Multimeric t cell modulatory polypeptides and methods of use thereof

Abstract

The present disclosure provides T cell modulatory multimeric polypeptides comprising an immunomodulatory polypeptide and comprising an epitope presented Wilms tumor peptide. The T cell modulatory multimeric polypeptides are useful for modulating the activity of T cells and are useful for modulating an immune response in an individual.

Description

[0001] Cross-references

[0002] This application claims priority to 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, the disclosures of which are incorporated herein by reference in their entirety.

[0003] By referencing the sequence list provided in a text file and incorporating...

[0004] The sequence list 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

[0005] Adaptive immune responses involve the binding of T cell receptors (TCRs) on the surface of T cells to non-covalently presented small peptide antigens on the surface of antigen-presenting cells (APCs) via the major histocompatibility complex (MHC; also known as the human leukocyte antigen (HLA) complex in humans). This binding represents a targeting mechanism of the immune system and is an essential molecular interaction for T cell regulation (activation or inhibition) and effector function. Following epitope-specific cell targeting, the targeted T cells are activated by binding costimulatory proteins found on APCs to their corresponding costimulatory proteins on T cells. Driving T cell specificity and activation or inhibition requires two signals: epitope / TCR binding and binding of APC costimulatory proteins to T cell costimulatory proteins. TCRs are specific to a given epitope; however, costimulatory proteins are not epitope-specific and are instead generally expressed on all T cells or large subsets of T cells. Summary of the Invention

[0006] This disclosure provides a T-cell regulatory multimer (TMMP) comprising an immunomodulatory polypeptide and an epitope-presenting Wilms tumor peptide. The T-cell regulatory multimer is suitable for modulating T-cell activity and for modulating immune responses in an individual. Attached Figure Description

[0007] Figure 1A-1F This is a schematic depiction of various TMMPs disclosed herein.

[0008] Figures 2A-2F A schematic depiction of various disulfide-linked TMMPs disclosed herein.

[0009] Figures 3A-3E Provide the amino acid sequence of the WT-1 peptide. Figures 3A-3EThe sequences are listed in SEQ ID NO: 399-403.

[0010] Figures 4A-4E The amino acid sequence of an exemplary polypeptide chain of the TMMP disclosed herein is provided. Figures 4A-4E The sequences of exemplary polypeptide chains are listed in SEQ ID NO: 405-409. Epitope sequences are listed below: Figure 4D : CMTWNQMNL (SEQ IDNO:266); Figure 4E : CYTWNQMNL (SEQ ID NO:267).

[0011] Figures 5A-5H Provide the amino acid sequence of the immunoglobulin Fc polypeptide. Figure 5A-5G The sequences are listed in SEQ ID NO: 410-421. Figure 5H The sequence described is listed in SEQ ID NO:487.

[0012] Figure 6 Multiple amino acid sequence alignments are provided for β-2 microglobulin (β2M) precursors (i.e., including the leader sequence) from the following animals: Homo sapiens (NP_004039.1; SEQ ID NO: 19), Pantroglodytes (NP_001009066.1; SEQ ID NO: 19), Macaca mulatta (NP_001040602.1; SEQ ID NO: 20), Bos taurus (NP_776318.1; SEQ ID NO: 21), and Mus musculus (NP_033865.2; SEQ ID NO: 22). Amino acids 1-20 are signal peptides.

[0013] Figures 7A-7C Provide allele 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 amino acid sequence of the full-length human HLA heavy chain; allele B 0702 (SEQ ID NO: 27) ( Figure 7BThe amino acid sequence of the full-length human HLA heavy chain; and the full-length human HLA-C heavy chain (SEQ ID NO: 28) Figure 7C The amino acid sequence of ).

[0014] Figure 8 This document provides alignments of eleven mature class I MHC heavy chain amino acid sequences without leader sequences, transmembrane domains, or intracellular domains. From top to bottom: SEQ ID NO: 41-51.

[0015] Figures 9A-9B Provide HLA-A heavy chain amino acid sequence ( Figure 9A ) and the consensus sequence ( Figure 9B The comparison was performed using SEQ ID NO: 29.

[0016] Figures 10A-10B Provide HLA-B heavy chain amino acid sequence ( Figure 10A ; respectively SEQ ID NO: 207-213) and common sequence ( Figure 10B The comparison was performed using SEQ ID NO: 30.

[0017] Figure 11A-11B Provide HLA-C heavy chain amino acid sequence ( Figure 11A ; respectively SEQ ID NO: 214-222) and common sequence ( Figure 11B The comparison was performed using SEQ ID NO: 31.

[0018] Figure 12 Provide the common amino acid sequences of each of the HLA-E, HLA-F, and HLA-G heavy chains (SEQ ID NO: 32-34, respectively). The positions of each amino acid (aa) are indicated by sequentially numbered "X" residues; the positions of amino acids 84, 139, and 236 are double-underlined.

[0019] Figure 13 The comparison of common amino acid sequences of HLA-A (SEQ ID NO: 29), -B (SEQ ID NO: 30), -C (SEQ ID NO: 31), -E (SEQ ID NO: 32), -F (SEQ ID NO: 33) and -G (SEQ ID NO: 34) is provided.

[0020] Figure 14A-14J The amino acid sequence of the polypeptide chain of TMMP linked by disulfide bonds is provided in this disclosure. Figure 14A-14I The sequences of the polypeptide chains are listed in SEQ ID NO: 422-430. The epitope sequences are listed below: 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 14J (SEQ ID NO: 486).

[0021] Figure 15 Expression and stability data of the TMMP containing WT1 (37-45) epitopes disclosed herein are described.

[0022] Figure 16 Expression and stability data of the TMMP containing WT1 (126-134) epitopes disclosed herein are described.

[0023] Figures 17A-17D A schematic depiction of the TMMP linked by disulfide bonds is provided in this disclosure.

[0024] Figures 18A-18C A schematic depiction of an example configuration of a disulfide-linked TMMP provided in this disclosure is given.

[0025] Figure 19 A schematic depiction of examples of the location of immunomodulatory peptides in the TMMP of this disclosure.

[0026] Figure 20A-20R The amino acid sequence of an exemplary polypeptide chain of the TMMP disclosed herein is provided. Figure 20A-20R The sequences of exemplary polypeptide chains are listed in SEQ ID NO: 431-448. Epitope sequences are listed below: 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 200: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).

[0027] Figure 21 Depicting the presence of WT1 peptide epitopes and HLA-A The role of TMMP in the heavy chain of 02 on antigen-specific CD8+ T cell amplification.

[0028] Figure 22 The role of TMMP containing the WT1 peptide epitope in the expansion of WT1-specific CD8+ T cells from total PBMCs during an 8-day restimulation culture following a 10-day induction culture.

[0029] Figure 23 The production of TNF-α and IFN-γ is depicted by WT1-specific CD8+ T cells containing WT1 37-45 TMMPs with G2C or R12C / G2C architectures.

[0030] Figure 24 The production of TNF-α and IFN-γ is depicted by WT1-specific CD8+ T cells with WT1 126-134 TMMP amplification and an R12C / G2C architecture.

[0031] Figure 25 Describe the role of disulfide bonds in IL-2-driven immune cell activation.

[0032] Figure 26 The effects of TMMP containing a variant IL-2 as an immunomodulatory peptide on CGLL-2 proliferation were described compared to proleukine.

[0033] Figure 27 Depicts the binding of “1715 + 2380” TMPP to various Fc receptors.

[0034] Figure 28 Depicting the presence of WT1 peptide epitopes 235-243 (M236Y) and HLA-A 24-chain TMMPs target antigen-specific CD8 + The role of T cell expansion.

[0035] Figure 29 Depicting the presence of WT1 peptide epitopes 239-247 (Q240Y) and HLA-A 24-chain TMMPs target antigen-specific CD8 + The role of T cell expansion.

[0036] Figure 30 Describe TMMP-induced CD69 expression with an IL-2 peptide engineered at position 1 or 3, peptide epitopes WT1 235-243 (M236Y), HLA-A24 heavy chain, and G2C or R12C / G2C disulfide framework.

[0037] Figure 31 The effects of TMMP (with an IL-2 peptide engineered at position 1 or 3, peptide epitopes WT1 239-247 (Q240Y), an HLA-A24 heavy chain, and a G2C or R12C / G2C disulfide framework) on CD69 expression were described compared with adelin and recombinant human IL-2 (rhIL-2).

[0038] Figure 32 The effects of TMMP (with an IL-2 peptide engineered at position 1 or 3, peptide epitopes WT1 235-243 (M236Y), an HLA-A24 heavy chain, and a G2C or R12C / G2C disulfide framework) on CTLL-2 proliferation were described compared to adrenaline.

[0039] Figure 33 The effects of TMMP (with an IL-2 peptide engineered at position 1 or 3, peptide epitopes WT1 239-247 (Q240Y), an HLA-A24 heavy chain, and a G2C or R12C / G2C disulfide framework) on CTLL-2 proliferation were described compared to adrenaline.

[0040] Figure 34 Depicts the binding of “3425 + 3529” TMPP to various Fc receptors.

[0041] Figures 35A-35F The present disclosure provides an exemplary polypeptide chain amino acid sequence of TMMP, wherein the polypeptide chain comprises the WT-1 peptide SMTWNQMNL (SEQ ID NO:451).

[0042] Figures 36A-36F The present disclosure provides an exemplary polypeptide chain amino acid sequence of TMMP, wherein the polypeptide chain comprises the WT-1 peptide GCMTWNQMNL (SEQ ID NO:452).

[0043] Figures 37A-37F The present disclosure provides an exemplary polypeptide chain amino acid sequence of TMMP, wherein the polypeptide chain comprises the WT-1 peptide SYTWNQMNL (SEQ ID NO:453).

[0044] Figures 38A-38F The present disclosure provides an exemplary polypeptide chain amino acid sequence of TMMP, wherein the polypeptide chain comprises the WT-1 peptide GCYTWNQMNL (SEQ ID NO:454).

[0045] Figure 39 Describing WT1 37-45 Specific CD8 + The expansion of T cells into unpromoted PBMCs, wherein the expansion is caused by the TMMP (“CUE-102 / A02 WT1”) disclosed herein. 37-45 IST”) induces.

[0046] Figures 40A-40B Describing WT1 37-45 Specific CD8 + T cells from WT1 37-45 Peptide-induced amplification of PBMCs, wherein the amplification is induced by the TMMP (“CUE-102 / A02 WT1”) disclosed herein. 37-45 IST”) induces.

[0047] Figures 41A-41B Depicting the TMMP (“CUE-102 / A02 WT1”) disclosed herein 37-45 IST) or the production of TNF-α, IL-6 and IFN-γ induced by wild-type IL-2, as well as the upregulation of CD69.

[0048] Figures 42A-42B The TMMP (“CUE-102 / A02 WT1”) described in this disclosure 37-45 WT1 amplification from peptide-induced PBMCs in the presence of IST) 37-45 Peptide-specific CD8 + T cell CTL activity against peptide-presenting target cells.

[0049] Figure 43 Depicting WT1 peptide epitopes WT1 235-243 (C235S; M236Y) and HLA-A 24-chain TMMPs target antigen-specific CD8 + The role of T cell expansion.

[0050] Figure 44 Depicting the WT1 peptide epitopes WT1 239-247 (Q240Y) and HLA-A 24-chain TMMPs target antigen-specific CD8 + The role of T cell expansion.

[0051] Figure 45Describing the effect of TMMP containing WT1 peptide epitopes 37-45 on antigen-specific CD8 in naive HLA-A2 (AAD) transgenic mice + The role of T cell expansion.

[0052] Figures 46A-46D Provide the amino acid sequence of the HLA-E heavy chain.

[0053] Figures 47A-47D Provide the amino acid sequence of the HLA-G heavy chain.

[0054] definition

[0055] As used interchangeably in this document, the terms “polynucleotide” and “nucleic acid” refer to polymeric forms of nucleotides of any length, namely ribonucleotides or deoxyribonucleotides. Therefore, this term includes, but is not limited to, single-stranded, double-stranded, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers containing purine and pyrimidine bases or other naturally occurring, chemically or biochemically modified, non-natural, or derivatized nucleotide bases.

[0056] The terms “peptide,” “polypeptide,” and “protein” are used interchangeably herein and refer to a polymeric form of amino acids of any length, which may include coding and non-coding amino acids, chemically or biochemically modified or derived amino acids, and polypeptides having a modified peptide backbone. Furthermore, as used herein, “polypeptide” refers to modifications of the native sequence, such as deletions, additions, and substitutions (generally conserved in nature as known to those skilled in the art), provided the protein retains the desired activity. These modifications may be intentional, such as by site-directed mutagenesis, or may be accidental, such as by mutations in the host producing the protein, or by errors resulting from polymerase chain reaction (PCR) amplification or other recombinant DNA methods. References herein to specific residues or residue numbers in a known polypeptide should be understood to refer to the amino acid at that position in the wild-type polypeptide. With regard to the extent to which the sequence of a wild-type polypeptide is altered by the addition or deletion of one or more amino acids, those skilled in the art will understand that references to specific residues or residue numbers will be altered accordingly to refer to the same specific amino acid in the altered polypeptide, which should be understood to be located at the altered position number. For example, if a class I MHC polypeptide is altered by adding an amino acid to its N-terminus, then a reference to position 84 or a specific residue at position 84 will be understood to indicate the amino acid at position 85 on the altered polypeptide. Similarly, a reference here to the substitution of a specific amino acid at a specific position, such as Y84, should be understood to refer to the substitution of the amino acid at position 84 in the wild-type polypeptide. The Y84C substitution should therefore be understood as a Cys residue substituting for a Tyr residue present in the wild-type sequence. If, for example, a wild-type polypeptide is altered to change the amino acid at position 84 from its wild-type amino acid to a substitute amino acid, then the substitution of the amino acid at position 84 should be understood as a substitution of the substitute amino acid. If, in this case, the polypeptide is also altered by the addition or deletion of one or more amino acids, then a reference to substitution should be understood to refer to the substitution of the substitute amino acid at the altered position number. A reference to a polypeptide, such as a class I MHC polypeptide, that is “not naturally occurring Cys residue” means that the polypeptide contains a Cys residue at a position where Cys is absent in the corresponding wild-type polypeptide. This can be achieved through conventional protein engineering, where a cysteine ​​residue replaces an amino acid present in the wild-type sequence.

[0057] A percentage of "sequence identity" between a polynucleotide or polypeptide and another polynucleotide or polypeptide means that, during alignment, the percentages of bases or amino acids are the same and in the same relative positions when comparing the two sequences. Sequence identity can be determined in many different ways. To determine sequence identity, various convenient methods and computer programs (e.g., BLAST, T-COFFEE, MUSCLE, MAFFT, etc.) available on the World Wide Web can be used to align sequences. These websites include ncbi.nlm.nili.gov / BLAST, ebi.ac.uk / Tools / msa / tcoffee / , ebi.ac.uk / Tools / msa / muscle / , and mafft.cbrc.jp / alignment / software / . See, for example, Altschul et al. (1990), J.Mol.Bioi.215:403-10. Unless otherwise stated, the BLAST computer program is used to determine sequence identity.

[0058] The term "conserved amino acid substitution" refers to the interchangeability of amino acid residues with similar side chains in a protein. For example, an amino acid group with aliphatic side chains consists of glycine, alanine, valine, leucine, and isoleucine; an amino acid group with aliphatic hydroxyl side chains consists of serine and threonine; an amino acid group with amide-containing side chains consists of asparagine and glutamine; an amino acid group with aromatic side chains consists of phenylalanine, tyrosine, and tryptophan; an amino acid group with basic side chains consists of lysine, arginine, and histidine; an amino acid group with acidic side chains consists of glutamic acid and aspartic acid; and an amino acid group with sulfur-containing side chains consists of cysteine ​​and methionine. Exemplary conserved amino acid substituents are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine-glycine, and asparagine-glutamine.

[0059] As used herein, the term "immunologic synapse" or "immune synapse" generally refers to the natural interface between two interacting immune cells in an adaptive immune response, including, for example, the interface between antigen-presenting cells (APCs) or target cells and effector cells such as lymphocytes, effector T cells, natural killer cells, etc. Immunologic synapses between APCs and T cells are typically initiated through interactions between T cell antigen receptors and major histocompatibility complex molecules, as described, for example, in Bromley et al., Annu Rev Immunol. 2001;19:375-96, the contents of which are incorporated herein by reference in their entirety.

[0060] "T cells" include all types of CD3-expressing immune cells, including T helper cells (CD4+). +Cells), cytotoxic T cells (CD8) + T cells, T-regulatory cells (Tregs), and NK-T cells.

[0061] As used herein, the term "immunomodulatory polypeptide" (also known as "co-stimulatory polypeptide") includes polypeptides on antigen-presenting cells (APCs) (e.g., dendritic cells, B cells, etc.) that specifically bind to homologous co-immunomodulatory polypeptides on T cells, thereby providing a signal that, in addition to the primary signal provided by, for example, the binding of the TCR / CD3 complex to a peptide-loaded major histocompatibility complex (MHC) polypeptide, mediates T cell responses, including but not limited to responses such as proliferation, activation, and differentiation. Immunomodulatory peptides may include, but are not limited to, CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L, Fas ligand (FasL), inducible co-stimulatory ligand (ICOS-L), intracellular adhesion molecule (ICAM), CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, HVEM, lymphotoxin β receptor, 3 / TR6, ILT3, ILT4, HVEM, agonists or antibodies that bind to Toll ligand receptors, and ligands that specifically bind to B7-H3.

[0062] As mentioned above, "immunomodulatory peptides" (also referred to as "MODs" in this article) specifically bind to homologous coimmunomodulatory peptides on T cells.

[0063] The "immunomodulatory domain" ("MOD") of the disclosed TMMP binds to a homologous coimmunomodulatory polypeptide that can be present on target T cells.

[0064] As used in this article, the term "in vivo" refers to any process or procedure that occurs within the body.

[0065] As used in this article, “in vitro” means any process or procedure that occurs outside the body.

[0066] As used in this article, "heterogeneous" means that nucleotides or polypeptides are not found in natural nucleic acids or proteins, respectively.

[0067] As used herein, “recombinant” refers to the product of various combinations of cloning, restriction, polymerase chain reaction (PCR), and / or ligation steps of a specific nucleic acid (DNA or RNA), which produce constructs having structural coding or non-coding sequences distinguishable from endogenous nucleic acids found in natural systems. DNA sequences encoding polypeptides can be assembled from cDNA fragments or a series of synthetic oligonucleotides to provide synthetic nucleic acids capable of expression from recombinant transcription units contained in cellular or cell-free transcription and translation systems.

[0068] The terms “recombinant expression vector” or “DNA construct” are used interchangeably herein to refer to a DNA molecule containing a vector and at least one insert. Recombinant expression vectors are typically generated for the purpose of expressing and / or propagating inserts or for the purpose of constructing other recombinant nucleotide sequences. Inserts may or may not be ligated to a promoter sequence and may or may not be ligated to a DNA regulatory sequence.

[0069] As used herein, the term "affinity" refers to the equilibrium constant of the reversible binding of two reagents (e.g., antibody and antigen) and is expressed as the dissociation constant (Ka). D As used herein, the term "affinity" refers to the resistance of a complex of two or more reagents to dissociation upon dilution. The terms "immunoreactivity" and "preferential binding" are used interchangeably herein with respect to antibody and / or antigen-binding fragments.

[0070] As used herein, the term "binding" (e.g., regarding the binding of TMMP to peptides on T cells, such as T cell receptors) refers to a non-covalent interaction between two molecules. Non-covalent binding refers to the direct association between two molecules due to interactions such as electrostatics, hydrophobicity, ionicity, and / or hydrogen bonding (including interactions such as salt bridges and water bridges). "Affinity" refers to the strength of the non-covalent binding; increased binding affinity is associated with a lower K0. D Related. "Specific binding" generally refers to the binding of a ligand to the portion that serves as its designated binding site or receptor. "Non-specific binding" generally refers to the binding of a ligand to a portion 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.

[0071] The terms “treatment” and “treating” are used herein to generally mean achieving a desired pharmacological and / or physiological effect. This effect may be preventative in terms of complete or partial prevention of a disease or its symptoms, and / or therapeutic in terms of partial or complete cure of the disease and / or adverse effects caused by the disease. As used herein, “treatment” covers any treatment of a disease or symptom in mammals and includes: (a) preventing the occurrence of a disease or symptom in a subject who may be predisposed to acquiring the disease or symptom but has not yet been diagnosed with the disease; (b) suppressing the disease or symptom, i.e., preventing its development; and / or (c) alleviating the disease, i.e. causing disease remission. Therapeutic agents may be administered before, during, or after the onset of a disease or injury. Particular attention is paid to the treatment of developing diseases in which said treatment stabilizes or alleviates unwanted clinical symptoms in the patient. Such treatment is ideally administered before complete loss of function in the diseased tissue. Targeted therapies will ideally be administered during the symptomatic phase of the disease and, in some cases, after the symptomatic phase of the disease.

[0072] The terms “individual,” “subject,” “host,” and “patient” are used interchangeably in this document and refer to any mammalian subject requiring diagnosis, treatment, or therapy. Mammals include, for example, humans, non-human primates, rodents (e.g., rats; mice), rabbits (e.g., rabbits), ungulates (e.g., cows, sheep, pigs, horses, goats, etc.).

[0073] Unless otherwise stated, the term "generally" is intended to cover both "completely" and "mostly but not completely". For example, an Ig Fc that "generally does not induce cell lysis" means an Ig Fc that does not induce cell lysis at all or to a large extent.

[0074] As used herein, the term “about” when used with a quantity indicates that the quantity can vary within 10% of the specified quantity. For example, “about 100” means a quantity of 90-110. When “about” is used in the context of a range, when used with respect to the lower limit of the range, “about” means that the lower limit includes quantities 10% lower than the lower limit of the range, and when used with respect to the upper limit of the range, the upper limit includes quantities 10% higher than the upper limit of the range. For example, “about 100 to about 1000” means a range extending from 90 to 1100.

[0075] Before further describing this disclosure, it should be understood that the invention is not limited to the specific embodiments described, and therefore the embodiments are of course subject to variation. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting, as the scope of this disclosure is limited only by the scope of the appended claims.

[0076] When numerical ranges are provided, it should be understood that all interpolations between the upper and lower limits of that range up to one-tenth of the lower limit (unless the context explicitly states otherwise), as well as any other stated values ​​or interpolations within that range, are included within the scope of this invention. The upper and lower limits of these smaller ranges may be independently included in the smaller range and also cover any explicitly excluded limits within the stated range that fall within this disclosure. When a stated range includes one or both limits, the range excluding any one or both of those included limits may also be included within this disclosure.

[0077] 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 pertains. While any methods and materials similar to or equivalent to those described herein may be used to practice or test this disclosure, preferred methods and materials are described hereafter. All publications mentioned herein are incorporated by reference to disclose and describe methods and / or materials associated with the content referenced in those publications.

[0078] It is important to note that, unless the context explicitly states otherwise, as used herein and in the appended claims, the singular forms “a” and “the” include multiple indicators. Thus, for example, reference to “T-cell regulatory multimer polypeptide” includes multiple such polypeptides, and reference to “immunomodulatory polypeptide” includes reference to one or more immunomodulatory polypeptides and their equivalents known to those skilled in the art. Further attention should be paid to the scope of the claims that can be drafted to exclude any optional elements. Therefore, this statement is intended to serve as a basis for the use of exclusionary terms such as “unique,” ​​“only,” or the use of negative limiting terms in connection with elements describing the scope of the claim.

[0079] It should be understood that certain features of this disclosure described in individual embodiments for clarity may also be provided in combination in a single embodiment. Conversely, various features of the invention described in individual embodiments for brevity may also be provided individually or in any suitable sub-combination. All combinations of embodiments relevant to this invention are expressly included in this invention and are disclosed herein as individually and expressly disclosed herein. Furthermore, all sub-combinations of various embodiments and their elements are also expressly included in this disclosure and are disclosed herein as individually and expressly disclosed herein.

[0080] The publications discussed herein are provided only because their disclosures predate the filing date of this application. Nothing herein should be construed as an admission that this disclosure is not entitled to precedence over this publication due to prior inventions. Furthermore, the provided disclosure date may differ from the actual disclosure date, which may require independent verification. Detailed Implementation

[0081] This disclosure provides a T-cell regulatory multimer polypeptide comprising an immunomodulatory polypeptide (“MOD”) and an epitope-presenting Wilms' tumor-1 (WT-1) peptide. TMMP is suitable for modulating T-cell activity and for modulating immune responses in an individual.

[0082] T cell-regulated multimeric polypeptide

[0083] This disclosure provides a T-cell regulatory multimer polypeptide (TMMP) 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. This disclosure also provides a TMMP that is a heterodimer comprising: a) a first polypeptide comprising a first MHC polypeptide; and b) a second polypeptide comprising a second MHC polypeptide, wherein the first polypeptide or the second polypeptide comprises an epitope (e.g., an epitope-presenting peptide); wherein the first polypeptide and / or the second polypeptide comprises one or more MODs that may be the same or different; and optionally an Ig Fc polypeptide or a non-Ig scaffold. The TMMP of this disclosure is also referred to herein as "the multimer polypeptide of this disclosure" or "synTac". In some cases, the peptide epitope present in the TMMP of this disclosure is a WT-1 peptide.

[0084] This 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 polypeptide and / or the second polypeptide comprises at least one (i.e., one or more) MODs. Optionally, the first polypeptide or the 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 exhibiting a reduced affinity for a homologous co-immunomodulatory polypeptide (“co-MOD”) compared to the affinity of the corresponding wild-type MOD for the co-MOD. The epitope present in the TMMP binds to the T cell receptor (TCR) on T cells 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). TMMP binds to first T cells with at least 25% higher affinity than it binds to second T cells, wherein the first T cells express a homologous co-MOD and a TCR that binds to the epitope with an affinity of at least 100 μM on their surface, and wherein the second T cells express a homologous co-MOD on their surface but do not express 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 cases, the peptide epitope present in TMMP is the WT-1 peptide.

[0085] This disclosure provides a TMMP, wherein the TMMP is:

[0086] 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 that presents an epitope to T cells); wherein the first polypeptide and / or the second polypeptide comprises one or more MODs that may be the same or different, and wherein 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 wherein the first polypeptide or the second polypeptide optionally comprises an Ig Fc polypeptide or a non-Ig scaffold; or

[0087] 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; wherein the first polypeptide and / or the second polypeptide comprises one or more MODs that may be the same or different.

[0088] At least one of the aforementioned MODs is a variant of a wild-type MOD, wherein the variant MOD contains substitutions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids compared to the amino acid sequence of the corresponding wild-type MOD.

[0089] At least one of the said one or more MODs is a variant MOD that exhibits a reduced affinity for a homologous co-MOD compared to the affinity of the corresponding wild-type MOD for the homologous co-MOD, and wherein the epitopes are at least 10 -7 M binds to the TCR on T cells with affinity such that: i) the affinity of the TMMP peptide to a first T cell is at least 25% higher than the affinity of TMMP to a second T cell, wherein the first T cell expresses homologous co-MOD and at least 10 -7 M has an affinity for binding epitopes to TCRs, and the second T cells express homologous co-MOD on their surface, but do not express at least 10 on their surface. -7 The affinity of M for the TCR of the epitope; and / or ii) when measured by biolayer interferometry, the ratio of the binding affinity of control TMMP to homologous co-MOD to the binding affinity of TMMP containing wild-type MOD variants to homologous co-MOD ranges from 1.5:1 to 10. 61. The control group comprises wild-type MOD; and the variant MOD contains 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 corresponding wild-type MOD.

[0090] The first polypeptide or the second polypeptide may optionally contain an Ig Fc polypeptide or a non-Ig scaffold; or

[0091] C) A heterodimer comprising: a) a first polypeptide comprising, in order from N-terminus to C-terminus: i) an epitope; ii) a first MHC polypeptide; and b) a second polypeptide comprising, in order from N-terminus to C-terminus: i) a second MHC polypeptide; and ii) an optional Ig Fc polypeptide or a non-Ig scaffold, wherein the TMMP comprises one or more MODs that may be the same or different, wherein at least one of the one or more MODs 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 both the C-terminus of the first polypeptide and the N-terminus of the second polypeptide, and wherein 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

[0092] Optionally, at least one of said one or more MODs is a variant MOD that exhibits a reduced affinity for a homologous co-MOD compared to the affinity of the corresponding wild-type MOD for the homologous co-MOD, and wherein the epitopes are at least 10 -7 M binds to the TCR on T cells with an affinity such that: i) the affinity of the TMMP to the first T cell is at least 25% higher than the affinity of the TMMP to the second T cell, wherein the first T cell expresses homologous co-MOD and at least 10 -7 M has an affinity for binding epitopes to TCRs, and the second T cells express homologous co-MOD on their surface, but do not express at least 10 on their surface. -7 The affinity of M for the TCR of the epitope; and / or ii) when measured by biolayer interferometry, the ratio of the binding affinity of control TMMP to homologous co-MOD to the binding affinity of TMMP containing wild-type MOD variants to homologous co-MOD ranges from 1.5:1 to 10. 61. The control group comprises a wild-type MOD; and the variant MOD contains 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 corresponding wild-type MOD. In some cases, the peptide epitope present in the TMMP is the WT-1 peptide.

[0093] This disclosure provides a TMMP comprising: a) a first polypeptide comprising, in order from N-terminus to C-terminus: i) an epitope; ii) a first MHC polypeptide; and b) a second polypeptide comprising, in order from N-terminus to C-terminus: i) a second MHC polypeptide; and ii) optionally an Ig Fc polypeptide or a non-Ig scaffold. The TMMP comprises one or more MODs, wherein at least one of the one or more MODs 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 both 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 exhibiting a reduced affinity for a homologous co-MOD compared to the affinity of the corresponding wild-type MOD for the homologous co-MOD. The epitope present in the TMMP binds to the T cell receptor (TCR) on T cells 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). TMMP binds to a first T cell with an affinity at least 25% higher than that to a second T cell, wherein the first T cell expresses a homologous co-MOD and a TCR that binds to the epitope with an affinity of at least 100 μM on its surface, and wherein the second T cell expresses a homologous co-MOD on its surface but does not express 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) on its surface.

[0094] The affinity of a MOD present in TMMP to its homologous co-MOD is at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least greater than 95% less than the affinity of the corresponding wild-type MOD to its homologous co-MOD.

[0095] The reduced affinity of MODs for their homologous co-MODs, combined with the epitope's affinity for TCRs, provides enhanced selectivity for TMMPs. For example, the TMMPs of this disclosure selectively bind to first T cells compared to second T cells, the first T cells exhibiting: i) TCRs specific to epitopes present in the TMMP; and ii) co-MODs binding to MODs present in the TMMP, while the second T cells exhibiting: i) TCRs specific to epitopes other than those present in the TMMP; and ii) co-MODs binding to MODs present in the TMMP. For example, the affinity of the TMMP of this disclosure to a first T cell 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 times, at least 2.5 times, at least 5 times, at least 10 times, at least 15 times, at least 20 times, at least 25 times, at least 50 times, at least 100 times, or greater than 100 times.

[0096] In some cases, TMMP, when administered to an individual in need, induces both epitope-specific and epitope-nonspecific T-cell responses. In other words, in some cases, TMMP, when administered to an individual in need, induces an epitope-specific T-cell response by modulating the activity of a first T cell exhibiting: i) a TCR specific to the epitope present in the TMMP; ii) a co-MOD bound to a MOD present in the TMMP; and induces an epitope-nonspecific T-cell response by modulating the activity of a second T cell exhibiting: i) a TCR specific to an epitope other than the epitope present in the TMMP; and ii) a co-MOD bound to a MOD present in the TMMP. The ratio of epitope-specific T-cell response to epitope-nonspecific T-cell response 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 approximately 2:1 to approximately 5:1, approximately 5:1 to approximately 10:1, approximately 10:1 to approximately 15:1, approximately 15:1 to approximately 20:1, approximately 20:1 to approximately 25:1, approximately 25:1 to approximately 50:1, or approximately 50:1 to approximately 100:1, or greater than 100:1. "Regulating T cell activity" may include one or more of the following: i) activating cytotoxicity (e.g., CD8+). + ii) Inducing cytotoxicity (e.g., CD8+) T cells; + iii) Induction of cytotoxicity (e.g., CD8+) in T cells; +iv) T cell production and release of cytotoxins (e.g., perforin; granzyme; granulosin); iv) inhibition of the activity of autoreactive T cells; etc.

[0097] The reduced affinity of MODs for their homologous co-MODs, combined with the epitope's affinity for TCRs, provides enhanced selectivity for TMMPs. Thus, for example, TMMPs bind to first T cells with a higher affinity than to second T cells, exhibiting: i) a TCR specific to the epitope present in the TMMP; and ii) a co-MOD bound to the MOD present in the TMMP, while the second T cells exhibit: i) a TCR specific to epitopes other than those present in the TMMP; and ii) a co-MOD bound to the MOD present in the TMMP.

[0098] The binding affinity between MOCs and their homologous co-MODs can be determined by biolayer interferometry (BLI) using purified MODs and purified homologous co-MODs. The binding affinity between TMMPs and their homologous co-MODs can also be determined by BLI using purified TMMPs and homologous co-MODs. 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.

[0099] BLI assays can be performed using an Octet RED 96 (Pal FortéBio) instrument or a similar instrument. A TMMP (e.g., the TMMP of this disclosure; a control TMMP (wherein the control TMMP contains a wild-type immunomodulatory peptide)) is immobilized on an insoluble support (“biosensor”). The immobilized TMMP is the “target”. Immobilization can be achieved by immobilizing a capture antibody onto the insoluble support, wherein the capture antibody immobilizes the TMMP. For example, immobilization can be achieved by immobilizing an anti-Fc antibody (e.g., anti-human IgG Fc) onto the insoluble support, wherein the immobilized anti-Fc antibody binds to and immobilizes the TMMP (wherein the TMMP contains an IgFc peptide). A co-immunomodulatory peptide is applied to the immobilized TMMP at several different concentrations, and the instrument’s reaction is recorded. Assays are performed in a liquid medium containing 25 mM HEPES pH 6.8, 5% polyethylene glycol 6000, 50 mM KCl, 0.1% bovine serum albumin, and 0.02% Tween 20 nonionic detergent. The binding of the co-immunomodulatory peptide to immobilized TMMP was performed at 30°C. As a positive control for binding affinity, an anti-class I MHC monoclonal antibody could be used. For example, the anti-HLA class I monoclonal antibody W6 / 32 (American Type Culture Collection, HB-95; Parham et al. (1979)) could be used. J. Immunol .123:342), which has 7 nM K D A standard curve can be generated using serial dilutions of anti-class I MHC monoclonal antibodies. The co-immunomodulatory peptide or anti-class I MHC mAb is the "analyte". BLI analysis measures the interference pattern of white light reflected from two surfaces: i) the immobilized peptide ("target"); and ii) the internal reference layer. Changes in the number of molecules ("analytes"; e.g., co-immunomodulatory peptides; anti-HLA antibodies) bound to the biosensor tip cause a shift in the interference pattern; this shift in the interference pattern can be measured instantaneously. Two kinetic terms describing the affinity of the target / analyte interaction are the association constant (…). k a ) and dissociation constant ( k d The ratio of these two terms (). k d / a The affinity constant K is generated. D .

[0100] BLI assays were performed in multi-well plates. To perform the assay, the plate layout and assay steps were defined, and the biosensor was assigned in the Octet Data Acquisition software. The biosensor assembly was hydrated. The hydrated biosensor assembly and assay plate were equilibrated on the Octet instrument for 10 minutes. Once data was acquired, it was loaded into the Octet Data Analysis software. In the processing window, data were processed by specifying reference subtraction, y-axis alignment, inter-step correction, and Savitzky-Golay filtering. In the analysis window, data were analyzed by specifying the analysis (association and dissociation), selecting the curve fitting model (1:1), the fitting method (global), and the target window (in seconds). The quality of the fit was evaluated. If within a 3-fold range, the Kt of each data point (analyte concentration) was evaluated. D The value is taken as the average. K D The error value should be within approximately one order of magnitude of the affinity constant; R 2 The value should be higher than 0.95. See, for example, Abdiche et al. (2008). J. Anal.Biochem. 377:209.

[0101] Unless otherwise stated herein, the affinity of the TMMP of this disclosure for homologous coimmunomodulatory peptides or the affinity of the control TMMP (wherein the control TMMP contains wild-type immunomodulatory peptides) for homologous coimmunomodulatory peptides is determined using the BLI assay as described above.

[0102] In some cases, i) the binding affinity of the control TMMP (wherein the control comprises a wild-type immunomodulatory peptide) to the homologous coimmunomodulatory peptide is equal to ii) the binding affinity ratio of the TMMP comprising a wild-type immunomodulatory peptide variant of this disclosure to the homologous coimmunomodulatory peptide, when measured by BLI (as described above), is at least 1.5:1, at least 2:1, at least 5:1, at least 10:1, at least 15:1, at least 20:1, at least 25:1, at least 50:1, at least 100:1, at least 500:1, at least 10 2 1. At least 5 x 10 2 1. At least 10 3 1. At least 5 x 10 3 1. At least 10 4 1. At least 10 5 1, or at least 10 61. In some cases, i) the binding affinity of the control TMMP (wherein the control comprises a wild-type immunomodulatory peptide) to the homologous coimmunomodulatory peptide is equal to ii) the binding affinity of the TMMP comprising a wild-type immunomodulatory peptide variant of this disclosure to the homologous coimmunomodulatory peptide, when measured by BLI, ranges from 1.5:1 to 10. 6 Within :1, for example, 1.5:1 to 10:1, 10:1 to 50:1, 50:1 to 10 2 1, 10 2 :1 to 10 3 1, 10 3 :1 to 10 4 1, 10 4 :1 to 10 5 :1 or 10 5 :1 to 10 6 :1.

[0103] As an example, when the control TMMP contains a wild-type IL-2 peptide, and when the TMMP of this disclosure contains a variant IL-2 peptide (with an amino acid sequence containing 1 to 10 amino acid substitutions relative to the wild-type IL-2 peptide) as an immunomodulatory peptide, i) the binding affinity of the control TMMP to the IL-2 receptor (i.e., the homologous co-immunomodulatory peptide) is equal to ii) the binding affinity ratio of the TMMP of this disclosure to the IL-2 receptor, when measured by BLI, is at least 1.5:1, at least 2:1, at least 5:1, at least 10:1, at least 15:1, at least 20:1, at least 25:1, at least 50:1, at least 100:1, at least 500:1, at least 10 2 1. At least 5 x 10 2 1. At least 10 3 1. At least 5 x 10 3 1. At least 10 4 1. At least 10 5 1, or at least 10 6 1. In some cases, when the control TMMP contains a wild-type IL-2 peptide, and when the TMMP of this disclosure contains a variant IL-2 peptide (with an amino acid sequence containing 1 to 10 amino acid substitutions relative to the wild-type IL-2 peptide) as an immunomodulatory peptide, i) the binding affinity of the control TMMP to the IL-2 receptor (i.e., the homologous co-immunomodulatory peptide) is the same as ii) the binding affinity of the TMMP of this disclosure to the IL-2 receptor, when measured by BLI, ranges from 1.5:1 to 10. 6 Within :1, for example, 1.5:1 to 10:1, 10:1 to 50:1, 50:1 to 10 2 1, 10 2 :1 to 10 31, 10 3 :1 to 10 4 1, 10 4 :1 to 10 5 :1 or 10 5 :1 to 10 6 :1.

[0104] The binding affinity of TMMP to target T cells disclosed herein can be measured according to the procedure described in the published PCT application WO 2019 / 051091 published on March 14, 2019. See

[0063] .

[0105] In some cases, when measured as described in the preceding paragraphs, the TMMP of this disclosure exhibits selective binding to target T cells compared to the binding of TMMP library members to control T cells containing: i) a homologous co-immunomodulatory polypeptide that binds to the parent wild-type immunomodulatory polypeptide; and ii) a T cell receptor that binds to epitopes other than those present in the TMMP library members.

[0106] Dimerization™MP

[0107] The TMMP disclosed herein can be dimerized; that is, the present disclosure provides multimeric polypeptides comprising dimers of the TMMP disclosed herein. Therefore, the present disclosure provides a TMMP comprising: A) a first heterodimer, the first heterodimer comprising: a) a first polypeptide, the first polypeptide comprising: i) a peptide epitope; and ii) a first MHC polypeptide; and b) a second polypeptide, the second polypeptide comprising: i) a second MHC polypeptide, wherein the first heterodimer comprises one or more MODs; and B) a second heterodimer, the second heterodimer comprising: a) a first polypeptide, the first polypeptide comprising: i) a peptide epitope; and ii) a first MHC polypeptide; and b) a second polypeptide, the second polypeptide comprising: i) a second MHC polypeptide, wherein the second heterodimer comprises one or more MODs, and wherein the first heterodimer and the second heterodimer are covalently linked to each other. In some cases, the amino acid sequences of the two TMMPs are identical. In some cases, the first heterodimer and the second heterodimer are covalently linked to each other via the C-terminal regions of the second polypeptide of the first heterodimer and the second polypeptide of the second heterodimer. In other cases, the first heterodimer and the second heterodimer are covalently linked to each other via the C-terminal amino acid of the second polypeptide of the first heterodimer and the C-terminal region of the second polypeptide of the second heterodimer; for example, in some cases, the C-terminal amino acid of the second polypeptide of the first heterodimer and the C-terminal region of the second polypeptide of the second heterodimer are linked to each other directly or via a linker. The linker may be a peptide linker. The peptide linker may have a length of 1 amino acid to 200 amino acids (e.g., 1 amino acid (aa) to 5 aa, 5 aa to 10 aa, 10 aa to 25 aa, 25 aa to 50 aa, 50 aa to 100 aa, 100 aa to 150 aa, or 150 aa to 200 aa). In some cases, the peptide epitopes of the first heterodimer and the second heterodimer contain the same amino acid sequence. In some cases, the first MHC polypeptide of both the first and second heterodimers is a class I MHC β2-microglobulin, and the second MHC polypeptide of both the first and second heterodimers is a class I MHC heavy chain. In some cases, the MOD of the first heterodimer and the MOD of the second heterodimer contain the same amino acid sequence. In some cases, the MOD of the first and second heterodimers are variant MODs containing 1 to 10 amino acid substitutions compared to the corresponding parent wild-type MOD, and these 1 to 10 amino acid substitutions result in reduced affinity binding between the variant immunomodulatory polypeptide and the homologous coimmunomodulatory polypeptide.In some cases, the first heterodimer immunomodulatory peptide and the second heterodimer immunomodulatory peptide 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 peptides, MODs, and peptide epitopes are described below.

[0108] MHC peptides

[0109] As described above, the TMMP of this disclosure includes MHC peptides. For the purposes of this disclosure, the term "major histocompatibility complex (MHC) peptide" is intended to include MHC peptides from various species, including human MHC (also known as human leukocyte antigen (HLA)) peptides, rodent (e.g., mouse, rat, etc.) MHC peptides, and MHC peptides from other mammalian species (e.g., rabbits, non-human primates, canines, felines, ungulates (e.g., horse, cattle, sheep, goats, etc.). The term "MHC peptide" is intended to include class I MHC peptides (e.g., β-2 microglobulin and class I MHC heavy chains).

[0110] In some cases, the first MHC polypeptide is a class I MHC β2M (β2M) polypeptide, and the second MHC polypeptide is a class I MHC heavy chain (H chain) (“MHC-H”). In other cases, the first MHC polypeptide is a class I MHC heavy chain polypeptide, and the second MHC polypeptide is a β2M polypeptide. In some cases, both the β2M and MHC-H chains are of human origin; that is, the MHC-H chain is an HLA heavy chain or a variant thereof. Unless otherwise expressly stated, the TMMPs of this disclosure do not include a membrane anchoring domain (transmembrane domain) of a class I MHC heavy chain or are sufficient to anchor the resulting TMMP to cells expressing it. For example,A portion of the class I MHC heavy chain in eukaryotic cells (such as mammalian cells). In some cases, the class I MHC heavy chain present in the TMMP of this disclosure does not include the signal peptide, transmembrane domain, or intracellular domain (cytoplasmic tail) associated with the native class I MHC heavy chain. Thus, for example, in some cases, the class I MHC heavy chain present in the TMMP of this disclosure includes only the α1, α2, and α3 domains of the class I MHC heavy chain. In some cases, the class I MHC heavy chain present in the TMMP has a length of about 270 amino acids (aa) to about 290 aa. In some cases, the heavy chains of type I MHC present in TMMP have lengths 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.

[0111] In some cases, the MHC peptides of TMMP are human MHC peptides, also known as human leukocyte antigen ("HLA") peptides. In other cases, the MHC peptides of TMMP are class I HLA peptides, such as β2-microglobulin peptides or class I HLA heavy chain peptides. Class I HLA heavy chain peptides include HLA-A heavy chain peptides, HLA-B heavy chain peptides, HLA-C heavy chain peptides, HLA-E heavy chain peptides, HLA-F heavy chain peptides, and HLA-G heavy chain peptides.

[0112] Type I MHC heavy chains

[0113] In some cases, the class I MHC heavy chain polypeptides present in TMMP contain all or part of the amino acid sequence (e.g., 50, 75, 100, 150, 200, or 250 consecutive amino acids) of any human HLA heavy chain polypeptide depicted in Figures 7-13, 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. In some cases, the class I MHC 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 cases, class I MHC heavy chain polypeptides present in TMMP contain 1-30, 1-5, 5-10, 10-15, 15-20, 20-25, or 25-30 amino acid insertions, deletions, and / or substitutions (except for those positions indicated as variable in the heavy chain common sequence) of any of the amino acid sequences depicted in Figures 7-13. In some cases, class I MHC heavy chains do not include transmembrane domains or cytoplasmic domains. As an example, class I MHC heavy chain polypeptides in TMMP may contain the same amino acid insertions, deletions, and / or substitutions as shown in Figures 7-13. Figure 7A , 7B The amino acid sequence of any of the human HLA-A heavy chain polypeptides described in 7C, from amino acid 25 to 300 (lacking all or substantially all of the leader sequence, transmembrane sequence and cytoplasmic sequence) or from amino acid 25 to 365 (lacking the leader 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.

[0114] Figure 7A , 7B The amino acid sequence of the human leukocyte antigen (HLA) class I heavy chain polypeptide is provided by 7C. The signal sequence amino acids 1-24 are bolded and underlined. Figure 7A Item 3A.1 is the HLA-A heavy chain (HLA-A 01:01:01:01 or A 0101)(NCBI Login NP_001229687.1) SEQ ID NO:23; Entry 3A.2 from HLA-A 1101 SEQ ID NO:24; Entry 3A.3 from HLA-A 2402 SEQ ID NO:25 and entry 3A.4 from HLA-A 3303 SEQ ID NO:26. Figure 7B Provide HLA-B sequence 07:02:01 (HLA-B 0702) NCBI GenBank login NP_005505.2 (see also GenBank login AUV50118.1.). Figure 7C Provide HLA-C sequence 0701 (GenBank login NP_001229971.1) (HLA-C 07:01:01:01 or HLA-Cw 070101, HLA-Cw 07 See GenBank login CAO78194.1).

[0115] Figure 8 This provides alignments of eleven mature class I MHC heavy chain amino acid sequences without leader sequences, transmembrane domains, or intracellular domains. The aligned sequences include human HLA-A, HLA-B, and HLA-C, mouse H2K protein sequences, three HLA-A variants (var.1, var.2C, and var.2CP), and three human HLA-A variants (HLA-A...). 1101; HLA-A 2402; and HLA-A 3303). Positions (84 and 139 of the mature protein) are indicated in the alignment, where cysteine ​​residues can be introduced (e.g., by substitution) to form disulfide bonds to stabilize the MHC H chain-β2M complex. Position 236 (the position of the mature polypeptide) is also shown in the alignment, which can be substituted with cysteine ​​residues to form interchain disulfide bonds with β2M. For example (at aa 12). Arrows appear above those positions and bold the residues. The seventh HLA-A sequence shown in alignment (var. 2c) represents the sequence of variant 2 with C residues substituted at positions 84, 139, and 236. Box flanking residues 84, 139, and 236 represent groups of five amino acids on either side of those six groups of five residues, denoted as 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 independently selected from 1 to 5 amino acid substitutions: (i) any naturally occurring amino acid or (ii) any naturally occurring amino acid other than proline or glycine.

[0116] about Figure 9A In some cases: i) aac1 (amino acid cluster 1) may be the amino acid sequence GTLRG (SEQ ID NO: 287) or have one or two amino acids missing or replaced by other naturally occurring amino acids. For example ii) The sequence L is replaced by I, V, A or F; ii) aac2 (amino acid cluster 2) may be the amino acid sequence YNQSE (SEQ ID NO:288) or have one or two amino acids missing or replaced by other naturally occurring amino acids. For example, The sequence is N replaced by Q, Q replaced by N, and / or E replaced by D; iii) aac3 (amino acid cluster 3) may be the amino acid sequence TAADM (SEQ ID NO:289) or have one or two amino acids missing or replaced by other naturally occurring amino acids. For example, The sequence is T replaced by S, A replaced by G, D replaced by E, and / or M replaced by L, V, or I; iv) aac4 (amino acid cluster 4) may be the amino acid sequence AQTTK (SEQ ID NO:290) or have one or two amino acids missing or replaced by other naturally occurring amino acids. For example, A sequence in which A is replaced by G, Q by N, or T by S, and / or K by R or Q; v) aac5 (amino acid cluster 5) may be an amino acid sequence VETRP (SEQ ID NO: 291) or have one or two amino acids missing or replaced by other naturally occurring amino acids. For example, A sequence in which V is replaced by I or L, E by D, T by S, and / or R by K; and / or vi) aac6 (amino acid cluster 6) may be an amino acid sequence GDGTF (SEQ ID NO:292) or have one or two amino acids missing or replaced by other naturally occurring amino acids. For example, A sequence in which D is permuted by E, T by S, or F by L, W, or Y.

[0117] Figure 9-11 provides an alignment of mature HLA class I heavy chain amino acid sequences (without leader sequences, transmembrane domains, or intracellular domains). Figure 9A The amino acid sequence compared is the HLA-A1 heavy chain of the following alleles: A 0101, A 0201, A 0301, A 1101, A 2301, A 2402, A 2407, A 3303 and A 3401. Figure 10AThe amino acid sequence compared is the HLA-B class I heavy chain of the following alleles: B 0702, B 0801, B 1502, B 3802, B 4001, B 4601 and B 5301. Figure 11A The amino acid sequence compared is the HLA-C class I heavy chain of the following alleles: C 0102, C 0303, C 0304, C 0401, C 0602, C 0701, C 0801 and C 1502. Positions (84 and 139 of the mature protein) are indicated in the alignment, where cysteine ​​residues can be introduced (e.g., by substitution) to form disulfide bonds to stabilize the HLA H chain-β2M complex. Position 236 (the position of the mature polypeptide) is also shown in the alignment, which can be substituted with cysteine ​​residues to form interchain disulfide bonds with β2M. For example (at aa 12). Flanking residues 84, 139, and 236 show groups of five amino acids on any side of those six groups of five residues, denoted as 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 independently selected from 1 to 5 amino acid substitutions: (i) any naturally occurring amino acid or (ii) any naturally occurring amino acid other than proline or glycine.

[0118] Figure 9A , 10A 11A provides the amino acid sequence alignments of mature HLA-A, HLA-B, and HLA-C class I heavy chains, respectively. It also provides the sequence of the extracellular portion of the mature protein (without a leader sequence, transmembrane domain, or intracellular domain). For example... Figure 8 The description also shows the positions of aa residues 84, 139, and 236 and their flanking residues (aac1 to aac6), which may be independently selected from 1 to 5 amino acid substitutions: (i) any naturally occurring amino acid or (ii) any naturally occurring amino acid other than proline or glycine. Figure 9B , 10B and 11B provide respectively in Figure 9A , 10A The common amino acid sequences of the HLA-A, HLA-B, and HLA-C sequences provided in 11A. The common sequences display the variable amino acid positions as sequentially numbered "X" residues and double-underline the positions of amino acids 84, 139, and 236.

[0119] about Figure 9A In some cases: i) aac1 (amino acid cluster 1) may be the amino acid sequence GTLRG (SEQ ID NO: 287) or have one or two amino acids missing or replaced by other naturally occurring amino acids. For example ii) The sequence L is replaced by I, V, A or F; ii) aac2 (amino acid cluster 2) may be the amino acid sequence YNQSE (SEQ ID NO:288) or have one or two amino acids missing or replaced by other naturally occurring amino acids. For example, The sequence is N replaced by Q, Q replaced by N, and / or E replaced by D; iii) aac3 (amino acid cluster 3) may be the amino acid sequence TAADM (SEQ ID NO:289) or have one or two amino acids missing or replaced by other naturally occurring amino acids. For example, The sequence is T replaced by S, A replaced by G, D replaced by E, and / or M replaced by L, V, or I; iv) aac4 (amino acid cluster 4) may be the amino acid sequence AQTTK (SEQ ID NO:290) or have one or two amino acids missing or replaced by other naturally occurring amino acids. For example, A sequence in which A is replaced by G, Q by N, or T by S, and / or K by R or Q; v) aac5 (amino acid cluster 5) may be an amino acid sequence VETRP (SEQ ID NO: 291) or have one or two amino acids missing or replaced by other naturally occurring amino acids. For example, A sequence in which V is replaced by I or L, E by D, T by S, and / or R by K; and / or vi) aac6 (amino acid cluster 6) may be an amino acid sequence GDGTF (SEQ ID NO:292) or have one or two amino acids missing or replaced by other naturally occurring amino acids. For example, A sequence in which D is permuted by E, T by S, or F by L, W, or Y.

[0120] about Figure 10A In some cases: i) aac1 (amino acid cluster 1) may be an amino acid sequence RNLRG (SEQ ID NO: 293) or have one or two amino acids missing or replaced by other naturally occurring amino acids. For exampleii) aac2 (amino acid cluster 2) may be an amino acid sequence YNQSE (SEQ ID NO:288) or one or two amino acids may be missing or replaced by other naturally occurring amino acids. For example iii) aac3 (amino acid cluster 3) may be the amino acid sequence TAADT (SEQ ID NO:294) or have one or two amino acids missing or replaced by other naturally occurring amino acids. For example The sequence is: (i) the first T is replaced by S; and / or A is replaced by G; and / or D is replaced by E; and / or the second T is replaced by S; iv) aac4 (amino acid cluster 4) may be the amino acid sequence AQITQ (SEQ ID NO:295) or one or two amino acids may be missing or replaced by other naturally occurring amino acids. For example The sequence is: A is replaced by G; and / or the first Q is replaced by N; and / or I is replaced by L or V; and / or T is replaced by S; and / or the second Q is replaced by N; v) aac5 (amino acid cluster 5) may be the amino acid sequence VETRP (SEQ ID NO:291) or have one or two amino acids missing or replaced by other naturally occurring amino acids. For example The sequence is one in which V is replaced by I or L, E by D, T by S, and / or R by K; and / or vi) aac6 (amino acid cluster 6) may be an amino acid sequence GDRTF (SEQ ID NO:296) or have one or two amino acids missing or replaced by other naturally occurring amino acids. For example A sequence in which D is permuted by E; and / or T is permuted by S; and / or R is permuted by K or H; and / or F is permuted by L, W or Y.

[0121] about Figure 11A In some cases: i) aac1 (amino acid cluster 1) may be an amino acid sequence RNLRG (SEQ ID NO: 293) or have one or two amino acids missing or replaced by other naturally occurring amino acids. For example The sequence is defined as follows: (i) N is replaced by K; and / or L is replaced by A or I; and / or the second R is replaced by H; and / or G is replaced by T or S. The sequence is also defined as follows: (ii) aac2 (amino acid cluster 2) may be the amino acid sequence YNQSE (SEQ ID NO:288) or one or two amino acids may be missing or replaced by other naturally occurring amino acids. For exampleiii) aac3 (amino acid cluster 3) may be the amino acid sequence TAADT (SEQ ID NO:294) or have one or two amino acids missing or replaced by other naturally occurring amino acids. For example The sequence is: (i) the first T is replaced by S; and / or A is replaced by G; and / or D is replaced by E; and / or the second T is replaced by S; iv) aac4 (amino acid cluster 4) may be the amino acid sequence AQITQ (SEQ ID NO:295) or one or two amino acids may be missing or replaced by other naturally occurring amino acids. For example The sequence is: A is replaced by G; and / or the first Q is replaced by N; and / or I is replaced by L; and / or the second Q is replaced by N or K. v) aac5 (amino acid cluster 5) may be an amino acid sequence VETRP (SEQ ID NO: 291) or one or two amino acids may be missing or replaced by other naturally occurring amino acids. For example The sequence is one in which V is replaced by I or L, E by D, T by S, and / or R by K or H; and / or vi) aac6 (amino acid cluster 6) may be an amino acid sequence GDGTF (SEQ ID NO:292) or have one or two amino acids missing or replaced by other naturally occurring amino acids. For example A sequence in which D is permuted by E; and / or T is permuted by S; and / or F is permuted by L, W or Y.

[0122] HLA-A

[0123] In some cases, TMMP contains an HLA-A heavy chain peptide. The HLA-A heavy chain peptide sequence or a portion thereof that may be incorporated into the TMMP of this disclosure includes, but is not limited to, the following alleles: A 0101, A 0201, A 0301, A 1101, A 2301, A 2402, A 2407, A 3303 and A 3401, which is incomplete or substantially complete Figure 9A Alignment is performed in the case of leader sequences, transmembrane sequences, and cytoplasmic sequences. Any of those alleles may be contained at positions 84, 139, and / or 236 (e.g., ...). Figure 9AMutations selected from the following at one or more positions (as shown): 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). Alternatively, an HLA-A sequence may be used that has 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 with all or part of the sequence of those HLA-A alleles (e.g., 50, 75, 100, 150, 200, or 250 consecutive amino acids). For example It may contain 1-25, 1-5, 5-10, 10-15, 15-20, 20-25, or 25-30 amino acid insertions, deletions, and / or substitutions.

[0124] In some cases, TMMP contains an HLA-A heavy chain polypeptide that includes the following HLA-A common amino acid sequence: Where X1 is F, Y, S, or T; X2 is K or R; X3 is Q, G, E, or R; X4 is N or E; X5 is R or G; X6 is N or K; X7 is M or V; X8 is H or Q; X9 is T or I; X10 is D or H; X11 is A, V, or E; X12 is N or D; X13 is G or R; X14 is T or I; X15 is L or A; X16 is R or L; X17 is G or R; X18 is A or D; X19 is I, L, or V; X20 is I, R, or M; X21 is F or Y; X22 is S or P; 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; 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.

[0125] As an example, a class I MHC heavy chain polypeptide of TMMP may contain 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 with the following human HLA-A heavy chain amino acid sequence: GSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDGETRKVKAHSQTHRVDLGTLRGYYNQSEAGSHTVQRMYGCDVGSDWRFLRGYHQYAYDGKDYIALKEDLRSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGTCVEWLRRYLENGKETLQRTDAPKTHMTHHAVSDHEATLRCWALSFYPAEITLTWQRDGEDQTQDTELVETRPAGDGTFQKWAAVVVPSGQEQRYTCHVQHEGLPKPLTLRWEP (SEQ ID NO:44).

[0126] In some cases, HLA-A heavy chain peptides suitable for inclusion in TMMP contain the following amino acid sequence: GSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDGETRKVKAHSQTHRVDLGTLRGYYNQSEAGSHTVQRMYGCDVGSDWRFLRGYHQYAYDGKDYIALKEDLRSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGTCVEWLRRYLENGKETLQRTDAPKTHMTHHAVSDHEATLRCWALSFYPAEITLTWQRDGEDQTQDTELVETRPAGDGTFQKWAAVVVPSGQEQRYTCHVQHEGLPKPLTLRWEP (SEQ ID NO:44). This HLA-A heavy chain peptide is also known as "HLA-A "0201" or simply "HLA-A02". In some cases, the C-terminal Pro is not included in the TMMP. For example, in some cases, the HLA-A02 peptide suitable for inclusion in the TMMP contains the following amino acid sequence: GSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDGETRKVKAHSQTHRVDLGTLRGYYNQSEAGSHTVQRMYGCDVGSDWRFLRGYHQYAYDGKDYIALKEDLRSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGTCVEWLRRYLENGKETLQRTDAPKTHMTHHAVSDHEATLRCWALSFYPAEITLTWQRDGEDQTQDTELVETRPAGDGTFQKWAAVVVPSGQEQRYTCHVQHEGLPKPLTLRWE (SEQ ID NO:449).

[0127] HLA-A (Y84C; A236C)

[0128] In some cases, HLA-A heavy chain peptides suitable for inclusion in TMMP contain 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 with the following human HLA-A heavy chain (Y84C; A236C): GSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDGETRKVKAHSQTHRVD LGTLRGCYNQSEAGSHTVQRMYGCDVGSDWRFLRGYHQYAYDGKDYIALKEDLRSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGTCVEWLRRYLENGKETLQRTDAPKTHMTHHAVSDHEATLRCWALSFYPAEITLTWQRDGEDQTQDTELVETRPCGDGTFQKWAAVVVPSGQEQRYTCHVQHEGLPKPLTLRWE (SEQ ID NO:488), wherein amino acid 84 is Cys and amino acid 236 is Cys.

[0129] HLA-A (Y84A; A236C)

[0130] In some cases, class I MHC heavy chain peptides contain Y84A and A236C substitutions. For example, in some cases, class I MHC heavy chain peptides contain an amino acid sequence that has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity with the following human HLA-A heavy chain (Y84A; A236C): GSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDGETRKVKAHSQTHRVDLGTLRG A YNQSEAGSHTVQRMYGCDVGSDWRFLRGYHQYAYDGKDYIALKEDLRSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGTCVEWLRRYLENGKETLQRTDAPKTHMTHHAVSDHEATLRCWALSFYPAEITLTWQRDGEDQTQDTELVETRP C GDGTFQKWAAVVVPSGQEQRYTCHVQHEGLPKPLTLRWEP (SEQ ID NO:48), wherein amino acid 84 is Ala and amino acid 236 is Cys. In some cases, Cys-236 forms an interchain disulfide bond with Cys-12 of the variant β2M polypeptide containing an R12C substitution.

[0131] In some cases, HLA-A heavy chain peptides suitable for inclusion in TMMP are HLA-A02 (Y84A; A236C) peptides containing the following amino acid sequence: GSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDGETRKVKAHSQTHRVDLGTLRG A YNQSEAGSHTVQRMYGCDVGSDWRFLRGYHQYAYDGKDYIALKEDLRSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGTCVEWLRRYLENGKETLQRTDAPKTHMTHHAVSDHEATLRCWALSFYPAEITLTWQRDGEDQTQDTELVETRP C GDGTFQKWAAVVVPSGQEQRYTCHVQHEGLPKPLTLRWEP (SEQ ID NO: 48).

[0132] In some cases, HLA-A heavy chain peptides suitable for inclusion in TMMP are HLA-A02 (Y84A; A236C) peptides containing the following amino acid sequence: GSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDGETRKVKAHSQTHRVDLGTLRG A YNQSEAGSHTVQRMYGCDVGSDWRFLRGYHQYAYDGKDYIALKEDLRSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGTCVEWLRRYLENGKETLQRTDAPKTHMTHHAVSDHEATLRCWALSFYPAEITLTWQRDGEDQTQDTELVETRP C GDGTFQKWAAVVVPSGQEQRYTCHVQHEGLPKPLTLRWE (SEQID NO:46).

[0133] HLA-A (Y84C; A139C)

[0134] In some cases, class I MHC heavy chain peptides contain Y84C and A139C substitutions. For example, in some cases, class I MHC heavy chain peptides contain an amino acid sequence that has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity with the following human HLA-A heavy chain (Y84C; A139C): GSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDGETRKVKAHSQTHRVDLGTLRG C YNQSEAGSHTVQRMYGCDVGSDWRFLRGYHQYAYDGKDYIALKEDLRSWTAADM C AQTTKHKWEAAHVAEQLRAYLEGTCVEWLRRYLENGKETLQRTDAPKTHMTHHAVSDHEATLRCWALSFYPAEITLTWQRDGEDQTQDTELVETRPAGDGTFQKWAAVVVPSGQEQRYTCHVQHEGLPKPLTLRWEP (SEQ ID NO:299), wherein amino acid 84 is Cys and amino acid 139 is Cys. In some cases, Cys-84 and Cys-139 form an intrachain disulfide bond.

[0135] HLA-A11 (HLA-A 1101)

[0136] As a non-limiting example, a class I MHC heavy chain polypeptide of TMMP may contain 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 with the following human HLA-A11 heavy chain amino acid sequence: GSHSMRYFYTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDQETRNVKAQSQTDRVDLGTLRGYYNQSEDGSHTIQIMYGCDVGPDGRFLRGYRQDAYDGKDYIALNEDLRSWTAADMAAQITKRKWEAAHAAEQQRAYLEGTCVEWLRRYLENGKETLQRTDPPKTHMTHHPISDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLRWE (SEQ ID NO:300). This type of MHC heavy chain can be prominent in Asian populations, which include individual groups of people of Asian descent.

[0137] HLA-A11 (Y84A; A236C)

[0138] As a non-limiting example, in some cases, class I MHC heavy chain polypeptides are HLA-A11 alleles containing Y84A and A236C substitutions. For example, in some cases, class I MHC heavy chain polypeptides contain 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 with the following human HLA-A A11 heavy chain (Y84A; A236C): GSHSMRYFYTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDQETRNVKAQSQTDRVDLGTLRG A YNQSEDGSHTIQIMYGCDVGPDGRFLRGYRQDAYDGKDYIALNEDLRSWTAADMAAQITKRKWEAAHAAEQQRAYLEGTCVEWLRRYLENGKETLQRTDPPKTHMTHHPISDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRP CGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLRWE (SEQ ID NO:301), wherein amino acid 84 is Ala and amino acid 236 is Cys. In some cases, Cys-236 forms an interchain disulfide bond with Cys-12 of the variant β2M polypeptide containing an R12C substitution.

[0139] HLA-A11 (Y84C; A236C)

[0140] In some cases, class I MHC heavy chain polypeptides present in TMMP contain amino acid sequences that have 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 with the following human HLA-A A11 heavy chain (Y84C; A236C): GSHSMRYFYTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDQETRNVKAQSQTDRVDLGTLRG C YNQSEDGSHTIQIMYGCDVGPDGRFLRGYRQDAYDGKDYIALNEDLRSWTAADMAAQITKRKWEAAHAAEQQRAYLEGTCVEWLRRYLENGKETLQRTDPPKTHMTHHPISDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRP C GDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLRWE (SEQ ID NO:500), wherein amino acid 84 is Cys and amino acid 236 is Cys.

[0141] HLA-A24 (HLA-A 2402)

[0142] As a non-limiting example, the class I MHC heavy chain polypeptide of TMMP may contain human HLA-A24 (also known as HLA-A) 2402) The following amino acid sequences have 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: GSHSMRYFSTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDEETGKVKAHSQTDRENLRIALRYYNQSEAGSHTLQMMFGCDVGSDGRFLRGYHQYAYDGKDYIALKEDLRSW TAADMAAQITKRKWEAAHVAEQQRAYLEGTCVDGLRRYLENGKETLQRTDPPKTHMTHHPISDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLRWEPSSQPTVPIVGIIAGLVLLGAVITGAVVAAVMWRRNSSDRKGGSYSQAASSDSAQGSDVSLTACKV (SEQ ID NO:302). This type I MHC heavy chain can be prominent in Asian populations, including individual groups of Asian descent. In some cases, amino acid 84 is Ala. In some cases, amino acid 84 is Cys. In some cases, amino acid 236 is Cys. In some cases, amino acid 84 is Ala and amino acid 236 is Cys. In some cases, amino acid 84 is Cys and amino acid 236 is Cys.

[0143] In some cases, the class I MHC heavy chain peptide of TMMP may contain human HLA-A24 (also known as HLA-A) 2402) The heavy chain 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: GSHSMRYFSTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDEETGKVKAHSQTDRENLRIALR Y YNQSEAGSHTLQMMFGCDVGSDGRFLRGYHQYAYDGKDYIALKEDLRSWTAADMAAQITKRKWEAAHVAEQQRAYLEGTCVDGLRRYLENGKETLQRTDPPKTHMTHHPISDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRP AGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLRWE (SEQ ID NO:455), wherein amino acid 84 is Tyr and amino acid 236 is Ala (amino acids 84 and 236 are in bold and underlined); and wherein the class I MHC heavy chain has a length of approximately 275 amino acids.

[0144] In some cases, the class I MHC heavy chain peptide of TMMP may contain human HLA-A24 (also known as HLA-A) 2402) The heavy chain 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: GSHSMRYFSTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDEETGKVKAHSQTDRENLRIALR A YNQSEAGSHTLQMMFGCDVGSDGRFLRGYHQYAYDGKDYIALKEDLRSWTAADMAAQITKRKWEAAHVAEQQRAYLEGTCVDGLRRYLENGKETLQRTDPPKTHMTHHPISDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRP A GDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLRWE (SEQ ID NO:456), wherein amino acid 84 is Ala and amino acid 236 is Ala (amino acids 84 and 236 are in bold and underlined); and wherein the class I MHC heavy chain has a length of approximately 275 amino acids.

[0145] In some cases, the class I MHC heavy chain peptide of TMMP may contain human HLA-A24 (also known as HLA-A) 2402) The heavy chain 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: GSHSMRYFSTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDEETGKVKAHSQTDRENLRIALR YYNQSEAGSHTLQMMFGCDVGSDGRFLRGYHQYAYDGKDYIALKEDLRSWTAADMAAQITKRKWEAAHVAEQQRAYLEGTCVDGLRRYLENGKETLQRTDPPKTHMTHHPISDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRP C GDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLRWE (SEQ ID NO:457), wherein amino acid 84 is Tyr and amino acid 236 is Cys (amino acids 84 and 236 are in bold and underlined); and wherein the class I MHC heavy chain has a length of approximately 275 amino acids.

[0146] In some cases, the class I MHC heavy chain peptide of TMMP may contain human HLA-A24 (also known as HLA-A) 2402) The heavy chain 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: GSHSMRYFSTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDEETGKVKAHSQTDRENLRIALR A YNQSEAGSHTLQMMFGCDVGSDGRFLRGYHQYAYDGKDYIALKEDLRSWTAADMAAQITKRKWEAAHVAEQQRAYLEGTCVDGLRRYLENGKETLQRTDPPKTHMTHHPISDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRP C GDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLRWE (SEQ ID NO:458), wherein amino acid 84 is ALA and amino acid 236 is CYS (amino acids 84 and 236 are in bold and underlined); and wherein the class I MHC heavy chain has a length of approximately 275 amino acids.

[0147] In some cases, class I MHC heavy chain peptides may contain human HLA-A24 (also known as HLA-A) 2402) The heavy chain 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: GSHSMRYFSTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDEETGKVKAHSQTDRENLRIALR C YNQSEAGSHTLQMMFGCDVGSDGRFLRGYHQYAYDGKDYIALKEDLRSWTAADMAAQITKRKWEAAHVAEQQRAYLEGTCVDGLRRYLENGKETLQRTDPPKTHMTHHPISDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRP A GDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLRWE(SEQ ID NO:459), wherein amino acid 84 is CYS and amino acid 236 is ALA (amino acids 84 and 236 are in bold and underlined); and the class I MHC heavy chain has a length of approximately 275 amino acids.

[0148] In some cases, the class I MHC heavy chain peptide of TMMP may contain human HLA-A24 (also known as HLA-A) 2402) The heavy chain 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: GSHSMRYFSTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDEETGKVKAHSQTDRENLRIALR C YNQSEAGSHTLQMMFGCDVGSDGRFLRGYHQYAYDGKDYIALKEDLRSWTAADMAAQITKRKWEAAHVAEQQRAYLEGTCVDGLRRYLENGKETLQRTDPPKTHMTHHPISDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRP C GDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLRWE (SEQ ID NO:346), wherein amino acid 84 is CYS and amino acid 236 is CYS (amino acids 84 and 236 are in bold and underlined); and wherein the class I MHC heavy chain has a length of approximately 275 amino acids.

[0149] HLA-A33 (HLA-A 3303)

[0150] As a non-limiting example, a class I MHC heavy chain polypeptide of TMMP may contain 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 with the following human HLA-A33 heavy chain amino acid sequence: GSHSMRYFTTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDRNTRNVKAHSQIDRVDLGTLRGYYNQSEAGSHTIQMMYGCDVGSDGRFLRGYQQD AYDGKDYIALNEDLRSWTAADMAAQITQRKWEAARVAEQLRAYLEGTCVEWLRRYLENGKETLQRTDPPKTHMTHHAVSDHEATLRCWALSFYPAEITLTWQRDGEDQTQDTELVETRPAGDGTFQKWASVVVPSGQEQRYTCHVQHEGLPKPLTLRWEPSSQPTIPIVGIIAGLVLFGAVFAGAVVAAVRWRRKSSDRKGGSYSQAASSDSAQGSDMSLTACKV (SEQ ID NO:303). This type I MHC heavy chain can be prominent in Asian populations, including individual groups of Asian descent. In some cases, amino acid 84 is Ala. In some cases, amino acid 84 is Cys. In some cases, amino acid 236 is Cys. In some cases, amino acid 84 is Ala and amino acid 236 is Cys. In some cases, amino acid 84 is Cys and amino acid 236 is Cys.

[0151] HLA-B

[0152] In some cases, TMMP contains HLA-B heavy chain peptides. HLA-B heavy chain peptide sequences or portions thereof that can be incorporated into TMMP include, but are not limited to, the following alleles: B 0702, B 0801, B 1502, B 3802, B 4001, B 4601 and B 5301, which is incomplete or largely complete Figure 10AAlignment is performed in the case of leader sequences, transmembrane sequences, and cytoplasmic sequences. Any of those alleles may be contained at positions 84, 139, and / or 236 (e.g., ...). Figure 10A Mutations selected from the following at one or more positions (as shown): 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). Alternatively, an HLA-B polypeptide containing 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 with all or part of the sequence of those HLA-B alleles (e.g., 50, 75, 100, 150, 200, or 250 consecutive amino acids) may also be used. For example It may contain 1-25, 1-5, 5-10, 10-15, 15-20, 20-25, or 25-30 amino acid insertions, deletions, and / or substitutions.

[0153] In some cases, TMMP contains an HLA-B heavy chain polypeptide that includes the following HLA-B common amino acid sequence:

[0154] GSHSMRYF X1 T X2X3 SRPGRGEPRFI X4 VGYVDDT X5 FVRFDSDA X6 SPR X7X8 PRAPWIEQEGPEYWDR X9 TQ X10X11 KT X12X13 TQ X14 Y X15X16 NL X17X18X19X20 YYNQSEAGSH X21X22QX23 MYGCDLGPDGRLLRGHDQSAYDGKDYIALNEDL X24 SWTAADTAAQI X25 QRK X26 EAAR X27 AEQ X28 R X29 YLEG X30 CVEWLRRYLENGK X31X32 L X33 RADPPKTHVTHHP X34SDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRPAGDRTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLRWEP (SEQ ID NO:30), wherein X1 is H, Y, or D; X2 is A or S; X3 is M or V; X4 is A, S, or T; X5 is Q or L; X6 is A or T; X7 is E, MK, or T; X8 is A or T; X9 is E or N; X10 is I or K; X11 is Y, F, S, or C; X12 is N or Q; X13 is A or T; X14 is D or Y; X15 is E or V; X16 is S or N; X17 is T or N. 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.

[0155] As an example, a class I MHC heavy chain polypeptide of TMMP may contain 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 with the following human HLA-B heavy chain amino acid sequence: GSHSMRYFYTSVSRPGRGEPRFISVGYVDDTQFVRFDSDAASPREEPRAPWIEQEGPEYWDRNTQIYKAQAQTDRESLRNLRGYYNQSEAGSHTLQSMYGCDVGPDGRLLRGHDQYAYDGKDYIALNEDLRSWTAADTAAQITQRKWEAAREAEQRRAYLEGECVEWLRRYLENGKDKLERADPPKTHVTHHPISDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRPAGDRTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLRWEP (SEQ ID NO:207).

[0156] HLA-B (Y84A; A236C)

[0157] As a non-limiting example, in some cases, a class I MHC heavy chain polypeptide is an HLA-B polypeptide containing Y84A and A236C substitutions. For example, in some cases, a class I MHC heavy chain polypeptide contains 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 with the following human HLA-B heavy chain (Y84A; A236C): GSHSMRYFYTSVSRPGRGEPRFISVGYVDDTQFVRFDSDAASPREEPRAPWIEQEGPEYWDRNTQIYKAQAQTDRESLRNLRG A YNQSEAGSHTLQSMYGCDVGPDGRLLRGHDQYAYDGKDYIALNEDLRSWTAADTAAQITQRKWEAAREAEQRRAYLEGECVEWLRRYLENGKDKLERADPPKTHVTHHPISDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRP C GDRTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLRWEP (SEQ ID NO:305), wherein amino acid 84 is Ala and amino acid 236 is Cys. In some cases, Cys-236 forms an interchain disulfide bond with Cys-12 of the variant β2M polypeptide containing an R12C substitution.

[0158] HLA-B (Y84C; A139C)

[0159] In some cases, class I MHC heavy chain peptides contain Y84C and A139C substitutions. For example, in some cases, class I MHC heavy chain peptides contain an amino acid sequence that has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity with the following human HLA-B heavy chain (Y84C; A139C): GSHSMRYFYTSVSRPGRGEPRFISVGYVDDTQFVRFDSDAASPREEPRAPWIEQEGPEYWDRNTQIYKAQAQTDRESLRNLRG C YNQSEAGSHTLQSMYGCDVGPDGRLLRGHDQYAYDGKDYIALNEDLRSWTAADT CAQITQRKWEAAREAEQRRAYLEGECVEWLRRYLENGKDKLERADPPKTHVTHHPISDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRPAGDRTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLRWEP (SEQ ID NO:306), wherein amino acid 84 is Cys and amino acid 139 is Cys. In some cases, Cys-84 and Cys-139 form an intrachain disulfide bond.

[0160] HLA-B 0702

[0161] As an example, in some cases, class I MHC heavy chain peptides present in TMMP contain Figure 10A HLA-B in The amino acid sequence of 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 with all or part of that sequence (e.g., 50, 75, 100, 150, 200, or 250 consecutive amino acids). For example It may contain 1-25, 1-5, 5-10, 10-15, 15-20, 20-25, or 25-30 amino acid insertions, deletions, and / or substitutions. In some cases, the HLA-B heavy chain peptide of the TMMP disclosed herein is related to... Figure 8 HLA-B or sequence markers in Figure 10A The "B" in the mark When 0702 has less than 100% identity, it may contain mutations selected from one or more of positions 84, 139, and / or 236: a tyrosine-to-alanine substitution at position 84 (Y84A); a tyrosine-to-cysteine ​​substitution at position 84 (Y84C); an alanine-to-cysteine ​​substitution at position 139 (A139C); and an alanine-to-cysteine ​​substitution at position 236 (A236C). In some cases, the HLA-B heavy chain polypeptide of the TMMP of this disclosure contains Y84A and A236C substitutions. In some cases, the HLA-B of the TMMP of this disclosure... The 0702 heavy chain polypeptide contains Y84C and A139C substitutions. In some cases, the HLA-B heavy chain polypeptide of the TMMP disclosed herein contains Y84C, A139C, and A236C substitutions.

[0162] HLA-C

[0163] In some cases, TMMP contains an HLA-C heavy chain polypeptide. HLA-C heavy chain polypeptides or portions thereof that may be incorporated into the TMMP of this disclosure include, but are not limited to, the following alleles: C 0102, C 0303, C 0304, C 0401, C 0602, C 0701, C 0801 and C 1502, which is incomplete or substantially complete Figure 11A Alignment is performed in the case of leader sequences, transmembrane sequences, and cytoplasmic sequences. Any of those alleles may be contained at positions 84, 139, and / or 236 (e.g., ...). Figure 11A Mutations selected from the following at one or more of the positions shown: a tyrosine-to-alanine substitution at position 84 (Y84A); a tyrosine-to-cysteine ​​substitution at position 84 (Y84C); an alanine-to-cysteine ​​substitution at position 139 (A139C); and an alanine-to-cysteine ​​substitution at position 236 (A236C). Alternatively, an HLA-C polypeptide containing 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 with all or part of the sequence of those HLA-C alleles (e.g., 50, 75, 100, 150, 200, or 250 consecutive amino acids) may also be used. For example It may contain 1-25, 1-5, 5-10, 10-15, 15-20, 20-25, or 25-30 amino acid insertions, deletions, and / or substitutions.

[0164] In some cases, TMMP contains an HLA-C heavy chain polypeptide that includes the following HLA-C common amino acid sequence: Where 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; X19 is L or V; X20 is Y or H; X21 is D or N; X22 is Y, F, S or L; X23 is L or W; X24 is E, A or T; X25 is R, L or W; X26 is L or T; X27 is E or K; X28 is E or K; X29 is H or P; X30 is R or V; X31 is W or R; X32 is V or M; X33 is E or Q; X34 is M or V; X35 is P or Q; X36 is R or S; and X37 is P or G.

[0165] As an example, a class I MHC heavy chain polypeptide of TMMP may contain 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 with the following human HLA-C heavy chain amino acid sequence: CSHSMRYFDTAVSRPGRGEPRFISVGYVDDTQFVRFDSDAASPRGEPRAPWVEQEGPEYWDRETQNYKRQAQADRVSLRNLRGYYNQSEDGSHTLQRMYGCDLGPDGRLLRGYDQSAYDGKDYIALNEDLRSWTAADTAAQITQRKLEAARAAEQLRAYLEGTCVEWLRRYLENGKETLQRAEPPKTHVTHHPLSDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRPAGDGTFQKWAAVVVPSGQEQRYTCHMQHEGLQEPLTLSWEP (SEQ ID) NO:219).

[0166] HLA-C (Y84A; A236C)

[0167] As a non-limiting example, in some cases, a class I MHC heavy chain polypeptide is an HLA-C polypeptide comprising substitutions for Y84A and A236C. For instance, in some cases, a class I MHC heavy chain polypeptide comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity with the following human HLA-C heavy chain (Y84A; A236C): CSHSMRYFDTAVSRPGRGEPRFISVGYVDDTQFVRFDSDAASPRGEPRAPWVEQEGPEYWDRETQNYKRQAQADRVSLRNLRG A YNQSEDGSHTLQRMYGCDLGPDGRLLRGYDQSAYDGKDYIALNEDLRSWTAADTAAQITQRKLEAARAAEQLRAYLEGTCVEWLRRYLENGKETLQRAEPPKTHVTHHPLSDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRP C GDGTFQKWAAVVVPSGQEQRYTCHMQHEGLQEPLTLSWEP (SEQ ID NO:308), wherein amino acid 84 is Ala and amino acid 236 is Cys. In some cases, Cys-236 forms an interchain disulfide bond with Cys-12 of the variant β2M polypeptide containing an R12C substitution.

[0168] HLA-C (Y84C; A139C)

[0169] In some cases, class I MHC heavy chain peptides contain Y84C and A139C substitutions. For example, in some cases, class I MHC heavy chain peptides contain an amino acid sequence that has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity with the following human HLA-C heavy chain (Y84C; A139C): CSHSMRYFDTAVSRPGRGEPRFISVGYVDDTQFVRFDSDAASPRGEPRAPWVEQEGPEYWDRETQNYKRQAQADRVSLRNLRG C YNQSEDGSHTLQRMYGCDLGPDGRLLRGYDQSAYDGKDYIALNEDLRSWTAADT CAQITQRKLEAARAAEQLRAYLEGTCVEWLRRYLENGKETLQRAEPPKTHVTHHPLSDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRPAGDGTFQKWAAVVVPSGQEQRYTCHMQHEGLQEPLTLSWEP (SEQ ID NO:397), wherein amino acid 84 is Cys and amino acid 139 is Cys. In some cases, Cys-84 and Cys-139 form an intrachain disulfide bond.

[0170] HLA-C 0701

[0171] In some cases, the class I MHC heavy chain peptides of TMMP contain Figure 11A HLA-C in The amino acid sequence of 0701 ( Figure 8 The HLA-C marker in the sequence 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 with all or part of the sequence (e.g., 50, 75, 100, 150, 200, or 250 consecutive amino acids). For example It may contain 1-25, 1-5, 5-10, 10-15, 15-20, 20-25, or 25-30 amino acid insertions, deletions, and / or substitutions. In some cases, the HLA-C heavy chain peptide in TMMP is related to... Figure 11A HLA-C sequence markers in When 0701 has less than 100% identity, it may contain mutations selected from one or more of positions 84, 139, and / or 236, namely: a tyrosine-to-alanine substitution at position 84 (Y84A); a tyrosine-to-cysteine ​​substitution at position 84 (Y84C); an alanine-to-cysteine ​​substitution at position 139 (A139C); and an alanine-to-cysteine ​​substitution at position 236 (A236C). In some cases, the HLA-C heavy chain polypeptide of TMMP contains both Y84A and A236C substitutions. In some cases, the HLA-C of T-cell-MMP or its epitope conjugates... The 0701 heavy chain polypeptide contains Y84C and A139C substitutions. In some cases, the HLA-C heavy chain polypeptide of the TMMP disclosed herein contains Y84C, A139C, and A236C substitutions.

[0172] Non-classical HLA-E, HLA-F, and HLA-G type I MHC heavy chains

[0173] In some cases, TMMPs contain non-classical class I MHC heavy chain polypeptides. Non-classical HLA heavy chain polypeptides or portions thereof that may be incorporated into the TMMPs of this disclosure include, but are not limited to, those of the HLA-E, HLA-F, and HLA-G alleles. The amino acid sequences of the HLA-E, HLA-F, and HLA-G heavy chain polypeptides (as well as the HLA-A, HLA-B, and HLA-C alleles) are available at hla.alleles.org / nomenclature / index.html, the European Institute for Bioinformatics (www.ebiac.uk, part of the European Laboratory for Molecular Biology (EMBL)), and the National Center for Biotechnology Information (www.ncbi.nlmnih.gov).

[0174] Suitable non-restricted examples of HLA-E alleles include, but are not limited to, HLA-E. 0101 (HLA-E 01:01:01:01), HLA-E 01:03(HLA-E 01:03:01:01), HLA-E 01:04, HLA-E 01:05, HLA-E 01:06, HLA-E 01:07, HLA-E 01:09 and HLA-E 01:10. Suitable, non-restrictive examples of HLA-F alleles include, but are not limited to, HLA-F. 0101 (HLA-F 01:01:01:01), HLA-F 01:02, HLA-F 01:03(HLA-F 01:03:01:01), HLA-F 01:04, HLA-F 01:05 and HLA-F 01:06. Suitable, non-restrictive examples of HLA-G alleles include, but are not limited to, HLA-G. 0101 (HLA-G 01:01:01:01), HLA-G 01:02, HLA-G 01:03(HLA-G 01:03:01:01), HLA-G 01:04 (HLA-G 01:04:01:01), HLA-G 01:06, HLA-G 01:07, HLA-G 01:08, HLA-G 01:09: HLA-G 01:10, HLA-G 01:10, HLA-G 01:11, HLA-G 01:12, HLA-G 01:14, HLA-G 01:15, HLA-G 01:16, HLA-G 01:17, HLA-G 01:18: HLA-G 01:19, HLA-G 01:20 and HLA-G 01:22. The common sequences of all or substantially all of the HLA E, HLA-F, and HLA-G alleles, without leader sequences, transmembrane sequences, and cytoplasmic sequences, are provided. Figure 12 In, and with Figure 13 The common sequences of the HLA-A, HLA-B, and HLA-C alleles mentioned above were compared.

[0175] Figures 46A-46D The appropriate amino acid sequence of the HLA-E heavy chain polypeptide is provided, in which Figure 46A HLA-E is provided The amino acid sequence of 01:01 (wild type); Figure 46B HLA-E with Y84C and A2346C replacements is provided. The amino acid sequence is 01:01; Figure 46C HLA-E is provided The amino acid sequence of 01:03 (wild type); and Figure 46D HLA-E with Y84C and A2346C replacements is provided. The amino acid sequence of 01:03.

[0176] Figure 47A -7D provides the amino acid sequence of a suitable HLA-G heavy chain polypeptide, in which Figure 47A HLA-G is provided The amino acid sequence of 01:01 (wild type); Figure 47BHLA-G is available with Y84C and A2346C replacements. The amino acid sequence is 01:01; Figure 47C HLA-G is provided The amino acid sequence of 01:04 (wild type); and Figure 47D HLA-G is available with Y84C and A2346C replacements. The amino acid sequence of 01:04.

[0177] Figure 12 Provide the common sequences of each of HLA-E, HLA-F and HLA-G, where the variable aa positions are indicated by sequentially numbered "X" residues and the positions of aa 84, 139 and 236 are double-underlined.

[0178] Figure 13 The alignments of common amino acid sequences for HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, and HLA-G are provided in Figures 9-13. Variable residues in each sequence are listed as "X", with their sequence numbers removed. Figure 8 As shown, the positions of the five amino acid clusters of aa 84, 139 and 236 and their flanks are indicated. These five amino acid clusters can be independently selected from 1 to 5 amino acid substitutions of: (i) any naturally occurring amino acid or (ii) any naturally occurring amino acid other than proline or glycine.

[0179] Any of the HLA-E, HLA-F, and / or HLA-G alleles mentioned above may contain, for example: Figure 13 The shown concordant sequence has substitutions at one or more of positions 84, 139, and / or 236. In some cases, these substitutions may be selected from: position 84 tyrosine to alanine (Y84A) or cysteine ​​(Y84C), or in the HLA-F case, R84A or R84C substitution; position 139 alanine to cysteine ​​(A139C), or in the HLA-F case, V139C substitution; and position 236 alanine to cysteine ​​substitution (A236C). Alternatively, substitutions may be made with... Figure 13 All or part of any of the listed common sequences (e.g., 50, 75, 100, 150, 200, or 250 consecutive amino acids) have 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 in the HLA-E, HLA-F, and / or HLA-G sequences. For exampleIn addition to the variable residue variations listed herein, these sequences may also contain 1-25, 1-5, 5-10, 10-15, 15-20, 20-25, or 25-30 amino acid insertions, deletions, and / or substitutions.

[0180] Mouse H2K

[0181] In some cases, class I MHC heavy chain peptides present in TMMP contain mouse H2K (SEQ ID NO:45) ( Figure 8 The amino acid sequence of mouse H2K 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 with all or part of that sequence (e.g., 50, 75, 100, 150, 200, or 250 consecutive amino acids). For example It may contain 1-25, 1-5, 5-10, 10-15, 15-20, 20-25, or 25-30 amino acid insertions, deletions, and / or substitutions. In some cases, the mouse H2K heavy chain peptide in TMMP is related to... Figure 8 When the mouse H2K sequence marker has less than 100% identity, it may contain mutations selected from one or more of positions 84, 139, and / or 236: tyrosine to alanine at position 84 (Y84A); tyrosine to cysteine ​​at position 84 (Y84C); alanine to cysteine ​​at position 139 (A139C); and alanine to cysteine ​​substitution at position 236 (A236C). In some cases, the mouse H2K heavy chain polypeptide of TMMP contains Y84A and A236C substitutions. In some cases, the mouse H2K heavy chain polypeptide of TMMP of this disclosure contains Y84C and A139C substitutions. In some cases, the mouse H2K heavy chain polypeptide of TMMP of this disclosure contains Y84C, A139C, and A236C substitutions.

[0182] Exemplary combinations

[0183] Table 1 below presents various combinations of type I MHC heavy chain sequence modifications that can be incorporated into the TMMP of this disclosure.

[0184] Table 1

[0185]

[0186]

[0187] The sequence identity range is the MHC-H polypeptide sequence incorporated into TMMP relative to... Figure 8-13The permissible range of sequence identity for the corresponding portions of the sequence listed in the table does not include variable residues in the shared sequence.

[0188] β-2 microglobulin

[0189] The β2-microglobulin (β2M) polypeptide of TMMP disclosed herein can be human β2M polypeptide, non-human primate β2M polypeptide, mouse β2M polypeptide, etc. In some cases, the β2M polypeptide contains... Figure 6 The β2M polypeptide described herein has an amino acid sequence with 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. In some cases, the β2M polypeptide contains amino acids that are identical to... Figure 6 The amino acid sequences 21 to 119 of the β2M amino acid sequence described herein have amino acid sequence identity of at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%.

[0190] In some cases, suitable β2M peptides contain the following amino acid sequence:

[0191] IQRTPKIQVY SCHPAENGKS NFLNCYVSGF HPSDIEVDLLKNGERIEKVE HSDLSFSKDWSFYLLYYTEF TPTEKDEYAC RVNHVTLSQP KIVKWDRDM (SEQ ID NO:311); and the HLA class I heavy chain polypeptide contains the following amino acid sequence:

[0192] GSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDGETRKVKAHSQTHRVDL(aa1){C}(aa2)AGSHTVQRMYGCDVGSDWRFLRGYHQYAYDGKDYIALKEDLRSW(aa3){C}(aa4))HKWEAAHVAEQLRAYLEGTCVEWLRRYLENGKETLQRTDAPKTHMTHHAVSDHEATLRCWALSFYPAEITLTWQRDGEDQTQDTEL(aa5)(C)(aa6)QKWAAVVVPSGQEQRYTCHVQHEGLPKPLTLRWEP (SEQ ID NO:309), wherein the cysteine ​​residue indicated by {C} forms a disulfide bond between the α1 and α2-1 helices and the (C) residue forms a disulfide bond with the β2M polypeptide cysteine ​​at position 12. In the above sequences, "aa1" represents "amino acid cluster 1"; "aa2" represents "amino acid cluster 2"; "aa3" represents "amino acid cluster 3"; "aa4" represents "amino acid cluster 4"; "aa5" represents "amino acid cluster 5"; and "aa6" represents "amino acid cluster 6"; see, for example, Figure 10. aa1, aa2, aa3, aa4, aa5, and aa6 are selected and independently chosen as 1-5 amino acid residues each time they appear, wherein the amino acid residues i) are independently selected from any naturally occurring (e.g., encoding) amino acid or ii) any naturally occurring amino acid other than proline or glycine.

[0193] In some cases, the MHC polypeptide contains a single amino acid substitution relative to a reference MHC polypeptide (which may be a wild-type MHC polypeptide), wherein the single amino acid substitution is a cysteine ​​(Cys) residue replacing one amino acid. When such cysteine ​​residues are present in the MHC polypeptide of the first polypeptide of the TMMP disclosed herein, they can form disulfide bonds with cysteine ​​residues present in the second polypeptide chain of the TMMP.

[0194] In some cases, the first MHC peptide in the first polypeptide of TMMP and / or the second MHC peptide in the second polypeptide of TMMP includes an amino acid substitution to replace an amino acid with a cysteine, wherein the substituted cysteine ​​in the first MHC peptide forms a disulfide bond with the cysteine ​​in the second MHC peptide, or wherein the substituted cysteine ​​in the first MHC peptide forms a disulfide bond with the substituted cysteine ​​in the second MHC peptide.

[0195] For example, in some cases, one of the following residue pairs in HLA β2-microglobulin and HLA class I heavy chain is replaced by a cysteine ​​residue (where the residue number is the mature polypeptide): 1) β2M residue 12, HLA class I heavy chain residue 236; 2) β2M residue 12, HLA class I heavy chain residue 237; 3) β2M residue 8, HLA class I heavy chain residue 234; 4) β2M residue 10, HLA class I heavy chain residue 235; 5) β2M residue 24, HLA class I heavy chain residue 236; 6) β2M residue 28, HLA class I heavy chain residue 232; 7) β2M residue 98, HLA class I heavy chain residue 192; 8) β2M residue 99, HLA class I heavy chain residue 234; 9) β2M residue 3, HLA class I heavy chain residue 120; 10) β2M residue 31, HLA class I heavy chain residue 120; 11) β2M residue 31, HLA class I heavy chain residue 120. 11) β2M residue 53, HLA class I heavy chain residue 35; 12) β2M residue 60, HLA class I heavy chain residue 96; 13) β2M residue 60, HLA class I heavy chain residue 122; 14) β2M residue 63, HLA class I heavy chain residue 27; 15) β2M residue Arg3, HLA class I heavy chain residue Gly120; 16) β2M residue His31, HLA class I heavy chain residue Gln96; 17) β2M residue Asp53, HLA class I heavy chain residue Arg35; 18) β2M residue Trp60, HLA class I heavy chain residue Gln96; 19) β2M residue Trp60, HLA class I heavy chain residue Asp122; 20) β2M residue Tyr63, HLA class I heavy chain residue Gly122; 21) Class I heavy chain residue Tyr27; 22) β2M residue Lys6, HLA Class I heavy chain residue Glu232; 23) β2M residue Gln8, HLA Class I heavy chain residue Arg234; 24) β2M residue Tyr10, HLA Class I heavy chain residue Pro235; 25) β2M residue Ser11, HLA Class I heavy chain residue Gln242; 26) β2M residue Asn24, HLA Class I heavy chain residue Ala236; 27) β2M residue Ser28, HLA Class I heavy chain residue Glu232; 28) β2M residue Asp98, HLA Class I heavy chain residue His192; and 29) β2M residue Met99, HLA Class I heavy chain residue Arg234. The amino acid numbering of MHC / HLA class I heavy chains is based on mature MHC / HLA class I heavy chains, which do not have a signal peptide. For example, in some cases, residue 236 of the mature HLA-A amino acid sequence is replaced by Cys. In some cases, residue 236 of the mature HLA-B amino acid sequence is replaced by Cys. In some cases, residue 236 of the mature HLA-C amino acid sequence is replaced by Cys.In some cases, ... Figure 6 Residue 32 of the amino acid sequence depicted in the figure (corresponding to Arg-12 of mature β2M) is replaced by Cys.

[0196] In some cases, the β2M polypeptide contains the following amino acid sequence: IQRTPKIQVY S R HPAENGKSNFLNCYVSGF HPSDIEVDLLKNGERIEKVE HSDLSFSKDW SFYLLYYTEF TPTEKDEYAC RVNHVTLSQPKIVKWDRDM (SEQ ID NO:310). In some cases, the β2M polypeptide contains the following amino acid sequence: IQRTPKIQVYS C HPAENGKS NFLNCYVSGF HPSDIEVDLLKNGERIEKVE HSDLSFSKDW SFYLLYYTEF TPTEKDEYACRVNHVTLSQP KIVKWDRDM (SEQ ID NO: 311).

[0197] In some cases, HLA class I heavy chain peptides contain HLA-A. 2402 amino acid sequence: GSHSMRYFSTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDEETGKVKAHSQTDRENLRIALR Y YNQSEAGSHTLQMMFGCDVGSDGRFLRGYHQYAYDGKDYIALKEDLRSWTAADMAAQITKRKWEAAHVAEQQRAYLEGTCVDGLRRYLENGKETLQRTDPPKTHMTHHPISDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRP A GDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLRWE (SEQ ID NO:455).

[0198] In some cases, HLA class I heavy chain peptides contain HLA-A. 2402 amino acid sequence: GSHSMRYFSTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDEETGKVKAHSQTDRENLRIALR AYNQSEAGSHTLQMMFGCDVGSDGRFLRGYHQYAYDGKDYIALKEDLRSWTAADMAAQITKRKWEAAHVAEQQRAYLEGTCVDGLRRYLENGKETLQRTDPPKTHMTHHPISDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRP A GDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLRWE (SEQ ID NO:456).

[0199] In some cases, HLA class I heavy chain peptides contain the following amino acid sequence: GSHSMRYFSTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDEETGKVKAHSQTDRENLRIALR C YNQSEAGSHTLQMMFGCDVGSDGRFLRGYHQYAYDGKDYIALKEDLRSWTAADMAAQITKRKWEAAHVAEQQRAYLEGTCVDGLRRYLENGKETLQRTDPPKTHMTHHPISDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRP A GDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLRWE (SEQ ID NO:459).

[0200] In some cases, HLA class I heavy chain peptides contain the following amino acid sequence: GSHSMRYFSTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDEETGKVKAHSQTDRENLRIALR Y YNQSEAGSHTLQMMFGCDVGSDGRFLRGYHQYAYDGKDYIALKEDLRSWTAADMAAQITKRKWEAAHVAEQQRAYLEGTCVDGLRRYLENGKETLQRTDPPKTHMTHHPISDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRP C GDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLRWE (SEQ ID NO:457).

[0201] In some cases, HLA class I heavy chain peptides contain the following amino acid sequence: GSHSMRYFSTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDEETGKVKAHSQTDRENLRIALR A YNQSEAGSHTLQMMFGCDVGSDGRFLRGYHQYAYDGKDYIALKEDLRSWTAADMAAQITKRKWEAAHVAEQQRAYLEGTCVDGLRRYLENGKETLQRTDPPKTHMTHHPISDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRP C GDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLRWE (SEQ ID NO:458).

[0202] In some cases, HLA class I heavy chain peptides contain the following amino acid sequence: GSHSMRYFSTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDEETGKVKAHSQTDRENLRIALR C YNQSEAGSHTLQMMFGCDVGSDGRFLRGYHQYAYDGKDYIALKEDLRSWTAADMAAQITKRKWEAAHVAEQQRAYLEGTCVDGLRRYLENGKETLQRTDPPKTHMTHHPISDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRP C GDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLRWE (SEQ ID NO:346).

[0203] In some cases, HLA class I heavy chain peptides contain the following amino acid sequence: GSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDGETRKVKAHSQTHRVDLGTLRGYYNQSEAGSHTVQRMYGCDVGSDWRFLRGYHQYAYDGKDYIALKEDLRSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGTCVEWLRRYLENGKETLQRTDAPKTHMTHHAVSDHEATLRCWALSFYPAEITLTWQRDGEDQTQDTELVETRPA GDGTFQKWAAVVVPSGQEQRYTCHVQHEGLPKPLTLRWEP (SEQ ID NO:44).

[0204] In some cases, HLA class I heavy chain peptides contain the following amino acid sequence: GSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDGETRKVKAHSQTHRVDLGTLRGYYNQSEAGSHTVQRMYGCDVGSDWRFLRGYHQYAYDGKDYIALKEDLRSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGTCVEWLRRYLENGKETLQRTDAPKTHMTHHAVSDHEATLRCWALSFYPAEITLTWQRDGEDQTQDTELVETRP C GDGTFQKWAAVVVPSGQEQRYTCHVQHEGLPKPLTLRWEP (SEQ ID NO:312).

[0205] In some cases, HLA class I heavy chain peptides contain the following amino acid sequence: GSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDGETRKVKAHSQTHRVDLGTLRG A YNQSEAGSHTVQRMYGCDVGSDWRFLRGYHQYAYDGKDYIALKEDLRSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGTCVEWLRRYLENGKETLQRTDAPKTHMTHHAVSDHEATLRCWALSFYPAEITLTWQRDGEDQTQDTELVETRP C GDGTFQKWAAVVVPSGQEQRYTCHVQHEGLPKPLTLRWE (SEQ ID NO:46).

[0206] In some cases, β2M peptides contain the following amino acid sequence:

[0207] IQRTPKIQVY S CHPAENGKS NFLNCYVSGF HPSDIEVDLLKNGERIEKVE HSDLSFSKDWSFYLLYYTEF TPTEKDEYAC RVNHVTLSQP KIVKWDRDM (SEQ ID NO:311); and the HLA class I heavy chain polypeptide of TMMP contains the following amino acid sequence:

[0208] GSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDGETRKVKAHSQTHRVDLGTLRGYYNQSEAGSHTVQRMYGCDVGSDWRFLRGYHQYA YDGKDYIALKEDLRSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGTCVEWLRRYLENGKETLQRTDAPKTHMTHHAVSDHEATLRCWALSFYPAEITLTWQRDGEDQTQDTELVETRP C GDGTFQKWAAVVVPSGQEQRYTCHVQHEGLPKPLTLRWEP (SEQ ID NO:312), wherein the underlined and bolded Cys residues form disulfide bonds with each other in TMMP.

[0209] In some cases, β2M peptides contain the following amino acid sequence:

[0210] IQRTPKIQVY S C HPAENGKS NFLNCYVSGF HPSDIEVDLLKNGERIEKVE HSDLSFSKDWSFYLLYYTEF TPTEKDEYAC RVNHVTLSQP KIVKWDRDM (SEQ ID NO:311); and the HLA class I heavy chain polypeptide of TMMP contains the following amino acid sequence:

[0211] GSHSMRYFSTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDEETGKVKAHSQTDRENLRIALR YYNQSEAGSHTLQMMFGCDVGSDGRFLRGYHQYAYDGKDYIALKEDLRSWTAADMAAQITKRKWEAAHVAEQQRAYLEGTCVDGLRRYLENGKETLQRTDPPKTHMTHHPISDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRP C GDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLRWE (SEQ ID NO:457), wherein the Cys residue at amino acid 236 in the HLA class I heavy chain polypeptide and the Cys residue at residue 12 in the β2M polypeptide form a disulfide bond with each other in TMMP.

[0212] In some cases, the β2M polypeptide contains the following amino acid sequence: IQRTPKIQVYS C HPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDM (SEQ ID NO: 311).

[0213] In some cases, the first and second polypeptides of TMMP are linked to each other via disulfide bonds by: i) Cys residues present in the linker connecting the peptide epitope in the first polypeptide chain to the β2M polypeptide; and ii) Cys residues present in the class I MHC heavy chain of the second polypeptide chain. In some cases, the Cys residues present in the class I MHC heavy chain are Cys introduced as a Y84C substitution. In some cases, the linker connecting the peptide epitope in the first polypeptide chain to the β2M polypeptide is GCGGS(G4S)n (SEQ ID NO:315), where n is 1, 2, 3, 4, 5, 6, 7, 8, or 9. For example, in some cases, the linker comprises the amino acid sequence GCGGSGGGGSGGGGSGGGGS (SEQ ID NO:316). As 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 schematically depicted in... Figures 2A-2F middle.

[0214] TMMP with multiple disulfide bonds

[0215] In some cases, the first polypeptide and the second polypeptide of the TMMP disclosed herein are linked to each other by at least two disulfide bonds (i.e., two interchain disulfide bonds). Examples of such multi-disulfide-bonded TMMPs are schematically depicted in... Figure 17A and17B and Figures 18A-18C In addition, when TMMP contains IgFc peptides, the heterodimeric TMMP can dimerize, allowing disulfide bonds to link the IgFc peptides in the two heterodimeric TMMPs. This arrangement is schematically depicted in [illustration missing]. Figure 17C and 17D In this section, disulfide bonds are indicated by dashed lines. Unless otherwise stated, the at least two disulfide bonds mentioned in the multi-disulfide-linked TMMPP in this section do not refer to the disulfide bonds linking the IgFc polypeptide in the dimerized TMMPP.

[0216] As described above, in some cases, the first and second polypeptides of TMMP are linked together by at least two disulfide bonds (i.e., two interchain disulfide bonds). For example, in some cases, the first and second polypeptides of TMMP are linked together by two interchain disulfide bonds. As another example, in some cases, the first and second polypeptides of TMMP are linked together by three interchain disulfide bonds. As yet another example, in some cases, the first and second polypeptides of the TMMP disclosed herein are linked together by four interchain disulfide bonds.

[0217] In some cases, when a peptide epitope in the first polypeptide of TMMP is linked to a β2M polypeptide via a linker containing Cys, at least one of at least two disulfide bonds connects the Cys in the linker to the Cys present in the class I MHC heavy chain of the second polypeptide.

[0218] In some cases, multi-disulfide-bonded TMMPs (e.g., didisulfide-bonded TMMPs) exhibit increased stability compared to control TMMPs containing only one of the at least two disulfide bonds. in vitro Stability. For example, in some cases, multi-disulfide-linked TMMPs (e.g., di-disulfide-linked TMMPs) exhibit at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 50%, at least 2 times, at least 5 times, or at least 10 times greater in vitro stability compared to control TMMPs that include only one of the at least two disulfide bonds.

[0219] Does the polydisulfide-linked TMMP (e.g., the didisulfide-linked TMMP) exhibit an increase compared to a control TMMP containing only one of the at least two disulfide bonds? in vitroStability can be determined by measuring the amount of disulfide-linked heterodimer TMMP present in the sample over time and / or under specified conditions and / or during TMMP purification.

[0220] For example, in some cases, when TMMPs are stored at 37°C for a period of time (e.g., about 1 week to about 2 weeks, about 2 weeks to about 4 weeks, or about 4 weeks to about 2 months), multi-disulfide-bonded TMMPs (e.g., didisulfide-bonded TMMPs) exhibit at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 50%, at least 2 times, at least 5 times, or at least 10 times greater than control TMMPs containing only one of the at least two disulfide bonds. in vitro Stability. For example, in some cases, at 37°C... in vitro The amount of disulfide-linked heterodimer TMMP remaining after storing multi-disulfide-linked TMMPs (e.g., di-disulfide-linked TMMPs) for 28 days was compared with that stored at 37°C. in vitro The amount of disulfide-linked heterodimer TMMP remaining after storing the control TMMP (including TMMP containing only one of the two disulfide bonds in a multi-disulfide-linked TMMP) 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 times, at least 5 times, or at least 10 times more.

[0221] As an example, it includes, Figure 14A and 14B The TMMPs depicted, with disulfide bonds linking peptides 1715 and 2380, exhibit a larger size compared to TMMPs containing peptides 2405 and 2380. in vitro Stability, in which peptide 2405 is described as Figure 14D In this context, such TMMPs contain only a single disulfide bond, wherein the single disulfide bond is formed between: i) Cys at the G2C junction between the epitope and β2M; and ii) Cys provided by substitution of Y84C in the heavy chain of type I MHC. As another example, TMMPs containing... Figure 14A and 14B The TMMPs depicted with disulfide bonds linking peptides 1715 and 2380 exhibit a larger size compared to TMMPs containing peptides 1380 and 2380. in vitro Stability, wherein peptide 1380 is described in Figure 14E In this context, such TMMPs contain only one disulfide bond, wherein the single disulfide bond is formed between: i) Cys provided by R12C substitution in the β2M polypeptide; and ii) Cys provided by A236C substitution in the class I MHC heavy chain.

[0222] In some cases, multi-disulfide bonded TMMPs exhibit increased [potential] compared to control TMMPs containing only one of the at least two disulfide bonds. in vivo Stability. For example, in some cases, multi-disulfide-bonded TMMPs exhibit at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 50%, at least 2 times, at least 5 times, or at least 10 times greater stability compared to control TMMPs containing only one of the at least two disulfide bonds. in vivo stability.

[0223] In some cases, the presence of two disulfide bonds in a polydisulfide-linked TMMP (e.g., a didisulfide-linked TMMP) results in an increased amount of disulfide-linked heterodimer TMMP compared to the amount of disulfide-linked heterodimer TMMP produced when the TMMP is a control TMMP containing only one of the at least two disulfide bonds. For example, polydisulfide-linked TMMPs (e.g., didisulfide-linked TMMPs) can... in vitro TMMP is produced in cultured mammalian cells, wherein the mammalian cells are cultured in a liquid cell culture medium. TMMP can be secreted into the cell culture medium. The cells can be lysed, generating cell lysates, and TMMP can be present in the cell lysates. TMMP can be purified from the cell culture medium and / or cell lysates. For example, when the TMMP contains an IgG1 Fc peptide, the cell culture medium and / or cell lysates can be contacted with immobilized protein A (e.g., the cell culture medium and / or cell lysates can be applied to a protein A column, wherein protein A is immobilized on beads). TMMP present in the cell culture medium and / or cell lysates binds to the immobilized protein A. After washing the column to remove unbound material, the bound TMMP is eluted, thereby generating a protein A eluate. The amount of disulfide-linked heterodimeric TMMP present in the protein A eluent 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% greater than the amount of disulfide-linked heterodimeric TMMP present in the protein A eluent when the TMMP is a control TMMP that includes only one of the at least two disulfide bonds present in a multi-disulfide-linked TMMP (e.g., a disulfide-linked TMMP). In some cases, the percentage of total TMMP protein in the eluent that is non-aggregated disulfide-linked heterodimeric TMMP is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%. The protein A eluent may undergo size exclusion chromatography (SEC) and / or one or more other additional purification steps.

[0224] In some cases, TMMP comprises at least one heterodimer comprising: a) a first polypeptide comprising: i) a WT1 peptide epitope, wherein the WT1 peptide is at least 4 amino acids long (e.g., 4 to 25 amino acids; for example, the WT1 peptide has a length of 4, 5, 6, 7, 8, 9, 10-15, 15-20, or 20-25 amino acids); and ii) a first MHC polypeptide; b) a second polypeptide comprising a second MHC polypeptide; and c) at least one MOD, wherein the first polypeptide and / or the second polypeptide comprises a MOD, and wherein the heterodimer comprises two disulfide bonds between the first polypeptide and the second polypeptide (i.e., the heterodimer comprises: i) a first disulfide bond connecting the first polypeptide and the second polypeptide; and ii) a second disulfide bond connecting the first polypeptide and the second polypeptide). In other words, the first Cys residue in the first polypeptide forms a disulfide bond (first disulfide bond) with the first Cys residue in the second polypeptide; and the second Cys residue in the first polypeptide forms a disulfide bond (second disulfide bond) with the second Cys residue in the second polypeptide.

[0225] In some cases, a 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 a class I MHC heavy chain polypeptide, wherein one or both of the first polypeptide and the second polypeptide comprise at least one MOD, wherein the TMMP comprises: a) a first disulfide bond between i) a Cys present in the linker between the peptide epitope and the β2M polypeptide; and ii) a first Cys introduced into the class I MHC heavy chain polypeptide; and b) at least one second disulfide bond between the first polypeptide and the second polypeptide, wherein the at least one second disulfide bond is between i) a Cys at the C-terminus of a Cys present in the linker in the first polypeptide; and ii) a Cys at the C-terminus of a first Cys introduced into the class I MHC heavy chain polypeptide in the second polypeptide.

[0226] In some cases, the first disulfide bond forming Cys residue and the second disulfide bond forming Cys residue in the first or second polypeptide of TMMP are spaced about 10 to about 200 amino acids apart. For example, in some cases, the first disulfide bond forming Cys residue and the second disulfide bond forming Cys residue in the first or second polypeptide of TMMP are spaced about 10 amino acids (aa) apart, ranging from 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, about 70 aa to about 80 aa, about 80 aa to about 90 aa, about 90 aa to about 100 aa, about 100 aa to about 110 aa, about 110 aa to about 120 aa, about 120 aa to about 130 aa, about 130 aa to about 140 aa, about 140 aa to about 150 aa, about 150 aa to about 160 aa, about 160 aa to about 160 aa. aa to about 170 aa, about 170 aa to about 180 aa, about 180 aa to about 190 aa, or about 190 aa to about 200 aa.

[0227] As an example, in some cases, the first disulfide bond forming Cys residue and the second disulfide bond forming Cys residue in the first polypeptide of TMMP are spaced apart 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 at 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) at the C-terminus of the first disulfide bond forming Cys residue in the first polypeptide. In some cases, the second disulfide bond in the first polypeptide forms at a position 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 aa at the C-terminus of the Cys residue. In some cases, the second disulfide bond in the first polypeptide forms at a position 15 aa at the C-terminus of the Cys residue. In some cases, the second disulfide bond in the first polypeptide forms at a position 20 aa at the C-terminus of the Cys residue. In some cases, the second disulfide bond in the first polypeptide forms at a position 25 aa at the C-terminus of the Cys residue.

[0228] In some cases, the first disulfide bond-forming Cys residue and the second disulfide bond-forming Cys residue in the second polypeptide of the TMMP disclosed herein are spaced about 140 to about 160 amino acids apart. For example, in some cases, the second disulfide bond-forming Cys residue in the second polypeptide is located about 140 to about 160 amino acids from the C-terminus of the first disulfide bond-forming Cys residue in the second polypeptide. In some cases, the second disulfide bond in the second polypeptide forms a Cys residue at the C-terminus of the Cys residue, specifically at amino acids 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, or 160.

[0229] A polydisulfide-linked TMMP (e.g., a didisulfide-linked TMMP) may comprise: a) a first polypeptide comprising: i) a WT1 peptide (e.g., a WT1 peptide having 4 to about 25 amino acids); and ii) a first MHC polypeptide comprising a peptide linker between the WT1 peptide and the first MHC polypeptide, wherein the peptide linker comprises a Cys residue, and wherein the first MHC polypeptide is a β2M polypeptide comprising an amino acid substitution introducing a Cys residue; b) and a second polypeptide comprising a second MHC polypeptide, wherein the second MHC polypeptide is a class I heavy chain comprising HLA-A-based... 0201 ( Figure 9A The first polypeptide and / or the second polypeptide contain the following: (i) amino acid number (as depicted in the diagram) or the Y84C substitution and A236C substitution at the corresponding position in another type I heavy chain allele, wherein the TMMP comprises a disulfide bond between a Cys residue in the peptide linker and an amino acid position 84 of the type I heavy chain or a Cys residue at the corresponding position in another type I heavy chain allele; and (ii) at least one MOD, wherein the first polypeptide and / or the second polypeptide comprises the at least one MOD. Examples are schematically depicted in the diagram. Figure 17A and Figure 17B middle.

[0230] In some cases, the peptide linker comprises the amino acid sequence GCGGS (SEQ ID NO:318). In some cases, the peptide linker comprises the amino acid sequence GCGGS(GGGGS)n (SEQ ID NO:319), where n is an integer from 1 to 10. In some cases, the peptide linker comprises the amino acid sequence GCGGS(GGGGS)n (SEQ ID NO:398), where n is 1. In some cases, the peptide linker comprises the amino acid sequence GCGGS(GGGGS)n (SEQ ID NO:320), where n is 2. In some cases, the peptide linker comprises the amino acid sequence GCGGS(GGGGS)n (SEQ ID NO:321), where n is 3. In some cases, the peptide linker comprises the amino acid sequence GCGGS(GGGGS)n (SEQ ID NO:322), where n is 4. In some cases, the peptide linker comprises the amino acid sequence GCGGS(GGGGS)n (SEQ ID NO:323), where n is 5. In some cases, the peptide linker comprises the amino acid sequence GCGGS(GGGGS)n (SEQ ID NO: 324), where n is 6. In some cases, the peptide linker comprises the amino acid sequence GCGGS(GGGGS)n (SEQ ID NO: 325), where n is 7. In some cases, the peptide linker comprises the amino acid sequence GCGGS(GGGGS)n (SEQ ID NO: 326), where n is 8. In some cases, the peptide linker comprises the amino acid sequence GCGGS(GGGGS)n (SEQ ID NO: 327), where n is 9. In some cases, the peptide linker comprises the amino acid sequence GCGGS(GGGGS)n (SEQ ID NO: 328), where n is 10.

[0231] In some cases, the peptide linker comprises the amino acid sequence CGGGS (SEQ ID NO: 329). In some cases, the peptide linker comprises the amino acid sequence CGGGS(GGGGS)n (SEQ ID NO: 330), where n is an integer from 1 to 10. In some cases, the peptide linker comprises the amino acid sequence CGGGS(GGGGS)n (SEQ ID NO: 331), where n is 1. In some cases, the peptide linker comprises the amino acid sequence CGGGS(GGGGS)n (SEQ ID NO: 332), where n is 2. In some cases, the peptide linker comprises the amino acid sequence CGGGS(GGGGS)n (SEQ ID NO: 333), where n is 3. In some cases, the peptide linker comprises the amino acid sequence CGGGS(GGGGS)n (SEQ ID NO: 334), where n is 4. In some cases, the peptide linker comprises the amino acid sequence CGGGS(GGGGS)n (SEQ ID NO: 335), where n is 5. In some cases, the peptide linker comprises the amino acid sequence CGGGS(GGGGS)n (SEQ ID NO:336), where n is 6. In some cases, the peptide linker comprises the amino acid sequence CGGGS(GGGGS)n (SEQ ID NO:337), where n is 7. In some cases, the peptide linker comprises the amino acid sequence CGGGS(GGGGS)n (SEQ ID NO:338), where n is 8. In some cases, the peptide linker comprises the amino acid sequence CGGGS(GGGGS)n (SEQ ID NO:339), where n is 9. In some cases, the peptide linker comprises the amino acid sequence CGGGS(GGGGS)n (SEQ ID NO:340), where n is 10.

[0232] The following includes HLA-A based 0201 ( Figure 9A Non-restrictive examples of class I MHC heavy chains with amino acid numbers (as depicted in the text) or with Y84C and A236C substitutions at the corresponding positions in another class I heavy chain allele.

[0233] HLA-A

[0234] In some cases, a multi-disulfide-linked TMMP (e.g., a didisulfide-linked TMMP) comprises: a) a first polypeptide comprising: i) a WT1 peptide (e.g., a WT1 peptide having 4 to about 25 amino acids); and ii) a first MHC polypeptide comprising a peptide linker between the WT1 peptide and the first MHC polypeptide, wherein the peptide linker comprises a Cys residue, and wherein the first MHC polypeptide is a β2M polypeptide comprising an amino acid substitution introducing a Cys residue; and b) a second polypeptide comprising HLA-A. Class I MHC heavy chains, wherein the heavy chain comprises 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 with the following amino acid sequence: GSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDGETRKVKAHSQTHRVDLGTLRGCYNQSEAGSHTVQRMYGCDVGSDWRFLRGYHQYAYDGKDYIALKEDLRSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGTCVEWLRRYLENGKETLQRTDAPKTHMTHHAVSDHEATLRCWALSFYPAEITLTWQRDGEDQTQDTELVETRPCGDGTFQKWAAVVVPSGQEQRYTCHVQHEGLPKPLTLRWEP (SEQ ID) (SEQ ID NO:342), wherein amino acid 84 is Cys and amino acid 236 is Cys; and c) at least one MOD, wherein the first polypeptide and / or the second polypeptide comprises the at least one MOD. In some cases, the peptide linker comprises the amino acid sequence GCGGS (SEQ ID NO:318). In some cases, the peptide linker comprises the amino acid sequence GCGGS(GGGGS)n (SEQ ID NO:319), wherein n is an integer from 1 to 10. In some cases, the β2M polypeptide comprises an R12C substitution. For example, the β2M polypeptide may comprise an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity with the following amino acid sequence: IQRTPKIQVYSCHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDM (SEQ ID NO:311), wherein amino acid 12 is Cys.The at least one MOD may be a polypeptide that exerts an activating / stimulating effect on target T cells or an inhibitory / suppressive effect on target T cells. For example, the at least one MOD may be a cytokine (e.g., IL2 peptide, IL7 peptide, IL12 peptide, IL15 peptide, IL17 peptide, IL21 peptide, IL27 peptide, IL-23 peptide, TGFβ peptide, etc.; and includes all family members, such as IL17A, IL-17B, IL-17C, IL-17D, IL-17E, IL-17F, IL-17E), 4-1BBL peptide, ICOS-L peptide, OX-40L peptide, CD80 peptide, CD86 peptide (CD80 and CD86 are also referred to as B7-1 and B7-2, respectively), CD40 peptide, CD70 peptide, JAG1 (CD339) peptide, ICAM (CD540 peptide, PD-L1 peptide, FasL peptide, PD-L2 peptide, PD-1H (VISTA) peptide, ICOS-L) peptide, etc. (CD275) peptide, GITRL peptide, HVEM peptide, CXCL10 peptide, CXCL9 peptide, CXCL11 peptide, CXCL13 peptide, and CX3CL1 peptide, galactoglobulin-9 peptide, CD83 peptide, CD30L peptide, HLA-G peptide, MICA peptide, MICB peptide, HVEM (CD270) peptide, lymphotoxin β receptor peptide, 3 / TR6 peptide, ILT3 peptide, ILT4 peptide, CXCL10 peptide, CXCL9 peptide, CXCL11 peptide, CXCL13 peptide, or CX3CL1 peptide. These MODs can be wild-type peptides or variants of wild-type peptides. In some cases, MODs are activating (“stimulatory”) immunomodulatory peptides; for example, MODs can produce an activating / stimulatory effect on T cells. Examples of activating MODs include, for example, CD80, CD86, 4-1BBL, OX40L, CD70, ICOS-L, CD40, and ICAM. (CD54), IL2, IL7, IL12, IL15, IL17, IL21, IL27, IL23, GITRL, TGFβ, and lymphotoxin β receptor. In some cases, MODs are inhibitory (“inhibitory”) MODs; for example, MODs can produce inhibitory / inhibitory effects on T cells. Examples of inhibitory MODs include, for example, PD-1H, PD-L1, PD-L2, TGFβ, FasL, HVEM, galactolectin-9, ILT3, and ILT4. Depending on the situation, TGFβ peptides can produce activating / stimulatory or inhibitory / inhibitory effects.

[0235] In some cases, a multi-disulfide-linked TMMP (e.g., a didisulfide-linked TMMP) comprises: a) a first polypeptide comprising: i) a WT1 peptide (e.g., a WT1 peptide having 4 to about 25 amino acids); and ii) a first MHC polypeptide comprising a peptide linker between the WT1 peptide and the first MHC polypeptide, wherein the peptide linker comprises a Cys residue, and wherein the first MHC polypeptide is a β2M polypeptide comprising an amino acid substitution introducing a Cys residue; and b) a second polypeptide comprising an HLA-A I class MHC 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 with the following amino acid sequence: GSHSMRYFSTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDEETGKVKAHSQTDRENLRIALR C YNQSEAGSHTLQMMFGCDVGSDGRFLRGYHQYAYDGKDYIALKEDLRSWTAADMAAQITKRKWEAAHVAEQQRAYLEGTCVDGLRRYLENGKETLQRTDPPKTHMTHHPISDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRP CGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLRWE (SEQ ID NO:346), wherein amino acid 84 is Cys and amino acid 236 is Cys; and c) at least one MOD, wherein the first polypeptide and / or the second polypeptide comprises the at least one immunomodulatory polypeptide. In some cases, the peptide linker comprises the amino acid sequence GCGGS (SEQ ID NO:318). In some cases, the peptide linker comprises the amino acid sequence GCGGS(GGGGS)n (SEQ ID NO:319), wherein n is an integer from 1 to 10. In some cases, the β2M polypeptide comprises an R12C substitution. For example, the β2M polypeptide may comprise an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity with the following amino acid sequence: IQRTPKIQVYSCHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDM (SEQ ID NO:311), wherein amino acid 12 is Cys. The at least one MOD may be a polypeptide that exerts an activating / stimulatory effect on target T cells or an inhibitory / suppressive effect on target T cells.For example, the at least one MOD may be a cytokine (e.g., IL2 peptide, IL7 peptide, IL12 peptide, IL15 peptide, IL17 peptide, IL21 peptide, IL27 peptide, IL-23 peptide, TGFβ peptide, etc.; and includes all family members, such as IL17A, IL-17B, IL-17C, IL-17D, IL-17E, IL-17F, IL-17E), 4-1BBL peptide, ICOS-L peptide, OX-40L peptide, CD80 peptide, CD86 peptide (CD80 and CD86 are also referred to as B7-1 and B7-2, respectively), CD40 peptide, CD70 peptide, JAG1 (CD339) peptide, ICAM (CD540 peptide, PD-L1 peptide, FasL peptide, PD-L2 peptide, PD-1H (VISTA) peptide, ICOS-L) peptide, etc. (CD275) peptide, GITRL peptide, HVEM peptide, CXCL10 peptide, CXCL9 peptide, CXCL11 peptide, CXCL13 peptide, and CX3CL1 peptide, galactoglobulin-9 peptide, CD83 peptide, CD30L peptide, HLA-G peptide, MICA peptide, MICB peptide, HVEM (CD270) peptide, lymphotoxin β receptor peptide, 3 / TR6 peptide, ILT3 peptide, ILT4 peptide, CXCL10 peptide, CXCL9 peptide, CXCL11 peptide, CXCL13 peptide, and CX3CL1 peptide. These MODs can be wild-type peptides or variants of wild-type peptides. Among them, the following MODs can produce activating / stimulatory effects: CD80, CD86, 4-1BBL, OX40L, CD70, ICOS-L, CD40, and 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 inhibitory / inhibitory effects: PD-1H, PD-L1, PD-L2, TGFβ, FasL, HVEM, galactolectin-9, ILT3, and ILT4. Depending on the case, the TGFβ peptide can produce activating / stimulatory or inhibitory / inhibitory effects. In some cases, at least one of the MODs is a variant with reduced affinity, as described elsewhere herein. In some cases, the first or second peptide comprises an Ig Fc peptide.

[0236] In some cases, the at least one MOD is an affinity-reduced variant, as described elsewhere herein. In some cases, the first or second polypeptide comprises an Ig Fc polypeptide.

[0237] In some cases, polydisulfide-linked TMMPs (e.g., didisulfide-linked TMMPs) contain HLA-A class I heavy chain peptides. In some cases, the HLA-A heavy chain peptides present in the polydisulfide-linked TMMPs (e.g., didisulfide-linked TMMPs) of this disclosure contain HLA-A class I heavy chain peptides. Figure 9A HLA-A as depicted in 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 The 3401 amino acid sequence has an amino acid sequence with at least 95%, at least 98%, or at least 99% amino acid sequence identity, wherein the HLA-A heavy chain polypeptide contains Y84C and A236C substitutions.

[0238] HLA-A 0101 (Y84C; A236C)

[0239] In some cases, HLA-A heavy chain peptides present in multi-disulfide-linked TMMPs (e.g., di-disulfide-linked TMMPs) contain HLA-A... The 0101 (Y84C; A236C) amino acid sequence has at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity:

[0240] GSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQKMEPRAPWIEQEGPEYWDQETRNMKAHSQTDRANLGTLRGCYNQSEDGSHTIQIMYGCDVGPDGRFLRGYRQDAYDGKDYIALNEDLRSWTAAD MAAQITKRKWEAVHAAEQRRVYLEGRCVDGLRRYLENGKETLQRTDPPKTHMTHHPISDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRPCGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLRWE (SEQ ID NO:343), wherein amino acid 84 is Cys and amino acid 236 is Cys.

[0241] HLA-A 0201 (Y84C; A236C)

[0242] In some cases, HLA-A heavy chain peptides present in multi-disulfide-linked TMMPs (e.g., di-disulfide-linked TMMPs) contain HLA-A... 0201 (Y84C; A236C) has an amino acid sequence with at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity: GSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDGETRKVKAHSQTHRVDLGTLRGCYNQSEAGSHTVQRMYGCDVGSDWRFLRGYHQYAYDGKDYIALKEDLRSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGTCVEWLRRYLENGKETLQRTDAPKTHMTHHAVSDHEATLRCWALSFYPAEITLTWQRDGEDQTQDTELVETRPCGDGTFQKWAAVVVPSGQEQRYTCHVQHEGLPKPLTLRWEP (SEQ ID NO:342), wherein amino acid 84 is Cys and amino acid 236 is Cys.

[0243] HLA-A 0202 (Y84C; A236C)

[0244] In some cases, HLA-A heavy chain peptides present in multi-disulfide-linked TMMPs (e.g., di-disulfide-linked TMMPs) contain HLA-A... 0202 (Y84C; A236C) has an amino acid sequence with at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity: GSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDGETRKVKAHSQTHRVDLGTLRGCYNQSEAGSHTVQRMYGCDVGSDWRFLRGYHQYAYDGKDYIALKEDLRSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGTCVEWLRRYLENGKETLQRTDAPKTHMTHHAVSDHEATLRCWALSFYPAEITLTWQRDGEDQTQDTELVETRPCGDGTFQKWAAVVVPSGQEQRYTCHVQHEGLPKPLTLRWE (SEQ ID NO:341), wherein amino acid 84 is Cys and amino acid 236 is Cys.

[0245] HLA-A 1101 (Y84C; A236C)

[0246] In some cases, HLA-A heavy chain peptides present in multi-disulfide-linked TMMPs (e.g., di-disulfide-linked TMMPs) contain HLA-A... The amino acid sequence 1101 (Y84C; A236C) has at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity: GSHSMRYFYTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDQETRNVKAQSQTDRVDLGTLRGCYNQSEDGSHTIQIMYGCDVGPDGRFLRGYRQDAYDGKDYIALNEDLRSWTAADMAAQITKRKWEAAHAAEQQRAYLEGRCVEWLRRYLENGKETLQRTDPPKTHMTHHPISDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRPCGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLRWE (SEQ ID NO:344), wherein amino acid 84 is Cys and amino acid 236 is Cys.

[0247] HLA-A 2301 (Y84C; A236C)

[0248] In some cases, HLA-A heavy chain peptides present in multi-disulfide-linked TMMPs (e.g., di-disulfide-linked TMMPs) contain HLA-A... The amino acid sequence 2301 (Y84C; A236C) has at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity: GSHSMRYFSTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDEETGKVKAHSQTDRENLRIALRCYNQSEAGSHTLQMMFGCDVGSDGRFLRGYHQYAYDGKDYIALKEDLRSWTAADMAAQITQRKWEAARVAEQLRAYLEGTCVDGLRRYLENGKETLQRTDPPKTHMTHHPISDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRPCGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLRWE (SEQ ID NO:345), wherein amino acid 84 is Cys and amino acid 236 is Cys.

[0249] HLA-A 2402 (Y84C; A236C)

[0250] In some cases, HLA-A heavy chain peptides present in multi-disulfide-linked TMMPs (e.g., di-disulfide-linked TMMPs) contain HLA-A... The amino acid sequence 2402 (Y84C; A236C) has at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity: GSHSMRYFSTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDEETGKVKAHSQTDRENLRIALRCYNQSEAGSHTLQMMFGCDVGSDGRFLRGYHQYAYDGKDYIALKEDLRSWTAADMAAQITKRKWEAAHVAEQQRAYLEGTCVDGLRRYLENGKETLQRTDPPKTHMTHHPISDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRPCGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLRWE (SEQ ID NO:346), wherein amino acid 84 is Cys and amino acid 236 is Cys.

[0251] HLA-A 2407 (Y84C; A236C)

[0252] In some cases, HLA-A heavy chain peptides present in multi-disulfide-linked TMMPs (e.g., di-disulfide-linked TMMPs) contain HLA-A... The amino acid sequence 2407 (Y84C; A236C) has at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity: GSHSMRYFSTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDEETGKVKAQSQTDRENLRIALRCYNQSEAGSHTLQMMFGCDVGSDGRFLRGYHQYAYDGKDYIALKEDLRSWTAADMAAQITKRKWEAAHVAEQQRAYLEGTCVDGLRRYLENGKETLQRTDPPKTHMTHHPISDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRPCGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLRWE (SEQ ID NO:347), wherein amino acid 84 is Cys and amino acid 236 is Cys.

[0253] HLA-A 3303 (Y84C; A236C)

[0254] In some cases, HLA-A heavy chain peptides present in multi-disulfide-linked TMMPs (e.g., di-disulfide-linked TMMPs) contain HLA-A... The amino acid sequence 3303 (Y84C; A236C) has at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity:

[0255] GSHSMRYFTTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDRNTRNVKAHSQIDRVDLGTLRGCYNQSEAGSHTIQMMYGCDVGSDGRFLRGYQQDAYDGKDYIALNEDLRSWTAAD MAAQITQRKWEAARVAEQLRAYLEGTCVEWLRRYLENGKETLQRTDPPKTHMTHHAVSDHEATLRCWALSFYPAEITLTWQRDGEDQTQDTELVETRPCGDGTFQKWASVVVPSGQEQRYTCHVQHEGLPKPLTLRWE (SEQ ID NO:348), wherein amino acid 84 is Cys and amino acid 236 is Cys.

[0256] HLA-A 3401 (Y84C; A236C)

[0257] In some cases, HLA-A heavy chain peptides present in multi-disulfide-linked TMMPs (e.g., di-disulfide-linked TMMPs) contain HLA-A... The amino acid sequence 3401 (Y84C; A236C) has at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity:

[0258] GSHSMRYFYTSVSRPGRGEPFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDRNTRKVKAQSQTDRVDLGTLRGCYNQSEDGSHTIQRMYGCDVGPDGRFLRGYQQDAYDGKDYIALNEDLRSWTAAD MAAQITQRKWETAHEAEQWRAYLEGTCVEWLRRYLENGKETLQRTDAPKTHMTHHAVSDHEATLRCWALSFYPAEITLTWQRDGEDQTQDTELVETRPCGDGTFQKWASVVVPSGQEQRYTCHVQHEGLPKPLTLRWE (SEQ ID NO:349), wherein amino acid 84 is Cys and amino acid 236 is Cys.

[0259] HLA-B

[0260] In some cases, a multi-disulfide-linked TMMP (e.g., a didisulfide-linked TMMP) comprises: a) a first polypeptide comprising: i) a WT1 peptide (e.g., a WT1 peptide having 4 to about 25 amino acids); and ii) a first MHC polypeptide comprising a peptide linker between the WT1 peptide and the first MHC polypeptide, wherein the peptide linker comprises a Cys residue, and wherein the first MHC polypeptide is a β2M polypeptide comprising an amino acid substitution introducing a Cys residue; and b) a second polypeptide comprising HLA-B. Class I MHC heavy chains, wherein the heavy chain comprises 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 with the following amino acid sequence: GSHSMRYFYTSVSRPGRGEPRFISVGYVDDTQFVRFDSDAASPREEPRAPWIEQEGPEYWDRNTQIYKAQAQTDRESLRNLRGCYNQSEAGSHTLQSMYGCDVGPDGRLLRGHDQYAYDGKDYIALNEDLRSWTAADTAAQITQRKWEAAREAEQRRAYLEGECVEWLRRYLENGKDKLERADPPKTHVTHHPISDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRPCGDRTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLRWEP (SEQ ID) (SEQ ID NO:350), wherein amino acid 84 is Cys and amino acid 236 is Cys; and c) at least one MOD, wherein the first polypeptide and / or the second polypeptide comprises the at least one MOD. In some cases, the peptide linker comprises the amino acid sequence GCGGS (SEQ ID NO:318). In some cases, the peptide linker comprises the amino acid sequence GCGGS(GGGGS)n (SEQ ID NO:319), wherein n is an integer from 1 to 10. In some cases, the β2M polypeptide comprises an R12C substitution. For example, the β2M polypeptide may comprise an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity with the following amino acid sequence: IQRTPKIQVYSCHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDM (SEQ ID NO:311), wherein amino acid 12 is Cys. The at least one MOD may be a polypeptide that exerts an activating / stimulating effect on target T cells or an inhibitory / suppressive effect on target T cells.For example, the at least one MOD may be a cytokine (e.g., IL2 peptide, IL7 peptide, IL12 peptide, IL15 peptide, IL17 peptide, IL21 peptide, IL27 peptide, IL-23 peptide, TGFβ peptide, etc.; and includes all family members, such as IL17A, IL-17B, IL-17C, IL-17D, IL-17E, IL-17F, IL-17E), 4-1BBL peptide, ICOS-L peptide, OX-40L peptide, CD80 peptide, CD86 peptide (CD80 and CD86 are also referred to as B7-1 and B7-2, respectively), CD40 peptide, CD70 peptide, JAG1 (CD339) peptide, ICAM (CD540 peptide, PD-L1 peptide, FasL peptide, PD-L2 peptide, PD-1H) peptide, PD-1H peptide, etc. (VISTA) peptide, ICOS-L (CD275) peptide, GITRL peptide, HVEM peptide, CXCL10 peptide, CXCL9 peptide, CXCL11 peptide, CXCL13 peptide, and CX3CL1 peptide, galactoglobulin-9 peptide, CD83 peptide, CD30L peptide, HLA-G peptide, MICA peptide, MICB peptide, HVEM (CD270) peptide, lymphotoxin β receptor peptide, 3 / TR6 peptide, ILT3 peptide, ILT4 peptide, CXCL10 peptide, CXCL9 peptide, CXCL11 peptide, CXCL13 peptide, or CX3CL1 peptide. These MODs can be wild-type peptides or variants of wild-type peptides. In some cases, MODs are activating (“stimulatory”) immunomodulatory peptides; for example, MODs can produce activating / stimulatory effects on T cells. Examples of activating MODs include, for example, CD80, CD86, 4-1BBL, OX40L, CD70, ICOS-L, CD40, and ICA. M(CD54), IL2, IL7, IL12, IL15, IL17, IL21, IL27, IL23, GITRL, TGFβ, and lymphotoxin β receptor. In some cases, immunomodulatory peptides are inhibitory (“inhibitory”) MODs; for example, MODs can produce inhibitory / inhibitory effects on T cells. Examples of inhibitory MODs include, for example, PD-1H, PD-L1, PD-L2, TGFβ, FasL, HVEM, galactolectin-9, ILT3, and ILT4. Depending on the situation, TGFβ peptides can produce activating / stimulatory or inhibitory / inhibitory effects.

[0261] In some cases, the at least one MOD is an affinity-reduced variant, as described elsewhere herein. In some cases, the first or second polypeptide comprises an Ig Fc polypeptide.

[0262] In some cases, multi-disulfide-linked TMMPs (e.g., didisulfide-linked TMMPs) contain HLA-B class I heavy chain peptides. In some cases, the HLA-B heavy chain peptides present in multi-disulfide-linked TMMPs (e.g., didisulfide-linked TMMPs) contain HLA-B class I heavy chain peptides. Figure 10A HLA-B as depicted in 0702, HLA-B 0801, HLA-B 1502, HLA-B 3802, HLA-B 4001, HLA-B 4601 or HLA-B The 5301 amino acid sequence has an amino acid sequence with at least 95%, at least 98%, or at least 99% amino acid sequence identity, wherein the HLA-B heavy chain polypeptide contains Y84C and A236C substitutions.

[0263] HLA-B 0702 (Y84C; A236C)

[0264] In some cases, HLA-B heavy chain peptides present in multi-disulfide-linked TMMPs (e.g., di-disulfide-linked TMMPs) contain HLA-B... The amino acid sequence 0702 (Y84C; A236C) has at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity:

[0265] GSHSMRYFYTSVSRPGRGEPRFISVGYVDDTQFVRFDSDAASPREEPRAPWIEQEGPEYWDRNTQIYKAQAQTDRESLRNLRGCYNQSEAGSHTLQSMYGCDVGPDGRLLRGHDQYAYDGKDYIALNEDLRSWTAAD TAAQITQRKWEAAREAEQRRAYLEGECVEWLRRYLENGKDKLERADPPKTHVTHHPISDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRPCGDRTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLRWE (SEQ ID NO:350), wherein amino acid 84 is Cys and amino acid 236 is Cys.

[0266] HLA-B 0801 (Y84C; A236C)

[0267] In some cases, HLA-B heavy chain peptides present in multi-disulfide-linked TMMPs (e.g., di-disulfide-linked TMMPs) contain HLA-B... The amino acid sequence 0801 (Y84C; A236C) has at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity:

[0268] GSHSMRYFDTAMSRPGRGEPRISVGYVDDTQFVRFDSDAASPREEPRAPWIEQEGPEYWDRNTQIFKTNTQTDRESLRNLRGCYNQSEAGSHTLQSMYGCDVGPDGRLLRGHNQYAYDGKDYIALNEDLRSWTAAD TAAQITQRKWEAARVAEQDRAYLEGTCVEWLRRYLENGKDTLERADPPKTHVTHHPISDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRPCGDRTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLRWE (SEQ ID NO:351), wherein amino acid 84 is Cys and amino acid 236 is Cys.

[0269] HLA-B 1502 (Y84C; A236C)

[0270] In some cases, HLA-B heavy chain peptides present in multi-disulfide-linked TMMPs (e.g., di-disulfide-linked TMMPs) contain HLA-B... The amino acid sequence 1502 (Y84C; A236C) has at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity:

[0271] GSHSMRYFYTAMSRPGRGEPRFIAVGYVDDTQFVRFDSDAASPRMAPRAPWIEQEGPEYWDRNTQISKTNTQTYRESLRNLRGCYNQSEAGSHIIQRMYGCDVGPDGRLLRGYDQSAYDGKDYIALNEDLSSWTAAD TAAQITQRKWEAAREAEQLRAYLEGLCVEWLRRYLENGKETLQRADPPKTHVTHHPISDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRPCGDRTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLRWE (SEQ ID NO:352), wherein amino acid 84 is Cys and amino acid 236 is Cys.

[0272] HLA-B 3802 (Y84C; A236C)

[0273] In some cases, HLA-B heavy chain peptides present in multi-disulfide-linked TMMPs (e.g., di-disulfide-linked TMMPs) contain HLA-B... The amino acid sequence 3802 (Y84C; A236C) has at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity:

[0274] GSHSMRYFYTSVSRPGRGEPRFISVGYVDDTQFVRFDSDAASPREEPRAPWIEQEGPEYWDRNTQICKTNTQTYRENLRTALRCYNQSEAGSHTLQRMYGCDVGPDGRLLRGHNQFAYDGKDYIALNEDLSSWTAAD TAAQITQRKWEAARVAEQLRTYLEGTCVEWLRRYLENGKETLQRADPPKTHVTHHPISDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRPCGDRTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLRWE (SEQ ID NO:353), wherein amino acid 84 is Cys and amino acid 236 is Cys.

[0275] HLA-B 4001 (Y84C; A2346C)

[0276] In some cases, HLA-B heavy chain peptides present in multi-disulfide-linked TMMPs (e.g., di-disulfide-linked TMMPs) contain HLA-B... The amino acid sequence 4001 (Y84C; A236C) has at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity:

[0277] GSHSMRYFHTAMSRPGRGEPRFITVGYVDDTLFVRFDSDATSPRKEPRAPWIEQEGPEYWDRETQISKTNTQTYRESLRNLRGCYNQSEAGSHTLQRMYGCDVGPDGRLLRGHNQYAYDGKDYIALNEDLRSWTAAD TAAQISQRKLEAARVAEQLRAYLEGECVEWLRRYLENGKDKLERADPPKTHVTHHPISDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRPCGDRTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLRWE (SEQ ID NO:354), wherein amino acid 84 is Cys and amino acid 236 is Cys.

[0278] HLA-B 4601 (Y84C; A236C)

[0279] In some cases, HLA-B heavy chain peptides present in multi-disulfide-linked TMMPs (e.g., di-disulfide-linked TMMPs) contain HLA-B... The amino acid sequence 4601 (Y84C; A236C) has at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity:

[0280] GSHSMRYFYTAMSRPGRGEPRFIAVGYVDDTQFVRFDSDAASPRMAPRAPWIEQEGPEYWDRETQKYKRQAQTDRVSLRNLRGCYNQSEAGSHTLQRMYGCDVGPDGRLLRGHDQSAYDGKDYIALNEDLSSWTAAD TAAQITQRKWEAAREAEQWRAYLEGLCVEWLRRYLENGKETLQRADPPKTHVTHHPISDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRPCGDRTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLRWE (SEQ ID NO:355), wherein amino acid 84 is Cys and amino acid 236 is Cys.

[0281] HLA-B 5301 (Y84C; A236C)

[0282] In some cases, HLA-B heavy chain peptides present in multi-disulfide-linked TMMPs (e.g., di-disulfide-linked TMMPs) contain HLA-B... The amino acid sequence 5301 (Y84C; A236C) has at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity:

[0283] GSHSMRYFYTAMSRPGRGEPRFIAVGYVDDTQFVRFDSDAASPRTEPRAPWIEQEGPEYWDRNTQIFKTNTQTYRENLRIALRCYNQSEAGSHIIQRMYGCDLGPDGRLLRGHDQSAYDGKDYIALNEDLSSWTAAD TAAQITQRKWEAARVAEQLRAYLEGLCVEWLRRYLENGKETLQRADPPKTHVTHHPVSDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRPCGDRTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLRWE (SEQ ID NO:356), wherein amino acid 84 is Cys and amino acid 236 is Cys.

[0284] HLA-C

[0285] In some cases, a multi-disulfide-linked TMMP (e.g., a didisulfide-linked TMMP) comprises: a) a first polypeptide comprising: i) a WT-1 peptide (e.g., a 1 WT-1 peptide having 4 to about 25 amino acids); and ii) a first MHC polypeptide comprising a peptide linker between the WT-1 peptide and the first MHC polypeptide, wherein the peptide linker comprises a Cys residue, and wherein the first MHC polypeptide is a β2M polypeptide comprising an amino acid substitution introducing a Cys residue; and b) a second polypeptide comprising HLA-C. Class I MHC heavy chains, wherein the heavy chain comprises 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 with the following amino acid sequence: CSHSMRYFDTAVSRPGRGEPRFISVGYVDDTQFVRFDSDAASPRGEPRAPWVEQEGPEYWDRETQNYKRQAQADRVSLRNLRGCYNQSEDGSHTLQRMYGCDLGPDGRLLRGYDQSAYDGKDYIALNEDLRSWTAADTAAQITQRKLEAARAAEQLRAYLEGTCVEWLRRYLENGKETLQRAEPPKTHVTHHPLSDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRPCGDGTFQKWAAVVVPSGQEQRYTCHMQHEGLQEPLTLSWEP (SEQ ID) (SEQ ID NO:357), wherein amino acid 84 is Cys and amino acid 236 is Cys; and c) at least one MOD, wherein the first polypeptide and / or the second polypeptide comprises the at least one MOD. In some cases, the peptide linker comprises the amino acid sequence GCGGS (SEQ ID NO:318). In some cases, the peptide linker comprises the amino acid sequence GCGGS(GGGGS)n (SEQ ID NO:319), wherein n is an integer from 1 to 10. In some cases, the β2M polypeptide comprises an R12C substitution. For example, the β2M polypeptide may comprise an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity with the following amino acid sequence: IQRTPKIQVYSCHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDM (SEQ ID NO:311), wherein amino acid 12 is Cys. The at least one MOD may be a polypeptide that exerts an activating / stimulating effect on target T cells or an inhibitory / suppressive effect on target T cells.For example, the at least one MOD may be a cytokine (e.g., IL2 peptide, IL7 peptide, IL12 peptide, IL15 peptide, IL17 peptide, IL21 peptide, IL27 peptide, IL-23 peptide, TGFβ peptide, etc.; and includes all family members, such as IL17A, IL-17B, IL-17C, IL-17D, IL-17E, IL-17F, IL-17E), 4-1BBL peptide, ICOS-L peptide, OX-40L peptide, CD80 peptide, CD86 peptide (CD80 and CD86 are also referred to as B7-1 and B7-2, respectively), CD40 peptide, CD70 peptide, JAG1 (CD339) peptide, ICAM (CD540 peptide, PD-L1 peptide, FasL peptide, PD-L2 peptide, PD-1H (VISTA) peptide, ICOS-L) peptide, etc. (CD275) peptide, GITRL peptide, HVEM peptide, CXCL10 peptide, CXCL9 peptide, CXCL11 peptide, CXCL13 peptide, and CX3CL1 peptide, galactoglobulin-9 peptide, CD83 peptide, CD30L peptide, HLA-G peptide, MICA peptide, MICB peptide, HVEM(CD270) peptide, lymphotoxin β receptor peptide, 3 / TR6 peptide, ILT3 peptide, ILT4 peptide, CXCL10 peptide, CXCL9 peptide, CXCL11 peptide, CXCL13 peptide, or CX3CL1 peptide. These MODs can be wild-type peptides or variants of wild-type peptides. In some cases, immunomodulatory peptides are activating (“stimulatory”) MODs; for example, MODs can activate T cells. / Stimulating effect. Examples of activated immunomodulatory peptides include, for example, 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 cases, MODs are inhibitory (“inhibitory”) MODs; for example, MODs can produce an inhibitory / inhibitory effect on T cells. Examples of inhibitory MODs include, for example, PD-1H, PD-L1, PD-L2, TGFβ, FasL, HVEM, galactolectin-9, ILT3, and ILT4. Depending on the situation, TGFβ peptides can produce an activating / stimulatory or inhibitory / inhibitory effect.

[0286] In some cases, the at least one MOD is an affinity-reduced variant, as described elsewhere herein. In some cases, the first or second polypeptide comprises an Ig Fc polypeptide.

[0287] In some cases, polydisulfide-linked TMMPs (e.g., didisulfide-linked TMMPs) contain HLA-C class I heavy chain peptides. In some cases, the HLA-C heavy chain peptides present in polydisulfide-linked TMMPs (e.g., didisulfide-linked TMMPs) contain HLA-C class I heavy chain peptides. Figure 11A HLA-C as depicted in 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 1502 amino acid sequence has an amino acid sequence identity of at least 95%, at least 98%, or at least 99%, wherein the HLA-C heavy chain polypeptide contains Y84C and A236C substitutions.

[0288] HLA-C 01:02 (Y84C; A236C)

[0289] In some cases, HLA-C heavy chain peptides present in multi-disulfide-linked TMMPs (e.g., di-disulfide-linked TMMPs) contain HLA-C... 01:02 (Y84C; A236C) amino acid sequences having at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity:

[0290] CSHSMKYFFTSVSRPGRGEPRISVGYVDDTQFVRFDSDAASPRGEPRAPWVEQEGPEYWDRETQKYKRQAQTDRVSLRNLRGCYNQSEAGSHTLQWMCGCDLGPDGRLLRGYDQYAYDGKDYIALNEDLRSWTAADT AAQITQRKWEAAREAEQRRAYLEGTCVEWLRRYLENGKETLQRAEHPKTHVTHHPVSDHEATLRCWALGFYPAEITLTWQWDGEDQTQDTELVETRPCGDGTFQKWAAVMVPSGEEQRYTCHVQHEGLPEPLTLRWEP (SEQ ID NO:358), wherein amino acid 84 is Cys and amino acid 236 is Cys.

[0291] HLA-C 0303 (Y84C; A236C)

[0292] In some cases, HLA-C heavy chain peptides present in multi-disulfide-linked TMMPs (e.g., di-disulfide-linked TMMPs) contain HLA-C... 03:03 (Y84C; A236C) amino acid sequences having at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity:

[0293] GSHSMRYFYTAVSRPGRGEPHFIAVGYVDDTQFVRFDSDAASPRGEPRAPWVEQEGPEYWDRETQKYKRQAQTDRVSLRNLRGCYNQSEARSHIIQRMYGCDVGPDGRLLRGYDQYAYDGKDYIALNEDLRSWTAADT AAQITQRKWEAAREAEQLRAYLEGLCVEWLRRYLKNGKETLQRAEHPKTHVTHHPVSDHEATLRCWALGFYPAEITLTWQWDGEDQTQDTELVETRPCGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLTLRWEP (SEQ ID NO:359), wherein amino acid 84 is Cys and amino acid 236 is Cys.

[0294] HLA-C 0304 (Y84C; A236C)

[0295] In some cases, HLA-C heavy chain peptides present in multi-disulfide-linked TMMPs (e.g., di-disulfide-linked TMMPs) contain HLA-C... 03:04 (Y84C; A236C) amino acid sequences having at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity:

[0296] GSHSMRYFYTAVSRPGRGEPHFIAVGYVDDTQFVRFDSDAASPRGEPRAPWVEQEGPEYWDRETQKYKRQAQTDRVSLRNLRGCYNQSEAGSHIIQRMYGCDVGPDGRLLRGYDQYAYDGKDYIALNEDLRSWTAADT AAQITQRKWEAAREAEQLRAYLEGLCVEWLRRYLKNGKETLQRAEHPKTHVTHHPVSDHEATLRCWALGFYPAEITLTWQWDGEDQTQDTELVETRPCGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLTLRWEP (SEQ ID NO:360), wherein amino acid 84 is Cys and amino acid 236 is Cys.

[0297] HLA-C 0401 (Y84C; A236C)

[0298] In some cases, HLA-C heavy chain peptides present in multi-disulfide-linked TMMPs (e.g., di-disulfide-linked TMMPs) contain HLA-C... 04:01 (Y84C; A236C) amino acid sequences having at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity:

[0299] GSHSMRYFSTSVSWPGRGEPRFIAVGYVDDTQFVRFDSDAASPRGEPREPWVEQEGPEYWDRETQKYKRQAQADRVNLRKLRGCYNQSEDGSHTLQRMFGCDLGPDGRLLRGYNQFAYDGKDYIALNEDLRSWTAADT AAQITQRKWEAAREAEQRRAYLEGTCVEWLRRYLENGKETLQRAEHPKTHVTHHPVSDHEATLRCWALGFYPAEITLTWQWDGEDQTQDTELVETRPCGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLTLRWKP (SEQ ID NO:361), wherein amino acid 84 is Cys and amino acid 236 is Cys.

[0300] HLA-C 0602 (Y84C; A236C)

[0301] In some cases, HLA-C heavy chain peptides present in multi-disulfide-linked TMMPs (e.g., di-disulfide-linked TMMPs) contain HLA-C... 06:02 (Y84C; A236C) amino acid sequences having at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity:

[0302] CSHSMRYFDTAVSRPGRGEPRISVGYVDDTQFVRFDSDAASPRGEPRAPWVEQEGPEYWDRETQKYKRQAQADRVNLRKLRGCYNQSEDGSHTLQWMYGCDLGPDGRLLRGYDQSAYDGKDYIALNEDLRSWTAADT AAQITQRKWEAAREAEQWRAYLEGTCVEWLRRYLENGKETLQRAEHPKTHVTHHPVSDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRPCGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLTLRWEP (SEQ ID NO:362), wherein amino acid 84 is Cys and amino acid 236 is Cys.

[0303] HLA-C 0701 (Y84C; A236C)

[0304] In some cases, HLA-C heavy chain peptides present in multi-disulfide-linked TMMPs (e.g., di-disulfide-linked TMMPs) contain HLA-C... 07:01 (Y84C; A236C) amino acid sequences having at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity:

[0305] CSHSMRYFDTAVSRPGRGEPRISVGYVDDTQFVRFDSDAASPRGEPRAPWVEQEGPEYWDRETQNYKRQAQADRVSLRNLRGCYNQSEDGSHTLQRMYGCDLGPDGRLLRGYDQSAYDGKDYIALNEDLRSWTAADT AAQITQRKLEAARAAEQLRAYLEGTCVEWLRRYLENGQRAEPPKTHVTHHPLSDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRPCGDGTFQKWAAVVVPSGQEQRYTCHMQHEGLQEPLTLSWEP (SEQ ID NO:357), wherein amino acid 84 is Cys and amino acid 236 is Cys.

[0306] HLA-C 0702 (Y84C; A236C)

[0307] In some cases, HLA-C heavy chain peptides present in multi-disulfide-linked TMMPs (e.g., di-disulfide-linked TMMPs) contain HLA-C... 07:02 (Y84C; A236C) amino acid sequences having at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity:

[0308] CSHSMRYFDTAVSRPGRGEPRISVGYVDDTQFVRFDSDAASPRGEPRAPWVEQEGPEYWDRETQKYKRQAQADRVSLRNLRGCYNQSEDGSHTLQRMSGCDLGPDGRLLRGYDQSAYDGKDYIALNEDLRSWTAADT AAQITQRKLEAARAAEQLRAYLEGTCVEWLRRYLENGQRAEPPKTHVTHHPLSDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRPCGDGTFQKWAAVVVPSGQEQRYTCHMQHEGLQEPLTLSWEP (SEQ ID NO:404), wherein amino acid 84 is Cys and amino acid 236 is Cys.

[0309] HLA-C 0801 (Y84C; A236C)

[0310] In some cases, HLA-C heavy chain peptides present in multi-disulfide-linked TMMPs (e.g., di-disulfide-linked TMMPs) contain HLA-C... 08:01 (Y84C; A236C) amino acid sequences having at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity:

[0311] CSHSMRYFYTAVSRPGRGEPFIAVGYVDDTQFVQFDSDAASPRGEPRAPWVEQEGPEYWDRETQKYKRQAQTDRVSLRNLRGCYNQSEAGSHTLQRMYGCDLGPDGRLLRGYNQFAYDGKDYIALNEDLRSWTAADT AAQITQRKWEAARTAEQLRAYLEGTCVEWLRRYLENGKKTLQRAEHPKTHVTHHPVSDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRPCGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLTLRWGP (SEQ ID NO:363), wherein amino acid 84 is Cys and amino acid 236 is Cys.

[0312] HLA-C 1502 (Y84C; A236C)

[0313] In some cases, HLA-C heavy chain peptides present in multi-disulfide-linked TMMPs (e.g., di-disulfide-linked TMMPs) contain HLA-C... 15:02 (Y84C; A236C) amino acid sequences having at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity:

[0314] CSHSMRYFYTAVSRPGRGEPHFIAVGYVDDTQFVRFDSDAASPRGEPRAPWVEQEGPEYWDRETQNYKRQAQTDRVNLRKLRGCYNQSEAGSHIIQRMYGCDLGPDGRLLRGHDQLAYDGKDYIALNEDLRSWTAADT AAQITQRKWEAAREAEQLRAYLEGTCVEWLRRYLENGKETLQRAEHPKTHVTHHPVSDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRPCGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLTLRWEP (SEQ ID NO:364), wherein amino acid 84 is Cys and amino acid 236 is Cys.

[0315] scaffold peptides

[0316] TMMP may contain an Fc peptide or another suitable scaffold peptide.

[0317] Suitable scaffold peptides include antibody-based scaffold peptides and non-antibody-based scaffolds. Non-antibody-based scaffolds include, for example, albumin, XTEN (extended recombinant) peptides, transferrin, Fc receptor peptides, and elastin-like peptides (see, for example, Hassouneh et al. (2012)). Methods: Enzymol. 502:215; for example, polypeptides containing a pentapeptide repeat unit (Val-Pro-Gly-X-Gly; SEQ ID NO:59), where X is any amino acid other than proline, albumin-binding polypeptides, filamentous polypeptides (see, for example, Valluzzi et al. (2002)). Philos Trans R Soc Lond B Biol Sci. 357:165), filamentous elastin-like polypeptide (SELP; see, for example, Megeed et al. (2002)). Adv Drug Deliv Rev (e.g., 54:1075). Suitable XTEN peptides include, for example, those disclosed in WO 2009 / 023270, WO 2010 / 091122, WO 2007 / 103515, US 2010 / 0189682 and US 2009 / 0092582; see also Schellenberger et al. (2009). Nat Biotechnol .27:1186). Suitable albumin peptides include, for example, human serum albumin.

[0318] In some cases, suitable scaffold peptides will be peptides with extended half-lives. Therefore, in some situations, suitable scaffold peptides increase the half-life of TMMP compared to control TMMP lacking a scaffold peptide. in vivo Half-life (e.g., serum half-life). For example, in some cases, the scaffold peptide increases the half-life of TMMP compared to control TMMP lacking the scaffold peptide. in vivo The half-life (e.g., serum half-life) increased 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 greater than 100-fold. As an example, in some cases, the Fc peptide increased the half-life of TMMP compared to control TMMP lacking the Fc peptide. body Inside The half-life (e.g., serum half-life) increased 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 times, at least about 2.5 times, at least about 5 times, at least about 10 times, at least about 25 times, at least about 50 times, at least about 100 times, or more than 100 times.

[0319] Fc polypeptide

[0320] In some cases, the first and / or second polypeptide chains of the TMMP disclosed herein contain an Fc polypeptide. The Fc polypeptide of the TMMP may be human IgG1 Fc, human IgG2 Fc, human IgG3 Fc, human IgG4 Fc, etc. In some cases, the Fc polypeptide contains... Figure 5A-5G The amino acid sequence of the Fc region depicted in 5H has 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. In some cases, the Fc polypeptide contains amino acid sequences that are identical to... Figure 5A-5G The amino acid sequence of the Fc region depicted in 5H has 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. In some cases, the Fc region contains amino acid sequences that are identical to... Figure 5A The human IgG1 Fc polypeptide depicted has an amino acid sequence with 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. In some cases, the Fc region contains amino acids that are identical to those in the original text. Figure 5AThe human IgG1 Fc polypeptide depicted has an amino acid sequence with 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; and contains an N77 substitution; for example, the Fc polypeptide contains an N77A substitution. In some cases, the Fc polypeptide contains an amino acid sequence with an N77A substitution. Figure 5A The human IgG2 Fc polypeptide described herein has an amino acid sequence with 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; for example, the Fc polypeptide contains amino acids that are identical to those described herein. Figure 5A The amino acid sequence 99-325 of the human IgG2 Fc polypeptide depicted has 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. In some cases, the Fc polypeptide contains amino acids that are identical to those in the original text. Figure 5A The human IgG3 Fc polypeptide described herein has an amino acid sequence with 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; for example, the Fc polypeptide contains amino acids that are identical to those described herein. Figure 5A The amino acid sequence 19-246 of the human IgG3 Fc polypeptide depicted has 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. In some cases, the Fc polypeptide contains amino acid sequences that are identical to those described in the original text. Figure 5B The human IgM Fc polypeptide described herein has an amino acid sequence with 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; for example, the Fc polypeptide contains amino acids that are identical to those described herein. Figure 5B The amino acid sequence 1-276 of the human IgM Fc polypeptide described in the image has 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. In some cases, the Fc polypeptide contains amino acid sequences that are identical to those described in the image. Figure 5C The human IgA Fc polypeptide described herein has an amino acid sequence with 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; for example, the Fc polypeptide contains amino acids that are identical to those described herein. Figure 5CThe amino acid sequence of the human IgA Fc polypeptide described herein has amino acid sequence identity of 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%.

[0321] In some cases, Fc peptides contain... Figure 5C The human IgG4 Fc polypeptide depicted has an amino acid sequence with 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. In some cases, the Fc polypeptide contains amino acids that are identical to those described above. Figure 5C The amino acid sequence of the human IgG4 Fc polypeptide described herein has amino acid sequence identity of 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%.

[0322] In some cases, the IgG4 Fc polypeptide contains the following amino acid sequence: PPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:365).

[0323] In some cases, the Fc peptide present in TMMP contains Figure 5A The amino acid sequence depicted in [the image / image] (human IgG1 Fc). In some cases, the Fc polypeptide present in TMMP contains [the following information / content]. Figure 5A The amino acid sequence depicted (human IgG1 Fc) is an exception, with N297 substitutions made with amino acids other than asparagine. Figure 5A The amino acid sequence depicted in [the image / image] is N77. In some cases, the Fc polypeptide present in TMMP contains [the amino acid sequence / image]. Figure 5C The amino acid sequence depicted (containing N297A-substituted human IgG1 Fc, said substitution is) Figure 5A The amino acid sequence depicted in [the image / image] is N77. In some cases, the Fc polypeptide present in TMMP contains [the amino acid sequence / image]. Figure 5AThe amino acid sequence depicted in the figure (human IgG1 Fc) is an exception, with L234 substitutions made with amino acids other than leucine. Figure 5A The amino acid sequence depicted in L14). In some cases, the Fc polypeptide present in TMMP contains Figure 5A The amino acid sequence described in the figure (human IgG1 Fc) is an exception, with L235 substitutions made with amino acids other than leucine. Figure 5A The amino acid sequence depicted in the image is L15. In some cases, the IgG1 Fc polypeptide contains... Figure 5A The C-terminal Lys is depicted in the image. In other cases, the IgG1 Fc peptide does not include... Figure 5A The C-terminal Lys is depicted in the text.

[0324] In some cases, the Fc peptide present in TMMP contains Figure 5E The amino acid sequence depicted in [the text]. In some cases, Fc polypeptides contain [the text is incomplete and requires further context]. Figure 5E The amino acid sequence is depicted, but without a C-terminal Lys. In some cases, the Fc polypeptide present in TMMP contains... Figure 5F The amino acid sequence depicted in [the text]. In some cases, Fc polypeptides contain [the text is incomplete and requires further context]. Figure 5F The amino acid sequence is depicted, but without a C-terminal Lys. In some cases, the Fc polypeptide present in TMMP contains... Figure 5G The amino acid sequence depicted (containing L234A and L235A substitutions in human IgG1 Fc, these substitutions correspond to...) Figure 5G The amino acid sequences depicted are located at positions 14 and 15. In some cases, Fc polypeptides contain... Figure 5G The amino acid sequence is depicted, but without a C-terminal Lys. In some cases, the Fc polypeptide present in TMMP contains... Figure 5A The amino acid sequence depicted (human IgG1 Fc) is an exception, with P331 substitutions made with amino acids other than proline. Figure 5A The amino acid sequence depicted in the diagram is P111; in some cases, the substitution is a P331S substitution. In some cases, the Fc polypeptide present in TMMP contains... Figure 5A The amino acid sequence depicted in the figure (human IgG1 Fc) is exceptional, except for the substitutions at L234 and L235 with amino acids other than leucine. Figure 5A The amino acid sequences depicted in the diagram are L14 and L15. In some cases, the Fc polypeptide present in TMMP contains... Figure 5A The amino acid sequence depicted in the figure (human IgG1 Fc) is exceptional, except for the substitutions at L234 and L235 with amino acids other than leucine. Figure 5AThe amino acid sequences depicted in the diagram (L14 and L15) and P331 substitutions with amino acids other than proline ( Figure 5A The amino acid sequence depicted in P111). In some cases, the Fc polypeptide present in TMMP contains Figure 5E The amino acid sequence depicted (including L234F, L235E, and P331S substitutions (corresponding to...) Figure 5E The amino acid sequence depicted in the image contains amino acid positions 14, 15, and 111 (human IgG1 Fc). In some cases, the Fc polypeptide present in TMMP is a peptide containing L234A and L235A substitutions (with Ala substitutions). Figure 5A The IgG1 Fc polypeptide, as depicted in the amino acid sequence (substitution of L14 and L15), is a variant of the IgG1 Fc polypeptide. Figure 5G The description.

[0325] In some cases, the Fc polypeptide present in TMMP has the following amino acid sequence: DKTHTCPPCPAPE AA GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:489); and has a length of 226 amino acids.

[0326] connector

[0327] The TMMP disclosed herein may include one or more adapters, wherein the one or more adapters are between one or more of the following: i) a class I MHC peptide and an IgFc peptide, wherein this adapter is referred to herein as “L1”; ii) a MOD and a class I MHC peptide, wherein this adapter is referred to herein as “L2”; iii) a first MOD and a second MOD, wherein this adapter is referred to herein as “L3”; iv) a peptide antigen (“epitope”) and a class I MHC peptide; v) a class I MHC peptide and a dimerized peptide (e.g., a first or second member of a dimerized pair); and vi) a dimerized peptide (e.g., a first or second member of a dimerized pair) and an IgFc peptide.

[0328] As used herein, the phrase “peptide linker between any two components of TMMP” refers to a peptide linker between any two adjacent polypeptides within TMMP. For example, as used herein, the phrase “peptide linker between any two components of TMMP” refers to a peptide linker between one or more of the following: i) a peptide with a β2M polypeptide; ii) a β2M polypeptide with a class I MHC heavy chain polypeptide; iii) a class I MHC heavy chain polypeptide with an Ig Fc polypeptide; iv) a class I MHC heavy chain polypeptide with a MOD; v) an Ig Fc polypeptide with a MOD; and vi) a first MOD with a second MOD.

[0329] Suitable linkers (also called "spacers") can be readily selected and can have any of a number of suitable lengths, such as 1 to 25 amino acids, 3 to 20 amino acids, 2 to 15 amino acids, 3 to 12 amino acids, including 4 to 10 amino acids, 5 to 9 amino acids, 6 to 8 amino acids, or 7 to 8 amino acids. Suitable linker lengths can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids. In some cases, the linker has a length of 25 to 50 amino acids, for example, lengths of 25 to 30, 30 to 35, 35 to 40, 40 to 45, or 45 to 50 amino acids.

[0330] An exemplary connector includes a glycine polymer (G). n Glycine-serine polymers (including, for example, (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 can be used; both Gly and Ser are relatively unstructured and therefore can act as neutral linkers between components. Glycine polymers can be used; glycine is significantly more readily accessible into the phi-psi space than alanine and is much less restricted than residues with longer side chains (see Scheraga, Rev. Computational Chem.11173-142 (1992)). Exemplary adapters may comprise amino acid sequences, including but not limited to GGSG (SEQ ID NO:368), GGSGG (SEQ ID NO:369), GGSSG (SEQ ID NO:370), GGSGG (SEQ ID NO:371), GGGSG (SEQ ID NO:372), GSSSG (SEQ ID NO:373), etc. Exemplary adapters may comprise, for example, Gly(Ser4)n (SEQ ID NO:374), where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some cases, the adapter comprises the amino acid sequence (GSSSS)n (SEQ ID NO:375), where n is 4. In some cases, the adapter comprises the amino acid sequence (GSSSS)n (SEQ ID NO:376), where n is 5. In some cases, the adapter comprises the amino acid sequence (GGGGS)n (SEQ ID NO:377), where n is 1. In some cases, the linker contains the amino acid sequence (GGGGS)n (SEQ ID NO:378), where n is 2. In some cases, the linker contains the amino acid sequence (GGGGS)n (SEQ ID NO:379), where n is 3. In some cases, the linker contains the amino acid sequence (GGGGS)n (SEQ ID NO:380), where n is 4. In some cases, the linker contains the amino acid sequence (GGGGS)n (SEQ ID NO:381), where n is 5. In some cases, the linker contains the amino acid sequence (GGGGS)n (SEQ ID NO:382), where n is 6. In some cases, the linker contains the amino acid sequence (GGGGS)n (SEQ ID NO:383), where n is 7; in some cases, the linker contains the amino acid sequence (GGGGS)n (SEQ ID NO:384), where n is 8; in some cases, the linker contains the amino acid sequence (GGGGS)n (SEQ ID NO:385), where n is 9; in some cases, the linker contains the amino acid sequence (GGGGS)n (SEQ ID NO:386), where n is 10. In some cases, the linker contains the amino acid sequence AAAGG (SEQ ID NO:283).

[0331] In some cases, the cysteine ​​residues included in the linker polypeptide of the first polypeptide of TMMP can form disulfide bonds with the cysteine ​​residues of the second polypeptide of TMMP. In some cases, for example, a suitable linker contains the amino acid sequence G. CGGSGGGGGSGGGGS (SEQ ID NO:317). As another example, a suitable adapter may comprise 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 cases, the adapter comprises the amino acid sequence GCGGSGGGGSGGGGSGGGGS (SEQ ID NO:316). As another example, the adapter comprises the amino acid sequence GCGGSGGGGSGGGGS (SEQ ID NO:317).

[0332] Epitope

[0333] In some cases, the epitope present in the TMMP (the peptide presenting one or more epitopes) is the WT-1 peptide, for example, the WT-1 peptide that presents the epitope to the TCR along with the MHC. The amino acid sequence of the WT-1 isotype is presented in... Figures 3A-3E In this document, a WT-1 peptide presenting one or more epitopes is referred to as a "WT-1 peptide" or "WT-1 epitope". In some cases, the WT-1 epitope present in the TMMP of this disclosure may be related to... Figures 3A-3E A peptide consisting 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) of amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity with any of the described WT-1 amino acid sequences. In some cases, the WT-1 epitope present in the TMMP of this disclosure may be a peptide with... Figure 3A The WT-1 amino acid sequence described herein is a peptide consisting 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) with at least 90%, 95%, 98%, 99%, or 100% amino acid sequence identity. In some cases, the WT-1 epitope present in TMMP may be a peptide with... Figure 3A The WT-1 amino acid sequence described herein is a peptide consisting 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 amino acid sequence identity of at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity. In some cases, the WT-1 epitope present in the TMMP of this disclosure may be a peptide with... Figure 3CThe WT-1 amino acid sequence described herein is a peptide consisting 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) with at least 90%, 95%, 98%, 99%, or 100% amino acid sequence identity. In some cases, the WT-1 epitope present in TMMP may be a peptide with... Figure 3A The WT-1 amino acid sequence described herein is a peptide consisting 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 amino acid sequence identity of at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity. In some cases, the WT-1 epitope present in the TMMP of this disclosure may be a peptide with... Figure 3E The WT-1 amino acid sequence described herein is a peptide consisting 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 amino acid sequence identity of at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity. In some cases, the WT-1 epitope in the TMMP is 6 amino acids long. In some cases, the WT-1 epitope in the TMMP is 7 amino acids long. In some cases, the WT-1 epitope in the TMMP is 8 amino acids long. In some cases, the WT-1 epitope in the TMMP is 9 amino acids long. In some cases, the WT-1 epitope in the TMMP is 10 amino acids long. In some cases, the WT-1 epitope in the TMMP is 11 amino acids long. In some cases, the WT-1 epitope in the TMMP is 6 to 25 amino acids long. In some cases, the WT-1 epitope in TMMP is 6 to 20 amino acids long. In some cases, the WT-1 epitope in TMMP is 7 to 25 amino acids long. In some cases, the WT-1 epitope in TMMP is 7 to 20 amino acids long. In some cases, the WT-1 epitope in TMMP is at least 4 amino acids long, at least 6 amino acids long, or at least 7 amino acids long.

[0334] Epitopes present in TMMPs can have a length of approximately 4 to approximately 25 amino acids, for example, epitopes can have a length of 4 amino acids (aa) to 10 aa, 10 aa to 15 aa, 15 aa to 20 aa, or 20 aa to 25 aa. For example, epitopes present in TMMPs can have a length of 4 amino acids (aa), 5 aa, 6 aa, 7 aa, 8 aa, 9 aa, 10 aa, 11 aa, 12 aa, 13 aa, 14 aa, 15 aa, 16 aa, 17 aa, 18 aa, 19 aa, 20 aa, 21 aa, 22 aa, 23 aa, 24 aa, or 25 aa. In some cases, epitopes present in TMMPs have a length of 5 to 10 amino acids, for example, 5 aa, 6 aa, 7 aa, 8 aa, 9 aa, or 10 aa.

[0335] The WT-1 epitope present in TMMP is a T cell-specific peptide; that is, the epitope is specifically bound by WT-1 epitope-specific T cells. Epitope-specific T cells bind to epitopes with a reference amino acid sequence, but generally do not bind to epitopes with different amino acid sequences. For example, if an epitope-specific T cell binds to an epitope with a reference amino acid sequence and also binds to an epitope with a different amino acid sequence, if any, its affinity is less than 10. -6 M, less than 10 -5 M or less than 10 -4 M. Epitope-specific T cells can bind to epitopes that have a specific affinity for them, with an affinity of at least 10. -7 M, at least 10 -8 M, at least 10 -9 M or at least 10 -10 M.

[0336] Examples of WT-1 peptides suitable for inclusion in TMMP include, but are not limited to, 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), WNLMNLGATLKGVAA (SEQ ID NO:232), MNLGATLK (SEQ ID NO:233), MTWNYMNLGATLKGV (SEQID 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:242), 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 IDNO:250), KRPFMCAYPGCNK (SEQ ID NO:251), KRPFMCAYPGCYK (SEQ ID NO:252), FMCAYPGCY(SEQ ID NO:253), FMCAYPGCK (SEQ ID NO:254), KRPFMCAYPGCNKRY (SEQ IDThe following peptides are present in TMMP: 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 cases, the WT-1 peptide present in TMMP is CMTWNQMN (SEQ ID NO:261). In some cases, the WT-1 peptide present in TMMP is CYTWNQMNL (SEQ ID NO:262).

[0337] In some cases, the WT-1 peptide present in TMMP presents HLA-A. 2402 Restrictive Epitope. HLA-A Submission WT-1 peptides with the 2402 restriction epitope include, for example, 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).

[0338] In some cases, the WT-1 peptide present in TMMP presents HLA-A. 0201 Restrictive Epitope. HLA-A Presentation. WT-1 peptides with the 0201 restriction epitope include, for example, 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).

[0339] In some cases, the WT-1 peptide present in TMMP presents HLA-A. The 2402 restriction epitope does not have an N-terminal Cys. For example, when the WT-1 peptide contains an N-terminal Cys, the N-terminal Cys can be replaced by Ser. As another example, when the WT-1 peptide contains an N-terminal Cys, Gly can be added to the N-terminus. For example, the WT-1 peptide present in TMMP may contain the amino acid sequence X1X2X3TWNQMNL (SEQ ID NO:460) or X2X3TWNQMNL (SEQ ID NO:461), wherein X1, X2, and X3 are each independently any amino acid, provided that the N-terminal amino acid is not Cys, and wherein the WT-1 peptide epitope has a length of 9 to 25 amino acids. In some of these embodiments, the WT-1 peptide has a length of 9 or 10 amino acids. Examples of WT-1 peptides suitable for inclusion in 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 cases, the WT-1 peptide present in the TMMP of this disclosure has the amino acid sequence SMTWNQMNL (SEQ ID NO:451) and is 9 amino acids in length. In some cases, the WT-1 peptide present in the TMMP has the amino acid sequence GCMTWNQMNL (SEQ ID NO:452) and is 10 amino acids in length. In some cases, the WT-1 peptide present in the TMMP has the amino acid sequence SYTWNQMNL (SEQ ID NO:453) and is 9 amino acids in length. In some cases, the WT-1 peptide present in the TMMP has the amino acid sequence GCYTWNQMNL (SEQ ID NO:454) and is 10 amino acids in length.

[0340] HLA / peptide binding assay

[0341] Whether a given peptide (e.g., the WT-1 peptide) binds to class I HLA (comprising the HLA heavy chain and β2M peptide) and whether it can effectively present epitopes to the TCR when bound to the HLA complex can be determined using any of a number of known methods. Assays include binding assays and T cell activation assays.

[0342] Cell-based binding assays

[0343] As an example, cell-based peptide-inducible stability assays can be used to determine peptide-HLA class I binding. In this assay, the target peptide is allowed to bind to TAP-deficient cells, i.e., cells with defective transporters associated with antigen processing (TAP) mechanisms and therefore fewer class I molecules on their surface. Such cells include, for example, the human T2 cell line (T2 (174 x CEM.T2; USTC No. CRL-1992). Henderson et al. (1992) Science 255:1264. Without TAP-mediated transport of the cytoplasmic peptide-deficient type I to the endoplasmic reticulum, the assembled class I complex is structurally unstable and only transiently retained on the cell surface. However, when T2 cells are cultured with exogenous peptides capable of binding class I, the surface peptide-HLA class I complex is stable and detectable by flow cytometry using, for example, pan-anti-class I monoclonal antibodies. The addition of peptides to stabilize the peptide-HLA complex on the cell surface and consequently extend its lifespan validates its identity. Analysis can be performed using flow cytometry, for example, with a pan-HLA class I antibody containing a fluorescent label. Binding of the peptide to various allelic forms of the HLA H chain can be tested by genetically modifying T2 cells to express the target allele of the HLA H chain.

[0344] The following are the T2 assay results for peptides and HLA-A. Non-limiting examples of the use of 0201 in combination. T2 cells are washed in cell culture medium and concentrated to 10... 6 Cells / ml. The target peptide was prepared in cell culture medium and serially diluted to provide concentrations of 200 μM, 100 μM, 20 μM, and 2 μM. Cells were mixed 1:1 with each peptide dilution to obtain a final volume of 200 μL and final peptide concentrations of 100 μM, 50 μM, 10 μM, and 1 μM. A HLA A 0201-binding peptide, GILGFVFTL (SEQ ID NO: 395), and non-HLA A 0201-Restricted peptide, HPVGEADYF (SEQ ID NO: 396) (HLA-B Cells (3501) were included as positive and negative controls, respectively. The cell / peptide mixture was maintained at 37°C in 5% CO2 for ten minutes; then incubated overnight at room temperature. Cells were subsequently incubated at 37°C for 2 hours and stained with fluorescently labeled anti-human HLA antibody. Cells were then washed twice with phosphate-buffered saline and analyzed using flow cytometry. Binding strength was measured using the mean fluorescence intensity (MFI) of the anti-HLA antibody staining.

[0345] Biochemical binding assay

[0346] The binding of HLA peptides (complexes of HLA heavy chain peptides and β2M peptides) to target peptides can be tested in a cell-free in vitro assay system. For example, a labeled reference peptide (e.g., fluorescently labeled) is bound to an HLA peptide (complex of HLA heavy chain peptides and β2M peptides) to form an HLA-reference peptide complex. The ability of the target test peptide 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.

[0347] As another example, the target peptide can be cultured with HLA molecules (a complex of the HLA heavy chain and β2M peptide), and the stability of the HLA / peptide complex can be measured via immunoassay. The ability of the target peptide to stabilize the HLA molecule is compared to the ability of a control peptide to present a known T-cell epitope. Stability is determined based on the presence or absence of the native conformation of the HLA / peptide complex as detected using an anti-HLA antibody. See, for example, Westrop et al. (2009). J. Immunol.Methods 341:76; Steinitz et al. (2012) Blood 119:4073; and US Patent No. 9,205,144.

[0348] T cell activation assay

[0349] Whether a given peptide binds to class I HLA (including the HLA heavy chain and β2M peptide) and whether it can effectively present epitopes to the TCR when bound to the HLA complex can be determined by assessing the T cell response to the peptide-HLA complex. Measurable T cell responses include, for example, interferon-γ (IFNγ) production and cytotoxic activity.

[0350] ELISPOT measurement

[0351] Suitable assays include, for example, enzyme-linked immunospot (ELISPOT) assays. In this assay, CD8 is measured after antigen-presenting cells (APCs) that present a complex of the target peptide with HLA class I are subjected to measurement. + IFNγ is produced by T cells. Antibodies against IFNγ are immobilized on the wells of a multi-well plate. APCs are added to the wells and incubated with the target peptide for a period of time, allowing the peptide to bind to HLA class I on the surface of the APCs. CD8 receptors specific to this peptide are then activated. + T cells were added to the wells, and the plate was incubated for approximately 24 hours. The wells were then washed, and any IFNγ bound to the immobilized anti-IFNγ antibody was detected using a detectable labeled anti-IFNγ antibody. A colorimetric assay could be used. For example, the detectable labeled anti-IFNγ antibody could be a biotin-labeled anti-IFNγ antibody, which could be detected using, for example, streptavidin conjugated to alkaline phosphatase. A BCIP / NBT (5-bromo-4-chloro-3-indolyl phosphate / nitroblue tetrazolium) solution was added to develop the assay. The presence of IFNγ-secreting T cells was identified by the staining. Negative controls included APCs that had not been exposed to the peptide. APCs expressing various HLA H chain alleles could be used to determine whether the target peptide effectively bound to an HLA class I molecule containing a specific HLA H chain.

[0352] Cytotoxicity assay

[0353] Whether a given peptide binds to a specific HLA class I H chain and whether it can effectively present an epitope to the TCR when bound to an HLA class I complex containing an H chain can also be determined using cytotoxicity assays. Cytotoxicity assays involve reacting target cells with cytotoxic CD8+. + T cells are cultured together. Target cells display a peptide / HLA class I complex containing the target peptide and an HLA class I molecule containing the HLA H chain to be tested on their surface. Target cells can be radiolabeled, for example... 51 Cr radiolabeling. Whether target cells effectively present epitopes to cytotoxic CD8. + TCR on T cells, thus controlled by CD8 + T cell-induced cytotoxic activity against target cells is measured by... 51 Cr is determined from the release of lysed target cells. Specific cytotoxicity can be calculated as the amount of cytotoxic activity in the presence of the peptide minus the amount of cytotoxic activity in the absence of the peptide.

[0354] Detection of antigen-specific T cells using peptide-HLA tetramer

[0355] As another example, fluorescent or heavy metal tags are used to generate multimers (e.g., tetramers) of peptide-HLA complexes. These multimers can then be used to identify and quantify specific T cells via flow cytometry (FACS) or cytotoxicity-to-fluidization (CyTOF). The 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.

[0356] Immunomodulatory peptides ("MOD")

[0357] In some cases, the MODs present in TMMP are wild-type (“wt”) MODs. As mentioned above, in other cases, the MODs present in TMMP are variants of the wt MOD, exhibiting a reduced affinity for co-MODs compared to the corresponding wild-type MODs. Suitable MODs exhibiting reduced affinity for co-MODs can differ from the wild-type MOD by 1 to 20 amino acids (aa). For example, in some cases, the amino acid sequence of variant MODs present in TMMP differs from the corresponding wild-type MOD by 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 aa. As another example, in some cases, the amino acid sequence of the variant MOD present in TMMP differs 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.

[0358] As described above, the MOD may contain a variant of the wt immunomodulatory peptide that exhibits reduced binding to its 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 TMMP may bind to its co-MOD with an affinity for the co-MOD that is at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or less than 95% less than the corresponding wild-type MOD. Exemplary pairs of immunomodulatory peptides and homologous co-immunomodulatory peptides include, but are not limited to:

[0359] a) 4-1BBL (immunomodulatory peptide) and 4-1BB (homologous co-immunomodulatory peptide);

[0360] b) PD-L1 (immunomodulatory peptide) and PD1 (homogeneous co-immunomodulatory peptide);

[0361] c) IL-2 (immunomodulatory peptide) and IL-2 receptor (homologous co-immunomodulatory peptide);

[0362] d) CD80 (immunomodulatory peptide) and CD86 (homologous co-immunomodulatory peptide);

[0363] e) CD86 (immunomodulatory peptide) and CD28 (homologous co-immunomodulatory peptide);

[0364] f) OX40L (CD252) (immunomodulatory peptide) and OX40 (CD134) (homologous co-immunomodulatory peptide);

[0365] g) Fas ligand (immunomodulatory polypeptide) and Fas (homologous co-immunomodulatory polypeptide);

[0366] h) ICOS-L (immunomodulatory peptide) and ICOS (homological co-immunomodulatory peptide);

[0367] i) ICAM (immunomodulatory peptide) and LFA-1 (homologous co-immunomodulatory peptide);

[0368] j) CD30L (immunomodulatory peptide) and CD30 (homologous co-immunomodulatory peptide);

[0369] k) CD40 (immunomodulatory peptide) and CD40L (homologous co-immunomodulatory peptide);

[0370] l) CD83 (immunomodulatory peptide) and CD83L (homologous co-immunomodulatory peptide);

[0371] m) HVEM (CD270) (immunomodulatory peptide) and CD160 (homologous co-immunomodulatory peptide);

[0372] n) JAG1 (CD339) (immunomodulatory peptide) and Notch (homologous co-immunomodulatory peptide);

[0373] o) JAG1 (immunomodulatory peptide) and CD46 (homologous co-immunomodulatory peptide);

[0374] p) CD80 (immunomodulatory peptide) and CTLA4 (homological co-immunomodulatory peptide);

[0375] q) CD86 (immunomodulatory peptide) and CTLA4 (homological co-immunomodulatory peptide); and

[0376] r) CD70 (immunomodulatory peptide) and CD27 (homologous co-immunomodulatory peptide).

[0377] like Figure 19 As illustrated, a MOD (i.e., one or more MODs) can exist at any of the multiple locations in the TMMP. Figure 19 The locations of the two copies of the variant IL-2 peptide are depicted; however, the MOD can be any of the various MODs described herein. Figure 19 As described, MOD can be: 1) at the N-terminus of the class I MHC heavy chain; 2) at the C-terminus of the class I MHC heavy chain and the N-terminus of the Ig Fc polypeptide; in other words, between the class I MHC heavy chain and the Ig Fc polypeptide; 3) at the C-terminus of the Ig Fc polypeptide; 4) at the N-terminus of the peptide epitope; or 5) at the C-terminus of the β2M polypeptide.

[0378] PD-L1 – Wild Type and Variants

[0379] The MOD present in TMMP can be a wild-type PD-L1 peptide or a variant PD-L1 peptide.

[0380] In some cases, the MOD present in TMMP is a PD-L1 peptide. In some cases, the PD-L1 peptide of TMMP contains 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 with the PD-L1 amino acid sequence listed in SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3. The PD-L1 variant MOD is described in PCT application WO 2019 / 051091 published on March 14, 2019. See

[00157] -

[00169] .

[0381] In some cases, the variant PD-L1 peptide exhibits a reduced binding affinity to PD-1 (e.g., a PD-1 peptide comprising the amino acid sequence listed in SEQ ID NO:3) compared to the binding affinity of the PD-L1 peptide comprising the amino acid sequence listed in SEQ ID NO:1 or SEQ ID NO:2. For example, in some cases, the binding affinity of the variant PD-L1 peptide of this disclosure to PD-1 (e.g., a PD-1 peptide comprising the amino acid sequence listed in SEQ ID NO:3) is at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, less than 50%, less than 55%, less than 60%, less than 65%, less than 70%, less than 75%, less than 80%, less than 85%, less than 90%, less than 95%, or less than 95%.

[0382] CD80 – Wild type and variants

[0383] The MOD present in TMMP can be a wild-type CD80 peptide or a variant CD80 peptide. The CD80 variant MOD is described in the published PCT application WO 2019 / 051091, published on March 14, 2019. See

[00170] -

[00196] .

[0384] In some cases, the MOD present in TMMP is a CD80 polypeptide. In some cases, the CD80 polypeptide of TMMP contains 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 with the amino acid sequences listed in SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7.

[0385] In some cases, the variant CD80 peptide exhibits a reduced binding affinity for CD28 compared to the binding affinity of the CD80 peptide containing the amino acid sequence listed in SEQ ID NO:4 for CD28. For example, in some cases, the binding affinity of the variant CD80 peptide for CD28 is at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, less than 50%, less than 55%, less than 60%, less than 65%, less than 70%, less than 75%, less than 80%, less than 85%, less than 90%, less than 95%, or less than 95%.

[0386] CD86 – Wild type and variants

[0387] The MOD present in TMMP can be a wild-type CD86 peptide or a variant CD86 peptide. The CD80 variant MOD is described in the published PCT application WO 2019 / 051091, published on March 14, 2019. See

[00197] -

[00228] .

[0388] In some cases, the MOD present in TMMP is a CD86 polypeptide. In some cases, the CD86 polypeptide of TMMP contains 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 with the CD86 amino acid sequence listed in SEQ ID NO:8 or SEQ ID NO:9.

[0389] 4-1BBL – Wild type and variants

[0390] The MOD present in TMMP can be a wild-type 4-1BBL peptide or a variant 4-1BBL peptide. The 4-1BBL variant MOD is described in the published PCT application WO 2019 / 051091, published on March 14, 2019. See

[00229] -

[00324] .

[0391] In some cases, the MOD present in TMMP is a 4-1BBL polypeptide. In some cases, the 4-1BBL polypeptide of TMMP contains 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 with the 4-1BBL amino acid sequence listed in SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, or SEQ ID NO:14.

[0392] In some cases, the variant 4-1BBL peptide exhibits a reduced binding affinity for 4-1BB compared to the binding affinity of the 4-1BBL peptide comprising the amino acid sequence listed in one of SEQ ID NO:10-13. For example, in some cases, when measured under the same conditions, the binding affinity of the variant 4-1BBL peptide of this disclosure for 4-1BB is at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or less than 95%.

[0393] IL-2 variants

[0394] In some cases, the variant MOD present in the TMMP of this disclosure is a variant IL-2 polypeptide. Wild-type IL-2 binds to the IL-2 receptor (IL-2R), which is a heterotrimeric polypeptide comprising IL-2Rα, IL-2Rβ and IL-2Rγ.

[0395] The amino acid sequence of wild-type IL-2 can be as follows: APTSSSTKKT QLQL EH LLL D LQMILNGINNYKNPKLTRML T F KF Y MPKKA TELKHLQCLEEELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSETTFMCEYADE TATIVEFLNRWITFC Q SIIS TLT (SEQ ID NO:15).

[0396] Wild-type IL2 binds to the IL2 receptor (IL2R) on the cell surface. In some cases, the IL2 receptor is a heterotrimeric polypeptide containing 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, IL2Rβ, and IL-2Rγ may be as follows.

[0397] Human IL-2Rα: ELCDDDPPE IPHATFKAMA YKEGTMLNCE CKRGFRRIKS GSLYMLCTGNSSHSSWDNQC QCTSSATRNT TKQVTPQPEE QKERKTTEMQ SPMQPVDQAS LPGHCREPPP WENEATERIYHFVVGQMVYY QCVQGYRALH RGPAESVCKM THGKTRWTQP QLICTGEMET SQFPGEEEKPQ ASPEGRPESETSCLVTTDF QIQTEMAATM ETSIFTTEYQ VAVAGCVFLL ISVLLLSGLT WQRRQRKSRR TI (SEQ IDNO:16).

[0398] Human IL-2Rβ: VNG TSQFTCFYNS RANISCVWSQ DGALQDTSCQ VHAWPDRRRW NQTCELLPVSQASWACNLIL GAPDSQKLTT VDIVTLRVLC REGVRWRVMA IQDFKPFENL RLMAPISLQV VHVETHRCNISWEISQASHY FERHLEFEAR TLSPGHTWEE APLLTLKQKQ EWICLETLTP DTQYEFQVRV KPLQGEFTTWSPWSQPLAFR TKPAALGKDT IPWLGHLLVG LSGAFGFIIL VYLLINCRNT GPWLKKVLKC NTPDPSKFFSQLSSEHGGDV QKWLSSPFPS SSFSPGGLAP EISPLEVLER DKVTQLLLQQ DKVPEPASLS SNHSLTSCFTNQGYFFFHLP DALEIEACQV YFTYDPYSEE DPDEGVAGAP TGSSPQPLQP LSGEDDAYCT FPSRDDLLLFSPSLLGGPSP PSTAPGGSGA GEERMPPSLQ ERVPRDWDPQ PLGPPTPGVP DLVDFQPPPE LVLREAGEEVPDAGPREGVS FPWSRPPGQG EFRALNARLP LNTDAYLSLQ ELQGQDPTHL V (SEQ ID NO:17).

[0399] Human IL-2Rγ: LNTTILTP NGNEDTTADF FLTTMPTDSL SVSTLPLPEV QCFVFNVEYMNCTWNSSSEP QPTNLTLHYW YKNSDNDKVQ KCSHYLFSEE ITSGCQLQKK EIHLYQTFVV QLQDPREPRRQATQMLKLQN LVIPWAPENL TLHKLSESQL ELNWNNRFLN HCLEHLVQYR TDWDHSWTEQ SVDYRHKFSLPSVDGQKRYT FRVRSRFNPL CGSAQHWSEW SHPIHWGSNT SKENPFLFAL EAVVISVGSM GLIISLLCVYFWLERTMPRI PTLKNLEDLV TEYHGNFSAW SGVSKGLAES LQPDYSERLC LVSEIPPKGG ALGEGPGASPCNQHSPYWAP PCYTLKPET (SEQ ID NO:18).

[0400] In some cases, when TMMP contains a variant IL-2 peptide, “homologous co-MOD” refers to an IL-2R containing a peptide comprising the amino acid sequences SEQ ID NO:16, 17, and 18.

[0401] In some cases, the variant IL-2 peptide exhibits a reduced binding affinity for IL-2R compared to the binding affinity of the IL-2 peptide comprising the amino acid sequence listed in SEQ ID NO:15. For example, in some cases, when measured under the same conditions, the binding affinity of the variant IL-2 peptide to IL-2R is at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or less than 95%.

[0402] In some cases, the variant IL-2 peptide has a binding affinity for IL-2R ranging from 100 nM to 100 μM. As another example, in some cases, the binding affinity of the variant IL-2 peptide to IL-2R (e.g., IL-2R comprising a peptide containing the amino acid sequence listed in SEQ ID NO:16-18) is about 100 nM to 150 nM, about 150 nM to about 200 nM, about 200 nM to about 250 nM, about 250 nM to about 300 nM, about 300 nM to about 350 nM, about 350 nM to about 400 nM, about 400 nM to about 500 nM, about 500 nM to about 600 nM, about 600 nM to about 700 nM, about 700 nM to about 800 nM, about 800 nM to about 900 nM, about 900 nM to about 1 μM to 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. 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.

[0403] In some cases, the variant IL-2 peptide has a single amino acid substitution compared to the IL-2 amino acid sequence listed in SEQ ID NO:15. In some cases, the variant IL-2 peptide has 2 to 10 amino acid substitutions compared to the IL-2 amino acid sequence listed in SEQ ID NO:15. In some cases, the variant IL-2 peptide has 2 amino acid substitutions compared to the IL-2 amino acid sequence listed in SEQ ID NO:15. In some cases, the variant IL-2 peptide has 3 amino acid substitutions compared to the IL-2 amino acid sequence listed in SEQ ID NO:15. In some cases, the variant IL-2 peptide has 4 amino acid substitutions compared to the IL-2 amino acid sequence listed in SEQ ID NO:15. In some cases, the variant IL-2 peptide has 5 amino acid substitutions compared to the IL-2 amino acid sequence listed in SEQ ID NO:15. In some cases, the variant IL-2 peptide has 6 amino acid substitutions compared to the IL-2 amino acid sequence listed in SEQ ID NO:15. In some cases, the variant IL-2 peptide has 7 amino acid substitutions compared to the IL-2 amino acid sequence listed in SEQ ID NO:15. In some cases, the variant IL-2 peptide has 8 amino acid substitutions compared to the IL-2 amino acid sequence listed in SEQ ID NO:15. In some cases, the variant IL-2 peptide has 9 amino acid substitutions compared to the IL-2 amino acid sequence listed in SEQ ID NO:15. In some cases, the variant IL-2 peptide has 10 amino acid substitutions compared to the IL-2 amino acid sequence listed in SEQ ID NO:15.

[0404] Suitable IL-2 variants include a polypeptide comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity with any of the following amino acid sequences:

[0405] APTSSSTKKT QLQLEHLLLD LQMILNGINN YKNPKLTRML T XKFYMPKKA TELKHLQCLEEELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNR WITFCQSIISTLT (SEQ ID NO:181), where X is any amino acid except Phe. In some cases, X is Ala. In some cases, X is Met. In some cases, X is Pro. In some cases, X is Ser. In some cases, X is Thr. In some cases, X is Trp. In some cases, X is Tyr. In some cases, X is Val. In some cases, X is His;

[0406] APTSSSTKKT QLQLEHLLL X LQMILNGINN YKNPKLTRML TFKFYMPKKA TELKHLQCLEEELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNR WITFCQSIISTLT (SEQ ID NO:182), where X is any amino acid except Asp. In some cases, X is Ala;

[0407] APTSSSTKKT QLQL X HLLLD LQMILNGINN YKNPKLTRML TFKFYMPKKA TELKHLQCLEEELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNR WITFCQSIISTLT (SEQ ID NO:183), where X is any amino acid except Glu. In some cases, X is Ala.

[0408] APTSSSTKKT QLQLE XLLLD LQMILNGINN YKNPKLTRML TFKFYMPKKA TELKHLQCLEEELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNR WITFCQSIISTLT (SEQ ID NO:184), where X is any amino acid except His. In some cases, X is Ala. In some cases, X is Thr. In some cases, X is Asn. In some cases, X is Cys. In some cases, X is Gln. In some cases, X is Met. In some cases, X is Val. In some cases, X is Trp;

[0409] APTSSSTKKT QLQLE X LLLD LQMILNGINN YKNPKLTRML TFKFYMPKKA TELKHLQCLEEELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNR WITFCQSIISTLT (SEQ ID NO:185), where X is any amino acid except His. In some cases, X is Ala. In some cases, X is Arg. In some cases, X is Asn. In some cases, X is Asp. In some cases, X is Cys. In some cases, X is Glu. In some cases, X is Gln. In some cases, X is Gly. In some cases, X is Ile. In some cases, X is Lys. In some cases, X is Leu. In some cases, X is Met. In some cases, X is Phe. In some cases, X is Pro. In some cases, X is Ser. In some cases, X is Thr. In some cases, X is Tyr. In some cases, X is Trp. In some cases, X is Val.

[0410] APTSSSTKKT QLQLEHLLLLD LQMILNGINN YKNPKLTRML TFKF X MPKKA TELKHLQCLEEELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNR WITFCQSIISTLT (SEQ ID NO:186), where X is any amino acid except Tyr. In some cases, X is Ala;

[0411] APTSSSTKKT QLQLEHLLLD LQMILNGINN YKNPKLTRML TFKFYMPKKA TELKHLQCLEEELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNR WITFC X SIISTLT (SEQ ID NO:187), where X is any amino acid except Gln. In some cases, X is Ala;

[0412] APTSSSTKKT QLQLE X1 LLLD LQMILNGINN YKNPKLTRML T X2 KFYMPKKA TELKHLQCLEEELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNR WITFCQSIISTLT (SEQ ID NO:188), wherein X1 is any amino acid except His, and X2 is any amino acid except Phe. In some cases, X1 is Ala. In some cases, X2 is Ala. In some cases, X1 is Ala; and X2 is Ala. In some cases, X1 is Thr; and X2 is Ala;

[0413] APTSSSTKKT QLQLEHLLL X1 LQMILNGINN YKNPKLTRML T X2 KFYMPKKA TELKHLQCLEEELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNR WITFCQSIISTLT (SEQ ID NO:189), wherein X1 is any amino acid except Asp; and X2 is any amino acid except Phe. In some cases, X1 is Ala. In some cases, X2 is Ala. In some cases, X1 is Ala; and X2 is Ala;

[0414] APTSSSTKKT QLQL X1 HLLL X2 LQMILNGINN YKNPKLTRML T X3KFYMPKKA TELKHLQCLEEELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNR WITFCQSIISTLT (SEQ ID NO:190), wherein X1 is any amino acid except Glu; X2 is any amino acid except Asp; and X3 is any amino acid except Phe. In some cases, X1 is Ala. In some cases, X2 is Ala. In some cases, X3 is Ala. In some cases, X1 is Ala; X2 is Ala; and X3 is Ala;

[0415] APTSSSTKKT QLQLE X1 LLL X2 LQMILNGINN YKNPKLTRML T X3 KFYMPKKA TELKHLQCLEEELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNR WITFCQSIISTLT (SEQ ID NO:191), wherein X1 is any amino acid except His; X2 is any amino acid except Asp; and X3 is any amino acid except Phe. In some cases, X1 is Ala. In some cases, X2 is Ala. In some cases, X3 is Ala. In some cases, X1 is Ala; X2 is Ala; and X3 is Ala;

[0416] APTSSSTKKT QLQLEHLLL X1 LQMILNGINN YKNPKLTRML T X2 KFYMPKKA TELKHLQCLEEELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNR WITFC X3 SIISTLT (SEQ ID NO:192), wherein X1 is any amino acid except Asp; X2 is any amino acid except Phe; and X3 is any amino acid except Gln. In some cases, X1 is Ala. In some cases, X2 is Ala. In some cases, X3 is Ala. In some cases, X1 is Ala; X2 is Ala; and X3 is Ala;

[0417] APTSSSTKKT QLQLEHLLLX1 LQMILNGINN YKNPKLTRML T X2 KF X3 MPKKA TELKHLQCLEEELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNR WITFCQSIISTLT (SEQ ID NO:193), wherein X1 is any amino acid except Asp; X2 is any amino acid except Phe; and X3 is any amino acid except Tyr. In some cases, X1 is Ala. In some cases, X2 is Ala. In some cases, X3 is Ala. In some cases, X1 is Ala; X2 is Ala; and X3 is Ala;

[0418] APTSSSTKKT QLQLE X1 LLL X2 LQMILNGINN YKNPKLTRML T X3 KF X4 MPKKA TELKHLQCLEEELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNR WITFCQSIISTLT (SEQ ID NO:194), wherein X1 is any amino acid except His; X2 is any amino acid except Asp; X3 is any amino acid except Phe; and X4 is any amino acid except Tyr. In some cases, X1 is Ala. In some cases, X2 is Ala. In some cases, X3 is Ala. In some cases, X4 is Ala. In some cases, X1 is Ala; X2 is Ala; X3 is Ala; and X4 is Ala;

[0419] APTSSSTKKT QLQLEHLLL X1 LQMILNGINN YKNPKLTRML T X2 KF X3 MPKKA TELKHLQCLEEELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNR WITFC X4SIISTLT (SEQ ID NO:195), wherein X1 is any amino acid except Asp; X2 is any amino acid except Phe; X3 is any amino acid except Tyr; and X4 is any amino acid except Gln. In some cases, X1 is Ala. In some cases, X2 is Ala. In some cases, X3 is Ala. In some cases, X4 is Ala. In some cases, X1 is Ala; X2 is Ala; X3 is Ala; and X4 is Ala;

[0420] APTSSSTKKT QLQLE X1 LLL X2 LQMILNGINN YKNPKLTRML T X3 KF X4 MPKKA TELKHLQCLEEELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNR WITFC X5 SIISTLT (SEQ ID NO:196), wherein X1 is any amino acid except His; X2 is any amino acid except Asp; X3 is any amino acid except Phe; X4 is any amino acid except Tyr; and X5 is any amino acid except Gln. In some cases, X1 is Ala. In some cases, X2 is Ala. In some cases, X3 is Ala. In some cases, X4 is Ala. In some cases, X5 is Ala. In some cases, X1 is Ala; X2 is Ala; X3 is Ala; X4 is Ala; X5 is Ala; and

[0421] APTSSSTKKT QLQLE X1 LLLD LQMILNGINN YKNPKLTRML T X2 KFYMPKKA TELKHLQCLEEELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNR WITFC X3 SIISTLT (SEQ ID NO:197), wherein X1 is any amino acid except His; X2 is any amino acid except Phe; and X3 is any amino acid except Gln. In some cases, X1 is Ala. In some cases, X2 is Ala. In some cases, X3 is Ala. In some cases, X1 is Ala; X2 is Ala; and X3 is Ala.

[0422] In some cases, suitable variant IL-2 peptides contain an amino acid sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to the following amino acid sequence: APTSSSTKKT QLQLEALLLDLQMILNGINN YKNPKLTRML TAKFYMPKKA TELKHLQCLEEELKPLEEVL NLAQSKNFHL RPRDLISNINVIVLELKGSE TTFMCEYADE TATIVEFLNRWITFCQSIIS TLT (SEQ ID NO:490), meaning the variant IL-2 peptide has the amino acid sequence of wild-type IL-2 but with H16A and F42A substitutions (shown in bold). Alternatively, the aforementioned sequence can be used, but with substitutions other than Ala at H16 and / or F42; for example, H16T can be used instead of H16A. In some cases, the variant IL-2 polypeptide present in TMP contains the following amino acid sequence: APTSSSTKKT QLQLEALLLD LQMILNGINNYKNPKLTRML TAKFYMPKKA TELKHLQCLEEELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSETTFMCEYADE TATIVEFLNRWITFCQSIIS TLT (SEQ ID NO:490). In some cases, the variant IL-2 polypeptide present in TMMP contains the following amino acid sequence: APTSSSTKKT QLQLETLLLD LQMILNGINN YKNPKLTRMLTAKFYMPKKA TELKHLQCLEEELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADETATIVEFLNRWITFCQSIIS TLT (SEQ ID NO:491). In some cases, M contains two copies of this variant IL-2 polypeptide.

[0423] Additional peptides

[0424] In addition to the peptides described above, the peptides of the TMMP disclosed herein may also include one or more peptides. Suitable additional peptides include epitope tags and affinity domains. The one or more additional peptides may be located at the N-terminus of the TMMP peptide chain, at the C-terminus of the TMMP peptide chain, or within the TMMP peptide chain.

[0425] Epitope label

[0426] Suitable epitope tags include, but are not limited to, hemagglutinins (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), etc.

[0427] Affinity domain

[0428] The affinity domain includes a peptide sequence that can interact with a binding partner, such as a binding partner immobilized on a solid support for identification or purification. DNA sequences encoding multiple consecutive single amino acids, such as histidines, when fused to the expressed protein, can be used to purify recombinant proteins in one step by binding with a resin column such as nickel agarose via high affinity. Exemplary affinity domains are provided in published PCT application WO 2019 / 051091, published on March 14, 2019. See

[00355] .

[0429] Drug conjugates

[0430] The polypeptide chain of the TMMP disclosed herein may contain a small molecule drug attached (e.g., covalently attached) to the polypeptide chain. For example, when the TMMP of this disclosure contains an Fc polypeptide, the Fc polypeptide may contain a covalently attached small molecule drug. In some cases, the small molecule drug is a cancer chemotherapeutic agent, such as a cytotoxic agent. Such drug conjugates and suitable chemotherapeutic agents are disclosed in the published PCT application WO 2019 / 051091, published on March 14, 2019. See

[00356] -

[00363] .

[0431] Exemplary TMMP

[0432] The TMMP of this 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 polypeptide and / or the second polypeptide comprises an MOD. Therefore, in some cases, the TMMP of this disclosure 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 cases, the 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 cases, 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 cases, the at least one MOD is a wild-type immunomodulatory polypeptide. In other cases, the at least one MOD is a variant MOD exhibiting a reduced affinity for the co-immunomodulatory polypeptide compared to the affinity of the corresponding wild-type MOD for the co-immunomodulatory polypeptide. In some cases, TMMP comprises two MODs having the same amino acid sequence.

[0433] In some cases, TMMP comprises: a) a first polypeptide, which comprises, from N-terminus to C-terminus,: i) a WT-1 peptide epitope; ii) a first MHC polypeptide; and iii) at least one MOD; and b) a second polypeptide, which comprises, from N-terminus to C-terminus,: i) a second MHC polypeptide; and ii) an Ig Fc polypeptide. In some cases, the first MHC polypeptide is a β2M polypeptide; and the second MHC polypeptide is an HLA heavy chain polypeptide. In some cases, the HLA heavy chain polypeptide is an HLA-A24 polypeptide. In some cases, the HLA heavy chain polypeptide is an HLA-A24 polypeptide with a Y84C substitution. In some cases, the HLA heavy chain polypeptide is an HLA-A24 polypeptide with a Y84C substitution and Ala at position 236. In some cases, the HLA heavy chain polypeptide is an HLA-A24 polypeptide with a Y84A substitution. In some cases, the HLA heavy chain polypeptide is an HLA-A24 polypeptide with Y84A and A236C substitutions. In some cases, the HLA heavy chain polypeptide is an HLA-A24 polypeptide with Y84C and A236C substitutions. In some cases, the β2M polypeptide contains Arg (R12) at position 12. In some cases, the β2M polypeptide contains an R12C substitution. In some cases, the HLA heavy chain polypeptide is an HLA-A24 polypeptide with an A236C substitution. In some cases, the first polypeptide comprises, from N-terminus to C-terminus: i) a WT-1 peptide epitope; ii) a first MHC polypeptide; and iii) two MODs having the same amino acid sequence. In some cases, the Ig Fc polypeptide is a human IgG1 Fc polypeptide. In some cases, the Ig Fc polypeptide is an IgG1 Fc polypeptide containing L234A and L235A substitutions. In some cases, the first polypeptide and the second polypeptide are linked to each other by disulfide bonds. In some cases, the MOD is a variant IL-2 polypeptide containing H16A and F42A substitutions. In some cases, the MOD is a variant IL-2 polypeptide containing H16T and F42A substitutions. In some cases, the peptide linker is between one or more of the following: i) a second MHC polypeptide with an Ig Fc polypeptide; ii) an epitope with a first MHC polypeptide; iii) a first MHC polypeptide with a MOD; and (when the TMMP contains two MODs on the first polypeptide chain) iv) between two MODs. In some cases, the peptide linker contains the amino acid sequence AAAGG (SEQ ID NO: 283). In some cases, the peptide linker contains the amino acid sequence (GGGGS)n (SEQ ID NO: 284), where n is an integer from 1 to 10 (e.g., where n is 2, 3, or 4). In some cases, the peptide linker contains the amino acid sequence GCGGS(GGGGS)n (SEQ ID NO: 319), where n is an integer from 1 to 9 (e.g., where n is 2, 3, or 4).In some cases, the WT-1 peptide epitope is CMTWNQMN (SEQ ID NO: 261). In some cases, the WT-1 peptide epitope is CYTWNQMNL (SEQ ID NO: 262). In some cases, the WT-1 peptide epitope is SMTWNQMNL (SEQ ID NO: 451). In some cases, the WT-1 peptide epitope is GCMTWNQMNL (SEQ ID NO: 452). In some cases, the WT-1 peptide epitope is SYTWNQMNL (SEQ ID NO: 453). In some cases, the WT-1 peptide epitope is GCYTWNQMNL (SEQ ID NO: 454).

[0434] In some cases, TMMP comprises: a) a first polypeptide, which comprises, from N-terminus to C-terminus,: i) a WT-1 peptide epitope; and ii) a first MHC polypeptide; and b) a second polypeptide, which comprises, from N-terminus to C-terminus,: i) at least one MOD; ii) a second MHC polypeptide; and iii) an Ig Fc polypeptide. In some cases, the first MHC polypeptide is a β2M polypeptide; and the second MHC polypeptide is an HLA heavy chain polypeptide. In some cases, the HLA heavy chain polypeptide is an HLA-A24 polypeptide. In some cases, the HLA heavy chain polypeptide is an HLA-A24 polypeptide with an A236C substitution. In some cases, the HLA heavy chain polypeptide is an HLA-A24 polypeptide with a Y84C substitution. In some cases, the HLA heavy chain polypeptide is an HLA-A24 polypeptide with a Y84C substitution and an Ala at position 236. In some cases, the HLA heavy chain polypeptide is an HLA-A24 polypeptide with a Y84A substitution. In some cases, the HLA heavy chain polypeptide is an HLA-A24 polypeptide with Y84A and A236C substitutions. In some cases, the HLA heavy chain polypeptide is an HLA-A24 polypeptide with Y84C and A236C substitutions. In some cases, the β2M polypeptide contains Arg (R12) at position 12. In some cases, the β2M polypeptide contains an R12C substitution. In some cases, the second polypeptide contains, from N-terminus to C-terminus: i) two MODs having the same amino acid sequence; ii) a second MHC polypeptide; and iii) an Ig Fc polypeptide. In some cases, the Ig Fc polypeptide is a human IgG1 Fc polypeptide. In some cases, the Ig Fc polypeptide is an IgG1 Fc polypeptide containing L234A and L235A substitutions. In some cases, the first and second polypeptides are linked to each other by disulfide bonds. In some cases, the MOD is a variant IL-2 polypeptide containing H16A and F42A substitutions. In some cases, the MOD is a variant IL-2 polypeptide containing H16T and F42A substitutions. In some cases, the peptide linker is between one or more of the following: i) a second MHC polypeptide with an Ig Fc polypeptide; ii) an epitope with a first MHC polypeptide; iii) a first MHC polypeptide with a MOD; and (when TMMP contains two MODs on the second polypeptide chain) iv) between two MODs. In some cases, the peptide linker contains the amino acid sequence AAAGG (SEQ ID NO: 283). In some cases, the peptide linker contains the amino acid sequence (GGGGS)n (SEQ ID NO: 284), where n is an integer from 1 to 10 (e.g., where n is 2, 3, or 4). In some cases, the peptide linker contains the amino acid sequence GCGGS(GGGGS)n (SEQ ID NO: 319), where n is an integer from 1 to 9 (e.g., where n is 2, 3, or 4).In some cases, the WT-1 peptide epitope is CMTWNQMN (SEQ ID NO: 261). In some cases, the WT-1 peptide epitope is CYTWNQMNL (SEQ ID NO: 262). In some cases, the WT-1 peptide epitope is SMTWNQMNL (SEQ ID NO: 451). In some cases, the WT-1 peptide epitope is GCMTWNQMNL (SEQ ID NO: 452). In some cases, the WT-1 peptide epitope is SYTWNQMNL (SEQ ID NO: 453). In some cases, the WT-1 peptide epitope is GCYTWNQMNL (SEQ ID NO: 454).

[0435] In some cases, TMMP comprises: a) a first polypeptide, which comprises, from N-terminus to C-terminus,: i) a WT-1 peptide epitope; and ii) a first MHC polypeptide; and b) a second polypeptide, which comprises, from N-terminus to C-terminus,: i) a second MHC polypeptide; ii) an Ig Fc polypeptide; and iii) at least one MOD. In some cases, the first MHC polypeptide is a β2M polypeptide; and the second MHC polypeptide is an HLA heavy chain polypeptide. In some cases, the HLA heavy chain polypeptide is an HLA-A24 polypeptide. In some cases, the HLA heavy chain polypeptide is an HLA-A24 polypeptide with an A236C substitution. In some cases, the HLA heavy chain polypeptide is an HLA-A24 polypeptide with a Y84C substitution. In some cases, the HLA heavy chain polypeptide is an HLA-A24 polypeptide with a Y84C substitution and an Ala at position 236. In some cases, the HLA heavy chain polypeptide is an HLA-A24 polypeptide with a Y84A substitution. In some cases, the HLA heavy chain polypeptide is an HLA-A24 polypeptide with Y84A and A236C substitutions. In some cases, the HLA heavy chain polypeptide is an HLA-A24 polypeptide with Y84C and A236C substitutions. In some cases, the β2M polypeptide contains Arg (R12) at position 12. In some cases, the β2M polypeptide contains an R12C substitution. In some cases, the second polypeptide comprises, from N-terminus to C-terminus: i) a second MHC polypeptide; ii) an Ig Fc polypeptide; and iii) two MODs having the same amino acid sequence. In some cases, the Ig Fc polypeptide is a human IgG1 Fc polypeptide. In some cases, the Ig Fc polypeptide is an IgG1 Fc polypeptide containing L234A and L235A substitutions. In some cases, the first and second polypeptides are linked to each other by disulfide bonds. In some cases, the MOD is a variant IL-2 polypeptide containing H16A and F42A substitutions. In some cases, the MOD is a variant IL-2 polypeptide containing H16T and F42A substitutions. In some cases, the peptide linker is between one or more of the following: i) a second MHC polypeptide with an Ig Fc polypeptide; ii) an epitope with a first MHC polypeptide; iii) an Ig Fc polypeptide with a MOD; and (when the TMMP contains both MODs on the second polypeptide chain) iv) between these two MODs. In some cases, the peptide linker contains the amino acid sequence AAAGG (SEQ ID NO: 283). In some cases, the peptide linker contains the amino acid sequence (GGGGS)n (SEQ ID NO: 284), where n is an integer from 1 to 10 (e.g., where n is 2, 3, or 4).In some cases, the peptide linker comprises the amino acid sequence GCGGS(GGGGS)n (SEQ ID NO: 319), where n is an integer from 1 to 9 (e.g., where n is 2, 3, or 4). In some cases, the WT-1 peptide epitope is CMTWNQMN (SEQ ID NO: 261). In some cases, the WT-1 peptide epitope is CYTWNQMNL (SEQ ID NO: 262). In some cases, the WT-1 peptide epitope is SMTWNQMNL (SEQ ID NO: 451). In some cases, the WT-1 peptide epitope is GCMTWNQMNL (SEQ ID NO: 452). In some cases, the WT-1 peptide epitope is SYTWNQMNL (SEQ ID NO: 453). In some cases, the WT-1 peptide epitope is GCYTWNQMNL (SEQ ID NO: 454).

[0436] In some cases, 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 cases, the first MHC polypeptide is a β2M polypeptide; and the second MHC polypeptide is an HLA heavy chain polypeptide. In some cases, the HLA heavy chain polypeptide is an HLA-A24 polypeptide. In some cases, the HLA heavy chain polypeptide is an HLA-A24 polypeptide with an A236C substitution. In some cases, the Ig Fc polypeptide is a human IgG1 Fc polypeptide. In some cases, the Ig Fc polypeptide is an IgG1 Fc polypeptide containing L234A and L235A substitutions. In some cases, the first polypeptide and the second polypeptide are linked to each other by disulfide bonds. In some cases, the MOD is a variant IL-2 polypeptide containing H16A and F42A substitutions. In some cases, MOD is a variant IL-2 polypeptide containing H16T and F42A substitutions. In some cases, the WT-1 peptide epitope is CMTWNQMN (SEQ ID NO: 261). In some cases, the WT-1 peptide epitope is CYTWNQMNL (SEQ ID NO: 262).

[0437] In some cases, TMMP comprises: a) a first polypeptide, which comprises, from N-terminus to C-terminus,: i) at least one MOD; ii) a WT-1 peptide epitope; and iii) a first MHC polypeptide; and b) a second polypeptide, which comprises, from N-terminus to C-terminus,: i) a second MHC polypeptide; and ii) an Ig Fc polypeptide. In some cases, the first MHC polypeptide is a β2M polypeptide; and the second MHC polypeptide is an HLA heavy chain polypeptide. In some cases, the HLA heavy chain polypeptide is an HLA-A24 polypeptide. In some cases, the HLA heavy chain polypeptide is an HLA-A24 polypeptide with an A236C substitution. In some cases, the first polypeptide comprises, from N-terminus to C-terminus,: i) two MODs having the same amino acid sequence; ii) a WT-1 peptide epitope; and iii) a first MHC polypeptide. In some cases, the Ig Fc polypeptide is a human IgG1 Fc polypeptide. In some cases, the Ig Fc polypeptide is an IgG1 Fc polypeptide containing L234A and L235A substitutions. In some cases, the first polypeptide and the second polypeptide are linked to each other by disulfide bonds. In some cases, the MOD is a variant IL-2 polypeptide containing H16A and F42A substitutions. In some cases, the MOD is a variant IL-2 polypeptide containing H16T and F42A substitutions. In some cases, the peptide linker is between one or more of the following: 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 TMMP contains two MODs on the first polypeptide chain) iv) between two MODs. In some cases, the peptide linker contains the amino acid sequence AAAGG (SEQ ID NO: 283). In some cases, the peptide linker contains the amino acid sequence (GGGGS)n (SEQ ID NO: 284), where n is an integer from 1 to 10 (e.g., where n is 2, 3, or 4). In some cases, the WT-1 peptide epitope is CMTWNQMN (SEQ ID NO: 261). In other cases, the WT-1 peptide epitope is CYTWNQMNL (SEQ ID NO: 262).

[0438] In some cases, 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 cases, the first MHC polypeptide is a β2M polypeptide; and the second MHC polypeptide is an HLA heavy chain polypeptide. In some cases, the HLA heavy chain polypeptide is an HLA-A24 polypeptide. In some cases, the HLA heavy chain polypeptide is an HLA-A24 polypeptide with an A236C substitution. In some cases, the second polypeptide comprises, from N-terminus to C-terminus: i) a second MHC polypeptide; ii) two MODs having the same amino acid sequence; and iii) an Ig Fc polypeptide. In some cases, the Ig Fc polypeptide is a human IgG1 Fc polypeptide. In some cases, the Ig Fc polypeptide is an IgG1 Fc polypeptide containing L234A and L235A substitutions. In some cases, the first polypeptide and the second polypeptide are linked to each other by disulfide bonds. In some cases, the MOD is a variant IL-2 polypeptide containing H16A and F42A substitutions. In some cases, the MOD is a variant IL-2 polypeptide containing H16T and F42A substitutions. In some cases, the peptide linker is between one or more of the following: 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 (when TMMP contains both MODs on the second polypeptide chain) iv) between these two MODs. In some cases, the peptide linker contains the amino acid sequence AAAGG (SEQ ID NO: 283). In some cases, the peptide linker contains the amino acid sequence (GGGGS)n (SEQ ID NO: 284), where n is an integer from 1 to 10 (e.g., where n is 2, 3, or 4). In some cases, the WT-1 peptide epitope is CMTWNQMN (SEQ ID NO: 261). In other cases, the WT-1 peptide epitope is CYTWNQMNL (SEQ ID NO: 262).

[0439] In some cases, TMMP comprises: a) a first polypeptide, which comprises, from N-terminus to C-terminus, at least one MOD; ii) a WT-1 peptide epitope; and iii) a first MHC polypeptide; and b) a second polypeptide, which comprises, from N-terminus to C-terminus, at least one MHC polypeptide; and ii) an Ig Fc polypeptide. In some cases, the first MHC polypeptide is a β2M polypeptide; and the second MHC polypeptide is an HLA heavy chain polypeptide. In some cases, the HLA heavy chain polypeptide is an HLA-A24 polypeptide. In some cases, the HLA heavy chain polypeptide is an HLA-A24 polypeptide with an A236C substitution. In some cases, the HLA heavy chain polypeptide is an HLA-A24 polypeptide with a Y84C substitution. In some cases, the HLA heavy chain polypeptide is an HLA-A24 polypeptide with a Y84C substitution and an Ala at position 236. In some cases, the HLA heavy chain polypeptide is an HLA-A24 polypeptide with a Y84A substitution. In some cases, the HLA heavy chain polypeptide is an HLA-A24 polypeptide with Y84A and A236C substitutions. In some cases, the HLA heavy chain polypeptide is an HLA-A24 polypeptide with Y84C and A236C substitutions. In some cases, the β2M polypeptide contains Arg (R12) at position 12. In some cases, the β2M polypeptide contains an R12C substitution. In some cases, the first polypeptide contains, from N-terminus to C-terminus: i) two MODs having the same amino acid sequence; ii) a WT-1 peptide epitope; and iii) a first MHC polypeptide. In some cases, the Ig Fc polypeptide is a human IgG1 Fc polypeptide. In some cases, the Ig Fc polypeptide is an IgG1 Fc polypeptide containing L234A and L235A substitutions. In some cases, the first polypeptide and the second polypeptide are linked to each other by disulfide bonds. In some cases, the MOD is a variant IL-2 polypeptide containing H16A and F42A substitutions. In some cases, the MOD is a variant IL-2 polypeptide containing H16T and F42A substitutions. In some cases, the peptide linker is between one or more of the following: i) a second MHC polypeptide and an Ig Fc polypeptide; ii) an epitope and a first MHC polypeptide; iii) a MOD and an epitope; and (when the TMMP contains two immunomodulatory polypeptides on the first polypeptide chain) iv) between two MODs. In some cases, the peptide linker contains the amino acid sequence AAAGG (SEQ ID NO: 283). In some cases, the peptide linker contains the amino acid sequence (GGGGS)n (SEQ ID NO: 284), where n is an integer from 1 to 10 (e.g., where n is 2, 3, or 4). In some cases, the peptide linker contains the amino acid sequence GCGGS(GGGGS)n (SEQ ID NO: 319), where n is an integer from 1 to 9 (e.g., where n is 2, 3, or 4).In some cases, the WT-1 peptide epitope is CMTWNQMN (SEQ ID NO: 261). In some cases, the WT-1 peptide epitope is CYTWNQMNL (SEQ ID NO: 262). In some cases, the WT-1 peptide epitope is SMTWNQMNL (SEQ ID NO: 451). In some cases, the WT-1 peptide epitope is GCMTWNQMNL (SEQ ID NO: 452). In some cases, the WT-1 peptide epitope is SYTWNQMNL (SEQ ID NO: 453). In some cases, the WT-1 peptide epitope is GCYTWNQMNL (SEQ ID NO: 454).

[0440] In some cases, TMMP comprises: a) a first polypeptide comprising, in order from N-terminus to C-terminus: i) a WT-1 peptide epitope; and ii) a first MHC polypeptide; and b) a second polypeptide comprising, in order from N-terminus to C-terminus: i) a second MHC polypeptide; ii) at least one MOD; and iii) an Ig Fc polypeptide. In some cases, the first MHC polypeptide is a β2M polypeptide; and the second MHC polypeptide is an HLA heavy chain polypeptide. In some cases, the HLA heavy chain polypeptide is an HLA-A24 polypeptide. In some cases, the HLA heavy chain polypeptide is an HLA-A24 polypeptide with an A236C substitution. In some cases, the HLA heavy chain polypeptide is an HLA-A24 polypeptide with a Y84C substitution. In some cases, the HLA heavy chain polypeptide is an HLA-A24 polypeptide with a Y84C substitution and an Ala at position 236. In some cases, the HLA heavy chain polypeptide is an HLA-A24 polypeptide with a Y84A substitution. In some cases, the HLA heavy chain polypeptide is an HLA-A24 polypeptide with Y84A and A236C substitutions. In some cases, the HLA heavy chain polypeptide is an HLA-A24 polypeptide with Y84C and A236C substitutions. In some cases, the β2M polypeptide contains Arg (R12) at position 12. In some cases, the β2M polypeptide contains an R12C substitution. In some cases, the second polypeptide comprises, from N-terminus to C-terminus: i) a second MHC polypeptide; ii) two MODs having the same amino acid sequence; and iii) an Ig Fc polypeptide. In some cases, the Ig Fc polypeptide is a human IgG1 Fc polypeptide. In some cases, the Ig Fc polypeptide is an IgG1 Fc polypeptide containing L234A and L235A substitutions. In some cases, the first and second polypeptides are linked to each other by disulfide bonds. In some cases, the MOD is a variant IL-2 polypeptide containing H16A and F42A substitutions. In some cases, the MOD is a variant IL-2 polypeptide containing H16T and F42A substitutions. In some cases, the peptide linker is between one or more of the following: i) a second MHC polypeptide with the MOD; ii) the MOD with an Ig Fc polypeptide; iii) an epitope with a first MHC polypeptide; iii) a first MHC polypeptide with the MOD; and (when the TMMP contains both MODs on the second polypeptide chain) iv) between these two MODs. In some cases, the peptide linker contains the amino acid sequence AAAGG (SEQ ID NO:283). In some cases, the peptide linker contains the amino acid sequence (GGGGS)n (SEQ ID NO:284), where n is an integer from 1 to 10 (e.g., where n is 2, 3, or 4).In some cases, the peptide linker comprises the amino acid sequence GCGGS(GGGGS)n (SEQ ID NO:319), where n is an integer from 1 to 9 (e.g., where n is 2, 3, or 4). In some cases, the WT-1 peptide epitope is CMTWNQMN (SEQ ID NO:261). In some cases, the WT-1 peptide epitope is CYTWNQMNL (SEQ ID NO:262). In some cases, the WT-1 peptide epitope is SMTWNQMNL (SEQ ID NO:451). In some cases, the WT-1 peptide epitope is GCMTWNQMNL (SEQ ID NO:452). In some cases, the WT-1 peptide epitope is SYTWNQMNL (SEQ ID NO:453). In some cases, the WT-1 peptide epitope is GCYTWNQMNL (SEQ ID NO:454).

[0441] As mentioned above and as Figure 19 As illustrated, MODs (i.e., one or more MODs) may be present at any of the multiple locations of the TMMP of this disclosure. Figure 19 The locations of the two copies of the variant IL-2 peptide are depicted; however, the MOD can be any of the various MODs described herein. Figure 19As described, the MOD may be: 1) at the N-terminus of the class I MHC heavy chain (position 1); 2) at the C-terminus of the class I MHC heavy chain and the N-terminus of the Ig Fc polypeptide; in other words, between the class I MHC heavy chain and the Ig Fc polypeptide (position 2); 3) at the C-terminus of the Ig Fc polypeptide (position 3); 4) at the N-terminus of the peptide epitope (position 4); or 5) at 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 at the N-terminus of the class I MHC heavy chain; for example, wherein the TMMP comprises: a) a first polypeptide, which comprises, in order from N-terminus to C-terminus: i) a peptide epitope (e.g., WT-1 peptide); and ii) a β2M polypeptide; and b) a second polypeptide, which comprises, in order 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 MOD on the same polypeptide chain as the class I MHC heavy chain, but at the C-terminus of the class I MHC heavy chain rather than the C-terminus of the polypeptide chain; for example, wherein TMMP comprises: a) a first polypeptide, which comprises, in order from N-terminus to C-terminus: i) a peptide epitope (e.g., WT-1 peptide); and ii) a β2M polypeptide; and b) a second polypeptide, which comprises, in order from N-terminus to C-terminus: i) a class I MHC heavy chain polypeptide; ii) one or more MODs; and iii) an IgFc polypeptide. "Position 3" refers to the position of MOD on the same polypeptide chain as the class I MHC heavy chain and at the C-terminus of the polypeptide chain; for example, wherein TMMP comprises: a) a first polypeptide, which comprises, in order from N-terminus to C-terminus: i) a peptide epitope (e.g., WT-1 peptide); and ii) a β2M polypeptide; and b) a second polypeptide, which comprises, in order 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 MOD on the same polypeptide chain as the β2M polypeptide and at the peptide epitope and the N-terminus of the β2M polypeptide; for example, wherein TMMP comprises: a) a first polypeptide, the first polypeptide comprising, from N-terminus to C-terminus: i) one or more immunomodulatory polypeptides; ii) a peptide epitope (e.g., WT-1 peptide); and iii) the 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 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, wherein TMMP comprises: a) a first polypeptide, which comprises, in order from N-terminus to C-terminus: i) a peptide epitope (e.g., WT-1 peptide); and ii) a β2M polypeptide; and iii) one or more MODs; and b) a second polypeptide, which comprises a class I MHC heavy chain polypeptide (e.g., a second polypeptide, which comprises, in order from N-terminus to C-terminus: i) a class I MHC heavy chain polypeptide; and ii) an Ig Fc polypeptide).

[0442] In addition, as discussed above and as Figures 18A-18C Schematic illustration: The first and second polypeptide chains of TMMP may be linked by one or more disulfide bonds. For example, TMMP may comprise: a) a first polypeptide chain comprising a β2M polypeptide with an R12C substitution; and b) a second polypeptide chain comprising a class I MHC heavy chain polypeptide with an A236C substitution, such that a disulfide bond is formed between a Cys at position 12 of the β2M polypeptide in the first polypeptide chain and a Cys at position 236 of the class I MHC heavy chain polypeptide in the second polypeptide chain. As another example, TMMP may comprise: a) a first polypeptide comprising, in order from N-terminus to C-terminus: i) a peptide epitope; ii) a peptide linker comprising GCGGS (G4S). n (SEQ ID NO: 315) sequence, where n is 1, 2, or 3; and iii) β2M polypeptide; and b) a second polypeptide comprising a class I MHC heavy chain polypeptide with Y84C substitution, such that a disulfide bond is formed between Cys in the peptide linker of the first polypeptide chain and Cys at position 84 of the class I MHC heavy chain polypeptide of the second polypeptide chain. In other examples, TMMP may comprise: a) a first polypeptide comprising, in order from N-terminus to C-terminus: i) a peptide epitope; ii) a peptide linker comprising GCGGS (G4S). n(SEQ ID NO:315) sequence, where n is 1, 2, or 3; and iii) 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, such that: i) a first disulfide bond is formed between a Cys at the peptide linker of the first polypeptide chain and a Cys at position 84 of the class I MHC heavy chain polypeptide of the second polypeptide chain; and ii) a second disulfide bond is formed between a Cys at position 12 of the β2M polypeptide of the first polypeptide chain and a Cys at position 236 of the class I MHC heavy chain polypeptide of the second polypeptide chain. For simplicity, the first disulfide bond is referred to as "G2C / Y84C"; and the second disulfide bond is referred to as "R12C / A236C". TMMP may include: a) G2C / Y84C disulfide bonds without R12C / A236C disulfide bonds; b) R12C / A236C disulfide bonds without G2C / Y84C disulfide bonds; or c) G2C / Y84C disulfide bonds and R12C / A236C disulfide bonds.

[0443] TMMP may include: a) a G2C / Y84C disulfide bond without an R12C / A236C disulfide bond; and b) at least one immunomodulatory polypeptide at position 1. TMMP may include: a) a G2C / Y84C disulfide bond without an R12C / A236C disulfide bond; and b) at least one MOD at position 2. TMMP of this disclosure may include: a) a G2C / Y84C disulfide bond without an R12C / A236C disulfide bond; and b) at least one MOD at position 3. TMMP may include: a) a G2C / Y84C disulfide bond without an R12C / A236C disulfide bond; and b) at least one MOD at position 4. TMMP may include: a) a G2C / Y84C disulfide bond without an R12C / A236C disulfide bond; and b) at least one MOD at position 5.

[0444] TMMP may include: a) an R12C / A236C disulfide bond without a G2C / Y84C disulfide bond; and at least one MOD at position 1. TMMP may include: a) an R12C / A236C disulfide bond without a G2C / Y84C disulfide bond; and at least one MOD at position 2. TMMP may include: a) an R12C / A236C disulfide bond without a G2C / Y84C disulfide bond; and b) at least one MOD at position 3. The TMMP of this disclosure may include: a) an R12C / A236C disulfide bond without a G2C / Y84C disulfide bond; and at least one immunomodulatory polypeptide at position 4. TMMP may include: a) an R12C / A236C disulfide bond without a G2C / Y84C disulfide bond; and at least one MOD at position 5.

[0445] The TMMP may include: a) a G2C / Y84C disulfide bond and an R12C / A236C disulfide bond; and b) at least one MOD at position 1. The TMMP may include: a) a G2C / Y84C disulfide bond and an R12C / A236C disulfide bond; and b) at least one MOD at position 2. The TMMP may include: a) a G2C / Y84C disulfide bond and an R12C / A236C disulfide bond; and b) at least one MOD at position 3. The TMMP of this disclosure may include: a) a G2C / Y84C disulfide bond and an R12C / A236C disulfide bond; and b) at least one MOD at position 4. The TMMP may include: a) a G2C / Y84C disulfide bond and an R12C / A236C disulfide bond; and b) at least one MOD at position 5.

[0446] Non-limiting examples of the amino acid sequences of the first and second polypeptide chains of TMMP disclosed herein are provided. Figure 4A -4K and Figure 20A-20R middle.

[0447] In some cases, TMMP comprises: a) a first polypeptide chain, the first polypeptide chain comprising, as Figure 4D The depicted amino acid sequence is designated as "2752"; and b) a second polypeptide chain, the second polypeptide chain comprising as follows Figure 4C The amino acid sequence indicated by “3159” is depicted.

[0448] In some cases, TMMP comprises: a) a first polypeptide chain, the first polypeptide chain comprising, as Figure 4E The depicted amino acid sequence is indicated as "2753"; and b) a second polypeptide chain, the second polypeptide chain comprising as follows Figure 4C The described amino acid sequence is designated "3159". This type of TMMP contains: a) as... Figure 19 The MOD at position 3 is depicted; and b) the R12C / A236C disulfide bond (instead of the G2C / Y84C disulfide bond).

[0449] In some cases, TMMP comprises: a) a first polypeptide chain, the first polypeptide chain comprising, as Figure 4D The depicted amino acid sequence is designated as "2752"; and b) a second polypeptide chain, the second polypeptide chain comprising as follows Figure 4B The amino acid sequence indicated by “2750” is depicted.

[0450] In some cases, TMMP comprises: a) a first polypeptide chain, the first polypeptide chain comprising, as Figure 4E The depicted amino acid sequence is indicated as "2753"; and b) a second polypeptide chain, the second polypeptide chain comprising as follows Figure 4BThe described amino acid sequence is designated "2750". This type of TMMP contains: a) as... Figure 19 The MOD at position 1 is depicted; and b) the R12C / A236C disulfide bond (instead of the G2C / Y84C disulfide bond).

[0451] In some cases, TMMP comprises: a) a first polypeptide chain, the first polypeptide chain comprising, as Figure 4D The depicted amino acid sequence is designated as "2752"; and b) a second polypeptide chain, the second polypeptide chain comprising as follows Figure 4A The amino acid sequence indicated by “3158” is depicted.

[0452] In some cases, TMMP comprises: a) a first polypeptide chain, the first polypeptide chain comprising, as Figure 4E The depicted amino acid sequence is indicated as "2753"; and b) a second polypeptide chain, the second polypeptide chain comprising as follows Figure 4A The amino acid sequence indicated by “3158” is depicted.

[0453] In some cases, TMMP comprises: a) a first polypeptide chain, the first polypeptide chain comprising, as Figure 14B The depicted amino acid sequence is indicated as "2380"; and b) a second polypeptide chain, the second polypeptide chain comprising as follows Figure 14A The amino acid sequence indicated by “1715” is depicted.

[0454] In some cases, TMMP comprises: a) a first polypeptide chain comprising, in order from N-terminus to C-terminus: i) a WT-1 peptide with the sequence VLDFAPPGA (SEQ ID NO:259); ii) a linker having the amino acid sequence GCGGSGGGGSGGGGS (SEQ ID NO:317); iii) a β2M polypeptide comprising Cys at position 12 (e.g., β2M having the amino acid sequence listed in SEQ ID NO:311); and b) a second polypeptide chain comprising, in order from N-terminus to C-terminus: i) a variant IL-2 polypeptide comprising H16A and F42A substitutions (i.e., containing Ala at positions 16 and 42, e.g., the amino acid sequence listed in SEQ ID NO:188, where X1 is Ala and X2 is Ala); ii) a (GGGGS)4 linker; iii) Variant IL-2 peptides containing H16A and F42A substitutions (i.e., containing Ala at positions 16 and 42, such as the amino acid sequence listed in SEQ ID NO:188, where X1 is Ala and X2 is Ala); iv) (GGGGS)4 linker; v) HLA A0202 heavy chain containing Cys at positions 84 and 236 (e.g., HLA heavy chain peptides containing the amino acid sequence listed in SEQ ID NO:341); vi) AAAGG linker; and vii) Ig Fc peptides. In some cases, the Ig Fc peptide contains... Figure 5A-5G The amino acid sequence of the Fc region depicted in 5H has at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity. In some cases, the Ig Fc peptide is a variant Ig Fc peptide that contains one or more sequence variations relative to the wild-type peptide, wherein the ability of the Ig Fc peptide to induce cell lysis via complement-dependent cytotoxicity (CDC) and / or antibody-dependent cytotoxicity (ADCC) is reduced or substantially eliminated. In some cases, the Ig Fc peptide is a variant human IgG1 Fc peptide that contains L234A and / or L235A substitutions ( Figure 5H The amino acid sequences depicted are L14 and L15. In some cases, Ig Fc peptides contain... Figure 5H The amino acid sequence described in the diagram and listed in SEQ ID NO:487.

[0455] In some cases, TMMP comprises: a) a first polypeptide chain comprising, in order 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 GCGGSGGGGSGGGGS (SEQ ID NO: 317); and iii) a β2M polypeptide comprising Cys at position 12 (e.g., β2M having the amino acid sequence listed in SEQ ID NO: 311); and b) a second polypeptide chain comprising, in order from N-terminus to C-terminus: i) a variant IL-2 polypeptide comprising H16A and F42A substitutions (i.e., comprising Ala at positions 16 and 42, e.g., the amino acid sequence listed in SEQ ID NO: 188, wherein X1 is Ala and X2 is Ala); ii) (GGGGS)4 linker; iii) variant IL-2 peptides containing H16A and F42A substitutions (i.e., containing Ala at positions 16 and 42, such as the amino acid sequence listed in SEQ ID NO:188, where X1 is Ala and X2 is Ala); iv) (GGGGS)4 linker; v) HLA A0202 heavy chain peptides containing Cys at positions 84 and 236 (e.g., HLA heavy chain peptides containing the amino acid sequence listed in SEQ ID NO:341); vi) AAAGG linker; and vii) Ig Fc peptides containing Ala at positions 14 and 15 and lacking C-terminal Lys (e.g., containing... Figure 5H The Ig Fc polypeptide depicted in SEQ ID NO:487). For example, in some cases, TMMP comprises: a) a first polypeptide chain containing, for example, the amino acid sequence listed in SEQ ID NO:487. Figure 14B The depicted amino acid sequence is indicated as "2380"; and b) a second polypeptide chain, the second polypeptide chain comprising as follows Figure 14J The amino acid sequence indicated by “1715” is depicted. Figure 14J The construct described in the document (“1715 without C-terminal Lys”) is also referred to as “1715Δ” in this document.

[0456] In some cases, TMMP comprises: a) a first polypeptide chain, the first polypeptide chain comprising, as Figure 14B The depicted amino acid sequence is designated as "2381"; and b) a second polypeptide chain, the second polypeptide chain comprising as follows Figure 14A The depicted date is "1715" or as... Figure 14J The depicted amino acid sequence is indicated by 1715Δ and is listed in SEQ ID NO:486.

[0457] In some cases, TMMP comprises: a) a first polypeptide chain, the first polypeptide chain comprising, as Figure 14BThe depicted amino acid sequence is indicated as "2380"; and b) a second polypeptide chain, the second polypeptide chain comprising as follows Figure 14D The amino acid sequence indicated by “2405” is depicted.

[0458] In some cases, TMMP comprises: a) a first polypeptide chain, the first polypeptide chain comprising, as Figure 14B The depicted amino acid sequence is designated as "2381"; and b) a second polypeptide chain, the second polypeptide chain comprising as follows Figure 14D The depicted amino acid sequence is designated "2405". In some cases, TMMP comprises: a) a first polypeptide chain containing, as shown in the image below. Figure 14F The depicted amino acid sequence is indicated as "2762"; and b) a second polypeptide chain, the second polypeptide chain comprising as follows Figure 14D The amino acid sequence indicated by “2405” is depicted.

[0459] In some cases, TMMP comprises: a) a first polypeptide chain, the first polypeptide chain comprising, as Figure 14B The depicted amino acid sequence is indicated as "2380"; and b) a second polypeptide chain, the second polypeptide chain comprising as follows Figure 14E The amino acid sequence indicated by "1380" is depicted.

[0460] In some cases, TMMP comprises: a) a first polypeptide chain, the first polypeptide chain comprising, as Figure 14B The depicted amino acid sequence is designated as "2381"; and b) a second polypeptide chain, the second polypeptide chain comprising as follows Figure 14E The amino acid sequence indicated by "1380" is depicted.

[0461] In some cases, TMMP comprises: a) a first polypeptide chain, the first polypeptide chain comprising, as Figure 20A The depicted amino acid sequence is designated as "3592"; and b) a second polypeptide chain, the second polypeptide chain comprising as follows Figure 20H The described amino acid sequence is designated as "3188". This type of TMMP contains: a) as... Figure 19 The MOD at position 1 is depicted; and b) both the G2C / Y84C disulfide bond and the R12C / A236C disulfide bond.

[0462] In some cases, TMMP comprises: a) a first polypeptide chain, the first polypeptide chain comprising, as Figure 20B The depicted amino acid sequence is indicated as "3425"; and b) a second polypeptide chain, the second polypeptide chain comprising as follows Figure 20H The described amino acid sequence is designated as "3188". This type of TMMP contains: a) as... Figure 19The MOD at position 3 is depicted; and b) both the G2C / Y84C disulfide bond and the R12C / A236C disulfide bond.

[0463] In some cases, TMMP comprises: a) a first polypeptide chain, the first polypeptide chain comprising, as Figure 20C The depicted amino acid sequence is indicated as "3196"; and b) a second polypeptide chain, the second polypeptide chain comprising as follows Figure 20I The described amino acid sequence is designated as "3604". This type of TMMP contains: a) as... Figure 19 The MOD at position 5 is depicted; and b) both the G2C / Y84C disulfide bond and the R12C / A236C disulfide bond.

[0464] In some cases, TMMP comprises: a) a first polypeptide chain, the first polypeptide chain comprising, as Figure 20D The depicted amino acid sequence is indicated as "2764"; and b) a second polypeptide chain, the second polypeptide chain comprising as follows Figure 20J The described amino acid sequence is designated as "3603". This type of TMMP contains: a) as... Figure 19 The MOD at position 5 is depicted; and b) the R12C / A236C disulfide bond (instead of the G2C / Y84C disulfide bond).

[0465] In some cases, TMMP comprises: a) a first polypeptide chain, the first polypeptide chain comprising, as Figure 20E The depicted amino acid sequence is designated as "3593"; and b) a second polypeptide chain, the second polypeptide chain comprising as follows Figure 20K The described amino acid sequence is designated "3192". This type of TMMP contains: a) as... Figure 19 The MOD at position 1 is depicted; and b) the G2C / Y84C disulfide bond (instead of the R12C / A236C disulfide bond).

[0466] In some cases, TMMP comprises: a) a first polypeptide chain, the first polypeptide chain comprising, as Figure 20F The depicted amino acid sequence is designated as "3426"; and b) a second polypeptide chain, the second polypeptide chain comprising as follows Figure 20K The described amino acid sequence is designated "3192". This type of TMMP contains: a) as... Figure 19 The MOD at position 3 is depicted; and b) the G2C / Y84C disulfide bond (instead of the R12C / A236C disulfide bond).

[0467] In some cases, TMMP comprises: a) a first polypeptide chain, the first polypeptide chain comprising, as Figure 20GThe depicted amino acid sequence is indicated as "3197"; and b) a second polypeptide chain, the second polypeptide chain comprising as follows Figure 20L The described amino acid sequence is designated as "3605". This type of TMMP contains: a) as... Figure 19 The MOD at position 5 is depicted; and b) the G2C / Y84C disulfide bond (instead of the R12C / A236C disulfide bond).

[0468] In some cases, TMMP comprises: a) a first polypeptide chain, the first polypeptide chain comprising, as Figure 20A The depicted amino acid sequence is designated as "3592"; and b) a second polypeptide chain, the second polypeptide chain comprising as follows Figure 20M The depicted amino acid sequence is designated as "3529". This type of TMMP contains: a) as... Figure 19 The MOD at position 1 is depicted; and b) both the G2C / Y84C disulfide bond and the R12C / A236C disulfide bond.

[0469] In some cases, TMMP comprises: a) a first polypeptide chain, the first polypeptide chain comprising, as Figure 20B The depicted amino acid sequence is indicated as "3425"; and b) a second polypeptide chain, the second polypeptide chain comprising as follows Figure 20M The depicted amino acid sequence is designated as "3529". This type of TMMP contains: a) as... Figure 19 The MOD is depicted at position 3; and b) both the G2C / Y84C disulfide bond and the R12C / A236C disulfide bond; and also includes the WT1 239-247 (Q240Y) epitope.

[0470] In some cases, TMMP comprises: a) a first polypeptide chain, the first polypeptide chain comprising, as Figure 20C The depicted amino acid sequence is indicated as "3196"; and b) a second polypeptide chain, the second polypeptide chain comprising as follows Figure 20N The described amino acid sequence is designated as "3709". This type of TMMP contains: a) as... Figure 19 The MOD at position 3 is depicted; and b) both the G2C / Y84C disulfide bond and the R12C / A236C disulfide bond.

[0471] In some cases, TMMP comprises: a) a first polypeptide chain, the first polypeptide chain comprising, as Figure 4B The depicted amino acid sequence is indicated as "2750"; and b) a second polypeptide chain, the second polypeptide chain comprising as follows Figure 200 The described amino acid sequence is designated as "3528". This type of TMMP contains: a) as... Figure 19The MOD at position 1 is depicted; and b) the R12C / A236C disulfide bond (instead of the G2C / Y84C disulfide bond).

[0472] In some cases, TMMP comprises: a) a first polypeptide chain, the first polypeptide chain comprising, as Figure 4C The depicted amino acid sequence is designated as “3159”; and b) a second polypeptide chain, the second polypeptide chain comprising as follows Figure 200 The described amino acid sequence is designated as "3528". This type of TMMP contains: a) as... Figure 19 The MOD at position 3 is depicted; and b) the R12C / A236C disulfide bond (instead of the G2C / Y84C disulfide bond).

[0473] In some cases, TMMP comprises: a) a first polypeptide chain, the first polypeptide chain comprising, as Figure 20D The depicted amino acid sequence is indicated as "2764"; and b) a second polypeptide chain, the second polypeptide chain comprising as follows Figure 20P The described amino acid sequence is designated as "3708". This type of TMMP contains: a) as... Figure 19 The MOD at position 5 is depicted; and b) the R12C / A236C disulfide bond (instead of the G2C / Y84C disulfide bond).

[0474] In some cases, TMMP comprises: a) a first polypeptide chain, the first polypeptide chain comprising, as Figure 20E The depicted amino acid sequence is designated as "3593"; and b) a second polypeptide chain, the second polypeptide chain comprising as follows Figure 20Q The described amino acid sequence is designated as "3530". This type of TMMP contains: a) as... Figure 19 The MOD at position 1 is depicted; and b) the G2C / Y84C disulfide bond (instead of the R12C / A236C disulfide bond).

[0475] In some cases, TMMP comprises: a) a first polypeptide chain, the first polypeptide chain comprising, as Figure 20F The depicted amino acid sequence is designated as "3426"; and b) a second polypeptide chain, the second polypeptide chain comprising as follows Figure 20Q The described amino acid sequence is designated as "3530". This type of TMMP contains: a) as... Figure 19 The MOD at position 3 is depicted; and b) the G2C / Y84C disulfide bond (instead of the R12C / A236C disulfide bond).

[0476] In some cases, TMMP comprises: a) a first polypeptide chain, the first polypeptide chain comprising, as Figure 20GThe depicted amino acid sequence is indicated as "3197"; and b) a second polypeptide chain, the second polypeptide chain comprising as follows Figure 20R The described amino acid sequence is designated "3710". This type of TMMP contains: a) as... Figure 19 The MOD at position 5 is depicted; and b) the G2C / Y84C disulfide bond (instead of the R12C / A236C disulfide bond).

[0477] In some cases, TMMP comprises: a) a first polypeptide chain, the first polypeptide chain comprising, as Figure 20F The depicted amino acid sequence is designated as "3426"; and b) a second polypeptide chain, the second polypeptide chain comprising as follows Figure 20M The depicted amino acid sequence is designated as "3529". This type of TMMP contains: a) as... Figure 19 The MOD is depicted at position 3; and b) the G2C / Y84C disulfide bond (instead of the R12C / A236C disulfide bond); and also contains the WT1 239-247 (Q240Y) epitope.

[0478] In some cases, TMMP comprises: a) a first polypeptide chain, the first polypeptide chain comprising, as Figure 20B The depicted amino acid sequence is indicated as "3425"; and b) a second polypeptide chain, the second polypeptide chain comprising as follows Figure 200 The described amino acid sequence is designated as "3528". This type of TMMP contains: a) as... Figure 19 The MOD at position 3 is depicted; and b) the R12C / A236C disulfide bond (instead of the G2C / Y84C disulfide bond); and also contains the WT1 239-247 (Q240Y) epitope.

[0479] In some cases, TMMP comprises: a) a first polypeptide chain, the first polypeptide chain comprising, as Figure 20B The depicted amino acid sequence is indicated as "3425"; and b) a second polypeptide chain, the second polypeptide chain comprising as follows Figure 20Q The described amino acid sequence is designated as "3530". This type of TMMP contains: a) as... Figure 19 The MOD is depicted at position 3; and b) the G2C / Y84C disulfide bond (instead of the R12C / A236C disulfide bond); and also contains the WT1 239-247 (Q240Y) epitope.

[0480] In some cases, TMMP comprises: a) a first polypeptide chain, the first polypeptide chain comprising, as Figure 4C The depicted amino acid sequence is designated as “3159”; and b) a second polypeptide chain, the second polypeptide chain comprising as follows Figure 20HThe described amino acid sequence is designated as "3188". This type of TMMP contains: a) as... Figure 19 The MOD at position 3 is depicted; and b) the R12C / A236C disulfide bond (instead of the G2C / Y84C disulfide bond); and also contains the WT1 235-243 (M236Y) epitope.

[0481] An exemplary TMMP with tabletop SMTWNQMNL (WT1 (235-243; C235S))

[0482] In some cases, TMMP comprises: a) a first polypeptide chain, the first polypeptide chain comprising, as Figure 35A The described amino acid sequence; and b) a second polypeptide chain, the second polypeptide chain comprising as follows Figure 4B The described amino acid sequence. This type of TMMP contains: a) as... Figure 19 The MOD at position 1 is depicted; and b) the R12C / A236C disulfide bond (instead of the G2C / Y84C disulfide bond).

[0483] In some cases, TMMP comprises: a) a first polypeptide chain, the first polypeptide chain comprising, as Figure 35A The described amino acid sequence; and b) a second polypeptide chain, the second polypeptide chain comprising as follows Figure 4C The described amino acid sequence. This type of TMMP contains: a) as... Figure 19 The MOD at position 3 is depicted; and b) the R12C / A236C disulfide bond (instead of the G2C / Y84C disulfide bond).

[0484] In some cases, TMMP comprises: a) a first polypeptide chain, the first polypeptide chain comprising, as Figure 35B The described amino acid sequence; and b) a second polypeptide chain, the second polypeptide chain comprising as follows Figure 20A The described amino acid sequence. This type of TMMP contains: a) as... Figure 19 The MOD at position 1 is depicted; and b) both the G2C / Y84C disulfide bond and the R12C / A236C disulfide bond.

[0485] In some cases, TMMP comprises: a) a first polypeptide chain, the first polypeptide chain comprising, as Figure 35B The described amino acid sequence; and b) a second polypeptide chain, the second polypeptide chain comprising as follows Figure 20B The described amino acid sequence. This type of TMMP contains: a) as... Figure 19 The MOD at position 3 is depicted; and b) both the G2C / Y84C disulfide bond and the R12C / A236C disulfide bond.

[0486] In some cases, TMMP comprises: a) a first polypeptide chain, the first polypeptide chain comprising, as Figure 35CThe described amino acid sequence; and b) a second polypeptide chain, the second polypeptide chain comprising as follows Figure 20E The described amino acid sequence. This type of TMMP contains: a) as... Figure 19 The MOD at position 1 is depicted; and b) the G2C / Y84C disulfide bond (instead of the R12C / A236C disulfide bond).

[0487] In some cases, TMMP comprises: a) a first polypeptide chain, the first polypeptide chain comprising, as Figure 35C The described amino acid sequence; and b) a second polypeptide chain, the second polypeptide chain comprising as follows Figure 20F The described amino acid sequence. This type of TMMP contains: a) as... Figure 19 The MOD at position 3 is depicted; and b) the G2C / Y84C disulfide bond (instead of the R12C / A236C disulfide bond).

[0488] In some cases, TMMP comprises: a) a first polypeptide chain, the first polypeptide chain comprising, as Figure 35D The described amino acid sequence; and b) a second polypeptide chain, the second polypeptide chain comprising as follows Figure 20G The described amino acid sequence. This type of TMMP contains: a) as... Figure 19 The MOD at position 5 is depicted; and b) the G2C / Y84C disulfide bond (instead of the R12C / A236C disulfide bond).

[0489] In some cases, TMMP comprises: a) a first polypeptide chain, the first polypeptide chain comprising, as Figure 35E The described amino acid sequence; and b) a second polypeptide chain, the second polypeptide chain comprising as follows Figure 20C The described amino acid sequence. This type of TMMP contains: a) as... Figure 19 The MOD at position 5 is depicted; and b) both the G2C / Y84C disulfide bond and the R12C / A236C disulfide bond.

[0490] In some cases, TMMP comprises: a) a first polypeptide chain, the first polypeptide chain comprising, as Figure 35F The described amino acid sequence; and b) a second polypeptide chain, the second polypeptide chain comprising as follows Figure 20D The described amino acid sequence. This type of TMMP contains: a) as... Figure 19 The MOD at position 5 is depicted; and b) the R12C / A236C disulfide bond (instead of the G2C / Y84C disulfide bond).

[0491] An exemplary TMMP with the epitope GCMTWNQMNL (WT1 (235-243; G-1))

[0492] In some cases, TMMP comprises: a) a first polypeptide chain, the first polypeptide chain comprising, as Figure 36A The described amino acid sequence; and b) a second polypeptide chain, the second polypeptide chain comprising as follows Figure 4B The described amino acid sequence. This type of TMMP contains: a) as... Figure 19 The MOD at position 1 is depicted; and b) the R12C / A236C disulfide bond (instead of the G2C / Y84C disulfide bond).

[0493] In some cases, TMMP comprises: a) a first polypeptide chain, the first polypeptide chain comprising, as Figure 36A The described amino acid sequence; and b) a second polypeptide chain, the second polypeptide chain comprising as follows Figure 4C The described amino acid sequence. This type of TMMP contains: a) as... Figure 19 The MOD at position 3 is depicted; and b) the R12C / A236C disulfide bond (instead of the G2C / Y84C disulfide bond).

[0494] In some cases, TMMP comprises: a) a first polypeptide chain, the first polypeptide chain comprising, as Figure 36B The described amino acid sequence; and b) a second polypeptide chain, the second polypeptide chain comprising as follows Figure 20A The described amino acid sequence. This type of TMMP contains: a) as... Figure 19 The MOD at position 1 is depicted; and b) both the G2C / Y84C disulfide bond and the R12C / A236C disulfide bond.

[0495] In some cases, TMMP comprises: a) a first polypeptide chain, the first polypeptide chain comprising, as Figure 36B The described amino acid sequence; and b) a second polypeptide chain, the second polypeptide chain comprising as follows Figure 20B The described amino acid sequence. This type of TMMP contains: a) as... Figure 19 The MOD at position 3 is depicted; and b) both the G2C / Y84C disulfide bond and the R12C / A236C disulfide bond.

[0496] In some cases, TMMP comprises: a) a first polypeptide chain, the first polypeptide chain comprising, as Figure 36C The described amino acid sequence; and b) a second polypeptide chain, the second polypeptide chain comprising as follows Figure 20E The described amino acid sequence. This type of TMMP contains: a) as... Figure 19 The MOD at position 1 is depicted; and b) the G2C / Y84C disulfide bond (instead of the R12C / A236C disulfide bond).

[0497] In some cases, TMMP comprises: a) a first polypeptide chain, the first polypeptide chain comprising, as Figure 36CThe described amino acid sequence; and b) a second polypeptide chain, the second polypeptide chain comprising as follows Figure 20F The described amino acid sequence. This type of TMMP contains: a) as... Figure 19 The MOD at position 3 is depicted; and b) the G2C / Y84C disulfide bond (instead of the R12C / A236C disulfide bond).

[0498] In some cases, TMMP comprises: a) a first polypeptide chain, the first polypeptide chain comprising, as Figure 36D The described amino acid sequence; and b) a second polypeptide chain, the second polypeptide chain comprising as follows Figure 20G The described amino acid sequence. This type of TMMP contains: a) as... Figure 19 The immunomodulatory peptide depicted at position 5; and b) the G2C / Y84C disulfide bond (instead of the R12C / A236C disulfide bond).

[0499] In some cases, TMMP comprises: a) a first polypeptide chain, the first polypeptide chain comprising, as Figure 36E The described amino acid sequence; and b) a second polypeptide chain, the second polypeptide chain comprising as follows Figure 20C The described amino acid sequence. This type of TMMP contains: a) as... Figure 19 The MOD at position 5 is depicted; and b) both the G2C / Y84C disulfide bond and the R12C / A236C disulfide bond.

[0500] In some cases, TMMP comprises: a) a first polypeptide chain, the first polypeptide chain comprising, as Figure 36F The described amino acid sequence; and b) a second polypeptide chain, the second polypeptide chain comprising as follows Figure 20D The described amino acid sequence. This type of TMMP contains: a) as... Figure 19 The MOD at position 5 is depicted; and b) the R12C / A236C disulfide bond (instead of the G2C / Y84C disulfide bond).

[0501] An exemplary TMMP with the tabletop SYTWNQMNL (WT1 (235-243; C235S; M236Y))

[0502] In some cases, TMMP comprises: a) a first polypeptide chain, the first polypeptide chain comprising, as Figure 37A The described amino acid sequence; and b) a second polypeptide chain, the second polypeptide chain comprising as follows Figure 4B The described amino acid sequence. This type of TMMP contains: a) as... Figure 19 The MOD at position 1 is depicted; and b) the R12C / A236C disulfide bond (instead of the G2C / Y84C disulfide bond).

[0503] In some cases, TMMP comprises: a) a first polypeptide chain, the first polypeptide chain comprising, as Figure 37A The described amino acid sequence; and b) a second polypeptide chain, the second polypeptide chain comprising as follows Figure 4C The described amino acid sequence. This type of TMMP contains: a) as... Figure 19 The MOD at position 3 is depicted; and b) the R12C / A236C disulfide bond (instead of the G2C / Y84C disulfide bond).

[0504] In some cases, TMMP comprises: a) a first polypeptide chain, the first polypeptide chain comprising, as Figure 37B The described amino acid sequence; and b) a second polypeptide chain, the second polypeptide chain comprising as follows Figure 20A The described amino acid sequence. This type of TMMP contains: a) as... Figure 19 The MOD at position 1 is depicted; and b) both the G2C / Y84C disulfide bond and the R12C / A236C disulfide bond.

[0505] In some cases, TMMP comprises: a) a first polypeptide chain, the first polypeptide chain comprising, as Figure 37B The described amino acid sequence; and b) a second polypeptide chain, the second polypeptide chain comprising as follows Figure 20B The described amino acid sequence. This type of TMMP contains: a) as... Figure 19 The MOD at position 3 is depicted; and b) both the G2C / Y84C disulfide bond and the R12C / A236C disulfide bond.

[0506] In some cases, TMMP comprises: a) a first polypeptide chain, the first polypeptide chain comprising, as Figure 37C The described amino acid sequence; and b) a second polypeptide chain, the second polypeptide chain comprising as follows Figure 20E The described amino acid sequence. This type of TMMP contains: a) as... Figure 19 The MOD at position 1 is depicted; and b) the G2C / Y84C disulfide bond (instead of the R12C / A236C disulfide bond).

[0507] In some cases, TMMP comprises: a) a first polypeptide chain, the first polypeptide chain comprising, as Figure 37C The described amino acid sequence; and b) a second polypeptide chain, the second polypeptide chain comprising as follows Figure 20F The described amino acid sequence. This type of TMMP contains: a) as... Figure 19 The MOD at position 3 is depicted; and b) the G2C / Y84C disulfide bond (instead of the R12C / A236C disulfide bond).

[0508] In some cases, TMMP comprises: a) a first polypeptide chain, the first polypeptide chain comprising, as Figure 37DThe described amino acid sequence; and b) a second polypeptide chain, the second polypeptide chain comprising as follows Figure 20G The described amino acid sequence. This type of TMMP contains: a) as... Figure 19 The MOD at position 5 is depicted; and b) the G2C / Y84C disulfide bond (instead of the R12C / A236C disulfide bond).

[0509] In some cases, TMMP comprises: a) a first polypeptide chain, the first polypeptide chain comprising, as Figure 37E The described amino acid sequence; and b) a second polypeptide chain, the second polypeptide chain comprising as follows Figure 20C The described amino acid sequence. This type of TMMP contains: a) as... Figure 19 The MOD at position 5 is depicted; and b) both the G2C / Y84C disulfide bond and the R12C / A236C disulfide bond.

[0510] In some cases, TMMP comprises: a) a first polypeptide chain, the first polypeptide chain comprising, as Figure 37F The described amino acid sequence; and b) a second polypeptide chain, the second polypeptide chain comprising as follows Figure 20D The described amino acid sequence. This type of TMMP contains: a) as... Figure 19 The MOD at position 5 is depicted; and b) the R12C / A236C disulfide bond (instead of the G2C / Y84C disulfide bond).

[0511] An exemplary TMMP with epitope GCYTWNQMNL (WT1 (235-243; G-1; M236Y))

[0512] In some cases, TMMP comprises: a) a first polypeptide chain, the first polypeptide chain comprising, as Figure 38A The described amino acid sequence; and b) a second polypeptide chain, the second polypeptide chain comprising as follows Figure 4B The described amino acid sequence. This type of TMMP contains: a) as... Figure 19 The MOD at position 1 is depicted; and b) the R12C / A236C disulfide bond (instead of the G2C / Y84C disulfide bond).

[0513] In some cases, TMMP comprises: a) a first polypeptide chain, the first polypeptide chain comprising, as Figure 38A The described amino acid sequence; and b) a second polypeptide chain, the second polypeptide chain comprising as follows Figure 4C The described amino acid sequence. This type of TMMP contains: a) as... Figure 19 The MOD at position 3 is depicted; and b) the R12C / A236C disulfide bond (instead of the G2C / Y84C disulfide bond).

[0514] In some cases, TMMP comprises: a) a first polypeptide chain, the first polypeptide chain comprising, as Figure 38B The described amino acid sequence; and b) a second polypeptide chain, the second polypeptide chain comprising as follows Figure 20A The described amino acid sequence. This type of TMMP contains: a) as... Figure 19 The MOD at position 1 is depicted; and b) both the G2C / Y84C disulfide bond and the R12C / A236C disulfide bond.

[0515] In some cases, TMMP comprises: a) a first polypeptide chain, the first polypeptide chain comprising, as Figure 38B The described amino acid sequence; and b) a second polypeptide chain, the second polypeptide chain comprising as follows Figure 20B The described amino acid sequence. This type of TMMP contains: a) as... Figure 19 The MOD at position 3 is depicted; and b) both the G2C / Y84C disulfide bond and the R12C / A236C disulfide bond.

[0516] In some cases, TMMP comprises: a) a first polypeptide chain, the first polypeptide chain comprising, as Figure 38C The described amino acid sequence; and b) a second polypeptide chain, the second polypeptide chain comprising as follows Figure 20E The described amino acid sequence. This type of TMMP contains: a) as... Figure 19 The MOD at position 1 is depicted; and b) the G2C / Y84C disulfide bond (instead of the R12C / A236C disulfide bond).

[0517] In some cases, TMMP comprises: a) a first polypeptide chain, the first polypeptide chain comprising, as Figure 38C The described amino acid sequence; and b) a second polypeptide chain, the second polypeptide chain comprising as follows Figure 20F The described amino acid sequence. This type of TMMP contains: a) as... Figure 19 The MOD at position 3 is depicted; and b) the G2C / Y84C disulfide bond (instead of the R12C / A236C disulfide bond).

[0518] In some cases, TMMP comprises: a) a first polypeptide chain, the first polypeptide chain comprising, as Figure 38D The described amino acid sequence; and b) a second polypeptide chain, the second polypeptide chain comprising as follows Figure 20G The described amino acid sequence. This type of TMMP contains: a) as... Figure 19 The MOD at position 5 is depicted; and b) the G2C / Y84C disulfide bond (instead of the R12C / A236C disulfide bond).

[0519] In some cases, TMMP comprises: a) a first polypeptide chain, the first polypeptide chain comprising, as Figure 38EThe described amino acid sequence; and b) a second polypeptide chain, the second polypeptide chain comprising as follows Figure 20C The described amino acid sequence. This type of TMMP contains: a) as... Figure 19 The MOD at position 5 is depicted; and b) both the G2C / Y84C disulfide bond and the R12C / A236C disulfide bond.

[0520] In some cases, TMMP comprises: a) a first polypeptide chain, the first polypeptide chain comprising, as Figure 38F The described amino acid sequence; and b) a second polypeptide chain, the second polypeptide chain comprising as follows Figure 20D The described amino acid sequence. This type of TMMP contains: a) as... Figure 19 The MOD at position 5 is depicted; and b) the R12C / A236C disulfide bond (instead of the G2C / Y84C disulfide bond).

[0521] Methods for generating multimeric T cell regulatory peptides

[0522] The published PCT application WO 2019 / 051091, published on March 14, 2019, provides a method for obtaining a TMMP containing one or more variant MODs, which have a lower affinity for the homologous co-MOD compared to the affinity of the corresponding parent wild-type immunomodulatory peptide for the co-immunomodulatory peptide. See

[00364] -

[00387] .

[0523] Nucleic acid

[0524] This disclosure provides a nucleic acid comprising a nucleotide sequence encoding the TMMP of this disclosure.

[0525] In some cases, individual polypeptide chains of TMMP are encoded in separate nucleic acids. In some cases, the entire polypeptide chain of TMMP is encoded in a single nucleic acid. In some cases, the first nucleic acid contains the nucleotide sequence encoding the first polypeptide of TMMP, and the second nucleic acid contains the nucleotide sequence encoding the second polypeptide of TMMP. In some cases, a single nucleic acid contains the nucleotide sequences encoding both the first polypeptide and the second polypeptide of TMMP.

[0526] Individual nucleic acids of individual polypeptide chains encoding multimeric polypeptides

[0527] This disclosure provides nucleic acids comprising a nucleotide sequence encoding TMMP. As described above, in some cases, individual polypeptide chains of TMMP are encoded in separate nucleic acids. In some cases, the nucleotide sequence encoding the individual polypeptide chain of TMMP is operatively linked to a transcriptional control element, such as a promoter, like a promoter that functions in eukaryotic cells, wherein said promoter may be a constitutive promoter or an inducible promoter.

[0528] This disclosure provides a first nucleic acid and a second nucleic acid, wherein the first nucleic acid comprises a nucleotide sequence encoding a first polypeptide of TMMP, wherein the first polypeptide comprises, from N-terminus to C-terminus: a) an epitope (e.g., a T-cell epitope); b) a first MHC polypeptide; and c) a MOD (e.g., a wild-type MOD as described above or a reduced-affinity variant MOD); and wherein the second nucleic acid comprises a nucleotide sequence encoding a second polypeptide of TMMP, wherein the second polypeptide comprises, from N-terminus to C-terminus: a) a second MHC polypeptide; and b) an Ig Fc polypeptide. Suitable T-cell epitopes, MHC polypeptides, MODs, and Ig Fc polypeptides are as described above. In some cases, the nucleotide sequences encoding the first and second polypeptides are operatively linked to a transcriptional control element. In some cases, the transcriptional control element is a promoter that functions in eukaryotic cells. In some cases, the nucleic acid is present in a separate expression vector.

[0529] This disclosure provides a first nucleic acid and a second nucleic acid, wherein the first nucleic acid comprises a nucleotide sequence encoding a first polypeptide encoding TMMP, wherein the first polypeptide comprises, from N-terminus to C-terminus: a) an epitope (e.g., a T-cell epitope); and b) a first MHC polypeptide; and wherein the second nucleic acid comprises a nucleotide sequence encoding a second polypeptide encoding TMMP, wherein the second polypeptide comprises, from N-terminus to C-terminus: a) a MOD (e.g., a wild-type MOD as described above or a reduced-affinity variant MOD); b) a second MHC polypeptide; and c) an Ig Fc polypeptide. Suitable T-cell epitopes, MHC polypeptides, MODs, and Ig Fc polypeptides are as described above. In some cases, the nucleotide sequences encoding the first and second polypeptides are operatively linked to a transcriptional control element. In some cases, the transcriptional control element is a promoter that functions in eukaryotic cells. In some cases, the nucleic acid is present in a separate expression vector.

[0530] Nucleic acids encoding two or more polypeptides present in a multimeric polypeptide.

[0531] This disclosure provides a nucleic acid comprising nucleotide sequences encoding at least a first polypeptide and a second polypeptide of TMMP. In some cases, when the TMMP of this disclosure comprises a first polypeptide, a second polypeptide, and a third polypeptide, the nucleic acid comprises nucleotide sequences encoding the first polypeptide, the second polypeptide, and the third polypeptide. In some cases, the nucleotide sequences encoding the first polypeptide and the second polypeptide of TMMP include a proteolytically cleavable linker inserted between the nucleotide sequences encoding the first polypeptide and the nucleotide sequences encoding the second polypeptide. In some cases, the nucleotide sequences encoding the first polypeptide and the second polypeptide of TMMP include an internal ribosome entry site (IRES) inserted between the nucleotide sequences encoding the first polypeptide and the nucleotide sequences encoding the second polypeptide. In some cases, the nucleotide sequences encoding the first polypeptide and the second polypeptide of TMMP include a ribosome jumping signal (or cis-acting hydrolase element, CHYSEL (SEQ ID NO: 394...

Claims

1. A T-cell regulatory multimer polypeptide comprising at least one heterodimer, wherein the at least one heterodimer comprises: a) A first polypeptide, the first polypeptide comprising, from the N-terminus to the C-terminus: i) Wilms' tumor-1 (WT-1) peptide epitope, wherein the amino acid sequence of the WT-1 peptide epitope is SEQ ID NO:259, SEQ ID NO:260, SEQ ID NO:262, SEQ ID NO:263 or SEQ ID NO:

264. ii) A linker containing Cys residues, wherein the linker is GCGGS(GGGGS)n, where n is 1, 2, 3, 4, 5, 6, 7, 8, or 9, and iii) A β-2 microglobulin (β2M) polypeptide comprising the amino acid sequence of SEQ ID NO:311, wherein the amino acid sequence comprises an R12C substitution, and b) A second polypeptide, the second polypeptide comprising, from the N-terminus to the C-terminus: i) Class I major histocompatibility complex (MHC) heavy chain peptides, wherein the Class I MHC heavy chain peptides are: (1) The substitution of Cys residue at position 84 and Cys residue at position 236 is included relative to any one of SEQ ID NO:41-44, 49-51, 198, 200, 202, 204 and 206. (2) Substitution of the Cys residue contained at position 84 relative to SEQ ID NO:46 or 48, or (3) Contains Cys residues at positions 84 and 236 as shown in SEQ ID NO:

47. ii) Immunoglobulin (Ig) Fc peptides, and iii) At least one activated immunomodulatory peptide (MOD), wherein the at least one activated immunomodulatory peptide comprises a variant IL-2 peptide containing H16A and F42A substitutions. or i) at least one activated immunomodulatory polypeptide (MOD), wherein the at least one activated immunomodulatory polypeptide comprises a variant IL-2 polypeptide containing H16A and F42A substitutions. ii) Class I major histocompatibility complex (MHC) heavy chain peptides, wherein the Class I MHC heavy chain peptides are: (1) The substitution of Cys residue at position 84 and Cys residue at position 236 is included relative to any one of SEQ ID NO:41-44, 49-51, 198, 200, 202, 204 and 206. (2) Substitution of the Cys residue contained at position 84 relative to SEQ ID NO:46 or 48, or (3) Contains Cys residues at positions 84 and 236 as shown in SEQ ID NO:

47. iii) Immunoglobulin (Ig) Fc polypeptide, The second polypeptide optionally includes one or more peptide linkers between the components of the second polypeptide. The heterodimer comprises a first disulfide bond linking (i) a Cys residue in the linker between the WT-1 peptide epitope and the β2M polypeptide and (ii) a first Cys residue at position 84 of the introduction of the class I MHC heavy chain polypeptide. The heterodimer comprises a second disulfide bond linking the β2M R12C Cys residue in the first polypeptide and the Cys residue at position 236 of the class I MHC heavy chain in the second polypeptide, and The Ig Fc polypeptide does not contain the C-terminal lysine shown in any of SEQ ID NO:410-413 and SEQ ID NO:416-421.

2. The T cell regulatory multimer polypeptide as described in claim 1, wherein: a) The first polypeptide comprises, from the N-terminus to the C-terminus: i) The WT-1 peptide epitope; ii) Connectors containing Cys; and iii) the β2M polypeptide; and b) The second polypeptide comprises, from the N-terminus to the C-terminus: i) at least one immunomodulatory polypeptide; ii) Optional connectors; iii) The Class I MHC heavy chain polypeptide; iv) Optional connectors; and v) The Ig Fc polypeptide.

3. The T-cell regulatory multimer polypeptide as described in claim 1, wherein: a) The first polypeptide comprises, from the N-terminus to the C-terminus: i) The WT-1 peptide epitope; ii) Connectors containing Cys; and iii) the β2M polypeptide; and b) The second polypeptide comprises, from the N-terminus to the C-terminus: i) The Class I MHC heavy chain polypeptide; ii) Optional connectors; iii) The Ig Fc polypeptide; iv) Optional connectors; and v) at least one immunomodulatory polypeptide.

4. The T-cell regulatory multimer polypeptide of claim 2 or 3, wherein when the T-cell regulatory multimer polypeptide (TMMP) comprises more than one immunomodulatory polypeptide, the TMMP comprises a linker between the first MOD and the second MOD.

5. The T-cell regulatory multimer polypeptide of claim 1, wherein the multimer polypeptide comprises two variant IL-2 polypeptides, each of the two variant IL-2 polypeptides comprising H16A and F42A substitutions, and wherein the two variant IL-2 polypeptides are identical and tandem.

6. The T-cell regulatory multimer polypeptide according to any one of claims 1 to 3, wherein the Ig Fc polypeptide comprises (i) the amino acid sequence shown in any one of SEQ ID NO:418 to SEQ ID NO:421 and without a C-terminal lysine, or (ii) the amino acid sequence shown in SEQ ID NO:

487.

7. The T cell regulatory multimer polypeptide according to any one of claims 1 to 3, wherein the class I MHC heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 500, SEQ ID NO: 346, or SEQ ID NO:

488.

8. The T-cell regulatory multimer polypeptide of claim 1, wherein the first polypeptide of the at least one heterodimer has the amino acid sequence shown in SEQ ID NO: 423; and the second polypeptide of the at least one heterodimer has the amino acid sequence shown in SEQ ID NO:

486.

9. The T-cell regulatory multimer polypeptide according to any one of claims 1 to 3 and 8, wherein the multimer polypeptide comprises two copies of the at least one heterodimer, and wherein the two copies of the heterodimer are covalently linked by one or more disulfide bonds between the IgFc polypeptides of the two heterodimers.

10. The T-cell regulatory multimer polypeptide of claim 9, wherein the multimer polypeptide comprises a first heterodimer and a second heterodimer. The first polypeptide of the first heterodimer and the first polypeptide of the second heterodimer both have the amino acid sequence shown in SEQ ID NO: 423; The second polypeptide of the first heterodimer and the second polypeptide of the second heterodimer both have the amino acid sequence shown in SEQ ID NO: 486; and The first heterodimer and the second heterodimer are covalently bonded by two disulfide bonds formed between the IgFc polypeptide of the first heterodimer and the IgFc polypeptide of the second heterodimer.

11. A composition comprising one or more nucleic acids encoding a first polypeptide and a second polypeptide as described in any one of claims 1 to 10, which are T-cell regulatory multimers.

12. A composition comprising one or more expression vectors, said expression vectors comprising one or more nucleic acids as described in claim 11.

13. Use of a pharmaceutical composition comprising the T-cell regulatory multimer polypeptide according to any one of claims 1 to 10 in the preparation of a medicament for treating cancer in patients, wherein the cancer expresses the WT-1 protein, wherein the cancer is breast cancer, acute myeloid leukemia, myelodysplastic syndrome, non-small cell lung cancer, mesothelioma, or pancreatic cancer.

14. The use according to claim 13, wherein the treatment further comprises administering one or more checkpoint inhibitors to the patient.

15. The use according to claim 14, wherein the 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.

16. The use according to claim 15, wherein the checkpoint inhibitor is an antibody specific to PD-1, PD-L1, or CTLA4.

17. The use according to claim 15 or 16, wherein the one or more checkpoint inhibitors are selected from the group consisting of: nivolumab, pembrolizumab, pildizumab, AMP-224, MPDL3280A, MDX-1105, MEDI-4736, ipilimumab, trimemumab, IMP321, MGA271, BMS-986016, lirelurumab, urerlurumab, PF-05082566, IPH2101, MEDI-6469, CP-870,893, mogliflozin, vararilurumab, acimetab, galiliximab, AMP-514, AUNP 12, edomomod, NLG-919, INCB024360, KN035, and combinations thereof.

18. A method for selectively delivering an immunomodulatory polypeptide to target T cells, the method comprising contacting a mixed population of T cells in vitro with a T cell regulatory multimer polypeptide as claimed in any one of claims 1 to 10, wherein the mixed population of T cells comprises the target T cells and non-target T cells, wherein the target T cells are specific for a WT-1 epitope present in the T cell regulatory multimer polypeptide, and wherein the contact delivers one or more immunomodulatory polypeptides present in the T cell regulatory multimer polypeptide to the target T cells.

19. A method for detecting the presence of target T cells binding to the WT-1 epitope in a mixed T cell population obtained from an individual, the method comprising: a) Contacting the mixed T cell population in vitro with the T cell regulatory multimer polypeptide as described in any one of claims 1 to 10, wherein the T cell regulatory multimer polypeptide comprises the WT-1 epitope; and b) Detect the activation and / or proliferation of T cells in response to the contact, wherein activated and / or proliferating T cells indicate the presence of the target T cells.

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