Method for regulating an immune response

Abstract

The present disclosure provides methods for modulating an immune response in an individual. The present disclosure provides methods of treatment. The present disclosure provides methods comprising administering to an individual a multimeric polypeptide (synTac) and an immune checkpoint inhibitor. The present disclosure provides methods comprising administering to an individual a multimeric polypeptide (synTac) that is undergoing treatment with an immune checkpoint inhibitor. [Selection diagram] None

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 62 / 471,832, filed Mar. 15, 2017, and U.S. Provisional Patent Application No. 62 / 521,009, filed Jun. 16, 2017, the entire disclosures of which are hereby incorporated by reference herein.

Background Art

[0002] Introduction The adaptive immune response requires the binding of a T - cell receptor (TCR) present on the surface of T cells to small peptide antigens non - covalently presented on the surface of antigen - presenting cells (APCs) by the major histocompatibility complex (MHC; in humans, also referred to as the human leukocyte antigen (HLA) complex). This binding represents the targeting mechanism of the immune system and is the molecular interaction necessary for the regulation (activation or suppression) and effector functions of T cells. Following epitope - specific cell targeting, the targeted T cells are activated through the binding of co - stimulatory proteins on the APCs to the corresponding co - stimulatory proteins on the T cells. For T - cell specificity and activation or suppression, signals from both epitope / TCR binding and the binding of APC co - stimulatory proteins to T - cell co - stimulatory proteins are required. The TCR is specific for a particular epitope, while co - stimulatory proteins are not epitope - specific but rather are widely expressed on all T cells or large subsets of T cells.

Summary of the Invention

[0003] Summary The present disclosure provides methods of modulating an individual's immune response. The present disclosure provides methods of treatment. The present disclosure provides methods comprising administering to an individual a multimeric polypeptide (synTac) and an immune checkpoint inhibitor. The present disclosure provides methods comprising administering a multimeric polypeptide (synTac) to an individual undergoing treatment with an immune checkpoint inhibitor. [The present invention 1001] A method of modulating an immune response in an individual in need thereof, comprising administering to the individual a multimeric polypeptide and an immune checkpoint inhibitor, wherein the multimeric polypeptide a) in order from the N-terminus to the C-terminus, i) an epitope, ii) a first major histocompatibility complex (MHC) polypeptide and a first polypeptide, b) in order from the N-terminus to the C-terminus, i) a second MHC polypeptide, and ii) optionally, an immunoglobulin (Ig) Fc polypeptide or a non-Ig backbone and a second polypeptide and, the multimeric polypeptide comprises one or more immunomodulatory polypeptides, and the one or more immunomodulatory polypeptides are 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) located at the C-terminus of the first polypeptide and the N-terminus of the second polypeptide, wherein the administering modulates the immune response in the individual, the method. [The present invention 1002] wherein the multimeric polypeptide a) in order from the N-terminus to the C-terminus, i) an epitope, ii) a first MHC polypeptide, and iii) an immunomodulatory domain and a first polypeptide, b) in order from the N-terminus to the C-terminus, i) a second MHC polypeptide, and ii) an Ig Fc polypeptide and a second polypeptide and, the method of the present invention 1001. [The present invention 1003] wherein the multimeric polypeptide a) in order from the N-terminus to the C-terminus, i) an epitope, and ii) a first MHC polypeptide and a first polypeptide, b) in order from the N-terminus to the C-terminus, i) an immunomodulatory domain, iii) a second MHC polypeptide, and ii) an immunoglobulin (Ig) Fc polypeptide and a second polypeptide and, the method of the present invention 1001. [The present invention 1004] wherein the multimeric polypeptide a) in order from the N-terminus to the C-terminus, i) an epitope, and ii) a first MHC polypeptide and a first polypeptide, b) in order from the N-terminus to the C-terminus, i) a second MHC polypeptide, and ii) an Ig Fc polypeptide, and iii) an immunomodulatory domain and a second polypeptide and, the method of the present invention 1001. [The present invention 1005] wherein the multimeric polypeptide a) in order from the N-terminus to the C-terminus, i) an epitope, and ii) a first MHC polypeptide A first polypeptide comprising b) In the order from the N-terminus to the C-terminus i) a second MHC polypeptide, and ii) an immunomodulatory domain A second polypeptide comprising The method of the present invention 1001 comprising [The present invention 1006] The multimeric polypeptide is a) In the order from the N-terminus to the C-terminus i) an epitope, and ii) a first MHC polypeptide A first polypeptide comprising b) In the order from the N-terminus to the C-terminus i) an immunomodulatory domain, and ii) a second MHC polypeptide A second polypeptide comprising The method of the present invention 1001 comprising [The present invention 1007] The multimeric polypeptide is a) In the order from the N-terminus to the C-terminus i) an epitope, ii) a first MHC polypeptide, and iii) an immunomodulatory domain A first polypeptide comprising b) In the order from the N-terminus to the C-terminus i) a second MHC polypeptide A second polypeptide comprising The method of the present invention 1001 comprising [The present invention 1008] The non-Ig backbone of the multimeric polypeptide is an XTEN polypeptide, a transferrin polypeptide, an Fc receptor polypeptide, an elastin-like polypeptide, a silk-like polypeptide, or a silk-elastin-like polypeptide, the method of the present invention 1001. [The present invention 1009] The first MHC polypeptide of the multimeric polypeptide is a β2-microglobulin polypeptide, and the second MHC polypeptide is an MHC class I heavy chain polypeptide, the method according to any one of the present inventions 1001 to 1008. [The present invention 1010] The β2-microglobulin polypeptide of the multimeric polypeptide comprises an amino acid sequence having at least 85% amino acid sequence identity to one of the amino acid sequences described in FIG. 6, the method of the present invention 1009. [The present invention 1011] The MHC class I heavy chain polypeptide of the multimeric polypeptide is a heavy chain of HLA-A, HLA-B or HLA-C, the method of the present invention 1009. [The present invention 1012] The MHC class I heavy chain polypeptide comprises an amino acid sequence having at least 85% amino acid sequence identity to the amino acid sequence described in one of FIGS. 5A to 5C, the method of the present invention 1011. [The present invention 1013] Any of the methods of the present invention 1001 to 1008, wherein the first MHC polypeptide of the multimeric polypeptide is an α-chain polypeptide of MHC class II and the second MHC polypeptide is a β-chain polypeptide of MHC class II. [The present invention 1014] Any of the methods of the present invention 1001 to 1013, wherein the epitope is a T cell epitope. [The present invention 1015] Any of the methods of the present invention 1001 to 1007, wherein the multimeric polypeptide of the multimeric polypeptide comprises an Fc polypeptide, and the Ig Fc polypeptide is an IgG1 Fc polypeptide, an IgG2 Fc polypeptide, an IgG3 Fc polypeptide, an IgG4 Fc polypeptide, an IgA Fc polypeptide, or an IgM Fc polypeptide. [The present invention 1016] The method of the present invention 1015, wherein the Ig Fc polypeptide comprises an amino acid sequence having at least 85% amino acid sequence identity to the amino acid sequence shown in FIGS. 4A to 4C. [The present invention 1017] Any of the methods of the present invention 1001 to 1016, wherein the first polypeptide and the second polypeptide of the multimeric polypeptide are non-covalently associated. [The present invention 1018] Any of the methods of the present invention 1001 to 1016, wherein the first polypeptide and the second polypeptide of the multimeric polypeptide are covalently linked. [The present invention 1019] The method of the present invention 1013, wherein the covalent bond is by a disulfide bond. [The present invention 1020] The method of the present invention 1019, wherein the first MHC polypeptide of the multimeric polypeptide, or the linker between the epitope and the first MHC polypeptide, comprises an amino acid substitution that results in a first Cys residue, the second MHC polypeptide of the multimeric polypeptide comprises an amino acid substitution that results in a second Cys residue, and the disulfide linkage is between the first Cys residue and the second Cys residue. [The present invention 1021] Any of the methods of the present invention 1001 to 1008, wherein the multimeric polypeptide comprises a linker between the epitope and the first MHC polypeptide, between the immunomodulatory polypeptide and the MHC polypeptide, or between the MHC polypeptide and the Ig Fc. [The present invention 1022] The immunomodulatory polypeptide of the multimeric polypeptide is selected from 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, and a PD-L2 polypeptide, and is any of the methods of the present invention from 1001 to 1008. [The present invention 1023] The method according to any one of the present inventions 1001 to 1022, wherein the multimeric polypeptide comprises two or more immunomodulatory polypeptides. [The present invention 1024] The method of the present invention 1023, wherein the two or more immunomodulatory polypeptides are arranged tandemly. [The present invention 1025] The immunomodulatory polypeptide is selected from a 4-1BBL polypeptide, a CD80 polypeptide, a CD86 polypeptide, an IL-2 polypeptide, a B7-1 polypeptide, a B7-2 polypeptide, an ICOS-L polypeptide, an OX-40L polypeptide, a CD86 polypeptide, a PD-L1 polypeptide, a FasL polypeptide, and a PD-L2 polypeptide, and is any of the methods of the present invention from 1001 to 1024. [The present invention 1026] The immunomodulatory polypeptide is a mutant immunomodulatory polypeptide, has one or more amino acid substitutions as compared with the native form of the immunomodulatory polypeptide, and the mutant immunomodulatory polypeptide exhibits a decrease in binding affinity for a costimulatory polypeptide to which the native form of the immunomodulatory polypeptide binds, and is any of the methods of the present invention from 1001 to 1025. [The present invention 1027] The immunomodulatory polypeptide is a mutant IL-2 polypeptide comprising an amino acid sequence having at least 85% amino acid sequence identity to that set forth in SEQ ID NO: 1, the mutant IL-2 polypeptide has one or more amino acid substitutions as compared with that set forth in SEQ ID NO: 1, the mutant IL-2 polypeptide exhibits a decrease in binding affinity for an IL-2 receptor (IL2R) comprising α, β, and γ polypeptides having the amino acid sequences shown in FIGS. 3A to 3C as compared with the binding affinity of the IL-2 amino acid sequence set forth in one of SEQ ID NO: 1 for the IL2R, and is any of the methods of the present invention from 1001 to 1025. [The present invention 1028] The method of the present invention 1027, wherein the mutant IL-2 polypeptide comprises one or more substitutions among E15, H16, D20, F42, Y45 and Q126. [The present invention 1029] wherein the mutant IL-2 polypeptide a) substitution of F42 and D20, b) substitution of F42 and H16, c) substitution of F42, D20 and Y45, or d) substitution of F42, H16 and Q126 The method of the present invention 1028, comprising. [The present invention 1030] wherein the immunomodulatory polypeptide is a mutant 4-1BBL immunomodulatory polypeptide, wherein the mutant 4-1BBL immunomodulatory polypeptide exhibits a decrease in binding affinity compared to the binding affinity of the 4-1BB polypeptide having the amino acid sequence shown in FIG. 37 to the 4-1BBL amino acid sequence shown in FIG. 36A, and / or wherein the mutant 4-1BBL immunomodulatory polypeptide exhibits an increase in production level by mammalian cells compared to the production level of the 4-1BBL amino acid sequence shown in FIG. 36A, any method of the present invention 1001-1021. [The present invention 1031] The method of the present invention 1030, wherein the mutant 4-1BBL immunomodulatory polypeptide comprises one substitution among amino acids 91, 92, 94-115, 117-126, 128-132, 144-153, 155-158, 184-187, 189-191, 193-195, 197, 210-219, 221-224, 226, 228-231, 233 and 234 based on the amino acid numbering described in FIG. 36A. [The present invention 1032] The method of the present invention 1030 or 1031, wherein the mutant immunomodulatory polypeptide exhibits a binding affinity of less than 50% of the binding affinity exhibited by the 4-1BBL amino acid sequence shown in FIG. 36A or described in one of SEQ ID NOs: 213-215 with respect to the 4-1BB polypeptide. [The present invention 1033] The method of any one of the present invention 1001-1032, wherein the multimeric polypeptide comprises an Ig Fc polypeptide comprising one or more amino acid substitutions selected from N297A, L234A, L235A, L234F, L235E and P331S. [The present invention 1034] wherein the Ig Fc polypeptide a) N297A substitution, b) L234A substitution and L235A substitution, c) L234F substitution and L235E substitution, or d) L234F substitution, L235E substitution and P331S substitution The method of the present invention 1033, comprising. [The present invention 1035] Any method according to any one of inventions 1001 to 1034, wherein the epitope of the multimeric polypeptide comprises the amino acid sequence YMLDLQPETT (SEQ ID NO: 77). [Invention 1036] Any method according to any one of inventions 1001 to 1035, wherein the β2-microglobulin polypeptide of the multimeric polypeptide comprises the amino acid sequence shown in FIG. 34A. [Invention 1037] Any method according to any one of inventions 1001 to 1036, wherein the heavy chain polypeptide of the major histocompatibility complex (MHC) of the multimeric polypeptide comprises the amino acid sequence shown in FIG. 34C. [Invention 1038] Any method according to any one of inventions 1001 to 1037, wherein the immune checkpoint inhibitor is an antibody specific for the immune checkpoint inhibitor. [Invention 1039] The method of invention 1038, wherein the antibody is a monoclonal antibody. [Invention 1040] The method of invention 1038 or 1039, wherein the antibody comprises at least one humanized light chain and / or heavy chain framework region. [Invention 1041] The method of invention 1038, wherein the antibody comprises an Fc polypeptide, and the Ig Fc polypeptide is an IgG1 Fc polypeptide, an IgG2 Fc polypeptide, an IgG3 Fc polypeptide, an IgG4 Fc polypeptide, an IgA Fc polypeptide, or an IgM Fc polypeptide. [Invention 1042] The method of invention 1038, wherein the antibody is an Fv fragment, a nanobody, or a Fab fragment. [Invention 1043] Any method according to any one of inventions 1038 to 1042, wherein the immune checkpoint inhibitor is an antibody specific for an immune checkpoint inhibitor selected from CD27, CD28, CD40, CD122, CD96, CD73, CD47, OX40, GITR, CSF1R, JAK, PI3Kδ, PI3Kγ, TAM, arginase, CD137 (also known as 4-1BB), ICOS, A2AR, B7-H3, B7-H4, BTLA, CTLA-4, LAG3, TIM3, VISTA, CD96, TIGIT, CD122, PD-1, PD-L1, and PD-L2. [Invention 1044] Any method according to any one of inventions 1038 to 1042, wherein the immune checkpoint inhibitor is an antibody specific for PD1. [Invention 1045] The method of invention 1044, wherein the antibody is pembrolizumab, nivolumab, pidilizumab, or BMS-39886. [The present invention 1046] The method according to any one of inventions 1038 to 1042, wherein the immune checkpoint inhibitor is an antibody specific to PD-L1. [The present invention 1047] The method according to invention 1046, wherein the antibody is durvalumab, atezolizumab, KN035 or avelumab. [The present invention 1048] The method according to any one of inventions 1038 to 1042, wherein the immune checkpoint inhibitor is an antibody specific to CTLA4. [The present invention 1049] The method according to invention 1048, wherein the antibody is ipilimumab or tremelimumab. [The present invention 1050] The method according to any one of inventions 1001 to 1049, wherein the multimeric polypeptide and the immune checkpoint inhibitor are administered by the same administration route. [The present invention 1051] The method according to any one of inventions 1001 to 1049, wherein the multimeric polypeptide and the immune checkpoint inhibitor are administered by different administration routes. [The present invention 1052] The method according to any one of inventions 1001 to 1051, wherein the multimeric polypeptide is administered by an administration route selected from subcutaneous, intravenous, near the tumor and intramuscular. [The present invention 1053] The method according to any one of inventions 1001 to 1051, wherein the immune checkpoint inhibitor is administered by an administration route selected from subcutaneous, intravenous, near the tumor, and intramuscular. [The present invention 1054] The method according to any one of inventions 1001 to 1053, wherein the individual is a human.

Brief Description of the Drawings

[0004] [Figure 1] Various embodiments of the T cell regulatory multimer polypeptides are shown schematically. In these embodiments, disulfide bonds are formed between MHC (e.g., HLA) polypeptides present in separate polypeptides. [Figure 2] The amino acid sequence of wild-type human IL-2 is shown (Figure 2A), and the amino acid sequences of mutant IL-2 polypeptides are shown (Figures 2B - 2Q). [Figure 3] The amino acid sequences of the α-chain (Figure 3A), β-chain (Figure 3B), and γ-chain (Figure 3C) of the IL-2 receptor are shown. [Figure 4] The amino acid sequence of the immunoglobulin Fc polypeptide is shown. [Figure 5] The amino acid sequence of the human leukocyte antigen (HLA) class I heavy chain polypeptide is shown. The signal sequence is underlined. [Figure 6] Multiple amino acid sequence alignments of β-2 microglobulin (β2M) precursors (i.e., including the leader sequence) from Homo sapiens (NP_004039.1; SEQ ID NO: 95), Pan troglodytes (NP_001009066.1; SEQ ID NO: 195), Macaca mulatta (NP_001040602.1; SEQ ID NO: 96), Bos taurus (NP_776318.1; SEQ ID NO: 97), and Mus musculus (NP_033865.2; SEQ ID NO: 98) are shown. Amino acids 1 - 20 are the signal peptide. [Figure 7] Production of the IL-2 / synTac of the present disclosure (「Cue-IL-2-a」 and 「Cue-IL-2-b」) after transient transfection is shown. Figure 7A shows the unpurified yield, and Figure 7B shows the purified product. [Figure 8]Shows the production of IL-2 / synTac of the present disclosure. The IL-2 polypeptide is present on the light chain (a polypeptide chain containing the light chain of the MHC class I molecule (e.g., β2M)) or on the heavy chain (a polypeptide chain containing the heavy chain of the MHC class I molecule). [Figure 9] Shows the expression levels of IL-2 / syn-Tac. IL-2 is either wild-type (wt) or contains various combinations of F42A, D20K, Q126A, E15A, Y45A, and H16A. [Figure 10] Shows the expression of IL-2 / synTac of the present disclosure. IL-2 is present in 1 copy (1X), 2 copies (2X), or 3 copies (3X) in synTac. [Figure 11] Shows the in vitro stimulation of antigen-specific CD8+ T cells and non-specific CD8+ T cells by IL-2 / synTac of the present disclosure. The IL-2 variant containing the substitutions of F42A and H16A is present in 2 copies in synTac. [Figure 12] Shows the binding of IL-2 / synTac to specific (lymphocytic choriomeningitis virus; LCMV) or non-specific (OT1; recognizing ovalbumin) CD8+ T cells. [Figure 13] Shows the IL-2 / synTac-mediated signal transduction in antigen-specific (LCMV) or non-specific (BL6) CD8+ T cells. [Figure 14] Shows the percentage of phosphorylated signal transducer and activator of transcription 5 (pSTAT5)-positive cells after stimulation of CD8+ antigen-specific (LCMV) cells or non-specific (BL6) cells with IL-2 / synTac of the present disclosure at various IL-2 / synTac concentrations. [Figure 15] Shows the in vivo activity of IL-2 / synTac of the present disclosure. The left panel shows the fold change in the number of antigen-specific CD8+ T cells after administration of phosphate-buffered saline (PBS), recombinant IL-2 (rIL-2), or IL-2 / synTac of the present disclosure. The right panel shows the antigen-specific response and non-antigen-specific response after administration of PBS, rIL-2, or IL-2 / synTac of the present disclosure. [Figure 16]Shows the effects of increasing dosage (Figure 16A) and administration route (Figure 16B). [Figure 17] Shows the effect of the copy number of IL-2 on its in vivo efficacy against tumors. [Figure 18] Shows the serum half-life of IL-2 / synTac after intraperitoneal administration of the IL-2 / synTac of the present disclosure at a dose of 10 mg / kg. [Figure 19] Shows the stability of IL-2 / synTac 2 hours after intraperitoneal administration of the IL-2 / synTac of the present disclosure at a dose of 10 mg / kg. [Figure 20] Shows the size exclusion chromatography data for IL-2 / synTac after maintaining the IL-2 / synTac of the present disclosure at 4°C or 37°C for 5 days. [Figure 21] Shows the amino acid sequence of the heavy chain of IL-2 / synTac of the present disclosure containing a leader peptide. The IL-2 / synTac heavy chain contains an IgG1 Fc with an N297A substitution. [Figure 22] Shows the amino acid sequence of the heavy chain of IL-2 / synTac of the present disclosure without a leader peptide. The IL-2 / synTac heavy chain contains an IgG1 Fc with an N297A substitution. [Figure 23] Shows the nucleotide sequence encoding the IL-2 / synTac heavy chain shown in Figure 21 (Figure 23A) and an explanation of the sequence (Figure 23B). [Figure 24] Shows the amino acid sequence of the heavy chain of IL-2 / synTac containing a leader peptide. The IL-2 / synTac heavy chain contains an IgG1 Fc with L234A and L235A substitutions. [Figure 25] Shows the amino acid sequence of the heavy chain of IL-2 / synTac without a leader peptide. The IL-2 / synTac heavy chain contains an IgG1 Fc with L234A and L235A substitutions. [Figure 26] Shows the nucleotide sequence encoding the IL-2 / synTac heavy chain shown in Figure 24 (Figure 26A) and an explanation of the sequence (Figure 26B). [Figure 27]The amino acid sequence of the heavy chain of IL-2 / synTac, including the leader peptide, is shown. The IL-2 / synTac heavy chain contains an IgG1 Fc with substitutions of L234F, L235E, and P331S. [Figure 28] The amino acid sequence of the heavy chain of IL-2 / synTac, without the leader peptide, is shown. The IL-2 / synTac heavy chain contains an IgG1 Fc with substitutions of L234F, L235E, and P331S. [Figure 29] The nucleotide sequence encoding the IL-2 / synTac heavy chain shown in Figure 27 (Figure 29A) and the description of the sequence (Figure 29B) are shown. [Figure 30] The amino acid sequence of the light chain of IL-2 / synTac, including the leader peptide, is shown. The IL-2 / synTac light chain contains the human papillomavirus (HPV) E7 epitope. [Figure 31] The amino acid sequence of the light chain of IL-2 / synTac, without the leader peptide, is shown. The IL-2 / synTac light chain contains the HPV E7 epitope. [Figure 32] The nucleotide sequence encoding the IL-2 / synTac light chain shown in Figure 30 is shown. [Figure 33] The amino acid sequences of wild-type human IgG1 Fc (Figure 33A), IgG1 Fc with substitutions of L234F, L235E, and P331S (Figure 33B), IgG1 Fc with substitution of N297A (Figure 33C), and IgG1 Fc with substitutions of L234A and L235A (Figure 33D) are shown. [Figure 34] The amino acid sequences of β2-microglobulin (R12C) polypeptide (Figure 34A), mutant IL-2 (H16A;F42A) polypeptide (Figure 34B), and class I MHC-H chain A0201 (Y84A;A236C) (Figure 34C) are shown. [Figure 35] The synergistic effect of IL-2 / synTac and anti-PD1 antibody on the reduction of tumor volume is shown. [Figure 36] The amino acid sequence of 4-1BBL (Figure 36A) and examples of mutant 4-1BBL polypeptides (Figure 36B - 36IIII) are shown. [Figure 37] Shows the amino acid sequence of 4-1BB. [Figure 38] Shows interferon-gamma (IFN-γ) secretion by target cells contacted with the synTac polypeptide according to an embodiment of the present disclosure for 3 days (Figure 38A) or 5 days (Figure 8B). [Figure 39] Shows interleukin-2 (IL-2) secretion by target cells contacted with the synTac polypeptide according to an embodiment of the present disclosure for 3 days (Figure 39A) or 5 days (Figure 9B). [Figure 40] Shows interleukin-6 (IL-6) secretion by target cells contacted with the synTac polypeptide according to an embodiment of the present disclosure for 3 days (Figure 40A) or 5 days (Figure 40B). [Figure 41] Shows tumor necrosis factor-alpha (TNFα) secretion by target cells contacted with the synTac polypeptide according to an embodiment of the present disclosure for 3 days (Figure 41A) or 5 days (Figure 41B). [Figure 42] Shows interleukin-10 (IL-10) secretion by target cells contacted with the synTac polypeptide according to an embodiment of the present disclosure for 3 days (Figure 42A) or 5 days (Figure 42B). [Figure 43] Shows interleukin-17A (IL-17A) secretion by target cells contacted with the synTac polypeptide according to an embodiment of the present disclosure for 3 days (Figure 43A) or 5 days (Figure 43B). [Figure 44] Shows interleukin-4 (IL-4) secretion by target cells contacted with the synTac polypeptide according to an embodiment of the present disclosure for 3 days (Figure 44A) or 5 days (Figure 44B). [Figure 45] Shows the proliferation of target cells contacted with the synTac polypeptide according to an embodiment of the present disclosure. [Figure 46] Shows the viability of target cells contacted with the synTac polypeptide according to an embodiment of the present disclosure. [Figure 47] Shows the expression levels of various synTac polypeptides produced in CHO cells. [Figure 48]Shows the in vivo effect of the synTac polypeptide of the present disclosure on tumor volume. [Figure 49] Shows the effects of co - administration of various doses of 4 - 1BBL / synTac and anti - PD1 antibody on tumor burden and the percentage of granzyme B+ tumor - infiltrating lymphocytes (TIL). [Figure 50] Shows the amino acid sequence of the PD - L1 polypeptide. [Figure 51] Shows the amino acid sequence of the CD80 polypeptide. [Figure 52] Shows the amino acid sequence of the ICOS - L polypeptide. [Figure 53] Shows the amino acid sequence of the OX40L polypeptide. [Figure 54] Shows the amino acid sequence of the PD - L2 polypeptide. [Figure 55] Shows the amino acid sequence of the CD86 (B7 - 2) polypeptide. [Figure 56] Shows the amino acid sequence of the Fas ligand (FAS - L) polypeptide.

Mode for Carrying Out the Invention

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

[0006] The terms "peptide", "polypeptide", and "protein" are used interchangeably herein and refer to polymeric forms of amino acids of any length, including coded and non-coded amino acids, chemically or biochemically modified amino acids, or derivatized amino acids, and polypeptides having modified peptide backbones.

[0007] A polynucleotide or polypeptide having a certain percentage of "sequence identity" to another polynucleotide or polypeptide means that, when the two sequences are aligned and compared, that percentage of bases or amino acids is the same and in the same relative positions. Sequence identity can be determined in a number of different ways. To determine sequence identity, the sequences can be aligned using a variety of convenient methods and computer programs (e.g., BLAST, T-COFFEE, MUSCLE, MAFFT, etc.), which are available through websites on the World Wide Web including ncbi.nlm.nili.gov / BLAST, ebi.ac.uk / Tools / msa / tcoffee / , ebi.ac.uk / Tools / msa / muscle / , mafft.cbrc.jp / alignment / software / . See, for example, Altschul et al. (1990), J. Mol. Bioi. 215:403-10.

[0008] The term "conservative amino acid substitution" refers to the interchangeability of amino acid residues in proteins with 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] "Binding," as used herein (e.g., with respect to binding of a T cell regulatory multimeric polypeptide to a polypeptide (e.g., a T cell receptor) on a T cell), refers to a non-covalent interaction. Binding interactions generally occur in the 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 binding, and an increase in binding affinity is characterized by a K D correlates with a decrease in

[0010] As used herein, the terms "immunological synapse" or "immune synapse" generally refer to the natural interface between two immune cells that interact in an adaptive immune response, including, for example, the interface between an antigen-presenting cell (APC) or target cell and an effector cell, such as a lymphocyte, effector T cell, natural killer cell, etc. The immunological synapse between an APC and a T cell is generally initiated by the interaction of the 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 disclosure of this document is hereby incorporated by reference in its entirety.

[0011] "T cell" includes all types of immune cells expressing CD3, including T helper cells (CD4 + cells), cytotoxic T cells (CD8 + cells), regulatory T cells (Tregs) and NK-T cells.

[0012] "Costimulatory polypeptide" (also referred to herein as "immunomodulatory polypeptide") when the term is used herein, by specifically binding to a cognate costimulatory polypeptide on a T cell (also referred to herein as "cognate immunocoregulatory polypeptide"), in addition to a primary signal (e.g., that brought about by the binding of a TCR / CD3 complex to a major histocompatibility complex (MHC) polypeptide loaded with a peptide), provides a signal that mediates a T cell response (including, but not limited to, proliferation, activation, differentiation, etc.), and includes a polypeptide on an antigen-presenting cell (APC) (e.g., dendritic cell, B cell, etc.). Costimulatory ligands 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 β receptor, 3 / TR6, ILT3, ILT4, HVEM, an agonist or antibody that binds to a Toll ligand receptor, and a ligand that specifically binds to B7-H3. Also, costimulatory ligands include, inter alia, an antibody that specifically binds to a costimulatory molecule present on a T cell, including, but not limited to, a ligand that specifically binds to CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, LIGHT, NKG2C, B7-H3, and CD83.

[0013] The "regulatory domain" ("MOD") of a T cell regulatory multimer polypeptide includes an IL-2 polypeptide such as a costimulatory polypeptide, e.g., a mutant IL-2 polypeptide.

[0014] "Heterologous" when used herein means a nucleotide or polypeptide not found in an unmodified nucleic acid or protein, respectively.

[0015] As used herein, "recombinant" means the product of various combinations of the steps of cloning, restriction, polymerase chain reaction (PCR) and / or ligation that result in a construct in which a particular nucleic acid (DNA or RNA) has a structural coding or non-coding sequence distinguishable from the endogenous nucleic acid found in nature. A DNA sequence encoding a polypeptide can be assembled from cDNA fragments or a series of synthetic oligonucleotides, and a synthetic nucleic acid capable of expression from a recombinant transcription unit contained in a cell or cell-free transcription / translation system can be obtained.

[0016] The terms "recombinant expression vector" or "DNA construct" are used interchangeably herein to refer to a DNA molecule that contains a vector and one insert. Recombinant expression vectors are usually made for the expression and / or propagation of an insert(s) or for the construction of 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 DNA control sequences.

[0017] The terms “antibody” and “immunoglobulin” include antibodies or immunoglobulins of any isotype, antibody fragments 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, bispecific antibodies, multispecific antibodies, and fusion proteins that include the antigen-binding (also referred to herein as antigen-binding) portion of an antibody and a non-antibody protein. The term also encompasses 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 are 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, but other production methods known to those of skill in the art can also be used (e.g., antibodies derived from antibody phage display libraries). Antibodies can be monovalent or divalent. An antibody can be an Ig monomer, which is a “Y-shaped” molecule consisting of four polypeptide chains, two heavy chains and two light chains linked by disulfide bonds.

[0018] As used herein, the term "humanized antibody" refers to an antibody that contains portions of antibodies of different origins, wherein at least one portion contains an amino acid sequence of human origin. For example, a humanized antibody can include a portion derived from a non-human immunoglobulin having the required specificity, such as a mouse, and a portion derived from a human immunoglobulin sequence (e.g., a chimeric immunoglobulin), and these can be chemically joined together by conventional techniques (e.g., synthesis) or produced as a continuous polypeptide using genetic engineering techniques (e.g., DNA encoding the protein portion of a chimeric antibody can be expressed to generate a continuous polypeptide chain). Another example of a humanized antibody is an antibody containing one or more antibody chains that include CDRs derived from a non-human antibody and framework regions derived from a human light chain and / or heavy chain (e.g., CDR-grafted antibodies with or without framework modifications). 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 to Cabilly et al., European Patent No. 0,125,023B1 to Cabilly et al., U.S. Patent No. 4,816,397 to Boss et al., European Patent No. 0,120,694B1 to Boss et al., WO86 / 01533 to Neuberger, M.S. et al., European Patent No. 0,194,276B1 to Neuberger, M.S. et al., U.S. Patent No. 5,225,539 to Winter, European Patent No. 0,239,400B1 to Winter, European Patent Application No. 0,519,596A1 to Padlan, E.A. et al. Also, for single-chain antibodies, see U.S. Patent No. 4,946,778 to Ladner et al., U.S. Patent No. 5,476,786 to Huston, and Bird, R.E. et al., Science, 242:423-426 (1988).

[0019] For example, humanized antibodies can be prepared and produced using synthetic nucleic acids and / or recombinant nucleic acids to prepare a gene (e.g., cDNA) encoding the desired humanized chain. For example, a nucleic acid (e.g., DNA) sequence encoding a humanized variable region can be modified and constructed using the PCR mutagenesis method with a DNA sequence encoding a human or humanized chain, such as a DNA template of a previously humanized variable region (e.g., Kamman, M., et al., Nucl. Acids Res., 17:5404 (1989)); Sato, K., et al., Cancer Research, 53:851-856 (1993); Daugherty, B. L. et al., Nucleic Acids Res., 19(9):2471-2476 (1991); and Lewis, A. P. and J. S. Crowe, Gene, 101:297-302 (1991)). Mutants can also be easily prepared using these methods or other suitable methods. For example, mutagenesis can be performed on the cloned variable region, and a sequence encoding a mutation with the desired specificity can be selected (e.g., from a phage library; e.g., see U.S. Patent No. 5,514,548 to Krebber et al.; WO93 / 06213 published Apr. 1, 1993 by Hoogenboom et al.).

[0020] An "antibody fragment" includes a portion of an intact antibody, e.g., the antigen-binding region 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 (dAb; Holt et al. (2003) Trends Biotechnol. 21:484); single-chain antibody molecules; and multispecific antibodies formed from antibody fragments. Digestion of an antibody with papain produces two identical antigen-binding fragments called "Fab" fragments, each having a single antigen-binding site, and a residual "Fc" fragment, a notation reflecting the ability to crystallize readily. Pepsin treatment yields an F(ab')2 fragment that has two antigen-binding sites and can still cross-link antigens.

[0021] "Fv" is the smallest antibody fragment containing the complete antigen recognition binding site. This region is composed of a dimer of one heavy chain variable domain and one light chain variable domain, which are non-covalently and tightly associated. In this configuration, the three CDRs of each variable domain interact to define the antigen binding site on the surface of the V H -V L -dimer. Overall, six CDRs confer antigen binding specificity to the antibody. However, even a single variable domain (or half of the Fv containing only three CDRs specific for the antigen), although having a lower affinity than the complete binding site, has the ability to recognize and bind the antigen.

[0022] The "Fab" fragment also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. The Fab fragment and the Fab' fragment differ in that several residues containing one or more cysteines derived from the antibody hinge region are added to the carboxyl terminus of the heavy chain CH1 domain. Fab'-SH is the notation in this specification for Fab' in which the cysteine residue(s) of the constant domain have free thiol groups. The F(ab')2 antibody fragment was originally prepared as a pair of Fab' fragments having hinge cysteines between the fragments. Other chemical couplings of antibody fragments are also known.

[0023] The "light chain" of an antibody (immunoglobulin) derived from any vertebrate species can be assigned to one of two distinct types, called κ and λ, based on the amino acid sequence of their constant domains. Immunoglobulins can be assigned to different classes according to the amino acid sequence of the constant domains of their heavy chains. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and some of these classes can be further classified into subclasses (isotypes), for example, IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. Subclasses can be further divided into types, for example, IgG2a and IgG2b.

[0024] The "single-chain Fv", "sFv", or "scFv" antibody fragment contains the V H domain and the V L domain, and these domains are present in a single polypeptide chain. In some embodiments, the Fv polypeptide further contains a polypeptide linker between the V H domain and the V L domain, which enables the sFv to form a structure desirable for antigen binding. For an overview of sFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenberg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0025] The term "diabody" refers to a small antibody fragment having two antigen-binding sites, in which the heavy-chain variable domain (V L ) linked to the light-chain variable domain (V H ) is included in the same polypeptide chain (V H -V L ). By using a short linker such that no pairing occurs between the two domains on the same chain, these domains are paired with the complementary domains of another chain to form two antigen-binding sites. Diabodies are more fully described, for example, in EP404,097; WO93 / 11161; and Hollinger et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448.

[0026] As used herein, the term "affinity" refers to the equilibrium constant of the reversible binding of two agents (e.g., an antibody and an antigen), the dissociation constant (K D) It is represented by. The affinity is at least more than 1-fold, at least more than 2-fold, at least more than 3-fold, at least more than 4-fold, at least more than 5-fold, at least more than 6-fold, at least more than 7-fold, at least more than 8-fold, at least more than 9-fold, at least more than 10-fold, at least more than 20-fold, at least more than 30-fold, at least more than 40-fold, at least more than 50-fold, at least more than 60-fold, at least more than 70-fold, at least more than 80-fold, at least more than 90-fold, at least more than 100-fold, or at least more than 1,000-fold, or more, than the affinity of the antibody for an unrelated amino acid sequence. The affinity of the antibody for the target protein can be, for example, from about 100 nanomolar (nM) to about 0.1 nM, from about 100 nM to about 1 picomolar (pM), or from about 100 nM to about 1 femtomolar (fM) or more. As used herein, the term "avidity" refers to the dissociation resistance of a complex of two or more agents after dilution. The terms "immunoreactivity" and "preferential binding" are used interchangeably herein with respect to antibodies and / or antigen-binding fragments.

[0027] The term "binding" refers to the direct association between two molecules, for example, by covalent, electrostatic, hydrophobic, and ionic and / or hydrogen bond interactions (including interactions such as salt bridges and hydrogen bridges). "Specific binding" is at least about 10 -7 M or more, for example, 5 × 10 -7 M, 10 -8 M, 5 × 10 -8 M or more. "Nonspecific binding" refers to a binding having an affinity of less than about 10 -7 M, for example, 10 -6 M, 10 -5 M, 10 -4 M, etc.

[0028] As used herein, the terms "CDR" or "complementary determining region" are intended to mean the non - contiguous antigen - binding sites found within the variable regions of both the heavy and light chain polypeptides. The CDRs are described by Kabat et al., J. Biol. Chem. 252:6609 - 6616 (1977); Kabat et al., U.S. Dept. of Health and Human Services, "Sequences of proteins of immunological interest" (1991) (also referred to herein as Kabat 1991), by Chothia et al., J. Mol. Biol. 196:901 - 917 (1987) (also referred to herein as Chothia 1987); and by MacCallum et al., J. Mol. Biol. 262:732 - 745 (1996), where the definitions include overlaps or subsets of amino acid residues when compared to each other. However, the application of any definition referring to the CDRs of an antibody or grafted antibody or its variants is intended to be within the scope of the terms defined and used herein. The amino acid residues encompassing the CDRs as defined by each of the above - mentioned references are described in the following table for comparison. The CDRs listed in Table 2 are defined according to Kabat 1991. Table: Definition of CDR TIFF0007717440000001.tif34136 1 Residue numbering follows the notation of Kabat et al. cited above. 2 Residue numbering follows the notation of Chothia et al. cited above. 3 Residue numbering follows the notation of MacCallum et al. cited above.

[0029] As used herein, the terms "CDR-L1", "CDR-L2" and "CDR-L3" each refer to the first, second and third CDRs in the light chain variable region. As used herein, the terms "CDR-H1", "CDR-H2" and "CDR-H3" each refer to the first, second and third CDRs in the heavy chain variable region. As used herein, the terms "CDR-1", "CDR-2" and "CDR-3" each refer to the first, second and third CDRs in the variable region of either chain.

[0030] As used herein, the term "framework", when used with respect to an antibody variable region, is intended to mean all amino acid residues within the variable region of the antibody other than the CDR regions. 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 amino acids other than the CDRs. As used herein, the term "framework region" is intended to mean each domain of the framework separated by the CDRs.

[0031] The terms "treatment", "treating", etc. are used herein generally to mean obtaining a desired pharmacological and / or physiological effect. The effect can 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 an adverse effect that may result from the disease. "Treatment" as used herein encompasses any treatment of a disease or condition in a mammal, including (a) prevention of the onset of a disease or condition in a subject who may be predisposed to the disease or condition but has not yet been diagnosed as having it, (b) inhibition of a disease or condition, i.e., preventing its occurrence, or (c) alleviation of a disease, i.e., inducing regression of the disease. A therapeutic agent can be administered before, during, or after the occurrence of a disease or injury. Treatment of an ongoing disease is particularly targeted, in which case the treatment stabilizes or reduces the patient's undesirable clinical symptoms. Such treatment is preferably carried out before the function in the affected tissue is completely lost. The treatment method of the subject is preferably administered during the symptomatic period of the disease and, in some cases, after the symptomatic period of the disease.

[0032] The terms "individual", "subject", "host", and "patient" are used interchangeably herein and refer to any mammalian subject for which a diagnosis, treatment, or therapy is desired. Mammals include, for example, humans, non-human primates, rodents (e.g., rats, mice), lagomorphs (e.g., rabbits), ungulates (e.g., cows, sheep, pigs, horses, goats, etc.).

[0033] Before further describing the present invention, it is to be understood that the present invention is not limited to the specific embodiments described, which can, of course, be diverse in themselves. 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 is limited only by the appended claims.

[0034] When a range of values is recited, unless otherwise expressly specified, intervening values between the upper and lower limits of that range in increments of one tenth of the lower limit, and any other specified value or intervening value within the specified range, are understood to be included within the invention. Upper and lower limits of these narrower ranges may independently be included within the narrower ranges, and also within the invention even if any specific limit value within the specified range is excluded. Where the specified range includes one or both of the limit values, the range excluding one or both of the included limit values is also included herein.

[0035] Unless defined otherwise, 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 be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited.

[0036] 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, reference to "a mutant IL-2 polypeptide" includes a plurality of such polypeptides, reference to "a class I HLA heavy chain polypeptide" includes reference to one or more class I HLA heavy chain polypeptides and equivalents thereof known to those of skill in the art, and the like. It should also be noted that the claims may be drafted to exclude any optional element. Thus, this description is intended to serve as a basis for use of the exclusive terms, such as "only", "solely", etc., or use of "negative" limitations in connection with the recitation of claim elements.

[0037] For clarity, some features of the invention are described in the context of separate embodiments, but it is also recognized that they can be provided in combination as one embodiment. Conversely, to avoid complication, various features of the invention are described in the context of one embodiment, but these may also be provided separately or in any suitable partial combination. All combinations of embodiments of the invention are clearly embraced by the invention and are disclosed herein as if each and every combination were individually and explicitly disclosed. In addition, all partial combinations of the various embodiments and their elements are also clearly embraced by the invention and are disclosed herein as if each and every such partial combination were individually and explicitly disclosed herein.

[0038] The publications discussed herein are described only with respect to their disclosure prior to the filing date of the present application. Nothing in this specification should be construed as an admission that the invention is not entitled to antedate such publication by virtue of prior invention. Further, the publication dates noted may be different from the actual publication dates, which may need to be independently confirmed.

[0039] Detailed Description The present disclosure provides a method of treatment comprising administering to a subject in need thereof a T cell regulatory multimer polypeptide (a “synTac” multimer polypeptide) and at least one additional therapeutic agent. In some cases, the at least one additional therapeutic agent is an immune checkpoint inhibitor. In some cases, the immune checkpoint inhibitor is an antibody specific for an immune checkpoint. The present disclosure provides a method comprising administering to a subject a multimer polypeptide (synTac) and an immune checkpoint inhibitor. The present disclosure provides a method comprising administering to a subject receiving treatment with an immune checkpoint inhibitor a multimer polypeptide (synTac).

[0040] The "T cell regulatory multimeric polypeptide" is also referred to herein as "synTac polypeptide" or "synTac multimeric polypeptide" or simply "synTac". The synTac polypeptide contains a regulatory domain. In some cases, the regulatory domain contains a wild-type amino acid sequence, for example, an amino acid sequence found in a naturally occurring regulatory polypeptide. In some cases, the regulatory domain is a mutant regulatory domain, and the mutant regulatory domain exhibits a reduced binding affinity for an immunomodulatory polypeptide as compared to the affinity of the wild-type regulatory domain for the immunomodulatory polypeptide. The synTac polypeptide can regulate the activity of the targeted T cells. The synTac polypeptide containing a mutant regulatory domain results in an improvement in the specificity of the target cells.

[0041] In some cases, the disclosed method of treatment comprises administering to an individual in need thereof a synTac and an immune checkpoint inhibitor. In some cases, the synTac and the immune checkpoint inhibitor provide a synergistic effect as compared to the effect(s) of synTac (monotherapy) or the immune checkpoint inhibitor alone (monotherapy) when administered alone.

[0042] The combination of synTac and immune checkpoint inhibitors is, in some cases, more effective than the additive effect of synTac administered as monotherapy or an immune checkpoint inhibitor administered as monotherapy. For example, in some cases, the synergistic effect of synTac and immune checkpoint inhibitors enables the use of low doses of synTac or immune checkpoint inhibitors and / or a reduction in the frequency of their administration to individuals who require synTac or immune checkpoint inhibitors. The ability to use the therapeutic agent (synTac or immune checkpoint inhibitor) at a low dose and / or administer the agent less frequently can reduce the toxicity or other adverse side effects that may accompany the administration of the therapeutic agent in monotherapy without reducing the effectiveness of the therapeutic agent in the treatment. In addition, the synergistic effect of synTac and immune checkpoint inhibitors can improve the clinical benefit compared to the clinical benefit obtained with synTac monotherapy or immune checkpoint inhibitor monotherapy. Examples of clinical benefit include, for example, a reduction in tumor burden in an individual, a reduction in the number of cancer cells in an individual, an increase in the survival time of an individual, an increase in the remission time, and the like. Finally, the synergistic effect of synTac and immune checkpoint inhibitors can reduce the harmful or undesirable side effects associated with synTac monotherapy or immune checkpoint inhibitor monotherapy.

[0043] immune checkpoint inhibitor Exemplary immune checkpoint inhibitors include inhibitors that target immune checkpoint polypeptides such as CD27, CD28, CD40, CD122, CD96, CD73, CD47, OX40, GITR, CSF1R, JAK, PI3Kδ, PI3Kγ, TAM, arginase, CD137 (also known as 4-1BB), ICOS, A2AR, B7-H3, B7-H4, BTLA, CTLA-4, LAG3, TIM3, VISTA, CD96, TIGIT, CD122, PD-1, PD-L1, and PD-L2. In some cases, the immune checkpoint polypeptide is a stimulatory checkpoint molecule selected from CD27, CD28, CD40, ICOS, OX40, GITR, CD122, and CD137. In some cases, the immune checkpoint polypeptide is an inhibitory checkpoint molecule selected from A2AR, B7-H3, B7-H4, BTLA, CTLA-4, IDO, KIR, LAG3, PD-1, TIM3, CD96, TIGIT, and VISTA.

[0044] In some cases, the immune checkpoint inhibitor is an antibody specific for the immune checkpoint. In some cases, the anti-immune checkpoint antibody is a monoclonal antibody. In some cases, the anti-immune checkpoint antibody is humanized or deimmunized so that the antibody does not substantially elicit a human immune response. In some cases, the anti-immune checkpoint antibody is a humanized monoclonal antibody. In some cases, the anti-immune checkpoint antibody is a deimmunized monoclonal antibody. In some cases, the anti-immune checkpoint antibody is a fully human monoclonal antibody. In some cases, the anti-immune checkpoint antibody inhibits the binding of the immune checkpoint polypeptide to the ligand of the immune checkpoint polypeptide. In some cases, the anti-immune checkpoint antibody inhibits the binding of the immune checkpoint polypeptide to the receptor of the immune checkpoint polypeptide.

[0045] Antibodies that are specific for immune checkpoints and function as immune checkpoint inhibitors, such as monoclonal antibodies, are known in the art. See, for example, Wurz et al. (2016) Ther. Adv. Med. Oncol. 8:4; and Naidoo et al. (2015) Ann. Oncol. 26:2375.

[0046] Suitable anti-immune checkpoint antibodies include, but are not limited to, nivolumab (Bristol-Myers Squibb), pembrolizumab (Merck), pidilizumab (Curetech), AMP-224 (GlaxoSmithKline / Amplimmune), MPDL3280A (Roche), MDX-1105 (Medarex, Inc. / Bristol Myer Squibb), MEDI-4736 (Medimmune / AstraZeneca), alefacept (Merck Serono), ipilimumab (YERVOY (Bristol-Myers Squibb)), tremelimumab (Pfizer), pidilizumab (CureTech, Ltd.), IMP321 (Immutep S.A.), MGA271 (Macrogenics), BMS-986016 (Bristol-Meyers Squibb), lirilumab (Bristol-Myers Squibb), urelumab (Bristol-Myers Squibb), PF-05082566 (Pfizer), IPH2101 (Innate Pharma / Bristol-Myers Squibb), MEDI-6469 (MedImmune / AZ), CP-870,893 (Genentech), mogamulizumab (Kyowa HakkoKirin), balstilimab (CelIDex Therapeutics), avelumab (EMD Serono), galiximab (Biogen Idec), AMP-514 (Amplimmune / AZ), AUNP 12 (Aurigene and PierreFabre), indoximod (NewLink Genetics), NLG-919 (NewLink Genetics), INCB024360 (Incyte), KN035, and combinations thereof.

[0047] Suitable anti-LAG3 antibodies include, for example, BMS-986016 and LAG525. Suitable anti-GITR antibodies include, for example, TRX518, MK-4166, INCAGN01876 and MK-1248. Suitable anti-OX40 antibodies include, for example, MEDI0562, INCAGN01949, GSK2831781, GSK-3174998, MOXR-0916, PF-04518600 and LAG525. Suitable anti-VISTA antibodies are described, for example, in WO2015 / 097536.

[0048] Suitable dosages of the anti-immune checkpoint antibodies are from about 1 mg / kg to about 2400 mg / kg per day, for example, from about 1 mg / kg to about 1200 mg / kg per day, including from about 50 mg / kg to about 1200 mg / kg per day. Other representative dosages of the agent include about 5 mg / kg, 10 mg / kg, 15 mg / kg, 20 mg / kg, 25 mg / kg, 30 mg / kg, 35 mg / kg, 40 mg / kg, 45 mg / kg, 50 mg / kg, 60 mg / kg, 70 mg / kg, 80 mg / kg, 90 mg / kg, 100 mg / kg, 125 mg / kg, 150 mg / kg, 175 mg / kg, 200 mg / kg, 250 mg / kg, 300 mg / kg, 400 mg / kg, 500 mg / kg, 600 mg / kg, 700 mg / kg, 800 mg / kg, 900 mg / kg, 1000 mg / kg, 1100 mg / kg, 1200 mg / kg, 1300 mg / kg, 1400 mg / kg, 1500 mg / kg, 1600 mg / kg, 1700 mg / kg, 1800 mg / kg, 1900 mg / kg, 2000 mg / kg, 2100 mg / kg, 2200 mg / kg and 2300 mg / kg per day. The effective dosage of the antibody may be administered as 2, 3, 4, 5, 6 or more sub-dosages, administered separately at appropriate intervals throughout the day.

[0049] Anti-PD-1 antibody In some cases, the immune checkpoint inhibitor is an anti-PD-1 antibody.

[0050] Suitable anti-PD-1 antibodies include, for example, nivolumab, pembrolizumab (also known as MK-3475), pidilizumab, SHR-1210, PDR001, and AMP-224. In some cases, the anti-PD-1 monoclonal antibody is nivolumab, pembrolizumab, or PDR001. Suitable anti-PD1 antibodies are described in U.S. Patent Application Publication No. 2017 / 0044259. For pidilizumab, see, for example, Rosenblatt et al. (2011) J. Immunother. 34:409-18.

[0051] In some cases, the anti-PD1 antibody is pembrolizumab. The amino acid sequence of the heavy chain of pembrolizumab is TIFF0007717440000002.tif52151. The amino acid sequence of the heavy chain variable (VH) region is underlined.

[0052] The amino acid sequence of the light chain of pembrolizumab is TIFF0007717440000003.tif26151. The amino acid sequence of the light chain variable (VL) region is underlined.

[0053] In some cases, the anti-PD-1 antibody comprises the VH and VL regions of pembrolizumab. In some cases, the anti-PD-1 antibody comprises the CDRs of the heavy and light chains of pembrolizumab.

[0054] In some cases, the anti-PD-1 antibody is nivolumab (also known as MDX-1106 or BMS-936558). See, for example, Topalian et al. (2012) N. Engl. J. Med. 366:2443-2454; and U.S. Patent No. 8,008,449). The amino acid sequence of the heavy chain of nivolumab is TIFF0007717440000004.tif53150.

[0055] The amino acid sequence of the light chain of nivolumab is It is TIFF0007717440000005.tif26151.

[0056] In some cases, the anti-PD-1 antibody comprises the CDRs of the heavy and light chains of nivolumab.

[0057] Anti-CTLA4 antibody In some cases, the anti-CTLA-4 antibody is ipilimumab or tremelimumab. For tremelimumab, see, for example, Ribas et al. (2013) J. Clin. Oncol. 31:616-22.

[0058] In some cases, the anti-CTLA-4 antibody is ipilimumab. The amino acid sequence of the heavy chain of ipilimumab is It is TIFF0007717440000006.tif52151. The amino acid sequence of the VH region is underlined.

[0059] The amino acid sequence of the light chain of ipilimumab is It is TIFF0007717440000007.tif26151. The amino acid sequence of the VL region is underlined.

[0060] In some cases, the anti-CTLA4 antibody comprises the VH and VL regions of ipilimumab. In some cases, the anti-CTLA4 antibody comprises the CDRs of the heavy and light chains of ipilimumab.

[0061] Anti-PD-L1 antibody In some cases, the immune checkpoint inhibitor is an anti-PD-L1 monoclonal antibody. In some cases, the anti-PD-L1 monoclonal antibody is BMS-935559, MEDI4736, MPDL3280A (also known as RG7446), KN035, or MSB0010718C. In some embodiments, the anti-PD-L1 monoclonal antibody is MPDL3280A (atezolizumab) or MEDI4736 (durvalumab). For durvalumab, see, for example, WO2011 / 066389. For atezolizumab, see, for example, U.S. Patent No. 8,217,149.

[0062] In some cases, the anti-PD-L1 antibody is atezolizumab. The amino acid sequence of the heavy chain of atezolizumab is TIFF0007717440000008.tif52151.

[0063] The amino acid sequence of the light chain of atezolizumab is TIFF0007717440000009.tif26151.

[0064] In some cases, the anti-PD-L1 antibody comprises the CDRs of the heavy and light chains of atezolizumab.

[0065] In some cases, the anti-PDL1 antibody is KN035, a fully humanized anti-PD-L1 single-domain antibody fused to a human IgG1 Fc polypeptide. Zhang et al. (2017) Cell Discov. 3:17004; and WO2017 / 020801. The single-domain antibody portion of KN035 may comprise the amino acid sequence: TIFF0007717440000010.tif17151, and in the sequence, the underlined amino acids are CDR1, CDR2, and CDR3.

[0066] T cell regulatory multimer polypeptide (synTac) Multimeric (e.g., heterodimeric, heterotrimeric) polypeptides suitable for use in the methods of the present disclosure are described below. The multimeric polypeptide is a T cell regulatory polypeptide and is also referred to herein as a "T cell regulatory multimeric polypeptide" or "synTac" ("immunological synapse for T cell activation").

[0067] The T cell regulatory multimer polypeptide comprises: a) a first polypeptide, in the order from the N-terminus to the C-terminus, i) an epitope, ii) a first major histocompatibility complex (MHC) polypeptide; and b) a second polypeptide, in the order from the N-terminus to the C-terminus, i) a second MHC polypeptide, and ii) optionally, an immunoglobulin (Ig) Fc polypeptide or a non-Ig backbone. The multimer polypeptide contains one or more immunomodulatory ("MOD") domains, and the one or more immunomodulatory domains are located 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. In some cases, the T cell multimer polypeptide comprises: a) a first polypeptide, in the order from the N-terminus to the C-terminus, i) an epitope, ii) a first MHC polypeptide, and iii) an immunomodulatory domain; and b) a second polypeptide, in the order from the N-terminus to the C-terminus, i) a second MHC polypeptide, and ii) an Ig Fc polypeptide. In some cases, the T cell multimer polypeptide comprises: a) a first polypeptide, in the order from the N-terminus to the C-terminus, i) an epitope, and ii) a first MHC polypeptide; and b) a second polypeptide, in the order from the N-terminus to the C-terminus, i) an immunomodulatory domain, iii) a second MHC polypeptide, and ii) an Ig Fc polypeptide. In some cases, the T cell multimer polypeptide comprises: a) a first polypeptide, in the order from the N-terminus to the C-terminus, i) an epitope, and ii) a first MHC polypeptide; and b) a second polypeptide, in the order from the N-terminus to the C-terminus, i) a second MHC polypeptide, ii) an Ig Fc polypeptide, and iii) an immunomodulatory ("MOD") domain. In some cases, the T cell multimer polypeptide comprises: a) a first polypeptide, in the order from the N-terminus to the C-terminus, i) an epitope, and ii) a first MHC polypeptide; and b) a second polypeptide, in the order from the N-terminus to the C-terminus, i) a second MHC polypeptide, and ii) an immunomodulatory domain.In some cases, the T cell multimer polypeptide comprises: a) a first polypeptide comprising, in order from the N-terminus to the C-terminus, i) an epitope, and ii) a first MHC polypeptide; and b) a second polypeptide comprising, in order from the N-terminus to the C-terminus, i) an immunomodulatory domain, and ii) a second MHC polypeptide. In some cases, the T cell multimer polypeptide comprises: a) a first polypeptide comprising, in order from the N-terminus to the C-terminus, i) an epitope, ii) a first MHC polypeptide, and iii) an immunomodulatory domain; and b) a second polypeptide comprising, in order from the N-terminus to the C-terminus, i) a second MHC polypeptide.

[0068] In some cases, the multimer polypeptide comprises a non-Ig backbone. For example, in some cases, the non-Ig backbone is an XTEN polypeptide, a transferrin polypeptide, an Fc receptor polypeptide, an elastin-like polypeptide, a silk-like polypeptide, or a silk-elastin-like polypeptide.

[0069] In some cases, the first MHC polypeptide is a β2-microglobulin (β2M) polypeptide and the second MHC polypeptide is an MHC class I heavy chain polypeptide. Suitable β2-M polypeptides include amino acid sequences having 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 the β2M polypeptide shown in FIG. 6. In some cases, the MHC class I heavy chain polypeptide is a heavy chain of HLA-A, HLA-B or HLA-C. In some cases, the MHC class I heavy chain polypeptide includes an amino acid sequence having 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 set forth in one of FIGS. 5A-5C. In some cases, the first MHC polypeptide is an MHC class II α-chain polypeptide and the second MHC polypeptide is an MHC class II β-chain polypeptide.

[0070] Epitopes present in the multimeric polypeptide can be T cell epitopes.

[0071] In some cases, the multimeric polypeptide comprises an Ig Fc polypeptide. In some cases, the Ig Fc polypeptide is an IgG1 Fc polypeptide, an IgG2 Fc polypeptide, an IgG3 Fc polypeptide, an IgG4 Fc polypeptide, an IgA Fc polypeptide, or an IgM Fc polypeptide. In some cases, the Ig Fc polypeptide comprises an amino acid sequence having 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 sequences shown in FIGS. 4A - 4C.

[0072] The first polypeptide and the second polypeptide of the multimeric polypeptide can associate non - covalently. The first polypeptide and the second polypeptide of the multimeric polypeptide can be covalently linked. The first polypeptide and the second polypeptide of the multimeric polypeptide can be covalently linked, where the covalent bond is by a disulfide bond. In some cases, the first MHC polypeptide, or the linker between the epitope and the first MHC polypeptide, comprises an amino acid substitution that results in a first Cys residue, the second MHC polypeptide comprises an amino acid substitution that results in a second Cys residue, and the disulfide linkage is between the first Cys residue and the second Cys residue.

[0073] The multimeric polypeptide can include a linker in one or more of between the epitope and the first MHC polypeptide, between two copies of the immunomodulatory ("MOD") polypeptide, between the immunomodulatory polypeptide and the second MHC polypeptide, and between the second MHC polypeptide and the Ig Fc polypeptide.

[0074] Immune regulatory polypeptides suitable for inclusion in T cell multimer polypeptides include, but are not limited to, 4-1BBL polypeptide, B7-1 polypeptide, B7-2 polypeptide, ICOS-L polypeptide, OX-40L polypeptide, CD80 polypeptide, CD86 polypeptide, IL-2 polypeptide, PD-L1 polypeptide, FasL polypeptide, and PD-L2 polypeptide.

[0075] The multimer polypeptide can include two or more immune regulatory polypeptides. The multimer polypeptide can include two immune regulatory polypeptides. In some cases, the two immune regulatory polypeptides are tandemly arranged. The multimer polypeptide can include three immune regulatory polypeptides. In some cases, the three immune regulatory polypeptides are tandemly arranged.

[0076] The multimer polypeptide can include a third polypeptide, and the third polypeptide includes an amino acid sequence having at least 90% amino acid sequence identity to the immune regulatory polypeptide of the first polypeptide or the second polypeptide, and includes an immune regulatory polypeptide. In some cases, the third polypeptide is covalently linked to the first polypeptide.

[0077] Examples of suitable multimer polypeptides are described in WO2017 / 151940; WO2017 / 201210; and PCT / US2017 / 067663. The disclosures of WO2017 / 151940, WO2017 / 201210 and PCT / US2017 / 067663 are incorporated herein by reference.

[0078] MHC polypeptide As described above, the multimeric polypeptides of the present disclosure include MHC polypeptides. For the purposes of the present disclosure, the term "major histocompatibility complex (MHC) polypeptide" includes MHC polypeptides of humans (also referred to as human leukocyte antigens (HLA)), MHC polypeptides of rodents (e.g., mice, rats, etc.), and MHC polypeptides of other mammalian species (e.g., lagomorphs, non-human primates, canids, felids, ungulates (e.g., horses, cows, sheep, goats, etc.), etc.), and is intended to include MHC polypeptides of various species. The term "MHC polypeptide" is intended to include class I MHC polypeptides (e.g., β-2 microglobulin and MHC class I heavy chain) and MHC class II polypeptides (e.g., MHC class II α polypeptide and MHC class II β polypeptide).

[0079] As described above, in some embodiments of the multimeric polypeptides of the present disclosure, the first and second MHC polypeptides are class I MHC polypeptides. For example, in some cases, the first MHC polypeptide is an MHC class I β2-microglobulin (β2M) polypeptide, and the second MHC polypeptide is an MHC class I heavy chain (H chain). In other cases, the first and second MHC polypeptides are class II MHC polypeptides. For example, in some cases, the first MHC polypeptide is an MHC class II α-chain polypeptide, and the second MHC polypeptide is an MHC class II β-chain polypeptide. In other cases, the first polypeptide is an MHC class II β-chain polypeptide, and the second MHC polypeptide is an MHC class II α-chain polypeptide.

[0080] In some cases, the MHC polypeptide of the multimeric polypeptide of the present disclosure is a human MHC polypeptide, and the human MHC polypeptide is also referred to as a "human leukocyte antigen" ("HLA") polypeptide. In some cases, the MHC polypeptide of the multimeric polypeptide of the present disclosure 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. In some cases, the MHC polypeptide of the multimeric polypeptide of the present disclosure is a class II HLA polypeptide, such as a class II HLA α chain or a class II HLA β chain. MHC class II polypeptides include MCH class II DP α and β polypeptides, DM α and β polypeptides, DOA α and β polypeptides, DOB α and β polypeptides, DQ α and β polypeptides, and DR α and β polypeptides.

[0081] In some cases, the MHC class I heavy chain polypeptide of the multimeric polypeptide may include an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% amino acid sequence identity to the amino acid sequence shown in one of FIGS. 5A-5C.

[0082] HLA-A As an example, the MHC class I heavy chain polypeptide of the multimeric polypeptide may include an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% amino acid sequence identity to the following human HLA-A heavy chain amino acid sequence: TIFF0007717440000011.tif32151.

[0083] HLA-A(Y84A;A236C) In some cases, the MHC class I heavy chain polypeptide comprises the substitutions Y84A and A236C. For example, in some cases, the MHC class I heavy chain polypeptide has the following human HLA-A heavy chain (Y84A; A236C) amino acid sequence: It may comprise an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% amino acid sequence identity to TIFF0007717440000012.tif32151 (in the sequence, amino acid 84 is Ala and amino acid 236 is Cys). In some cases, Cys-236 forms an interchain disulfide bond with Cys-12 of a mutant β2M polypeptide comprising the substitution R12C.

[0084] HLA-A(Y84C;A139C) In some cases, the MHC class I heavy chain polypeptide comprises the substitutions Y84C and A139C. For example, in some cases, the MHC class I heavy chain polypeptide has the following human HLA-A heavy chain (Y84C; A139C) amino acid sequence: It may comprise an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% amino acid sequence identity to TIFF0007717440000013.tif32151 (in the sequence, amino acid 84 is Cys and amino acid 139 is Cys). In some cases, Cys-84 forms an interchain disulfide bond with Cys-139.

[0085] HLA-A A11(HLA-A11) As a non-limiting example, the MHC class I heavy chain polypeptide of the multimeric polypeptide has the following human HLA-A A11 (also referred to as "HLA-A11") heavy chain amino acid sequence: It may 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 TIFF0007717440000014.tif32151. Such MHC class I heavy chains may be prominent in the Asian population, including populations of Asian individuals.

[0086] HLA-A A11(Y84A;A236C) As a non-limiting example, in some cases, the MHC class I heavy chain polypeptide is the HLA-A A11 allele containing the substitutions Y84A and A236C. For example, in some cases, the MHC class I heavy chain polypeptide has the following human HLA-A A11 heavy chain (Y84A;A236C) amino acid sequence: It may 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 TIFF0007717440000015.tif32151 (in the sequence, amino acid 84 is Ala and amino acid 236 is Cys). In some cases, Cys-236 forms an interchain disulfide bond with Cys-12 of a mutant β2M polypeptide containing the substitution R12C.

[0087] HLA-B As another example, the MHC class I heavy chain polypeptide of the multimeric polypeptide has the following human HLA-B heavy chain amino acid sequence: It may 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 TIFF0007717440000016.tif32151.

[0088] HLA-B(Y84A;A236C) As a non-limiting example, in some cases, the MHC class I heavy chain polypeptide is an HLA-B polypeptide comprising the substitutions Y84A and A236C. For example, in some cases, the MHC class I heavy chain polypeptide has the following human HLA-B heavy chain (Y84A; A236C) amino acid sequence: TIFF0007717440000017.tif33151 (in the sequence, amino acid 84 is Ala and amino acid 236 is Cys) and 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 thereto. In some cases, Cys-236 forms an interchain disulfide bond with Cys-12 of a mutant β2M polypeptide comprising the substitution R12C.

[0089] HLA-B(Y84C;A139C) In some cases, the MHC class I heavy chain polypeptide comprises the substitutions Y84C and A139C. For example, in some cases, the MHC class I heavy chain polypeptide has the following human HLA-B heavy chain (Y84C; A139C) amino acid sequence: TIFF0007717440000018.tif32151 (in the sequence, amino acid 84 is Cys and amino acid 139 is Cys) and 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 thereto. In some cases, Cys-84 forms an interchain disulfide bond with Cys-139.

[0090] HLA-C As another example, the MHC class I heavy chain polypeptide of the multimeric polypeptide has the following human HLA-C heavy chain amino acid sequence: It may 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 TIFF0007717440000019.tif32152.

[0091] HLA-C(Y84A;A236C) As a non-limiting example, in some cases, the MHC class I heavy chain polypeptide is an HLA-C polypeptide comprising the substitutions Y84A and A236C. For example, in some cases, the MHC class I heavy chain polypeptide has the following human HLA-C heavy chain (Y84A;A236C) amino acid sequence: It may 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 TIFF0007717440000020.tif32152 (in the sequence, amino acid 84 is Ala and amino acid 236 is Cys). In some cases, Cys-236 forms an interchain disulfide bond with Cys-12 of a mutant β2M polypeptide comprising the substitution R12C.

[0092] HLA-C(Y84C;A139C) In some cases, the MHC class I heavy chain polypeptide comprises the substitutions Y84C and A139C. For example, in some cases, the MHC class I heavy chain polypeptide has the following human HLA-C heavy chain (Y84C;A139C) amino acid sequence: It may 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 TIFF0007717440000021.tif32152 (in the sequence, amino acid 84 is Cys and amino acid 139 is Cys). In some cases, Cys-84 forms an interchain disulfide bond with Cys-139.

[0093] In some cases, the MHC class I heavy chain polypeptide of the multimeric polypeptide may comprise an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% amino acid sequence identity to the amino acid sequence shown in one of FIGS. 3A-3C.

[0094] As an example, the MHC class I heavy chain polypeptide of the multimeric polypeptide may comprise an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% amino acid sequence identity to amino acids 25-365 of the amino acid sequence of the human HLA-A heavy chain polypeptide shown in FIG. 3A.

[0095] As another example, the MHC class I heavy chain polypeptide of the multimeric polypeptide may comprise an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% amino acid sequence identity to amino acids 25-362 of the amino acid sequence of the human HLA-B heavy chain polypeptide shown in FIG. 3B.

[0096] As another example, the MHC class I heavy chain polypeptide of the multimeric polypeptide may comprise an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% amino acid sequence identity to amino acids 25-362 of the amino acid sequence of the human HLA-C heavy chain polypeptide shown in FIG. 3C.

[0097] As another example, the MHC class I heavy chain polypeptide of the multimeric polypeptide may comprise an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% amino acid sequence identity to the following amino acid sequence: TIFF0007717440000022.tif32151。

[0098] The β2-microglobulin (β2M) polypeptide of the multimeric polypeptide can be a human β2M polypeptide, a non-human primate β2M polypeptide, a murine β2M polypeptide, etc. In some cases, the β2M polypeptide comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% amino acid sequence identity to the β2M amino acid sequence shown in FIG. 6. In some cases, the β2M polypeptide comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% amino acid sequence identity to amino acids 21-119 of the β2M amino acid sequence shown in FIG. 6.

[0099] In some cases, the MHC polypeptide comprises a single amino acid substitution as compared to a reference MHC polypeptide (the reference MHC polypeptide can be a wild-type MHC polypeptide), and the single amino acid substitution is one that substitutes one amino acid with a cysteine (Cys) residue. Such a cysteine residue, when present in the MHC polypeptide of the first polypeptide of the multimeric polypeptide of the present disclosure, can form a disulfide bond with a cysteine residue present in the second polypeptide chain of the multimeric polypeptide of the present disclosure.

[0100] In some cases, the first MHC polypeptide in the first polypeptide of the multimeric polypeptide and / or the second MHC polypeptide in the second polypeptide of the multimeric polypeptide comprises an amino acid substitution that replaces one amino acid with cysteine, and the substituted cysteine in the first MHC polypeptide forms a disulfide bond with the cysteine in the second MHC polypeptide, or 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.

[0101] For example, in some cases, one of the following residue pairs in HLA β2-microglobulin and the HLA class I heavy chain is replaced 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 Arg3, HLA class I heavy chain residue Gly120; 16) β2M residue His31, HLA class I heavy chain residue Gln96; 17) β2M residue Asp53, HLA class I heavy chain residue Arg35; 18) β2M residue Trp60, HLA class I heavy chain residue Gln96; 19) β2M residue Trp60, HLA class I heavy chain residue Asp122; 20) β2M residue Tyr63, HLA class I heavy chain residue Tyr27; 21) β2M residue Lys6, HLA class I heavy chain residue Glu232; 22) β2M residue Gln8, HLA class I heavy chain residue Arg234; 23) β2M residue Tyr10, HLA class I heavy chain residue Pro235; 24) β2M residue Ser11, HLA class I heavy chain residue Gln242; 25) β2M residue Asn24, HLA class I heavy chain residue Ala236; 26) β2M residue Ser28, HLA class I heavy chain residue Glu232; 27) β2M residue Asp98, HLA class I heavy chain residue His192; and 28) β2M residue Met99, HLA class I heavy chain residue Arg234. The amino acid numbering of the MHC / HLA class I heavy chain is based on the mature MHC / HLA class I heavy chain without the signal peptide.For example, in the amino acid sequence containing the signal peptide shown in FIG. 5A, Gly120 is Gly144, Gln96 is Gln120, and so on. In some cases, the β2M polypeptide contains an R12C substitution, and the HLA class I heavy chain contains an A236C substitution. In such cases, a disulfide bond is formed between Cys-12 of the β2M polypeptide and Cys-236 of the HLA class I heavy chain. For example, in some cases, residue 236 of the mature HLA-A amino acid sequence (i.e., residue 260 of the amino acid sequence shown in FIG. 5A) is substituted with Cys. In some cases, residue 236 of the mature HLA-B amino acid sequence (i.e., residue 260 of the amino acid sequence shown in FIG. 5B) is substituted with Cys. In some cases, residue 236 of the mature HLA-C amino acid sequence (i.e., residue 260 of the amino acid sequence shown in FIG. 5C) is substituted with Cys. In some cases, amino acid sequence residue 32 shown in FIG. 6 (corresponding to Arg-12 of mature β2M) is substituted with Cys.

[0102] In some cases, the β2M polypeptide has the amino acid sequence: TIFF0007717440000023.tif18131. In some cases, the β2M polypeptide has the amino acid sequence: TIFF0007717440000024.tif18128.

[0103] In some cases, the HLA class I heavy chain polypeptide has the amino acid sequence: TIFF0007717440000025.tif32151.

[0104] In some cases, the HLA class I heavy chain polypeptide has the amino acid sequence: TIFF0007717440000026.tif39151.

[0105] In some cases, the HLA class I heavy chain polypeptide has the amino acid sequence: It includes TIFF0007717440000027.tif32151.

[0106] In some cases, the β2M polypeptide has the following amino acid sequence: It includes TIFF0007717440000028.tif19145, and the HLA class I heavy chain polypeptide of the multimeric polypeptide of the present disclosure has the following amino acid sequence: It includes TIFF0007717440000029.tif39151, and the underlined Cys residues form disulfide bonds with each other within the multimeric polypeptide.

[0107] In some cases, the β2M polypeptide has the amino acid sequence: It includes TIFF0007717440000030.tif12150.

[0108] Scaffold polypeptide The T cell regulatory multimeric polypeptide includes an Fc polypeptide or another suitable scaffold polypeptide.

[0109] Suitable scaffold polypeptides include antibody-based scaffold polypeptides and non-antibody-based scaffolds. Non-antibody-based scaffolds include, for example, albumin, XTEN (extended recombinant) polypeptides, transferrin, Fc receptor polypeptides, elastin-like polypeptides (see, e.g., Hassouneh et al. (2012) Methods Enzymol. 502:215; e.g., polypeptides containing pentapeptide repeat units of (Val-Pro-Gly-X-Gly; SEQ ID NO: 212), where X is any amino acid other than proline), albumin-binding polypeptides, silk-like polypeptides (see, e.g., Valluzzi et al. (2002) Philos Trans R Soc Lond B Biol Sci. 357:165), silk-elastin-like polypeptides (SELP; see, e.g., Megeed et al. (2002) Adv Drug Deliv Rev. 54:1075), and the like. Suitable XTEN polypeptides include, for example, those disclosed in 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.

[0110] Suitable backbone polypeptides are, in some cases, half-life extended polypeptides. Thus, in some cases, suitable backbone polypeptides increase the in vivo half-life (e.g., serum half-life) of the multimeric polypeptide as compared to a control multimeric polypeptide that does not have a backbone polypeptide. For example, in some cases, the backbone polypeptide increases the in vivo half-life (e.g., serum half-life) of the multimeric polypeptide 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 as compared to a control multimeric polypeptide that does not have a backbone polypeptide. As an example, in some cases, the Fc polypeptide increases the in vivo half-life (e.g., serum half-life) of the multimeric polypeptide 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 as compared to a control multimeric polypeptide that does not have an Fc polypeptide.

[0111] Fc polypeptide In some cases, the first and / or second polypeptide chains of the multimeric polypeptide comprise an Fc polypeptide. The Fc polypeptide of the multimeric polypeptide can be, for example, human IgG1 Fc, human IgG2 Fc, human IgG3 Fc, human IgG4 Fc, etc. In some cases, the Fc polypeptide comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% or 100% amino acid sequence identity to the amino acid sequence of the Fc region shown in FIGS. 4A - C. In some cases, 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. 4A. In some cases, 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. 4A and also comprises a substitution at N77, for example, the Fc polypeptide comprises a substitution of N77A. In some cases, the Fc polypeptide comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% or 100% amino acid sequence identity to the human IgG2 Fc polypeptide shown in FIG. 4A, for example, the Fc polypeptide comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% or 100% amino acid sequence identity to amino acids 99 - 325 of the human IgG2 Fc polypeptide shown in FIG. 4A.In some cases, the Fc polypeptide comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% or 100% amino acid sequence identity to the human IgG3 Fc polypeptide shown in Figure 4A. For example, the Fc polypeptide comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% or 100% amino acid sequence identity to amino acids 19-246 of the human IgG3 Fc polypeptide shown in Figure 4A. In some cases, the Fc polypeptide comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% or 100% amino acid sequence identity to the human IgM Fc polypeptide shown in Figure 4B. 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 4B. In some cases, the Fc polypeptide comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% or 100% amino acid sequence identity to the human IgA Fc polypeptide shown in Figure 4C. 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 4C.

[0112] In some cases, the Fc polypeptide present in the multimeric polypeptide comprises the amino acid sequence shown in Figure 33A (human IgG1 Fc). In some cases, the Fc polypeptide present in the multimeric polypeptide of the present disclosure comprises the amino acid sequence shown in Figure 33A (human IgG1 Fc), except for substitutions with amino acids other than asparagine at N297. In some cases, the Fc polypeptide present in the multimeric polypeptide of the present disclosure comprises the amino acid sequence shown in Figure 33C (human IgG1 Fc containing the N297A substitution). In some cases, the Fc polypeptide present in the multimeric polypeptide of the present disclosure comprises the amino acid sequence shown in Figure 33A (human IgG1 Fc), except for substitutions with amino acids other than leucine at L234. In some cases, the Fc polypeptide present in the multimeric polypeptide of the present disclosure comprises the amino acid sequence shown in Figure 33A (human IgG1 Fc), except for substitutions with amino acids other than leucine at L235. In some cases, the Fc polypeptide present in the multimeric polypeptide of the present disclosure comprises the amino acid sequence shown in Figure 33D (human IgG1 Fc containing the L234A substitution and the L235A substitution). In some cases, the Fc polypeptide present in the multimeric polypeptide of the present disclosure comprises the amino acid sequence shown in Figure 33A (human IgG1 Fc), except for substitutions with amino acids other than proline at P331, and in some cases, the substitution is the P331S substitution. In some cases, the Fc polypeptide present in the multimeric polypeptide of the present disclosure comprises the amino acid sequence shown in Figure 33A (human IgG1 Fc), except for substitutions with amino acids other than leucine at L234 and L235. In some cases, the Fc polypeptide present in the multimeric polypeptide of the present disclosure comprises the amino acid sequence shown in Figure 33A (human IgG1 Fc), except for substitutions with amino acids other than leucine at L234 and L235 and substitutions with amino acids other than proline at P331. In some cases, the Fc polypeptide present in the multimeric polypeptide of the present disclosure comprises the amino acid sequence shown in Figure 33B (human IgG1 Fc containing the L234F, L235E, and P331S substitutions).In some cases, the Fc polypeptide present in the multimeric polypeptide is an IgG1 Fc polypeptide comprising the substitutions L234A and L235A.

[0113] Linker The multimeric polypeptide can include, for example, a linker peptide inserted between an epitope and an MHC polypeptide, between an MHC polypeptide and an immunomodulatory polypeptide, between an MHC polypeptide and an Ig Fc polypeptide, between a first immunomodulatory polypeptide and a second immunomodulatory polypeptide, or between a second immunomodulatory polypeptide and a third immunomodulatory polypeptide.

[0114] For example, the multimeric polypeptide can include, for example, a linker peptide inserted between an epitope and an MHC polypeptide, between an MHC polypeptide and an immunomodulatory polypeptide, between an MHC polypeptide and an Ig Fc polypeptide, between a first mutant IL-2 polypeptide and a second mutant IL-2 polypeptide, or between a second mutant IL-2 polypeptide and a third mutant IL-2 polypeptide. As another example, the multimeric polypeptide can include, for example, a linker peptide inserted between an epitope and an MHC polypeptide, between an MHC polypeptide and an immunomodulatory polypeptide, between an MHC polypeptide and an Ig Fc polypeptide, between a first mutant 4-1BBL polypeptide and a second mutant 4-1BBL polypeptide, or between a second mutant 4-1BBL polypeptide and a third mutant 4-1BBL polypeptide.

[0115] Suitable linkers (also referred to as "spacers") can be readily selected and can be any of a number of suitable lengths, including 1 to 25 amino acids, 3 to 20 amino acids, 2 to 15 amino acids, 3 to 12 amino acids, etc., as well as 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.

[0116] Exemplary linkers include glycine polymers (G) n , glycine-serine polymers (e.g., (GS) n , (GSGGS) n (SEQ ID NO: 210) and (GGGS) n (SEQ ID NO: 211) (wherein in the sequence, n is at least one integer), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers can be used, and since both Gly and Ser are relatively unstructured, they can function as intermediate tethers between components. Glycine polymers can be used, and glycine can utilize significantly more Φ-Ψ space than alanine and is less restricted than residues with long side chains (see Scheraga, Rev. Computational Chem. 11173-142 (1992)).

[0117] Exemplary linkers can include amino acid sequences including, but not limited to, GGSG (SEQ ID NO: 65), GGSGG (SEQ ID NO: 66), GSGSG (SEQ ID NO: 67), GSGGG (SEQ ID NO: 68), GGGSG (SEQ ID NO: 69), GSSSG (SEQ ID NO: 70), and the like. Exemplary linkers can include, for example, Gly(Ser4)n, where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some cases, the linker includes the amino acid sequence (GSSSS)n (SEQ ID NO: 71), where n is 4. In some cases, the linker includes the amino acid sequence (GSSSS)n (SEQ ID NO: 72), where n is 5. In some cases, the linker includes the amino acid sequence (GGGGS)n (SEQ ID NO: 205), where n is 1. In some cases, the linker includes the amino acid sequence (GGGGS)n (SEQ ID NO: 206), where n is 2. In some cases, the linker includes the amino acid sequence (GGGGS)n (SEQ ID NO: 207), where n is 3. In some cases, the linker includes the amino acid sequence (GGGGS)n (SEQ ID NO: 208), where n is 4. In some cases, the linker includes the amino acid sequence (GGGGS)n (SEQ ID NO: 209), where n is 5. In some cases, the linker includes the amino acid sequence AAAGG (SEQ ID NO: 73).

[0118] In some cases, the linker polypeptide present in the first polypeptide of the multimeric polypeptide of the present disclosure can include a cysteine residue that can form a disulfide bond with a cysteine residue present in the second polypeptide of the multimeric polypeptide of the present disclosure. In some cases, for example, a suitable linker includes the amino acid sequence TIFF0007717440000031.tif6128 is included.

[0119] Epitope The epitopes (peptides that provide one or more epitopes) present in the multimeric polypeptides of the present disclosure can have a length of about 4 amino acids to about 25 amino acids. For example, the epitopes can have a length of 4 amino acids (aa) to 10 aa, 10 aa to 15 aa, 15 aa to 20 aa, or 20 aa to 25 aa. For example, the epitopes present in the multimeric polypeptides of the present disclosure can have a length of 4 amino acids (aa), 5 aa, 6 aa, 7 aa, 8 aa, 9 aa, 10 aa, 11 aa, 12 aa, 13 aa, 14 aa, 15 aa, 16 aa, 17 aa, 18 aa, 19 aa, 20 aa, 21 aa, 22 aa, 23 aa, 24 aa, or 25 aa. In some cases, the epitopes present in the multimeric polypeptides of the present disclosure have a length of 5 amino acids to 10 amino acids, for example, 5 aa, 6 aa, 7 aa, 8 aa, 9 aa, or 10 aa.

[0120] The epitopes present in the multimeric polypeptides of the present disclosure are specifically bound by T cells, that is, the epitopes are specifically bound by epitope-specific T cells. Epitope-specific T cells bind to epitopes having a reference amino acid sequence, but do not substantially bind to epitopes different from the reference amino acid sequence. For example, epitope-specific T cells bind to an epitope having a reference amino acid sequence, and even if they bind to an epitope different from the reference amino acid sequence, it is -6 less than 10 -5 M, 10 -4 less than M, or 10 -7 less than M affinity. Epitope-specific T cells can bind to specific epitopes with an affinity of at least 10 -8 M, at least 10 -9 M, at least 10 -10 M, or at least 10

[0121] Suitable epitopes include, but are not limited to, epitopes present in cancer-related antigens. Cancer-related antigens include, but are not limited to, folate receptor alpha, carbonic anhydrase IX (CAIX), CD19, CD20, CD22, CD30, CD33, CD44v7 / 8, carcinoembryonic antigen (CEA), epithelial glycoprotein-2 (EGP-2), epithelial glycoprotein-40 (EGP-40), folate-binding protein (FBP), fetal acetylcholine receptor, ganglioside antigen GD2, Her2 / neu, IL-13R-a2, kappa light chain, LeY, L1 cell adhesion molecule, melanoma-associated antigen (MAGE), MAGE-A1, mesothelin, MUC1, NKG2D ligand, tumor fetal 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, for example, Vigneron et al. (2013) Cancer Immunity 13:15; and Vigneron (2015) BioMed Res. Int’l Article ID 948501. In some cases, the epitope is a human papillomavirus E7 antigen epitope. See, for example, Ramos et al. (2013) J. Immunother. 36:66.

[0122] In some cases, the epitope is HPV16E7 / 82-90 (LLMGTLGIV; SEQ ID NO:75). In some cases, the epitope is HPV16E7 / 86-93 (TLGIVCPI; SEQ ID NO:76). In some cases, the epitope is HPV16E7 / 11-20 (YMLDLQPETT; SEQ ID NO:77). In some cases, the epitope is HPV16E7 / 11-19 (YMLDLQPET; SEQ ID NO:78). For further suitable HPV epitopes, see, for example, Ressing et al. ((1995) J. Immunol. 154:5934).

[0123] Immunomodulatory polypeptide Suitable immunomodulatory polypeptides include, but are not limited to, IL-2 polypeptide, 4-1BBL polypeptide, B7-1 polypeptide, B7-2 polypeptide, ICOS-L polypeptide, OX-40L polypeptide, CD80 polypeptide, CD86 polypeptide, PD-L1 polypeptide, FasL polypeptide, and PD-L2 polypeptide.

[0124] In some cases, the immunomodulatory polypeptide comprises 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 with the amino acid sequence of the PD-L1 polypeptide shown in FIG. 50A or FIG. 50B.

[0125] In some cases, the immunomodulatory polypeptide comprises 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 with the amino acid sequence of the CD80 polypeptide shown in FIG. 51.

[0126] In some cases, the immunomodulatory polypeptide comprises 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 with the amino acid sequence of the ICOS-L polypeptide shown in FIG. 51.

[0127] In some cases, the immunomodulatory polypeptide comprises 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 with the amino acid sequence of the OX40L polypeptide shown in FIG. 53.

[0128] In some cases, the immunomodulatory polypeptide comprises 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 with the amino acid sequence of the PD-L2 polypeptide shown in FIG. 54.

[0129] In some cases, the immunomodulatory polypeptide comprises 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 with the amino acid sequence of the CD86 polypeptide shown in FIG. 55.

[0130] In some cases, the immunomodulatory polypeptide comprises 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 with the amino acid sequence of the FAS-L polypeptide shown in FIG. 56.

[0131] In some cases, the immunomodulatory polypeptide present in the synTac exhibits a reduced binding affinity compared to the binding affinity of the wild-type immunomodulatory polypeptide for the same allogeneic immunomodulatory polypeptide expressed on the surface of T cells. In some cases, where the synTac contains an immunomodulatory polypeptide with reduced affinity, the synTac polypeptide exhibits a reduced binding to the allogeneic immunomodulatory polypeptide expressed on the surface of T cells. For example, in some cases, a synTac polypeptide containing an immunomodulatory polypeptide with reduced affinity binds to the allogeneic immunomodulatory polypeptide with a binding affinity that is at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% lower than the binding affinity of a control synTac polypeptide containing the wild-type immunomodulatory polypeptide for the same allogeneic immunomodulatory polypeptide.

[0132] Determination of binding affinity The binding affinity between an immunomodulatory polypeptide and its cognate immunocoregulatory polypeptide can be determined by the Biolayer Interference (BLI) method using the purified immunomodulatory polypeptide and the purified cognate immunocoregulatory polypeptide. The binding affinity between the synTac of the present disclosure and its cognate immunocoregulatory polypeptide can also be determined by BLI using the purified synTac and the cognate immunocoregulatory polypeptide. The BLI method is well known to those skilled in the art. See, for example, Lad et al. (2015) J. Biomol. Screen. 20(4):498-507; and Shah and Duncan (2014) J. Vis. Exp. 18:e51383. The specific and relative binding affinities between the immunomodulatory polypeptide and its cognate immunocoregulatory polypeptide, or between the synTac and its cognate immunocoregulatory polypeptide, as described in the present disclosure, can be determined using the following procedure.

[0133] To determine the binding affinity between synTac of the present disclosure and its cognate immunomodulatory polypeptide, a BLI assay can be performed as follows using an Octet RED 96 (Pal ForteBio) instrument or a similar instrument. To determine the binding affinity of a T cell regulatory multimer polypeptide (e.g., synTac of the present disclosure; or a control T cell regulatory multimer polypeptide, where the control T cell regulatory multimer polypeptide includes a wild-type immunomodulatory polypeptide), the T cell regulatory multimer polypeptide is immobilized on an insoluble support ("biosensor"). The immobilized T cell regulatory multimer polypeptide is the "target". Immobilization can be performed by immobilizing a capture antibody on the insoluble support, and the capture antibody immobilizes the T cell regulatory multimer polypeptide. For example, immobilization can be performed by immobilizing an anti-Fc (e.g., anti-human IgG Fc) antibody on the insoluble support, and the immobilized anti-Fc antibody binds to and immobilizes a T cell regulatory multimer polypeptide (where the T cell regulatory multimer polypeptide includes an IgFc polypeptide). The immunomodulatory polypeptide is applied to the immobilized T cell regulatory multimer polypeptide at several different concentrations, and the response of the instrument 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 nonionic surfactant. Binding of the immunomodulatory polypeptide to the immobilized T cell regulatory multimer polypeptide 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 DThe anti-HLA class I monoclonal antibody W6 / 32 (American Type Culture Collection No. HB-95; Parham et al. (1979) J. Immunol. 123:342) having [the relevant property] can be used. Serial dilutions of the anti-MHC class I monoclonal antibody can be used to generate a standard curve. The immunomodulatory polypeptide or the anti-MHC class I mAb is the "analyte". BLI analyzes the interference wave of white light reflected from two surfaces of i) the immobilized polypeptide ("target") and ii) the internal reference layer. A change in the number of molecules ("analyte"; e.g., immunomodulatory polypeptide, anti-HLA antibody) binding to the biosensor chip causes a shift in the interference wave, and this shift in the interference wave can be measured in real time. Two kinetic terms that describe the affinity of the target / analyte interaction are the association constant (k a ) and the dissociation constant (k d ). The ratio of these two terms (k d / a ) gives the affinity constant K D .

[0134] As described above, the determination of the binding affinity between an immunomodulatory polypeptide (e.g., IL-2 or an IL-2 variant) and its cognate immunocoregulatory polypeptide (e.g., IL-2R) can also be determined by BLI. The assay is similar to that described above for the synTac multimeric polypeptide. The BLI assay can be performed as follows using an Octet RED 96 (Pal ForteBio) instrument or a similar instrument. An immunomodulatory polypeptide (e.g., a mutant IL-2 polypeptide of the present disclosure) and a control immunomodulatory polypeptide (where the control immunomodulatory polypeptide includes a wild-type immunomodulatory polypeptide, e.g., wild-type IL-2), which are components of the synTac of the present disclosure, are immobilized on an insoluble support (“biosensor”). The immunomodulatory polypeptide is the “target”. The immobilization can be performed by immobilizing a capture antibody on the insoluble support, and the capture antibody immobilizes the immunomodulatory polypeptide. For example, if the target is fused to an immunoaffinity tag (e.g., FLAG, human IgG Fc), the immobilization can be performed by immobilizing an appropriate antibody against the immunoaffinity tag (e.g., anti-human IgG Fc) on the insoluble support, and the immobilized antibody binds to and immobilizes the immunomodulatory polypeptide (where the immunomodulatory polypeptide includes an IgFc polypeptide). The immunocoregulatory polypeptide (or polypeptides) is applied at several different concentrations to the immobilized immunomodulatory polypeptide, and the response of the instrument is recorded. Alternatively, the immunocoregulatory polypeptide (or polypeptides) is immobilized on the biosensor (e.g., against an IL-2 receptor heterotrimer, e.g., a monomeric subunit, a heterodimeric subcomplex, or a complete heterotrimer), and the immunomodulatory polypeptide is applied at several different concentrations to the immobilized immunocoregulatory polypeptide(s), and the response of the instrument 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 nonionic surfactant. The binding of the immunocoregulatory polypeptide to the immobilized immunomodulatory polypeptide 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 Kd of 7 nM. D The anti-HLA class I monoclonal antibody W6 / 32 (American Type Culture Collection No. HB-95; Parham et al. (1979) J. Immunol. 123:342) having a Kd of D can be used. Serial dilutions of the anti-MHC class I monoclonal antibody can be used to generate a standard curve. The immunomodulatory polypeptide or anti-MHC class I mAb is the "analyte". BLI analyzes the interference wave of white light reflected from two surfaces of i) the immobilized polypeptide ("target") and ii) the internal reference layer. A change in the number of molecules ("analyte"; e.g., immunomodulatory polypeptide, anti-HLA antibody) binding to the biosensor chip causes a shift in the interference wave, and this shift in the interference wave can be measured in real time. Two kinetic terms that describe the affinity of the target / analyte interaction are the association constant (k a ) and the dissociation constant (k d ). The ratio of these two terms (k d / a ) gives the affinity constant K D . Thus, by determining both the binding affinity of a wild-type immunomodulatory polypeptide (e.g., IL-2) for its receptor (e.g., IL-2R) and the binding affinity of a mutant immunomodulatory polypeptide (e.g., an IL-2 variant disclosed herein) for its cognate immunomodulatory polypeptide (e.g., its receptor) (e.g., IL-2R), it becomes possible to determine the relative binding affinity of the mutant immunomodulatory polypeptide for the cognate immunomodulatory polypeptide compared to the wild-type immunomodulatory polypeptide. That is, it can be determined whether the binding affinity of the mutant immunomodulatory polypeptide for its receptor (its cognate immunomodulatory polypeptide) is decreased compared to the binding affinity of the wild-type immunomodulatory polypeptide for the same cognate immunomodulatory polypeptide, and if so, what percentage decrease in binding affinity from the wild-type immunomodulatory polypeptide.

[0135] The BLI assay is performed in a multi-well plate. To perform the assay, determine the plate layout, determine the assay steps, and assign the biosensor to the Octet data acquisition software. Hydrate the biosensor assembly. Equilibrate the hydrated biosensor assembly and the assay plate on the Octet instrument for 10 minutes. Once data is acquired, load the acquired data into the Octet data analysis software. Perform data processing by specifying methods for reference subtraction, y-axis alignment, stepwise correction, and Savitzky-Golay filtering in the processing window. Perform data analysis by specifying the steps to analyze (association and dissociation), selecting the curve fitting model (1:1), the fitting method (global), and the window of interest (in seconds) in the analysis window. Evaluate the quality of the fit. The K D values of each data trace (analyte concentration) can be averaged if they are within a three-fold range. K D error values must be within 10-fold of the affinity constant value, and R 2 values should be greater than 0.95. See, for example, Abdiche et al. (2008) J. Anal. Biochem. 377:209.

[0136] In some cases, the ratio of i) the binding affinity of a control T cell regulatory multimer polypeptide (where the control includes a wild-type immunoregulatory polypeptide, e.g., wild-type IL-2) for a cognate immunocoregulatory polypeptide (e.g., IL-2R), to ii) the binding affinity of a T cell regulatory multimer polypeptide of the present disclosure comprising a mutant of a wild-type immunoregulatory polypeptide (e.g., mutant IL-2) for a cognate immunocoregulatory polypeptide (e.g., IL-2R), 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 :1. In some cases, i) the binding affinity of a control T cell regulatory multimer polypeptide (where the control includes a wild-type immunoregulatory polypeptide) for a homologous immunocoregulatory polypeptide, and ii) the binding affinity of the T cell regulatory multimer polypeptide of the present disclosure comprising a mutant of the wild-type immunoregulatory polypeptide for a homologous immunocoregulatory polypeptide, when measured by BLI, is 1.5:1 to 10 6 :1. For example, 1.5:1 to 10:1, 10:1 to 50:1, 50:1 to 10 2 :1. 10 2 :1 to 10 3 :1. 10 3 :1 to 10 4 :1. 10 4 :1 to 10 5 :1. Or 10 5 :1 to 10 6 :1. within the range of.

[0137] In some cases, i) the binding affinity of a control immunoregulatory polypeptide (where the control includes a wild-type immunoregulatory polypeptide, for example, wild-type IL-2) for a homologous immunocoregulatory polypeptide (for example, IL-2R), and ii) the binding affinity of the immunoregulatory polypeptide of the present disclosure comprising a mutant of the wild-type immunoregulatory polypeptide (for example, mutant IL-2) for a homologous immunocoregulatory polypeptide (for example, IL-2R), when measured by BLI (as described above), is at least 1.5:1, at least 2:1, at least 5:1, at least 10:1, at least 15:1, at least 20:1, at least 25:1, at least 50:1, at least 100:1, at least 500:1, at least 10 2 :1. At least 5×10 2 :1. At least 10 3 :1. At least 5×10 3 :1. At least 10 4 :1. At least 105 : 1, or at least 10 6 : 1. In some cases, i) the binding affinity of a control immunomodulatory polypeptide (where the control includes a wild-type immunomodulatory polypeptide) for a cognate immunocoregulatory polypeptide, and ii) the binding affinity of an immunomodulatory polypeptide of the present disclosure comprising a mutant of a wild-type immunomodulatory polypeptide for a cognate immunocoregulatory polypeptide, the ratio of which, when measured by BLI, is 1.5:1 to 10 6 : 1, for example, 1.5:1 to 10:1, 10:1 to 50:1, 50:1 to 10 2 : 1, 10 2 : 1 to 10 3 : 1, 10 3 : 1 to 10 4 : 1, 10 4 : 1 to 10 5 : 1, or 10 5 : 1 to 10 6 : within the range of 1.

[0138] IL-2 / synTac In some cases, the multimeric polypeptide comprises wild-type (native) IL-2 as a regulatory domain. In some cases, the multimeric polypeptide comprises a mutant IL-2 polypeptide as a regulatory domain.

[0139] A T cell regulatory multimeric polypeptide comprising an IL-2 polypeptide as a regulatory ("MOD") domain is also referred to as "IL-2 / synTac", "IL-2 / synTac polypeptide" or "IL-2 / multimeric polypeptide".

[0140] In some cases, the IL-2 / synTac polypeptide comprises a wild-type IL-2 polypeptide. In some cases, the synTac polypeptide comprises one copy of the wild-type IL-2 polypeptide. In some cases, the synTac polypeptide comprises two copies of the wild-type IL-2 polypeptide. In some cases, the synTac polypeptide comprises three copies of the wild-type IL-2 polypeptide. In some cases, the wild-type IL-2 polypeptide comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2A. The wild-type amino acid sequence of the human IL2 polypeptide can be as follows: TIFF0007717440000032.tif19132。

[0141] In some cases, the synTac polypeptide comprises a mutant IL-2 polypeptide. The mutant IL-2 polypeptide present in the multimeric polypeptide exhibits a reduced binding affinity for IL2R as compared to the binding affinity of wild-type IL-2 for IL2R. The multimeric polypeptide comprising the mutant IL-2 polypeptide also exhibits a reduced binding affinity for IL2R as compared to that of a control multimeric polypeptide comprising wild-type IL-2 for IL2R (e.g., IL2R comprising α, β and γ polypeptides comprising the amino acid sequences (mature form) shown in FIGS. 3A-3C).

[0142] In some cases, the IL-2 / synTac polypeptide exhibits a reduced binding affinity for the IL2R as compared to the binding affinity of the IL2 polypeptide containing the amino acid sequence shown in FIG. 2A for the IL2R. For example, in some cases, the IL-2 / synTac polypeptide binds to the IL2R with a binding affinity lower than that of a control synTac polypeptide containing the IL2 polypeptide containing the amino acid sequence shown in FIG. 2A for the IL2R containing the α, β, and γ polypeptides containing the amino acid sequences (mature form) shown in FIGS. 3A-3C. For example, in some cases, the IL-2 / synTac polypeptide is at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% lower in binding affinity than the binding affinity of a control synTac polypeptide containing the IL-2 polypeptide containing the amino acid sequence shown in FIG. 2A for the IL2R (e.g., the IL2R containing the α, β, and γ polypeptides containing the amino acid sequences (mature form) shown in FIGS. 3A-3C), and binds to the IL2R.

[0143] In some cases, the IL-2 / synTac polypeptide has a binding affinity for IL2R of 100 nM to about 100 μM. In some cases, the IL-2 / synTac polypeptide has a binding affinity for IL2R of about 100 nM to 500 nM. For example, in some cases, the IL-2 / synTac polypeptide has a binding affinity for IL2R (e.g., IL2R comprising α, β and γ polypeptides including the amino acid sequences (mature form) shown in FIGS. 3A-3C) of about 100 nM to about 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 450 nM, or about 450 nM to about 500 nM. In some cases, the IL-2 / synTac polypeptide has a binding affinity for IL2R (e.g., IL2R comprising α, β and γ polypeptides including the amino acid sequences (mature form) shown in FIGS. 3A-3C) of about 500 nM to 1 μM. For example, in some cases, the IL-2 / synTac polypeptide has a binding affinity for IL2R (e.g., IL2R comprising α, β and γ polypeptides including the amino acid sequences (mature form) shown in FIGS. 3A-3C) of 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, or about 900 nM to about 1 μM. In some cases, the IL-2 / synTac polypeptide has a binding affinity for IL2R (e.g., IL2R comprising α, β and γ polypeptides including the amino acid sequences (mature form) shown in FIGS. 3A-3C) of about 1 μM to 10 μM. For example, in some cases, the IL-2 / synTac polypeptide has a binding affinity for IL2R (e.g., IL2R comprising α, β and γ polypeptides including the amino acid sequences (mature form) shown in FIGS. 3A-3C) of about 1 μM to 2 μM, about 2 μM to about 3 μM, about 3 μM to about 4 μM, about 4 μM to about 5 μM, about 5 μM to about 6 μM, about 6 μM to about 7 μM, about 7 μM to about 8 μM, about 8 μM to about 9 μM, or about 9 μM to about 10 μM.In some cases, the IL-2 / synTac polypeptide has a binding affinity of about 10 μM to 100 μM for the IL2R (e.g., the IL2R comprising the α, β, and γ polypeptides including the amino acid sequences (mature form) shown in FIGS. 3A-3C). For example, in some cases, the IL-2 / synTac polypeptide has a binding affinity of about 10 μM to about 20 μM, about 20 μM to about 30 μM, about 30 μM to about 40 μM, about 40 μM to about 50 μM, about 50 μM to about 60 μM, about 60 μM to about 70 μM, about 70 μM to about 80 μM, about 80 μM to about 90 μM, or about 90 μM to about 100 μM for the IL2R (e.g., the IL2R comprising the α, β, and γ polypeptides including the amino acid sequences (mature form) shown in FIGS. 3A-3C).

[0144] The mutant IL2 polypeptide present in the IL-2 / synTac polypeptide may have one amino acid substitution as compared to the wild-type IL2 polypeptide (e.g., the IL2 polypeptide comprising the amino acid sequence shown in FIG. 2A or set forth in SEQ ID NO: 1). In some cases, the mutant IL2 polypeptide present in the IL-2 / synTac polypeptide has 2 to 10 amino acid substitutions as compared to the wild-type IL2 polypeptide (e.g., the IL2 polypeptide comprising the amino acid sequence shown in FIG. 2A or set forth in SEQ ID NO: 1). In some cases, the mutant IL2 polypeptide present in the synTac polypeptide of the present disclosure has two amino acid substitutions as compared to the wild-type IL2 polypeptide (e.g., the IL2 polypeptide comprising the amino acid sequence shown in FIG. 2A or set forth in SEQ ID NO: 1). In some cases, the mutant IL2 polypeptide present in the synTac polypeptide of the present disclosure has three amino acid substitutions as compared to the wild-type IL2 polypeptide (e.g., the IL2 polypeptide comprising the amino acid sequence shown in FIG. 2A or set forth in SEQ ID NO: 1). In some cases, the mutant IL2 polypeptide present in the synTac polypeptide of the present disclosure has four amino acid substitutions as compared to the wild-type IL2 polypeptide (e.g., the IL2 polypeptide comprising the amino acid sequence shown in FIG. 2A or set forth in SEQ ID NO: 1). In some cases, the mutant IL2 polypeptide present in the synTac polypeptide of the present disclosure has five amino acid substitutions as compared to the wild-type IL2 polypeptide (e.g., the IL2 polypeptide comprising the amino acid sequence shown in FIG. 2A or set forth in SEQ ID NO: 1). In some cases, the mutant IL2 polypeptide present in the synTac polypeptide of the present disclosure has six amino acid substitutions as compared to the wild-type IL2 polypeptide (e.g., the IL2 polypeptide comprising the amino acid sequence shown in FIG. 2A or set forth in SEQ ID NO: 1). In some cases, the mutant IL2 polypeptide present in the synTac polypeptide of the present disclosure has seven amino acid substitutions as compared to the wild-type IL2 polypeptide (e.g., the IL2 polypeptide comprising the amino acid sequence shown in FIG. 2A or set forth in SEQ ID NO: 1).In some cases, the mutant IL2 polypeptide present in the synTac polypeptide of the present disclosure has eight amino acid substitutions compared to the wild-type IL2 polypeptide (e.g., the IL2 polypeptide comprising the amino acid sequence shown in FIG. 2A or set forth in SEQ ID NO: 1). In some cases, the mutant IL2 polypeptide present in the synTac polypeptide of the present disclosure has nine amino acid substitutions compared to the wild-type IL2 polypeptide (e.g., the IL2 polypeptide comprising the amino acid sequence shown in FIG. 2A or set forth in SEQ ID NO: 1). In some cases, the mutant IL2 polypeptide present in the synTac polypeptide of the present disclosure has ten amino acid substitutions compared to the wild-type IL2 polypeptide (e.g., the IL2 polypeptide comprising the amino acid sequence shown in FIG. 2A or set forth in SEQ ID NO: 1).

[0145] In some cases, the multimeric polypeptides of the present disclosure include a first polypeptide and a second polypeptide. The first polypeptide, in order from the amino terminus (N-terminus) to the carboxyl terminus (C-terminus), comprises a) an epitope (e.g., a T cell epitope), b) a first major histocompatibility complex (MHC) polypeptide, and c) an immunomodulatory polypeptide (e.g., a mutant IL2 polypeptide of the present disclosure). The second polypeptide, in order from the N-terminus to the C-terminus, comprises a) a second MHC polypeptide and b) an immunoglobulin (Ig) Fc polypeptide. In other cases, the multimeric polypeptides of the present disclosure include a first polypeptide and a second polypeptide. The first polypeptide, in order from the N-terminus to the C-terminus, comprises a) an epitope (e.g., a T cell epitope) and b) a first MHC polypeptide. The second polypeptide, in order from the N-terminus to the C-terminus, comprises a) an immunomodulatory polypeptide (e.g., a mutant IL2 polypeptide of the present disclosure), b) a second MHC polypeptide, and c) an Ig Fc polypeptide. In some cases, the first and second MHC polypeptides are class I MHC polypeptides. For example, in some cases, the first MHC polypeptide is an MHC class I β2-microglobulin (B2M or β2M) polypeptide, and the second MHC polypeptide is an MHC class I heavy chain (H chain), or the first MHC polypeptide is an MHC class I H chain, and the second MHC polypeptide is an MHC class I β2M polypeptide. In other cases, the first and second MHC polypeptides are class II MHC polypeptides. For example, in some cases, the first MHC polypeptide is an MHC class II α-chain polypeptide, and the second MHC polypeptide is an MHC class II β-chain polypeptide. In other cases, the first polypeptide is an MHC class II β-chain polypeptide, and the second MHC polypeptide is an MHC class II α-chain polypeptide. In some cases, the multimeric polypeptide includes two or more immunomodulatory polypeptides, and at least one of the immunomodulatory polypeptides is a mutant IL2 immunomodulatory polypeptide of the present disclosure.When the multimeric polypeptide of the present disclosure contains two or more immunomodulatory polypeptides, in some cases, the two or more immunomodulatory polypeptides are present in the same polypeptide chain and can be arranged in tandem. When the multimeric polypeptide of the present disclosure contains two or more immunomodulatory polypeptides, in some cases, the two or more immunomodulatory polypeptides are present in separate polypeptides. In some cases, the multimeric polypeptide of the present disclosure is a heterodimer. In some cases, the multimeric polypeptide of the present disclosure is a trimeric polypeptide.

[0146] In some cases, the multimeric polypeptide comprises: a first polypeptide comprising, in order from the N-terminus to the C-terminus, i) an epitope, and ii) a first MHC polypeptide; and a second polypeptide comprising, in order from the N-terminus to the C-terminus, i) a second MHC polypeptide, ii) an Ig Fc polypeptide, and iii) an immunomodulatory domain (e.g., a mutant IL2 polypeptide of the present disclosure). In some cases, the multimeric polypeptide of the present disclosure comprises: a first polypeptide comprising, in order from the N-terminus to the C-terminus, i) an epitope, and ii) a first MHC polypeptide; and a second polypeptide comprising, in order from the N-terminus to the C-terminus, i) a second MHC polypeptide, and ii) an immunomodulatory domain (e.g., a mutant IL2 polypeptide of the present disclosure). In some cases, the multimeric polypeptide of the present disclosure comprises: a first polypeptide comprising, in order from the N-terminus to the C-terminus, i) an epitope, and ii) a first MHC polypeptide; and a second polypeptide comprising, in order from the N-terminus to the C-terminus, i) an immunomodulatory domain (e.g., a mutant IL2 polypeptide of the present disclosure), and ii) a second MHC polypeptide. In some cases, the multimeric polypeptide of the present disclosure comprises: a first polypeptide comprising, in order from the N-terminus to the C-terminus, i) an epitope, ii) a first MHC polypeptide, and iii) an immunomodulatory domain (e.g., a mutant IL2 polypeptide of the present disclosure); and a second polypeptide comprising, in order from the N-terminus to the C-terminus, i) a second MHC polypeptide. In some cases, when the multimeric polypeptide of the present disclosure comprises a non-Ig backbone, the non-Ig backbone is an XTEN peptide, a transferrin polypeptide, an Fc receptor polypeptide, an elastin-like polypeptide, a silk-like polypeptide, or a silk-elastin-like polypeptide.

[0147] In some cases, the multimeric polypeptides of the present disclosure are monovalent. In some cases, the multimeric polypeptides of the present disclosure are multivalent. In some cases, the multivalent multimeric polypeptides of the present disclosure comprise an immunoglobulin Fc polypeptide in one of the first or second polypeptides. For example, depending on the Fc polypeptide present in the multimeric polypeptide of the present disclosure, the multimeric polypeptide can be a homodimer, in which case two molecules of the multimeric polypeptide are present in the homodimer, and the two molecules of the multimeric polypeptide can be disulfide-linked to each other via, for example, the Fc polypeptides present in the two molecules. As another example, the multimeric polypeptides of the present disclosure can comprise a multimeric polypeptide of 3, 4, or 5 molecules, in which case each molecule of the multimeric polypeptide can be disulfide-linked to each other via, for example, the Fc polypeptides present in each molecule.

[0148] In some cases, the multimeric polypeptide comprises: a) a first polypeptide comprising, in order from the N-terminus to the C-terminus, i) an epitope, ii) a β2M polypeptide, and iii) a mutant IL2 polypeptide of the present disclosure; and b) a second polypeptide comprising, in order from the N-terminus to the C-terminus, i) a class I MHC heavy chain, and ii) an Fc polypeptide. In some cases, the multimeric polypeptide of the present disclosure comprises: a) a first polypeptide comprising, in order from the N-terminus to the C-terminus, i) an epitope, and ii) a β2M polypeptide; and b) a second polypeptide comprising, in order from the N-terminus to the C-terminus, i) a mutant IL2 polypeptide of the present disclosure, ii) a class I MHC heavy chain, and iii) an Fc polypeptide. In some cases, the multimeric polypeptide of the present disclosure comprises: a) a first polypeptide comprising, in order from the N-terminus to the C-terminus, i) an epitope, ii) a β2M polypeptide, iii) a first mutant IL2 polypeptide of the present disclosure, iv) a second mutant IL2 polypeptide of the present disclosure, and v) a third mutant IL2 polypeptide of the present disclosure; and b) a second polypeptide comprising, in order from the N-terminus to the C-terminus, i) a class I MHC heavy chain, and ii) an Fc polypeptide. In some cases, the first, second, and third mutant IL2 polypeptides have the same amino acid sequence. In some cases, the first, second, and third mutant IL2 polypeptides have different amino acid sequences from each other. In some cases, the multimeric polypeptide of the present disclosure comprises: a) a first polypeptide comprising, in order from the N-terminus to the C-terminus, i) an epitope, and ii) a β2M polypeptide; and b) a second polypeptide comprising, in order from the N-terminus to the C-terminus, i) a first mutant IL2 polypeptide of the present disclosure, ii) a second mutant IL2 polypeptide of the present disclosure, iii) a third mutant IL2 polypeptide of the present disclosure, iv) a class I MHC heavy chain, and v) an Fc polypeptide. In some cases, the first, second, and third mutant IL2 polypeptides have the same amino acid sequence. In some cases, the first, second, and third mutant IL2 polypeptides have different amino acid sequences from each other.

[0149] Substitution of F42 In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in Figure 2B, and amino acid 42 is an amino acid other than phenylalanine, for example, amino acid 42 is Gly, Ala, Val, Leu, Ile, Pro, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu. In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in Figure 2B, and amino acid 42 is Ala, Gly, Val, Leu or Ile. In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in Figure 2B, and amino acid 42 is Ala. In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in Figure 2B, and amino acid 42 is Gly. In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in Figure 2B, and amino acid 42 is Val. In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in Figure 2B, and amino acid 42 is Leu.In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2B, and amino acid 42 is Ile. In some cases, there is one copy of the mutant IL-2 polypeptide in the multimeric polypeptide of the present disclosure. In some cases, the multimeric polypeptide of the present disclosure comprises two copies of the mutant IL-2 polypeptide, for example, the two copies are arranged tandemly without a linker between the two copies, or are arranged tandemly and separated by a linker peptide. In some cases, the multimeric polypeptide of the present disclosure comprises three copies of the mutant IL-2 polypeptide, for example, the three copies are arranged tandemly without a linker between the three copies, or are arranged tandemly and separated by a linker peptide. In some cases, when the IL-2 / synTac of the present disclosure comprises an HLA class I heavy chain and β2M, the IL-2 polypeptide(s) is / are on the polypeptide chain comprising the HLA class I heavy chain. In some cases, when the IL-2 / synTac of the present disclosure comprises an HLA class I heavy chain and β2M, the IL-2 polypeptide(s) is / are on the polypeptide chain comprising the β2M polypeptide. In some cases, the mutant IL-2 polypeptide, or synTac comprising the polypeptide, has a binding affinity for IL2R of about 100 nM to 150 nM, about 150 nM to about 200 nM, about 200 nM to about 250 nM, about 250 nM to about 300 nM, about 300 nM to about 350 nM, about 350 nM to about 400 nM, about 400 nM to about 500 nM, about 500 nM to about 600 nM, about 600 nM to about 700 nM, about 700 nM to about 800 nM, about 800 nM to about 900 nM, about 900 nM to about 1 μM, 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. In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure has a length of 133 amino acids.

[0150] Replacement of Y45 In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2F, wherein amino acid 45 is an amino acid other than tyrosine. For example, amino acid 45 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu. In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2F, wherein amino acid 45 is Ala, Gly, Val, Leu or Ile. In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2F, wherein amino acid 45 is Ala. In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2F, wherein amino acid 45 is Gly. In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2F, wherein amino acid 45 is Val. In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2F, wherein amino acid 45 is Leu.In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in Figure 2F, and amino acid 45 is Ile. In some cases, there is one copy of the mutant IL-2 polypeptide in the multimeric polypeptide of the present disclosure. In some cases, the multimeric polypeptide of the present disclosure comprises two copies of the mutant IL-2 polypeptide. For example, the two copies are arranged tandemly without a linker between the two copies, or are arranged tandemly and separated by a linker peptide. In some cases, the multimeric polypeptide of the present disclosure comprises three copies of the mutant IL-2 polypeptide. For example, the three copies are arranged tandemly without a linker between the three copies, or are arranged tandemly and separated by a linker peptide. In some cases, when the IL-2 / synTac of the present disclosure comprises an HLA class I heavy chain and β2M, the IL-2 polypeptide(s) is / are on the polypeptide chain comprising the HLA class I heavy chain. In some cases, when the IL-2 / synTac of the present disclosure comprises an HLA class I heavy chain and β2M, the IL-2 polypeptide(s) is / are on the polypeptide chain comprising the β2M polypeptide. In some cases, the mutant IL-2 polypeptide, or synTac comprising the polypeptide, has a binding affinity for IL2R of about 100 nM to 150 nM, about 150 nM to about 200 nM, about 200 nM to about 250 nM, about 250 nM to about 300 nM, about 300 nM to about 350 nM, about 350 nM to about 400 nM, about 400 nM to about 500 nM, about 500 nM to about 600 nM, about 600 nM to about 700 nM, about 700 nM to about 800 nM, about 800 nM to about 900 nM, about 900 nM to about 1 μM, 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. In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure has a length of 133 amino acids.

[0151] Replacement of Q126 In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2G, and amino acid 126 is an amino acid other than glutamine. For example, amino acid 126 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Lys, Arg, His, Asp or Glu. In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2G, and amino acid 126 is Ala, Gly, Val, Leu or Ile. In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2G, and amino acid 126 is Ala. In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2G, and amino acid 126 is Gly. In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2G, and amino acid 126 is Val. In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2G, and amino acid 126 is Leu.In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2G, and amino acid 126 is Ile. In some cases, there is one copy of the mutant IL-2 polypeptide in the multimeric polypeptide of the present disclosure. In some cases, the multimeric polypeptide of the present disclosure comprises two copies of the mutant IL-2 polypeptide. For example, the two copies are arranged tandemly without a linker between the two copies, or are arranged tandemly and separated by a linker peptide. In some cases, the multimeric polypeptide of the present disclosure comprises three copies of the mutant IL-2 polypeptide. For example, the three copies are arranged tandemly without a linker between the three copies, or are arranged tandemly and separated by a linker peptide. In some cases, when the IL-2 / synTac of the present disclosure comprises an HLA class I heavy chain and β2M, the IL-2 polypeptide(s) is / are on the polypeptide chain comprising the HLA class I heavy chain. In some cases, when the IL-2 / synTac of the present disclosure comprises an HLA class I heavy chain and β2M, the IL-2 polypeptide(s) is / are on the polypeptide chain comprising the β2M polypeptide. In some cases, the mutant IL-2 polypeptide, or synTac comprising the polypeptide, has a binding affinity for IL2R of about 100 nM to 150 nM, about 150 nM to about 200 nM, about 200 nM to about 250 nM, about 250 nM to about 300 nM, about 300 nM to about 350 nM, about 350 nM to about 400 nM, about 400 nM to about 500 nM, about 500 nM to about 600 nM, about 600 nM to about 700 nM, about 700 nM to about 800 nM, about 800 nM to about 900 nM, about 900 nM to about 1 μM, 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. In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure has a length of 133 amino acids.

[0152] Replacement of F42 and H16 In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2H, amino acid 42 is an amino acid other than phenylalanine, for example, amino acid 42 is Gly, Ala, Val, Leu, Ile, Pro, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu, amino acid 16 is an amino acid other than histidine, for example, amino acid 16 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, Asp or Glu. In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2H, amino acid 42 is Ala, Gly, Val, Leu or Ile, and amino acid 16 is Ala, Gly, Val, Leu or Ile. In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2H, amino acid 42 is Ala, and amino acid 16 is Ala. In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2H, amino acid 42 is Ala, and amino acid 16 is Gly.In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2H, wherein amino acid 42 is Val and amino acid 16 is Ala. In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2H, wherein amino acid 42 is Leu and amino acid 16 is Ala. In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2H, wherein amino acid 42 is Ile and amino acid 16 is Ala. In some cases, there is one copy of the mutant IL-2 polypeptide in the multimeric polypeptide of the present disclosure. In some cases, the multimeric polypeptide of the present disclosure comprises two copies of the mutant IL-2 polypeptide. For example, the two copies are arranged tandemly without a linker between the two copies or are arranged tandemly and separated by a linker peptide. In some cases, the multimeric polypeptide of the present disclosure comprises three copies of the mutant IL-2 polypeptide. For example, the three copies are arranged tandemly without a linker between the three copies or are arranged tandemly and separated by a linker peptide. In some cases, when the IL-2 / synTac of the present disclosure comprises an HLA class I heavy chain and β2M, the IL-2 polypeptide(s) is / are on the polypeptide chain comprising the HLA class I heavy chain. In some cases, when the IL-2 / synTac of the present disclosure comprises an HLA class I heavy chain and β2M, the IL-2 polypeptide(s) is / are on the polypeptide chain comprising the β2M polypeptide.In some cases, the multimeric polypeptide of the present disclosure comprises two copies of an IL-2 variant comprising the substitutions F42A and H16A, the multimeric polypeptide comprises an HLA class I heavy chain and a β2M polypeptide, and the two copies of IL-2(F42A, H16A) are on a polypeptide chain comprising the HLA class I heavy chain. In some cases, the mutant IL-2 polypeptide, or a synTac comprising the polypeptide, has a binding affinity for IL2R of about 100 nM to 150 nM, about 150 nM to about 200 nM, about 200 nM to about 250 nM, about 250 nM to about 300 nM, about 300 nM to about 350 nM, about 350 nM to about 400 nM, about 400 nM to about 500 nM, about 500 nM to about 600 nM, about 600 nM to about 700 nM, about 700 nM to about 800 nM, about 800 nM to about 900 nM, about 900 nM to about 1 μM, 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. In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure has a length of 133 amino acids. In some cases, the mutant IL-2 polypeptide comprises the amino acid sequence shown in FIG. 34B (including the substitutions H16A and F42A).

[0153] Substitutions of F42 and D20 In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in Figure 2I, wherein amino acid 42 is an amino acid other than phenylalanine, for example, amino acid 42 is Gly, Ala, Val, Leu, Ile, Pro, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu, and amino acid 20 is an amino acid other than aspartic acid, for example, amino acid 20 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His or Glu. In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in Figure 2I, wherein amino acid 42 is Ala, Gly, Val, Leu or Ile, and amino acid 20 is Ala, Gly, Val, Leu or Ile. In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in Figure 2I, wherein amino acid 42 is Ala, Gly, Val, Leu or Ile, and amino acid 20 is Asn, Gln, Lys, Arg or His. In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in Figure 2I, wherein amino acid 42 is Ala, and amino acid 20 is Ala.In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2I, wherein amino acid 42 is Ala and amino acid 20 is Gly. In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2I, wherein amino acid 42 is Val and amino acid 20 is Ala. In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2I, wherein amino acid 42 is Leu and amino acid 20 is Ala. In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2I, wherein amino acid 42 is Ile and amino acid 20 is Ala. In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2I, wherein amino acid 42 is Ala and amino acid 20 is Asn. In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2I, wherein amino acid 42 is Ala and amino acid 20 is Gln.In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2I, wherein amino acid 42 is Ala and amino acid 20 is Lys. In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2I, wherein amino acid 42 is Ala and amino acid 20 is Arg. In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2I, wherein amino acid 42 is Ala and amino acid 20 is His. In some cases, there is one copy of the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure. In some cases, the multimeric polypeptide of the present disclosure comprises two copies of the mutant IL-2 polypeptide, for example, the two copies are arranged tandemly without a linker between the two copies or are arranged tandemly and separated by a linker peptide. In some cases, the multimeric polypeptide of the present disclosure comprises three copies of the mutant IL-2 polypeptide, for example, the three copies are arranged tandemly without a linker between the three copies or are arranged tandemly and separated by a linker peptide. In some cases, when the IL-2 / synTac of the present disclosure comprises an HLA class I heavy chain and β2M, the IL-2 polypeptide(s) is / are on the polypeptide chain comprising the HLA class I heavy chain. In some cases, when the IL-2 / synTac of the present disclosure comprises an HLA class I heavy chain and β2M, the IL-2 polypeptide(s) is / are on the polypeptide chain comprising the β2M polypeptide.In some cases, the mutant IL-2 polypeptide, or the synTac comprising the polypeptide, has a binding affinity for IL2R of about 100 nM to 150 nM, about 150 nM to about 200 nM, about 200 nM to about 250 nM, about 250 nM to about 300 nM, about 300 nM to about 350 nM, about 350 nM to about 400 nM, about 400 nM to about 500 nM, about 500 nM to about 600 nM, about 600 nM to about 700 nM, about 700 nM to about 800 nM, about 800 nM to about 900 nM, about 900 nM to about 1 μM, 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. In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure has a length of 133 amino acids.

[0154] Substitutions of F42, D20 and E15 In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2J, wherein amino acid 42 is an amino acid other than phenylalanine, for example, amino acid 42 is Gly, Ala, Val, Leu, Ile, Pro, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu; amino acid 20 is an amino acid other than aspartic acid, for example, amino acid 20 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His or Glu; and amino acid 15 is an amino acid other than glutamic acid, for example, amino acid 15 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His or Asp. In some cases, the mutant IL-2 polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2J, wherein amino acid 42 is Ala, Gly, Val, Leu or Ile; amino acid 20 is Ala, Gly, Val, Leu or Ile; and amino acid 15 is Ala, Gly, Val, Leu or Ile. In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2J, wherein amino acid 42 is Ala, Gly, Val, Leu or Ile; amino acid 20 is Asn, Gln, Lys, Arg or His; and amino acid 15 is Ala, Gly, Val, Leu or Ile.In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2J, wherein amino acid 42 is Ala, amino acid 20 is Ala, and amino acid 15 is Ala. In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2J, wherein amino acid 42 is Ala, amino acid 20 is Gly, and amino acid 15 is Gly. In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2J, wherein amino acid 42 is Val, amino acid 20 is Ala, and amino acid 15 is Gly. In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2J, wherein amino acid 42 is Leu, amino acid 20 is Ala, and amino acid 15 is Gly. In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2J, wherein amino acid 42 is Ile, amino acid 20 is Ala, and amino acid 15 is Ala.In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2J, wherein amino acid 42 is Ala, amino acid 20 is Asn, and amino acid 15 is Ala. In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2I, wherein amino acid 42 is Ala, amino acid 20 is Gln, and amino acid 15 is Ala. In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2J, wherein amino acid 42 is Ala, amino acid 20 is Lys, and amino acid 15 is Ala. In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2J, wherein amino acid 42 is Ala, amino acid 20 is Arg, and amino acid 15 is Ala. In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2I, wherein amino acid 42 is Ala, amino acid 20 is His, and amino acid 15 is Ala. In some cases, there is one copy of the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure.In some cases, the multimeric polypeptides of the present disclosure comprise two copies of a mutant IL-2 polypeptide. For example, the two copies are arranged tandemly without a linker between the two copies, or are arranged tandemly and separated by a linker peptide. In some cases, when the IL-2 / synTac of the present disclosure comprises an HLA class I heavy chain and β2M, the IL-2 polypeptide(s) is / are on the polypeptide chain comprising the HLA class I heavy chain. In some cases, when the IL-2 / synTac of the present disclosure comprises an HLA class I heavy chain and β2M, the IL-2 polypeptide(s) is / are on the polypeptide chain comprising the β2M polypeptide. In some cases, the multimeric polypeptides of the present disclosure comprise three copies of a mutant IL-2 polypeptide. For example, the three copies are arranged tandemly without a linker between the three copies, or are arranged tandemly and separated by a linker peptide. In some cases, the mutant IL-2 polypeptide, or a synTac comprising the polypeptide, has a binding affinity for IL2R of about 100 nM to 150 nM, about 150 nM to about 200 nM, about 200 nM to about 250 nM, about 250 nM to about 300 nM, about 300 nM to about 350 nM, about 350 nM to about 400 nM, about 400 nM to about 500 nM, about 500 nM to about 600 nM, about 600 nM to about 700 nM, about 700 nM to about 800 nM, about 800 nM to about 900 nM, about 900 nM to about 1 μM, 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. In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptides of the present disclosure has a length of 133 amino acids.

[0155] Substitutions at F42, D20 and H16 In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2K, amino acid 42 is an amino acid other than phenylalanine, for example, amino acid 42 is Gly, Ala, Val, Leu, Ile, Pro, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu, amino acid 20 is an amino acid other than aspartic acid, for example, amino acid 20 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His or Glu, and amino acid 16 is an amino acid other than histidine, for example, amino acid 16 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, Asp or Glu. In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2K, amino acid 42 is Ala, Gly, Val, Leu or Ile, amino acid 20 is Ala, Gly, Val, Leu or Ile, and amino acid 16 is Ala, Gly, Val, Leu or Ile. In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2K, amino acid 42 is Ala, Gly, Val, Leu or Ile, amino acid 20 is Asn, Gln, Lys, Arg or His, and amino acid 16 is Ala, Gly, Val, Leu or Ile.In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2K, wherein amino acid 42 is Ala, amino acid 20 is Ala, and amino acid 16 is Ala. In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2K, wherein amino acid 42 is Ala, amino acid 20 is Gly, and amino acid 16 is Gly. In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2K, wherein amino acid 42 is Val, amino acid 20 is Ala, and amino acid 16 is Gly. In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2K, wherein amino acid 42 is Leu, amino acid 20 is Ala, and amino acid 16 is Gly. In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2K, wherein amino acid 42 is Ile, amino acid 20 is Ala, and amino acid 16 is Ala.In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2K, wherein amino acid 42 is Ala, amino acid 20 is Asn, and amino acid 16 is Ala. In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2K, wherein amino acid 42 is Ala, amino acid 20 is Gln, and amino acid 16 is Ala. In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2K, wherein amino acid 42 is Ala, amino acid 20 is Lys, and amino acid 16 is Ala. In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2K, wherein amino acid 42 is Ala, amino acid 20 is Arg, and amino acid 16 is Ala. In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2K, wherein amino acid 42 is Ala, amino acid 20 is His, and amino acid 16 is Ala. In some cases, there is one copy of the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure.In some cases, the multimeric polypeptide of the present disclosure comprises two copies of the mutant IL-2 polypeptide. For example, the two copies are arranged tandemly without a linker between the two copies, or are arranged tandemly and separated by a linker peptide. In some cases, the multimeric polypeptide of the present disclosure comprises three copies of the mutant IL-2 polypeptide. For example, the three copies are arranged tandemly without a linker between the three copies, or are arranged tandemly and separated by a linker peptide. In some cases, when the IL-2 / synTac of the present disclosure comprises an HLA class I heavy chain and β2M, the IL-2 polypeptide(s) is / are on the polypeptide chain comprising the HLA class I heavy chain. In some cases, when the IL-2 / synTac of the present disclosure comprises an HLA class I heavy chain and β2M, the IL-2 polypeptide(s) is / are on the polypeptide chain comprising the β2M polypeptide. In some cases, the mutant IL-2 polypeptide, or the synTac comprising the polypeptide, has a binding affinity for IL2R of about 100 nM to 150 nM, about 150 nM to about 200 nM, about 200 nM to about 250 nM, about 250 nM to about 300 nM, about 300 nM to about 350 nM, about 350 nM to about 400 nM, about 400 nM to about 500 nM, about 500 nM to about 600 nM, about 600 nM to about 700 nM, about 700 nM to about 800 nM, about 800 nM to about 900 nM, about 900 nM to about 1 μM, 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. In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure has a length of 133 amino acids.

[0156] Substitutions at F42, D20 and Q126 In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in Figure 2L, wherein amino acid 42 is an amino acid other than phenylalanine, for example, amino acid 42 is Gly, Ala, Val, Leu, Ile, Pro, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu, amino acid 20 is an amino acid other than aspartic acid, for example, amino acid 20 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His or Glu, and amino acid 126 is an amino acid other than glutamine, for example, amino acid 126 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Lys, Arg, His, Asp or Glu. In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in Figure 2L, wherein amino acid 42 is Ala, Gly, Val, Leu or Ile, amino acid 20 is Ala, Gly, Val, Leu or Ile, and amino acid 126 is Ala, Gly, Val, Leu or Ile. In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in Figure 2L, wherein amino acid 42 is Ala, Gly, Val, Leu or Ile, amino acid 20 is Asn, Gln, Lys, Arg or His, and amino acid 126 is Ala, Gly, Val, Leu or Ile.In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2L, wherein amino acid 42 is Ala, amino acid 20 is Ala, and amino acid 126 is Ala. In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2L, wherein amino acid 42 is Ala, amino acid 20 is Gly, and amino acid 126 is Gly. In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2L, wherein amino acid 42 is Val, amino acid 20 is Ala, and amino acid 126 is Gly. In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2L, wherein amino acid 42 is Leu, amino acid 20 is Ala, and amino acid 126 is Gly. In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2L, wherein amino acid 42 is Ile, amino acid 20 is Ala, and amino acid 126 is Ala.In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in Figure 2L, wherein amino acid 42 is Ala, amino acid 20 is Asn, and amino acid 126 is Ala. In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in Figure 2L, wherein amino acid 42 is Ala, amino acid 20 is Gln, and amino acid 126 is Ala. In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in Figure 2L, wherein amino acid 42 is Ala, amino acid 20 is Lys, and amino acid 126 is Ala. In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in Figure 2L, wherein amino acid 42 is Ala, amino acid 20 is Arg, and amino acid 126 is Ala. In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in Figure 2L, wherein amino acid 42 is Ala, amino acid 20 is His, and amino acid 126 is Ala. In some cases, there is one copy of the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure.In some cases, the multimeric polypeptides of the present disclosure comprise two copies of a mutant IL-2 polypeptide, for example, the two copies are arranged tandemly without a linker between the two copies or are arranged tandemly and separated by a linker peptide. In some cases, the multimeric polypeptides of the present disclosure comprise three copies of a mutant IL-2 polypeptide, for example, the three copies are arranged tandemly without a linker between the three copies or are arranged tandemly and separated by a linker peptide. In some cases, when the IL-2 / synTac of the present disclosure comprises an HLA class I heavy chain and β2M, the IL-2 polypeptide(s) is / are on the polypeptide chain comprising the HLA class I heavy chain. In some cases, when the IL-2 / synTac of the present disclosure comprises an HLA class I heavy chain and β2M, the IL-2 polypeptide(s) is / are on the polypeptide chain comprising the β2M polypeptide. In some cases, the mutant IL-2 polypeptide, or synTac comprising the polypeptide, has a binding affinity for IL2R of about 100 nM to 150 nM, about 150 nM to about 200 nM, about 200 nM to about 250 nM, about 250 nM to about 300 nM, about 300 nM to about 350 nM, about 350 nM to about 400 nM, about 400 nM to about 500 nM, about 500 nM to about 600 nM, about 600 nM to about 700 nM, about 700 nM to about 800 nM, about 800 nM to about 900 nM, about 900 nM to about 1 μM, 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. In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptides of the present disclosure has a length of 133 amino acids.

[0157] Substitutions at F42, D20 and Y45 In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2M, wherein amino acid 42 is an amino acid other than phenylalanine, for example, amino acid 42 is Gly, Ala, Val, Leu, Ile, Pro, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu; amino acid 20 is an amino acid other than aspartic acid, for example, amino acid 20 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His or Glu; and amino acid 45 is an amino acid other than tyrosine, for example, amino acid 45 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu. In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2M, wherein amino acid 42 is Ala, Gly, Val, Leu or Ile; amino acid 20 is Ala, Gly, Val, Leu or Ile; and amino acid 45 is Ala, Gly, Val, Leu or Ile. In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2M, wherein amino acid 42 is Ala, Gly, Val, Leu or Ile; amino acid 20 is Asn, Gln, Lys, Arg or His; and amino acid 45 is Ala, Gly, Val, Leu or Ile.In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in Figure 2M, wherein amino acid 42 is Ala, amino acid 20 is Ala, and amino acid 45 is Ala. In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in Figure 2M, wherein amino acid 42 is Ala, amino acid 20 is Gly, and amino acid 45 is Gly. In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in Figure 2M, wherein amino acid 42 is Val, amino acid 20 is Ala, and amino acid 45 is Gly. In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in Figure 2M, wherein amino acid 42 is Leu, amino acid 20 is Ala, and amino acid 45 is Gly. In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in Figure 2M, wherein amino acid 42 is Ile, amino acid 20 is Ala, and amino acid 45 is Ala.In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2M, wherein amino acid 42 is Ala, amino acid 20 is Asn, and amino acid 45 is Ala. In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2M, wherein amino acid 42 is Ala, amino acid 20 is Gln, and amino acid 45 is Ala. In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2M, wherein amino acid 42 is Ala, amino acid 20 is Lys, and amino acid 45 is Ala. In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2M, wherein amino acid 42 is Ala, amino acid 20 is Arg, and amino acid 45 is Ala. In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2M, wherein amino acid 42 is Ala, amino acid 20 is His, and amino acid 45 is Ala. In some cases, there is one copy of the mutant IL-2 polypeptide in the multimeric polypeptide of the present disclosure.In some cases, the multimeric polypeptides of the present disclosure comprise two copies of the mutant IL-2 polypeptide. For example, the two copies are arranged tandemly without a linker between the two copies, or are arranged tandemly and separated by a linker peptide. In some cases, the multimeric polypeptides of the present disclosure comprise three copies of the mutant IL-2 polypeptide. For example, the three copies are arranged tandemly without a linker between the three copies, or are arranged tandemly and separated by a linker peptide. In some cases, when the IL-2 / synTac of the present disclosure comprises an HLA class I heavy chain and β2M, the IL-2 polypeptide(s) is / are on the polypeptide chain comprising the HLA class I heavy chain. In some cases, when the IL-2 / synTac of the present disclosure comprises an HLA class I heavy chain and β2M, the IL-2 polypeptide(s) is / are on the polypeptide chain comprising the β2M polypeptide. In some cases, the mutant IL-2 polypeptide, or the synTac comprising the polypeptide, has a binding affinity for IL2R of about 100 nM to 150 nM, about 150 nM to about 200 nM, about 200 nM to about 250 nM, about 250 nM to about 300 nM, about 300 nM to about 350 nM, about 350 nM to about 400 nM, about 400 nM to about 500 nM, about 500 nM to about 600 nM, about 600 nM to about 700 nM, about 700 nM to about 800 nM, about 800 nM to about 900 nM, about 900 nM to about 1 μM, 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. In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptides of the present disclosure has a length of 133 amino acids.

[0158] Substitutions at F4, D20, Y45 and H16 In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2N, amino acid 42 is an amino acid other than phenylalanine, for example, amino acid 42 is Gly, Ala, Val, Leu, Ile, Pro, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu, amino acid 20 is an amino acid other than aspartic acid, for example, amino acid 20 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His or Glu, amino acid 45 is an amino acid other than tyrosine, for example, amino acid 45 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu, amino acid 16 is an amino acid other than histidine, for example, amino acid 16 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, Asp or Glu. In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2N, amino acid 42 is Ala, Gly, Val, Leu or Ile, amino acid 20 is Ala, Gly, Val, Leu or Ile, amino acid 45 is Ala, Gly, Val, Leu or Ile, and amino acid 16 is Ala, Gly, Val, Leu or Ile.In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2N, wherein amino acid 42 is Ala, Gly, Val, Leu or Ile, amino acid 20 is Asn, Gln, Lys, Arg or His, amino acid 45 is Ala, Gly, Val, Leu or Ile, and amino acid 16 is Ala, Gly, Val, Leu or Ile. In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2N, wherein amino acid 42 is Ala, amino acid 20 is Ala, amino acid 45 is Ala, and amino acid 16 is Ala. In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2N, wherein amino acid 42 is Ala, amino acid 20 is Gly, amino acid 45 is Gly, and amino acid 16 is Ala. In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2N, wherein amino acid 42 is Val, amino acid 20 is Ala, amino acid 45 is Gly, and amino acid 16 is Ala. In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2N, wherein amino acid 42 is Leu, amino acid 20 is Ala, amino acid 45 is Gly, and amino acid 16 is Val.In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2N, wherein amino acid 42 is Ile, amino acid 20 is Ala, amino acid 45 is Ala, and amino acid 16 is Gly. In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2N, wherein amino acid 42 is Ala, amino acid 20 is Asn, amino acid 45 is Ala, and amino acid 16 is Ala. In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2N, wherein amino acid 42 is Ala, amino acid 20 is Gln, amino acid 45 is Ala, and amino acid 16 is Ala. In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2N, wherein amino acid 42 is Ala, amino acid 20 is Lys, amino acid 45 is Ala, and amino acid 16 is Ala. In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2N, wherein amino acid 42 is Ala, amino acid 20 is Arg, amino acid 45 is Ala, and amino acid 16 is Ala.In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2N, wherein amino acid 42 is Ala, amino acid 20 is His, amino acid 45 is Ala, and amino acid 16 is Ala. In some cases, there is one copy of the mutant IL-2 polypeptide in the multimeric polypeptide of the present disclosure. In some cases, the multimeric polypeptide of the present disclosure comprises two copies of the mutant IL-2 polypeptide. For example, the two copies are arranged tandemly without a linker between the two copies, or are arranged tandemly and separated by a linker peptide. In some cases, the multimeric polypeptide of the present disclosure comprises three copies of the mutant IL-2 polypeptide. For example, the three copies are arranged tandemly without a linker between the three copies, or are arranged tandemly and separated by a linker peptide. In some cases, when the IL-2 / synTac of the present disclosure comprises an HLA class I heavy chain and β2M, the IL-2 polypeptide(s) is / are on the polypeptide chain comprising the HLA class I heavy chain. In some cases, when the IL-2 / synTac of the present disclosure comprises an HLA class I heavy chain and β2M, the IL-2 polypeptide(s) is / are on the polypeptide chain comprising the β2M polypeptide. In some cases, the mutant IL-2 polypeptide, or synTac comprising the polypeptide, has a binding affinity for IL2R of about 100 nM to 150 nM, about 150 nM to about 200 nM, about 200 nM to about 250 nM, about 250 nM to about 300 nM, about 300 nM to about 350 nM, about 350 nM to about 400 nM, about 400 nM to about 500 nM, about 500 nM to about 600 nM, about 600 nM to about 700 nM, about 700 nM to about 800 nM, about 800 nM to about 900 nM, about 900 nM to about 1 μM, 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.In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure has a length of 133 amino acids.

[0159] Substitutions of F42, D20, Y45 and Q126 In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2O, wherein amino acid 42 is an amino acid other than phenylalanine, for example, amino acid 42 is Gly, Ala, Val, Leu, Ile, Pro, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu, amino acid 20 is an amino acid other than aspartic acid, for example, amino acid 20 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His or Glu, amino acid 45 is an amino acid other than tyrosine, for example, amino acid 45 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu, and amino acid 126 is an amino acid other than glutamine, for example, amino acid 126 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Lys, Arg, His, Asp or Glu. In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2O, wherein amino acid 42 is Ala, Gly, Val, Leu or Ile, amino acid 20 is Ala, Gly, Val, Leu or Ile, amino acid 45 is Ala, Gly, Val, Leu or Ile, and amino acid 126 is Ala, Gly, Val, Leu or Ile.In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in Figure 2O, amino acid 42 is Ala, Gly, Val, Leu or Ile, amino acid 20 is Asn, Gln, Lys, Arg or His, amino acid 45 is Ala, Gly, Val, Leu or Ile, and amino acid 126 is Ala, Gly, Val, Leu or Ile. In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in Figure 2O, amino acid 42 is Ala, amino acid 20 is Ala, amino acid 45 is Ala, and amino acid 126 is Ala. In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in Figure 2O, amino acid 42 is Ala, amino acid 20 is Gly, amino acid 45 is Gly, and amino acid 126 is Ala. In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in Figure 2O, amino acid 42 is Val, amino acid 20 is Ala, amino acid 45 is Gly, and amino acid 126 is Ala.In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in Figure 2O, wherein amino acid 42 is Leu, amino acid 20 is Ala, amino acid 45 is Gly, and amino acid 126 is Val. In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in Figure 2O, wherein amino acid 42 is Ile, amino acid 20 is Ala, amino acid 45 is Ala, and amino acid 126 is Gly. In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in Figure 2O, wherein amino acid 42 is Ala, amino acid 20 is Asn, amino acid 45 is Ala, and amino acid 126 is Ala. In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in Figure 2O, wherein amino acid 42 is Ala, amino acid 20 is Gln, amino acid 45 is Ala, and amino acid 126 is Ala. In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in Figure 2O, wherein amino acid 42 is Ala, amino acid 20 is Lys, amino acid 45 is Ala, and amino acid 126 is Ala.In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2O, wherein amino acid 42 is Ala, amino acid 20 is Arg, amino acid 45 is Ala, and amino acid 126 is Ala. In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2O, wherein amino acid 42 is Ala, amino acid 20 is His, amino acid 45 is Ala, and amino acid 126 is Ala. In some cases, there is one copy of the mutant IL-2 polypeptide in the multimeric polypeptide of the present disclosure. In some cases, the multimeric polypeptide of the present disclosure comprises two copies of the mutant IL-2 polypeptide, for example, the two copies are arranged tandemly without a linker between the two copies, or are arranged tandemly and separated by a linker peptide. In some cases, the multimeric polypeptide of the present disclosure comprises three copies of the mutant IL-2 polypeptide, for example, the three copies are arranged tandemly without a linker between the three copies, or are arranged tandemly and separated by a linker peptide. In some cases, when the IL-2 / synTac of the present disclosure comprises an HLA class I heavy chain and β2M, the IL-2 polypeptide(s) is / are on the polypeptide chain comprising the HLA class I heavy chain. In some cases, when the IL-2 / synTac of the present disclosure comprises an HLA class I heavy chain and β2M, the IL-2 polypeptide(s) is / are on the polypeptide chain comprising the β2M polypeptide.In some cases, the mutant IL-2 polypeptide, or the synTac comprising the polypeptide, has a binding affinity for IL2R of about 100 nM to 150 nM, about 150 nM to about 200 nM, about 200 nM to about 250 nM, about 250 nM to about 300 nM, about 300 nM to about 350 nM, about 350 nM to about 400 nM, about 400 nM to about 500 nM, about 500 nM to about 600 nM, about 600 nM to about 700 nM, about 700 nM to about 800 nM, about 800 nM to about 900 nM, about 900 nM to about 1 μM, 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. In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure has a length of 133 amino acids.

[0160] Substitutions of F42, D20, Y45, H16, and Q126 In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in Figure 2P, wherein amino acid 42 is an amino acid other than phenylalanine, for example, amino acid 42 is Gly, Ala, Val, Leu, Ile, Pro, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu, amino acid 20 is an amino acid other than aspartic acid, for example, amino acid 20 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His or Glu, amino acid 45 is an amino acid other than tyrosine, for example, amino acid 45 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu, amino acid 126 is an amino acid other than glutamine, for example, amino acid 126 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Lys, Arg, His, Asp or Glu, and amino acid 16 is an amino acid other than histidine, for example, amino acid 16 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, Asp or Glu.In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in Figure 2P, wherein amino acid 42 is Ala, Gly, Val, Leu or Ile, amino acid 20 is Ala, Gly, Val, Leu or Ile, amino acid 45 is Ala, Gly, Val, Leu or Ile, amino acid 126 is Ala, Gly, Val, Leu or Ile, and amino acid 16 is Ala, Gly, Val, Leu or Ile. In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in Figure 2P, wherein amino acid 42 is Ala, Gly, Val, Leu or Ile, amino acid 20 is Asn, Gln, Lys, Arg or His, amino acid 45 is Ala, Gly, Val, Leu or Ile, amino acid 126 is Ala, Gly, Val, Leu or Ile, and amino acid 16 is Ala, Gly, Val, Leu or Ile. In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in Figure 2P, wherein amino acid 42 is Ala, amino acid 20 is Ala, amino acid 45 is Ala, amino acid 126 is Ala, and amino acid 16 is Ala. In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in Figure 2P, wherein amino acid 42 is Ala, amino acid 20 is Gly, amino acid 45 is Gly, amino acid 126 is Ala, and amino acid 16 is Ala.In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in Figure 2P, wherein amino acid 42 is Val, amino acid 20 is Ala, amino acid 45 is Gly, amino acid 126 is Ala, and amino acid 16 is Ala. In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in Figure 2P, wherein amino acid 42 is Leu, amino acid 20 is Ala, amino acid 45 is Gly, amino acid 126 is Val, and amino acid 16 is Ala. In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in Figure 2P, wherein amino acid 42 is Ile, amino acid 20 is Ala, amino acid 45 is Ala, amino acid 126 is Gly, and amino acid 16 is Ala. In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in Figure 2P, wherein amino acid 42 is Ala, amino acid 20 is Asn, amino acid 45 is Ala, amino acid 126 is Ala, and amino acid 16 is Ala.In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in Figure 2P, wherein amino acid 42 is Ala, amino acid 20 is Gln, amino acid 45 is Ala, amino acid 126 is Ala, and amino acid 16 is Ala. In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in Figure 2P, wherein amino acid 42 is Ala, amino acid 20 is Lys, amino acid 45 is Ala, amino acid 126 is Ala, and amino acid 16 is Ala. In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in Figure 2P, wherein amino acid 42 is Ala, amino acid 20 is Arg, amino acid 45 is Ala, amino acid 126 is Ala, and amino acid 16 is Ala. In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in Figure 2P, wherein amino acid 42 is Ala, amino acid 20 is His, amino acid 45 is Ala, amino acid 126 is Ala, and amino acid 16 is Ala. In some cases, there is one copy of the mutant IL-2 polypeptide in the multimeric polypeptide of the present disclosure. In some cases, the multimeric polypeptide of the present disclosure contains two copies of the mutant IL-2 polypeptide, for example, the two copies are arranged tandemly without a linker between the two copies or are arranged tandemly and separated by a linker peptide.In some cases, the multimeric polypeptide of the present disclosure comprises three copies of a mutant IL-2 polypeptide, for example, the three copies are arranged tandemly without a linker between the three copies, or are arranged tandemly and separated by a linker peptide. In some cases, when the IL-2 / synTac of the present disclosure comprises an HLA class I heavy chain and β2M, the IL-2 polypeptide(s) is / are on a polypeptide chain comprising the HLA class I heavy chain. In some cases, when the IL-2 / synTac of the present disclosure comprises an HLA class I heavy chain and β2M, the IL-2 polypeptide(s) is / are on a polypeptide chain comprising the β2M polypeptide. In some cases, the mutant IL-2 polypeptide, or synTac comprising the polypeptide, has a binding affinity for IL2R of about 100 nM to 150 nM, about 150 nM to about 200 nM, about 200 nM to about 250 nM, about 250 nM to about 300 nM, about 300 nM to about 350 nM, about 350 nM to about 400 nM, about 400 nM to about 500 nM, about 500 nM to about 600 nM, about 600 nM to about 700 nM, about 700 nM to about 800 nM, about 800 nM to about 900 nM, about 900 nM to about 1 μM, 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. In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure has a length of 133 amino acids.

[0161] Substitutions at F42, Q126, and H16 In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2Q, wherein amino acid 42 is an amino acid other than phenylalanine, for example, amino acid 42 is Gly, Ala, Val, Leu, Ile, Pro, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu; amino acid 126 is an amino acid other than glutamine, for example, amino acid 126 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Lys, Arg, His, Asp or Glu; and amino acid 16 is an amino acid other than histidine, for example, amino acid 16 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, Asp or Glu. In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2Q, wherein amino acid 42 is Ala, Gly, Val, Leu or Ile; amino acid 126 is Ala, Gly, Val, Leu or Ile; and amino acid 16 is Ala, Gly, Val, Leu or Ile. In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2Q, wherein amino acid 42 is Ala, Gly, Val, Leu or Ile; amino acid 126 is Asn, Gln, Lys, Arg or His; and amino acid 16 is Ala, Gly, Val, Leu or Ile.In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in Figure 2Q, wherein amino acid 42 is Ala, amino acid 126 is Ala, and amino acid 16 is Ala. In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in Figure 2Q, wherein amino acid 42 is Ala, amino acid 126 is Gly, and amino acid 16 is Gly. In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in Figure 2Q, wherein amino acid 42 is Val, amino acid 126 is Ala, and amino acid 16 is Gly. In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in Figure 2Q, wherein amino acid 42 is Leu, amino acid 126 is Ala, and amino acid 16 is Gly. In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in Figure 2Q, wherein amino acid 42 is Ile, amino acid 126 is Ala, and amino acid 16 is Ala.In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2Q, amino acid 42 is Ala, amino acid 126 is Asn, and amino acid 16 is Ala. In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2Q, amino acid 42 is Ala, amino acid 126 is Ala, and amino acid 16 is Ala. In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2Q, amino acid 42 is Ala, amino acid 126 is Lys, and amino acid 16 is Ala. In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2Q, amino acid 42 is Ala, amino acid 126 is Arg, and amino acid 16 is Ala. In some cases, the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 2Q, amino acid 42 is Ala, amino acid 126 is His, and amino acid 16 is Ala. In some cases, there is one copy of the mutant IL-2 polypeptide present in the multimeric polypeptide of the present disclosure.In some cases, the multimeric polypeptides of the present disclosure contain two copies of a mutant IL-2 polypeptide. For example, the two copies are arranged tandemly without a linker between the two copies, or are arranged tandemly and separated by a linker peptide. In some cases, the multimeric polypeptides of the present disclosure contain three copies of a mutant IL-2 polypeptide. For example, the three copies are arranged tandemly without a linker between the three copies, or are arranged tandemly and separated by a linker peptide. In some cases, when the IL-2 / synTac of the present disclosure contains an HLA class I heavy chain and β2M, the IL-2 polypeptide(s) is / are on a polypeptide chain containing the HLA class I heavy chain. In some cases, when the IL-2 / synTac of the present disclosure contains an HLA class I heavy chain and β2M, the IL-2 polypeptide(s) is / are on a polypeptide chain containing the β2M polypeptide. In some cases, the mutant IL-2 polypeptide, or synTac containing the mutant IL-2 polypeptide, has a binding affinity for IL2R of about 100 nM to 150 nM, about 150 nM to about 200 nM, about 200 nM to about 250 nM, about 250 nM to about 300 nM, about 300 nM to about 350 nM, about 350 nM to about 400 nM, about 400 nM to about 500 nM, about 500 nM to about 600 nM, about 600 nM to about 700 nM, about 700 nM to about 800 nM, about 800 nM to about 900 nM, about 900 nM to about 1 μM, 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. In some cases, the mutant IL-2 polypeptide has a length of 133 amino acids.

[0162] 4-1BBL In some cases, a synTac suitable for use in the methods of the present disclosure contains a 4-1BBL polypeptide as an immunomodulatory domain(s). Suitable 4-1BBL immunomodulatory domains include wild-type 4-1BBL immunomodulatory domains and mutant 4-1BBL immunomodulatory domains.

[0163] The wild-type human 4-1BBL amino acid sequence is shown in Figure 36A. The tumor necrosis factor (TNF) homology domain (THD) of human 4-1BBL comprises amino acids 81-254, amino acids 80-254, or amino acids 80-246 of the amino acid sequence shown in Figure 36A. Thus, the wild-type amino acid sequence of the THD of human 4-1BBL can be, for example, one of SEQ ID NOs: 213-215 below. TIFF0007717440000033.tif78150

[0164] Wild-type 4-1BBL binds to 4-1BB (CD137). The amino acid sequence of 4-1BB is shown in Figure 37. The mutant 4-1BBL polypeptides of the present disclosure bind to 4-1BB with a lower affinity compared to the binding of wild-type 4-1BBL to 4-1BB.

[0165] Mutant 4-1BBL polypeptides include those having an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 95% or at least 99% amino acid sequence identity to the corresponding wild-type 4-1BBL polypeptide, and having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids, or more than 15 amino acids different compared to the corresponding wild-type 4-1BBL polypeptide. In some cases, the amino acid sequence of the mutant 4-1BBL polypeptide differs from the wild-type 4-1BBL polypeptide by only 1 amino acid. In some cases, the amino acid sequence of the mutant 4-1BBL polypeptide differs from the wild-type 4-1BBL polypeptide by only 2 amino acids. In some cases, the amino acid sequence of the mutant 4-1BBL polypeptide differs from the wild-type 4-1BBL polypeptide by only 3 amino acids. In some cases, the amino acid sequence of the mutant 4-1BBL polypeptide differs from the wild-type 4-1BBL polypeptide by only 4 amino acids. In some cases, the amino acid sequence of the mutant 4-1BBL polypeptide differs from the wild-type 4-1BBL polypeptide by only 5 amino acids.

[0166] In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure exhibits a reduced binding affinity for 4-1BB as compared to the binding affinity of the 4-1BBL polypeptide containing the amino acid sequence shown in FIG. 36A for 4-1BB. For example, in some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure binds to 4-1BB with a binding affinity lower than that of the 4-1BBL polypeptide containing the amino acid sequence shown in FIG. 36A for the 4-1BB polypeptide containing the amino acid sequence shown in FIG. 37. For example, in some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure has a binding affinity for 4-1BB (e.g., the 4-1BB polypeptide containing the amino acid sequence shown in FIG. 37) that is at least 10% lower, at least 15% lower, at least 20% lower, at least 25% lower, at least 30% lower, at least 35% lower, at least 40% lower, at least 45% lower, at least 50% lower, at least 55% lower, at least 60% lower, at least 65% lower, at least 70% lower, at least 75% lower, at least 80% lower, at least 85% lower, at least 90% lower, at least 95% lower, or more than 95% lower than the binding affinity of the 4-1BBL polypeptide containing the amino acid sequence shown in FIG. 36A for 4-1BB.

[0167] In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure exhibits a decreased binding affinity for 4-1BB as compared to the binding affinity of the 4-1BBL polypeptide containing the amino acid sequence shown in SEQ ID NO: 213 for 4-1BB. For example, in some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure binds to 4-1BB with a binding affinity lower than that of the 4-1BBL polypeptide containing the amino acid sequence shown in SEQ ID NO: 213 for the 4-1BB polypeptide containing the amino acid sequence shown in one of FIGS. 37A-37C. For example, in some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure is at least 10% lower, at least 15% lower, at least 20% lower, at least 25% lower, at least 30% lower, at least 35% lower, at least 40% lower, at least 45% lower, at least 50% lower, at least 55% lower, at least 60% lower, at least 65% lower, at least 70% lower, at least 75% lower, at least 80% lower, at least 85% lower, at least 90% lower, at least 95% lower, or more than 95% lower in binding affinity than the binding affinity of the 4-1BBL polypeptide containing the amino acid sequence shown in SEQ ID NO: 213 for 4-1BB (e.g., the 4-1BB polypeptide containing the amino acid sequence shown in FIG. 37), and binds to 4-1BB.

[0168] In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure has a binding affinity for 4-1BB of 100 nM to 100 μM. As another example, in some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure has a binding affinity for 4-1BB (e.g., a 4-1BB polypeptide comprising the amino acid sequence shown in FIG. 37) of about 100 nM to 150 nM, about 150 nM to about 200 nM, about 200 nM to about 250 nM, about 250 nM to about 300 nM, about 300 nM to about 350 nM, about 350 nM to about 400 nM, about 400 nM to about 500 nM, about 500 nM to about 600 nM, about 600 nM to about 700 nM, about 700 nM to about 800 nM, about 800 nM to about 900 nM, about 900 nM to about 1 μM, 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.

[0169] In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure exhibits an increase in production in mammalian host cells as compared to the production of a control multimeric polypeptide comprising a wild-type 4-1BBL polypeptide (e.g., a 4-1BBL polypeptide comprising the amino acid sequence shown in FIG. 36A or described in SEQ ID NO: 213) in the same mammalian host cells. For example, in some cases, when the mutant 4-1BBL polypeptide present in the multimeric polypeptide is expressed in mammalian host cells, it is produced in an amount that is 25% to about 50% more, about 50% to about 75% more, about 75% to about 2-fold more, about 2-fold to about 5-fold more, about 5-fold to about 10-fold more, about 10-fold to about 20-fold more, about 20-fold to about 30-fold more, about 30-fold to about 40-fold more, about 40-fold to about 50-fold more, about 50-fold to about 75-fold more, about 75-fold to about 100-fold more, or more than 100-fold more than the amount of a control multimeric polypeptide comprising a wild-type 4-1BBL polypeptide (e.g., a 4-1BBL polypeptide comprising the amino acid sequence shown in FIG. 36A or described in SEQ ID NO: 213) produced in the same mammalian host cells.

[0170] In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide is produced in mammalian host cells in an amount of about 50 mg / L to about 75 mg / L, about 75 mg / L to about 100 mg / L, about 100 mg / L to about 150 mg / L, about 150 mg / L to about 200 mg / L, about 200 mg / L to about 250 mg / L, about 250 mg / L to about 500 mg / L, or more than 500 mg / L. In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide is produced in mammalian host cells in an amount of about 10 mg / L to about 15 mg / L, about 15 mg / L to about 20 mg / L, about 20 mg / L to about 25 mg / L, about 25 mg / L to about 30 mg / L, about 35 mg / L to about 40 mg / L, about 40 mg / L to about 45 mg / L, or about 45 mg / L to about 50 mg / L.

[0171] The mutant 4-1BBL polypeptide present in the multimeric polypeptide may have one amino acid substitution as compared to the wild-type 4-1BBL polypeptide (e.g., the 4-1BBL polypeptide comprising the amino acid sequence shown in FIG. 36A or set forth in SEQ ID NO: 213). In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure has 2 to 10 amino acid substitutions as compared to the wild-type 4-1BBL polypeptide (e.g., the 4-1BBL polypeptide comprising the amino acid sequence shown in FIG. 36A or set forth in SEQ ID NO: 213). In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure has two amino acid substitutions as compared to the wild-type 4-1BBL polypeptide (e.g., the 4-1BBL polypeptide comprising the amino acid sequence shown in FIG. 36A or set forth in SEQ ID NO: 213). In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure has three amino acid substitutions as compared to the wild-type 4-1BBL polypeptide (e.g., the 4-1BBL polypeptide comprising the amino acid sequence shown in FIG. 36A or set forth in SEQ ID NO: 213). In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure has four amino acid substitutions as compared to the wild-type 4-1BBL polypeptide (e.g., the 4-1BBL polypeptide comprising the amino acid sequence shown in FIG. 36A or set forth in SEQ ID NO: 213). In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure has five amino acid substitutions as compared to the wild-type 4-1BBL polypeptide (e.g., the 4-1BBL polypeptide comprising the amino acid sequence shown in FIG. 36A or set forth in SEQ ID NO: 213). In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure has six amino acid substitutions as compared to the wild-type 4-1BBL polypeptide (e.g., the 4-1BBL polypeptide comprising the amino acid sequence shown in FIG. 36A or set forth in SEQ ID NO: 213).In some cases, the mutant 4-1BBL polypeptide of the present disclosure has seven amino acid substitutions compared to the wild-type 4-1BBL polypeptide (e.g., the 4-1BBL polypeptide comprising the amino acid sequence shown in FIG. 36A or set forth in SEQ ID NO: 213). In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure has eight amino acid substitutions compared to the wild-type 4-1BBL polypeptide (e.g., the 4-1BBL polypeptide comprising the amino acid sequence shown in FIG. 36A or set forth in SEQ ID NO: 213). In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure has nine amino acid substitutions compared to the wild-type 4-1BBL polypeptide (e.g., the 4-1BBL polypeptide comprising the amino acid sequence shown in FIG. 36A or set forth in SEQ ID NO: 213). In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure has ten amino acid substitutions compared to the wild-type 4-1BBL polypeptide (e.g., the 4-1BBL polypeptide comprising the amino acid sequence shown in FIG. 36A or set forth in SEQ ID NO: 213).

[0172] In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure has 11 to 50 amino acid substitutions compared to the wild-type 4-1BBL polypeptide (e.g., the 4-1BBL polypeptide comprising the amino acid sequence shown in FIG. 36A or set forth in SEQ ID NO: 213). For example, in some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure has 11 to 15, 15 to 20, 20 to 25, 25 to 30, 30 to 35, 35 to 40, 40 to 45, or 45 to 50 amino acid substitutions compared to the wild-type 4-1BBL polypeptide (e.g., the 4-1BBL polypeptide comprising the amino acid sequence shown in FIG. 36A or set forth in SEQ ID NO: 213).

[0173] Suitable mutant 4-1BBL polypeptides that may be included in the multimeric polypeptides of the present disclosure include those described above.

[0174] 4-1BBL containing a substitution of K127 In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptides of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 36B, wherein amino acid 127 (designated "x") is an amino acid other than lysine; for example, amino acid 127 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp or Glu. In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptides of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 213, wherein amino acid 47 is an amino acid other than lysine; for example, amino acid 47 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp or Glu.

[0175] In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 214 and comprises an amino acid substitution at K48. In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 214 and comprises an amino acid substitution at K48. In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 215 and comprises an amino acid substitution at K48. In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 215 and comprises an amino acid substitution at K48.

[0176] K127+M91 substitution In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 36B, wherein i) amino acid 127 (shown as "x") is an amino acid other than lysine, for example, amino acid 127 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp or Glu, and ii) is an amino acid substitution of M91, and amino acid 91 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Asn, Gln, Lys, Arg, His, Asp or Glu. In some cases, amino acid 127 is Ala and amino acid 91 is Ala. In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 213, wherein i) amino acid 47 is an amino acid other than lysine, for example, amino acid 47 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp or Glu, and ii) amino acid 11 is other than methionine, for example, amino acid 11 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Asn, Gln, Lys, Arg, His, Asp or Glu. In some cases, amino acid 47 is Ala and amino acid 11 is Ala.

[0177] K127+F92 substitution In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptides of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 36B, wherein i) amino acid 127 (designated "x") is an amino acid other than lysine, for example, amino acid 127 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp or Glu, and ii) is an amino acid substitution at F92, and amino acid 92 is Gly, Ala, Val, Leu, Ile, Pro, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu. In some cases, amino acid 127 is Ala and amino acid 92 is Ala. In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptides of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 213, wherein i) amino acid 47 is an amino acid other than lysine, for example, amino acid 47 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp or Glu, and ii) amino acid 12 is other than phenylalanine, for example, amino acid 12 is Gly, Ala, Val, Leu, Ile, Pro, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu. In some cases, amino acid 47 is Ala and amino acid 12 is Ala.

[0178] K127+Q94 substitution In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 36B, wherein i) amino acid 127 (designated as "x") is an amino acid other than lysine, for example, amino acid 127 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp or Glu, and ii) it is an amino acid substitution at Q94, and amino acid 94 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Lys, Arg, His, Asp or Glu. In some cases, amino acid 127 is Ala and amino acid 94 is Ala. In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 213, wherein i) amino acid 47 is an amino acid other than lysine, for example, amino acid 47 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp or Glu, and ii) amino acid 14 is other than glutamine, for example, amino acid 14 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Lys, Arg, His, Asp or Glu. In some cases, amino acid 47 is Ala and amino acid 14 is Ala.

[0179] K127+L95 substitution In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 36B, wherein i) amino acid 127 (designated as "x") is an amino acid other than lysine, for example, amino acid 127 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp or Glu, and ii) is an amino acid substitution at L95, and amino acid 95 is Gly, Ala, Val, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu. In some cases, amino acid 127 is Ala and amino acid 95 is Ala. In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 213, wherein i) amino acid 47 is an amino acid other than lysine, for example, amino acid 47 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp or Glu, and ii) amino acid 15 is other than leucine, for example, amino acid 15 is Gly, Ala, Val, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu. In some cases, amino acid 47 is Ala and amino acid 15 is Ala.

[0180] K127+V96 substitution In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 36B, wherein i) amino acid 127 (designated "x") is an amino acid other than lysine, for example, amino acid 127 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp or Glu, and ii) is an amino acid substitution at V96, and amino acid 96 is Gly, Ala, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu. In some cases, amino acid 127 is Ala and amino acid 96 is Ala. In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 213, wherein i) amino acid 47 is an amino acid other than lysine, for example, amino acid 47 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp or Glu, and ii) amino acid 16 is other than valine, for example, amino acid 16 is Gly, Ala, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu. In some cases, amino acid 47 is Ala and amino acid 16 is Ala.

[0181] K127+Q98 substitution In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 36B, wherein i) amino acid 127 (designated "x") is an amino acid other than lysine, for example, amino acid 127 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp or Glu, and ii) is an amino acid substitution at Q98, and amino acid 98 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Lys, Arg, His, Asp or Glu. In some cases, amino acid 127 is Ala and amino acid 98 is Ala. In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 213, wherein i) amino acid 47 is an amino acid other than lysine, for example, amino acid 47 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp or Glu, and ii) amino acid 18 is other than glutamine, for example, amino acid 18 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Lys, Arg, His, Asp or Glu. In some cases, amino acid 47 is Ala and amino acid 18 is Ala.

[0182] K127+N99 substitution In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 36B, wherein i) amino acid 127 (designated as "x") is an amino acid other than lysine, for example, amino acid 127 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp or Glu, and ii) it is an amino acid substitution at N99, and amino acid 99 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Gln, Lys, Arg, His, Asp or Glu. In some cases, amino acid 127 is Ala and amino acid 99 is Ala. In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 213, wherein i) amino acid 47 is an amino acid other than lysine, for example, amino acid 47 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp or Glu, and ii) amino acid 19 is other than asparagine, for example, amino acid 19 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Gln, Lys, Arg, His, Asp or Glu. In some cases, amino acid 47 is Ala and amino acid 19 is Ala.

[0183] K127+V100 substitution In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 36B, wherein i) amino acid 127 (designated as "x") is an amino acid other than lysine, for example, amino acid 127 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp or Glu, and ii) it is an amino acid substitution at V100, and amino acid 100 is Gly, Ala, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu. In some cases, amino acid 127 is Ala and amino acid 100 is Ala. In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 213, wherein i) amino acid 47 is an amino acid other than lysine, for example, amino acid 47 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp or Glu, and ii) amino acid 20 is other than valine, for example, amino acid 20 is Gly, Ala, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu. In some cases, amino acid 47 is Ala and amino acid 20 is Ala.

[0184] K127+L101 substitution In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 36B, wherein i) amino acid 127 (designated "x") is an amino acid other than lysine, for example, amino acid 127 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp or Glu, and ii) is an amino acid substitution of L101, and amino acid 101 is Gly, Ala, Val, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu. In some cases, amino acid 127 is Ala and amino acid 101 is Ala. In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 213, wherein i) amino acid 47 is an amino acid other than lysine, for example, amino acid 47 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp or Glu, and ii) amino acid 21 is other than leucine, for example, amino acid 21 is Gly, Ala, Val, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu. In some cases, amino acid 47 is Ala and amino acid 21 is Ala.

[0185] K127+L102 substitution In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 36B, wherein i) amino acid 127 (designated as "x") is an amino acid other than lysine, for example, amino acid 127 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp or Glu, and ii) it is an amino acid substitution of L102, and amino acid 102 is Gly, Ala, Val, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu. In some cases, amino acid 127 is Ala and amino acid 102 is Ala. In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 213, wherein i) amino acid 47 is an amino acid other than lysine, for example, amino acid 47 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp or Glu, and ii) amino acid 22 is other than leucine, for example, amino acid 22 is Gly, Ala, Val, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu. In some cases, amino acid 47 is Ala and amino acid 22 is Ala.

[0186] K127+I103 substitution In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 36B, wherein i) amino acid 127 (designated as "x") is an amino acid other than lysine, for example, amino acid 127 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp or Glu, and ii) it is an amino acid substitution of I103, and amino acid 103 is Gly, Ala, Val, Leu, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu. In some cases, amino acid 127 is Ala and amino acid 103 is Ala. In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 213, wherein i) amino acid 47 is an amino acid other than lysine, for example, amino acid 47 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp or Glu, and ii) amino acid 23 is other than isoleucine, for example, amino acid 23 is Gly, Ala, Val, Leu, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu. In some cases, amino acid 47 is Ala and amino acid 23 is Ala.

[0187] K127+D104 substitution In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptides of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 36B, wherein i) amino acid 127 (designated "x") is an amino acid other than lysine, for example, amino acid 127 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp or Glu, and ii) is an amino acid substitution at D104, and amino acid 104 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His or Glu. In some cases, amino acid 127 is Ala and amino acid 104 is Ala. In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptides of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 213, wherein i) amino acid 47 is an amino acid other than lysine, for example, amino acid 47 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp or Glu, and ii) amino acid 24 is other than aspartic acid, for example, amino acid 24 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His or Glu. In some cases, amino acid 47 is Ala and amino acid 24 is Ala.

[0188] K127+G105 substitution In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 36B, wherein i) amino acid 127 (designated as "x") is an amino acid other than lysine, for example, amino acid 127 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp or Glu, and ii) it is an amino acid substitution at G105, and amino acid 105 is Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu. In some cases, amino acid 127 is Ala and amino acid 105 is Ala. In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 213, wherein i) amino acid 47 is an amino acid other than lysine, for example, amino acid 47 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp or Glu, and ii) amino acid 25 is other than glycine, for example, amino acid 25 is Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu. In some cases, amino acid 47 is Ala and amino acid 25 is Ala.

[0189] K127+P106 substitution In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 36B, wherein i) amino acid 127 (designated as "x") is an amino acid other than lysine, for example, amino acid 127 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp or Glu, and ii) it is an amino acid substitution at P106, and amino acid 106 is Gly, Ala, Val, Leu, Ile, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu. In some cases, amino acid 127 is Ala and amino acid 106 is Ala. In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 213, wherein i) amino acid 47 is an amino acid other than lysine, for example, amino acid 47 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp or Glu, and ii) amino acid 26 is other than proline, for example, amino acid 26 is Gly, Ala, Val, Leu, Ile, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu. In some cases, amino acid 47 is Ala and amino acid 26 is Ala.

[0190] K127+L107 substitution In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 36B, wherein i) amino acid 127 (designated as "x") is an amino acid other than lysine, for example, amino acid 127 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp or Glu, and ii) it is an amino acid substitution at L107, and amino acid 107 is Gly, Ala, Val, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu. In some cases, amino acid 127 is Ala and amino acid 107 is Ala. In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 213, wherein i) amino acid 47 is an amino acid other than lysine, for example, amino acid 47 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp or Glu, and ii) amino acid 27 is other than leucine, for example, amino acid 27 is Gly, Ala, Val, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu. In some cases, amino acid 47 is Ala and amino acid 27 is Ala.

[0191] K127+S108 substitution In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 36B, wherein i) amino acid 127 (designated as "x") is an amino acid other than lysine, for example, amino acid 127 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp or Glu, and ii) is an amino acid substitution at S108, and amino acid 108 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu. In some cases, amino acid 127 is Ala and amino acid 108 is Ala. In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 213, wherein i) amino acid 47 is an amino acid other than lysine, for example, amino acid 47 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp or Glu, and ii) amino acid 28 is other than serine, for example, amino acid 28 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu. In some cases, amino acid 47 is Ala and amino acid 28 is Ala.

[0192] K127+W109 substitution In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 36B, wherein i) amino acid 127 (designated "x") is an amino acid other than lysine, for example, amino acid 127 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp or Glu, and ii) is an amino acid substitution of W109, and amino acid 109 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu. In some cases, amino acid 127 is Ala and amino acid 109 is Ala. In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 213, wherein i) amino acid 47 is an amino acid other than lysine, for example, amino acid 47 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp or Glu, and ii) amino acid 29 is other than tryptophan, for example, amino acid 29 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu. In some cases, amino acid 47 is Ala and amino acid 29 is Ala.

[0193] K127+Y110 substitution In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 36B, wherein i) amino acid 127 (designated as "x") is an amino acid other than lysine, for example, amino acid 127 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp or Glu, and ii) it is an amino acid substitution at Y110, and amino acid 110 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu. In some cases, amino acid 127 is Ala and amino acid 110 is Ala. In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 213, wherein i) amino acid 47 is an amino acid other than lysine, for example, amino acid 47 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp or Glu, and ii) amino acid 30 is other than tyrosine, for example, amino acid 30 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu. In some cases, amino acid 47 is Ala and amino acid 30 is Ala.

[0194] K127+S111 substitution In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 36B, wherein i) amino acid 127 (designated as "x") is an amino acid other than lysine, for example, amino acid 127 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp or Glu, and ii) is an amino acid substitution of S111, and amino acid 111 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu. In some cases, amino acid 127 is Ala and amino acid 111 is Ala. In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 213, wherein i) amino acid 47 is an amino acid other than lysine, for example, amino acid 47 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp or Glu, and ii) amino acid 31 is other than serine, for example, amino acid 31 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu. In some cases, amino acid 47 is Ala and amino acid 31 is Ala.

[0195] K127+D112 substitution In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 36B, wherein i) amino acid 127 (designated as "x") is an amino acid other than lysine, for example, amino acid 127 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp or Glu, and ii) it is an amino acid substitution of D112, and amino acid 112 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His or Glu. In some cases, amino acid 127 is Ala and amino acid 112 is Ala. In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 213, wherein i) amino acid 47 is an amino acid other than lysine, for example, amino acid 47 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp or Glu, and ii) amino acid 32 is other than aspartic acid, for example, amino acid 32 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His or Glu. In some cases, amino acid 47 is Ala and amino acid 32 is Ala.

[0196] K127+P113 substitution In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 36B, wherein i) amino acid 127 (designated as "x") is an amino acid other than lysine, for example, amino acid 127 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp or Glu, and ii) is an amino acid substitution at P113, and amino acid 113 is Gly, Ala, Val, Leu, Ile, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu. In some cases, amino acid 127 is Ala and amino acid 113 is Ala. In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 213, wherein i) amino acid 47 is an amino acid other than lysine, for example, amino acid 47 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp or Glu, and ii) amino acid 33 is other than proline, for example, amino acid 33 is Gly, Ala, Val, Leu, Ile, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu. In some cases, amino acid 47 is Ala and amino acid 33 is Ala.

[0197] K127+G114 substitution In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 36B, wherein i) amino acid 127 (shown as "x") is an amino acid other than lysine, for example, amino acid 127 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp or Glu, and ii) is an amino acid substitution of G114, and amino acid 114 is Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu. In some cases, amino acid 127 is Ala and amino acid 114 is Ala. In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 213, wherein i) amino acid 47 is an amino acid other than lysine, for example, amino acid 47 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp or Glu, and ii) amino acid 34 is other than glycine, for example, amino acid 34 is Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu. In some cases, amino acid 47 is Ala and amino acid 34 is Ala.

[0198] K127+L115 substitution In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 36B, wherein i) amino acid 127 (designated as "x") is an amino acid other than lysine, for example, amino acid 127 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp or Glu, and ii) it is an amino acid substitution of L115, and amino acid 115 is Gly, Ala, Val, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu. In some cases, amino acid 127 is Ala and amino acid 115 is Ala. In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 213, wherein i) amino acid 47 is an amino acid other than lysine, for example, amino acid 47 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Arg, His, Asp or Glu, and ii) amino acid 35 is other than leucine, for example, amino acid 35 is Gly, Ala, Val, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu. In some cases, amino acid 47 is Ala and amino acid 35 is Ala.

[0199] 4-1BBL containing the substitution of Q227 In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 36D, wherein amino acid 227 (designated "x") is an amino acid other than glutamine, for example, amino acid 227 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Lys, Arg, His, Asp or Glu. In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 213, wherein amino acid 147 is other than glutamine, for example, amino acid 147 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Lys, Arg, His, Asp or Glu.

[0200] In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 214 and comprises an amino acid substitution at Q148. In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 214 and comprises an amino acid substitution at Q148. In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 215 and comprises an amino acid substitution at Q148. In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 215 and comprises an amino acid substitution at Q148.

[0201] 4-1BBL containing substitution of M91 In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 36E, wherein amino acid 91 (designated "x") is an amino acid other than methionine, for example, amino acid 91 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Asn, Gln, Lys, Arg, His, Asp or Glu. In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 213, wherein amino acid 11 is other than methionine, for example, amino acid 11 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Asn, Gln, Lys, Arg, His, Asp or Glu.

[0202] In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 214 and comprises an amino acid substitution at M12. In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 214 and comprises an amino acid substitution at M12. In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 215 and comprises an amino acid substitution at M12. In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 215 and comprises an amino acid substitution at M12.

[0203] 4-1BBL containing the substitution of F92 In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 36F, and amino acid 92 (designated "x") is an amino acid other than phenylalanine. For example, amino acid 92 is Gly, Ala, Val, Leu, Ile, Pro, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu. In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 213, and amino acid 12 is other than phenylalanine. For example, amino acid 12 is Gly, Ala, Val, Leu, Ile, Pro, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu.

[0204] In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 214 and comprises an amino acid substitution at F13. In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 214 and comprises an amino acid substitution at F13. In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 215 and comprises an amino acid substitution at F13. In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 215 and comprises an amino acid substitution at F13.

[0205] 4-1BBL comprising substitution of Q94 In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 36G, wherein amino acid 94 (designated as "x") is an amino acid other than glutamine, for example, amino acid 94 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Lys, Arg, His, Asp or Glu. In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 213, wherein amino acid 14 is other than glutamine, for example, amino acid 14 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Lys, Arg, His, Asp or Glu.

[0206] In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 214 and comprises an amino acid substitution at Q15. In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 214 and comprises an amino acid substitution at Q15. In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 215 and comprises an amino acid substitution at Q15. In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 215 and comprises an amino acid substitution at Q15.

[0207] 4-1BBL containing the substitution of L95 In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 36H, and amino acid 95 (designated "x") is an amino acid other than leucine, for example, amino acid 95 is Gly, Ala, Val, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu. In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 213, and amino acid 15 is other than leucine, for example, amino acid 15 is Gly, Ala, Val, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu.

[0208] In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 214 and comprises an amino acid substitution at L16. In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 214 and comprises an amino acid substitution at L16. In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 215 and comprises an amino acid substitution at L16. In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 215 and comprises an amino acid substitution at L16.

[0209] 4-1BBL containing a substitution of V96 In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 36I, wherein amino acid 96 (designated as "x") is an amino acid other than valine, for example, amino acid 96 is Gly, Ala, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu. In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 213, wherein amino acid 16 is other than valine, for example, amino acid 16 is Gly, Ala, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu.

[0210] In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 2

[0211] 4-1BBL containing the substitution of Q98 In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 36J, and amino acid 98 (designated as "x") is an amino acid other than glutamine. For example, amino acid 98 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Lys, Arg, His, Asp or Glu. In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 213, and amino acid 18 is other than glutamine. For example, amino acid 18 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Lys, Arg, His, Asp or Glu.

[0212] In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 214 and comprises an amino acid substitution at Q19. In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 214 and comprises an amino acid substitution at Q19. In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 215 and comprises an amino acid substitution at Q19. In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 215 and comprises an amino acid substitution at Q19.

[0213] 4-1BBL containing a substitution at N99 In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 36K, wherein amino acid 99 (designated as "x") is an amino acid other than asparagine. For example, amino acid 99 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Gln, Lys, Arg, His, Asp or Glu. In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 213, wherein amino acid 19 is other than asparagine. For example, amino acid 19 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Gln, Lys, Arg, His, Asp or Glu.

[0214] In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 214 and includes an amino acid substitution at N20. In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 214 and includes an amino acid substitution at N20. In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 215 and includes an amino acid substitution at N20. In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 215 and includes an amino acid substitution at N20.

[0215] 4-1BBL containing the substitution of V100 In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 36L, and amino acid 100 (designated "x") is an amino acid other than valine, for example, amino acid 100 is Gly, Ala, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu. In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 213, and amino acid 20 is other than valine, for example, amino acid 20 is Gly, Ala, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu.

[0216] In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 214 and comprises an amino acid substitution at V21. In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 214 and comprises an amino acid substitution at V21. In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 215 and comprises an amino acid substitution at V21. In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 215 and comprises an amino acid substitution at V21.

[0217] 4-1BBL containing a substitution at L101 In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 36M, wherein amino acid 101 (designated as "x") is an amino acid other than leucine, for example, amino acid 101 is Gly, Ala, Val, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu. In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 213, wherein amino acid 21 is other than leucine, for example, amino acid 21 is Gly, Ala, Val, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu.

[0218] In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 214 and includes an amino acid substitution at L22. In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 214 and includes an amino acid substitution at L22. In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 215 and includes an amino acid substitution at L22. In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 215 and includes an amino acid substitution at L22.

[0219] 4-1BBL containing the substitution of L102 In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptides of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 36N, wherein amino acid 102 (designated "x") is an amino acid other than leucine, for example, amino acid 102 is Gly, Ala, Val, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu. In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptides of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 213, wherein amino acid 22 is other than leucine, for example, amino acid 22 is Gly, Ala, Val, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu.

[0220] In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 214 and comprises an amino acid substitution at L23. In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 214 and comprises an amino acid substitution at L23. In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 215 and comprises an amino acid substitution at L23. In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 215 and comprises an amino acid substitution at L23.

[0221] 4-1BBL containing a substitution of I103 In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 36O, wherein amino acid 103 (designated as "x") is an amino acid other than isoleucine, for example, amino acid 103 is Gly, Ala, Val, Leu, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu. In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 213, wherein amino acid 23 is other than isoleucine, for example, amino acid 23 is Gly, Ala, Val, Leu, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu.

[0222] In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 214 and includes an amino acid substitution at I24. In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 214 and includes an amino acid substitution at I24. In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 215 and includes an amino acid substitution at I24. In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 215 and includes an amino acid substitution at I24.

[0223] 4-1BBL comprising a substitution of D104 In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 36P, wherein amino acid 104 (designated "x") is an amino acid other than aspartic acid, for example, amino acid 104 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His or Glu. In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 213, wherein amino acid 24 is other than aspartic acid, for example, amino acid 24 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His or Glu.

[0224] In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 214 and includes an amino acid substitution at D25. In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 214 and includes an amino acid substitution at D25. In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 215 and includes an amino acid substitution at D25. In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 215 and includes an amino acid substitution at D25.

[0225] 4-1BBL containing a substitution at G105 In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in FIG. 36Q, wherein amino acid 105 (designated as "x") is an amino acid other than glycine, for example, amino acid 105 is Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu. In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 213, wherein amino acid 25 is other than glycine, for example, amino acid 25 is Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu.

[0226] In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 214 and comprises an amino acid substitution at G26. In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 214 and comprises an amino acid substitution at G26. In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 215 and comprises an amino acid substitution at G26. In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptide of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 215 and comprises an amino acid substitution at G26.

[0227] 4-1BBL containing substitution of P106 In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptides of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in Figure 36R, wherein amino acid 106 (designated "x") is an amino acid other than proline, for example, amino acid 106 is Gly, Ala, Val, Leu, Ile, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu. In some cases, the mutant 4-1BBL polypeptide present in the multimeric polypeptides of the present disclosure comprises an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 213, wherein amino acid 26 is other than proline, for example, amino acid 26 is Gly, Ala, Val, Leu, ...

Claims

1. A medicament comprising a multimeric polypeptide for regulating an immune response in an individual in need thereof, the multimeric polypeptide comprising a heterodimeric polypeptide, the heterodimeric polypeptide comprising, a) in the order from the N-terminus to the C-terminus, i) an HPV16 E7 epitope peptide comprising the amino acid sequence YMLDLQPETT (SEQ ID NO: 77), and ii) a β2-microglobulin (β2M) polypeptide comprising a first polypeptide, and b) in the order from the N-terminus to the C-terminus, i) a mutant IL-2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 49, ii) an MHC class I heavy chain polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 50, and iii) an immunoglobulin (Ig) Fc polypeptide comprising a second polypeptide and, one or more linkers may be inserted between one or more components of the first and second polypeptides, A medicament, characterized in that it is used in combination with an immune checkpoint inhibitor.

2. The medicament according to claim 1, characterized in that the immune checkpoint inhibitor and the multimeric polypeptide are administered simultaneously or at different times.

3. The medicament according to claim 1 or 2, wherein the Ig Fc polypeptide contained in the multimeric polypeptide comprises substitutions of L234A and L235A.

4. The medicament according to any one of claims 1 to 3, wherein the β2M polypeptide comprises an amino acid sequence having at least 95% amino acid sequence identity to the amino acids at positions 21 to 119 of any of the amino acid sequences shown in SEQ ID NOs: 95, 195, 96, 97 and 98.

5. The medicament according to any one of claims 1 to 4, wherein the multimeric polypeptide comprises a disulfide bond connecting a Cys residue in the β2M polypeptide and a Cys residue in the MHC class I heavy chain polypeptide.

6. The medicament according to claim 5, wherein the disulfide bond connects Cys at amino acid residue 12 of the β2M polypeptide and Cys at amino acid residue 236 of the MHC class I heavy chain polypeptide.

7. The medicament according to any one of claims 1 to 6, wherein the multimeric polypeptide comprises a linker between the epitope peptide and the β2M polypeptide.

8. i) the β2-microglobulin (β2M) polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 48; ii) the MHC class I heavy chain polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 50; and iii) the multimeric polypeptide comprises a disulfide bond linking Cys at position 12 of the amino acid residues of the β2M polypeptide and Cys at position 236 of the amino acid residues of the MHC class I heavy chain polypeptide. The medicament according to claim 1. **Claim 9** i) the second polypeptide comprises two copies of the mutant IL-2 polypeptide; ii) the Ig Fc polypeptide comprises an amino acid sequence having at least 95% amino acid sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs: 44 to 47; The medicament according to claim 8. **Claim 10** the first polypeptide comprises a linker between the epitope peptide and the β2-microglobulin polypeptide; and the second polypeptide a) between the first mutant IL-2 polypeptide and the second mutant IL-2 polypeptide; b) between the mutant IL-2 polypeptide and the MHC class I heavy chain polypeptide; and c) between the MHC class I heavy chain polypeptide and the Ig Fc polypeptide independently comprises a peptide linker selected from. The medicament according to claim 9. **Claim 11** Each peptide linker is (GGGGS) ３ , (GGGGS) ４ 11. The pharmaceutical composition of claim 10, wherein the hydroxyl group is independently selected from the group consisting of , and AAAGG. **Claim 12** the heterodimeric polypeptide a) in the order from the N-terminus to the C-terminus, i) an HPV16 E7 epitope peptide comprising the amino acid sequence YMLDLQPETT (SEQ ID NO: 77); ii) a linker comprising the amino acid sequence GGGGSGGGGGSGGGGGS (SEQ ID NO: 207); and iii) a β2-microglobulin polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 48 comprising a first polypeptide, and b) a second polypeptide comprising an amino acid sequence selected from the amino acid sequence set forth in SEQ ID NO: 33, the amino acid sequence set forth in SEQ ID NO: 36, and the amino acid sequence set forth in SEQ ID NO:

39. The medicament according to any one of claims 7 to 11. **Claim 13** The multimeric polypeptide is a homodimer comprising two copies of the heterodimeric polypeptide, and the homodimer comprises one or more disulfide bonds that link the Ig Fc polypeptide of one heterodimeric polypeptide to the Ig Fc polypeptide of another heterodimeric polypeptide. The pharmaceutical according to any one of claims 1 to 12.

14. The immune checkpoint inhibitor is an antibody that binds to a polypeptide selected from the group consisting of CD27, CD28, CD40, CD122, CD96, CD73, CD47, OX40, GITR, CSF1R, JAK, PI3Kδ, PI3Kγ, TAM, arginase, CD137, ICOS, A2AR, B7-H3, B7-H4, BTLA, CTLA-4, LAG3, TIM3, VISTA, CD96, TIGIT, CD122, PD-1, PD-L1, and PD-L2. The pharmaceutical according to any one of claims 1 to 13.

15. The immune checkpoint inhibitor is an antibody specific for PD-L1, CTLA4, or TIGIT. The pharmaceutical according to any one of claims 1 to 14.

16. The immune checkpoint inhibitor is an antibody specific for PD-1. The pharmaceutical according to any one of claims 1 to 15.

17. The immune checkpoint inhibitor is pembrolizumab. The pharmaceutical according to claim 16.

18. The immune checkpoint inhibitor is nivolumab. The pharmaceutical according to claim 16.

19. Use of the multimeric polypeptide in the manufacture of a pharmaceutical comprising the multimeric polypeptide for modulating an immune response in an individual, wherein the multimeric polypeptide comprises a heterodimeric polypeptide, the heterodimeric polypeptide comprises: a) In the order from the N-terminus to the C-terminus, i) an HPV16E7 epitope peptide comprising the amino acid sequence YMLDLQPETT (SEQ ID NO: 77), and ii) a β2-microglobulin (β2M) polypeptide comprising a first polypeptide, and b) In the order from the N-terminus to the C-terminus, i) a mutant IL-2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 49, ii) an MHC class I heavy chain polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 50, and iii) an immunoglobulin (Ig) Fc polypeptide comprising a second polypeptide and One or more linkers may be inserted between one or more components of the first and second polypeptides. Use, characterized by being used in combination with an immune checkpoint inhibitor.

20. Use according to claim 19, characterized in that the immune checkpoint inhibitor and the multimeric polypeptide are administered simultaneously or at different times.

21. Use according to claim 19 or 20, characterized in that the IgG1 Fc polypeptide comprised in the multimeric polypeptide comprises the substitutions L234A and L235A.

22. Use according to any one of claims 19 to 21, characterized in that the β2-microglobulin (β2M) polypeptide comprises an amino acid sequence having at least 95% amino acid sequence identity to the amino acids at positions 21 to 119 of any of the amino acid sequences shown in SEQ ID NOs: 95, 195, 96, 97 and 98.

23. Use according to any one of claims 19 to 22, characterized in that the multimeric polypeptide comprises a disulfide bond connecting a Cys residue in the β2M polypeptide and a Cys residue in the MHC class I heavy chain polypeptide.

24. Use according to claim 23, characterized in that the disulfide bond connects the Cys at amino acid residue 12 of the β2M polypeptide to the Cys bond at amino acid residue 236 of the MHC class I heavy chain polypeptide.

25. Use according to any one of claims 19 to 24, characterized in that the multimeric polypeptide comprises a linker between the epitope peptide and the β2M polypeptide.

26. i) the β2-microglobulin polypeptide comprises the amino acid sequence shown in SEQ ID NO: 48, ii) the MHC class I heavy chain polypeptide comprises the amino acid sequence shown in SEQ ID NO: 50, and iii) the multimeric polypeptide comprises a disulfide bond connecting the Cys at amino acid residue 12 of the β2M polypeptide to the Cys at amino acid residue 236 of the MHC class I heavy chain polypeptide, Use according to claim 19.

27. i) the second polypeptide comprises two copies of a mutant IL-2 polypeptide, and ii) the Ig Fc polypeptide comprises an amino acid sequence having at least 95% amino acid sequence identity to the amino acid sequence shown in any of SEQ ID NOs: 44 to 47. Use according to claim 26.

28. The first polypeptide includes a linker between the epitope peptide and the β2-microglobulin polypeptide, and the second polypeptide a) between the first mutant IL-2 polypeptide and the second mutant IL-2 polypeptide, b) between the mutant IL-2 polypeptide and the MHC class I heavy chain polypeptide, and c) between the MHC class I heavy chain polypeptide and the Ig Fc polypeptide independently includes a peptide linker selected from, The use according to claim 27.

29. each of said peptide linkers being independently selected from (GGGGGS) ３ , (GGGGGS) ４ , and AAAGG, the use according to claim 28

30. The heterodimer polypeptide a) in the order from the N-terminus to the C-terminus, i) an HPV16 E7 epitope peptide comprising the amino acid sequence YMLDLQPETT (SEQ ID NO: 77), ii) a linker comprising the amino acid sequence GGGGSGGGGGSGGGGG (SEQ ID NO: 207), and iii) a β2-microglobulin polypeptide comprising the amino acid sequence shown in SEQ ID NO: 48 including a first polypeptide, and b) a second polypeptide comprising an amino acid sequence selected from the amino acid sequence shown in SEQ ID NO: 33, the amino acid sequence shown in SEQ ID NO: 36, and the amino acid sequence shown in SEQ ID NO: 39, The use according to any one of claims 25 to 29.

31. The multimer polypeptide is a homodimer comprising two copies of the heterodimer polypeptide, and the homodimer comprises one or more disulfide bonds connecting the Ig Fc polypeptide of one heterodimer polypeptide and the Ig Fc polypeptide of another heterodimer polypeptide. The use according to any one of claims 19 to 30.

32. The immune checkpoint inhibitor is an antibody that binds to a polypeptide selected from the group consisting of CD27, CD28, CD40, CD122, CD96, CD73, CD47, OX40, GITR, CSF1R, JAK, PI3Kδ, PI3Kγ, TAM, arginase, CD137, ICOS, A2AR, B7-H3, B7-H4, BTLA, CTLA-4, LAG3, TIM3, VISTA, CD96, TIGIT, CD122, PD-1, PD-L1 and PD-L2. The use according to any one of claims 19 to 31.

33. The use according to any one of claims 19 to 32, wherein the immune checkpoint inhibitor is an antibody specific for PD-L1, CTLA4 or TIGIT.

34. The use according to any one of claims 19 to 32, wherein the immune checkpoint inhibitor is an antibody specific for PD-1.

35. The use according to claim 34, wherein the immune checkpoint inhibitor is pembrolizumab.

36. The use according to claim 34, wherein the immune checkpoint inhibitor is nivolumab.

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