T cell modulating multimeric polypeptides and methods of use thereof

Abstract

The present disclosure provides T cell regulatory multimeric polypeptides (TMMPs) comprising (i) an optional immune modulating polypeptide, such as a variant IL-2 polypeptide, (ii) a class I HLA major histocompatibility complex (MHC) polypeptide (a class I HLA heavy chain polypeptide and a β2 microglobulin polypeptide), (iii) a peptide that presents an epitope to a T cell receptor and forms a peptide-MHC complex (pMHC) with the class I MHC polypeptide, (iv) a tumor targeting polypeptide, and (v) an optional Ig Fc polypeptide or other scaffold. Such TMMPs are useful for modulating the activity of T cells, modulating the immune response in an individual, and "redirecting" a patient's repertoire of antiviral T cells to attack and kill cancer cells.

Description

[Technical field]

[0001] cross reference This application claims the benefit of U.S. Provisional Patent Application No. 63 / 283,028, filed November 24, 2021, U.S. Provisional Patent Application No. 63 / 309,288, filed February 11, 2022, and U.S. Provisional Patent Application No. 63 / 338,131, filed May 4, 2022, which are incorporated by reference in their entireties herein.

[0002] Incorporation by Reference of Electronically Submitted Materials The Sequence Listing is provided herein as Sequence Listing XML "CUEB-145WO_SEQUENCE_LIST" created on November 17, 2022 and having a size of 1,190,000 bytes. The contents of the Sequence Listing XML are incorporated herein by reference in their entirety. [Background technology]

[0003] preface The adaptive immune response involves the binding of the T cell receptor (TCR) present on the surface of a T cell to small peptide antigens non-covalently presented by the major histocompatibility complex (MHC, also called human leukocyte antigen (HLA) complex in humans) on the surface of an antigen-presenting cell (APC). This binding represents the targeting mechanism of the immune system and is a molecular interaction essential for T cell regulation (activation or inhibition) and effector function. Following epitope-specific cell targeting, the targeted T cell is activated by binding of costimulatory proteins found on the APC with the corresponding costimulatory proteins on the T cell. Both signals, epitope / TCR binding and binding of APC costimulatory proteins to T cell costimulatory proteins, are required to drive T cell specificity and activation or inhibition. While TCRs are specific for a given epitope, costimulatory proteins are not epitope specific, but instead are generally expressed on all T cells or on large T cell subsets. Summary of the Invention

[0004] overview The present disclosure provides T cell regulatory multimeric polypeptides (TMMPs) that include an immunomodulatory polypeptide, a class I HLA polypeptide (a class I HLA heavy chain polypeptide and a β2 microglobulin polypeptide), a peptide that presents an epitope to a T cell receptor, and a tumor targeting polypeptide. The TMMPs of the present disclosure are useful for modulating the activity of T cells, modulating the immune response in an individual, and "redirecting" a patient's repertoire of antiviral T cells to attack and kill cancer cells. [Brief description of the drawings]

[0005] [Figure 1A] 1A-1B provide the amino acid sequence of a wild-type human β2M polypeptide (SEQ ID NO:1) (FIG. 1A) and the amino acid sequence of a β2M polypeptide having an R12C substitution (SEQ ID NO:2) (FIG. 1B). [Figure 1B] Same as above.

[0006] [Figure 2A] 2A-2M provide the amino acid sequences of immunoglobulin Fc polypeptides (SEQ ID NOs:3-15, respectively). [Figure 2B] Same as above. [Figure 2C] Same as above. [Figure 2D] Same as above. [Figure 2E] Same as above. [Figure 2F] Same as above. [Figure 2G] Same as above. [Figure 2H] Same as above. [Figure 2I] Same as above. [Figure 2J] Same as above. [Figure 2K] Same as above. [Figure 2L] Same as above. [Figure 2M] Same as above.

[0007] [Figure 3A]Figures 3A-3E provide the amino acid sequence of wild-type HLA-A*0201 (SEQ ID NO: 16) (Figure 3A) and variants (SEQ ID NOs: 17-20, respectively) (Figures 3B-3E). [Figure 3B] Same as above. [Figure 3C] Same as above. [Figure 3D] Same as above. [Figure 3E] Same as above.

[0008] [Figure 4A] Figures 4A-4E provide the amino acid sequence of wild-type HLA-A*1101 (SEQ ID NO: 21) (Figure 4A) and variants (SEQ ID NOs: 22-25, respectively) (Figures 4B-4E). [Figure 4B] Same as above. [Figure 4C] Same as above. [Figure 4D] Same as above. [Figure 4E] Same as above.

[0009] [Figure 5A] Figures 5A-5E provide the amino acid sequence of wild-type HLA-A*2402 (SEQ ID NO: 26) (Figure 5A) and variants (SEQ ID NOs: 27-30, respectively) (Figures 5B-5E). [Figure 5B] Same as above. [Figure 5C] Same as above. [Figure 5D] Same as above. [Figure 5E] Same as above.

[0010] [Figure 6A] Figures 6A-6E provide the amino acid sequence of wild-type HLA-A*3303 (SEQ ID NO: 31) (Figure 6A) and variants (SEQ ID NOs: 32-35, respectively) (Figures 6B-6E). [Figure 6B] Same as above. [Figure 6C] Same as above. [Figure 6D] Same as above. [Figure 6E] Same as above.

[0011] [Figure 7A-1] Figures 7A-7B provide an alignment of the HLA-A heavy chain amino acid sequences (Figure 7A, SEQ ID NOs: 36-44, respectively) and the consensus sequence (Figure 7B, SEQ ID NO: 45). [Figure 7A-2] Same as above. [Figure 7A-3] Same as above. [Figure 7B] Same as above.

[0012] [Figure 8A-1] Figures 8A-8B provide an alignment of the HLA-B heavy chain amino acid sequences (Figure 8A, SEQ ID NOs: 46-52, respectively) and the consensus sequence (Figure 8B, SEQ ID NO: 53). [Figure 8A-2] Same as above. [Figure 8B] Same as above.

[0013] [Figure 9A-1] Figures 9A-9B provide an alignment of the HLA-C heavy chain amino acid sequences (Figure 9A, SEQ ID NOs: 54-61, respectively) and the consensus sequence (Figure 9B, SEQ ID NO: 62). [Figure 9A-2] Same as above. [Figure 9A-3] Same as above. [Figure 9B] Same as above.

[0014] [Figure 10] Figure 10 provides the consensus amino acid sequences for each of the HLA-E, -F, and -G heavy chains (SEQ ID NOs:63-65, respectively). Variable amino acid (aa) positions are shown as consecutively numbered "X" residues, with amino acid positions 84, 139, and 236 double underlined.

[0015] [Figure 11-1]Figure 11 provides an alignment of the consensus amino acid sequences of HLA-A (SEQ ID NO:45), -B (SEQ ID NO:53), -C (SEQ ID NO:62), -E (SEQ ID NO:63), -F (SEQ ID NO:64), and -G (SEQ ID NO:65). [Figure 11-2] Same as above.

[0016] [Figure 12A] Figures 12A-12D provide the amino acid sequences of wild-type IL-2 polypeptide (SEQ ID NO:66) (Figure 12A), IL-2Rα (SEQ ID NO:67) (Figure 12B), IL-2Rβ (SEQ ID NO:68) (Figure 12C), and IL-2Rγ (SEQ ID NO:69) (Figure 12D). [Figure 12B] Same as above. [Figure 12C] Same as above. [Figure 12D] Same as above.

[0017] [Figure 13A] Figures 13A-13K provide the amino acid sequences of SARS-CoV-2 encoded polypeptides (SEQ ID NOs:70-94). [Figure 13B] Same as above. [Figure 13C] Same as above. [Figure 13D] Same as above. [Figure 13E-1] Same as above. [Figure 13E-2] Same as above. [Figure 13F] Same as above. [Figure 13G] Same as above. [Figure 13H] Same as above. [Figure 13I] Same as above. [Figure 13J] Same as above. [Figure 13K] Same as above.

[0018] [Figure 14-1] FIG. 14 provides CD8 T cell epitopes and primary HLA class I heavy chain allele restriction of SARS-CoV-2 encoded polypeptides. [Figure 14-2] Same as above. [Figure 14-3] Same as above. [Figure 14-4] Same as above. [Figure 14-5] Same as above. [Figure 14-6] Same as above. [Figure 14-7] Same as above. [Figure 14-8] Same as above. [Figure 14-9] Same as above. [Figure 14-10] Same as above. [Figure 14-11] Same as above. [Figure 14-12] Same as above. [Figure 14-13] Same as above. [Figure 14-14] Same as above. [Figure 14-15] Same as above. [Figure 14-16] Same as above. [Figure 14-17] Same as above. [Figure 14-18] Same as above. [Figure 14-19] Same as above. [Figure 14-20] Same as above. [Figure 14-21] Same as above. [Figure 14-22] Same as above. [Figure 14-23] Same as above. [Figure 14-24] Same as above. [Figure 14-25] Same as above.

[0019] [Figure 15-1] FIG. 15 provides CD8 T cell epitopes and HLA class I heavy chain allele restriction of SARS-CoV-2 encoded polypeptides. [Figure 15-2] Same as above. [Figure 15-3] Same as above. [Figure 15-4] Same as above. [Figure 15-5] Same as above. [Figure 15-6] Same as above. [Figure 15-7] Same as above. [Figure 15-8] Same as above. [Figure 15-9] Same as above. [Figure 15-10] Same as above. [Figure 15-11] Same as above. [Figure 15-12] Same as above. [Figure 15-13] Same as above. [Figure 15-14] Same as above. [Figure 15-15] Same as above. [Figure 15-16] Same as above. [Figure 15-17] Same as above. [Figure 15-18] Same as above. [Figure 15-19] Same as above. [Figure 15-20] Same as above. [Figure 15-21] Same as above.

[0020] [Figure 16A] Figures 16A-16D provide the amino acid sequences of the HLA-E heavy chains (SEQ ID NOs: 837-840, respectively). [Figure 16B] Same as above. [Figure 16C] Same as above. [Figure 16D] Same as above.

[0021] [Figure 17A] Figures 17A-17D provide the amino acid sequences of the HLA-G heavy chains (SEQ ID NOs:841-844, respectively). [Figure 17B] Same as above. [Figure 17C] Same as above. [Figure 17D] Same as above.

[0022] [Figure 18A] 18A-18D provide schematic diagrams of TMMP. [Figure 18B] Same as above. [Figure 18C] Same as above. [Figure 18D] Same as above.

[0023] [Figure 19-1] FIG. 19 provides the amino acid sequences and HLA class I heavy chain allele restriction of SARS-CoV-2 peptides. [Figure 19-2] Same as above.

[0024] [Figure 20A] Figures 20A-20J provide amino acid sequences of exemplary TMMP "heavy chain" constructs (Figures 20A (SEQ ID NO:867), 20C (SEQ ID NO:868), 20E (SEQ ID NO:869), 20G (SEQ ID NO:870), and 20I (SEQ ID NO:871)) and descriptions (Figures 20B, 20D, 20F, 20H, and 20J). [Figure 20B] Same as above. [Figure 20C] Same as above. [Figure 20D] Same as above. [Figure 20E] Same as above. [Figure 20F] Same as above. [Figure 20G] Same as above. [Figure 20H] Same as above. [Figure 20I] Same as above. [Figure 20J] Same as above.

[0025] [Figure 21A] 21A-21E provide amino acid sequences and descriptions of exemplary TMMP "light chain" constructs. [Figure 21B] Same as above. [Figure 21C] Same as above. [Figure 21D] Same as above. [Figure 21E] Same as above.

[0026] [Figure 22A] 22A-22H provide the amino acid sequences of exemplary anti-mesothelin scFvs. [Figure 22B] Same as above. [Figure 22C] Same as above. [Figure 22D] Same as above. [Figure 22E] Same as above. [Figure 22F] Same as above. [Figure 22G] Same as above. [Fig. 22H] Same as above.

[0027] [Figure 23A] Figures 23A-23D provide the amino acid sequences of exemplary anti-TROP-2 scFvs. [Figure 23B] Same as above. [Figure 23C] Same as above. [Figure 23D] Same as above.

[0028] [Figure 24A] 24A-24B provide the amino acid sequences of exemplary "mAb fusion" constructs. [Figure 24B] Same as above.

[0029] [Figure 25A] 25A-25B provide the amino acid sequences of exemplary "mAb fusion" constructs. [Figure 25B] Same as above.

[0030] [Figure 26A] 26A-26B provide the amino acid sequences of exemplary "mAb fusion" constructs. [Figure 26B] Same as above.

[0031] [Figure 27A] 27A-27B provide the amino acid sequences of exemplary anti-CD28 scFvs. [Figure 27B] Same as above.

[0032] [Figure 28A] 28A-28C provide schematic diagrams of TMMPs showing various possible MOD positions and various possible TTP positions. [Figure 28B] Same as above. [Figure 28C] Same as above.

[0033] [Figure 29] Figure 29 shows PBMC reactivity to SARS-CoV-2 HLA-A*0201 peptide YLQPRTFLL (SEQ ID NO: 218).

[0034] [Diagram 30] Figure 30 shows the proliferation of SARS-CoV-2 (SCV2)-reactive CD8+ T cells.

[0035] [Figure 31A] Figures 31A-31D show the effect of various constructs on the activation of SARS-CoV2-specific T cells present in a population of PBMCs (Figures 31A and 31B) and PBMC-mediated killing of target B cells (Figures 31C and 31D). [Figure 31B] Same as above. [Figure 31C] Same as above. [Figure 31D] Same as above.

[0036] [Figure 32A] Figures 32A-32G provide the amino acid sequences of the polypeptide chains of an exemplary TMMP, with Figure 32A providing the amino acid sequence of a polypeptide designated "4518" (SEQ ID NO: 867), Figure 32B providing the amino acid sequence of a polypeptide designated "4700" (SEQ ID NO: 883), and Figures 32C-32G providing the amino acid sequences of components of the 4518 and 4700 polypeptides. [Figure 32B] Same as above. [Figure 32C] Same as above. [Fig. 32D] Same as above. [Figure 32E] Same as above. [Fig. 32F] Same as above. [Fig. 32G] Same as above.

[0037] [Diagram 33] FIG. 33 provides the amino acid sequence of a polypeptide designated "4656" (SEQ ID NO:910).

[0038] [Diagram 34] FIG. 34 provides the amino acid sequence of a polypeptide designated "3534" (SEQ ID NO:911).

[0039] [Diagram 35] FIG. 35 provides the amino acid sequence of the polypeptide designated "1717" (SEQ ID NO:912).

[0040] [Figure 36A] Figures 36A-36B show the effect of various constructs on the proliferation of SARS-CoV2-specific CD8+ T cells (Figure 36A) or CMV-specific CD8+ T cells present in a population of PBMCs. [Figure 36B] Same as above.

[0041] [Figure 37A] 37A-37B show the effect of various constructs on killing of CD19+ tumor cells. [Figure 37B] Same as above.

[0042] [Figure 38A] Figures 38A-38L provide the amino acid sequences of various polypeptides (Figures 38A-38H) suitable for inclusion in a TMMP (SEQ ID NOs: 1209-1216, respectively) and the amino acid sequences of specific components of the polypeptides (SEQ ID NOs: 872, 18, 877, and 878, respectively) (Figures 38I-38L). [Figure 38B] Same as above. [Figure 38C] Same as above. [Figure 38D] Same as above. [Figure 38E] Same as above. [Figure 38F] Same as above. [Figure 38G] Same as above. [Fig. 38H] Same as above. [Fig. 38I] Same as above. [Figure 38J] Same as above. [Figure 38K]Same as above. [Figure 38L] Same as above.

[0043] [Figure 39A] Figures 39A-39B provide examples of amino acid sequences of polypeptides suitable for inclusion in a TMMP (SEQ ID NOs: 1217 and 1218, respectively). [Figure 39B] Same as above.

[0044] [Figure 40A] Figures 40A-40B provide examples of amino acid sequences of polypeptides suitable for inclusion in a TMMP (SEQ ID NOs: 1219 and 1218, respectively). [Figure 40B] Same as above.

[0045] [Figure 41A] Figures 41A-41C provide examples of amino acid sequences of polypeptides suitable for inclusion in a TMMP (SEQ ID NOs: 870, 1220, and 883, respectively). [Figure 41B] Same as above. [Figure 41C] Same as above.

[0046] [Figure 42A] Figures 42A-42C provide examples of amino acid sequences of polypeptides suitable for inclusion in a TMMP (SEQ ID NOs:1221-1223, respectively). [Figure 42B] Same as above. [Figure 42C] Same as above.

[0047] [Figure 43A] Figures 43A and 43B provide the amino acid sequences of constructs 1715 (Figure 43A) (SEQ ID NO: 1224) and 1717 (Figure 43B) (SEQ ID NO: 912). [Figure 43B] Same as above.

[0048] [Diagram 44] FIG. 44 shows the effect of linker length on TMMP-mediated targeted tumor cell killing.

[0049] [Diagram 45] FIG. 45 shows the effect of variant IL-2 polypeptide copy number on TMMP-mediated targeted tumor cell killing.

[0050] [Diagram 46] FIG. 46 shows TMMP-mediated anti-viral T cell proliferation. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0051] definition The terms "polynucleotide" and "nucleic acid" are used interchangeably herein to refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, the term includes, but is not limited to, single-, double-, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers that contain purine and pyrimidine bases, or other natural, chemically or biochemically modified non-natural or derivatized nucleotide bases.

[0052] The terms "peptide", "polypeptide", and "protein" are used interchangeably herein to refer to polymeric forms of amino acids of any length, and may include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides with modified peptide backbones. Furthermore, as used herein, "polypeptide" refers to a protein containing modifications such as deletions, additions, and substitutions (generally of a conservative nature as known to those of skill in the art) to the native sequence, so long as the protein maintains the desired activity. These modifications may be deliberate, such as site-directed mutagenesis, or accidental, such as mutations of the host producing the protein, or errors due to polymerase chain reaction (PCR) amplification or other recombinant DNA methods. Reference herein to a particular residue or residue number in a known polypeptide will be understood to refer to the amino acid at that position in the wild-type polypeptide. To the extent that the sequence of the wild-type polypeptide is modified by either the addition or deletion of one or more amino acids, one of skill in the art will understand that reference to a particular residue or residue number will be correspondingly altered to refer to the same particular amino acid in the modified polypeptide, but at the altered position number. For example, if an MHC class I polypeptide is modified by adding an amino acid to the N-terminus, reference to position 84 or a particular residue at position 84 will be understood to refer to the amino acid at position 85 on the modified polypeptide. Similarly, reference herein to a substitution of a particular amino acid at a particular position, e.g., Y84, will be understood to refer to the substitution of that amino acid for the amino acid at position 84 of the wild-type polypeptide. Thus, a Y84C substitution will be understood to be a substitution of a Cys residue for the Tyr residue present in the wild-type sequence. For example, if a wild-type polypeptide is modified to change the amino acid at position 84 from its wild-type amino acid to an alternative amino acid, the substitution of the amino acid at position 84 will be understood to refer to the substitution of the alternative amino acid. If such a polypeptide is also modified by the addition or deletion of one or more amino acids, reference to a substitution will be understood to refer to the substitution of the alternative amino acid at the altered position number.Reference to a "non-native Cys residue" in a polypeptide, e.g., an MHC class I polypeptide, means that the polypeptide contains a Cys residue at a position where there is no Cys in the corresponding wild-type polypeptide. This can be accomplished through routine protein engineering, in which a cysteine ​​is substituted for an amino acid that occurs in the wild-type sequence.

[0053] A polynucleotide or polypeptide has a certain percentage of "sequence identity" to another polynucleotide or polypeptide, meaning that when the two sequences are compared and aligned, that percentage of bases or amino acids are the same and in the same relative positions. Sequence identity can be measured using a number of different methods. To determine sequence identity, sequences may be aligned using a variety of convenient methods and computer programs (e.g., BLAST, T-COFFEE, MUSCLE, MAFFT, etc.) available on the World Wide Web, including sites such as ncbi.nlm.nili.gov / BLAST, ebi.ac.uk / Tools / msa / tcoffee / , ebi.ac.uk / Tools / msa / muscle / , mafft.cbrc.jp / alignment / software / . See, e.g., Altschul et al. (1990), J. Bioi. 215:403-10. Unless otherwise indicated, "sequence identity" referred to herein is determined using BLAST (Basic Local Alignment Search Tool) as described in Altschul et al. (1990) J. Mol. Biol. 215:403.

[0054] The term "conservative amino acid substitution" refers to the interchangeability of amino acid residues with similar side chains in proteins. For example, the group of amino acids with aliphatic side chains consists of glycine, alanine, valine, leucine, and isoleucine, the group of amino acids with aliphatic hydroxyl side chains consists of serine and threonine, the group of amino acids with amide-containing side chains consists of asparagine and glutamine, the group of amino acids with aromatic side chains consists of phenylalanine, tyrosine, and tryptophan, the group of amino acids with basic side chains consists of lysine, arginine, and histidine, the group of amino acids with acidic side chains consists of glutamic acid and aspartic acid, and the group of amino acids with sulfur-containing side chains consists of cysteine ​​and methionine. Exemplary conservative amino acid substitution groups are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine-glycine, and asparagine-glutamine.

[0055] As used herein, the term "immunological synapse" or "immune synapse" generally refers to the natural interface between two interacting immune cells of the adaptive immune response, including, for example, the interface between an antigen-presenting cell (APC) or target cell and an effector cell, e.g., a lymphocyte, an effector T cell, a natural killer cell, etc. The immunological synapse between an APC and a T cell is generally initiated by the interaction of a T cell antigen receptor with a major histocompatibility complex molecule, as described, for example, in Bromley et al., Annu Rev Immunol. (2001) 19:375-96, the entire disclosure of which is incorporated herein by reference.

[0056] "T cells" are helper T cells (CD4 + cells), cytotoxic T cells (CD8 + These include all types of immune cells that express CD3, including T cell regulators (TCRs), regulatory T cells (Tregs), and NK-T cells.

[0057] As used herein, the term "immunomodulatory polypeptide" (also referred to herein as "MOD") refers to a polypeptide that specifically binds to a cognate costimulatory polypeptide on a T cell, thereby providing signals that mediate T cell responses, including, but not limited to, proliferation, activation, differentiation, etc., in addition to the primary signal provided by, for example, binding of the TCR / CD3 complex to a peptide-loaded major histocompatibility complex (MHC) polypeptide. MODs discussed herein can include, but are not limited to, wild-type or wild-type variant polypeptides such as cytokines (e.g., IL-2), CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L, Fas ligand (FasL), inducible costimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM), CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, HVEM, lymphotoxin beta receptor, 3 / TR6, ILT3, ILT4, HVEM, agonists or antibodies that bind to the Toll ligand receptor, and ligands that specifically bind to B7-H3. MODs of TMMPs can bind to cognate costimulatory polypeptides (i.e., "co-MODs") present on target T cells.

[0058] As used herein, the term "in vivo" refers to any process or procedure that occurs inside the body.

[0059] As used herein, the term "in vitro" refers to any process or procedure that occurs outside the body.

[0060] As used herein, "heterologous" means a nucleotide or polypeptide that is not found in the native nucleic acid or protein, respectively.

[0061] As used herein, "recombinant" means that a particular nucleic acid (DNA or RNA) is the product of various combinations of cloning, restriction, polymerase chain reaction (PCR) and / or ligation steps that result in a construct having structural coding or non-coding sequences distinguishable from endogenous nucleic acids present in natural systems. A DNA sequence encoding a polypeptide may be assembled from cDNA fragments or a series of synthetic oligonucleotides to provide a synthetic nucleic acid expressible from a recombinant transcription unit contained within a cell or in a cell-free transcription and translation system.

[0062] The terms "recombinant expression vector" or "DNA construct" are used interchangeably herein to refer to a DNA molecule that includes a vector and at least one insert. Recombinant expression vectors are usually constructed for the purpose of expressing and / or propagating an insert or for the construction of other recombinant nucleotide sequences. The insert may or may not be operably linked to a promoter sequence and may or may not be operably linked to a DNA regulatory sequence.

[0063] As used herein, the term "affinity" refers to the equilibrium constant for the reversible binding of two substances (e.g., an antibody and an antigen), and the dissociation constant (K D ) As used herein, the term "affinity" refers to the resistance of a complex of two or more substances to dissociation after dilution. The terms "immunoreactive" and "preferentially bind" are used interchangeably herein with respect to antibodies and / or antigen-binding fragments.

[0064] As used herein (e.g., with respect to binding of a TMMP to a polypeptide on a T cell (e.g., a T cell receptor)), the term "binding" refers to a non-covalent interaction between two molecules. Non-covalent binding refers to a direct association between two molecules, for example, by electrostatic, hydrophobic, ionic, and / or hydrogen bonding interactions, including interactions such as salt bridges and water bridges. "Affinity" refers to the strength of the non-covalent bond, and increased binding affinity is indicated by a lower KD "Specific binding" generally refers to the binding of a ligand to a moiety that is its designated binding site or receptor. "Non-specific binding" generally refers to the binding of a ligand to a moiety other than its designated binding site or receptor. As used herein, "covalently binding" or "covalent bond" means the formation of one or more covalent chemical bonds between two distinct molecules.

[0065] As used herein, the terms "treatment", "treating" and the like generally refer to obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic in terms of completely or partially preventing a disease or its symptoms, and / or may be therapeutic in terms of partially or completely curing a disease and / or the deleterious effects caused by the disease. As used herein, "treatment" encompasses any treatment of a disease or condition in a mammal, (a) preventing the onset of a disease or condition in a subject who is susceptible to that disease or condition, or who may not be susceptible but has not yet been diagnosed as having it; (b) inhibiting the disease or one or more symptoms associated with the disease, e.g., preventing its onset, and / or (c) relieving the disease, i.e., causing regression of the disease; Therapeutic agents may be administered before, during, and / or after the onset of disease or injury. Of particular interest is the treatment of ongoing disease, which stabilizes or reduces the undesirable clinical symptoms of the patient. Such treatment is preferably administered before complete loss of function of the affected tissue. The therapy will preferably be administered during, and in some cases after, the symptomatic stage of the disease.

[0066] The terms "individual," "subject," "host," and "patient" are used interchangeably herein to refer to any mammalian subject for whom diagnosis, treatment, or therapy is desired. Mammals include, for example, humans, non-human primates, rodents (e.g., rats, mice), lagomorphs (e.g., rabbits), ungulates (e.g., cows, sheep, pigs, horses, goats, etc.), and the like. Unless otherwise indicated, the terms "individual," "subject," "host," and "patient" refer to humans.

[0067] The terms "antibody" and "immunoglobulin" include antibodies or immunoglobulins of any isotype, fragments of antibodies that retain specific binding to an antigen, including, but not limited to, Fab, Fv, scFv, and Fd fragments, chimeric antibodies, humanized antibodies, single chain antibodies (scAbs), single domain antibodies (dAbs), single domain heavy chain antibodies, single domain light chain antibodies, nanobodies, bispecific antibodies, multispecific antibodies, and fusion proteins comprising an antigen-binding (also referred to herein as antigen-binding) portion of an antibody and a non-antibody protein. Antibodies can be detectably labeled, for example, with a radioisotope, an enzyme that generates a detectable product, a fluorescent protein, and the like. Antibodies can be further conjugated to other moieties, such as, for example, members of a specific binding pair, such as biotin (a member of the biotin-avidin specific binding pair). The terms include Fab', Fv, F(ab') and F(ab') fragments that retain specific binding to an antigen. 2 , and / or other antibody fragments, as well as monoclonal antibodies. As used herein, a monoclonal antibody is an antibody produced by the same group of cells, all of which were produced from a single cell by repeated cell replication. That is, a clone of cells produces only a single antibody species. Monoclonal antibodies can be produced using hybridoma production techniques, although other production methods known to those skilled in the art can also be used (e.g., antibodies derived from antibody phage display libraries). Antibodies can be monovalent or bivalent. Antibodies can be Ig monomers, which are "Y-shaped" molecules consisting of four polypeptide chains, two heavy chains and two light chains connected by disulfide bonds.

[0068] The term "nanobody" (Nb), as used herein, refers to the smallest antigen-binding fragment or single variable domain (V) derived from a naturally occurring heavy chain antibody. HH ), known to those skilled in the art. They are derived from the heavy chain-only antibodies found in the Camelidae family (see Hamers-Casterman et al. (1993) Nature 363:446; Desmyter et al. (1996) Nature Structural Biol. 3:803; and Desmyter et al. (2015) Curr. Opin. Struct. Biol. 32:1), in which the "Camelidae" family are found immunoglobulins that lack light polypeptide chains. "Camelidae" includes Old World Camelidae (Camelus bactrianus and Camelus dromedarius) and New World Camelidae (e.g., Alpaca (Llama paccos), Llama glama, Guanaco (Llama guanicoe), and Vicuña (Llama vicugna). Single variable domain heavy chain antibodies are referred to herein as nanobodies or V HH These are called antibodies.

[0069] An "antibody fragment" includes a portion of an intact antibody, such as the antigen-binding or variable region of an intact antibody. Examples of antibody fragments include Fab, Fab', F(ab') 2 antibody fragments, as well as Fv fragments; diabodies; linear antibodies (Zapata et al., Protein Eng. 8(10):1057-1062 (1995)); domain antibodies (dAbs; Holt et al. (2003) Trends Biotechnol. 21:484); single-chain antibody molecules; and multispecific antibodies formed from antibody fragments. Papain digestion of antibodies produces two identical antigen-binding fragments called "Fab" fragments, each with a single antigen-binding site, and a remaining "Fc" fragment, a name reflecting the ability to crystallize readily. Pepsin treatment produces an F(ab') fragment that has two antigen-binding sites and is still capable of cross-linking antigen. 2 Produces fragments.

[0070] An "Fv" is the minimum antibody fragment that contains a complete antigen recognition and binding site. This region consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association. It is in this configuration that the three CDRs of each variable domain interact to define an antigen binding site on the surface of the VH-VL dimer. Together, the six CDRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although with a lower affinity than the entire binding site.

[0071] "Fab" fragments also contain the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab fragments differ from Fab' fragments by the addition of a few residues at the carboxyl terminus of the heavy chain CH1 domain including one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine ​​residues of the constant domains bear a free thiol group. F(ab')2 antibody fragments were originally produced as pairs of Fab' fragments that have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0072] The "light chains" of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequences of their constant domains. Depending on the amino acid sequences of the constant domains of their heavy chains, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these classes can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The subclasses can be further divided into types, e.g., IgG2a and IgG2b.

[0073] "Single-chain Fv" or "sFv" or "scFv" antibody fragments are fragments of the V H and V L domains, and these domains are present in a single polypeptide chain. In some embodiments, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains that enables the sFv to form the desired structure for antigen binding. For a review of sFvs, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0074] The term "diabody" refers to a small antibody fragment with two antigen-binding sites, which consists of a heavy chain variable domain (VH) (VL) connected to a light chain variable domain (VL) in the same polypeptide chain. H -V L (Diabodies include diabodies, which are complementary to the diabodies described in US Pat. No. 5,993,311; and Hollinger et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448.) By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains on another chain, creating two antigen-binding sites. Diabodies are described in more detail in, for example, EP 404,097; WO 93 / 11161; and Hollinger et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448.

[0075] As used herein, the term "CDR" or "complementarity determining region" is intended to mean the non-contiguous antigen binding sites found within the variable regions of both heavy and light chain polypeptides. CDRs are described by Kabat et al. (1977) J. Biol. Chem. 252:6609; Kabat et al., USDept. of Health and Human Services, "Sequences of proteins of immunological interest" (1991) (also referred to herein as Kabat 1991) by Chothia et al. (1987) J. Mol. Biol. 196:901 (also referred to herein as Chothia 1987), and MacCallum et al. (1996) J. Mol. Biol. 262:732, and the definitions include overlapping or subsets of amino acid residues when compared with each other. Nevertheless, application of either definition to refer to the CDRs of an antibody or grafted antibody or variants thereof is intended to be within the scope of the term as defined and used herein. The amino acid residues encompassing the CDRs as defined by each of the above cited documents are set forth in Table 1 below for comparison.

[0076] Table 1: CDR definitions TIFF2024541334000002.tif38137 1 Residue numbering follows the nomenclature of Kabat et al., 1991, supra. 2 Residue numbering follows the nomenclature of Chothia et al., supra. 3 Residue numbering follows the nomenclature of MacCallum et al., supra.

[0077] As used herein, the terms "CDR-L1", "CDR-L2", and "CDR-L3" refer to the first, second, and third CDRs in the light chain variable region, respectively. The terms "CDR-L1", "CDR-L2", and "CDR-L3" may be used interchangeably with "VL CDR1", "VL CDR2", and "VL CDR3", respectively. As used herein, the terms "CDR-H1", "CDR-H2", and "CDR-H3" refer to the first, second, and third CDRs in the heavy chain variable region, respectively. The terms "CDR-H1", "CDR-H2", and "CDR-H3" may be used interchangeably with "VH CDR1", "VH CDR2", and "VH CDR3", respectively. As used herein, the terms "CDR-1", "CDR-2", and "CDR-3" refer to the first, second, and third CDRs in the variable region of either chain, respectively.

[0078] As used herein, the term "framework", when used in reference to an antibody variable region, is intended to mean all amino acid residues outside the CDR regions in the variable region of an antibody. Variable region frameworks are generally discontinuous amino acid sequences of about 100-120 amino acids in length, but are intended to refer only to those amino acids outside the CDRs. As used herein, the term "framework region" is intended to mean each domain of the framework separated by the CDRs.

[0079] Unless otherwise indicated, the term "substantially" is intended to encompass both "entirely" and "largely, but not entirely." For example, an Ig Fc that "substantially does not induce ADCC" means an Ig Fc that does not induce ADCC at all or that does not induce ADCC for the most part.

[0080] As used herein, the term "about" used in connection with an amount indicates that the amount may vary by 10% of the amount stated. For example, "about 100" means an amount of 90 to 110. When about is used in the context of a range, "about" used in connection with a lower amount in a range means that the lower amount includes an amount 10% lower than the lower amount in the range, and "about" used in connection with a higher amount in a range means that the higher amount includes an amount 10% higher than the upper amount in the range. For example, about 100 to about 1000 means that the range extends from 90 to 1100.

[0081] As used herein, the term "MHC heavy chain polypeptide" refers collectively to the domains of the MHC heavy chain polypeptide present in the TMMP. For example, the MHC heavy chain polypeptide can include an α1 domain, an α2 domain, and an α3 domain.

[0082] Before the present disclosure is further described, it is to be understood that this disclosure is not limited to particular embodiments described, as such may, of course, vary. It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope will be limited only by the appended claims.

[0083] Where a range of numerical values ​​is provided, it is understood that each intervening value between the upper and lower limits of that range, to one-tenth of the unit of the lower limit unless the context clearly dictates otherwise, and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these narrower ranges may be independently included in the narrower range, and are also encompassed within the disclosure, subject to any specifically excluded value in the stated range. Where an explicit range includes one or both of those upper and lower limits, ranges excluding either or both of those included upper and lower limits are also encompassed within the disclosure.

[0084] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs.Although any method and material similar or equivalent to the methods and materials described herein can be used in the practice or testing of this disclosure, the preferred methods and materials are described below.All publications mentioned herein are incorporated by reference to disclose and describe the methods and / or materials in connection with the content in which they are cited.

[0085] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, a reference to a "T cell modulating polypeptide" includes a plurality of such polypeptides, a reference to an "immunomodulating polypeptide" includes a reference to one or more immunomodulating polypeptides and equivalents thereof known to those skilled in the art, and so forth. It is further noted that the claims may be drafted to exclude any element. Thus, this statement is intended to serve as a predicate for use of exclusive terminology, such as "solely," "only," or the use of a "negative" limitation in connection with the recitation of claim elements.

[0086] It is to be understood that certain features of the present disclosure that are described in the context of individual embodiments for clarity may also be provided in combination within a single embodiment. Conversely, various features of the present disclosure that are described in the context of a single embodiment for brevity may also be provided separately or in any suitable subcombination. All combinations of the embodiments belonging to the present disclosure are expressly embraced by the present disclosure and are disclosed herein as if all combinations were individually and expressly disclosed. In addition, all subcombinations of the various embodiments and elements thereof are also expressly embraced by the present disclosure and are disclosed herein as if all such subcombinations were individually and expressly disclosed herein.

[0087] Publications discussed herein are provided solely for their disclosure. Nothing herein should be construed as an admission that the present disclosure is not entitled to antedate such publications. Further, the dates of publication provided may be different from the actual publication dates, which may need to be independently confirmed.

[0088] Detailed Description The present disclosure relates to (i) an optional immune modulating polypeptide, such as a variant IL-2 polypeptide; (ii) Class I HLA major histocompatibility complex (MHC) polypeptides (Class I HLA heavy chain polypeptides and β2 microglobulin polypeptides); (iii) a peptide that presents an epitope to a T cell receptor and forms a peptide-MHC complex (pMHC) with a class I MHC polypeptide; (iv) a tumor targeting polypeptide, and (v) an optional Ig Fc polypeptide or other scaffold; The present invention provides a T cell regulatory multimeric polypeptide (TMMP) comprising:

[0089] TMMPs are useful for modulating the activity of T cells and for modulating immune responses in individuals. Importantly, the TMMPs disclosed herein are useful for "redirecting" a patient's repertoire of anti-pathogenic (e.g., anti-viral) T cells to attack and kill cancer cells. The TMMP achieves this result by (i) binding to the patient's cancer cells via the TMMP's tumor targeting polypeptide (TTP), which is specific for and binds to antigens on the cancer cells, and (ii) presenting a pMHC that mimics the pMHC on the surface of pathogen-infected cells, e.g., cells infected with viruses, bacteria, or other microorganisms that can cause disease. In this way, the TMMP effectively "paints" the cancer cells, making them look like pathogen-infected cells. The "painted" cancer cells become susceptible to being attacked and killed by the patient's existing repertoire of anti-pathogenic T cells that have T cell receptors (TCRs) that recognize and bind the pMHC presented by the TMMP.

[0090] For example, a TMMP may have a TTP that binds to an antigen on a cancer cell, and a pMHC that mimics the pMHC of a cell infected with a virus, such as the SARS-CoV-2 virus. Thus, the patient's existing repertoire of T cells directed against SARS-CoV-2 virus-infected cells may be redirected to the patient's cancer cells, and the T cells may be activated via binding of the TCR of the T cells to the pMHC of the TMMP. Activation of the T cells may lead to the release of their cytotoxic components and killing of the cancer cells. In such cases, the patient's repertoire of T cells specific for the SARS-CoV-2 virus may be the result of one or more of a previous SARS-CoV-2 infection, vaccination with a vaccine that elicits T cells with a TCR specific for the pMHC of the TMMP, and / or previous treatment with a T cell regulatory protein designed to prime and / or increase the patient's T cells with a TCR that specifically binds to the pMHC of the TMMP.

[0091] The TMMPs disclosed herein are useful for any patient with a hematological cancer or solid tumor, including those whose cancer has evaded the immune system due to HLA loss, which prevents the patient's own cancer-specific T cells from recognizing and killing the cancer cells.

[0092] T-CELL REGULATORY MULTIMERIC POLYPEPTIDES The present disclosure provides T cell regulatory multimeric polypeptides (TMMPs) comprising a) a first polypeptide and b) a second polypeptide, the TMMP comprising a peptide epitope (defined below), a first major histocompatibility complex (MHC) polypeptide, a second MHC polypeptide, one or more immune modulating polypeptides, an immunoglobulin (Ig) Fc polypeptide or a non-Ig scaffold, and a tumor targeting polypeptide (TTP).

[0093] In some examples, the TMMP comprises a heterodimer (comprising two distinct and separate polypeptide chains) comprising a) a first polypeptide and b) a second polypeptide, the TMMP comprising a peptide epitope, a first MHC polypeptide, a second MHC polypeptide, one or more immunomodulatory polypeptides, an Ig Fc polypeptide or a non-Ig scaffold, and a TTP. The TMMP may comprise a single heterodimer. The TMMP may comprise two copies of the heterodimer, i.e., the TMMP may be a homodimer of the heterodimer. The two copies of the heterodimer may be linked to each other, for example, by a disulfide bond (e.g., a disulfide bond between the Ig Fc polypeptides present in the heterodimer).

[0094] In some examples, the TMMP comprises one or more heterodimers, each heterodimer comprising a) a first polypeptide comprising a first MHC polypeptide and b) a second polypeptide comprising a second MHC polypeptide, the first polypeptide or the second polypeptide comprising a peptide epitope (defined below), the first polypeptide and / or the second polypeptide comprising one or more immunomodulatory polypeptides, which may be the same or different, and an Ig Fc polypeptide or a non-Ig scaffold. The first or second polypeptide also comprises TTP. In some examples, the TTP is at the C-terminus of the IgFc polypeptide or the non-Ig scaffold. In some examples, the TTP is at the N-terminus of the polypeptide comprising the Ig Fc polypeptide. In some examples, the TTP is at the C-terminus of the polypeptide comprising the peptide epitope.

[0095] A peptide epitope present in a TMMP presents a SARS-CoV-2 peptide (e.g., a SARS-CoV-2 encoded peptide). As used herein, the term "peptide epitope" refers to a peptide that, when complexed with an MHC polypeptide, presents an epitope to a T cell receptor (TCR). When complexed with an MHC polypeptide, a peptide epitope can present one or more epitopes to one or more TCRs.

[0096] In some examples, the TMMP comprises i) a SARS-CoV-2 peptide, and iii) a TTP that targets a cancer-associated antigen. Such a TMMP binds to a cancer cell that expresses the cancer-associated antigen targeted by the TTP. The TMMP regulates the activity of a T cell specific for a viral epitope present in the TMMP. For example, in some examples, the TMMP increases the proliferation and / or cytotoxic activity of a T cell specific for a viral epitope present in the TMMP. Upon contact with a T cell specific for a viral epitope present in the TMMP, the cytotoxic activity of the T cell against a cancer cell that expresses the cancer-associated antigen targeted by the TTP present in the TMMP can be increased.

[0097] TMMPs containing pMHC bearing SARS-CoV-2 epitopes are particularly useful because the high infection rate of SARS-CoV-2 worldwide and the availability of a commercially available vaccine mean that most patients already have, or can be induced to have through vaccination, a repertoire of T cells capable of recognizing pMHC bearing SARS-CoV-2 epitopes. However, as discussed above, the pMHC of the TMMP may alternatively present peptide epitopes from other viruses or pathogenic microorganisms for which the patient has a pre-existing repertoire of T cells. For example, the pMHC may present epitopes that would be recognized by T cells resulting from administration of commonly available vaccines that provide immunity to pathogens such as tetanus, influenza, or other available vaccines. Many commercially available vaccines exist against pathogens such as, for example, cholera, dengue fever, diphtheria, hepatitis, hemophilus influenza type b (Hib), human papillomavirus (HPV), influenza, Japanese encephalitis, malaria, measles, meningococcal meningitis, mumps, whooping cough, pneumococcal disease, polio infection, rabies, rotavirus, rubella, tetanus, tick-borne encephalitis, tuberculosis, typhoid fever, chickenpox, and yellow fever, with many other diseases in development such as, for example, enterotoxigenic E. coli, group B streptococcus (GBS), herpes simplex virus, HIV-1, malaria, gonorrhea, non-typhoidal salmonellosis, norovirus, paratyphoid, respiratory syncytial virus (RSV), schistosomiasis, erythroderma, group A streptococcus (GAS), tuberculosis, and other influenza vaccines. Alternatively (or additionally), one or more doses of a T cell modulating polypeptide may be administered to the patient prior to administration of the TMMP to induce CD8 +They can prime and activate and / or induce proliferation (and thereby increase the number) of T cells. See, for example, the T cell modulating polypeptides described in WO 2021 / 195108 (Cue Biopharma, Inc.), published on September 30, 2021, WO 2021 / 195411 (Cue Biopharma, Inc.), published on September 30, 2021, and WO 2022 / 015880 (Cue Biopharma, Inc.), published on January 20, 2022.

[0098] In some examples, the TMMP comprises one or more heterodimers, each heterodimer being a) i) a peptide epitope (SARS-CoV-2 peptide); and ii) a first MHC polypeptide; and A first polypeptide comprising: b) a second polypeptide comprising a second MHC polypeptide; c) one or more immunomodulatory polypeptides, wherein the first and / or second polypeptide comprises at least one (i.e., one or more) immunomodulatory polypeptide; d) an Ig Fc polypeptide or a non-Ig scaffold, wherein the first and / or second polypeptide comprises an Ig Fc polypeptide or a non-Ig scaffold; and e) a polypeptide that targets cancer cells (Tumor Targeting Polypeptide, "TTP"); Includes. In some examples, at least one of the one or more immunomodulatory polypeptides comprises the amino acid sequence of a wild-type immunomodulatory polypeptide. In some examples, at least one of the one or more immunomodulatory polypeptides is a variant immunomodulatory polypeptide that binds to a cognate co-immunomodulatory polypeptide but exhibits reduced affinity compared to the affinity of the corresponding wild-type immunomodulatory polypeptide for the cognate co-immunomodulatory polypeptide. In some examples, the peptide epitopes present in the TMMP represent infectious disease-associated epitopes (e.g., virally encoded peptides).

[0099] TMMPs contain tumor-targeting polypeptides, ie, polypeptides that target cancer-associated epitopes displayed on the surface of cancer cells.

[0100] In some examples, the TMMP is: A) a) a first polypeptide comprising a first MHC polypeptide; b) a second polypeptide comprising a second MHC polypeptide; and wherein the first polypeptide or the second polypeptide comprises a SARS-CoV-2 peptide, the first polypeptide and / or the second polypeptide comprise one or more immunomodulatory polypeptides, which may be the same or different, at least one of the one or more immunomodulatory polypeptides may be a wild-type immunomodulatory polypeptide or a variant of a wild-type immunomodulatory polypeptide, the first polypeptide and / or the second polypeptide comprises an Ig Fc polypeptide or a non-Ig scaffold, and the first and / or the second polypeptide comprises TTP; or B) a) a first polypeptide comprising a first MHC polypeptide; b) a second polypeptide comprising a second MHC polypeptide; and wherein the first polypeptide or the second polypeptide comprises a SARS-CoV-2 peptide, and the first polypeptide and / or the second polypeptide comprises one or more immunomodulatory polypeptides, which may be the same or different; at least one of the one or more immunomodulatory polypeptides is a variant of a wild-type immunomodulatory polypeptide; at least one of the one or more immunomodulatory polypeptides is a variant immunomodulatory polypeptide that exhibits reduced affinity for a cognate immunomodulatory polypeptide compared to the affinity of a corresponding wild-type immunomodulatory polypeptide for the cognate immunomodulatory polypeptide; a heterodimer, wherein the first polypeptide and / or the second polypeptide comprises an Ig Fc polypeptide or a non-Ig scaffold, and the first and / or second polypeptide comprises TTP; or C) a) From the N-terminus to the C-terminus: i) a SARS-CoV-2 peptide; and ii) a first MHC polypeptide; and and a first polypeptide comprising: b) In order from the N-terminus to the C-terminus, i) a second MHC polypeptide; and ii) optionally an immunoglobulin (Ig) Fc polypeptide or a non-Ig scaffold; and a second polypeptide comprising wherein the TMMP comprises one or more immunomodulatory polypeptides, which may be the same or different, and at least one of the one or more immunomodulatory polypeptides is located A) at the C-terminus of a first polypeptide, B) at the N-terminus of a second polypeptide, C) at the C-terminus of the second polypeptide, or D) at the C-terminus of the first polypeptide and the N-terminus of the second polypeptide, and at least one of the one or more immunomodulatory polypeptides may be a wild-type immunomodulatory polypeptide or a variant of a wild-type immunomodulatory polypeptide; Optionally, the heterodimer, wherein at least one of the one or more immunomodulatory polypeptides is a variant immunomodulatory polypeptide that exhibits lower affinity for a cognate cognate immunomodulatory polypeptide compared to the affinity of the corresponding wild-type immunomodulatory polypeptide for the cognate cognate immunomodulatory polypeptide.

[0101] In some instances, the immunomodulatory polypeptide present in the TMMP comprises a wild-type (naturally occurring) amino acid sequence.

[0102] In some instances, an immunomodulatory polypeptide present in a TMMP binds to a cognate immunomodulatory polypeptide with a lower affinity compared to the affinity of the corresponding wild-type immunomodulatory polypeptide for the cognate immunomodulatory polypeptide.

[0103] The reduced affinity of an immunomodulatory polypeptide for its cognate co-immunomodulatory polypeptide, combined with the affinity of an epitope for a TCR, provides enhanced selectivity of the TMMP, e.g., a TMMP selectively binds to a first T cell that presents both i) a TCR specific for an epitope present in the TMMP and ii) a co-immunomodulatory polypeptide that binds to an immunomodulatory polypeptide present in the TMMP, compared to binding to a second T cell that presents i) a TCR specific for an epitope other than the epitope present in the TMMP, and ii) a co-immunomodulatory polypeptide that binds to the immunomodulatory polypeptide present in the TMMP.

[0104] In some examples, the TMMP, when administered to an individual in need thereof, induces both epitope-specific and epitope-nonspecific T cell responses. In other words, in some examples, the TMMP, when administered to an individual in need thereof, induces an epitope-specific T cell response by modulating the activity of a first T cell that presents both i) a TCR specific for a peptide epitope present in the TMMP and ii) a co-immunomodulatory polypeptide that binds to an immunomodulatory polypeptide present in the TMMP, and induces an epitope-nonspecific T cell response by modulating the activity of a second T cell that presents i) a TCR specific for an epitope other than the epitope present in the TMMP and ii) a co-immunomodulatory polypeptide that binds to an immunomodulatory polypeptide present in the TMMP. The ratio of epitope-specific 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 response to epitope-nonspecific T cell response is about 2:1 to about 5:1, about 5:1 to about 10:1, about 10:1 to about 15:1, about 15:1 to about 20:1, about 20:1 to about 25:1, about 25:1 to about 50:1, or about 50:1 to about 100:1, or more than 100:1. Examples of "regulating the activity" of T cells include: i) cytotoxicity (e.g., CD8 + ) activating T cells; ii) cytotoxicity (e.g., CD8 + iii) inducing cytotoxic activity of T cells; and iv) cytotoxicity (e.g., CD8 + ) inducing the production and release of cytotoxins (e.g., perforin, granzymes, granulysin) by T cells; iv) suppressing the activity of autoreactive T cells, etc. As used herein, selectively modulating refers to inducing an epitope-specific T cell response that is greater than an epitope-nonspecific T cell response.

[0105] The reduced affinity of an immunomodulatory polypeptide for its cognate co-immunomodulatory polypeptide, combined with the affinity of an epitope for a TCR, provides enhanced selectivity for a TMMP. Thus, for example, a TMMP binds with higher affinity to a first T cell presenting both i) a TCR specific for an epitope present in the TMMP, and ii) a co-immunomodulatory polypeptide that binds to an immunomodulatory polypeptide present in the TMMP, compared to the affinity with which a TMMP binds to a second T cell presenting i) a TCR specific for an epitope other than the epitope present in the TMMP, and ii) a co-immunomodulatory polypeptide that binds to an immunomodulatory polypeptide present in the TMMP.

[0106] The binding affinity between an immunomodulatory polypeptide and its cognate co-immunomodulatory polypeptide can be determined by biolayer interferometry (BLI) as described in WO 2020 / 243315 (Cue Biopharma, Inc.).

[0107] Dimerized TMMP TMMPs may be dimerized, i.e., the present disclosure provides multimeric polypeptides comprising dimers of TMMPs. Thus, the present disclosure provides TMMPs comprising: A) a) i) a peptide epitope; and ii) a first major histocompatibility complex (MHC) polypeptide; and and a first polypeptide comprising: b) i) a second MHC polypeptide and a second polypeptide comprising a first heterodimer comprising one or more immunomodulatory polypeptides; and B) a) i) a peptide epitope; and ii) a first MHC polypeptide; and and a first polypeptide comprising: b) i) a second MHC polypeptide and a second polypeptide comprising a second polypeptide comprising one or more immunomodulatory polypeptides, wherein the first heterodimer and the second heterodimer are covalently linked to each other. In some cases, the two TMMPs are identical to each other in amino acid sequence. In some cases, the first heterodimer and the second heterodimer are covalently linked to each other via the C-terminal region of the second polypeptide of the first heterodimer and the C-terminal region of the second polypeptide of the second heterodimer. In some 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 either directly or via a linker. The linker may be a peptide linker. The peptide linker can 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 examples, the peptide epitope of the first heterodimer and the peptide epitope of the second heterodimer comprise the same amino acid sequence. In some examples, the first MHC polypeptide of the first and second heterodimers is MHC class I β2-microglobulin and the second MHC polypeptide of the first and second heterodimers is MHC class I heavy chain. In some examples, the immunomodulatory polypeptide of the first heterodimer and the immunomodulatory polypeptide of the second heterodimer comprise the same amino acid sequence. In some examples, the first heterodimeric immunomodulatory polypeptide and the second heterodimeric immunomodulatory polypeptide are variant immunomodulatory polypeptides that comprise between 1 and 10 amino acid substitutions compared to the corresponding parent wild-type immunomodulatory polypeptide, wherein the 1 to 10 amino acid substitutions result in reduced affinity binding of the variant immunomodulatory polypeptide to a cognate co-immunomodulatory polypeptide.In some examples, the first heterodimeric immunomodulatory polypeptide and the second heterodimeric immunomodulatory polypeptide are each independently selected from the group consisting of IL-2, 4-1BBL, PD-L1, CD80, CD86, ICOS-L, OX-40L, FasL, JAG1 (CD339), TGFβ, CD70, and ICAM. Examples of suitable MHC polypeptides, immunomodulatory polypeptides, and peptide epitopes are described below. The first and / or second polypeptide comprises i) an Ig Fc polypeptide or a non-Ig scaffold, and ii) a tumor targeting polypeptide.

[0108] Multiple disulfide bond TMMP In some examples, the first and second polypeptides of the TMMP are linked together by at least two disulfide bonds (i.e., two interchain disulfide bonds). Examples of such multiple disulfide bonded TMMPs are shown diagrammatically in Figures 18A-18C. Additionally, when the TMMP includes an IgFc polypeptide, the heterodimeric TMMP can be dimerized such that a disulfide bond links the IgFc polypeptides in the two heterodimeric TMMPs. Such an arrangement is shown diagrammatically in Figure 18D, with the disulfide bonds represented by dashed lines. Unless otherwise indicated, the at least two disulfide bonds described in the multiple disulfide bonded TMMPs in this section do not refer to the disulfide bonds linking the IgFc polypeptides in the dimerized TMMP.

[0109] As described above, in some cases, the first and second polypeptides of the 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 the TMMP are linked together by two interchain disulfide bonds. As another example, in some cases, the first and second polypeptides of the TMMP are linked together by three interchain disulfide bonds. As another example, in some cases, the first and second polypeptides of the TMMP are linked together by four interchain disulfide bonds.

[0110] In some examples, when a peptide epitope in a first polypeptide of a TMMP is linked to a β2M polypeptide by a linker that includes a Cys, at least one of the at least two disulfide bonds links a Cys in the linker to a Cys of an MHC class I heavy chain in a second polypeptide. In some examples, when a peptide epitope in a first polypeptide of a TMMP is linked to an MHC class I heavy chain polypeptide by a linker, at least one of the at least two disulfide bonds links a Cys in the linker to a Cys in a β2M polypeptide present in the second polypeptide.

[0111] In some examples, the T cell regulatory multimeric polypeptide comprises: a) i) a peptide epitope; and ii) a first MHC polypeptide; and A first polypeptide comprising: b) a second polypeptide comprising a second MHC polypeptide; c) at least one immunomodulatory polypeptide, wherein the first and / or second polypeptide comprises at least one immunomodulatory polypeptide; d) an Ig Fc polypeptide or a non-Ig scaffold, wherein the first and / or second polypeptide comprises an Ig Fc polypeptide or a non-Ig scaffold; and e) TTP, wherein the first and / or second polypeptide comprises TTP; and at least one heterodimer comprising A heterodimer comprises at least two disulfide bonds (e.g., two disulfide bonds) between a first polypeptide and a second polypeptide (e.g., a heterodimer comprises i) a first disulfide bond linking the first polypeptide and the second polypeptide, and ii) a second disulfide bond linking the first polypeptide and the second polypeptide). In other words, the first polypeptide includes a first Cys residue that forms a disulfide bond (first disulfide bond) with a first Cys residue of the second polypeptide, and the first polypeptide includes a second Cys residue that forms a disulfide bond (second disulfide bond) with a second Cys residue of the second polypeptide.

[0112] In some instances, TMMP may be a) From the N-terminus to the C-terminus: i) a peptide epitope; and ii) a peptide linker; and iii) a β2M polypeptide; and a first polypeptide comprising: b) MHC class I heavy chain polypeptide A second polypeptide comprising wherein one or both of the first and second polypeptides comprises at least one immunomodulatory polypeptide; TMMP is a) i) a Cys present in the linker between the peptide epitope and the β2M polypeptide; ii) a first Cys introduced into the MHC class I heavy chain polypeptide; and and a first disulfide bond between b) a first polypeptide and a second polypeptide at least a second disulfide bond between: i) a Cys in the first polypeptide that is C-terminal to a Cys present in the linker; and ii) a Cys in the second polypeptide that is C-terminal to a first Cys introduced into the MHC class I heavy chain polypeptide. As noted above, the TMMP also comprises i) an Ig Fc polypeptide or a non-Ig scaffold, and ii) a TTP.

[0113] A multiple disulfide bond TMMP (e.g., a double disulfide bond TMMP) can be prepared, for example, by a) i) a peptide epitope; and ii) a first MHC polypeptide; and a first polypeptide comprising a peptide linker between the peptide and a first MHC polypeptide, the peptide linker comprising a Cys residue, and the first MHC polypeptide being a β2M polypeptide comprising an amino acid substitution introducing a Cys residue; b) a second polypeptide comprising a second MHC polypeptide, the second MHC polypeptide being HLA-A * a class I heavy chain comprising a Y84C substitution and an A236C substitution based on the amino acid numbering of β2M polypeptide 0201 (shown in FIG. 7A ), or at the corresponding positions of another class I heavy chain allele, wherein TMMP comprises a disulfide bond between a Cys residue in the peptide linker and a Cys residue at amino acid position 84 of the class I heavy chain or at the corresponding position of another class I heavy chain allele, and wherein TMMP comprises a disulfide bond between the introduced Cys residue in the β2M polypeptide and a Cys at amino acid position 236 of the class I heavy chain or at the corresponding position of another class I heavy chain allele; and c) at least one immunomodulatory polypeptide, wherein the first and / or second polypeptide is at least one immunomodulatory polypeptide As noted above, the TMMP may also include i) an Ig Fc polypeptide or a non-Ig scaffold, and ii) a TTP.

[0114] SARS-COV-2 Peptides As described above, TMMPs are typically at least about four amino acids in length and contain a betacoronavirus (e.g., SARS-CoV-2) peptide that presents a SARS-CoV-2 epitope to T cells within an MHC / peptide complex (e.g., an HLA / peptide complex).

[0115] The SARS-CoV-2 peptides present in the TMMP can have a length of at least 4 amino acids, for example, 4 amino acids to about 25 amino acids (e.g., including within the ranges of lengths of 4 amino acids (aa), 5aa, 6aa, 7aa, 8aa, 9aa, 10aa, 11aa, 12aa, 13aa, 14aa, 15aa, 16aa, 17aa, 18aa, 19aa, 20aa, 21aa, 22aa, 23aa, 24aa, or 25aa, 4-20 amino acids, 6-18 amino acids, 8-15 amino acids, 8-12 amino acids, 9-11 amino acids, 9-10 amino acids, 5-10 amino acids, 10-15 amino acids, 10-20 amino acids, and 15-25 amino acids), for example, 9, 10, 11, 12, 13, or 14 amino acids in length.

[0116] The SARS-CoV-2 epitopes present in the TMMP are peptides that are specifically bound by T cells, i.e., the epitopes are specifically bound by epitope-specific T cells. Epitope-specific T cells bind to an epitope having a reference amino acid sequence, but do not substantially bind to an epitope that differs from the reference amino acid sequence. For example, epitope-specific T cells bind to an epitope having a reference amino acid sequence, but do not substantially bind to an epitope that differs from the reference amino acid sequence, for example, 10 to 20% of the epitopes present in the TMMP. -6 Less than M, 10 -5 Less than M or 10 -4An epitope-specific T cell can bind to the epitope for which it is specific with an affinity of at least 10 M. -7 M, at least 10 -8 M, at least 10 -9 M, or at least 10 -10 It can bind with an affinity of M.

[0117] The peptide epitope present in the TMMP is a peptide of a betacoronavirus encoded polypeptide. In some examples, the peptide epitope is a SARS-CoV-2 peptide (i.e., a peptide of a SARS-CoV-2 encoded polypeptide). In some examples, the peptide epitope is a SARS-CoV-2 peptide derived from a SARS-CoV-2 encoded surface glycoprotein. In some examples, the peptide epitope is a SARS-CoV-2 peptide derived from a SARS-CoV-2 encoded membrane glycoprotein. In some examples, the peptide epitope is a SARS-CoV-2 peptide derived from a SARS-CoV-2 encoded nucleocapsid phosphoprotein.

[0118] In some examples, the peptides present in the TMMP are peptides between 4 amino acids (aa) and 25 aa in length (e.g., 4 aa, 5 aa, 6 aa, 7, aa, 8 aa, 9 aa, 10 aa, 11 aa, 12 aa, 13 aa, 14 aa, 15 aa, 16 aa, 17 aa, 18 aa, 19 aa, 20 aa, 21 aa, 22 aa, 23 aa, 24 aa, or 25 aa in length) of a polypeptide comprising an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to any one of the SARS-CoV-2 polypeptides shown in Figures 13A-13J. In some examples, the peptide present in the TMMP is a peptide of 4 amino acids (aa) to 25 aa in length (e.g., 4 aa, 5 aa, 6 aa, 7 aa, 8 aa, 9 aa, 10 aa, 11 aa, 12 aa, 13 aa, 14 aa, 15 aa, 16 aa, 17 aa, 18 aa, 19 aa, 20 aa, 21 aa, 22 aa, 23 aa, 24 aa, or 25 aa in length) of one of the polypeptides shown in Figures 13A-13J.

[0119] In some examples, the peptide present in the TMMP is any one of the peptides shown in Figure 14. In some examples, the peptide present in the TMMP is any one of the peptides shown in Figure 15.

[0120] In some examples, the peptide epitope is a SARS-CoV-2 peptide derived from a SARS-CoV-2 encoded surface glycoprotein. In some examples, the peptides present in the TMMP are peptides between 4 amino acids (aa) and 25 aa in length (e.g., 4 aa, 5 aa, 6 aa, 7, aa, 8 aa, 9 aa, 10 aa, 11 aa, 12 aa, 13 aa, 14 aa, 15 aa, 16 aa, 17 aa, 18 aa, 19 aa, 20 aa, 21 aa, 22 aa, 23 aa, 24 aa, or 25 aa in length) of a polypeptide comprising an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the SARS-CoV-2 surface glycoprotein shown in FIG. 13J. In some examples, the peptides present in the TMMP are peptides of the SARS-CoV-2 surface glycoprotein shown in Figure 13J that are 4 amino acids (aa) to 25aa in length (e.g., 4aa, 5aa, 6aa, 7aa, 8aa, 9aa, 10aa, 11aa, 12aa, 13aa, 14aa, 15aa, 16aa, 17aa, 18aa, 19aa, 20aa, 21aa, 22aa, 23aa, 24aa, or 25aa in length).

[0121] In some examples, the peptide epitope is a SARS-CoV-2 peptide derived from a SARS-CoV-2 encoded surface glycoprotein, and the SARS-CoV-2 is the omicron variant (also known as the B1.1.529 variant). In some examples, the peptides present in the TMMP are peptides between 4 amino acids (aa) and 25 aa in length (e.g., 4 aa, 5 aa, 6 aa, 7, aa, 8 aa, 9 aa, 10 aa, 11 aa, 12 aa, 13 aa, 14 aa, 15 aa, 16 aa, 17 aa, 18 aa, 19 aa, 20 aa, 21 aa, 22 aa, 23 aa, 24 aa, or 25 aa in length) of a polypeptide comprising an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the SARS-CoV-2 surface glycoprotein shown in FIG. 13K. As shown in FIG. 13K, the spike glycoprotein of the omicron variant contains the following changes: A67V, del69-70, T95I, del142-144, Y145D, del211, L212I, ins214EPE, G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T4 78K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K, L981F, substitutions in bold are within the receptor binding domain (RBD), "ins" indicates insertion and "del" indicates deletion. In some examples, the peptides present in the TMMP are peptides of the SARS-CoV-2 surface glycoprotein shown in Figure 13K that are 4 amino acids (aa) to 25aa in length (e.g., 4aa, 5aa, 6aa, 7aa, 8aa, 9aa, 10aa, 11aa, 12aa, 13aa, 14aa, 15aa, 16aa, 17aa, 18aa, 19aa, 20aa, 21aa, 22aa, 23aa, 24aa, or 25aa in length).

[0122] In some examples, the peptide present in the TMMP is a SARS-CoV-2 peptide derived from a SARS-CoV-2 encoded surface glycoprotein peptide and is selected from the group consisting of: NLTTRTQL (SEQ ID NO: 533), LPPAYTNSF (SEQ ID NO: 590), KVFRSSVLH (SEQ ID NO: 288), LPFFSNVTW (SEQ ID NO: 591), PFFSNVTWF (SEQ ID NO: 388), RFDNPVLPF (SEQ ID NO: 389), LPFNDGVYF (SEQ ID NO: 475), GVYFASTEK (SEQ ID NO: 289), TEKSNIIRGW (SEQ ID NO: 3 ... No. 711), TLDSKTQSL (SEQ ID NO: 534), GVYYHKNNK (SEQ ID NO: 290), YYHKNNKSW (SEQ ID NO: 412), VYSSANNCTF (SEQ ID NO: 390), FEYVSQPFL (SEQ ID NO: 661), EYVSQPFLM (SEQ ID NO: 391), FVFKNIDGY (SEQ ID NO: 592), TPINLVRDL (SEQ ID NO: 476), LPQGFSAL (SEQ ID NO: 477), LPIGINITRF (SEQ ID NO: 593), INITRFQTL (SEQ ID NO: 535), LLALHRSYL (SEQ ID NO: 536), WTAGAAAYY ( SEQ ID NO: 145), YYVGYLQPRTF (SEQ ID NO: 392), YLQPRTFLL (SEQ ID NO: 218), YLQPRTFL (SEQ ID NO: 537), SETKCTLKSF (SEQ ID NO: 712), TLKSFTVEK (SEQ ID NO: 291), QPTESIVRF (SEQ ID NO: 594), RFPNITNLCPF (SEQ ID NO: 413), GEVFNATRF (SEQ ID NO: 662), NATRFASVY (SEQ ID NO: 595), LYNSASFSTF (SEQ ID NO: 393), NSASFSTFK (SEQ ID NO: 329), RQIAPGQTGK (SEQ ID NO: 292), KIA DYNYKL (SEQ ID NO: 219), NYNYLYRLF (SEQ ID NO: 394), RLFRKSNLK (SEQ ID NO: 293), KPFERDISTEI (SEQ ID NO: 478), YFPLQSYGF (SEQ ID NO: 395), QPYRVVVL (SEQ ID NO: 479), PYRVVVLSF (SEQ ID NO: 396), GPKKSTNLV (SEQ ID NO: 480), TSNQVAVLY (SEQ ID NO: 146), VYSTGSNVF (SEQ ID NO: 397), AEHVNNSY (SEQ ID NO: 721), IPIGAGICASY (SEQ ID NO: 596), SPRRARSVA (SEQ ID NO: 481),VASQSIIAY (SEQ ID NO: 597), SIIAYTMSL (SEQ ID NO: 220), LGAENSVAY (SEQ ID NO: 598), AYSNNSIAIPTNF (SEQ ID NO: 414), IPTNFTISV (SEQ ID NO: 482), TEILPVSMTK (SEQ ID NO: 330), QEVFAQVKQIY (SEQ ID NO: 713), KQIYKTPPIK (SEQ ID NO: 294), IYKTPPIKDF (SEQ ID NO: 398), LLFNKVTL A (SEQ ID NO: 221), TLADAGFIK (SEQ ID NO: 295), LADAGFIKQY (SEQ ID NO: 147), ADAGFIKQY (SEQ ID NO: 714), VLPPLLTDEMIAQY (SEQ ID NO: 148), IPFAMQMAY (SEQ ID NO: 599), SSTASALGK (SEQ ID NO: 331), VLNDILSRL (SEQ ID NO: 222), RLDKVEAEV (SEQ ID NO: 223), VEAEVQIDRL (SEQ ID NO: 663), AEVQIDRLI (SEQ ID NO: 664), LITGRLQSL (SEQ ID NO: 538), RLQSLQTYV (SEQ ID NO: 224), AEIRASANL (SEQ ID NO: 665), ASANLAATK (SEQ ID NO: 296), HLMSFPQSA (SEQ ID NO: 225), FPQSAPHGVVF (SEQ ID NO: 600), APHGVVFL (SEQ ID NO: 483), VTYVPAQEK (SEQ ID NO: 297), TYVPAQEKNF (SEQ ID NO: 39 9), REGVFVSNGTHW (SEQ ID NO: 715), GTHWFVTQR (SEQ ID NO: 332), TVYDPLQPELDSFK (SEQ ID NO: 333), KEIDRLNEV (SEQ ID NO: 666), QELGKYEQYIKW (SEQ ID NO: 716), YEQYIKWPW (SEQ ID NO: 717), QYIKWPWYI (SEQ ID NO: 400), FIAGLIAIV (SEQ ID NO: 226), and SEPVLKGVKL (SEQ ID NO: 484).

[0123] In some examples, the peptide epitope is a SARS-CoV-2 peptide derived from a SARS-CoV-2 encoded membrane glycoprotein. In some examples, the peptides present in the TMMP are peptides between 4 amino acids (aa) and 25 aa in length (e.g., 4 aa, 5 aa, 6 aa, 7, aa, 8 aa, 9 aa, 10 aa, 11 aa, 12 aa, 13 aa, 14 aa, 15 aa, 16 aa, 17 aa, 18 aa, 19 aa, 20 aa, 21 aa, 22 aa, 23 aa, 24 aa, or 25 aa in length) of a polypeptide comprising an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the SARS-CoV-2 membrane glycoprotein shown in FIG. 13D. In some examples, the peptides present in the TMMP are peptides of 4 amino acids (aa) to 25aa in length (e.g., 4aa, 5aa, 6aa, 7aa, 8aa, 9aa, 10aa, 11aa, 12aa, 13aa, 14aa, 15aa, 16aa, 17aa, 18aa, 19aa, 20aa, 21aa, 22aa, 23aa, 24aa, or 25aa in length) of the SARS-CoV-2 membrane glycoprotein shown in FIG. 13D.

[0124] In some examples, the peptide present in the TMMP is a SARS-CoV-2 peptide derived from a SARS-CoV-2 encoded membrane glycoprotein peptide and is selected from the group consisting of GTITVEELK (SEQ ID NO: 302), EELKKLLEQW (SEQ ID NO: 671), KLLEQWNLV (SEQ ID NO: 155), FAYANRNRF (SEQ ID NO: 544), YANRNRFLY (SEQ ID NO: 545), SYFIASFRLF (SEQ ID NO: 338), RLFARTRSM (SEQ ID NO: 491), VPLHGTIL (SEQ ID NO: 427), SELVIGAVIL (SEQ ID NO: 602), HLRIAGHHL (SEQ ID NO: 492), RIAGHHLGR (SEQ ID NO: 233), KEITVATSRTL (SEQ ID NO: 603), ATSRTLSYYK (SEQ ID NO: 303), ASQRVAGDSGFAAY (SEQ ID NO: 101), and VAGDSGFAAY (SEQ ID NO: 102).

[0125] In some examples, the peptide epitope is a SARS-CoV-2 peptide derived from the SARS-CoV-2 encoded nucleocapsid phosphoprotein. In some examples, the peptides present in the TMMP are peptides between 4 amino acids (aa) and 25 aa in length (e.g., 4 aa, 5 aa, 6 aa, 7, aa, 8 aa, 9 aa, 10 aa, 11 aa, 12 aa, 13 aa, 14 aa, 15 aa, 16 aa, 17 aa, 18 aa, 19 aa, 20 aa, 21 aa, 22 aa, 23 aa, 24 aa, or 25 aa in length) of a polypeptide comprising an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the SARS-CoV-2 nucleocapsid phosphoprotein shown in FIG. 13F. In some examples, the peptides present in the TMMP are peptides of 4 amino acids (aa) to 25aa in length (e.g., 4aa, 5aa, 6aa, 7aa, 8aa, 9aa, 10aa, 11aa, 12aa, 13aa, 14aa, 15aa, 16aa, 17aa, 18aa, 19aa, 20aa, 21aa, 22aa, 23aa, 24aa, or 25aa in length) of the SARS-CoV-2 nucleocapsid phosphoprotein shown in FIG. 13F.

[0126] In some examples, the peptide epitope is a SARS-CoV-2 peptide derived from the SARS-CoV-2 encoded nucleocapsid phosphoprotein and is selected from the group consisting of LPNNTASWF (SEQ ID NO:567), KFPRGQGVPI (SEQ ID NO:454), NTNSSPDDQIGYY (SEQ ID NO:119), SPRWYFYYL (SEQ ID NO:455), LLLDRLNQL (SEQ ID NO:192), KAYNVTQAF (SEQ ID NO:568), QELIRQGTDYKHW (SEQ ID NO:690), ASAFFGMSR (SEQ ID NO:315), SRIGMEVTPSGTW (SEQ ID NO:691), GMEVTPSGTWL (SEQ ID NO:692), TPSGTWLTY (SEQ ID NO:569), AYKTFPPTEPK (SEQ ID NO:316), and LPAADLDDF (SEQ ID NO:570).

[0127] In some examples, the peptide epitope is a SARS-CoV-2 peptide shown in Figure 19. In some examples, the peptide is an HLA-A peptide shown in Figure 19. * 01:01 restricted peptide. In some examples, the peptide is an HLA-A 01:01 restricted peptide as shown in FIG. * 02:01 restricted peptide. In some examples, the peptide is an HLA-A 02:01 restricted peptide as shown in FIG. * In some examples, the peptide is an HLA-A 11:01 restricted peptide. * In some examples, the peptide is an HLA-B 24:01 restricted peptide. * 07:02 is a restricted peptide. The peptides of Figure 19 may be paired with the HLA alleles shown in Figure 19 or with different HLA alleles.

[0128] In some examples, the peptide epitope is RLQSLQTYV (SEQ ID NO: 224). In some examples, the peptide epitope is YLQPRTFLL (SEQ ID NO: 218).

[0129] In some examples, the peptide epitope may be presented in a complex between a β2M polypeptide and an HLA-E polypeptide. As one non-limiting example, in some examples, the peptide epitope is a peptide of a SARS-CoV-2 Nsp13 (helicase) polypeptide (see, e.g., FIG. 13C). In some examples, the peptide epitope is VMPLSAPTL (SEQ ID NO: 914). In some examples, the TMMP comprises an HLA-E heavy chain polypeptide and comprises the peptide epitope VMPLSAPTL (SEQ ID NO: 914).

[0130] MHC Polypeptides As discussed above, TMMPs include MHC polypeptides. For purposes of this disclosure, the term "major histocompatibility complex (MHC) polypeptide" is meant to include MHC polypeptides of various species, including human MHC (also referred to as human leukocyte antigen (HLA)) polypeptides, rodent (e.g., mouse, rat, etc.) MHC polypeptides, and MHC polypeptides of other mammals (e.g., lagomorphs, non-human primates, canines, felines, ungulates (e.g., horses, cows, sheep, goats, etc.)). The term "MHC polypeptide" is meant to include class I MHC polypeptides (e.g., beta-2 microglobulin and MHC class I heavy chains).

[0131] In some examples, the first MHC polypeptide is an MHC class I beta 2 microglobulin (β2M) polypeptide and the second MHC polypeptide is an MHC class I heavy chain (H chain) ("MHC-H"). In other examples, the first MHC polypeptide is an MHC class I heavy chain polypeptide and the second MHC polypeptide is a β2M polypeptide. In some examples, both the β2M and the MHC-H chain are of human origin, i.e., the MHC-H chain is an HLA heavy chain, or a variant thereof. Unless otherwise specified, the TMMP does not include the membrane anchor domain (transmembrane region) of the MHC class I heavy chain, or a portion of the MHC class I heavy chain sufficient to anchor the resulting TMMP to the cell in which it is expressed (e.g., a eukaryotic cell such as a mammalian cell). In some examples, the MHC class I heavy chain present in the TMMP does not include a signal peptide, transmembrane domain, or intracellular domain (cytoplasmic tail) associated with a native MHC class I heavy chain. Thus, for example, in some instances, the MHC class I heavy chain present in the TMMP comprises only the α1, α2, and α3 domains of the MHC class I heavy chain polypeptide. In some instances, the MHC class I heavy chain present in the TMMP has a length of about 270 amino acids (aa) to about 290 aa. In some instances, the MHC class I heavy chain present in the TMMP has a length of 270aa, 271aa, 272aa, 273aa, 274aa, 275aa, 276aa, 277aa, 278aa, 279aa, 280aa, 281aa, 282aa, 283aa, 284aa, 285aa, 286aa, 287aa, 288aa, 289aa, or 290aa.

[0132] MHC class I heavy chain In some examples, the MHC class I heavy chain polypeptide present in the TMMP comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to all or a portion (e.g., 50, 75, 100, 150, 200, or 250 contiguous amino acids) of the amino acid sequence of any of the human HLA heavy chain polypeptides shown in Figures 3-11. In some examples, the MHC class I heavy chain has a length of 270aa, 271aa, 272aa, 273aa, 274aa, 275aa, 276aa, 277aa, 278aa, 279aa, 280aa, 281aa, 282aa, 283aa, 284aa, 285aa, 286aa, 287aa, 288aa, 289aa, or 290aa. In some examples, the MHC class I heavy chain has a length of 270aa to 280aa. In some examples, the MHC class I heavy chain polypeptide present in the TMMP comprises 1-30, 1-5, 5-10, 10-15, 15-20, 20-25, or 25-30 amino acid insertions, deletions, and / or substitutions (in addition to those positions shown to be variable in the heavy chain consensus sequence) of any one of the amino acid sequences shown in Figures 3-11. As noted above, MHC class I heavy chains typically do not include transmembrane or cytoplasmic domains.

[0133] In some examples, the MHC polypeptide of the TMMP is a human MHC polypeptide, which is also referred to as a "human leukocyte antigen" ("HLA") polypeptide. In some examples, the MHC polypeptide of the TMMP is a class I HLA polypeptide, such as a β2-microglobulin polypeptide, or a class I HLA heavy chain polypeptide. Class I HLA heavy chain polypeptides include HLA-A heavy chain polypeptides, HLA-B heavy chain polypeptides, HLA-C heavy chain polypeptides, HLA-E heavy chain polypeptides, HLA-F heavy chain polypeptides, and HLA-G heavy chain polypeptides.

[0134] In some examples, the TMMP comprises an HLA-A heavy chain polypeptide. HLA-A heavy chain peptide sequences or portions thereof that can be incorporated into the TMMP include, but are not limited to, the following alleles: A * 0101, A * 0201, A * 0301, A * 1101, A * 2301, A * 2402, A * 2407, A * 3303, and A * 3401. Any of these alleles may contain a mutation at one or more of positions 84, 139, and 236 (as shown in Figures 3-6, 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). Further, all or a portion of the sequence of those HLA-A alleles (e.g., 50, 75, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3401). HLA-A sequences having at least 75% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%) or 100% amino acid sequence identity over 0, 200, or 250 contiguous amino acids (e.g., may contain 1-25, 1-5, 5-10, 10-15, 15-20, 20-25, or 25-30 amino acid insertions, deletions, and / or substitutions). Some examples are provided below.

[0135] HLA-A02 In some examples, the MHC class I heavy chain polypeptide of the TMMP comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence shown in FIG. 3A. In some examples, an HLA-A heavy chain polypeptide suitable for inclusion in a TMMP comprises the amino acid sequence shown in FIG. 3A. This HLA-A heavy chain polypeptide is referred to as "HLA-A *As described in WO 2018 / 119114 and WO 2020 / 132138, the TMMP can include one or more mutations from wild-type HLA-A02, including providing a Cys residue capable of forming a disulfide bond, such as (i) between the β2M and the MHC class I heavy chain, and / or (ii) between the MHC heavy chain and the linker connecting the peptide epitope to the β2M polypeptide, and / or (iii) an intrachain disulfide bond within the MHC class I heavy chain polypeptide.

[0136] To facilitate the formation of such disulfide bonds, one or more non-naturally occurring Cys residues can be provided in the heavy chain polypeptide. For example, the MHC class I heavy chain polypeptide comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following: A) The HLA-A02 (Y84C; A236C) amino acid sequence shown in FIG. 3C, in which amino acid 84 is Cys and amino acid 236 is Cys. In some examples, Cys-236 forms an interchain disulfide bond with Cys-12 of a variant β2M polypeptide that includes an R12C substitution: or B) The HLA-A02(Y84C;A236C) amino acid sequence shown in FIG. 3D, in which amino acid 84 is Ala and amino acid 236 is Cys. In some examples, Cys-236 forms an interchain disulfide bond with Cys-12 of a variant β2M polypeptide that includes an R12C substitution: or C) HLA-A02(Y84C;A139C) amino acid sequence shown in FIG. 3E, in which amino acid 84 is Cys and amino acid 139 is Cys.

[0137] As described above, in some examples, the MHC class I heavy chain polypeptide comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the HLA-A02 (Y84A; A236 wild type) amino acid sequence shown in FIG. 3B, wherein amino acid 84 is Tyr and amino acid 236 is Ala.

[0138] HLA-A11(HLA-A * 1101) In some examples, the MHC class I heavy chain polypeptide of the TMMP comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence shown in FIG. 4A. In some examples, an HLA-A heavy chain polypeptide suitable for inclusion in a TMMP comprises the amino acid sequence shown in FIG. 4A. This HLA-A heavy chain polypeptide is referred to as "HLA-A * Variants include, for example, MHC class I heavy chain polypeptides that include an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following: A) HLA-A11 (Y84A; A236 wild type) amino acid sequence shown in FIG. 4B, in which amino acid 84 is Tyr and amino acid 236 is Ala; B) HLA-A11(Y84C;A236C) amino acid sequence shown in FIG. 4C, in which amino acid 84 is Cys and amino acid 236 is Cys; C) The HLA-A11 (Y84A;A236C) amino acid sequence shown in FIG. 4D, in which amino acid 84 is Ala and amino acid 236 is Cys. In some instances, Cys-236 forms an interchain disulfide bond with Cys-12 of a variant β2M polypeptide that includes an R12C substitution: and D) The HLA-A11 (Y84C;A139C) amino acid sequence shown in Figure 4E, in which amino acid 84 is Cys and amino acid 139 is Cys. In some instances, Cys-84 forms an intrachain disulfide bond with Cys-139.

[0139] HLA-A24(HLA-A * 2402) In some examples, the MHC class I heavy chain polypeptide of the TMMP comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence shown in FIG. 5A. In some examples, an HLA-A heavy chain polypeptide suitable for inclusion in a TMMP comprises the amino acid sequence shown in FIG. 5A. This HLA-A heavy chain polypeptide is referred to as "HLA-A * Variants include, for example, MHC class I heavy chain polypeptides that include an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to: A) HLA-A24 (Y84A; A236 wild type) amino acid sequence shown in FIG. 5B, in which amino acid 84 is Tyr and amino acid 236 is Ala; B) HLA-A24(Y84C;A236C) amino acid sequence shown in FIG. 5C, in which amino acid 84 is Cys and amino acid 236 is Cys; C) The HLA-A24 (Y84A; A236C) amino acid sequence shown in FIG. 5D, in which amino acid 84 is Ala and amino acid 236 is Cys. In some instances, Cys-236 forms an interchain disulfide bond with Cys-12 of a variant β2M polypeptide that includes an R12C substitution: and D) The HLA-A24(Y84C;A139C) amino acid sequence shown in Figure 5E, in which amino acid 84 is Cys and amino acid 139 is Cys. In some instances, Cys-84 forms an intrachain disulfide bond with Cys-139.

[0140] HLA-A33(HLA-A * 3303) In some examples, the MHC class I heavy chain polypeptide of the TMMP comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence shown in FIG. 6A. In some examples, an HLA-A heavy chain polypeptide suitable for inclusion in a TMMP comprises the amino acid sequence shown in FIG. 6A. This HLA-A heavy chain polypeptide is referred to as "HLA-A * Variants include, for example, MHC class I heavy chain polypeptides that include an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following: A) HLA-A24 (Y84A; A236 wild type) amino acid sequence shown in FIG. 6B, in which amino acid 84 is Tyr and amino acid 236 is Ala; B) HLA-A24(Y84C;A236C) amino acid sequence shown in FIG. 6C, in which amino acid 84 is Cys and amino acid 236 is Cys; C) The HLA-A24 (Y84A; A236C) amino acid sequence shown in FIG. 6D, in which amino acid 84 is Ala and amino acid 236 is Cys. In some instances, Cys-236 forms an interchain disulfide bond with Cys-12 of a variant β2M polypeptide that includes an R12C substitution: and D) The HLA-A24(Y84C;A139C) amino acid sequence shown in Figure 6E, in which amino acid 84 is Cys and amino acid 139 is Cys. In some instances, Cys-84 forms an intrachain disulfide bond with Cys-139.

[0141] Figures 7-9 provide alignments of mature HLA class I heavy chain amino acid sequences (excluding leader sequences or transmembrane or intracellular domains). The amino acid sequences in Figure 7A are HLA-A class I heavy chains of the following alleles: A * 0101, A * 0201, A * 0301, A * 1101, A * 2301, A * 2402, A * 2407, A * 3303, and A * 3401. The amino acid sequence in FIG. 8A 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. The amino acid sequence in FIG. 9A 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) where cysteine ​​residues may be introduced (e.g., by substitution) for the formation of disulfide bonds to stabilize the HLA H chain-β2M complex are shown in the alignment. Also shown in the alignment is position 236 (of the mature polypeptide), which may be replaced (e.g., at aa12) by a cysteine ​​residue capable of forming an interchain disulfide bond with β2M. Boxes adjacent to residues 84, 139, and 236 indicate groups of five amino acids on either side of those sextuplets of five residues, designated aac1 ("amino acid cluster 1"), aac2 ("amino acid cluster 2"), aac3 ("amino acid cluster 3"), aac4 ("amino acid cluster 4"), aac5 ("amino acid cluster 5"), and aac6 ("amino acid cluster 6"), which may be replaced with (i) any naturally occurring amino acid or (ii) one to five amino acids independently selected from any naturally occurring amino acid except proline or glycine.

[0142] Figures 7A, 8A, and 9A provide alignments of the amino acid sequences of mature HLA-A, HLA-B, and HLA-C class I heavy chains, respectively. Sequences are provided for the extracellular portion of the mature proteins (excluding leader sequences or transmembrane or intracellular domains). Also shown are the positions of aa residues 84, 139, and 236, as well as the positions of the adjacent residues (aac1-aac6) that may be replaced with (i) any naturally occurring amino acid or (ii) one to five amino acids independently selected from any naturally occurring amino acid except proline or glycine. Figures 7B, 8B, and 9B provide consensus amino acid sequences for the HLA-A, -B, and -C sequences provided in Figures 7A, 8A, and 9A, respectively. The consensus sequences show the variable amino acid positions as consecutively numbered "X" residues, with amino acid positions 84, 139, and 236 double underlined.

[0143] With reference to FIG. 7A, in some examples: i) aac1 (amino acid cluster 1) may be the amino acid sequence GTLRG (SEQ ID NO: 915) or a sequence thereof in which one or two amino acids have been deleted or substituted with other naturally occurring amino acids (e.g., L is replaced with I, V, A, or F); ii) aac2 (amino acid cluster 2) may be the amino acid sequence YNQSE (SEQ ID NO: 916) or a sequence thereof in which one or two amino acids have been deleted or substituted with other naturally occurring amino acids (e.g., N is replaced by Q, Q is replaced by N, and / or E is replaced by D); iii) aac3 (amino acid cluster 3) may be the amino acid sequence TAADM (SEQ ID NO: 917) or a sequence thereof in which one or two amino acids have been deleted or substituted with other naturally occurring amino acids (e.g., T is replaced by S, A is replaced by G, D is replaced by E, and / or M is replaced by L, V, or I); iv) aac4 (amino acid cluster 4) may be the amino acid sequence AQTTK (SEQ ID NO: 918) or a sequence thereof in which one or two amino acids have been deleted or substituted with other naturally occurring amino acids (e.g., A is replaced by G, Q is replaced by N, or T is replaced by S, and / or K is replaced by R or Q); v) aac5 (amino acid cluster 5) may be the amino acid sequence VETRP (SEQ ID NO: 919) or a sequence thereof in which one or two amino acids have been deleted or substituted with other naturally occurring amino acids (e.g., V is replaced by I or L, E is replaced by D, T is replaced by S, and / or R is replaced by K), and / or vi) aac6 (amino acid cluster 6) may be the amino acid sequence GDGTF (SEQ ID NO: 920) or a sequence thereof in which one or two amino acids have been deleted or substituted with other naturally occurring amino acids (e.g., D is replaced with E, T is replaced with S, or F is replaced with L, W, or Y).

[0144] With reference to FIG. 8A, in some examples: i) aac1 (amino acid cluster 1) may be the amino acid sequence RNLRG (SEQ ID NO:921) or a sequence thereof in which one or two amino acids have been deleted or substituted with other naturally occurring amino acids (e.g., N is replaced by T or I, and / or L is replaced by A, and / or the second R is replaced by L, and / or G is replaced by R); ii) aac2 (amino acid cluster 2) may be the amino acid sequence YNQSE (SEQ ID NO: 916) or a sequence thereof in which one or two amino acids have been deleted or substituted with other naturally occurring amino acids (e.g., N is replaced by Q, Q is replaced by N, and / or E is replaced by D); iii) aac3 (amino acid cluster 3) may be the amino acid sequence TAADT (SEQ ID NO: 922) or a sequence thereof in which one or two amino acids have been deleted or substituted with other naturally occurring amino acids (e.g., the first T is replaced 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: 923) or a sequence thereof in which one or two amino acids have been deleted or substituted with other naturally occurring amino acids (e.g., A is replaced 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: 919) or a sequence thereof in which one or two amino acids have been deleted or substituted with other naturally occurring amino acids (e.g., V is replaced by I or L, E is replaced by D, T is replaced by S, and / or R is replaced by K), and / or vi) aac6 (amino acid cluster 6) may be the amino acid sequence GDRTF (SEQ ID NO: 924) or a sequence thereof in which one or two amino acids have been deleted or substituted with other naturally occurring amino acids (e.g., D is replaced by E, and / or T is replaced by S, and / or R is replaced by K or H, and / or F is replaced by L, W, or Y).

[0145] With reference to FIG. 9A, in some examples: i) aac1 (amino acid cluster 1) may be the amino acid sequence RNLRG (SEQ ID NO:921) or a sequence thereof in which one or two amino acids have been deleted or, if substituted, with another naturally occurring amino acid (e.g., N is replaced by K, and / or L is replaced by A or I, and / or the second R is replaced by H, and / or G is replaced by T or S); ii) aac2 (amino acid cluster 2) may be the amino acid sequence YNQSE (SEQ ID NO: 916) or a sequence thereof in which one or two amino acids have been deleted or substituted with other naturally occurring amino acids (e.g., N is replaced by Q, Q is replaced by N, and / or E is replaced by D); iii) aac3 (amino acid cluster 3) may be the amino acid sequence TAADT (SEQ ID NO: 922) or a sequence thereof in which one or two amino acids have been deleted or substituted with other naturally occurring amino acids (e.g., the first T is replaced by S, and / or A is replaced by S, 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: 923) or a sequence thereof in which one or two amino acids have been deleted or substituted with other naturally occurring amino acids (e.g., A is replaced by G, and / or the first Q is replaced by N, and / or I is replaced by L, and / or the second Q is replaced by N or K), v) aac5 (amino acid cluster 5) may be the amino acid sequence VETRP (SEQ ID NO: 919) or a sequence thereof in which one or two amino acids have been deleted or substituted with other naturally occurring amino acids (e.g., V is replaced by I or L, E is replaced by D, T is replaced by S, and / or R is replaced by K or H), and / or vi) aac6 (amino acid cluster 6) may be the amino acid sequence GDGTF (SEQ ID NO: 920) or a sequence thereof in which one or two amino acids have been deleted or substituted with other naturally occurring amino acids (e.g., D is replaced by E, and / or T is replaced by S, and / or F is replaced by L, W, or Y).

[0146] Nonclassical HLA-E, -F, and -G MHC class I heavy chains In some examples, the TMMP comprises a non-classical MHC class I heavy chain polypeptide. Non-classical HLA heavy chain polypeptides or portions thereof that may be incorporated into the TMMP include, but are not limited to, HLA-E, HLA-F, and HLA-G alleles. The amino acid sequences of HLA-E, -F, and -G heavy chain polypeptides (and HLA-A, -B, and -C alleles) can be found on the World Wide Web at hla.alleles.org / nomenclature / index.html, at the European Bioinformatics Institute, part of the European Molecular Biology Laboratory (EMBL) (www(dot)ebi(dot)ac(dot)uk), and at the National Center for Biotechnology Information (www(dot)ncbi(dot)nlm(dot)nih(dot)gov).

[0147] Non-limiting examples of suitable HLA-E alleles include HLA-E * 0101(HLA-E * 01:01:01:01), HLA-E * 01:03(HLA-E * 01:03:01:01), HLA-E * 01:04, HLA-E * 01:05, HLA-E * 01:06, HLA-E * 01:07, HLA-E * 01:09, and HLA-E * Among these, the isoform HLA-E * 0101 and HLA-E * 01.03 are particularly noteworthy because they are highly prevalent alleles and differ by only one amino acid (Arg or Gly at position 107). For example, the amino acid sequences of suitable HLA-E heavy chain polypeptides are provided in Figures 16A-16D, where Figure 16A shows the amino acid sequence of HLA-E * FIG. 16B provides the amino acid sequence of HLA-E 01:01 (wild type) with Y84C and A2346C substitutions. * 16C provides the amino acid sequence of HLA-E 01:01; *FIG. 16D provides the amino acid sequence of HLA-E 01:03 (wild type) with Y84C and A2346C substitutions. * 01:03. In some examples, therefore, the MHC class I heavy chain polypeptide of TMMP can comprise an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence shown in Figures 16A, 16B, 16C, or 16D.

[0148] Non-limiting examples of suitable HLA-F alleles include HLA-F * 0101(HLA-F * 01:01:01:01), HLA-F * 01:02, HLA-F * 01:03 (HLA-F * 01:03:01:01), HLA-F * 01:04, HLA-F * 01:05, and HLA-F * 01:06. Non-limiting examples of suitable HLA-G alleles include, but are not limited to, the following: 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. Of these, the isoform HLA-G * 0101(HLA-G * 01:01:01:01) and HLA-G * 01:04 (HLA-G * 01:04:01:01) are of particular note because they are highly prevalent alleles. For example, the amino acid sequences of suitable HLA-G heavy chain polypeptides are provided in Figures 17A-17D. * FIG. 17B provides the amino acid sequence of HLA-G 01:01 (wild type) with Y84C and A2346C substitutions. * 17C provides the amino acid sequence of HLA-G * FIG. 17D provides the amino acid sequence of HLA-G 01:04 (wild type) with Y84C and A2346C substitutions. * 01:04. In some examples, therefore, the MHC class I heavy chain polypeptide of TMMP comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence shown in Figure 17A, 17B, 17C, or 17D.

[0149] The consensus sequences for those HLA E, HLA-F, and HLA-G alleles, lacking all or substantially all of the leader, transmembrane, and cytoplasmic sequences, are provided in FIG. 10 and aligned with the consensus sequences for the HLA-A, HLA-B, and HLA-C alleles described above in FIG. 11.

[0150] FIG. 10 shows the consensus sequences for HLA-E, HLA-F, and HLA-G, respectively, with variable aa positions shown as consecutively numbered "X" residues, with positions aa 84, 139, and 236 double underlined.

[0151] Figure 11 provides an alignment of the consensus amino acid sequences of HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, and HLA-G shown in Figures 7-11. Variable residues in each sequence are listed as "X" with sequential numbering removed. Positions aa84, 139, and 236 are shown with five amino acid clusters adjacent to them that can be replaced with (i) any naturally occurring amino acid, or (ii) one to five amino acids independently selected from any naturally occurring amino acid except proline or glycine.

[0152] Any of the above HLA-E, HLA-F, and / or HLA-G alleles may include substitutions at one or more of positions 84, 139, and / or 236, as shown in Figure 11 for the consensus sequence. In some examples, the substitutions may be selected from the following: a tyrosine to alanine (Y84A) or cysteine ​​(Y84C), or for HLA-F, an R84A or R84C substitution at position 84; an alanine to cysteine ​​(A139C), or for HLA-F, V139C; and an alanine to cysteine ​​substitution at position 236 (A236C). In addition, HLA-E, -F, and / or -G sequences having at least 75% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%) or 100% amino acid sequence identity to all or a portion (e.g., 50, 75, 100, 150, 200, or 250 contiguous amino acids) of the respective consensus sequences set forth in FIG. 11 may also be used (e.g., the sequences may include insertions, deletions, and / or substitutions of 1-25, 1-5, 5-10, 10-15, 15-20, 20-25, or 25-30 amino acids in addition to changes at the variable residues listed in the figure).

[0153] Beta-2 microglobulin The β2-microglobulin (β2M) polypeptide of the TMMP can be a human β2M polypeptide, a non-human primate β2M polypeptide, a mouse β2M polypeptide, etc. In some examples, the β2M polypeptide comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence set forth in Figure 1A (wild-type human β2M). In some examples, the β2M polypeptide present in the TMMP comprises the amino acid sequence shown in Figure 1A (wild-type human β2M).

[0154] In some examples, the MHC polypeptide present in the TMMP comprises a single amino acid substitution compared to a reference MHC polypeptide (which may be a wild-type MHC polypeptide), where the single amino acid substitution replaces an amino acid with a cysteine ​​(Cys) residue. Such a cysteine ​​residue can form a disulfide bond with a natural or non-natural cysteine ​​residue present in the MHC heavy chain of the TMMP. As used herein, reference to a "non-natural Cys residue" in an MHC class I polypeptide means that the polypeptide comprises a Cys residue at a position where there is no Cys in the corresponding wild-type polypeptide. This can be achieved through routine protein engineering, where a cysteine ​​is replaced with an amino acid occurring in the wild-type sequence.

[0155] In some examples, the β2M polypeptide present in the TMMP comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 100% amino acid sequence identity to the amino acid sequence shown in Figure 1B, where amino acid 12 is Cys, i.e., the β2M comprises a non-naturally occurring Cys at position 12 as a result of an R12C substitution. In some examples, the β2M polypeptide present in the TMMP comprises the amino acid sequence shown in Figure 1B.

[0156] Immunomodulating Polypeptides In some examples, the immunomodulatory polypeptide ("MOD") present in the TMMP is a wild-type ("wt") MOD. As noted above, in other examples, the MOD present in the TMMP is a variant of a wild-type MOD that has reduced affinity for the co-MOD compared to the affinity of the corresponding wild-type MOD for the co-MOD. A suitable MOD that exhibits reduced affinity for the co-MOD may have between one amino acid (aa) and 20 aa differences from the wild-type MOD.

[0157] As discussed above, a MOD can include a variant of a wild-type immunomodulatory polypeptide that can exhibit reduced binding to the co-MOD, including, for example, reduced binding to one or more chains or domains of the co-MOD. For example, a variant MOD present in a TMMP can bind to the co-MOD with an affinity that is at least 10% less, at least 15% less, at least 20% less, at least 25% less, at least 30% less, at least 35% less, at least 40% less, at least 45% less, at least 50% less, at least 55% less, at least 60% less, at least 65% less, at least 70% less, at least 75% less, at least 80% less, at least 85% less, at least 90% less, at least 95% less, or greater than 95% less than the affinity of the corresponding wild-type MOD for the co-MOD.

[0158] Exemplary pairs of MODs and their co-MODs include, but are not limited to, those set forth in Table 2 below.

[0159] (Table 2) TIFF2024541334000003.tif105128

[0160] One or more MODs may be present in a TMMP at any of a variety of locations. For example, in some instances, a MOD (or multiple MODs) may be present: 1) at the C-terminus of the MHC class I heavy chain and the N-terminus of the Ig Fc polypeptide, in other words, between the MHC class I heavy chain polypeptide and the Ig Fc polypeptide, 2) at the C-terminus of the Ig Fc polypeptide, or 3) at the N-terminus of a peptide epitope.

[0161] For example, immune modulating polypeptides and variants, including lower affinity variants, of PD-L1, CD80, CD86, 4-1BBL, and IL-2, are described in published literature, such as, for example, published WO 2020132138A1 and WO 2019 / 051091, the disclosures regarding MODs and specific variant MODs of PD-L1, CD80, CD86, 4-1BBL, IL-2 are expressly incorporated herein by reference, in particular paragraphs

[0260] to

[0455] of WO 2020132138A1 and paragraphs

[0157] to

[0352] of WO 2019 / 051091.

[0162] Of particular note is MOD, a variant of the cytokine IL-2. Wild-type IL-2 binds to the IL-2 receptor (IL-2R) on the surface of T cells. Wild-type IL-2 has a strong affinity for IL-2R and binds to activate most or virtually all CD8+ T cells. For this reason, synthetic forms of wild-type IL-2, such as the drug aldesleukin (trademark Proleukin®), are known to have severe side effects when administered to humans for the treatment of cancer, because IL-2 indiscriminately activates both target and non-target T cells.

[0163] The IL-2 receptor is, in some instances, a heterotrimeric polypeptide comprising an alpha chain (IL-2Rα, also referred to as CD25), a beta chain (IL-2Rβ, also referred to as CD122), and a gamma chain (IL-2Rγ, also referred to as CD132). The amino acid sequences of human IL-2, human IL-2Rα, IL2Rβ, and IL-2Rγ are known. See, for example, the above-mentioned published International Publication Nos. WO2020132138A1 and WO2019 / 051091. For example, a wild-type IL-2 polypeptide may have the amino acid sequence shown in FIG. 12A. The amino acid sequences of human IL-2Rα, human IL-2Rβ, and human IL-2Rγ are shown in FIG. 12B, FIG. 12C, and FIG. 12D, respectively. In some examples, the human IL-2Rα, human IL-2Rβ, and human IL-2Rγ polypeptides are in the mature form (lacking the signal peptide).

[0164] In some examples, the IL-2 variant MODs of the present disclosure exhibit reduced binding to IL-2Rα, thereby minimizing or substantially reducing activation of Tregs by the IL-2 variant. Alternatively, or additionally, in some examples, such that the IL-2 variant MODs exhibit reduced overall affinity for IL-2R, the IL-2 variant MODs of the present disclosure exhibit reduced binding to IL-2Rβ. In some examples, such that the IL-2 variant polypeptides exhibit reduced overall affinity for IL-2R, the IL-2 variant MODs of the present disclosure exhibit both properties, i.e., reduced or substantially no binding to IL-2Rα and reduced binding to IL-2Rβ. For example, IL-2 variants with substitutions at H16 and F42 exhibited reduced binding to IL-2Rα and IL-2Rβ. See Quayle et al., Clin Cancer Res;26(8)April 15,2020, which discloses that the binding affinity of IL-2 polypeptides with H16A and F42A substitutions for human IL-2Rα and IL-2Rβ was reduced by 110-fold and 3-fold, respectively, compared to wild-type IL2 binding, primarily due to faster off-rates for each of these interactions. TMMPs containing such variants, including variants that exhibit reduced binding to IL-2Rα and IL-2Rβ, have demonstrated the ability to preferentially bind and activate IL-2 receptors on T cells that contain a target TCR specific for a peptide epitope on the TMMP, and are therefore less likely to deliver IL-2 to non-target T cells, i.e., T cells that do not contain a TCR that specifically binds to a peptide epitope on the TMMP. That is, binding of the IL-2 variant MOD to its costimulatory polypeptide on T cells is substantially driven by binding of the MHC epitope moiety rather than binding of IL-2. In some examples, the IL-2 variant MODs of the present disclosure exhibit reduced binding to IL-2Rγ, which may be in addition to reduced binding to IL-2Rα and / or IL-2Rβ.

[0165] Thus, suitable IL-2 variant MODs include polypeptides comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the wild-type IL-2 amino acid sequence shown in Figure 12A, as well as having one or more amino acid differences from the wild-type IL-2 amino acid sequence shown in Figure 12A that provide reduced binding to IL-2Rα, IL-2Rβ, and / or IL-2Rγ. In some examples, such variant IL-2 polypeptides of the present disclosure exhibit reduced binding affinity to IL-2R compared to the binding affinity of an IL-2 polypeptide comprising the wild-type IL-2 amino acid sequence shown in Figure 12A. For example, in some examples, the variant IL-2 polypeptide binds to IL-2R with a binding affinity that is at least 10% lower, at least 15% lower, at least 20% lower, at least 25% lower, at least 30% lower, at least 35% lower, at least 40% lower, at least 45% lower, at least 50% lower, at least 55% lower, at least 60% lower, at least 65% lower, at least 70% lower, at least 75% lower, at least 80% lower, at least 85% lower, at least 90% lower, at least 95% lower, or greater than 95% lower than the binding affinity of an IL-2 polypeptide comprising a wild-type IL-2 amino acid sequence shown in FIG. 12A to IL-2R (e.g., an IL-2R comprising an amino acid sequence shown in FIGS. 12B-12D or a polypeptide comprising a mature form of the amino acid sequence shown in FIGS. 12B-12D) when assayed under the same conditions.

[0166] Mutations that can reduce binding of IL-2 to IL-2Rα include substitutions at one or more of the following amino acids: R38, F42, K43, Y45, E62, P65, E68, V69, and L72. For example, an IL-2 variant can include substitutions at one, two, three, four, five, or more of the foregoing amino acids. Exemplary substitutions include the following from Table 3:

[0167] (Table 3) TIFF2024541334000004.tif53165

[0168] As mentioned above, substitution of F42 with an amino acid other than Phe, e.g., Ala, has been shown to substantially reduce binding of IL-2 variants to IL-2Rα. See Quayle et al., Clin Cancer Res 2020;26:1953-64, who reported that F42A substitution caused a 110-fold decrease in binding to IL-2Rα. Thus, binding to IL-2Rα is substantially eliminated by this mutation.

[0169] Mutations that can reduce binding of IL-2 to IL-2Rβ include substitutions at one or more of the following amino acids: E15, H16, L19, D20, D84, S87, N88, V91, I92. For example, an IL-2 variant can include substitutions at one, two, three, four, five, or more of the foregoing amino acids. Exemplary substitutions include the following from Table 4:

[0170] (Table 4) TIFF2024541334000005.tif58164

[0171] As mentioned above, substitution of H16 with an amino acid other than His, e.g., Ala, has been shown to reduce binding of IL-2 variants to IL-2Rβ. See Quayle et al., Clin Cancer Res 2020;26:1953-64, who reported that H16A substitution caused a 3-fold decrease in binding to IL-2Rβ.

[0172] Some exemplary combinations of mutations that reduce binding of IL-2 variant polypeptides to IL-2Rα and IL-2Rβ include the following from Table 5:

[0173] (Table 5) TIFF2024541334000006.tif62163TIFF2024541334000007.tif234163TIFF2024541334000008.tif234163 TIFF2024541334000009.tif245163TIFF2024541334000010.tif189164TIFF2024541334000011.tif204164

[0174] Exemplary IL-2 variant polypeptide amino acid sequences include amino acid sequences having at least about 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to any one of the following IL-2 variant amino acid sequences (I)-(XVIII): TIFF2024541334000012.tif17154 sequence, X 1 is any amino acid other than Arg, e.g., Ala, Asp, or Glu; TIFF2024541334000013.tif17156 sequence, X 1 is any amino acid other than Phe, e.g., Ala or Lys, and in some instances, X 1 may alternatively be Gly, Val, Leu, Ile, Arg, Asn, Cys, Glu, Gln, Ile, Lys, Met, Phe, Pro, Ser, Tyr, Trp or Val; TIFF2024541334000014.tif17155Array, X 1 is any amino acid other than Lys, e.g., Glu; TIFF2024541334000015.tif17155Array, X 1 is any amino acid other than Tyr; TIFF2024541334000016.tif17156 sequence, X 1 is any amino acid other than Glu, e.g., Gln; TIFF2024541334000017.tif17156 sequence, X 1 is any amino acid other than Pro; TIFF2024541334000018.tif17156 sequence, X 1 is any amino acid other than Glu; TIFF2024541334000019.tif17155Array, X 1 is any amino acid other than Val; TIFF2024541334000020.tif17156 sequence, X 1 is any amino acid other than Leu; TIFF2024541334000021.tif17154 sequence, X 1 is any amino acid other than Glu, e.g., Ala; TIFF2024541334000022.tif17154 sequence, X 1 is any amino acid other than His, e.g., Ala, Glu, Thr, or Asp, and in some instances, X 1 is Gly, Val, Leu, Ile, Arg, Asn, Cys, Glu, Gln, Ile, Lys, Met, Phe, Pro, Ser, Tyr, Trp or Val, TIFF2024541334000023.tif17154 sequence, X 1 is any amino acid other than Leu; TIFF2024541334000024.tif17154 sequence, X 1 is any amino acid other than Asp, e.g., Asn; TIFF2024541334000025.tif17155Array, X 1 is any amino acid other than Asp, e.g., His, Lys, or Arg; TIFF2024541334000026.tif17157 sequence, X 1 is any amino acid other than Ser; TIFF2024541334000027.tif17155Array, X 1 is any amino acid other than Asn, e.g., Ser, Ala, Gly, Arg, Thr or Asp; TIFF2024541334000028.tif17156 sequence, X 1 is any amino acid other than Val, e.g., Glu, Ala, or Thr; and TIFF2024541334000029.tif17158 sequence, X 1 is any amino acid other than Ile, for example Ala.

[0175] In any of the above IL-2 variant amino acid sequences (I) to (XVIII), the amino acid sequence may contain one or more additional mutations that reduce binding of the IL-2 variant to IL-2Rα and / or IL-2Rβ (compared to the binding affinity of wild-type IL-2 to IL-2Rα and / or IL-2Rβ).

[0176] Determination of binding affinity may be carried out by known methods.

[0177] In some examples, a suitable variant IL-2 polypeptide has the following amino acid sequence: TIFF2024541334000030.tif17155. That is, the variant IL-2 polypeptide has the amino acid sequence of wild-type IL-2 but has H16 and F42 substitutions (X 1 and X 2 , shown in bold). As noted above, the histidine at position 16 can be substituted, for example, with Ala, Glu, Thr, or Asp, although any of the following substitutions can also be used: Gly, Val, Leu, Ile, Arg, Asn, Cys, Glu, Gln, Ile, Lys, Met, Phe, Pro, Ser, Tyr, or Trp. The phenylalanine at position 42 can be substituted, for example, with Ala or Lys, although any of the following substitutions can also be used: Met, Pro, Ser, Thr, Trp, Tyr, or Val.

[0178] In some examples, the IL-2 polypeptide present in the TMMP has the following amino acid sequence: Contains TIFF2024541334000031.tif17153.

[0179] In some examples, the IL-2 polypeptide present in the TMMP has the following amino acid sequence: TIFF2024541334000032.tif17153. In some examples, the TMMP comprises two copies of such a variant IL-2 polypeptide.

[0180] In some examples, the IL-2 polypeptide present in the TMMP has the following amino acid sequence: TIFF2024541334000033.tif17153. In some examples, the TMMP comprises two copies of such a variant IL-2 polypeptide.

[0181] In some examples, the IL-2 polypeptide present in the TMMP has the following amino acid sequence: TIFF2024541334000034.tif17153. In some examples, the TMMP comprises two copies of such a variant IL-2 polypeptide.

[0182] As a further example of a MOD, in some instances, the MOD present in TMMP is a 4-1BBL polypeptide. In some instances, the 4-1BBL polypeptide of TMMP has the following 4-1BBL amino acid sequence: The amino acid sequence has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF2024541334000035.tif26164.

[0183] In some examples, the MOD present in the TMMP is a CD80 polypeptide. In some examples, the CD80 polypeptide of the TMMP has the following CD80 amino acid sequence: The amino acid sequence has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF2024541334000036.tif31158.

[0184] In some examples, the MOD present in the TMMP is a CD86 polypeptide. In some examples, the CD86 polypeptide of the TMMP has the following CD86 amino acid sequence: It includes an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF2024541334000037.tif25166.

[0185] Anti-CD28 Polypeptides In some examples, the immunomodulatory polypeptide present in the TMMP is an anti-CD28 antibody (e.g., anti-CD28 scFv, anti-CD28 nanobody, anti-CD28 diabody, etc.). The ability of anti-CD28 antibodies to act as superagonists, agonists, or antagonists of CD28 activity has been described. See, for example, Poirier et al., (2012) Amer. J. of Transplantation, "CD28-specific Immunomodulating Antibodies: What Can Be Learned From Experimental Models?" 12:1682-1690. Of particular note are anti-CD28 antibodies that act as agonists or superagonists.

[0186] Thus, in some examples, the TMMP comprises an anti-CD28 antibody as at least one of the one or more immune-modulating polypeptides. In some examples, the TMMP comprises a) an anti-CD28 antibody, and b) one or more IL-2 polypeptides as the one or more immune-modulating polypeptides. In some examples, the TMMP comprises a) an anti-CD28 antibody, and b) one or more IL-2 polypeptides as the one or more immune-modulating polypeptides, where the one or more IL-2 polypeptides are variant IL-2 polypeptides as described above (e.g., an IL-2 polypeptide comprising an H16A and F42A substitution, an IL-2 polypeptide comprising an H16T and F42A substitution, an IL-2 polypeptide comprising an H16T and F42T substitution, and an IL-2 polypeptide comprising an H16A and F42T substitution, etc.). In some examples, the TMMP comprises a) an anti-CD28 antibody, and b) two copies of an IL-2 polypeptide comprising an H16A and F42A substitution as the one or more immune-modulating polypeptides. Examples of anti-CD28 VL and VH polypeptides are provided below. In some examples, the TMMP comprises, as one or more immunomodulatory polypeptides, a) an anti-CD28 antibody, and b) two copies of an IL-2 polypeptide comprising an H16T and F42A substitution. In some examples, the TMMP comprises, as one or more immunomodulatory polypeptides, a) an anti-CD28 antibody, and b) one, two, or three copies of a 4-1BBL polypeptide. In some examples, the TMMP comprises, as one or more immunomodulatory polypeptides, a) an anti-CD28 antibody, and b) three copies of a 4-1BBL polypeptide comprising a wild-type amino acid sequence. In some examples, the TMMP comprises, as one or more immunomodulatory polypeptides, a) an anti-CD28 antibody, and b) three copies of a variant 4-1BBL polypeptide. The two different immunomodulatory polypeptides can be present at any of several different positions in the first or second polypeptide of the TMMP heterodimer. For example, in some instances, the anti-CD28 antibody is at the C-terminus of a first polypeptide and the second immune modulating polypeptide (e.g., an IL-2 polypeptide (e.g., a variant IL-2 polypeptide), a 4-1BBL polypeptide, etc.) is at the N-terminus of a second polypeptide.As another example, in some instances, the anti-CD28 antibody is at the N-terminus of the second polypeptide and the second immune modulating polypeptide (e.g., an IL-2 polypeptide (e.g., a variant IL-2 polypeptide), a 4-1BBL polypeptide, etc.) is at the C-terminus of the second polypeptide.

[0187] In some examples, an anti-CD28 antibody suitable for inclusion as an immunomodulatory polypeptide in a TMMP is a) the amino acid sequence: VL CDR1, VL CDR2, and VL CDR3 present in the light chain variable region (VL) comprising TIFF2024541334000038.tif13166; and b) the amino acid sequence: VH CDR1, CDR2, and CDR3 present in the heavy chain variable region (VH) containing TIFF2024541334000039.tif18163 Includes. In some cases, V H and V L CDRs are as defined by Kabat (see, e.g., Table 1, above, and Kabat 1991). H and V L The CDRs are as defined by Chothia (see, e.g., Table 1, above, and Chothia 1987). In some examples, the VH CDRs are: DYGVH (SEQ ID NO: 931) (VH CDR1), VIWAGGGTNYNSALMS (SEQ ID NO: 932) (VH CDR2), and DKGYSYYYSMDY (SEQ ID NO: 933) (VH CDR3).

[0188] In some examples, an anti-CD28 antibody suitable for inclusion as an immunomodulatory polypeptide in a TMMP is a) the amino acid sequence: a VL region comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF2024541334000040.tif11165; and b) the amino acid sequence: A VH region comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF2024541334000041.tif18164. Includes.

[0189] In some instances, anti-CD28 antibodies suitable for inclusion as an immunomodulatory polypeptide in a TMMP include, in order from N-terminus to C-terminus: a) the amino acid sequence: a VL region comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF2024541334000042.tif12165; b) a peptide linker, and c) the amino acid sequence: A VH region comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF2024541334000043.tif19165. It is an scFv comprising: In some examples, the peptide linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO: 934), where n is an integer from 1 to 10 (e.g., n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). In some examples, the peptide linker comprises the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 875) and has a length of 15 amino acids.

[0190] In some instances, anti-CD28 antibodies suitable for inclusion as an immunomodulatory polypeptide in a TMMP include, in order from N-terminus to C-terminus: a) the amino acid sequence: a VH region comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF2024541334000044.tif19165; b) a peptide linker, and c) the amino acid sequence: A VL region comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF2024541334000045.tif11164. It is an scFv comprising: In some examples, the peptide linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO: 934), where n is an integer from 1 to 10 (e.g., n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). In some examples, the peptide linker comprises the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 875) and has a length of 15 amino acids.

[0191] Location of immunomodulatory polypeptides and TTP An immunomodulatory polypeptide (i.e., one or more immunomodulatory polypeptides) may be present in a TMMP at any of a variety of positions. For example, an immunomodulatory polypeptide may be present: 1) at the N-terminus of the MHC class I heavy chain (position 1), 2) at the C-terminus of the MHC class I heavy chain and the N-terminus of the Ig Fc polypeptide, in other words, between the MHC class I heavy chain and the Ig Fc polypeptide (position 2), 3) 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). Similarly, a TTP may be present in a TMMP at any of a variety of positions. Non-limiting examples of various MOD positions and various TTP positions are shown in Figures 28A-28C.

[0192] Of the embodiments shown in Figures 28A-28C, configurations in which MOD is at position 1 or position 3 and TTP is at position 1 or position 3, e.g., (MOD position 1, TTP position 3) and (MOD position 3, TTP position 1), may be more commonly used. As noted above, in any of the above embodiments, one or more of the individual components of the first polypeptide in each of the TMMP combinations (e.g., peptide epitopes, β2M polypeptides, TTP, etc.) are linked together by one or more linkers (e.g., peptide linkers described elsewhere herein). In any of the above embodiments, one or more of the individual components of the second polypeptide in each of the TMMP combinations (e.g., MHC class I heavy chains, immunomodulatory polypeptides, Ig Fc polypeptides, etc.) are linked together by one or more linkers (e.g., peptide linkers described elsewhere herein).

[0193] Scaffold Polypeptides The TMMP may comprise an Fc polypeptide or another suitable scaffold polypeptide.

[0194] Suitable scaffold polypeptides include antibody-based scaffold polypeptides and non-antibody-based scaffolds.Non-antibody-based scaffolds include, for example, albumin, XTEN (extended recombinant) polypeptide, transferrin, Fc receptor polypeptide, elastin-like polypeptide (see, for example, Hassouneh et al. (2012) Methods Enzymol. 502:215; for example, the polypeptide that comprises pentapeptide repeat unit (Val-Pro-Gly-X-Gly; SEQ ID NO: 935), where X is any amino acid other than proline), albumin-binding polypeptide, silk-like polypeptide (see, for example, Valluzzi et al. (2002) Philos Trans R Soc Lond B Biol Sci. 357:165), silk-elastin-like polypeptide (SELP; see, for example, Megeed et al. (2002) Adv Drug Deliv Rev. 54:1075) and the like. Suitable XTEN polypeptides include, for example, those disclosed in WO 2009 / 023270, WO 2010 / 091122, WO 2007 / 103515, U.S. Patent Application Publication No. 2010 / 0189682, and WO 2009 / 0092582; see also Schellenberger et al. (2009) Nat Biotechnol. 27:1186). Suitable albumin polypeptides include, for example, human serum albumin.

[0195] In some cases, suitable scaffold polypeptides are polypeptides that extend half-life. Thus, in some cases, suitable scaffold polypeptides increase the in vivo half-life (e.g., serum half-life) of TMMPs compared to control TMMPs that lack scaffold polypeptides. For example, in some cases, scaffold polypeptides increase the in vivo half-life (e.g., serum half-life) of TMMPs by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 50%, at least about 2-fold, at least about 2.5-fold, at least about 5-fold, at least about 10-fold, at least about 25-fold, at least about 50-fold, at least about 100-fold, or more than 100-fold compared to control TMMPs that lack scaffold polypeptides. By way of example, in some instances, the Fc polypeptide increases the in vivo half-life (e.g., serum half-life) of the TMMP by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 50%, at least about 2-fold, at least about 2.5-fold, at least about 5-fold, at least about 10-fold, at least about 25-fold, at least about 50-fold, at least about 100-fold, or more than 100-fold, compared to a control TMMP lacking the Fc polypeptide.

[0196] Fc Polypeptides In some examples, the TMMP comprises an Ig Fc polypeptide. The Ig Fc polypeptide is also referred to herein as "Fc polypeptide". The Ig Fc polypeptide of the TMMP can be human IgG1 Fc, human IgG2 Fc, human IgG3 Fc, human IgG4 Fc, etc., or a variant of a wild-type Ig Fc polypeptide. The variant includes a natural variant, a non-natural variant, and a combination thereof. For example, the Ig Fc can be a variant of an Fc polypeptide, such as human IgG1 Fc, which has a substantially reduced ability to affect complement-dependent cytotoxicity (CDC) or antibody-dependent cellular cytotoxicity (ADCC). See, for example, the variant human IgG1 Fc polypeptide of FIG. 2B and FIG. 2D).

[0197] In some examples, the Fc polypeptide present in the TMMP comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to an Fc amino acid sequence shown in any one of Figures 2A-2M.

[0198] In some examples, the Fc polypeptide present in the TMMP is an IgG1 Fc polypeptide or a variant of an IgG1 Fc polypeptide. For example, in some examples, the Fc polypeptide present in the TMMP comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the human IgG1 Fc polypeptide shown in Figure 2A. As another example, in some examples, the Fc polypeptide present in the TMMP comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the Fc polypeptide shown in Figure 2B, and the Ig Fc polypeptide comprises an Ala at position 14 and an Ala at position 15. In any of the above embodiments, the Ig Fc polypeptide can have an N77 substitution, i.e., the Ig Fc polypeptide can have an amino acid other than Asn at position 77, and in some examples, the Ig Fc polypeptide has Ala at position 77. In some examples, the Fc polypeptide present in the TMMP comprises the amino acid sequence shown in Figure 2A. In some examples, the Fc polypeptide present in the TMMP comprises the amino acid sequence shown in Figure 2B.

[0199] In some examples, the Fc polypeptide present in the TMMP is an IgG1 Fc polypeptide, or a variant of an IgG1 Fc polypeptide, including naturally occurring variants, non-naturally occurring variants, and combinations thereof. For example, in some examples, the Fc polypeptide present in the TMMP comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the human IgG1 Fc polypeptide shown in Figure 2C, wherein the Ig Fc polypeptide comprises Glu at position 136 and Met at position 138. As another example, in some cases, the Fc polypeptide present in the TMMP comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the human IgG1 Fc polypeptide shown in FIG. 2D, and the Ig Fc polypeptide comprises Ala at positions 14 and 15, and the Fc polypeptide comprises Glu at position 136 and Met at position 138. In any of the above embodiments, the Ig Fc polypeptide can have an N77 substitution, i.e., the Ig Fc polypeptide can have an amino acid other than Asn at position 77, and in some cases, the Ig Fc polypeptide has Ala at position 77. In some cases, the Fc polypeptide present in the TMMP comprises the amino acid sequence shown in FIG. 2C. In some cases, the Fc polypeptide present in the TMMP comprises the amino acid sequence shown in FIG. 2D.

[0200] In some examples, the Fc polypeptide present in the TMMP comprises the amino acid sequence shown in FIG. 2E (human IgG1 Fc comprising an L234F substitution, an L235E substitution, and a P331S substitution, where L234 corresponds to amino acid 14 of the amino acid sequence shown in FIG. 2A, L235 corresponds to amino acid 15 of the amino acid sequence shown in FIG. 2E, and P331 corresponds to amino acid 111 of the amino acid sequence shown in FIG. 2E). In some examples, the Fc polypeptide present in the TMMP comprises the amino acid sequence shown in FIG. 2F, comprising an N279A substitution (amino acid sequence of N77A shown in FIG. 2F). The substitution at N297 leads to the removal of glycosylation, resulting in an antibody sequence with reduced complement component 1q ("C1q") binding compared to the wild-type protein, thus reducing complement-dependent cytotoxicity (CDC). In some examples, the Fc polypeptide present in the TMMP comprises a substitution at K322. K322 (e.g., K322A) substitutions show a substantial reduction in FcγR binding affinity and antibody-dependent cell-mediated cytotoxicity (ADCC), while C1q binding and CDC function are substantially or completely eliminated.

[0201] In some examples, the Fc polypeptide comprises an amino acid sequence having at least 70%, 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 identity to the human IgG2 Fc polypeptide shown in FIG. 2G, e.g., the Fc polypeptide comprises an amino acid sequence having at least 70%, 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 identity to amino acids 99-325 of the human IgG2 Fc polypeptide shown in FIG. 2G (e.g., an Ig Fc polypeptide has a length of about 227 amino acids). In some examples, the Fc polypeptide comprises an amino acid sequence having at least 70%, 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 identity to the human IgG3 Fc polypeptide shown in FIG. 2H, e.g., the Fc polypeptide comprises an amino acid sequence having at least 70%, 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 identity to amino acids 19-246 of the human IgG3 Fc polypeptide shown in FIG. 2H (e.g., an Ig Fc polypeptide has a length of about 228 amino acids). In some examples, the Fc polypeptide comprises an amino acid sequence having at least 70%, 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 identity to the human IgM Fc polypeptide shown in FIG. 2J, e.g., the Fc polypeptide comprises an amino acid sequence having at least 70%, 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 identity to amino acids 1-276 of the human IgM Fc polypeptide shown in FIG. 2J.In some examples, the Fc polypeptide comprises an amino acid sequence having at least 70%, 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 identity to the human IgA Fc polypeptide shown in FIG. 2K, e.g., the Fc polypeptide comprises an amino acid sequence having at least 70%, 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 identity to amino acids 1-234 of the human IgA Fc polypeptide shown in FIG. 2K.

[0202] In some examples, the Fc polypeptide comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the human IgG4 Fc polypeptide shown in Figure 2M. In some examples, the Fc polypeptide comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to amino acids 100-327 of the human IgG4 Fc polypeptide shown in Figure 2M (e.g., an Ig Fc polypeptide has a length of about 228 amino acids).

[0203] In some examples, the IgG4 Fc polypeptide has the following amino acid sequence: Contains TIFF2024541334000046.tif25164.

[0204] Linker As discussed above, a TMMP can include one or more peptide linkers, i.e., a linker comprising a contiguous stretch of two or more amino acids, where the one or more linkers are between one or more components of the TMMP. For example, a TMMP can include one or more peptide linkers, i.e., a linker comprising a contiguous stretch of two or more amino acids, where the one or more linkers are between one or more components of the TMMP. i) an MHC class I heavy chain polypeptide and an Ig Fc polypeptide; ii) MOD and an MHC class I polypeptide; iii) a first MOD and a second MOD; iv) peptides and MHC class I polypeptides; v) peptides and β2M polypeptides; vi) TTP and an MHC class I heavy chain polypeptide; vii) TTP and an Ig Fc polypeptide, and viii) TTPs and MODs; between one or more of the following:

[0205] As used herein, the term "optional peptide linker between any two of the components of a TMMP" refers to a peptide linker between any two adjacent polypeptides within a TMMP. For example, as used herein, the term "optional peptide linker between any two of the components of a TMMP" refers to a peptide linker between one or more of the following: ) an MHC class I heavy chain polypeptide and an Ig Fc polypeptide; ii) MOD and an MHC class I polypeptide; iii) a first MOD and a second MOD; iv) peptides and MHC class I polypeptides; v) peptides and β2M polypeptides; vi) TTP and an MHC class I heavy chain polypeptide; vii) TTP and an Ig Fc polypeptide, and viii) TTPs and MODs. As discussed below, the linker can be a) a flexible peptide linker, including a short flexible peptide linker, or b) a rigid peptide linker.

[0206] Suitable linkers (also referred to as "spacers") can be readily selected and can be any of several suitable lengths, such as 1 to 25 amino acids, 3 to 20 amino acids, 2 to 15 amino acids, 3 to 12 amino acids, 4 to 10 amino acids, 5 to 9 amino acids, 6 to 8 amino acids, or 7 to 8 amino acids. Suitable linkers can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids in length. In some examples, the linker has a length of 25 to 50 amino acids, e.g., 25 to 30, 30 to 35, 35 to 40, 40 to 45, or 45 to 50 amino acids.

[0207] Flexible Peptide Linkers Exemplary flexible peptide linkers include glycine polymers (G) n (SEQ ID NO: 946), glycine-serine polymers (e.g., (GS) n (SEQ ID NO: 937), (GSGGS) n (SEQ ID NO: 938), (GGGGS)n (SEQ ID NO: 934), and (GGGS) n(SEQ ID NO: 939), where n is at least one integer and can be an integer between 1 and 10), glycine-alanine polymers, alanine-serine polymers, and other flexible peptide linkers known in the art. Glycine and glycine-serine polymers can be used; both Gly and Ser are relatively unstructured and can therefore function as neutral tethers between components. Glycine polymers can be used; glycine has access to significantly more phi-psi space than alanine and is significantly less restricted than residues with longer side chains (see Scheraga, Rev. Computational Chem. 11173-142 (1992)). Exemplary linkers can include amino acid sequences including, but not limited to, GGSG (SEQ ID NO: 940), GGSGG (SEQ ID NO: 941), GSGSG (SEQ ID NO: 942), GSGGG (SEQ ID NO: 943), GGGSG (SEQ ID NO: 944), GSSSG (SEQ ID NO: 945), and the like.

[0208] Exemplary flexible peptide linkers include, for example, (GGGGS)n (SEQ ID NO:934), also referred to as "G4S" linkers), where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some examples, the linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO:934), where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some examples, the linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO:947), where n is 2. In some examples, the linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO:875), where n is 3. In some examples, the linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO:876), where n is 4. In some examples, the linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO:948), where n is 7. In some examples, the linker comprises the amino acid sequence AAAGG (SEQ ID NO:874). Also suitable is a linker having the amino acid sequence AAAGG (SEQ ID NO: 874). In the TMMP of the disclosure, the β2M polypeptide may be linked to the MHC heavy chain polypeptide by a (GGGGS)n (SEQ ID NO: 934) linker, where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, e.g., n=3, n=4, or n=7.

[0209] As used in this disclosure, a "short flexible peptide linker" refers to a flexible peptide linker that contains fewer than 15 amino acids, i.e., 2-14 amino acids. For example, a short flexible peptide linker can contain 2-4 amino acids, 2-5 amino acids, 3-6 amino acids, 4-8 amino acids, 5-10 amino acids, or 10-14 amino acids. Included within this range are flexible peptide linkers that contain 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 amino acids.

[0210] Rigid Peptide Linkers In some instances, the peptide linker is a rigid peptide linker. As used herein, the term "rigid peptide linker" refers to a linker that comprises a contiguous stretch of two or more amino acids that effectively separates protein domains by maintaining a substantially fixed distance / spatial separation between the domains, thereby reducing or substantially eliminating unfavorable interactions between such domains. For example, a rigid linker may be placed between either MOD or TTP when they are at position 1 and / or position 3 described above. Rigid peptide linkers are known in the art and generally adopt a relatively well-defined conformation when in solution. Rigid peptide linkers include those that have a specific secondary and / or tertiary structure in solution, and are typically of sufficient length to impart a secondary or tertiary structure to the linker. Rigid peptide linkers include proline-rich peptide linkers and peptide linkers that have an inflexible helical structure, such as an α-helical structure. Rigid peptide linkers are described, for example, in Chen et al. (2013) Adv. Drug Deliv. Rev. 65:1357; and Klein et al. (2014) Protein Engineering, Design & Selection 27:325.

[0211] Examples of rigid peptide linkers include, for example, (EAAAK)n (SEQ ID NO: 949), A(EAAAK)nA (SEQ ID NO: 950), A(EAAAK)nALEA(EAAAK)nA (SEQ ID NO: 951), (Lys-Pro)n (SEQ ID NO: 952), (Glu-Pro)n (SEQ ID NO: 953), (Thr-Pro-Arg)n (SEQ ID NO: 954), and (Ala-Pro)n (SEQ ID NO: 955), where n is an integer from 1 to 20 (e.g., n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20). Non-limiting examples of suitable rigid peptide linkers, including EAAAK (SEQ ID NO: 956), (EAAAK) 2 (SEQ ID NO:958), (EAAAK) 3(SEQ ID NO: 959), A(EAAAK) 4 ALEA(EAAAK) 4 A (SEQ ID NO: 960), and AEAAAKEAAAKA (SEQ ID NO: 961). Non-limiting examples of suitable rigid peptide linkers that contain (AP)n include APAP (SEQ ID NO: 962, also referred to herein as "(AP)2"), APAPAPAP (SEQ ID NO: 963, also referred to herein as "(AP)4"), APAPAPAPAPAP (SEQ ID NO: 964, also referred to herein as "(AP)6"), APAPAPAPAPAPAPAP (SEQ ID NO: 965, also referred to herein as "(AP)8"), and APAPAPAPAPAPAPAPAPAP (SEQ ID NO: 955, also referred to herein as "(AP)10"). Non-limiting examples of suitable rigid peptide linkers comprising (KP)n include KPKP (SEQ ID NO:967, also referred to herein as "(KP)2"), KPKPKPKP (SEQ ID NO:968, also referred to herein as "(KP)4"), KPKPKPKPKPKP (SEQ ID NO:969, also referred to herein as "(KP)6"), KPKPKPKPKPKPKPKP (SEQ ID NO:970, also referred to herein as "(KP)8"), and KPKPKPKPKPKPKPKPKPKP (SEQ ID NO:952, also referred to herein as "(KP)10"). Non-limiting examples of suitable rigid peptide linkers comprising (EP)n include EPEP (SEQ ID NO:972, also referred to herein as "(EP)2"), EPEPEPEPEP (SEQ ID NO:973, also referred to herein as "(EP)4"), EPEPEPEPEPEPEP (SEQ ID NO:974, also referred to herein as "(EP)6"), EPEPEPEPEPEPEPEP (SEQ ID NO:975, also referred to herein as "(EP)8"), and EPEPEPEPEPEPEPEPEPEPEP (SEQ ID NO:953, also referred to herein as "(EP)10").

[0212] Cysteine-Containing Linkers As described above, in some cases, the linker peptide in the first polypeptide chain of the TMMP may contain a cysteine ​​residue that can form an interchain disulfide bond with a cysteine ​​residue present in the second polypeptide chain. For example, as described above, in some cases, the TMMP, or dimerized TMMP, such as a homodimer, contains a linker between the peptide epitope and the β2M polypeptide that contains a cysteine ​​residue that forms an interchain disulfide bond with a cysteine ​​residue in the MHC class I heavy chain polypeptide present in the other chain of the TMMP. For example, in some cases, the TMMP, or dimerized TMMP, such as a homodimer, contains a cysteine-containing linker between the peptide epitope and the β2M polypeptide, and the cysteine ​​residue in the linker forms an interchain disulfide bond with a cysteine ​​residue at amino acid 236 (e.g., formed by A236C substitution) in the MHC class I heavy chain polypeptide present in the TMMP.

[0213] In some examples, the peptide linker between the SARS-CoV-2 peptide and the β2M polypeptide comprises the amino acid sequence GCGGS (SEQ ID NO: 977). In some examples, the peptide linker between the SARS-CoV-2 peptide and the β2M polypeptide comprises the amino acid sequence GCGGS(GGGGS)n (SEQ ID NO: 978), where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, e.g., 1, 2, or 3. In some examples, the peptide linker between the peptide and the β2M polypeptide comprises the amino acid sequence GCGGS(GGGGS)n (SEQ ID NO: 882), where n is 2.

[0214] In some examples, the peptide linker between the SARS-CoV-2 peptide and the β2M polypeptide comprises the amino acid sequence CGGGS (SEQ ID NO: 979). In some examples, the peptide linker comprises the amino acid sequence CGGGS(GGGGS)n (SEQ ID NO: 980), where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, e.g., 1, 2, or 3.

[0215] In some examples, the peptide linker between the SARS-CoV-2 peptide and the β2M polypeptide comprises the amino acid sequence GGCGS (SEQ ID NO: 981). In some examples, the peptide linker comprises the amino acid sequence GGCGS(GGGGS)n (SEQ ID NO: 982), where n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, e.g., 1, 2, or 3.

[0216] In some examples, the peptide linker between the SARS-CoV-2 peptide and the β2M polypeptide comprises the amino acid sequence GGGCS (SEQ ID NO: 983). In some examples, the peptide linker comprises the amino acid sequence GGGCS(GGGGS)n (SEQ ID NO: 984), where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, e.g., 1, 2, or 3.

[0217] In some examples, the peptide linker between the SARS-CoV-2 peptide and the β2M polypeptide comprises the amino acid sequence GGGGC (SEQ ID NO: 985). In some examples, the peptide linker comprises the amino acid sequence GGGGC(GGGGS)n (SEQ ID NO: 986), where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, e.g., 1, 2, or 3.

[0218] Linker between TTP and MHC class I heavy chain polypeptide As discussed below, the TTP is located at the N-terminus of the MHC heavy chain polypeptide and the linker between them is about 12 amino acids or less in length, e.g., 10 amino acids or less (e.g., (GGGGS) 2 It has been found that the cytotoxic effect of TMMP is greater when the linker between them is about 12 amino acids or less in length (e.g., (GGGGS) (SEQ ID NO: 947) linker), 8 amino acids or less, 7 amino acids or less, 6 amino acids or less, or 5 amino acids or less in length (e.g., GGGGS (SEQ ID NO: 873) linker). In some cases, the linker between them is about 12 amino acids or less in length. In some cases, the linker between them is 10 amino acids or less in length (e.g., (GGGGS) 2(SEQ ID NO:947) linker). In some cases, the linker between them is 8 amino acids or less in length. In some cases, the linker between them is 7 amino acids or less in length. In some cases, the linker between them is 6 amino acids or less in length. In some cases, the linker between them is 5 amino acids or less in length (e.g., GGGGS (SEQ ID NO:873) linker).

[0219] Dimerized TMMP As mentioned above, in some cases, the TMMP can form a dimer. That is, the present disclosure provides a polypeptide comprising a dimer of two TMMPs. Thus, the present disclosure provides a protein that is a dimerized TMMP comprising two TMMPs covalently linked to each other. The covalent bond of the dimer may be one or more disulfide bonds between an Ig Fc polypeptide in a first TMMP and an Ig Fc polypeptide in a second TMMP. By way of example only, the Ig Fc may be a variant of a human IgG1 Fc polypeptide, the variant having a substantially reduced ability to affect complement-dependent cytotoxicity (CDC) or antibody-dependent cellular cytotoxicity (ADCC) (e.g., the human IgG1 Fc polypeptide of FIG. 2B or FIG. 2D). When the TMMP comprises an Ig Fc polypeptide, the TMMP will typically self-assemble into a dimer by spontaneously forming a disulfide bond with the IgG1 Fc polypeptide of another TMMP. Thus, for example, the Ig Fc polypeptides in the first TMMP and the second TMMP may be linked to each other by one or more disulfide bonds. In most cases, the two TMMPs will be identical to each other in amino acid sequence and will contain an Ig Fc polypeptide that spontaneously forms one or more disulfide bonds, thereby forming a dimerized TMMP that is a homodimer.

[0220] Thus, the present disclosure provides a protein comprising a) a first TMMP and b) a second TMMP, which may optionally be identical to the first TMMP, wherein the first and second TMMP are covalently bonded to each other. The covalent bond may be a disulfide bond between an Ig Fc polypeptide in the first TMMP and an Ig Fc polypeptide in the second TMMP.

[0221] Optionally, the Ig Fc polypeptide of each TMMP may contain an interspecies dimerization sequence, such as, for example, a "knobs-in-holes" sequence that allows for selective dimerization of two different TMMPs. The interspecies binding sequences favor the formation of heterodimers with their cognate polypeptide sequences (i.e., the interspecies sequence and its corresponding interspecies sequence), particularly those based on Ig Fc sequence variants. Such interspecies polypeptide sequences include knobs-in-holes and knobs-in-hole sequences that promote the formation of one or more disulfide bonds. For example, one interspecies binding pair includes the T366Y and Y407T mutant pair at the CH3 domain interface of IgG1, or the corresponding residues in other immunoglobulins. See Ridgway et al., Protein Engineering 9:7, 617-621 (1996). A second interspecies binding pair includes a knob formed by a T366W substitution and a hole formed by a triple substitution of T366S, L368A, and Y407V on a complementary Ig Fc sequence. See Xu et al. mAbs 7:1, 231-242 (2015). Another interspecies binding pair has a first Fc polypeptide with Y349C, T366S, L368A, and Y407V substitutions, and a second Ig Fc polypeptide with S354C and T366W substitutions (a disulfide bond can be formed between Y349C and S354C). See, e.g., Brinkmann and Konthermann, mAbs 9:2, 182-212 (2015). The Ig Fc polypeptide sequence can be stabilized by the formation of disulfide bonds (e.g., hinge region disulfide bonds) between the Ig Fc polypeptides, with or without knob-in-hole modifications. Thus, in some instances, the dimerized TMMP can be a heterodimer comprising two TMMP chains that are not identical in amino acid sequence.

[0222] Interspecies dimerization sequences may also be used to allow the TMMP to be linked to non-TMMP molecules that can provide additional functionality to the TMMP. For example, the TMMP may be linked to a molecule that contains a polypeptide that binds to a cancer-associated antigen (e.g., an antibody or binding fragment thereof, such as an scFv), thereby allowing the TMMP to localize to tissues that contain the cancer-associated antigen.

[0223] Tumor targeting polypeptide (TTP) As mentioned above, the TMMP comprises, in the first and / or second polypeptide, a tumor targeting polypeptide (TTP), i.e., a polypeptide specific for a cancer-associated epitope. A "cancer-associated" epitope is an epitope present in a cancer-associated antigen. In some examples, the TTP is an antibody. In some examples, the TTP is a single-chain T-cell receptor (scTCR).

[0224] target In some cases, the TTP present in the TMMP targets a cancer-associated antigen. In some cases, the target of the TTP is a peptide / HLA (pHLA) complex on the surface of a cancer cell, and the peptide can be a cancer-associated peptide (e.g., a peptide fragment of a cancer-associated antigen).

[0225] Cancer-associated antigens Cancer-associated antigens that can be targeted by tumor targeting polypeptides present in TMMPs include, for example, NY-ESO (New York Esophageal Squamous Cell Carcinoma 1), MART-1 (Melanoma Antigen Recognized by T Cells 1, also known as Melan-A), HPV (Human Papilloma Virus) E6, BCMA (B-cell maturation antigen), CD123, CD133, CD171, CD19, CD20, CD22, CD30, CD33, CD38, CD138, CEA (Carcinoembryonic Antigen), EGFR (Epidermal Growth Factor Receptor), EGFRvIII (Epidermal Growth Factor Receptor Variant III), EpCAM (Epithelial Cell Adhesion Molecule), EphA2 (Ephrin Type A Receptor 2), Disialogan, and / or other tumour-associated antigens. These include gliosid GD2, GPC3 (glypican-3), HER2, IL13Ralpha2 (interleukin-13 receptor subunit alpha-2), LeY (difucosylated type 2 blood-associated antigen), MAGE-A3 (melanoma-associated antigen 3), melanoma glycoprotein, mesothelin, MUC1 (mucin 1), MUC16 (mucin-16), myelin, NKG2D (natural killer group 2D), ligand, PSMA (prostate-specific membrane antigen), and ROR1 (type I receptor tyrosine kinase-like orphan receptor).

[0226] Cancer-associated antigens that can be targeted by TTPs present in TMMPs include 17-1A antigen, alpha-fetoprotein (AFP), alpha-actinin-4, A3, antigen specific for the A33 antibody, ART-4, B7, Ba733, BAGE, bcl-2, bcl-6, BCMA, BrE3-antigen, CA125, CAMEL, CAP-1, carbonic anhydrase IX (CAIX), CASP-8 / m, CCL19, CCL21, CD1, CD1a, CD2, CD3, CD4, CD5, CD8, CD11A. , CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD29, CD30, CD32b, CD33, CD37, CD38, CD40, CD40L, CD44, CD45, CD46, CD5 2, CD54, CD55, CD59, CD64, CD66a-e, CD67, CD70, CD70L, CD74, CD79a, CD79b, CD80, CD83, CD95, CD123, CD126, CD132, CD133, CD138, CD1 47, CD154, CD171, CDC27, CDK-4 / m, CDKN2A, CEA, CEACAM5, CEACAM6, claudins (e.g., claudin-1, claudin-10, claudin-18 (e.g., claudin-18, isoform 2)), complement factors (e.g., C3, C3a, C3b, C5a, and C5), colon-specific antigen-p (CSAp), c-Met, CTLA-4, CXCR4, CXCR7, CXCL12, DAM, Dickkopf-related protein ( DKK), ED-B fibronectin, epidermal growth factor receptor (EGFR), EGFRvIII, EGP-1 (TROP-2), EGP-2, ELF2-M, Ep-CAM, EphA2, EphA3, fibroblast activation protein (FAP), fibroblast growth factor (FGF), Flt-1, Flt-3, folate binding protein, folate receptor, G250 antigen, gangliosides (such as GC2, GD3, and GM2), GAGE, GD2, gp100, GPC3, GRO-13, HLA-DR, HM1.24, human chorionic gonadotropin (HCG) and its subunits, HER2, HER3, HMGB-1, hypoxia-inducible factor (HIF-1), HIF-1a, HSP70-2M, HST-2, Ia, IFN-gamma, IFN-alpha, IFN-beta, IFN-X, IL-4R, IL-6R, IL-13R, IL13Ralpha2, IL-15R, IL-17R, IL-18R, IL-2, IL-6, IL-8, IL-12, IL-15, IL-17, IL-18, IL-23, IL-25, ILGF, ILGF-1R, insulin-like growth factor-1 (IGF-1), IGF-1R, integrin αVβ3, integrin α5β1, KC4-antigen, killer cell immunoglobulin-like receptor (KIR), Kras, KS-1 antigen, KS1-4, LDR / FUT, Le. gamma , macrophage migration inhibitory factor (MIF), MAGE, MAGE-3, MART-1, MART-2, mCRP, MCP-1, melanoma glycoprotein, mesothelin, MIP-1A, MIP-1B, MIF, mucins (such as MUC1, MUC2, MUC3, MUC4, MUC5ac, MUC13, MUC16, MUM-1 / 2, and MUM-3), NCA66, NCA95, NCA90, nectin-4, NY-ESO-1, PAM4 antigen, pancreatic cancer mucins, PD-1, PD-L These include PD-1 receptor, placental growth factor, p53, PLAGL2, prostatic acid phosphatase, PSA, PRAME, PSMA, P1GF, RSS, RANTES, SAGE, 5100, survivin, survivin-2B, T101, TAC, TAG-72, tenascin, Thomson-Friedenreich antigen, Tn antigen, TNF-α, tumor necrosis antigen, TRAG-3, TRAIL receptor, vascular endothelial growth factor (VEGF), VEGF receptor (VEGFR), and WT-1.

[0227] In some examples, the cancer-associated antigen is an antigen associated with blood cancer.Examples of such antigens include but are not limited to BCMA, C5, CD19, CD20, CD22, CD25, CD30, CD33, CD38, CD40, CD45, CD52, CD56, CD66, CD74, CD79a, CD79b, CD80, CD138, CTLA-4, CXCR4, DKK, EphA3, GM2, HLA-DR beta, integrin αVβ3, IGF-R1, IL6, KIR, PD-1, PD-L1, TRAILR1, TRAILR2, transferrin receptor and VEGF. In some examples, the cancer associated antigen is an antigen expressed by malignant B cells, such as CD19, CD20, CD22, CD25, CD38, CD40, CD45, CD74, CD80, CTLA-4, IGF-R1, IL6, PD-1, TRAILR2, or VEGF.

[0228] In some examples, the cancer-associated antigen is an antigen associated with a solid tumor. Examples of such antigens include CAIX, cadherin, CEA, c-MET, CTLA-4, EGFR family members, EpCAM, EphA3, FAP, folate binding protein, FR-alpha, gangliosides (such as GC2, GD3, and GM2), HER2, HER3, IGF-1R, integrin αVβ3, integrin α5β1, Le gamma , Liv1, mesothelin, mucin, NaPi2b, PD-1, PD-L1, PD-1 receptor, pgA33, PSMA, RANKL, ROR1, TAG-72, tenascin, TRAILR1, TRAILR2, VEGF, VEGFR, and others listed above.

[0229] Peptide / HLA complexes In some examples, the target of the TTP is a peptide / HLA (pHLA) complex on the surface of a cancer cell, and the peptide can be a cancer-associated peptide (e.g., a peptide fragment of a cancer-associated antigen). Cancer-associated peptides are known in the art. In some examples, the cancer-associated peptide ... fragment of an HLA-A *It binds to an HLA complex containing the 0201 heavy chain and the β2M polypeptide.

[0230] In some cases, epitopes present on pHLA on the surface of cancer cells are associated with HLA-A * 0101, A * 0201, A * 0301, A * 1101, A * 2301, A * 2402, A * 2407, A * 3303, and / or A * 3401. In some cases, epitopes present on pHLA on the surface of cancer cells bind to HLA-B * 0702, B * 0801, B * 1502, B * 3802, B * 4001, B * 4601, and / or B * 5301. In some cases, epitopes present on pHLA on the surface of cancer cells are associated with C * 0102, C * 0303, C * 0304, C * 0401, C * 0602, C * 0701, C * 702, C * 0801, and / or C * It binds to HLA complexes containing HLA heavy chains such as 1502.

[0231] In some examples, the epitope is a cancer-associated epitope of any one of the following cancer-associated antigens: MUC1 polypeptide, LMP2 polypeptide, epidermal growth factor receptor (EGFR) vIII polypeptide, HER-2 / neu polypeptide, melanoma antigen family A, 3 (MAGE) polypeptide, or a combination thereof. A3) polypeptide, p53 polypeptide, mutant p53 polypeptide, NY-ESO-1 polypeptide, folate hydrolase (prostate specific membrane antigen; PSMA) polypeptide, carcinoembryonic antigen (CEA) polypeptide, claudin polypeptide (e.g., claudin-1, claudin-10, claudin-18 (e.g., claudin-18, isoform 2)), nectin-4 polypeptide, melanoma antigen recognized by T cells (melanA / MART1) polypeptide, Ras polypeptide, gp100 polypeptide, proteinase 3 (PR1) polypeptide, bcr-abl polypeptide, tyrosinase polypeptide, survivin polypeptide, prostate specific antigen (PSA) polypeptide, hTERT polypeptide, sarcoma metastasis breakpoint polypeptide, synovial sarcoma X (SSX) breakpoint polypeptide, EphA2 polypeptide, acid phosphatase, prostate (PAP) polypeptide, melanoma inhibitor of apoptosis (ML-IAP), epithelial cell adhesion molecule (EpCAM) polypeptide, ERG (TMPRSS2 ETS fusion) polypeptide, NA17 polypeptide, paired box-3 (PAX3) polypeptide, anaplastic lymphoma kinase (ALK) polypeptide, androgen receptor polypeptide, cyclin B1 polypeptide, N-myc proto-oncogene (MYCN) polypeptide, Ras homolog gene family member C (RhoC) polypeptide, tyrosinase-related protein-2 (TRP-2) polypeptide, mesothelin polypeptide, prostate stem cell antigen (PSCA) polypeptide, melanoma associated antigen-1 (MAGE A1) polypeptide, cytochrome P450 1B1 (CYP1B1) polypeptide, placenta-specific protein 1 (PLAC1) polypeptide, BORIS polypeptide (also known as CCCTC-binding factor or CTCF), ETV6-AML polypeptide, breast cancer antigen NY-BR-1 polypeptide (also known as ankyrin repeat domain-containing protein 30A), regulator of G protein signaling (RGS5) polypeptide, squamous cell carcinoma cell antigen recognized by T cells (SART3) polypeptide, carbonic anhydrase IX polypeptide, paired box-5 (PAX5) polypeptide, OY-TES1 (testis antigen, also known as acrosin-binding protein) polypeptide, sperm protein 17 polypeptide, lymphoid cell-specific protein-tyrosine kinase (LCK) polypeptide, high molecular weight melanoma-associated antigen (HMW-MAA), A-kinase anchoring protein-4 (AKAP-4), synovial sarcoma X breakpoint 2 (SS X2) polypeptide, X antigen family member 1 (XAGE1) polypeptide, B7 homolog 3 (B7H3, also known as CD276) polypeptide, legumain polypeptide (LGMN1, also known as asparaginyl endopeptidase), tyrosine kinase-2 with Ig and EGF homology domains (Tie-2, also known as angiopoietin-1 receptor) polypeptide, P antigen family member 4 (PAGE4) polypeptide, vascular endothelial growth factor receptor 2 (VEGF2) polypeptide, MAD-CT-1 polypeptide, fibroblast activation protein (FAP) polypeptide, platelet-derived growth factor receptor beta (PDGFβ) polypeptide, MAD-CT-2 polypeptide, Fos-related antigen-1 (FOSL) polypeptide, human papilloma virus (HPV) antigen, alpha-fetoprotein (AFP) antigen, and Wilms' tumor-1 (WT1) antigen.

[0232] For example, in some instances, the TTP present in TMMP is a) HLA heavy chain (e.g., HLA-A * 0201 heavy chain or HLA-A * WT-1 peptide bound to an HLA complex containing the β2M polypeptide and the β2 heavy chain; b) an HPV peptide bound to an HLA complex comprising a class I HLA heavy chain and a β2M polypeptide; c) a mesothelin peptide bound to an HLA complex comprising a class I HLA heavy chain and a β2M polypeptide; d) a Her2 peptide bound to an HLA complex comprising a class I HLA heavy chain and a β2M polypeptide; or e) a BCMA peptide bound to an HLA complex comprising a class I HLA heavy chain and a β2M polypeptide; Bind to.

[0233] In some examples, the cancer associated peptide has the following mesothelin amino acid sequence: A peptide of a mesothelin polypeptide having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF2024541334000047.tif66163. For example, mesothelin peptides present in the pHLA complex include i) KLLGPHVEGL (SEQ ID NO: 988), ii) HLA-A * It may be AFYPGYLCSL (SEQ ID NO: 989), iii) VLPLTVAEV (SEQ ID NO: 990), iv) ELAVALAQK (SEQ ID NO: 991), v) ALQGGGPPY (SEQ ID NO: 992), vi) FYPGYLCSL (SEQ ID NO: 993), vii) LYPKARLAF (SEQ ID NO: 994), viii) LLFLLFSLGWVGPSR (SEQ ID NO: 995), ix) VNKGHEMSPQAPRRP (SEQ ID NO: 996), x) FMKLRTDAVLPLTVA (SEQ ID NO: 997), or xi) DAALLATQMD (SEQ ID NO: 998), which is capable of binding to 2402 / β2M.

[0234] In some examples, the cancer associated peptide has the following Her2 (receptor tyrosine-protein kinase erbB2) amino acid sequence: A peptide of a Her2 polypeptide having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF2024541334000048.tif118163.

[0235] In some examples, the cancer associated peptide has the following BCMA amino acid sequence: A peptide of a BCMA polypeptide having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF2024541334000049.tif24153.

[0236] In some examples, the cancer associated peptide has the following WT-1 amino acid sequence: A peptide of a WT-1 polypeptide having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF2024541334000050.tif57162.

[0237] Non-limiting examples of WT-1 peptides include RMFPNAPYL (SEQ ID NO: 1002), CMTWNQMN (SEQ ID NO: 1003), CYTWNQMNL (SEQ ID NO: 1004), CMTWNQMNLGATLKG (SEQ ID NO: 1005), WNQMNLGATLKGVAA (SEQ ID NO: 1006), CMTWNYMNLGATLKG (SEQ ID NO: 1007), WNYMNLGATLKGVAA (SEQ ID NO: 1008), MTWNQMNLGATLKGV (SEQ ID NO: 1009), TWNQMNLGATLKGVA (SEQ ID NO: 1010), CMTWNL MNLGATLKG (SEQ ID NO: 1011), MTWNLMNLGATLKGV (SEQ ID NO: 1012), TWNLMNLGATLKGVA (SEQ ID NO: 1013), WNLMNLGATLKGVAA (SEQ ID NO: 1014), MNLGATLK (SEQ ID NO: 1015), MTWNYMNLGATLKGV (SEQ ID NO: 1016), TWNYMNLGATLKGVA (SEQ ID NO: 1017), CMTWNQMNLGATLKGVA (SEQ ID NO: 1018), CMTWNLMNLGATLKGVA (SEQ ID NO: 1019), CMTWNYMNLGATLKGV A (SEQ ID NO: 1020), GYLRNPTAC (SEQ ID NO: 1021), GALRNPTAL (SEQ ID NO: 1022), YALRNPTAC (SEQ ID NO: 1023), GLLRNPTAC (SEQ ID NO: 1024), RYRPHPGAL (SEQ ID NO: 1025), YQRPHPGAL (SEQ ID NO: 1026), RLRPHPGAL (SEQ ID NO: 1027), RIRPHPGAL (SEQ ID NO: 1028), QFPNHSFKHEDPMGQ (SEQ ID NO: 1029), HSFKHEDPY (SEQ ID NO: 1030), QFPNHSFKHEDPM (SEQ ID NO: 1031) , QFPNHSFKHEDPY (SEQ ID NO: 1032), KRPFMCAYPGCNK (SEQ ID NO: 1033), KRPFMCAYPGCYK (SEQ ID NO: 1034), FMCAYPGCY (SEQ ID NO: 1035), FMCAYPGCK (SEQ ID NO: 1036), KRPFMCAYPGCNKRY (SEQ ID NO: 1037), SEKRPFMCAYPGCNK (SEQ ID NO: 1038), KRPFMCAYPGCYKRY (SEQ ID NO: 1039), NLMNLGATL (SEQ ID NO: 1040), and NYMNLGATL (SEQ ID NO: 1041).

[0238] In some examples, the cancer-associated peptide is a peptide of a human papilloma virus (HPV) polypeptide that has at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity with the HPV polypeptide.The HPV peptide can be a peptide of an HPV E6 polypeptide or an HPV E7 polypeptide.The HPV epitope can be an epitope of any of various HPV genotypes, including, for example, HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59, HPV68, HPV73, or HPV82. Non-limiting examples of HPV peptides include: E6 18-26 (KLPQLCTEL; SEQ ID NO:1042); E6 26-34 (LQTTIHDII; SEQ ID NO:1043); E6 49-57 (VYDFAFRDL; SEQ ID NO:1044); E6 52-60 (FAFRDLCIV; SEQ ID NO:1045); E6 75-83 (KFYSKISEY; SEQ ID NO:1046); E6 80-88 (ISEYRHYCY; SEQ ID NO:1047); E7 7-15 (TLHEYMLDL; SEQ ID NO:1048); E7 11-19 (YMLDLQPET; SEQ ID NO:1049); E7 44-52 (QAEPDRAHY; SEQ ID NO:1050); E7 49-57 (RAHYNIVTF (SEQ ID NO:1051); E7 61-69 (CDSTLRLCV; SEQ ID NO:1052); and E7 67-76 (LCVQSTHVDI; SEQ ID NO:1053); E7 82-90 (LLMGTLGIV; SEQ ID NO:1054); E7 86-93 (TLGIVCPI; SEQ ID NO:1055); and E7 92-93 (LLMGTLGIVCPI; SEQ ID NO:1056).

[0239] In some examples, the cancer associated peptide has the following Claudin-18 (isoform 2) (CLDN 18.2) amino acid sequence: The cancer-associated peptide is a peptide of a claudin polypeptide having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF2024541334000051.tif31164. In some examples, the cancer-associated peptide is a peptide of a claudin polypeptide having the amino acid sequence TEDEVQSYPSKHDYV (SEQ ID NO: 1058) (and having a length of about 15 amino acids) or EVQSYPSKHDYV (SEQ ID NO: 1059) (and having a length of about 12 amino acids).

[0240] In some examples, the cancer-associated peptide is a peptide of trophoblast cell surface antigen-2 (Trop-2) polypeptide. Trop-2 (also known as epithelial glycoprotein-1, gastrointestinal tumor-associated antigen GA733-1, membrane component staining 1 surface marker-1, and tumor-associated calcium signal transducer-2) is a transmembrane glycoprotein that is upregulated in many cancer types and is the protein product of the TACSTD2 gene. In some examples, the cancer-associated peptide is a peptide of the following TROP-2 amino acid sequence: A peptide of a TROP-2 polypeptide having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF2024541334000052.tif31166.

[0241] antibody As mentioned above, in some cases, the TTP present in TMMP is an antibody.In some cases, the TTP is an antibody specific to a cancer-associated antigen.In some cases, the TTP is an antibody specific to a peptide / HLA complex on the surface of cancer cells, and the peptide can be a cancer-associated peptide (e.g., a peptide of a cancer-associated antigen).

[0242] Non-limiting examples of cancer-associated antigen targeting antibodies that can be included in a TMMP include avituzumab (anti-CD51), LL1 (anti-CD74), LL2 or RFB4 (anti-CD22), veltuzumab (hA20, anti-CD20), binutuzu (anti-CD20), binutuzumab (GA101, anti-CD20), daratuzumab (anti-CD38), lambrolizumab (anti-PD-1 receptor), nivolumab (anti-PD-1 receptor), ipilimumab (anti-CTLA-4), RS7 (anti-TROP-2), PAM4 or KC4 (both anti-mucins), MN-14 (anti-CEA), MN-15 or MN-3 (anti-CEACAM6), Mu-9 (anti-colon specific antigen-p), Immu 31 (anti-alpha-fetoprotein), R1 (anti-IGF-1R), A19 (anti-CD19), TAG-72 (e.g., CC49), Tn, J591 or HuJ591 (anti-PSMA), AB-PG1-XG1-026 (anti-PSMA dimer), D2 / B (anti-PSMA), G250 (anti-carbonic anhydrase IX), L243 (anti-HLA-DR), alemtuzumab (anti-CD52), oportuzumab (anti-EpCAM), bevacizumab (anti-V These include, but are not limited to, EGF), cetuximab (anti-EGFR), gemtuzumab (anti-CD33), ibritumomab tiuxetan (anti-CD20), panitumumab (anti-EGFR), tositumomab (anti-CD20), PAM4 (also known as clivatuzumab; anti-mucin), trastuzumab (anti-HER2), pertuzumab (anti-HER2), polatuzumab (anti-CD79b), and anetuzumab (anti-mesothelin).

[0243] In some examples, the tumor targeting polypeptide is a single chain antibody. In some examples, the tumor targeting polypeptide is an scFv. In some examples, the tumor targeting polypeptide is a nanobody (also referred to as a single domain antibody (sdAb)). In some examples, the tumor targeting polypeptide is a heavy chain nanobody. In some examples, the tumor targeting polypeptide is a light chain nanobody.

[0244] The VH and VL amino acid sequences of various tumor antigen-binding antibodies are known in the art, as are the light and heavy chain CDRs of such antibodies.See, for example, Ling et al. (2018) Frontiers Immunol.9:469; International Publication No. 2005 / 012493; US Patent Application Publication No. 2019 / 0119375; US Patent Application Publication No. 2013 / 0066055.The following are non-limiting examples of tumor antigen-binding antibodies:

[0245] anti-Her2 In some instances, the anti-Her2 antibody a) the amino acid sequence: a light chain comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF2024541334000053.tif24163; and b) the amino acid sequence: a heavy chain comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF2024541334000054.tif51164. Includes.

[0246] In some examples, the anti-Her2 antibody comprises a light chain variable region (VL) present in the light chain amino acid sequence provided above and a heavy chain variable region (VH) present in the heavy chain amino acid sequence provided above. For example, the anti-Her2 antibody comprises a) the amino acid sequence: a VL comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF2024541334000055.tif11164; and b) the amino acid sequence: A VH comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF2024541334000056.tif17167. Includes. In some examples, the anti-Her2 antibody is comprised, in order from N-terminus to C-terminus: a) the amino acid sequence: a VH comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF2024541334000057.tif17167; and b) a linker, and c) the amino acid sequence: A VL comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF2024541334000058.tif11164. Includes. Suitable linkers are described elsewhere herein and include, for example, (GGGGS)n (SEQ ID NO:934), where n is an integer from 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10).

[0247] In some examples, the anti-Her2 antibody comprises a VL CDR1, a VL CDR2, and a VL CDR3 present in the light chain amino acid sequence provided above, and a VH CDR1, a CDR2, and a CDR3 present in the heavy chain amino acid sequence provided above. H and V L CDRs are as defined by Kabat (see, e.g., Table 1, above, and Kabat 1991). H and V L The CDRs are as defined by Chothia (see, eg, Table 1, above, and Chothia 1987).

[0248] For example, an anti-Her2 antibody may comprise a VL CDR1 having the amino acid sequence RASQDVNTAVA (SEQ ID NO: 1067), a VL CDR2 having the amino acid sequence SASFLY (SEQ ID NO: 1068), a VL CDR3 having the amino acid sequence QQHYTTPP (SEQ ID NO: 1069), a VH CDR1 having the amino acid sequence GFNIKDTY (SEQ ID NO: 1070), a VH CDR2 having the amino acid sequence IYPTNGYT (SEQ ID NO: 1071), and a VH CDR3 having the amino acid sequence SRWGGDGFYAMDY (SEQ ID NO: 1072).

[0249] In some examples, the anti-Her2 antibody is an scFv antibody. For example, the anti-Her2 scFv has the following amino acid sequence: It 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 to TIFF2024541334000059.tif24163.

[0250] As another example, in some instances, the anti-Her2 antibody a) the amino acid sequence: a light chain variable region (VL) comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF2024541334000060.tif24163; and b) the amino acid sequence: A heavy chain variable region (VH) comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF2024541334000061.tif51164. Includes.

[0251] In some examples, the anti-Her2 antibody comprises a VL present in the light chain amino acid sequence provided above and a VH present in the heavy chain amino acid sequence provided above. For example, the anti-Her2 antibody comprises a) the amino acid sequence: a VL comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF2024541334000062.tif11163; and b) the amino acid sequence: A VH comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF2024541334000063.tif17164. Includes.

[0252] In some examples, the anti-Her2 antibody comprises a VL CDR1, a VL CDR2, and a VL CDR3 present in the light chain amino acid sequence provided above, and a VH CDR1, a CDR2, and a CDR3 present in the heavy chain amino acid sequence provided above. H and V L CDRs are as defined by Kabat (see, e.g., Table 1, above, and Kabat 1991). H and V L The CDRs are as defined by Chothia (see, eg, Table 1, above, and Chothia 1987).

[0253] For example, an anti-HER2 antibody may comprise a VL CDR1 having the amino acid sequence KASQDVSIGVA (SEQ ID NO: 1078), a VL CDR2 having the amino acid sequence SASYRY (SEQ ID NO: 1079), a VL CDR3 having the amino acid sequence QQYYIYPY (SEQ ID NO: 1080), a VH CDR1 having the amino acid sequence GFTFTDYTMD (SEQ ID NO: 1081), a VH CDR2 having the amino acid sequence ADVNPNSGGSIYNQRFKG (SEQ ID NO: 1082), and a VH CDR3 having the amino acid sequence ARNLGPSFYFDY (SEQ ID NO: 1083).

[0254] In some examples, the anti-Her2 antibody is an scFv. For example, in some examples, the anti-Her2 scFv has the following amino acid sequence: It includes an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF2024541334000064.tif31164.

[0255] anti-CD19 Anti-CD19 antibodies are known in the art, and the VH and VL, or VH and VL CDRs, of any anti-CD19 antibody can be used in the TMMP. See, e.g., WO 2005 / 012493.

[0256] In some examples, the anti-CD19 antibody comprises a VL CDR1 comprising the amino acid sequence KASQSVDYDGDSYLN (SEQ ID NO: 1084), a VL CDR2 comprising the amino acid sequence DASNLVS (SEQ ID NO: 1085), and a VL CDR3 comprising the amino acid sequence QQSTEDPWT (SEQ ID NO: 1086). In some examples, the anti-CD19 antibody comprises a VH CDR1 comprising the amino acid sequence SYWMN (SEQ ID NO: 1087), a VH CDR2 comprising the amino acid sequence QIWPGDGDTNYNGKFKG (SEQ ID NO: 1088), and a VH CDR3 comprising the amino acid sequence RETTTVGRYYYAMDY (SEQ ID NO: 1089). In some examples, the anti-CD19 antibody comprises a VL CDR1 comprising the amino acid sequence KASQSVDYDGDSYLN (SEQ ID NO: 1084), a VL CDR2 comprising the amino acid sequence DASNLVS (SEQ ID NO: 1085), a VL CDR3 comprising the amino acid sequence QQSTEDPWT (SEQ ID NO: 1086), a VH CDR1 comprising the amino acid sequence SYWMN (SEQ ID NO: 1087), a VH CDR2 comprising the amino acid sequence QIWPGDGDTNYNGKFKG (SEQ ID NO: 1088), and a VH CDR3 comprising the amino acid sequence RETTTVGRYYYAMDY (SEQ ID NO: 1089).

[0257] In some examples, the anti-CD19 antibody is an scFv. For example, in some examples, the anti-CD19 scFv has the following amino acid sequence: It includes an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF2024541334000065.tif31168.

[0258] Anti-mesothelin Anti-mesothelin antibodies are known in the art, and the VH and VL or VH and VL CDRs of any anti-mesothelin antibody can be used in the TMMP. See US Patent Application Publication No. 2019 / 0000944, WO 2009 / 045957, WO 2014 / 031476, U.S. Patent No. 8,460,660, US Patent Application Publication No. 2013 / 0066055, and WO 2009 / 068204. In some examples, the TTP is an anti-mesothelin scFv or anti-mesothelin nanobody that includes the VH and VL CDRs present in any one of the amino acid sequences set forth in Figures 22A-22H. In some examples, the TTP is an anti-mesothelin scFv that includes the amino acid sequence set forth in any one of Figures 22A-22H.

[0259] In some instances, the anti-mesothelin antibody a) the amino acid sequence: a light chain comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF2024541334000066.tif23163; and b) the amino acid sequence: a heavy chain comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF2024541334000067.tif51164. Includes.

[0260] In some examples, the anti-mesothelin antibody comprises a VL present in the light chain amino acid sequence provided above and a VH present in the heavy chain amino acid sequence provided above. For example, the anti-mesothelin antibody comprises a) the amino acid sequence: a VL comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF2024541334000068.tif11162; and b) the amino acid sequence: A VH comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF2024541334000069.tif17163. may include.

[0261] In some examples, the anti-mesothelin antibody comprises a VL CDR1, a VL CDR2, and a VL CDR3 present in the light chain amino acid sequence provided above, and a VH CDR1, a CDR2, and a CDR3 present in the heavy chain amino acid sequence provided above. H and V L CDRs are as defined by Kabat (see, e.g., Table 1, above, and Kabat 1991). H and V L The CDRs are as defined by Chothia (see, eg, Table 1, above, and Chothia 1987).

[0262] For example, an anti-mesothelin antibody may comprise a VL CDR1 having the amino acid sequence TGTSSDIGGYNSVS (SEQ ID NO: 1095), a VL CDR2 having the amino acid sequence LMIYGVNNRPS (SEQ ID NO: 1096), a VL CDR3 having the amino acid sequence SSYDIESATP (SEQ ID NO: 1097), a VH CDR1 having the amino acid sequence GYSFTSYWIG (SEQ ID NO: 1098), a VH CDR2 having the amino acid sequence WMGIIDPGDSRTRYSP (SEQ ID NO: 1099), and a VH CDR3 having the amino acid sequence GQLYGGTYMDG (SEQ ID NO: 1100).

[0263] The anti-mesothelin antibody can be an scFv. As one non-limiting example, the anti-mesothelin scFv can include the following amino acid sequence: In the TIFF2024541334000070.tif31164 sequence, the VH CDR1, CDR2, and CDR3 are underlined and the VL CDR1, CDR2, and CDR3 are shown in bold and underlined.

[0264] As one non-limiting example, the anti-mesothelin scFv can comprise the following amino acid sequence: In the TIFF2024541334000071.tif31164 sequence, the VH CDR1, CDR2, and CDR3 are underlined and the VL CDR1, CDR2, and CDR3 are shown in bold and underlined.

[0265] In some instances, an anti-mesothelin antibody suitable for inclusion in a TMMP is a) the amino acid sequence: VL CDR1, VL CDR2, and VL CDR3 present in the light chain variable region (VL) comprising TIFF2024541334000072.tif11162; and b) the amino acid sequence: VH CDR1, CDR2, and CDR3 present in the heavy chain variable region (VH) containing TIFF2024541334000073.tif17163 Includes. In some cases, V H and V L CDRs are as defined by Kabat (see, e.g., Table 1, above, and Kabat 1991). H and V L The CDRs are as defined by Chothia (see, e.g., Table 1, above, and Chothia 1987). See, e.g., BMS6A5.

[0266] In some instances, an anti-mesothelin antibody suitable for inclusion in a TMMP is a) the amino acid sequence: A VL region comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF2024541334000074.tif11162; and b) the amino acid sequence: A VH region comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF2024541334000075.tif17163. Includes.

[0267] In some instances, anti-mesothelin antibodies suitable for inclusion in a TMMP include, in order from N-terminus to C-terminus: a) the amino acid sequence: a VH region comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF2024541334000076.tif17163; b) a peptide linker, and c) the amino acid sequence: A VL region comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF2024541334000077.tif11162. It is an scFv comprising: In some examples, the peptide linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO: 934), where n is an integer from 1 to 10 (e.g., n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). In some examples, the peptide linker comprises the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 875) and has a length of 15 amino acids.

[0268] In some instances, anti-mesothelin antibodies suitable for inclusion in a TMMP include, in order from N-terminus to C-terminus: a) the amino acid sequence: a VL region comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF2024541334000078.tif11162; b) a peptide linker, and c) the amino acid sequence: A VH region comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF2024541334000079.tif17163. It is an scFv comprising: In some examples, the peptide linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO: 934), where n is an integer from 1 to 10 (e.g., n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). In some examples, the peptide linker comprises the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 875) and has a length of 15 amino acids.

[0269] In some instances, an anti-mesothelin antibody suitable for inclusion in a TMMP is a) the amino acid sequence: VL CDR1, VL CDR2, and VL CDR3 present in the light chain variable region (VL) comprising TIFF2024541334000080.tif11162, and b) the amino acid sequence: VH CDR1, CDR2, and CDR3 present in the heavy chain variable region (VH) containing TIFF2024541334000081.tif11169 Includes. In some cases, V H and V L CDRs are as defined by Kabat (see, e.g., Table 1, above, and Kabat 1991). H and V LThe CDRs are as defined by Chothia (see, e.g., Table 1, above, and Chothia 1987). See, e.g., amatuximab.

[0270] In some instances, an anti-mesothelin antibody suitable for inclusion in a TMMP is a) the amino acid sequence: a VL region comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF2024541334000082.tif11162; and b) the amino acid sequence: A VH region comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF2024541334000083.tif11168. Includes.

[0271] In some instances, anti-mesothelin antibodies suitable for inclusion in a TMMP include, in order from N-terminus to C-terminus: a) the amino acid sequence: a VL region comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF2024541334000084.tif11162; b) a peptide linker, and c) the amino acid sequence: A VH region comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF2024541334000085.tif11167. It is an scFv comprising: In some examples, the peptide linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO: 934), where n is an integer from 1 to 10 (e.g., n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). In some examples, the peptide linker comprises the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 875) and has a length of 15 amino acids.

[0272] In some instances, anti-mesothelin antibodies suitable for inclusion in a TMMP include, in order from N-terminus to C-terminus: a) the amino acid sequence: a VH region comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF2024541334000086.tif17164; b) a peptide linker, and c) the amino acid sequence: A VL region comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF2024541334000087.tif11162. It is an scFv comprising: In some examples, the peptide linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO: 934), where n is an integer from 1 to 10 (e.g., n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). In some examples, the peptide linker comprises the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 875) and has a length of 15 amino acids.

[0273] In some instances, an anti-mesothelin antibody suitable for inclusion in a TMMP is a) the amino acid sequence: VL CDR1, VL CDR2, and VL CDR3 present in the light chain variable region (VL) comprising TIFF2024541334000088.tif11162, and b) the amino acid sequence: VH CDR1, CDR2, and CDR3 present in the heavy chain variable region (VH) containing TIFF2024541334000089.tif17163 Includes. In some cases, V H and V L CDRs are as defined by Kabat (see, e.g., Table 1, above, and Kabat 1991). H and V L The CDRs are as defined by Chothia (see, eg, Table 1, above, and Chothia 1987).

[0274] In some instances, an anti-mesothelin antibody suitable for inclusion in a TMMP is a) the amino acid sequence: a VL region comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF2024541334000090.tif11162; and b) the amino acid sequence: A VH region comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF2024541334000091.tif17163. Includes.

[0275] In some instances, anti-mesothelin antibodies suitable for inclusion in a TMMP include, in order from N-terminus to C-terminus: a) the amino acid sequence: a VL region comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF2024541334000092.tif11162; b) a peptide linker, and c) the amino acid sequence: A VH region comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF2024541334000093.tif17163. It is an scFv comprising: In some examples, the peptide linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO: 934), where n is an integer from 1 to 10 (e.g., n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). In some examples, the peptide linker comprises the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 875) and has a length of 15 amino acids.

[0276] In some instances, anti-mesothelin antibodies suitable for inclusion in a TMMP include, in order from N-terminus to C-terminus: a) the amino acid sequence: a VH region comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF2024541334000094.tif17163; b) a peptide linker, and c) the amino acid sequence: A VL region comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF2024541334000095.tif11162. It is an scFv comprising: In some examples, the peptide linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO: 934), where n is an integer from 1 to 10 (e.g., n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). In some examples, the peptide linker comprises the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 875) and has a length of 15 amino acids.

[0277] In some instances, an anti-mesothelin antibody suitable for inclusion in a TMMP is a) the amino acid sequence: VL CDR1, VL CDR2, and VL CDR3 present in the light chain variable region (VL) comprising TIFF2024541334000096.tif11163; and b) the amino acid sequence: VH CDR1, CDR2, and CDR3 present in the heavy chain variable region (VH) containing TIFF2024541334000097.tif17168 Includes. In some cases, V H and V L CDRs are as defined by Kabat (see, e.g., Table 1, above, and Kabat 1991). H and V L The CDRs are as defined by Chothia (see, e.g., Table 1, above, and Chothia 1987). See, e.g., RG7787.

[0278] In some instances, an anti-mesothelin antibody suitable for inclusion in a TMMP is a) the amino acid sequence: a VL region comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF2024541334000098.tif11163; and b) the amino acid sequence: A VH region comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF2024541334000099.tif17168. Includes.

[0279] In some instances, anti-mesothelin antibodies suitable for inclusion in a TMMP include, in order from N-terminus to C-terminus: a) the amino acid sequence: a VL region comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF2024541334000100.tif11163; b) a peptide linker, and c) the amino acid sequence: A VH region comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF2024541334000101.tif17168. In some examples, the peptide linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO:934), where n is an integer from 1 to 10 (e.g., n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). In some examples, the peptide linker comprises the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 875) and has a length of 15 amino acids.

[0280] In some instances, anti-mesothelin antibodies suitable for inclusion in a TMMP include, in order from N-terminus to C-terminus: a) the amino acid sequence: a VH region comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF2024541334000102.tif17168; b) a peptide linker, and c) the amino acid sequence: A VL region comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF2024541334000103.tif11163. It is an scFv comprising: In some examples, the peptide linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO: 934), where n is an integer from 1 to 10 (e.g., n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). In some examples, the peptide linker comprises the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 875) and has a length of 15 amino acids.

[0281] In some examples, the anti-mesothelin scFv comprises the following amino acid sequence: In the TIFF2024541334000104.tif24164 sequence, the VH sequence is italicized, the (GGGGS)3 (SEQ ID NO:875) linker is bold and underlined, and the VL sequence is underlined.

[0282] In some examples, the anti-mesothelin scFv comprises the following amino acid sequence: In the TIFF2024541334000105.tif24164 sequence, the VL sequence is underlined, the (GGGGS)3 (SEQ ID NO:875) linker is bold and underlined, and the VL sequence is italicized.

[0283] In some examples, the anti-mesothelin scFv comprises the following amino acid sequence: In the TIFF2024541334000106.tif24164 sequence, the VH sequence is italicized, the (GGGGS)3 (SEQ ID NO:875) linker is bold and underlined, and the VL sequence is underlined.

[0284] In some examples, the anti-mesothelin scFv comprises the following amino acid sequence: In the TIFF2024541334000107.tif24162 sequence, the VL sequence is underlined, the (GGGGS)3 (SEQ ID NO:875) linker is bold and underlined, and the VL sequence is underlined.

[0285] In some examples, the anti-mesothelin scFv comprises the following amino acid sequence: In the TIFF2024541334000108.tif24164 sequence, the VH sequence is italicized, the (GGGGS)3 (SEQ ID NO:875) linker is bold and underlined, and the VL sequence is underlined.

[0286] In some examples, the anti-mesothelin scFv comprises the following amino acid sequence: In the TIFF2024541334000109.tif24164 sequence, the VL sequence is underlined, the (GGGGS)3 (SEQ ID NO:875) linker is bold and underlined, and the VL sequence is italicized.

[0287] In some examples, the anti-mesothelin scFv comprises the following amino acid sequence: In the TIFF2024541334000110.tif24164 sequence, the VH sequence is italicized, the (GGGGS)3 (SEQ ID NO:875) linker is bold and underlined, and the VL sequence is underlined.

[0288] In some examples, the anti-mesothelin scFv comprises the following amino acid sequence: In the TIFF2024541334000111.tif24163 sequence, the VL sequence is underlined, the (GGGGS)3 (SEQ ID NO:875) linker is bold and underlined, and the VL sequence is italicized.

[0289] anti-PSMA Prostate-specific membrane antigen (PSMA) (also known as folate hydrolase 1 (FOLH1), membrane glutamate carboxypeptidase, and N-acetylated alpha-linked acidic dipeptidase 1) is upregulated in cancer cells of the prostate gland and is used as a diagnostic and prognostic indicator of prostate cancer.

[0290] Anti-PSMA antibodies are known in the art, and the VH and VL, or VH and VL CDRs, of any anti-PSMA antibody can be used in TMMP. See, e.g., U.S. Patent No. 10,179,819 and U.S. Patent Application Publication No. 2021 / 0277141.

[0291] anti-CD22 CD22 (also known as B lymphocyte cell adhesion molecule, sialic acid-binding Ig-like lectin 2, or SIGLEC2) is a sialic acid-binding adhesion molecule that is primarily restricted to the B cell lineage and expressed on most B lineage malignancies.

[0292] Anti-CD22 antibodies are known in the art, and the VH and VL or VH and VL CDRs of any anti-CD22 antibody can be used in TMMP. See, for example, Xiao et al. (2009) Mabs 1:297 (depicting the fully human anti-CD22 m971 scFv), and US Patent Publication No. 2020 / 0147134. Examples of anti-CD22 antibodies include epratuzumab and inotuzumab. See, for example, Lenoard et al. (2007) Oncogene 26:3704 and US Patent No. 5,789,554 (depicting epratuzumab), and DiJoseph et al. (2007) Leukemia 21:2240 (depicting inotuzumab).

[0293] For example, anti-CD22 antibodies i) a heavy chain variable region (VH) CDR1 having the following amino acid sequence: GDSVSSNSAA (SEQ ID NO: 1110); ii) a VH CDR2 having the following amino acid sequence: TYYRSKWYN (SEQ ID NO: 1111), iii) a VH CDR3 having the following amino acid sequence: AREVTGDLEDAFDI (SEQ ID NO: 1112); iv) a light chain variable region (VL) CDR1 having the following amino acid sequence: QTIWSY (SEQ ID NO: 1113); v) a VL CDR2 having the following amino acid sequence: AAS (Ala-Ala-Ser), and vi) a VL CDR3 having the following amino acid sequence: QSYSIPQT (SEQ ID NO: 1114); may include.

[0294] anti-TROP-2 Trophoblast cell surface antigen 2 (Trop-2) (also known as epithelial glycoprotein-1, gastrointestinal tumor-associated antigen GA733-1, membrane component staining 1 surface marker-1, and tumor-associated calcium signal transducer-2) is a transmembrane glycoprotein that is upregulated in many cancer types and is the protein product of the TACSTD2 gene.

[0295] In some examples, the TTP is an anti-TROP-2 scFv or anti-TROP-2 nanobody that comprises the VH and VL CDRs present in any one of the amino acid sequences set forth in Figures 23A-23D. In some examples, the TTP is an anti-TROP-2 scFv that comprises the amino acid sequence set forth in any one of Figures 23A-23D.

[0296] Anti-TROP-2 antibodies are known in the art, and the VH and VL or VH and VL CDRs of any anti-TROP-2 antibody can be used in TMMP. See, for example, U.S. Patent No. 7,238,785. In some examples, the anti-TROP-2 antibody comprises i) light chain CDR sequence CDR1 (KASQDVSIAVA, SEQ ID NO: 1115), CDR2 (SASYRYT, SEQ ID NO: 1116), and CDR3 (QQHYITPLT, SEQ ID NO: 1117), and ii) heavy chain CDR sequence CDR1 (NYGMN, SEQ ID NO: 1118), CDR2 (WINTYTGEPTYTDDFKG, SEQ ID NO: 1119), and CDR3 (GGFGSSYWYFDV, SEQ ID NO: 1120).

[0297] In some examples, the anti-TROP-2 antibody comprises i) heavy chain CDR sequences CDR1 (TAGMQ, SEQ ID NO: 1121), CDR2 (WINTHSGVPKYAEDFKG, SEQ ID NO: 1122), and CDR3 (SGFGSSYWYFDV, SEQ ID NO: 1123), and ii) light chain CDR sequences CDR1 (KASQDVSTAVA, SEQ ID NO: 1124), CDR2 (SASYRYT, SEQ ID NO: 1116), and CDR3 (QQHYITPLT, SEQ ID NO: 1117).

[0298] In some examples, an anti-TROP2 antibody suitable for inclusion in a TMMP is a) the amino acid sequence: VL CDR1, VL CDR2, and VL CDR3 present in the light chain variable region (VL) comprising TIFF2024541334000112.tif11162, and b) the amino acid sequence: VH CDR1, CDR2, and CDR3 present in the heavy chain variable region (VH) containing TIFF2024541334000113.tif17170 Includes. In some cases, V H and V L CDRs are as defined by Kabat (see, e.g., Table 1, above, and Kabat 1991). H and V L The CDRs are as defined by Chothia (see, eg, Table 1, above, and Chothia 1987).

[0299] In some examples, an anti-TROP-2 antibody suitable for inclusion in a TMMP is a) the amino acid sequence: a VL region comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF2024541334000114.tif11162; and b) the amino acid sequence: A VH region comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF2024541334000115.tif17163. Includes.

[0300] In some examples, anti-TROP-2 antibodies suitable for inclusion in TMMP include, in order from N-terminus to C-terminus: a) the amino acid sequence: a VL region comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF2024541334000116.tif11162; b) a peptide linker, and c) the amino acid sequence: A VH region comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF2024541334000117.tif17169. It is an scFv comprising: In some examples, the peptide linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO: 934), where n is an integer from 1 to 10 (e.g., n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). In some examples, the peptide linker comprises the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 875) and has a length of 15 amino acids.

[0301] In some examples, anti-TROP-2 antibodies suitable for inclusion in TMMP include, in order from N-terminus to C-terminus: a) the amino acid sequence: a VH region comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF2024541334000118.tif17169; b) a peptide linker, and c) the amino acid sequence: A VL region comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF2024541334000119.tif11162. It is an scFv comprising: In some examples, the peptide linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO: 934), where n is an integer from 1 to 10 (e.g., n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). In some examples, the peptide linker comprises the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 875) and has a length of 15 amino acids.

[0302] In some examples, an anti-TROP2 antibody suitable for inclusion in a TMMP is a) the amino acid sequence: VL CDR1, VL CDR2, and VL CDR3 present in the light chain variable region (VL) comprising TIFF2024541334000120.tif11162, and b) the amino acid sequence: VH CDR1, CDR2, and CDR3 present in the heavy chain variable region (VH) containing TIFF2024541334000121.tif17169 Includes. In some cases, V H and V L CDRs are as defined by Kabat (see, e.g., Table 1, above, and Kabat 1991). H and V L The CDRs are as defined by Chothia (see, eg, Table 1, above, and Chothia 1987).

[0303] In some examples, an anti-TROP-2 antibody suitable for inclusion in a TMMP is a) the amino acid sequence: a VL region comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF2024541334000122.tif11162; and b) the amino acid sequence: A VH region comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF2024541334000123.tif17169. Includes.

[0304] In some examples, anti-TROP-2 antibodies suitable for inclusion in TMMP include, in order from N-terminus to C-terminus: a) the amino acid sequence: a VL region comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF2024541334000124.tif11162; b) a peptide linker, and c) the amino acid sequence: A VH region comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF2024541334000125.tif17169. It is an scFv comprising: In some examples, the peptide linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO: 934), where n is an integer from 1 to 10 (e.g., n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). In some examples, the peptide linker comprises the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 875) and has a length of 15 amino acids.

[0305] In some examples, anti-TROP-2 antibodies suitable for inclusion in TMMP include, in order from N-terminus to C-terminus: a) the amino acid sequence: a VH region comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF2024541334000126.tif17169; b) a peptide linker, and c) the amino acid sequence: A VL region comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF2024541334000127.tif11162. It is an scFv comprising: In some examples, the peptide linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO: 934), where n is an integer from 1 to 10 (e.g., n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). In some examples, the peptide linker comprises the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 875) and has a length of 15 amino acids.

[0306] anti-BCMA Anti-BCMA (B cell maturation antigen) antibodies are known in the art, and the VH and VL, or VH and VL CDRs, of any anti-BCMA antibody can be used in the TMMP. See, e.g., WO 2014 / 089335, U.S. Patent Application Publication No. 2019 / 0153061, and WO 2017 / 093942.

[0307] In some instances, the anti-BCMA antibody a) the amino acid sequence: a light chain comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF2024541334000128.tif24163; and b) the amino acid sequence: a heavy chain comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF2024541334000129.tif50164. Includes.

[0308] In some examples, the anti-BCMA antibody comprises a VL present in the light chain amino acid sequence provided above and a VH present in the heavy chain amino acid sequence provided above. For example, the anti-BCMA antibody comprises a) the amino acid sequence: a VL comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF2024541334000130.tif17157; and b) the amino acid sequence: A VH comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF2024541334000131.tif17163. may include.

[0309] In some examples, the anti-BCMA antibody comprises a VL CDR1, a VL CDR2, and a VL CDR3 present in the light chain amino acid sequence provided above, and a VH CDR1, a CDR2, and a CDR3 present in the heavy chain amino acid sequence provided above. H and V L CDRs are as defined by Kabat (see, e.g., Table 1, above, and Kabat 1991). H and V L The CDRs are as defined by Chothia (see, eg, Table 1, above, and Chothia 1987).

[0310] For example, an anti-BCMA antibody may comprise a VL CDR1 having the amino acid sequence SSNIGSNT (SEQ ID NO: 1133), a VL CDR2 having the amino acid sequence NYH, a VL CDR3 having the amino acid sequence AAWDDSLNGWV (SEQ ID NO: 1134), a VH CDR1 having the amino acid sequence GFTGFGDYA (SEQ ID NO: 1135), a VH CDR2 having the amino acid sequence SRSKAYGGTT (SEQ ID NO: 1136), and a VH CDR3 having the amino acid sequence ASSGYSSGWTPFDY (SEQ ID NO: 1137).

[0311] The anti-BCMA antibody may be an scFv. As one non-limiting example, the anti-BCMA scFv has the following amino acid sequence: TIFF2024541334000132.tif31169 may be included.

[0312] As another example, the anti-BCMA scFv may have the following amino acid sequence: TIFF2024541334000133.tif31164 may be included.

[0313] In some examples, the anti-BCMA antibody may comprise a VL CDR1 having the amino acid sequence SASQDISNYLN (SEQ ID NO: 1140), a VL CDR2 having the amino acid sequence YTSNLHS (SEQ ID NO: 1141), a VL CDR3 having the amino acid sequence QQYRKLPWT (SEQ ID NO: 1142), a VH CDR1 having the amino acid sequence NYWMH (SEQ ID NO: 1143), a VH CDR2 having the amino acid sequence ATYRGHSDTYYNQKFKG (SEQ ID NO: 1144), and a VH CDR3 having the amino acid sequence GAIYNGYDVLDN (SEQ ID NO: 1145).

[0314] In some instances, the anti-BCMA antibody a) the amino acid sequence: a light chain comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF2024541334000134.tif11162. Includes.

[0315] In some instances, the anti-BCMA antibody a) the amino acid sequence: a heavy chain comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF2024541334000135.tif17162. Includes.

[0316] In some instances, the anti-BCMA antibody (e.g., the antibody referred to in the literature as belantamab) The amino acid sequence: light chain, including TIFF2024541334000136.tif11162, and The amino acid sequence: Heavy chain containing TIFF2024541334000137.tif17162 Includes.

[0317] In some examples, the anti-BCMA antibody has a cancer chemotherapeutic agent linked to the antibody. For example, in some examples, the anti-BCMA antibody is GSK2857916 (belantamab-mafodotin), and monomethyl auristatin F (MMAF) is linked to the anti-BCMA antibody belantamab via a maleimidocaproyl linker.

[0318] anti-MUC1 In some cases, the TTP present in the TMMP is an antibody specific to MUC1. For example, the TTP can be specific to the MUC1 polypeptide present on cancer cells. In some cases, the TTP is specific to a truncated form of MUC1, see, for example, Fessler et al. (2009) Breast Cancer Res. 118: 113. In some cases, the TTP is an antibody specific to glycosylated MUC1 peptide, see, for example, Naito et al. (2017) ACS Omega 2: 7493 and U.S. Patent No. 10,017,580.

[0319] As one non-limiting example, the TTP can be a single chain Fv specific for MUC1.See, for example, Singh et al.(2007) Mol.Cancer Ther.6:562, Thie et al.(2011) PLoSOne 6:e15921, Imai et al.(2004) Leukemia 18:676, Posey et al.(2016) Immunity 44:1444, EP 3130607, EP 3164418, WO 2002 / 044217, and US 2018 / 0112007.In some examples, the TTP is a scFv specific for the MUC1 peptide VTSAPDTRPAPGSTAPPAHG (SEQ ID NO:1148). In some examples, the TTP is an scFv specific for the MUC1 peptide SNIKFRPGSVVVQLTLAFREGTINVHDVETQFNQYKTEAASRY (SEQ ID NO: 1149). In some examples, the TTP is an scFv specific for the MUC1 peptide SVVVQLTLAFREGTINVHDVETQFNQYKTEAASRY (SEQ ID NO: 1150). In some examples, the TTP is an scFv specific for the MUC1 peptide LAFREGTINVHDVETQFNQY (SEQ ID NO: 1151). In some examples, the TTP is an scFv specific for the MUC1 peptide SNIKFRPGSVVVQLTLAAFREGTIN (SEQ ID NO: 1152).

[0320] As an example, an anti-MUC1 antibody may comprise a VH CDR1 having the amino acid sequence RYGMS (SEQ ID NO: 1153), a VH CDR2 having the amino acid sequence TISGGGTYIYYPDSVKG (SEQ ID NO: 1154), a VH CDR3 having the amino acid sequence DNYGRNYDYGMDY (SEQ ID NO: 1155), a VL CDR1 having the amino acid sequence SATSSVSYIH (SEQ ID NO: 1156), a VL CDR2 having the amino acid sequence STSNLAS (SEQ ID NO: 1157), and a VL CDR3 having the amino acid sequence QQRSSSPFT (SEQ ID NO: 1158). See, e.g., U.S. Patent Application Publication No. 2018 / 0112007.

[0321] As another example, an anti-MUC1 antibody can comprise a VH CDR1 having the amino acid sequence GYAMS (SEQ ID NO: 1159), a VH CDR2 having the amino acid sequence TISSGGTYIYYPDSVKG (SEQ ID NO: 1160), a VH CDR3 having the amino acid sequence LGGDNYYEYFDV (SEQ ID NO: 1161), a VL CDR1 having the amino acid sequence RASKSVSTSGYSYMH (SEQ ID NO: 1162), a VL CDR2 having the amino acid sequence LASNLES (SEQ ID NO: 1163), and a VL CDR3 having the amino acid sequence QHSRELPFT (SEQ ID NO: 1164). See, e.g., U.S. Patent Application Publication No. 2018 / 0112007.

[0322] As another example, an anti-MUC1 antibody can comprise a VH CDR1 having the amino acid sequence DYAMN (SEQ ID NO: 1165), a VH CDR2 having the amino acid sequence VISTFSGNINFNQKFKG (SEQ ID NO: 1166), a VH CDR3 having the amino acid sequence SDYYGPYFDY (SEQ ID NO: 1167), a VL CDR1 having the amino acid sequence RSSQTIVHSNGNTYLE (SEQ ID NO: 1168), a VL CDR2 having the amino acid sequence KVSNRFS (SEQ ID NO: 1169), and a VL CDR3 having the amino acid sequence (FQGSHVPFT (SEQ ID NO: 1170). See, e.g., U.S. Patent Application Publication No. 2018 / 0112007.

[0323] As another example, an anti-MUC1 antibody can comprise a VH CDR1 having the amino acid sequence GYAMS (SEQ ID NO: 1159), a VH CDR2 having the amino acid sequence TISSGGTYIYYPDSVKG (SEQ ID NO: 1160), a VH CDR3 having the amino acid sequence LGGDNYYEY (SEQ ID NO: 1171), a VL CDR1 having the amino acid sequence TASKSVSTSGYSYMH (SEQ ID NO: 1172), a VL CDR2 having the amino acid sequence LVSNLES (SEQ ID NO: 1173), and a VL CDR3 having the amino acid sequence QHIRELTRSE (SEQ ID NO: 1174). See, e.g., U.S. Patent Application Publication No. 2018 / 0112007.

[0324] anti-MUC16 In some examples, the TTP present in the TMMP is an antibody specific to MUC16 (also known as CA125). For example, see Yin et al. (2002) Int. J. Cancer 98:737. For example, the TTP can be specific to MUC16 polypeptide present on cancer cells. For example, see US Patent Application Publication No. 2018 / 0118848 and US Patent Application Publication No. 2018 / 0112008. In some examples, the MUC16-specific TTP is an scFv. In some examples, the MUC16-specific TTP is a nanobody.

[0325] As an example, an anti-MUC16 antibody may comprise a VH CDR1 having the amino acid sequence GFTFSNYY (SEQ ID NO: 1175), a VH CDR2 having the amino acid sequence ISGRGSTI (SEQ ID NO: 1176), a VH CDR3 having the amino acid sequence VKDRGGYSPY (SEQ ID NO: 1177), a VL CDR1 having the amino acid sequence QSISTY (SEQ ID NO: 1178), a VL CDR2 having the amino acid sequence TAS, and a VL CDR3 having the amino acid sequence QQSYSTPPIT (SEQ ID NO: 1179). See, e.g., U.S. Patent Application Publication No. 2018 / 0118848.

[0326] Anti-claudin-18.2 In some examples, the TPP present in the TMMP is an antibody specific to claudin-18 isoform 2 ("claudin-18.2"). For example, see International Publication No. WO 2013 / 167259. In some examples, the claudin-18.2 specific TTP is an scFv. In some examples, the claudin-18.2 specific TTP is a nanobody. In some examples, the TPP present in the TMMP is an antibody specific to TEDEVQSYPSKHDYV (SEQ ID NO: 1058) or EVQSYPSKHDYV (SEQ ID NO: 1059).

[0327] As an example, an anti-claudin-18.2 antibody may include a VH CDR1 having the amino acid sequence GYTFTDYS (sequence number 1180), a VH CDR2 having the amino acid sequence INTETGVP (sequence number 1181), a VH CDR3 having the amino acid sequence ARRTGFDY (sequence number 1182), a VL CDR1 having the amino acid sequence KNLLHSDGITY (sequence number 1183), a VL CDR2 having the amino acid sequence RVS, and a VL CDR3 having the amino acid sequence VQVLELPFT (sequence number 1184).

[0328] As another example, an anti-claudin-18.2 antibody may comprise a VH CDR1 having the amino acid sequence GFTFSSYA (SEQ ID NO: 1185), a VH CDR2 having the amino acid sequence ISDGGSYS (SEQ ID NO: 1186), a VH CDR3 having the amino acid sequence ARDSYYDNSYVRDY (SEQ ID NO: 1187), a VL CDR1 having the amino acid sequence QDINTF (SEQ ID NO: 1188), a VL CDR2 having the amino acid sequence RTN, and a VL CDR3 having the amino acid sequence LQYDEFPLT (SEQ ID NO: 1189).

[0329] mAb fusion In embodiments, the TMMP may comprise a heterodimer, a) the first polypeptide of the heterodimer is i) a single-chain polypeptide comprising a peptide epitope (e.g., a viral epitope), a β2M polypeptide, and an MHC class I heavy chain polypeptide; ii) an antibody heavy chain polypeptide; and Including, b) the second polypeptide of the heterodimer comprises an antibody light chain polypeptide; Either the first polypeptide or the second polypeptide comprises one or more immunomodulatory polypeptides, e.g., on the N-terminus of an antibody light chain polypeptide, on the C-terminus of an antibody light chain polypeptide, or on the C-terminus of an antibody heavy chain polypeptide. Therefore, TMMP is a) i) peptide epitopes (e.g. viral epitopes); ii) β2M polypeptide; iii) an MHC class I heavy chain polypeptide, and iv) an antibody heavy chain polypeptide (e.g., an antibody heavy chain comprising, in order from N-terminus to C-terminus, i) a VH polypeptide, ii) an Ig CH1 polypeptide, and iii) an IgG Fc polypeptide). and a first polypeptide comprising: b) i) one or more immunomodulatory polypeptides, and ii) an antibody light chain polypeptide (e.g., an antibody light chain comprising, in order from N-terminus to C-terminus, i) a VL polypeptide, and an Ig kappa light chain constant region polypeptide); and a second polypeptide comprising The heterodimer may comprise TMMP is a) i) peptide epitopes (e.g. viral epitopes); ii) β2M polypeptide; iii) an MHC class I heavy chain polypeptide; iv) an antibody heavy chain polypeptide (e.g., an antibody heavy chain comprising, in order from N-terminus to C-terminus, i) a VH polypeptide, ii) an Ig CH1 polypeptide, and iii) an IgG Fc polypeptide); and v) one or more immunomodulatory polypeptides and a first polypeptide comprising: b) an antibody light chain polypeptide (e.g., an antibody light chain comprising, in order from N-terminus to C-terminus, i) a VL polypeptide, and an Ig kappa light chain constant region polypeptide); and a second polypeptide comprising The heterodimer may comprise TMMP is a) i) peptide epitopes (e.g. viral epitopes); ii) β2M polypeptide; iii) an MHC class I heavy chain polypeptide, and iv) an antibody heavy chain polypeptide (e.g., an antibody heavy chain comprising, in order from N-terminus to C-terminus, i) a VH polypeptide, ii) an Ig CH1 polypeptide, and iii) an IgG Fc polypeptide). and a first polypeptide comprising: b) i) an antibody light chain polypeptide (e.g., an antibody light chain comprising, in order from N-terminus to C-terminus, i) a VL polypeptide, and an Ig kappa light chain constant region polypeptide); and ii) one or more immunomodulatory polypeptides and a second polypeptide comprising The heterodimer may comprise Such heterodimers can dimerize (e.g., form homodimers of two heterodimers by formation of disulfide bonds formed between the antibody heavy chain polypeptides of each heterodimer). Such TMMPs are referred to herein as "monoclonal antibody fusions" or "mAb fusions." The VH and VL polypeptides are on separate polypeptides of the heterodimer, and together, the VH and VL polypeptides bind to a cancer epitope, thus constituting a TTP.

[0330] The following are non-limiting examples of mAb fusions.

[0331] As an example, the TMMP may comprise a) a first polypeptide comprising the amino acid sequence shown in Figure 24A, and b) a second polypeptide comprising the amino acid sequence shown in Figure 24B. As an example, the SARS-CoV-2 peptide may have the amino acid sequence YLQPRTFLL (SEQ ID NO:218).

[0332] As another example, the TMMP can include a) a first polypeptide comprising the amino acid sequence shown in Figure 25A, and b) a second polypeptide comprising the amino acid sequence shown in Figure 25B. As an example, the SARS-CoV-2 peptide can have the amino acid sequence YLQPRTFLL (SEQ ID NO:218).

[0333] As another example, the TMMP can include a) a first polypeptide comprising the amino acid sequence shown in Figure 26A, and b) a second polypeptide comprising the amino acid sequence shown in Figure 26B. As an example, the SARS-CoV-2 peptide can have the amino acid sequence YLQPRTFLL (SEQ ID NO:218).

[0334] Single-chain T-cell receptor As mentioned above, in some cases, the TTP present in TMMP is scTCR. TTP is scTCR specific to peptide / HLA complex on the surface of cancer cell, and peptide can be cancer-associated peptide (e.g., peptide of cancer-associated antigen). Amino acid sequence of scTCR specific to cancer-associated peptide bound to HLA complex is known in the art. For example, see US Patent Application Publication No. 2019 / 0135914, US Patent Application Publication No. 2019 / 0062398, and US Patent Application Publication No. 2018 / 0371049.

[0335] An scTCR comprises an alpha chain variable region (Vα) and a beta chain variable region (Vβ) covalently linked via a suitable peptide linker sequence. For example, Vα can be covalently linked to Vβ via a suitable peptide linker (L) sequence fused to the C-terminus of Vα and the N-terminus of Vβ. An scTCR can have the structure of Vα-L-Vβ. An scTCR can have the structure of Vβ-L-Vα. An scTCR can also comprise a constant domain (also referred to as a constant region). In some instances, the scTCR is, in order from N-terminus to C-terminus: i) a TCR alpha chain variable domain polypeptide, ii) a peptide linker; iii) a TCR β chain variable domain polypeptide, and iv) a TCR β chain constant region extracellular domain polypeptide; Includes. In some instances, the scTCR is, in order from N-terminus to C-terminus: i) a TCR β chain variable domain polypeptide, ii) a peptide linker; iii) a TCR alpha chain variable domain polypeptide, and iv) TCR α chain constant region extracellular domain polypeptide; Includes.

[0336] The amino acid sequence of scTCR specific for peptide / HLA complex, where the peptide is a cancer-associated peptide, is known in the art.See, for example, US Patent Application Publication No. 2019 / 0135914, US Patent Application Publication No. 2019 / 0062398, US Patent Application Publication No. 2018 / 0371049, US Patent Application Publication No. 2019 / 0144563, and US Patent Application Publication No. 2019 / 0119350.

[0337] For example, scTCR is a TCR that targets HLA-A * The scTCR may be specific for a NY-ESO epitope, such as the SLLMWITQC (SEQ ID NO: 1190) peptide, bound to an HLA complex comprising the 0201 heavy chain and the β2M polypeptide. i) the amino acid sequence: and a TCR α chain variable region comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF2024541334000138.tif11163, wherein amino acid 20 can be V or A; amino acid 51 can be Q, P, S, T, or M; amino acid 52 can be S, P, F, or G; amino acid 53 can be S, W, H, or T; amino acid 94 can be P, H, or A; and amino acid 95 can be T, L, M, A, Q, Y, or Y. , E, I, F, V, N, G, S, D, or R; amino acid 96 can be S, L, T, Y, I, Q, V, E, A, W, R, G, H, D, or K; amino acid 97 can be G, D, N, V, S, T, or A; amino acid 98 can be G, P, H, S, T, W, or A; amino acid 99 can be S, T, Y, D, H, V, N, E, G, Q, K, A, I, or R; amino acid 100 can be Y, F, M, or D; amino acid 101 can be I, P, T, or M; and amino acid 103 can be T or A, and ii) the amino acid sequence: and wherein amino acid 70 can be T or I; amino acid 94 can be Y, N, or F; amino acid 95 can be V or L; and amino acid 97 can be N, G, or D. For example, in some instances, the scTCR has i) the following amino acid sequence: The TCR alpha chain variable region, comprising TIFF2024541334000140.tif11162, and the following amino acid sequence: TIFF2024541334000141.tif11162, including the TCR β chain variable region.

[0338] As another example, the scTCR can be used to detect an HPV epitope (e.g., the amino acid sequence YIIFVYIPL (HPV16 E5 63-71 ;SEQ ID NO: 1195), KLPQLCTEL (HPV16 E6 11-19 ; SEQ ID NO: 1042), TIHEIILECV (HPV16 E6; SEQ ID NO: 1196), YMLDLQPET (HPV16 E7 11-19 ;SEQ ID NO: 1049), TLGIVCPI (HPV16 E7 86-93 ) (SEQ ID NO: 1055), KCIDFYSRI (HPV18 E6 67-75 ;SEQ ID NO: 1197), or FQQLFLNTL (HPV18 E7 86-94 ;SEQ ID NO: 1198). In one example, such a scTCR may be specific for i) the amino acid sequence: a TCR α chain variable region comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF2024541334000142.tif17164; and ii) the amino acid sequence: a TCR beta chain variable region comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF2024541334000143.tif17164; Includes.

[0339] Contrast agents In some instances, the TTP of the TMMP comprises an imaging agent or radiolabel, where the imaging agent facilitates imaging of the tumor to which the TMMP is bound.

[0340] Suitable agents include computed tomography (CT), positron emission tomography (PET), and single photon emission computed tomography (SPECT) radiotracers. Suitable PET / SPECT imaging agents include, for example, positron emitters, e.g., 11 C. 13 N, 18 F, 82 Ru, and 15 O. Iodinated CT contrast agents can be used. Suitable contrast agents include gadolinium (Gd), dysprosium, and iron. Gd chelates such as Gd diethylenetriaminepentaacetic acid (GdDTPA), Gd tetraazacyclododecanetetraacetic acid (GdDOTA), polylysine-Gd chelate, and derivatives thereof can be used. Suitable radioisotopes include: 123 I (iodine), 18 F (fluorine), 99 Tc (technetium), 111 In (indium), and 67 Ga (gallium) is an example.

[0341] HLA / peptide binding assay Whether a given peptide (e.g., a peptide containing an epitope) binds to class I HLA (including HLA heavy chain and β2M polypeptide), and whether it can effectively present the epitope to TCR when bound to the HLA complex, can be determined using any of several well-known methods. Assays include binding assays and T cell activation assays. See, for example, published International Publication No. WO 2020 / 243315 (Cue Biopharma, Inc.).

[0342] Exemplary TMMPs TMMP is a) i) a SARS-CoV-2 peptide; and ii) a first MHC polypeptide; and A first polypeptide comprising: b) a second polypeptide comprising a second MHC polypeptide; c) at least one immunomodulatory polypeptide, wherein the first and / or second polypeptide comprises an immunomodulatory polypeptide; d) an Ig Fc polypeptide or a non-Ig scaffold, wherein the first and / or second polypeptide comprises an Ig Fc polypeptide or a non-Ig scaffold; and e) a tumor targeting polypeptide (TTP), wherein the first and / or second polypeptide comprises TTP; The compound comprises at least one heterodimer comprising: These components may be arranged in any of a variety of configurations, see, for example, Figures 18A-18C and 28A-28C for non-limiting examples of such configurations.

[0343] Therefore, in some instances, TMMP may: a) i) a SARS-CoV-2 peptide; and ii) a first MHC class I polypeptide; and iii) at least one immunomodulatory polypeptide; A first polypeptide comprising: b) a second polypeptide comprising a second MHC class I polypeptide; c) an Ig Fc polypeptide or a non-Ig scaffold, wherein the first and / or second polypeptide comprises an Ig Fc polypeptide or a non-Ig scaffold; and d) TTP, wherein the first and / or second polypeptide comprises TTP; The compound comprises at least one heterodimer comprising: In another example, TMMP is a) a first polypeptide comprising i) a SARS-CoV-2 peptide and ii) a first MHC class I polypeptide; b) a second polypeptide comprising i) a second MHC class I polypeptide and ii) at least one immunomodulatory polypeptide; c) an Ig Fc polypeptide or a non-Ig scaffold, wherein the first and / or second polypeptide comprises an Ig Fc polypeptide or a non-Ig scaffold; and d) TTP, wherein the first and / or second polypeptide comprises TTP; The compound comprises at least one heterodimer comprising: In some instances, TMMP may be a) a first polypeptide comprising i) a SARS-CoV-2 peptide, ii) a first MHC class I polypeptide, and iii) at least one immunomodulatory polypeptide; b) a second polypeptide comprising i) a second MHC class I polypeptide and ii) at least one immunomodulatory polypeptide; c) an Ig Fc polypeptide or a non-Ig scaffold, wherein the first and / or second polypeptide comprises an Ig Fc polypeptide or a non-Ig scaffold; and d) TTP, wherein the first and / or second polypeptide comprises TTP; The compound comprises at least one heterodimer comprising: In some examples, at least one immunomodulatory polypeptide is a wild-type immunomodulatory polypeptide. In other examples, at least one immunomodulatory polypeptide is a variant immunomodulatory polypeptide that exhibits a lower affinity for a co-immunomodulatory polypeptide compared to the affinity of the corresponding wild-type immunomodulatory polypeptide for the co-immunomodulatory polypeptide. In some examples, the TMMP comprises two immunomodulatory polypeptides, and the two immunomodulatory polypeptides have the same amino acid sequence. In some examples, the TTP is an antibody specific for a cancer-associated antigen, e.g., a cancer-associated antigen present on the surface of a cancer cell. In some examples, the TTP is an scFv or nanobody. In some examples, the TTP is an antibody specific for a cancer-associated peptide / HLA complex (i.e., an HLA heavy chain and a β2M polypeptide) present on the surface of a cancer cell. In some examples, the TTP is a single-chain T cell receptor (scTCR) specific for a cancer-associated antigen, e.g., a cancer-associated antigen present on the surface of a cancer cell.

[0344] For example, in some instances, TMMP may be a) From the N-terminus to the C-terminus: i) SARS-CoV-2 peptides; ii) an optional linker, and iii) β2M polypeptide and a first polypeptide comprising: b) In order from the N-terminus to the C-terminus, i) TTPs, ii) an optional linker; iii) an MHC class I heavy chain polypeptide; iv) an optional linker; v) an Ig Fc polypeptide; vi) an optional linker, and vii) MOD and a second polypeptide comprising Includes. In some instances, TMMP may be a) From the N-terminus to the C-terminus: i) SARS-CoV-2 peptides; ii) a linker, and iii) β2M polypeptide and a first polypeptide comprising: b) In order from the N-terminus to the C-terminus, i) TTPs, ii) a linker, iii) an MHC class I heavy chain polypeptide; iv) a linker, v) an Ig Fc polypeptide; vi) a linker, and vii) MOD and a second polypeptide comprising Includes. In some instances, TMMP may be a) From the N-terminus to the C-terminus: i) SARS-CoV-2 peptides; ii) a linker, and iii) β2M polypeptide and a first polypeptide comprising: b) In order from the N-terminus to the C-terminus, i) TTPs, ii) a linker, iii) an MHC class I heavy chain polypeptide; iv) a linker, v) an Ig Fc polypeptide; vi) a linker; vii) the first MOD, viii) a linker, and ix) Second MOD and a second polypeptide comprising Includes. In some examples, the HLA heavy chain comprises the amino acid sequence HLA-A shown in any of Figures 3A-3E, 4A-4E, 5A-5E, and 6A-6E. * 0201, HLA-A * 1101, HLA-A * 2402, or HLA-A *3303. In some examples, the HLA heavy chain comprises an amino acid sequence having at least 95% amino acid sequence identity to an HLA-E amino acid sequence set forth in any one of Figures 16A-16D. In some examples, the HLA heavy chain comprises an amino acid sequence having at least 95% amino acid sequence identity to an HLA-G amino acid sequence set forth in any one of Figures 17A-17D. In some examples, the HLA heavy chain polypeptide comprises an amino acid sequence having at least 95% amino acid sequence identity to an HLA-A amino acid sequence set forth in any one of Figures 17A-17D. * In some examples, the HLA heavy chain polypeptide is an HLA-A 0201 polypeptide. * In some examples, the HLA heavy chain polypeptide is an HLA-A 2402 polypeptide. * In some examples, the HLA heavy chain polypeptide is an HLA-A 1101 polypeptide. *3303 polypeptide. In some examples, the HLA heavy chain polypeptide is an HLA-E polypeptide. In some examples, the HLA heavy chain polypeptide is an HLA-G polypeptide. In some examples, the Ig Fc polypeptide is a human IgG1 Fc polypeptide. In some examples, the Ig Fc polypeptide is an IgG1 Fc polypeptide comprising an L234A and an L235A substitution. In some examples, the first and second polypeptides are disulfide bonded to each other. In some examples, the first and second polypeptides are linked to each other by two disulfide bonds. In some examples, the immunomodulatory polypeptide comprises a wild-type amino acid sequence, and in other examples, the immunomodulatory polypeptide is a variant, e.g., as described above. In some examples, the immunomodulatory polypeptide is a 4-1BBL polypeptide, e.g., a variant 4-1BBL polypeptide. In some examples, the immunomodulatory polypeptide is a variant IL-2 polypeptide. In some examples, the immunomodulatory polypeptide is a variant IL-2 polypeptide comprising an H16A and an F42A substitution. In some examples, the immune modulating polypeptide is a variant IL-2 polypeptide comprising H16T and F42A substitutions. In some examples, the second polypeptide comprises a linker between the TTP and the MHC class I heavy chain, the linker having a length of less than about 25 amino acids, less than about 20 amino acids, less than about 15 amino acids, less than about 10 amino acids, less than about 8 amino acids, or less than about 6 amino acids. In some examples, the second polypeptide comprises a rigid peptide linker between the Ig Fc polypeptide and the MOD. In some examples, the TTP is an antibody specific to a cancer-associated antigen, e.g., a cancer-associated antigen present on the surface of a cancer cell. In some examples, the TTP is an scFv or nanobody. In some examples, the TTP is an antibody specific to a cancer-associated peptide / HLA complex (i.e., an HLA heavy chain and a β2M polypeptide) present on the surface of a cancer cell. In some examples, the TTP is an scTCR specific to a cancer-associated antigen, e.g., a cancer-associated antigen present on the surface of a cancer cell. In some examples, the SARS-CoV-2 peptide comprises the amino acid sequence YLQPRTFLL (SEQ ID NO: 218) and has a length of 9 amino acids.In some examples, the SARS-CoV-2 peptide has the amino acid sequence shown in Figure 19. In some examples, the SARS-CoV-2 peptide includes the amino acid sequence GTHWFVTQR (SEQ ID NO: 332) and has a length of 9 amino acids. In some examples, the SARS-CoV-2 peptide includes the amino acid sequence QYIKWPWYI (SEQ ID NO: 400) and has a length of 9 amino acids.

[0345] In some instances, the TMMP is an HLA-A * HLA-A having an amino acid sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence of the 0201 polypeptide. * 201 polypeptide. In some examples, the TMMP comprises an IgG1 Fc polypeptide having an amino acid sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence of the IgG1 Fc polypeptide shown in FIG. 20B. In some examples, the TMMP comprises an IL-2 polypeptide having an amino acid sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence of the IL-2 polypeptide shown in FIG. 20B.

[0346] In some instances, TMMP may be associated with HLA-A, as shown in FIG. * HLA-A having an amino acid sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence of the 1101 polypeptide. *1101 polypeptide. In some examples, the TMMP comprises an IgG1 Fc polypeptide having an amino acid sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence of the IgG1 Fc polypeptide shown in Figure 20D. In some examples, the TMMP comprises an IL-2 polypeptide having an amino acid sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence of the IL-2 polypeptide shown in Figure 20D.

[0347] In some instances, TMMP is an HLA-A * HLA-A having an amino acid sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence of the 2402 polypeptide. * 2402 polypeptide. In some examples, the TMMP comprises an IgG1 Fc polypeptide having an amino acid sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence of the IgG1 Fc polypeptide shown in Figure 20F. In some examples, the TMMP comprises an IL-2 polypeptide having an amino acid sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence of the IL-2 polypeptide shown in Figure 20F.

[0348] In some instances, TMMP may be associated with HLA-E as shown in FIG. *HLA-E having an amino acid sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence of the HLA-E polypeptide. * 20H. In some examples, the TMMP comprises an IgG1 Fc polypeptide having an amino acid sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence of the IgG1 Fc polypeptide shown in FIG. 20H. In some examples, the TMMP comprises an IL-2 polypeptide having an amino acid sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence of the IL-2 polypeptide shown in FIG. 20H.

[0349] In some instances, TMMP binds to HLA-E as shown in FIG. * HLA-E having an amino acid sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence of the polypeptide. *20J. In some examples, the TMMP comprises an IgG1 Fc polypeptide having an amino acid sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence of the IgG1 Fc polypeptide shown in FIG. 20J. In some examples, the TMMP comprises an IL-2 polypeptide having an amino acid sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence of the IL-2 polypeptide shown in FIG. 20J.

[0350] As one non-limiting example, the TMMP comprises a) a first polypeptide comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in FIG. 20A, and b) a second polypeptide comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in FIG. 21A. As one non-limiting example, the TMMP comprises a) a first polypeptide comprising an amino acid sequence depicted in FIG. 20A, and b) a second polypeptide comprising an amino acid sequence depicted in FIG. 21A. In some examples, (Xn) is a SARS-CoV-2 peptide having one of the amino acid sequences depicted in FIG. 19. In some examples, the SARS-CoV-2 peptide comprises the amino acid sequence GTHWFVTQR (SEQ ID NO: 332) and has a length of 9 amino acids. In some examples, the SARS-CoV-2 peptide comprises the amino acid sequence QYIKWPWYI (SEQ ID NO: 400) and has a length of 9 amino acids.

[0351] In some examples, the TMMP comprises a) a first polypeptide comprising the amino acid sequence depicted in FIG. 20A, except that the first polypeptide comprises an anti-Her2 scFv instead of an anti-CD19 scFv, and b) a second polypeptide comprising the amino acid sequence depicted in FIG. 21A. In some examples, (Xn) is a SARS-CoV-2 peptide having one of the amino acid sequences depicted in FIG. 19. In some examples, the SARS-CoV-2 peptide comprises the amino acid sequence GTHWFVTQR (SEQ ID NO: 332) and has a length of 9 amino acids. In some examples, the SARS-CoV-2 peptide comprises the amino acid sequence QYIKWPWYI (SEQ ID NO: 400) and has a length of 9 amino acids.

[0352] In some examples, the TMMP comprises a) a first polypeptide comprising the amino acid sequence depicted in FIG. 20A, except that the first polypeptide comprises an anti-CD22 scFv instead of an anti-CD19 scFv, and b) a second polypeptide comprising the amino acid sequence depicted in FIG. 21A. In some examples, (Xn) is a SARS-CoV-2 peptide having one of the amino acid sequences depicted in FIG. 19. In some examples, the SARS-CoV-2 peptide comprises the amino acid sequence GTHWFVTQR (SEQ ID NO: 332) and has a length of 9 amino acids. In some examples, the SARS-CoV-2 peptide comprises the amino acid sequence QYIKWPWYI (SEQ ID NO: 400) and has a length of 9 amino acids.

[0353] In some examples, the TMMP comprises a) a first polypeptide comprising the amino acid sequence depicted in FIG. 20A, except that the first polypeptide comprises an anti-TROP-2 scFv instead of an anti-CD19 scFv, and b) a second polypeptide comprising the amino acid sequence depicted in FIG. 21A. In some examples, (Xn) is a SARS-CoV-2 peptide having one of the amino acid sequences depicted in FIG. 19. In some examples, the SARS-CoV-2 peptide comprises the amino acid sequence GTHWFVTQR (SEQ ID NO: 332) and has a length of 9 amino acids. In some examples, the SARS-CoV-2 peptide comprises the amino acid sequence QYIKWPWYI (SEQ ID NO: 400) and has a length of 9 amino acids.

[0354] In some examples, the TMMP comprises a) a first polypeptide comprising the amino acid sequence depicted in FIG. 20A, except that the first polypeptide comprises an anti-mesothelin scFv instead of an anti-CD19 scFv, and b) a second polypeptide comprising the amino acid sequence depicted in FIG. 21A. In some examples, (Xn) is a SARS-CoV-2 peptide having one of the amino acid sequences depicted in FIG. 19. In some examples, the SARS-CoV-2 peptide comprises the amino acid sequence GTHWFVTQR (SEQ ID NO: 332) and has a length of 9 amino acids. In some examples, the SARS-CoV-2 peptide comprises the amino acid sequence QYIKWPWYI (SEQ ID NO: 400) and has a length of 9 amino acids.

[0355] In some examples, the TMMP comprises a) a first polypeptide comprising the amino acid sequence depicted in FIG. 20A, except that the first polypeptide comprises an anti-BCMA scFv instead of an anti-CD19 scFv, and b) a second polypeptide comprising the amino acid sequence depicted in FIG. 21A. In some examples, (Xn) is a SARS-CoV-2 peptide having one of the amino acid sequences depicted in FIG. 19. In some examples, the SARS-CoV-2 peptide comprises the amino acid sequence GTHWFVTQR (SEQ ID NO: 332) and has a length of 9 amino acids. In some examples, the SARS-CoV-2 peptide comprises the amino acid sequence QYIKWPWYI (SEQ ID NO: 400) and has a length of 9 amino acids.

[0356] As one non-limiting example, a TMMP comprises a) a first polypeptide comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Figure 20A, and b) a second polypeptide comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Figure 21 B. In some examples, a TMMP comprises a) a first polypeptide comprising the amino acid sequence depicted in Figure 20A, and b) a second polypeptide comprising the amino acid sequence depicted in Figure 21B.

[0357] As one non-limiting example, a TMMP comprises a) a first polypeptide comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Figure 20A, and b) a second polypeptide comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Figure 21 C. In some examples, a TMMP comprises a) a first polypeptide comprising the amino acid sequence depicted in Figure 20A, and b) a second polypeptide comprising the amino acid sequence depicted in Figure 21C.

[0358] As one non-limiting example, a TMMP comprises a) a first polypeptide comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Figure 20A, and b) a second polypeptide comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Figure 21 D. In some examples, a TMMP comprises a) a first polypeptide comprising the amino acid sequence depicted in Figure 20A, and b) a second polypeptide comprising the amino acid sequence depicted in Figure 21D.

[0359] As one non-limiting example, a TMMP comprises a) a first polypeptide comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Figure 20E, and b) a second polypeptide comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Figure 21 B. In some examples, a TMMP comprises a) a first polypeptide comprising the amino acid sequence depicted in Figure 20E, and b) a second polypeptide comprising the amino acid sequence depicted in Figure 21B.

[0360] As one non-limiting example, a TMMP comprises a) a first polypeptide comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Figure 20D, and b) a second polypeptide comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Figure 21 C. In some examples, a TMMP comprises a) a first polypeptide comprising the amino acid sequence depicted in Figure 20C, and b) a second polypeptide comprising the amino acid sequence depicted in Figure 21C.

[0361] As one non-limiting example, a TMMP comprises a) a first polypeptide comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Figure 20E, and b) a second polypeptide comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in Figure 21 D. In some examples, a TMMP comprises a) a first polypeptide comprising the amino acid sequence depicted in Figure 20E, and b) a second polypeptide comprising the amino acid sequence depicted in Figure 21D.

[0362] As described above, in some examples, the TMMP comprises an HLA-E heavy chain polypeptide. In some examples, the TMMP comprises a SARS-CoV-2 peptide having the amino acid sequence YLQPRTLFL (SEQ ID NO: 887). In some examples, the TMMP comprises a SARS-CoV-2 peptide having the amino acid sequence YLQPRTLIL (SEQ ID NO: 1201). In some examples, the TMMP comprises a SARS-CoV-2 peptide having the amino acid sequence YLQPRTLVL (SEQ ID NO: 1202). As one non-limiting example, the TMMP comprises a) a first polypeptide comprising the amino acid sequence shown in FIG. 20G, and b) a second polypeptide comprising the amino acid sequence shown in FIG. 21E. As another non-limiting example, the TMMP comprises a) a first polypeptide comprising the amino acid sequence shown in FIG. 20I, and b) a second polypeptide comprising the amino acid sequence shown in FIG. 21E. In some examples, the TMMP has a TTP other than an anti-CD19 scFv. For example, in some instances, a TMMP comprises a) a first polypeptide comprising the amino acid sequence shown in Figure 20G, except that the first polypeptide comprises an anti-mesothelin scFv, an anti-CD22 scFv, or other anti-cancer related scFv instead of an anti-CD19 scFv, and b) a second polypeptide comprising the amino acid sequence shown in Figure 21 E. As another example, in some instances, a TMMP comprises a) a first polypeptide comprising the amino acid sequence shown in Figure 20I, except that the first polypeptide comprises an anti-mesothelin scFv instead of an anti-CD19 scFv, and b) a second polypeptide comprising the amino acid sequence shown in Figure 21 E.

[0363] As one non-limiting example, TMMP may be a) i) a SARS-CoV2 peptide having the amino acid sequence YLQPRTFLL (SEQ ID NO: 218); ii) a linker comprising the amino acid sequence GCGGS(GGGS)2 (SEQ ID NO: 1203), and iii) the β2M amino acid sequence: a β2M polypeptide comprising an amino acid sequence having at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF2024541334000144.tif11162, wherein amino acid 12 is Cys; and a first polypeptide comprising: b) i) the anti-CD19 scFv amino acid sequence: an anti-CD19 scFv comprising an amino acid sequence having at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF2024541334000145.tif31169; ii) a linker, such as a linker having the sequence GGGGS (SEQ ID NO: 873); iii) The following HLA-A * 0201 amino acid sequence: HLA-A comprising an amino acid sequence having at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF2024541334000146.tif31164. * 0201 polypeptide, in which amino acid 84 is Cys and amino acid 236 is Cys; iv) a linker, e.g., a linker having the sequence AAAGG (SEQ ID NO: 874); v) the IgG Fc amino acid sequence: an IgG Fc polypeptide comprising an amino acid sequence having at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF2024541334000147.tif24163, wherein amino acid 14 is Ala and amino acid 15 is Ala; vi) a linker, e.g., a linker having the amino acid sequence (GGGGS)3 (SEQ ID NO:875); vii) a first MOD having the following variant IL-2 amino acid sequence: a first MOD comprising an amino acid sequence having at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF2024541334000148.tif17163, e.g., wherein amino acid 16 is Ala and amino acid 42 is Ala; viii) a linker, e.g., a linker having the amino acid sequence (GGGGS)4 (SEQ ID NO:876), and ix) a second MOD having the following variant IL-2 amino acid sequence: a second MOD comprising an amino acid sequence having at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF2024541334000149.tif17163, e.g., wherein amino acid 16 is Ala and amino acid 42 is Ala; and a second polypeptide comprising Includes. As an example, in some instances, the TMMP comprises a) a first polypeptide comprising an amino acid sequence having at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity with the amino acid sequence of the "4700" construct depicted in Figure 32B, and b) a second polypeptide comprising an amino acid sequence having at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity with the amino acid sequence of the "4518" construct depicted in Figure 32A. In some instances, the TMMP comprises a) a first polypeptide comprising the amino acid sequence of the "4700" construct depicted in Figure 32B, and b) a second polypeptide comprising the amino acid sequence of the "4518" construct depicted in Figure 32A.

[0364] In some examples, the TMMP comprises a) a first polypeptide comprising an amino acid sequence having at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity with the amino acid sequence of the "4700" construct depicted in Figure 32B, and b) a second polypeptide comprising an amino acid sequence having at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity with the amino acid sequence of the "4728" construct depicted in Figure 38A. In some examples, the TMMP comprises a) a first polypeptide comprising the amino acid sequence of the "4700" construct depicted in Figure 32B, and b) a second polypeptide comprising the amino acid sequence of the "4728" construct depicted in Figure 38A.

[0365] In some examples, the TMMP comprises a) a first polypeptide comprising an amino acid sequence having at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity with the amino acid sequence of the "4700" construct depicted in Figure 32B, and b) a second polypeptide comprising an amino acid sequence having at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity with the amino acid sequence of the "4730" construct depicted in Figure 38B. In some examples, the TMMP comprises a) a first polypeptide comprising the amino acid sequence of the "4700" construct depicted in Figure 32B, and b) a second polypeptide comprising the amino acid sequence of the "4730" construct depicted in Figure 38B.

[0366] In some examples, the TMMP comprises a) a first polypeptide comprising an amino acid sequence having at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity with the amino acid sequence of the "4700" construct depicted in Figure 32B, and b) a second polypeptide comprising an amino acid sequence having at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity with the amino acid sequence of the "4701" construct depicted in Figure 38C. In some examples, the TMMP comprises a) a first polypeptide comprising the amino acid sequence of the "4700" construct depicted in Figure 32B, and b) a second polypeptide comprising the amino acid sequence of the "4701" construct depicted in Figure 38C.

[0367] In some examples, the TMMP comprises a) a first polypeptide comprising an amino acid sequence having at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity with the amino acid sequence of the "4700" construct depicted in Figure 32B, and b) a second polypeptide comprising an amino acid sequence having at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity with the amino acid sequence of the "4729" construct depicted in Figure 38D. In some examples, the TMMP comprises a) a first polypeptide comprising the amino acid sequence of the "4700" construct depicted in Figure 32B, and b) a second polypeptide comprising the amino acid sequence of the "4729" construct depicted in Figure 38D.

[0368] In some examples, the TMMP comprises a) a first polypeptide comprising an amino acid sequence having at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity with the amino acid sequence of the "4700" construct depicted in Figure 32B, and b) a second polypeptide comprising an amino acid sequence having at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity with the amino acid sequence of the "4731" construct depicted in Figure 38E. In some examples, the TMMP comprises a) a first polypeptide comprising the amino acid sequence of the "4700" construct depicted in Figure 32B, and b) a second polypeptide comprising the amino acid sequence of the "4731" construct depicted in Figure 38E.

[0369] In some examples, the TMMP comprises a) a first polypeptide comprising an amino acid sequence having at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity with the amino acid sequence of the "4700" construct depicted in Figure 32B, and b) a second polypeptide comprising an amino acid sequence having at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity with the amino acid sequence of the "4776" construct depicted in Figure 38F. In some examples, the TMMP comprises a) a first polypeptide comprising the amino acid sequence of the "4700" construct depicted in Figure 32B, and b) a second polypeptide comprising the amino acid sequence of the "4776" construct depicted in Figure 38F.

[0370] In some examples, the TMMP comprises a) a first polypeptide comprising an amino acid sequence having at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity with the amino acid sequence of the "4700" construct depicted in Figure 32B, and b) a second polypeptide comprising an amino acid sequence having at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity with the amino acid sequence of the "4777" construct depicted in Figure 38G. In some examples, the TMMP comprises a) a first polypeptide comprising the amino acid sequence of the "4700" construct depicted in Figure 32B, and b) a second polypeptide comprising the amino acid sequence of the "4777" construct depicted in Figure 38G.

[0371] In some examples, the TMMP comprises a) a first polypeptide comprising an amino acid sequence having at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity with the amino acid sequence of the "4700" construct depicted in Figure 32B, and b) a second polypeptide comprising an amino acid sequence having at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity with the amino acid sequence of the "4778" construct depicted in Figure 38H. In some examples, the TMMP comprises a) a first polypeptide comprising an amino acid sequence of the "4700" construct depicted in Figure 32B, and b) a second polypeptide comprising an amino acid sequence of the "4778" construct depicted in Figure 38H.

[0372] In some examples, the TMMP comprises a) a first polypeptide comprising an amino acid sequence having at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity with the amino acid sequence of the "4700" construct depicted in Figure 32B, and b) a second polypeptide comprising an amino acid sequence having at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity with the amino acid sequence of the "4519" construct depicted in Figure 42A. In some examples, the TMMP comprises a) a first polypeptide comprising the amino acid sequence of the "4700" construct depicted in Figure 32B, and b) a second polypeptide comprising the amino acid sequence of the "4519" construct depicted in Figure 42A.

[0373] In some examples, the TMMP comprises a) a first polypeptide comprising an amino acid sequence having at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity with the amino acid sequence of the "4700" construct depicted in Figure 32B, and b) a second polypeptide comprising an amino acid sequence having at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity with the amino acid sequence of the "4520" construct depicted in Figure 42B. In some examples, the TMMP comprises a) a first polypeptide comprising the amino acid sequence of the "4700" construct depicted in Figure 32B, and b) a second polypeptide comprising the amino acid sequence of the "4520" construct depicted in Figure 42B.

[0374] In some examples, the TMMP comprises a) a first polypeptide comprising an amino acid sequence having at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity with the amino acid sequence of the "4700" construct depicted in Figure 32B, and b) a second polypeptide comprising an amino acid sequence having at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity with the amino acid sequence of the "4337" construct depicted in Figure 42C. In some examples, the TMMP comprises a) a first polypeptide comprising the amino acid sequence of the "4700" construct depicted in Figure 32B, and b) a second polypeptide comprising the amino acid sequence of the "4337" construct depicted in Figure 42C.

[0375] In some instances, TMMP may be a) i) a peptide epitope having a length of 4 amino acids to 25 amino acids (e.g., 6 to 12 amino acids, 8 to 12 amino acids, etc.); ii) a linker having the amino acid sequence GCGGS(GGGGS)2 (SEQ ID NO: 882), and iii) a β2M polypeptide comprising an amino acid sequence having at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the β2M amino acid sequence shown in FIG. 32G. and a first polypeptide comprising: b) the structure: A second polypeptide of TIFF2024541334000150.tif44163, in which, in the sequence, [X] is an scFv specific for a cancer-associated antigen, or a nanobody specific for a cancer-associated antigen, [Y] is an HLA heavy chain polypeptide, and [Z] is an Ig Fc polypeptide; and X 1 is an amino acid other than His, and X 2 is an amino acid other than Phe; and Includes. Such TMMPs are shown in Figures 40A and 40B. In some examples, the Ig Fc polypeptide is a variant of an Fc polypeptide, such as human IgG1 Fc, which variant has a substantially reduced ability to affect CDC or ADCC.

[0376] In some instances, TMMP may be a) i) a peptide epitope having a length of 4 amino acids to 25 amino acids (e.g., 6 to 12 amino acids, 8 to 12 amino acids, etc.); ii) a linker having the amino acid sequence GCGGS(GGGGS)2 (SEQ ID NO: 882), and iii) a β2M polypeptide comprising an amino acid sequence having at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the β2M amino acid sequence set forth in FIG. 32G. and a first polypeptide comprising: b) i) an scFv polypeptide or nanobody specific for a cancer-associated antigen; ii) a linker having the sequence GGGGS (SEQ ID NO: 873); iii) HLA heavy chain polypeptides; iv) a linker having the sequence AAAGG (SEQ ID NO: 874); v) an Ig Fc polypeptide comprising an amino acid sequence having at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the Ig Fc amino acid sequence set forth in Figure 38K; vi) a linker having the sequence (GGGGS)3 (SEQ ID NO:875); vii) a variant IL-2 polypeptide comprising an amino acid sequence having at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the variant IL-2 amino acid sequence set forth in Figure 38L, wherein amino acid 16 is other than His (e.g., amino acid 16 is Ala) and amino acid 42 is other than Phe (e.g., amino acid 42 is Ala); viii) a linker having the sequence (GGGGS)4 (SEQ ID NO:876), and ix) a variant IL-2 polypeptide comprising an amino acid sequence having at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the variant IL-2 amino acid sequence set forth in Figure 38L, wherein amino acid 16 is other than His (e.g., amino acid 16 is Ala) and amino acid 42 is other than Phe (e.g., amino acid 42 is Ala); and a second polypeptide comprising Includes. Such TMMPs are shown in Figures 40A and 40B. In some examples, the Ig Fc polypeptide is a variant of an Fc polypeptide, such as human IgG1 Fc, which variant has a substantially reduced ability to affect CDC or ADCC, for example, in some examples, the variant Ig Fc polypeptide includes Ala (instead of Leu) at position 14 and Ala (instead of Leu) at position 15, based on the amino acid numbering shown in Figure 38K. In some examples, the peptide epitope is a SARS-CoV-2 peptide. In some examples, the HLA heavy chain polypeptide is an HLA-A heavy chain polypeptide. * 0201 polypeptide, e.g., an HLA polypeptide comprising an amino acid sequence having at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence shown in FIG. 3A. In some examples, the HLA heavy chain polypeptide is an HLA-A polypeptide comprising an amino acid sequence having at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence shown in FIG. 3A. * In some examples, the HLA heavy chain polypeptide is an HLA-A 0201 polypeptide (e.g., as shown in FIG. 3C). * 1101 polypeptide, e.g., an HLA polypeptide comprising an amino acid sequence having at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence shown in FIG. 4A. In some examples, the HLA heavy chain polypeptide is an HLA-A polypeptide comprising an amino acid sequence having at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence shown in FIG. 4A. * In some examples, the HLA heavy chain polypeptide is an HLA-A 1101 polypeptide (e.g., as shown in FIG. 4C). *2402 polypeptide, e.g., an HLA polypeptide comprising an amino acid sequence having at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence shown in Figure 5A. In some examples, the HLA heavy chain polypeptide is an HLA-A polypeptide comprising an amino acid sequence having at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence shown in Figure 5A. * In some examples, the HLA heavy chain polypeptide is an HLA-A 2402 polypeptide (e.g., as shown in FIG. 5C). * 3303 polypeptide, e.g., an HLA polypeptide comprising an amino acid sequence having at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence shown in Figure 6A. In some examples, the HLA heavy chain polypeptide is an HLA-A polypeptide comprising an amino acid sequence having at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence shown in Figure 6A. *3303 polypeptide (e.g., as shown in FIG. 6C). In some examples, the HLA heavy chain polypeptide is an HLA-E polypeptide. In some examples, the HLA-E polypeptide comprises an amino acid sequence having at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to an HLA-E amino acid sequence set forth in any one of FIGS. 16A-16D. In some examples, the HLA-E polypeptide comprises a Y84C and an A236C substitution (e.g., as shown in FIG. 16B and FIG. 16D). In some examples, the HLA heavy chain polypeptide is an HLA-E polypeptide and the peptide epitope is a SARS-CoV2 peptide having the amino acid sequence VMPLSAPTL (SEQ ID NO: 914). In some examples, the HLA heavy chain polypeptide is an HLA-E polypeptide and the peptide epitope is a SARS-CoV2 peptide having the amino acid sequence YLQPRTFLL (SEQ ID NO: 218). In some examples, the scFv polypeptide or nanobody specifically binds to CD19. In some examples, the scFv polypeptide or nanobody specifically binds to mesothelin. In some examples, the scFv polypeptide or nanobody specifically binds to TROP-2. In some examples, the scFv polypeptide or nanobody specifically binds to CD28. In some examples, the scFv polypeptide or nanobody specifically binds to Her2. In some examples, the scFv polypeptide or nanobody specifically binds to PSMA. In some examples, the scFv polypeptide or nanobody specifically binds to CD22. In some examples, the scFv polypeptide or nanobody specifically binds to BCMA. In some examples, the scFv polypeptide or nanobody specifically binds to MUC1. In some examples, the scFv polypeptide or nanobody specifically binds to MUC16. In some examples, the scFv polypeptide or nanobody specifically binds to claudin-18.2. In some examples, the TMMP comprises an scFv. As one non-limiting example, in some embodiments, the TMMP comprises a) a first polypeptide comprising the amino acid sequence shown in FIG. 41B, and b) a second polypeptide comprising the amino acid sequence shown in FIG. 41A.As another non-limiting example, in some instances, the TMMP comprises a) a first polypeptide comprising the amino acid sequence shown in FIG. 41C, and b) a second polypeptide comprising the amino acid sequence shown in FIG. 41A.

[0377] Although embodiments involving SARS-CoV-2 are exemplified herein, as noted above, peptide epitopes from other pathogens may be employed in TMMPs that also include one or more MODs. For example, other suitable epitopes include, but are not limited to, epitopes present in infectious disease pathogens, such as virally encoded polypeptides or epitopes presented by bacteria. Examples of viral infectious agents include, for example, adenoviruses, adeno-associated viruses, alphaviruses (togaviruses), eastern equine encephalitis virus, eastern equine encephalomyelitis virus, Venezuelan equine encephalomyelitis vaccine strain TC-83, western equine encephalomyelitis virus, arenaviruses, lymphocytic choroiditis virus (non-neurotropic strains), Tacaribe virus complex, Bunyaviruses, Bunyamwera virus, Rift Valley fever virus vaccine strain MP-12, chikungunya virus, calciviruses, coronaviruses, cowpox virus, flaviviruses (togaviruses) group B arboviruses, dengue virus serotypes 1, 2, 3, and 4, yellow fever virus vaccine strain 17D, hepatitis A, B, C, D, and E viruses, cytomegaloviruses, and the like. Viruses, Epstein-Barr virus, Eastern equine encephalitis virus, Herpes simplex types 1 and 2, Herpes zoster, Human herpesviruses 6 and 7, Hepatitis C virus (HVC), Hepatitis B virus (HBV), Influenza viruses A, B, and C, Papovaviruses, Newcastle disease virus, Measles virus, Mumps virus, Parainfluenza viruses 1, 2, 3, and 4, Polyomaviruses (JC virus, BK virus), Respiratory syncytial virus, Human parvovirus (B19), Coxsackieviruses A and B, Echoviruses, Polioviruses, Rhinoviruses, Variola minor virus, Variola major virus, Whitepox reovirus Reoviruses), Coltiviruses, human rotaviruses, and Orbiviruses (Colorado tick fever viruses), Rabies viruses, Vesicular stomatitis viruses, Rubiviruses (rubella), Semliki Forest viruses, Saint Louis encephalitis viruses, Venezuelan equine encephalitis viruses, Venezuelan equine encephalomyelitis viruses, Arenaviruses (also known as South American hemorrhagic fever viruses),Tick-borne encephalitis virus complex, including Flexal, Lymphocytic choriomeningitis virus (LCM) (neurotropic strains), Hantavirus, including Hantaan virus, Rift Valley fever virus, Japanese encephalitis virus, Yellow fever virus, Monkeypox virus, Human immunodeficiency virus (HIV) types 1 and 2, Human T-cell lymphotropic virus (HTLV) types 1 and 2, Simian immunodeficiency virus (SIV), Vesicular stomatitis virus, Guanarito virus, Lassa fever virus, Junin virus, Machupo virus, Sabia, Crimean-Congo hemorrhagic fever virus, Ebola virus, Marburg virus, Central European tick-borne encephalitis, Far Eastern tick-borne encephalitis, Hanzalova, Hypr, Kumlinge, Kyasanur Forest disease, Omsk hemorrhagic fever, and Russian spring-summer encephalitis virus (flavi), simian herpesvirus (herpes B or monkey B virus), long-tailed monkey herpesvirus type 1 (herpes B virus), equine morbillivirus (hendra and hendra-like viruses), Nipah virus, variola major virus (smallpox virus), variola minor virus (smallpox), African swine fever virus, African horse sickness virus, Akabane virus, avian influenza virus (highly pathogenic), bluetongue virus, camelpox virus, classical swine fever virus, cowrie aluminium (heartwater disease), foot and mouth disease virus, goat pox virus, Japanese encephalitis virus, lumpy skin disease virus, malignant catarrhal fever virus, Menangle virus, Newcastle disease virus (VVND), vesicular stomatitis virus (adventitious), and Zika virus. Antigens encoded by such viruses are known in the art, and peptide epitopes suitable for use in the TMMPs of the present disclosure may include peptides from any known viral antigen. In some examples, the viral epitope is an epitope present in a viral antigen encoded by a virus other than SARS-CoV-2 that infects a large portion of the human population, such as, for example, cytomegalovirus (CMV), Epstein-Barr virus (EBV), human papilloma virus, adenovirus, and the like. For example,See published International Publication No. WO 2020 / 243315 (Cue Biopharma, Inc.), paragraphs

[0321] -

[0338] . Thus, the present disclosure expressly contemplates TMMPs that include epitopes other than SARS-CoV-2 epitopes.

[0378] TMMP without immunomodulatory polypeptides The present disclosure provides a TMMP comprising a) a first polypeptide and b) a second polypeptide, the TMMP comprising a peptide epitope, a first MHC polypeptide, a second MHC polypeptide, an immunoglobulin (Ig) Fc polypeptide or a non-Ig scaffold, and a TTP, the TMMP comprising no immunomodulatory polypeptide (e.g., no MOD listed in Table 2, e.g., no IL-2 polypeptide, no 4-1BBL polypeptide, etc.). Such a TMMP may also be referred to herein as a "MOD-less TMMP." The components of a MOD-less TMMP (e.g., peptide epitope, first MHC polypeptide, second MHC polypeptide, Ig Fc polypeptide or a non-Ig scaffold, and the TTP) may be arranged in a variety of configurations. The components (other than the MOD) are as described above.

[0379] In some examples, the present disclosure provides: a) i) a SARS-CoV-2 peptide having a length of about 4 amino acids to about 25 amino acids, for example 8 to 12 amino acids; β2M polypeptide; A first polypeptide comprising: b) a second polypeptide, an MHC class I heavy chain polypeptide; c) an Ig Fc polypeptide or a non-Ig scaffold, wherein the first or second polypeptide comprises an Ig Fc polypeptide or a non-Ig scaffold; and d) TTP, wherein the first and / or second polypeptide comprises TTP, and the protein does not comprise an immunomodulatory polypeptide; In one embodiment, at least one heterodimer comprising: One or more of the individual components of the first and / or second polypeptides may be linked together by one or more linkers. Suitable SARS-CoV-2 peptides, MHC class I heavy chain polypeptides, and Ig Fc polypeptides are described above. Such proteins may also be referred to as "MOD-less TMMPs."

[0380] In some examples, the MODless TMMP is dimerized. Thus, the present disclosure provides a protein that is a dimerized MODless TMMP, comprising two MODless TMMPs covalently linked to each other. The covalent bond of the dimer may be one or more disulfide bonds between an Ig Fc polypeptide in a first MODless TMMP and an Ig Fc polypeptide in a second MODless TMMP.

[0381] In some cases, MOD-less TMMP a) (i) a peptide epitope, wherein the peptide epitope is a SARS-CoV-2 peptide, and (ii) a first MHC polypeptide, wherein the first MHC polypeptide is a β2M polypeptide; and a first polypeptide comprising: b) (i) TTPs; (ii) a second MHC polypeptide, wherein the second MHC polypeptide is an MHC class I heavy chain polypeptide, and (iii) an Ig Fc polypeptide, and a second polypeptide comprising Includes. The peptide linker is (i) between a peptide epitope and a β2M polypeptide; (ii) between an MHC class I heavy chain polypeptide and TTP; (iii) between an MHC class I heavy chain polypeptide and an Ig Fc polypeptide may be placed in one or more of the following: In some examples, the first polypeptide comprises, in order from N-terminus to C-terminus: (i) a SARS-CoV-2 peptide, and (ii) β2M polypeptide Includes. In some examples, the first polypeptide comprises, in order from N-terminus to C-terminus: (i) a SARS-CoV-2 peptide, (ii) a peptide linker, the peptide linker optionally comprising a Cys residue, and (iii) β2M polypeptide, Includes. In some examples, the second polypeptide comprises, in order from N-terminus to C-terminus: (i) TTPs; (ii) an optional linker; (iii) an MHC class I heavy chain polypeptide, (iv) an optional linker, and (v) an Ig Fc polypeptide, Includes. In some examples, the second polypeptide comprises, in order from N-terminus to C-terminus: (i) an MHC class I heavy chain polypeptide, (ii) an optional linker; (iii) an Ig Fc polypeptide, (iv) an optional linker, and (v) TTPs, Includes. In some examples, the second polypeptide comprises, in order from N-terminus to C-terminus: (i) an MHC class I heavy chain polypeptide, (ii) an optional linker; (iii) TTPs, (iv) an optional linker, and (v) an Ig Fc polypeptide, Includes. In some examples, the β2M polypeptide comprises a Cys residue, for example, a Cys residue at position 12 according to the amino acid numbering of the β2M amino acid sequence shown in Figure 1B. In some examples, the MHC class I heavy chain polypeptide comprises a Cys residue at position 84 and / or a Cys residue at position 236.

[0382] The following are non-limiting examples of MOD-less TMMPs:

[0383] In some cases, MOD-less TMMPs a) (i) a SARS-CoV-2 peptide, and (ii) the β2M amino acid sequence: a β2M 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 to TIFF2024541334000151.tif11162, wherein amino acid 12 is Cys; and a first polypeptide comprising: b) (i) an anti-CD19 scFv 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 to the anti-CD19 scFv polypeptide amino acid sequence depicted in Figure 32C; (ii) HLA-A as shown in FIG. * an MHC class I heavy chain 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 to the 0201(Y84C;A236C) amino acid sequence, wherein amino acid 84 is Cys and amino acid 236 is Cys; and (iii) an Ig Fc 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 to the Ig Fc(L14A, L15A) amino acid sequence depicted in Figure 32E, wherein amino acid 14 is Ala and amino acid 15 is Ala; and a second polypeptide comprising Includes. In some examples, the first polypeptide comprises a Cys-containing peptide linker between the SARS-CoV-2 peptide and the β2M polypeptide. As an example, in some examples, the first polypeptide comprises a peptide linker between the SARS-CoV-2 peptide and the β2M polypeptide of the following amino acid sequence: GCGGSGGGSGGGGGS (SEQ ID NO: 882). In some examples, the SARS-CoV-2 peptide comprises the amino acid sequence YLQPRTFLL (SEQ ID NO: 218). In some examples, the first polypeptide comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence of the polypeptide designated "4700" and depicted in FIG. 32B. In some examples, the first polypeptide comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence of the polypeptide designated "4656" and depicted in FIG. 33.

[0384] In some cases, MOD-less TMMP a) (i) a SARS-CoV-2 peptide, and (ii) the β2M amino acid sequence: a β2M 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 to TIFF2024541334000152.tif11162, wherein amino acid 12 is Cys; and a first polypeptide comprising: b) (i) an anti-CD19 scFv 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 to the anti-CD19 scFv polypeptide amino acid sequence depicted in Figure 32C; (ii) an MHC class I heavy chain 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 to the HLA-E(Y84C;A236C) amino acid sequence depicted in Figure 16B or Figure 16D, wherein amino acid 84 is Cys and amino acid 236 is Cys; (iii) an Ig Fc 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 to the Ig Fc(L14A, L15A) amino acid sequence depicted in Figure 32E, wherein amino acid 14 is Ala and amino acid 15 is Ala; and a second polypeptide comprising Includes. In some examples, the first polypeptide comprises a Cys-containing peptide linker between the SARS-CoV-2 peptide and the β2M polypeptide. As an example, in some examples, the first polypeptide comprises a peptide linker between the SARS-CoV-2 peptide and the β2M polypeptide of the following amino acid sequence: GCGGSGGGSGGGGGS (SEQ ID NO: 882). In some examples, the SARS-CoV-2 peptide comprises the amino acid sequence VMPLSAPTL (SEQ ID NO: 914).

[0385] Although MOD-less embodiments involving SARS-CoV-2 are exemplified herein, one of skill in the art will readily appreciate that other peptide epitopes may be used in MOD-less TMMPs. For example, other suitable epitopes include, but are not limited to, epitopes present in infectious disease pathogens, such as epitopes presented by virally encoded polypeptides. Examples of viral infectious agents include, for example, adenoviruses, adeno-associated viruses, alphaviruses (togaviruses), eastern equine encephalitis virus, eastern equine encephalomyelitis virus, Venezuelan equine encephalomyelitis vaccine strain TC-83, western equine encephalomyelitis virus, arenaviruses, lymphocytic choroiditis virus (non-neurotropic strains), Tacaribe virus complex, Bunyaviruses, Bunyamwera virus, Rift Valley fever virus vaccine strain MP-12, chikungunya virus, calciviruses, coronaviruses, cowpox virus, flaviviruses (togaviruses) group B arboviruses, dengue virus serotypes 1, 2, 3, and 4, yellow fever virus vaccine strain 17D, hepatitis A, B, C, D, and E viruses, cytomegaloviruses, and the like. Viruses, Epstein-Barr virus, Eastern equine encephalitis virus, Herpes simplex types 1 and 2, Herpes zoster, Human herpesviruses 6 and 7, Hepatitis C virus (HVC), Hepatitis B virus (HBV), Influenza viruses A, B, and C, Papovaviruses, Newcastle disease virus, Measles virus, Mumps virus, Parainfluenza viruses 1, 2, 3, and 4, Polyomaviruses (JC virus, BK virus), Respiratory syncytial virus, Human parvovirus (B19), Coxsackieviruses A and B, Echoviruses, Polioviruses, Rhinoviruses, Variola minor virus, Variola major virus, Whitepox reovirus Reoviruses), Coltiviruses, human rotaviruses, and Orbiviruses (Colorado tick fever viruses), Rabies viruses, Vesicular stomatitis viruses, Rubiviruses (rubella), Semliki Forest viruses, Saint Louis encephalitis viruses, Venezuelan equine encephalitis viruses, Venezuelan equine encephalomyelitis viruses, Arenaviruses (also known as South American hemorrhagic fever viruses),Tick-borne encephalitis virus complex, including Flexal, Lymphocytic choriomeningitis virus (LCM) (neurotropic strains), Hantavirus, including Hantaan virus, Rift Valley fever virus, Japanese encephalitis virus, Yellow fever virus, Monkeypox virus, Human immunodeficiency virus (HIV) types 1 and 2, Human T-cell lymphotropic virus (HTLV) types 1 and 2, Simian immunodeficiency virus (SIV), Vesicular stomatitis virus, Guanarito virus, Lassa fever virus, Junin virus, Machupo virus, Sabia, Crimean-Congo hemorrhagic fever virus, Ebola virus, Marburg virus, Central European tick-borne encephalitis, Far Eastern tick-borne encephalitis, Hanzalova, Hypr, Kumlinge, Kyasanur Forest disease, Omsk hemorrhagic fever, and Russian spring-summer encephalitis virus (flavi), simian herpesvirus (herpes B or monkey B virus), long-tailed monkey herpesvirus type 1 (herpes B virus), equine morbillivirus (hendra and hendra-like viruses), Nipah virus, variola major virus (smallpox virus), variola minor virus (smallpox), African swine fever virus, African horse sickness virus, Akabane virus, avian influenza virus (highly pathogenic), bluetongue virus, camelpox virus, classical swine fever virus, cowrie aluminium (heartwater disease), foot and mouth disease virus, goat pox virus, Japanese encephalitis virus, lumpy skin disease virus, malignant catarrhal fever virus, Menangle virus, Newcastle disease virus (VVND), vesicular stomatitis virus (adventitious), and Zika virus. Antigens encoded by such viruses are known in the art, and peptide epitopes suitable for use in the TMMP of the present disclosure may include peptides from any known viral antigen. In some examples, the viral epitope is an epitope present in a viral antigen encoded by a virus that infects a large portion of the human population, including, for example, cytomegalovirus (CMV), Epstein-Barr virus (EBV), human papilloma virus, adenovirus, and the like. See, for example, paragraphs

[0321] -

[0338] of published International Publication No. WO 2020 / 243315 (Cue Biopharma, Inc.). Thus, the present disclosure providesExplicitly contemplates MOD-less TMMPs that contain epitopes other than SARS-CoV-2 epitopes.

[0386] For example, in some cases, MOD-less TMMPs may: a) (i) a viral peptide epitope, e.g., the peptide epitope is a CMV peptide, and (ii) a first MHC polypeptide, wherein the first MHC polypeptide is a β2M polypeptide; and a first polypeptide comprising: b) (i) TTPs; (ii) a second MHC polypeptide, wherein the second MHC polypeptide is an MHC class I heavy chain polypeptide, and (iii) an Ig Fc polypeptide, and a second polypeptide comprising Includes. The peptide linker is (i) between a peptide epitope and a β2M polypeptide; (ii) between an MHC class I heavy chain polypeptide and TTP; (iii) between an MHC class I heavy chain polypeptide and an Ig Fc polypeptide may be placed in one or more of the following: In some examples, the first polypeptide comprises, in order from N-terminus to C-terminus: (i) a CMV peptide, and (ii) β2M polypeptide Includes. In some examples, the first polypeptide comprises, in order from N-terminus to C-terminus: (i) a viral peptide epitope, e.g., a CMV peptide epitope; (ii) a peptide linker, the peptide linker comprising a Cys residue, and (iii) β2M polypeptide, Includes. In some examples, the second polypeptide comprises, in order from N-terminus to C-terminus: (i) TTPs; (ii) an optional linker; (iii) an MHC class I heavy chain polypeptide, (iv) an optional linker, and (v) an Ig Fc polypeptide, Includes. In some examples, the second polypeptide comprises, in order from N-terminus to C-terminus: (i) an MHC class I heavy chain polypeptide, (ii) an optional linker; (iii) an Ig Fc polypeptide, (iv) an optional linker, and (v) TTPs, Includes. In some examples, the second polypeptide comprises, in order from N-terminus to C-terminus: (i) an MHC class I heavy chain polypeptide, (ii) an optional linker; (iii) TTPs, (iv) an optional linker, and (v) an Ig Fc polypeptide, Includes. In some examples, the β2M polypeptide comprises a Cys residue, for example, a Cys residue at position 12 according to the amino acid numbering of the β2M amino acid sequence shown in Figure 1B. In some examples, the MHC class I heavy chain polypeptide comprises a Cys residue at position 84 and / or a Cys residue at position 236. In some examples, the peptide epitope is a CMV peptide having the amino acid sequence NLVPMVATV (SEQ ID NO:913).

[0387] In some cases, MOD-less TMMP a) (i) a CMV peptide, and (ii) the β2M amino acid sequence: a β2M 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 to TIFF2024541334000153.tif11162, wherein amino acid 12 is Cys; and a first polypeptide comprising: b) (i) an anti-CD19 scFv 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 to the anti-CD19 scFv polypeptide amino acid sequence depicted in Figure 32C; (ii) HLA-A as shown in FIG. * an MHC class I heavy chain 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 to the 0201(Y84C;A236C) amino acid sequence, wherein amino acid 84 is Cys and amino acid 236 is Cys; and (iii) an Ig Fc 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 to the Ig Fc(L14A, L15A) amino acid sequence depicted in Figure 32E, wherein amino acid 14 is Ala and amino acid 15 is Ala; and a second polypeptide comprising Includes. In some examples, the CMV peptide has the amino acid sequence NLVPMVATV (SEQ ID NO: 913). In some examples, the first polypeptide comprises a Cys-containing peptide linker between the CMV peptide and the β2M polypeptide. As an example, in some examples, the first polypeptide comprises a peptide linker between the CMV peptide and the β2M polypeptide with the following amino acid sequence: GCGGSGGGGSGGGGS (SEQ ID NO: 882). In some examples, the first polypeptide comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence of the polypeptide designated "1717" and depicted in FIG. 35. In some examples, the first polypeptide comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence of the polypeptide designated "4656" and depicted in FIG. 33.

[0388] nucleic acid The present disclosure provides a nucleic acid comprising a nucleotide sequence encoding TMMP.The present disclosure provides a nucleic acid comprising a nucleotide sequence encoding TMMP.

[0389] The present disclosure provides a nucleic acid comprising a nucleotide sequence encoding a TMMP. In some examples, each polypeptide chain of a TMMP is encoded within an individual nucleic acid. In some examples, all polypeptide chains of a TMMP are encoded within a single nucleic acid. In some examples, a first nucleic acid comprises a nucleotide sequence encoding a first polypeptide of a TMMP, and a second nucleic acid comprises a nucleotide sequence encoding a second polypeptide of a TMMP. In some examples, a single nucleic acid comprises a nucleotide sequence encoding a first polypeptide of a TMMP and a second polypeptide of a TMMP.

[0390] Individual nucleic acids encoding individual polypeptide chains of a multimeric polypeptide The present disclosure provides a nucleic acid comprising the nucleotide sequence encoding TMMP.As mentioned above, in some examples, each polypeptide chain of TMMP is encoded in each nucleic acid.In some examples, the nucleotide sequence encoding each polypeptide chain of TMMP is functionally linked to a transcriptional control element, such as a promoter, for example, a promoter that is functional in eukaryotic cells, and the promoter can be a constitutive promoter or an inducible promoter.

[0391] For example, the disclosure provides a first nucleic acid and a second nucleic acid, The first nucleic acid comprises a nucleotide sequence encoding a first polypeptide of TMMP, the first polypeptide comprising, in order from N-terminus to C-terminus: a) peptide epitopes, b) a first MHC polypeptide, and c) Immunomodulatory polypeptides (e.g., the low affinity variants described above) Including, The second nucleic acid comprises a nucleotide sequence encoding a second polypeptide of TMMP, the second polypeptide comprising, in order from N-terminus to C-terminus: a) a second MHC polypeptide, b) an Ig Fc polypeptide, and c) TTP Includes. Suitable peptide epitopes, MHC polypeptides, immunomodulatory polypeptides, Ig Fc polypeptides and TTPs are as described above.In some cases, the nucleotide sequence encoding the first polypeptide and the nucleotide sequence encoding the second polypeptide are functionally linked to a transcriptional control element.In some cases, the transcriptional control element is a promoter that is functional in eukaryotic cells.In some cases, the nucleic acid is present in each expression vector.

[0392] The disclosure provides a first nucleic acid and a second nucleic acid, The first nucleic acid comprises a nucleotide sequence encoding a first polypeptide of TMMP, the first polypeptide comprising, in order from N-terminus to C-terminus: a) peptide epitopes and b) a first MHC polypeptide Including, The second nucleic acid comprises a nucleotide sequence encoding a second polypeptide of TMMP, the second polypeptide comprising, in order from N-terminus to C-terminus: a) an immunomodulatory polypeptide (e.g., the low affinity variant forms described above); b) a second MHC polypeptide, c) an Ig Fc polypeptide, and d) TTP Includes. Suitable peptide epitopes, MHC polypeptides, immunomodulatory polypeptides and Ig Fc polypeptides are as described above.In some cases, the nucleotide sequence encoding the first polypeptide and the nucleotide sequence encoding the second polypeptide are operably linked to a transcriptio...

Claims

1. a) i) a peptide epitope having a length of about 4 amino acids to about 25 amino acids, and ii) a first major histocompatibility complex (MHC) polypeptide, wherein the first MHC polypeptide is a β2 microglobulin (β2M) polypeptide. and a first polypeptide comprising: b) a second polypeptide comprising a second MHC polypeptide, wherein the second MHC polypeptide is an MHC class I heavy chain polypeptide; and c) an immunoglobulin (Ig) Fc polypeptide or non-Ig scaffold, wherein the first or second polypeptide comprises an Ig Fc polypeptide or non-Ig scaffold; and d) a targeting polypeptide that specifically binds to an antigen selected from CD19, BCMA, CD20, CD22, CD38, CD79a, CD79b, or CD138, wherein the first and / or second polypeptide comprises a targeting polypeptide; and At least one heterodimer comprising 1. A T cell modulatory multimeric polypeptide (TMMP) comprising: TMMP does not contain immunomodulatory polypeptides, One or more of the individual components of the first and / or second polypeptides in each of the above TMMP combinations may be linked together by one or more independently selected peptide linkers. T-cell regulatory multimeric polypeptides (TMMPs).

2. a1) The first polypeptide comprises, in order from the N-terminus to the C-terminus: i) a peptide epitope; and ii) a β2M polypeptide, and b1) the second polypeptide comprises, in order from the N-terminus to the C-terminus: i) a targeting polypeptide; and ii) an MHC class I heavy chain polypeptide; and iv) an Ig Fc polypeptide; or a2) the first polypeptide comprises, in order from the N-terminus to the C-terminus: i) a peptide epitope; and ii) a β2M polypeptide, and b2) the second polypeptide comprises, in order from the N-terminus to the C-terminus: i) an MHC class I heavy chain polypeptide; and ii) an Ig Fc polypeptide; and iii) a targeting polypeptide; or a3) the first polypeptide comprises, in order from the N-terminus to the C-terminus: i) a peptide epitope; and ii) a β2M polypeptide, and b3) the second polypeptide comprises, in order from the N-terminus to the C-terminus: i) an MHC class I heavy chain polypeptide; and ii) a targeting polypeptide; and iii) an Ig Fc polypeptide; and ii) a β2M polypeptide; One or more of the individual components of the first and / or second polypeptides in each of the above TMMP combinations may be linked together by one or more independently selected peptide linkers. The TMMP according to claim 1.

3. The first polypeptide comprises, in order from the N-terminus to the C-terminus: i) a peptide epitope; and ii) a peptide linker; and iii) a β2M polypeptide, and The second polypeptide comprises, in order from the N-terminus to the C-terminus: i) a targeting polypeptide; and ii) a peptide linker; and iii) an MHC class I heavy chain polypeptide; and iv) a peptide linker; and iv) an Ig Fc polypeptide; the peptide linker between the targeting polypeptide and the MHC class I heavy chain polypeptide comprises a length of 10 amino acids or less; The TMMP described in claim 2.

4. The TMMP described in claim 3, wherein the second polypeptide comprises an Ig Fc polypeptide, the Ig Fc polypeptide is an IgG1 Fc polypeptide, and the Ig Fc comprises one or more amino acid substitutions that reduce or substantially eliminate antibody-dependent cell-mediated cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC).

5. The TMMP described in claim 4, wherein the targeting polypeptide is an antibody or an antibody fragment.

6. The TMMP described in claim 5, wherein the peptide epitope has a length of 8 to 14 amino acids, and the first polypeptide and the second polypeptide are covalently bonded to each other via one or more disulfide bonds.

7. The first and second polypeptides are covalently linked to each other via at least two disulfide bonds; a) the first disulfide bond is i) a Cys present in the linker between the peptide epitope and the β2M polypeptide; ii) a Cys residue at position 84 of the MHC class I heavy chain polypeptide; and Between and b) the second disulfide bond is i) a Cys residue at position 12 of the β2M polypeptide; ii) a Cys residue at position 236 of the MHC class I heavy chain polypeptide; and Between The TMMP described in claim 6.

8. The TMMP described in claim 3, wherein the peptide linker is 5 amino acids or less in length.

9. A TMMP described in any one of claims 1 to 8, wherein the peptide epitope is a peptide from a viral antigen.

10. The TMMP described in claim 9, wherein the peptide epitope is a peptide from a viral antigen encoded by a virus selected from cytomegalovirus (CMV), Epstein-Barr virus (EBV), human papillomavirus, and adenovirus.

11. A TMMP described in any one of claims 1 to 8, wherein the targeting polypeptide selectively binds to CD19.

12. a) the first polypeptide comprises, in order from the N-terminus to the C-terminus: i) a SARS-CoV-2 peptide, wherein the SARS-CoV-2 peptide comprises the amino acid sequence YLQPRTFLL (SEQ ID NO:218); ii) a peptide linker; and iii) a β2M polypeptide, wherein the β2M polypeptide comprises an amino acid sequence having at least 95% amino acid sequence identity to the β2M amino acid sequence set forth in SEQ ID NO:2; and and b) the second polypeptide comprises, in order from N-terminus to C-terminus: i) a targeting polypeptide, wherein the targeting polypeptide is an anti-CD19 single chain Fv antibody; ii) a peptide linker; and iii) an MHC class I heavy chain polypeptide, wherein the MHC class I heavy chain polypeptide comprises an amino acid sequence having at least 95% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 18; and iv) a peptide linker; and v) an Ig Fc polypeptide, wherein the Ig Fc polypeptide comprises an amino acid sequence having at least 95% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 877; and Including, The TMMP described in claim 11. Claim 13: i) the β2M polypeptide contains a Cys at position 12; ii) the anti-CD19 single chain Fv antibody comprises an amino acid sequence having at least 95% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 872; iii) the MHC class I heavy chain comprises a Cys at position 84 and a Cys at position 236; iv) the Ig Fc polypeptide comprises Ala at position 14 and Ala at position 15; The TMMP described in claim 12.

14. The method of claim 1, wherein: a) the first polypeptide comprises an amino acid sequence having at least 95% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO:883; and b) the second polypeptide comprises an amino acid sequence having at least 95% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 910; The TMMP described in claim 13.

15. The TMMP described in claim 14, wherein the first polypeptide has the amino acid sequence set forth in SEQ ID NO: 883 and the second polypeptide has the amino acid sequence set forth in SEQ ID NO:

910.

16. The TMMP described in claim 11, wherein the peptide epitope is an epitope of a cytomegalovirus (CMV) polypeptide.

17. The TMMP described in claim 16, wherein the peptide epitope has the amino acid sequence NLVPMVATV (sequence number 913) and is 9 amino acids in length.

18. The method of claim 18, wherein: a) the first polypeptide comprises an amino acid sequence having at least 95% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO:912; and b) the second polypeptide comprises an amino acid sequence having at least 95% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 910; TMMP according to claim 16.

19. The TMMP described in claim 16, wherein the first polypeptide has the amino acid sequence set forth in SEQ ID NO: 912 and the second polypeptide has the amino acid sequence set forth in SEQ ID NO:

910.

20. A TMMP that is a dimer comprising two heterodimers described in any one of claims 1 to 19, wherein the two heterodimers are covalently bonded to each other by one or more disulfide bonds between the Ig Fc polypeptides present in the first and second heterodimers.

21. A pharmaceutical composition comprising the TMMP described in any one of claims 1 to 20.

22. A nucleic acid comprising a nucleotide sequence encoding the first polypeptide and the second polypeptide according to any one of claims 1 to 19, and b) first and second nucleic acids, i) a first nucleic acid comprising a nucleotide sequence encoding a first polypeptide according to any one of claims 1 to 19; and ii) a second nucleic acid comprising a nucleotide sequence encoding a second polypeptide according to any one of claims 1 to 19; a first and a second nucleic acid; A composition comprising:

23. A method for producing TMMP, comprising the steps of culturing in a culture medium a host cell genetically modified with one or more nucleic acids encoding the first and second polypeptides of TMMP described in any one of claims 1 to 20, and isolating TMMP from the genetically modified host cell and / or the culture medium.

24. The pharmaceutical composition of claim 21 for use in treating an autoimmune disease.

25. The pharmaceutical composition of claim 21 for use in treating cancer.

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