Multimer t-cell regulatory polypeptide, and method for using the same

Abstract

To provide a t-cell regulatory multimer polypeptide effective for regulating the activity of a T-cell and for regulating immune response in an individual, a method for producing the polypeptide, and a pharmaceutical composition including the T-cell regulatory multimer polypeptide.SOLUTION: Provided is a T-cell regulatory multimer polypeptide that includes an immunoregulatory polypeptide and also includes an epitope-presenting Wilms tumor peptide.SELECTED DRAWING: Figure 1A

Description

[Technical Field]

[0001] cross reference This application claims the benefit of U.S. Provisional Patent Application No. 63 / 023,834, filed May 12, 2020, and U.S. Provisional Patent Application No. 63 / 041,451, filed June 19, 2020, which applications are incorporated herein by reference in their entireties.

[0002] Incorporation by reference of sequence listing provided as a text file The Sequence Listing is provided as a text file "CUEB-133WO_SEQ_LIST2_ST25.txt" created on May 7, 2021, and is 800 KB in size. The contents of the text file are incorporated herein by reference in their entirety. [Background technology]

[0003] introduction The adaptive immune response is driven by the binding of T cell receptors (TCRs) present on the surface of T cells to small peptide antigens noncovalently presented on the surface of antigen-presenting cells (APCs) via the major histocompatibility complex (MHC, also known in humans as the human leukocyte antigen (HLA) complex). This binding represents the targeting mechanism of the immune system and is a molecular interaction essential for T cell regulation (activation or suppression) and effector function. Following epitope-specific cell targeting, the targeted T cell is activated by the binding of costimulatory proteins present on the APC with corresponding costimulatory proteins on the T cell. Both signals—epitope / TCR binding and the binding of APC costimulatory proteins with T cell costimulatory proteins—are required to drive T cell specificity and activation or suppression. While TCRs are specific for a given epitope, costimulatory proteins are not epitope-specific; instead, they are generally expressed on all T cells or many T cell subsets. Summary of the Invention

[0004] overview The present disclosure provides T cell regulatory multimeric polypeptides (TMMPs) comprising immunomodulatory polypeptides and including epitope-presenting Wilms tumor peptides. The T cell regulatory multimeric polypeptides are useful for modulating the activity of T cells and for modulating immune responses in individuals. [Brief explanation of the drawings]

[0005] [Figure 1A] FIG. 1 is a schematic diagram of various TMMPs of the present disclosure. [Figure 1B] FIG. 1 is a schematic diagram of various TMMPs of the present disclosure. [Figure 1C] FIG. 1 is a schematic diagram of various TMMPs of the present disclosure. [Figure 1D] FIG. 1 is a schematic diagram of various TMMPs of the present disclosure. [Figure 1E] FIG. 1 is a schematic diagram of various TMMPs of the present disclosure. [Figure 1F] FIG. 1 is a schematic diagram of various TMMPs of the present disclosure. [Figure 2A] FIG. 1 is a schematic diagram of various disulfide-linked TMMPs of the present disclosure. [Figure 2B] FIG. 1 is a schematic diagram of various disulfide-linked TMMPs of the present disclosure. [Figure 2C] FIG. 1 is a schematic diagram of various disulfide-linked TMMPs of the present disclosure. [Figure 2D] FIG. 1 is a schematic diagram of various disulfide-linked TMMPs of the present disclosure. [Figure 2E] FIG. 1 is a schematic diagram of various disulfide-linked TMMPs of the present disclosure. [Figure 2F] FIG. 1 is a schematic diagram of various disulfide-linked TMMPs of the present disclosure. [Figure 3A] The amino acid sequences of WT-1 polypeptides are shown in Figures 3A to 3E, which are set forth as SEQ ID NOs: 399 to 403, respectively. [Figure 3B] The amino acid sequences of WT-1 polypeptides are shown in Figures 3A to 3E, which are set forth as SEQ ID NOs: 399 to 403, respectively. [Figure 3C]The amino acid sequences of WT-1 polypeptides are shown in Figures 3A to 3E, which are set forth as SEQ ID NOs: 399 to 403, respectively. [Figure 3D] The amino acid sequences of WT-1 polypeptides are shown in Figures 3A to 3E, which are set forth as SEQ ID NOs: 399 to 403, respectively. [Figure 3E] The amino acid sequences of WT-1 polypeptides are shown in Figures 3A to 3E, which are set forth as SEQ ID NOs: 399 to 403, respectively. [Figure 4A] 4A-4E show the amino acid sequences of exemplary polypeptide chains of TMMPs of the present disclosure. The sequences of the exemplary polypeptide chains of Figures 4A-4E are set forth in SEQ ID NOs: 405-409, respectively. [Figure 4B] 4A-4E show the amino acid sequences of exemplary polypeptide chains of TMMPs of the present disclosure. The sequences of the exemplary polypeptide chains of Figures 4A-4E are set forth in SEQ ID NOs: 405-409, respectively. [Figure 4C] 4A-4E show the amino acid sequences of exemplary polypeptide chains of TMMPs of the present disclosure. The sequences of the exemplary polypeptide chains of Figures 4A-4E are set forth in SEQ ID NOs: 405-409, respectively. [Figure 4D] The amino acid sequences of exemplary polypeptide chains of TMMPs of the present disclosure are shown. The sequences of the exemplary polypeptide chains of Figures 4A-4E are set forth in SEQ ID NOs: 405-409, respectively. The epitope sequence is shown below: Figure 4D: CMTWNQMNL (SEQ ID NO: 266). [Figure 4E] The amino acid sequences of exemplary polypeptide chains of TMMPs of the present disclosure are shown. The sequences of the exemplary polypeptide chains of Figures 4A-4E are set forth in SEQ ID NOs: 405-409, respectively. The epitope sequence is shown below: Figure 4E: CYTWNQMNL (SEQ ID NO: 267). [Figure 5A] The amino acid sequences of immunoglobulin Fc polypeptides are shown in SEQ ID NOs: 410 to 421, respectively, in Figures 5A to 5G. [Figure 5B] The amino acid sequences of immunoglobulin Fc polypeptides are shown in SEQ ID NOs: 410 to 421, respectively, in Figures 5A to 5G. [Figure 5C]The amino acid sequences of immunoglobulin Fc polypeptides are shown in SEQ ID NOs: 410 to 421, respectively, in Figures 5A to 5G. [Figure 5D] The amino acid sequences of immunoglobulin Fc polypeptides are shown in SEQ ID NOs: 410 to 421, respectively, in Figures 5A to 5G. [Figure 5E] The amino acid sequences of immunoglobulin Fc polypeptides are shown in SEQ ID NOs: 410 to 421, respectively, in Figures 5A to 5G. [Figure 5F] The amino acid sequences of immunoglobulin Fc polypeptides are shown in SEQ ID NOs: 410 to 421, respectively, in Figures 5A to 5G. [Figure 5G] The amino acid sequences of immunoglobulin Fc polypeptides are shown in SEQ ID NOs: 410 to 421, respectively, in Figures 5A to 5G. [Figure 5H] The sequence shown in Figure 5H is set forth in SEQ ID NO:487. [Figure 6]

[0023] Figure 1 shows a multiple amino acid sequence alignment of the beta-2 microglobulin (β2M) precursor (i.e., including the leader sequence) from human (Homo sapiens) (NP_004039.1; SEQ ID NO: 19), chimpanzee (Pan troglodytes) (NP_001009066.1; SEQ ID NO: 19), rhesus monkey (Macaca mulatta) (NP_001040602.1; SEQ ID NO: 20), cow (Bos taurus) (NP_776318.1; SEQ ID NO: 21), and mouse (Mus musculus) (NP_033865.2; SEQ ID NO: 22). Amino acids 1-20 are the signal peptide. [Figure 7A] The amino acid sequences of the full-length human HLA heavy chains of alleles A*0101 (SEQ ID NO: 23), A*1101 (SEQ ID NO: 24), A*2402 (SEQ ID NO: 25), and A*3303 (SEQ ID NO: 26) (Figure 7A), the full-length human HLA heavy chain of allele B*0702 (SEQ ID NO: 27) (Figure 7B), and the full-length human HLA-C heavy chain (SEQ ID NO: 28) (Figure 7C) are shown. [Figure 7B]The amino acid sequences of the full-length human HLA heavy chains of alleles A*0101 (SEQ ID NO: 23), A*1101 (SEQ ID NO: 24), A*2402 (SEQ ID NO: 25), and A*3303 (SEQ ID NO: 26) (Figure 7A), the full-length human HLA heavy chain of allele B*0702 (SEQ ID NO: 27) (Figure 7B), and the full-length human HLA-C heavy chain (SEQ ID NO: 28) (Figure 7C) are shown. [Figure 7C] The amino acid sequences of the full-length human HLA heavy chains of alleles A*0101 (SEQ ID NO: 23), A*1101 (SEQ ID NO: 24), A*2402 (SEQ ID NO: 25), and A*3303 (SEQ ID NO: 26) (Figure 7A), the full-length human HLA heavy chain of allele B*0702 (SEQ ID NO: 27) (Figure 7B), and the full-length human HLA-C heavy chain (SEQ ID NO: 28) (Figure 7C) are shown. [Figure 8-1] The figure shows an alignment of 11 types of mature MHC class I heavy chain amino acid sequences, excluding the leader sequence, transmembrane domain, and intracellular domain, which are SEQ ID NOs: 41 to 51 from top to bottom. [Figure 8-2] The figure shows an alignment of 11 types of mature MHC class I heavy chain amino acid sequences, excluding the leader sequence, transmembrane domain, and intracellular domain, which are SEQ ID NOs: 41 to 51 from top to bottom. [Figure 9A-1] 1 shows an alignment of HLA-A heavy chain amino acid sequences. [Figure 9A-2] 1 shows an alignment of HLA-A heavy chain amino acid sequences. [Figure 9B] The consensus sequence for the HLA-A heavy chain (SEQ ID NO: 29) is shown. [Figure 10A-1] 1 shows an alignment of HLA-B heavy chain amino acid sequences (SEQ ID NOs: 207 to 213, respectively). [Figure 10A-2] 1 shows an alignment of HLA-B heavy chain amino acid sequences (SEQ ID NOs: 207 to 213, respectively). [Figure 10B] The consensus sequence for the HLA-B heavy chain (SEQ ID NO: 30) is shown. [Figure 11A-1] 1 shows an alignment of HLA-C heavy chain amino acid sequences (SEQ ID NOs: 214 to 222, respectively). [Figure 11A-2]1 shows an alignment of HLA-C heavy chain amino acid sequences (SEQ ID NOs: 214 to 222, respectively). [Figure 11B] The consensus sequence for the HLA-C heavy chain (SEQ ID NO: 31) is shown. [Figure 12] Consensus amino acid sequences for each of the HLA-E, HLA-F, and HLA-G heavy chains (SEQ ID NOS: 32-34, respectively) are shown. Variable amino acid (aa) positions are indicated as consecutively numbered "X" residues. Amino acid positions 84, 139, and 236 are double underlined. [Figure 13] An alignment of the consensus amino acid sequences of HLA-A (sequence number 29), HLA-B (sequence number 30), HLA-C (sequence number 31), HLA-E (sequence number 32), HLA-F (sequence number 33), and HLA-G (sequence number 34) is shown. [Figure 14A]

[0049] Figures 14A-14I show the amino acid sequences of the polypeptide chains of the double-disulfide-bonded TMMP of the present disclosure. The sequences of the polypeptide chains in Figures 14A-14I are set forth in SEQ ID NOs: 422-430, respectively. The epitope sequences are shown as follows: Figure 14B: VLDFAPPGA (SEQ ID NO: 259), Figure 14C: RMFPNAPYL (SEQ ID NO: 260), Figure 14F: VLDFAPPGA (SEQ ID NO: 259), Figure 14G: RMFPNAPYL (SEQ ID NO: 260), Figure 14H: YMFPNAPYL (SEQ ID NO: 264), Figure 14I: YMFPNAPYL (SEQ ID NO: 264), and Figure 14J (SEQ ID NO: 486). [Figure 14B]

[0049] Figures 14A-14I show the amino acid sequences of the polypeptide chains of the double-disulfide-bonded TMMP of the present disclosure. The sequences of the polypeptide chains in Figures 14A-14I are set forth in SEQ ID NOs: 422-430, respectively. The epitope sequences are shown as follows: Figure 14B: VLDFAPPGA (SEQ ID NO: 259), Figure 14C: RMFPNAPYL (SEQ ID NO: 260), Figure 14F: VLDFAPPGA (SEQ ID NO: 259), Figure 14G: RMFPNAPYL (SEQ ID NO: 260), Figure 14H: YMFPNAPYL (SEQ ID NO: 264), Figure 14I: YMFPNAPYL (SEQ ID NO: 264), and Figure 14J (SEQ ID NO: 486). [Figure 14C]

[0049] Figures 14A-14I show the amino acid sequences of the polypeptide chains of the double-disulfide-bonded TMMP of the present disclosure. The sequences of the polypeptide chains in Figures 14A-14I are set forth in SEQ ID NOs: 422-430, respectively. The epitope sequences are shown as follows: Figure 14B: VLDFAPPGA (SEQ ID NO: 259), Figure 14C: RMFPNAPYL (SEQ ID NO: 260), Figure 14F: VLDFAPPGA (SEQ ID NO: 259), Figure 14G: RMFPNAPYL (SEQ ID NO: 260), Figure 14H: YMFPNAPYL (SEQ ID NO: 264), Figure 14I: YMFPNAPYL (SEQ ID NO: 264), and Figure 14J (SEQ ID NO: 486). [Figure 14D]

[0049] Figures 14A-14I show the amino acid sequences of the polypeptide chains of the double-disulfide-bonded TMMP of the present disclosure. The sequences of the polypeptide chains in Figures 14A-14I are set forth in SEQ ID NOs: 422-430, respectively. The epitope sequences are shown as follows: Figure 14B: VLDFAPPGA (SEQ ID NO: 259), Figure 14C: RMFPNAPYL (SEQ ID NO: 260), Figure 14F: VLDFAPPGA (SEQ ID NO: 259), Figure 14G: RMFPNAPYL (SEQ ID NO: 260), Figure 14H: YMFPNAPYL (SEQ ID NO: 264), Figure 14I: YMFPNAPYL (SEQ ID NO: 264), and Figure 14J (SEQ ID NO: 486). [Figure 14E]

[0049] Figures 14A-14I show the amino acid sequences of the polypeptide chains of the double-disulfide-bonded TMMP of the present disclosure. The sequences of the polypeptide chains in Figures 14A-14I are set forth in SEQ ID NOs: 422-430, respectively. The epitope sequences are shown as follows: Figure 14B: VLDFAPPGA (SEQ ID NO: 259), Figure 14C: RMFPNAPYL (SEQ ID NO: 260), Figure 14F: VLDFAPPGA (SEQ ID NO: 259), Figure 14G: RMFPNAPYL (SEQ ID NO: 260), Figure 14H: YMFPNAPYL (SEQ ID NO: 264), Figure 14I: YMFPNAPYL (SEQ ID NO: 264), and Figure 14J (SEQ ID NO: 486). [Figure 14F]

[0049] Figures 14A-14I show the amino acid sequences of the polypeptide chains of the double-disulfide-bonded TMMP of the present disclosure. The sequences of the polypeptide chains in Figures 14A-14I are set forth in SEQ ID NOs: 422-430, respectively. The epitope sequences are shown as follows: Figure 14B: VLDFAPPGA (SEQ ID NO: 259), Figure 14C: RMFPNAPYL (SEQ ID NO: 260), Figure 14F: VLDFAPPGA (SEQ ID NO: 259), Figure 14G: RMFPNAPYL (SEQ ID NO: 260), Figure 14H: YMFPNAPYL (SEQ ID NO: 264), Figure 14I: YMFPNAPYL (SEQ ID NO: 264), and Figure 14J (SEQ ID NO: 486). [Figure 14G]

[0049] Figures 14A-14I show the amino acid sequences of the polypeptide chains of the double-disulfide-bonded TMMP of the present disclosure. The sequences of the polypeptide chains in Figures 14A-14I are set forth in SEQ ID NOs: 422-430, respectively. The epitope sequences are shown as follows: Figure 14B: VLDFAPPGA (SEQ ID NO: 259), Figure 14C: RMFPNAPYL (SEQ ID NO: 260), Figure 14F: VLDFAPPGA (SEQ ID NO: 259), Figure 14G: RMFPNAPYL (SEQ ID NO: 260), Figure 14H: YMFPNAPYL (SEQ ID NO: 264), Figure 14I: YMFPNAPYL (SEQ ID NO: 264), and Figure 14J (SEQ ID NO: 486). [Figure 14H]

[0049] Figures 14A-14I show the amino acid sequences of the polypeptide chains of the double-disulfide-bonded TMMP of the present disclosure. The sequences of the polypeptide chains in Figures 14A-14I are set forth in SEQ ID NOs: 422-430, respectively. The epitope sequences are shown as follows: Figure 14B: VLDFAPPGA (SEQ ID NO: 259), Figure 14C: RMFPNAPYL (SEQ ID NO: 260), Figure 14F: VLDFAPPGA (SEQ ID NO: 259), Figure 14G: RMFPNAPYL (SEQ ID NO: 260), Figure 14H: YMFPNAPYL (SEQ ID NO: 264), Figure 14I: YMFPNAPYL (SEQ ID NO: 264), and Figure 14J (SEQ ID NO: 486). [Figure 14I]

[0049] Figures 14A-14I show the amino acid sequences of the polypeptide chains of the double-disulfide-bonded TMMP of the present disclosure. The sequences of the polypeptide chains in Figures 14A-14I are set forth in SEQ ID NOs: 422-430, respectively. The epitope sequences are shown as follows: Figure 14B: VLDFAPPGA (SEQ ID NO: 259), Figure 14C: RMFPNAPYL (SEQ ID NO: 260), Figure 14F: VLDFAPPGA (SEQ ID NO: 259), Figure 14G: RMFPNAPYL (SEQ ID NO: 260), Figure 14H: YMFPNAPYL (SEQ ID NO: 264), Figure 14I: YMFPNAPYL (SEQ ID NO: 264), and Figure 14J (SEQ ID NO: 486). [Figure 14J]

[0049] Figures 14A-14I show the amino acid sequences of the polypeptide chains of the double-disulfide-bonded TMMP of the present disclosure. The sequences of the polypeptide chains in Figures 14A-14I are set forth in SEQ ID NOs: 422-430, respectively. The epitope sequences are shown as follows: Figure 14B: VLDFAPPGA (SEQ ID NO: 259), Figure 14C: RMFPNAPYL (SEQ ID NO: 260), Figure 14F: VLDFAPPGA (SEQ ID NO: 259), Figure 14G: RMFPNAPYL (SEQ ID NO: 260), Figure 14H: YMFPNAPYL (SEQ ID NO: 264), Figure 14I: YMFPNAPYL (SEQ ID NO: 264), and Figure 14J (SEQ ID NO: 486). [Figure 15] 1 shows expression and stability data for the WT1(37-45) epitope-containing TMMP of the present disclosure. [Figure 16] 1 shows expression and stability data for TMMPs containing the WT1 (126-134) epitope of the present disclosure. [Figure 17A] 1 shows a schematic diagram of a double disulfide bond TMMP of the present disclosure. [Figure 17B] 1 shows a schematic diagram of a double disulfide bond TMMP of the present disclosure. [Figure 17C] 1 shows a schematic diagram of a double disulfide bond TMMP of the present disclosure. [Figure 17D] 1 shows a schematic diagram of a double disulfide bond TMMP of the present disclosure. [Figure 18-1] 1 shows a schematic diagram of an example of a disulfide-bonded TMMP configuration of the present disclosure. [Figure 18-2] 1 shows a schematic diagram of an example of a disulfide-bonded TMMP configuration of the present disclosure. [Figure 19] 1 shows a schematic diagram of an example of the location of an immunomodulatory polypeptide in a TMMP of the present disclosure. [Figure 20A]

[0039] Figures 20A-20R show the amino acid sequences of exemplary polypeptide chains of TMMPs of the present disclosure. The sequences of exemplary polypeptide chains in Figures 20A-20R are set forth in SEQ ID NOs: 431-448, respectively. The epitope sequences are shown as follows: Figure 20H: CYTWNQMNL (SEQ ID NO: 262), Figure 20I: CYTWNQMNL (SEQ ID NO: 262), Figure 20J: CYTWNQMNL (SEQ ID NO: 262), Figure 20K: CYTWNQMNL (SEQ ID NO: 262), Figure 20L: CYTWNQMNL (SEQ ID NO: 262), Figure 20M: NYMNLGATL (SEQ ID NO: 263), Figure 20N: NYMNLGATL (SEQ ID NO: 263), Figure 20O: NYMNLGATL (SEQ ID NO: 263), Figure 20P: NYMNLGATL (SEQ ID NO: 263), Figure 20Q: NYMNLGATL (SEQ ID NO: 263), Figure 20R: NYMNLGATL (SEQ ID NO: 263). [Figure 20B]

[0039] Figures 20A-20R show the amino acid sequences of exemplary polypeptide chains of TMMPs of the present disclosure. The sequences of exemplary polypeptide chains in Figures 20A-20R are set forth in SEQ ID NOs: 431-448, respectively. The epitope sequences are shown as follows: Figure 20H: CYTWNQMNL (SEQ ID NO: 262), Figure 20I: CYTWNQMNL (SEQ ID NO: 262), Figure 20J: CYTWNQMNL (SEQ ID NO: 262), Figure 20K: CYTWNQMNL (SEQ ID NO: 262), Figure 20L: CYTWNQMNL (SEQ ID NO: 262), Figure 20M: NYMNLGATL (SEQ ID NO: 263), Figure 20N: NYMNLGATL (SEQ ID NO: 263), Figure 20O: NYMNLGATL (SEQ ID NO: 263), Figure 20P: NYMNLGATL (SEQ ID NO: 263), Figure 20Q: NYMNLGATL (SEQ ID NO: 263), Figure 20R: NYMNLGATL (SEQ ID NO: 263). [Figure 20C]

[0039] Figures 20A-20R show the amino acid sequences of exemplary polypeptide chains of TMMPs of the present disclosure. The sequences of exemplary polypeptide chains in Figures 20A-20R are set forth in SEQ ID NOs: 431-448, respectively. The epitope sequences are shown as follows: Figure 20H: CYTWNQMNL (SEQ ID NO: 262), Figure 20I: CYTWNQMNL (SEQ ID NO: 262), Figure 20J: CYTWNQMNL (SEQ ID NO: 262), Figure 20K: CYTWNQMNL (SEQ ID NO: 262), Figure 20L: CYTWNQMNL (SEQ ID NO: 262), Figure 20M: NYMNLGATL (SEQ ID NO: 263), Figure 20N: NYMNLGATL (SEQ ID NO: 263), Figure 20O: NYMNLGATL (SEQ ID NO: 263), Figure 20P: NYMNLGATL (SEQ ID NO: 263), Figure 20Q: NYMNLGATL (SEQ ID NO: 263), Figure 20R: NYMNLGATL (SEQ ID NO: 263). [Figure 20D]

[0039] Figures 20A-20R show the amino acid sequences of exemplary polypeptide chains of TMMPs of the present disclosure. The sequences of exemplary polypeptide chains in Figures 20A-20R are set forth in SEQ ID NOs: 431-448, respectively. The epitope sequences are shown as follows: Figure 20H: CYTWNQMNL (SEQ ID NO: 262), Figure 20I: CYTWNQMNL (SEQ ID NO: 262), Figure 20J: CYTWNQMNL (SEQ ID NO: 262), Figure 20K: CYTWNQMNL (SEQ ID NO: 262), Figure 20L: CYTWNQMNL (SEQ ID NO: 262), Figure 20M: NYMNLGATL (SEQ ID NO: 263), Figure 20N: NYMNLGATL (SEQ ID NO: 263), Figure 20O: NYMNLGATL (SEQ ID NO: 263), Figure 20P: NYMNLGATL (SEQ ID NO: 263), Figure 20Q: NYMNLGATL (SEQ ID NO: 263), Figure 20R: NYMNLGATL (SEQ ID NO: 263). [Figure 20E]

[0039] Figures 20A-20R show the amino acid sequences of exemplary polypeptide chains of TMMPs of the present disclosure. The sequences of exemplary polypeptide chains in Figures 20A-20R are set forth in SEQ ID NOs: 431-448, respectively. The epitope sequences are shown as follows: Figure 20H: CYTWNQMNL (SEQ ID NO: 262), Figure 20I: CYTWNQMNL (SEQ ID NO: 262), Figure 20J: CYTWNQMNL (SEQ ID NO: 262), Figure 20K: CYTWNQMNL (SEQ ID NO: 262), Figure 20L: CYTWNQMNL (SEQ ID NO: 262), Figure 20M: NYMNLGATL (SEQ ID NO: 263), Figure 20N: NYMNLGATL (SEQ ID NO: 263), Figure 20O: NYMNLGATL (SEQ ID NO: 263), Figure 20P: NYMNLGATL (SEQ ID NO: 263), Figure 20Q: NYMNLGATL (SEQ ID NO: 263), Figure 20R: NYMNLGATL (SEQ ID NO: 263). [Figure 20F]

[0039] Figures 20A-20R show the amino acid sequences of exemplary polypeptide chains of TMMPs of the present disclosure. The sequences of exemplary polypeptide chains in Figures 20A-20R are set forth in SEQ ID NOs: 431-448, respectively. The epitope sequences are shown as follows: Figure 20H: CYTWNQMNL (SEQ ID NO: 262), Figure 20I: CYTWNQMNL (SEQ ID NO: 262), Figure 20J: CYTWNQMNL (SEQ ID NO: 262), Figure 20K: CYTWNQMNL (SEQ ID NO: 262), Figure 20L: CYTWNQMNL (SEQ ID NO: 262), Figure 20M: NYMNLGATL (SEQ ID NO: 263), Figure 20N: NYMNLGATL (SEQ ID NO: 263), Figure 20O: NYMNLGATL (SEQ ID NO: 263), Figure 20P: NYMNLGATL (SEQ ID NO: 263), Figure 20Q: NYMNLGATL (SEQ ID NO: 263), Figure 20R: NYMNLGATL (SEQ ID NO: 263). [Figure 20G]

[0039] Figures 20A-20R show the amino acid sequences of exemplary polypeptide chains of TMMPs of the present disclosure. The sequences of exemplary polypeptide chains in Figures 20A-20R are set forth in SEQ ID NOs: 431-448, respectively. The epitope sequences are shown as follows: Figure 20H: CYTWNQMNL (SEQ ID NO: 262), Figure 20I: CYTWNQMNL (SEQ ID NO: 262), Figure 20J: CYTWNQMNL (SEQ ID NO: 262), Figure 20K: CYTWNQMNL (SEQ ID NO: 262), Figure 20L: CYTWNQMNL (SEQ ID NO: 262), Figure 20M: NYMNLGATL (SEQ ID NO: 263), Figure 20N: NYMNLGATL (SEQ ID NO: 263), Figure 20O: NYMNLGATL (SEQ ID NO: 263), Figure 20P: NYMNLGATL (SEQ ID NO: 263), Figure 20Q: NYMNLGATL (SEQ ID NO: 263), Figure 20R: NYMNLGATL (SEQ ID NO: 263). [Figure 20H]

[0039] Figures 20A-20R show the amino acid sequences of exemplary polypeptide chains of TMMPs of the present disclosure. The sequences of exemplary polypeptide chains in Figures 20A-20R are set forth in SEQ ID NOs: 431-448, respectively. The epitope sequences are shown as follows: Figure 20H: CYTWNQMNL (SEQ ID NO: 262), Figure 20I: CYTWNQMNL (SEQ ID NO: 262), Figure 20J: CYTWNQMNL (SEQ ID NO: 262), Figure 20K: CYTWNQMNL (SEQ ID NO: 262), Figure 20L: CYTWNQMNL (SEQ ID NO: 262), Figure 20M: NYMNLGATL (SEQ ID NO: 263), Figure 20N: NYMNLGATL (SEQ ID NO: 263), Figure 20O: NYMNLGATL (SEQ ID NO: 263), Figure 20P: NYMNLGATL (SEQ ID NO: 263), Figure 20Q: NYMNLGATL (SEQ ID NO: 263), Figure 20R: NYMNLGATL (SEQ ID NO: 263). [Figure 20I]

[0039] Figures 20A-20R show the amino acid sequences of exemplary polypeptide chains of TMMPs of the present disclosure. The sequences of exemplary polypeptide chains in Figures 20A-20R are set forth in SEQ ID NOs: 431-448, respectively. The epitope sequences are shown as follows: Figure 20H: CYTWNQMNL (SEQ ID NO: 262), Figure 20I: CYTWNQMNL (SEQ ID NO: 262), Figure 20J: CYTWNQMNL (SEQ ID NO: 262), Figure 20K: CYTWNQMNL (SEQ ID NO: 262), Figure 20L: CYTWNQMNL (SEQ ID NO: 262), Figure 20M: NYMNLGATL (SEQ ID NO: 263), Figure 20N: NYMNLGATL (SEQ ID NO: 263), Figure 20O: NYMNLGATL (SEQ ID NO: 263), Figure 20P: NYMNLGATL (SEQ ID NO: 263), Figure 20Q: NYMNLGATL (SEQ ID NO: 263), Figure 20R: NYMNLGATL (SEQ ID NO: 263). [Figure 20J]

[0039] Figures 20A-20R show the amino acid sequences of exemplary polypeptide chains of TMMPs of the present disclosure. The sequences of exemplary polypeptide chains in Figures 20A-20R are set forth in SEQ ID NOs: 431-448, respectively. The epitope sequences are shown as follows: Figure 20H: CYTWNQMNL (SEQ ID NO: 262), Figure 20I: CYTWNQMNL (SEQ ID NO: 262), Figure 20J: CYTWNQMNL (SEQ ID NO: 262), Figure 20K: CYTWNQMNL (SEQ ID NO: 262), Figure 20L: CYTWNQMNL (SEQ ID NO: 262), Figure 20M: NYMNLGATL (SEQ ID NO: 263), Figure 20N: NYMNLGATL (SEQ ID NO: 263), Figure 20O: NYMNLGATL (SEQ ID NO: 263), Figure 20P: NYMNLGATL (SEQ ID NO: 263), Figure 20Q: NYMNLGATL (SEQ ID NO: 263), Figure 20R: NYMNLGATL (SEQ ID NO: 263). [Figure 20K]

[0039] Figures 20A-20R show the amino acid sequences of exemplary polypeptide chains of TMMPs of the present disclosure. The sequences of exemplary polypeptide chains in Figures 20A-20R are set forth in SEQ ID NOs: 431-448, respectively. The epitope sequences are shown as follows: Figure 20H: CYTWNQMNL (SEQ ID NO: 262), Figure 20I: CYTWNQMNL (SEQ ID NO: 262), Figure 20J: CYTWNQMNL (SEQ ID NO: 262), Figure 20K: CYTWNQMNL (SEQ ID NO: 262), Figure 20L: CYTWNQMNL (SEQ ID NO: 262), Figure 20M: NYMNLGATL (SEQ ID NO: 263), Figure 20N: NYMNLGATL (SEQ ID NO: 263), Figure 20O: NYMNLGATL (SEQ ID NO: 263), Figure 20P: NYMNLGATL (SEQ ID NO: 263), Figure 20Q: NYMNLGATL (SEQ ID NO: 263), Figure 20R: NYMNLGATL (SEQ ID NO: 263). [Figure 20L]

[0039] Figures 20A-20R show the amino acid sequences of exemplary polypeptide chains of TMMPs of the present disclosure. The sequences of exemplary polypeptide chains in Figures 20A-20R are set forth in SEQ ID NOs: 431-448, respectively. The epitope sequences are shown as follows: Figure 20H: CYTWNQMNL (SEQ ID NO: 262), Figure 20I: CYTWNQMNL (SEQ ID NO: 262), Figure 20J: CYTWNQMNL (SEQ ID NO: 262), Figure 20K: CYTWNQMNL (SEQ ID NO: 262), Figure 20L: CYTWNQMNL (SEQ ID NO: 262), Figure 20M: NYMNLGATL (SEQ ID NO: 263), Figure 20N: NYMNLGATL (SEQ ID NO: 263), Figure 20O: NYMNLGATL (SEQ ID NO: 263), Figure 20P: NYMNLGATL (SEQ ID NO: 263), Figure 20Q: NYMNLGATL (SEQ ID NO: 263), Figure 20R: NYMNLGATL (SEQ ID NO: 263). [Figure 20M]

[0039] Figures 20A-20R show the amino acid sequences of exemplary polypeptide chains of TMMPs of the present disclosure. The sequences of exemplary polypeptide chains in Figures 20A-20R are set forth in SEQ ID NOs: 431-448, respectively. The epitope sequences are shown as follows: Figure 20H: CYTWNQMNL (SEQ ID NO: 262), Figure 20I: CYTWNQMNL (SEQ ID NO: 262), Figure 20J: CYTWNQMNL (SEQ ID NO: 262), Figure 20K: CYTWNQMNL (SEQ ID NO: 262), Figure 20L: CYTWNQMNL (SEQ ID NO: 262), Figure 20M: NYMNLGATL (SEQ ID NO: 263), Figure 20N: NYMNLGATL (SEQ ID NO: 263), Figure 20O: NYMNLGATL (SEQ ID NO: 263), Figure 20P: NYMNLGATL (SEQ ID NO: 263), Figure 20Q: NYMNLGATL (SEQ ID NO: 263), Figure 20R: NYMNLGATL (SEQ ID NO: 263). [Figure 20N]

[0039] Figures 20A-20R show the amino acid sequences of exemplary polypeptide chains of TMMPs of the present disclosure. The sequences of exemplary polypeptide chains in Figures 20A-20R are set forth in SEQ ID NOs: 431-448, respectively. The epitope sequences are shown as follows: Figure 20H: CYTWNQMNL (SEQ ID NO: 262), Figure 20I: CYTWNQMNL (SEQ ID NO: 262), Figure 20J: CYTWNQMNL (SEQ ID NO: 262), Figure 20K: CYTWNQMNL (SEQ ID NO: 262), Figure 20L: CYTWNQMNL (SEQ ID NO: 262), Figure 20M: NYMNLGATL (SEQ ID NO: 263), Figure 20N: NYMNLGATL (SEQ ID NO: 263), Figure 20O: NYMNLGATL (SEQ ID NO: 263), Figure 20P: NYMNLGATL (SEQ ID NO: 263), Figure 20Q: NYMNLGATL (SEQ ID NO: 263), Figure 20R: NYMNLGATL (SEQ ID NO: 263). [Figure 20O]

[0039] Figures 20A-20R show the amino acid sequences of exemplary polypeptide chains of TMMPs of the present disclosure. The sequences of exemplary polypeptide chains in Figures 20A-20R are set forth in SEQ ID NOs: 431-448, respectively. The epitope sequences are shown as follows: Figure 20H: CYTWNQMNL (SEQ ID NO: 262), Figure 20I: CYTWNQMNL (SEQ ID NO: 262), Figure 20J: CYTWNQMNL (SEQ ID NO: 262), Figure 20K: CYTWNQMNL (SEQ ID NO: 262), Figure 20L: CYTWNQMNL (SEQ ID NO: 262), Figure 20M: NYMNLGATL (SEQ ID NO: 263), Figure 20N: NYMNLGATL (SEQ ID NO: 263), Figure 20O: NYMNLGATL (SEQ ID NO: 263), Figure 20P: NYMNLGATL (SEQ ID NO: 263), Figure 20Q: NYMNLGATL (SEQ ID NO: 263), Figure 20R: NYMNLGATL (SEQ ID NO: 263). [Figure 20P]

[0039] Figures 20A-20R show the amino acid sequences of exemplary polypeptide chains of TMMPs of the present disclosure. The sequences of exemplary polypeptide chains in Figures 20A-20R are set forth in SEQ ID NOs: 431-448, respectively. The epitope sequences are shown as follows: Figure 20H: CYTWNQMNL (SEQ ID NO: 262), Figure 20I: CYTWNQMNL (SEQ ID NO: 262), Figure 20J: CYTWNQMNL (SEQ ID NO: 262), Figure 20K: CYTWNQMNL (SEQ ID NO: 262), Figure 20L: CYTWNQMNL (SEQ ID NO: 262), Figure 20M: NYMNLGATL (SEQ ID NO: 263), Figure 20N: NYMNLGATL (SEQ ID NO: 263), Figure 20O: NYMNLGATL (SEQ ID NO: 263), Figure 20P: NYMNLGATL (SEQ ID NO: 263), Figure 20Q: NYMNLGATL (SEQ ID NO: 263), Figure 20R: NYMNLGATL (SEQ ID NO: 263). [Figure 20Q]

[0039] Figures 20A-20R show the amino acid sequences of exemplary polypeptide chains of TMMPs of the present disclosure. The sequences of exemplary polypeptide chains in Figures 20A-20R are set forth in SEQ ID NOs: 431-448, respectively. The epitope sequences are shown as follows: Figure 20H: CYTWNQMNL (SEQ ID NO: 262), Figure 20I: CYTWNQMNL (SEQ ID NO: 262), Figure 20J: CYTWNQMNL (SEQ ID NO: 262), Figure 20K: CYTWNQMNL (SEQ ID NO: 262), Figure 20L: CYTWNQMNL (SEQ ID NO: 262), Figure 20M: NYMNLGATL (SEQ ID NO: 263), Figure 20N: NYMNLGATL (SEQ ID NO: 263), Figure 20O: NYMNLGATL (SEQ ID NO: 263), Figure 20P: NYMNLGATL (SEQ ID NO: 263), Figure 20Q: NYMNLGATL (SEQ ID NO: 263), Figure 20R: NYMNLGATL (SEQ ID NO: 263). [Figure 20R]

[0039] Figures 20A-20R show the amino acid sequences of exemplary polypeptide chains of TMMPs of the present disclosure. The sequences of exemplary polypeptide chains in Figures 20A-20R are set forth in SEQ ID NOs: 431-448, respectively. The epitope sequences are shown as follows: Figure 20H: CYTWNQMNL (SEQ ID NO: 262), Figure 20I: CYTWNQMNL (SEQ ID NO: 262), Figure 20J: CYTWNQMNL (SEQ ID NO: 262), Figure 20K: CYTWNQMNL (SEQ ID NO: 262), Figure 20L: CYTWNQMNL (SEQ ID NO: 262), Figure 20M: NYMNLGATL (SEQ ID NO: 263), Figure 20N: NYMNLGATL (SEQ ID NO: 263), Figure 20O: NYMNLGATL (SEQ ID NO: 263), Figure 20P: NYMNLGATL (SEQ ID NO: 263), Figure 20Q: NYMNLGATL (SEQ ID NO: 263), Figure 20R: NYMNLGATL (SEQ ID NO: 263). [Figure 21-1] 1 shows the effect of TMMP having a WT1 peptide epitope and an HLA-A*02 heavy chain on the proliferation of antigen-specific CD8+ T cells. [Figure 21-2] 1 shows the effect of TMMP having a WT1 peptide epitope and an HLA-A*02 heavy chain on the proliferation of antigen-specific CD8+ T cells. [Figure 22-1]1 shows the effect of TMMP bearing a WT1 peptide epitope on the proliferation of WT1-specific CD8+ T cells derived from total PBMCs over the course of a 10-day priming culture followed by an 8-day restimulation culture. [Figure 22-2] 1 shows the effect of TMMP bearing a WT1 peptide epitope on the proliferation of WT1-specific CD8+ T cells derived from total PBMCs over the course of a 10-day priming culture followed by an 8-day restimulation culture. [Figure 22-3] 1 shows the effect of TMMP bearing a WT1 peptide epitope on the proliferation of WT1-specific CD8+ T cells derived from total PBMCs over the course of a 10-day priming culture followed by an 8-day restimulation culture. [Figure 23] 1 shows the production of TNF-α and IFN-γ by WT1-specific CD8+ T cells grown in TMMP carrying WT1 37-45, which has either a G2C or R12C / G2C framework. [Figure 24] 1 shows the production of TNF-α and IFN-γ by WT1-specific CD8+ T cells grown in TMMP containing WT1 126-134 having the R12C / G2C framework. [Figure 25] 1 shows the effect of disulfide bonds on IL-2-driven immune cell activation. [Figure 26] The effect of variant IL-2 bearing TMMP as an immunomodulatory polypeptide on CGLL-2 proliferation is shown in comparison with Proleukin. [Figure 27-1] Binding of "1715+2380" TMPP to various Fc receptors is shown. [Figure 27-2] Binding of "1715+2380" TMPP to various Fc receptors is shown. [Figure 27-3] Binding of "1715+2380" TMPP to various Fc receptors is shown. [Figure 27-4] Binding of "1715+2380" TMPP to various Fc receptors is shown. [Figure 28]1 shows the effect of TMMP containing the WT1 peptide epitope 235-243 (M236Y) and the HLA-A*24 heavy chain on the proliferation of antigen-specific CD8+ T cells. [Figure 29-1] 1 shows the effect of TMMP containing the WT1 peptide epitope 239-247 (Q240Y) and the HLA-A*24 heavy chain on antigen-specific CD8+ T cell proliferation. [Figure 29-2] 1 shows the effect of TMMP containing the WT1 peptide epitope 239-247 (Q240Y) and the HLA-A*24 heavy chain on antigen-specific CD8+ T cell proliferation. [Figure 30] 1 shows induction of CD69 expression by IL-2 polypeptides modified at positions 1 or 3, the peptide epitope WT1 235-243 (M236Y), the HLA-A24 heavy chain, and TMMP having a G2C or R12C / G2C disulfide framework. [Figure 31-1] 1 shows the effect of TMMP (an IL-2 polypeptide modified at positions 1 or 3, having the peptide epitope WT1 239-247 (Q240Y), an HLA-A24 heavy chain, and a G2C or R12C / G2C disulfide framework) on CD69 expression compared to Proleukin and recombinant human IL-2 (rhIL-2). [Figure 31-2] 1 shows the effect of TMMP (an IL-2 polypeptide modified at positions 1 or 3, having the peptide epitope WT1 239-247 (Q240Y), an HLA-A24 heavy chain, and a G2C or R12C / G2C disulfide framework) on CD69 expression compared to Proleukin and recombinant human IL-2 (rhIL-2). [Figure 32] 1 shows the effect of TMMP (an IL-2 polypeptide modified at positions 1 or 3, having the peptide epitope WT1 235-243 (M236Y), an HLA-A24 heavy chain, and a G2C or R12C / G2C disulfide framework) on CTLL-2 proliferation compared to Proleukin. [Figure 33]1 shows the effect of TMMP (an IL-2 polypeptide modified at positions 1 or 3, having the peptide epitope WT1 239-247 (Q240Y), an HLA-A24 heavy chain, and a G2C or R12C / G2C disulfide framework) on CTLL-2 proliferation compared to Proleukin. [Figure 34-1] Binding of "3425+3529" TMPP to various Fc receptors is shown. [Figure 34-2] Binding of "3425+3529" TMPP to various Fc receptors is shown. [Figure 34-3] Binding of "3425+3529" TMPP to various Fc receptors is shown. [Figure 34-4] Binding of "3425+3529" TMPP to various Fc receptors is shown. [Figure 35A] 1 shows the amino acid sequence of an exemplary polypeptide chain of a TMMP of the present disclosure, which comprises the WT-1 peptide SMTWNQMNL (SEQ ID NO: 451). [Figure 35B] 1 shows the amino acid sequence of an exemplary polypeptide chain of a TMMP of the present disclosure, which comprises the WT-1 peptide SMTWNQMNL (SEQ ID NO: 451). [Figure 35C] 1 shows the amino acid sequence of an exemplary polypeptide chain of a TMMP of the present disclosure, which comprises the WT-1 peptide SMTWNQMNL (SEQ ID NO: 451). [Figure 35D] 1 shows the amino acid sequence of an exemplary polypeptide chain of a TMMP of the present disclosure, which comprises the WT-1 peptide SMTWNQMNL (SEQ ID NO: 451). [Figure 35E] 1 shows the amino acid sequence of an exemplary polypeptide chain of a TMMP of the present disclosure, which comprises the WT-1 peptide SMTWNQMNL (SEQ ID NO: 451). [Figure 35F] 1 shows the amino acid sequence of an exemplary polypeptide chain of a TMMP of the present disclosure, which comprises the WT-1 peptide SMTWNQMNL (SEQ ID NO: 451). [Figure 36A]1 shows the amino acid sequence of an exemplary polypeptide chain of a TMMP of the present disclosure, which comprises the WT-1 peptide GCMTWNQMNL (SEQ ID NO: 452). [Figure 36B] 1 shows the amino acid sequence of an exemplary polypeptide chain of a TMMP of the present disclosure, which comprises the WT-1 peptide GCMTWNQMNL (SEQ ID NO: 452). [Figure 36C] 1 shows the amino acid sequence of an exemplary polypeptide chain of a TMMP of the present disclosure, which comprises the WT-1 peptide GCMTWNQMNL (SEQ ID NO: 452). [Figure 36D] 1 shows the amino acid sequence of an exemplary polypeptide chain of a TMMP of the present disclosure, which comprises the WT-1 peptide GCMTWNQMNL (SEQ ID NO: 452). [Figure 36E] 1 shows the amino acid sequence of an exemplary polypeptide chain of a TMMP of the present disclosure, which comprises the WT-1 peptide GCMTWNQMNL (SEQ ID NO: 452). [Figure 36F] 1 shows the amino acid sequence of an exemplary polypeptide chain of a TMMP of the present disclosure, which comprises the WT-1 peptide GCMTWNQMNL (SEQ ID NO: 452). [Figure 37A] 1 shows the amino acid sequence of an exemplary polypeptide chain of a TMMP of the present disclosure, which comprises the WT-1 peptide SYTWNQMNL (SEQ ID NO: 453). [Figure 37B] 1 shows the amino acid sequence of an exemplary polypeptide chain of a TMMP of the present disclosure, which comprises the WT-1 peptide SYTWNQMNL (SEQ ID NO: 453). [Figure 37C] 1 shows the amino acid sequence of an exemplary polypeptide chain of a TMMP of the present disclosure, which comprises the WT-1 peptide SYTWNQMNL (SEQ ID NO: 453). [Figure 37D] 1 shows the amino acid sequence of an exemplary polypeptide chain of a TMMP of the present disclosure, which comprises the WT-1 peptide SYTWNQMNL (SEQ ID NO: 453). [Figure 37E]1 shows the amino acid sequence of an exemplary polypeptide chain of a TMMP of the present disclosure, which comprises the WT-1 peptide SYTWNQMNL (SEQ ID NO: 453). [Figure 37F] 1 shows the amino acid sequence of an exemplary polypeptide chain of a TMMP of the present disclosure, which comprises the WT-1 peptide SYTWNQMNL (SEQ ID NO: 453). [Figure 38A] 1 shows the amino acid sequence of an exemplary polypeptide chain of a TMMP of the present disclosure, which polypeptide chain includes the WT-1 peptide GCYTWNQMNL (SEQ ID NO: 454). [Figure 38B] 1 shows the amino acid sequence of an exemplary polypeptide chain of a TMMP of the present disclosure, which polypeptide chain includes the WT-1 peptide GCYTWNQMNL (SEQ ID NO: 454). [Figure 38C] 1 shows the amino acid sequence of an exemplary polypeptide chain of a TMMP of the present disclosure, which polypeptide chain includes the WT-1 peptide GCYTWNQMNL (SEQ ID NO: 454). [Figure 38D] 1 shows the amino acid sequence of an exemplary polypeptide chain of a TMMP of the present disclosure, which polypeptide chain includes the WT-1 peptide GCYTWNQMNL (SEQ ID NO: 454). [Figure 38E] 1 shows the amino acid sequence of an exemplary polypeptide chain of a TMMP of the present disclosure, which polypeptide chain includes the WT-1 peptide GCYTWNQMNL (SEQ ID NO: 454). [Figure 38F] 1 shows the amino acid sequence of an exemplary polypeptide chain of a TMMP of the present disclosure, which polypeptide chain includes the WT-1 peptide GCYTWNQMNL (SEQ ID NO: 454). [Figure 39-1] This shows the proliferation of WT137-45-specific CD8+ T cells derived from unstimulated PBMCs, which was induced by the TMMP of the present disclosure ("CUE-102 / A02 WT137-45 IST"). [Figure 39-2]This shows the proliferation of WT137-45-specific CD8+ T cells derived from unstimulated PBMCs, which was induced by the TMMP of the present disclosure ("CUE-102 / A02 WT137-45 IST"). [Figure 40A-1] This shows the proliferation of WT137-45-specific CD8+ T cells derived from PBMCs stimulated with WT137-45 peptide, and this proliferation was induced by the TMMP of the present disclosure ("CUE-102 / A02 WT137-45 IST"). [Figure 40A-2] This shows the proliferation of WT137-45-specific CD8+ T cells derived from PBMCs stimulated with WT137-45 peptide, and this proliferation was induced by the TMMP of the present disclosure ("CUE-102 / A02 WT137-45 IST"). [Figure 40B] This shows the proliferation of WT137-45-specific CD8+ T cells derived from PBMCs stimulated with WT137-45 peptide, and this proliferation was induced by the TMMP of the present disclosure ("CUE-102 / A02 WT137-45 IST"). [Figure 41A] 1 shows the production of TNF-α, IL-6, and IFN-γ, and upregulation of CD69 induced by a TMMP of the disclosure ("CUE-102 / A02 WT137-45 IST") or wild-type IL-2. [Figure 41B] 1 shows the production of TNF-α, IL-6, and IFN-γ, and upregulation of CD69 induced by a TMMP of the disclosure ("CUE-102 / A02 WT137-45 IST") or wild-type IL-2. [Figure 42A-1] 1 shows the CTL activity of WT137-45 peptide-specific CD8+ T cells expanded from peptide-stimulated PBMCs in the presence of a TMMP of the present disclosure ("CUE-102 / A02 WT137-45 IST") against peptide-presenting target cells. [Figure 42A-2] 1 shows the CTL activity of WT137-45 peptide-specific CD8+ T cells expanded from peptide-stimulated PBMCs in the presence of a TMMP of the present disclosure ("CUE-102 / A02 WT137-45 IST") against peptide-presenting target cells. [Figure 42B] 1 shows the CTL activity of WT137-45 peptide-specific CD8+ T cells expanded from peptide-stimulated PBMCs in the presence of a TMMP of the present disclosure ("CUE-102 / A02 WT137-45 IST") against peptide-presenting target cells. [Figure 43] 1 shows the effect of TMMP containing the WT1 peptide epitope WT1 235-243 (C235S;M236Y) and the HLA-A*24 heavy chain on antigen-specific CD8+ T cell proliferation. [Figure 44] 1 shows the effect of TMMP containing the WT1 peptide epitope WT1 239-247 (Q240Y) and the HLA-A*24 heavy chain on antigen-specific CD8+ T cell proliferation. [Figure 45] 1 shows the effect of TMMP containing the WT1 peptide epitope 37-45 on antigen-specific CD8+ T cell proliferation in naive HLA-A2 (AAD) transgenic mice. [Figure 46A] The amino acid sequence of the HLA-E heavy chain is shown. [Figure 46B] The amino acid sequence of the HLA-E heavy chain is shown. [Figure 46C] The amino acid sequence of the HLA-E heavy chain is shown. [Figure 46D] The amino acid sequence of the HLA-E heavy chain is shown. [Figure 47A] The amino acid sequence of the HLA-G heavy chain is shown. [Figure 47B] The amino acid sequence of the HLA-G heavy chain is shown. [Figure 47C] The amino acid sequence of the HLA-G heavy chain is shown. [Figure 47D] The amino acid sequence of the HLA-G heavy chain is shown. DETAILED DESCRIPTION OF THE INVENTION

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

[0007] The terms "peptide," "polypeptide," and "protein" are used interchangeably herein to refer to polymeric forms of amino acids of any length, and may include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides with modified peptide backbones. Furthermore, as used herein, "polypeptide" refers to proteins containing modifications, such as deletions, additions, and substitutions (generally conservative in nature, as known to those of skill in the art) relative to the native sequence, so long as the protein maintains a desired activity. These modifications may be intentional, such as through site-directed mutagenesis, or accidental, such as through mutations of the host producing the protein or errors in polymerase chain reaction (PCR) amplification or other recombinant DNA methods. Reference herein to a particular residue or residue number in a known polypeptide is understood to refer to the amino acid at that position in the wild-type polypeptide. Since the sequence of a wild-type polypeptide is altered by either the addition or deletion of one or more amino acids, those skilled in the art will understand that a reference to a particular residue or residue number is correspondingly altered to refer to the same specific amino acid (understood as the residue at the altered position number) in the altered polypeptide. For example, if an MHC class I polypeptide is modified by adding one amino acid to the N-terminus, a reference to position 84 or to a specific residue at position 84 is understood to refer to the amino acid at position 85 of the modified polypeptide. Similarly, a reference herein to a specific amino acid substitution at a specific position, e.g., Y84, is understood to refer to the substitution of that amino acid for the amino acid at position 84 of the wild-type polypeptide. Thus, a Y84C substitution is understood to be the substitution of a Cys residue for the Tyr residue present in the wild-type sequence. For example, if a wild-type polypeptide is modified such that the amino acid at position 84 is changed from its wild-type amino acid to an alternative amino acid, the substitution of the amino acid at position 84 is understood to refer to the substitution of the alternative amino acid.In such cases, when a polypeptide is similarly modified by the addition or deletion of one or more amino acids, reference to a substitution is understood to refer to the substitution of an alternative amino acid at the changed position number. Reference to a "non-naturally occurring Cys residue" in a polypeptide, e.g., an MHC class I polypeptide, means that the polypeptide contains a Cys residue at a position where no Cys is present in the corresponding wild-type polypeptide. This can be achieved by conventional protein modification, substituting an amino acid present in the wild-type sequence with cysteine.

[0008] A polynucleotide or polypeptide has a certain percent "sequence identity" to another polynucleotide or polypeptide, meaning that the percentage of bases or amino acids are the same and in the same relative positions when the two sequences are aligned and compared. Sequence identity can be measured using a number of different methods. To measure sequence identity, sequences may be aligned using a variety of convenient methods and computer programs (e.g., BLAST, T-COFFEE, MUSCLE, MAFFT, etc.) available on the world wide web at sites including ncbi.nlm.nili.gov / BLAST, ebi.ac.uk / Tools / msa / tcoffee / , ebi.ac.uk / Tools / msa / muscle / , and mafft.cbrc.jp / alignment / software / . See, e.g., Altschul et al. (1990), J. Mol. Biol. 215:403-10. Unless otherwise specified, sequence identity is determined using the BLAST computer program.

[0009] The term "conservative amino acid substitution" refers to the interchangeability of amino acid residues in proteins that have similar side chains. For example, the group of amino acids with aliphatic side chains consists of glycine, alanine, valine, leucine, and isoleucine; the group of amino acids with aliphatic hydroxyl side chains consists of serine and threonine; the group of amino acids with amide-containing side chains consists of asparagine and glutamine; the group of amino acids with aromatic side chains consists of phenylalanine, tyrosine, and tryptophan; the group of amino acids with basic side chains consists of lysine, arginine, and histidine; the group of amino acids with acidic side chains consists of glutamic acid and aspartic acid; and the group of amino acids with sulfur-containing side chains consists of cysteine ​​and methionine. Exemplary conservative amino acid substitution groups are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine-glycine, and asparagine-glutamine.

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

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

[0012] As used herein, the term "immunomodulating polypeptide" (also referred to as "costimulatory polypeptide") includes polypeptides on antigen-presenting cells (APCs) (e.g., dendritic cells, B cells, etc.) that specifically bind to a cognate co-immunomodulating polypeptide on a T cell, thereby providing signals that mediate T cell responses, including, but not limited to, proliferation, activation, differentiation, etc., in addition to the primary signal provided, for example, by binding of the TCR / CD3 complex to a peptide-binding major histocompatibility complex (MHC) polypeptide. Immunomodulatory polypeptides include, but are not limited to, CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L, Fas ligand (FasL), inducible costimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM), CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, HVEM, lymphotoxin beta receptor, 3 / TR6, ILT3, ILT4, HVEM, agonists or antibodies that bind to Toll ligand receptors, and ligands that specifically bind to B7-H3.

[0013] As described above, an "immunomodulating polypeptide" (also referred to herein as a "MOD") specifically binds to a cognate co-immunomodulating polypeptide on a T cell.

[0014] The "immunomodulatory domain" ("MOD") of the TMMP of the present disclosure binds to a cognate co-immunomodulatory polypeptide that may be present on a target T cell.

[0015] As used herein, the term "in vivo" refers to any process or procedure that takes place inside the body.

[0016] As used herein, the term "in vitro" refers to any process or procedure that occurs outside the body.

[0017] As used herein, "heterologous" means a nucleotide or polypeptide that is not found in naturally occurring nucleic acids or proteins, respectively.

[0018] As used herein, "recombinant" means that a particular nucleic acid (DNA or RNA) is the product of various combinations of cloning, restriction, polymerase chain reaction (PCR), and / or ligation steps that result in a construct having structural coding or non-coding sequences that are distinguishable from endogenous nucleic acids present in natural systems. A DNA sequence encoding a polypeptide may be assembled from cDNA fragments or a series of synthetic oligonucleotides to obtain a synthetic nucleic acid capable of expression by a recombinant transcription unit contained within a cellular or cell-free transcription and translation system.

[0019] The terms "recombinant expression vector" or "DNA construct" are used interchangeably herein to refer to a DNA molecule comprising a vector and at least one insert. Recombinant expression vectors are typically constructed for the purpose of expressing and / or propagating an insert(s) or for constructing other recombinant nucleotide sequences. The insert(s) may or may not be operably linked to a promoter sequence and may or may not be operably linked to a DNA regulatory sequence.

[0020] As used herein, the term "affinity" refers to the equilibrium constant for the reversible binding of two substances (e.g., an antibody and an antigen), and also refers to the dissociation constant (K D ) As used herein, the term "avidity" refers to the resistance of a complex of two or more substances to dissociation after dilution. The terms "immunoreactive" and "preferentially bind" are used interchangeably herein with respect to antibodies and / or antigen-binding fragments.

[0021] As used herein (e.g., with respect to binding of TMMP to a polypeptide (e.g., a T cell receptor) on a T cell), the term "binding" refers to a non-covalent interaction between two molecules. Non-covalent binding refers to a direct association between two molecules, for example, through electrostatic, hydrophobic, ionic, and / or hydrogen-bonding interactions, including interactions such as salt bridges and hydrogen bridges. "Affinity" refers to the strength of the non-covalent bond; a high binding affinity is associated with a low K D "Specific binding" usually refers to the binding of a ligand to a moiety that is its designated binding site or receptor. "Non-specific binding" usually refers to the binding of a ligand to a moiety other than its designated binding site or receptor. As used herein, "covalent binding" or "covalent bond" refers to the formation of one or more covalent chemical bonds between two different molecules.

[0022] As used herein, the terms "treatment," "treating," and the like generally refer to obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic, in terms of completely or partially preventing a disease or its symptoms, and / or therapeutic, in terms of partially or completely curing a disease and / or side effects resulting from the disease. As used herein, "treatment" encompasses any treatment of a disease or condition in a mammal, including (a) preventing the onset of the disease or condition in a subject susceptible to, but not yet diagnosed with, the disease or condition; (b) suppressing the disease or condition, i.e., arresting its development; and / or (c) alleviating the disease, i.e., causing regression of the disease. Therapeutic agents may be administered before, during, or after the onset of a disease or disorder. Treatment of an ongoing disease (wherein treatment stabilizes or suppresses undesirable clinical symptoms in a patient) is particularly advantageous. Such treatment is desirably performed before complete loss of function of the affected tissue. The therapeutic agent is desirably administered during, and optionally after, the symptomatic stage of the disease.

[0023] The terms "individual," "subject," "host," and "patient" are used interchangeably herein to refer to any mammalian subject for whom diagnosis, treatment, or therapy is desired. Mammals include, for example, humans, non-human primates, rodents (e.g., rats, mice), lagomorphs (e.g., rabbits), and ungulates (e.g., cows, sheep, pigs, horses, goats, etc.).

[0024] Unless otherwise specified, the term "substantially" is intended to encompass both "completely" and "largely, but not completely." For example, an Ig Fc that "does not substantially induce cell lysis" means an Ig Fc that does not induce cell lysis at all or induces little cell lysis.

[0025] As used herein, the term "about" when used in reference to a quantity indicates that the quantity may vary by 10% of the stated amount. For example, "about 100" means an amount of 90 to 110. When "about" is used in reference to a range, "about" when used in reference to the lower limit of the range means that the lower limit includes an amount 10% lower than the lower limit of the range, and "about" when used in reference to the upper limit of the range means that the upper limit includes an amount 10% higher than the upper limit of the range. For example, about 100 to about 1000 means that the range spans 90 to 1100.

[0026] Before the present disclosure is further described, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.

[0027] When a range of numerical values ​​is provided, it is understood that each intervening value (to one-tenth of the unit of the lower limit, unless the context clearly dictates otherwise) between the upper and lower limits of that range, and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these narrower ranges may independently be included in the narrower range and are also encompassed within the disclosure, subject to any specifically excluded value in the stated range. When an stated range includes one or both of those upper and lower limits, ranges excluding either or both of those included limits are also encompassed within the disclosure.

[0028] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, the preferred methods and materials are described below. All publications mentioned herein are incorporated by reference to disclose and describe the methods and / or materials in connection with the content in which the publications are cited.

[0029] It should be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, a reference to "T cell modulatory multimeric polypeptides" includes a plurality of such polypeptides; a reference to "immunomodulating polypeptides" includes a reference to one or more immunomodulating polypeptides and equivalents thereof known to those skilled in the art; and so forth. It should be further noted that the claims may be drafted to exclude any element. Accordingly, this statement intends that the use of exclusive terminology, such as "solely," "only," or the use of a "negative" limitation in connection with the recitation of claim elements, serves as antecedent.

[0030] It is to be understood that certain features of the present disclosure that are, for clarity, described in the context of individual embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the present invention that are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination. All combinations of embodiments belonging to the present invention are expressly embraced by the present invention and are disclosed herein as if all combinations were individually and specifically disclosed. In addition, all subcombinations of the various embodiments and elements thereof are also expressly embraced by the present disclosure and are disclosed herein as if all such subcombinations were individually and specifically disclosed herein.

[0031] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein should be construed as an admission that the present disclosure is not entitled to antedate such publication. Further, the dates of publication provided may be different from the actual publication dates, which may need to be independently confirmed.

[0032] Detailed Description The present disclosure provides T cell modulatory multimeric polypeptides comprising immunomodulatory polypeptides ("MODs") and including epitope-presenting Wilms' tumor-1 (WT-1) peptides. TMMPs are useful for modulating T cell activation and for modulating immune responses in individuals.

[0033] T cell regulatory multimeric polypeptides The present disclosure provides T cell regulatory multimeric polypeptides (TMMPs) comprising a) a first polypeptide and b) a second polypeptide, wherein the TMMP comprises an epitope, a first major histocompatibility complex (MHC) polypeptide, a second MHC polypeptide, one or more MODs, and, optionally, an immunoglobulin (Ig) Fc polypeptide or a non-Ig scaffold. The present disclosure also provides TMMPs, wherein the TMMP is a heterodimer comprising a) a first polypeptide comprising the first MHC polypeptide and b) a second polypeptide comprising the second MHC polypeptide, wherein the first polypeptide or the second polypeptide comprises an epitope (e.g., a peptide presenting the epitope), and the first polypeptide and / or the second polypeptide comprise one or more MODs, which may be the same or different, and, optionally, an Ig Fc polypeptide or a non-Ig scaffold. The TMMPs of the present disclosure are also referred to herein as "multimeric polypeptides of the present disclosure" or "synTac." In some examples, the peptide epitope present in a TMMP of the present disclosure is a WT-1 peptide.

[0034] The present disclosure provides a TMMP comprising a heterodimeric polypeptide comprising: a) a first polypeptide comprising i) a peptide epitope and ii) a first MHC polypeptide; b) a second polypeptide comprising a second MHC polypeptide; and c) at least one MOD, wherein the first and / or second polypeptide comprises at least one (i.e., one or more) MOD. Optionally, the first or second polypeptide comprises an Ig Fc polypeptide or a non-Ig scaffold. At least one of the one or more MODs is a variant MOD that exhibits reduced affinity for a cognate co-immunomodulatory polypeptide ("co-MOD") compared to the affinity of the corresponding wild-type MOD for the cognate co-MOD. The epitope present in the TMMP binds to a T cell receptor (TCR) on a T cell with an affinity of at least 100 μM (e.g., at least 10 μM, at least 1 μM, at least 100 nM, at least 10 nM, or at least 1 nM). The TMMP binds to a first T cell with an affinity that is at least 25% higher than the affinity with which the TMMP binds to a second T cell, the first T cell expresses on its surface a cognate co-MOD and a TCR that binds to the epitope with an affinity of at least 100 μM, and the second T cell expresses a cognate co-MOD on its surface but does not express on its surface a TCR that binds to the epitope with an affinity of at least 100 μM (e.g., at least 10 μM, at least 1 μM, at least 100 nM, at least 10 nM, or at least 1 nM). In some examples, the peptide epitope present in the TMMP is a WT-1 peptide.

[0035] The present disclosure provides a TMMP, the TMMP comprising: A) a heterodimer comprising: a) a first polypeptide comprising a first MHC polypeptide; and b) a second polypeptide comprising a second MHC polypeptide, wherein the first polypeptide or the second polypeptide comprises an epitope (e.g., a peptide presenting the epitope); the first polypeptide and / or the second polypeptide comprises one or more MODs, which may be the same or different, and at least one of the one or more MODs may be a wild-type MOD or a variant of a wild-type MOD, wherein the variant MOD comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid substitutions compared to the amino acid sequence of the corresponding wild-type MOD; and the first polypeptide or the second polypeptide optionally comprises an Ig Fc polypeptide or a non-Ig scaffold; B) A heterodimer comprising: a) a first polypeptide comprising a first MHC polypeptide; and b) a second polypeptide comprising a second MHC polypeptide, wherein the first polypeptide or the second polypeptide comprises an epitope; and the first polypeptide and / or the second polypeptide comprise one or more MODs, which may be the same or different, wherein at least one of the one or more MODs is a variant of a wild-type MOD, wherein the variant MOD comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid substitutions compared to the amino acid sequence of the corresponding wild-type MOD; and at least one of the one or more MODs is a variant MOD that exhibits reduced affinity for a cognate co-MOD compared to the affinity of the corresponding wild-type MOD for the cognate co-MOD; and the epitope is at least 10 times more selectively targeted to a TCR on a T cell. -7 M, whereby i) the TMMP polypeptide binds to a first T cell with an affinity that is at least 25% higher than the affinity with which TMMP binds to a second T cell, and the first T cell has on its surface a cognate co-MOD and an epitope of at least 10 -7The second T cell expresses a TCR that binds with an affinity of M, and the second T cell expresses on its surface a cognate co-MOD but with at least 10 affinity to the epitope. -7 and / or ii) the ratio of the binding affinity of a control TMMP (control containing wild-type MOD) to the cognate co-MOD to the binding affinity of a TMMP containing a variant of wild-type MOD to the cognate co-MOD is 1.5:1–10, as measured by biolayer interferometry. 6 :1, wherein the variant MOD comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid substitutions compared to the amino acid sequence of the corresponding wild-type MOD, and the first polypeptide or the second polypeptide optionally comprises an Ig Fc polypeptide or a non-Ig scaffold), or C) A heterodimer comprising: a) a first polypeptide, the first polypeptide comprising, in order from N-terminus to C-terminus: i) an epitope; and ii) a first MHC polypeptide; and b) a second polypeptide, the first polypeptide comprising, in order from N-terminus to C-terminus: i) a second MHC polypeptide; and ii) optionally, a second polypeptide comprising an Ig Fc polypeptide or a non-Ig scaffold; wherein the TMMP comprises one or more MODs, which may be the same or different, at least one of the one or more MODs being A) at the C-terminus of the first polypeptide, B) at the N-terminus of the second polypeptide, C) at the C-terminus of the second polypeptide, or D) at the C-terminus of the first polypeptide and the N-terminus of the second polypeptide, wherein at least one of the one or more MODs is a wild-type MOD or may be a variant of a wild-type MOD, wherein the variant MOD comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid substitutions compared to the amino acid sequence of the corresponding wild-type MOD, and optionally at least one of the one or more MODs is a variant MOD that exhibits reduced affinity for a cognate co-MOD compared to the affinity of the corresponding wild-type MOD for the cognate co-MOD, and the epitope is at least 10 -7 M, whereby i) TMMP binds to a first T cell with an affinity that is at least 25% higher than the affinity with which TMMP binds to a second T cell, and the first T cell has on its surface a cognate co-MOD and an epitope of at least 10 -7 The second T cell expresses a TCR that binds with an affinity of M, and the second T cell expresses on its surface a cognate co-MOD but with at least 10 affinity to the epitope. -7 and / or ii) the ratio of the binding affinity of a control TMMP (control containing wild-type MOD) to the cognate co-MOD to the binding affinity of a TMMP containing a variant of wild-type MOD to the cognate co-MOD is 1.5:1–10, as measured by biolayer interferometry. 6:1, and the variant MOD comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid substitutions compared to the amino acid sequence of the corresponding wild-type MOD. In some examples, the peptide epitope present in the TMMP is a WT-1 peptide.

[0036] The present disclosure provides a TMMP comprising: a) a first polypeptide comprising, in N-terminal to C-terminal order: i) an epitope and ii) a first MHC polypeptide; and b) a second polypeptide comprising, in N-terminal to C-terminal order: i) a second MHC polypeptide and ii) optionally, an Ig Fc polypeptide or a non-Ig scaffold. The TMMP comprises one or more MODs, at least one of which is A) at the C-terminus of the first polypeptide, B) at the N-terminus of the second polypeptide, C) at the C-terminus of the second polypeptide, or D) at the C-terminus of the first polypeptide and the N-terminus of the second polypeptide. At least one of the one or more MODs is a variant MOD that exhibits reduced affinity for a cognate co-MOD compared to the affinity of a corresponding wild-type MOD for the cognate co-MOD. The epitope present in the TMMP binds to a T cell receptor (TCR) on a T cell with an affinity of at least 100 μM (e.g., at least 10 μM, at least 1 μM, at least 100 nM, at least 10 nM, or at least 1 nM). The TMMP binds to a first T cell with an affinity that is at least 25% higher than the affinity with which the TMMP binds to a second T cell, the first T cell expresses on its surface a cognate co-MOD and a TCR that binds to the epitope with an affinity of at least 100 μM, and the second T cell expresses a cognate co-MOD on its surface but does not express on its surface a TCR that binds to the epitope with an affinity of at least 100 μM (e.g., at least 10 μM, at least 1 μM, at least 100 nM, at least 10 nM, or at least 1 nM).

[0037] A MOD present in a TMMP binds to its cognate co-MOD with an affinity that is at least 10% lower, at least 15% lower, at least 20% lower, at least 25% lower, at least 30% lower, at least 35% lower, at least 40% lower, at least 45% lower, at least 50% lower, at least 55% lower, at least 60% lower, at least 65% lower, at least 70% lower, at least 75% lower, at least 80% lower, at least 85% lower, at least 90% lower, at least 95% lower, or greater than 95% lower than the affinity of the corresponding wild-type MOD for the cognate co-MOD.

[0038] The combination of the low affinity of a MOD for its cognate co-MOD and the affinity of the epitope for the TCR results in improved selectivity of the TMMP. For example, a TMMP of the present disclosure selectively binds to a first T cell that presents both i) a TCR specific for an epitope present in the TMMP and ii) a co-MOD that binds to the MOD present in the TMMP, compared to binding to a second T cell that presents i) a TCR specific for an epitope other than the epitope present in the TMMP and ii) a co-MOD that binds to the MOD present in the TMMP. For example, a TMMP of the disclosure binds to a first T cell with an affinity that is at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 2-fold, at least 2.5-fold, at least 5-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 50-fold, at least 100-fold, or greater than 100-fold greater than the affinity with which a TMMP of the disclosure binds to a second T cell.

[0039] In some examples, when administered to an individual in need thereof, the TMMP induces both epitope-specific and non-epitope-specific T cell responses. In other words, in some examples, when administered to an individual in need thereof, the TMMP induces an epitope-specific T cell response by modulating the activation of a first T cell that presents both i) a TCR specific for an epitope present in the TMMP and ii) a co-MOD that binds to a MOD present in the TMMP, and induces an epitope-non-specific T cell response by modulating the activation of a second T cell that presents i) a TCR specific for an epitope other than the epitope present in the TMMP and ii) a co-MOD that binds to a MOD present in the TMMP. The ratio of epitope-specific to epitope-non-specific T cell responses is at least 2:1, at least 5:1, at least 10:1, at least 15:1, at least 20:1, at least 25:1, at least 50:1, or at least 100:1. The ratio of epitope-specific T cell responses to epitope-nonspecific T cell responses is about 2:1 to about 5:1, about 5:1 to about 10:1, about 10:1 to about 15:1, about 15:1 to about 20:1, about 20:1 to about 25:1, about 25:1 to about 50:1, or about 50:1 to about 100:1, or more than 100:1. Examples of "regulating activation" of T cells include: i) cytotoxicity (e.g., CD8 + 1) activating T cells; 2) cytotoxic (e.g., CD8 + ) inducing T cell cytotoxic activity; iii) cytotoxicity (e.g., CD8 + ) inducing the production and release of cytotoxins (e.g., perforin, granzymes, granulysin) by T cells; and iv) suppressing the activation of autoreactive T cells.

[0040] The combination of the low affinity of a MOD for its cognate co-MOD and the affinity of the epitope for the TCR results in improved selectivity of the TMMP. Thus, for example, a TMMP binds to a first T cell presenting both i) a TCR specific for an epitope present in the TMMP and ii) a co-MOD that binds to the MOD present in the TMMP with a higher avidity than the avidity with which a TMMP of the present disclosure binds to a second T cell presenting i) a TCR specific for an epitope other than the epitope present in the TMMP and ii) a co-MOD that binds to the MOD present in the TMMP.

[0041] The binding affinity between MOC and its cognate co-MOD can be measured by biolayer interferometry (BLI) using purified MOD and purified co-MOD. The binding affinity between TMMP and its cognate co-MOD can be measured by BLI using purified TMMP and cognate co-MOD. BLI methods are well known to those skilled in the art. See, for example, Lad et al. (2015) J. Biomol. Screen. 20(4):498-507; and Shah and Duncan (2014) J. Vis. Exp. 18:e51383.

[0042] BLI assays can be performed using an Octet RED 96 (Pal ForteBio) instrument or similar instrument as follows: TMMP (e.g., a TMMP of the present disclosure, a control TMMP (wherein the control TMMP contains a wild-type immunomodulatory polypeptide)) is immobilized on an insoluble support ("biosensor"). The immobilized TMMP is the "target." Immobilization can be achieved by immobilizing a capture antibody on the insoluble support, which immobilizes the TMMP. For example, immobilization can be achieved by immobilizing an anti-Fc (e.g., anti-human IgG Fc) antibody on the insoluble support, which binds to and immobilizes the TMMP (wherein the TMMP contains an IgFc polypeptide). Several different concentrations of a co-immunomodulatory polypeptide are added to the immobilized TMMP, and the instrument's response is recorded. The assay is performed in a liquid medium containing 25 mM HEPES pH 6.8, 5% poly(ethylene glycol) 6000, 50 mM KCl, 0.1% bovine serum albumin, and 0.02% Tween 20 non-ionic surfactant. Binding of the co-immunomodulatory polypeptide to immobilized TMMP is performed at 30°C. As a positive control for binding affinity, an anti-MHC class I monoclonal antibody may be used. For example, a K of 7 nM D Anti-HLA class I monoclonal antibody W6 / 32 (American Type Culture Collection No. HB-95; Parham et al. (1979) J. Immunol. 123:342) having the formula: (Illegible) may be used. A standard curve may be generated using serial dilutions of the anti-MHC class I monoclonal antibody. The co-immunomodulatory polypeptide, i.e., anti-MHC class I mAb, is the "analyte." BLI analyzes the interference pattern of white light reflected from two surfaces: i) the immobilized polypeptide ("target") and ii) an internal reference layer. Changes in the number of molecules ("analytes," e.g., co-immunomodulatory polypeptide, anti-HLA antibody) bound to the biosensor chip result in a shift in the interference pattern, which may be measured in real time. Two kinetic terms describing the affinity of the target / analyte interaction are the binding rate constant (k a ) and dissociation constant (k d ) The ratio of these two terms (kd / a ) to determine the affinity constant (K D ) is required.

[0043] BLI assays are performed using multiwell plates. To run the assay, use Octet Data Acquisition software to define the plate layout, define the assay steps, and assign biosensors. Hydrate the biosensor assembly. Equilibrate the hydrated biosensor assembly and assay plate on the Octet instrument for 10 minutes. Once data is acquired, load the acquired data into Octet Data Analysis software. Process the data in the Processing window by specifying the methods for reference subtraction, y-axis alignment, inter-step correction, and Savitzky-Golay filtering. Analyze the data in the Analysis window by specifying the analysis steps (association and dissociation) and selecting the curve fitting model (1:1), fitting method (global), and window of interest (seconds). Assess the quality of the fit. If the K of each data record is within a 3-fold range, D The values ​​(analyte concentrations) may be averaged. D The error value must be within one order of magnitude of the affinity constant value, R 2 The value should be greater than 0.95. See, e.g., Abdiche et al. (2008) J. Anal. Biochem. 377:209.

[0044] Unless otherwise specified herein, the affinity of a TMMP of the present disclosure for a cognate co-immunomodulatory polypeptide, or the affinity of a control TMMP (wherein the control TMMP comprises a wild-type immunomodulatory polypeptide) for a cognate co-immunomodulatory polypeptide, is measured using BLI as described above.

[0045] In some examples, the ratio of i) the binding affinity of a control TMMP (wherein the control comprises a wild-type immunomodulatory polypeptide) to the cognate co-immunomodulatory polypeptide and ii) the binding affinity of a TMMP of the disclosure comprising a variant form of a wild-type immunomodulatory polypeptide to the cognate co-immunomodulatory polypeptide, as measured by BLI (described above), is at least 1.5:1, at least 2:1, at least 5:1, at least 10:1, at least 15:1, at least 20:1, at least 25:1, at least 50:1, at least 100:1, at least 500:1, at least 10 2 :1, at least 5 × 10 2 :1, at least 10 3 :1, at least 5 × 10 3 :1, at least 10 4 :1, at least 10 5 :1, or at least 10 6 In some examples, the ratio of i) the binding affinity of a control TMMP (wherein the control comprises a wild-type immunomodulatory polypeptide) to the cognate co-immunomodulatory polypeptide and ii) the binding affinity of a TMMP of the disclosure comprising a variant of the wild-type immunomodulatory polypeptide to the cognate co-immunomodulatory polypeptide is 1.5:1 to 10, as measured by BLI. 6 :1, for example, 1.5:1 to 10:1, 10:1 to 50:1, 50:1 to 10 2 :1, 10 2 :1~10 3 :1, 10 3 :1~10 4 :1, 10 4 :1~10 5 :1 or 10 5 :1~10 6 :1 range.

[0046] In one example, the immunomodulatory polypeptides include a control TMMP that comprises a wild-type IL-2 polypeptide and a TMMP of the disclosure that comprises a variant IL-2 polypeptide (comprising 1-10 amino acid substitutions compared to the amino acid sequence of the wild-type IL-2 polypeptide), and the ratio of i) the binding affinity of the control TMMP to the IL-2 receptor (i.e., the cognate co-immunomodulatory polypeptide) to ii) the binding affinity of the TMMP of the disclosure to the IL-2 receptor is at least 1.5:1, at least 2:1, at least 5:1, at least 10:1, at least 15:1, at least 20:1, at least 25:1, at least 50:1, at least 100:1, at least 500:1, at least 10 2 :1, at least 5 × 10 2 :1, at least 10 3 :1, at least 5 × 10 3 :1, at least 10 4 :1, at least 10 5 :1, or at least 10 6 In some examples, the immunomodulatory polypeptides include a control TMMP that includes a wild-type IL-2 polypeptide and a TMMP of the disclosure that includes a variant IL-2 polypeptide (comprising 1-10 amino acid substitutions compared to the amino acid sequence of the wild-type IL-2 polypeptide), and the ratio of i) the binding affinity of the control TMMP to the IL-2 receptor (i.e., the cognate co-immunomodulatory polypeptide) to ii) the binding affinity of the TMMP of the disclosure to the IL-2 receptor is 1.5:1-10 as measured by BLI. 6 :1, for example, 1.5:1 to 10:1, 10:1 to 50:1, 50:1 to 10 2 :1, 10 2 :1~10 3 :1, 10 3 :1~10 4 :1, 10 4 :1~10 5 :1 or 10 5 :1~10 6 :1 range.

[0047] The binding affinity of the TMMPs of the present disclosure for target T cells can be measured according to the procedures described in published PCT application WO2019 / 051091, published March 14, 2019. See

[0063] .

[0048] In some examples, when measured as described in the immediately preceding paragraph, the TMMPs of the present disclosure exhibit selective binding to target T cells compared to binding of a TMMP library member to a control T cell comprising i) a cognate co-immunomodulatory polypeptide that binds to the parent wild-type immunomodulatory polypeptide and ii) a T cell receptor that binds to an epitope other than the epitope present in the TMMP library member.

[0049] Dimerized TMMP The TMMPs of the present disclosure may be dimerized, meaning that the present disclosure provides multimeric polypeptides comprising dimers of the TMMPs of the present disclosure. Thus, the present disclosure provides a TMMP comprising A) a first heterodimer and B) a second heterodimer, wherein A) the first heterodimer comprises a) a first polypeptide and b) a second polypeptide, wherein a) the first polypeptide comprises i) a peptide epitope and ii) a first MHC polypeptide, and b) the second polypeptide comprises i) a second MHC polypeptide (wherein the first heterodimer comprises one or more MODs); and B) the second heterodimer comprises a) the first polypeptide and b) a second polypeptide, wherein a) the first polypeptide comprises i) a peptide epitope and ii) the first MHC polypeptide, and b) the second polypeptide comprises i) a second MHC polypeptide (wherein the second heterodimer comprises one or more MODs), whereby the first heterodimer and the second heterodimer are covalently linked to each other. In some examples, the two TMMPs are identical to each other in amino acid sequence. In some examples, the first heterodimer and the second heterodimer are covalently linked to each other via the C-terminal region of the second polypeptide of the first heterodimer and the C-terminal region of the second polypeptide of the second heterodimer. In some examples, the first heterodimer and the second heterodimer are covalently linked to each other via the C-terminal amino acid of the second polypeptide of the first heterodimer and the C-terminal region of the second polypeptide of the second heterodimer; for example, in some examples, the C-terminal amino acid of the second polypeptide of the first heterodimer and the C-terminal region of the second polypeptide of the second heterodimer are linked to each other either directly or via a linker. The linker may be a peptide linker. The peptide linker may have a length of 1 to 200 amino acids (e.g., 1 to 5 amino acids (aa), 5 to 10 amino acids, 10 to 25 amino acids, 25 to 50 amino acids, 50 to 100 amino acids, 100 to 150 amino acids, or 150 to 200 amino acids). In some examples, the peptide epitope of the first heterodimer and the peptide epitope of the second heterodimer comprise the same amino acid sequence.In some examples, the first MHC polypeptide of the first and second heterodimers is MHC class I β2-microglobulin, and the second MHC polypeptide of the first and second heterodimers is an MHC class I heavy chain. In some examples, the MOD of the first heterodimer and the MOD of the second heterodimer comprise identical amino acid sequences. In some examples, the MOD(s) of the first heterodimer and the MOD(s) of the second heterodimer are variant MODs comprising 1 to 10 amino acid substitutions compared to a corresponding parent wild-type MOD, wherein the 1 to 10 amino acid substitutions result in a lower binding affinity of the variant immunomodulatory polypeptide to a cognate co-immunomodulatory polypeptide. In some examples, the first heterodimeric immunomodulatory polypeptide and the second heterodimeric immunomodulatory polypeptide are each independently selected from the group consisting of IL-2, 4-1BBL, PD-L1, CD80, CD86, ICOS-L, OX-40L, FasL, JAG1 (CD339), TGFβ, CD70, and ICAM. Examples of suitable MHC polypeptides, MODs, and peptide epitopes are described below.

[0050] MHC polypeptides As described above, the TMMPs of the present disclosure include MHC polypeptides. For purposes of this disclosure, the term "major histocompatibility complex (MHC) polypeptide" is intended to include various types of MHC polypeptides, including human MHC (also known as human leukocyte antigen (HLA)) polypeptides, rodent (e.g., mouse, rat, etc.) MHC polypeptides, and MHC polypeptides of other mammalian species (e.g., lagomorphs, non-human primates, canines, felines, and ungulates (e.g., horses, cattle, sheep, goats, etc.)). The term "MHC polypeptide" is intended to include class I MHC polypeptides (e.g., beta-2 microglobulin and MHC class I heavy chains).

[0051] In some examples, the first MHC polypeptide is an MHC class I β2M (β2M) polypeptide, and the second MHC polypeptide is an MHC class I heavy chain (H chain) ("MHC-H"). In other examples, the first MHC polypeptide is an MHC class I heavy chain polypeptide, and the second MHC polypeptide is a β2M polypeptide. In some examples, both the β2M chain and the MHC-H chain are human, i.e., the MHC-H chain is an HLA heavy chain or a variant thereof. Unless otherwise specified, the TMMPs of the present disclosure do not include the membrane-anchoring domain (transmembrane region) of the MHC class I heavy chain, or a portion of the MHC class I heavy chain sufficient to anchor the resulting TMMP in a cell that expresses it (e.g., a eukaryotic cell, such as a mammalian cell). In some examples, the MHC class I heavy chain present in the TMMPs of the present disclosure does not include the signal peptide, transmembrane domain, or intracellular domain (cytoplasmic tail) associated with a naturally occurring MHC class I heavy chain. Thus, for example, in some instances, the MHC class I heavy chain present in the TMMP comprises only the α1, α2, and α3 domains of the MHC class I heavy chain. In some instances, the MHC class I heavy chain present in the TMMP of the present disclosure has a length of about 270 amino acids (aa) to about 290 aa. In some instances, the MHC class I heavy chain present in the TMMP has a length of 270 aa, 271 aa, 272 aa, 273 aa, 274 aa, 275 aa, 276 aa, 277 aa, 278 aa, 279 aa, 280 aa, 281 aa, 282 aa, 283 aa, 284 aa, 285 aa, 286 aa, 287 aa, 288 aa, 289 aa, or 290 aa.

[0052] In some examples, the MHC polypeptide of the TMMP is a human MHC polypeptide, which is also referred to as a "human leukocyte antigen" ("HLA") polypeptide. In some examples, the MHC polypeptide of the TMMP is a class I HLA polypeptide, such as a β2-microglobulin polypeptide or a class I HLA heavy chain polypeptide. Class I HLA heavy chain polypeptides include HLA-A heavy chain polypeptides, HLA-B heavy chain polypeptides, HLA-C heavy chain polypeptides, HLA-E heavy chain polypeptides, HLA-F heavy chain polypeptides, and HLA-G heavy chain polypeptides.

[0053] MHC class I heavy chain In some examples, the MHC class I heavy chain polypeptide present in the TMMP comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to all or a portion (e.g., 50, 75, 100, 150, 200, or 250 consecutive amino acids) of the amino acid sequence of any of the human HLA heavy chain polypeptides shown in Figures 7-13. In some examples, the MHC class I heavy chain has a length of 270 aa, 271 aa, 272 aa, 273 aa, 274 aa, 275 aa, 276 aa, 277 aa, 278 aa, 279 aa, 280 aa, 281 aa, 282 aa, 283 aa, 284 aa, 285 aa, 286 aa, 287 aa, 288 aa, 289 aa, or 290 aa. In some examples, the MHC class I heavy chain polypeptide present in the TMMP comprises 1-30, 1-5, 5-10, 10-15, 15-20, 20-25, or 25-30 amino acid insertions, deletions, and / or substitutions of any one of the amino acid sequences shown in Figures 7-13 (in addition to those positions indicated as variable in the heavy chain consensus sequence). In some examples, the MHC class I heavy chain does not include a transmembrane or cytoplasmic domain. As an example, the MHC class I heavy chain polypeptide of the TMMP can include an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% amino acid sequence identity to amino acids 25-300 (lacking all or substantially all of the leader sequence, transmembrane sequence, and cytoplasmic sequence) or amino acids 25-365 (lacking the leader) of the human HLA-A heavy chain polypeptide shown in any one of Figures 7A, 7B, and 7C.

[0054] Figures 7A, 7B, and 7C provide the amino acid sequence of the human leukocyte antigen (HLA) class I heavy chain polypeptide. Amino acids 1-24, the signal sequence, are shown in bold and underlined. Entry 3A.1 in Figure 7A is the amino acid sequence of the HLA-A heavy chain (HLA-A * 01:01:01:01 or A *0101) (NCBI accession number NP_001229687.1) and SEQ ID NO: 23; entry 3A.2 is HLA-A * 1101 is derived from SEQ ID NO: 24, and entry 3A.3 is derived from HLA-A * 2402 is derived from SEQ ID NO: 25, and entry 3A.4 is derived from HLA-A * 3303 is derived from SEQ ID NO: 26. Figure 7B shows the sequence HLA-B * 07:02:01(HLA-B * 0702) NCBI GenBank accession number NP_005505.2 (see also GenBank accession number AUV50118.1). Figure 7C shows the sequence HLA-C * 0701 (GenBank accession number NP_001229971.1) (HLA-C * 07:01:01:01 or HLA-Cw * 070101, HLA-Cw * 07, see GenBank accession number CAO78194.1).

[0055] Figure 8 shows an alignment of 11 mature MHC class I heavy chain amino acid sequences (not including their leader sequences or transmembrane or intracellular domains). The aligned sequences include human HLA-A, HLA-B, and HLA-C, mouse H2K protein sequence, three variants of HLA-A (variant 1, variant 2C, and variant 2CP), and three human HLA-A variants (HLA-A * 1101, HLA-A * 2402, and HLA-A *3303). Shown in the alignment are positions (84 and 139 of the mature protein) where cysteine ​​residues can be introduced (e.g., by substitution) to form disulfide bonds that stabilize the MHC chain-β2M complex. Also shown in the alignment is position 236 (of the mature polypeptide) which can be substituted with a cysteine ​​residue (e.g., at aa 12) that can form an interchain disulfide bond with β2M. Arrows are shown above each of these positions, and the residues are in bold. The seventh HLA-A sequence shown in the alignment (variant 2c) shows the sequence of variant 2 with C residue substitutions at positions 84, 139, and 236. The boxes flanking residues 84, 139, and 236 indicate groups of five amino acids on either side of six sets of five residues that may be substituted with (i) any naturally occurring amino acid, or (ii) one to five amino acids independently selected from any naturally occurring amino acid except proline or glycine, denoted aac1 ("amino acid cluster 1"), aac2 ("amino acid cluster 2"), aac3 ("amino acid cluster 3"), aac4 ("amino acid cluster 4"), aac5 ("amino acid cluster 5"), and aac6 ("amino acid cluster 6").

[0056] With reference to FIG. 8 , in some examples, i) aac1 (amino acid cluster 1) may be the amino acid sequence GTLRG (SEQ ID NO: 287) or a sequence in which one or two amino acids have been deleted or substituted with other naturally occurring amino acids (e.g., L has been substituted with I, V, A, or F); ii) aac2 (amino acid cluster 2) may be the amino acid sequence YNQSE (SEQ ID NO: 288) or a sequence in which one or two amino acids have been deleted or substituted with other naturally occurring amino acids (e.g., N has been substituted with Q, Q has been substituted with N, and / or E has been substituted with D); iii) aac3 (amino acid cluster 3) may be the amino acid sequence TAADM (SEQ ID NO: 289) or a sequence in which one or two amino acids have been deleted or substituted with other naturally occurring amino acids (e.g., T has been substituted with S, A has been substituted with G, D has been substituted with E, and / or M has been substituted with L, V, or I); and iv) aac4 (amino acid cluster 4) may be the amino acid sequence YNQSE (SEQ ID NO: 289) or a sequence in which one or two amino acids have been deleted or substituted with other naturally occurring amino acids (e.g., T has been substituted with S, A has been substituted with G, D has been substituted with E, and / or M has been substituted with L, V, or I). aac4) may be the amino acid sequence AQTTK (SEQ ID NO: 290) or a sequence in which one or two amino acids have been deleted or substituted with other naturally occurring amino acids (e.g., A is substituted with G, Q is substituted with N, T is substituted with S, and / or K is substituted with R or Q); v) aac5 (amino acid cluster 5) may be the amino acid sequence VETRP (SEQ ID NO: 291) or a sequence in which one or two amino acids have been deleted or substituted with other naturally occurring amino acids (e.g., V is substituted with I or L, E is substituted with D, T is substituted with S, and / or R is substituted with K); and / or vi) aac6 (amino acid cluster 6) may be the amino acid sequence GDGTF (SEQ ID NO: 292) or a sequence in which one or two amino acids have been deleted or substituted with other naturally occurring amino acids (e.g., D is substituted with E, T is substituted with S, or F is substituted with L, W, or Y).

[0057] Figures 9-11 show alignments of mature HLA class I heavy chain amino acid sequences (not including leader sequences or transmembrane or intracellular domains). The aligned amino acid sequences in Figure 9A are HLA-A class I heavy chains of the following alleles: A * 0101, A * 0201, A * 0301, A * 1101, A * 2301, A * 2402, A * 2407, A * 3303 and A * 3401. The aligned amino acid sequences in Figure 10A are the HLA-B class I heavy chains of the following alleles: B * 0702, B * 0801, B * 1502, B * 3802, B * 4001, B * 4601 and B * 5301. The aligned amino acid sequences in Figure 11A are the HLA-C class I heavy chains of the following alleles: C * 0102, C * 0303, C * 0304, C * 0401, C * 0602, C * 0701, C * 0801 and C *1502. The alignment shows positions (84 and 139 of the mature protein) where cysteine ​​residues can be introduced (e.g., by substitution) to form disulfide bonds to stabilize the HLA H chain-β2M complex. The alignment also shows position 236 (of the mature polypeptide), which can be substituted (e.g., at aa 12) by a cysteine ​​residue capable of forming an interchain disulfide bond with β2M. The boxes adjacent to residues 84, 139, and 236 indicate five amino acid groups on either side of six sets of five residues, designated aac1 (for "amino acid cluster 1"), aac2 (for "amino acid cluster 2"), aac3 (for "amino acid cluster 3"), aac4 (for "amino acid cluster 4"), aac5 (for "amino acid cluster 5"), and aac6 (for "amino acid cluster 6"), which may be substituted by one to five amino acids independently selected from (i) natural amino acids or (ii) natural amino acids excluding proline or glycine.

[0058] Figures 9A, 10A, and 11A show alignments of the amino acid sequences of mature HLA-A, HLA-B, and HLA-C class I heavy chains, respectively. The sequences represent the extracellular portions of the mature proteins (excluding leader sequences, transmembrane domains, or intracellular domains). Also shown are aa residues 84, 139, and 236, as well as their adjacent residues (aac1-aac6), which can be substituted with (i) any naturally occurring amino acid or (ii) one to five amino acids independently selected from naturally occurring amino acids excluding proline or glycine, as described in Figure 8. Figures 9B, 10B, and 11B show consensus amino acid sequences for the HLA-A, HLA-B, and HLA-C sequences shown in Figures 9A, 10A, and 11A, respectively. In the consensus sequences, variable amino acid positions are indicated as consecutively numbered "X" residues, with amino acid positions 84, 139, and 236 double-underlined.

[0059] With reference to FIG. 9A , in some examples, i) aac1 (amino acid cluster 1) may be the amino acid sequence GTLRG (SEQ ID NO: 287) or a sequence from which one or two amino acids have been deleted or substituted with other naturally occurring amino acids (e.g., L has been substituted with I, V, A, or F); ii) aac2 (amino acid cluster 2) may be the amino acid sequence YNQSE (SEQ ID NO: 288) or a sequence from which one or two amino acids have been deleted or substituted with other naturally occurring amino acids (e.g., N has been substituted with Q, Q has been substituted with N, and / or E has been substituted with D); iii) aac3 (amino acid cluster 3) may be the amino acid sequence TAADM (SEQ ID NO: 289) or a sequence from which one or two amino acids have been deleted or substituted with other naturally occurring amino acids (e.g., T has been substituted with S, A has been substituted with G, D has been substituted with E, and / or M has been substituted with L, V, or I); and iv) aac4 (amino acid cluster 4) may be the amino acid sequence AQTTK (SEQ ID NO: 290) or a sequence from which one or two amino acids have been deleted or substituted with other naturally occurring amino acids (e.g., A has been replaced with G, Q has been replaced with N, T has been replaced with S, and / or K has been replaced with R or Q); v) aac5 (amino acid cluster 5) may be the amino acid sequence VETRP (SEQ ID NO: 291) or a sequence from which one or two amino acids have been deleted or substituted with other naturally occurring amino acids (e.g., V has been replaced with I or L, E has been replaced with D, T has been replaced with S, and / or R has been replaced with K); and / or vi) aac6 (amino acid cluster 6) may be the amino acid sequence GDGTF (SEQ ID NO: 292) or a sequence from which one or two amino acids have been deleted or substituted with other naturally occurring amino acids (e.g., D has been replaced with E, T has been replaced with S, or F has been replaced with L, W, or Y).

[0060] With reference to FIG. 10A , in some examples, i) aac1 (amino acid cluster 1) may be the amino acid sequence RNLRG (SEQ ID NO: 293) or a sequence from which one or two amino acids have been deleted or substituted with other natural amino acids (e.g., N replaced with T or I, and / or L replaced with A, and / or a second R replaced with L, and / or G replaced with R), and ii) aac2 (amino acid cluster 2) may be the amino acid sequence YNQSE (SEQ ID NO: 288) or a sequence from which one or two amino acids have been deleted or substituted with other natural amino acids (e.g., N replaced with T or I, and / or L replaced with A, and / or a second R replaced with L, and / or G replaced with R). aac3 (amino acid cluster 3) may be the amino acid sequence TAADT (SEQ ID NO: 294) or a sequence in which one or two amino acids have been deleted or substituted with other natural amino acids (e.g., replacing N with Q, replacing Q with N, and / or replacing E with D); iii) aac3 (amino acid cluster 3) may be the amino acid sequence TAADT (SEQ ID NO: 294) or a sequence in which one or two amino acids have been deleted or substituted with other natural amino acids (e.g., replacing the first T with S, and / or replacing A with G, and / or replacing D with E, and / or replacing the second T with S); iv) aac4 (amino acid cluster 4) may be the amino acid sequence TAADT (SEQ ID NO: 294) or a sequence in which one or two amino acids have been deleted or substituted with other natural amino acids (e.g., replacing the first T with S, and / or replacing A with G, and / or replacing D with E, and / or replacing the second T with S); aac4) may be the amino acid sequence AQITQ (SEQ ID NO: 295) or a sequence obtained by deleting one or two amino acids from the sequence or substituting them with other natural amino acids (e.g., A is substituted with G, and / or the first Q is substituted with N, and / or I is substituted with L or V, and / or T is substituted with S, and / or the second Q is substituted with N); v) aac5 (amino acid cluster 5) may be the amino acid sequence VETRP (SEQ ID NO: 291) or a sequence obtained by deleting one or two amino acids from the sequence or substituting them with other natural amino acids (e.g., A is substituted with G, and / or the first Q is substituted with N, and / or I is substituted with L or V, and / or T is substituted with S, and / or the second Q is substituted with N); may be a sequence in which other naturally occurring amino acids are substituted (e.g., V is substituted with I or L, E is substituted with D, T is substituted with S, and / or R is substituted with K); and / or vi) aac6 (amino acid cluster 6) may be the amino acid sequence GDRTF (SEQ ID NO: 296) or a sequence in which one or two amino acids have been deleted from that sequence or substituted with other naturally occurring amino acids (e.g., D is substituted with E, and / or T is substituted with S, and / or R is substituted with K or H, and / or F is substituted with L, W, or Y).

[0061] With reference to FIG. 11A , in some examples: i) aac1 (amino acid cluster 1) may be the amino acid sequence RNLRG (SEQ ID NO: 293) or a sequence from which one or two amino acids have been deleted or substituted with other naturally occurring amino acids (e.g., N is replaced with K, and / or L is replaced with A or I, and / or R is replaced with H, and / or G is replaced with T or S); ii) aac2 (amino acid cluster 2) may be the amino acid sequence YNQSE (SEQ ID NO: 288) or a sequence from which one or two amino acids have been deleted or substituted with other naturally occurring amino acids (e.g., N is replaced with K, and / or L is replaced with A or I, and / or R is replaced with H, and / or G is replaced with T or S); iii) aac3 (amino acid cluster 3) may be the amino acid sequence TAADT (SEQ ID NO: 294) or a sequence in which one or two amino acids have been deleted or substituted with other natural amino acids (e.g., the first T has been replaced with S, and / or the A has been replaced with G, and / or the D has been replaced with E, and / or the second T has been replaced with S). iv) aac4 (amino acid cluster 4) may be the amino acid sequence AQITQ (SEQ ID NO: 295) or a sequence obtained by deleting one or two amino acids from the sequence or substituting them with other natural amino acids (e.g., substituting A with G, and / or substituting the first Q with N, and / or substituting I with L, and / or substituting the second Q with N or K), and v) aac5 (amino acid cluster 5) may be the amino acid sequence VETRP (SEQ ID NO: 291) or a sequence obtained by deleting one or two amino acids from the sequence. and / or vi) aac6 (amino acid cluster 6) may be the amino acid sequence GDGTF (SEQ ID NO: 292) or a sequence in which one or two amino acids have been deleted or substituted with other naturally occurring amino acids (e.g., D has been substituted with E, and / or T has been substituted with S, and / or F has been substituted with L, W, or Y).

[0062] HLA-A In some examples, the TMMP comprises an HLA-A heavy chain polypeptide. HLA-A heavy chain peptide sequences or portions thereof that can be incorporated into the TMMP of the present disclosure include, but are not limited to, allele A * 001, A * 0201, A * 0301, A * 1101, A * 2301, A * 2402, A * 2407, A * 3303 and A * 3401, which are aligned in Figure 9A without all or substantially all of the leader, transmembrane, and cytoplasmic sequences. Any of these alleles may contain a mutation at one or more of positions 84, 139, and / or 236 (shown in Figure 9A) selected from the following: tyrosine to alanine at position 84 (Y84A), tyrosine to cysteine ​​at position 84 (Y84C), alanine to cysteine ​​at position 139 (A139C), and alanine to cysteine ​​substitution at position 236 (A236C). Additionally, HLA-A sequences having at least 75% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%) or 100% amino acid sequence identity to all or a portion (e.g., 50, 75, 100, 150, 200, or 250 consecutive amino acids) of the sequence of an HLA-A allele may also be used (e.g., which may include insertions, deletions, and / or substitutions of 1 to 25, 1 to 5, 5 to 10, 10 to 15, 15 to 20, 20 to 25, or 25 to 30 amino acids).

[0063] In some instances, the TMMP has the following HLA-A consensus amino acid sequence: TIFF2025186334000002.tif42165 (SEQ ID NO: 29) (X1 is F, Y, S, or T; X2 is K or R; X3 is Q, G, E, or R; X4 is N or E; X5 is R or G; X6 is N or K; X7 is M or V; X8 is H or Q; X9 is T or I; and X10 is D or H. X11 is A, V, or E, X12 is N or D, X13 is G or R, X14 is T or I, X15 is L or A, X16 is R or L, X17 is G or R, X18 is A or D, X19 is I, L, or V, X20 is I, R, or M, X21 is F or Y, and X22 is S or P. X23 is W or G, X24 is R, H, or Q, X25 is D or Y, X26 is N or K, X27 is T or I, X28 is K or Q, X29 is R or H, X30 is A or T, X31 is A or V, X32 is H or R, X33 is R, L, Q, or W, and X34 is V or A. X35 is D or E, X36 is R or T, X37 is D or E, X38 is W or G, X39 is P or A, X40 is P or A, X41 is V or I, X42 is S or G, X43 is A or S, X44 is Q or E, and X45 is P or L.

[0064] As an example, the MHC class I heavy chain polypeptide of TMMP has the following human HLA-A heavy chain 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 TIFF2025186334000003.tif42165 (SEQ ID NO: 44).

[0065] In some instances, an HLA-A heavy chain polypeptide suitable for inclusion in a TMMP has the following amino acid sequence: TIFF2025186334000004.tif42165 (SEQ ID NO: 44). This HLA-A heavy chain polypeptide is "HLA-A * In some instances, the C-terminal Pro is not included in the TMMP. For example, in some instances, an HLA-A02 polypeptide suitable for inclusion in a TMMP has the following amino acid sequence: TIFF2025186334000005.tif42165 (sequence number 449).

[0066] HLA-A(Y84C, A236C) In some examples, an HLA-A heavy chain polypeptide suitable for inclusion in a TMMP has the following human HLA-A heavy chain (Y84C, A236C) amino acid sequence: The present invention relates to 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 TIFF2025186334000006.tif48165 (SEQ ID NO: 488) (amino acid 84 is Cys and amino acid 236 is Cys).

[0067] HLA-A (Y84A, A236C) In some instances, the MHC class I heavy chain polypeptide comprises a Y84A substitution and an A236C substitution. For example, in some instances, the MHC class I heavy chain polypeptide has the following human HLA-A heavy chain (Y84A, A236C) amino acid sequence: The variant 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 TIFF2025186334000007.tif42165 (SEQ ID NO:48) (amino acid 84 is Ala and amino acid 236 is Cys). In some examples, Cys-236 forms an interchain disulfide bond with Cys-12 of a variant β2M polypeptide comprising an R12C substitution.

[0068] In some examples, an HLA-A heavy chain polypeptide suitable for inclusion in a TMMP is an HLA-A02 (Y84A, A236C) polypeptide comprising the following amino acid sequence: TIFF2025186334000008.tif42165 (SEQ ID NO: 48).

[0069] In some examples, an HLA-A heavy chain polypeptide suitable for inclusion in a TMMP is an HLA-A02 (Y84A, A236C) polypeptide comprising the following amino acid sequence: TIFF2025186334000009.tif42165 (SEQ ID NO: 46).

[0070] HLA-A(Y84C, A139C) In some instances, the MHC class I heavy chain polypeptide comprises a Y84C substitution and an A139C substitution. For example, in some instances, the MHC class I heavy chain polypeptide has the following human HLA-A heavy chain (Y84C, A139C) amino acid sequence: The amino acid sequence has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF2025186334000010.tif42165 (SEQ ID NO: 299) (amino acid 84 is Cys and amino acid 139 is Cys). In some examples, Cys-84 forms an intrachain disulfide bond with Cys-139.

[0071] HLA-A11 (HLA-A * 1101) As one non-limiting example, the MHC class I heavy chain polypeptide of TMMP has the following human HLA-A11 heavy chain amino acid sequence: TIFF2025186334000011.tif42165 (SEQ ID NO: 300). Such MHC class I heavy chains may be prominent in Asian populations, including populations of individuals of Asian descent.

[0072] HLA-A11(Y84A, A236C) As one non-limiting example, in some instances, the MHC class I heavy chain polypeptide is an HLA-A11 allele that includes a Y84A substitution and an A236C substitution. For example, in some instances, the MHC class I heavy chain polypeptide has the following human HLA-A11 heavy chain (Y84A, A236C) amino acid sequence: The variant 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 TIFF2025186334000012.tif42165 (SEQ ID NO: 301) (amino acid 84 is Ala and amino acid 236 is Cys). In some examples, Cys-236 forms an interchain disulfide bond with Cys-12 of a variant β2M polypeptide comprising an R12C substitution.

[0073] HLA-A11 (Y84C, A236C) In some instances, the MHC class I heavy chain polypeptide present in the TMMP has the following human HLA-A A11 heavy chain (Y84C, A236C) amino acid sequence: The present invention relates to 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 TIFF2025186334000013.tif35165 (SEQ ID NO: 301) (amino acid 84 is Cys and amino acid 236 is Cys).

[0074] HLA-A24 (HLA-A * 2402) As one non-limiting example, the MHC class I heavy chain polypeptide of TMMP has the following human HLA-A24 heavy chain amino acid sequence: * Also known as 2402), The MHC class I heavy chain may comprise an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF2025186334000014.tif42165 (SEQ ID NO: 302). Such an MHC class I heavy chain may be prominent in Asian populations, including populations of individuals of Asian descent. In some examples, amino acid 84 is Ala. In some examples, amino acid 84 is Cys. In some examples, amino acid 236 is Cys. In some examples, amino acid 84 is Ala and amino acid 236 is Cys. In some examples, amino acid 84 is Cys and amino acid 236 is Cys.

[0075] In some instances, the MHC class I heavy chain polypeptide of the TMMP is associated with the following human HLA-A24 (HLA-A * 2402) heavy chain amino acid sequence, and the MHC class I heavy chain has a length of about 275 amino acids.

[0076] In some instances, the MHC class I heavy chain polypeptide of the TMMP is associated with the following human HLA-A24 (HLA-A * 2402) heavy chain amino acid sequence, and the MHC class I heavy chain has a length of about 275 amino acids.

[0077] In some instances, the MHC class I heavy chain polypeptide of the TMMP is associated with the following human HLA-A24 (HLA-A * 2402) heavy chain amino acid sequence, and the MHC class I heavy chain has a length of about 275 amino acids.

[0078] In some instances, the MHC class I heavy chain polypeptide of the TMMP is associated with the following human HLA-A24 (HLA-A * 2402) heavy chain amino acid sequence, and the MHC class I heavy chain has a length of about 275 amino acids.

[0079] In some instances, the MHC class I heavy chain polypeptide of the TMMP is associated with the following human HLA-A24 (HLA-A * 2402) heavy chain amino acid sequence, and the MHC class I heavy chain has a length of about 275 amino acids.

[0080] In some instances, the MHC class I heavy chain polypeptide of the TMMP is associated with the following human HLA-A24 (HLA-A * 2402) heavy chain amino acid sequence, and the MHC class I heavy chain has a length of about 275 amino acids.

[0081] HLA-A33 (HLA-A * 3303) As one non-limiting example, the MHC class I heavy chain polypeptide of TMMP has the following human HLA-A33 heavy chain amino acid sequence: The MHC class I heavy chain may comprise an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF2025186334000021.tif42165 (SEQ ID NO: 303). Such an MHC class I heavy chain may be prominent in Asian populations, including populations of individuals of Asian descent. In some examples, amino acid 84 is Ala. In some examples, amino acid 84 is Cys. In some examples, amino acid 236 is Cys. In some examples, amino acid 84 is Ala and amino acid 236 is Cys. In some examples, amino acid 84 is Cys and amino acid 236 is Cys.

[0082] HLA-B In some instances, the TMMP comprises an HLA-B heavy chain polypeptide. HLA-B heavy chain peptide sequences or portions thereof that may be incorporated into the TMMP include, but are not limited to, alleles: B * 0702, B * 0801, B * 1502, B * 3802, B * 4001, B * 4601 and B *5301, which are aligned in Figure 10A without all or substantially all of the leader, transmembrane, and cytoplasmic sequences. Any of these alleles may contain a mutation at one or more of positions 84, 139, and / or 236 (shown in Figure 10A) selected from the following: tyrosine to alanine at position 84 (Y84A), tyrosine to cysteine ​​at position 84 (Y84C), alanine to cysteine ​​at position 139 (A139C), and alanine to cysteine ​​substitution at position 236 (A236C). Additionally, HLA-B polypeptides comprising an amino acid sequence having at least 75% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%) or 100% amino acid sequence identity to all or a portion (e.g., 50, 75, 100, 150, 200, or 250 contiguous amino acids) of the sequence of an HLA-B allele may also be used (e.g., which may include insertions, deletions, and / or substitutions of 1 to 25, 1 to 5, 5 to 10, 10 to 15, 15 to 20, 20 to 25, or 25 to 30 amino acids).

[0083] In some instances, the TMMP has the following HLA-B consensus amino acid sequence: TIFF2025186334000022.tif42165 (SEQ ID NO: 30) (X1 is H, Y, or D, X2 is A or S, X3 is M or V, X4 is A, S, or T, X5 is Q or L, X6 is A or T, X7 is E, M, K, or T, X8 is A or T, X9 is E or N, X10 is I or K, X11 is Y, F, S, or C, X12 is N or Q, X13 is A or T, X14 is D or Y, X15 is E or V, X16 is S or N, X17 is T, X18 is A or L, X19 is L or R, X20 is R or G, X21 is T or I, X22 is L or I, X23 is R or S, X24 is R or S, X25 is S or T, X26 is L or W, X27 is E or V, X28 is R, D, L, or W, X29 is A or T, X30 is L, E, or T, X31 is E or D, X32 is K or T, X33 is E or Q, and X34 is I or V.

[0084] As an example, the MHC class I heavy chain polypeptide of TMMP has the following human HLA-B heavy chain 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 TIFF2025186334000023.tif42165 (SEQ ID NO: 207).

[0085] HLA-B (Y84A, A236C) As one non-limiting example, in some instances, the MHC class I heavy chain polypeptide is an HLA-B polypeptide that includes a Y84A substitution and an A236C substitution. For example, in some instances, the MHC class I heavy chain polypeptide has the following human HLA-B heavy chain (Y84A, A236C) amino acid sequence: The variant 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 TIFF2025186334000024.tif42165 (SEQ ID NO: 305) (amino acid 84 is Ala and amino acid 236 is Cys). In some examples, Cys-236 forms an interchain disulfide bond with Cys-12 of a variant β2M polypeptide comprising an R12C substitution.

[0086] HLA-B (Y84C, A139C) In some instances, the MHC class I heavy chain polypeptide comprises a Y84C substitution and an A139C substitution. For example, in some instances, the MHC class I heavy chain polypeptide has the following human HLA-B heavy chain (Y84C, A139C) amino acid sequence: The amino acid sequence has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF2025186334000025.tif42165 (SEQ ID NO: 306) (amino acid 84 is Cys and amino acid 139 is Cys). In some examples, Cys-84 forms an intrachain disulfide bond with Cys-139.

[0087] HLA-B * 0702 As an example, in some instances, the MHC class I heavy chain polypeptide present in the TMMP is HLA-B *0702 (SEQ ID NO: 207), or a sequence having at least 75% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%) or 100% amino acid sequence identity to all or a portion (e.g., 50, 75, 100, 150, 200, or 250 contiguous amino acids) of that sequence (e.g., which may include 1-25, 1-5, 5-10, 10-15, 15-20, 20-25, or 25-30 amino acid insertions, deletions, and / or substitutions). In some examples, the HLA-B heavy chain polypeptide of a TMMP of the present disclosure comprises the sequence labeled HLA-B in FIG. 8 or "B" in FIG. 10A. * If a sequence has less than 100% identity to the sequence labeled "0702," it may contain a mutation at one or more of positions 84, 139, and / or 236 selected from the following: a tyrosine to alanine substitution at position 84 (Y84A), a tyrosine to cysteine ​​substitution at position 84 (Y84C), an alanine to cysteine ​​at position 139 (A139C), and an alanine to cysteine ​​substitution at position 236 (A236C). In some examples, the HLA-B heavy chain polypeptide of the TMMP of the present disclosure contains the Y84A and A236C substitutions. In some cases, the HLA-B heavy chain polypeptide of the TMMP of the present disclosure contains the Y84A and A236C substitutions. * The 0702 heavy chain polypeptide comprises Y84C and A139C substitutions. In some examples, the HLA-B heavy chain polypeptide of a TMMP of the present disclosure comprises Y84C, A139C, and A236C substitutions.

[0088] HLA-C In some examples, the TMMP comprises an HLA-C heavy chain polypeptide. HLA-C heavy chain polypeptides or portions thereof that can be incorporated into the TMMP of the present disclosure include, but are not limited to, alleles: C * 0102, C * 0303, C * 0304, C * 0401, C * 0602, C * 0701, C * 0801 and C *1502, which align without including all or substantially all of the leader, transmembrane, and cytoplasmic sequences in Figure 11 A. Any of these alleles may contain a mutation at one or more of positions 84, 139, and / or 236 (shown in Figure 11A) selected from the following: a tyrosine to alanine substitution at position 84 (Y84A), a tyrosine to cysteine ​​substitution at position 84 (Y84C), an alanine to cysteine ​​substitution at position 139 (A139C), or an alanine to cysteine ​​substitution at position 236 (A236C). Additionally, HLA-C polypeptides comprising an amino acid sequence having at least 75% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%) or 100% amino acid sequence identity to all or a portion (e.g., 50, 75, 100, 150, 200, or 250 consecutive amino acids) of the sequence of an HLA-C allele may also be used (e.g., which may include insertions, deletions, and / or substitutions of 1 to 25, 1 to 5, 5 to 10, 10 to 15, 15 to 20, 20 to 25, or 25 to 30 amino acids).

[0089] In some instances, the TMMP has the following HLA-C consensus amino acid sequence: TIFF2025186334000026.tif42165 (SEQ ID NO: 31) (X1 is C or G, X2 is R or K, X3 is F, Y, S, or D, X4 is R or W, X5 is H or R, X6 is A or S, X7 is Q or R, X8 is A or E, X9 is N or K, X10 is T or A, X11 is S or N, X12 is N or K, X13 is A or D, X14 is G or R, X15 is T or I, X16 is L or I, X17 is W or R, X18 is C, Y, F, or S, and X19 is X21 is L or V, X20 is Y or H, X21 is D or N, X22 is Y, F, S, or L, X23 is L or W, X24 is E, A, or T, X25 is R, L, or W, X26 is L or T, X27 is E or K, X28 is E or K, X29 is H or P, X30 is R or V, X31 is W or R, X32 is V or M, X33 is E or Q, X34 is M or V, X35 is P or Q, X36 is R or S, and X37 is P or G.

[0090] As an example, the MHC class I heavy chain polypeptide of TMMP has the following human HLA-C heavy chain 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 TIFF2025186334000027.tif42165 (SEQ ID NO: 219).

[0091] HLA-C (Y84A, A236C) As one non-limiting example, in some instances, the MHC class I heavy chain polypeptide is an HLA-C polypeptide that includes a Y84A substitution and an A236C substitution. For example, in some instances, the MHC class I heavy chain polypeptide has the following human HLA-C heavy chain (Y84A, A236C) amino acid sequence: The variant 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 TIFF2025186334000028.tif42165 (SEQ ID NO: 308) (amino acid 84 is Ala and amino acid 236 is Cys). In some examples, Cys-236 forms an interchain disulfide bond with Cys-12 of a variant β2M polypeptide comprising an R12C substitution.

[0092] HLA-C (Y84C, A139C) In some instances, the MHC class I heavy chain polypeptide comprises a Y84C substitution and an A139C substitution. For example, in some instances, the MHC class I heavy chain polypeptide has the following human HLA-C heavy chain (Y84C, A139C) amino acid sequence: The amino acid sequence has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF2025186334000029.tif36165 (SEQ ID NO: 397) (amino acid 84 is Cys and amino acid 139 is Cys). In some examples, Cys-84 forms an intrachain disulfide bond with Cys-139.

[0093] HLA-C * 0701 In some instances, the MHC class I heavy chain polypeptide of the TMMP is HLA-C *8), or an amino acid sequence having at least 75% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%) or 100% amino acid sequence identity to all or a portion (e.g., 50, 75, 100, 150, 200, or 250 contiguous amino acids) of that sequence (e.g., which may include insertions, deletions, and / or substitutions of 1-25, 1-5, 5-10, 10-15, 15-20, 20-25, or 25-30 amino acids). In some examples, the HLA-C heavy chain polypeptide of TMMP is HLA-C heavy chain polypeptide of TMMP in FIG. 11A. * If it has less than 100% identity to the sequence labeled 0701, it may contain a mutation at one or more of positions 84, 139, and / or 236 selected from the following: a tyrosine to alanine substitution at position 84 (Y84A), a tyrosine to cysteine ​​substitution at position 84 (Y84C), an alanine to cysteine ​​substitution at position 139 (A139C), an alanine to cysteine ​​substitution at position 236 (A236C). In some examples, the HLA-C heavy chain polypeptide of the T-cell MMP contains the Y84A and A236C substitutions. In some examples, the HLA-C heavy chain polypeptide of the T-cell MMP contains the Y84A and A236C substitutions. * The 0701 heavy chain polypeptide or epitope complex thereof comprises Y84C and A139C substitutions. In some examples, the HLA-C heavy chain polypeptide of a TMMP of the present disclosure comprises Y84C, A139C, and A236C substitutions.

[0094] Non-classical HLA-E, HLA-F, and HLA-G MHC class I heavy chains In some examples, the TMMP comprises a non-classical MHC class I heavy chain polypeptide. Non-classical HLA heavy chain polypeptides, or portions thereof, that can be incorporated into the TMMP of the present disclosure include, but are not limited to, those of HLA-E, HLA-F, and HLA-G alleles. The amino acid sequences of HLA-E, HLA-F, and HLA-G heavy chain polypeptides (and HLA-A, HLA-B, and HLA-C alleles) can be found on the World Wide Web at hla.alleles.org / nomenclature / index.html, at the European Bioinformatics Institute (www.ebi.ac.uk), which is part of the European Molecular Biology Laboratory (EMBL), and at the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov).

[0095] Non-limiting examples of suitable HLA-E alleles include HLA-E * 0101(HLA-E * 01:01:01:01), HLA-E * 01:03(HLA-E * 01:03:01:01), HLA-E * 01:04, HLA-E * 01:05, HLA-E * 01:06, HLA-E * 01:07, HLA-E * 01:09, and HLA-E * Non-limiting examples of suitable HLA-F alleles include, but are not limited to, HLA-F 01:10. * 0101(HLA-F * 01:01:01:01), HLA-F * 01:02, HLA-F * 01:03(HLA-F * 01:03:01:01), HLA-F * 01:04, HLA-F * 01:05, and HLA-F *Non-limiting examples of suitable HLA-G alleles include, but are not limited to, HLA-G 01:06. * 0101(HLA-G * 01:01:01:01), HLA-G * 01:02, HLA-G * 01:03(HLA-G * 01:03:01:01), HLA-G * 01:04(HLA-G * 01:04:01:01), HLA-G * 01:06, HLA-G * 01:07, HLA-G * 01:08, HLA-G * 01:09:HLA-G * 01:10, HLA-G * 01:10, HLA-G * 01:11, HLA-G * 01:12, HLA-G * 01:14, HLA-G * 01:15, HLA-G * 01:16, HLA-G * 01:17, HLA-G * 01:18:HLA-G * 01:19, HLA-G * 01:20, and HLA-G * 01:22. The consensus sequences for those HLA E, HLA-F, and HLA-G alleles that lack all or substantially all of the leader, transmembrane, and cytoplasmic sequences are shown in Figure 12 and aligned with the consensus sequences for the HLA-A, HLA-B, and HLA-C alleles described above in Figure 13.

[0096] The amino acid sequences of suitable HLA-E heavy chain polypeptides are shown in Figures 46A-46D. * Figure 46B shows the amino acid sequence of HLA-E 01:01 (wild type) with Y84C and A2346C substitutions. * Figure 46C shows the amino acid sequence of HLA-E 01:01. *Figure 46D shows the amino acid sequence of HLA-E 01:03 (wild type) with Y84C and A2346C substitutions. * The amino acid sequence of 01:03 is shown.

[0097] The amino acid sequences of suitable HLA-G heavy chain polypeptides are shown in Figures 47A-47D. * Figure 47B shows the amino acid sequence of 01:01 (wild type), and Figure 47C shows the amino acid sequence of HLA-G with Y84C and A2346C substitutions. * Figure 47C shows the amino acid sequence of HLA-G * Figure 47D shows the amino acid sequence of 01:04 (wild type), and Figure 47E shows the amino acid sequence of HLA-G with Y84C and A2346C substitutions. * The amino acid sequence of 01:04 is shown.

[0098] Figure 12 shows the consensus sequences for each of HLA-E, HLA-F, and HLA-G, with variable aa positions shown as consecutively numbered "X" residues, with positions aas 84, 139, and 236 double underlined.

[0099] Figure 13 shows an alignment of the consensus amino acid sequences of HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, and HLA-G shown in Figures 9-13. Variable residues in each sequence are indicated as "X" with the consecutive numbers omitted. Also shown are positions aas 84, 139, and 236, along with their adjacent five amino acid clusters, which may be substituted with (i) any naturally occurring amino acid or (ii) one to five amino acids independently selected from any naturally occurring amino acid except proline or glycine, as shown in Figure 8.

[0100] Any of the above HLA-E, HLA-F, and / or HLA-G alleles may include substitutions at one or more of positions 84, 139, and / or 236, as shown for the consensus sequence in Figure 13. In some examples, the substitutions may be selected from the following: a tyrosine to alanine (Y84A) or cysteine ​​(Y84C), or in the case of HLA-F, an R84A or R84C substitution at position 84, an alanine to cysteine ​​(A139C), or in the case of HLA-F, V139C, at position 139, and an alanine to cysteine ​​substitution at position 236 (A236C). Additionally, HLA-E, HLA-F, and / or HLA-G sequences having at least 75% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%) or 100% amino acid sequence identity to all or a portion (e.g., 50, 75, 100, 150, 200, or 250 contiguous amino acids) of any of the consensus sequences set forth in FIG. 13 may also be used (e.g., the sequences may include insertions, deletions, and / or substitutions of 1-25, 1-5, 5-10, 10-15, 15-20, 20-25, or 25-30 amino acids in addition to changes at the variable residues set forth in the figure).

[0101] Mouse H2K In some examples, the MHC class I heavy chain polypeptide present in the TMMP comprises the amino acid sequence of mouse H2K (SEQ ID NO: 45) (mouse H2K in FIG. 8), or a sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to all or a portion (e.g., 50, 75, 100, 150, 200, or 250 contiguous amino acids) of that sequence (e.g., which may include insertions, deletions, and / or substitutions of 1-25, 1-5, 5-10, 10-15, 15-20, 20-25, or 25-30 amino acids). In some examples, if the mouse H2K heavy chain polypeptide of TMMP has less than 100% identity to the sequence labeled Mouse H2K in FIG. 8 , it may contain a mutation selected from the following at one or more of positions 84, 139, and / or 236: tyrosine to alanine at position 84 (Y84A), tyrosine to cysteine ​​at position 84 (Y84C), alanine to cysteine ​​at position 139 (A139C), and alanine to cysteine ​​at position 236 (A236C). In some examples, the mouse H2K heavy chain polypeptide of TMMP contains Y84A and A236C substitutions. In some examples, the mouse H2K heavy chain polypeptide of TMMP of the present disclosure contains Y84C and A139C substitutions. In some examples, the mouse H2K heavy chain polypeptide of TMMP of the present disclosure contains Y84C, A139C, and A236C substitutions.

[0102] Combination examples Table 1 below shows various combinations of MHC class I heavy chain sequence modifications that can be incorporated into the TMMPs of the present disclosure.

[0103] [Table 1] TIFF2025186334000031.tif189165

[0104] Beta-2 microglobulin The β2-microglobulin (β2M) polypeptide of the TMMP of the present disclosure can be a human β2M polypeptide, a non-human primate β2M polypeptide, a mouse β2M polypeptide, etc. In some examples, the β2M polypeptide comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the β2M amino acid sequence set forth in Figure 6. In some examples, the β2M polypeptide comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to amino acids 21-119 of the β2M amino acid sequence set forth in Figure 6.

[0105] In some instances, a suitable β2M polypeptide has the following amino acid sequence: TIFF2025186334000032.tif16165 (SEQ ID NO: 311), and the HLA class I heavy chain polypeptide comprises the following amino acid sequence: TIFF2025186334000033.tif43165 (SEQ ID NO: 309) (the cysteine ​​residue indicated by {C} forms a disulfide bond between the α1 helix and the α2-1 helix, and the (C) residue forms a disulfide bond with the β2M polypeptide cysteine ​​at position 12). In the above sequence, "aa1" is "amino acid cluster 1," "aa2" is "amino acid cluster 2," "aa3" is "amino acid cluster 3," "aa4" is "amino acid cluster 4," "aa5" is "amino acid cluster 5," and "aa6" is "amino acid cluster 6," see, e.g., FIG. 10 ). Each occurrence of aa1, aa2, aa3, aa4, aa5, and aa6 is independently selected to be 1 to 5 amino acid residues, which are either i) independently selected from any naturally occurring (e.g., encoded) amino acid, or ii) proline is any naturally occurring amino acid except glycine.

[0106] In some examples, the MHC polypeptide comprises a single amino acid substitution compared to a reference MHC polypeptide (which may be a wild-type MHC polypeptide), where the single amino acid substitution replaces an amino acid with a cysteine ​​(Cys) residue that, when present in an MHC polypeptide of a first polypeptide of a TMMP of the present disclosure, can form a disulfide bond with a cysteine ​​residue present in a second polypeptide chain of the TMMP.

[0107] In some examples, the first MHC polypeptide within the first polypeptide of the TMMP and / or the second MHC polypeptide within the second polypeptide of the TMMP comprises an amino acid substitution substituting an amino acid with a cysteine, wherein the substituted cysteine ​​in the first MHC polypeptide forms a disulfide bond with the cysteine ​​in the second MHC polypeptide, the cysteine ​​in the first MHC polypeptide forms a disulfide bond with the substituted cysteine ​​in the second MHC polypeptide, or the substituted cysteine ​​in the first MHC polypeptide forms a disulfide bond with the substituted cysteine ​​in the second MHC polypeptide.

[0108] For example, in some instances, the following pairs of residues in HLA β2-microglobulin and HLA class I heavy chains are present: 1) β2M residue 12, HLA class I heavy chain residue 236; 2) β2M residue 12, HLA class I heavy chain residue 237; 3) β2M residue 8, HLA class I heavy chain residue 234; 4) β2M residue 10, HLA class I heavy chain residue 235; 5) β2M residue 24, HLA class I heavy chain residue 236; 6) β2M residue 28, HLA class I heavy chain residue 232; 7) β2M residue 98, HLA class I heavy chain residue residue 192, 8) β2M residue 99, HLA class I heavy chain residue 234, 9) β2M residue 3, HLA class I heavy chain residue 120, 10) β2M residue 31, HLA class I heavy chain residue 96, 11) β2M residue 53, HLA class I heavy chain residue 35, 12) β2M residue 60, HLA class I heavy chain residue 96, 13) β2M residue 60, HLA class I heavy chain residue 122, 14) β2M residue 63, HLA class I heavy chain residue 27, 15) β2M residue Arg3, HLA class I heavy chain residue Gly120, 16) β2M residue Hi s31, HLA class I heavy chain residue Gln96, 17) β2M residue Asp53, HLA class I heavy chain residue Arg35, 18) β2M residue Trp60, HLA class I heavy chain residue Gln96, 19) β2M residue Trp60, HLA class I heavy chain residue Asp122, 20) β2M residue Tyr63, HLA class I heavy chain residue Tyr27, 21) β2M residue Lys6, HLA class I heavy chain residue Glu232, 22) β2M residue Gln8, HLA class I heavy chain residue Arg234, 23) β2M residue Tyr1 One of the following residues is substituted with cysteine: 1) HLA class I heavy chain residue Pro235; 2) β2M residue Ser11; 2) HLA class I heavy chain residue Gln242; 25) β2M residue Asn24; 26) β2M residue Ser28; 27) β2M residue Asp98; 28) β2M residue Met99; and 30) HLA class I heavy chain residue Arg234 (residue numbers refer to the mature polypeptide). The amino acid numbering of the MHC / HLA class I heavy chain is based on the mature MHC / HLA class I heavy chain without the signal peptide. For example, in some cases, residue 236 of the mature HLA-A amino acid sequence is substituted with Cys.In some examples, residue 236 of the mature HLA-B amino acid sequence is substituted with Cys. In some examples, residue 236 of the mature HLA-C amino acid sequence is substituted with Cys. In some examples, residue 32 of the amino acid sequence set forth in Figure 6 (corresponding to Arg-12 of mature β2M) is substituted with Cys.

[0109] In some instances, the β2M polypeptide has the amino acid sequence: TIFF2025186334000034.tif22165 (SEQ ID NO: 310). In some examples, the β2M polypeptide comprises the amino acid sequence: Contains TIFF2025186334000035.tif22165 (sequence number 311).

[0110] In some instances, the HLA class I heavy chain polypeptide is HLA-A * 2402 amino acid sequence, Contains TIFF2025186334000036.tif35165 (sequence number 455).

[0111] In some instances, the HLA class I heavy chain polypeptide is HLA-A * 2402 amino acid sequence, Contains TIFF2025186334000037.tif36165 (sequence number 456).

[0112] In some instances, the HLA class I heavy chain polypeptide has the amino acid sequence: Contains TIFF2025186334000038.tif34165 (sequence number 459).

[0113] In some instances, the HLA class I heavy chain polypeptide has the amino acid sequence: Contains TIFF2025186334000039.tif33165 (sequence number 457).

[0114] In some instances, the HLA class I heavy chain polypeptide has the amino acid sequence: Contains TIFF2025186334000040.tif36165 (sequence number 458).

[0115] In some instances, the HLA class I heavy chain polypeptide has the amino acid sequence: Contains TIFF2025186334000041.tif34165 (sequence number 346).

[0116] In some instances, the HLA class I heavy chain polypeptide has the amino acid sequence: Contains TIFF2025186334000042.tif43165 (SEQ ID NO: 44).

[0117] In some instances, the HLA class I heavy chain polypeptide has the amino acid sequence: Contains TIFF2025186334000043.tif43165 (sequence number 312).

[0118] In some instances, the HLA class I heavy chain polypeptide has the amino acid sequence: Contains TIFF2025186334000044.tif43165 (SEQ ID NO: 46).

[0119] In some instances, the β2M polypeptide has the amino acid sequence: TIFF2025186334000045.tif15165 (SEQ ID NO: 311), The HLA class I heavy chain polypeptide of TMMP has the following amino acid sequence: TIFF2025186334000046.tif43165 (SEQ ID NO: 312) (the underlined and bold Cys residues form disulfide bonds with each other within TMMP).

[0120] In some instances, the β2M polypeptide has the amino acid sequence: TIFF2025186334000047.tif16165 (SEQ ID NO: 311), The HLA class I heavy chain polypeptide of TMMP has the following amino acid sequence: TIFF2025186334000048.tif33165 (SEQ ID NO: 457) (the Cys residue at amino acid 236 of the HLA class I heavy chain polypeptide and the Cys at residue 12 of the β2M polypeptide form a disulfide bond with each other within TMMP).

[0121] In some instances, the β2M polypeptide has the amino acid sequence: Contains TIFF2025186334000049.tif17165 (sequence number 311).

[0122] In some examples, the first and second polypeptides of TMMP are disulfide-bonded to each other via i) a Cys residue present in the linker connecting the peptide epitope in the first polypeptide chain and the β2M polypeptide, and ii) a Cys residue present in the MHC class I heavy chain in the second polypeptide chain. In some examples, the Cys residue present in the MHC class I heavy chain is a Cys introduced as a Y84C substitution. In some examples, the linker connecting the peptide epitope in the first polypeptide chain and the β2M polypeptide is GCGGS(G4S)n (SEQ ID NO: 315) (n is 1, 2, 3, 4, 5, 6, 7, 8, or 9). For example, in some examples, the linker comprises the amino acid sequence GCGGSGGGGSGGGGSGGGGS (SEQ ID NO: 316). In another example, the linker comprises the amino acid sequence GCGGSGGGGSGGGGS (SEQ ID NO: 317). Examples of disulfide-linked first and second polypeptides of TMMP are shown schematically in Figures 2A-2F.

[0123] Multi-disulfide bond TMMP In some examples, the first and second polypeptides of the TMMPs of the present disclosure are linked to one another by at least two disulfide bonds (i.e., two interchain disulfide bonds). Examples of such multiply disulfide-linked TMMPs are shown schematically in Figures 17A and 17B and Figures 18A-18C. Furthermore, when a TMMP includes an IgFc polypeptide, the heterodimeric TMMP can be dimerized, with a disulfide bond linking the IgFc polypeptides in the two heterodimeric TMMPs. Such an arrangement is shown schematically in Figures 17C and 17D, where the disulfide bonds are represented by dashed lines. Unless otherwise specified, the reference to at least two disulfide bonds in the multiply disulfide-linked TMMPPs in this section does not refer to the disulfide bond linking the IgFc polypeptides of the dimerized TMMP.

[0124] As described above, in some examples, the first and second polypeptides of the TMMP are linked to each other by at least two disulfide bonds (i.e., two interchain disulfide bonds). For example, in some examples, the first and second polypeptides of the TMMP are linked to each other by two interchain disulfide bonds. As another example, in some examples, the first and second polypeptides of the TMMP are linked to each other by three interchain disulfide bonds. As another example, in some examples, the first and second polypeptides of the TMMP of the present disclosure are linked to each other by four interchain disulfide bonds.

[0125] In some examples, when a peptide epitope in a first polypeptide of TMMP is linked to a β2M polypeptide by a linker containing Cys, at least one of the at least two disulfide bonds links a Cys in the linker to a Cys of an MHC class I heavy chain in a second polypeptide. In some examples, when a peptide epitope in a first polypeptide of TMMP is linked to an MHC class I heavy chain polypeptide by a linker, at least one of the at least two disulfide bonds links a Cys in the linker to a Cys in a β2M polypeptide present in the second polypeptide.

[0126] In some examples, a multiple disulfide bond TMMP (e.g., a double disulfide bond TMMP) exhibits increased stability compared to a control TMMP containing only one of at least two disulfide bonds. In some examples, a multiple disulfide bond TMMP (e.g., a double disulfide bond TMMP) exhibits increased in vitro stability compared to a control TMMP containing only one of at least two disulfide bonds. For example, in some examples, a multiple disulfide bond TMMP (e.g., a double disulfide bond TMMP) exhibits at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 50%, at least 2-fold, at least 5-fold, or at least 10-fold increased in vitro stability compared to a control TMMP containing only one of at least two disulfide bonds.

[0127] Whether a multiply disulfide-bonded TMMP (e.g., a double-disulfide-bonded TMMP) exhibits increased in vitro stability compared to a control TMMP containing only one of the at least two disulfide bonds can be determined by measuring the amount of disulfide-bonded heterodimeric TMMP present in a sample over time and / or under specific conditions and / or during purification of the TMMP.

[0128] For example, in some instances, a multiple disulfide bonded TMMP (e.g., a double disulfide bonded TMMP) exhibits at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 50%, at least 2-fold, at least 5-fold, or at least 10-fold greater in vitro stability than a control TMMP containing only one of the at least two disulfides when the TMMP is stored at 37°C for a period of time (e.g., from about 1 week to about 2 weeks, from about 2 weeks to about 4 weeks, or from about 4 weeks to about 2 months). For example, in some instances, the amount of disulfide-linked heterodimer TMMP remaining after in vitro storage of a multiply disulfide-linked TMMP (e.g., a double-disulfide-linked TMMP) at 37°C for 28 days is at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 50%, at least 2-fold, at least 5-fold, or at least 10-fold greater than the amount of disulfide-linked heterodimer TMMP remaining after in vitro storage of a control TMMP (a TMMP containing only one of the at least two disulfide bonds present in the multiply disulfide-linked TMMP) at 37°C for 28 days.

[0129] As an example, as shown in Figures 14A and 14B, a double-disulfide-bonded TMMP comprising polypeptides 1715 and 2380 exhibits higher in vitro stability than a TMMP comprising polypeptides 2405 and 2380 (polypeptide 2405 is shown in Figure 14D), which contains only one disulfide bond, the single disulfide bond being formed between i) a Cys in the G2C linker between the epitope and β2M and ii) a Cys provided by the Y84C substitution in the MHC class I heavy chain. As another example, as shown in Figures 14A and 14B, a double-disulfide-bonded TMMP comprising polypeptides 1715 and 2380 exhibits higher in vitro stability than a TMMP comprising polypeptides 1380 and 2380 (polypeptide 1380 is shown in Figure 14E), which contains only one disulfide bond, the single disulfide bond being formed between i) a Cys provided by the R12C substitution in the β2M polypeptide and ii) a Cys provided by the A236C substitution in the MHC class I heavy chain.

[0130] In some instances, the multiple disulfide bond TMMP exhibits greater in vivo stability than a control TMMP containing only one of the at least two disulfide bonds, e.g., in some instances, the multiple disulfide bond TMMP exhibits at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 50%, at least 2-fold, at least 5-fold, or at least 10-fold greater in vivo stability than a control TMMP containing only one of the at least two disulfide bonds.

[0131] In some examples, the presence of two disulfide bonds in a multiply disulfide-bonded TMMP (e.g., a double-disulfide-bonded TMMP) increases the amount of disulfide-bonded heterodimeric TMMP produced compared to the amount of disulfide-bonded heterodimeric TMMP produced when the TMMP is a control TMMP containing only one of the at least two disulfide bonds. For example, a multiply disulfide-bonded TMMP (e.g., a double-disulfide-bonded TMMP) can be produced in mammalian cells during in vitro cell culture, where the mammalian cells are cultured in a liquid cell culture medium. The TMMP can be secreted into the cell culture medium. The cells can be lysed to produce a cell lysate, and the TMMP can be present in the cell lysate. The TMMP can be purified from the cell culture medium and / or the cell lysate. For example, if the TMMP contains an IgG1 Fc polypeptide, the cell culture medium and / or the cell lysate can be contacted with immobilized Protein A (e.g., the cell culture medium and / or the cell lysate can be applied to a Protein A column in which Protein A is immobilized on beads). TMMP present in the cell culture medium and / or cell lysate binds to the immobilized Protein A. After washing the column to remove unbound material, the bound TMMP is eluted to produce a Protein A eluate. The amount of disulfide-linked heterodimeric TMMP present in the Protein A eluate is at least 0.5%, at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, or at least 10% higher than the amount of disulfide-linked heterodimeric TMMP present in the Protein A eluate when the TMMP is a control TMMP containing only one of the at least two disulfide bonds present in a multi-disulfide-linked TMMP (e.g., a double-disulfide-linked TMMP). In some examples, the percentage of total TMMP protein in the eluate that is unaggregated disulfide-linked heterodimeric TMMP is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%.The Protein A eluate may be subjected to size exclusion chromatography (SEC) and / or one or more other additional purification steps.

[0132] In some examples, the TMMP comprises at least one heterodimer comprising: a) i) a WT1 peptide epitope (the WT1 peptide has a length of at least 4 amino acids (e.g., 4 to 25 amino acids, e.g., the WT1 peptide has a length of 4, 5, 6, 7, 8, 9, 10 to 15, 15 to 20, or 20 to 25 amino acids)), and ii) a first polypeptide comprising a first MHC polypeptide, b) a second polypeptide comprising a second MHC polypeptide, and c) at least one MOD (the first and / or second polypeptide comprises a MOD, and the heterodimer comprises two disulfide bonds between the first and second polypeptides (i.e., the heterodimer comprises i) a first disulfide bond linking the first and second polypeptides, and ii) a second disulfide bond linking the first and second polypeptides). In other words, the first polypeptide contains a first Cys residue that forms a disulfide bond (first disulfide bond) with a first Cys residue of the second polypeptide, and the first polypeptide contains a second Cys residue that forms a disulfide bond (second disulfide bond) with a second Cys residue of the second polypeptide.

[0133] In some examples, the TMMP comprises a) a first polypeptide comprising, in order from N-terminus to C-terminus, i) a peptide epitope, ii) a peptide linker, and iii) a β2M polypeptide; and b) a second polypeptide comprising an MHC class I heavy chain polypeptide, wherein one or both of the first and second polypeptides comprise at least one MOD, wherein the TMMP comprises a) a first disulfide bond between i) a Cys present in the linker between the Cys peptide epitope and the β2M polypeptide and ii) a first Cys introduced into the MHC class I heavy chain polypeptide, and b) at least a second disulfide bond between the first and second polypeptides, wherein the at least second disulfide bond is between i) a Cys in the first polypeptide C-terminal to the Cys present in the linker and ii) a Cys in the second polypeptide C-terminal to the first Cys introduced into the MHC class I heavy chain polypeptide.

[0134] In some examples, the first and second disulfide bond-forming Cys residues in the first or second polypeptide of TMMP are separated from each other by about 10 to about 200 amino acids. For example, in some examples, the first and second disulfide bond-forming Cys residues in the first or second polypeptide of TMMP are separated from each other by about 10 amino acids (aa) to about 15 aa, about 15 aa to about 20 aa, about 20 aa to about 25 aa, about 25 aa to about 30 aa, about 30 aa to about 40 aa, about 40 aa to about 50 aa, about 50 aa to about 60 aa, about 60 aa to about 70 aa, or about 70 aa to about 80 aa. , about 80aa to about 90aa, about 90aa to about 100aa, about 100aa to about 110aa, about 110aa to about 120aa, about 120aa to about 130aa, about 130aa to about 140aa, about 140aa to about 150aa, about 150aa to about 160aa, about 160aa to about 170aa, about 170aa to about 180aa, about 180aa to about 190aa, or about 190aa to about 200aa.

[0135] For example, in some cases, the first and second disulfide bond-forming Cys residues in the first polypeptide of TMMP are separated from each other by about 10 to about 80 amino acid residues. For example, in some cases, the second disulfide bond-forming Cys residue in the first polypeptide is located about 10 to about 80 amino acids (e.g., about 10 amino acids (aa) to about 15 aa, about 15 aa to about 20 aa, about 20 aa to about 25 aa, about 25 aa to about 30 aa, about 30 aa to about 40 aa, about 40 aa to about 50 aa, about 50 aa to about 60 aa, about 60 aa to about 70 aa, or about 70 aa to about 80 aa) C-terminal to the first disulfide bond-forming Cys residue in the first polypeptide. In some examples, the second disulfide bond-forming Cys residue in the first polypeptide is 10 aa, 11 aa, 12 aa, 13 aa, 14 aa, 15 aa, 16 aa, 17 aa, 18 aa, 19 aa, 20 aa, 21 aa, 22 aa, 23 aa, 24 aa, or 25 aa C-terminal to the first disulfide bond-forming Cys residue in the first polypeptide. In some examples, the second disulfide bond-forming Cys residue in the first polypeptide is 15 aa C-terminal to the first disulfide bond-forming Cys residue in the first polypeptide. In some examples, the second disulfide bond-forming Cys residue in the first polypeptide is 20 aa C-terminal to the first disulfide bond-forming Cys residue in the first polypeptide. In some examples, the second disulfide bond-forming Cys residue of the first polypeptide is 25 aa C-terminal to the first disulfide bond-forming Cys residue of the first polypeptide.

[0136] In some examples, the first and second disulfide bond-forming Cys residues in the second polypeptide of a TMMP of the present disclosure are separated from each other by about 140 to about 160 amino acids. For example, in some examples, the second disulfide bond-forming Cys residue of the second polypeptide is about 140 to about 160 amino acids C-terminal to the first disulfide bond-forming Cys residue of the second polypeptide. In some examples, the second disulfide bond-forming Cys residue of the second polypeptide is 140 amino acids (aa), 141aa, 142aa, 143aa, 144aa, 145aa, 146aa, 147aa, 148aa, 149aa, 150aa, 151aa, 152aa, 153aa, 154aa, 155aa, 156aa, 157aa, 158aa, 159aa, or 160aa C-terminal to the first disulfide bond-forming Cys residue in the second polypeptide.

[0137] The multiple disulfide-bonded TMMP (e.g., double disulfide-bonded TMMP) includes a) a first polypeptide comprising i) a WT1 peptide (e.g., a WT1 peptide of 4 to about 25 amino acids) and ii) a first MHC polypeptide (the first polypeptide comprises a peptide linker between the WT1 peptide and the first MHC polypeptide, the peptide linker comprising a Cys residue, and the first MHC polypeptide comprising a β2M polypeptide comprising an amino acid substitution introducing a Cys residue); b) a second polypeptide comprising a β2M polypeptide and a second MHC polypeptide (the second MHC polypeptide comprises an HLA-A *and (c) a Class I heavy chain containing a Y84C substitution and an A236C substitution based on amino acid numbering of β2M polypeptide (as shown in Figure 9A), or a substitution at the corresponding position in another Class I heavy chain allele, wherein the TMMP has a disulfide bond between a Cys residue in the peptide linker and a Cys residue at amino acid position 84 of the Class I heavy chain or the corresponding position in another Class I heavy chain allele, and the TMMP has a disulfide bond between a Cys residue introduced into the β2M polypeptide and a Cys residue at amino acid position 236 of the Class I heavy chain or the corresponding position in another Class I heavy chain allele), and (d) at least one MOD, wherein the first and / or second polypeptides contain at least one MOD. Examples are shown schematically in Figures 17A and 17B.

[0138] In some examples, the peptide linker comprises the amino acid sequence GCGGS (SEQ ID NO: 318). In some examples, the peptide linker comprises the amino acid sequence GCGGS(GGGGS)n (SEQ ID NO: 319), wherein n is an integer between 1 and 10. In some examples, the peptide linker comprises the amino acid sequence GCGGS(GGGGS)n (SEQ ID NO: 398), wherein n is 1. In some examples, the peptide linker comprises the amino acid sequence GCGGS(GGGGS)n (SEQ ID NO: 320), wherein n is 2. In some examples, the peptide linker comprises the amino acid sequence GCGGS(GGGGS)n (SEQ ID NO: 321), wherein n is 3. In some examples, the peptide linker comprises the amino acid sequence GCGGS(GGGGS)n (SEQ ID NO: 322), wherein n is 4. In some examples, the peptide linker comprises the amino acid sequence GCGGS(GGGGS)n (SEQ ID NO: 323), wherein n is 5. In some examples, the peptide linker comprises the amino acid sequence GCGGS(GGGGS)n (SEQ ID NO: 324), wherein n is 6. In some examples, the peptide linker comprises the amino acid sequence GCGGS(GGGGS)n (SEQ ID NO: 325), wherein n is 7. In some examples, the peptide linker comprises the amino acid sequence GCGGS(GGGGS)n (SEQ ID NO: 326), wherein n is 8. In some examples, the peptide linker comprises the amino acid sequence GCGGS(GGGGS)n (SEQ ID NO: 327), wherein n is 9. In some examples, the peptide linker comprises the amino acid sequence GCGGS(GGGGS)n (SEQ ID NO: 328), wherein n is 10.

[0139] In some examples, the peptide linker comprises the amino acid sequence CGGGS (SEQ ID NO: 329). In some examples, the peptide linker comprises the amino acid sequence CGGGS(GGGGS)n (SEQ ID NO: 330), wherein n is an integer between 1 and 10. In some examples, the peptide linker comprises the amino acid sequence CGGGS(GGGGS)n (SEQ ID NO: 331), wherein n is 1. In some examples, the peptide linker comprises the amino acid sequence CGGGS(GGGGS)n (SEQ ID NO: 332), wherein n is 2. In some examples, the peptide linker comprises the amino acid sequence CGGGS(GGGGS)n (SEQ ID NO: 333), wherein n is 3. In some examples, the peptide linker comprises the amino acid sequence CGGGS(GGGGS)n (SEQ ID NO: 334), wherein n is 4. In some examples, the peptide linker comprises the amino acid sequence CGGGS(GGGGS)n (SEQ ID NO: 335), wherein n is 5. In some examples, the peptide linker comprises the amino acid sequence CGGGS(GGGGS)n (SEQ ID NO: 336), wherein n is 6. In some examples, the peptide linker comprises the amino acid sequence CGGGS(GGGGS)n (SEQ ID NO: 337), wherein n is 7. In some examples, the peptide linker comprises the amino acid sequence CGGGS(GGGGS)n (SEQ ID NO: 338), wherein n is 8. In some examples, the peptide linker comprises the amino acid sequence CGGGS(GGGGS)n (SEQ ID NO: 339), wherein n is 9. In some examples, the peptide linker comprises the amino acid sequence CGGGS(GGGGS)n (SEQ ID NO: 340), wherein n is 10.

[0140] The following are HLA-A * 9A-9C are non-limiting examples of MHC class I heavy chains containing the Y84C and A236C substitutions based on the amino acid numbering of 0201 (shown in FIG. 9A), or substitutions at the corresponding positions in another class I heavy chain allele.

[0141] HLA-A In some examples, a multiply disulfide-bonded TMMP (e.g., a double-disulfide-bonded TMMP) comprises: a) a first polypeptide comprising: i) a WT1 peptide (e.g., a WT1 peptide of 4 amino acids to about 25 amino acids); and ii) a first MHC polypeptide (the first polypeptide comprises a peptide linker between the WT1 peptide and the first MHC polypeptide, the peptide linker comprising a Cys residue, and the first MHC polypeptide being a β2M polypeptide comprising an amino acid substitution introducing a Cys residue); and b) the following amino acid sequence: and c) a second polypeptide comprising an HLA-A MHC class I heavy chain comprising an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF2025186334000050.tif38165 (SEQ ID NO: 342) (amino acid 84 is Cys and amino acid 236 is Cys), and at least one MOD, wherein the first and / or second polypeptide comprises at least one MOD. In some examples, the peptide linker comprises the amino acid sequence GCGGS (SEQ ID NO: 318). In some examples, the peptide linker comprises the amino acid sequence GCGGS(GGGGS)n (SEQ ID NO: 319), where n is an integer between 1 and 10. In some examples, the β2M polypeptide comprises an R12C substitution. For example, the β2M polypeptide may comprise the following amino acid sequence: The at least one MOD can be a polypeptide that confers an activating / stimulatory effect on target T cells or a suppressive / inhibitory effect on target T cells. For example, at least one MOD is a cytokine (e.g., an IL2 polypeptide, an IL7 polypeptide, an IL12 polypeptide, an IL15 polypeptide, an IL17 polypeptide, an IL21 polypeptide, an IL27 polypeptide, an IL-23 polypeptide, a TGFβ polypeptide, etc., including all family members, e.g., IL17A, IL-17B, IL-17C, IL-17D, IL-17E, IL-17F, IL-17E), a 4-1BBL polypeptide, an ICOS-L polypeptide, an OX-40L polypeptide, a CD80 polypeptide, a CD86 polypeptide (CD80 and CD86 are also known as B7-1 and B7-2, respectively), a CD40 polypeptide, a CD70 polypeptide, a JAG1 (CD339) polypeptide, an ICAM (CD540 polypeptide), a PD-L1 polypeptide, a FasL polypeptide, a PD-L2 polypeptide, The MOD may be a wild-type polypeptide, a PD-1H (VISTA) polypeptide, an ICOS-L (CD275) polypeptide, a GITRL polypeptide, an HVEM polypeptide, a CXCL10 polypeptide, a CXCL9 polypeptide, a CXCL11 polypeptide, a CXCL13 polypeptide, and a CX3CL1 polypeptide, a galectin-9 polypeptide, a CD83 polypeptide, a CD30L polypeptide, an HLA-G polypeptide, a MICA polypeptide, a MICB polypeptide, an HVEM (CD270) polypeptide, a lymphotoxin beta receptor polypeptide, a 3 / TR6 polypeptide, an ILT3 polypeptide, an ILT4 polypeptide, a CXCL10 polypeptide, a CXCL9 polypeptide, a CXCL11 polypeptide, a CXCL13 polypeptide, or a CX3CL1 polypeptide. These MODs may be wild-type polypeptides or variant forms of the wild-type polypeptides.In some examples, a MOD is an activating ("stimulatory") MOD, e.g., the MOD can produce an activating / stimulatory effect on T cells. Examples of activating MODs include, e.g., CD80, CD86, 4-1BBL, OX40L, CD70, ICOS-L, CD40, ICAM (CD54), IL2, IL7, IL12, IL15, IL17, IL21, IL27, IL23, GITRL, TGFβ, and lymphotoxin β receptor. In some examples, a MOD is an inhibitory ("inhibitory") MOD, e.g., the MOD can produce an inhibitory / inhibitory effect on T cells. Examples of inhibitory MODs include, e.g., PD-1H, PD-L1, PD-L2, TGFβ, FasL, HVEM, galectin-9, ILT3, and ILT4. TGFβ polypeptides can produce either an activating / stimulatory effect or an inhibitory / inhibitory effect, depending on the context.

[0142] In some examples, the multiple disulfide-bonded TMMP (e.g., the double disulfide-bonded TMMP) comprises a) a first polypeptide comprising i) a WT1 peptide (e.g., a WT1 peptide of 4 amino acids to about 25 amino acids), and ii) a first MHC polypeptide (the first polypeptide comprises a peptide linker between the WT1 peptide and the first MHC polypeptide, the peptide linker comprising a Cys residue, and the first MHC polypeptide being a β2M polypeptide comprising an amino acid substitution introducing a Cys residue), and b) a second polypeptide comprising an HLA-A MHC class I heavy chain comprising an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: TIFF2025186334000052.tif35165 (SEQ ID NO: 346) (amino acid 84 is Cys and amino acid 236 is Cys), and c) at least one MOD (the first and / or second polypeptides comprise at least one immunomodulatory polypeptide). In some examples, the peptide linker comprises the amino acid sequence GCGGS (SEQ ID NO: 318). In some examples, the peptide linker comprises the amino acid sequence GCGGS(GGGGS)n (SEQ ID NO: 319), where n is an integer between 1 and 10. In some examples, the β2M polypeptide comprises an R12C substitution. For example, the β2M polypeptide can comprise an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: TIFF2025186334000053.tif16165 (SEQ ID NO: 311) (amino acid 12 is Cys). The at least one MOD can be a polypeptide that exerts an activating / stimulatory effect on target T cells or a suppressive / inhibitory effect on target T cells. For example, at least one MOD is a cytokine (e.g., an IL2 polypeptide, an IL7 polypeptide, an IL12 polypeptide, an IL15 polypeptide, an IL17 polypeptide, an IL21 polypeptide, an IL27 polypeptide, an IL-23 polypeptide, a TGFβ polypeptide, etc., including all family members, e.g., IL17A, IL-17B, IL-17C, IL-17D, IL-17E, IL-17F, IL-17E), a 4-1BBL polypeptide, an ICOS-L polypeptide, an OX-40L polypeptide, a CD80 polypeptide, a CD86 polypeptide (CD80 and CD86 are also known as B7-1 and B7-2, respectively), a CD40 polypeptide, a CD70 polypeptide, a JAG1 (CD339) polypeptide, an ICAM (CD540 polypeptide), a PD-L1 polypeptide, a FasL polypeptide, a PD-L2 polypeptide, The MOD may be a wild-type polypeptide, a PD-1H (VISTA) polypeptide, an ICOS-L (CD275) polypeptide, a GITRL polypeptide, an HVEM polypeptide, a CXCL10 polypeptide, a CXCL9 polypeptide, a CXCL11 polypeptide, a CXCL13 polypeptide, a CX3CL1 polypeptide, a galectin-9 polypeptide, a CD83 polypeptide, a CD30L polypeptide, an HLA-G polypeptide, a MICA polypeptide, a MICB polypeptide, an HVEM (CD270) polypeptide, a lymphotoxin beta receptor polypeptide, a 3 / TR6 polypeptide, an ILT3 polypeptide, an ILT4 polypeptide, a CXCL10 polypeptide, a CXCL9 polypeptide, a CXCL11 polypeptide, a CXCL13 polypeptide, and a CX3CL1 polypeptide. These MODs may be wild-type polypeptides or variant forms of the wild-type polypeptides.Among these, the following MODs can produce an activating / stimulatory effect: CD80, CD86, 4-1BBL, OX40L, CD70, ICOS-L, CD40, ICAM (CD54), IL2, IL7, IL12, IL15, IL17, IL21, IL27, IL23, GITRL, TGFβ, lymphotoxin β receptor, 3 / TR6, ILT3, ILT4, CXCL10, CXCL9, CXCL11, CXCL13, and CX3CL1. Among these, the following MODs can produce an inhibitory / inhibitory effect: PD-1H, PD-L1, PD-L2, TGFβ, FasL, HVEM, galectin-9, ILT3, and ILT4. TGFβ polypeptides can produce either an activating / stimulatory effect or an inhibitory / inhibitory effect, depending on the context. In some examples, at least one MOD is a variant with reduced affinity, as described elsewhere herein. In some examples, the first or second polypeptide comprises an Ig Fc polypeptide.

[0143] In some examples, at least one MOD is a variant with reduced affinity, as described elsewhere herein. In some examples, the first or second polypeptide comprises an Ig Fc polypeptide.

[0144] In some examples, the multiply disulfide-bonded TMMP (e.g., double disulfide-bonded TMMP) comprises an HLA-A class I heavy chain polypeptide. In some examples, the HLA-A heavy chain polypeptide present in the multiply disulfide-bonded TMMP (e.g., double disulfide-bonded TMMP) of the present disclosure comprises an HLA-A class I heavy chain polypeptide shown in FIG. * 0101, HLA-A * 0201, HLA-A * 0202, HLA-A * 1101, HLA-A * 2301, HLA-A * 2402, HLA-A * 2407, HLA-A * 3303, or HLA-A *3401, and the HLA-A heavy chain polypeptide comprises a Y84C and an A236C substitution.

[0145] HLA-A * 0101(Y84C, A236C) In some instances, the HLA-A heavy chain polypeptide present in the multiply disulfide-bonded TMMP (e.g., the double disulfide-bonded TMMP) is * 0101 (Y84C, A236C) amino acid sequence: TIFF2025186334000054.tif36165 (SEQ ID NO: 343), wherein amino acid 84 is Cys and amino acid 236 is Cys.

[0146] HLA-A * 0201(Y84C, A236C) In some instances, the HLA-A heavy chain polypeptide present in the multiply disulfide-bonded TMMP (e.g., the double disulfide-bonded TMMP) is selected from the group consisting of: * 0201 (Y84C, A236C) amino acid sequence: TIFF2025186334000055.tif36165 (SEQ ID NO: 342), wherein amino acid 84 is Cys and amino acid 236 is Cys.

[0147] HLA-A * 0202(Y84C, A236C) In some instances, the HLA-A heavy chain polypeptide present in the multiply disulfide-bonded TMMP (e.g., the double disulfide-bonded TMMP) is * 0202 (Y84C, A236C) amino acid sequence: TIFF2025186334000056.tif36165 (SEQ ID NO: 341), wherein amino acid 84 is Cys and amino acid 236 is Cys.

[0148] HLA-A * 1101(Y84C, A236C) In some instances, the HLA-A heavy chain polypeptide present in the multiply disulfide-bonded TMMP (e.g., the double disulfide-bonded TMMP) is * 1101(Y84C, A236C) amino acid sequence: TIFF2025186334000057.tif36165 (SEQ ID NO: 344), wherein amino acid 84 is Cys and amino acid 236 is Cys.

[0149] HLA-A * 2301(Y84C, A236C) In some instances, the HLA-A heavy chain polypeptide present in the multiply disulfide-bonded TMMP (e.g., the double disulfide-bonded TMMP) is * 2301(Y84C, A236C) amino acid sequence: TIFF2025186334000058.tif36165 (SEQ ID NO: 345), wherein amino acid 84 is Cys and amino acid 236 is Cys.

[0150] HLA-A * 2402(Y84C, A236C) In some instances, the HLA-A heavy chain polypeptide present in the multiply disulfide-bonded TMMP (e.g., the double disulfide-bonded TMMP) is * 2402 (Y84C, A236C) amino acid sequence: TIFF2025186334000059.tif36165 (SEQ ID NO: 346), wherein amino acid 84 is Cys and amino acid 236 is Cys.

[0151] HLA-A * 2407(Y84C, A236C) In some instances, the HLA-A heavy chain polypeptide present in the multiply disulfide-bonded TMMP (e.g., the double disulfide-bonded TMMP) is * 2407(Y84C, A236C) amino acid sequence: TIFF2025186334000060.tif36165 (SEQ ID NO: 347), wherein amino acid 84 is Cys and amino acid 236 is Cys.

[0152] HLA-A * 3303(Y84C, A236C) In some instances, the HLA-A heavy chain polypeptide present in the multiply disulfide-bonded TMMP (e.g., the double disulfide-bonded TMMP) is * 3303 (Y84C, A236C) amino acid sequence: TIFF2025186334000061.tif36165 (SEQ ID NO: 348), wherein amino acid 84 is Cys and amino acid 236 is Cys.

[0153] HLA-A * 3401(Y84C, A236C) In some instances, the HLA-A heavy chain polypeptide present in the multiply disulfide-bonded TMMP (e.g., the double disulfide-bonded TMMP) is * 3401 (Y84C, A236C) amino acid sequence: TIFF2025186334000062.tif36165 (SEQ ID NO: 349), wherein amino acid 84 is Cys and amino acid 236 is Cys.

[0154] HLA-B In some examples, a multiply disulfide-bonded TMMP (e.g., a double-disulfide-bonded TMMP) comprises: a) a first polypeptide comprising: i) a WT1 peptide (e.g., a WT1 peptide of 4 amino acids to about 25 amino acids); and ii) a first MHC polypeptide (the first polypeptide comprises a peptide linker between the WT1 peptide and the first MHC polypeptide, the peptide linker comprising a Cys residue, and the first MHC polypeptide being a β2M polypeptide comprising an amino acid substitution introducing a Cys residue); and b) the following amino acid sequence: and c) a second polypeptide comprising an HLA-B MHC class I heavy chain comprising an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF2025186334000063.tif36165 (SEQ ID NO: 350) (wherein amino acid 84 is Cys and amino acid 236 is Cys), and at least one MOD, wherein the first and / or second polypeptide comprises at least one MOD. In some examples, the peptide linker comprises the amino acid sequence GCGGS (SEQ ID NO: 318). In some examples, the peptide linker comprises the amino acid sequence GCGGS(GGGGS)n (SEQ ID NO: 319), where n is an integer between 1 and 10. In some examples, the β2M polypeptide comprises an R12C substitution. For example, the β2M polypeptide may comprise the following amino acid sequence: The at least one MOD can comprise an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF2025186334000064.tif15165 (SEQ ID NO: 311), where amino acid 12 is Cys. The at least one MOD can be a polypeptide that confers an activating / stimulatory effect on target T cells or a suppressive / inhibitory effect on target T cells. For example, at least one MOD is a cytokine (e.g., an IL2 polypeptide, an IL7 polypeptide, an IL12 polypeptide, an IL15 polypeptide, an IL17 polypeptide, an IL21 polypeptide, an IL27 polypeptide, an IL-23 polypeptide, a TGFβ polypeptide, etc., including all family members, e.g., IL17A, IL-17B, IL-17C, IL-17D, IL-17E, IL-17F, IL-17E), a 4-1BBL polypeptide, an ICOS-L polypeptide, an OX-40L polypeptide, a CD80 polypeptide, a CD86 polypeptide (CD80 and CD86 are also known as B7-1 and B7-2, respectively), a CD40 polypeptide, a CD70 polypeptide, a JAG1 (CD339) polypeptide, an ICAM (CD540 polypeptide), a PD-L1 polypeptide, a FasL polypeptide, a PD-L2 polypeptide, The MOD may be a wild-type polypeptide, a PD-1H (VISTA) polypeptide, an ICOS-L (CD275) polypeptide, a GITRL polypeptide, an HVEM polypeptide, a CXCL10 polypeptide, a CXCL9 polypeptide, a CXCL11 polypeptide, a CXCL13 polypeptide, and a CX3CL1 polypeptide, a galectin-9 polypeptide, a CD83 polypeptide, a CD30L polypeptide, an HLA-G polypeptide, a MICA polypeptide, a MICB polypeptide, an HVEM (CD270) polypeptide, a lymphotoxin beta receptor polypeptide, a 3 / TR6 polypeptide, an ILT3 polypeptide, an ILT4 polypeptide, a CXCL10 polypeptide, a CXCL9 polypeptide, a CXCL11 polypeptide, a CXCL13 polypeptide, or a CX3CL1 polypeptide. These MODs may be wild-type polypeptides or variant forms of the wild-type polypeptides.In some examples, a MOD is an activating ("stimulatory") immunomodulatory polypeptide; e.g., a MOD can produce an activating / stimulatory effect on T cells. Examples of activating MODs include, e.g., CD80, CD86, 4-1BBL, OX40L, CD70, ICOS-L, CD40, ICAM (CD54), IL2, IL7, IL12, IL15, IL17, IL21, IL27, IL23, GITRL, TGFβ, and lymphotoxin β receptor. In some examples, an immunomodulatory polypeptide is an inhibitory ("inhibitory") MOD; e.g., a MOD can produce an inhibitory / inhibitory effect on T cells. Examples of inhibitory MODs include, e.g., PD-1H, PD-L1, PD-L2, TGFβ, FasL, HVEM, galectin-9, ILT3, and ILT4. TGFβ polypeptides can produce either an activating / stimulatory effect or an inhibitory / inhibitory effect, depending on the context.

[0155] In some examples, at least one MOD is a variant with reduced affinity, as described elsewhere herein. In some examples, the first or second polypeptide comprises an Ig Fc polypeptide.

[0156] In some examples, the multiply disulfide-bonded TMMP (e.g., double disulfide-bonded TMMP) comprises an HLA-B class I heavy chain polypeptide. In some examples, the HLA-B heavy chain polypeptide present in the multiply disulfide-bonded TMMP (e.g., double disulfide-bonded TMMP) comprises an HLA-B class I heavy chain polypeptide shown in FIG. 10A. * 0702, HLA-B * 0801, HLA-B * 1502, HLA-B * 3802, HLA-B * 4001, HLA-B * 4601, or HLA-B * The HLA-B heavy chain polypeptide comprises an amino acid sequence having at least 95%, at least 98%, or at least 99% amino acid sequence identity to the 5301 amino acid sequence, and the HLA-B heavy chain polypeptide comprises a Y84C and an A236C substitution.

[0157] HLA-B* 0702(Y84C, A236C) In some instances, the HLA-B heavy chain polypeptide present in the multiply disulfide-bonded TMMP (e.g., the double disulfide-bonded TMMP) is * 0702 (Y84C, A236C) amino acid sequence: TIFF2025186334000065.tif37165 (SEQ ID NO: 350), wherein amino acid 84 is Cys and amino acid 236 is Cys.

[0158] HLA-B * 0801(Y84C, A236C) In some instances, the HLA-B heavy chain polypeptide present in the multiply disulfide-bonded TMMP (e.g., the double disulfide-bonded TMMP) is * 0801 (Y84C, A236C) amino acid sequence: TIFF2025186334000066.tif37165 (SEQ ID NO: 351), wherein amino acid 84 is Cys and amino acid 236 is Cys.

[0159] HLA-B * 1502(Y84C, A236C) In some instances, the HLA-B heavy chain polypeptide present in the multiply disulfide-bonded TMMP (e.g., the double disulfide-bonded TMMP) is * 1502 (Y84C, A236C) amino acid sequence: TIFF2025186334000067.tif37165 (SEQ ID NO: 352), wherein amino acid 84 is Cys and amino acid 236 is Cys.

[0160] HLA-B* 3802(Y84C, A236C) In some instances, the HLA-B heavy chain polypeptide present in the multiply disulfide-bonded TMMP (e.g., the double disulfide-bonded TMMP) is * 3802 (Y84C, A236C) amino acid sequence: TIFF2025186334000068.tif37165 (SEQ ID NO: 353), wherein amino acid 84 is Cys and amino acid 236 is Cys.

[0161] HLA-B * 4001(Y84C, A2346C) In some instances, the HLA-B heavy chain polypeptide present in the multiply disulfide-bonded TMMP (e.g., the double disulfide-bonded TMMP) is * 4001 (Y84C, A236C) amino acid sequence: TIFF2025186334000069.tif37165 (SEQ ID NO: 354), wherein amino acid 84 is Cys and amino acid 236 is Cys.

[0162] HLA-B * 4601(Y84C, A236C) In some instances, the HLA-B heavy chain polypeptide present in the multiply disulfide-bonded TMMP (e.g., the double disulfide-bonded TMMP) is * 4601 (Y84C, A236C) amino acid sequence: TIFF2025186334000070.tif37165 (SEQ ID NO: 355), wherein amino acid 84 is Cys and amino acid 236 is Cys.

[0163] HLA-B* 5301(Y84C, A236C) In some instances, the HLA-B heavy chain polypeptide present in the multiply disulfide-bonded TMMP (e.g., the double disulfide-bonded TMMP) is * 5301(Y84C, A236C) amino acid sequence: TIFF2025186334000071.tif37165 (SEQ ID NO: 356), wherein amino acid 84 is Cys and amino acid 236 is Cys.

[0164] HLA-C In some examples, a multiply disulfide-bonded TMMP (e.g., a double-disulfide-bonded TMMP) comprises: a) a first polypeptide comprising: i) a WT-1 peptide (e.g., a WT-1 peptide of 4 amino acids to about 25 amino acids); and ii) a first MHC polypeptide (the first polypeptide comprises a peptide linker between the WT-1 peptide and the first MHC polypeptide, the peptide linker comprising a Cys residue, and the first MHC polypeptide being a β2M polypeptide comprising an amino acid substitution introducing a Cys residue); and b) the following amino acid sequence: and c) a second polypeptide comprising an HLA-C MHC class I heavy chain comprising an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF2025186334000072.tif37165 (SEQ ID NO: 357) (wherein amino acid 84 is Cys and amino acid 236 is Cys), and at least one MOD, wherein the first and / or second polypeptide comprises at least one MOD. In some examples, the peptide linker comprises the amino acid sequence GCGGS (SEQ ID NO: 318). In some examples, the peptide linker comprises the amino acid sequence GCGGS(GGGGS)n (SEQ ID NO: 319), where n is an integer between 1 and 10. In some examples, the β2M polypeptide comprises an R12C substitution. For example, the β2M polypeptide may comprise the following amino acid sequence: The at least one MOD can comprise an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF2025186334000073.tif16165 (SEQ ID NO: 311), where amino acid 12 is Cys. The at least one MOD can be a polypeptide that confers an activating / stimulatory effect on target T cells or a suppressive / inhibitory effect on target T cells. For example, at least one MOD is a cytokine (e.g., an IL2 polypeptide, an IL7 polypeptide, an IL12 polypeptide, an IL15 polypeptide, an IL17 polypeptide, an IL21 polypeptide, an IL27 polypeptide, an IL-23 polypeptide, a TGFβ polypeptide, etc., including all family members, e.g., IL17A, IL-17B, IL-17C, IL-17D, IL-17E, IL-17F, IL-17E), a 4-1BBL polypeptide, an ICOS-L polypeptide, an OX-40L polypeptide, a CD80 polypeptide, a CD86 polypeptide (CD80 and CD86 are also known as B7-1 and B7-2, respectively), a CD40 polypeptide, a CD70 polypeptide, a JAG1 (CD339) polypeptide, an ICAM (CD540 polypeptide), a PD-L1 polypeptide, a FasL polypeptide, a PD-L2 polypeptide, The MOD may be a wild-type polypeptide, a PD-1H (VISTA) polypeptide, an ICOS-L (CD275) polypeptide, a GITRL polypeptide, an HVEM polypeptide, a CXCL10 polypeptide, a CXCL9 polypeptide, a CXCL11 polypeptide, a CXCL13 polypeptide, and a CX3CL1 polypeptide, a galectin-9 polypeptide, a CD83 polypeptide, a CD30L polypeptide, an HLA-G polypeptide, a MICA polypeptide, a MICB polypeptide, an HVEM (CD270) polypeptide, a lymphotoxin beta receptor polypeptide, a 3 / TR6 polypeptide, an ILT3 polypeptide, an ILT4 polypeptide, a CXCL10 polypeptide, a CXCL9 polypeptide, a CXCL11 polypeptide, a CXCL13 polypeptide, or a CX3CL1 polypeptide. These MODs may be wild-type polypeptides or variant forms of the wild-type polypeptides.In some examples, the immunomodulatory polypeptide is an activating ("stimulatory") MOD, e.g., the MOD can produce an activating / stimulatory effect on T cells. Examples of activating immunomodulatory polypeptides include, e.g., CD80, CD86, 4-1BBL, OX40L, CD70, ICOS-L, CD40, ICAM (CD54), IL2, IL7, IL12, IL15, IL17, IL21, IL27, IL23, GITRL, TGFβ, and lymphotoxin β receptor. In some examples, the MOD is an inhibitory ("inhibitory") MOD, e.g., the MOD can produce an inhibitory / inhibitory effect on T cells. Examples of inhibitory MODs include, e.g., PD-1H, PD-L1, PD-L2, TGFβ, FasL, HVEM, galectin-9, ILT3, and ILT4. TGFβ polypeptides can produce either an activating / stimulatory effect or an inhibitory / inhibitory effect, depending on the context.

[0165] In some examples, at least one MOD is a variant with reduced affinity, as described elsewhere herein. In some examples, the first or second polypeptide comprises an Ig Fc polypeptide.

[0166] In some examples, the multiply disulfide-bonded TMMP (e.g., double disulfide-bonded TMMP) comprises an HLA-C class I heavy chain polypeptide. In some examples, the HLA-C heavy chain polypeptide present in the multiply disulfide-bonded TMMP (e.g., double disulfide-bonded TMMP) comprises an HLA-C class I heavy chain polypeptide shown in FIG. 11A. * 0102, HLA-C * 0303, HLA-C * 0304, HLA-C * 0401, HLA-C * 0602, HLA-C * 0701, HLA-C * 0702, HLA-C * 0801, or HLA-C * The HLA-C heavy chain polypeptide comprises an amino acid sequence having at least 95%, at least 98%, or at least 99% amino acid sequence identity to the 1502 amino acid sequence, and the HLA-C heavy chain polypeptide comprises a Y84C and an A236C substitution.

[0167] HLA-C * 01:02 (Y84C, A236C) In some instances, the HLA-C heavy chain polypeptide present in the multiply disulfide-bonded TMMP (e.g., the double disulfide-bonded TMMP) is * 01:02 (Y84C, A236C) amino acid sequence: TIFF2025186334000074.tif34165 (SEQ ID NO: 358), wherein amino acid 84 is Cys and amino acid 236 is Cys.

[0168] HLA-C * 0303(Y84C, A236C) In some instances, the HLA-C heavy chain polypeptide present in the multiply disulfide-bonded TMMP (e.g., the double disulfide-bonded TMMP) is * 03:03 (Y84C, A236C) amino acid sequence: TIFF2025186334000075.tif34165 (SEQ ID NO: 359), wherein amino acid 84 is Cys and amino acid 236 is Cys.

[0169] HLA-C * 0304(Y84C, A236C) In some instances, the HLA-C heavy chain polypeptide present in the multiply disulfide-bonded TMMP (e.g., the double disulfide-bonded TMMP) is * 03:04 (Y84C, A236C) amino acid sequence: TIFF2025186334000076.tif34165 (SEQ ID NO: 360), wherein amino acid 84 is Cys and amino acid 236 is Cys.

[0170] HLA-C * 0401(Y84C, A236C) In some instances, the HLA-C heavy chain polypeptide present in the multiply disulfide-bonded TMMP (e.g., the double disulfide-bonded TMMP) is * 04:01 (Y84C, A236C) amino acid sequence: TIFF2025186334000077.tif34165 (SEQ ID NO: 361), wherein amino acid 84 is Cys and amino acid 236 is Cys.

[0171] HLA-C * 0602(Y84C, A236C) In some instances, the HLA-C heavy chain polypeptide present in the multiply disulfide-bonded TMMP (e.g., the double disulfide-bonded TMMP) is * 06:02 (Y84C, A236C) amino acid sequence: TIFF2025186334000078.tif34165 (SEQ ID NO: 362), wherein amino acid 84 is Cys and amino acid 236 is Cys.

[0172] HLA-C * 0701(Y84C, A236C) In some instances, the HLA-C heavy chain polypeptide present in the multiply disulfide-bonded TMMP (e.g., the double disulfide-bonded TMMP) is * 07:01 (Y84C, A236C) amino acid sequence: TIFF2025186334000079.tif34165 (SEQ ID NO: 357), wherein amino acid 84 is Cys and amino acid 236 is Cys.

[0173] HLA-C * 0702(Y84C, A236C) In some instances, the HLA-C heavy chain polypeptide present in the multiply disulfide-bonded TMMP (e.g., the double disulfide-bonded TMMP) is * 07:02 (Y84C, A236C) amino acid sequence: TIFF2025186334000080.tif34165 (SEQ ID NO: 404), wherein amino acid 84 is Cys and amino acid 236 is Cys.

[0174] HLA-C * 0801(Y84C, A236C) In some instances, the HLA-C heavy chain polypeptide present in the multiply disulfide-bonded TMMP (e.g., the double disulfide-bonded TMMP) is * 08:01 (Y84C, A236C) amino acid sequence: TIFF2025186334000081.tif34165 (SEQ ID NO: 363), wherein amino acid 84 is Cys and amino acid 236 is Cys.

[0175] HLA-C * 1502(Y84C, A236C) In some instances, the HLA-C heavy chain polypeptide present in the multiply disulfide-bonded TMMP (e.g., the double disulfide-bonded TMMP) is *15:02 (Y84C, A236C) amino acid sequence: TIFF2025186334000082.tif34165 (SEQ ID NO: 364), wherein amino acid 84 is Cys and amino acid 236 is Cys.

[0176] Scaffold Polypeptide The TMMP can include an Fc polypeptide, or can include another suitable scaffold polypeptide.

[0177] Suitable scaffold polypeptides include antibody-based scaffold polypeptides and non-antibody-based scaffolds. Non-antibody-based scaffolds include, for example, albumin, XTEN (extended recombinant) polypeptides, transferrin, Fc receptor polypeptides, elastin-like polypeptides (see, for example, Hassouneh et al. (2012) Methods Enzymol. 502:215, e.g., a polypeptide comprising a pentapeptide repeat unit of (Val-Pro-Gly-X-Gly, SEQ ID NO: 59) (wherein X is any amino acid except proline)), albumin-binding polypeptides, silk-like polypeptides (see, for example, Valluzzi et al. (2002) Philos Trans R Soc Lond B Biol Sci. 357:165), and silk-elastin-like polypeptides (SELPs, see, for example, Megeed et al. (2002) Adv Drug Deliv Rev. 54:1075). Suitable XTEN polypeptides include, for example, those disclosed in WO2009 / 023270, WO2010 / 091122, WO2007 / 103515, US2010 / 0189682, and US2009 / 0092582, see also Schellenberger et al. (2009) Nat Biotechnol. 27:1186. Suitable albumin polypeptides include, for example, human serum albumin.

[0178] In some cases, a suitable scaffold polypeptide is a half-life extending polypeptide. Thus, in some cases, a suitable scaffold polypeptide extends the in vivo half-life (e.g., serum half-life) of a TMMP compared to a control TMMP lacking the scaffold polypeptide. For example, in some cases, a scaffold polypeptide extends the in vivo half-life (e.g., serum half-life) of a TMMP by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 50%, at least about 2-fold, at least about 2.5-fold, at least about 5-fold, at least about 10-fold, at least about 25-fold, at least about 50-fold, at least about 100-fold, or more than 100-fold compared to a control TMMP lacking the scaffold polypeptide. As an example, in some instances, the Fc polypeptide extends the in vivo half-life (e.g., serum half-life) of the TMMP by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 50%, at least about 2-fold, at least about 2.5-fold, at least about 5-fold, at least about 10-fold, at least about 25-fold, at least about 50-fold, at least about 100-fold, or more than 100-fold, compared to a control TMMP lacking the Fc polypeptide.

[0179] Fc polypeptide In some examples, the first and / or second polypeptide chain of the TMMP comprises an Fc polypeptide. The Fc polypeptide of the TMMP of the present disclosure may be human IgG1 Fc, human IgG2 Fc, human IgG3 Fc, human IgG4 Fc, etc. In some examples, the Fc polypeptide comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to the amino acid sequence of the Fc region set forth in Figures 5A-5G or 5H. In some examples, the Fc region comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to the human IgG1 Fc polypeptide set forth in Figure 5A. In some examples, the Fc region comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to the human IgG1 Fc polypeptide depicted in FIG. 5A and comprises a substitution at N77, e.g., the Fc polypeptide comprises an N77A substitution. In some examples, the Fc polypeptide comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to the human IgG2 Fc polypeptide depicted in FIG. 5A, for example, the Fc polypeptide comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to amino acids 99 to 325 of the human IgG2 Fc polypeptide depicted in FIG. 5A.In some examples, the Fc polypeptide comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to the human IgG3 Fc polypeptide depicted in FIG. 5A, for example, the Fc polypeptide comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to amino acids 19 to 246 of the human IgG3 Fc polypeptide depicted in FIG. 5A. In some examples, the Fc polypeptide comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to the human IgM Fc polypeptide depicted in Figure 5B, for example, the Fc polypeptide comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to amino acids 1-276 of the human IgM Fc polypeptide depicted in Figure 5B. In some examples, the Fc polypeptide comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to the human IgA Fc polypeptide depicted in Figure 5C, for example, the Fc polypeptide comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to amino acids 1-234 of the human IgA Fc polypeptide depicted in Figure 5C.

[0180] In some examples, the Fc polypeptide comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to the human IgG4 Fc polypeptide shown in Figure 5C. In some examples, the Fc polypeptide comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to amino acids 100 to 327 of the human IgG4 Fc polypeptide shown in Figure 5C.

[0181] In some examples, the IgG4 Fc polypeptide has the following amino acid sequence: Contains TIFF2025186334000083.tif26165 (sequence number 365).

[0182] In some examples, the Fc polypeptide present in the TMMP comprises the amino acid sequence set forth in Figure 5A (human IgG1 Fc). In some examples, the Fc polypeptide present in the TMMP comprises the amino acid sequence set forth in Figure 5A (human IgG1 Fc) except for the substitution of N297 (N77 in the amino acid sequence shown in Figure 5A) with an amino acid other than asparagine. In some examples, the Fc polypeptide present in the TMMP comprises the amino acid sequence set forth in Figure 5C (human IgG1 Fc comprising an N297A substitution (N77 in the amino acid sequence shown in Figure 5A)). In some examples, the Fc polypeptide present in the TMMP comprises the amino acid sequence set forth in Figure 5A (human IgG1 Fc) except for the substitution of L234 (L14 in the amino acid sequence shown in Figure 5A) with an amino acid other than leucine. In some examples, the Fc polypeptide present in the TMMP comprises the amino acid sequence set forth in Figure 5A (human IgG1 Fc) except for the substitution of L235 (L15 in the amino acid sequence shown in Figure 5A) with an amino acid other than leucine. In some examples, the IgG1 Fc polypeptide comprises the C-terminal Lys shown in Figure 5 A. In other examples, the IgG1 Fc polypeptide does not comprise the C-terminal Lys shown in Figure 5A.

[0183] In some examples, the Fc polypeptide present in the TMMP comprises the amino acid sequence set forth in Figure 5E. In some examples, the Fc polypeptide comprises the amino acid sequence shown in Figure 5E, but does not include a C-terminal Lys. In some examples, the Fc polypeptide present in the TMMP comprises the amino acid sequence set forth in Figure 5F. In some examples, the Fc polypeptide comprises the amino acid sequence shown in Figure 5F, but does not include a C-terminal Lys. In some examples, the Fc polypeptide present in the TMMP comprises the amino acid sequence set forth in Figure 5G (human IgG1 Fc comprising L234A and L235A substitutions corresponding to positions 14 and 15 of the amino acid sequence shown in Figure 5G). In some examples, the Fc polypeptide comprises the amino acid sequence set forth in Figure 5G, but does not include a C-terminal Lys. In some examples, the Fc polypeptide present in the TMMP comprises the amino acid sequence set forth in Figure 5A (human IgG1 Fc), except for the substitution of P331 (P111 of the amino acid sequence shown in Figure 5A) with an amino acid other than proline, and in some examples, the substitution is a P331S substitution. In some examples, the Fc polypeptide present in the TMMP comprises the amino acid sequence set forth in FIG. 5A (human IgG1 Fc), except for the substitution of L234 and L235 (L14 and L15 in the amino acid sequence shown in FIG. 5A) with an amino acid other than leucine. In some examples, the Fc polypeptide present in the TMMP comprises the amino acid sequence set forth in FIG. 5A (human IgG1 Fc), except for the substitution of L234 and L235 (L14 and L15 in the amino acid sequence shown in FIG. 5A) with an amino acid other than leucine and the substitution of P331 (P111 in the amino acid sequence shown in FIG. 5A) with an amino acid other than proline. In some examples, the Fc polypeptide present in the TMMP comprises the amino acid sequence set forth in FIG. 5E (human IgG1 Fc comprising an L234F substitution, an L235E substitution, and a P331S substitution corresponding to amino acid positions 14, 15, and 111 in the amino acid sequence shown in FIG. 5E). In some examples, the Fc polypeptide present in the TMMP is an IgG1 Fc polypeptide containing an L234A substitution and an L235A substitution (substitution of L14 and L15 with Ala in the amino acid sequence shown in FIG. 5A), as shown in FIG. 5G.

[0184] In some instances, the Fc polypeptide present in the TMMP has the amino acid sequence TIFF2025186334000084.tif27165 (SEQ ID NO: 489) and has a length of 226 amino acids.

[0185] Linker A TMMP of the present disclosure may include one or more linkers, the one or more linkers being located at one or more of: i) between the MHC class I polypeptide and the Ig Fc polypeptide (such a linker is referred to herein as "L1"); ii) between the MOD and the MHC class I polypeptide (such a linker is referred to herein as "L2"); iii) between the first MOD and the second MOD (such a linker is referred to herein as "L3"); iv) between the peptide antigen ("epitope") and the MHC class I polypeptide; v) between the MHC class I polypeptide and the dimerization polypeptide (e.g., the first or second member of the dimerization pair); and vi) between the dimerization polypeptide (e.g., the first or second member of the dimerization pair) and the Ig Fc polypeptide.

[0186] As used herein, the phrase "peptide linker between any two components of a TMMP" refers to a peptide linker between any two adjacent polypeptides within a TMMP. For example, as used herein, the phrase "peptide linker between any two components of a TMMP" refers to a peptide linker between one or more of: i) a peptide and a β2M polypeptide, ii) a β2M polypeptide and an MHC class I heavy chain polypeptide, iii) an MHC class I heavy chain polypeptide and an Ig Fc polypeptide, iv) an MHC class I heavy chain polypeptide and a MOD, v) an Ig Fc polypeptide and a MOD, and vi) a first MOD and a second MOD.

[0187] Suitable linkers (also called "spacers") can be readily selected and may be any of a number of suitable lengths, e.g., 1 to 25 amino acids, 3 to 20 amino acids, 2 to 15 amino acids, 3 to 12 amino acids, etc., including 4 to 10 amino acids, 5 to 9 amino acids, 6 to 8 amino acids, or 7 to 8 amino acids. Suitable linkers may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids in length. In some examples, the linker is 25 to 50 amino acids in length, e.g., 25 to 30, 30 to 35, 35 to 40, 40 to 45, or 45 to 50 amino acids in length.

[0188] Exemplary linkers include glycine polymers (G) n , glycine-serine polymers (e.g., (GS) n , (GSGGS) n (SEQ ID NO: 366), and (GGGS) n(SEQ ID NO: 367), where n is an integer of at least 1), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers may also be used, with both Gly and Ser being relatively free and therefore able to function as intermediate tethers between components. Glycine polymers may also be used, with glycine having significantly more access to the φ-ψ space than alanine and being much less restricted than residues with longer side chains (see Scheraga, Rev. Computational Chem. 11173-142 (1992)). Exemplary linkers may include amino acid sequences including, but not limited to, GGSG (SEQ ID NO: 368), GGSGG (SEQ ID NO: 369), GSGSG (SEQ ID NO: 370), GSGGG (SEQ ID NO: 371), GGGSG (SEQ ID NO: 372), GSSSG (SEQ ID NO: 373), and the like. Exemplary linkers may include, for example, Gly(Ser4)n (SEQ ID NO: 374) (n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). In some examples, the linker comprises the amino acid sequence (GSSSS)n (SEQ ID NO: 375) (n is 4). In some examples, the linker comprises the amino acid sequence (GSSSS)n (SEQ ID NO: 376) (n is 5). In some examples, the linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO: 377) (n is 1). In some examples, the linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO: 378) (n is 2). In some examples, the linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO: 379) (n is 3). In some examples, the linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO: 380) (n is 4). In some examples, the linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO:381) (n is 5). In some examples, the linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO:382) (n is 6). In some examples, the linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO:383) (n is 7). In some examples, the linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO:384) (n is 8).In some examples, the linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO:385), where n is 9. In some examples, the linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO:386), where n is 10. In some examples, the linker comprises the amino acid sequence AAAGG (SEQ ID NO:283).

[0189] In some instances, a linker polypeptide present in a first polypeptide of TMMP comprises a cysteine ​​residue capable of forming a disulfide bond with a cysteine ​​residue present in a second polypeptide of TMMP. In some instances, for example, a suitable linker has the amino acid sequence TIFF2025186334000085.tif10165 (SEQ ID NO: 317). In another example, a suitable linker may include the amino acid sequence GCGGS(G4S)n (SEQ ID NO: 315), where n is 1, 2, 3, 4, 5, 6, 7, 8, or 9. For example, in some examples, the linker includes the amino acid sequence GCGGSGGGGSGGGGSGGGGS (SEQ ID NO: 316). In another example, the linker includes the amino acid sequence GCGGSGGGGSGGGGS (SEQ ID NO: 317).

[0190] epitope In some examples, the epitope (peptide presenting one or more epitopes) present in a TMMP is a WT-1 peptide, e.g., a WT-1 peptide that presents an epitope to a TCR in conjunction with an MHC. The amino acid sequences of WT-1 isoforms are shown in Figures 3A-3E. WT-1 peptides presenting one or more epitopes are referred to herein as "WT-1 peptides" or "WT-1 epitopes." In some examples, a WT-1 epitope present in a TMMP of the present disclosure may be a peptide of 4 to 25 consecutive amino acids (e.g., 4 amino acids (aa), 5 aa, 6 aa, 7 aa, 8 aa, 9 aa, 10-15 aa, 15-20 aa, or 20-25 aa) having an amino acid sequence that shares at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity with the WT-1 amino acid sequence shown in any one of Figures 3A-3E. In some examples, a WT-1 epitope present in a TMMP of the present disclosure may be a peptide of 4 to 25 consecutive amino acids (e.g., 4 amino acids (aa), 5 amino acids, 6 amino acids, 7 amino acids, 8 amino acids, 9 amino acids, 10 to 15 amino acids, 15 to 20 amino acids, or 20 to 25 amino acids) having an amino acid sequence that shares at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity with the WT-1 amino acid sequence shown in FIG. 3A. In some examples, a WT-1 epitope present in a TMMP of the present disclosure may be a peptide of 4 to 25 consecutive amino acids (e.g., 4 amino acids (aa), 5 amino acids, 6 amino acids, 7 amino acids, or 8 amino acids). In some examples, the WT-1 epitope present in TMMP may be a peptide of 4 to 25 consecutive amino acids (e.g., 4 amino acids (aa), 5 aa, 6 aa, 7 aa, 8 aa, 9 aa, 10-15 aa, 15-20 aa, or 20-25 aa) having an amino acid sequence that has at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the WT-1 amino acid sequence shown in Figure 3B.In some examples, a WT-1 epitope present in a TMMP of the present disclosure may be a peptide of 4 to 25 consecutive amino acids (e.g., 4 amino acids (aa), 5 aa, 6 aa, 7 aa, 8 aa, 9 aa, 10-15 aa, 15-20 aa, or 20-25 aa) having an amino acid sequence that has at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the WT-1 amino acid sequence shown in FIG. 3C . In some examples, the WT-1 epitope present in TMMP may be a peptide of 4 to 25 consecutive amino acids (e.g., 4 amino acids (aa), 5 aa, 6 aa, 7 aa, 8 aa, 9 aa, 10-15 aa, 15-20 aa, or 20-25 aa) having an amino acid sequence that has at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the WT-1 amino acid sequence shown in Figure 3D. In some examples, a WT-1 epitope present in a TMMP of the present disclosure may be a peptide of 4 to 25 consecutive amino acids (e.g., 4 amino acids (aa), 5 aa, 6 aa, 7 aa, 8 aa, 9 aa, 10 to 15 aa, 15 to 20 aa, or 20 to 25 aa) having an amino acid sequence that has at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the WT-1 amino acid sequence shown in FIG. 3E. In some examples, a WT-1 epitope present in a TMMP is 6 amino acids in length. In some examples, a WT-1 epitope present in a TMMP is 7 amino acids in length. In some examples, a WT-1 epitope present in a TMMP is 8 amino acids in length. In some examples, a WT-1 epitope present in a TMMP is 9 amino acids in length. In some examples, a WT-1 epitope present in a TMMP is 10 amino acids in length. In some examples, the WT-1 epitope present in TMMP is 11 amino acids in length. In some examples, the WT-1 epitope present in TMMP is 6 to 25 amino acids in length. In some examples, the WT-1 epitope present in TMMP is 6 to 20 amino acids in length. In some examples, the WT-1 epitope present in TMMP is 7 to 25 amino acids in length.In some examples, the WT-1 epitope present in the TMMP is 7 to 20 amino acids in length, hi some examples, the WT-1 epitope present in the TMMP is at least 4 amino acids in length, at least 6 amino acids in length, or at least 7 amino acids in length.

[0191] Epitopes present in TMMP can be about 4 amino acids to about 25 amino acids in length, for example, epitopes can be 4 amino acids (aa) to 10 aa, 10 aa to 15 aa, 15 aa to 20 aa, or 20 aa to 25 aa in length. For example, epitopes present in TMMP can be 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 length. In some examples, epitopes present in TMMP have a length of 5 to 10 amino acids, for example, 5 aa, 6 aa, 7 aa, 8 aa, 9 aa, or 10 aa.

[0192] A WT-1 epitope present in a TMMP is a peptide to which T cells specifically bind, i.e., the epitope is bound by WT-1 epitope-specific T cells. Epitope-specific T cells bind to an epitope having a reference amino acid sequence but do not substantially bind to an epitope different from the reference amino acid sequence. For example, epitope-specific T cells bind to an epitope having a reference amino acid sequence and do not substantially bind to an epitope different from the reference amino acid sequence, even if they do so at all. -6 Under M, 10 -5 Less than M or 10 -4 Epitope-specific T cells bind with an affinity of less than M. Epitope-specific T cells bind with an affinity of less than M. -7 M, at least 10 -8 M, at least 10 -9 M, or at least 10 -10 It can bind to an epitope for which it is specific with an affinity of M.

[0193] Examples of WT-1 peptides suitable for inclusion in a TMMP include CMTWNQMNLGATLKG (SEQ ID NO: 223), WNQMNLGATLKGVAA (SEQ ID NO: 224), CMTWNYMNLGATLKG (SEQ ID NO: 225), WNYMNLGATLKGVAA (SEQ ID NO: 226), MTWNQMNLGATLKGV (SEQ ID NO: 227), TWNQMNLGATLKGVA (SEQ ID NO: 228), CMTWNLMNLGATLKG (SEQ ID NO: 229), MTWNLMNLGATLKGV (SEQ ID NO: 230), TWNLMNLGATLKGVA (SEQ ID NO: 231), and TWNLMNLGATLKGVA (SEQ ID NO: 232). 1), WNLMNLGATLKGVAA (SEQ ID NO: 232), MNLGATLK (SEQ ID NO: 233), MTWNYMNLGATLKGV (SEQ ID NO: 234), TWNYMNLGATLKGVA (SEQ ID NO: 235), CMTWNQMNLGATLKGVA (SEQ ID NO: 236), CMTWNLMNLGATLKGVA (SEQ ID NO: 237), CMTWNYMNLGATLKGVA (SEQ ID NO: 238), GYLRNPTAC (SEQ ID NO: 239), GALRNPTAL (SEQ ID NO: 240), YALRNPTAC (SEQ ID NO: 241), GLLRNPTAC (SEQ ID NO: 2 42), RYRPHPGAL (SEQ ID NO: 243), YQRPHPGAL (SEQ ID NO: 244), RLRPHPGAL (SEQ ID NO: 245), RIRPHPGAL (SEQ ID NO: 246), QFPNHSFKHEDPMGQ (SEQ ID NO: 247), HSFKHEDPY (SEQ ID NO: 248), QFPNHSFKHEDPM (SEQ ID NO: 249), QFPNHSFKHEDPY (SEQ ID NO: 250), KRPFMCAYPGCNK (SEQ ID NO: 251), KRPFMCAYPGCYK (SEQ ID NO: 252), FMCAYPGCY (SEQ ID NO: 253), FMCAYPGCK (SEQ ID NO: 254), KRPFMCAYPGCNKRY (SEQ ID NO: 255), SEKRPFMCAYPGCNK (SEQ ID NO: 256), KRPFMCAYPGCYKRY (SEQ ID NO: 257), NLMNLGATL (SEQ ID NO: 258), VLDFAPPGA (SEQ ID NO: 259), RMFPNAPYL (SEQ ID NO: 260), CMTWNQMN (SEQ ID NO: 261), CYTWNQMNL (SEQ ID NO: 262), NYMNLGATL (SEQ ID NO: 263), YMFPNAPYL (SEQ ID NO: 264), SLGEQQYSV (SEQ ID NO: 265), CMTWNQMNL (SEQ ID NO: 266),and NQMNLGATL (SEQ ID NO: 267). In some examples, the WT-1 peptide present in TMMP is CMTWNQMN (SEQ ID NO: 261). In some examples, the WT-1 peptide present in TMMP is CYTWNQMNL (SEQ ID NO: 262).

[0194] In some examples, the WT-1 peptide present in the TMMP of the present disclosure is a peptide that binds to HLA-A * Presents the 2402 restricted epitope. HLA-A * Examples of WT-1 peptides displaying the 2402-restricted epitope include CMTWNQMN (SEQ ID NO: 261), NYMNLGATL (SEQ ID NO: 263) (WT-1 239-247, Q240Y), CYTWNQMNL (SEQ ID NO: 262) (WT-1 235-243), CMTWNQMNL (SEQ ID NO: 266) (WT-1 235-243), NQMNLGATL (SEQ ID NO: 267) (WT-1 239-247), and NLMNLGATL (SEQ ID NO: 258) (WT-1 239-247, Q240L).

[0195] In some cases, the WT-1 peptide present in TMMP binds to HLA-A * Presents the 0201 restricted epitope. HLA-A * Examples of WT-1 peptides displaying the O201-restricted epitope include VLDFAPPGA (SEQ ID NO: 259) (WT-1 37-45), RMFPNAPYL (SEQ ID NO: 260) (WT-1 126-134), YMFPNAPYL (SEQ ID NO: 264) (WT-1 126-134, R126Y), SLGEQQYSV (SEQ ID NO: 265) (WT-1 187-195), and NLMNLGATL (SEQ ID NO: 258) (WT-1 239-247, Q240L).

[0196] In some cases, WT-1 peptides present in TMMP bind to HLA-A *The WT-1 peptide presents a 2402-restricted epitope and does not have an N-terminal Cys. For example, if the WT-1 peptide contains an N-terminal Cys, the N-terminal Cys can be replaced with Ser. As another example, if the WT-1 peptide contains an N-terminal Cys, Gly can be added to the N-terminus. For example, a WT-1 peptide present in TMMP can have the amino acid sequence X1X2X3TWNQMNL (SEQ ID NO: 460) or X2X3TWNQMNL (SEQ ID NO: 461), where X1, X2, and X3 are each independently any amino acid, provided that the N-terminal amino acid is not Cys, and the WT-1 peptide epitope is 9 to 25 amino acids in length. In some of these embodiments, the WT-1 peptide is 9 or 10 amino acids in length. Examples of WT-1 peptides suitable for inclusion in a TMMP include, but are not limited to, SMTWNQMNL (SEQ ID NO: 451), GCMTWNQMNL (SEQ ID NO: 452), SYTWNQMNL (SEQ ID NO: 453), or GCYTWNQMNL (SEQ ID NO: 454). In some examples, the WT-1 peptide present in a TMMP of the present disclosure has the amino acid sequence SMTWNQMNL (SEQ ID NO: 451) and is 9 amino acids in length. In some examples, the WT-1 peptide present in a TMMP has the amino acid sequence GCMTWNQMNL (SEQ ID NO: 452) and is 10 amino acids in length. In some examples, the WT-1 peptide present in a TMMP has the amino acid sequence SYTWNQMNL (SEQ ID NO: 453) and is 9 amino acids in length. In some examples, the WT-1 peptide present in a TMMP has the amino acid sequence GCYTWNQMNL (SEQ ID NO: 454) and is 10 amino acids in length.

[0197] HLA / peptide binding assay Whether a given peptide (e.g., a WT-1 peptide) can bind to class I HLA (containing an HLA heavy chain and a β2M polypeptide) and effectively present an epitope to TCR when bound to an HLA complex can be determined using any of a number of well-known methods, including binding assays and T cell activation assays.

[0198] Cell-based binding assays As an example, peptide-HLA class I binding can be determined using a cell-based peptide-induced stabilization assay, in which a peptide of interest is able to bind to TAP-deficient cells, i.e., cells that lack the antigen peptide transporter (TAP) machinery and therefore have few surface class I molecules. Such cells include, for example, the human T2 cell line (T2(174×CEM.T2; American Type Culture Collection (ATCC) number CRL-1992). Henderson et al. (1992) Science 255:1264. Class I complexes assembled without efficient TAP-dependent transport of cytosolic peptides into the endoplasmic reticulum are structurally unstable and are only transiently retained on the cell surface. However, when T2 cells are incubated with exogenous peptides capable of binding to class I, surface peptide-HLA class I complexes are stabilized and can be detected, for example, by flow cytometry using a pan-anti-class I monoclonal antibody. The stabilization of peptide-HLA complexes on the cell surface by the addition of peptide and the resulting extended lifespan confirm their identity. For example, if the pan-HLA class I antibody contains a fluorescent label, analysis can be performed using flow cytometry. By genetically modifying T2 cells to express the desired allelic HLA H chain, peptide binding to various allelic forms of the HLA H chain can be tested.

[0199] The following are HLA A * This is a non-limiting example of the use of the T2 assay to assess peptide binding to 0201. T2 cells were washed with cell culture medium and incubated for 10 min. 6 Concentrate to cells / ml. Prepare peptides of interest in cell culture medium and serially dilute to concentrations of 200 μM, 100 μM, 20 μM, and 2 μM. Mix cells 1:1 with each peptide dilution to a final volume of 200 μL and final peptide concentrations of 100 μM, 50 μM, 10 μM, and 1 μM. HLA A *0201-binding peptide GILGFVFTL (SEQ ID NO: 395), and non-HLA A * The 0201 restricted peptide HPVGEADYF (SEQ ID NO: 396) (HLA-B * 3501) are included as positive and negative controls, respectively. The cell / peptide mixture is kept at 37°C, 5% CO2 for 10 minutes, then incubated at room temperature overnight. The cells are then incubated at 37°C for 2 hours and stained with fluorescently labeled anti-human HLA antibodies. The cells are washed twice with phosphate-buffered saline and analyzed using flow cytometry. The mean fluorescence intensity (MFI) of the anti-HLA antibody staining is used to measure the strength of binding.

[0200] Biochemical binding assays In a cell-free in vitro assay system, an HLA polypeptide (an HLA heavy chain polypeptide complexed with a β2M polypeptide) can be tested for binding to a peptide of interest. For example, a labeled (e.g., fluorescently labeled) reference peptide can be bound to an HLA polypeptide (an HLA heavy chain polypeptide complexed with a β2M polypeptide) to form an HLA-reference peptide complex. The ability of the test peptide of interest to displace the labeled reference peptide from the HLA-reference peptide complex is tested. The relative binding affinity is calculated as the amount of test peptide required to displace the bound reference peptide. See, for example, van der Burg et al. (1995) Human Immunol. 44:189.

[0201] As another example, a peptide of interest can be incubated with an HLA molecule (HLA heavy chain complexed with a β2M polypeptide), and the stabilization of the HLA / peptide complex can be measured in an immunoassay format. The ability of the peptide of interest to stabilize the HLA molecule is compared to the ability of a control peptide that presents a known T cell epitope. Detection of stabilization is based on the presence or absence of an HLA / peptide complex in its native conformation, detected using an anti-HLA antibody. See, e.g., Westrop et al. (2009) J. Immunol. Methods 341:76; Steinitz et al. (2012) Blood 119:4073; and U.S. Patent No. 9,205,144.

[0202] T cell activation assay When a given peptide binds to class I HLA (including an HLA heavy chain and a β2M polypeptide) and binds to an HLA complex, whether the peptide can effectively present an epitope to a TCR can be determined by evaluating the T cell response to the peptide-HLA complex. Examples of T cell responses that can be measured include interferon-γ (IFNγ) production and cytotoxic activity.

[0203] ELISPOT assay Suitable assays include, for example, enzyme-linked immunospot (ELISPOT) assays. + IFNγ production by T cells is measured using antigen-presenting cells (APCs) that present a peptide of interest complexed with HLA class I. Antibodies against IFNγ are immobilized in the wells of a multi-well plate. APCs are added to the wells and incubated with the peptide of interest for a period of time, allowing the peptide to bind to HLA class I on the surface of the APCs. +T cells are added to the wells, and the plate is incubated for approximately 24 hours. The wells are then washed, and IFNγ bound to the immobilized anti-IFNγ antibody is detected using a detectably labeled anti-IFNγ antibody. Colorimetric analysis can be used. For example, the detectably labeled anti-IFNγ antibody can be a biotin-labeled anti-IFNγ antibody, which can be detected using, for example, alkaline phosphatase-conjugated streptavidin. BCIP / NBT (5-bromo-4-chloro-3-indolylphosphate / nitroblue tetrazolium) solution is added to develop the assay. The presence of IFNγ-secreting T cells is identified by a colored spot. A negative control includes APCs not contacted with peptide. APCs expressing various HLA H chain alleles can be used to determine whether a peptide of interest effectively binds to an HLA class I molecule containing a specific HLA H chain.

[0204] Cytotoxicity assay Cytotoxicity assays can also be used to determine whether a given peptide can bind to a specific HLA class I heavy chain and effectively present the epitope to the TCR when bound to an HLA class I complex containing the heavy chain. Cytotoxicity assays involve target cells expressing cytotoxic CD8 + The target cells present on their surface a peptide / HLA class I complex containing the peptide of interest and an HLA class I molecule containing the HLA heavy chain to be tested. The target cells are then incubated with, for example, 51 Cr can be used to radiolabel target cells. + Effectively presenting epitopes to the TCR on T cells, thereby targeting CD8 + Whether or not T cells induce cytotoxic activity against target cells was determined by measuring the amount of cytotoxic activity from lysed target cells. 51 Specific cytotoxicity can be calculated as the amount of cytotoxic activity in the presence of peptide minus the amount of cytotoxic activity in the absence of peptide.

[0205] Detection of antigen-specific T cells by peptide-HLA tetramers In another example, multimers (e.g., tetramers) of peptide-HLA complexes are generated with fluorescent or heavy metal tags. The multimers can then be used to identify and quantify specific T cells via flow cytometry (FACS) or mass cytometry (CyTOF). Detection of epitope-specific T cells provides direct evidence that peptide-bound HLA molecules can bind to specific TCRs on a subset of antigen-specific T cells. See, for example, Klenerman et al. (2002) Nature Reviews Immunol. 2:263.

[0206] Immunomodulatory Polypeptides ("MODs") In some examples, the MOD present in the TMMP is a wild-type ("wt") MOD. As noted above, in other examples, the MOD present in the TMMP is a variant of the wtMOD that has reduced affinity for the co-MOD compared to the affinity of the corresponding wild-type MOD for the co-MOD. Suitable MODs that exhibit reduced affinity for the co-MOD may differ from the wild-type MOD by 1 amino acid (aa) to 20 aa. For example, in some examples, the variant MOD present in the TMMP differs in amino acid sequence from the corresponding wild-type MOD by 1 aa, 2 aa, 3 aa, 4 aa, 5 aa, 6 aa, 7 aa, 8 aa, 9 aa, or 10 aa. As another example, in some examples, the variant MOD present in the TMMP differs in amino acid sequence from the corresponding wild-type MOD by 11 aa, 12 aa, 13 aa, 14 aa, 15 aa, 16 aa, 17 aa, 18 aa, 19 aa, or 20 aa.

[0207] As noted above, a MOD can include a variant of a wt immunomodulatory polypeptide that can exhibit reduced binding to the co-MOD, including, for example, reduced binding to one or more chains or domains of the co-MOD. For example, a variant MOD present in a TMMP can bind to the co-MOD with an affinity that is at least 10% lower, at least 15% lower, at least 20% lower, at least 25% lower, at least 30% lower, at least 35% lower, at least 40% lower, at least 45% lower, at least 50% lower, at least 55% lower, at least 60% lower, at least 65% lower, at least 70% lower, at least 75% lower, at least 80% lower, at least 85% lower, at least 90% lower, at least 95% lower, or greater than 95% lower than the affinity of the corresponding wild-type MOD for the co-MOD. Exemplary pairs of immunomodulatory polypeptides and homologous co-immunomodulatory polypeptides include: a) 4-1BBL (immunomodulating polypeptide) and 4-1BB (cognate immunomodulating polypeptide); b) PD-L1 (immunomodulatory polypeptide) and PD1 (cognate immunomodulatory polypeptide); c) IL-2 (immunomodulating polypeptide) and IL-2 receptor (cognate immunomodulating polypeptide); d) CD80 (immunomodulating polypeptide) and CD86 (cognate immunomodulating polypeptide); e) CD86 (immunomodulating polypeptide) and CD28 (cognate immunomodulating polypeptide); f) OX40L (CD252) (immunomodulatory polypeptide) and OX40 (CD134) (cognate immunomodulatory polypeptide); g) Fas ligand (an immunomodulatory polypeptide) and Fas (a cognate immunomodulatory polypeptide); h) ICOS-L (immunomodulating polypeptide) and ICOS (cognate immunomodulating polypeptide); i) ICAM (immunomodulating polypeptide) and LFA-1 (cognate coimmunomodulating polypeptide); j) CD30L (immunomodulating polypeptide) and CD30 (cognate immunomodulating polypeptide); k) CD40 (immunomodulating polypeptide) and CD40L (cognate immunomodulating polypeptide); l) CD83 (immunomodulating polypeptide) and CD83L (cognate immunomodulating polypeptide); m) HVEM (CD270) (immunomodulating polypeptide) and CD160 (cognate immunomodulating polypeptide); n) JAG1 (CD339) (immunomodulatory polypeptide) and Notch (cognate immunomodulatory polypeptide); o) JAG1 (immunomodulatory polypeptide) and CD46 (cognate coimmunomodulatory polypeptide); p) CD80 (immunomodulating polypeptide) and CTLA4 (cognate immunomodulating polypeptide); q) CD86 (immunomodulating polypeptide) and CTLA4 (cognate immunomodulating polypeptide), and r) CD70 (immunomodulating polypeptide) and CD27 (cognate immunomodulating polypeptide); These include, but are not limited to:

[0208] As shown schematically in Figure 19, a MOD (i.e., one or more MODs) can be present at any of a variety of positions in a TMMP. While Figure 19 shows the location of two copies of a variant IL-2 polypeptide, the MOD can be any of a variety of MODs as described herein. As shown in Figure 19, the MOD can be 1) N-terminal of the MHC class I heavy chain, 2) C-terminal of the MHC class I heavy chain and N-terminal of the Ig Fc polypeptide, in other words, between the MHC class I heavy chain and the Ig Fc polypeptide, 3) C-terminal of the Ig Fc polypeptide, 4) N-terminal of the peptide epitope, or 5) C-terminal of the β2M polypeptide.

[0209] PD-L1 - wild type and variant The MOD present in the TMMP can be a wild-type PD-L1 polypeptide or a variant PD-L1 polypeptide.

[0210] In some examples, the MOD present in the TMMP is a PD-L1 polypeptide. In some examples, the PD-L1 polypeptide of the TMMP 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 PD-L1 amino acid sequence set forth in SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3. Variant MODs of PD-L1 are described in published PCT application WO2019 / 051091, published March 14, 2019. See

[0157] -

[0169] .

[0211] In some examples, variant PD-L1 polypeptides exhibit reduced binding affinity to PD-1 (e.g., a PD-1 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 3) compared to the binding affinity of a PD-L1 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2. For example, in some examples, variant PD-L1 polypeptides of the present disclosure bind to PD-1 (e.g., a PD-1 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 3) with an affinity that is at least 10% less, at least 15% less, at least 20% less, at least 25% less, at least 30% less, at least 35% less, at least 40% less, at least 45% less, at least 50% less, at least 55% less, at least 60% less, at least 65% less, at least 70% less, at least 75% less, at least 80% less, at least 85% less, at least 90% less, at least 95% less, or greater than 95% less than the binding affinity of a PD-L1 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

[0212] CD80 - wild type and variant forms The MOD present in the TMMP can be a wild-type CD80 polypeptide or a variant CD80 polypeptide. CD80 variant MODs are described in published PCT application WO2019 / 051091, published March 14, 2019. See

[0170] -

[0196] .

[0213] In some examples, the MOD present in the TMMP is a CD80 polypeptide. In some examples, the CD80 polypeptide of the TMMP comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequences set forth in SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7.

[0214] In some examples, the variant CD80 polypeptide exhibits reduced binding affinity to CD28 compared to the binding affinity to CD28 of a CD80 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 4. For example, in some examples, the variant CD80 polypeptide binds to CD28 with an affinity that is at least 10% lower, at least 15% lower, at least 20% lower, at least 25% lower, at least 30% lower, at least 35% lower, at least 40% lower, at least 45% lower, at least 50% lower, at least 55% lower, at least 60% lower, at least 65% lower, at least 70% lower, at least 75% lower, at least 80% lower, at least 85% lower, at least 90% lower, at least 95% lower, or more than 95% lower than the binding affinity to CD28 of a CD80 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 4 (e.g., a CD28 polypeptide comprising the amino acid sequence set forth in one of SEQ ID NOs: 5, 6, or 7).

[0215] CD86 - wild type and variant The MOD present in the TMMP can be a wild-type CD86 polypeptide or a variant CD86 polypeptide. CD80 variant MODs are described in published PCT application WO2019 / 051091, published March 14, 2019. See

[0197] -

[0228] .

[0216] In some examples, the MOD present in the TMMP is a CD86 polypeptide. In some examples, the CD86 polypeptide of the TMMP comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the CD86 amino acid sequence set forth in SEQ ID NO:8 or SEQ ID NO:9.

[0217] 4-1BBL - wild type and variants The MOD present in the TMMP can be a wild-type 4-1BBL polypeptide or a variant 4-1BBL polypeptide. Variant MODs of 4-1BBL are described in published PCT application WO2019 / 051091, published March 14, 2019. See

[0229] -

[0324] .

[0218] In some examples, the MOD present in TMMP is a 4-1BBL polypeptide. In some examples, the 4-1BBL polypeptide of TMMP 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 4-1BBL amino acid sequence set forth in SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, or SEQ ID NO:14.

[0219] In some examples, variant 4-1BBL polypeptides exhibit reduced binding affinity for 4-1BB compared to the binding affinity of a 4-1BBL polypeptide comprising the amino acid sequence set forth in one of SEQ ID NOs: 10-13. For example, in some examples, variant 4-1BBL polypeptides of the present disclosure bind to 4-1BB with a binding affinity that is at least 10% lower, at least 15% lower, at least 20% lower, at least 25% lower, at least 30% lower, at least 35% lower, at least 40% lower, at least 45% lower, at least 50% lower, at least 55% lower, at least 60% lower, at least 65% lower, at least 70% lower, at least 75% lower, at least 80% lower, at least 85% lower, at least 90% lower, at least 95% lower, or more than 95% lower than the binding affinity of a 4-1BBL polypeptide comprising the amino acid sequence set forth in one of SEQ ID NOs: 10-13 for a 4-1BB polypeptide (e.g., a 4-1BB polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 14) when assayed under identical conditions.

[0220] IL-2 variant In some examples, the variant MOD present in the TMMP of the present disclosure is a variant IL-2 polypeptide. Wild-type IL-2 binds to the IL-2 receptor (IL-2R), a heterotrimeric polypeptide comprising IL-2Rα, IL-2Rβ, and IL-2Rγ.

[0221] The wild-type IL-2 amino acid sequence is as follows: TIFF2025186334000086.tif25165 (SEQ ID NO: 15) may be.

[0222] Wild-type IL-2 binds to the IL-2 receptor (IL-2R) on the surface of cells. The IL-2 receptor is, in some instances, a heterotrimeric polypeptide comprising an α chain (IL-2Rα, also known as CD25), a β chain (IL-2Rβ, also known as CD122), and a γ chain (IL-2Rγ, also known as CD132). The amino acid sequences of human IL-2Rα, human IL-2Rβ, and human IL-2Rγ are listed below. Human IL-2Rα: TIFF2025186334000087.tif37165 (SEQ ID NO: 16), Human IL-2Rβ: TIFF2025186334000088.tif66165 (SEQ ID NO: 17), Human IL-2Rγ: TIFF2025186334000089.tif56165 (SEQ ID NO: 18), may be.

[0223] In some examples, when the TMMP comprises a variant IL-2 polypeptide, the "cognate co-MOD" is an IL-2R comprising a polypeptide comprising the amino acid sequence of SEQ ID NOs: 16, 17, and 18.

[0224] In some examples, the variant IL-2 polypeptide exhibits a lower binding affinity to IL-2R compared to the binding affinity of an IL-2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 15. For example, in some examples, the variant IL-2 polypeptide binds to IL-2R with a binding affinity that is at least 10% lower, at least 15% lower, at least 20% lower, at least 25% lower, at least 30% lower, at least 35% lower, at least 40% lower, at least 45% lower, at least 50% lower, at least 55% lower, at least 60% lower, at least 65% lower, at least 70% lower, at least 75% lower, at least 80% lower, at least 85% lower, at least 90% lower, at least 95% lower, or more than 95% lower than the binding affinity of an IL-2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 15 to IL-2R (e.g., an IL-2R comprising a polypeptide comprising an amino acid sequence set forth in SEQ ID NOs: 16-18), when assayed under identical conditions.

[0225] In some examples, the variant IL-2 polypeptide has a binding affinity to IL-2R of 100 nM to 100 μM. In other examples, the variant IL-2 polypeptide has a binding affinity to IL-2R of about 100 nM to 150 nM, about 150 nM to about 200 nM, about 200 nM to about 250 nM, about 250 nM to about 300 nM, about 300 nM to about 350 nM, about 350 nM to about 400 nM, or about 400 nM to about 500 nM to IL-2R (e.g., an IL-2R comprising a polypeptide comprising the amino acid sequence set forth in SEQ ID NOs: 16 to 18). M, 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.

[0226] In some examples, the variant IL-2 polypeptide has a single amino acid substitution compared to the IL-2 amino acid sequence set forth in SEQ ID NO: 15. In some examples, the variant IL-2 polypeptide has 2 to 10 amino acid substitutions compared to the IL-2 amino acid sequence set forth in SEQ ID NO: 15. In some examples, the variant IL-2 polypeptide has two amino acid substitutions compared to the IL-2 amino acid sequence set forth in SEQ ID NO: 15. In some examples, the variant IL-2 polypeptide has three amino acid substitutions compared to the IL-2 amino acid sequence set forth in SEQ ID NO: 15. In some examples, the variant IL-2 polypeptide has four amino acid substitutions compared to the IL-2 amino acid sequence set forth in SEQ ID NO: 15. In some examples, the variant IL-2 polypeptide has five amino acid substitutions compared to the IL-2 amino acid sequence set forth in SEQ ID NO: 15. In some examples, the variant IL-2 polypeptide has six amino acid substitutions compared to the IL-2 amino acid sequence set forth in SEQ ID NO: 15. In some examples, the variant IL-2 polypeptide has seven amino acid substitutions compared to the IL-2 amino acid sequence set forth in SEQ ID NO: 15. In some examples, the variant IL-2 polypeptide has 8 amino acid substitutions compared to the IL-2 amino acid sequence set forth in SEQ ID NO: 15. In some examples, the variant IL-2 polypeptide has 9 amino acid substitutions compared to the IL-2 amino acid sequence set forth in SEQ ID NO: 15. In some examples, the variant IL-2 polypeptide has 10 amino acid substitutions compared to the IL-2 amino acid sequence set forth in SEQ ID NO: 15.

[0227] Suitable IL-2 variants include those having the following amino acid sequence: TIFF2025186334000090.tif24165 (SEQ ID NO: 181) (X is any amino acid other than Phe) (in some examples, Xi is Ala. In some examples, Xi is Met. In some examples, Xi is Pro. In some examples, Xi is Ser. In some examples, Xi is Thr. In some examples, Xi is Trp. In some examples, Xi is Tyr. In some examples, Xi is Val. In some examples, Xi is His), TIFF2025186334000091.tif24165 (SEQ ID NO: 182) (wherein X is any amino acid except Asp) (in some examples, X is Ala); TIFF2025186334000092.tif24165 (SEQ ID NO: 183) (wherein X is any amino acid other than Glu) (in some examples, X is Ala); TIFF2025186334000093.tif24165 (SEQ ID NO: 184) (wherein Xi is any amino acid other than His) (in some instances, Xi is Ala. In some instances, Xi is Thr. In some instances, Xi is Asn. In some instances, Xi is Cys. In some instances, Xi is Gln. In some instances, Xi is Met. In some instances, Xi is Val. In some instances, Xi is Trp), TIFF2025186334000094.tif24165 (SEQ ID NO: 185) (X is any amino acid other than His) (in some cases, Xi is Ala. In some cases, Xi is Arg. In some cases, Xi is Asn. In some cases, Xi is Asp. In some cases, Xi is Cys. In some cases, Xi is Glu. In some cases, Xi is Gln. In some cases, Xi is Gly. In some cases, Xi is Ile. In some cases, Xi is Lys. In some cases, Xi is Leu. In some cases, Xi is Met. In some cases, Xi is Phe. In some cases, Xi is Pro. In some cases, Xi is Ser. In some cases, Xi is Thr. In some cases, Xi is Tyr. In some cases, Xi is Trp. In some cases, Xi is Val), TIFF2025186334000095.tif24165 (SEQ ID NO: 186) (wherein X is any amino acid except Tyr) (in some examples, X is Ala), TIFF2025186334000096.tif24165 (SEQ ID NO: 187) (wherein X is any amino acid except Gln) (in some instances, X is Ala); TIFF2025186334000097.tif24165 (SEQ ID NO: 188) (X1 is any amino acid other than His, and X2 is any amino acid other than Phe) (in some examples, X1 is Ala. In some examples, X2 is Ala. In some examples, X1 is Ala and X2 is Ala. In some examples, X1 is Thr and X2 is Ala), TIFF2025186334000098.tif24165 (SEQ ID NO: 189) (X1 is any amino acid other than Asp and X2 is any amino acid other than Phe) (in some examples, X1 is Ala. In some examples, X2 is Ala. In some examples, X1 is Ala and X2 is Ala), TIFF2025186334000099.tif24165 (SEQ ID NO: 190) (X1 is any amino acid other than Glu, X2 is any amino acid other than Asp, and X3 is any amino acid other than Phe) (in some examples, X1 is Ala. In some examples, X2 is Ala. In some examples, X3 is Ala. In some examples, X1 is Ala, X2 is Ala, and X3 is Ala), TIFF2025186334000100.tif24165 (SEQ ID NO: 191) (X1 is any amino acid other than His, X2 is any amino acid other than Asp, and X3 is any amino acid other than Phe) (in some examples, X1 is Ala. In some examples, X2 is Ala. In some examples, X3 is Ala. In some examples, X1 is Ala, X2 is Ala, and X3 is Ala), TIFF2025186334000101.tif24165 (SEQ ID NO: 192) (X1 is any amino acid other than Asp, X2 is any amino acid other than Phe, and X3 is any amino acid other than Gln) (in some examples, X1 is Ala. In some examples, X2 is Ala. In some examples, X3 is Ala. In some examples, X1 is Ala, X2 is Ala, and X3 is Ala), TIFF2025186334000102.tif24165 (SEQ ID NO: 193) (X1 is any amino acid other than Asp, X2 is any amino acid other than Phe, and X3 is any amino acid other than Tyr) (in some examples, X1 is Ala. In some examples, X2 is Ala. In some examples, X3 is Ala. In some examples, X1 is Ala, X2 is Ala, and X3 is Ala), TIFF2025186334000103.tif24165 (SEQ ID NO: 194) (X1 is any amino acid other than His, X2 is any amino acid other than Asp, X3 is any amino acid other than Phe, and X4 is any amino acid other than Tyr) (in some examples, X1 is Ala. In some examples, X2 is Ala. In some examples, X3 is Ala. In some examples, X4 is Ala. In some examples, X1 is Ala, X2 is Ala, X3 is Ala, and X4 is Ala), TIFF2025186334000104.tif24165 (SEQ ID NO: 195) (X1 is any amino acid other than Asp, X2 is any amino acid other than Phe, X3 is any amino acid other than Tyr, and X4 is any amino acid other than Gln) (in some examples, X1 is Ala. In some examples, X2 is Ala. In some examples, X3 is Ala. In some examples, X4 is Ala. In some examples, X1 is Ala, X2 is Ala, X3 is Ala, and X4 is Ala), TIFF2025186334000105.tif24165 (SEQ ID NO: 196) (X1 is any amino acid other than His, X2 is any amino acid other than Asp, X3 is any amino acid other than Phe, X4 is any amino acid other than Tyr, and X5 is any amino acid other than Gln) (in some examples, X1 is Ala. In some examples, X2 is Ala. In some examples, X3 is Ala. In some examples, X4 is Ala. In some examples, X5 is Ala. In some examples, X1 is Ala, X2 is Ala, X3 is Ala, X4 is Ala, and X5 is Ala), and TIFF2025186334000106.tif24165 (SEQ ID NO: 197) (X1 is any amino acid other than His, X2 is any amino acid other than Phe, and X3 is any amino acid other than Gln) (in some examples, X1 is Ala. In some examples, X2 is Ala. In some examples, X3 is Ala. In some examples, X1 is Ala, X2 is Ala, and X3 is Ala), and polypeptides comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to any one of:

[0228] In some examples, a suitable variant IL-2 polypeptide has the amino acid sequence: TIFF2025186334000107.tif21165 (SEQ ID NO:490), i.e., the variant IL-2 polypeptide has the amino acid sequence of wild-type IL-2 but with H16A and F42A substitutions (shown in bold). Alternatively, sequences having substitutions other than Ala at H16 and / or F42 in the foregoing sequences may be used, e.g., H16T may be used in place of H16A. In some examples, the variant IL-2 polypeptide present in TMP has the amino acid sequence: TIFF2025186334000108.tif22165 (SEQ ID NO: 490). In some examples, the variant IL-2 polypeptide present in the TMMP has the amino acid sequence: TIFF2025186334000109.tif22165 (SEQ ID NO: 491). In some examples, M comprises two copies of such a variant IL-2 polypeptide.

[0229] Another polypeptide The polypeptide chain of the TMMP of the present disclosure may contain one or more polypeptides in addition to the above-mentioned polypeptides. Suitable additional polypeptides include epitope tags and affinity domains. The one or more additional polypeptides may be contained at the N-terminus of the polypeptide chain of the TMMP, at the C-terminus of the polypeptide chain of the TMMP, or within the polypeptide chain of the TMMP.

[0230] epitope tag Suitable epitope tags include, but are not limited to, hemagglutinin (HA, e.g., YPYDVPDYA (SEQ ID NO: 271)), FLAG (e.g., DYKDDDDK (SEQ ID NO: 272)), c-myc (e.g., EQKLISEEDL (SEQ ID NO: 273)), and the like.

[0231] Affinity Domain Affinity domains include peptide sequences capable of interacting with a binding partner, such as peptide sequences immobilized on a solid support, useful for identification or purification. DNA sequences encoding multiple consecutive single amino acids (e.g., histidines), when fused to an expressed protein, can be used for one-step purification of recombinant proteins by high-affinity binding to a resin column such as nickel sepharose. Exemplary affinity domains are described in published PCT application WO2019 / 051091, published March 14, 2014. See

[0355] .

[0232] Drug conjugates The polypeptide chain of a TMMP of the present disclosure may include a small molecule drug conjugated (e.g., covalently bonded) to the polypeptide chain. For example, if a TMMP of the present disclosure includes an Fc polypeptide, the Fc polypeptide may include a covalently bonded small molecule drug. In some examples, the small molecule drug is a cancer chemotherapeutic agent, e.g., a cytotoxic drug. Disclosure of such drug conjugates and suitable chemotherapeutic agents is provided in published PCT application WO2019 / 051091, published March 14, 2019. See

[0356] -

[0363] .

[0233] Exemplary TMMPs A TMMP of the present disclosure comprises at least one heterodimer comprising: a) a first polypeptide comprising i) a WT-1 peptide epitope and ii) a first MHC polypeptide; b) a second polypeptide comprising a second MHC polypeptide; and c) at least one MOD, wherein the first and / or second polypeptide comprises a MOD. Thus, in some examples, a TMMP comprises at least one heterodimer comprising: a) a first polypeptide comprising i) a WT-1 peptide epitope, ii) a first MHC polypeptide, and iii) at least one MOD; and b) a second polypeptide comprising a second MHC polypeptide. In other examples, a TMMP comprises at least one heterodimer comprising: a) a first polypeptide comprising i) a WT-1 peptide epitope and ii) a first MHC polypeptide; and b) a second polypeptide comprising i) a second MHC polypeptide, and ii) at least one MOD. In some examples, the TMMP comprises at least one heterodimer comprising: a) a first polypeptide comprising i) a WT-1 peptide epitope, ii) a first MHC polypeptide, and iii) at least one MOD; and b) a second polypeptide comprising i) a second MHC polypeptide and ii) at least one MOD. In some examples, at least one MOD is a wild-type immunomodulatory polypeptide. In other examples, at least one MOD is a variant MOD that exhibits lower affinity for a co-immunomodulatory polypeptide compared to the affinity of a corresponding wild-type MOD for the co-immunomodulatory polypeptide. In some examples, the TMMP comprises two MODs, wherein the two MODs have the same amino acid sequence.

[0234] In some examples, the TMMP comprises: a) a first polypeptide comprising, in N-terminal to C-terminal order: i) a WT-1 peptide epitope, ii) a first MHC polypeptide, and iii) at least one MOD; and b) a second polypeptide comprising, in N-terminal to C-terminal order: i) a second MHC polypeptide and ii) an Ig Fc polypeptide. In some examples, the first MHC polypeptide is a β2M polypeptide, and the second MHC polypeptide is an HLA heavy chain polypeptide. In some examples, the HLA heavy chain polypeptide is an HLA-A24 polypeptide. In some examples, the HLA heavy chain polypeptide is an HLA-A24 polypeptide with a Y84C substitution. In some examples, the HLA heavy chain polypeptide is an HLA-A24 polypeptide with a Y84C substitution and an Ala at position 236. In some examples, the HLA heavy chain polypeptide is an HLA-A24 polypeptide with a Y84A substitution. In some examples, the HLA heavy chain polypeptide is an HLA-A24 polypeptide with a Y84A substitution and an A236C substitution. In some examples, the HLA heavy chain polypeptide is an HLA-A24 polypeptide having a Y84C substitution and an A236C substitution. In some examples, the β2M polypeptide includes an Arg at position 12 (R12). In some examples, the β2M polypeptide includes an R12C substitution. In some examples, the first polypeptide includes, in N-terminal to C-terminal order: i) a WT-1 peptide epitope, ii) a first MHC polypeptide, and iii) two MODs, wherein the two MODs have the same amino acid sequence. In some examples, the Ig Fc polypeptide is a human IgG1 Fc polypeptide. In some examples, the Ig Fc polypeptide is an IgG1 Fc polypeptide including L234A and L235A substitutions. In some examples, the first and second polypeptides are disulfide-bonded to each other. In some examples, the MOD is a variant IL-2 polypeptide including H16A and F42A substitutions. In some examples, the MOD is a variant IL-2 polypeptide including H16T and F42A substitutions.In some examples, a peptide linker is present between one or more of: i) the second MHC polypeptide and the Ig Fc polypeptide; ii) the epitope and the first MHC polypeptide; iii) the first MHC polypeptide and the MOD; and (if the TMMP comprises two MODs on the first polypeptide chain) iv) between the two MODs. In some examples, the peptide linker comprises the amino acid sequence AAAGG (SEQ ID NO: 283). In some examples, the peptide linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO: 284), where n is an integer between 1 and 10 (e.g., n is 2, 3, or 4). In some examples, the peptide linker comprises the amino acid sequence GCGGS(GGGGS)n (SEQ ID NO: 319), where n is an integer between 1 and 9 (e.g., n is 2, 3, or 4). In some examples, the WT-1 peptide epitope is CMTWNQMN (SEQ ID NO: 261). In some examples, the WT-1 peptide epitope is CYTWNQMNL (SEQ ID NO: 262). In some examples, the WT-1 peptide epitope is SMTWNQMNL (SEQ ID NO: 451). In some examples, the WT-1 peptide epitope is GCMTWNQMNL (SEQ ID NO: 452). In some examples, the WT-1 peptide epitope is SYTWNQMNL (SEQ ID NO: 453). In some examples, the WT-1 peptide epitope is GCYTWNQMNL (SEQ ID NO: 454).

[0235] In some examples, the TMMP comprises a) a first polypeptide comprising, in N-terminal to C-terminal order: i) a WT-1 peptide epitope; and ii) a first MHC polypeptide; and b) a second polypeptide comprising, in N-terminal to C-terminal order: i) at least one MOD; ii) a second MHC polypeptide; and iii) an Ig Fc polypeptide. In some examples, the first MHC polypeptide is a β2M polypeptide, and the second MHC polypeptide is an HLA heavy chain polypeptide. In some examples, the HLA heavy chain polypeptide is an HLA-A24 polypeptide. In some examples, the HLA heavy chain polypeptide is an HLA-A24 polypeptide having an A236C substitution. In some examples, the HLA heavy chain polypeptide is an HLA-A24 polypeptide having a Y84C substitution. In some examples, the HLA heavy chain polypeptide is an HLA-A24 polypeptide having a Y84C substitution and an Ala at position 236. In some examples, the HLA heavy chain polypeptide is an HLA-A24 polypeptide having a Y84A substitution. In some examples, the HLA heavy chain polypeptide is an HLA-A24 polypeptide having a Y84A substitution and an A236C substitution. In some examples, the HLA heavy chain polypeptide is an HLA-A24 polypeptide having a Y84C substitution and an A236C substitution. In some examples, the β2M polypeptide comprises an Arg at position 12 (R12). In some examples, the β2M polypeptide comprises an R12C substitution. In some examples, the second polypeptide comprises, from N-terminus to C-terminus, i) two MODs (the two MODs have the same amino acid sequence), ii) a second MHC polypeptide, and iii) an Ig Fc polypeptide. In some examples, the Ig Fc polypeptide is a human IgG1 Fc polypeptide. In some examples, the Ig Fc polypeptide is an IgG1 Fc polypeptide comprising L234A and L235A substitutions. In some examples, the first and second polypeptides are disulfide bonded to each other. In some examples, the MOD is a variant IL-2 polypeptide comprising H16A and F42A substitutions. In some examples, the MOD is a variant IL-2 polypeptide that includes H16T and F42A substitutions.In some examples, a peptide linker is present between one or more of: i) the second MHC polypeptide and the Ig Fc polypeptide; ii) the epitope and the first MHC polypeptide; iii) the first MHC polypeptide and the MOD; and (if the TMMP comprises two MODs on the second polypeptide chain) iv) between the two MODs. In some examples, the peptide linker comprises the amino acid sequence AAAGG (SEQ ID NO: 283). In some examples, the peptide linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO: 284), where n is an integer between 1 and 10 (e.g., n is 2, 3, or 4). In some examples, the peptide linker comprises the amino acid sequence GCGGS(GGGGS)n (SEQ ID NO: 319), where n is an integer between 1 and 9 (e.g., n is 2, 3, or 4). In some examples, the WT-1 peptide epitope is CMTWNQMN (SEQ ID NO: 261). In some examples, the WT-1 peptide epitope is CYTWNQMNL (SEQ ID NO: 262). In some examples, the WT-1 peptide epitope is SMTWNQMNL (SEQ ID NO: 451). In some examples, the WT-1 peptide epitope is GCMTWNQMNL (SEQ ID NO: 452). In some examples, the WT-1 peptide epitope is SYTWNQMNL (SEQ ID NO: 453). In some examples, the WT-1 peptide epitope is GCYTWNQMNL (SEQ ID NO: 454).

[0236] In some examples, the TMMP comprises: a) a first polypeptide comprising, in N-terminal to C-terminal order: i) a WT-1 peptide epitope and ii) a first MHC polypeptide; and b) a second polypeptide comprising, in N-terminal to C-terminal order: i) a second MHC polypeptide, ii) an Ig Fc polypeptide, and iii) at least one MOD. In some examples, the first MHC polypeptide is a β2M polypeptide, and the second MHC polypeptide is an HLA heavy chain polypeptide. In some examples, the HLA heavy chain polypeptide is an HLA-A24 polypeptide. In some examples, the HLA heavy chain polypeptide is an HLA-A24 polypeptide with an A236C substitution. In some examples, the HLA heavy chain polypeptide is an HLA-A24 polypeptide with a Y84C substitution. In some examples, the HLA heavy chain polypeptide is an HLA-A24 polypeptide with a Y84C substitution and an Ala at position 236. In some examples, the HLA heavy chain polypeptide is an HLA-A24 polypeptide with a Y84A substitution. In some examples, the HLA heavy chain polypeptide is an HLA-A24 polypeptide having a Y84A substitution and an A236C substitution. In some examples, the HLA heavy chain polypeptide is an HLA-A24 polypeptide having a Y84C substitution and an A236C substitution. In some examples, the β2M polypeptide comprises an Arg at position 12 (R12). In some examples, the β2M polypeptide comprises an R12C substitution. In some examples, the second polypeptide comprises, in N-terminal to C-terminal order: i) a second MHC polypeptide, ii) an Ig Fc polypeptide, and iii) two MODs, wherein the two MODs have the same amino acid sequence. In some examples, the Ig Fc polypeptide is a human IgG1 Fc polypeptide. In some examples, the Ig Fc polypeptide is an IgG1 Fc polypeptide comprising L234A and L235A substitutions. In some examples, the first and second polypeptides are disulfide bonded to each other. In some examples, the MOD is a variant IL-2 polypeptide comprising H16A and F42A substitutions. In some examples, the MOD is a variant IL-2 polypeptide that includes H16T and F42A substitutions.In some examples, a peptide linker is present between one or more of: i) the second MHC polypeptide and the Ig Fc polypeptide; ii) the epitope and the first MHC polypeptide; iii) the Ig Fc polypeptide and the MOD; and (if the TMMP comprises two MODs on the second polypeptide chain) iv) between two immunomodulatory polypeptides. In some examples, the peptide linker comprises the amino acid sequence AAAGG (SEQ ID NO: 283). In some examples, the peptide linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO: 284), where n is an integer between 1 and 10 (e.g., n is 2, 3, or 4). In some examples, the peptide linker comprises the amino acid sequence GCGGS(GGGGS)n (SEQ ID NO: 319), where n is an integer between 1 and 9 (e.g., n is 2, 3, or 4). In some examples, the WT-1 peptide epitope is CMTWNQMN (SEQ ID NO: 261). In some examples, the WT-1 peptide epitope is CYTWNQMNL (SEQ ID NO: 262). In some examples, the WT-1 peptide epitope is SMTWNQMNL (SEQ ID NO: 451). In some examples, the WT-1 peptide epitope is GCMTWNQMNL (SEQ ID NO: 452). In some examples, the WT-1 peptide epitope is SYTWNQMNL (SEQ ID NO: 453). In some examples, the WT-1 peptide epitope is GCYTWNQMNL (SEQ ID NO: 454).

[0237] In some examples, the TMMP comprises: a) a first polypeptide comprising, from N-terminus to C-terminus, i) a WT-1 peptide epitope and ii) a first MHC polypeptide; and b) a second polypeptide comprising, from N-terminus to C-terminus, i) at least one MOD, ii) a second MHC polypeptide, and iii) an Ig Fc polypeptide. In some examples, the first MHC polypeptide is a β2M polypeptide, and the second MHC polypeptide is an HLA heavy chain polypeptide. In some examples, the HLA heavy chain polypeptide is an HLA-A24 polypeptide. In some examples, the HLA heavy chain polypeptide is an HLA-A24 polypeptide having an A236C substitution. In some examples, the Ig Fc polypeptide is a human IgG1 Fc polypeptide. In some examples, the Ig Fc polypeptide is an IgG1 Fc polypeptide having L234A and L235A substitutions. In some examples, the first and second polypeptides are disulfide-bonded to each other. In some examples, the MOD is a variant IL-2 polypeptide comprising an H16A and F42A substitution. In some examples, the MOD is a variant IL-2 polypeptide comprising an H16T and F42A substitution. In some examples, the WT-1 peptide epitope is CMTWNQMN (SEQ ID NO: 261). In some examples, the WT-1 peptide epitope is CYTWNQMNL (SEQ ID NO: 262).

[0238] In some examples, the TMMP comprises: a) a first polypeptide comprising, in N- to C-terminal order: i) at least one MOD; ii) a WT-1 peptide epitope; and iii) a first MHC polypeptide; and b) a second polypeptide comprising, in N- to C-terminal order: i) a second MHC polypeptide; and ii) an Ig Fc polypeptide. In some examples, the first MHC polypeptide is a β2M polypeptide, and the second MHC polypeptide is an HLA heavy chain polypeptide. In some examples, the HLA heavy chain polypeptide is an HLA-A24 polypeptide. In some examples, the HLA heavy chain polypeptide is an HLA-A24 polypeptide with an A236C substitution. In some examples, the first polypeptide comprises, in N- to C-terminal order: i) two MODs (the two MODs have the same amino acid sequence); ii) a WT-1 peptide epitope; and iii) the first MHC polypeptide. In some examples, the Ig Fc polypeptide is a human IgG1 Fc polypeptide. In some examples, the Ig Fc polypeptide is an IgG1 Fc polypeptide comprising L234A and L235A substitutions. In some examples, the first and second polypeptides are disulfide bonded to each other. In some examples, the MOD is a variant IL-2 polypeptide comprising H16A and F42A substitutions. In some examples, the MOD is a variant IL-2 polypeptide comprising H16T and F42A substitutions. In some examples, a peptide linker is present between one or more of: i) the second MHC polypeptide and the Ig Fc polypeptide, ii) the epitope and the first MHC polypeptide, iii) the MOD and the epitope, and (if the TMMP comprises two MODs on the first polypeptide chain) iv) between the two MODs. In some examples, the peptide linker comprises the amino acid sequence AAAGG (SEQ ID NO: 283). In some examples, the peptide linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO: 284), where n is an integer between 1 and 10 (e.g., n is 2, 3, or 4). In some examples, the WT-1 peptide epitope is CMTWNQMN (SEQ ID NO: 261). In some examples, the WT-1 peptide epitope is CYTWNQMNL (SEQ ID NO: 262).

[0239] In some examples, the TMMP comprises: a) a first polypeptide comprising, from N-terminus to C-terminus, i) a WT-1 peptide epitope and ii) a first MHC polypeptide; and b) a second polypeptide comprising, from N-terminus to C-terminus, i) a second MHC polypeptide, ii) at least one MOD, and iii) an Ig Fc polypeptide. In some examples, the first MHC polypeptide is a β2M polypeptide, and the second MHC polypeptide is an HLA heavy chain polypeptide. In some examples, the HLA heavy chain polypeptide is an HLA-A24 polypeptide. In some examples, the HLA heavy chain polypeptide is an HLA-A24 polypeptide with an A236C substitution. In some examples, the second polypeptide comprises, from N-terminus to C-terminus, i) a second MHC polypeptide, ii) two MODs (the two MODs have the same amino acid sequence), and iii) an Ig Fc polypeptide. In some examples, the Ig Fc polypeptide is a human IgG1 Fc polypeptide. In some examples, the Ig Fc polypeptide is an IgG1 Fc polypeptide comprising L234A and L235A substitutions. In some examples, the first and second polypeptides are disulfide bonded to each other. In some examples, the MOD is a variant IL-2 polypeptide comprising H16A and F42A substitutions. In some examples, the MOD is a variant IL-2 polypeptide comprising H16T and F42A substitutions. In some examples, a peptide linker is present between one or more of: i) the second MHC polypeptide and the MOD; ii) the MOD and the Ig Fc polypeptide; iii) the epitope and the first MHC polypeptide; iii) the first MHC polypeptide and the MOD; and (if the TMMP comprises two MODs on the second polypeptide chain) iv) between the two MODs. In some examples, the peptide linker comprises the amino acid sequence AAAGG (SEQ ID NO: 283). In some examples, the peptide linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO: 284), where n is an integer between 1 and 10 (e.g., n is 2, 3, or 4). In some examples, the WT-1 peptide epitope is CMTWNQMN (SEQ ID NO: 261). In some examples, the WT-1 peptide epitope is CYTWNQMNL (SEQ ID NO: 262).

[0240] In some examples, the TMMP comprises: a) a first polypeptide comprising, in N-terminal to C-terminal order: i) at least one MOD; ii) a WT-1 peptide epitope; and iii) a first MHC polypeptide; and b) a second polypeptide comprising, in N-terminal to C-terminal order: i) a second MHC polypeptide; and ii) an Ig Fc polypeptide. In some examples, the first MHC polypeptide is a β2M polypeptide, and the second MHC polypeptide is an HLA heavy chain polypeptide. In some examples, the HLA heavy chain polypeptide is an HLA-A24 polypeptide. In some examples, the HLA heavy chain polypeptide is an HLA-A24 polypeptide having an A236C substitution. In some examples, the HLA heavy chain polypeptide is an HLA-A24 polypeptide having a Y84C substitution. In some examples, the HLA heavy chain polypeptide is an HLA-A24 polypeptide having a Y84C substitution and an Ala at position 236. In some examples, the HLA heavy chain polypeptide is an HLA-A24 polypeptide having a Y84A substitution. In some examples, the HLA heavy chain polypeptide is an HLA-A24 polypeptide having a Y84A substitution and an A236C substitution. In some examples, the HLA heavy chain polypeptide is an HLA-A24 polypeptide having a Y84C substitution and an A236C substitution. In some examples, the β2M polypeptide comprises an Arg at position 12 (R12). In some examples, the β2M polypeptide comprises an R12C substitution. In some examples, the first polypeptide comprises, from N-terminus to C-terminus, i) two MODs (the two MODs have the same amino acid sequence), ii) a WT-1 peptide epitope, and iii) a first MHC polypeptide. In some examples, the Ig Fc polypeptide is a human IgG1 Fc polypeptide. In some examples, the Ig Fc polypeptide is an IgG1 Fc polypeptide comprising an L234A and an L235A substitution. In some examples, the first and second polypeptides are disulfide-bonded to each other. In some examples, the MOD is a variant IL-2 polypeptide comprising an H16A and an F42A substitution. In some examples, the MOD is a variant IL-2 polypeptide comprising an H16T and an F42A substitution.In some examples, a peptide linker is present between one or more of: i) the second MHC polypeptide and the Ig Fc polypeptide; ii) the epitope and the first MHC polypeptide; iii) the MOD and the epitope; and (when the TMMP comprises two immunomodulatory polypeptides on the first polypeptide chain) iv) between two MODs. In some examples, the peptide linker comprises the amino acid sequence AAAGG (SEQ ID NO: 283). In some examples, the peptide linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO: 284), where n is an integer between 1 and 10 (e.g., n is 2, 3, or 4). In some examples, the peptide linker comprises the amino acid sequence GCGGS(GGGGS)n (SEQ ID NO: 319), where n is an integer between 1 and 9 (e.g., n is 2, 3, or 4). In some examples, the WT-1 peptide epitope is CMTWNQMN (SEQ ID NO: 261). In some examples, the WT-1 peptide epitope is CYTWNQMNL (SEQ ID NO: 262). In some examples, the WT-1 peptide epitope is SMTWNQMNL (SEQ ID NO: 451). In some examples, the WT-1 peptide epitope is GCMTWNQMNL (SEQ ID NO: 452). In some examples, the WT-1 peptide epitope is SYTWNQMNL (SEQ ID NO: 453). In some examples, the WT-1 peptide epitope is GCYTWNQMNL (SEQ ID NO: 454).

[0241] In some examples, the TMMP comprises: a) a first polypeptide comprising, in N-terminal to C-terminal order: i) a WT-1 peptide epitope and ii) a first MHC polypeptide; and b) a second polypeptide comprising, in N-terminal to C-terminal order: i) a second MHC polypeptide, ii) at least one MOD, and iii) an Ig Fc polypeptide. In some examples, the first MHC polypeptide is a β2M polypeptide, and the second MHC polypeptide is an HLA heavy chain polypeptide. In some examples, the HLA heavy chain polypeptide is an HLA-A24 polypeptide. In some examples, the HLA heavy chain polypeptide is an HLA-A24 polypeptide having an A236C substitution. In some examples, the HLA heavy chain polypeptide is an HLA-A24 polypeptide having a Y84C substitution. In some examples, the HLA heavy chain polypeptide is an HLA-A24 polypeptide having a Y84C substitution and an Ala at position 236. In some examples, the HLA heavy chain polypeptide is an HLA-A24 polypeptide having a Y84A substitution. In some examples, the HLA heavy chain polypeptide is an HLA-A24 polypeptide having a Y84A substitution and an A236C substitution. In some examples, the HLA heavy chain polypeptide is an HLA-A24 polypeptide having a Y84C substitution and an A236C substitution. In some examples, the β2M polypeptide comprises an Arg at position 12 (R12). In some examples, the β2M polypeptide comprises an R12C substitution. In some examples, the second polypeptide comprises, from N-terminus to C-terminus, i) a second MHC polypeptide, ii) two MODs (the two MODs have the same amino acid sequence), and iii) an Ig Fc polypeptide. In some examples, the Ig Fc polypeptide is a human IgG1 Fc polypeptide. In some examples, the Ig Fc polypeptide is an IgG1 Fc polypeptide comprising an L234A and an L235A substitution. In some examples, the first and second polypeptides are disulfide bonded to each other. In some examples, the MOD is a variant IL-2 polypeptide comprising an H16A and an F42A substitution. In some examples, the MOD is a variant IL-2 polypeptide comprising an H16T and an F42A substitution.In some examples, a peptide linker is present between one or more of: i) the second MHC polypeptide and a MOD; ii) a MOD and an Ig Fc polypeptide; iii) an epitope and a first MHC polypeptide; iii) a first MHC polypeptide and a MOD; and (if the TMMP comprises two MODs on the second polypeptide chain) iv) between two MODs. In some examples, the peptide linker comprises the amino acid sequence AAAGG (SEQ ID NO: 283). In some examples, the peptide linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO: 284), where n is an integer between 1 and 10 (e.g., n is 2, 3, or 4). In some examples, the peptide linker comprises the amino acid sequence GCGGS(GGGGS)n (SEQ ID NO: 319), where n is an integer between 1 and 9 (e.g., n is 2, 3, or 4). In some examples, the WT-1 peptide epitope is CMTWNQMN (SEQ ID NO: 261). In some examples, the WT-1 peptide epitope is CYTWNQMNL (SEQ ID NO: 262). In some examples, the WT-1 peptide epitope is SMTWNQMNL (SEQ ID NO: 451). In some examples, the WT-1 peptide epitope is GCMTWNQMNL (SEQ ID NO: 452). In some examples, the WT-1 peptide epitope is SYTWNQMNL (SEQ ID NO: 453). In some examples, the WT-1 peptide epitope is GCYTWNQMNL (SEQ ID NO: 454).

[0242] As described above and shown schematically in Figure 19, a MOD (i.e., one or more MODs) can be present in a TMMP of the present disclosure at any of a variety of positions. While Figure 19 shows the location of two copies of a variant IL-2 polypeptide, the MOD can be any of a variety of MODs, as described herein. As shown in Figure 19, the MOD can be 1) the N-terminus of the MHC class I heavy chain (position 1), 2) the C-terminus of the MHC class I heavy chain and the N-terminus of the Ig Fc polypeptide, in other words, between the MHC class I heavy chain and the Ig Fc polypeptide (position 2), 3) the C-terminus of the Ig Fc polypeptide (position 3), 4) the N-terminus of the peptide epitope (position 4), or 5) the C-terminus of the β2M polypeptide (position 5). "Position 1" refers to the position of the MOD on the same polypeptide chain as the class I MHC heavy chain and on the N-terminus of the class I MHC heavy chain, e.g., in this case, the TMMP comprises a) a first polypeptide comprising, from N-terminus to C-terminus, i) a peptide epitope (e.g., a WT-1 peptide) and ii) a β2M polypeptide, and b) a second polypeptide comprising, from N-terminus to C-terminus, i) one or more MODs and ii) a class I MHC heavy chain polypeptide. "Position 2" refers to the position of a MOD on the same polypeptide chain as a class I MHC heavy chain that is C-terminal to the class I MHC heavy chain but not at the C-terminus of the polypeptide chain; for example, in this case, a TMMP comprises a) a first polypeptide comprising, from N-terminus to C-terminus, i) a peptide epitope (e.g., a WT-1 peptide) and ii) a β2M polypeptide, and b) a second polypeptide comprising, from N-terminus to C-terminus, i) a class I MHC heavy chain polypeptide, ii) one or more MODs, and iii) an Ig Fc polypeptide. "Position 3" refers to the position of the MOD on the same polypeptide chain as the class I MHC heavy chain and at the C-terminus of the polypeptide chain, e.g., in this case, the TMMP comprises a) a first polypeptide comprising, from N-terminus to C-terminus, i) a peptide epitope (e.g., a WT-1 peptide) and ii) a β2M polypeptide, and b) a second polypeptide comprising, from N-terminus to C-terminus, i) a class I MHC heavy chain polypeptide, ii) an Ig Fc polypeptide, and iii) one or more MODs."Position 4" refers to the position of a MOD on the same polypeptide chain as the β2M polypeptide and at the peptide epitope and N-terminus of the β2M polypeptide; for example, in this case, a TMMP comprises a) a first polypeptide comprising, from N-terminus to C-terminus, i) one or more MODs, ii) a peptide epitope (e.g., a WT-1 peptide), and iii) a β2M polypeptide; and b) a second polypeptide comprising a Class I MHC heavy chain polypeptide (e.g., a second polypeptide comprising, from N-terminus to C-terminus, i) a Class I MHC heavy chain polypeptide and ii) an Ig Fc polypeptide). "Position 5" refers to the position of a MOD on the same polypeptide chain as the β2M polypeptide and at the C-terminus of the β2M polypeptide (e.g., the C-terminus of the polypeptide chain). For example, in this case, the TMMP comprises: a) a first polypeptide comprising, in order from N-terminus to C-terminus: i) a peptide epitope (e.g., a WT-1 peptide), ii) a β2M polypeptide, and iii) one or more MODs; and b) a second polypeptide comprising a class I MHC heavy chain polypeptide (e.g., a second polypeptide comprising, in order from N-terminus to C-terminus: i) a class I MHC heavy chain polypeptide and ii) an Ig Fc polypeptide).

[0243] Furthermore, as discussed above and shown schematically in Figures 18A-18C, the first and second polypeptide chains of a TMMP can be linked by one or more disulfide bonds. For example, a TMMP can include a) a first polypeptide chain comprising a β2M polypeptide having an R12C substitution, and b) a second polypeptide chain comprising a Class I MHC heavy chain polypeptide having an A236C substitution, thereby forming a disulfide bond between Cys at position 12 of the β2M polypeptide of the first polypeptide chain and Cys at position 236 of the Class I MHC heavy chain polypeptide of the second polypeptide chain. As another example, a TMMP can include, from N- to C-terminus, i) a peptide epitope and ii) a GCGGS (G4S) peptide. n(SEQ ID NO: 315) (n is 1, 2, or 3), and iii) a first polypeptide comprising a β2M polypeptide, and b) a second polypeptide comprising a Class I MHC heavy chain polypeptide with a Y84C substitution, thereby forming a disulfide bond between Cys in the peptide linker of the first polypeptide chain and Cys at position 84 of the Class I MHC heavy chain polypeptide of the second polypeptide chain. In another example, the TMMP comprises, a) from N- to C-terminus, i) a peptide epitope, and ii) a GCGGS (G4S) n (SEQ ID NO:315) (n is 1, 2, or 3); iii) a first polypeptide comprising a β2M polypeptide having an R12C substitution; and b) a second polypeptide comprising a Class I MHC heavy chain polypeptide having a Y84C substitution and an A236C substitution, thereby forming i) a first disulfide bond between Cys of the peptide linker of the first polypeptide chain and Cys at position 84 of the Class I MHC heavy chain polypeptide of the second polypeptide chain, and ii) a second disulfide bond between Cys at position 12 of the β2M polypeptide of the first polypeptide chain and Cys at position 236 of the Class I MHC heavy chain polypeptide of the second polypeptide chain. For simplicity, the first disulfide bond will be referred to as "G2C / Y84C" and the second disulfide bond will be referred to as "R12C / A236C." The TMMP can have a) a G2C / Y84C disulfide bond and no R12C / A236C disulfide bond, b) an R12C / A236C disulfide bond and no G2C / Y84C disulfide bond, or c) a G2C / Y84C disulfide bond and an R12C / A236C disulfide bond.

[0244] A TMMP can a) include a G2C / Y84C disulfide bond and not include an R12C / A236C disulfide bond, and b) include at least one immunomodulatory polypeptide at position 1. A TMMP can a) include a G2C / Y84C disulfide bond and not include an R12C / A236C disulfide bond, and b) include at least one MOD at position 2. A TMMP of the present disclosure can a) include a G2C / Y84C disulfide bond and not include an R12C / A236C disulfide bond, and b) include at least one MOD at position 3. A TMMP can a) include a G2C / Y84C disulfide bond and not include an R12C / A236C disulfide bond, and b) include at least one MOD at position 4. The TMMP can a) contain a G2C / Y84C disulfide bond and not contain a R12C / A236C disulfide bond, and b) contain at least one MOD at position 5.

[0245] A TMMP can include a) an R12C / A236C disulfide bond, no G2C / Y84C disulfide bond, and at least one MOD at position 1. A TMMP can include a) an R12C / A236C disulfide bond, no G2C / Y84C disulfide bond, and at least one MOD at position 2. A TMMP can include a) an R12C / A236C disulfide bond, no G2C / Y84C disulfide bond, and at least one MOD at position 3. A TMMP of the present disclosure can include a) an R12C / A236C disulfide bond, no G2C / Y84C disulfide bond, and at least one immunomodulatory polypeptide at position 4. The TMMP can include: a) an R12C / A236C disulfide bond, but not a G2C / Y84C disulfide bond, and at least one MOD at position 5.

[0246] A TMMP can include a) a G2C / Y84C disulfide bond and a R12C / A236C disulfide bond, and b) at least one MOD at position 1. A TMMP can include a) a G2C / Y84C disulfide bond and a R12C / A236C disulfide bond, and b) at least one MOD at position 2. A TMMP can include a) a G2C / Y84C disulfide bond and a R12C / A236C disulfide bond, and b) at least one MOD at position 3. A TMMP of the present disclosure can include a) a G2C / Y84C disulfide bond and a R12C / A236C disulfide bond, and b) at least one MOD at position 4. The TMMP can include a) a G2C / Y84C disulfide bond and a R12C / A236C disulfide bond, and b) at least one MOD at position 5.

[0247] Non-limiting examples of amino acid sequences of the first and second polypeptide chains of TMMPs of the present disclosure are shown in Figures 4A-4K and Figures 20A-20R.

[0248] In some examples, the TMMP comprises a) a first polypeptide chain comprising an amino acid sequence designated "2752" as shown in Figure 4D, and b) a second polypeptide chain comprising an amino acid sequence designated "3159" as shown in Figure 4C.

[0249] In some examples, the TMMP comprises a) a first polypeptide chain comprising the amino acid sequence designated "2753" as shown in Figure 4E, and b) a second polypeptide chain comprising the amino acid sequence designated "3159" as shown in Figure 4C. Such a TMMP comprises a) a MOD on position 3 as shown in Figure 19, and b) an R12C / A236C disulfide bond (but does not comprise a G2C / Y84C disulfide bond).

[0250] In some instances, the TMMP comprises a) a first polypeptide chain comprising an amino acid sequence designated "2752" as shown in Figure 4D, and b) a second polypeptide chain comprising an amino acid sequence designated "2750" as shown in Figure 4B.

[0251] In some examples, a TMMP comprises a) a first polypeptide chain comprising an amino acid sequence designated "2753" as shown in Figure 4E, and b) a second polypeptide chain comprising an amino acid sequence designated "2750" as shown in Figure 4B. Such a TMMP comprises a) a MOD at position 1 as shown in Figure 19, and b) an R12C / A236C disulfide bond (but does not comprise a G2C / Y84C disulfide bond).

[0252] In some instances, the TMMP comprises a) a first polypeptide chain comprising an amino acid sequence designated "2752," as shown in FIG. 4D, and b) a second polypeptide chain comprising an amino acid sequence designated "3158," as shown in FIG. 4A.

[0253] In some instances, the TMMP comprises a) a first polypeptide chain comprising an amino acid sequence designated "2753," as shown in FIG. 4E, and b) a second polypeptide chain comprising an amino acid sequence designated "3158," as shown in FIG. 4A.

[0254] In some instances, the TMMP comprises a) a first polypeptide chain comprising an amino acid sequence designated "2380," as shown in FIG. 14B, and b) a second polypeptide chain comprising an amino acid sequence designated "1715," as shown in FIG. 14A.

[0255] In some examples, the TMMP comprises a) a first polypeptide chain comprising, from N-terminus to C-terminus: i) a WT-1 peptide having the sequence VLDFAPPGA (SEQ ID NO: 259); ii) a linker having the amino acid sequence GCGGSGGGSGGGGS (SEQ ID NO: 317); and iii) a β2M polypeptide comprising a Cys at position 12 (e.g., a β2M having the amino acid sequence set forth in SEQ ID NO: 311); and b) a first polypeptide chain comprising, from N-terminus to C-terminus: i) a variant IL-2 polypeptide comprising H16A and F42A substitutions ii) a (GGGGS) linker; iii) a variant IL-2 polypeptide comprising H16A and F42A substitutions (i.e., the amino acid sequence set forth in SEQ ID NO: 188 (X1 is Ala and X2 is Ala)), iv) a (GGGGS) linker; v) an HLA A0202 heavy chain comprising Cys at positions 84 and 236 (e.g., an HLA heavy chain polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 341); vi) an AAAGG linker; and vii) a second polypeptide chain comprising an Ig Fc polypeptide. In some examples, the Ig Fc polypeptide comprises an amino acid sequence having at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to the amino acid sequence of the Fc region shown in Figures 5A-5G or 5H. In some examples, the Ig Fc polypeptide is a variant Ig Fc polypeptide comprising one or more sequence changes compared to the wild-type polypeptide, such that the ability of the Ig Fc polypeptide to induce cell lysis via complement-dependent cytotoxicity (CDC) and / or antibody-dependent cellular cytotoxicity (ADCC) is reduced or substantially eliminated. In some examples, the Ig Fc polypeptide is a variant human IgG1 Fc polypeptide comprising an L234A and / or L235A substitution (L14 and L15 in the amino acid sequence shown in Figure 5H). In some examples, the Ig Fc polypeptide comprises the amino acid sequence shown in Figure 5H and set forth in SEQ ID NO:487.

[0256] In some examples, the TMMP comprises a) a first polypeptide chain comprising, from N-terminus to C-terminus: i) a WT-1 peptide of the sequence VLDFAPPGA (SEQ ID NO: 259); ii) a linker having the amino acid sequence GCGGSGGGSGGGGGS (SEQ ID NO: 317); and iii) a β2M polypeptide comprising a Cys at position 12 (e.g., a β2M having the amino acid sequence set forth in SEQ ID NO: 311); and b) a first polypeptide chain comprising, from N-terminus to C-terminus: i) a variant IL-2 polypeptide comprising H16A and F42A substitutions (e.g., a β2M having the amino acid sequence set forth in SEQ ID NO: 311). ii) a (GGGGS) linker; iii) a variant IL-2 polypeptide comprising H16A and F42A substitutions (i.e., an amino acid sequence set forth in SEQ ID NO: 188 (X1 is Ala and X2 is Ala) comprising Ala at positions 16 and 42; iv) a (GGGGS) linker; v) an HLA A0202 heavy chain comprising Cys at positions 84 and 236 (e.g., an HLA heavy chain polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 341); vi) an AAAGG linker; and vii) an Ig Fc polypeptide comprising Ala at positions 14 and 15 and lacking a C-terminal Lys (e.g., an Ig Fc polypeptide shown in Figure 5H and comprising the amino acid sequence set forth in SEQ ID NO: 487). For example, in some instances, a TMMP comprises a) a first polypeptide chain comprising an amino acid sequence designated "2380," as shown in Figure 14B, and b) a second polypeptide chain comprising an amino acid sequence designated "1715 without C-terminal Lys," as shown in Figure 14J. The construct shown in Figure 14J ("1715 without C-terminal Lys") is also referred to herein as "1715Δ."

[0257] In some instances, the TMMP comprises a) a first polypeptide chain comprising an amino acid sequence designated "2381," as shown in FIG. 14B, and b) a second polypeptide chain comprising an amino acid sequence designated "1715," as shown in FIG. 14A, or 1715Δ, as shown in FIG. 14J and set forth in SEQ ID NO: 486.

[0258] In some instances, the TMMP comprises a) a first polypeptide chain comprising an amino acid sequence designated "2380," as shown in Figure 14B, and b) a second polypeptide chain comprising an amino acid sequence designated "2405," as shown in Figure 14D.

[0259] In some examples, the TMMP comprises a) a first polypeptide chain comprising an amino acid sequence designated "2381," as shown in Figure 14B, and b) a second polypeptide chain comprising an amino acid sequence designated "2405," as shown in Figure 14D. In some examples, the TMMP comprises a) a first polypeptide chain comprising an amino acid sequence designated "2762," as shown in Figure 14F, and b) a second polypeptide chain comprising an amino acid sequence designated "2405," as shown in Figure 14D.

[0260] In some instances, the TMMP comprises a) a first polypeptide chain comprising an amino acid sequence designated "2380," as shown in FIG. 14B, and b) a second polypeptide chain comprising an amino acid sequence designated "1380," as shown in FIG. 14E.

[0261] In some instances, the TMMP comprises a) a first polypeptide chain comprising an amino acid sequence designated "2381," as shown in FIG. 14B, and b) a second polypeptide chain comprising an amino acid sequence designated "1380," as shown in FIG. 14E.

[0262] In some examples, a TMMP comprises a) a first polypeptide chain comprising an amino acid sequence designated "3592," as shown in Figure 20A, and b) a second polypeptide chain comprising an amino acid sequence designated "3188," as shown in Figure 20H. Such a TMMP comprises a) a MOD on position 1, as shown in Figure 19, and b) both a G2C / Y84C disulfide bond and a R12C / A236C disulfide bond.

[0263] In some examples, a TMMP comprises a) a first polypeptide chain comprising an amino acid sequence designated "3425," as shown in Figure 20B, and b) a second polypeptide chain comprising an amino acid sequence designated "3188," as shown in Figure 20H. Such a TMMP comprises a) a MOD on position 3, as shown in Figure 19, and b) both a G2C / Y84C disulfide bond and a R12C / A236C disulfide bond.

[0264] In some examples, a TMMP comprises a) a first polypeptide chain comprising an amino acid sequence designated "3196," as shown in Figure 20C, and b) a second polypeptide chain comprising an amino acid sequence designated "3604," as shown in Figure 20I. Such a TMMP comprises a) a MOD on position 5, as shown in Figure 19, and b) both a G2C / Y84C disulfide bond and a R12C / A236C disulfide bond.

[0265] In some examples, the TMMP comprises a) a first polypeptide chain comprising an amino acid sequence designated "2764," as shown in Figure 20D, and b) a second polypeptide chain comprising an amino acid sequence designated "3603," as shown in Figure 20J. Such a TMMP comprises a) a MOD at position 5, as shown in Figure 19, and b) an R12C / A236C disulfide bond (but does not comprise a G2C / Y84C disulfide bond).

[0266] In some examples, the TMMP comprises a) a first polypeptide chain comprising an amino acid sequence designated "3593," as shown in Figure 20E, and b) a second polypeptide chain comprising an amino acid sequence designated "3192," as shown in Figure 20K. Such a TMMP comprises a) a MOD at position 1, as shown in Figure 19, and b) a G2C / Y84C disulfide bond (but does not comprise an R12C / A236C disulfide bond).

[0267] In some examples, the TMMP comprises a) a first polypeptide chain comprising an amino acid sequence designated "3426," as shown in Figure 20F, and b) a second polypeptide chain comprising an amino acid sequence designated "3192," as shown in Figure 20K. Such a TMMP comprises a) a MOD on position 3, as shown in Figure 19, and b) a G2C / Y84C disulfide bond (but does not comprise an R12C / A236C disulfide bond).

[0268] In some examples, the TMMP comprises a) a first polypeptide chain comprising an amino acid sequence designated "3197," as shown in Figure 20G, and b) a second polypeptide chain comprising an amino acid sequence designated "3605," as shown in Figure 20L. Such a TMMP comprises a) a MOD on position 5, as shown in Figure 19, and b) a G2C / Y84C disulfide bond (but not an R12C / A236C disulfide bond).

[0269] In some examples, a TMMP comprises a) a first polypeptide chain comprising an amino acid sequence designated "3592," as shown in Figure 20A, and b) a second polypeptide chain comprising an amino acid sequence designated "3529," as shown in Figure 20M. Such a TMMP comprises a) a MOD at position 1, as shown in Figure 19, and b) both a G2C / Y84C disulfide bond and a R12C / A236C disulfide bond.

[0270] In some examples, the TMMP comprises a) a first polypeptide chain comprising an amino acid sequence designated "3425" as shown in Figure 20B, and b) a second polypeptide chain comprising an amino acid sequence designated "3529" as shown in Figure 20M. Such a TMMP comprises a) a MOD at position 3 as shown in Figure 19, and b) both a G2C / Y84C disulfide bond and a R12C / A236C disulfide bond, and further comprises the WT1 239-247 (Q240Y) epitope.

[0271] In some examples, the TMMP comprises a) a first polypeptide chain comprising an amino acid sequence designated "3196," as shown in Figure 20C, and b) a second polypeptide chain comprising an amino acid sequence designated "3709," as shown in Figure 20N. Such a TMMP comprises a) a MOD on position 3, as shown in Figure 19, and b) both a G2C / Y84C disulfide bond and a R12C / A236C disulfide bond.

[0272] In some examples, the TMMP comprises a) a first polypeptide chain comprising an amino acid sequence designated "2750," as shown in Figure 4B, and b) a second polypeptide chain comprising an amino acid sequence designated "3528," as shown in Figure 20O. Such a TMMP comprises a) a MOD at position 1, as shown in Figure 19, and b) an R12C / A236C disulfide bond (but does not comprise a G2C / Y84C disulfide bond).

[0273] In some examples, the TMMP comprises a) a first polypeptide chain comprising an amino acid sequence designated "3159," as shown in Figure 4C, and b) a second polypeptide chain comprising an amino acid sequence designated "3528," as shown in Figure 20O. Such a TMMP comprises a) a MOD on position 3, as shown in Figure 19, and b) an R12C / A236C disulfide bond (but does not comprise a G2C / Y84C disulfide bond).

[0274] In some examples, the TMMP comprises a) a first polypeptide chain comprising an amino acid sequence designated "2764," as shown in Figure 20D, and b) a second polypeptide chain comprising an amino acid sequence designated "3708," as shown in Figure 20P. Such a TMMP comprises a) a MOD at position 5, as shown in Figure 19, and b) an R12C / A236C disulfide bond (but does not comprise a G2C / Y84C disulfide bond).

[0275] In some examples, the TMMP comprises a) a first polypeptide chain comprising an amino acid sequence designated "3593," as shown in Figure 20E, and b) a second polypeptide chain comprising an amino acid sequence designated "3530," as shown in Figure 20Q. Such a TMMP comprises a) a MOD at position 1, as shown in Figure 19, and b) a G2C / Y84C disulfide bond (but does not comprise an R12C / A236C disulfide bond).

[0276] In some examples, the TMMP comprises a) a first polypeptide chain comprising an amino acid sequence designated "3426," as shown in Figure 20F, and b) a second polypeptide chain comprising an amino acid sequence designated "3530," as shown in Figure 20Q. Such a TMMP comprises a) a MOD on position 3, as shown in Figure 19, and b) a G2C / Y84C disulfide bond (but does not comprise an R12C / A236C disulfide bond).

[0277] In some examples, the TMMP comprises a) a first polypeptide chain comprising an amino acid sequence designated "3197," as shown in Figure 20G, and b) a second polypeptide chain comprising an amino acid sequence designated "3710," as shown in Figure 20R. Such a TMMP comprises a) a MOD on position 5, as shown in Figure 19, and b) a G2C / Y84C disulfide bond (but not an R12C / A236C disulfide bond).

[0278] In some examples, the TMMP comprises a) a first polypeptide chain comprising an amino acid sequence designated "3426," as shown in Figure 20F, and b) a second polypeptide chain comprising an amino acid sequence designated "3529," as shown in Figure 20M. Such a TMMP comprises a) a MOD at position 3, as shown in Figure 19, and b) a G2C / Y84C disulfide bond (but not an R12C / A236C disulfide bond), and also comprises the WT1 239-247 (Q240Y) epitope.

[0279] In some examples, the TMMP comprises a) a first polypeptide chain comprising an amino acid sequence designated "3425," as shown in Figure 20B, and b) a second polypeptide chain comprising an amino acid sequence designated "3528," as shown in Figure 20O. Such a TMMP comprises a) a MOD at position 3, as shown in Figure 19, and b) an R12C / A236C disulfide bond (but not a G2C / Y84C disulfide bond), and also comprises the WT1 239-247 (Q240Y) epitope.

[0280] In some examples, the TMMP comprises a) a first polypeptide chain comprising an amino acid sequence designated "3425," as shown in Figure 20B, and b) a second polypeptide chain comprising an amino acid sequence designated "3530," as shown in Figure 20Q. Such a TMMP comprises a) a MOD at position 3, as shown in Figure 19, and b) a G2C / Y84C disulfide bond (but not an R12C / A236C disulfide bond), and also comprises the WT1 239-247 (Q240Y) epitope.

[0281] In some examples, the TMMP comprises a) a first polypeptide chain comprising an amino acid sequence designated "3159," as shown in FIG. 4C, and b) a second polypeptide chain comprising an amino acid sequence designated "3188," as shown in FIG. 20H. Such a TMMP comprises a) a MOD at position 3, as shown in FIG. 19, and b) an R12C / A236C disulfide bond (but not a G2C / Y84C disulfide bond), and also comprises the WT1 235-243 (M236Y) epitope.

[0282] An exemplary TMMP having the epitope SMTWNQMNL (WT1(235-243, C235S)) In some examples, the TMMP comprises a) a first polypeptide chain comprising the amino acid sequence shown in Figure 35A, and b) a second polypeptide chain comprising the amino acid sequence shown in Figure 4B. Such a TMMP comprises a) a MOD at position 1, as shown in Figure 19, and b) an R12C / A236C disulfide bond (but does not comprise a G2C / Y84C disulfide bond).

[0283] In some examples, the TMMP comprises a) a first polypeptide chain comprising the amino acid sequence shown in Figure 35A, and b) a second polypeptide chain comprising the amino acid sequence shown in Figure 4C. Such a TMMP comprises a) a MOD at position 3, as shown in Figure 19, and b) an R12C / A236C disulfide bond (but does not comprise a G2C / Y84C disulfide bond).

[0284] In some examples, the TMMP comprises a) a first polypeptide chain comprising the amino acid sequence shown in Figure 35B, and b) a second polypeptide chain comprising the amino acid sequence shown in Figure 20A. Such a TMMP comprises a) a MOD at position 1, as shown in Figure 19, and b) both a G2C / Y84C disulfide bond and a R12C / A236C disulfide bond.

[0285] In some examples, the TMMP comprises a) a first polypeptide chain comprising the amino acid sequence shown in Figure 35B, and b) a second polypeptide chain comprising the amino acid sequence shown in Figure 20B. Such a TMMP comprises a) a MOD at position 3, as shown in Figure 19, and b) both a G2C / Y84C disulfide bond and a R12C / A236C disulfide bond.

[0286] In some examples, the TMMP comprises a) a first polypeptide chain comprising the amino acid sequence shown in Figure 35C, and b) a second polypeptide chain comprising the amino acid sequence shown in Figure 20E. Such a TMMP comprises a) a MOD at position 1, as shown in Figure 19, and b) a G2C / Y84C disulfide bond (but does not comprise an R12C / A236C disulfide bond).

[0287] In some examples, the TMMP comprises a) a first polypeptide chain comprising the amino acid sequence shown in Figure 35C, and b) a second polypeptide chain comprising the amino acid sequence shown in Figure 20F. Such a TMMP comprises a) a MOD at position 3, as shown in Figure 19, and b) a G2C / Y84C disulfide bond (but does not comprise an R12C / A236C disulfide bond).

[0288] In some examples, the TMMP comprises a) a first polypeptide chain comprising the amino acid sequence shown in Figure 35D, and b) a second polypeptide chain comprising the amino acid sequence shown in Figure 20G. Such a TMMP comprises a) a MOD at position 5, as shown in Figure 19, and b) a G2C / Y84C disulfide bond (but does not include an R12C / A236C disulfide bond).

[0289] In some examples, the TMMP comprises a) a first polypeptide chain comprising the amino acid sequence shown in Figure 35E, and b) a second polypeptide chain comprising the amino acid sequence shown in Figure 20C. Such a TMMP comprises a) a MOD at position 5, as shown in Figure 19, and b) both a G2C / Y84C disulfide bond and a R12C / A236C disulfide bond.

[0290] In some examples, the TMMP comprises a) a first polypeptide chain comprising the amino acid sequence shown in Figure 35F, and b) a second polypeptide chain comprising the amino acid sequence shown in Figure 20D. Such a TMMP comprises a) a MOD at position 5, as shown in Figure 19, and b) an R12C / A236C disulfide bond (but does not include a G2C / Y84C disulfide bond).

[0291] An exemplary TMMP having the epitope GCMTWNQMNL (WT1(235-243, G-1)) In some examples, the TMMP comprises a) a first polypeptide chain comprising the amino acid sequence shown in Figure 36A, and b) a second polypeptide chain comprising the amino acid sequence shown in Figure 4B. Such a TMMP comprises a) a MOD at position 1, as shown in Figure 19, and b) an R12C / A236C disulfide bond (but does not comprise a G2C / Y84C disulfide bond).

[0292] In some examples, the TMMP comprises a) a first polypeptide chain comprising the amino acid sequence shown in Figure 36A, and b) a second polypeptide chain comprising the amino acid sequence shown in Figure 4C. Such a TMMP comprises a) a MOD at position 3, as shown in Figure 19, and b) an R12C / A236C disulfide bond (but does not comprise a G2C / Y84C disulfide bond).

[0293] In some examples, a TMMP comprises a) a first polypeptide chain comprising the amino acid sequence shown in Figure 36B, and b) a second polypeptide chain comprising the amino acid sequence shown in Figure 20A. Such a TMMP comprises a) a MOD at position 1, as shown in Figure 19, and b) both a G2C / Y84C disulfide bond and a R12C / A236C disulfide bond.

[0294] In some examples, the TMMP comprises a) a first polypeptide chain comprising the amino acid sequence shown in Figure 36B, and b) a second polypeptide chain comprising the amino acid sequence shown in Figure 20B. Such a TMMP comprises a) a MOD at position 3, as shown in Figure 19, and b) both a G2C / Y84C disulfide bond and a R12C / A236C disulfide bond.

[0295] In some examples, the TMMP comprises a) a first polypeptide chain comprising the amino acid sequence shown in Figure 36C, and b) a second polypeptide chain comprising the amino acid sequence shown in Figure 20E. Such a TMMP comprises a) a MOD at position 1, as shown in Figure 19, and b) a G2C / Y84C disulfide bond (but does not comprise an R12C / A236C disulfide bond).

[0296] In some examples, the TMMP comprises a) a first polypeptide chain comprising the amino acid sequence shown in Figure 36C, and b) a second polypeptide chain comprising the amino acid sequence shown in Figure 20F. Such a TMMP comprises a) a MOD at position 3, as shown in Figure 19, and b) a G2C / Y84C disulfide bond (but does not comprise an R12C / A236C disulfide bond).

[0297] In some examples, the TMMP comprises a) a first polypeptide chain comprising the amino acid sequence shown in Figure 36D, and b) a second polypeptide chain comprising the amino acid sequence shown in Figure 20G. Such a TMMP comprises a) a MOD at position 5, as shown in Figure 19, and b) a G2C / Y84C disulfide bond (but does not include an R12C / A236C disulfide bond).

[0298] In some examples, the TMMP comprises a) a first polypeptide chain comprising the amino acid sequence shown in Figure 36E, and b) a second polypeptide chain comprising the amino acid sequence shown in Figure 20C. Such a TMMP comprises a) a MOD at position 5, as shown in Figure 19, and b) both a G2C / Y84C disulfide bond and a R12C / A236C disulfide bond.

[0299] In some examples, the TMMP comprises a) a first polypeptide chain comprising the amino acid sequence shown in Figure 36F, and b) a second polypeptide chain comprising the amino acid sequence shown in Figure 20D. Such a TMMP comprises a) a MOD at position 5, as shown in Figure 19, and b) an R12C / A236C disulfide bond (but does not include a G2C / Y84C disulfide bond).

[0300] An exemplary TMMP having the epitope SYTWNQMNL (WT1(235-243, C235S, M236Y)) In some examples, the TMMP comprises a) a first polypeptide chain comprising the amino acid sequence shown in Figure 37A, and b) a second polypeptide chain comprising the amino acid sequence shown in Figure 4B. Such a TMMP comprises a) a MOD at position 1, as shown in Figure 19, and b) an R12C / A236C disulfide bond (but does not comprise a G2C / Y84C disulfide bond).

[0301] In some examples, the TMMP comprises a) a first polypeptide chain comprising the amino acid sequence shown in Figure 37A, and b) a second polypeptide chain comprising the amino acid sequence shown in Figure 4C. Such a TMMP comprises a) a MOD at position 3, as shown in Figure 19, and b) an R12C / A236C disulfide bond (but does not comprise a G2C / Y84C disulfide bond).

[0302] In some examples, a TMMP comprises a) a first polypeptide chain comprising the amino acid sequence shown in Figure 37B, and b) a second polypeptide chain comprising the amino acid sequence shown in Figure 20A. Such a TMMP comprises a) a MOD at position 1, as shown in Figure 19, and b) both a G2C / Y84C disulfide bond and a R12C / A236C disulfide bond.

[0303] In some examples, the TMMP comprises a) a first polypeptide chain comprising the amino acid sequence shown in Figure 37B, and b) a second polypeptide chain comprising the amino acid sequence shown in Figure 20B. Such a TMMP comprises a) a MOD at position 3, as shown in Figure 19, and b) both a G2C / Y84C disulfide bond and a R12C / A236C disulfide bond.

[0304] In some examples, the TMMP comprises a) a first polypeptide chain comprising the amino acid sequence shown in Figure 37C, and b) a second polypeptide chain comprising the amino acid sequence shown in Figure 20E. Such a TMMP comprises a) a MOD at position 1, as shown in Figure 19, and b) a G2C / Y84C disulfide bond (but does not comprise an R12C / A236C disulfide bond).

[0305] In some examples, the TMMP comprises a) a first polypeptide chain comprising the amino acid sequence shown in Figure 37C, and b) a second polypeptide chain comprising the amino acid sequence shown in Figure 20F. Such a TMMP comprises a) a MOD at position 3, as shown in Figure 19, and b) a G2C / Y84C disulfide bond (but does not comprise an R12C / A236C disulfide bond).

[0306] In some examples, the TMMP comprises a) a first polypeptide chain comprising the amino acid sequence shown in Figure 37D, and b) a second polypeptide chain comprising the amino acid sequence shown in Figure 20G. Such a TMMP comprises a) a MOD at position 5, as shown in Figure 19, and b) a G2C / Y84C disulfide bond (but does not include an R12C / A236C disulfide bond).

[0307] In some examples, the TMMP comprises a) a first polypeptide chain comprising the amino acid sequence shown in Figure 37E, and b) a second polypeptide chain comprising the amino acid sequence shown in Figure 20C. Such a TMMP comprises a) a MOD at position 5, as shown in Figure 19, and b) both a G2C / Y84C disulfide bond and a R12C / A236C disulfide bond.

[0308] In some examples, the TMMP comprises a) a first polypeptide chain comprising the amino acid sequence shown in Figure 37F, and b) a second polypeptide chain comprising the amino acid sequence shown in Figure 20D. Such a TMMP comprises a) a MOD at position 5, as shown in Figure 19, and b) an R12C / A236C disulfide bond (but does not include a G2C / Y84C disulfide bond).

[0309] An exemplary TMMP having the epitope GCYTWNQMNL (WT1(235-243, G-1, M236Y)) In some examples, the TMMP comprises a) a first polypeptide chain comprising the amino acid sequence shown in Figure 38A, and b) a second polypeptide chain comprising the amino acid sequence shown in Figure 4B. Such a TMMP comprises a) a MOD at position 1, as shown in Figure 19, and b) an R12C / A236C disulfide bond (but does not comprise a G2C / Y84C disulfide bond).

[0310] In some examples, the TMMP comprises a) a first polypeptide chain comprising the amino acid sequence shown in Figure 38A, and b) a second polypeptide chain comprising the amino acid sequence shown in Figure 4C. Such a TMMP comprises a) a MOD at position 3, as shown in Figure 19, and b) an R12C / A236C disulfide bond (but does not comprise a G2C / Y84C disulfide bond).

[0311] In some examples, a TMMP comprises a) a first polypeptide chain comprising the amino acid sequence shown in Figure 38B, and b) a second polypeptide chain comprising the amino acid sequence shown in Figure 20A. Such a TMMP comprises a) a MOD at position 1, as shown in Figure 19, and b) both a G2C / Y84C disulfide bond and a R12C / A236C disulfide bond.

[0312] In some examples, the TMMP comprises a) a first polypeptide chain comprising the amino acid sequence shown in Figure 38B, and b) a second polypeptide chain comprising the amino acid sequence shown in Figure 20B. Such a TMMP comprises a) a MOD at position 3, as shown in Figure 19, and b) both a G2C / Y84C disulfide bond and a R12C / A236C disulfide bond.

[0313] In some examples, the TMMP comprises a) a first polypeptide chain comprising the amino acid sequence shown in Figure 38C, and b) a second polypeptide chain comprising the amino acid sequence shown in Figure 20E. Such a TMMP comprises a) a MOD at position 1, as shown in Figure 19, and b) a G2C / Y84C disulfide bond (but does not comprise an R12C / A236C disulfide bond).

[0314] In some examples, the TMMP comprises a) a first polypeptide chain comprising the amino acid sequence shown in Figure 38C, and b) a second polypeptide chain comprising the amino acid sequence shown in Figure 20F. Such a TMMP comprises a) a MOD at position 3, as shown in Figure 19, and b) a G2C / Y84C disulfide bond (but does not comprise an R12C / A236C disulfide bond).

[0315] In some examples, the TMMP comprises a) a first polypeptide chain comprising the amino acid sequence shown in Figure 38D, and b) a second polypeptide chain comprising the amino acid sequence shown in Figure 20G. Such a TMMP comprises a) a MOD at position 5, as shown in Figure 19, and b) a G2C / Y84C disulfide bond (but does not include an R12C / A236C disulfide bond).

[0316] In some examples, the TMMP comprises a) a first polypeptide chain comprising the amino acid sequence shown in Figure 38E, and b) a second polypeptide chain comprising the amino acid sequence shown in Figure 20C. Such a TMMP comprises a) a MOD at position 5, as shown in Figure 19, and b) both a G2C / Y84C disulfide bond and a R12C / A236C disulfide bond.

[0317] In some examples, the TMMP comprises a) a first polypeptide chain comprising the amino acid sequence shown in Figure 38F, and b) a second polypeptide chain comprising the amino acid sequence shown in Figure 20D. Such a TMMP comprises a) a MOD ...

Claims

1. A T cell modulatory multimeric polypeptide (TMMP) comprising at least one heterodimer, wherein said at least one heterodimer is a) i) a Wilms tumor-1 (WT-1) peptide epitope having the amino acid sequence VLDFAPPGA (SEQ ID NO: 259); ii) a β2 microglobulin (β2M) polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 311; and iii) a linker between the WT-1 peptide epitope and the β2M polypeptide, the linker comprising the amino acid sequence set forth in SEQ ID NO: 317; and a first polypeptide comprising: b) i) a major histocompatibility complex (MHC) class I heavy chain polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 341; ii) an immunoglobulin (Ig) Fc polypeptide; and iii) two variant IL-2 immunomodulatory polypeptides; a second polypeptide comprising Including, the heterodimer comprises a first disulfide bond formed between (i) a Cys residue in a linker between the WT-1 peptide epitope and the β2M polypeptide, and (ii) a Cys residue at position 84 of the MHC class I heavy chain polypeptide; the heterodimer comprises a second disulfide bond formed between the Cys residue at position 12 of the β2M polypeptide and the Cys residue at position 236 of the MHC class I heavy chain polypeptide; the IgFc polypeptide does not comprise a C-terminal lysine; each of the variant IL-2 polypeptides exhibits reduced affinity for the IL-2 receptor compared to the affinity of a wild-type IL-2 polypeptide for the IL-2 receptor; and The TMMP, wherein the two variant IL-2 polypeptides have the same amino acid sequence and comprise the amino acid sequence set forth in SEQ ID NO: 188, wherein the amino acid at position 16 is an amino acid other than His and the amino acid at position 42 is an amino acid other than Phe.

2. a) the first polypeptide comprises, in order from N-terminus to C-terminus: i) the WT-1 peptide epitope; ii) the linker, and iii) the β2M polypeptide and b) the second polypeptide comprises, in order from N-terminus to C-terminus: i) the first variant IL-2 polypeptide; ii) a peptide linker; iii) the second variant IL-2 polypeptide; iv) a peptide linker; v) the MHC class I heavy chain polypeptide; vi) a peptide linker, and vii) the IgFc polypeptide Including, The T cell regulatory multimeric polypeptide of claim 1.

3. 2. The T cell regulatory multimeric polypeptide of claim 1, wherein each of said variant IL-2 polypeptides comprises i) an H16A substitution and an F42A substitution, or ii) an H16T substitution and an F42A substitution.

4. below: a) in order from N-terminus to C-terminus: i) a Wilms tumor-1 (WT-1) peptide having the amino acid sequence VLDFAPPGA (SEQ ID NO: 259); ii) a linker having the amino acid sequence GCGGSGGGGSGGGGS (SEQ ID NO: 317); iii) a β2-microglobulin polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 311; and a first polypeptide comprising: b) in order from the N-terminus to the C-terminus: i) a variant IL-2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 188, １ is Ala, and X ２ is Ala; and ii) a (GGGGS) linker; and iii) a variant IL-2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 188, wherein X １ is Ala, and X ２ is Ala; and iv) a major histocompatibility complex (MHC) class I heavy chain polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 341; and v) a linker comprising the amino acid sequence AAAGG; vi) an immunoglobulin (Ig) Fc polypeptide; and a second polypeptide comprising At least one heterodimer comprising 1. A T cell modulatory multimeric polypeptide (TMMP) comprising: the heterodimer comprises a first disulfide bond formed between (i) a Cys residue in a linker between the WT-1 peptide epitope and the β2M polypeptide, and (ii) a Cys residue at position 84 of the MHC class I heavy chain polypeptide; the heterodimer comprises a second disulfide bond formed between the Cys residue at position 12 of the β2M polypeptide and the Cys residue at position 236 of the MHC class I heavy chain polypeptide; and the IgFc polypeptide does not contain a C-terminal lysine; T-cell regulatory multimeric polypeptides (TMMPs).

5. the Ig Fc polypeptide is a variant human Ig Fc polypeptide comprising a L234A and / or a L235A substitution (L14 and L15 in the amino acid sequence set forth in Figure 5H (SEQ ID NO:487)); the ability of said Ig Fc polypeptide to induce cell lysis via complement-dependent cytotoxicity (CDC) and / or antibody-dependent cellular cytotoxicity (ADCC) is reduced or eliminated; The TMMP according to claim 4.

6. the first polypeptide comprises construct 2380 having the amino acid sequence set forth in FIG. 14B (SEQ ID NO:423); The second polypeptide comprises construct 1715Δ having the amino acid sequence set forth in FIG. 14J (SEQ ID NO:486); TMMP according to claim 4 or 5.

7. A TMMP comprising a homodimer of the heterodimer of any one of claims 4 to 6, wherein the first and second heterodimers are covalently linked by one or more disulfide bonds between the IgFc polypeptides of the first and second heterodimers.

8. A composition comprising one or more nucleic acids encoding the first and second polypeptides according to any one of claims 4 to 6.

9. An expression vector comprising the nucleic acid of claim 8.

10. A genetically modified host cell that has been genetically modified with the nucleic acid of claim 8 or the expression vector of claim 9.

11. 1. A method for producing a T cell regulatory multimeric polypeptide (TMMP), comprising:

11. The method of claim 10, comprising culturing the genetically modified host cell in vitro in a culture medium under conditions such that the host cell synthesizes TMMP. The manufacturing method.

12. 8. A T cell regulatory multimeric polypeptide according to any one of claims 4 to 7 for use in a method of treatment, comprising: the method comprises selectively modulating the activity of T cells specific for Wilms' tumor-1 (WT-1) epitopes by contacting the T cells with the T cell modulatory multimeric polypeptide; the contact selectively modulates the activity of the WT-1 epitope-specific T cells. T cell regulatory multimeric polypeptides.

13. A pharmaceutical composition for use in a method for treating patients with WT-1-associated cancer, comprising the T cell regulatory multimeric polypeptide of any one of claims 4 to 7.

14. 14. The pharmaceutical composition of claim 13, wherein the cancer is acute myeloid leukemia, myeloma, ovarian cancer, pancreatic cancer, non-small cell lung cancer, colorectal cancer, breast cancer, Wilms' tumor, mesothelioma, soft tissue sarcoma, neuroblastoma, or nephroblastoma.

15. 15. The pharmaceutical composition of claim 13 or 14, further comprising one or more immune checkpoint inhibitors.

16. 16. The pharmaceutical composition of claim 15, wherein 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.

17. The pharmaceutical composition of claim 16, wherein the immune checkpoint inhibitor is an antibody specific for PD-1, PD-L1, LAG3, TIGIT, or CTLA4.

18. 18. The pharmaceutical composition of claim 16 or 17, wherein the one or more immune checkpoint inhibitors are selected from the group consisting of nivolumab, pembrolizumab, pidiluzumab, AMP-224, MPDL3280A, MDX-1105, MEDI-4736, allerumab, ipilimumab, tremelimumab, pidilizumab, IMP321, MGA271, BMS-986016, lirilumab, urelumab, PF-05082566, IPH2101, MEDI-6469, CP-870,893, mogamulizumab, varlilumab, avelumab, galiximab, AMP-514, AUNP12, indoximod, NLG-919, INCB024360, KN035, and combinations thereof.

19. The pharmaceutical composition of claim 17, wherein the immune checkpoint inhibitor is an antibody specific for PD-1.

20. 20. The pharmaceutical composition of claim 19, wherein the immune checkpoint inhibitor is nivolumab or pembrolizumab.

21. The pharmaceutical composition of claim 17, wherein the immune checkpoint inhibitor is an antibody specific for PD-L1.

22. 22. The pharmaceutical composition of claim 21, wherein the immune checkpoint inhibitor is atezolizumab or durvalumab.

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