer-associated peptide / MHC complex present on the surface of cancer cells. [The present invention 1020] The T cell regulatory multimeric polypeptide of any of claims 1001 to 1014, wherein the tumor targeting polypeptide is a single-chain T cell receptor specific for a cancer-associated antigen on the surface of cancer cells. [The present invention 1021] The T cell regulatory multimeric polypeptide of any of claims 1001 to 1020, wherein the first polypeptide and the second polypeptide are covalently bonded to each other. [The present invention 1022] The T cell regulatory multimeric polypeptide of the present invention 1021, wherein said covalent bond is via a disulfide bond. [The present invention 1023] 1022. The T cell regulatory multimeric polypeptide of the present invention, wherein the β2M polypeptide and the MHC heavy chain polypeptide are linked by a disulfide bond linking a Cys residue in the β2M polypeptide and a Cys residue in the MHC heavy chain polypeptide. [The present invention 1024] 1023. The T cell regulatory multimeric polypeptide of the present invention, wherein a Cys at amino acid residue 12 of said β2M polypeptide is disulfide-bonded to a Cys at amino acid residue 236 of said MHC heavy chain polypeptide. [The present invention 1025] 1022. The T cell regulatory multimeric polypeptide of the present invention, wherein the first polypeptide chain comprises a linker between said peptide epitope and said β2M polypeptide, and said disulfide bond connects a Cys present in said linker with a Cys of said MHC heavy chain polypeptide. [The present invention 1026] 1022. The T cell regulatory multimeric polypeptide of the invention, wherein the first polypeptide chain comprises a linker between said peptide epitope and said β2M polypeptide, and said disulfide bond connects a Cys substituted for Gly2 in said linker with a Cys substituted for Tyr84 of said MHC heavy chain polypeptide. [The present invention 1027] 1027. The T cell regulatory multimeric polypeptide of any of claims 1001 to 1026, wherein said first and said second polypeptides are covalently bound to each other via at least two disulfide bonds. [The present invention 1028] (a) the first disulfide bond is (i) a Cys present in the linker between the peptide epitope and a first MHC class I polypeptide that is a β2M polypeptide; (ii) a Cys residue introduced via a Y84C substitution into a second MHC class I polypeptide that is an MHC class I heavy chain polypeptide; Located between (b) the second disulfide bond is (i) a Cys residue introduced into the β2M polypeptide via an R12C substitution; and (ii) a Cys residue introduced into the MHC class I heavy chain polypeptide via an A236C substitution; Between The T cell regulatory multimeric polypeptide of the present invention 1027. [The present invention 1029] The T cell regulatory multimeric polypeptide of the present invention 1028, wherein said linker comprises the amino acid sequence GCGGS. [The present invention 1030] 1029. The T cell regulatory multimeric polypeptide of the present invention, wherein the linker comprises the amino acid sequence GCGGS(GGGGS)n, where n is an integer of 1 to 10. [The present invention 1031] The T cell regulatory multimeric polypeptide of any of claims 1001 to 1030, wherein the peptide epitope has a length of about 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, including within the ranges of 4 to 20 aa, 6 to 18 aa, 8 to 15 aa, 8 to 12 aa, 5 to 10 aa, 10 to 15 aa, 15 to 20 aa, 10 to 20 aa, or 15 to 25 aa). [The present invention 1032] the first or second MHC polypeptide (a) an amino acid sequence having at least 95% amino acid sequence identity to the HLA-A*0101, HLA-A*0201, HLA-A*0201, HLA-A*1101, HLA-A*2301, HLA-A*2402, HLA-A*2407, HLA-A*3303, or HLA-A*3401 amino acid sequence shown in Figure 7A; or (b) an amino acid sequence having at least 95% amino acid sequence identity to the HLA-B*0702, HLA-B*0801, HLA-B*1502, HLA-B*3802, HLA-B*4001, HLA-B*4601, or HLA-B*5301 amino acid sequence shown in Figure 8A; or (c) an amino acid sequence having at least 95% amino acid sequence identity to HLA-C*0102, HLA-C*0303, HLA-C*0304, HLA-C*0401, HLA-C*0602, HLA-C*0701, HLA-C*0702, HLA-C*0801, or HLA-C*1502 shown in Figure 9A. Including, A T cell regulatory multimeric polypeptide according to any one of 1001 to 1031 of the present invention. [The present invention 1033] A T cell regulatory multimeric polypeptide of any of claims 1001 to 1032, wherein the first MHC polypeptide is a β2M polypeptide and the second MHC polypeptide comprises an amino acid sequence having at least 95% amino acid sequence identity to an HLA-A*2402 polypeptide. [The present invention 1034] The T cell regulatory multimeric polypeptide of any of claims 1001 to 1032, wherein the first MHC polypeptide is a β2M polypeptide and the second MHC polypeptide is an HLA-A*1101 polypeptide. [This invention 1035] A T cell regulatory multimeric polypeptide of any of claims 1001 to 1032, wherein the first MHC polypeptide is a β2M polypeptide and the second MHC polypeptide comprises an amino acid sequence having at least 95% amino acid sequence identity to an HLA-A*3303 polypeptide. [The present invention 1036] The T cell regulatory multimeric polypeptide of any of claims 1001 to 1032, wherein the first MHC polypeptide is a β2M polypeptide and the second MHC polypeptide comprises an amino acid sequence having at least 95% amino acid sequence identity to an HLA-A*0201 polypeptide. [This invention 1037] the immunomodulatory polypeptide (i) H16A and F42A substitutions, or (ii) H16T and F42A substitutions a variant IL-2 polypeptide comprising A T cell regulatory multimeric polypeptide according to any one of 1001 to 1036 of the present invention. [The present invention 1038] The T cell regulatory multimeric polypeptide of any of claims 1001 to 1037, wherein the epitope is a cancer-associated epitope. [This invention 1039] The T cell regulatory multimeric polypeptide of any one of claims 1001 to 1037, wherein the epitope is a peptide of a viral antigen. [The present invention 1040] The T cell regulatory multimeric polypeptide of the present invention 1039, wherein said viral antigen is a cytomegalovirus (CMV) polypeptide. [The present invention 1041] The T cell regulatory multimeric polypeptide of the present invention 1040, wherein said CMV polypeptide is a CMV pp65 polypeptide. [The present invention 1042] 1041. The T cell regulatory multimeric polypeptide of the present invention, wherein said peptide has the amino acid sequence NLVPMVATV and has a length of 9 amino acids. [This invention 1043] the multimeric polypeptide comprises a first and a second heterodimer; The T cell regulatory multimeric polypeptide of any of claims 1001 to 1042, wherein the first and second heterodimers are covalently linked by one or more disulfide bonds between the Ig Fc polypeptides of the first and second heterodimers. [This invention 1044] A nucleic acid comprising a nucleotide sequence encoding a first or second polypeptide of any one of the present inventions 1001 to 1043, wherein the first or second polypeptide comprises at least one immunomodulatory polypeptide. [This invention 1045] An expression vector comprising the nucleic acid of the present invention. [The present invention 1046] A method for selectively regulating the activity of epitope-specific T cells, the method comprising contacting the T cells with any of the T cell regulatory multimeric polypeptides of the present invention 1001 to 1043, wherein the contacting selectively regulates the activity of the epitope-specific T cells. [This invention 1047] A method for treating a patient with cancer, comprising administering to the patient an effective amount of a pharmaceutical composition comprising any one of the T cell regulatory multimeric polypeptides of the present invention 1001 to 1043. [This invention 1048] 1047. The method of claim 1047, wherein said cancer is carcinoma, sarcoma, melanoma, leukemia, or lymphoma. [This invention 1049] The method of any one of claims 1047 to 1048, wherein said administration is intramuscular, intravenous, peritumoral, or intratumoral. [The present invention 1050] A method for modulating an immune response in an individual, said method comprising administering to said individual an effective amount of any of the T cell regulatory multimeric polypeptides of inventions 1001 to 1043; administering induces an epitope-specific T cell response and an epitope-non-specific T cell response; The method, wherein the ratio of the epitope-specific T cell response to the epitope-non-specific T cell response is at least 2:1. [Brief explanation of the drawings]

[0004] [Figure 1A] 1A-1J are schematic diagrams of various TMMPs 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 1G] See legend to Figure 1A. [Figure 1H] See legend to Figure 1A. [Figure 1I] See legend to Figure 1A. [Figure 1J] See legend to Figure 1A. [Figure 2A] 2A-2F are schematic diagrams of various disulfide-linked TMMPs 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 3-1] 3A-3G provide the amino acid sequences of immunoglobulin Fc polypeptides, which are set forth in SEQ ID NOs: 19-30. [Figure 3-2] See description of Figure 3-1. [Figure 3-3] See description of Figure 3-1. [Figure 3-4] See description of Figure 3-1. [Figure 4] Multiple amino acid sequence alignments of beta-2 microglobulin (β2M) precursors (i.e., including the leader sequence) from Homo sapiens (NP_004039.1; SEQ ID NO: 31), chimpanzee (Pan troglodytes) (NP_001009066.1; SEQ ID NO: 31), rhesus monkey (Macaca mulatta) (NP_001040602.1; SEQ ID NO: 32), cow (Bos taurus) (NP_776318.1; SEQ ID NO: 33), and mouse (Mus musculus) (NP_033865.2; SEQ ID NO: 34) are provided. Amino acids 1-20 are the signal peptide. [Figure 5A] Figures 5A-5C provide 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 7A), the full-length human HLA heavy chain of allele B*0702 (SEQ ID NO: 39) (Figure 7B), and the full-length human HLA-C heavy chain (SEQ ID NO: 40) (Figure 7C). [Figure 5B] See legend to Figure 5A. [Figure 5C] See legend to Figure 5A. [Figure 6-1]6 provides an alignment of 11 mature MHC class I heavy chain amino acid sequences, excluding the leader sequence, transmembrane domain, and intracellular domain. The sequences are SEQ ID NOs: 41 to 51, from top to bottom. [Figure 6-2] See description of Figure 6-1. [Figure 7A-1] Figures 7A-7B provide an alignment of HLA-A heavy chain amino acid sequences (Figure 7A; SEQ ID NOs: 52-60, respectively) and the consensus sequence (Figure 7B: 61). [Figure 7A-2] See legend to Figure 7A-1. [Figure 7B] See legend to Figure 7A-1. [Figure 8A-1] 8A-8B provide an alignment of the HLA-B heavy chain amino acid sequences (FIG. 8A; SEQ ID NOs: 62-68, respectively) and the consensus sequence (FIG. 8B; SEQ ID NO: 69). [Figure 8A-2] See legend to Figure 8A-1. [Figure 8B] See legend to Figure 8A-1. [Figure 9A-1] 9A-9B provide an alignment of HLA-C heavy chain amino acid sequences (FIG. 9A; SEQ ID NOs: 70-78, respectively) and the consensus sequence (FIG. 9B; SEQ ID NO: 79). [Figure 9A-2] See legend to Figure 9A-1. [Figure 9B] See legend to Figure 9A-1. [Figure 10-1] Figure 10 provides the consensus amino acid sequences for each of the HLA-E, HLA-F, and HLA-G heavy chains (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 10-2] See description of Figure 10-1. [Figure 11-1]Figure 11 provides an alignment of the consensus amino acid sequences of HLA-A (SEQ ID NO: 83), HLA-B (SEQ ID NO: 84), HLA-C (SEQ ID NO: 85), HLA-E (SEQ ID NO: 86), HLA-F (SEQ ID NO: 87), and HLA-G (SEQ ID NO: 88). [Figure 11-2] See description of Figure 11-1. [Figure 12A] 12A-12D provide schematic diagrams of the multiply disulfide-bonded TMMPs 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] 13A-13F provide the amino acid sequences of examples of the first and second polypeptides of the TMMP of the present disclosure. The sequences are SEQ ID NOs: 89-94, respectively. [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 14A] 14A-14C provide the amino acid sequences of examples of polypeptides that may be included in the TMMPs of the present disclosure. The sequences are SEQ ID NOs: 95-97. [Figure 14B] See legend to Figure 14A. [Figure 14C] See legend to Figure 14A. [Figure 15A] 15A-15B provide the amino acid sequences of examples of polypeptides that may be included in the TMMPs of the present disclosure. The sequences are SEQ ID NOs: 561-562, respectively. [Figure 15B] See legend to Figure 15A. [Figure 16A]16A-16B show the effect of TMMP according to an embodiment of the present disclosure on CD8+ cell lytic activity. [Figure 16B] See legend to Figure 16A. 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.

[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.

[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 (including, for example, 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, including helper T cells (CD4 + cells), cytotoxic T cells (CD8 + These include T-cells, T regulatory cells (Tregs), and NK-T cells.

[0011] As used herein, the term "immunomodulatory polypeptide" (also referred to as "costimulatory polypeptide") includes polypeptides on antigen-presenting cells (APCs) (e.g., dendritic cells, B cells, etc.) that specifically bind to a related co-immunomodulatory polypeptide on a T cell, thereby providing signals that mediate T cell responses, including, but not limited to, proliferation, activation, differentiation, etc., in addition to the primary signal provided, for example, by binding of the TCR / CD3 complex to a peptide-binding major histocompatibility complex (MHC) polypeptide. Immunomodulatory polypeptides include, but are not limited to, 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 B7-H3.

[0012] As described above, an "immunomodulating polypeptide" (also referred to herein as a "MOD") specifically binds to a related co-immunomodulating polypeptide on a T cell.

[0013] The "immunomodulatory domain" ("MOD") of the TMMP of the present disclosure binds to a related co-immunomodulatory polypeptide that may be present on the target T cell.

[0014] As used herein, "heterologous" means a nucleotide or polypeptide that is not found in naturally occurring nucleic acids or proteins, 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 a TMMP to a polypeptide (e.g., a T cell receptor) on a T cell), the term "binding" refers to a non-covalent interaction between two molecules. Non-covalent binding refers to a direct association between two molecules, for example, through electrostatic, hydrophobic, ionic, and / or hydrogen-bonding interactions, including interactions such as salt bridges and hydrogen 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 "Non-specific binding" generally refers to binding with an affinity of about 10 M or greater. -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, 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 the disease to regress. Therapeutic agents may be administered before, during, or after the onset of a disease or injury. 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 therapy is desirably administered during, and in some cases after, the symptomatic stage of the disease.

[0020] 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.).

[0021] The terms "antibody" and "immunoglobulin" include antibodies or immunoglobulins of any isotype, fragments of antibodies that retain specific binding to an antigen (including, but not limited to, Fab, Fv, scFv, and Fd fragments, chimeric antibodies, humanized antibodies, single-chain antibodies (scAbs), single-domain antibodies (dAbs), single-domain heavy-chain antibodies, single-domain light-chain antibodies, nanobodies, diabodies, multispecific antibodies, and fusion proteins comprising an antibody and an antigen-binding (also referred to herein as antigen binding) portion of a non-antibody protein). Antibodies can be detectably labeled, for example, with a radioisotope, an enzyme that generates a detectable product, a fluorescent protein, etc. Antibodies can be further conjugated to other moieties, such as a member of a specific binding pair, e.g., biotin (a member of the biotin-avidin specific binding pair). The terms also encompass Fab', Fv, F(ab')2, and / or other antibody fragments that retain specific binding to an antigen, as well as monoclonal antibodies. As used herein, a monoclonal antibody is an antibody produced by the same group of cells, all of which were produced from a single cell by repeated cell replication. That is, a clone of cells produces only a single antibody species. Monoclonal antibodies can be produced using hybridoma production techniques, although other production methods known to those skilled in the art can also be used (e.g., antibodies derived from antibody phage display libraries). Antibodies can be monovalent or bivalent. Antibodies can be Ig monomers, which are "Y-shaped" molecules consisting of four polypeptide chains: two heavy chains and two light chains connected by disulfide bonds.

[0022] As used herein, the term "humanized immunoglobulin" refers to an immunoglobulin containing portions of immunoglobulins of different origins, at least one portion of which contains an amino acid sequence of human origin. For example, a humanized antibody can contain portions derived from immunoglobulins of non-human origin, e.g., from immunoglobulin sequences of murine and human origin (e.g., chimeric immunoglobulins), which have the requisite specificity, chemically combined by conventional techniques (e.g., synthetically), or prepared as a contiguous polypeptide using genetic engineering techniques (e.g., DNA encoding the protein portions of a chimeric antibody can be expressed to produce a contiguous polypeptide chain). Another example of a humanized immunoglobulin is an immunoglobulin containing one or more immunoglobulin chains that contain complementarity-determining regions (CDRs) derived from an antibody of non-human origin and framework regions derived from light and / or heavy chains of human origin (e.g., CDR-grafted antibodies, with or without framework changes). Chimeric or CDR-grafted single-chain antibodies are also encompassed by the term humanized immunoglobulin. See, e.g., U.S. Patent No. 4,816,567, European Patent No. 0,125,023B1, U.S. Patent No. 4,816,397, European Patent No. 0,120,694B1, WO 86 / 01533, European Patent No. 0,194,276B1, U.S. Patent No. 5,225,539, European Patent No. 0,239,400B1, and European Patent Application No. 0,519,596A1. Regarding single chain antibodies, see also U.S. Patent No. 4,946,778, U.S. Patent No. 5,476,786, and Bird et al. (1988) Science 242:423.

[0023] The term "nanobody" (Nb), as used herein, refers to the smallest antigen-binding fragment or single variable domain (V) derived from a naturally occurring heavy chain antibody. HH) and are known to those skilled in the art. They are derived from heavy chain-only antibodies found in camelids (Hamers-Casterman et al. (1993) Nature 363:446, Desmyter et al. (1996) Nature Structural Biol. 3:803, and Desmyter et al. (2015) Curr. Opin. Struct. Biol. 32:1). In the "Camelidae" family, immunoglobulins that lack light chain polypeptides are found. "Camelidae" consists of Old World camelids (Camelus bactrianus and Camelus dromedarius) and New World camelids (e.g., Llama paccos, Llama glama, Llama guanicoe, and Llama vicugna). Single variable domain heavy chain antibodies are herein referred to as nanobodies or V HH They are called antibodies.

[0024] "Antibody fragments" include portions of intact antibodies, such as the antigen-binding or variable region of an intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments, diabodies, linear antibodies (Zapata et al. Protein Eng. 8(10):1057-1062 (1995)), domain antibodies (dAbs; Holt et al. (2003) Trends Biotechnol. 21:484), single-chain antibody molecules, and multispecific antibodies formed from antibody fragments. Papain digestion of an antibody produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site, and a remaining "Fc" fragment, a name reflecting its ability to readily crystallize. Pepsin treatment yields an F(ab')2 fragment that has two antigen-binding sites and is still capable of cross-linking antigen.

[0025] An "Fv" is the minimum antibody fragment that contains a complete antigen-recognition and antigen-binding site. This region consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association. It is in this configuration that the three CDRs of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. Together, the six CDRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although with a lower affinity than the entire binding site.

[0026] "Fab" fragments also contain the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab fragments differ from Fab' fragments by the addition of a few residues at the carboxyl terminus of the heavy chain CH1 domain including one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine ​​residue(s) of the constant domains bear a free thiol group. F(ab')2 antibody fragments were originally produced as pairs of Fab' fragments that have hinge cysteines between them. Other chemical linkages of antibody fragments are also known.

[0027] The "light chains" of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequence of their constant domains. Depending on the amino acid sequence of the constant domains of their heavy chains, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these classes can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The subclasses can be further divided into types, e.g., IgG2a and IgG2b.

[0028] "Single-chain Fv" or "sFv" or "scFv" antibody fragments are fragments of the V of an antibody. H and VL domains, and these domains are present in a single polypeptide chain. In some embodiments, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains that enables the sFv to form the desired structure for antigen binding. For a review of sFvs, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0029] The term "diabody" refers to a small antibody fragment with two antigen-binding sites, which consists of a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain (V H- V L (Diabodies generally comprise a diabody having a complementary domain and a complementary domain.) By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains on another chain and create two antigen-binding sites. Diabodies are described more fully in, for example, EP 404,097, WO 93 / 11161, and Hollinger et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448.

[0030] As used herein, the term "CDR" or "complementarity-determining region" is intended to mean the noncontiguous antigen-binding sites found within the variable regions of both heavy and light chain polypeptides. CDRs are described by Kabat et al. (1977) J. Biol. Chem. 252:6609, Kabat et al., US Dept. of Health and Human Services, "Sequences of proteins of immunological interest" (1991) (also referred to herein as Kabat 1991), Chothia et al. (1987) J. Mol. Biol. 196:901 (also referred to herein as Chothia 1987), and MacCallum et al. (1996) J. Mol. Biol. 262:732 (the definitions include overlapping or subsets of amino acid residues when compared with each other). Nevertheless, application of either definition to refer to the CDRs of an antibody or grafted antibody or variant thereof is intended to be within the scope of the term as defined and used herein. The amino acid residues that encompass the CDRs as defined by each of the above cited references are set forth below in Table 2 for comparison.

[0031] (Table 2) CDR definitions TIFF0007762068000001.tif34136 1 Residue numbering follows the nomenclature of Kabat et al., 1991 (supra). 2 Residue numbering follows the nomenclature of Chothia et al. (supra). 3 Residue numbering follows the nomenclature of MacCallum et al. (supra).

[0032] As used herein, the terms "CDR-L1," "CDR-L2," and "CDR-L3" refer to the first, second, and third CDRs, respectively, in a light chain variable region. As used herein, the terms "CDR-H1," "CDR-H2," and "CDR-H3" refer to the first, second, and third CDRs, respectively, in a heavy chain variable region. As used herein, the terms "CDR-1," "CDR-2," and "CDR-3" refer to the first, second, and third CDRs, respectively, in the variable region of either chain.

[0033] As used herein, the term "framework," when used in reference to an antibody variable region, is intended to refer to all amino acid residues outside the CDR regions within the variable region of an antibody. The variable region framework is generally a discontinuous amino acid sequence about 100 to 120 amino acids in length, but is intended to refer only to those amino acids outside the CDRs. As used herein, the term "framework region" is intended to refer to each domain of the framework separated by the CDRs.

[0034] Before the present invention is further described, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting, as the scope of the present invention will be limited only by the appended claims.

[0035] 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 invention. The upper and lower limits of these narrower ranges may independently be included in the narrower range and are also encompassed within the invention, subject to any specifically excluded value in the stated range. When a 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 invention.

[0036] 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 invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are 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.

[0037] 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 "T cell modulatory multimeric polypeptides" includes a plurality of such polypeptides; a reference to "immunomodulating polypeptides" 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.

[0038] It is to be understood that certain features of the invention that are, for clarity, described in the context of individual embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention 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 embodiments belonging to the invention are expressly embraced by the invention 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 invention and are disclosed herein as if all such subcombinations were individually and specifically disclosed herein.

[0039] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates, which may need to be independently confirmed.

[0040] Detailed Description The present disclosure provides T cell regulatory multimeric polypeptides, including immunomodulatory polypeptides and including epitope-presenting peptides. TMMPs are useful for modulating T cell activation and for modulating immune responses in individuals.

[0041] T cell regulatory multimeric polypeptides The present disclosure provides T cell modulatory multimeric polypeptides (TMMPs) comprising a) a first polypeptide and b) a second polypeptide, the TMMP comprising a peptide epitope (defined below), a first major histocompatibility complex (MHC) polypeptide, a second MHC polypeptide, one or more immune-modulating polypeptides, an immunoglobulin (Ig) Fc polypeptide or a non-Ig scaffold, and a tumor-targeting polypeptide (TTP).

[0042] The present disclosure provides a TMMP, 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 peptide epitope (defined below), and the first polypeptide and / or the second polypeptide comprises one or more immunomodulatory polypeptides, which may be the same or different, and an Ig Fc polypeptide or a non-Ig scaffold. The first or second polypeptide also comprises a tumor-targeting polypeptide. In some examples, the tumor-targeting polypeptide is C-terminal to the Ig Fc polypeptide or non-Ig scaffold. The TMMP of the present disclosure is also referred to herein as the "multimeric polypeptide of the present disclosure" or "synTac."

[0043] As used herein, the term "peptide epitope" means a peptide that, when complexed with an MHC polypeptide, presents an epitope to a T cell receptor (TCR). Peptide epitopes have a length of at least four amino acids, e.g., from 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, including lengths ranging from 4 to 20 aa, 6 to 18 aa, 8 to 15 aa, 8 to 12 aa, 5 to 10 aa, 10 to 15 aa, 15 to 20 aa, 10 to 20 aa, or 15 to 25 aa). When complexed with an MHC polypeptide, peptide epitopes can present one or more epitopes to one or more TCRs. In some examples, peptide epitopes present in the TMMPs of the present disclosure represent cancer-associated epitopes. In some examples, peptide epitopes present in the TMMPs of the present disclosure represent infectious disease-associated epitopes (e.g., virally encoded peptides).

[0044] In some examples, the TMMP of the present disclosure comprises i) a viral epitope (e.g., a virally encoded peptide) and iii) a TTP that targets a cancer-associated antigen. Such a TMMP binds to cancer cells expressing the cancer-associated antigen targeted by the TTP. The TMMP regulates the activity of T cells specific for the viral epitope present in the TMMP. For example, in some examples, the TMMP increases the proliferation and / or cytotoxic activity of T cells specific for the viral epitope present in the TMMP. Contact with T cells specific for the viral epitope present in the TMMP can increase the cytotoxic activity of the T cells against cancer cells expressing the cancer-associated antigen targeted by the TTP present in the TMMP.

[0045] The present disclosure provides TMMPs comprising a) a heterodimeric polypeptide comprising: i) a first polypeptide comprising a peptide epitope and ii) a first MHC polypeptide; b) a second polypeptide comprising a second MHC polypeptide; c) at least one immunomodulatory polypeptide (the first and / or second polypeptides comprise at least one (i.e., one or more) immunomodulatory polypeptide); d) an Ig Fc polypeptide or a non-Ig scaffold (the first and / or second polypeptides comprise an Ig Fc polypeptide or a non-Ig scaffold); and e) a polypeptide that targets cancer cells (tumor targeting polypeptide, "TTP"). In some examples, at least one of the one or more immunomodulatory polypeptides is a variant immunomodulatory polypeptide that exhibits reduced affinity for a related co-immunomodulatory polypeptide compared to the affinity of the corresponding wild-type immunomodulatory polypeptide for the related co-immunomodulatory polypeptide. The epitope present in the TMMP of the present disclosure binds to a T cell receptor (TCR) on a T cell with an affinity of at least 100 μM (e.g., at least 10 μM, at least 1 μM, at least 100 nM, at least 10 nM, or at least 1 nM). The TMMP of the present disclosure binds to a first T cell with an affinity that is at least 25% higher than the affinity with which the TMMP binds to a second T cell, the first T cell expressing on its surface a relevant co-immunomodulatory polypeptide and a TCR that binds to the epitope with an affinity of at least 100 μM, and the second T cell expressing a relevant co-immunomodulatory polypeptide on its surface but not a TCR that binds to the epitope with an affinity of at least 100 μM (e.g., at least 10 μM, at least 1 μM, at least 100 nM, at least 10 nM, or at least 1 nM). In some examples, the peptide epitope present in the TMMP of the present disclosure represents a cancer-associated epitope. In some examples, peptide epitopes present within the TMMPs of the present disclosure represent infectious disease-associated epitopes (eg, virally encoded peptides).

[0046] The TMMPs of the present disclosure comprise tumor-targeting polypeptides, i.e., polypeptides that target cancer-associated epitopes displayed on the surface of cancer cells.

[0047] The present disclosure provides a TMMP, the TMMP comprising: A) a heterodimer comprising: a) a first polypeptide comprising a first MHC polypeptide; and b) a second polypeptide comprising a second MHC polypeptide, wherein the first polypeptide or the second polypeptide comprises a peptide epitope; the first polypeptide and / or the second polypeptide comprise one or more immunomodulatory polypeptides which may be the same or different; 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; the first polypeptide and / or the second polypeptide comprises an Ig Fc polypeptide or a non-Ig scaffold; and the first and / or second polypeptide comprises a tumor-targeting polypeptide; or B) A heterodimer comprising: a) a first polypeptide comprising a first MHC polypeptide; and b) a second polypeptide comprising a second MHC polypeptide, wherein the first polypeptide or the second polypeptide comprises an epitope, and the first polypeptide and / or the second polypeptide comprises one or more immunomodulatory polypeptides, which may be the same or different; at least one of the one or more immunomodulatory polypeptides is a variant of a wild-type immunomodulatory polypeptide, wherein the variant immunomodulatory polypeptide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid substitutions compared to the amino acid sequence of the corresponding wild-type immunomodulatory polypeptide; and a variant immunomodulatory polypeptide, wherein at least one of the one or more immunomodulatory domains exhibits reduced affinity for an associated co-immunomodulatory polypeptide compared to the affinity of the corresponding wild-type immunomodulatory polypeptide for the associated co-immunomodulatory polypeptide, and the epitope is selected from at least 10 -7 M, whereby i) the TMMP polypeptide binds to the first T cell with an affinity that is at least 25% higher than the affinity with which the TMMP binds to the second T cell, and the first T cell has on its surface a relevant co-immunomodulating polypeptide and at least 10 -7 and a second T cell expresses on its surface a TCR that binds to an epitope with an affinity of at least 10 M, and a related co-immunomodulatory polypeptide, but on its surface -7 and / or ii) the ratio of the binding affinity of a control TMMP (the control comprising a wild-type immunomodulatory polypeptide) for the relevant co-immunomodulatory polypeptide to the binding affinity of a TMMP comprising a variant form of the wild-type immunomodulatory polypeptide for the relevant co-immunomodulatory polypeptide is 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; the first polypeptide and / or the second polypeptide comprises an Ig Fc polypeptide or a non-Ig scaffold, and the first and / or second polypeptide comprises a tumor-targeting polypeptide, or C) A heterodimer comprising: a) a first polypeptide comprising, in N-terminal to C-terminal order: i) an epitope; 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 TMMP 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 / are located at: A) the C-terminus of the first polypeptide; B) A) at the N-terminus of the second polypeptide; B) at the C-terminus of the second polypeptide; or C) at the C-terminus of the first polypeptide and the N-terminus of the second polypeptide, wherein 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; Optionally, at least one of the one or more immunomodulatory domains is a variant immunomodulatory polypeptide that exhibits reduced affinity for an associated co-immunomodulatory polypeptide compared to the affinity of a corresponding wild-type immunomodulatory polypeptide for the associated co-immunomodulatory polypeptide, and the epitope is at least 10 -7 M, whereby i) TMMP binds to a first T cell with an affinity that is at least 25% higher than the affinity with which TMMP binds to a second T cell, and the first T cell has on its surface a relevant co-immunomodulatory polypeptide and at least 10 -7 and a second T cell expresses on its surface a TCR that binds to an epitope with an affinity of at least 10 M, and a related co-immunomodulatory polypeptide, but on its surface -7and / or ii) the ratio of the binding affinity of a control TMMP (the control comprising a wild-type immunomodulatory polypeptide) for the relevant co-immunomodulatory polypeptide to the binding affinity of a TMMP comprising a variant form of the wild-type immunomodulatory polypeptide for the relevant co-immunomodulatory polypeptide is 1.5:1-10, as measured by biolayer interferometry. 6 :1, and 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. The first polypeptide and / or the second polypeptide comprise an Ig Fc polypeptide or a non-Ig scaffold, and the first and / or second polypeptide comprise a tumor-targeting polypeptide. In some examples, the epitope present in the TMMP of the present disclosure represents a cancer-associated epitope. In some examples, the epitope present in the TMMP of the present disclosure represents an infectious disease-associated epitope (e.g., a virally encoded peptide).

[0048] The present disclosure provides a TMMP comprising: a) a first polypeptide comprising, in order from N-terminus to C-terminus: i) an epitope, ii) a first MHC polypeptide; and b) a second polypeptide comprising, in order from N-terminus to C-terminus: i) a second MHC polypeptide, ii) an Ig Fc polypeptide or a non-Ig scaffold, and iii) a tumor-targeting polypeptide. In some examples, the TMMP of the disclosure comprises one or more immunomodulatory polypeptides, wherein at least one of the one or more immunomodulatory polypeptides is at: A) the C-terminus of the first polypeptide, B) the N-terminus of the second polypeptide, C) the C-terminus of the second polypeptide, or D) the C-terminus of the first polypeptide and the N-terminus of the second polypeptide. At least one of the one or more immunomodulatory polypeptides is a variant immunomodulatory polypeptide that exhibits reduced affinity for a related co-immunomodulatory polypeptide compared to the affinity of a corresponding wild-type immunomodulatory polypeptide for the related co-immunomodulatory polypeptide. An epitope present in a TMMP of the present disclosure binds to a T cell receptor (TCR) on a T cell with an affinity of at least 100 μM (e.g., at least 10 μM, at least 1 μM, at least 100 nM, at least 10 nM, or at least 1 nM). The TMMP of the present disclosure binds to a first T cell with an affinity that is at least 25% higher than the affinity with which the TMMP binds to a second T cell, the first T cell expressing on its surface a relevant co-immunomodulatory polypeptide and a TCR that binds to the epitope with an affinity of at least 100 μM, and the second T cell expressing a relevant co-immunomodulatory polypeptide on its surface but not a TCR that binds to the epitope with an affinity of at least 100 μM (e.g., at least 10 μM, at least 1 μM, at least 100 nM, at least 10 nM, or at least 1 nM).

[0049] In some examples, epitopes present in the TMMP of the present disclosure may bind to a TCR on a T cell in a manner that is approximately 10 -4 M ~ approx. 5x10 -4 M, about 5x10 -4 M~about 10 -5 M, about 10 -5 M~5x10 -5 M, about 5x10-5 M~10 -6 M, about 10 -6 M ~ approx. 5x10 -6 M, about 5x10 -6 M~about 10 -7 M, about 10 -7 M ~ approx. 5x10 -7 M, about 5x10 -7 M~about 10 -8 M, or about 10 -8 M~about 10 -9 Expressed another way, in some examples, an epitope present in a TMMP of the present disclosure binds to a 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.

[0050] In some examples, the immunomodulatory polypeptides present in the TMMPs of the present disclosure comprise wild-type (naturally occurring) amino acid sequences.

[0051] In some examples, an immunomodulatory polypeptide present in a TMMP of the present disclosure binds to its associated co-immunomodulatory polypeptide with an affinity that is at least 10% less, at least 15% less, at least 20% less, at least 25% less, at least 30% less, at least 35% less, at least 40% less, at least 45% less, at least 50% less, at least 55% less, at least 60% less, at least 65% less, at least 70% less, at least 75% less, at least 80% less, at least 85% less, at least 90% less, at least 95% less, or more than 95% less than the affinity of the corresponding wild-type immunomodulatory polypeptide for the associated co-immunomodulatory polypeptide.

[0052] In some examples, variant immunomodulatory polypeptides present in a TMMP of the disclosure have a binding affinity for a related co-immunomodulatory polypeptide of between 1 nM and 100 nM or between 100 nM and 100 μM. For example, in some examples, variant immunomodulatory polypeptides present in a TMMP of the disclosure have a binding affinity for a related co-immunomodulatory polypeptide of between about 100 nM and 150 nM, between about 150 nM and 200 nM, between about 200 nM and 250 nM, between about 250 nM and 300 nM, between about 300 nM and 350 nM, between about 350 nM and 400 nM, between about 400 nM and 500 nM, between about 500 nM and 600 nM. In some instances, a variant immunomodulatory polypeptide present in a TMMP 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 a related co-immunomodulatory polypeptide.

[0053] The combination of the low affinity of an immunomodulatory polypeptide for its related co-immunomodulatory polypeptide and the affinity of the epitope for a TCR results in improved selectivity for the TMMPs of the present disclosure. For example, a TMMP of the present disclosure selectively binds to a first T cell that presents both i) a TCR specific for an epitope present in the TMMP and ii) a co-immunomodulatory polypeptide that binds to the immunomodulatory polypeptide present in the TMMP, compared to binding to a second T cell that presents i) a TCR specific for an epitope other than the epitope present in the TMMP and ii) a co-immunomodulatory polypeptide that binds to the immunomodulatory polypeptide present in the TMMP. For example, a TMMP 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 greater than the affinity with which it binds to a second T cell.

[0054] In some examples, the TMMP of the present disclosure, when administered to an individual in need thereof, induces both epitope-specific and non-epitope-specific T cell responses. In other words, in some examples, the TMMP of the present disclosure, when administered to an individual in need thereof, 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 TMMP and ii) a co-immunomodulatory polypeptide that binds to an immunomodulatory polypeptide present in the TMMP, 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 epitope present in the TMMP and ii) a co-immunomodulatory polypeptide that binds to an immunomodulatory polypeptide present in the TMMP. The ratio of epitope-specific to epitope-non-specific 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. "Modulating the activation" of T cells can include one or more of the following: i) activating cytotoxic (e.g., CD8+) T cells, ii) inducing the cytotoxic activity of cytotoxic (e.g., CD8+) T cells, iii) inducing the production and release of cytotoxins (e.g., perforin, granzyme, granulysin) by cytotoxic (e.g., CD8+) T cells, and iv) suppressing the activation of autoreactive T cells.

[0055] The combination of the low affinity of an immunomodulatory polypeptide for its related co-immunomodulatory polypeptide with the affinity of the epitope for a TCR results in improved selectivity for the TMMPs of the present disclosure. Thus, for example, a TMMP of the present disclosure binds to a first T cell that presents both i) a TCR specific for an epitope present in the TMMP and ii) a co-immunomodulatory polypeptide that binds to the immunomodulatory polypeptide present in the TMMP with a higher avidity than the avidity with which the TMMP 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 TMMP and ii) a co-immunomodulatory polypeptide that binds to the immunomodulatory polypeptide present in the TMMP.

[0056] The binding affinity between an immunomodulatory polypeptide and its associated co-immunomodulatory polypeptide can be determined by biolayer interferometry (BLI) using purified immunomodulatory polypeptides and purified associated co-immunomodulatory polypeptides. The binding affinity between a TMMP and its associated co-immunomodulatory polypeptide can be determined by BLI using purified TMMP and associated co-immunomodulatory polypeptides. 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.

[0057] BLI assays can be performed using an Octet RED 96 (Pal ForteBio) instrument or a similar instrument as follows: TMMP (e.g., a TMMP of the present disclosure, a control TMMP (the control TMMP contains a wild-type immunomodulatory polypeptide)) is immobilized on an insoluble support ("biosensor"). The immobilized TMMP is the "target." Immobilization can be achieved by immobilizing a capture antibody on the insoluble support, which immobilizes the TMMP. For example, immobilization can be achieved by immobilizing an anti-Fc (e.g., anti-human IgG Fc) antibody on the insoluble support, which binds to and immobilizes the TMMP (the TMMP contains an IgFc polypeptide). A co-immunomodulatory polypeptide is applied to the immobilized TMMP at several different concentrations, 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 the co-immunomodulatory polypeptide to the immobilized TMMP 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, α-heptane-2, ... a) and dissociation constant (k d ) These two terms (k d / a ) is the affinity constant K D This results in:

[0058] BLI assays are performed in multiwell plates. To run the assay, the plate layout is defined, the assay steps are defined, and the biosensors are assigned in the Octet Data Acquisition software. The biosensor assembly is hydrated. The hydrated biosensor assembly and assay plate are equilibrated on the Octet instrument for 10 minutes. Once the data is acquired, it is loaded into the Octet Data Analysis software. The data is processed in the Processing window, specifying the method of reference subtraction, y-axis alignment, inter-step correction, and Savitzky-Golay filtering. The data is analyzed in the Analysis window, specifying the steps to be analyzed (association and dissociation), the curve fit model (1:1), the fitting method (global), and the window of interest (in seconds). The quality of the fit is assessed. The K for each data trace (analyte concentration) is calculated. D Values ​​can be averaged within a factor of three. D The error value should be within one order of magnitude of the affinity constant value. 2 The value should be greater than 0.95. See, e.g., Abdiche et al. (2008) J. Anal. Biochem. 377:209.

[0059] Unless otherwise specified herein, the affinity of a TMMP of the present disclosure for a related co-immunomodulatory polypeptide, or the affinity of a control TMMP (wherein the control TMMP comprises a wild-type immunomodulatory polypeptide) for a related co-immunomodulatory polypeptide, is determined using BLI as described above.

[0060] In some examples, the ratio of i) the binding affinity of a control TMMP (wherein the control comprises a wild-type immunomodulatory polypeptide) to the relevant co-immunomodulatory polypeptide to ii) the binding affinity of a TMMP of the disclosure comprising a variant form of the wild-type immunomodulatory polypeptide to the relevant co-immunomodulatory polypeptide, as measured by BLI (as described above), is at least 1.5:1, at least 2:1, at least 5:1, at least 10:1, at least 15:1, at least 20:1, at least 25:1, at least 50:1, at least 100:1, at least 500:1, at least 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 TMMP (wherein the control comprises a wild-type immunomodulatory polypeptide) to the relevant co-immunomodulatory polypeptide to ii) the binding affinity of a TMMP of the disclosure comprising a variant version of the wild-type immunomodulatory polypeptide to the relevant co-immunomodulatory 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.

[0061] As an example, if a control TMMP comprises a wild-type IL-2 polypeptide and a TMMP of the present disclosure comprises a variant IL-2 polypeptide (comprising 1 to 10 amino acid substitutions relative to the amino acid sequence of the wild-type IL-2 polypeptide) as the immunomodulatory polypeptide, the ratio of i) the binding affinity of the control TMMP to the IL-2 receptor (i.e., the relevant co-immunomodulatory polypeptide) to ii) the binding affinity of the TMMP of the present disclosure to the IL-2 receptor, as measured by BLI, is at least 1.5:1, at least 2:1, at least 5:1, at least 10:1, at least 15:1, at least 20:1, at least 25:1, at least 50:1, at least 100:1, at least 500:1, at least 10 2 :1, at least 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, when a control TMMP comprises a wild-type IL-2 polypeptide and a TMMP of the present disclosure comprises a variant IL-2 polypeptide (comprising 1-10 amino acid substitutions relative to the amino acid sequence of the wild-type IL-2 polypeptide) as the immunomodulatory polypeptide, the ratio of i) the binding affinity of the control TMMP to the IL-2 receptor (i.e., the relevant co-immunomodulatory polypeptide) to ii) the binding affinity of the TMMP of the present disclosure to the IL-2 receptor is 1.5:1-10 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.

[0062] As another example, if a control TMMP comprises a wild-type PD-L1 polypeptide and a TMMP of the disclosure comprises a variant PD-L1 polypeptide (comprising 1-10 amino acid substitutions relative to the amino acid sequence of the wild-type PD-L1 polypeptide) as the immunomodulatory polypeptide, then the ratio of i) the binding affinity of the control TMMP to the PD-1 polypeptide (i.e., the relevant co-immunomodulatory polypeptide) to ii) the binding affinity of the TMMP of the disclosure to the PD-1 polypeptide, as measured by BLI, is at least 1.5:1, at least 2:1, at least 5:1, at least 10:1, at least 15:1, at least 20:1, at least 25:1, at least 50:1, at least 100:1, at least 500:1, at least 10 2 :1, at least 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.

[0063] As another example, when a control TMMP comprises a wild-type CD80 polypeptide and a TMMP of the disclosure comprises a variant CD80 polypeptide (comprising 1-10 amino acid substitutions relative to the amino acid sequence of the wild-type CD80 polypeptide) as the immunomodulatory polypeptide, the ratio of i) the binding affinity of the control TMMP to a CTLA4 polypeptide (i.e., a related co-immunomodulatory polypeptide) to ii) the binding affinity of the TMMP of the disclosure to a CTLA4 polypeptide, as measured by BLI, is at least 1.5:1, at least 2:1, at least 5:1, at least 10:1, at least 15:1, at least 20:1, at least 25:1, at least 50:1, at least 100:1, at least 500:1, at least 10 2 :1, at least 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 106 :1.

[0064] As another example, when a control TMMP comprises a wild-type CD80 polypeptide and a TMMP of the disclosure comprises a variant CD80 polypeptide (comprising 1 to 10 amino acid substitutions relative to the amino acid sequence of the wild-type CD80 polypeptide) as the immunomodulatory polypeptide, the ratio of i) the binding affinity of the control TMMP to a CD28 polypeptide (i.e., a related co-immunomodulatory polypeptide) to ii) the binding affinity of the TMMP of the disclosure to a CD28 polypeptide, as measured by BLI, is at least 1.5:1, at least 2:1, at least 5:1, at least 10:1, at least 15:1, at least 20:1, at least 25:1, at least 50:1, at least 100:1, at least 500:1, at least 10 2 :1, at least 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] As another example, if a control TMMP comprises a wild-type 4-1BBL polypeptide and a TMMP of the disclosure comprises a variant 4-1BBL polypeptide (comprising 1 to 10 amino acid substitutions relative to the amino acid sequence of the wild-type 4-1BBL polypeptide) as the immunomodulatory polypeptide, the ratio of i) the binding affinity of the control TMMP for the 4-1BB polypeptide (i.e., the related co-immunomodulatory polypeptide) to ii) the binding affinity of the TMMP of the disclosure for the 4-1BB polypeptide, as measured by BLI, is at least 1.5:1, at least 2:1, at least 5:1, at least 10:1, at least 15:1, at least 20:1, at least 25:1, at least 50:1, at least 100:1, at least 500:1, at least 10 2 :1, at least 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] As another example, when a control TMMP comprises a wild-type CD86 polypeptide and a TMMP of the disclosure comprises a variant CD86 polypeptide (comprising 1 to 10 amino acid substitutions relative to the amino acid sequence of the wild-type CD86 polypeptide) as the immunomodulatory polypeptide, the ratio of i) the binding affinity of the control TMMP to a CD28 polypeptide (i.e., a related co-immunomodulatory polypeptide) to ii) the binding affinity of the TMMP of the disclosure to a CD28 polypeptide, as measured by BLI, is at least 1.5:1, at least 2:1, at least 5:1, at least 10:1, at least 15:1, at least 20:1, at least 25:1, at least 50:1, at least 100:1, at least 500:1, at least 10 2 :1, at least 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] The binding affinity of a TMMP of the present disclosure for target T cells can be measured by the following method: A) contacting a TMMP of the present disclosure with target T cells that express i) a related co-immunomodulatory polypeptide that binds to the parent wild-type immunomodulatory polypeptide and ii) a T cell receptor that binds to the epitope on their surface (the TMMP comprises an epitope tag, such that the TMMP binds to the target T cells); B) contacting the TMMP bound to the target T cells with a fluorescently labeled binding agent (e.g., a fluorescently labeled antibody) that binds to the epitope tag to generate a TMMP / target T cell / binding agent complex; C) measuring the mean fluorescence intensity (MFI) of the TMMP / target T cell / binding agent complex using flow cytometry. The epitope tag can be, for example, a FLAG tag, a hemagglutinin tag, a c-myc tag, a poly(histidine) tag, etc. The MFI measured over a range of concentrations of the TMMP library members provides an indication of affinity. The MFI measured over a range of concentrations of the TMMP library members provides an indication of the half-maximal effective concentration (EC 50 In some examples, the EC of the TMMP of the present disclosure on target T cells is 50 is in the nM range, and the EC 50 (Control T cells express on their surface i) a related co-immunomodulatory 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 TMMP) are in the μM range. In some examples, the EC 50 EC of TMMP 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 TMMP of the present disclosure relative to control T cells 50 EC of TMMP on target T cells 50 The ratio of TMMP to TMMP indicates the selectivity of TMMP.

[0068] In some examples, when measured as described in the preceding paragraph, the TMMPs of the present disclosure exhibit selective binding to target T cells compared to binding of a TMMP library member to a control T cell that comprises i) a related co-immunomodulatory polypeptide that binds to a parent wild-type immunomodulatory polypeptide and ii) a T cell receptor that binds to an epitope other than the epitope present in the TMMP library member.

[0069] Dimerized TMMP The TMMPs of the present disclosure can be dimerized. That is, the present disclosure provides multimeric polypeptides comprising dimers of the TMMPs of the present disclosure. Thus, the present disclosure provides a TMMP comprising: A) a first heterodimer (the first heterodimer comprises one or more immunomodulatory polypeptides) comprising: a) a first polypeptide comprising i) a peptide epitope and ii) a first major histocompatibility complex (MHC) polypeptide; and b) i) a second polypeptide comprising a second MHC polypeptide; and B) a second heterodimer (the second heterodimer comprises one or more immunomodulatory polypeptides, the second heterodimer comprising: a) a peptide epitope and ii) the first MHC polypeptide; and b) i) a second polypeptide comprising the second MHC polypeptide; the second heterodimer comprises one or more immunomodulatory polypeptides, and the first heterodimer and the second heterodimer are covalently linked to each other. In some examples, the two TMMPs are identical to each other in amino acid sequence. 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 examples, 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 either directly or via a linker. The linker may be a peptide linker. The peptide linker can have a length of 1 to 200 amino acids (e.g., 1 to 5 amino acids (aa), 5 to 10 amino acids, 10 to 25 amino acids, 25 to 50 amino acids, 50 to 100 amino acids, 100 to 150 amino acids, or 150 to 200 amino acids). In some examples, the peptide epitope of the first heterodimer and the peptide epitope of the second heterodimer comprise the same amino acid sequence. In some examples, 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 identical amino acid sequences. In some examples, the first heterodimeric immunomodulatory polypeptide and the second heterodimeric immunomodulatory polypeptide are variant immunomodulatory polypeptides comprising 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 related co-immunomodulatory polypeptide. In some examples, the first heterodimeric immunomodulatory polypeptide and the second heterodimeric immunomodulatory polypeptide are each independently selected from the group consisting of IL-2, 4-1BBL, PD-L1, CD80, CD86, ICOS-L, OX-40L, FasL, JAG1 (CD339), TGFβ, CD70, and ICAM. Examples of suitable MHC polypeptides, immunomodulatory polypeptides, and peptide epitopes are described below. The first and / or second polypeptide comprises i) an Ig Fc polypeptide or a non-Ig scaffold and ii) a tumor targeting polypeptide.

[0070] MHC polypeptides As noted above, the TMMPs of the present disclosure include MHC polypeptides. For purposes of this disclosure, the term "major histocompatibility complex (MHC) polypeptide" is meant to include MHC polypeptides of various species, including human MHC (also referred to as human leukocyte antigen (HLA) polypeptides), rodent (e.g., mouse, rat, etc.) MHC polypeptides, and MHC polypeptides of other mammalian species (e.g., rabbit, non-human primate, dog, cat, ungulate (e.g., horse, cow, sheep, goat, etc.)). The term "MHC polypeptide" is meant to include class I MHC polypeptides (e.g., beta-2 microglobulin and MHC class I heavy chains).

[0071] 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 and the MHC-H chain are of human origin. That is, the MHC-H chain is an HLA heavy chain, or a variant thereof. Unless otherwise specified, the TMMPs of the present disclosure do not include the membrane anchor 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 TMMP to the cell in which it is expressed (e.g., a eukaryotic cell such as a mammalian cell). In some examples, the MHC class I heavy chain present in the TMMP of the present disclosure does not include the signal peptide, transmembrane domain, or intracellular domain (cytoplasmic tail) associated with a native MHC class I heavy chain. Thus, for example, in some instances, the MHC class I heavy chain present in a TMMP of the present disclosure comprises only the α1, α2, and α3 domains of the MHC class I heavy chain. In some instances, the MHC class I heavy chain present in a TMMP of the present disclosure has a length of about 270 amino acids (aa) to about 290 aa. In some instances, the MHC class I heavy chain present in a TMMP of the present disclosure has a length of 270 aa, 271 aa, 272 aa, 273 aa, 274 aa, 275 aa, 276 aa, 277 aa, 278 aa, 279 aa, 280 aa, 281 aa, 282 aa, 283 aa, 284 aa, 285 aa, 286 aa, 287 aa, 288 aa, 289 aa, or 290 aa.

[0072] In some examples, the MHC polypeptide of the TMMP 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 TMMP 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.

[0073] MHC class I heavy chain In some examples, the MHC class I heavy chain polypeptide present in a TMMP of the present disclosure comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to all or a portion (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 TMMP 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 (in addition to those positions shown to be variable in the heavy chain consensus sequence) of any one of the amino acid sequences shown in Figures 7-13. 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 TMMP 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.

[0074] Figures 5A, 5B, and 5C provide the amino acid sequences of human leukocyte antigen (HLA) class I heavy chain polypeptides. The signal sequence, amino acids 1-24, is bold and underlined. Figure 5A entries: 3A.1 is the HLA-A heavy chain (HLA-A*01:01:01:01 or A*0101) (NCBI accession NP_001229687.1), SEQ ID NO: 35; entry 3A.2 is from HLA-A*1101 SEQ ID NO: 36; entry 3A.3 is from HLA-A*2402 SEQ ID NO: 37; and entry 3A.4 is from HLA-A*3303 SEQ ID NO: 38. Figure 5B provides the sequence HLA-B*07:02:01 (HLA-B*0702) NCBI GenBank accession NP_005505.2 (see also GenBank accession AUV50118.1). Figure 5C provides HLA-C*0701 (GenBank accession NP_001229971.1) (HLA-C*07:01:01:01 or HLA-Cw*070101, HLA-Cw*07 (see GenBank accession CAO78194.1).

[0075] Figure 6 provides an alignment of 11 mature MHC class I heavy chain amino acid sequences, excluding leader sequences, transmembrane domains, or intracellular domains. The aligned sequences are human HLA-A, HLA-B, and HLA-C, the mouse H2K protein sequence, three variants of HLA-A (var. 1, var. 2C, and var. 2CP), and three human HLA-A variants (HLA-A*1101, HLA-A*2402, and HLA-A*3303). The alignment also shows positions (84 and 139 of the mature protein) where cysteine ​​residues can be introduced (e.g., by substitution) for disulfide bond formation to stabilize the MHC H chain-β2M complex. The alignment also shows position 236 (of the mature polypeptide), which can be substituted (e.g., at aa 12) with a cysteine ​​residue capable of forming an interchain disulfide bond with β2M. Arrows are displayed above each of these positions, and the residues are in bold. The seventh HLA-A sequence shown in the alignment (var. 2c) represents the sequence of variant 2, with substitutions of C residues at positions 84, 139, and 236. The boxes adjacent to residues 84, 139, and 236 indicate the five amino acid groups on either side of these six sets of five residues, designated aac1 ("amino acid cluster 1"), aac2 ("amino acid cluster 2"), aac3 ("amino acid cluster 3"), aac4 ("amino acid cluster 4"), aac5 ("amino acid cluster 5"), and aac6 ("amino acid cluster 6"), which may be replaced 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.

[0076] 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 thereof in which one or two amino acids have been deleted or substituted with other naturally occurring amino acids (e.g., L is replaced with I, V, A, or F); ii) aac2 (amino acid cluster 2) may be the amino acid sequence YNQSE (SEQ ID NO: 99) or a sequence thereof in which one or two amino acids have been deleted or substituted with other naturally occurring amino acids (e.g., N is replaced with Q, Q is replaced with N, and / or E is replaced with D); iii) aac3 (amino acid cluster 3) may be the amino acid sequence TAADM (SEQ ID NO: 100) or a sequence thereof in which one or two amino acids have been deleted or substituted with other naturally occurring amino acids (e.g., T is replaced with S, A is replaced with G, D is replaced with E, and / or M is replaced with L, V, or I); iv) aac4 (amino acid cluster 5) may be the amino acid sequence YNQSE (SEQ ID NO: 99) or a sequence thereof in which one or two amino acids have been deleted or substituted with other naturally occurring amino acids (e.g., T is replaced with S, A is replaced with G, D is replaced with E, and / or M is replaced with L, V, or I); aac4) may be the amino acid sequence AQTTK (SEQ ID NO: 101) or a sequence thereof in which one or two amino acids have been deleted or substituted with other naturally occurring amino acids (e.g., A is replaced with G, Q is replaced with N, or T is replaced with S, and / or K is replaced with R or Q); v) aac5 (amino acid cluster 5) may be the amino acid sequence VETRP (SEQ ID NO: 102) or a sequence thereof in which one or two amino acids have been deleted or substituted with other naturally occurring amino acids (e.g., V is replaced with I or L, E is replaced with D, T is replaced with S, and / or R is replaced with K); and / or vi) aac6 (amino acid cluster 6) may be the amino acid sequence GDGTF (SEQ ID NO: 103) or a sequence thereof in which one or two amino acids have been deleted or substituted with other naturally occurring amino acids (e.g., D is replaced with E, T is replaced with S, or F is replaced with L, W, or Y).

[0077] Figures 7-9 provide alignments of mature HLA class I heavy chain amino acid sequences (excluding leader sequences, transmembrane domains, 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 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 for 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) for disulfide bond formation 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) with a cysteine ​​residue capable of forming an interchain disulfide bond with β2M. The boxes adjacent residues 84, 139, and 236 indicate the five amino acid groups on either side of these six sets of five residues, designated aac1 ("amino acid cluster 1"), aac2 ("amino acid cluster 2"), aac3 ("amino acid cluster 3"), aac4 ("amino acid cluster 4"), aac5 ("amino acid cluster 5"), and aac6 ("amino acid cluster 6"), which may be replaced 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.

[0078] Figures 7A, 8A, and 9A provide alignments of the amino acid sequences of mature HLA-A, HLA-B, and HLA-C class I heavy chains, respectively. Sequences are provided for the extracellular portions of the mature proteins (excluding leader sequences or transmembrane or intracellular domains). Also shown are the positions of aa residues 84, 139, and 236, as well as their adjacent residues (aac1 through aac6), which can be replaced 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 described in Figure 6. Figures 7B, 8B, and 9B provide consensus amino acid sequences for the HLA-A, HLA-B, and HLA-C sequences provided in Figures 7A, 8A, and 9A, respectively. The consensus sequences show variable amino acid positions as consecutively numbered "X" residues, with amino acid positions 84, 139, and 236 double-underlined.

[0079] 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 thereof in which one or two amino acids have been deleted or substituted with other naturally occurring amino acids (e.g., L is replaced with I, V, A, or F); ii) aac2 (amino acid cluster 2) may be the amino acid sequence YNQSE (SEQ ID NO: 99) or a sequence thereof in which one or two amino acids have been deleted or substituted with other naturally occurring amino acids (e.g., N is replaced with Q, Q is replaced with N, and / or E is replaced with D); iii) aac3 (amino acid cluster 3) may be the amino acid sequence TAADM (SEQ ID NO: 100) or a sequence thereof in which one or two amino acids have been deleted or substituted with other naturally occurring amino acids (e.g., T is replaced with S, A is replaced with G, D is replaced with E, and / or M is replaced with L, V, or I); iv) aac4 (amino acid class Cluster 4) may be the amino acid sequence AQTTK (SEQ ID NO: 101) or a sequence thereof in which one or two amino acids have been deleted or substituted with other naturally occurring amino acids (e.g., A is replaced with G, Q is replaced with N, or T is replaced with S, and / or K is replaced with R or Q); v) aac5 (amino acid cluster 5) may be the amino acid sequence VETRP (SEQ ID NO: 102) or a sequence thereof in which one or two amino acids have been deleted or substituted with other naturally occurring amino acids (e.g., V is replaced with I or L, E is replaced with D, T is replaced with S, and / or R is replaced with K); and / or vi) aac6 (amino acid cluster 6) may be the amino acid sequence GDGTF (SEQ ID NO: 103) or a sequence thereof in which one or two amino acids have been deleted or substituted with other naturally occurring amino acids (e.g., D is replaced with E, T is replaced with S, or F is replaced with L, W, or Y).

[0080] 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 thereof in which one or two amino acids have been deleted or substituted with other naturally occurring amino acids (e.g., N is replaced with T or I, and / or L is replaced with A, and / or a second R is replaced with L, and / or G is replaced with R), ii) aac2 (amino acid cluster 2) may be the amino acid sequence YNQSE (SEQ ID NO: 99), or a sequence thereof in which one or two amino acids have been deleted or substituted with other naturally occurring amino acids (e.g., N is replaced with T or I, and / or L is replaced with A, and / or a second R is replaced with L, and / or G is replaced with R), aac3 (amino acid cluster 3) may be the amino acid sequence TAADT (SEQ ID NO: 105) or a sequence thereof in which one or two amino acids have been deleted or substituted with other natural amino acids (e.g., the first T is replaced with S, and / or the A is replaced with G, and / or the D is replaced with E, and / or the second T is replaced with S, and / or the third T is replaced with G, and / or the fourth T is replaced with E, and / or the fourth ... iv) aac4 (amino acid cluster 4) may be the amino acid sequence AQITQ (SEQ ID NO: 106) or a sequence thereof in which one or two amino acids are deleted or substituted with other naturally occurring amino acids (e.g., A is replaced with G, and / or the first Q is replaced with N, and / or I is replaced with L or V, and / or T is replaced with S, and / or the second Q is replaced with N); v) aac5 (amino acid cluster 5) may be the amino acid sequence VETRP (SEQ ID NO: 102), or a sequence thereof in which one or two amino acids have been deleted or substituted with other naturally occurring amino acids (e.g., V is replaced by I or L, E is replaced by D, T is replaced by S, and / or R is replaced by K); and / or vi) aac6 (amino acid cluster 6) may be the amino acid sequence GDRTF (SEQ ID NO: 107), or a sequence thereof in which one or two amino acids have been deleted or substituted with other naturally occurring amino acids (e.g., D is replaced by E,and / or T is replaced by S, and / or R is replaced by K or H, and / or F is replaced by L, W, or Y).

[0081] 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 thereof in which one or two amino acids have been deleted or substituted with other naturally occurring amino acids (e.g., N is replaced by K, and / or L is replaced by A or I, and / or the second R is replaced by H, and / or G is replaced by T or S), ii) aac2 (amino acid cluster 2) may be the amino acid sequence YNQSE (SEQ ID NO: 99), or a sequence thereof in which one or two amino acids have been deleted or substituted with other naturally occurring amino acids (e.g., N is replaced by K, and / or L is replaced by A or I, and / or the second R is replaced by H, and / or G is replaced by T or S), iii) aac3 (amino acid cluster 3) may be the amino acid sequence TAADT (SEQ ID NO: 105) or a sequence thereof in which one or two amino acids have been deleted or substituted with other naturally occurring amino acids (e.g., N is replaced with Q, Q is replaced with N, and / or E is replaced with D); iii) aac4 (amino acid cluster 4) may be the amino acid sequence TAADT (SEQ ID NO: 105) or a sequence thereof in which one or two amino acids have been deleted or substituted with other naturally occurring amino acids (e.g., the first T is replaced with S, and / or A is replaced with G, and / or D is replaced with E, and / or wherein the second T is replaced by S); iv) aac4 (amino acid cluster 4) may be the amino acid sequence AQITQ (SEQ ID NO: 106) or a sequence thereof in which one or two amino acids are deleted or substituted with other natural amino acids (e.g., A is replaced by G, and / or the first Q is replaced by N, and / or I is replaced by L, and / or the second Q is replaced by N or K); v) aac5 (amino acid cluster 5) may be the amino acid sequence VETRP (SEQ ID NO: 102) or one or more may be the amino acid sequence GDGTF (SEQ ID NO: 103) or a sequence thereof in which one or two amino acids have been deleted or substituted with other naturally occurring amino acids (e.g., V is replaced by I or L, E is replaced by D, T is replaced by S, and / or R is replaced by K or H); and / or vi) aac6 (amino acid cluster 6) may be the amino acid sequence GDGTF (SEQ ID NO: 103) or a sequence thereof in which one or two amino acids have been deleted or substituted with other naturally occurring amino acids (e.g., D is replaced by E, and / or T is replaced by S,and / or F may be replaced by L, W, or Y).

[0082] HLA-A In some examples, the TMMPs of the present disclosure comprise HLA-A heavy chain polypeptides. HLA-A heavy chain peptide sequences or portions thereof that can be incorporated into the TMMPs of the present disclosure include, but are not limited to, the following alleles: A*0101, A*0201, A*0301, A*1101, A*2301, A*2402, A*2407, A*3303, and A*3401, aligned without all or substantially all of the leader, transmembrane, and cytoplasmic sequences of Figure 7A. 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: a tyrosine to alanine substitution at position 84 (Y84A), a tyrosine to cysteine ​​substitution at position 84 (Y84C), an alanine to cysteine ​​substitution at position 139 (A139C), and an alanine to cysteine ​​substitution at position 236 (A236C). In addition, 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 to 25, 1 to 5, 5 to 10, 10 to 15, 15 to 20, 20 to 25, or 25 to 30 amino acids).

[0083] In some examples, the TMMP of the present disclosure has the following HLA-A consensus amino acid sequence: TIFF0007762068000002.tif38164 (X1 is F, Y, S, or T, X2 is K or R, X3 is Q, G, E, or R, X4 is N or E, X5 is R or G, X6 is N or K, X7 is M or V, X8 is H or Q, X9 is T or I, X10 is D or H, X11 is A, V, or E, X12 is N or D, X13 is G or R, X14 is T or I, X15 is L or A, X16 is R or L, X17 is G or R, X18 is A or D, X19 is I, L, or V, X20 is I, R, or M, X21 is F or Y, X22 is S or P, and X 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).

[0084] By way of example, the MHC class I heavy chain polypeptide of TMMP can comprise an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following human HLA-A heavy chain amino acid sequence: TIFF0007762068000003.tif31164.

[0085] In some examples, an HLA-A heavy chain polypeptide suitable for inclusion in a TMMP of the present disclosure comprises the following amino acid sequence: TIFF0007762068000004.tif31164. This HLA-A heavy chain polypeptide is also referred to as "HLA-A*0201" or simply "HLA-A02." In some instances, the C-terminal Pro is not included in the TMMP of the present disclosure. For example, in some instances, an HLA-A02 polypeptide suitable for inclusion in the TMMP of the present disclosure comprises the following amino acid sequence: TIFF0007762068000005.tif31164.

[0086] 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 following human HLA-A heavy chain (Y84A, A236C) amino acid sequence: TIFF0007762068000006.tif31164, in which 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.

[0087] In some examples, an HLA-A heavy chain polypeptide suitable for inclusion in a TMMP of the present disclosure is an HLA-A02(Y84A;A236C) polypeptide, comprising the following amino acid sequence: TIFF0007762068000007.tif31164.

[0088] In some examples, an HLA-A heavy chain polypeptide suitable for inclusion in a TMMP of the present disclosure is an HLA-A02(Y84A;A236C) polypeptide, comprising the following amino acid sequence: TIFF0007762068000008.tif31164.

[0089] 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 following human HLA-A heavy chain (Y84C, A139C) amino acid sequence: In the sequence TIFF0007762068000009.tif31164, amino acid 84 is Cys and amino acid 139 is Cys. In some instances, Cys-84 forms an intrachain disulfide bond with Cys-139.

[0090] HLA-A11 (HLA-A*1101) As one non-limiting example, the MHC class I heavy chain polypeptide of TMMP can comprise an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following human HLA-A11 heavy chain amino acid sequence: TIFF0007762068000010.tif31164. Such MHC class I heavy chains may be prominent in Asian populations, including populations of individuals of Asian descent.

[0091] HLA-A11(Y84A;A236C) As one non-limiting example, in some instances, the MHC class I heavy chain polypeptide is an HLA-A11 allele that includes a Y84A substitution and an A236C substitution. For example, in some instances, the MHC class I heavy chain polypeptide includes an amino acid sequence that has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following human HLA-A A11 heavy chain (Y84A;A236C) amino acid sequence: TIFF0007762068000011.tif31164, in which 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.

[0092] HLA-A24 (HLA-A*2402) As one non-limiting example, the MHC class I heavy chain polypeptide of the TMMP of the present disclosure can comprise an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following human HLA-A24 heavy chain amino acid sequence: TIFF0007762068000012.tif37164. Such MHC class I heavy chains may 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.

[0093] HLA-A33 (HLA-A*3303) As one non-limiting example, the MHC class I heavy chain polypeptide of the TMMP of the present disclosure can comprise an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following human HLA-A33 heavy chain amino acid sequence: TIFF0007762068000013.tif37164. Such MHC class I heavy chains may 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.

[0094] HLA-B In some examples, the TMMPs of the present disclosure comprise an HLA-B heavy chain polypeptide. HLA-B heavy chain peptide sequences or portions thereof that can be incorporated into the TMMPs of the present disclosure include, but are not limited to, the following alleles: B*0702, B*0801, B*1502, B*3802, B*4001, B*4601, and B*5301, aligned without all or substantially all of the leader, transmembrane, and cytoplasmic sequences of Figure 8A. 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: a tyrosine to alanine substitution at position 84 (Y84A), a tyrosine to cysteine ​​substitution at position 84 (Y84C), an alanine to cysteine ​​substitution at position 139 (A139C), and an alanine to cysteine ​​substitution at position 236 (A236C). In addition, 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).

[0095] In some examples, the TMMP of the present disclosure comprises an HLA-B heavy chain polypeptide comprising the following HLA-B consensus amino acid sequence: TIFF0007762068000014.tif37164, in which X1 is H, Y, or D, X2 is A or S, X3 is M or V, X4 is A, S, or T, X5 is Q or L, X6 is A or T, X7 is E, MK, or T, X8 is A or T, X9 is E or N, X10 is I or K, X11 is Y, F, S, or C, X12 is N or Q, X13 is A or T, X14 is D or Y, X15 is E or V, and X16 is S or N. , X17 is T, N, or I, X18 is A or L, X19 is L or R, X20 is R or G, X21 is T or I, X22 is L or I, X23 is R or S, X24 is R or S, X25 is S or T, X26 is L or W, X27 is E or V, X28 is R, D, L, or W, X29 is A or T, X30 is L, E, or T, X31 is E or D, X32 is K or T, X33 is E or Q, and X34 is I or V.

[0096] By way of example, an MHC class I heavy chain polypeptide of a TMMP of the present disclosure can comprise an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following human HLA-B heavy chain amino acid sequence: TIFF0007762068000015.tif31164.

[0097] 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 following human HLA-B heavy chain (Y84A, A236C) amino acid sequence: TIFF0007762068000016.tif31164, in which 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.

[0098] 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 following human HLA-B heavy chain (Y84C, A139C) amino acid sequence: In the sequence TIFF0007762068000017.tif31164, amino acid 84 is Cys and amino acid 139 is Cys. In some instances, Cys-84 forms an intrachain disulfide bond with Cys-139.

[0099] HLA-B*0702 By way of example, in some instances, an MHC class I heavy chain polypeptide present in a TMMP of the present disclosure comprises the amino acid sequence of HLA-B*0702 (SEQ ID NO: 62) of FIG. 8A, 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 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, if the HLA-B heavy chain polypeptide of the TMMP of the present disclosure has less than 100% identity to the sequence labeled HLA-B in FIG. 6 or the sequence labeled "B*0702" in FIG. 8A, 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 TMMP of the present disclosure contains Y84A and A236C substitutions. In some examples, the HLA-B*0702 heavy chain polypeptide of the TMMP of the present disclosure contains Y84C and A139C substitutions. In some examples, the HLA-B heavy chain polypeptide of the TMMP of the present disclosure comprises Y84C, A139C, and A236C substitutions.

[0100] HLA-C In some examples, the TMMPs of the present disclosure comprise HLA-C heavy chain polypeptides. HLA-C heavy chain peptide sequences or portions thereof that can be incorporated into the TMMPs of the present disclosure include, but are not limited to, the following alleles: C*0102, C*0303, C*0304, C*0401, C*0602, C*0701, C*0801, and C*1502, aligned without all or substantially all of the leader, transmembrane, and cytoplasmic sequences of 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), and an alanine to cysteine ​​substitution at position 236 (A236C). In addition, 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).

[0101] In some examples, the TMMP of the present disclosure comprises an HLA-C heavy chain polypeptide comprising the following HLA-C consensus amino acid sequence: TIFF0007762068000018.tif38164, in which X1 is C or G, X2 is R or K, X3 is F, Y, S, or D, X4 is R or W, X5 is H or R, X6 is A or S, X7 is Q or R, X8 is A or E, X9 is N or K, X10 is T or A, X11 is S or N, X12 is N or K, X13 is A or D, X14 is G or R, X15 is T or I, X16 is L or I, X17 is W or R, and X18 is C, Y, F, or 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.

[0102] By way of example, the MHC class I heavy chain polypeptide of the TMMP of the present disclosure can comprise an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following human HLA-C heavy chain amino acid sequence: TIFF0007762068000019.tif31164.

[0103] 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 following human HLA-C heavy chain (Y84A, A236C) amino acid sequence: TIFF0007762068000020.tif31164, in which 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.

[0104] 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 following human HLA-C heavy chain (Y84C, A139C) amino acid sequence: In the sequence TIFF0007762068000021.tif31164, amino acid 84 is Cys and amino acid 139 is Cys. In some instances, Cys-84 forms an intrachain disulfide bond with Cys-139.

[0105] HLA-C*0701 In some examples, the MHC class I heavy chain polypeptide of a TMMP of the present disclosure comprises 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 the amino acid sequence of HLA-C*0701 of Figure 9A (labeled HLA-C in Figure 6), or 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, if the HLA-C heavy chain polypeptide of a TMMP of the present disclosure has less than 100% identity to the sequence labeled HLA-C*0701 in Figure 9A, 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), and an alanine to cysteine ​​substitution at position 236 (A236C). In some examples, the HLA-C heavy chain polypeptide of a TMMP of the present disclosure contains Y84A and A236C substitutions. In some examples, the HLA-C*0701 heavy chain polypeptide of a T-cell MMP or an epitope conjugate thereof contains Y84C and A139C substitutions. In some examples, the HLA-C heavy chain polypeptide of the TMMP of the present disclosure comprises Y84C, A139C, and A236C substitutions.

[0106] Non-classical HLA-E, HLA-F, and HLA-G MHC class I heavy chains In some examples, the TMMP of the present disclosure comprises a non-classical MHC class I heavy chain polypeptide. Non-classical HLA heavy chain polypeptides, or portions thereof, that can be incorporated into the TMMP 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).

[0107] Non-limiting examples of suitable HLA-E alleles include, but are not limited to, HLA-E*0101 (HLA-E*01:01:01:01), HLA-E*01:03 (HLA-E*01:03:01:01), HLA-E*01:04, HLA-E*01:05, HLA-E*01:06, HLA-E*01:07, HLA-E*01:09, and HLA-E*01:10. Non-limiting examples of suitable HLA-F alleles include, but are not limited to, HLA-F*0101 (HLA-F*01:01:01:01), HLA-F*01:02, HLA-F*01:03 (HLA-F*01:03:01:01), HLA-F*01:04, HLA-F*01:05, and HLA-F*01:06. Non-limiting examples of suitable HLA-G alleles include HLA-G*0101 (HLA-G*01:01:01:01), HLA-G*01:02, HLA-G*01:03 (HLA-G*01:03:01:01), HLA-G*01:04 (HLA-G*01:04:01:01), HLA-G*01:06, HLA-G*01:07, HLA-G*01:08, HLA-G*01:09, HLA-G*01:10, HLA-G*01:11, HLA-G*01:12, HLA-G*01:13, 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*01:19, HLA-G*01:19, HLA-G*01:20, HLA-G*01:21, HLA-G*01:22, HLA-G*01:23, HLA-G*01:24, HLA-G*01:25, HLA-G*01:26, HLA-G*01:27, HLA-G*01:28, HLA-G*01:29, HLA-G*01:30, HLA-G*01:31, HLA-G*01:32, HLA-G*01:33, HLA-G*01:34, HLA-G*01:35, HLA-G*01:36, HLA-G*01:37, HLA-G*01:38, HLA-G*01:39, HLA-G*0 *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. Consensus sequences for these HLA E, HLA-F, and HLA-G alleles, excluding all or substantially all of the leader, transmembrane, and cytoplasmic sequences, are provided in Figure 10 and aligned with the consensus sequences for the HLA-A, HLA-B, and HLA-C alleles described above in Figure 11.

[0108] 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, and positions aa 84, 139, and 236 double underlined.

[0109] Figure 11 provides 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 listed as "X" with sequential numbering removed. As shown in Figure 6, positions aa 84, 139, and 236 are indicated with their adjacent 5-amino acid clusters that can be replaced 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.

[0110] 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 listed in the figure).

[0111] Mouse H2K In some examples, an MHC class I heavy chain polypeptide present in a TMMP 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 TMMP 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 ​​substitution at position 236 (A236C). In some examples, a mouse H2K heavy chain polypeptide of a TMMP of the present disclosure contains a Y84A and A236C substitution. In some examples, a mouse H2K heavy chain polypeptide of a TMMP of the present disclosure contains a Y84C and A139C substitution. In some examples, the mouse H2K heavy chain polypeptide of a TMMP of the present disclosure comprises Y84C, A139C, and A236C substitutions.

[0112] Combination examples Table 1 below shows various combinations of MHC class I heavy chain sequence modifications that can be incorporated into the TMMPs of the present disclosure.

[0113] (Table 1) TIFF0007762068000022.tif101159TIFF0007762068000023.tif236161**Sequence identity range is the allowable range of sequence identity of the MHC-H polypeptide sequence incorporated into TMMP to the corresponding portion of the sequences listed in Figures 6-11 not counting variable residues in the consensus sequence.

[0114] Beta-2 microglobulin The β2-microglobulin (β2M) polypeptide of the TMMP of the present disclosure can be a human β2M polypeptide, a non-human primate β2M polypeptide, a 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 6. 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.

[0115] In some instances, a suitable β2M polypeptide has the following amino acid sequence: TIFF0007762068000024.tif11156, wherein the HLA class I heavy chain polypeptide has the following amino acid sequence: TIFF0007762068000025.tif31164 (the cysteine ​​residue indicated by {C} forms a disulfide bond between the α1 helix and the α2-1 helix, and the (C) residue forms a disulfide bond with the β2M polypeptide cysteine ​​at position 12). In the above sequence, "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.

[0116] 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 an amino acid with a cysteine ​​(Cys) residue, which, when present in the MHC polypeptide of a first polypeptide of a TMMP of the present disclosure, can form a disulfide bond with a cysteine ​​residue present in the second polypeptide chain of the TMMP of the present disclosure.

[0117] In some examples, a first MHC polypeptide within a first polypeptide of a TMMP of the present disclosure and / or a second MHC polypeptide within a second polypeptide of a TMMP of the present disclosure comprises an amino acid substitution replacing an amino acid with a cysteine, wherein the substituted cysteine ​​in the first MHC polypeptide forms a disulfide bond with the cysteine ​​in the second MHC polypeptide, the cysteine ​​in the first MHC polypeptide forms a disulfide bond with the substituted cysteine ​​in the second MHC polypeptide, or the substituted cysteine ​​in the first MHC polypeptide forms a disulfide bond with the substituted cysteine ​​in the second MHC polypeptide.

[0118] For example, in some instances, one of the following pairs of residues in HLA β2-microglobulin and HLA class I heavy chains is substituted with cysteine ​​(residue numbers are those of the mature polypeptide): 1) β2M residue 12, HLA class I heavy chain residue 236; 2) β2M residue 12, HLA class I heavy chain residue 237; 3) β2M residue 8, HLA class I heavy chain residue 234; 4) β2M residue 10, HLA class I heavy chain residue 235; 5) β2M residue 24, HLA class I heavy chain residue 236; 6) β2M residue 28, HLA class I heavy chain residue 232, 7) β2M residue 98, HLA class I heavy chain residue 192, 8) β2M residue 99, HLA class I heavy chain residue 234, 9) β2M residue 3, HLA class I heavy chain residue 120, 10) β2M residue 31, HLA class I heavy chain residue 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 A rg3, HLA class I heavy chain residue Gly120, 16) β2M residue His31, HLA class I heavy chain residue Gln96, 17) β2M residue Asp53, HLA class I heavy chain residue Arg35, 18) β2M residue Trp60, HLA class I heavy chain residue Gln96, 19) β2M residue Trp60, HLA class I heavy chain residue Asp122, 20) β2M residue Tyr63, HLA class I heavy chain residue Tyr27, 21) β2M residue Lys6, HLA class I heavy chain residue Glu232, 22) β2M residue 1) Gln8, HLA class I heavy chain residue Arg234; 2) β2M residue Tyr10, HLA class I heavy chain residue Pro235; 24) β2M residue Ser11, HLA class I heavy chain residue Gln242; 25) β2M residue Asn24, HLA class I heavy chain residue Ala236; 26) β2M residue Ser28, HLA class I heavy chain residue Glu232; 27) β2M residue Asp98, HLA class I heavy chain residue His192; and 28) β2M residue Met99, HLA class I heavy chain residue Arg234. The amino acid numbering of the MHC / HLA class I heavy chain is based on the mature MHC / HLA class I heavy chain without the signal peptide. For example, in some instances, 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.

[0119] In some examples, the β2M polypeptide comprises the amino acid sequence: TIFF0007762068000026.tif17155. In some examples, the β2M polypeptide comprises the amino acid sequence: TIFF0007762068000027.tif18155.

[0120] In some examples, the HLA class I heavy chain polypeptide comprises the amino acid sequence: TIFF0007762068000028.tif31164.

[0121] In some examples, the HLA class I heavy chain polypeptide comprises the amino acid sequence: TIFF0007762068000029.tif31164.

[0122] In some examples, the HLA class I heavy chain polypeptide comprises the amino acid sequence: TIFF0007762068000030.tif31164.

[0123] In some instances, the β2M polypeptide has the amino acid sequence: TIFF0007762068000031.tif11156, and the HLA class I heavy chain polypeptide of the TMMP of the present disclosure has the following amino acid sequence: TIFF0007762068000032.tif31164 (the underlined and bold Cys residues form disulfide bonds with each other in TMMP).

[0124] In some examples, the β2M polypeptide comprises the amino acid sequence: TIFF0007762068000033.tif11162.

[0125] In some examples, the first and second polypeptides of the TMMP of the present disclosure are disulfide-bonded to each other via i) a Cys residue present in the linker connecting the peptide epitope in the first polypeptide chain and the β2M polypeptide, and ii) a Cys residue present in the MHC class I heavy chain in the second polypeptide 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 in the first polypeptide chain and the β2M polypeptide is GCGGS(G4S)n (SEQ ID NO: 136), 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 GCGGSGGGGSGGGGS (SEQ ID NO: 138). Examples of disulfide-bonded first and second polypeptides of a TMMP of the present disclosure are shown schematically in Figures 2A-2F.

[0126] Multi-disulfide bond TMMP In some examples, the first and second polypeptides of the TMMPs of the present disclosure are linked to one another by at least two disulfide bonds (i.e., two interchain disulfide bonds). Examples of such multiply disulfide-linked TMMPs are shown schematically in Figures 12A and 12B. Additionally, when the TMMPs of the present disclosure include an IgFc polypeptide, the heterodimeric TMMP can be dimerized, with a disulfide bond linking the IgFc polypeptides in the two heterodimeric TMMPs. 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 multiply disulfide-linked TMMPPs in this section does not refer to the disulfide bonds linking the IgFc polypeptides of the dimerized TMMP.

[0127] As described above, in some examples, the first and second polypeptides of the TMMP of the present disclosure are linked to each other by at least two disulfide bonds (i.e., two interchain disulfide bonds). For example, in some examples, the first and second polypeptides of the TMMP of the present disclosure are linked to each other by two interchain disulfide bonds. As another example, in some examples, the first and second polypeptides of the TMMP of the present disclosure are linked to each other by three interchain disulfide bonds. As another example, in some examples, the first and second polypeptides of the TMMP of the present disclosure are linked to each other by four interchain disulfide bonds.

[0128] In some examples, when a peptide epitope in a first polypeptide of a TMMP 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 of an MHC class I heavy chain in a second polypeptide. In some examples, when a peptide epitope in a first polypeptide of a TMMP 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 a β2M polypeptide present in the second polypeptide.

[0129] In some examples, the multiple disulfide bond TMMPs of the present disclosure (e.g., double disulfide bond TMMPs) exhibit increased stability compared to control TMMPs containing only one of at least two disulfide bonds. In some examples, the multiple disulfide bond TMMPs of the present disclosure (e.g., double disulfide bond TMMPs) exhibit increased in vitro stability compared to control TMMPs containing only one of at least two disulfide bonds. For example, in some examples, the multiple disulfide bond TMMPs of the present disclosure (e.g., double disulfide bond TMMPs) 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 increased in vitro stability compared to control TMMPs containing only one of at least two disulfide bonds.

[0130] Whether a multi-disulfide bonded TMMP of the present disclosure exhibits increased in vitro stability compared to a control TMMP containing only one of the at least two disulfide bonds can be determined by measuring the amount of disulfide-bonded heterodimeric TMMP present in a sample over time and / or under particular conditions and / or during purification of the TMMP.

[0131] For example, in some instances, a multi-disulfide bonded TMMP (e.g., a double-disulfide bonded TMMP) of the present disclosure exhibits at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 50%, at least 2-fold, at least 5-fold, or at least 10-fold greater in vitro stability than a control TMMP containing only one of the at least two disulfides when the TMMP 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 TMMP remaining after in vitro storage of a multi-disulfide-linked TMMP (e.g., a double-disulfide-linked TMMP) of the present disclosure 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 TMMP remaining after in vitro storage of a control TMMP (a TMMP containing only one of the at least two disulfide bonds present in the multi-disulfide-linked TMMP) at 37°C for 28 days.

[0132] In some instances, the multiple disulfide bond TMMPs of the present disclosure exhibit greater in vivo stability than a control TMMP containing only one of the at least two disulfide bonds, e.g., in some instances, the multiple disulfide bond TMMPs of the present disclosure exhibit at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 50%, at least 2-fold, at least 5-fold, or at least 10-fold greater in vivo stability than a control TMMP containing only one of the at least two disulfide bonds.

[0133] In some examples, the presence of two disulfide bonds in a multiply disulfide-bonded TMMP of the present disclosure (e.g., a double-disulfide-bonded TMMP) increases the amount of disulfide-bonded heterodimeric TMMP produced compared to the amount of disulfide-bonded heterodimeric TMMP produced when the TMMP is a control TMMP containing only one of the at least two disulfide bonds. For example, a multiply disulfide-bonded TMMP of the present disclosure (e.g., a double-disulfide-bonded TMMP) 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 TMMP can be secreted into the cell culture medium. The cells can be lysed to produce a cell lysate, and the TMMP can be present in the cell lysate. The TMMP can be purified from the cell culture medium and / or the cell lysate. For example, when the TMMP comprises an IgG1 Fc polypeptide, the cell culture medium and / or cell lysate can be contacted with immobilized Protein A (e.g., the cell culture medium and / or cell lysate can be applied to a Protein A column in which Protein A has been immobilized on beads). TMMP 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 TMMP is eluted to produce a Protein A eluate. The amount of disulfide-linked heterodimeric TMMP present in the Protein A eluate is at least 0.5%, at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, or at least 10% higher than the amount of disulfide-linked heterodimeric TMMP present in the Protein A eluate when the TMMP is a control TMMP that contains only one of the at least two disulfide bonds present in a multiple-disulfide-linked TMMP (e.g., a double-disulfide-linked TMMP). In some examples, the percentage of total TMMP protein in the eluate that is non-aggregated disulfide-linked heterodimeric TMMP 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.

[0134] In some examples, the T cell modulatory multimeric polypeptides of the present disclosure comprise a) a first polypeptide comprising i) a peptide epitope and ii) a first MHC polypeptide; b) a second polypeptide comprising a second MHC polypeptide; c) at least one immunomodulatory polypeptide (the first and / or second polypeptide comprises at least one immunomodulatory polypeptide); and d) an Ig Fc polypeptide or a non-Ig scaffold (the first and / or second polypeptide comprises an Ig Fc polypeptide or a non-Ig scaffold). and e) TTP (the first and / or second polypeptide comprises TTP), wherein 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 comprises 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 comprises a second Cys residue that forms a disulfide bond (second disulfide bond) with a second Cys residue of the second polypeptide.

[0135] In some examples, a TMMP of the present disclosure comprises a) a first polypeptide comprising, in order from N-terminus to C-terminus, i) a peptide epitope, ii) a peptide linker, and iii) a β2M polypeptide; and b) a second polypeptide comprising 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 TMMP comprises a) a first disulfide bond between i) a Cys present in the linker between the Cys peptide epitope 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 C-terminal to the Cys present in the linker and ii) a Cys in the second polypeptide C-terminal to the first Cys introduced into the MHC class I heavy chain polypeptide. As mentioned above, the TMMP also comprises i) an Ig Fc polypeptide or a non-Ig scaffold, and ii) TTP.

[0136] In some examples, the first and second disulfide bond-forming Cys residues in the first or second polypeptide of a TMMP of the present disclosure are separated from each other by about 10 amino acids to about 200 amino acids. For example, in some examples, the first and second disulfide bond-forming Cys residues in the first or second polypeptide of a TMMP are separated from each other by about 10 amino acids (aa) to about 15 aa, about 15 aa to about 20 aa, about 20 aa to about 25 aa, about 25 aa to about 30 aa, about 30 aa to about 40 aa, about 40 aa to about 50 aa, about 50 aa to about 60 aa, about 60 aa to about 70 aa, or about 70 aa to about 80 aa. , about 80aa to about 90aa, about 90aa to about 100aa, about 100aa to about 110aa, about 110aa to about 120aa, about 120aa to about 130aa, about 130aa to about 140aa, about 140aa to about 150aa, about 150aa to about 160aa, about 160aa to about 170aa, about 170aa to about 180aa, about 180aa to about 190aa, or about 190aa to about 200aa.

[0137] For example, in some examples, the first and second disulfide bond-forming Cys residues in the first polypeptide of a TMMP of the present disclosure are separated from each other by about 10 to about 80 amino acid residues. For example, in some examples, the second disulfide bond-forming Cys residue in the first polypeptide is located about 10 to about 80 amino acids (e.g., about 10 amino acids (aa) to about 15 aa, about 15 aa to about 20 aa, about 20 aa to about 25 aa, about 25 aa to about 30 aa, about 30 aa to about 40 aa, about 40 aa to about 50 aa, about 50 aa to about 60 aa, about 60 aa to about 70 aa, or about 70 aa to about 80 aa) C-terminal to the first disulfide bond-forming Cys residue in the first polypeptide. In some examples, the second disulfide bond-forming Cys residue in the first polypeptide is 10 aa, 11 aa, 12 aa, 13 aa, 14 aa, 15 aa, 16 aa, 17 aa, 18 aa, 19 aa, 20 aa, 21 aa, 22 aa, 23 aa, 24 aa, or 25 aa C-terminal to the first disulfide bond-forming Cys residue in the first polypeptide. In some examples, the second disulfide bond-forming Cys residue in the first polypeptide is 15 aa C-terminal to the first disulfide bond-forming Cys residue in the first polypeptide. In some examples, the second disulfide bond-forming Cys residue in the first polypeptide is 20 aa C-terminal to the first disulfide bond-forming Cys residue in the first polypeptide. In some examples, the second disulfide bond-forming Cys residue of the first polypeptide is 25 aa C-terminal to the first disulfide bond-forming Cys residue of the first polypeptide.

[0138] As another example, in some instances, the first and second disulfide bond-forming Cys residues in the second polypeptide of a TMMP of the present disclosure are separated from each other by about 140 to about 160 amino acids, e.g., in some instances, the second disulfide bond-forming Cys residue of the second polypeptide is about 140 to about 160 amino acids C-terminal to the first disulfide bond-forming Cys residue of the second polypeptide. In some examples, the second disulfide bond-forming Cys residue of the second polypeptide is 140 amino acids (aa), 141aa, 142aa, 143aa, 144aa, 145aa, 146aa, 147aa, 148aa, 149aa, 150aa, 151aa, 152aa, 153aa, 154aa, 155aa, 156aa, 157aa, 158aa, 159aa, or 160aa C-terminal to the first disulfide bond-forming Cys residue in the second polypeptide.

[0139] A multiple disulfide bond TMMP (e.g., a double disulfide bond TMMP) of the present disclosure can include, for example, a) a first polypeptide comprising i) a peptide epitope; and ii) a first MHC polypeptide (the first polypeptide comprises a peptide linker between the peptide and the first MHC polypeptide, the peptide linker comprising a Cys residue, and the first MHC polypeptide being 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 a Y84C substitution and an A236C substitution based on the amino acid numbering of HLA-A*0201 (shown in FIG. 7A ). and a class I heavy chain containing a Cys substitution at amino acid position 84 of the class I heavy chain or a substitution at the corresponding position of another class I heavy chain allele, wherein the TMMP contains a disulfide bond between the introduced Cys residue of the peptide linker and a Cys residue at amino acid position 84 of the class I heavy chain or the corresponding position of another class I heavy chain allele, and the TMMP contains a disulfide bond between the introduced Cys residue of the β2M polypeptide and a Cys residue at amino acid position 236 of the class I heavy chain or the corresponding position of another class I heavy chain allele; and c) at least one immunomodulatory polypeptide (the first and / or second polypeptides comprise at least one immunomodulatory polypeptide). Examples are shown schematically in Figures 12A and 12B. As described above, the TMMP also contains i) an Ig Fc polypeptide or a non-Ig scaffold, and ii) TTP.

[0140] 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), wherein n is an integer between 1 and 10. In some examples, the peptide linker comprises the amino acid sequence GCGGS(GGGGS)n (SEQ ID NO: 140), wherein n is 1. In some examples, the peptide linker comprises the amino acid sequence GCGGS(GGGGS)n (SEQ ID NO: 140), wherein n is 2. In some examples, the peptide linker comprises the amino acid sequence GCGGS(GGGGS)n (SEQ ID NO: 140), wherein n is 3. In some examples, the peptide linker comprises the amino acid sequence GCGGS(GGGGS)n (SEQ ID NO: 140), wherein n is 4. In some examples, the peptide linker comprises the amino acid sequence GCGGS(GGGGS)n (SEQ ID NO: 140), wherein n is 5. In some examples, the peptide linker comprises the amino acid sequence GCGGS(GGGGS)n (SEQ ID NO: 140), where n is 6. In some examples, the peptide linker comprises the amino acid sequence GCGGS(GGGGS)n (SEQ ID NO: 140), where n is 7. In some examples, the peptide linker comprises the amino acid sequence GCGGS(GGGGS)n (SEQ ID NO: 140), where n is 8. In some examples, the peptide linker comprises the amino acid sequence GCGGS(GGGGS)n (SEQ ID NO: 140), where n is 9. In some examples, the peptide linker comprises the amino acid sequence GCGGS(GGGGS)n (SEQ ID NO: 140), where n is 10.

[0141] 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), wherein n is an integer between 1 and 10. In some examples, the peptide linker comprises the amino acid sequence CGGGS(GGGGS)n (SEQ ID NO: 142), wherein n is 1. In some examples, the peptide linker comprises the amino acid sequence CGGGS(GGGGS)n (SEQ ID NO: 142), wherein n is 2. In some examples, the peptide linker comprises the amino acid sequence CGGGS(GGGGS)n (SEQ ID NO: 142), wherein n is 3. In some examples, the peptide linker comprises the amino acid sequence CGGGS(GGGGS)n (SEQ ID NO: 142), wherein n is 4. In some examples, the peptide linker comprises the amino acid sequence CGGGS(GGGGS)n (SEQ ID NO: 142), wherein n is 5. In some examples, the peptide linker comprises the amino acid sequence CGGGS(GGGGS)n (SEQ ID NO: 142), where n is 6. In some examples, the peptide linker comprises the amino acid sequence CGGGS(GGGGS)n (SEQ ID NO: 142), where n is 7. In some examples, the peptide linker comprises the amino acid sequence CGGGS(GGGGS)n (SEQ ID NO: 142), where n is 8. In some examples, the peptide linker comprises the amino acid sequence CGGGS(GGGGS)n (SEQ ID NO: 142), where n is 9. In some examples, the peptide linker comprises the amino acid sequence CGGGS(GGGGS)n (SEQ ID NO: 142), where n is 10.

[0142] The following are non-limiting examples of MHC class I heavy chains containing the Y84C and A236C substitutions based on the amino acid numbering of HLA-A*0201 (shown in Figure 7A), or substitutions at the corresponding positions of another class I heavy chain allele:

[0143] HLA-A In some examples, a multiple disulfide bond TMMP (e.g., a double disulfide bond TMMP) of the present disclosure comprises: a) a first polypeptide comprising i) a peptide epitope; and ii) a first MHC polypeptide (the first polypeptide comprises a peptide linker between the peptide epitope and the first MHC polypeptide, the peptide linker comprising a Cys residue, and the first MHC polypeptide being a β2M polypeptide comprising an amino acid substitution introducing the Cys residue); and b) a second polypeptide comprising an HLA-A 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 the following amino acid sequence: TIFF0007762068000034.tif31164, wherein amino acid 84 is Cys and amino acid 236 is Cys; and c) at least one immunomodulatory polypeptide (the first and / or second polypeptides comprise 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), wherein n is an integer between 1 and 10. 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: TIFF0007762068000035.tif11162, in which amino acid 12 is Cys. The at least one immunomodulatory polypeptide can be a cytokine, a 4-1BBL polypeptide, a B7-1 polypeptide, a B7-2 polypeptide, an ICOS-L polypeptide, an OX-40L polypeptide, a CD80 polypeptide, a CD86 polypeptide, a PD-L1 polypeptide, a FasL polypeptide, or a PD-L2 polypeptide. In some examples, as described elsewhere herein, the at least one immunomodulatory polypeptide is a variant form with reduced affinity. As described above, the TMMP also includes i) an Ig Fc polypeptide or a non-Ig scaffold, and ii) a TTP.

[0144] In some examples, a multi-disulfide bond TMMP (e.g., a double-disulfide bond TMMP) of the present disclosure comprises an HLA-A class I heavy chain polypeptide. In some examples, the HLA-A heavy chain polypeptide present in a multi-disulfide bond TMMP (e.g., a double-disulfide bond TMMP) of the present disclosure comprises an amino acid sequence having at least 95%, at least 98%, or at least 99% amino acid sequence identity to the HLA-A*0101, HLA-A*0201, HLA-A*0202, HLA-A*1101, HLA-A*2301, HLA-A*2402, HLA-A*2407, HLA-A*3303, or HLA-A*3401 amino acid sequence shown in Figure 7A, and the HLA-A heavy chain polypeptide comprises a Y84C and an A236C substitution.

[0145] HLA-A*0101(Y84C;A236C) In some examples, the HLA-A heavy chain polypeptide present within a multi-disulfide bond TMMP (e.g., a double-disulfide bond TMMP) of the present disclosure comprises an amino acid sequence having at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following HLA-A*0101 (Y84C, A236C) amino acid sequence: TIFF0007762068000036.tif31164, in the sequence, amino acid 84 is Cys and amino acid 236 is Cys.

[0146] HLA-A*0201(Y84C;A236C) In some examples, the HLA-A heavy chain polypeptide present within a multi-disulfide bond TMMP (e.g., a double-disulfide bond TMMP) of the present disclosure comprises an amino acid sequence having at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following HLA-A*0201 (Y84C, A236C) amino acid sequence: TIFF0007762068000037.tif31164, in the sequence, amino acid 84 is Cys and amino acid 236 is Cys.

[0147] HLA-A*0202(Y84C;A236C) In some examples, the HLA-A heavy chain polypeptide present within a multi-disulfide bond TMMP (e.g., a double-disulfide bond TMMP) of the present disclosure comprises an amino acid sequence having at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following HLA-A*0202 (Y84C, A236C) amino acid sequence: TIFF0007762068000038.tif31164, in which amino acid 84 is Cys and amino acid 236 is Cys.

[0148] HLA-A*1101(Y84C;A236C) In some examples, the HLA-A heavy chain polypeptide present within a multi-disulfide bond TMMP (e.g., a double-disulfide bond TMMP) of the present disclosure comprises an amino acid sequence having at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following HLA-A*1101 (Y84C, A236C) amino acid sequence: TIFF0007762068000039.tif31162, in the sequence, amino acid 84 is Cys and amino acid 236 is Cys.

[0149] HLA-A*2301(Y84C;A236C) In some examples, the HLA-A heavy chain polypeptide present within a multi-disulfide bond TMMP (e.g., a double-disulfide bond TMMP) of the present disclosure comprises an amino acid sequence having at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following HLA-A*2301 (Y84C, A236C) amino acid sequence: TIFF0007762068000040.tif31164, in the sequence, amino acid 84 is Cys and amino acid 236 is Cys.

[0150] HLA-A*2402(Y84C;A236C) In some examples, the HLA-A heavy chain polypeptide present within a multi-disulfide bond TMMP (e.g., a double-disulfide bond TMMP) of the present disclosure comprises an amino acid sequence having at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following HLA-A*2402 (Y84C, A236C) amino acid sequence: TIFF0007762068000041.tif31164, in the sequence, amino acid 84 is Cys and amino acid 236 is Cys.

[0151] HLA-A*2407(Y84C;A236C) In some examples, the HLA-A heavy chain polypeptide present within a multi-disulfide bond TMMP (e.g., a double-disulfide bond TMMP) of the present disclosure comprises an amino acid sequence having at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following HLA-A*2407 (Y84C, A236C) amino acid sequence: TIFF0007762068000042.tif31164, in the sequence, amino acid 84 is Cys and amino acid 236 is Cys.

[0152] HLA-A*3303(Y84C;A236C) In some examples, the HLA-A heavy chain polypeptide present within a multi-disulfide bond TMMP (e.g., a double-disulfide bond TMMP) of the present disclosure comprises an amino acid sequence having at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following HLA-A*3303 (Y84C, A236C) amino acid sequence: TIFF0007762068000043.tif31163, in the sequence, amino acid 84 is Cys and amino acid 236 is Cys.

[0153] HLA-A*3401(Y84C;A236C) In some examples, the HLA-A heavy chain polypeptide present within a multi-disulfide bond TMMP (e.g., a double-disulfide bond TMMP) of the present disclosure comprises an amino acid sequence having at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following HLA-A*3401 (Y84C, A236C) amino acid sequence: TIFF0007762068000044.tif31164, in the sequence, amino acid 84 is Cys and amino acid 236 is Cys.

[0154] HLA-B In some examples, a multiple disulfide bond TMMP (e.g., a double disulfide bond TMMP) of the present disclosure comprises: a) a first polypeptide comprising i) a peptide epitope; and ii) a first MHC polypeptide (the first polypeptide comprises a peptide linker between the peptide epitope and the first MHC polypeptide, the peptide linker comprising a Cys residue, and the first MHC polypeptide being a β2M polypeptide comprising an amino acid substitution introducing the Cys residue); and b) a second polypeptide comprising an HLA-B 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 the following amino acid sequence: TIFF0007762068000045.tif31164, wherein amino acid 84 is Cys and amino acid 236 is Cys; and c) at least one immunomodulatory polypeptide (the first and / or second polypeptides comprise 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), wherein n is an integer between 1 and 10. 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: TIFF0007762068000046.tif11162, in which amino acid 12 is Cys. The at least one immunomodulatory polypeptide can be a cytokine, a 4-1BBL polypeptide, a B7-1 polypeptide, a B7-2 polypeptide, an ICOS-L polypeptide, an OX-40L polypeptide, a CD80 polypeptide, a CD86 polypeptide, a PD-L1 polypeptide, a FasL polypeptide, or a PD-L2 polypeptide. In some examples, as described elsewhere herein, the at least one immunomodulatory polypeptide is a variant form with reduced affinity. As described above, the TMMP also includes i) an Ig Fc polypeptide or a non-Ig scaffold, and ii) a TTP.

[0155] In some examples, a multi-disulfide bond TMMP of the present disclosure comprises an HLA-B class I heavy chain polypeptide. In some examples, an HLA-B heavy chain polypeptide present in a multi-disulfide bond TMMP (e.g., a double-disulfide bond TMMP) of the present disclosure comprises an amino acid sequence having at least 95%, at least 98%, or at least 99% amino acid sequence identity to the HLA-B*0702, HLA-B*0801, HLA-B*1502, HLA-B*3802, HLA-B*4001, HLA-B*4601, or HLA-B*5301 amino acid sequence shown in Figure 8A, and the HLA-B heavy chain polypeptide comprises a Y84C and an A236C substitution.

[0156] HLA-B*0702(Y84C, A236C) In some examples, the HLA-B heavy chain polypeptide present in a multiply disulfide-bonded TMMP (e.g., a double-disulfide-bonded TMMP) of the present disclosure comprises an amino acid sequence having at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following HLA-B*0702 (Y84C, A236C) amino acid sequence: TIFF0007762068000047.tif31164, in the sequence, amino acid 84 is Cys and amino acid 236 is Cys.

[0157] HLA-B*0801(Y84C, A236C) In some examples, the HLA-B heavy chain polypeptide present in a multiply disulfide-bonded TMMP (e.g., a double-disulfide-bonded TMMP) of the present disclosure comprises an amino acid sequence having at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following HLA-B*0801 (Y84C, A236C) amino acid sequence: TIFF0007762068000048.tif31164, in the sequence, amino acid 84 is Cys and amino acid 236 is Cys.

[0158] HLA-B*1502(Y84C, A236C) In some examples, the HLA-B heavy chain polypeptide present in a multiply disulfide-bonded TMMP (e.g., a double-disulfide-bonded TMMP) of the present disclosure comprises an amino acid sequence having at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following HLA-B*1502 (Y84C, A236C) amino acid sequence: TIFF0007762068000049.tif31163, in which amino acid 84 is Cys and amino acid 236 is Cys.

[0159] HLA-B*3802(Y84C, A236C) In some examples, the HLA-B heavy chain polypeptide present in a multiply disulfide-bonded TMMP (e.g., a double-disulfide-bonded TMMP) of the present disclosure comprises an amino acid sequence having at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following HLA-B*3802 (Y84C, A236C) amino acid sequence: TIFF0007762068000050.tif31163, in which amino acid 84 is Cys and amino acid 236 is Cys.

[0160] HLA-B*4001(Y84C, A2346C) In some examples, the HLA-B heavy chain polypeptide present in a multiply disulfide-bonded TMMP (e.g., a double-disulfide-bonded TMMP) of the present disclosure comprises an amino acid sequence having at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following HLA-B*4001 (Y84C, A236C) amino acid sequence: TIFF0007762068000051.tif31164, in which amino acid 84 is Cys and amino acid 236 is Cys.

[0161] HLA-B*4601(Y84C, A236C) In some examples, the HLA-B heavy chain polypeptide present in a multiply disulfide-bonded TMMP (e.g., a double-disulfide-bonded TMMP) of the present disclosure comprises an amino acid sequence having at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following HLA-B*4601 (Y84C, A236C) amino acid sequence: TIFF0007762068000052.tif31164, in the sequence, amino acid 84 is Cys and amino acid 236 is Cys.

[0162] HLA-B*5301(Y84C, A236C) In some examples, the HLA-B heavy chain polypeptide present in a multiply disulfide-bonded TMMP (e.g., a double-disulfide-bonded TMMP) of the present disclosure comprises an amino acid sequence having at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following HLA-B*5301 (Y84C, A236C) amino acid sequence: TIFF0007762068000053.tif31163, in the sequence, amino acid 84 is Cys and amino acid 236 is Cys.

[0163] HLA-C In some examples, a multiple disulfide bond TMMP (e.g., a double disulfide bond TMMP) of the present disclosure comprises: a) a first polypeptide comprising i) a peptide epitope; and ii) a first MHC polypeptide (the first polypeptide comprises a peptide linker between the peptide and the first MHC polypeptide, the peptide linker comprising a Cys residue, and the first MHC polypeptide being a β2M polypeptide comprising an amino acid substitution introducing the Cys residue); and b) a second polypeptide comprising 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 the following amino acid sequence: TIFF0007762068000054.tif31164, wherein amino acid 84 is Cys and amino acid 236 is Cys; and c) at least one immunomodulatory polypeptide (the first and / or second polypeptides comprise 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), wherein n is an integer between 1 and 10. 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: TIFF0007762068000055.tif11162, in which amino acid 12 is Cys. The at least one immunomodulatory polypeptide can be a cytokine, a 4-1BBL polypeptide, a B7-1 polypeptide, a B7-2 polypeptide, an ICOS-L polypeptide, an OX-40L polypeptide, a CD80 polypeptide, a CD86 polypeptide, a PD-L1 polypeptide, a FasL polypeptide, or a PD-L2 polypeptide. In some examples, as described elsewhere herein, the at least one immunomodulatory polypeptide is a variant form with reduced affinity. As described above, the TMMP also includes i) an Ig Fc polypeptide or a non-Ig scaffold, and ii) a TTP.

[0164] In some examples, a multi-disulfide bond TMMP (e.g., a double-disulfide bond TMMP) of the present disclosure comprises an HLA-C class I heavy chain polypeptide. In some examples, the HLA-C heavy chain polypeptide present in a multi-disulfide bond TMMP (e.g., a double-disulfide bond TMMP) of the present disclosure comprises an amino acid sequence having at least 95%, at least 98%, or at least 99% amino acid sequence identity to the HLA-C*0102, HLA-C*0303, HLA-C*0304, HLA-C*0401, HLA-C*0602, HLA-C*0701, HLA-C*0702, HLA-C*0801, or HLA-C*1502 amino acid sequence shown in Figure 9A, and the HLA-C heavy chain polypeptide comprises a Y84C and an A236C substitution.

[0165] HLA-C*01:02(Y84C;A236C) In some examples, the HLA-C heavy chain polypeptide present within a multi-disulfide bond TMMP (e.g., a double-disulfide bond TMMP) of the present disclosure comprises an amino acid sequence having at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following HLA-C*01:02 (Y84C, A236C) amino acid sequence: TIFF0007762068000056.tif31164, in the sequence, amino acid 84 is Cys and amino acid 236 is Cys.

[0166] HLA-C*0303(Y84C;A236C) In some examples, the HLA-C heavy chain polypeptide present within a multi-disulfide bond TMMP (e.g., a double-disulfide bond TMMP) of the present disclosure comprises an amino acid sequence having at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following HLA-C*03:03(Y84C, A236C) amino acid sequence: TIFF0007762068000057.tif31164, in which amino acid 84 is Cys and amino acid 236 is Cys.

[0167] HLA-C*0304(Y84C;A236C) In some examples, the HLA-C heavy chain polypeptide present within a multi-disulfide bond TMMP (e.g., a double-disulfide bond TMMP) of the present disclosure comprises an amino acid sequence having at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following HLA-C*03:04 (Y84C, A236C) amino acid sequence: TIFF0007762068000058.tif31164, in the sequence, amino acid 84 is Cys and amino acid 236 is Cys.

[0168] HLA-C*0401(Y84C;A236C) In some examples, the HLA-C heavy chain polypeptide present within a multi-disulfide bond TMMP (e.g., a double-disulfide bond TMMP) of the present disclosure comprises an amino acid sequence having at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following HLA-C*04:01 (Y84C, A236C) amino acid sequence: TIFF0007762068000059.tif31165, in the sequence, amino acid 84 is Cys and amino acid 236 is Cys.

[0169] HLA-C*0602(Y84C;A236C) In some examples, the HLA-C heavy chain polypeptide present within a multi-disulfide bond TMMP (e.g., a double-disulfide bond TMMP) of the present disclosure comprises an amino acid sequence having at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following HLA-C*06:02(Y84C, A236C) amino acid sequence: TIFF0007762068000060.tif31163, in the sequence, amino acid 84 is Cys and amino acid 236 is Cys.

[0170] HLA-C*0701(Y84C;A236C) In some examples, the HLA-C heavy chain polypeptide present within a multi-disulfide bond TMMP (e.g., a double-disulfide bond TMMP) of the present disclosure comprises an amino acid sequence having at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following HLA-C*07:01 (Y84C, A236C) amino acid sequence: TIFF0007762068000061.tif31162, in the sequence, amino acid 84 is Cys and amino acid 236 is Cys.

[0171] HLA-C*0702(Y84C;A236C) In some examples, the HLA-C heavy chain polypeptide present within a multi-disulfide bond TMMP (e.g., a double-disulfide bond TMMP) of the present disclosure comprises an amino acid sequence having at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following HLA-C*07:02(Y84C, A236C) amino acid sequence: TIFF0007762068000062.tif31164, in the sequence, amino acid 84 is Cys and amino acid 236 is Cys.

[0172] HLA-C*0801(Y84C;A236C) In some examples, the HLA-C heavy chain polypeptide present within a multi-disulfide bond TMMP (e.g., a double-disulfide bond TMMP) of the present disclosure comprises an amino acid sequence having at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following HLA-C*08:01 (Y84C, A236C) amino acid sequence: TIFF0007762068000063.tif31164, in which amino acid 84 is Cys and amino acid 236 is Cys.

[0173] HLA-C*1502(Y84C;A236C) In some examples, the HLA-C heavy chain polypeptide present within a multi-disulfide bond TMMP (e.g., a double-disulfide bond TMMP) of the present disclosure comprises an amino acid sequence having at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following HLA-C*15:02(Y84C, A236C) amino acid sequence: TIFF0007762068000064.tif31163, in the sequence, amino acid 84 is Cys and amino acid 236 is Cys.

[0174] Scaffold Polypeptide The TMMPs of the present disclosure include Fc polypeptides or non-antibody scaffold polypeptides.

[0175] Suitable scaffold polypeptides include antibody-based scaffold polypeptides and non-antibody-based scaffolds, such as albumin, XTEN (extended recombinant) polypeptides, transferrin, Fc receptor polypeptides, elastin-like polypeptides (see, e.g., Hassouneh et al. (2012) Methods Enzymol. 502:215, e.g., polypeptides comprising pentapeptide repeat units of (Val-Pro-Gly-X- Gly, SEQ ID NO: 174), where X is any amino acid except proline), albumin-binding polypeptides, silk-like polypeptides (see, e.g., Valluzzi et al. (2002) Philos Trans R Soc Lond B Biol Sci. 357:165), and silk-elastin-like polypeptides (SELPs; see, e.g., Megeed et al. (2002) Adv Drug Deliv Rev. 54:1075). 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.

[0176] In some cases, a suitable scaffold polypeptide is a polypeptide that extends half-life. Thus, in some cases, a suitable scaffold polypeptide increases the in vivo half-life (e.g., serum half-life) of a TMMP compared to a control TMMP that lacks the scaffold polypeptide. For example, in some cases, a scaffold polypeptide increases the in vivo half-life (e.g., serum half-life) of a TMMP by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 50%, at least about 2-fold, at least about 2.5-fold, at least about 5-fold, at least about 10-fold, at least about 25-fold, at least about 50-fold, at least about 100-fold, or more than 100-fold compared to a control TMMP that lacks the scaffold polypeptide. For example, in some instances, the Fc polypeptide increases the in vivo half-life (e.g., serum half-life) of the TMMP by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 50%, at least about 2-fold, at least about 2.5-fold, at least about 5-fold, at least about 10-fold, at least about 25-fold, at least about 50-fold, at least about 100-fold, or more than 100-fold, compared to a control TMMP lacking the Fc polypeptide.

[0177] Fc polypeptide In some examples, the first and / or second polypeptide chains of a TMMP of the present disclosure comprise an Fc polypeptide. The Fc polypeptide of a TMMP of the present disclosure can be a human IgG1 Fc, a human IgG2 Fc, a human IgG3 Fc, a 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 shown 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 shown in Figure 3A. 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 shown in FIG. 3A and comprises a substitution of N77, e.g., the Fc polypeptide comprises 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 shown 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 shown 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 shown 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 shown 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 shown 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 shown 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 shown 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 shown in Figure 3C.

[0178] 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 3C.

[0179] In some examples, the IgG4 Fc polypeptide comprises the following amino acid sequence: TIFF0007762068000065.tif24163.

[0180] In some examples, the Fc polypeptide present in the TMMP comprises the amino acid sequence shown in Figure 3A (human IgG1 Fc). In some examples, the Fc polypeptide present in the TMMP comprises the amino acid sequence shown in Figure 3A (human IgG1 Fc), except for the substitution of N297 (N77 in the amino acid sequence shown in Figure 3A) with an amino acid other than asparagine. In some examples, the Fc polypeptide present in the TMMP comprises the amino acid sequence shown in Figure 3C (human IgG1 Fc with an N297A substitution, which is N77 in the amino acid sequence shown in Figure 3A). In some examples, the Fc polypeptide present in the TMMP comprises 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. In some examples, the Fc polypeptide present in the TMMP comprises the amino acid sequence shown in Figure 3A (human IgG1 Fc), except for the substitution of L235 (L15 in the amino acid sequence shown in Figure 3A) with an amino acid other than leucine.

[0181] In some examples, the Fc polypeptide present in the TMMP comprises the amino acid sequence shown in Figure 3E. In some examples, the Fc polypeptide present in the TMMP comprises the amino acid sequence shown in Figure 3F. In some examples, the Fc polypeptide present in the TMMP comprises the amino acid sequence shown in Figure 5G (human IgG1 Fc comprising 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 the TMMP comprises the amino acid sequence shown in Figure 3A (human IgG1 Fc), except for the substitution of an amino acid other than proline at P331 (P111 of the amino acid sequence shown in Figure 3A), and in some examples, the substitution is a P331S substitution. In some examples, the Fc polypeptide present in the TMMP comprises the amino acid sequence shown in Figure 3A (human IgG1 Fc), except for the substitution of an amino acid 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 the TMMP comprises the amino acid sequence shown in FIG. 3A (human IgG1 Fc), except for substitutions of amino acids other than leucine at L234 and L235 (L14 and L15 in the amino acid sequence shown in FIG. 3A ) and substitutions of amino acids other than proline at P331 (P111 in the amino acid sequence shown in FIG. 3A ). In some examples, the Fc polypeptide present in the TMMP comprises the amino acid sequence shown in FIG. 3E (human IgG1 Fc comprising L234F, L235E, and P331S substitutions (corresponding to amino acids 14, 15, and 111 in the amino acid sequence shown in FIG. 3E )). In some examples, the Fc polypeptide present in the TMMP is an IgG1 Fc polypeptide comprising L234A and L235A substitutions (substitutions of Ala at L14 and L15 in the amino acid sequence shown in FIG. 3A ), as shown in FIG. 3G.

[0182] Ig Fc heavy chain CH2 and CH3 domains, such as those shown in Figures 3A-3G, can also function as dimerization or multimerization sequences (e.g., when the TMMP comprises two or more heterodimers). If asymmetric pairing between two Ig Fc polypeptides is desired, the Ig Fc polypeptides may incorporate knob-in-hole modifications, for example, in the CH3 domain. Such knob-in-hole pairs include the T366Y and Y407T mutant pair at the CH3 domain interface of IgG1, or the corresponding residues in another Ig Fc ("T366" corresponds to amino acid 146 of the IgG1 Fc amino acid sequence shown in Figure 3A, and "Y407" corresponds to amino acid 187 of the IgG1 Fc amino acid sequence shown in Figure 3A). See Ridgway et al., Protein Engineering 9:7, 617-621 (1996) (substitutions are indicated by the EU numbering scheme of Kabat et al. (1991)). Another knob-in-hole pairing involves the formation of a knob by a T366W substitution and the formation of a hole by the triple substitutions T366S, L368A, and Y407V on the complementary Fc polypeptide ("T366" corresponds to amino acid 146 of the IgG1 Fc amino acid sequence shown in Figure 3A, "L368" corresponds to amino acid 148 of the IgG1 Fc amino acid sequence shown in Figure 3A, and "Y407" corresponds to amino acid 187 of the IgG1 Fc amino acid sequence shown in Figure 3A). See Xu et al. mAbs 7:1, 231-242 (2015). For example, in some instances, a first TMMP heterodimer may comprise an IgG1 Fc polypeptide comprising a T366Y substitution (e.g., a T146Y substitution based on the IgG1 Fc amino acid sequence shown in Figure 3A), and a second TMMP heterodimer may comprise an IgG1 Fc polypeptide comprising a Y407T substitution (e.g., a Y187T substitution based on the IgG1 Fc amino acid sequence shown in Figure 3A).As another example, in some instances, a first TMMP heterodimer may comprise an IgG1 Fc polypeptide comprising a T366W substitution (e.g., a T146W substitution based on the IgG1 Fc amino acid sequence shown in Figure 3A), and a second TMMP heterodimer may comprise an IgG1 Fc polypeptide comprising a T366S substitution, an L368A substitution, and a Y407V substitution (e.g., a T146S substitution, an L148A substitution, and a Y187V substitution based on the IgG1 Fc amino acid sequence shown in Figure 3A). The Fc polypeptides, with or without knob-into-hole modifications, can be stabilized by the formation of disulfide bonds (e.g., hinge region disulfide bonds) between the Fc polypeptides.

[0183] Tumor targeting polypeptide (TTP) As described above, the TMMPs of the present disclosure include, as the first and / or second polypeptide, a tumor targeting polypeptide (TTP), i.e., a polypeptide specific for a cancer-associated epitope. A "cancer-associated" epitope is an epitope present in a cancer-associated antigen. In some examples, the TTP is an antibody. In some examples, the TTP is a single-chain T-cell receptor (scTCR).

[0184] target In some examples, the TTP present in the TMMP of the present disclosure targets a cancer-associated antigen. In some examples, the target of the TTP is a peptide / HLA (pHLA) complex on the surface of a cancer cell, and the peptide can be a cancer-associated peptide (e.g., a peptide fragment of a cancer-associated antigen).

[0185] Cancer-associated antigens Cancer-associated antigens that can be targeted by tumor-targeting polypeptides present in the TMPPs of the present disclosure include, for example, NY-ESO (New York Esophageal Squamous Cell Carcinoma 1), MART-1 (Melanoma antigen recognized by T cells 1, also known as Melan-A), HPV (human papillomavirus) E6, BCMA (B-cell maturation antigen), CD123, CD133, CD171, CD19, CD20, CD22, CD30, CD33, CEA (carcinoembryonic antigen), EGFR (epidermal growth factor receptor), EGFRvIII (epidermal growth factor receptor variant III), EpCAM (epithelial cell adhesion molecule), EphA2 (ephrin type A receptor 2), disialoganglioside These include GD2, GPC3 (glypican-3), HER2, IL13Ralpha2 (interleukin-13 receptor subunit alpha-2), LeY (difucosylated type 2 blood-associated antigen), MAGE-A3 (melanoma-associated antigen 3), melanoma glycoprotein, mesothelin, MUC1 (mucin 1), MUC16 (mucin-16), myelin, NKG2D (natural killer group 2D), ligand, PSMA (prostate-specific membrane antigen), and ROR1 (type I receptor tyrosine kinase-like orphan receptor).

[0186] Cancer-associated antigens that may be targeted by the TTPs present in the TMPPs of the present disclosure include 17-1A antigen, alpha-fetoprotein (AFP), alpha-actinin-4, A3, antigen specific for the A33 antibody, ART-4, B7, Ba 733, BAGE, bcl-2, bcl-6, BCMA, BrE3-antigen, CA125, CAMEL, CAP-1, carbonic anhydrase IX (CAIX), CASP-8 / m, CCL19, CCL21, CD1, CD1a, CD2, CD3, CD4, CD5, CD8, CD11A, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD29, CD30, CD32b, CD33, C D37, CD38, CD40, CD40L, CD44, CD45, CD46, CD52, CD54, CD55, CD59, CD64, CD66a-e, CD67, CD70, CD70L, CD74, CD79a, CD7 9b, CD80, CD83, CD95, CD123, CD126, CD132, CD133, CD138, CD147, CD154, CD171, CDC27, CDK-4 / m, CDKN2A, CEA, CEACAM5 , CEACAM6, claudins (e.g., claudin-1, claudin-10, claudin-18 (e.g., claudin-18, isoform 2)), complement factors (such as C3, C3a, C3b, C5a, and C5), colon-specific antigen-p (CSAp), c-Met, CTLA-4, CXCR4, CXCR7, CXCL12, DAM, Dickkopf-related protein (DKK), ED-B fibronectin, epidermal growth factor receptor activator (EGFR), and receptor (EGFR), EGFRvIII, EGP-1 (TROP-2), EGP-2, ELF2-M, Ep-CAM, EphA2, EphA3, fibroblast activation protein (FAP), fibroblast growth factor (FGF), Flt-1, Flt-3, folate binding protein, folate receptor, G250 antigen, gangliosides (such as GC2, GD3, and GM2), GAGE, GD2, gp100, GPC3, GRO-13, HLA-DR, and HM1.24, human chorionic gonadotropin (HCG) and its subunits, HER2, HER3, HMGB-1, hypoxia-inducible factor (HIF-1), HIF-1a, HSP70-2M, HST-2, Ia, IFN-gamma, IFN-alpha, IFN-beta, IFN-X, IL-4R, IL-6R, IL-13R, IL13Ralpha2, IL-15R, IL-17R, IL-18R, IL-2, IL-6, IL-8, IL-12, IL-15, IL-17, IL-18, IL-23, IL-25, ILGF, ILGF-1R, insulin-like growth factor-1 (IGF-1), IGF-1R, integrin αVβ3, integrin α5β1, KC4-antigen, killer cell immunoglobulin-like receptor (KIR), Kras, KS-1-antigen, KS1-4, LDR / FUT, Le. ガンマ , macrophage migration inhibitory factor (MIF), MAGE, MAGE-3, MART-1, MART-2, mCRP, MCP-1, melanoma glycoprotein, mesothelin, MIP-1A, MIP-1B, MIF, mucins (such as MUC1, MUC2, MUC3, MUC4, MUC5ac, MUC13, MUC16, MUM-1 / 2, and MUM-3), NCA66, NCA95, NCA90, nectin-4, NY-ESO-1, PAM4 antigen, pancreatic cancer mucin, PD-1, PD-L1, PD-1 receptor, placenta These include, but are not limited to, growth factors, p53, PLAGL2, prostatic acid phosphatase, PSA, PRAME, PSMA, P1GF, RSS, RANTES, SAGE, 5100, survivin, survivin-2B, T101, TAC, TAG-72, tenascin, Thomson-Friedenreich antigen, Tn antigen, TNF-alpha, tumor necrosis antigen, TRAG-3, TRAIL receptor, vascular endothelial growth factor (VEGF), VEGF receptor (VEGFR), and WT-1.

[0187] In some examples, the cancer-associated antigen is an antigen associated with a blood cancer. Examples of such antigens include, but are not limited to, BCMA, C5, CD19, CD20, CD22, CD25, CD30, CD33, CD38, CD40, CD45, CD52, CD56, CD66, CD74, CD79a, CD79b, CD80, CD138, CTLA-4, CXCR4, DKK, EphA3, GM2, HLA-DR beta, integrin αVβ3, IGF-R1, IL6, KIR, PD-1, PD-L1, TRAILR1, TRAILR2, transferrin receptor, and VEGF. In some examples, the cancer-associated antigen is an antigen expressed by malignant B cells, such as CD19, CD20, CD22, CD25, CD38, CD40, CD45, CD74, CD80, CTLA-4, IGF-R1, IL6, PD-1, TRAILR2, or VEGF.

[0188] In some instances, the cancer-associated antigen is an antigen associated with a solid tumor. Examples of such antigens include CAIX, cadherin, CEA, c-MET, CTLA-4, EGFR family members, EpCAM, EphA3, FAP, folate-binding protein, FR-alpha, gangliosides (such as GC2, GD3, and GM2), HER2, HER3, IGF-1R, integrin αVβ3, integrin α5β1, Le ガンマ , Liv1, mesothelin, mucin, NaPi2b, PD-1, PD-L1, PD-1 receptor, pgA33, PSMA, RANKL, ROR1, TAG-72, tenascin, TRAILR1, TRAILR2, VEGF, VEGFR, and others listed above.

[0189] Peptide / HLA complexes In some cases, the target of the TTP is a peptide / HLA (pHLA) complex on the surface of a cancer cell, and the peptide can be a cancer-associated peptide (e.g., a peptide fragment of a cancer-associated antigen). Cancer-associated peptides are known in the art. In some cases, the cancer-associated peptide binds to an HLA complex containing an HLA-A*0201 heavy chain and a β2M polypeptide.

[0190] In some examples, epitopes present in pHLA on the surface of cancer cells bind to HLA complexes containing HLA heavy chains such as HLA-A*0101, A*0201, A*0301, A*1101, A*2301, A*2402, A*2407, A*3303, and / or A*3401. In some examples, epitopes present in pHLA on the surface of cancer cells bind to HLA complexes containing HLA heavy chains such as HLA-B*0702, B*0801, B*1502, B*3802, B*4001, B*4601, and / or B*5301. In some examples, epitopes present in pHLA on the surface of cancer cells bind to HLA complexes including HLA heavy chains such as C*0102, C*0303, C*0304, C*0401, C*0602, C*0701, C*702, C*0801, and / or C*1502.

[0191] In some examples, the epitope is a cancer-associated epitope of any one of the following cancer-associated antigens: MUC1 polypeptide, LMP2 polypeptide, epidermal growth factor receptor (EGFR) vIII polypeptide, HER-2 / neu polypeptide, melanoma antigen family A, 3 (MAGE) A3) polypeptide, p53 polypeptide, mutant p53 polypeptide, NY-ESO-1 polypeptide, folate hydrolase (prostate-specific membrane antigen; PSMA) polypeptide, carcinoembryonic antigen (CEA) polypeptide, claudin polypeptide (e.g., claudin-1, claudin-10, claudin-18 (e.g., claudin-18, isoform 2)), nectin-4 polypeptide, melanoma antigen recognized by T cells (melanA / MART1) polypeptide, Ras polypeptide, gp100 polypeptide, proteinase 3 (PR1) polypeptide, bcr-abl polypeptide, tyrosinase polypeptide, survivin polypeptide, prostate-specific antigen (PSA) polypeptide, hTERT polypeptide, sarcoma metastasis breakpoint polypeptide, synovial sarcoma X (SSX) breakpoint polypeptide, EphA2 polypeptide, acid phosphatase, prostate (PAP) polypeptide, melanoma inhibitor of apoptosis (ML-IAP), epithelial cell adhesion molecule (EpCAM) polypeptide, ERG (TMPRSS2) ETS fusion) polypeptide, NA17 polypeptide, paired box-3 (PAX3) polypeptide, anaplastic lymphoma kinase (ALK) polypeptide, androgen receptor polypeptide, cyclin B1 polypeptide, N-myc proto-oncogene (MYCN) polypeptide, Ras homolog gene family member C (RhoC) polypeptide, tyrosinase-related protein-2 (TRP-2) polypeptide, mesothelin polypeptide, prostate stem cell antigen (PSCA) polypeptide, melanoma-associated antigen-1 (MAGE A1) polypeptide, cytochrome P450 1B1 (CYP1B1) polypeptide, placenta-specific protein 1 (PLAC1) polypeptide, BORIS polypeptide (also known as CCCTC-binding factor or CTCF), ETV6-AML polypeptide, breast cancer antigen NY-BR-1 polypeptide (also known as ankyrin repeat domain-containing protein 30A), regulator of G-protein signaling (RGS5) polypeptide, squamous cell carcinoma cell antigen recognized by T cells (SART3) polypeptide, carbonic anhydrase IX polypeptide, paired box-5 (PAX5) polypeptide, OY-TES1 (testis antigen, also known as acrosin-binding protein) polypeptide, sperm protein 17 polypeptide, lymphoid cell-specific protein-tyrosine kinase (LCK) polypeptide, high-molecular-weight melanoma-associated antigen (HMW-MAA), A-kinase anchor protein-4 (AKAP-4), synovial sarcoma X breakpoint 2 (SS) X2) polypeptide, X antigen family member 1 (XAGE1) polypeptide, B7 homolog 3 (B7H3, also known as CD276) polypeptide, legumain polypeptide (LGMN1, also known as asparaginyl endopeptidase), tyrosine kinase-2 with Ig and EGF homology domains (Tie-2, also known as angiopoietin-1 receptor) polypeptide, P antigen family member 4 (PAGE4) polypeptide, vascular endothelial growth factor receptor 2 (VEGF2) polypeptide, MAD-CT-1 polypeptide, fibroblast activation protein (FAP) polypeptide, platelet-derived growth factor receptor beta (PDGFβ) polypeptide, MAD-CT-2 polypeptide, Fos-related antigen-1 (FOSL) polypeptide, human papilloma virus (HPV) antigen, alpha-fetoprotein (AFP) antigen, and Wilms' tumor-1 (WT1) antigen.

[0192] For example, in some instances, the TTP present in the TMMP of the present disclosure binds to: a) a WT-1 peptide bound to an HLA complex comprising an HLA heavy chain (e.g., an HLA-A*0201 heavy chain or an HLA-A*2402 heavy chain) and a β2M polypeptide; b) an HPV peptide bound to an HLA complex comprising a class I HLA heavy chain and a β2M polypeptide; c) a mesothelin peptide bound to an HLA complex comprising a class I HLA heavy chain and a β2M polypeptide; d) a Her2 peptide bound to an HLA complex comprising a class I HLA heavy chain and a β2M polypeptide; or e) a BCMA peptide bound to an HLA complex comprising a class I HLA heavy chain and a β2M polypeptide.

[0193] In some examples, the cancer-associated peptide is a peptide of a mesothelin polypeptide having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following mesothelin amino acid sequence: For example, the mesothelin peptide present in the pHLA complex can be i) KLLGPHVEGL (SEQ ID NO: 526), ​​ii) AFYPGYLCSL (SEQ ID NO: 177) capable of binding to HLA-A*2402 / β2M, iii) VLPLTVAEV (SEQ ID NO: 178), iv) ELAVALAQK (SEQ ID NO: 179), v) ALQGGGPPY (SEQ ID NO: 180), vi) FYPGYLCSL (SEQ ID NO: 181), vii) LYPKARLAF (SEQ ID NO: 182), viii) LLFLLFSLGWVGPSR (SEQ ID NO: 183), ix) VNKGHEMSPQAPRRP (SEQ ID NO: 184), x) FMKLRTDAVLPLTVA (SEQ ID NO: 185), or xi) DAALLATQMD (SEQ ID NO: 186).

[0194] In some examples, the cancer-associated peptide is a peptide of a Her2 polypeptide having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following Her2 (receptor tyrosine-protein kinase erbB2) amino acid sequence: TIFF0007762068000067.tif118163.

[0195] In some examples, the cancer associated peptide is a peptide of a BCMA polypeptide having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following BCMA amino acid sequence: TIFF0007762068000068.tif24153.

[0196] In some examples, the cancer-associated peptide is a peptide of a WT-1 polypeptide having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following WT-1 amino acid sequence: TIFF0007762068000069.tif58162.

[0197] Non-limiting examples of WT-1 peptides include RMFPNAPYL (SEQ ID NO: 190), CMTWNQMN (SEQ ID NO: 191), CYTWNQMNL (SEQ ID NO: 192), CMTWNQMNLGATLKG (SEQ ID NO: 193), WNQMNLGATLKGVAA (SEQ ID NO: 194), CMTWNYMNLGATLKG (SEQ ID NO: 195), WNYMNLGATLKGVAA (SEQ ID NO: 196), MTWNQMNLGATLKGV (SEQ ID NO: 197), TWNQMNLGATLKGVA (SEQ ID NO: 198), CMTW NLMNLGATLKG (SEQ ID NO: 199), MTWNLMNLGATLKGV (SEQ ID NO: 200), TWNLMNLGATLKGVA (SEQ ID NO: 201), WNLMNLGATLKGVAA (SEQ ID NO: 202), MNLGATLK (SEQ ID NO: 203), MTWNYMNLGATLKGV (SEQ ID NO: 204), TWNYMNLGATLKGVA (SEQ ID NO: 205), CMTWNQMNLGATLKGVA (SEQ ID NO: 206), CMTWNLMNLGATLKGVA (SEQ ID NO: 207), CMTWNYMNLGATL KGVA (SEQ ID NO: 208), GYLRNPTAC (SEQ ID NO: 209), GALRNPTAL (SEQ ID NO: 210), YALRNPTAC (SEQ ID NO: 211), GLLRNPTAC (SEQ ID NO: 212), RYRPHPGAL (SEQ ID NO: 213), YQRPHPGAL (SEQ ID NO: 214), RLRPHPGAL (SEQ ID NO: 215), RIRPHPGAL (SEQ ID NO: 216), QFPNHSFKHEDPMGQ (SEQ ID NO: 217), HSFKHEDPY (SEQ ID NO: 218), QFPNHSFKHEDPM (SEQ ID NO: 219) 9), QFPNHSFKHEDPY (SEQ ID NO: 220), KRPFMCAYPGCNK (SEQ ID NO: 221), KRPFMCAYPGCYK (SEQ ID NO: 222), FMCAYPGCY (SEQ ID NO: 223), FMCAYPGCK (SEQ ID NO: 224), KRPFMCAYPGCNKRY (SEQ ID NO: 225), SEKRPFMCAYPGCNK (SEQ ID NO: 226), KRPFMCAYPGCYKRY (SEQ ID NO: 227), NLMNLGATL (SEQ ID NO: 228), and NYMNLGATL (SEQ ID NO: 229).

[0198] In some examples, the cancer-associated peptide is a peptide of a human papillomavirus (HPV) polypeptide that has at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity with the HPV polypeptide.The HPV peptide can be a peptide of an HPV E6 polypeptide or an HPV E7 polypeptide.The HPV epitope can be, for example, an epitope of any of various HPV genotypes, including HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59, HPV68, HPV73, or HPV82. Non-limiting examples of HPV peptides include E6 18-26 (KLPQLCTEL; SEQ ID NO:230), E6 26-34 (LQTTIHDII; SEQ ID NO:231), E6 49-57 (VYDFAFRDL; SEQ ID NO:232), E6 52-60 (FAFRDLCIV; SEQ ID NO:233), E6 75-83 (KFYSKISEY; SEQ ID NO:234), E6 80-88 (ISEYRHYCY; SEQ ID NO:235), E7 7-15 (TLHEYMLDL; SEQ ID NO:236), E7 11-19 (YMLDLQPET; SEQ ID NO:237), E7 44-52 (QAEPDRAHY; SEQ ID NO:238), E7 49-57 (RAHYNIVTF (SEQ ID NO:239), E7 61-69 (CDSTLRLCV; SEQ ID NO: 240), and E7 67-76 (LCVQSTHVDI; SEQ ID NO: 241), E7 82-90 (LLMGTLGIV; SEQ ID NO: 242), E7 86-93 (TLGIVCPI; SEQ ID NO: 243) and E7 92-93 (LLMGTLGIVCPI; SEQ ID NO: 244).

[0199] In some examples, the cancer-associated peptide is a peptide of a claudin polypeptide having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following claudin-18 (isoform 2) (CLDN18.2) amino acid sequence: TIFF0007762068000070.tif31164. In some cases, the cancer-associated peptide is a peptide of a claudin polypeptide having the amino acid sequence TEDEVQSYPSKHDYV (SEQ ID NO: 246) (and having a length of about 15 amino acids) or EVQSYPSKHDYV (SEQ ID NO: 247) (and having a length of about 12 amino acids).

[0200] antibody As described above, in some examples, the TTP present in the TMMP of the present disclosure is an antibody. In some examples, the TTP is an antibody specific to a cancer-associated antigen. In some examples, the TTP is an antibody specific to a peptide / HLA complex on the surface of a cancer cell, and the peptide can be a cancer-associated peptide (e.g., a peptide of a cancer-associated antigen).

[0201] Non-limiting examples of cancer-associated antigen-targeting antibodies that can be included in the TMMPs of the present disclosure include abituzumab (anti-CD51), LL1 (anti-CD74), LL2 or RFB4 (anti-CD22), veltuzumab (hA20, anti-CD20), rituximab (anti-CD20), obinutuzumab (GA101, anti-CD20), daratuzumab (anti-CD38), lambrolizumab (anti-PD-1 receptor), nivolumab (anti-PD-1 receptor), ipilimumab (anti-CTLA-4), RS7 (anti-TROP-2), PAM4 or KC4 (both anti-mucins), MN-14 (anti-CEA), MN-15 or MN-3 (anti-CEACAM6), Mu-9 (anti-colon specific antigen-p), Immunoglobulin G (IMG), and IFN-γ (anti-IL-1). 31 (anti-alpha-fetoprotein), R1 (anti-IGF-1R), A19 (anti-CD19), TAG-72 (e.g., CC49), Tn, J591 or HuJ591 (anti-PSMA), AB-PG1-XG1-026 (anti-PSMA dimer), D2 / B (anti-PSMA), G250 (anti-carbonic anhydrase IX), L243 (anti-HLA-DR), alemtuzumab (anti-CD52), oportuzumab (anti-EpCAM), bevacizumab (anti VEGF), cetuximab (anti-EGFR), gemtuzumab (anti-CD33), ibritumomab tiuxetan (anti-CD20), panitumumab (anti-EGFR), tositumomab (anti-CD20), PAM4 (also known as clivatuzumab; anti-mucin), trastuzumab (anti-HER2), pertuzumab (anti-HER2), polatuzumab (anti-CD79b), and anetuzumab (anti-mesothelin).

[0202] In some examples, the tumor targeting polypeptide is an antibody. In some examples, the tumor targeting polypeptide is a single-chain antibody. In some examples, the tumor targeting polypeptide is an scFv. In some examples, the tumor targeting polypeptide is a nanobody (also referred to as a single-domain antibody (sdAb)). In some examples, the tumor targeting polypeptide is a heavy-chain nanobody. In some examples, the tumor targeting polypeptide is a light-chain nanobody.

[0203] The VH and VL amino acid sequences of various tumor antigen-binding antibodies are known in the art, as are the light and heavy chain CDRs of such antibodies. See, for example, Ling et al. (2018) Frontiers Immunol. 9:469, WO2005 / 012493, US2019 / 0119375, and US2013 / 0066055. The following are non-limiting examples of tumor antigen-binding antibodies:

[0204] anti-Her2 In some examples, the anti-Her2 antibody comprises: a) a light chain comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: TIFF0007762068000071.tif24163, and b) a heavy chain comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: Includes TIFF0007762068000072.tif51164.

[0205] In some examples, an anti-Her2 antibody comprises a light chain variable region (VL) present in the light chain amino acid sequence provided above and a heavy chain variable region (VH) present in the heavy chain amino acid sequence provided above. For example, an anti-Her2 antibody may comprise: a) a VL comprising the following 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: DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO: 250); and b) a VL comprising the following 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: DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO: 250). ) A VH comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS (SEQ ID NO: 251). In some examples, the anti-Her2 antibody comprises, in order from N-terminus to C-terminus, a) a VH comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLV a) a TVSS (SEQ ID NO: 252); b) a linker; and c) a VL comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO: 253).Suitable linkers are described elsewhere herein and include, for example, (GGGGS)n (SEQ ID NO: 254), where n is an integer between 1 and 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10).

[0206] In some examples, the anti-Her2 antibody comprises a VL CDR1, a VL CDR2, and a VL CDR3 present in the light chain amino acid sequence provided above, and a VH CDR1, a CDR2, and a CDR3 present in the heavy chain amino acid sequence provided above. H and V L CDRs are as defined by Kabat (see, e.g., Table 2 above, and Kabat 1991). H and V L The CDRs are as defined by Chothia (see, eg, Table 2 above, and Chothia 1987).

[0207] For example, an anti-Her2 antibody may comprise a VL CDR1 having the amino acid sequence RASQDVNTAVA (SEQ ID NO: 255), a VL CDR2 having the amino acid sequence SASFLY (SEQ ID NO: 256), a VL CDR3 having the amino acid sequence QQHYTTPP (SEQ ID NO: 257), a VH CDR1 having the amino acid sequence GFNIKDTY (SEQ ID NO: 258), a VH CDR2 having the amino acid sequence IYPTNGYT (SEQ ID NO: 259), and a VH CDR3 having the amino acid sequence SRWGGDGFYAMDY (SEQ ID NO: 260).

[0208] In some examples, the anti-Her2 antibody is an scFv antibody. For example, the anti-Her2 scFv can include 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: EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO: 261).

[0209] As another example, in some instances, the anti-Her2 antibody comprises: a) a light chain variable region (VL) comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: DIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYIYPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 262), and b) a heavy chain variable region (VH) comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVRQAPGKGLEWVADVNPNSGGSIYNQRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQGTLV TVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPC PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 263).

[0210] In some examples, the anti-Her2 antibody comprises a VL present in the light chain amino acid sequence provided above and a VH present in the heavy chain amino acid sequence provided above. For example, the anti-Her2 antibody may comprise: a) a VL comprising the following 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:DIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYIYPYTFGQGTKVEIK (SEQ ID NO: 264); b) VH comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVRQAPGKGLEWVADVNPNSGGSIYNQRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQGTLVTVSS (SEQ ID NO: 265).

[0211] In some examples, the anti-Her2 antibody comprises a VL CDR1, a VL CDR2, and a VL CDR3 present in the light chain amino acid sequence provided above, and a VH CDR1, a CDR2, and a CDR3 present in the heavy chain amino acid sequence provided above. H and V L CDRs are as defined by Kabat (see, e.g., Table 2 above, and Kabat 1991). H and V L The CDRs are as defined by Chothia (see, eg, Table 2 above, and Chothia 1987).

[0212] For example, an anti-HER2 antibody may comprise a VL CDR1 having the amino acid sequence KASQDVSIGVA (SEQ ID NO: 266), a VL CDR2 having the amino acid sequence SASYRY (SEQ ID NO: 267), a VL CDR3 having the amino acid sequence QQYYIYPY (SEQ ID NO: 268), a VH CDR1 having the amino acid sequence GFTFTDYTMD (SEQ ID NO: 269), a VH CDR2 having the amino acid sequence ADVNPNSGGSIYNQRFKG (SEQ ID NO: 270), and a VH CDR3 having the amino acid sequence ARNLGPSFYFDY (SEQ ID NO: 271).

[0213] In some examples, the anti-Her2 antibody is an scFv. For example, in some examples, the anti-Her2 scFv comprises 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: EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO: 272).

[0214] anti-CD19 Anti-CD19 antibodies are known in the art, and the VH and VL, or VH and VL CDRs, of any anti-CD19 antibody can be used in the TMMPs of the present disclosure. See, e.g., WO2005 / 012493.

[0215] In some examples, the anti-CD19 antibody comprises a VL CDR1 comprising the amino acid sequence KASQSVDYDGDSYLN (SEQ ID NO: 273), a VL CDR2 comprising the amino acid sequence DASNLVS (SEQ ID NO: 274), and a VL CDR3 comprising the amino acid sequence QQSTEDPWT (SEQ ID NO: 275). In some examples, the anti-CD19 antibody comprises a VH CDR1 comprising the amino acid sequence SYWMN (SEQ ID NO: 276), a VH CDR2 comprising the amino acid sequence QIWPGDGDTNYNGKFKG (SEQ ID NO: 277), and a VH CDR3 comprising the amino acid sequence RETTTVGRYYYAMDY (SEQ ID NO: 278). In some examples, the anti-CD19 antibody comprises a VL CDR1 comprising the amino acid sequence KASQSVDYDGDSYLN (SEQ ID NO: 279), a VL CDR2 comprising the amino acid sequence DASNLVS (SEQ ID NO: 280), a VL CDR3 comprising the amino acid sequence QQSTEDPWT (SEQ ID NO: 281), a VH CDR1 comprising the amino acid sequence SYWMN (SEQ ID NO: 282), a VH CDR2 comprising the amino acid sequence QIWPGDGDTNYNGKFKG (SEQ ID NO: 283), and a VH CDR3 comprising the amino acid sequence RETTTVGRYYYAMDY (SEQ ID NO: 284).

[0216] In some examples, the anti-CD19 antibody is an scFv. For example, in some examples, the anti-CD19 scFv comprises 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: DIQLTQSPASLAVSLGQRATISCKASQSVDYDGDSYLNWYQQIPGQPPKLLIYDASNLVSGIPPRFSGSGSGTDFTLNIHPVEKVDAATYHCQQSTEDPWTFGGGTKLEIKGGGGSGGGGSGGGGSQVQLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIWPGDGDTNYNGKFKGKATLTADESSSTAYMQLSSLASEDSAVYFCARRETTTVGRYYYAMDYWGQGTTVTVS (SEQ ID NO: 285).

[0217] Antimesothelin Anti-mesothelin antibodies are known in the art, and the VH and VL, or VH and VL CDRs, of any anti-mesothelin antibody can be used in the TMMPs of the present disclosure. See, e.g., US2019 / 0000944, WO2009 / 045957, WO2014 / 031476, USPN8,460,660, US2013 / 0066055, and WO2009 / 068204.

[0218] In some examples, the anti-mesothelin antibody comprises: a) a light chain comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: DIALTQPASVSGSPGQSITISCTGTSSDIGGYNSVSWYQQHPGKAPKLMIYGVNNRPSGVSNRFSGSSKSGNTASLTISGLQAEDEADYYCSSYDIESATPVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKGDSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTESS (SEQ ID NO: 286); b) a heavy chain comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: QVELVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQAPGKGLEWMGIIDPGDSRTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARGQLYGGTYMDGWGQGTLV TVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPC PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 287).

[0219] In some examples, an anti-mesothelin antibody comprises a VL present in the light chain amino acid sequence provided above and a VH present in the heavy chain amino acid sequence provided above. For example, an anti-mesothelin antibody may comprise: a) a VL comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: DIALTQPASVSGSPGQSITISCTGTSSDIGGYNSVSWYQQHPGKAPKLMIYGVNNRPSGVSNRFSGSSKSGNTASLTISGLQAEDEADYYCSSYDIESATPVFGGGTK (SEQ ID NO: 288); ) A VH comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: qvelvqsgaevkkpgeslkisckgsgysftsywigwvrqapgkglewmgiidpgdsrtryspsfqgqvtisadksistaylqwsslkasdtamyycargqlyggtymdgwgqgtlvTvss (SEQ ID NO: 289).

[0220] In some examples, the anti-mesothelin antibody comprises a VL CDR1, a VL CDR2, and a VL CDR3 present in the light chain amino acid sequence provided above, and a VH CDR1, a CDR2, and a CDR3 present in the heavy chain amino acid sequence provided above. H and V L CDRs are as defined by Kabat (see, e.g., Table 2 above, and Kabat 1991). H and V L The CDRs are as defined by Chothia (see, eg, Table 2 above, and Chothia 1987).

[0221] For example, an anti-mesothelin antibody may comprise a VL CDR1 having the amino acid sequence TGTSSDIGGYNSVS (SEQ ID NO: 290), a VL CDR2 having the amino acid sequence LMIYGVNNRPS (SEQ ID NO: 291), a VL CDR3 having the amino acid sequence SSYDIESATP (SEQ ID NO: 292), a VH CDR1 having the amino acid sequence GYSFTSYWIG (SEQ ID NO: 293), a VH CDR2 having the amino acid sequence WMGIIDPGDSRTRYSP (SEQ ID NO: 294), and a VH CDR3 having the amino acid sequence GQLYGGTYMDG (SEQ ID NO: 295).

[0222] The anti-mesothelin antibody can be an scFv. As one non-limiting example, the anti-mesothelin scFv can comprise the following amino acid sequence: TIFF0007762068000073.tif31164, in the sequence, VH CDR1, CDR2, and CDR3 are underlined, and VL CDR1, CDR2, and CDR3 are shown in bold and underlined.

[0223] As one non-limiting example, an anti-mesothelin scFv can comprise the following amino acid sequence: TIFF0007762068000074.tif31164, in the sequence, VH CDR1, CDR2, and CDR3 are underlined, and VL CDR1, CDR2, and CDR3 are shown in bold and underlined.

[0224] anti-BCMA Anti-BCMA (B cell maturation antigen) antibodies are known in the art, and the VH and VL, or VH and VL CDRs, of any anti-BCMA antibody can be used in the TMMPs of the present disclosure. See, e.g., WO2014 / 089335, US2019 / 0153061, and WO2017 / 093942.

[0225] In some examples, the anti-BCMA antibody comprises: a) a light chain comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: QSVLTQPPSASGTPGQRVTISCSGSSSNIGSNTVNWYQQLPGTAPKLLIFNYHQRPSGVPDRFSGSKSGSSASLAISGLQSEDEADYYCAAWDDSLNGWVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPDSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS (SEQ ID NO: 298); b) a heavy chain comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: EVQLVESGGGLVKPGGSLRLSCAASGFTFGDYALSWFRQAPGKGLEWVGVSRSKAYGGTTDYAASVKGRFTISRDDSKSTAYLQMNSLKTEDTAVYYCASSGYSSGWTPFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV VTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 299).

[0226] In some examples, the anti-BCMA antibody comprises a VL present in the light chain amino acid sequence provided above and a VH present in the heavy chain amino acid sequence provided above. For example, the anti-BCMA antibody comprises a VL comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: QSVLTQPPSASGTPGQRVTISCSGSSSNIGSNTVNWYQQLPGTAPKLLIFNYHQRPSGVPDRFSGSKSGSSASLAISGLQSEDEADYYCAAWDDSLNGWVFGGGTKLTVLG (SEQ ID NO: 300); and B) a VH comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: EVQLVESGGGLVKPGGSLRLSCAASGFTFGDYALSWFRQAPGKGLEWVGVSRSKAYGGTTDYAASVKGRFTISRDDSKSTAYLQMNSLKTEDTAVYYCASSGYSSGWTPFDYWGQGTLVTVSSASTKGPSV (SEQ ID NO: 301).

[0227] In some examples, the anti-BCMA antibody comprises a VL CDR1, a VL CDR2, and a VL CDR3 present in the light chain amino acid sequence provided above, and a VH CDR1, a CDR2, and a CDR3 present in the heavy chain amino acid sequence provided above. H and V L CDRs are as defined by Kabat (see, e.g., Table 2 above, and Kabat 1991). H and V L The CDRs are as defined by Chothia (see, eg, Table 2 above, and Chothia 1987).

[0228] For example, an anti-BCMA antibody may comprise a VL CDR1 having the amino acid sequence SSNIGSNT (SEQ ID NO: 302), a VL CDR2 having the amino acid sequence NYH, a VL CDR3 having the amino acid sequence AAWDDSLNGWV (SEQ ID NO: 303), a VH CDR1 having the amino acid sequence GFTFGDYA (SEQ ID NO: 304), a VH CDR2 having the amino acid sequence SRSKAYGGTT (SEQ ID NO: 305), and a VH CDR3 having the amino acid sequence ASSGYSSGWTPFDY (SEQ ID NO: 306).

[0229] The anti-BCMA antibody may be an scFv. As one non-limiting example, the anti-BCMA scFv may comprise the following amino acid sequence: QVQLVQSGAEVKKPGSSVKVSCKASGGTFSNYWMHWVRQAPGQGLEWMGATYRGHSDTYYNQKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARGAIYNGYDVLDNWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKLLIYYTSNLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYRKLPWTFGQGTKLEIKR (SEQ ID NO: 307).

[0230] As another example, the anti-BCMA scFv may comprise the following amino acid sequence: DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKLLIYYTSNLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYRKLPWTFGQGTKLEIKRGGGGSGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGGTFSNYWMHWVRQAPGQGLEWMGATYRGHSDTYYNQKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARGAIYNGYDVLDNWGQGTLVTVSS (SEQ ID NO: 308).

[0231] In some examples, the anti-BCMA antibody may comprise a VL CDR1 having the amino acid sequence SASQDISNYLN (SEQ ID NO: 309), a VL CDR2 having the amino acid sequence YTSNLHS (SEQ ID NO: 310), a VL CDR3 having the amino acid sequence QQYRKLPWT (SEQ ID NO: 311), a VH CDR1 having the amino acid sequence NYWMH (SEQ ID NO: 312), a VH CDR2 having the amino acid sequence ATYRGHSDTYYNQKFKG (SEQ ID NO: 313), and a VH CDR3 having the amino acid sequence GAIYNGYDVLDN (SEQ ID NO: 314).

[0232] In some examples, the anti-BCMA antibody comprises a) a light chain comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKLLIYYTSNLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYRKLPWTFGQGTKLEIKR (SEQ ID NO: 315).

[0233] In some examples, the anti-BCMA antibody comprises a) a heavy chain comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: QVQLVQSGAEVKKPGSSVKVSCKASGGTFSNYWMHWVRQAPGQGLEWMGATYRGHSDTYYNQKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARGAIYDGYDVLDNWGQGTLVTVSS (SEQ ID NO: 316).

[0234] In some examples, an anti-BCMA antibody (e.g., the antibody referred to in the literature as belantamab) comprises a light chain comprising the following amino acid sequence: DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKLLIYYTSNLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYRKLPWTFGQGTKLEIKR (SEQ ID NO: 317) and a heavy chain comprising the following amino acid sequence: QVQLVQSGAEVKKPGSSVKVSCKASGGTFSNYWMHWVRQAPGQGLEWMGATYRGHSDTYYNQKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARGAIYDGYDVLDNWGQGTLVTVSS (SEQ ID NO: 318).

[0235] In some examples, the anti-BCMA antibody has a cancer chemotherapeutic agent linked to the antibody. For example, in some examples, the anti-BCMA antibody is GSK2857916 (belantamab-mafodotin), and monomethyl auristatin F (MMAF) is linked to the anti-BCMA antibody belantamab via a maleimidocaproyl linker.

[0236] anti-MUC1 In some examples, the TTP present in the TMMP of the present disclosure is an antibody specific for MUC1. For example, the TTP can be specific for a MUC1 polypeptide present on cancer cells. In some examples, the TTP is specific for a truncated form of MUC1, see, e.g., Fessler et al. (2009) Breast Cancer Res. Treat. 118:113. In some examples, the TTP is an antibody specific for a glycosylated MUC1 peptide, see, e.g., Naito et al. (2017) ACS Omega 2:7493 and US 10,017,580.

[0237] As one non-limiting example, the TTP can be a single-chain Fv specific for MUC1. See, e.g., Singh et al. (2007) Mol. Cancer Ther. 6:562, Thie et al. (2011) PLoS One 6:e15921, Imai et al. (2004) Leukemia 18:676, Posey et al. (2016) Immunity 44:1444, EP3130607, EP3164418, WO2002 / 044217, and US2018 / 0112007. In some examples, the TTP is an scFv specific for the MUC1 peptide VTSAPDTRPAPGSTAPPAHG (SEQ ID NO: 319). In some examples, the TTP is an scFv specific for the MUC1 peptide SNIKFRPGSVVVQLTLAFREGTINVHDVETQFNQYKTEAASRY (SEQ ID NO: 320). In some examples, the TTP is an scFv specific for the MUC1 peptide SVVVQLTLAFREGTINVHDVETQFNQYKTEAASRY (SEQ ID NO: 321). In some examples, the TTP is an scFv specific for the MUC1 peptide LAFREGTINVHDVETQFNQY (SEQ ID NO: 322). In some examples, the TTP is an scFv specific for the MUC1 peptide SNIKFRPGSVVVQLTLAAFREGTIN (SEQ ID NO: 323).

[0238] As an example, an anti-MUC1 antibody can comprise a VH CDR1 having the amino acid sequence RYGMS (SEQ ID NO: 324), a VH CDR2 having the amino acid sequence TISGGGTYIYYPDSVKG (SEQ ID NO: 325), a VH CDR3 having the amino acid sequence DNYGRNYDYGMDY (SEQ ID NO: 326), a VL CDR1 having the amino acid sequence SATSSVSYIH (SEQ ID NO: 327), a VL CDR2 having the amino acid sequence STSNLAS (SEQ ID NO: 328), and a VL CDR3 having the amino acid sequence QQRSSSPFT (SEQ ID NO: 329). See, e.g., US2018 / 0112007.

[0239] As another example, an anti-MUC1 antibody can comprise a VH CDR1 having the amino acid sequence GYAMS (SEQ ID NO: 330), a VH CDR2 having the amino acid sequence TISSGGTYIYYPDSVKG (SEQ ID NO: 331), a VH CDR3 having the amino acid sequence LGGDNYYEYFDV (SEQ ID NO: 332), a VL CDR1 having the amino acid sequence RASKSVSTSGYSYMH (SEQ ID NO: 333), a VL CDR2 having the amino acid sequence LASNLES (SEQ ID NO: 334), and a VL CDR3 having the amino acid sequence QHSRELPFT (SEQ ID NO: 335). See, e.g., US2018 / 0112007.

[0240] As another example, an anti-MUC1 antibody can comprise a VH CDR1 having the amino acid sequence DYAMN (SEQ ID NO: 336), a VH CDR2 having the amino acid sequence VISTFSGNINFNQKFKG (SEQ ID NO: 337), a VH CDR3 having the amino acid sequence SDYYGPYFDY (SEQ ID NO: 338), a VL CDR1 having the amino acid sequence RSSQTIVHSNGNTYLE (SEQ ID NO: 339), a VL CDR2 having the amino acid sequence KVSNRFS (SEQ ID NO: 340), and a VL CDR3 having the amino acid sequence (FQGSHVPFT (SEQ ID NO: 341). See, e.g., US2018 / 0112007.

[0241] As another example, an anti-MUC1 antibody can comprise a VH CDR1 having the amino acid sequence GYAMS (SEQ ID NO: 342), a VH CDR2 having the amino acid sequence TISSGGTYIYYPDSVKG (SEQ ID NO: 343), a VH CDR3 having the amino acid sequence LGGDNYYEY (SEQ ID NO: 344), a VL CDR1 having the amino acid sequence TASKSVSTSGYSYMH (SEQ ID NO: 345), a VL CDR2 having the amino acid sequence LVSNLES (SEQ ID NO: 346), and a VL CDR3 having the amino acid sequence QHIRELTRSE (SEQ ID NO: 347). See, e.g., US2018 / 0112007.

[0242] anti-MUC16 In some examples, the TTP present in the TMMP of the present disclosure is an antibody specific for MUC16 (also known as CA125). See, e.g., Yin et al. (2002) Int. J. Cancer 98:737. For example, the TTP can be specific for a MUC16 polypeptide present on cancer cells. See, e.g., US2018 / 0118848 and US2018 / 0112008. In some examples, the MUC16-specific TTP is an scFv. In some examples, the MUC16-specific TTP is a nanobody.

[0243] As an example, an anti-MUC16 antibody can include a VH CDR1 having the amino acid sequence GFTFSNYY (SEQ ID NO: 348), a VH CDR2 having the amino acid sequence ISGRGSTI (SEQ ID NO: 349), a VH CDR3 having the amino acid sequence VKDRGGYSPY (SEQ ID NO: 350), a VL CDR1 having the amino acid sequence QSISTY (SEQ ID NO: 351), a VL CDR2 having the amino acid sequence TAS, and a VL CDR3 having the amino acid sequence QQSYSTPPIT (SEQ ID NO: 352). See, e.g., US2018 / 0118848.

[0244] Anti-claudin-18.2 In some examples, the TTP present in the TMMP of the present disclosure is an antibody specific for claudin-18 isoform 2 ("claudin-18.2"). See, for example, WO2013 / 167259. In some examples, the claudin-18.2-specific TTP is an scFv. In some examples, the claudin-18.2-specific TTP is a nanobody. In some examples, the TPP present in the TMMP of the present disclosure is an antibody specific for TEDEVQSYPSKHDYV (SEQ ID NO: 246) or EVQSYPSKHDYV (SEQ ID NO: 247).

[0245] As an example, an anti-claudin 18.2 antibody may include a VH CDR1 having the amino acid sequence GYTFTDYS (SEQ ID NO: 563), a VH CDR2 having the amino acid sequence INTETGVP (SEQ ID NO: 564), a VH CDR3 having the amino acid sequence ARRTGFDY (SEQ ID NO: 565), a VL CDR1 having the amino acid sequence KNLLHSDGITY (SEQ ID NO: 566), a VL CDR2 having the amino acid sequence RVS, and a VL CDR3 having the amino acid sequence VQVLELPFT (SEQ ID NO: 567).

[0246] As another example, an anti-claudin 18.2 antibody may comprise a VH CDR1 having the amino acid sequence GFTFSSYA (SEQ ID NO: 568), a VH CDR2 having the amino acid sequence ISDGGSYS (SEQ ID NO: 569), a VH CDR3 having the amino acid sequence ARDSYYDNSYVRDY (SEQ ID NO: 570), a VL CDR1 having the amino acid sequence QDINTF (SEQ ID NO: 571), a VL CDR2 having the amino acid sequence RTN, and a VL CDR3 having the amino acid sequence LQYDEFPLT (SEQ ID NO: 572).

[0247] Single-chain T-cell receptor As described above, in some examples, the TTP present in the TMMP of the present disclosure is an scTCR. The TTP is an scTCR specific for a peptide / HLA complex on the surface of a cancer cell, and the peptide can be a cancer-associated peptide (e.g., a peptide of a cancer-associated antigen). The amino acid sequences of scTCRs specific for cancer-associated peptides bound to HLA complexes are known in the art. See, for example, US2019 / 0135914, US2019 / 0062398, and US2018 / 0371049.

[0248] An scTCR comprises an alpha chain variable region (Vα) and a beta chain variable region (Vβ) covalently linked via a suitable peptide linker sequence. For example, Vα can be covalently linked to Vβ via a suitable peptide linker (L) sequence fused to the C-terminus of Vα and the N-terminus of Vβ. An scTCR can have a Vα-L-Vβ structure. An scTCR can have a Vβ-L-Vα structure. An scTCR can also comprise a constant domain (also referred to as a constant region). In some examples, an scTCR comprises, from N-terminus to C-terminus, i) a TCR α chain variable domain polypeptide, ii) a peptide linker, iii) a TCR β chain variable domain polypeptide, and iv) a TCR β chain constant region extracellular domain polypeptide. In some examples, an scTCR comprises, from N-terminus to C-terminus, i) a TCR β chain variable domain polypeptide, ii) a peptide linker, iii) a TCR α chain variable domain polypeptide, and iv) a TCR α chain constant region extracellular domain polypeptide.

[0249] Amino acid sequences of scTCRs specific for peptide / HLA complexes, where the peptide is a cancer-associated peptide, are known in the art, see, e.g., US2019 / 0135914, US2019 / 0062398, US2018 / 0371049, US2019 / 0144563, and US2019 / 0119350.

[0250] For example, an scTCR can be specific for an NY-ESO epitope, such as the SLLMWITQC peptide, bound to an HLA complex comprising an HLA-A*0201 heavy chain and a β2M polypeptide. By way of example, such a scTCR can include: i) a TCR α chain variable region comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: MQEVTQIPAALSVPEGENLVLNCSFTDSAIYNLQWFRQDPGKGLTSLLLIQSSQREQTSGRLNASLDKSSGRSTLYIAASQPGDSATYLCAVRPTSGGSYIPTFGRGTSLIVHPY (SEQ ID NO: 353), in which amino acid 20 can be V or A; amino acid 51 can be Q, P, S, T, or M; amino acid 52 can be S, P, F, or G; amino acid 53 can be S, W, H, or T; amino acid 94 can be P, H, or A; amino acid 95 can be T, L, M, A, Q, Y, E, I, F, V, N, G, S, D, or R; amino acid 96 can be S, L, T, Y, I, Q, V, E, A, W, R, G, H, D, or K; amino acid 9 amino acid 98 can be G, P, H, S, T, W, or A; amino acid 99 can be S, T, Y, D, H, V, N, E, G, Q, K, A, I, or R; amino acid 100 can be Y, F, M, or D; amino acid 101 can be I, P, T, or M; and amino acid 103 can be T or A; ii) at least 90%, at least 95%, at least 98%, at least 99%, to the following amino acid sequence: or a TCR β chain variable region comprising an amino acid sequence with 100% amino acid sequence identity: MGVTQTPKFQVLKTGQSMTLQCAQDMNHEYMSWYRQDPGMGLRLIHYSVGAGITDQGEVPNGYNVSRSTTEDFPLRLLSAAPSQTSVYFCASSYVGNTGELFFGEGSRLTVL (SEQ ID NO: 354), in which amino acid 18 can be M or V; amino acid 50 can be G, V, or I; and amino acid 52 can be G or Q;amino acid 53 can be I, T, or M; amino acid 55 can be D or R; amino acid 56 can be Q or R; amino acid 70 can be T or I; amino acid 94 can be Y, N, or F; amino acid 95 can be V or L; and amino acid 97 can be N, G, or D). For example, in some examples, the scTCR may include i) a TCR alpha chain variable region comprising the following amino acid sequence: MQEVTQIPAALSVPEGENLVLNCSFTDSAIYNLQWFRQDPGKGLTSLLLIMSHQREQTSGRLNASLDKSSGRSTLYIAASQPGDSATYLCAVRPTSGGSYIPTFGRGTSLIVHPY (SEQ ID NO: 355), and a TCR beta chain variable region comprising the following amino acid sequence: MGVTQTPKFQVLKTGQSMTLQCAQDMNHEYMSWYRQDPGMGLRLIHYSVSAGITDQGEVPNGYNVSRSTTEDFPLRLLSAAPSQTSVYFCASSYVGNTGELFFGEGSRLTVL (SEQ ID NO: 356);

[0251] In another example, the scTCR can bind to an HPV epitope (e.g., the amino acid sequence YIIFVYIPL (HPV16 E5)) bound to an HLA complex containing an HLA heavy chain and a β2M polypeptide. 63-71 ;SEQ ID NO: 357), KLPQLCTEL (HPV16 E6 11-19 ; SEQ ID NO: 358), TIHEIILECV (HPV16 E6; SEQ ID NO: 359), YMLDLQPET (HPV16 E7 11-19 ;SEQ ID NO: 360), TLGIVCPI (HPV16 E7 86-93 ) (SEQ ID NO: 361), KCIDFYSRI (HPV18 E6 67-75 ;SEQ ID NO: 362), or FQQLFLNTL (HPV18 E7 86-94363))). By way of example, such a scTCR can be specific for i) a TCR α chain variable region comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: METLLGLLILQLQLQWVSSKQEVTQIPAALSVPEGENLVLNCSFTDSAIYNLQWFRQDPGKGLTSLLLIQSSQREQTSGRLNASLDKSSGRSTLYIAASQPGDSATYLCAVRETSGSRLTFGEGTQLTVNPD (sequence sequence number 364), and ii) a TCR β chain variable region comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence: mgirllcrvafcflavglvdvkvtqssrylvkrtgekvflecvqdmdhenmfwyrqdpglglrliyfsydvkmkekgdipegysvsrekkerfslilesastnqtsmylcassfwgrstdtqyfgpgtrltvl (SEQ ID NO: 365).

[0252] contrast agents In some instances, the TTP of the TMMP of the present disclosure comprises an imaging agent or radiolabel, where the imaging agent facilitates imaging of the tumor to which the TMMP binds.

[0253] Suitable agents include computed tomography (CT), positron emission tomography (PET), and single photon emission computed tomography (SPECT) radiotracers. Suitable PET / SPECT imaging agents include, for example, positron emitters, such as 11 C. 13 N, 18 F, 82 Ru, and 15Iodinated CT contrast agents can be used. Suitable contrast agents include gadolinium (Gd), dysprosium, and iron. Gd chelates such as Gd diethylenetriaminepentaacetic acid (GdDTPA), Gd tetraazacyclododecanetetraacetic acid (GdDOTA), polylysine-Gd chelate, and derivatives thereof can be used. Suitable radioisotopes include: 123 I (iodine), 18 F (fluorine), 99 Tc (technetium), 111 In (indium), and 67 Ga (gallium) is an example.

[0254] Linker A TMMP of the present disclosure may include one or more linkers between one or more of: i) an MHC class I polypeptide and an Ig Fc polypeptide (such a linker is referred to herein as "L1"); ii) an immunomodulatory polypeptide and an MHC class I polypeptide (such a linker is referred to herein as "L2"); iii) a first immunomodulatory polypeptide and a second immunomodulatory polypeptide (such a linker is referred to herein as "L3"); iv) a peptide antigen ("epitope") and an MHC class I polypeptide; v) an MHC class I polypeptide and a dimerization polypeptide (e.g., a first or second member of a dimerization pair); vi) a dimerization polypeptide (e.g., a first or second member of a dimerization pair) and an Ig Fc polypeptide; and vii) an Ig Fc polypeptide (or non-Ig scaffold) and a tumor targeting polypeptide.

[0255] Suitable linkers (also referred to as "spacers") can be readily selected and can be of any of several suitable lengths, such as 1 to 25 amino acids, 3 to 20 amino acids, 2 to 15 amino acids, 3 to 12 amino acids, 4 to 10 amino acids, 5 to 9 amino acids, 6 to 8 amino acids, or 7 to 8 amino acids. Suitable linkers can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids in length. In some examples, the linker has a length of 25 to 50 amino acids, e.g., 25 to 30, 30 to 35, 35 to 40, 40 to 45, or 45 to 50 amino acids.

[0256] Exemplary linkers include glycine polymers (G) n , glycine-serine polymers (e.g., (GS)n, (GSGGS) n (SEQ ID NO: 366) and (GGGS) nFlexible linkers include glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers can be used; both Gly and Ser are relatively unstructured and can therefore function as neutral tethers between components. Glycine polymers can also be used; glycine has significantly more access to phi-psi space than alanine and is significantly less restricted than residues with longer side chains (see Scheraga, Rev. Computational Chem. 11173-142 (1992)). Exemplary linkers can include amino acid sequences including, but not limited to, GGSG (SEQ ID NO: 368), GGSGG (SEQ ID NO: 369), GSGSG (SEQ ID NO: 370), GSGGG (SEQ ID NO: 371), GGGSG (SEQ ID NO: 372), GSSSG (SEQ ID NO: 373), and the like. Exemplary linkers can include, for example, Gly(Ser4)n (SEQ ID NO: 374), where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some examples, the linker includes the amino acid sequence (GSSSS)n (SEQ ID NO: 375), where n is 4. In some examples, the linker includes the amino acid sequence (GSSSS)n (SEQ ID NO: 376), where n is 5. In some examples, the linker includes the amino acid sequence (GGGGS)n (SEQ ID NO: 377), where n is 1. In some examples, the linker includes the amino acid sequence (GGGGS)n (SEQ ID NO: 378), where n is 2. In some examples, the linker includes the amino acid sequence (GGGGS)n (SEQ ID NO: 379), where n is 3. In some examples, the linker includes the amino acid sequence (GGGGS)n (SEQ ID NO: 380), where n is 4. In some examples, the linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO: 381), where n is 5. In some examples, the linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO: 382), where n is 6. In some examples, the linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO: 383), where n is 7. In some examples, the linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO: 384), where n is 8.In some examples, the linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO: 385), where n is 9. In some examples, the linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO: 386), where n is 10. In some examples, the linker comprises the amino acid sequence AAAGG (SEQ ID NO: 387).

[0257] In some instances, a linker polypeptide present in a first polypeptide of a TMMP 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 TMMP of the present disclosure. In some instances, for example, a suitable linker has the amino acid sequence TIFF0007762068000075.tif4128. As another example, a suitable linker can include the amino acid sequence GCGGS(G4S)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).

[0258] epitope The TMMP of the present disclosure comprises any of a variety of peptide epitopes. As described above, the peptide epitopes present in the TMMP of the present disclosure are peptides that, when complexed with an MHC polypeptide, present the epitope to a T cell receptor (TCR). Epitope-specific T cells bind to an epitope having a given amino acid sequence, i.e., a "reference" amino acid sequence, but do not substantially bind to an epitope that differs from the reference amino acid sequence. For example, epitope-specific T cells, if present, will bind to an epitope that differs from the reference amino acid sequence by 10 or more. -6 Under M, 10 -5 Less than M or 10 -4 An epitope-specific T cell binds with an affinity of at least 10 M to the reference amino acid sequence, i.e., the epitope for which it is specific. -7 M, at least 10 -8 M, at least 10 -9M, or at least 10 -10 It can bind with an affinity of M.

[0259] Epitopes of peptide epitopes within the scope of the present disclosure include, but are not limited to, epitopes present in cancer-associated antigens, viral epitopes (e.g., epitopes present in viral antigens), etc. Cancer-associated antigens are known in the art; see, for example, Cheever et al. (2009) Clin. Cancer Res. 15:5323. Cancer-associated antigens include, but are not limited to, alpha-folate receptor, carbonic anhydrase IX (CAIX), CD19, CD20, CD22, CD30, CD33, CD44v7 / 8, carcinoembryonic antigen (CEA), epithelial glycoprotein-2 (EGP-2), epithelial glycoprotein-40 (EGP-40), folate-binding protein (FBP), fetal acetylcholine receptor, ganglioside antigen GD2, Her2 / neu, IL-13R-α2, kappa light chain, LeY, L1 cell adhesion molecule, melanoma-associated antigen (MAGE), MAGE-A1, mesothelin, MUC1, NKG2D ligand, carcinoembryonic antigen (h5T4), prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), tumor-associated glycoprotein-72 (TAG-72), and vascular endothelial growth factor receptor-2 (VEGF-R2). See, e.g., Vigneron et al. (2013) Cancer Immunity 13:15 and Vigneron (2015) BioMed Res. Int'l Article ID 948501, as well as epidermal growth factor receptor (EGFR) vIII polypeptides (see, e.g., Wong et al. (1992) Proc. Natl. Acad. Sci. USA 89:2965 and Miao et al. (2014) PLoSOne 9:e94281).

[0260] In some examples, suitable peptide epitopes include a MUC1 polypeptide, an LMP2 polypeptide, an epidermal growth factor receptor (EGFR) vIII polypeptide, a HER-2 / neu polypeptide, a melanoma antigen family A, 3 (MAGE A3) polypeptide, a p53 polypeptide, a mutant p53 polypeptide, an NY-ESO-1 polypeptide, a folate hydrolase (prostate-specific membrane antigen; PSMA) polypeptide, a carcinoembryonic antigen (CEA) polypeptide, a melanoma antigen recognized by T cells (melanA / MART1) polypeptide, a Ras polypeptide, a gp100 polypeptide, a proteinase 3 (PR1) polypeptide, a bcr-abl polypeptide, a tyrosinase polypeptide, a survivin polypeptide, a prostate-specific antigen (PSA) polypeptide, an hTERT polypeptide, a sarcoma metastasis breakpoint polypeptide, a synovial sarcoma X (SSX) breakpoint polypeptide, an EphA2 polypeptide, an acid phosphatase, prostate (PAP) polypeptide, a melanoma inhibitor of apoptosis (ML-IAP), an epithelial cell adhesion molecule (EpCAM) polypeptide, an ERG (TMPRSS2 ... ETS fusion) polypeptide, NA17 polypeptide, paired box-3 (PAX3) polypeptide, anaplastic lymphoma kinase (ALK) polypeptide, androgen receptor polypeptide, cyclin B1 polypeptide, N-myc proto-oncogene (MYCN) polypeptide, Ras homolog gene family member C (RhoC) polypeptide, tyrosinase-related protein-2 (TRP-2) polypeptide, mesothelin polypeptide, prostate stem cell antigen (PSCA) polypeptide, melanoma-associated antigen-1 (MAGE A1) polypeptide, cytochrome P4501B1 (CYP1B1) polypeptide, placenta-specific protein 1 (PLAC1) polypeptide, BORIS polypeptide (also known as CCCTC-binding factor or CTCF), ETV6-AML polypeptide, breast cancer antigen NY-BR-1 polypeptide (also known as ankyrin repeat domain-containing protein 30A), regulator of G protein signaling (RGS5) polypeptide, squamous cell carcinoma cell antigen recognized by T cells (SART3) polypeptide, carbonic anhydrase IX polypeptide, paired box-5 (PAX5) polypeptide, OY-TES1 (testis antigen, also known as acrosin-binding protein) polypeptide, sperm protein 17 polypeptide, lymphoid cell-specific protein-tyrosine kinase (LCK) polypeptide, high-molecular-weight melanoma-associated antigen (HMW-MAA), A-kinase anchor protein-4 ( The antibody presents epitopes of AKAP-4, synovial sarcoma X breakpoint 2 (SSX2) polypeptide, X antigen family member 1 (XAGE1) polypeptide, B7 homolog 3 (B7H3, also known as CD276) polypeptide, legumain polypeptide (LGMN1, also known as asparaginyl endopeptidase), tyrosine kinase-2 with Ig and EGF homology domains (Tie-2, also known as angiopoietin-1 receptor) polypeptide, P antigen family member 4 (PAGE4) polypeptide, vascular endothelial growth factor receptor 2 (VEGF2) polypeptide, MAD-CT-1 polypeptide, fibroblast activation protein (FAP) polypeptide, platelet-derived growth factor receptor beta (PDGFβ) polypeptide, MAD-CT-2 polypeptide, or Fos-related antigen-1 (FOSL) polypeptide. In some cases, human papillomavirus (HPV) antigens are specifically excluded. In some cases, alpha-fetoprotein (AFP) antigens are specifically excluded. In some instances, the Wilms Tumor-1 (WT1) antigen is specifically excluded.

[0261] Amino acid sequences of cancer-associated antigens are known in the art, and include, for example, MUC1 (GenBank CAA56734), LMP2 (GenBank CAA47024), EGFRvIII (GenBank NP_001333870), HER-2 / neu (GenBank AAI67147), MAGE-A3 (GenBank AAH11744), p53 (GenBank BAC16799), NY-ESO-1 (GenBank CAA05908), PSMA (GenBank AAH25672), CEA (GenBank AAA51967), melan / MART1 (GenBank NP_005502), Ras (GenBank NP_001123914), gp100 (GenBank AAC60634), bcr-abl (GenBank AAB60388), tyrosinase (GenBank AAB60319), survivin (GenBank AAC51660), PSA (GenBank CAD54617), hTERT (GenBank BAC11010), SSX (GenBank NP_001265620), Eph2A (GenBank NP_004422), PAP (GenBank AAH16344), ML-IAP (GenBank AAH14475), EpCAM (GenBank NP_002345), ERG (TMPRSS2 ETS fusion) (GenBank ACA81385), PAX3 (GenBank AAI01301), ALK (GenBank NP_004295), androgen receptor (GenBank NP_000035), cyclin B1 (GenBank CAO99273), MYCN (GenBank NP_001280157), RhoC (GenBank AAH52808), TRP-2 (GenBank AAC60627), Mesothelin (GenBank AAH09272), PSCA (GenBank AAH65183), MAGE A1 (GenBank NP_004979), CYP1B1 (GenBank AAM50512), PLAC1 (GenBank AAG22596), BORIS (GenBank NP_001255969), ETV6 (GenBank NP_001978), NY-BR1 (GenBankNP_443723), SART3 (GenBank NP_055521), carbonic anhydrase IX (GenBank EAW58359), PAX5 (GenBank NP_057953), OY-TES1 (GenBank NP_115878), sperm protein 17 (GenBank AAK20878), LCK (GenBank NP_001036236), HMW-MAA (GenBank NP_001888), AKAP-4 (GenBank NP_003877), SSX2 (GenBank CAA60111), XAGE1 (GenBank NP_001091073; XP_001125834; XP_001125856; and XP_001125872), B7H3 (GenBank NP_001019907;XP_947368;XP_950958;XP_950960;XP_950962;XP_950963;XP_950965; and NP_000450), PAGE4 (GenBank NP_001305806), VEGFR2 (GenBank NP_002244), MAD-CT-1 (GenBank NP_005893 NP_056215), FAP (GenBank NP_004451), PDGFβ (GenBank NP_002600), MAD-CT-2(GenBank See, e.g., FOSL (GenBank NP_001138574) and FOSL (GenBank NP_005429). These polypeptides are also discussed in, e.g., Cheever et al. (2009) Clin. Cancer Res. 15:5323 and references cited therein, Wagner et al. (2003) J. Cell. Sci. 116:1653, Matsui et al. (1990) Oncogene 5:249, Zhang et al. (1996) Nature 383:168.

[0262] Suitable epitopes include, but are not limited to, epitopes present in infectious disease pathogens, such as epitopes presented by virus-encoded polypeptides. Examples of viral infectious pathogens include, for example, adenovirus, adeno-associated virus, alphavirus (togavirus), eastern equine encephalitis virus, eastern equine encephalomyelitis virus, Venezuelan equine encephalomyelitis vaccine strain TC-83, western equine encephalomyelitis virus, arenavirus, lymphocytic choroiditis virus (non-neurotropic strain), Tacaribe virus complex, bunyavirus, Bunyamwera virus, Rift Valley fever virus vaccine strain MP-12, chikungunya virus, calcivirus, coronavirus, cowpox virus, flavivirus (togavirus) group B arbovirus, dengue virus serotypes 1, 2, 3, and 4, yellow fever virus vaccine strain 17D, hepatitis A, B, C, D, and E. Viruses, cytomegalovirus, Epstein-Barr virus, Eastern equine encephalitis virus, herpes simplex types 1 and 2, varicella-zoster, human herpesvirus types 6 and 7, hepatitis C virus (HVC), hepatitis B virus (HBV), influenza viruses types A, B, and C, papovavirus, Newcastle disease virus, measles virus, mumps virus, parainfluenza viruses types 1, 2, 3, and 4, polyomaviruses (JC virus, BK virus), respiratory syncytial virus, human parvovirus (B19), coxsackieviruses A and B, echovirus, poliovirus, rhinovirus, smallpox (variola minor virus), smallpox (variola major virus), whitepox Reoviruses, Coltiviruses, human rotaviruses, and Orbiviruses (Colorado tick fever viruses), rabies virus, vesicular stomatitis virus, Rubivirus (rubella), Semliki Forest virus, Saint Louis encephalitis virus, Venezuelan equine encephalitis virus, Venezuelan equine encephalomyelitis virus, Arenaviruses (also known as South American hemorrhagic fever viruses), Flexal, lymphocytic choriomeningitis virus (LCM) (neurotropic strains), Hantaviruses including Hantaan virus, Rift Valley fever virus, Japanese encephalitis virus, yellow fever virus, monkeypox virus, human immunodeficiency virus (HIV) types 1 and 2,Human T-cell lymphotropic virus (HTLV) types 1 and 2, simian immunodeficiency virus (SIV), vesicular stomatitis virus, Guanarito virus, Lassa fever virus, Junin virus, Machupo virus, Sabia, Crimean-Congo hemorrhagic fever virus, Ebola virus, Marburg virus, Central European tick-borne encephalitis, Far Eastern tick-borne encephalitis, Hanzalova, Hyprus, Kumlinge, Kyasanur Forest disease, Omsk hemorrhagic fever, and Russian spring-summer encephalitis virus (Flavonitis), including tick-borne encephalitis virus complex (Flavonitis), simian herpesvirus (Herpes B or Simian B virus), and long-tailed monkey herpesvirus 1 (Herpes B virus). Antigens encoded by such viruses include equine morbillivirus (Hendra and Hendra-like viruses), Nipah virus, variola major virus (smallpox virus), variola minor virus (smallpox), African swine fever virus, African horse sickness virus, Akabane virus, avian influenza virus (highly pathogenic), bluetongue virus, camelpox virus, classical swine fever virus, cowrie aluminum (heartwater), foot and mouth disease virus, goatpox virus, Japanese encephalitis virus, lumpy skin disease virus, malignant catarrhal fever virus, Menangle virus, Newcastle disease virus (VVND), vesicular stomatitis virus (adventitious), and Zika virus. Antigens encoded by such viruses are known in the art, and peptide epitopes suitable for use in the TMMPs of the present disclosure can include peptides from any known viral antigen. In some examples, HPV antigens are specifically excluded. In some examples, HBV antigens are specifically excluded. In some instances, the viral epitope is an epitope present in a viral antigen encoded by a virus that infects a large portion of the human population, including, for example, cytomegalovirus (CMV), Epstein-Barr virus (EBV), human papillomavirus, adenovirus, etc.

[0263] In some examples, the epitope peptides present in the TMMP of the present disclosure present epitopes specific to HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, or HLA-G alleles. In one embodiment, the epitope peptides present in the TMMP present epitopes restricted to HLA-A*0101, A*0201, A*0301, A*1101, A*2301, A*2402, A*2407, A*3303, and / or A*3401. In one embodiment, the epitope peptides present in the TMMP present epitopes restricted to HLA-B*0702, B*0801, B*1502, B*3802, B*4001, B*4601, and / or B*5301. In one embodiment, the epitope peptides present in TMMP present epitopes restricted to C*0102, C*0303, C*0304, C*0401, C*0602, C*0701, C*702, C*0801, and / or C*1502.

[0264] CMV peptide epitopes In some examples, the TMMP of the present disclosure comprises a CMV peptide epitope, i.e., a peptide that, when in an MHC / peptide complex (e.g., an HLA / peptide complex), presents a CMV epitope (i.e., an epitope present in a CMV antigen) to a T cell. Like other peptide epitopes of the present disclosure, CMV peptide epitopes have a length of at least 4 amino acids, 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, including within the ranges of 4-20 aa, 6-18 aa, 8-15 aa, 8-12 aa, 5-10 aa, 10-15 aa, 15-20 aa, 10-20 aa, or 15-25 aa).

[0265] A given CMV epitope-specific T cell binds to an epitope having a reference amino acid sequence of the given CMV epitope, but does not substantially bind to an epitope that differs from the reference amino acid sequence. For example, a given CMV epitope-specific T cell binds to a CMV epitope having a reference amino acid sequence, and, if present, to an epitope that differs from the reference amino acid sequence, at least 10 -6 Under M, 10 -5 Less than M or 10 -4 A given CMV epitope-specific T cell binds with an affinity of at least 10 M to the epitope for which it is specific. -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.

[0266] In some examples, the CMV peptide epitope present in the TMMP of the present disclosure is a peptide from CMV pp65. In some examples, the CMV peptide epitope present in the TMMP of the present disclosure is a peptide from CMV gB (glycoprotein B).

[0267] For example, in some instances, a CMV peptide epitope present in a TMMP of the disclosure has a length of at least 4 amino acids, 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, including within the ranges of 4-20 aa, 6-18 aa, 8-15 aa, 8-12 aa, 5-10 aa, 10-15 aa, 15-20 aa, 10-20 aa, or 15-25 aa in length), and is in accordance with the following CMV peptide epitopes: A peptide of a CMV polypeptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the pp65 amino acid sequence: TIFF0007762068000076.tif64164.

[0268] As one non-limiting example, a CMV peptide epitope present in a TMMP of the present disclosure has the amino acid sequence NLVPMVATV (SEQ ID NO: 393) and is 9 amino acids in length.

[0269] In some examples, a CMV peptide epitope present in a TMMP of the disclosure has a length of at least 4 amino acids, 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, including within the ranges of 4-20 aa, 6-18 aa, 8-15 aa, 8-12 aa, 5-10 aa, 10-15 aa, 15-20 aa, 10-20 aa, or 15-25 aa in length), and is A peptide of a CMV polypeptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the gB amino acid sequence: (SEQ ID NO: 394).

[0270] In some examples, the CMV epitopes present in the TMMP of the present disclosure represent epitopes specific to HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, or HLA-G alleles. In some examples, the epitope peptides present in the TMMP represent epitopes restricted to HLA-A*0101, A*0201, A*0301, A*1101, A*2301, A*2402, A*2407, A*3303, and / or A*3401. In some examples, the CMV epitopes present in the TMMP of the present disclosure represent epitopes restricted to HLA-B*0702, B*0801, B*1502, B*3802, B*4001, B*4601, and / or B*5301. In some examples, the CMV epitopes present in a TMMP of the present disclosure represent epitopes restricted to C*0102, C*0303, C*0304, C*0401, C*0602, C*0701, C*702, C*0801, and / or C*1502. As an example, in some examples, a TMMP of the present disclosure comprises: a) a CMV peptide epitope having the amino acid sequence NLVPMVATV (SEQ ID NO: 395) and having a length of 9 amino acids; b) an HLA-A*0201 class I heavy chain polypeptide; and c) a β2M polypeptide.

[0271] In some examples, the TMMP of the present disclosure includes an scFv or nanobody specific to a Her2 polypeptide present on the surface of a cancer cell as a TTP, and a CMV peptide epitope as an epitope. In some examples, the CMV peptide is a peptide of a CMV pp65 polypeptide. In some examples, the CMV peptide epitope is a peptide of a CMV gB polypeptide. In some examples, the CMV peptide epitope has the amino acid sequence NLVPMVATV (SEQ ID NO: 395) and is 9 amino acids in length.

[0272] In some examples, the TMMP of the present disclosure includes an scFv or nanobody specific for a MUC1 polypeptide present on the surface of a cancer cell as the TTP, and a CMV peptide epitope as the epitope. In some examples, the CMV peptide epitope is a peptide of a CMV pp65 polypeptide. In some examples, the CMV peptide is a peptide of a CMV gB polypeptide. In some examples, the CMV peptide has the amino acid sequence NLVPMVATV (SEQ ID NO: 395) and is 9 amino acids in length.

[0273] In some examples, the TMMP of the present disclosure includes an scFv or nanobody specific to a WT1 polypeptide present on the surface of a cancer cell as a TTP, and a CMV peptide epitope as an epitope. In some examples, the CMV peptide epitope is a peptide of a CMV pp65 polypeptide. In some examples, the CMV peptide epitope is a peptide of a CMV gB polypeptide. In some examples, the CMV peptide epitope has the amino acid sequence NLVPMVATV (SEQ ID NO: 395) and is 9 amino acids in length.

[0274] In some examples, the TMMP of the present disclosure includes an scFv or nanobody specific for a mesothelin polypeptide present on the surface of a cancer cell as the TTP, and a CMV peptide epitope as the epitope. In some examples, the CMV peptide epitope is a peptide of a CMV pp65 polypeptide. In some examples, the CMV peptide epitope is a peptide of a CMV gB polypeptide. In some examples, the CMV peptide epitope has the amino acid sequence NLVPMVATV (SEQ ID NO: 395) and is 9 amino acids in length.

[0275] In some examples, the TMMP of the present disclosure includes an scFv or nanobody specific for a CD19 polypeptide present on the surface of a cancer cell as a TTP, and a CMV peptide epitope as an epitope. In some examples, the CMV peptide epitope is a peptide of a CMV pp65 polypeptide. In some examples, the CMV peptide epitope is a peptide of a CMV gB polypeptide. In some examples, the CMV peptide epitope has the amino acid sequence NLVPMVATV (SEQ ID NO: 395) and is 9 amino acids in length.

[0276] In some examples, the TMMP of the present disclosure includes an scFv or nanobody specific for a BCMA polypeptide present on the surface of a cancer cell as a TTP, and a CMV peptide epitope as an epitope. In some examples, the CMV peptide epitope is a peptide of a CMV pp65 polypeptide. In some examples, the CMV peptide epitope is a peptide of a CMV gB polypeptide. In some examples, the CMV peptide epitope has the amino acid sequence NLVPMVATV (SEQ ID NO: 395) and is 9 amino acids in length.

[0277] In some examples, the TMMP of the present disclosure includes an scFv or nanobody specific for a MUC16 polypeptide present on the surface of a cancer cell as a TTP, and a CMV peptide epitope as an epitope. In some examples, the CMV peptide epitope is a peptide of a CMV pp65 polypeptide. In some examples, the CMV peptide epitope is a peptide of a CMV gB polypeptide. In some examples, the CMV peptide epitope has the amino acid sequence NLVPMVATV (SEQ ID NO: 395) and is 9 amino acids in length.

[0278] HLA / peptide binding assay Whether a given peptide (e.g., a peptide containing an epitope) binds to class I HLA (comprising an HLA heavy chain and a β2M polypeptide) and whether, upon binding to the HLA complex, it is able to effectively present the epitope to a TCR can be determined using any of several well-known methods, including binding assays and T cell activation assays.

[0279] Cell-based binding assays As an example, a cell-based peptide-induced stabilization assay can be used to determine peptide-HLA class I binding. In this assay, a peptide of interest can bind to TAP-deficient cells, i.e., cells with a defect in the transporter associated with antigen processing (TAP) machinery, resulting in little or no binding to surface class I molecules. Such cells include, for example, the human T2 cell line (T2(174×CEM.T2; American Type Culture Collection (ATCC) No. CRL-1992). Henderson et al. (1992) Science 255:1264. Without efficient TAP-mediated transport of cytoplasmic peptides into the endoplasmic reticulum, assembled class I complexes are structurally unstable and are only transiently retained on the cell surface. However, incubation of T2 cells with exogenous peptides capable of binding to class I stabilizes surface peptide-HLA class I complexes, which can be detected by flow cytometry, for example, with a pan-anti-class I monoclonal antibody. The stabilization of peptide-HLA complexes on the cell surface by peptide addition and the resulting increased longevity verify their identity. For example, if the pan-HLA class I antibody contains a fluorescent label, analysis can be performed using flow cytometry. Binding of peptides to various allelic forms of HLA H chains can be tested by genetically modifying T2 cells to express the allelic HLA H chain of interest.

[0280] The following is a non-limiting example of the use of the T2 assay to assess peptide binding to HLA A*0201. T2 cells were washed with cell culture medium and incubated for 10 min. 6 The peptides were concentrated to 1000 cells / ml. The peptides of interest were prepared in cell culture medium and serially diluted to obtain concentrations of 200 μM, 100 μM, 20 μM, and 2 μM. Cells were mixed 1:1 with each peptide dilution to obtain a final volume of 200 μL and final peptide concentrations of 100 μM, 50 μM, 10 μM, and 1 μM. The HLA A*0201-binding peptide, GILGFVFTL (SEQ ID NO: 396), and the non-HLA A*0201-restricted peptide, HPVGEADYF (SEQ ID NO: 397) (HLA-B*3501) were included as positive and negative controls, respectively. The cell / peptide mixture was maintained at 37°C and 5% CO2 for 10 minutes, then incubated overnight at room temperature. The cells were then incubated at 37°C for 2 hours and stained with a fluorescently labeled anti-human HLA antibody. The cells were washed twice with phosphate-buffered saline and analyzed using flow cytometry. The mean fluorescence intensity (MFI) of the anti-HLA antibody staining is used to measure the strength of binding.

[0281] Biochemical binding assays HLA polypeptides (HLA heavy chain polypeptides complexed with β2M polypeptides) can be tested for binding to peptides of interest in a cell-free in vitro assay system. For example, a labeled reference peptide (e.g., fluorescently labeled) is bound to an HLA polypeptide (HLA heavy chain polypeptides complexed with β2M polypeptides) to form an HLA reference peptide complex. The ability of the test peptide of interest to displace the labeled reference peptide from the HLA reference peptide complex is tested. Relative binding affinity is calculated as the amount of test peptide required to displace the bound reference peptide. See, for example, van der Burg et al. (1995) Human Immunol. 44:189.

[0282] As another example, a peptide of interest can be incubated with an HLA molecule (HLA heavy chain complexed with a β2M polypeptide), and the stabilization of the HLA / peptide complex can be measured in an immunoassay format. The ability of the peptide of interest to stabilize the HLA molecule is compared to a control peptide that presents a known T cell epitope. Detection of stabilization is based on the presence or absence of the native conformation of the HLA / peptide complex, detected using an anti-HLA antibody. See, e.g., Westrop et al. (2009) J. Immunol. Methods 341:76; Steinitz et al. (2012) Blood 119:4073; and U.S. Patent No. 9,205,144.

[0283] T cell activation assay Whether a given peptide binds to class I HLA (including an HLA heavy chain and a β2M polypeptide) and whether it can effectively present an epitope to a TCR upon binding to the HLA complex can be determined by assessing T cell responses to the peptide-HLA complex. T cell responses that can be measured include, for example, interferon-gamma (IFNγ) production, cytotoxic activity, etc.

[0284] ELISPOT assay Suitable assays include, for example, enzyme-linked immunospot (ELISPOT) assays. + IFNγ production by T cells is measured by tracking it on antigen-presenting cells (APCs) that present a peptide of interest complexed with HLA class I. Antibodies against IFNγ are immobilized on the wells of a multiwell plate. APCs are added to the wells and incubated with the peptide of interest for a period of time so that the peptide binds to HLA class I on the surface of the APCs. CD8 specific for the peptide is expressed. +T cells are added to the wells and the plate is incubated for approximately 24 hours. The wells are then washed, and any IFNγ bound to the immobilized anti-IFNγ antibody is detected using a detectably labeled anti-IFNγ antibody. A colorimetric assay can be used. For example, the detectably labeled anti-IFNγ antibody can be a biotin-labeled anti-IFNγ antibody that can be detected using streptavidin conjugated to alkaline phosphatase. BCIP / NBT (5-bromo-4-chloro-3-indolylphosphate / nitroblue tetrazolium) solution is added and the assay is developed. The presence of IFNγ-secreting T cells is identified by colored spots. Negative controls include APCs not contacted with peptide. APCs expressing various HLA H chain alleles can be used to determine whether a peptide of interest effectively binds to HLA class I molecules containing a specific HLA H chain.

[0285] Cytotoxicity assay Cytotoxicity assays can also be used to determine whether a given peptide binds to a particular HLA class I heavy chain and, upon binding to an HLA class I complex containing the heavy chain, effectively presents the epitope to the TCR. + This involves incubating target cells with T cells. The target cells present on their surface a peptide / HLA class I complex containing the peptide of interest and an HLA class I molecule containing the HLA heavy chain to be tested. The target cells can be, for example, 51 The target cells can be radiolabeled with cytotoxic CD8 + Effectively presenting epitopes to TCRs on T cells, thereby targeting CD8 + Whether or not T cells induce cytotoxic activity against target cells is determined by the amount of lysed target cells. 51 Specific cytotoxicity can be calculated as the amount of cytotoxic activity in the presence of the peptide minus the amount of cytotoxic activity in the absence of the peptide.

[0286] Detection of antigen-specific T cells using peptide-HLA tetramers As 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.

[0287] Immunomodulatory Polypeptides In some examples, the immunomodulatory polypeptide present in a TMMP of the present disclosure is a wild-type immunomodulatory polypeptide. In other examples, the immunomodulatory polypeptide present in a TMMP of the present disclosure is a variant immunomodulatory polypeptide that has a lower affinity for a co-immunomodulatory polypeptide compared to the affinity of the corresponding wild-type immunomodulatory polypeptide for the co-immunomodulatory polypeptide. A suitable immunomodulatory domain that exhibits a lower affinity for a co-immunomodulatory domain may 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 TMMP 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 examples, the variant immunomodulatory polypeptides present in the TMMPs of the present disclosure have an amino acid sequence that differs by 11 aa, 12 aa, 13 aa, 14 aa, 15 aa, 16 aa, 17 aa, 18 aa, 19 aa, or 20 aa from the corresponding wild-type immunomodulatory polypeptide. As an example, in some examples, the variant immunomodulatory polypeptides present in the TMMPs of the present disclosure contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions compared to the corresponding reference (e.g., wild-type) immunomodulatory polypeptide. In some examples, the variant immunomodulatory polypeptides present in the TMMPs of the present disclosure contain a single amino acid substitution compared to the corresponding reference (e.g., wild-type) immunomodulatory polypeptide. In some examples, the variant immunomodulatory polypeptides present in the TMMPs of the present disclosure contain two amino acid substitutions (e.g., no more than two amino acid substitutions) compared to the corresponding reference (e.g., wild-type) immunomodulatory polypeptide. In some examples, a variant immunomodulatory polypeptide present in a TMMP of the present disclosure comprises three amino acid substitutions (e.g., no more than three amino acid substitutions) compared to a corresponding reference (e.g., wild-type) immunomodulatory polypeptide. In some examples, a variant immunomodulatory polypeptide present in a TMMP of the present disclosure comprises four amino acid substitutions (e.g., no more than four amino acid substitutions) compared to a corresponding reference (e.g., wild-type) immunomodulatory polypeptide.In some examples, a variant immunomodulatory polypeptide present in a TMMP of the present disclosure comprises five amino acid substitutions (e.g., five or fewer amino acid substitutions) compared to a corresponding reference (e.g., wild-type) immunomodulatory polypeptide. In some examples, a variant immunomodulatory polypeptide present in a TMMP of the present disclosure comprises six amino acid substitutions (e.g., six or fewer amino acid substitutions) compared to a corresponding reference (e.g., wild-type) immunomodulatory polypeptide. In some examples, a variant immunomodulatory polypeptide present in a TMMP of the present disclosure comprises seven amino acid substitutions (e.g., seven or fewer amino acid substitutions) compared to a corresponding reference (e.g., wild-type) immunomodulatory polypeptide. In some examples, a variant immunomodulatory polypeptide present in a TMMP of the present disclosure comprises eight amino acid substitutions (e.g., eight or fewer amino acid substitutions) compared to a corresponding reference (e.g., wild-type) immunomodulatory polypeptide. In some examples, a variant immunomodulatory polypeptide present in a TMMP of the present disclosure comprises nine amino acid substitutions (e.g., nine or fewer amino acid substitutions) compared to a corresponding reference (e.g., wild-type) immunomodulatory polypeptide. In some examples, a variant immunomodulatory polypeptide present in a TMMP of the present disclosure comprises 10 amino acid substitutions (e.g., 10 or fewer amino acid substitutions) compared to a corresponding reference (e.g., wild-type) immunomodulatory polypeptide.

[0288] In some examples, a variant immunomodulatory polypeptide present in a TMMP of the present disclosure comprises 11 amino acid substitutions (e.g., 11 or fewer amino acid substitutions) compared to a corresponding reference (e.g., wild-type) immunomodulatory polypeptide.

[0289] In some examples, a variant immunomodulatory polypeptide present in a TMMP of the present disclosure comprises 12 amino acid substitutions (e.g., 12 or fewer amino acid substitutions) compared to a corresponding reference (e.g., wild-type) immunomodulatory polypeptide.

[0290] In some examples, a variant immunomodulatory polypeptide present in a TMMP of the present disclosure comprises 13 amino acid substitutions (e.g., 13 or fewer amino acid substitutions) compared to a corresponding reference (e.g., wild-type) immunomodulatory polypeptide.

[0291] In some examples, a variant immunomodulatory polypeptide present in a TMMP of the present disclosure comprises 14 amino acid substitutions (e.g., 14 or fewer amino acid substitutions) compared to a corresponding reference (e.g., wild-type) immunomodulatory polypeptide.

[0292] In some examples, a variant immunomodulatory polypeptide present in a TMMP of the present disclosure comprises 15 amino acid substitutions (e.g., 15 or fewer amino acid substitutions) compared to a corresponding reference (e.g., wild-type) immunomodulatory polypeptide.

[0293] In some examples, a variant immunomodulatory polypeptide present in a TMMP of the present disclosure comprises 16 amino acid substitutions (e.g., 16 or fewer amino acid substitutions) compared to a corresponding reference (e.g., wild-type) immunomodulatory polypeptide.

[0294] In some examples, a variant immunomodulatory polypeptide present in a TMMP of the present disclosure comprises 17 amino acid substitutions (e.g., 17 or fewer amino acid substitutions) compared to a corresponding reference (e.g., wild-type) immunomodulatory polypeptide.

[0295] In some examples, a variant immunomodulatory polypeptide present in a TMMP of the present disclosure comprises 18 amino acid substitutions (e.g., 18 or fewer amino acid substitutions) compared to a corresponding reference (e.g., wild-type) immunomodulatory polypeptide.

[0296] In some examples, a variant immunomodulatory polypeptide present in a TMMP of the present disclosure comprises 19 amino acid substitutions (e.g., 19 or fewer amino acid substitutions) compared to a corresponding reference (e.g., wild-type) immunomodulatory polypeptide.

[0297] In some examples, a variant immunomodulatory polypeptide present in a TMMP of the present disclosure comprises 20 amino acid substitutions (e.g., 20 or fewer amino acid substitutions) compared to a corresponding reference (e.g., wild-type) immunomodulatory polypeptide.

[0298] As discussed above, variant immunomodulatory polypeptides suitable for inclusion in the TMMPs of the present disclosure exhibit reduced affinity for related co-immunomodulatory polypeptides compared to the affinity of the corresponding wild-type immunomodulatory polypeptide for the related co-immunomodulatory polypeptide.

[0299] Exemplary pairs of immunomodulatory polypeptides and related co-immunomodulatory polypeptides include, but are not limited to, the following: a) 4-1BBL (immunomodulating polypeptide) and 4-1BB (related co-immunomodulating polypeptide); b) PD-L1 (immunomodulatory polypeptide) and PD1 (related co-immunomodulatory polypeptide); c) IL-2 (an immunomodulatory polypeptide) and IL-2 receptor (a related co-immunomodulatory polypeptide); d) CD80 (immunomodulating polypeptide) and CD86 (related co-immunomodulating polypeptide); e) CD86 (immunomodulating polypeptide) and CD28 (related co-immunomodulating polypeptide); f) OX40L (CD252) (immunomodulating polypeptide) and OX40 (CD134) (related co-immunomodulating polypeptide); g) Fas ligand (an immunomodulatory polypeptide) and Fas (a related co-immunomodulatory polypeptide); h) ICOS-L (immunomodulating polypeptide) and ICOS (related co-immunomodulating polypeptide); i) ICAM (immunomodulating polypeptide) and LFA-1 (related co-immunomodulating polypeptide); j) CD30L (immunomodulating polypeptide) and CD30 (related co-immunomodulating polypeptide); k) CD40 (immunomodulating polypeptide) and CD40L (related co-immunomodulating polypeptide); l) CD83 (immunomodulating polypeptide) and CD83L (related co-immunomodulating polypeptide); m) HVEM (CD270) (immunomodulating polypeptide) and CD160 (related co-immunomodulating polypeptide); n) JAG1 (CD339) (immunomodulating polypeptide) and Notch (related co-immunomodulating polypeptide); o) JAG1 (an immunomodulatory polypeptide) and CD46 (a related co-immunomodulatory polypeptide); p) CD80 (immunomodulating polypeptide) and CTLA4 (related co-immunomodulating polypeptide); q) CD86 (an immunomodulatory polypeptide) and CTLA4 (a related co-immunomodulatory polypeptide), and r) CD70 (immunomodulating polypeptide) and CD27 (related co-immunomodulating polypeptide).

[0300] In some examples, variant immunomodulatory polypeptides present in a TMMP of the disclosure have a binding affinity for a related co-immunomodulatory polypeptide of between 100 nM and 100 μM. For example, in some examples, variant immunomodulatory polypeptides present in a TMMP of the disclosure have a binding affinity for a related co-immunomodulatory polypeptide of between about 100 nM and 150 nM, between about 150 nM and about 200 nM, between about 200 nM and about 250 nM, between about 250 nM and about 300 nM, between about 300 nM and about 350 nM, between about 350 nM and about 400 nM, between about 400 nM and about 500 nM, between about 500 nM and about It has a binding affinity of 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.

[0301] Variant immunomodulatory polypeptides present in TMMPs of the present disclosure exhibit low affinity for related co-immunomodulatory polypeptides. Similarly, TMMPs of the present disclosure that include variant immunomodulatory polypeptides exhibit low affinity for related co-immunomodulatory polypeptides. Thus, for example, a TMMP of the present disclosure that includes a variant immunomodulatory polypeptide has a binding affinity of 100 nM to 100 μM for related co-immunomodulatory polypeptides. For example, in some examples, a TMMP of the present disclosure that includes a variant immunomodulatory polypeptide has a binding affinity 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 related co-immunomodulatory polypeptides. The antibody has a binding affinity of 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.

[0302] PD-L1 variant As one non-limiting example, in some instances, the variant immunomodulatory polypeptide present in a TMMP of the disclosure is a variant PD-L1 polypeptide. Wild-type PD-L1 binds to PD1.

[0303] A wild-type human PD-L1 polypeptide may comprise the following amino acid sequence: MRIFAVFIFM TYWHLLNAFT VTVPKDLYVV EYGSNMTIEC KFPVEKQLDL AALIVYWEME DKNIIQFVHG EEDLKVQHSS YRQRARLLKD QLSLGNAALQ ITDVKLQDAG VYRCMISYGG ADYKRITVKV NAPYNKINQR ILVVDPVTSE HELTCQAEGY PKAEVIWTSS DHQVLSGKTT TTNSKREEKL FNVTSTLRIN TTTNEIFYCT FRRLDPEENH TAELVIPGNI LNVSIKICLT LSPST (SEQ ID NO: 1).

[0304] The wild-type human PD-L1 ectodomain may comprise the following amino acid sequence: FT VTVPKDLYVV EYGSNMTIEC KFPVEKQLDL AALIVYWEME DKNIIQFVHG EEDLKVQHSS YRQRARLLKD QLSLGNAALQ ITDVKLQDAG VYRCMISYGG ADYKRITVKV NAPYNKINQR ILVVDPVTSE HELTCQAEGY PKAEVIWTSS DHQVLSGKTT TTNSKREEKL FNVTSTLRIN TTTNEIFYCT FRRLDPEENH TAELVIPGNI LNVSIKI (SEQ ID NO: 2).

[0305] A wild-type PD-1 polypeptide may comprise the following amino acid sequence: PGWFLDSPDR PWNPPTFSPA LLVVTEGDNA TFTCSFSNTS ESFVLNWYRM SPSNQTDKLA AFPEDRSQPG QDCRFRVTQL PNGRDFHMSV VRARRNDSGT YLCGAISLAP KAQIKESLRA ELRVTERRAE VPTAHPSPSP RPAGQFQTLV VGVVGGLLGS LVLLVWVLAV ICSRAARGTI GARRTGQPLK EDPSAVPVFS VDYGELDFQW REKTPEPPVP CVPEQTEYAT IVFPSGMGTS SPARRGSADG PRSAQPLRPE DGHCSWPL (SEQ ID NO: 3). In some examples, when a TMMP of the disclosure comprises a variant PD-L1 polypeptide, the "related co-immunomodulatory polypeptide" is a PD-1 polypeptide comprising the amino acid sequence of SEQ ID NO: 3.

[0306] In some examples, variant PD-L1 polypeptides exhibit reduced binding affinity to PD-1 (e.g., a PD-1 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 3) compared to the binding affinity of a PD-L1 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2. For example, in some examples, variant PD-L1 polypeptides of the present disclosure bind to PD-1 (e.g., a PD-1 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 3) with a binding affinity that is at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% less, at least 55% less, at least 60% less, at least 65% less, at least 70% less, at least 75% less, at least 80% less, at least 85% less, at least 90% less, at least 95% less, or greater than 95% less than the binding affinity of a PD-L1 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2.

[0307] In some examples, variant PD-L1 polypeptides have a binding affinity to PD-1 of between 1 nM and 1 mM. In some examples, variant PD-L1 polypeptides of the present disclosure have a binding affinity to PD-1 of between 100 nM and 100 μM. As another example, in some examples, variant PD-L1 polypeptides have a binding affinity to PD-1 of between about 100 nM and 150 nM, between about 150 nM and about 200 nM, between about 200 nM and about 250 nM, between about 250 nM and about 300 nM, between about 300 nM and about 350 nM, between about 350 nM and about 400 nM, between about 400 nM and about 500 nM, between about It has a binding affinity of 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.

[0308] In some examples, the variant PD-L1 polypeptide has a single amino acid substitution compared to the PD-L1 amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2. In some examples, the variant PD-L1 polypeptide has between 2 and 10 amino acid substitutions compared to the PD-L1 amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2. In some examples, the variant PD-L1 polypeptide has two amino acid substitutions compared to the PD-L1 amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2. In some examples, the variant PD-L1 polypeptide has three amino acid substitutions compared to the PD-L1 amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2. In some examples, the variant PD-L1 polypeptide has four amino acid substitutions compared to the PD-L1 amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2. In some examples, the variant PD-L1 polypeptide has five amino acid substitutions compared to the PD-L1 amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2. In some examples, the variant PD-L1 polypeptide has six amino acid substitutions compared to the PD-L1 amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2. In some examples, the variant PD-L1 polypeptide has 7 amino acid substitutions compared to the PD-L1 amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2. In some examples, the variant PD-L1 polypeptide has 8 amino acid substitutions compared to the PD-L1 amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2. In some examples, the variant PD-L1 polypeptide has 9 amino acid substitutions compared to the PD-L1 amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2. In some examples, the variant PD-L1 polypeptide has 10 amino acid substitutions compared to the PD-L1 amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2.

[0309] Suitable PD-L1 variants include polypeptides comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: TIFF0007762068000077.tif24161, in which X is any amino acid other than Asp. In some examples, X is Ala. In some examples, X is Arg.

[0310] Suitable PD-L1 variants include polypeptides comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: TIFF0007762068000078.tif24161, in which X is any amino acid other than Ile. In some examples, X is Asp.

[0311] Suitable PD-L1 variants include polypeptides comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: TIFF0007762068000079.tif24161, in which X is any amino acid other than Glu. In some examples, X is Arg.

[0312] CD80 variant In some examples, the variant immunomodulatory polypeptide present in the TMMP of the present disclosure is a variant CD80 polypeptide. Wild-type CD80 binds to CD28. Wild-type CD80 also binds to CD86.

[0313] The wild-type amino acid sequence of the ectodomain of human CD80 may be as follows: VIHVTK EVKEVATLSC GHNVSVEELA QTRIYWQKEK KMVLTMMSGD MNIWPEYKNR TIFDITNNLS IVILALRPSD EGTYECVVLK YEKDAFKREH LAEVTLSVKA DFPTPSISDF EIPTSNIRRI ICSTSGGFPE PHLSWLENGE ELNAINTTVS QDPETELYAV SSKLDFNMTT NHSFMCLIKY GHLRVNQTFN WNTTKQEHFP DN (SEQ ID NO: 4).

[0314] The wild-type CD28 amino acid sequence can be: MLRLLLALNL FPSIQVTGNK ILVKQSPMLV AYDNAVNLSC KYSYNLFSRE FRASLHKGLD SAVEVCVVYG NYSQQLQVYS KTGFNCDGKL GNESVTFYLQ NLYVNQTDIY FCKIEVMYPP PYLDNEKSNG TIIHVKGKHL CPSPLFPGPS KPFWVLVVVG GVLACYSLLV TVAFIIFWVR SKRSRLLHSD YMNMTPRRPG PTRKHYQPYA PPRDFAAYRS (SEQ ID NO:5). In some examples, when a TMMP of the disclosure comprises a variant CD80 polypeptide, the "related co-immunomodulatory polypeptide" is a CD28 polypeptide comprising the amino acid sequence of SEQ ID NO:5.

[0315] The wild-type CD28 amino acid sequence can be: MLRLLLALNL FPSIQVTGNK ILVKQSPMLV AYDNAVNLSW KHLCPSPLFP GPSKPFWVLV VVGGVLACYS LLVTVAFIIF WVRSKRSRLL HSDYMNMTPR RPGPTRKHYQ PYAPPRDFAA YRS (SEQ ID NO: 6).

[0316] The wild-type CD28 amino acid sequence can be: MLRLLLALNL FPSIQVTGKH LCPSPLFPGP SKPFWVLVVV GGVLACYSLL VTVAFIIFWV RSKRSRLLHS DYMNMTPRRP GPTRKHYQPY APPRDFAAYR S (SEQ ID NO: 7).

[0317] In some examples, the variant CD80 polypeptide exhibits reduced binding affinity for CD28 compared to the binding affinity for CD28 of a CD80 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 4. For example, in some examples, the variant CD80 polypeptide binds to CD28 with a binding affinity that is at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% less, at least 55% less, at least 60% less, at least 65% less, at least 70% less, at least 75% less, at least 80% less, at least 85% less, at least 90% less, at least 95% less, or more than 95% less than the binding affinity of a CD80 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 4 for CD28 (e.g., a CD28 polypeptide comprising the amino acid sequence set forth in one of SEQ ID NOs: 5, 6, or 7).

[0318] In some examples, the variant CD80 polypeptide has a binding affinity for CD28 of 100 nM to 100 μM. As another example, in some examples, the variant CD80 polypeptide of the present disclosure has a binding affinity for CD28 (e.g., a CD28 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7) of about 100 nM to 150 nM, about 150 nM to about 200 nM, about 200 nM to about 250 nM, about 250 nM to about 300 nM, about 300 nM to about 350 nM, about 350 nM to about 400 nM, about 400 nM to about 500 nM, or about 500 nM to about 600 nM. The antibody has a binding affinity of about 1 μM to about 500 nM, about 500 nM to about 600 nM, about 600 nM to about 700 nM, about 700 nM to about 800 nM, about 800 nM to about 900 nM, about 900 nM to about 1 μM, 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.

[0319] In some examples, the variant CD80 polypeptide has a single amino acid substitution compared to the CD80 amino acid sequence set forth in SEQ ID NO: 4. In some examples, the variant CD80 polypeptide has two to ten amino acid substitutions compared to the CD80 amino acid sequence set forth in SEQ ID NO: 4. In some examples, the variant CD80 polypeptide has two amino acid substitutions compared to the CD80 amino acid sequence set forth in SEQ ID NO: 4. In some examples, the variant CD80 polypeptide has three amino acid substitutions compared to the CD80 amino acid sequence set forth in SEQ ID NO: 4. In some examples, the variant CD80 polypeptide has four amino acid substitutions compared to the CD80 amino acid sequence set forth in SEQ ID NO: 4. In some examples, the variant CD80 polypeptide has five amino acid substitutions compared to the CD80 amino acid sequence set forth in SEQ ID NO: 4. In some examples, the variant CD80 polypeptide has six amino acid substitutions compared to the CD80 amino acid sequence set forth in SEQ ID NO: 4. In some examples, the variant CD80 polypeptide has seven amino acid substitutions compared to the CD80 amino acid sequence set forth in SEQ ID NO: 4. In some examples, the variant CD80 polypeptide has 8 amino acid substitutions compared to the CD80 amino acid sequence set forth in SEQ ID NO: 4. In some examples, the variant CD80 polypeptide has 9 amino acid substitutions compared to the CD80 amino acid sequence set forth in SEQ ID NO: 4. In some examples, the variant CD80 polypeptide has 10 amino acid substitutions compared to the CD80 amino acid sequence set forth in SEQ ID NO: 4.

[0320] Suitable CD80 variants include polypeptides comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to any one of the following amino acid sequences: TIFF0007762068000080.tif24155, in which X is any amino acid other than Asn. In some instances, X is Ala; TIFF0007762068000081.tif24155, in which X is any amino acid other than Asn. In some instances, X is Ala; TIFF0007762068000082.tif24157, in which X is any amino acid other than Ile. In some instances, X is Ala; TIFF0007762068000083.tif24155, in which X is any amino acid other than Lys. In some instances, X is Ala; TIFF0007762068000084.tif24155, in which X is any amino acid other than Gln. In some instances, X is Ala; TIFF0007762068000085.tif24155, in which X is any amino acid other than Asp. In some instances, X is Ala; TIFF0007762068000086.tif24155, in which X is any amino acid other than Leu. In some instances, X is Ala; TIFF0007762068000087.tif24155, in which X is any amino acid other than Tyr. In some instances, X is Ala; TIFF0007762068000088.tif24155, in which X is any amino acid other than Gln. In some instances, X is Ala; TIFF0007762068000089.tif24155, in which X is any amino acid other than Met. In some instances, X is Ala; TIFF0007762068000090.tif24155, in which X is any amino acid other than Val. In some instances, X is Ala; TIFF0007762068000091.tif24157, in which X is any amino acid other than Ile. In some instances, X is Ala; TIFF0007762068000092.tif24155, in which X is any amino acid other than Tyr. In some instances, X is Ala; TIFF0007762068000093.tif24155, in which X is any amino acid other than Asp. In some instances, X is Ala; TIFF0007762068000094.tif24155, in which X is any amino acid other than Phe. In some instances, X is Ala; TIFF0007762068000095.tif24155, in which X is any amino acid other than Ser. In some examples, X is Ala; and TIFF0007762068000096.tif24155, in which X is any amino acid other than Pro. In some examples, X is Ala.

[0321] CD86 variant In some examples, the variant immunomodulatory polypeptide present in a TMMP of the present disclosure is a variant CD86 polypeptide. Wild-type CD86 binds to CD28. In some examples, when a TMMP of the present disclosure comprises a variant CD86 polypeptide, the "related co-immunomodulatory polypeptide" is a CD28 polypeptide comprising the amino acid sequence of SEQ ID NO:5.

[0322] The amino acid sequence of the complete ectodomain of wild-type human CD86 may be as follows: TIFF0007762068000097.tif24164.

[0323] The ami...

Claims

1. (a) in order from the N-terminus to the C-terminus: (i) a peptide epitope representing an epitope of a viral antigen, the peptide epitope having a length of 8 amino acids to 15 amino acids; and (ii) β2M polypeptide a first polypeptide comprising: (b) in order from the N-terminus to the C-terminus: (i) a polypeptide that binds to a cancer-associated antigen (cancer targeting polypeptide); (ii) an MHC class I heavy chain polypeptide; (iii) an immunoglobulin (Ig) Fc polypeptide or a non-Ig scaffold; and (iv) one or more activated immunomodulatory polypeptides, each of said activated immunomodulatory polypeptides is a variant IL-2 polypeptide comprising an amino acid sequence having at least 95% amino acid sequence identity to SEQ ID NO:534, wherein the amino acid at position 16 is other than His and the amino acid at position 42 is other than Phe, and each of said activated immunomodulatory polypeptides is a variant IL-2 polypeptide that binds to the IL-2 receptor (IL-2R) with a binding affinity that is at least 10% less than the binding affinity of wild-type IL-2 for the IL-2R. and a second polypeptide comprising Including, one or more independently selected linkers are disposed between one or more components of said first polypeptide and said second polypeptide; When the TMMP comprises two or more activated immunomodulatory polypeptides, the TMMP comprises one or more linkers between the activated immunomodulatory polypeptides; the first polypeptide and the second polypeptide are covalently bound to each other via at least one disulfide bond; Heterodimeric T-cell regulatory multimeric polypeptides (TMMPs) that bind to cancer-associated antigens.

2. The TMMP of claim 1 , wherein the Ig Fc polypeptide is an IgG1 Fc polypeptide.

3. The TMMP of claim 2, wherein the IgG1 Fc polypeptide comprises one or more amino acid substitutions selected from N77A, L14A, L15A, L14F, L15E, and P111S based on the numbering of SEQ ID NO:

19.

4. the variant IL-2 polypeptide comprises an amino acid sequence having at least 95% amino acid sequence identity to SEQ ID NO: 15; The variant IL-2 polypeptide is (i) an H16A substitution and an F42A substitution, or (ii) H16T substitution and F42A substitution Including, The TMMP according to claim 1.

5. the TMMP comprises at least a first and a second disulfide bond; the first disulfide bond is (i) a Cys residue in the linker between the peptide epitope and the β2M polypeptide; (ii) a Cys residue in the MHC class I heavy chain polypeptide; is formed between the second disulfide bond is a Cys residue in the β2M polypeptide; a Cys residue in the MHC class I heavy chain polypeptide; formed between The TMMP according to any one of claims 1 to 4.

6. The TMMP of any one of claims 1 to 5, wherein the peptide epitope represents an epitope of cytomegalovirus, Epstein-Barr virus, or coronavirus.

7. The TMMP of any one of claims 1 to 6, wherein the cancer targeting polypeptide is an antibody or antibody fragment that retains specific binding to an antigen.

8. The TMMP of any one of claims 1 to 7, wherein the cancer targeting polypeptide is specific for Her2, CD19, WT1, MUC1, BCMA, mesothelin, or a claudin polypeptide.

9. The TMMP is (a)(i) the peptide epitope; (ii) an optional linker, and (iii) the β2M polypeptide a first polypeptide comprising: (b)(i) the cancer targeting polypeptide; (ii) an optional linker; (iii) the MHC class I heavy chain polypeptide; (iv) an optional linker; (v) the Ig Fc polypeptide; (vi) an optional linker, and (vii) the one or more activating immunomodulatory polypeptides a second polypeptide comprising Including, When comprising two or more activating immunomodulatory polypeptides, the activating immunomodulatory polypeptides may comprise one or more independently selected linkers between said activating immunomodulatory polypeptides. TMMP according to any one of claims 1 to 8.

10. below: (i) the peptide epitope; (ii) an optional linker; (iii) the β2M polypeptide a first polypeptide comprising: below: (i) the cancer targeting polypeptide; (ii) an optional linker; (iii) the MHC class I heavy chain polypeptide; (iv) an optional linker; (v) the immunoglobulin (Ig) Fc polypeptide; (vi) an optional linker; (vii) the variant IL-2 polypeptide; (viii) an optional linker, and (ix) the variant IL-2 polypeptide a second polypeptide comprising 10. The TMMP of claim 9, comprising:

11. the TMMP comprises two activated immunomodulatory polypeptides in tandem and joined by a linker; the MHC class I heavy chain polypeptide comprises an HLA-A, HLA-B, HLA-C, or HLA-E heavy chain polypeptide; the TMMP comprises at least a first and a second disulfide bond; The first disulfide bond is first (i) a Cys residue in the linker between the peptide epitope and the β2M polypeptide; (ii) a Cys residue at position 84 in the MHC class I heavy chain polypeptide; is formed between the second disulfide bond is the Cys residue at position 12 of the β2M polypeptide; the Cys residue at position 236 of the MHC class I heavy chain polypeptide; is formed between the Ig Fc polypeptide is an IgG1 polypeptide comprising one or more amino acid substitutions selected from N77A, L14A, L15A, L14F, L15E, and P111S based on the numbering of SEQ ID NO: 19; the peptide epitope represents an epitope of cytomegalovirus, Epstein-Barr virus, or coronavirus; TMMP according to claim 9 or 10.

12. each of said activating immunomodulatory polypeptides is a variant IL-2 polypeptide comprising the amino acid sequence of SEQ ID NO: 534, wherein: X 1 is Ala and X ２ A TMMP comprising two heterodimers comprising the first polypeptide and the second polypeptide of claim 1, wherein is Ala.

13. A TMMP comprising two heterodimers described in claim 12, wherein the two heterodimers are covalently bonded to each other by one or more disulfide bonds between their Ig Fc polypeptides.

14. A TMMP comprising two heterodimers described in claim 12 or 13, wherein the peptide epitope represents an epitope of coronavirus or cytomegalovirus (CMV).

15. A composition comprising one or more nucleic acids comprising nucleotide sequences encoding the first and second polypeptides of the TMMP according to any one of claims 1 to 14.

16. A method for producing TMMP, comprising culturing a host cell genetically modified with a nucleotide sequence encoding the first and second polypeptides of TMMP described in any one of claims 1 to 14 under conditions such that the genetically modified host cell produces TMMP.

17. A pharmaceutical composition comprising the TMMP of any one of claims 1 to 14 for use in the treatment of cancer in a human patient.

18. The pharmaceutical composition comprising the TMMP of claim 17, wherein the cancer is a blood cancer.

19. The pharmaceutical composition comprising the TMMP of claim 17, wherein the cancer is a solid tumor.

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