T cell engaging polypeptides and methods of use thereof

Abstract

The present disclosure provides a single chain T cell engaging polypeptide (TEP) comprising a peptide epitope, a class I major histocompatibility complex polypeptide, one or more immune modulating polypeptides, an immunoglobulin Fc polypeptide or a non-immunoglobulin scaffold polypeptide, and a tumor targeting polypeptide. The present disclosure provides a method of treating cancer comprising administering an effective amount of the TEP to an individual in need thereof. TIFF2025515575000018.tif17170

Description

[Technical field]

[0001] cross reference This application claims the benefit of U.S. Provisional Patent Application No. 63 / 331,167, filed April 14, 2022, which is incorporated by reference in its entirety herein.

[0002] Incorporation by Reference of Electronically Submitted Materials The Sequence Listing is provided herein as Sequence Listing XML "CUEB-151WO_SEQ_LIST", created on April 10, 2023, and having a size of 1,203,609 bytes. The contents of the Sequence Listing XML are incorporated herein by reference in their entirety. [Background technology]

[0003] Introduction The adaptive immune response involves the engagement of the T cell receptor (TCR) present on the surface of a T cell with small peptide antigens non-covalently presented on the surface of an antigen-presenting cell (APC) by the major histocompatibility complex (MHC, also called the human leukocyte antigen (HLA) complex in humans). The complex of peptide antigen presented by HLA may be referred to as "pHLA" or "pMHC". This engagement represents the targeting mechanism of the immune system and is a molecular interaction essential for T cell regulation (activation or inhibition) and effector function.

[0004] One important function of T cells is to bind and kill cancer cells. T cells accomplish this function by binding of the TCR to the pHLA complex, which is expressed on the surface of cancer cells. When T cells bind to the pHLA complex, they can release cytotoxic drugs that kill the cancer cells. However, the ability of T cells to find and kill cancer cells is subject to certain limitations.

[0005] First, cancer patients must have a pre-existing repertoire of T cells specific for pHLA complexes on the surface of cancer cells. However, many cancer patients may not have a sufficiently large number of T cells specific for their cancer. In such cases, the ability of T cells to kill cancer cells is insufficient to prevent continued growth of the cancer, even when the patient is administered a checkpoint inhibitor, such as an anti-PD1 antibody, designed to increase the number of T cells in the patient. See, for example, Au et al., 2021, Cancer Cell 39, 1-22 (November 8, 2021) (Non-Patent Document 1).

[0006] Second, mutations in cancer cells can occur over time. One such mutation is "HLA loss," in which cancer cells stop presenting pHLA complexes on their surface. Once this occurs, T cells can no longer bind to and kill the cancer cells.

[0007] Thus, there is a need for anti-cancer therapeutics that can be effective, especially when patients do not have a sufficient pre-existing repertoire of cancer-specific T cells and / or when the cancer is subject to HLA loss. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Au et al., 2021, Cancer Cell 39, 1-22 (November 8, 2021) Summary of the Invention

[0009] overview The present disclosure provides single chain T cell engaging polypeptides (TEPs) comprising a peptide epitope, a class I major histocompatibility complex polypeptide, a scaffold component such as an immunoglobulin Fc polypeptide or a non-immunoglobulin scaffold polypeptide, one or more tumor targeting polypeptides, and, optionally, one or more immune modulating polypeptides. The present disclosure provides methods of treating cancer comprising administering the TEPs. [Brief description of the drawings]

[0010] [Figure 1] 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 2-1] 2A-2M provide the amino acid sequences of immunoglobulin Fc polypeptides (SEQ ID NOs: 3-15, respectively). [Figure 2-2] See description of Figure 2-1. [Figure 2-3] See description of Figure 2-1. [Figure 2-4] See description of Figure 2-1. [Figure 2-5] See description of Figure 2-1. [Figure 2-6] See description of Figure 2-1. [Figure 3-1] 3A-3E provide the amino acid sequence of wild-type HLA-A*0201 (SEQ ID NO: 16) (FIG. 3A), and the amino acid sequences of variants (SEQ ID NOs: 17-20, respectively) (FIGS. 3B-3E). [Figure 3-2] See description of Figure 3-1. [Figure 4-1] 4A-4E provide the amino acid sequence of wild-type HLA-A*1101 (SEQ ID NO: 21) (FIG. 4A), and the amino acid sequences of variants (SEQ ID NOs: 22-25, respectively) (FIGS. 4B-4E). [Figure 4-2] See description of Figure 4-1. [Figure 5-1]5A-5E provide the amino acid sequence of wild-type HLA-A*2402 (SEQ ID NO: 26) (FIG. 5A), and the amino acid sequences of variants (SEQ ID NOs: 27-30, respectively) (FIGS. 5B-5E). [Figure 5-2] See description of Figure 5-1. [Figure 6-1] 6A-6E provide the amino acid sequence of wild-type HLA-A*3303 (SEQ ID NO: 31) (FIG. 6A), and the amino acid sequences of variants (SEQ ID NOs: 32-35, respectively) (FIGS. 6B-6E). [Figure 6-2] See description of Figure 6-1. [Figure 7A] 7A-7B provide an alignment of the HLA-A heavy chain amino acid sequences (FIG. 7A, SEQ ID NOs: 36-44, respectively) and the consensus sequence (FIG. 7B, SEQ ID NO: 45). [Figure 7B] See legend to Figure 7A. [Figure 8A] 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 8B] See legend to Figure 8A. [Figure 9A] Figures 9A-9B provide an alignment of the HLA-C heavy chain amino acid sequences (Figure 9A, SEQ ID NOs: 54-58, 1217, and 59-61, respectively) and the consensus sequence (Figure 9B, SEQ ID NO: 62). [Figure 9B] See legend to Figure 9A. [Figure 10] 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. [Figure 11-1] 11A-11D provide the amino acid sequences of the HLA-E heavy chains (SEQ ID NOs:837-840, respectively). [Figure 11-2] See description of Figure 11-1. [Figure 12-1] 12A-12D provide the amino acid sequences of the HLA-G heavy chains (SEQ ID NOs:841-844, respectively). [Figure 12-2] See description of Figure 12-1. [Figure 13A] Figures 13A-13K provide the amino acid sequences of SARS-CoV-2 encoded polypeptides (SEQ ID NOs:70-94). [Figure 13B] See legend to Figure 13A. [Figure 13C] See legend to Figure 13A. [Figure 13D] See legend to Figure 13A. [Figure 13E-1] See legend to Figure 13A. [Figure 13E-2] See legend to Figure 13A. [Figure 13F] See legend to Figure 13A. [Figure 13G] See legend to Figure 13A. [Figure 13H] See legend to Figure 13A. [Figure 13I] See legend to Figure 13A. [Figure 13J] See legend to Figure 13A. [Figure 13K] See legend to Figure 13A. [Figure 14-1] FIG. 14 provides the CD8 T cell epitopes of SARS-CoV-2 encoded polypeptides and the primary HLA class I heavy chain allele restriction. [Figure 14-2] See description of Figure 14-1. [Figure 14-3] See description of Figure 14-1. [Figure 14-4] See description of Figure 14-1. [Figure 14-5] See description of Figure 14-1. [Figure 14-6] See description of Figure 14-1. [Figure 14-7] See description of Figure 14-1. [Figure 14-8] See description of Figure 14-1. [Figure 14-9] See description of Figure 14-1. [Figure 14-10] See description of Figure 14-1. [Figure 14-11] See description of Figure 14-1. [Figure 14-12] See description of Figure 14-1. [Figure 14-13] See description of Figure 14-1. [Figure 14-14] See description of Figure 14-1. [Figure 14-15] See description of Figure 14-1. [Figure 14-16] See description of Figure 14-1. [Figure 14-17] See description of Figure 14-1. [Figure 14-18] See description of Figure 14-1. [Figure 14-19] See description of Figure 14-1. [Figure 14-20] See description of Figure 14-1. [Figure 14-21] See description of Figure 14-1. [Figure 14-22] See description of Figure 14-1. [Figure 14-23] See description of Figure 14-1. [Figure 14-24] See description of Figure 14-1. [Figure 14-25] See description of Figure 14-1. [Figure 15-1] FIG. 15 provides the amino acid sequences of SARS-CoV-2 peptides and HLA class I heavy chain allele restrictions. [Figure 15-2] See description of Figure 15-1. [Figure 16A] Figures 16A-16D provide the amino acid sequences of wild-type IL-2 polypeptide (Figure 16A), IL-2Rα (SEQ ID NO: 67) (Figure 16B), IL-2Rβ (SEQ ID NO: 68) (Figure 16C), and IL-2Rγ (SEQ ID NO: 69) (Figure 16D). [Figure 16B] See legend to Figure 16A. [Figure 16C] See legend to Figure 16A. [Figure 16D] See legend to Figure 16A. [Figure 17-1] Figures 17A-17N provide the amino acid sequences of an exemplary anti-mesothelin scFv (SEQ ID NOs: 888-895) (Figures 17A-17H), an exemplary anti-TROP-2 scFv (SEQ ID NOs: 896-899) (Figures 17I-17L), and an exemplary anti-CD28 scFv (SEQ ID NOs: 908 and 909) (Figures 17M-17N). [Figure 17-2] See description of Figure 17-1. [Figure 17-3] See description of Figure 17-1. [Figure 17-4] See description of Figure 17-1. [Figure 17-5] See description of Figure 17-1. [Figure 17-6] See description of Figure 17-1. [Figure 17-7] See description of Figure 17-1. [Figure 18A] 18A-18D provide schematic diagrams of examples of TEP configurations. [Figure 18B] See legend to Figure 18A. [Figure 18C] See legend to Figure 18A. [Figure 18D] See legend to Figure 18A. [Figure 19A] Figures 19A-19G provide the amino acid sequences of the constructs shown in Figure 20. Figure 19A provides the amino acid sequence of an exemplary single-chain TEP. [Figure 19B] See legend to Figure 19A. [Figure 19C] See legend to Figure 19A. [Figure 19D] See legend to Figure 19A. [Figure 19E] See legend to Figure 19A. [Figure 19F] See legend to Figure 19A. [Figure 19G] See legend to Figure 19A. [Figure 20] FIG. 20 shows the effect of various constructs on killing of CD19+ tumor cells. [Figure 21A] 21A-21J provide the amino acid sequences of exemplary single-chain TEPs (SEQ ID NOs: 1207-1215, and 1218, respectively). [Figure 21B] See legend to Figure 21A. [Figure 21C] See legend to Figure 21A. [Figure 21D] See legend to Figure 21A. [Figure 21E] See legend to Figure 21A. [Figure 21F] See legend to Figure 21A. [Figure 21G] See legend to Figure 21A. [Fig. 21H] See legend to Figure 21A. [Figure 21I] See legend to Figure 21A. [Figure 21J] See legend to Figure 21A. [Figure 22A] 22A-22B provide schematic diagrams of examples of TEP configurations. [Figure 22B] See legend to Figure 22A. [Figure 23A] Figures 23A-23B provide the amino acid sequences of construct 4770 (SEQ ID NO: 1208) (Figure 23A), and construct 4771 (SEQ ID NO: 1216) (Figure 23B). [Figure 23B] See legend to Figure 23A. [Figure 24] Figure 24 shows the effect of different constructs on killing of CD19+ tumor cells. Diamonds: construct 4770, filled circles: construct 4771, triangles: medium only. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] 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.

[0012] 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 may be 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 a 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 one amino acid to the N-terminus, reference to position 84 or a specific 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 specific amino acid substitution at a specific position, for example 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 the substitution of a Cys residue for the Tyr residue present in the wild-type sequence. For example, if a wild-type polypeptide is modified 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.Where 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 an alternative amino acid at the altered position number. Reference to a "non-naturally occurring Cys residue" in a polypeptide, such as 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, where a cysteine ​​is replaced with an amino acid that occurs in the wild-type sequence.

[0013] A polynucleotide or polypeptide has a certain percentage of "sequence identity" to another polynucleotide or polypeptide, which means that when the two sequences are compared, that percentage of bases or amino acids are the same and in the same relative positions when aligned. Unless otherwise stated, to determine sequence identity, sequences are aligned using the computer program BLAST (BLAST+2.10.0, using default parameters), which is available on the World Wide Web at sites including blast.ncbi.nlm.nih.gov / Blast.cgi. Unless otherwise stated, to determine the positions of corresponding amino acids (e.g., when making specific substitutions), sequence comparisons are performed using Clustal Omega Version 1.2.2 (using default parameters), available on the World Wide Web at www.ebi.ac.uk / Tools / msa / clustalo / . Where a polypeptide sequence contains fewer amino acids (aa) or more aa than a reference sequence having a SEQ ID NO, the percent sequence identity of the polypeptide sequence to the reference SEQ ID NO sequence is determined by aligning and comparing the amino acids of the polypeptide sequence in the same relative positions as the aa in the reference SEQ ID NO, including the additional aa in the polypeptide sequence without reference to the additional aa in the reference SEQ ID NO (where the reference SEQ ID NO has more aa than the polypeptide sequence) or the additional aa in the polypeptide sequence (where the polypeptide sequence has more aa than the reference SEQ ID NO).

[0014] The term "conservative amino acid substitution" refers to the interchangeability in proteins of amino acid residues with similar side chains. For example, the group of amino acids with aliphatic side chains consists of glycine, alanine, valine, leucine, and isoleucine, the group of amino acids with aliphatic hydroxyl side chains consists of serine and threonine, the group of amino acids with amide-containing side chains consists of asparagine and glutamine, the group of amino acids with aromatic side chains consists of phenylalanine, tyrosine, and tryptophan, the group of amino acids with basic side chains consists of lysine, arginine, and histidine, the group of amino acids with acidic side chains consists of glutamic acid and aspartate, 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.

[0015] 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.

[0016] "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.

[0017] 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. The MOD of the TEP can bind to a cognate costimulatory polypeptide (i.e., a "co-MOD") present on the target T cell.

[0018] As used herein, the term "in vivo" refers to any process or procedure that occurs inside the body.

[0019] As used herein, the term "in vitro" refers to any process or procedure that occurs outside the body.

[0020] As used herein, "heterologous" means a nucleotide or polypeptide that is not found in the native nucleic acid or protein, respectively.

[0021] 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 constructs 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 cellular or cell-free transcription and translation system.

[0022] 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 typically 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.

[0023] As used herein, the term "affinity" refers to the equilibrium constant for the reversible binding of two agents (e.g., an antibody and an antigen), and the dissociation constant (K D ) As used herein, the term "avidity" refers to the resistance of a complex of two or more agents to dissociation after dilution. The terms "immunoreactive" and "preferentially bind" are used interchangeably herein with respect to antibodies and / or antigen-binding fragments.

[0024] As used herein (e.g., with respect to binding of a TEP 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, e.g., 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 K D "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" refers to the formation of one or more covalent chemical bonds between two different molecules.

[0025] 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 the disease or its symptoms, and / or may be therapeutic in terms of partially or completely curing the disease and / or the adverse effects caused by the disease. As used herein, "treatment" encompasses any treatment of a disease or condition in a mammal, including (a) preventing the onset of the disease or condition in a subject who may or may not be susceptible to the disease or condition, but has not yet been diagnosed as having it, (b) suppressing the disease or one or more symptoms associated with the disease, e.g., arresting its onset, and / or (c) relieving the disease, i.e., regressing the disease. Therapeutic agents may be administered before, during, and / or after the onset of the disease or injury. Of particular interest are treatments of ongoing diseases that stabilize or reduce the undesirable clinical symptoms of the patient. Such treatments are desirably performed before complete loss of function of the affected tissue. The therapy will desirably be administered during, and in some cases after, the symptomatic stage of the disease.

[0026] 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.), etc. Unless otherwise indicated, the terms "individual," "subject," "host," and "patient" refer to humans.

[0027] 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, as well as 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.

[0028] 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 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 the "Camelidae" family, immunoglobulins are found that lack light polypeptide chains. "Camelidae" includes Old World Camelidae (Camelus bactrianus and Camelus dromedarius) as well as New World Camelidae (e.g., Llama paccos, Llama glama, Llama guanicoe, and Llama vicugna). Single variable domain heavy chain antibodies are herein referred to as nanobodies or V HH These are called antibodies.

[0029] 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, 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 F(ab') fragments that have two antigen-binding sites and are still capable of cross-linking antigen. 2 Produces fragments.

[0030] "Fv" is the smallest antibody fragment that contains a complete antigen recognition and binding site. This region consists of a dimer of one heavy chain variable domain 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 to an antigen, albeit with a lower affinity than the entire binding site.

[0031] "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.

[0032] The "light chains" of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequence of their constant domains. Depending on the amino acid sequence of the constant domains of their heavy chains, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these classes can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The subclasses can be further divided into types, e.g., IgG2a and IgG2b.

[0033] "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).

[0034] 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 a type of diabody that includes a diabody having a complementary domain on another chain, and a diabody having a complementary domain on another chain.) By using a linker that is too short to allow pairing between the two domains on the same chain, these 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.

[0035] 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., US Dept. of Health and Human Services, "Sequences of proteins of immunological interest" (1991) (also referred to herein as Kabat 1991), Chothia et al. (1987) J. Mol. Biol. 196:901 (also referred to herein as Chothia 1987), and MacCallum et al. (1996) J. Mol. Biol. 262:732, 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.

[0036] Table 1: CDR definitions TIFF2025515575000002.tif33132 1 Residue numbering follows the nomenclature of Kabat et al., 1991 (supra). 2Residue numbering follows the nomenclature of Chothia et al. (supra). 3 Residue numbering follows the nomenclature of MacCallum et al. (supra).

[0037] 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.

[0038] 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 about 100-120 amino acids long, 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.

[0039] 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.

[0040] 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.

[0041] As used herein, the term "MHC heavy chain polypeptide" refers collectively to the domains of the MHC heavy chain polypeptide present in the TEP. For example, the MHC heavy chain polypeptide can include an α1 domain, an α2 domain, and an α3 domain.

[0042] 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.

[0043] 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. When 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.

[0044] 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.Any method and material similar or equivalent to the method and material described herein can be used in the practice or testing of this disclosure, but preferred methods and materials are described below.All publications mentioned herein are incorporated by reference to disclose and describe the method and / or material in the context of the publication cited.

[0045] As used herein and in 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" or a "T cell engaging 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.

[0046] 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.

[0047] 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.

[0048] Detailed Description The present disclosure provides a single chain T cell engaging polypeptide (TEP) comprising: i) a peptide epitope (defined below); ii) a first major histocompatibility complex (MHC) polypeptide; iii) a second MHC polypeptide; iv) a scaffold component, such as an immunoglobulin (Ig) Fc polypeptide or a non-Ig scaffold; v) a tumor targeting polypeptide (TTP); and, optionally, vi) one or more MODs. The present disclosure provides a composition comprising the single chain TEP. The present disclosure provides a nucleic acid comprising a nucleotide sequence encoding the TEP of the present disclosure. The present disclosure provides a method of treatment comprising administering an effective amount of the TEP to an individual in need thereof.

[0049] Single-chain T cell-engaging polypeptide The present disclosure provides a single-chain TEP comprising: i) a peptide epitope; ii) a first MHC polypeptide; iii) a second MHC polypeptide; iv) a scaffold component, such as an Ig Fc polypeptide or a non-Ig scaffold; v) a TTP; and, optionally, iv) one or more MODs (also referred to herein as "MODs"). In other words, the aforementioned components of the TEP are within a single polypeptide chain. The components of the TEP may be directly linked to each other, or the components may be linked to each other via independently selected peptide linkers. For example, the TEP of the present disclosure may comprise one or more independently selected linkers between any two TEP components, e.g., between one or more of o: i) the peptide epitope and the MHC polypeptide; ii) the MHC polypeptide and the MOD; iii) the first MHC polypeptide and the second MHC polypeptide; iv) the MOD and the Ig Fc polypeptide; v) the MHC polypeptide and the Ig Fc polypeptide; and vi) the first MOD and the second MOD.

[0050] As used herein, the term "peptide epitope" refers to a peptide that, when complexed with an MHC polypeptide, presents the epitope to a T cell receptor (TCR). A peptide epitope has a length of at least 4 amino acids, e.g., 4 amino acids to about 25 amino acids (e.g., 4 amino acids (aa), 5aa, 6aa, 7aa, 8aa, 9aa, 10aa, 11aa, 12aa, 13aa, 14aa, 15aa, 16aa, 17aa, 18aa, 19aa, 20aa, 21aa, 22aa, 23aa, 24aa, or 25aa, including in the range of 4-20aa, 6-18aa, 8-15aa, 8-12aa, 5-10aa, 10-15aa, 15-20aa, 10-20aa, or 15-25aa). When complexed with an MHC polypeptide, the peptide epitope can present one or more epitopes to one or more TCRs. In some cases, the peptide epitopes present in the TEP present infectious disease-associated epitopes (e.g., virally encoded peptides).

[0051] In some cases, the TEPs of the present disclosure include i) viral epitopes (e.g., virally encoded peptides), for example, viral epitopes for which cancer patients have pre-existing T cell repertoires, either through infection and / or vaccination, and iii) TTPs that target cancer-associated antigens. Such TEPs bind to cancer cells that express the cancer-associated antigens targeted by the TTPs. The TEPs can then bind to T cells specific for the viral epitopes, which can then result in the release of cytotoxins that can kill the cancer cells. In this way, the TEPs can "redirect" virus-specific T cells to kill the cancer cells.

[0052] A TEP comprising one or more MODs can modulate the activity of T cells specific to a viral epitope present in the TEP. For example, in some cases, the MOD-containing TEP increases the proliferation and / or cytotoxic activity of T cells specific to a viral epitope present in the TEP. Contact with a T cell specific to a viral epitope present in the MOD-containing TEP can increase the cytotoxic activity of the T cell against a cancer cell expressing a cancer-associated antigen targeted by the TTP present in the TEP.

[0053] In some cases, the peptide epitope present in the TEP represents a SARS-CoV-2 peptide (e.g., a SARS-CoV-2 encoded peptide). In some cases, the peptide epitope present in the TEP represents a cytomegalovirus (CMV) peptide (e.g., a CMV encoded peptide).

[0054] The components of a single-chain TEP can be arranged in any of a variety of configurations. Figures 18A-18D show schematic diagrams of non-limiting examples of such configurations. For example, in some cases, a single-chain TEP comprises, in order from N-terminus to C-terminus, a) a peptide epitope, b) a β2M polypeptide, c) an MHC class I heavy chain polypeptide, d) a TTP, e) an Ig Fc polypeptide, and, optionally, f) one or more MODs (see Figure 18A). As another example, in some cases, a single-chain TEP comprises, in order from N-terminus to C-terminus, a) a peptide epitope, b) a β2M polypeptide, c) an MHC class I heavy chain polypeptide, d) one or more optional MODs, e) an Ig Fc polypeptide, and f) a TTP (see Figure 18B). As another example, in some cases, the single chain TEP comprises, in order from N-terminus to C-terminus, a) a peptide epitope, b) a β2M polypeptide, c) an MHC class I heavy chain polypeptide, d) one or more optional MODs, e) TTP, and f) an Ig Fc polypeptide (see FIG. 18C). As another example, in some cases, the single chain TEP comprises, in order from N-terminus to C-terminus, a) a peptide epitope, b) a β2M polypeptide, c) an MHC class I heavy chain polypeptide, d) TTP, e) one or more optional MODs, and f) an Ig Fc polypeptide (see FIG. 18D). In any of the above embodiments, the TEP may comprise one or more independently selected peptide linkers between any two of the components of the TEP. For example, the TEP may include a peptide linker between one or more of: a) the peptide epitope and the β2M polypeptide, b) between the β2M polypeptide and the MHC class I heavy chain polypeptide, c) between the MHC class I heavy chain polypeptide and the TTP, d) between the TTP and the Ig Fc polypeptide, e) between the Ig Fc polypeptide and the MOD, f) between two MODs (when the TEP comprises two MODs in tandem), g) between the Ig Fc and the TTP, h) between the MHC class I heavy chain polypeptide and the MOD, and i) between the MOD and the TTP.

[0055] Epitope As discussed above, the TEPs of the present disclosure include peptide epitopes. Peptides (peptide epitopes) present in the TEP can have a length of at least 4 amino acids, for example, 4 amino acids to about 25 amino acids (e.g., lengths ranging from 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.

[0056] Suitable epitopes include, but are not limited to, epitopes present in infectious disease agents, such as viral infectious disease agents, or other infectious agents. Typically, the epitopes are epitopes for which cancer patients have, or are likely to have, a pre-existing T cell repertoire due to previous infection and / or vaccination.

[0057] Examples of viral infectious disease agents include 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 viruses, Rift Valley fever virus vaccine strain MP-12, chikungunya viruses, calciviruses, coronaviruses, cowpox viruses, and 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, Cytomegalovirus, Epstein-Barr virus, Eastern equine encephalitis virus, Herpes simplex types 1 and 2, Herpes zoster, Human herpesvirus types 6 and 7, Hepatitis C virus (HVC), Hepatitis B virus (HBV), Influenza viruses types A, B, and C, Papovaviruses, Newcastle disease virus, Measles virus, Mumps virus, Parainfluenza virus types 1 and 2, types 3 and 4, polyomaviruses (JC virus, BK virus), respiratory syncytial virus, human parvovirus (B19), coxsackieviruses A and B, echovirus, poliovirus, rhinovirus, smallpox (variola minor virus), smallpox (variola major virus), whitepox reovirus, Coltivirus, human rotavirus, and orbivirus (Colorado tick fever virus), rabies virus, vesicular stomatitis virus, Rubivirus (rubella), Semliki Forest virus, Saint Louis encephalitis virus, Venezuelan equine ... Elauma encephalomyelitis virus, Arenaviruses (also known as South American hemorrhagic fever viruses), Flexal, Lymphocytic choriomeningitis virus (LCM) (neurotropic strains), Hantaviruses including Hantaan virus, Rift Valley fever virus, Japanese encephalitis virus, Yellow fever virus, Monkeypox virus, Human immunodeficiency virus (HIV) types 1 and 2, Human T-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, Central European encephalitis (Hypr, Kumlinge), Kyasanur Forest disease, Omsk hemorrhagic fever, and tick-borne encephalitis virus complex (Flavi), which includes Russian spring-summer encephalitis virus, 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, and variola major. Viruses that are known to be infectious include variola 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, Codori aluminum (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 TEPs of the present disclosure may include peptides from any known viral antigen.

[0058] In some cases, a viral epitope is an epitope present in a viral antigen encoded by a virus that infects a large portion of the human population, including, for example, cytomegalovirus (CMV), Epstein-Barr virus (EBV), human papillomavirus, adenovirus, coronaviruses such as SARS-CoV-2, etc.

[0059] In some cases, the epitope peptides present in the TEPs of the present disclosure present epitopes specific for HLA-A, -B, -C, -E, -F, or -G alleles. In one embodiment, the epitope peptides present in the TEPs present epitopes specific for HLA-A, -B, -C, -E, -F, or -G alleles. * 0101, A * 0201, A * 0301, A *1101, A * 2301, A * 2402, A * 2407, A * 3303, and / or A * In one embodiment, the epitope peptide present in the TEP is restricted to HLA-B 3401. * 0702, B * 0801, B * 1502, B * 3802, B * 4001, B * 4601, and / or B * In one embodiment, the epitope peptide present in the TEP is C * 0102, C * 0303, C * 0304, C * 0401, C * 0602, C * 0701, C * 702, C * 0801, and / or C * 1502. In one embodiment, the epitope peptide present in the TEP is HLA-E, e.g., HLA-E * 0101, and HLA-E * The epitopes presented are restricted to HLA-E, such as HLA-E, which is a highly prevalent HLA-E allele, e.g., 01.03.

[0060] CMV peptide epitopes In some cases, the TEPs of the present disclosure include CMV peptide epitopes, i.e., peptides that, when in an MHC / peptide complex (e.g., an HLA / peptide complex), present a CMV epitope (i.e., an epitope present in a CMV antigen) to a T cell. As with other peptide epitopes of the present disclosure, the CMV peptide epitope has a length of at least 4 amino acids, e.g., from 4 amino acids to about 25 amino acids (e.g., lengths ranging from 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, including within the ranges of 4-20aa, 6-18aa, 8-15aa, 8-12aa, 5-10aa, 10-15aa, 15-20aa, 10-20aa, or 15-25aa).

[0061] A given CMV epitope-specific T cell binds to an epitope having a reference amino acid sequence for the given CMV epitope, but does not substantially bind to an epitope that differs from the reference amino acid sequence. For example, a CMV epitope-specific T cell binds to a CMV epitope having a reference amino acid sequence and does not substantially bind to an epitope that differs from the reference amino acid sequence, in total, more than 10 -6 Less than M, 10 -5 Less than M or 10 -4 CMV epitope-specific T cells bind with an affinity of at least 10 M to the epitope for which they are specific. -7 M, at least 10 -8 M, at least 10 -9 M, or at least 10 -10 It can bind with an affinity of M.

[0062] In some cases, the CMV peptide epitope present in the TEP of the present disclosure is a peptide from CMV pp65. In some cases, the CMV peptide epitope present in the TEP of the present disclosure is a peptide from CMV gB (glycoprotein B).

[0063] For example, in some cases, a CMV peptide epitope present in a TEP of the disclosure has a length of at least 4 amino acids, e.g., 4 amino acids to about 25 amino acids (e.g., 4 amino acids (aa), 5 aa, 6 aa, 7 aa, 8 aa, 9 aa, 10 aa, 11 aa, 12 aa, 13 aa, 14 aa, 15 aa, 16 aa, 17 aa, 18 aa, 19 aa, 20 aa, 21 aa, 22 aa, 23 aa, 24 aa, or 25 aa in length, including in the range of 4-20 aa, 6-18 aa, 8-15 aa, 8-12 aa, 5-10 aa, 10-15 aa, 15-20 aa, 10-20 aa, or 15-25 aa in length), and is in the range of 4 to 20 aa, 6-18 aa, 8-15 aa, 8-12 aa, 5-10 aa, 10-15 aa, 15-20 aa, 10-20 aa, or 15-25 aa in length, Amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the pp65 amino acid sequence: TIFF2025515575000003.tif62159.

[0064] As one non-limiting example, a CMV peptide epitope present within a TEP of the present disclosure has the amino acid sequence NLVPMVATV (SEQ ID NO:913) and is 9 amino acids in length.

[0065] In some cases, a CMV peptide epitope present in a TEP of the disclosure has a length of at least 4 amino acids, e.g., 4 amino acids to about 25 amino acids (e.g., a length of 4 amino acids (aa), 5 aa, 6 aa, 7 aa, 8 aa, 9 aa, 10 aa, 11 aa, 12 aa, 13 aa, 14 aa, 15 aa, 16 aa, 17 aa, 18 aa, 19 aa, 20 aa, 21 aa, 22 aa, 23 aa, 24 aa, or 25 aa, including a length of 4-20 aa, 6-18 aa, 8-15 aa, 8-12 aa, 5-10 aa, 10-15 aa, 15-20 aa, 10-20 aa, or 15-25 aa), and is in the range of: A peptide of a CMV polypeptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the gB amino acid sequence: (Sequence number 1220).

[0066] In some cases, the CMV epitopes present in the TEPs of the present disclosure present epitopes specific for HLA-A, -B, -C, -E, -F, or -G alleles. In some cases, the epitope peptides present in the TEPs present epitopes specific for HLA-A, -B, -C, -E, -F, or -G alleles. * 0101,A * 0201, A * 0301, A * 1101, A * 2301, A * 2402, A * 2407, A * 3303, and / or A * In some cases, the CMV epitopes present in the TEPs of the present disclosure are restricted to HLA-B 3401. * 0702, B * 0801, B * 1502, B * 3802, B * 4001, B * 4601, and / or B * 5301. In some cases, the CMV epitopes present in the TEPs of the present disclosure are * 0102, C * 0303, C * 0304, C * 0401, C * 0602, C * 0701, C * 702, C * 0801, and / or C * As an example, in some cases, the TEP of the present disclosure presents an epitope that is restricted to HLA-A 1502. * 0201 class I heavy chain polypeptide, and c) a β2M polypeptide.

[0067] In some cases, the TEP of the present disclosure comprises an scFv or nanobody specific to a Her2 polypeptide present on the surface of a cancer cell as the TTP, and comprises a CMV peptide epitope as the epitope. In some cases, the CMV peptide is a peptide of a CMV pp65 polypeptide. In some cases, the CMV peptide epitope is a peptide of a CMV gB polypeptide. In some cases, the CMV peptide epitope has the amino acid sequence NLVPMVATV (SEQ ID NO: 913) and has a length of 9 amino acids.

[0068] In some cases, the TEP of the present disclosure includes an scFv or nanobody specific to a MUC1 polypeptide present on the surface of a cancer cell as the TTP, and includes a CMV peptide epitope as the epitope. In some cases, the CMV peptide epitope is a peptide of a CMV pp65 polypeptide. In some cases, the CMV peptide is a peptide of a CMV gB polypeptide. In some cases, the CMV peptide has the amino acid sequence NLVPMVATV (SEQ ID NO: 913) and is 9 amino acids long.

[0069] In some cases, the TEP of the present disclosure comprises an scFv or nanobody specific to a WT1 polypeptide present on the surface of a cancer cell as the TTP, and comprises a CMV peptide epitope as the epitope. In some cases, the CMV peptide epitope is a peptide of a CMV pp65 polypeptide. In some cases, the CMV peptide epitope is a peptide of a CMV gB polypeptide. In some cases, the CMV peptide epitope has the amino acid sequence NLVPMVATV (SEQ ID NO: 913) and has a length of 9 amino acids.

[0070] In some cases, the TEP of the present disclosure comprises an scFv or nanobody specific to a mesothelin polypeptide present on the surface of a cancer cell as the TTP, and comprises a CMV peptide epitope as the epitope. In some cases, the CMV peptide epitope is a peptide of a CMV pp65 polypeptide. In some cases, the CMV peptide epitope is a peptide of a CMV gB polypeptide. In some cases, the CMV peptide epitope has the amino acid sequence NLVPMVATV (SEQ ID NO: 913) and has a length of 9 amino acids.

[0071] In some cases, the TEP of the present disclosure comprises an scFv or nanobody specific to a CD19 polypeptide present on the surface of a cancer cell as the TTP, and comprises a CMV peptide epitope as the epitope. In some cases, the CMV peptide epitope is a peptide of a CMV pp65 polypeptide. In some cases, the CMV peptide epitope is a peptide of a CMV gB polypeptide. In some cases, the CMV peptide epitope has the amino acid sequence NLVPMVATV (SEQ ID NO: 913) and has a length of 9 amino acids.

[0072] In some cases, the TEP of the present disclosure includes an scFv or nanobody specific to a BCMA polypeptide present on the surface of a cancer cell as the TTP and includes a CMV peptide epitope as the epitope. In some cases, the CMV peptide epitope is a peptide of a CMV pp65 polypeptide. In some cases, the CMV peptide epitope is a peptide of a CMV gB polypeptide. In some cases, the CMV peptide epitope has the amino acid sequence NLVPMVATV (SEQ ID NO: 913) and is 9 amino acids long.

[0073] In some cases, the TEP of the present disclosure comprises an scFv or nanobody specific to a MUC16 polypeptide present on the surface of a cancer cell as the TTP, and comprises a CMV peptide epitope as the epitope. In some cases, the CMV peptide epitope is a peptide of a CMV pp65 polypeptide. In some cases, the CMV peptide epitope is a peptide of a CMV gB polypeptide. In some cases, the CMV peptide epitope has the amino acid sequence NLVPMVATV (SEQ ID NO: 913) and has a length of 9 amino acids.

[0074] SARS-CoV-2 Peptides As described above, in some cases, the TEP comprises a betacoronavirus (e.g., SARS-CoV-2) peptide that is typically at least about four amino acids in length and presents a SARS-CoV-2 epitope to T cells within an MHC / peptide complex (e.g., an HLA / peptide complex).

[0075] The SARS-CoV-2 peptides present in the TEP can have a length of at least 4 amino acids, for example, 4 amino acids to about 25 amino acids (e.g., including lengths in the ranges 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.

[0076] A SARS-CoV-2 epitope present in a TEP is a peptide that is specifically bound by a T cell, i.e., the epitope is specifically bound by an epitope-specific T cell. An epitope-specific T cell binds to an epitope having a reference amino acid sequence, but does not substantially bind to an epitope that differs from the reference amino acid sequence. For example, an epitope-specific T cell binds to an epitope having a reference amino acid sequence, but does not substantially bind to an epitope that differs from the reference amino acid sequence, for example, 10 to 15 days after the first antigen binding. -6 Less than M, 10 -5 Less than M or 10 -4 An 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.

[0077] The peptide epitope present in the TEP is a peptide of a betacoronavirus encoded polypeptide. In some cases, the peptide epitope is a SARS-CoV-2 peptide (i.e., a peptide of a SARS-CoV-2 encoded polypeptide). In some cases, the peptide epitope is a SARS-CoV-2 peptide derived from a SARS-CoV-2 encoded surface glycoprotein. In some cases, the peptide epitope is a SARS-CoV-2 peptide derived from a SARS-CoV-2 encoded membrane glycoprotein. In some cases, the peptide epitope is a SARS-CoV-2 peptide derived from a SARS-CoV-2 encoded nucleocapsid phosphoprotein.

[0078] In some cases, the peptides present in the TEP 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, 25 aa in length) of a polypeptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to any one of the SARS-CoV-2 polypeptides shown in Figures 13A-13J. In some cases, the peptide present in the TEP is a peptide 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 one of the polypeptides shown in Figures 13A-13J.

[0079] In some cases, the peptide present in the TEP is any one of the peptides shown in Figure 14. In some cases, the peptide present in the TEP is any one of the peptides shown in Figure 15.

[0080] In some cases, the peptide epitope is a SARS-CoV-2 peptide derived from a SARS-CoV-2 encoded surface glycoprotein. In some cases, the peptide present in the TEP 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 a polypeptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the SARS-CoV-2 surface glycoprotein shown in FIG. 13J. In some cases, the peptides present in the TEP are peptides of the SARS-CoV-2 surface glycoprotein shown in Figure 13J that are between 4 amino acids (aa) and 25 aa 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).

[0081] In some cases, 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 cases, the peptides present in the TEP are polypeptides having an amino acid sequence with 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 that are 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). 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, T47 8K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K, L981F (bold substitutions are within the receptor binding domain (RBD), "ins" indicates insertion and "del" indicates deletion). In some cases, the peptides present in the TEP are peptides of the SARS-CoV-2 surface glycoprotein shown in Figure 13K that are between 4 amino acids (aa) and 25 aa 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).

[0082] In some cases, the peptide present in the TEP 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), TEKSNIIR GW (SEQ ID 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), WTAGAAA YY (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), K IADYNYKL (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).

[0083] In some cases, the peptide epitope is a SARS-CoV-2 peptide derived from a SARS-CoV-2 encoded membrane glycoprotein. In some cases, the peptide present in the TEP 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 a polypeptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the SARS-CoV-2 membrane glycoprotein shown in FIG. 13D. In some cases, the peptides present in the TEP are peptides of the SARS-CoV-2 membrane glycoprotein shown in FIG. 13D that are 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).

[0084] In some cases, the peptide present in the TEP 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).

[0085] In some cases, the peptide epitope is a SARS-CoV-2 peptide derived from the SARS-CoV-2 encoded nucleocapsid protein. In some cases, the peptide present in the TEP is a polypeptide having an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the SARS-CoV-2 nucleocapsid phosphoprotein shown in FIG. 13F, the peptide being 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). In some cases, the peptide present in the TEP is a peptide of the SARS-CoV-2 nucleocapsid phosphoprotein shown in FIG. 13F that is between 4 amino acids (aa) and 25 aa 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).

[0086] In some cases, the peptide epitope is a SARS-CoV-2 peptide derived from the SARS-CoV-2 encoded nucleocapsidin protein 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).

[0087] In some instances, the peptide epitope is a SARS-CoV-2 peptide, as shown in Figure 15. In some instances, the peptide is an HLA-A peptide, as shown in Figure 15. * 01:01 restricted peptide. In some cases, the peptide is an HLA-A * 02:01 is a restricted peptide. In some cases, the peptide is an HLA-A * 11:01 restricted peptide. In some cases, the peptide is an HLA-A * 24:01 restricted peptide. In some cases, the peptide is an HLA-B * 07:02 is a restricted peptide. The peptides of Figure 15 may be paired with the HLA alleles shown in Figure 15 or with different HLA alleles.

[0088] In some cases, the peptide epitope is RLQSLQTYV (SEQ ID NO: 224). In some cases, the peptide epitope is YLQPRTFLL (SEQ ID NO: 218). In some cases, the TEP comprises an HLA-A*02:01 heavy chain polypeptide and comprises the peptide epitope YLQPRTFLL (SEQ ID NO: 218).

[0089] In some cases, 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 cases, the peptide epitope is a peptide of a SARS-CoV-2 Nsp13 polypeptide (see, e.g., FIG. 13C). In some cases, the peptide epitope is VMPLSAPTL (SEQ ID NO: 914). In some cases, the TEP comprises an HLA-E heavy chain polypeptide and comprises the peptide epitope VMPLSAPTL (SEQ ID NO: 914).

[0090] MHC Polypeptides As discussed above, TEPs include MHC polypeptides. For purposes of this disclosure, the term "major histocompatibility complex (MHC) polypeptide" is meant to include MHC polypeptides of various species, including human MHC (also referred to as human leukocyte antigen (HLA)) polypeptides, rodent (e.g., mouse, rat, etc.) MHC polypeptides, and MHC polypeptides of other mammalian species (e.g., 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).

[0091] In some cases, 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 cases, 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 TEP does not include a 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 TEP to the cell in which it is expressed (e.g., a eukaryotic cell such as a mammalian cell). In some cases, the MHC class I heavy chain present in the TEP does not include a signal peptide, a transmembrane domain, or an intracellular domain (cytoplasmic tail) associated with a natural MHC class I heavy chain. Thus, for example, in some cases, the MHC class I heavy chain present in the TEP comprises only the α1, α2, and α3 domains of the MHC class I heavy chain polypeptide. In some cases, the MHC class I heavy chain present in the TEP has a length of about 270 amino acids (aa) to about 290 aa. In some cases, the MHC class I heavy chain present in the TEP 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.

[0092] MHC class I heavy chain In some cases, the MHC class I heavy chain polypeptide present in the TEP comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to 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 cases, 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 cases, the MHC class I heavy chain has a length of 270aa-280aa. In some cases, the MHC class I heavy chain polypeptide present in the TEP 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 a transmembrane domain or a cytoplasmic domain.

[0093] In some cases, the MHC polypeptide of the TEP is a human MHC polypeptide, which is also referred to as a "human leukocyte antigen" ("HLA") polypeptide. In some cases, the MHC polypeptide of the TEP 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.

[0094] In some cases, the TEP comprises an HLA-A heavy chain polypeptide. HLA-A heavy chain peptide sequences, or portions thereof, that can be incorporated into the TEP 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 can include a mutation at one or more of positions 84, 139, and 236 (as selected from 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) as shown in Figures 3-6). Additionally, HLA-A sequences may be used that have at least 75% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%) or 100% amino acid sequence identity to all or a portion (e.g., 50, 75, 100, 150, 200, or 250 contiguous amino acids) of the sequences of those HLA-A alleles (which may include, for example, 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.

[0095] HLA-A02 In some cases, the MHC class I heavy chain polypeptide of the TEP comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence shown in Figure 3A. In some cases, an HLA-A heavy chain polypeptide suitable for inclusion in a TEP comprises the amino acid sequence shown in Figure 3A. The HLA-A heavy chain polypeptide is referred to as "HLA-A *As described in WO 2018 / 119114 and WO 2020 / 132138, the TEP 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.

[0096] 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 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 cases, Cys-236 forms an interchain disulfide bond with Cys-12 of a variant β2M polypeptide that contains an R12C substitution; or B) The HLA-A02 (Y84A;A236C) amino acid sequence shown in FIG. 3D, in which amino acid 84 is Ala and amino acid 236 is Cys. In some cases, Cys-236 forms an interchain disulfide bond with Cys-12 of a 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.

[0097] As described above, in some cases, the MHC class I heavy chain polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the 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.

[0098] HLA-A11(HLA-A * 1101) In some cases, the MHC class I heavy chain polypeptide of the TEP comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence shown in Figure 4A. In some cases, an HLA-A heavy chain polypeptide suitable for inclusion in a TEP comprises the amino acid sequence shown in Figure 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 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) The 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 cases, Cys-236 forms an interchain disulfide bond with Cys-12 of a variant β2M polypeptide that includes an R12C substitution; and D) 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 cases, Cys-84 forms an intrachain disulfide bond with Cys-139.

[0099] HLA-A24(HLA-A * 2402) In some cases, the MHC class I heavy chain polypeptide of the TEP comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence shown in Figure 5A. In some cases, an HLA-A heavy chain polypeptide suitable for inclusion in a TEP comprises the amino acid sequence shown in Figure 5A. The 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 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. 5B, in which amino acid 84 is Tyr and amino acid 236 is Ala; B) The 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 cases, 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 cases, Cys-84 forms an intrachain disulfide bond with Cys-139.

[0100] HLA-A33(HLA-A * 3303) In some cases, the MHC class I heavy chain polypeptide of the TEP comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence shown in FIG. 6A. In some cases, an HLA-A heavy chain polypeptide suitable for inclusion in a TEP comprises the amino acid sequence shown in FIG. 6A. The 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 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) The 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 cases, 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 cases, Cys-84 forms an intrachain disulfide bond with Cys-139.

[0101] 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 can 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 can be substituted (e.g., at aa12) by a cysteine ​​residue capable of forming an interchain disulfide bond with β2M. The boxes adjacent to residues 84, 139, and 236 indicate groups of five amino acids on either side of those six sets of five residues, designated aac1 ("amino acid cluster 1"), aac2 ("amino acid cluster 2"), aac3 ("amino acid cluster 3"), aac4 ("amino acid cluster 4"), aac5 ("amino acid cluster 5"), and aac6 ("amino acid cluster 6"), that may be replaced with one to five amino acids independently selected from (i) any naturally occurring amino acid, or (ii) any naturally occurring amino acid except proline or glycine.

[0102] Figures 7A, 8A, and 9A provide alignments of amino acid sequences of mature HLA-A, -B, and -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, and 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.

[0103] With reference to FIG. 7A , in some cases, 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 with Q, Q is replaced with N, and / or E is replaced with D); iii) aac3 (amino acid cluster 3) may be the amino acid sequence TAADM (SEQ ID NO: 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 with S, A is replaced with G, D is replaced with E, and / or M is replaced with L, V, or I); iv) aac4 (amino acid cluster 4) may be the amino acid sequence YNQSE (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., T is replaced with S, A is replaced with G, D is replaced with E, and / or M is replaced with L, V, or I); 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., 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 by E, T is replaced by S, or F is replaced by L, W, or Y).

[0104] With reference to FIG. 8A , in some cases, 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 with T or I, and / or L is replaced with A, and / or a second R is replaced with L, and / or G is replaced with R), ii) aac2 (amino acid cluster 2) may be the amino acid sequence YNQSE (SEQ ID NO: 916), or or a sequence in which one or two amino acids have been deleted or substituted with other naturally occurring amino acids (e.g., N is replaced with Q, Q is replaced with N, and / or E is replaced with D); iii) aac3 (amino acid cluster 3) may be the amino acid sequence TAADT (SEQ ID NO: 922) or a sequence in which one or two amino acids have been deleted or substituted with other naturally occurring amino acids (e.g., the first T is replaced with S, and / or A is replaced with G, and / or D is replaced with E); and / or 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., 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 substituted with other naturally occurring amino acids (e.g., D is replaced with E, and / or T is replaced with S, and / or R is replaced with K or H, and / or F is replaced with L, W, or Y).

[0105] With reference to FIG. 9A , in some cases, 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 with K, and / or L is replaced with A or I, and / or the second R is replaced with H, and / or G is replaced with T or S); ii) aac2 (amino acid cluster 2) may be the amino acid sequence YNQSE (SEQ ID NO: 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 with Q, Q is replaced with N, and / or E is replaced with D); iii) aac3 (amino acid cluster 3) may be the amino acid sequence 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 with S, and / or A is replaced with G, and / or D is replaced with E); 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 with G, and / or the first Q is replaced with N, and / or I is replaced with L, and / or the second Q is replaced with N or K); v) aac5 (amino acid cluster 5) may be 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., 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) aac7 (amino acid cluster 7) may be the amino acid sequence GDGTF (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.,D is replaced by E, and / or T is replaced by S, and / or F is replaced by L, W, or Y).

[0106] Nonclassical HLA-E, -F, and -G MHC class I heavy chains In some cases, the TEP comprises a non-classical MHC class I heavy chain polypeptide. Non-classical HLA heavy chain polypeptides, or portions thereof, that can be incorporated into the TEP include, but are not limited to, those of HLA-E, -F, and -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 (www(dot)ebi(dot)ac(dot)uk), which is part of the European Molecular Biology Laboratory (EMBL), and at the National Center for Biotechnology Information (www(dot)ncbi(dot)nlm(dot)nih(dot)gov).

[0107] The consensus sequences for these HLA-E, -F, and -G alleles, free of all or substantially all of the leader, transmembrane, and cytoplasmic sequences, are provided in Figure 10. Figure 10 provides the consensus sequences for each of HLA-E, -F, and -G, with the variable aa positions shown as consecutively numbered "X" residues, with positions aa 84, 139, and 236 double underlined.

[0108] 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 11A-11D, where Figure 11A shows the amino acid sequence of HLA-E * FIG. 11B provides the amino acid sequence of HLA-E 01:01 (wild type), with Y84C and A2346C substitutions. * FIG. 11C provides the amino acid sequence of HLA-E 01:01. * FIG. 11D provides the amino acid sequence of HLA-E 01:03 (wild type), with Y84C and A2346C substitutions. * 01:03. In some cases, therefore, the MHC class I heavy chain polypeptide of the TEP comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence shown in Figure 11A, 11B, 11C, or 11D.

[0109] 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 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 * Among 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 12A-12D. * FIG. 12B provides the amino acid sequence of HLA-G 01:01 (wild type), with Y84C and A2346C substitutions. * 12C provides the amino acid sequence of HLA-G 01:01; * FIG. 12D provides the amino acid sequence of HLA-G 01:04 (wild type), with Y84C and A2346C substitutions. *01:04. In some cases, therefore, the MHC class I heavy chain polypeptide of the TEP comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence shown in Figure 12A, 12B, 12C, or 12D.

[0110] Beta-2 microglobulin The β2-microglobulin (β2M) polypeptide of the TEP can be a human β2M polypeptide, a non-human primate β2M polypeptide, a mouse β2M polypeptide, etc. In some cases, the β2M polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence set forth in Figure 1A (wild-type human β2M). In some cases, the β2M polypeptide present in the TEP comprises the amino acid sequence shown in Figure 1A (wild-type human β2M).

[0111] In some cases, the MHC polypeptide present in the TEP 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 TEP. 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.

[0112] In some cases, the β2M polypeptide present in the TEP comprises an amino acid sequence having 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-native Cys at position 12 as a result of an R12C substitution. In some cases, the β2M polypeptide present in the TEP comprises the amino acid sequence shown in Figure 1B.

[0113] Immunomodulating Polypeptides In some cases, the MOD ("MOD") optionally present in the TEP is a wild-type ("wt") MOD. As noted above, in other cases, the MOD present in the TEP is a variant of the wtMOD that has reduced affinity for the co-MOD compared to the affinity of the corresponding wild-type MOD for the co-MOD. 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.

[0114] As discussed above, a MOD can include a variant of a wtMOD 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 TEP can bind to the co-MOD with an affinity that is at least 10% lower, at least 15% lower, at least 20% lower, at least 25% lower, at least 30% lower, at least 35% lower, at least 40% lower, at least 45% lower, at least 50% lower, at least 55% lower, at least 60% lower, at least 65% lower, at least 70% lower, at least 75% lower, at least 80% lower, at least 85% lower, at least 90% lower, at least 95% lower, or greater than 95% lower than the affinity of the corresponding wild-type MOD for the co-MOD.

[0115] Exemplary pairs of MODs and their co-MODs include, but are not limited to, those set forth in Table 2 below.

[0116] (Table 2) TIFF2025515575000004.tif140128

[0117] One or more MODs may be present in a TEP at any of a variety of locations, for example, in some cases, a MOD (or 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, i.e., 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 the peptide epitope.

[0118] Immunomodulatory polypeptides and variants, including lower affinity variants, of PD-L1, CD80, CD86, 4-1BBL, and IL-2 are described in the published literature, such as, for example, published International Publication No. WO2020132138A1, and International Publication No. WO2019 / 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 International Publication No. WO2020132138A1, and paragraphs

[0157] to

[0352] of International Publication No. WO2019 / 051091.

[0119] 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.

[0120] The IL-2 receptor is a heterotrimeric polypeptide that, in some cases, includes an alpha chain (IL-2Rα, also called CD25), a beta chain (IL-2Rβ, also called CD122), and a gamma chain (IL-2Rγ, also called CD132). The amino acid sequences of human IL-2, human IL-2Rα, IL2Rβ, and IL-2Rγ are known. See, for example, published International Publication No. WO2020132138A1, and International Publication No. WO2019 / 051091, supra. 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 cases, the human IL-2Rα, human IL-2Rβ, and human IL-2Rγ polypeptides are mature forms (lacking the signal peptide).

[0121] In some cases, 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 cases, the IL-2 variant MODs of the present disclosure exhibit reduced binding to IL-2Rβ, such that the IL-2 variant MOD exhibits reduced overall affinity for IL-2R. In some cases, 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, such that the IL-2 variant polypeptide exhibits reduced overall affinity for IL-2R. For example, IL-2 variants having substitutions at H16 and F42 exhibit 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. TEPs 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 TEP, 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 TEP. That is, binding of the IL-2 variant MOD to its costimulatory polypeptide on a T cell is substantially driven by binding of the MHC epitope portion, rather than binding of IL-2. In some cases, the IL-2 variant MOD of the present disclosure exhibits reduced binding to IL-2Rγ. This reduced binding to IL-2Rγ may be in addition to reduced binding to IL-2Rα, and / or IL-2Rβ.

[0122] Thus, suitable IL-2 variant MODs include polypeptides that include an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the wild-type IL-2 amino acid sequence shown in Figure 12A, as well as polypeptides that have 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 cases, 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 that includes the wild-type IL-2 amino acid sequence shown in Figure 12A. For example, in some cases, the variant IL-2 polypeptide binds to IL-2R with a binding affinity that is at least 10% lower, at least 15% lower, at least 20% lower, at least 25% lower, at least 30% lower, at least 35% lower, at least 40% lower, at least 45% lower, at least 50% lower, at least 55% lower, at least 60% lower, at least 65% lower, at least 70% lower, at least 75% lower, at least 80% lower, at least 85% lower, at least 90% lower, at least 95% lower, or more than 95% lower than the binding affinity of an IL-2 polypeptide comprising a wild-type IL-2 amino acid sequence shown in FIG. 12A to IL-2R (e.g., an IL-2R including a polypeptide comprising an amino acid sequence shown in FIGS. 12B-12D, or a mature form of an amino acid sequence shown in FIGS. 12B-12D), when assayed under the same conditions.

[0123] In some cases, a suitable variant IL-2 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 following amino acid sequence: TIFF2025515575000005.tif17149, i.e., the variant IL-2 polypeptide, has the amino acid sequence of wild-type IL-2, but with H16A and F42A substitutions (shown in bold). Alternatively, the sequence described above can be employed, but with substitutions other than Ala at H16 and / or F42, e.g., H16T can be employed in place of H16A. In some cases, the variant IL-2 polypeptide present in the TEP comprises the following amino acid sequence: TIFF2025515575000006.tif17152. In some cases, the variant IL-2 polypeptide present in the TEP comprises the following amino acid sequence: TIFF2025515575000007.tif23153. In some cases, the TEP comprises two copies of such a variant IL-2 polypeptide.

[0124] As a further example of a MOD, in some cases, a MOD present in a TEP is a 4-1BBL polypeptide. In some cases, the 4-1BBL polypeptide of the TEP comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following 4-1BBL amino acid sequence: DPAGLLDLRQG MFAQLVAQNV LLIDGPLSWY SDPGLAGVSL TGGLSYKEDT KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPA (SEQ ID NO: 926).

[0125] In some cases, the MOD present in the TEP is a CD80 polypeptide. In some cases, the CD80 polypeptide of the TEP comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following CD80 amino acid sequence: VIHVTK EVKEVATLSC GHNVSVEELA QTRIYWQKEK KMVLTMMSGD MNIWPEYKNR TIFDITNNLS IVILALRPSD EGTYECVVLK YEKDAFKREH LAEVTLSVKA DFPTPSISDF EIPTSNIRRI ICSTSGGFPE PHLSWLENGE ELNAINTTVS QDPETELYAV SSKLDFNMTT NHSFMCLIKY GHLRVNQTFN WNTTKQEHFP DN (SEQ ID NO: 927).

[0126] In some cases, the MOD present in the TEP is a CD86 polypeptide. In some cases, the CD86 polypeptide of the TEP comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following CD86 amino acid sequence: APLKIQAYFNETADLPCQFANSQNQSLSELVVFWQDQENLVLNEVYLGKEKFDSVHSKYMNRTSFDSDSWTLRLHNLQIKDKGLYQCIIHHKKPTGMIRIHQMNSELSVLANFSQPEIVPISNITENVYINLTCSSIHGYPEPKKMSVLLRTKNSTIEYDGIMQKSQDNVTELYDVSISLSVSFPDVTSNMTIFCILETDKTRLLSSPFSIELEDPQPPPDHIP (SEQ ID NO: 928).

[0127] Anti-CD28 Polypeptides In some cases, the MOD present in the TEP is an anti-CD28 antibody (e.g., an anti-CD28 scFv, an anti-CD28 nanobody, an anti-CD28 diabody, etc.). The ability of anti-CD28 antibodies to act as superagonists, agonists, or antagonists of CD28 activity has been described. See, e.g., 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.

[0128] Thus, in some cases, the TEP comprises an anti-CD28 antibody as at least one of the one or more MODs. In some cases, when the TEP is a heterodimer comprising two different TEPs linked via a mutually specific binding sequence (as discussed below), the TEP may comprise two different MODs, e.g., a) an anti-CD28 antibody, and b) one or more IL-2 polypeptides. In some cases, the TEP comprises, as one or more MODs, a) an anti-CD28 antibody, and b) one or more IL-2 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 cases, the TEP comprises, as one or more MODs, a) an anti-CD28 antibody, and b) two copies of an IL-2 polypeptide comprising an H16A and F42A substitution. Examples of anti-CD28 VL and VH polypeptides are provided below. In some cases, the TEP comprises, as one or more MODs, a) an anti-CD28 antibody, and b) two copies of an IL-2 polypeptide comprising an H16T and F42A substitution. In some cases, the TEP comprises, as one or more MODs, a) an anti-CD28 antibody, and b) one, two, or three copies of a 4-1BBL polypeptide. In some cases, the TEP comprises, as one or more MODs, a) an anti-CD28 antibody, and b) three copies of a 4-1BBL polypeptide comprising a wild-type amino acid sequence. In some cases, the TEP comprises, as one or more MODs, a) an anti-CD28 antibody, and b) three copies of a variant 4-1BBL polypeptide. The two different MODs can be at any of a number of different positions on the first or second TEP of a TEP heterodimer, for example, in some cases, the anti-CD28 antibody is at the C-terminus of the first TEP and the second MOD (e.g., a variant IL-2 polypeptide, a 4-1BBL polypeptide, etc.) is at the N-terminus of the second TEP.As another example, in some cases, an anti-CD28 antibody is at the N-terminus of a first TEP and a second MOD (e.g., a variant IL-2 polypeptide, a 4-1BBL polypeptide, etc.) is at the C-terminus of a second TEP.

[0129] In some instances, an anti-CD28 antibody suitable for inclusion as a MOD in a TEP comprises a) a VL CDR1, VL CDR2, and VL CDR3 present in a light chain variable region (VL) comprising the following amino acid sequence: QWYQQKPGQPPKLLIFAASNVESGVPARFSGSGSGTNFSLNIHPVDEDDVAMYFCQQSRKVPYTFGGGTKLEIKR (SEQ ID NO: 929), and b) a VH CDR1, CDR2, and CDR3 present in a heavy chain variable region (VH) comprising the following amino acid sequence: QVKLQQSGPGLVTPSQSLSITCTVSGFSLSDYGVHWVRQSPGQGLEWLGVIWAGGGTNYNSALMSRKSISKDNSKSQVFLKMNSLQADDTAVYYCARDKGYSYYYSMDYWGQGTTVTVSS (SEQ ID NO: 930). 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 cases, 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).

[0130] In some instances, an anti-CD28 antibody suitable for inclusion as a MOD in a TEP comprises: a) 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 the following amino acid sequence: QWYQQKPGQPPKLLIFAASNVESGVPARFSGSGSGTNFSLNIHPVDEDDVAMYFCQQSRKVPYTFGGGTKLEIKR (SEQ ID NO: 929); and b) a VL region comprising: The VH region 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 below: QVKLQQSGPGLVTPSQSLSITCTVSGFSLSDYGVHWVRQSPGQGLEWLGVIWAGGGTNYNSALMSRKSISKDNSKSQVFLKMNSLQADDTAVYYCARDKGYSYYYSMDYWGQGTTVTVSS (SEQ ID NO: 930).

[0131] In some instances, an anti-CD28 antibody suitable for inclusion as a MOD in a TEP comprises, in order from N-terminus to C-terminus, a) a VL region that comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: QWYQQKPGQPPKLLIFAASNVESGVPARFSGSGSGTNFSLNIHPVDEDDVAMYFCQQSRKVPYTFGGGTKLEIKR (SEQ ID NO: 929); b) a peptide linker and c) 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 the following amino acid sequence: QVKLQQSGPGLVTPSQSLSITCTVSGFSLSDYGVHWVRQSPGQGLEWLGVIWAGGGTNYNSALMSRKSISKDNSKSQVFLKMNSLQADDTAVYYCARDKGYSYYYSMDYWGQGTTVTVSS (SEQ ID NO: 930). In some cases, the peptide linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO: 934), where n is an integer between 1 and 10 (e.g., n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). In some cases, the peptide linker comprises the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 875) and has a length of 15 amino acids.

[0132] In some instances, an anti-CD28 antibody suitable for inclusion in a MOD in a TEP comprises, in order from N-terminus to C-terminus, a) a VH region that comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: QVKLQQSGPGLVTPSQSLSITCTVSGFSLSDYGVHWVRQSPGQGLEWLGVIWAGGGTNYNSALMSRKSISKDNSKSQVFLKMNSLQADDTAVYYCA RDKGYSYYYSMDYWGQGTTVTVSS (SEQ ID NO: 930), b) a peptide linker, and c) 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 the following amino acid sequence: QWYQQKPGQPPKLLIFAASNVESGVPARFSGSGSGTNFSLNIHPVDEDDVAMYFCQQSRKVPYTFGGGTKLEIKR (SEQ ID NO: 929). In some cases, the peptide linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO: 934), where n is an integer between 1 and 10 (e.g., n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). In some cases, the peptide linker comprises the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 875) and has a length of 15 amino acids.

[0133] Location of immunomodulatory polypeptides and TTP When a TEP includes at least one MOD, the one or more MODs may be present in the TEP at any of a variety of positions. For example, the MOD 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, the TTP may be present in the TEP at any of a variety of positions. Non-limiting examples of various MOD positions and various TTP positions are shown in Figures 18A-18D. As mentioned above, the MOD is optional, and thus each of the constructs in Figures 18A-D may also not include a MOD.

[0134] Scaffolding components The TEP comprises a scaffold component, such as an Ig Fc polypeptide or other suitable polypeptide, or carrier capable of displaying both the pHLA and the TTP.

[0135] 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 an 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. For suitable XTEN polypeptides, see, e.g., WO 2009 / 023270, WO 2010 / 091122, WO 2007 / 103515, U.S. Patent Application Publication No. 2010 / 0189682, U.S. Patent No. 2009 / 0092582, and Schellenberger et al. (2009) Nat Biotechnol. 27:1186). Suitable albumin polypeptides include, for example, human serum albumin.

[0136] Other suitable scaffold components capable of displaying both pHLA and TTP include carriers such as lipid vesicles (e.g., liposomes) or micelles, nanoparticles, PEGylated proteins (including site-specific PEGylation), fibronectin-based scaffold proteins, or artificial antigen-presenting cells such as engineered red blood cells and enucleated cells (e.g., platelets).

[0137] A suitable scaffold polypeptide is, in some cases, a polypeptide that extends half-life. Thus, in some cases, a suitable scaffold polypeptide increases the in vivo half-life (e.g., serum half-life) of the TEP compared to a control TEP that lacks the scaffold polypeptide. For example, in some cases, the scaffold polypeptide increases the in vivo half-life (e.g., serum half-life) of the TEP 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 TEP that lacks the scaffold polypeptide. By way of example, in some cases, the Fc polypeptide increases the in vivo half-life (e.g., serum half-life) of the TEP by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 50%, at least about 2-fold, at least about 2.5-fold, at least about 5-fold, at least about 10-fold, at least about 25-fold, at least about 50-fold, at least about 100-fold, or more than 100-fold, as compared to a control TEP lacking the Fc polypeptide.

[0138] Fc Polypeptides In some cases, the TEP comprises an Ig Fc polypeptide. The Ig Fc polypeptide is also referred to herein as "Fc polypeptide". The Ig Fc polypeptide of the TEP 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. Variants include naturally occurring variants, non-naturally occurring variants, and combinations 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 cell-mediated cytotoxicity (ADCC). See, for example, the variant human IgG1 Fc polypeptide in Figure 2B and Figure 2D.

[0139] In some cases, the Fc polypeptide present in the TEP comprises an amino acid sequence having at least 70%, 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. In some cases, the Ig Fc polypeptide does not include a C-terminal Lys (e.g., does not include a C-terminal Lys present in a wild-type Ig Fc polypeptide).

[0140] In some cases, the Fc polypeptide present in the TEP is an IgG1 Fc polypeptide or a variant of an IgG1 Fc polypeptide. For example, in some cases, the Fc polypeptide present in the TEP comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the human IgG1 Fc polypeptide shown in FIG. 2A. As another example, in some cases, the Fc polypeptide present in the TEP comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the Fc polypeptide shown in FIG. 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 cases, the Ig Fc polypeptide has an Ala at position 77. In some cases, the Fc polypeptide present in the TEP comprises the amino acid sequence shown in Figure 2A. In some cases, the Fc polypeptide present in the TEP comprises the amino acid sequence shown in Figure 2B.

[0141] In some cases, the Fc polypeptide present in the TEP 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 cases, the Fc polypeptide present in the TEP comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the human IgG1 Fc polypeptide shown in Figure 2C, wherein the Ig Fc polypeptide comprises a Glu at position 136 and a Met at position 138. As another example, in some cases, the Fc polypeptide present in the TEP comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the 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 TEP comprises the amino acid sequence shown in FIG. 2C. In some cases, the Fc polypeptide present in the TEP comprises the amino acid sequence shown in FIG. 2D.

[0142] In some cases, the Fc polypeptide present in the TEP comprises the amino acid sequence shown in FIG. 2E (human IgG1 Fc with L234F, L235E, and P331S substitutions, 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 cases, the Fc polypeptide present in the TEP comprises the amino acid sequence shown in FIG. 2F, including an N279A substitution (amino acid sequence of N77A shown in FIG. 2F). The substitution at N297 leads to the removal of carbohydrate modifications, 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 cases, the Fc polypeptide present in the TEP 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), and C1q binding and CDC function are substantially or completely eliminated.

[0143] In some cases, the Fc polypeptide comprises an amino acid sequence that has at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to a human IgG2 Fc polypeptide shown in FIG. 2G, e.g., the Fc polypeptide comprises an amino acid sequence that has at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence 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 cases, the Fc polypeptide comprises an amino acid sequence that has at least 70%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to a human IgG3 Fc polypeptide shown in FIG. 2H, e.g., the Fc polypeptide comprises an amino acid sequence that has at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence 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 cases, the Fc polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to a human IgM Fc polypeptide shown in FIG. 2J, e.g., the Fc polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to amino acids 1-276 of the human IgM Fc polypeptide shown in FIG. 2J.In some cases, the Fc polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the human IgA Fc polypeptide shown in FIG. 2K, e.g., the Fc polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to amino acids 1-234 of the human IgA Fc polypeptide shown in FIG. 2K.

[0144] Linker As discussed above, the TEP can include one or more independently selected peptide linkers, i.e., the linker comprises a contiguous stretch of two or more amino acids, and the one or more linkers are between one or more components of the TEP. For example, the TEP can include one or more independently selected peptide linkers, i.e., the linker comprises a contiguous stretch of two or more amino acids, and the one or more linkers are between one or more of the following: i) an MHC class I heavy chain polypeptide and an Ig Fc polypeptide, ii) a MOD and an MHC class I polypeptide, iii) a first MOD and a second MOD, iv) a peptide and an MHC class I polypeptide, v) a peptide and a β2M polypeptide, vi) a TTP and an MHC class I heavy chain polypeptide, vii) a TTP and an Ig Fc polypeptide, and viii) a TTP and a MOD.

[0145] As used herein, the phrase "optional peptide linker between any two of the components of the TEP" refers to a peptide linker between any two adjacent polypeptides in the TEP. For example, as used herein, the phrase "optional peptide linker between any two of the components of the TEP" refers to a peptide linker between one or more of the following: i) MHC class I heavy chain polypeptide and Ig Fc polypeptide, ii) MOD and MHC class I polypeptide, iii) first MOD and second MOD, iv) peptide and MHC class I polypeptide, v) peptide and β2M polypeptide, vi) TTP and MHC class I heavy chain polypeptide, vii) TTP and Ig Fc polypeptide, and vii) TTP and MOD. As discussed below, the linker can be a) a flexible peptide linker, including a short flexible peptide linker, or b) a rigid peptide linker.

[0146] Suitable linkers (also referred to as "spacers") can be readily selected and can be any of a number of suitable lengths, such as 1 to 25 amino acids, 3 to 20 amino acids, 2 to 15 amino acids, 3 to 12 amino acids, 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 cases, 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.

[0147] 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 an integer of at least 1 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, where both Gly and Ser are relatively unstructured and therefore can function as neutral tethers between components. Glycine polymers can be used, where 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.

[0148] Exemplary flexible peptide linkers include, for example, (GGGGS) (also referred to as a "G4S" linker). n (SEQ ID NO:934), where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some cases, the linker is 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 cases, the linker comprises the amino acid sequence (GGGGS) n (SEQ ID NO:947), wherein n is 2. In some cases, the linker comprises the amino acid sequence (GGGGS) n (SEQ ID NO:875), wherein n is 3. In some cases, the linker comprises the amino acid sequence (GGGGS) n (SEQ ID NO:876), wherein n is 4. In some cases, the linker comprises the amino acid sequence (GGGGS) n(SEQ ID NO:948), where n is 7. In some cases, the linker comprises the amino acid sequence AAAGG (SEQ ID NO:874). A linker having the amino acid sequence AAAGG (SEQ ID NO:874) is also suitable. In the TEP of the present disclosure, the β2M polypeptide comprises the amino acid sequence (GGGGS) n (SEQ ID NO:934) may be connected to the MHC heavy chain polypeptide by a linker, where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, for example, n=3, n=4, or n=7.

[0149] As used in this disclosure, "short flexible peptide linker" means a flexible peptide linker that contains less 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.

[0150] Rigid Peptide Linkers In some cases, 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 can be interposed when either MOD or TTP is at position 1 and / or position 3 as 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 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.

[0151] Examples of rigid peptide linkers include, for example, (EAAAK) n (SEQ ID NO: 949), A(EAAAK) n A (SEQ ID NO: 950), A (EAAAK) n ALEA(EAAAK) n A (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 include EAAAK (SEQ ID NO: 956), (EAAAK) 2 (SEQ ID NO:958), (EAAAK) 3 (SEQ ID NO:959), A(EAAAK) 4 ALEA(EAAAK) 4A (SEQ ID NO:960), and AEAAAKEAAAKA (SEQ ID NO:961). Non-limiting examples of suitable rigid peptide linkers containing (AP)n include PAPAP (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:966, 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").

[0152] Cysteine-Containing Linkers As described above, in some cases, the linker peptide in the TEP can form an intrachain disulfide bond with a cysteine ​​residue present elsewhere in the polypeptide chain. For example, as described above, in some cases, the TEP, or a dimerized TEP, such as a homodimer or heterodimer, comprises a linker between the peptide epitope and the β2M polypeptide, which comprises a cysteine ​​residue that forms an intrachain disulfide bond with a cysteine ​​residue in the MHC class I heavy chain polypeptide present in the TEP. For example, in some cases, the TEP, or a dimerized TEP, such as a homodimer or heterodimer, comprises a cysteine-containing linker between the peptide epitope and the β2M polypeptide, and the cysteine ​​residue in the linker forms an intrachain 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 TEP.

[0153] In some cases, the peptide linker between the peptide and the β2M polypeptide comprises the amino acid sequence GCGGS (SEQ ID NO: 977). In some cases, the peptide linker between the 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 cases, the peptide linker between the peptide and the β2M polypeptide comprises the amino acid sequence GCGGS (GGGGS) n (SEQ ID NO: 882), wherein n is 2.

[0154] In some cases, the peptide linker between the peptide and the β2M polypeptide comprises the amino acid sequence CGGGS (SEQ ID NO: 979). In some cases, 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, for example, 1, 2, or 3.

[0155] In some cases, the peptide linker between the peptide and the β2M polypeptide comprises the amino acid sequence GGCGS (SEQ ID NO: 981). In some cases, 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, for example, 1, 2, or 3.

[0156] In some cases, the peptide linker between the peptide and the β2M polypeptide comprises the amino acid sequence GGGCS (SEQ ID NO: 983). In some cases, 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, for example, 1, 2, or 3.

[0157] In some cases, the peptide linker between the peptide and the β2M polypeptide comprises the amino acid sequence GGGGC (SEQ ID NO: 985). In some cases, 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, for example, 1, 2, or 3.

[0158] Dimerized TEP As mentioned above, in some cases, the TEP can form a dimer. That is, the present disclosure provides a polypeptide comprising a dimer of two TEPs. Thus, the present disclosure provides a protein that is a dimerized TEP comprising two TEPs 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 TEP and an Ig Fc polypeptide in a second TEP. 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 cell-mediated cytotoxicity (ADCC) (e.g., the human IgG1 Fc polypeptide of FIG. 2B or FIG. 2D). When the TEP comprises an Ig Fc polypeptide, the TEP will typically self-assemble into a dimer by spontaneously forming a disulfide bond with the IgG1 Fc polypeptide of another TEP. Thus, for example, the Ig Fc polypeptides in the first and second TEPs can be linked to each other by one or more disulfide bonds. Often, the two TEPs will be identical in amino acid sequence to each other and contain Ig Fc polypeptides that spontaneously form one or more disulfide bonds, thereby forming a dimerized TEP that is a homodimer.

[0159] Thus, the present disclosure provides a protein comprising a) a first TEP and b) a second TEP that may optionally be identical (e.g., identical in amino acid sequence) to the first TEP, wherein the first and second TEPs are covalently linked to each other. The covalent link may be a disulfide bond between an Ig Fc polypeptide in the first TEP and an Ig Fc polypeptide in the second TEP. When the first and second TEPs of a dimeric protein are identical to each other in amino acid sequence, such a dimeric protein may be referred to as a "homodimeric TEP."

[0160] Optionally, the Ig Fc polypeptide of each TEP may include an interspecies dimerization sequence, such as, for example, a "knobs-in-holes" sequence that allows for selective dimerization of two different TEPs (e.g., TEPs that differ from each other in amino acid sequence). The interspecies binding sequences favor the formation of heterodimers with their cognate polypeptide sequences (i.e., the interspecies sequences and their corresponding interspecies sequences), especially 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 cases, a dimerized TEP may be a heterodimer comprising two TEP chains that are not identical in amino acid sequence, and such a dimerized TEP may be referred to as a "heterodimeric TEP."

[0161] Interspecies dimerization sequences may also be used to allow the TEP to be linked to non-TEP molecules that can provide additional functionality to the TEP. For example, the TEP may be linked to a molecule that includes a polypeptide that binds to a cancer associated antigen (e.g., an antibody such as an scFv, or a binding fragment thereof), thereby allowing the TEP to localize to tissues that contain the cancer associated antigen.

[0162] As a non-limiting example, a heterodimeric TEP comprising a first TEP and a second TEP, the first TEP and the second TEP differing from each other in amino acid sequence, may comprise a) a first TEP comprising a first peptide epitope and b) a second TEP comprising a second peptide epitope, the first peptide epitope being a first SARS-CoV-2 peptide, the second peptide epitope comprising a second SARS-CoV-2 peptide that differs in amino acid sequence from the first SARS-CoV-2 peptide epitope. Furthermore, the first and second TEPs may comprise the same or different TTPs. As an example, the first SARS-CoV-2 peptide may be YLQPRTFLL (SEQ ID NO: 218) and the second SARS-CoV-2 peptide may be VMPLSAPTL (SEQ ID NO: 914). In some cases, the first TEP and the second TEP have the same MHC class I heavy chain polypeptide (e.g., the MHC class I heavy chain polypeptide in the first TEP has the same amino acid sequence as the MHC class I heavy chain polypeptide in the second TEP). In other cases, the first TEP and the second TEP have different MHC class I heavy chain polypeptides (e.g., the MHC class I heavy chain polypeptide in the first TEP has a different amino acid sequence than the MHC class I heavy chain polypeptide in the second TEP). For example, in some cases, the MHC class I heavy chain polypeptide of the first TEP is HLA-A * 0201 polypeptide (e.g., HLA-A shown in FIG. 3A *11A ) and the MHC class I heavy chain polypeptide of the second TEP may be an HLA-E polypeptide (e.g., a polypeptide comprising amino acids having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the HLA-E amino acid sequence shown in FIG. 11A ).

[0163] As an example, in some cases, the heterodimeric TEP comprises a) the peptide epitope YLQPRTFLL (SEQ ID NO: 218) and * 0201 polypeptide (e.g., HLA-A shown in FIG. 3A * 11A , and a) a peptide epitope VMPLSAPTL (SEQ ID NO:914) and a second TEP comprising an HLA-E polypeptide (e.g., a polypeptide comprising amino acids having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the HLA-E amino acid sequence shown in FIG. 11A ).

[0164] Tumor targeting polypeptide (TTP) As mentioned above, the TEPs of the present disclosure include tumor targeting polypeptides (TTPs), i.e., polypeptides specific for cancer-associated epitopes. A "cancer-associated" epitope is an epitope present in a cancer-associated antigen. In some cases, the TTP is an antibody. In some cases, the TTP is a single-chain T-cell receptor (scTCR).

[0165] target In some cases, the TTP present in the TEP 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 may be a cancer-associated peptide (e.g., a peptide fragment of a cancer-associated antigen).

[0166] Cancer-associated antigens Cancer-associated antigens that can be targeted by tumor targeting polypeptides present in TEP include, for example, NY-ESO (New York Esophageal Squamous Cell Carcinoma 1), MART-1 (Melanoma Antigen Recognized by T Cells 1, also known as Melan-A), HPV (Human Papillomavirus) E6, BCMA (B-cell maturation antigen), CD123, CD133, CD171, CD19, CD20, CD22, CD30, CD33, 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), disialo These include ganglioside 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).

[0167] Cancer-associated antigens present in the TEP that can be targeted by TTP include 17-1A antigen, alpha-fetoprotein (AFP), alpha-actinin-4, A3, antigen specific for the A33 antibody, ART-4, B7, Ba 733, BAGE, bcl-2, bcl-6, BCMA, BrE3-antigen, CA125, CAMEL, CAP-1, carbonic anhydrase IX (CAIX), CASP-8 / m, CCL19, CCL21, CD1, CD1a, CD2, CD3, CD4, CD5, CD8, CD11A, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD29, CD30, CD32b, CD33, CD3 7, CD38, CD40, CD40L, CD44, CD45, CD46, CD52, CD54, CD55, CD59, CD64, CD66a-e, CD67, CD70, CD70L, CD74, CD79a, CD79b , CD80, CD83, CD95, CD123, CD126, CD132, CD133, CD138, CD147, CD154, CD171, CDC27, CDK-4 / m, CDKN2A, CEA, CEACAM5, CE ACAM6, 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 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.

[0168] In some cases, 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 cases, 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.

[0169] In some cases, 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.

[0170] Peptide / HLA complexes In some cases, the target of the TTP is a peptide / HLA (pHLA) complex on the surface of a cancer cell, and the peptide may be a cancer-associated peptide (e.g., a peptide fragment of a cancer-associated antigen). Cancer-associated peptides are known in the art. In some cases, the cancer-associated peptide is a peptide / HLA (pHLA) complex on the surface of a cancer cell. *It binds to the HLA complex, which contains the 0201 heavy chain and the β2M polypeptide.

[0171] 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 * Binds to HLA complexes containing HLA heavy chains such as 1502.

[0172] In some instances, 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 melanoma antigen family B, 3 (MEGA) polypeptide. 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 (erythroblast transformation specific transcription factor; 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 (SSX2 ) polypeptide, X antigen family member 1 (XAGE1) polypeptide, B7 homolog 3 (B7H3, also known as CD276) polypeptide, legumain polypeptide (LGMN1, also known as asparaginyl endopeptidase), tyrosine kinase with Ig and EGF homology domains-2 (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 papillomavirus (HPV) antigen, alpha-fetoprotein (AFP) antigen, and Wilms' tumor-1 (WT1) antigen.

[0173] For example, in some cases, the TTP present in the TEP may be a) a TTP that binds to an HLA heavy chain (e.g., HLA-A * 0201 heavy chain, or HLA-A *a) a WT-1 peptide bound to an HLA complex comprising a class I HLA heavy chain and a β2M polypeptide; b) a 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.

[0174] In some cases, the cancer associated peptide is a peptide of a mesothelin polypeptide that has at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following mesothelin amino acid sequence: LAGE TGQEAAPLDG VLANPPNISS LSPRQLLGFP CAEVSGLSTE RVRELAVALA QKNVKLSTEQ LRCLAHRLSE PPEDLDALPL DLLLFLNPDA FSGPQACTRF FSRITKANVD LLPRGAPERQ RLLPAALACW GVRGSLLSEA DVRALGGLAC DLPGRFVAES AEVLLPRLVS CPGPLDQDQQ EAARAALQGG GPPYGPPSTW SVSTMDALRG LLPVLGQPII RSIPQGIVAA WRQRSSRDPS WRQPERTILR PRFRREVEKT ACPSGKKARE IDESLIFYKK WELEACVDAA LLATQMDRVN AIPFTYEQLD VLKHKLDELY PQGYPESVIQ HLGYLFLKMS PEDIRKWNVT SLETLKALLE VNKGHEMSPQ VATLIDRFVK GRGQLDKDTL DTLTAFYPGY LCSLSPEELS SVPPSSIWAV RPQDLDTCDP RQLDVLYPKA RLAFQNMNGS EYFVKIQSFL GGAPTEDLKA LSQQNVSMDL ATFMKLRTDA VLPLTVAEVQ KLLGPHVEGL KAEERHRPVR DWILRQRQDD LDTLGLGLQG GIPNGYLVLD LSMQEALSGT PCLLGPGPVL TVLALLLAST LA (SEQ ID NO: 987). For example, the mesothelin peptides present in the pHLA complex can be: i) KLLGPHVEGL (SEQ ID NO: 988), ii) AFYPGYLCSL (SEQ ID NO: 989) (HLA-A * 2402 / β2M), 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).

[0175] In some cases, the cancer associated peptide is a peptide of a Her2 polypeptide having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following Her2 (receptor tyrosine-protein kinase erbB2) amino acid sequence: MELAALCRWG LLLALLPPGA ASTQVCTGTD MKLRLPASPE THLDMLRHLY QGCQVVQGNL ELTYLPTNAS LSFLQDIQEV QGYVLIAHNQ VRQVPLQRLR IVRGTQLFED NYALAVLDNG DPLNNTTPVT GASPGGLREL QLRSLTEILK GGVLIQRNPQ LCYQDTILWK DIFHKNNQLA LTLIDTNRSR ACHPCSPMCK GSRCWGESSE DCQSLRTVC AGGCARCKGP LPTDCCHEQC AAGCTGPKHS DCLACLHFNH SGICELHCPA LVTYNTDTFE SMPNPEGRYT FGASCVTACP YNYLSTDVGS CTLVCPLHNQ EVTAEDGTQR CEKCSKPCAR VCYGLGMEHL REVRAVTSAN IQEFAGCKKI FGSLAFLPES FDGDPASNTA PLQPEQLQVF ETLEEITGYL YISAWPDSLP DLSVFQNLQV IRGRILHNGA YSLTLQGLGI SWLGLRSLRE LGSGLALIHH NTHLCFVHTV PWDQLFRNPH QALLHTANRP EDECVGEGLA CHQLCARGHC WGPGPTQCVN CSQFLRGQEC VEECRVLQGL PREYVNARHC LPCHPECQPQ NGSVTCFGPE ADQCVACAHY KDPPFCVARC PSGVKPDLSY MPIWKFPDEE GACQPCPINC THSCVDLDDK GCPAEQRASP LTSIISAVVG ILLVVVLGVV FGILIKRRQQ KIRKYTMRRL LQETELVEPL TPSGAMPNQA QMRILKETEL RKVKVLGSGA FGTVYKGIWI PDGENVKIPV AIKVLRENTS PKANKEILDE AYVMAGVGSP YVSRLLGICL TSTVQLVTQL MPYGCLLDHV RENRGRLGSQ DLLNWCMQIA KGMSYLEDVR LVHRDLAARN VLVKSPNHVK ITDFGLARLL DIDETEYHAD GGKVPIKWMA LESILRRRFT HQSDVWSYGVTVWELMTFGA KPYDGIPARE IPDLLEKGER LPQPPICTID VYMIMVKCWM IDSECRPRFR ELVSEFSRMA RDPQRFVVIQ NEDLGPASPL DSTFYRSLLE DDDMGDLVDA EEYLVPQQGF FCPDPAPGAG GMVHHRHRSS STRNM (SEQ ID NO: 999).

[0176] In some cases, the cancer associated peptide is a peptide of a BCMA polypeptide that has at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following BCMA amino acid sequence: MLQMAGQCSQ NEYFDSLLHA CIPCQLRCSS NTPPLTCQRY CNASVTNSVK GTNAILWTCL GLSLIISLAV FVLMFLLRKI SSEPLKDEFK NTGSGLLGMA NIDLEKSRTG DEIILPRGLE YTVEECTCED CIKSKPKVDS DHCFPLPAME EGATILVTTK TNDYCKSLPA ALSATEIEKS ISAR (sequence number 1000).

[0177] In some instances, the cancer associated peptide is a peptide of a WT-1 polypeptide that has at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following WT-1 amino acid sequence: MDFLLLQDPA STCVPEPASQ HTLRSGPGCL QQPEQQGVRD PGGIWAKLGA AEASAERLQG RRSRGASGSE PQQMGSDVRD LNALLPAVPS LGGGGGCALP VSGAAQWAPV LDFAPPGASA YGSLGGPAPP PAPPPPPPPP PHSFIKQEPS WGGAEPHEEQ CLSAFTVHFS GQFTGTAGAC RYGPFGPPPP SQASSGQARM FPNAPYLPSC LESQPAIRNQ GYSTVTFDGT PSYGHTPSHH AAQFPNHSFK HEDPMGQQGS LGEQQYSVPP PVYGCHTPTD SCTGSQALLL RTPYSSDNLY QMTSQLECMT WNQMNLGATL KGHSTGYESD NHTTPILCGA QYRIHTHGVF RGIQDVRRVP GVAPTLVRSA SETSEKRPFM CAYPGCNKRY FKLSHLQMHS RKHTGEKPYQ CDFKDCERRF SRSDQLKRHQ RRHTGVKPFQ CKTCQRKFSR SDHLKTHTRT HTGEKPFSCR WPSCQKKFAR SDELVRHHNM HQRNMTKLQL AL(SEQ ID NO: 1001).

[0178] 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), CMTWN LMNLGATLKG (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), CMTWNYMNLGATLKG VA (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), 1), 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).

[0179] In some cases, the cancer associated peptide is a peptide of a human papillomavirus (HPV) polypeptide having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to a 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 the various genotypes of HPV, 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; sequence number 1052), and E7 67-76 (LCVQSTHVDI; sequence number 1053); E7 82-90 (LLMGTLGIV; sequence number 1054); E7 86-93 (TLGIVCPI; sequence number 1055), and E7 92-93 (LLMGTLGIVCPI; sequence number 1056).

[0180] In some cases, the cancer associated peptide is a peptide of a claudin polypeptide having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following claudin-18 (isoform 2) (CLDN 18.2) amino acid sequence: MAVTACQGLG FVVSLIGIAG IIAATCMDQW STQDLYNNPV TAVFNYQGLW RSCVRESSGF TECRGYFTLL GLPAMLQAVR ALMIVGIVLG AIGLLVSIFA LKCIRIGSME DSAKANMTLT SGIMFIVSGL CAIAGVSVFA NMLVTNFWMS TANMYTGMGG MVQTVQTRYT FGAALFVGWV AGGLTLIGGV ​​MMCIACRGLA PEETNYKAVS YHASGHSVAY KPGGFKASTG FGSNTKNKKI YDGGARTEDE VQSYPSKHDY V (SEQ ID NO: 1057). In some cases, 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 EEVQSYPSKHDYV (SEQ ID NO: 1059) (and having a length of about 12 amino acids).

[0181] In some cases, 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 and protein product of the TACSTD2 gene that is upregulated in many cancer types. In some cases, the cancer associated peptide is 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 the following TROP-2 amino acid sequences: QDNCTCPTNK MTVCSPDGPG GRCQCRALGS GMAVDCSTLT SKCLLLKARM SAPKNARTLV RPSEHALVDN DGLYDPDCDP EGRFKARQCN QTSVCWCVNS VGVRRTDKGD LSLRCDELVR THHILIDLRH RPTAGAFNHS DLDAELRRLF RERYRLHPKF VAAVHYEQPT IQIELRQNTS QKAAGDVDIG DAAYYFERDI KGESLFQGRG GLDLRVRGEP LQVERTLIYY LDEIPPKFSM KRLTAGLIAV IVVVVVALVA GMAVLVITNR RKSGKYKKVE IKELGELRKE PSL (sequence number 1060).

[0182] antibody As described above, in some cases, the TTP present in the TEP 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 a cancer cell, and the peptide can be a cancer-associated peptide (e.g., a peptide of a cancer-associated antigen).

[0183] Non-limiting examples of cancer-associated antigen-targeting antibodies that can be included in the TEP 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 These include, but are not limited to, VEGF), cetuximab (anti-EGFR), gemtuzumab (anti-CD33), ibritumomab tiuxetan (anti-CD20), panitumumab (anti-EGFR), tositumomab (anti-CD20), PAM4 (also known as clivatuzumab, anti-mucin), trastuzumab (anti-HER2), pertuzumab (anti-HER2), polatuzumab (anti-CD79b), and anetuzumab (anti-mesothelin).

[0184] In some cases, the tumor targeting polypeptide is a single chain antibody. In some cases, the tumor targeting polypeptide is an scFv. In some cases, the tumor targeting polypeptide is a nanobody (also referred to as a single domain antibody (sdAb)). In some cases, the tumor targeting polypeptide is a heavy chain nanobody. In some cases, the tumor targeting polypeptide is a light chain nanobody.

[0185] 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:

[0186] anti-Her2 In some instances, the anti-Her2 antibody comprises a) a light chain comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 1061), and b) a heavy chain comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLV TVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPC Contains PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (sequence number 1062).

[0187] In some cases, 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) a VL comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO: 1063), and b) a VH comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: (SEQ ID NO: 1064). In some cases, the anti-Her2 antibody comprises, in order from N-terminus to C-terminus, a) a VH comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS (SEQ ID NO: 1065), b) a peptide linker, and c) a VL comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: (SEQ ID NO: 1066). Suitable linkers are described elsewhere herein, 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).

[0188] In some cases, 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).

[0189] 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).

[0190] In some cases, the anti-Her2 antibody is an scFv antibody. For example, in some cases, the anti-Her2 scFv comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO: 1073).

[0191] As another example, in some cases, the anti-Her2 antibody comprises a) a light chain variable region (VL) comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: and b) an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVRQAPGKGLEWVADVNPNSGGSIYNQRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQGTLV TVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPC PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (sequence number 1075).

[0192] In some instances, 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) an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: VL:DIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYIYPYTFGQGTKVEIK (SEQ ID NO: 1076); and b) comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: VH: EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVRQAPGKGLEWVADVNPNSGGSIYNQRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQGTLVTVSS (SEQ ID NO: 1077).

[0193] In some cases, 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).

[0194] 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).

[0195] In some cases, the anti-Her2 antibody is an scFv. For example, in some cases, the anti-Her2 scFv comprises an amino acid sequence that has at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (sequence number 1073).

[0196] 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 TEP. See, e.g., WO 2005 / 012493.

[0197] In some instances, 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 instances, 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 instances, 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).

[0198] In some cases, the anti-CD19 antibody is an scFv. For example, in some cases, the anti-CD19 scFv comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence:DIQLTQSPASLAVSLGQRATISCKASQSVDYDGDSYLNWYQQIPGQPPKLLIYDASNLVSGIPPRFSGSGSGTDFTLNIHPVEKVDAATYHCQQSTEDPWTFGGGTKLEIKGGGGSGGGGSGGGGSQVQLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIWPGDGDTNYNGKFKGKATLTADESSSTAYMQLSSLASEDSAVYFCARRETTTVGRYYYAMDYWGQGTTVTVS (SEQ ID NO: 1090).

[0199] 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 TEP. 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 cases, 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 cases, the TTP is an anti-mesothelin scFv that includes the amino acid sequence set forth in any one of Figures 22A-22H.

[0200] In some instances, the anti-mesothelin antibody comprises a) a light chain that comprises an amino acid sequence that has at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: DIALTQPASVSGSPGQSITISCTGTSSDIGGYNSVSWYQQHPGKAPKLMIYGVNNRPSGVSNRFSGSSKSGNTASLTISGLQAEDEADYYCSSYDIESATPVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKGDSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTESS (SEQ ID NO: 1091), and b) a heavy chain comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: QVELVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQAPGKGLEWMGIIDPGDSRTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARGQLYGGTYMDGWGQGTLV TVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPC Contains PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (sequence number 1092).

[0201] In some instances, an anti-mesothelin antibody comprises a VL present in the light chain amino acid sequence provided above and a VH present in the heavy chain amino acid sequence provided above. For example, an anti-mesothelin antibody may comprise a) a VL comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity with the amino acid sequence: DIALTQPASVSGSPGQSITISCTGTSSDIGGYNSVSWYQQHPGKAPKLMIYGVNNRPSGVSNRFSGSSKSGNTASLTISGLQAEDEADYYCSSYDIESATPVFGGGTK (SEQ ID NO: 1093), and b) 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 with the amino acid sequence: DIALTQPASVSGSPGQSITISCTGTSSDIGGYNSVSWYQQHPGKAPKLMIYGVNNRPSGVSNRFSGSSKSGNTASLTISGLQAEDEADYYCSSYDIESATPVFGGGTK (SEQ ID NO: 1093). ) 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 VH:QVELVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQAPGKGLEWMGIIDPGDSRTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARGQLYGGTYMDGWGQGTLVTVSS (SEQ ID NO: 1094).

[0202] In some cases, 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).

[0203] 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).

[0204] 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 sequence TIFF2025515575000008.tif23167, VH CDR1, CDR2, and CDR3 are underlined and VL CDR1, CDR2, and CDR3 are bold and underlined.

[0205] As one non-limiting example, the anti-mesothelin scFv can comprise the following amino acid sequence: In the sequence TIFF2025515575000009.tif30160, VH CDR1, CDR2, and CDR3 are underlined and VL CDR1, CDR2, and CDR3 are bold and underlined.

[0206] In some cases, an anti-mesothelin antibody suitable for inclusion in a TEP comprises a) a VL CDR1, VL CDR2, and VL CDR3 present in a light chain variable region (VL) comprising the following amino acid sequence: EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPIFTFGPGTKVDIK (SEQ ID NO: 1103), and b) a VH CDR1, CDR2, and CDR3 present in a heavy chain variable region (VH) comprising the following amino acid sequence: QMQLVESGGGVVQPGRSLRLSCTASGFTFSNNGMHWVRQAPGKGLEWVAVIWFDGMNKFYVDSVKGRFTISRDNSKNTLYLEMNSLRAEDTAIYYCAREGDGSGIYYYYGMDVWGQGTTVTVSS (SEQ ID NO: 1104). 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.

[0207] In some cases, an anti-mesothelin antibody suitable for inclusion in a TEP comprises a) a VL region that comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPIFTFGPGTKVDIK (SEQ ID NO: 1103). , b) a peptide linker, and c) 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 the following amino acid sequence: QMQLVESGGGVVQPGRSLRLSCTASGFTFSNNGMHWVRQAPGKGLEWVAVIWFDGMNKFYVDSVKGRFTISRDNSKNTLYLEMNSLRAEDTAIYYCAREGDGSGIYYYYGMDVWGQGTTVTVSS (SEQ ID NO: 1104).

[0208] In some instances, an anti-mesothelin antibody suitable for inclusion in a TEP comprises, in order from N-terminus to C-terminus, a) a VH region that comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: QMQLVESGGGVVQPGRSLRLSCTASGFTFSNNGMHWVRQAPGKGLEWVAVIWFDGMNKFYVDSVKGRFTISRDNSKNTLYLEMNSLRAEDTAIYYCAREGDGSGIYYYYGMDVWGQG TTVTVSS (SEQ ID NO: 1104), b) a peptide linker, and c) 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 the following amino acid sequence: EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPIFTFGPGTKVDIK (SEQ ID NO: 1103). In some cases, the peptide linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO: 934), where n is an integer between 1 and 10 (e.g., n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). In some cases, the peptide linker comprises the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 875) and has a length of 15 amino acids.

[0209] In some cases, an anti-mesothelin antibody suitable for inclusion in a TEP comprises, in order from N-terminus to C-terminus, a) an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: a) a VL region: EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPIFTFGPGTKVDIK (SEQ ID NO: 110 3), b) a peptide linker, and c) 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 the following amino acid sequence: QMQLVESGGGVVQPGRSLRLSCTASGFTFSNNGMHWVRQAPGKGLEWVAVIWFDGMNKFYVDSVKGRFTISRDNSKNTLYLEMNSLRAEDTAIYYCAREGDGSGIYYYYGMDVWGQGTTVTVSS (SEQ ID NO: 1104). In some cases, the peptide linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO: 934), where n is an integer between 1 and 10 (e.g., n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). In some cases, the peptide linker comprises the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 875) and has a length of 15 amino acids.

[0210] In some cases, an anti-mesothelin antibody suitable for inclusion in a TEP comprises a) a VL CDR1, VL CDR2, and VL CDR3 present in a light chain variable region (VL) comprising the following amino acid sequence: DIELTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPGRFSGSGSGNSYSLTISSVEAEDDATYYCQQWSKHPLTFGSGTKVEIK (SEQ ID NO: 1105), and b) a VH CDR1, CDR2, and CDR3 present in a heavy chain variable region (VH) comprising the following amino acid sequence: QVQLQQSGPELEKPGASVKISCKASGYSFTGYTMNWVKQSHGKSLEWIGLITPYNGASSYNQKFRGKATLTVDKSSSTAYMDLLSLTSEDSAVYFCARGGYDGRGFDYWGSGTPVTVSS (SEQ ID NO: 1106). 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., amatuximab.

[0211] In some cases, an anti-mesothelin antibody suitable for inclusion in a TEP comprises a) a VL region that comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: DIELTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPGRFSGSGSGNSYSLTISSVEAEDDATYYCQQWSKHPLTFGSGTKVEIK (SEQ ID NO: 1105) ), b) a peptide linker, and c) 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 the following amino acid sequence: QVQLQQSGPELEKPGASVKISCKASGYSFTGYTMNWVKQSHGKSLEWIGLITPYNGASSYNQKFRGKATLTVDKSSSTAYMDLLSLTSEDSAVYFCARGGYDGRGFDYWGSGTPVTVSS (SEQ ID NO: 1106).

[0212] In some cases, an anti-mesothelin antibody suitable for inclusion in a TEP comprises, in order from N-terminus to C-terminus, a) an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: a) a VL region: DIELTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPGRFSGSGSGNSYSLTISSVEAEDDATYYCQQWSKHPLTFGSGTKVEIK (SEQ ID NO: 11) 05), b) a peptide linker, and c) 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 the following amino acid sequence: QVQLQQSGPELEKPGASVKISCKASGYSFTGYTMNWVKQSHGKSLEWIGLITPYNGASSYNQKFRGKATLTVDKSSSTAYMDLLSLTSEDSAVYFCARGGYDGRGFDYWGSGTPVTVSS (SEQ ID NO: 1106). In some cases, the peptide linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO: 934), where n is an integer between 1 and 10 (e.g., n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). In some cases, the peptide linker comprises the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 875) and has a length of 15 amino acids.

[0213] In some instances, an anti-mesothelin antibody suitable for inclusion in a TEP comprises, in order from N-terminus to C-terminus, a) a VH region that comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: QVQLQQSGPELEKPGASVKISCKASGYSFTGYTMNWVKQSHGKSLEWIGLITPYNGASSYNQKFRGKATLTVDKSSSTAYMDLLSLTSEDSAVYFCARGGYDGRGFDYWGSGT PVTVSS (SEQ ID NO: 1106), b) a peptide linker, and c) 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 the following amino acid sequence: DIELTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPGRFSGSGSGNSYSLTISSVEAEDDATYYCQQWSKHPLTFGSGTKVEIK (SEQ ID NO: 1105). In some cases, the peptide linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO: 934), where n is an integer between 1 and 10 (e.g., n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). In some cases, the peptide linker comprises the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 875) and has a length of 15 amino acids.

[0214] In some cases, an anti-mesothelin antibody suitable for inclusion in a TEP comprises a) a VL CDR1, VL CDR2, and VL CDR3 present in a light chain variable region (VL) comprising the following amino acid sequence: DIALTQPASVSGSPGQSITISCTGTSSDIGGYNSVSWYQQHPGKAPKLMIYGVNNRPSGVSNRFSGSSKSGNTASLTISGLQAEDEADYYCSSYDIESATPVFGGGTKLTVLG (SEQ ID NO: 1108), and b) a VH CDR1, CDR2, and CDR3 present in a heavy chain variable region (VH) comprising the following amino acid sequence: QVELVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQAPGKGLEWMGIIDPGDSRTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARGQLYGGTYMDGWGQGTLVTVSS (SEQ ID NO: 1094). 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).

[0215] In some cases, an anti-mesothelin antibody suitable for inclusion in a TEP has a VL region that 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 the following amino acid sequence: DIALTQPASVSGSPGQSITISCTGTSSDIGGYNSVSWYQQHPGKAPKLMIYGVNNRPSGVSNRFSGSSKSGNTASLTISGLQAEDEADYYCSSYDIESATPVFGGGTKLTVLG (SEQ ID NO: 11). 07), b) a peptide linker, and c) 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 the following amino acid sequence: QVELVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQAPGKGLEWMGIIDPGDSRTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARGQLYGGTYMDGWGQGTLVTVSS (sequence number 1094).

[0216] In some instances, an anti-mesothelin antibody suitable for inclusion in a TEP comprises, in order from N-terminus to C-terminus, a) an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: a) a VL region: DIALTQPASVSGSPGQSITISCTGTSSDIGGYNSVSWYQQHPGKAPKLMIYGVNNRPSGVSNRFSGSSKSGNTASLTISGLQAEDEADYYCSSYDIESATPVFGGGTKLTVLG (SEQ ID NO: 1107), b) a peptide linker, and c) 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 the following amino acid sequence: QVELVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQAPGKGLEWMGIIDPGDSRTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARGQLYGGTYMDGWGQGTLVTVSS (SEQ ID NO: 1094). In some cases, the peptide linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO: 934), where n is an integer between 1 and 10 (e.g., n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). In some cases, the peptide linker comprises the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 875) and has a length of 15 amino acids.

[0217] In some instances, an anti-mesothelin antibody suitable for inclusion in a TEP comprises, in order from N-terminus to C-terminus, a) a VH region that comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: QVELVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQAPGKGLEWMGIIDPGDSRTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARGQLYGGTYMDGWGQGTLVT VSS (SEQ ID NO: 1094), b) a peptide linker, and c) 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 the following amino acid sequence: DIALTQPASVSGSPGQSITISCTGTSSDIGGYNSVSWYQQHPGKAPKLMIYGVNNRPSGVSNRFSGSSKSGNTASLTISGLQAEDEADYYCSSYDIESATPVFGGGTKLTVLG (SEQ ID NO: 1107). In some cases, 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 cases, the peptide linker comprises the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 875) and has a length of 15 amino acids.

[0218] In some cases, an anti-mesothelin antibody suitable for inclusion in a TEP comprises a) a VL CDR1, VL CDR2, and VL CDR3 present in a light chain variable region (VL) comprising the following amino acid sequence: DIQMTQSPSSLSASVGDRVTITCSASSSVSYMHWYQQKSGKAPKLLIYDTSKLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSKHPLTFGQGTKLEIK (SEQ ID NO: 1108), and b) a VH CDR1, CDR2, and CDR3 present in a heavy chain variable region (VH) comprising the following amino acid sequence: QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYTMNWVRQAPGQGLEWMGLITPYNGASSYNQKFRGKATMTVDTSTSTVYMELSSLRSEDTAVYYCARGGYDGRGFDYWGQGTLVTVSS (SEQ ID NO: 1109). 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.

[0219] In some cases, an anti-mesothelin antibody suitable for inclusion in a TEP comprises a) a VL region that comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: DIQMTQSPSSLSASVGDRVTITCSASSSVSYMHWYQQKSGKAPKLLIYDTSKLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSKHPLTFGQGTKLEIK (SEQ ID NO: 1108 ), b) a peptide linker, and c) 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 the following amino acid sequence: QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYTMNWVRQAPGQGLEWMGLITPYNGASSYNQKFRGKATMTVDTSTSTVYMELSSLRSEDTAVYYCARGGYDGRGFDYWGQGTLVTVSS (SEQ ID NO: 1109).

[0220] In some cases, an anti-mesothelin antibody suitable for inclusion in a TEP comprises, in order from N-terminus to C-terminus, a) an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: VL region: DIQMTQSPSSLSASVGDRVTITCSASSSVSYMHWYQQKSGKAPKLLIYDTSKLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSKHPLTFGQGTKLEIK (SEQ ID NO: 11) 08), b) a peptide linker, and c) 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 the following amino acid sequence: QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYTMNWVRQAPGQGLEWMGLITPYNGASSYNQKFRGKATMTVDTSTSTVYMELSSLRSEDTAVYYCARGGYDGRGFDYWGQGTLVTVSS (SEQ ID NO: 1109). In some cases, the peptide linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO: 934), where n is an integer between 1 and 10 (e.g., n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). In some cases, the peptide linker comprises the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 875) and has a length of 15 amino acids.

[0221] In some cases, an anti-mesothelin antibody suitable for inclusion in a TEP comprises, in order from N-terminus to C-terminus, a) a VH region that comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYTMNWVRQAPGQGLEWMGLITPYNGASSYNQKFRGKATMTVDTSTSTVYMELSSLRSEDTAVYYCARGGYDGRGFDYWGQGT The scFv comprises a) a VL region having an amino acid sequence with at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: DIQMTQSPSSLSASVGDRVTITCSASSSVSYMHWYQQKSGKAPKLLIYDTSKLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSKHPLTFGQGTKLEIK (SEQ ID NO: 1108). In some cases, the peptide linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO: 934), where n is an integer between 1 and 10 (e.g., n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). In some cases, the peptide linker comprises the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 875) and has a length of 15 amino acids.

[0222] In some instances, the anti-mesothelin scFv comprises the following amino acid sequence: TIFF2025515575000010.tif23159, in which the VH sequence is in italics, the (GGGGS)3 (SEQ ID NO:875) linker is in bold and underlined, and the VL sequence is underlined.

[0223] In some instances, the anti-mesothelin scFv comprises the following amino acid sequence: TIFF2025515575000011.tif23159, in the sequence the VL sequence is underlined, the (GGGGS)3 (SEQ ID NO:875) linker is bold and underlined, and the VL sequence is italicized.

[0224] In some instances, the anti-mesothelin scFv comprises the following amino acid sequence: TIFF2025515575000012.tif23160, in which the VH sequence is in italics, the (GGGGS)3 (SEQ ID NO:875) linker is in bold and underlined, and the VL sequence is underlined.

[0225] In some instances, the anti-mesothelin scFv comprises the following amino acid sequence: TIFF2025515575000013.tif23158, in the sequence the VL sequence is underlined, the (GGGGS)3 (SEQ ID NO:875) linker is bold and underlined, and the VL sequence is italicized.

[0226] In some instances, the anti-mesothelin scFv comprises the following amino acid sequence: TIFF2025515575000014.tif23160, in which the VH sequence is in italics, the (GGGGS)3 (SEQ ID NO:875) linker is in bold and underlined, and the VL sequence is underlined.

[0227] In some instances, the anti-mesothelin scFv comprises the following amino acid sequence: TIFF2025515575000015.tif23159, in the sequence the VL sequence is underlined, the (GGGGS)3 (SEQ ID NO:875) linker is bold and underlined, and the VL sequence is italicized.

[0228] In some instances, the anti-mesothelin scFv comprises the following amino acid sequence: TIFF2025515575000016.tif23159, in which the VH sequence is in italics, the (GGGGS)3 (SEQ ID NO:875) linker is in bold and underlined, and the VL sequence is underlined.

[0229] In some instances, the anti-mesothelin scFv comprises the following amino acid sequence: TIFF2025515575000017.tif23159, in the sequence the VL sequence is underlined, the (GGGGS)3 (SEQ ID NO:875) linker is bold and underlined, and the VL sequence is italicized.

[0230] 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.

[0231] 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 the TEP. See, e.g., U.S. Patent No. 10,179,819 and U.S. Patent Application Publication No. 2021 / 0277141.

[0232] 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.

[0233] Anti-CD22 antibodies are known in the art, and any anti-CD22 antibody VH and VL or VH and VL CDRs can be used in TEP. See, for example, Xiao et al. (2009) Mabs 1:297 (depicting 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).

[0234] For example, an anti-CD22 antibody may comprise: i) a heavy chain variable region (VH) CDR1 having the amino acid sequence: GDSVSSNSAA (SEQ ID NO:1110); ii) a VH CDR2 having the amino acid sequence: TYYRSKWYN (SEQ ID NO:1111); iii) a VH CDR3 having the amino acid sequence: AREVTGDLEDAFDI (SEQ ID NO:1112); iv) a light chain variable region (VL) CDR1 having the amino acid sequence: QTIWSY (SEQ ID NO:1113); v) a VL CDR2 having the amino acid sequence: AAS (Ala-Ala-Ser); and vi) a VL CDR3 having the amino acid sequence: QQSYSIPQT (SEQ ID NO:1114). anti-TROP-2

[0235] 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 and the protein product of the TACSTD2 gene that is upregulated in many cancer types.

[0236] In some cases, the TTP is an anti-TROP-2 scFv or an anti-TROP-2 nanobody that includes the VH and VL CDRs present in any one of the amino acid sequences set forth in Figures 23A-23D. In some cases, the TTP is an anti-TROP-2 scFv that includes the amino acid sequence set forth in any one of Figures 23A-23D.

[0237] 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 the TEP. See, for example, U.S. Patent No. 7,238,785. In some cases, the anti-TROP-2 antibody comprises i) light chain CDR sequences CDR1 (KASQDVSIAVA; SEQ ID NO: 1115), CDR2 (SASYRYT; SEQ ID NO: 1116), and CDR3 (QQHYITPLT; SEQ ID NO: 1117), and ii) heavy chain CDR sequences CDR1 (NYGMN; SEQ ID NO: 1118), CDR2 (WINTYTGEPTYTDDFKG; SEQ ID NO: 1119), and CDR3 (GGFGSSYWYFDV; SEQ ID NO: 1120).

[0238] In some cases, 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).

[0239] In some cases, an anti-TROP2 antibody suitable for inclusion in a TEP comprises a) a VL CDR1, VL CDR2, and VL CDR3 present in a light chain variable region (VL) comprising the following amino acid sequence: DIQLTQSPSSLSASVGDRVSITCKASQDVSIAVAWYQQKPGKAPKLLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDFAVYYCQQHYITPLTFGAGTKVEIK: (SEQ ID NO: 1125), and b) a VH CDR1, CDR2, and CDR3 present in a heavy chain variable region (VH) comprising the following amino acid sequence: QVQLQQSGSELKKPGASVKVSCKASGYTFTNYGMNWVKQAPGQGLKWMGWINTYTGEPTYTDDFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCARGGFGSSYWYFDVWGQGSLVTVSS (SEQ ID NO: 1125). 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).

[0240] In some instances, an anti-TROP-2 antibody suitable for inclusion in a TEP comprises a) 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 the following amino acid sequence: DIQLTQSPSSLSASVGDRVSITCKASQDVSIAVAWYQQKPGKAPKLLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDFAVYYCQQHYITPLTFGAGTKVEIK (SEQ ID NO: No. 1125), and b) 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 the following amino acid sequence: QVQLQQSGSELKKPGASVKVSCKASGYTFTNYGMNWVKQAPGQGLKWMGWINTYTGEPTYTDDFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCARGGFGSSYWYFDVWGQGSLVTVSS (SEQ ID NO: 1126).

[0241] In some instances, an anti-TROP-2 antibody suitable for inclusion in a TEP comprises, in order from N-terminus to C-terminus, a) a VL region that comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: DIQLTQSPSSLSASVGDRVSITCKASQDVSIAVAWYQQKPGKAPKLLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDFAVYYCQQHYITPLTFGAGTKVEIK (SEQ ID NO: 11) 25), b) a peptide linker, and c) 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 the following amino acid sequence: QVQLQQSGSELKKPGASVKVSCKASGYTFTNYGMNWVKQAPGQGLKWMGWINTYTGEPTYTDDFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCARGGFGSSYWYFDVWGQGSLVTVSS (SEQ ID NO: 1126). In some cases, the peptide linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO: 934), where n is an integer between 1 and 10 (e.g., n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). In some cases, the peptide linker comprises the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 875) and has a length of 15 amino acids.

[0242] In some cases, an anti-TROP-2 antibody suitable for inclusion in a TEP comprises, in order from N-terminus to C-terminus, a) a VH region that comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: QVQLQQSGSELKKPGASVKVSCKASGYTFTNYGMNWVKQAPGQGLKWMGWINTYTGEPTYTDDFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCARGGFGSSYWYFDVWGQG SLVTVSS (SEQ ID NO: 1126), b) a peptide linker, and c) 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 the following amino acid sequence: DIQLTQSPSSLSASVGDRVSITCKASQDVSIAVAWYQQKPGKAPKLLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDFAVYYCQQHYITPLTFGAGTKVEIK (SEQ ID NO: 1126). In some cases, the peptide linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO: 934), where n is an integer between 1 and 10 (e.g., n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). In some cases, the peptide linker comprises the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 875) and has a length of 15 amino acids.

[0243] In some cases, an anti-TROP2 antibody suitable for inclusion in a TEP comprises a) a VL CDR1, VL CDR2, and VL CDR3 present in a light chain variable region (VL) comprising the following amino acid sequence: DIQMTQSPSSLSASVGDRVTITCKASQDVSTAVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFAVYYCQQHYITPLTFGQGTKLEIK (SEQ ID NO: 1127), and b) a VH CDR1, CDR2, and CDR3 present in a heavy chain variable region (VH) comprising the following amino acid sequence: QVQLVQSGAEVKKPGASVKVSCKASGYTFTTAGMQWVRQAPGQGLEWMGWINTHSGVPKYAEDFKGRVTISADTSTSTAYLQLSSLKSEDTAVYYCARSGFGSSYWYFDVWGQGTLVTVSS (SEQ ID NO: 1128). 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).

[0244] In some instances, an anti-TROP-2 antibody suitable for inclusion in a TEP comprises a) 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 the following amino acid sequence: DIQMTQSPSSLSASVGDRVTITCKASQDVSTAVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFAVYYCQQHYITPLTFGQGTKLEIK (SEQ ID NO: No. 1127), and b) 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 the following amino acid sequence: QVQLVQSGAEVKKPGASVKVSCKASGYTFTTAGMQWVRQAPGQGLEWMGWINTHSGVPKYAEDFKGRVTISADTSTSTAYLQLSSLKSEDTAVYYCARSGFGSSYWYFDVWGQGTLVTVSS (SEQ ID NO: 1128).

[0245] In some cases, an anti-TROP-2 antibody suitable for inclusion in a TEP comprises, in order from N-terminus to C-terminus, a) an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: VL region: DIQMTQSPSSLSASVGDRVTITCKASQDVSTAVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFAVYYCQQHYITPLTFGQGTKLEIK (SEQ ID NO: 11 27), b) a peptide linker, and c) 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 the following amino acid sequence: QVQLVQSGAEVKKPGASVKVSCKASGYTFTTAGMQWVRQAPGQGLEWMGWINTHSGVPKYAEDFKGRVTISADTSTSTAYLQLSSLKSEDTAVYYCARSGFGSSYWYFDVWGQGTLVTVSS (SEQ ID NO: 1128). In some cases, the peptide linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO: 934), where n is an integer between 1 and 10 (e.g., n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). In some cases, the peptide linker comprises the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 875) and has a length of 15 amino acids.

[0246] In some cases, an anti-TROP-2 antibody suitable for inclusion in a TEP comprises, in order from N-terminus to C-terminus, a) a VH region that comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: QVQLVQSGAEVKKPGASVKVSCKASGYTFTTAGMQWVRQAPGQGLEWMGWINTHSGVPKYAEDFKGRVTISADTSTSTAYLQLSSLKSEDTAVYYCARSGFGSSYWYFDVWGQG A scFv comprising: a) a VL region comprising: TLVTVSS (SEQ ID NO: 1128); b) a peptide linker; and c) an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: DIQMTQSPSSLSASVGDRVTITCKASQDVSTAVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFAVYYCQQHYITPLTFGQGTKLEIK (SEQ ID NO: 1127). In some cases, the peptide linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO: 934), where n is an integer between 1 and 10 (e.g., n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). In some cases, the peptide linker comprises the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 875) and has a length of 15 amino acids.

[0247] 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 TEP. See, e.g., WO 2014 / 089335, U.S. Patent Application Publication No. 2019 / 0153061, and WO 2017 / 093942.

[0248] In some instances, the anti-BCMA antibody comprises a) a light chain comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: QSVLTQPPSASGTPGQRVTISCSGSSSNIGSNTVNWYQQLPGTAPKLLIFNYHQRPSGVPDRFSGSKSGSSASLAISGLQSEDEADYYCAAWDDSLNGWVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPDSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS (SEQ ID NO: 1129), and b) a heavy chain comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: EVQLVESGGGLVKPGGSLRLSCAASGFTFGDYALSWFRQAPGKGLEWVGVSRSKAYGGTTDYAASVKGRFTISRDDSKSTAYLQMNSLKTEDTAVYYCASSGYSSGWTPFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSS VVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (sequence number 1130).

[0249] In some instances, 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) a VL that comprises an amino acid sequence that has at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: QSVLTQPPSASGTPGQRVTISCSGSSSNIGSNTVNWYQQLPGTAPKLLIFNYHQRPSGVPDRFSGSKSGSSASLAISGLQSEDEADYYCAAWDDSLNGWVFGGGTKLTVLG (SEQ ID NO: 1131); and b) an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: Contains EVQLVESGGGLVKPGGSLRLSCAASGFTFGDYALSWFRQAPGKGLEWVGVSRSKAYGGTTDYAASVKGRFTISRDDSKSTAYLQMNSLKTEDTAVYYCASSGYSSGWTPFDYWGQGTLVTVSSASTKGPSV (sequence number 1132).

[0250] In some cases, 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).

[0251] 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).

[0252] The anti-BCMA antibody may be an scFv. As one non-limiting example, the anti-BCMA scFv may comprise the following amino acid sequence: QVQLVQSGAEVKKPGSSVKVSCKASGGTFSNYWMHWVRQAPGQGLEWMGATYRGHSDTYYNQKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARGAIYNGYDVLDNWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKLLIYYTSNLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYRKLPWTFGQGTKLEIKR (SEQ ID NO: 1138).

[0253] As another example, the anti-BCMA scFv may comprise the following amino acid sequence: DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKLLIYYTSNLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYRKLPWTFGQGTKLEIKRGGGGSGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGGTFSNYWMHWVRQAPGQGLEWMGATYRGHSDTYYNQKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARGAIYNGYDVLDNWGQGTLVTVSS (sequence number 1139).

[0254] In some cases, 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).

[0255] In some instances, the anti-BCMA antibody comprises a) a light chain comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKLLIYYTSNLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYRKLPWTFGQGTKLEIKR (SEQ ID NO: 1146).

[0256] In some cases, the anti-BCMA antibody comprises a) a heavy chain comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: QVQLVQSGAEVKKPGSSVKVSCKASGGTFSNYWMHWVRQAPGQGLEWMGATYRGHSDTYYNQKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARGAIYDGYDVLDNWGQGTLVTVSS (SEQ ID NO: 1147).

[0257] In some cases, an anti-BCMA antibody (e.g., an antibody referred to in the literature as belantamab) comprises a light chain comprising the following amino acid sequence: DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKLLIYYTSNLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYRKLPWTFGQGTKLEIKR (sequence number 1146), and a heavy chain comprising the following amino acid sequence: QVQLVQSGAEVKKPGSSVKVSCKASGGTFSNYWMHWVRQAPGQGLEWMGATYRGHSDTYYNQKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARGAIYDGYDVLDNWGQGTLVTVSS (sequence number 1147).

[0258] In some cases, the anti-BCMA antibody has a cancer chemotherapeutic agent linked to the antibody. For example, in some cases, 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.

[0259] anti-MUC1 In some cases, the TTP present in the TEP is an antibody specific to MUC1. For example, the TTP can be specific to a MUC1 polypeptide present on a cancer cell. In some cases, the TTP is specific to a truncated form of MUC1, see, for example, Fessler et al. (2009) Breast Cancer Res. Treat. 118:113. In some cases, the TTP is an antibody specific to a glycosylated MUC1 peptide, see, for example, Naito et al. (2017) ACS Omega 2:7493, and U.S. Patent No. 10,017,580.

[0260] As one non-limiting example, the TTP can be a single chain Fv specific to 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 cases, the TTP is a scFv specific to the MUC1 peptide VTSAPDTRPAPGSTAPPAHG (SEQ ID NO: 1148). In some cases, the TTP is an scFv specific for the MUC1 peptide SNIKFRPGSVVVQLTLAFREGTINVHDVETQFNQYKTEAASRY (SEQ ID NO: 1149). In some cases, the TTP is an scFv specific for the MUC1 peptide SVVVQLTLAFREGTINVHDVETQFNQYKTEAASRY (SEQ ID NO: 1150). In some cases, the TTP is an scFv specific for the MUC1 peptide LAFREGTINVHDVETQFNQY (SEQ ID NO: 1151). In some cases, the TTP is an scFv specific for the MUC1 peptide SNIKFRPGSVVVQLTLAAFREGTIN (SEQ ID NO: 1152).

[0261] 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.

[0262] 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.

[0263] 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.

[0264] 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.

[0265] anti-MUC16 In some cases, the TTP present in the TEP is an antibody specific for MUC16 (also known as CA125). See, e.g., Yin et al. (2002) Int. J. Cancer 98:737. For example, the TTP can be specific for a MUC16 polypeptide present on a cancer cell. See, e.g., U.S. Patent Application Publication No. 2018 / 0118848 and U.S. Patent Application Publication No. 2018 / 0112008. In some cases, the MUC16-specific TTP is an scFv. In some cases, the MUC16-specific TTP is a nanobody.

[0266] 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.

[0267] Anti-claudin-18.2 In some cases, the TPP present in the TEP is an antibody specific for claudin-18 isoform 2 ("claudin-18.2"). See, e.g., International Publication No. WO 2013 / 167259. In some cases, the claudin-18.2 specific TTP is an scFv. In some cases, the claudin-18.2 specific TTP is a nanobody. In some cases, the TPP present in the TEP is an antibody specific for TEDEVQSYPSKHDYV (SEQ ID NO: 1058), or EVQSYPSKHDYV (SEQ ID NO: 1059).

[0268] 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).

[0269] 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).

[0270] Single-chain T-cell receptor As mentioned above, in some cases, the TTP present in the TEP is a scTCR.The TTP is a scTCR specific to a peptide / HLA complex on the surface of a cancer cell, and the peptide can be a cancer-associated peptide (e.g., a peptide of a cancer-associated antigen).The amino acid sequence of the scTCR specific to a cancer-associated peptide bound to an HLA complex is known in the art.See, for example, US Patent Application Publication No. 2019 / 0135914, US Patent Application Publication No. 2019 / 0062398, and US Patent Application Publication No. 2018 / 0371049.

[0271] 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 cases, an scTCR comprises, in order from N-terminus to C-terminus, i) a TCR α chain variable domain polypeptide, ii) a peptide linker, iii) a TCR β chain variable domain polypeptide, and iv) a TCR β chain constant region extracellular domain polypeptide. In some cases, an scTCR comprises, in order from N-terminus to C-terminus, i) a TCR β chain variable domain polypeptide, ii) a peptide linker, iii) a TCR α chain variable domain polypeptide, and iv) a TCR α chain constant region extracellular domain polypeptide.

[0272] The amino acid sequence of a scTCR specific for a 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.

[0273] For example, scTCR is a TCR that targets HLA-A *NY-ESO epitope, such as SLLMWITQC peptide bound to an HLA complex comprising a 0201 heavy chain and a β2M polypeptide. By way of example, such a scTCR may comprise: i) a TCR α chain variable region comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: MQEVTQIPAALSVPEGENLVLNCSFTDSAIYNLQWFRQDPGKGLTSLLLIQSSQREQTSGRLNASLDKSSGRSTLYIAASQPGDSATYLCAVRPTSGGSYIPTFGRGTSLIVHPY (SEQ ID NO: 1191) (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, amino acid 95 can be T, L, M, A, Q, Y, E, I, F, V, N, G, S, D, or R, and amino acid 96 can be S, L, T, Y, I, Q, V, E, A, W, R, G, H, D, or K. wherein 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) at least 90% to the following amino acid sequence: A TCR beta chain variable region comprising an amino acid sequence having at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity: MGVTQTPKFQVLKTGQSMTLQCAQDMNHEYMSWYRQDPGMGLRLIHYSVGAGITDQGEVPNGYNVSRSTTEDFPLRLLSAAPSQTSVYFCASSYVGNTGELFFGEGSRLTVL (SEQ ID NO: 1192), in which amino acid 18 can be M, or V, and amino acid 50 can be G, V,or I, amino acid 52 can be G or Q, amino acid 53 can be I, T, or M, amino acid 55 can be D or R, amino acid 56 can be Q or R, amino acid 70 can be T or I, amino acid 94 can be Y, N, or F, amino acid 95 can be V or L, and amino acid 97 can be N, G, or D). For example, in some cases, the scTCR can include i) a TCR alpha chain variable region comprising the following amino acid sequence: MQEVTQIPAALSVPEGENLVLNCSFTDSAIYNLQWFRQDPGKGLTSLLLIMSHQREQTSGRLNASLDKSSGRSTLYIAASQPGDSATYLCAVRPTSGGSYIPTFGRGTSLIVHPY (SEQ ID NO: 1193), and a TCR beta chain variable region comprising the following amino acid sequence: MGVTQTPKFQVLKTGQSMTLQCAQDMNHEYMSWYRQDPGMGLRLIHYSVSAGITDQGEVPNGYNVSRSTTEDFPLRLLSAAPSQTSVYFCASSYVGNTGELFFGEGSRLTVL (SEQ ID NO: 1194).

[0274] 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; HPV peptide of SEQ ID NO: 1198). For example, such a scTCR may be specific for i) a TCR α chain variable region comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: METLLGLLILQLQLQWVSSKQEVTQIPAALSVPEGENLVLNCSFTDSAIYNLQWFRQDPGKGLTSLLLIQSSQREQTSGRLNASLDKSSGRSTLYIAASQPGDSATYLCAVRETSGSRLTFGEGTQLTVNPD (SEQ ID NO: 1199). and ii) 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 the following amino acid sequence: MGIRLLCRVAFCFLAVGLVDVKVTQSSRYLVKRTGEKVFLECVQDMDHENMFWYRQDPGLGLRLIYFSYDVKMKEKGDIPEGYSVSREKKERFSLILESASTNQTSMYLCASSFWGRSTDTQYFGPGTRLTVL (SEQ ID NO: 1200).

[0275] Contrast agents In some cases, the TEP's TTP comprises an imaging agent or radiolabel, where the imaging agent facilitates imaging of the tumor to which the TEP binds.

[0276] 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 15O. Iodinated CT contrast agents can be used. Suitable contrast agents include gadolinium (Gd), dysprosium, and iron. Gd chelates can be used, such as Gd diethylenetriaminepentaacetic acid (GdDTPA), Gd tetraazacyclododecanetetraacetic acid (GdDOTA), polylysine-Gd chelate, and derivatives thereof. Suitable radioisotopes include: 123 I (iodine), 18 F (fluorine), 99 Tc (technetium), 111 In (indium), and 67 Contains Ga (gallium).

[0277] HLA / peptide binding assay Whether a given peptide (e.g., a peptide containing an epitope) binds to class I HLA (including an HLA heavy chain and a β2M polypeptide) and, when bound to an HLA complex, can effectively present the epitope to a TCR can be determined using any of several well-known methods. Assays include binding assays and T cell activation assays. See, for example, published International Application No. 2020 / 243315 (Cue Biopharma, Inc.).

[0278] Exemplary TEPs FIG. 19A, and FIGS. 21A-21J, and FIGS. 23A-23B provide the amino acid sequences of exemplary single-chain TEPs.

[0279] For example, in some instances, the TEP comprises, in order from N-terminus to C-terminus, (a) a CMV peptide, e.g., a peptide having the amino acid sequence NLVPMVATV (SEQ ID NO: 913); (b) a β2M polypeptide comprising a Cys at position 12; (c) an HLA-A polypeptide comprising a Cys at position 84 and a Cys at position 236; *(d) a TTP, (e) an Ig Fc polypeptide, (f) a first variant IL-2 polypeptide comprising an Ala at position 16 and an Ala at position 42, and (g) a second variant IL-2 polypeptide comprising an Ala at position 16 and an Ala at position 42, wherein the TEP comprises a peptide linker between (a) and (b), between (b) and (c), between (c) and (d), between (e) and (f), and between (f) and (g). In some cases, the β2M 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 β2M amino acid sequence depicted in FIG. 1B. In some cases, the β2M polypeptide comprises an HLA-A polypeptide comprising ... * The 0201 polypeptide is HLA-A shown in FIG. *2B, wherein amino acid 14 is Ala and amino acid 15 is Ala. In some cases, the Ig Fc 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 shown in FIG. 2B, wherein amino acid 84 is Cys and amino acid 236 is Cys. In some cases, the TTP is an anti-CD19 scFv. In some cases, the Ig Fc 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 shown in FIG. 2B, wherein amino acid 14 is Ala and amino acid 15 is Ala. In some cases, the first and second variant IL-2 polypeptides comprise an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: APTSSSTKKTQLQLEALLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 878), in which amino acid 16 is Ala and amino acid 42 is Ala. In some cases, the TEP 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 depicted in FIG. 19A.

[0280] As another example, in some instances, the TEP comprises, in order from N-terminus to C-terminus, (a) a CMV peptide, e.g., a CMV peptide having the amino acid sequence NLVPMVATV (SEQ ID NO: 913); (b) a β2M polypeptide comprising a Cys at position 12; (c) an HLA-A polypeptide comprising a Cys at position 84 and a Cys at position 236; *0201 polypeptide, (d) a first variant IL-2 polypeptide comprising an Ala at position 16 and an Ala at position 42, (e) a second variant IL-2 polypeptide comprising an Ala at position 16 and an Ala at position 42, (d) an Ig Fc polypeptide, and (e) a TTP, wherein the TEP comprises a peptide linker between (a) and (b), between (b) and (c), between (c) and (d), between (e) and (f), and between (f) and (g). In some cases, the β2M 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 β2M amino acid sequence shown in FIG. 1B. In some cases, the β2M polypeptide comprises an HLA-A polypeptide comprising ... * The 0201 polypeptide is HLA-A shown in FIG. *2B, wherein amino acid 14 is Ala and amino acid 15 is Ala. In some cases, the Ig Fc 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 shown in FIG. 2B, wherein amino acid 84 is Cys and amino acid 236 is Cys. In some cases, the TTP is an anti-CD19 scFv. In some cases, the Ig Fc 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 shown in FIG. 2B, wherein amino acid 14 is Ala and amino acid 15 is Ala. In some cases, the first and second variant IL-2 polypeptides comprise an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: APTSSSTKKTQLQLEALLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 878), in which amino acid 16 is Ala and amino acid 42 is Ala. In some cases, the TEP 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 depicted in FIG. 21A.

[0281] As another example, in some cases, the TEP may comprise, in order from N-terminus to C-terminus, (a) a SARS-CoV-2 peptide, e.g., a peptide having the amino acid sequence YLQPRTFLL (SEQ ID NO: 218); (b) a β2M polypeptide comprising a Cys at position 12; (c) an HLA-A polypeptide comprising a Cys at position 84 and a Cys at position 236; *(d) a TTP, (e) an Ig Fc polypeptide, (f) a first variant IL-2 polypeptide comprising an Ala at position 16 and an Ala at position 42, and (g) a second variant IL-2 polypeptide comprising an Ala at position 16 and an Ala at position 42, wherein the TEP comprises a peptide linker between (a) and (b), between (b) and (c), between (c) and (d), between (e) and (f), and between (f) and (g). In some cases, the β2M 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 β2M amino acid sequence depicted in FIG. 1B. In some cases, the β2M polypeptide comprises an HLA-A polypeptide comprising ... * The 0201 polypeptide is HLA-A shown in FIG. *2B, wherein amino acid 14 is Ala and amino acid 15 is Ala. In some cases, the Ig Fc 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 shown in FIG. 2B, wherein amino acid 84 is Cys and amino acid 236 is Cys. In some cases, the TTP is an anti-CD19 scFv. In some cases, the Ig Fc 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 shown in FIG. 2B, wherein amino acid 14 is Ala and amino acid 15 is Ala. In some cases, the first and second variant IL-2 polypeptides comprise an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: APTSSSTKKTQLQLEALLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 878), in which amino acid 16 is Ala and amino acid 42 is Ala. In some cases, the TEP 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 depicted in FIG. 21B.

[0282] As another example, in some cases, the TEP may comprise, in order from N-terminus to C-terminus, (a) a SARS-CoV-2 peptide, e.g., a peptide having the amino acid sequence YLQPRTFLL (SEQ ID NO: 218); (b) a β2M polypeptide comprising a Cys at position 12; (c) an HLA-A polypeptide comprising a Cys at position 84 and a Cys at position 236; *0201 polypeptide, (d) a first variant IL-2 polypeptide comprising an Ala at position 16 and an Ala at position 42, (e) a second variant IL-2 polypeptide comprising an Ala at position 16 and an Ala at position 42, (d) an Ig Fc polypeptide, and (e) a TTP, wherein the TEP comprises a peptide linker between (a) and (b), between (b) and (c), between (c) and (d), between (e) and (f), and between (f) and (g). In some cases, the β2M 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 β2M amino acid sequence shown in FIG. 1B. In some cases, the β2M polypeptide comprises an HLA-A polypeptide comprising ... * The 0201 polypeptide is HLA-A shown in FIG. *2B, wherein amino acid 14 is Ala and amino acid 15 is Ala. In some cases, the Ig Fc 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 shown in FIG. 2B, wherein amino acid 84 is Cys and amino acid 236 is Cys. In some cases, the TTP is an anti-CD19 scFv. In some cases, the Ig Fc 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 shown in FIG. 2B, wherein amino acid 14 is Ala and amino acid 15 is Ala. In some cases, the first and second variant IL-2 polypeptides comprise an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: APTSSSTKKTQLQLEALLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO:878), in which amino acid 16 is Ala and amino acid 42 is Ala. In some cases, the TEP 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 depicted in FIG. 21C.

[0283] For example, in some instances, the TEP comprises, in order from N-terminus to C-terminus, (a) a CMV peptide, e.g., a peptide having the amino acid sequence NLVPMVATV (SEQ ID NO: 913); (b) a β2M polypeptide comprising a Cys at position 12; (c) an HLA-A polypeptide comprising a Cys at position 84 and a Cys at position 236; *(d) a TTP, (e) an Ig Fc polypeptide, (f) a first variant IL-2 polypeptide comprising an Ala at position 16 and an Ala at position 42, and (g) a second variant IL-2 polypeptide comprising an Ala at position 16 and an Ala at position 42, wherein the TEP comprises a peptide linker between (a) and (b), between (b) and (c), between (c) and (d), between (e) and (f), and between (f) and (g). In some cases, the β2M 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 β2M amino acid sequence depicted in FIG. 1B. In some cases, the β2M polypeptide comprises an HLA-A polypeptide comprising ... * The 0201 polypeptide is HLA-A shown in FIG. *17A , wherein amino acid 14 is Ala and amino acid 15 is Ala. In some cases, the TTP is an anti-mesothelin scFv. In some cases, the anti-mesothelin scFv comprises an amino acid sequence that has at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the Ig Fc amino acid sequence shown in FIG. 2B , wherein amino acid 14 is Ala and amino acid 15 is Ala. In some cases, the TTP is an anti-mesothelin scFv. In some cases, the anti-mesothelin scFv comprises an amino acid sequence that has at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the Ig Fc amino acid sequence shown in FIG. 2B , wherein amino acid 14 is Ala and amino acid 15 is Ala. In some cases, the first and second variant IL-2 polypeptides comprise an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: APTSSSTKKTQLQLEALLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 878), where amino acid 16 is Ala and amino acid 42 is Ala. In some cases, the TEP 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 depicted in FIG. 21D.

[0284] As another example, in some instances, the TEP comprises, in order from N-terminus to C-terminus, (a) a CMV peptide, e.g., a CMV peptide having the amino acid sequence NLVPMVATV (SEQ ID NO: 913); (b) a β2M polypeptide comprising a Cys at position 12; (c) an HLA-A polypeptide comprising a Cys at position 84 and a Cys at position 236; *0201 polypeptide, (d) a first variant IL-2 polypeptide comprising an Ala at position 16 and an Ala at position 42, (e) a second variant IL-2 polypeptide comprising an Ala at position 16 and an Ala at position 42, (d) an Ig Fc polypeptide, and (e) a TTP, wherein the TEP comprises a peptide linker between (a) and (b), between (b) and (c), between (c) and (d), between (e) and (f), and between (f) and (g). In some cases, the β2M 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 β2M amino acid sequence shown in FIG. 1B. In some cases, the β2M polypeptide comprises an HLA-A polypeptide comprising ... * The 0201 polypeptide is HLA-A shown in FIG. *17A , wherein amino acid 14 is Ala and amino acid 15 is Ala. In some cases, the TTP is an anti-mesothelin scFv. In some cases, the anti-mesothelin scFv comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the anti-mesothelin scFv amino acid sequence depicted in FIG. 17A . In some cases, the Ig Fc 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 depicted in FIG. 2B , wherein amino acid 14 is Ala and amino acid 15 is Ala. In some cases, the first and second variant IL-2 polypeptides comprise an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: APTSSSTKKTQLQLEALLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 878), in which amino acid 16 is Ala and amino acid 42 is Ala. In some cases, the TEP 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 depicted in FIG. 21E.

[0285] As another example, in some cases, the TEP may comprise, in order from N-terminus to C-terminus, (a) a SARS-CoV-2 peptide, e.g., a peptide having the amino acid sequence YLQPRTFLL (SEQ ID NO: 218); (b) a β2M polypeptide comprising a Cys at position 12; (c) an HLA-A polypeptide comprising a Cys at position 84 and a Cys at position 236; *(d) a TTP, (e) an Ig Fc polypeptide, (f) a first variant IL-2 polypeptide comprising an Ala at position 16 and an Ala at position 42, and (g) a second variant IL-2 polypeptide comprising an Ala at position 16 and an Ala at position 42, wherein the TEP comprises a peptide linker between (a) and (b), between (b) and (c), between (c) and (d), between (e) and (f), and between (f) and (g). In some cases, the β2M 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 β2M amino acid sequence depicted in FIG. 1B. In some cases, the β2M polypeptide comprises an HLA-A polypeptide comprising ... * The 0201 polypeptide is HLA-A shown in FIG. *17A , wherein amino acid 14 is Ala and amino acid 15 is Ala. In some cases, the TTP is an anti-mesothelin scFv. In some cases, the anti-mesothelin scFv comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the anti-mesothelin scFv amino acid sequence depicted in FIG. 17A . In some cases, the Ig Fc 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 depicted in FIG. 2B , wherein amino acid 14 is Ala and amino acid 15 is Ala. In some cases, the first and second variant IL-2 polypeptides comprise an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: APTSSSTKKTQLQLEALLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 878), in which amino acid 16 is Ala and amino acid 42 is Ala. In some cases, the TEP 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 depicted in FIG. 21F.

[0286] As another example, in some cases, the TEP may comprise, in order from N-terminus to C-terminus, (a) a SARS-CoV-2 peptide, e.g., a peptide having the amino acid sequence YLQPRTFLL (SEQ ID NO: 218); (b) a β2M polypeptide comprising a Cys at position 12; (c) an HLA-A polypeptide comprising a Cys at position 84 and a Cys at position 236; *0201 polypeptide, (d) a first variant IL-2 polypeptide comprising an Ala at position 16 and an Ala at position 42, (e) a second variant IL-2 polypeptide comprising an Ala at position 16 and an Ala at position 42, (d) an Ig Fc polypeptide, and (e) a TTP, wherein the TEP comprises a peptide linker between (a) and (b), between (b) and (c), between (c) and (d), between (e) and (f), and between (f) and (g). In some cases, the β2M 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 β2M amino acid sequence shown in FIG. 1B. In some cases, the β2M polypeptide comprises an HLA-A polypeptide comprising ... * The 0201 polypeptide is HLA-A shown in FIG. *17A , wherein amino acid 14 is Ala and amino acid 15 is Ala. In some cases, the TTP is an anti-mesothelin scFv. In some cases, the anti-mesothelin scFv comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the anti-mesothelin scFv amino acid sequence depicted in FIG. 17A . In some cases, the Ig Fc 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 depicted in FIG. 2B , wherein amino acid 14 is Ala and amino acid 15 is Ala. In some cases, the first and second variant IL-2 polypeptides comprise an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: APTSSSTKKTQLQLEALLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 878), in which amino acid 16 is Ala and amino acid 42 is Ala. In some cases, the TEP 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 depicted in FIG. 21G.

[0287] As another example, in some instances, the TEP comprises, in order from N-terminus to C-terminus, (a) a CMV peptide, e.g., a peptide having the amino acid sequence NLVPMVATV (SEQ ID NO: 913); (b) a β2M polypeptide comprising a Cys at position 12; (c) an HLA-E polypeptide comprising a Cys at position 84 and a Cys at position 236; *01:01 polypeptide, (d) TTP, (e) an Ig Fc polypeptide, (f) a first variant IL-2 polypeptide comprising an Ala at position 16 and an Ala at position 42, and (g) a second variant IL-2 polypeptide comprising an Ala at position 16 and an Ala at position 42, wherein the TEP comprises a peptide linker between (a) and (b), between (b) and (c), between (c) and (d), between (e) and (f), and between (f) and (g). In some cases, the β2M 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 β2M amino acid sequence depicted in FIG. 1B. In some cases, the β2M polypeptide comprises an HLA-E polypeptide comprising ... * The 01:01 polypeptide is identical to the HLA-E polypeptide shown in FIG. *2B, wherein amino acid 14 is Ala and amino acid 15 is Ala. In some cases, the Ig Fc 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 01:01 amino acid sequence, wherein amino acid 84 is Cys and amino acid 236 is Cys. In some cases, the TTP is an anti-CD19 scFv. In some cases, the Ig Fc 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 shown in FIG. 2B, wherein amino acid 14 is Ala and amino acid 15 is Ala. In some cases, the first and second variant IL-2 polypeptides comprise an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: APTSSSTKKTQLQLEALLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 878), in which amino acid 16 is Ala and amino acid 42 is Ala. In some cases, the TEP 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 depicted in FIG. 21H.

[0288] As another example, in some cases, the TEP comprises, in order from N-terminus to C-terminus, (a) a SARS-CoV-2 peptide, e.g., a peptide having the amino acid sequence YLQPRTFLL (SEQ ID NO: 218); (b) a β2M polypeptide comprising a Cys at position 12; (c) an HLA-E polypeptide comprising a Cys at position 84 and a Cys at position 236; *01:01 polypeptide, (d) TTP, (e) an Ig Fc polypeptide, (f) a first variant IL-2 polypeptide comprising an Ala at position 16 and an Ala at position 42, and (g) a second variant IL-2 polypeptide comprising an Ala at position 16 and an Ala at position 42, wherein the TEP comprises a peptide linker between (a) and (b), between (b) and (c), between (c) and (d), between (e) and (f), and between (f) and (g). In some cases, the β2M 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 β2M amino acid sequence depicted in FIG. 1B. In some cases, the β2M polypeptide comprises an HLA-E polypeptide comprising ... * The 01:01 polypeptide is associated with the HLA-E *2B, wherein amino acid 14 is Ala and amino acid 15 is Ala. In some cases, the Ig Fc 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 01:01 amino acid sequence, wherein amino acid 84 is Cys and amino acid 236 is Cys. In some cases, the TTP is an anti-CD19 scFv. In some cases, the Ig Fc 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 shown in FIG. 2B, wherein amino acid 14 is Ala and amino acid 15 is Ala. In some cases, the first and second variant IL-2 polypeptides comprise an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: APTSSSTKKTQLQLEALLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO:878), in which amino acid 16 is Ala and amino acid 42 is Ala. In some cases, the TEP 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 depicted in FIG.

[0289] As another example, in some instances, the TEP comprises, in order from N-terminus to C-terminus, (a) a SARS-CoV-2 peptide, e.g., a peptide having the amino acid sequence VMPLSAPTL (SEQ ID NO:914); (b) a β2M polypeptide comprising a Cys at position 12; (c) an HLA-E polypeptide comprising a Cys at position 84 and a Cys at position 236; *01:01 polypeptide, (d) TTP, (e) an Ig Fc polypeptide, (f) a first variant IL-2 polypeptide comprising an Ala at position 16 and an Ala at position 42, and (g) a second variant IL-2 polypeptide comprising an Ala at position 16 and an Ala at position 42, wherein the TEP comprises a peptide linker between (a) and (b), between (b) and (c), between (c) and (d), between (e) and (f), and between (f) and (g). In some cases, the β2M 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 β2M amino acid sequence depicted in FIG. 1B. In some cases, the β2M polypeptide comprises an HLA-E polypeptide comprising ... * The 01:01 polypeptide is associated with the HLA-E *2B, wherein amino acid 14 is Ala and amino acid 15 is Ala. In some cases, the Ig Fc 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 01:01 amino acid sequence, wherein amino acid 84 is Cys and amino acid 236 is Cys. In some cases, the TTP is an anti-CD19 scFv. In some cases, the Ig Fc 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 shown in FIG. 2B, wherein amino acid 14 is Ala and amino acid 15 is Ala. In some cases, the first and second variant IL-2 polypeptides comprise an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: APTSSSTKKTQLQLEALLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO:878), in which amino acid 16 is Ala and amino acid 42 is Ala. In some cases, the TEP 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 depicted in FIG. 21J.

[0290] As another example, in some cases, the TEP may comprise, in order from N-terminus to C-terminus, (a) a SARS-CoV-2 peptide, e.g., a peptide having the amino acid sequence YLQPRTFLL (SEQ ID NO: 218); (b) a β2M polypeptide comprising a Cys at position 12; (c) an HLA-A polypeptide comprising a Cys at position 84 and a Cys at position 236; *(d) a TTP, (e) an Ig Fc polypeptide, (f) a first variant IL-2 polypeptide comprising an Ala at position 16 and an Ala at position 42, and (g) a second variant IL-2 polypeptide comprising an Ala at position 16 and an Ala at position 42, wherein the TEP comprises a peptide linker between (a) and (b), between (b) and (c), between (c) and (d), between (e) and (f), and between (f) and (g). In some cases, the peptide linker between (a) and (b) comprises a Cys. In some cases, the peptide linker between (a) and (b) has the amino acid sequence GCGGS(GGGGS)2 (SEQ ID NO: 882). In some cases, the peptide linker between (b) and (c) has the amino acid sequence (GGGGS)3 (SEQ ID NO: 875). In some cases, the peptide linker between (c) and (d) is GGGGS (SEQ ID NO: 873). In some cases, the peptide linker between (d) and (e) is AAAGG (SEQ ID NO: 874). In some cases, the peptide linker between (e) and (f) is (GGGGS)3 (SEQ ID NO: 875). In some cases, the peptide linker between (e) and (f) is (AP)4. In some cases, the peptide linker between (f) and (g) is (GGGGS)4 (SEQ ID NO: 876). In some cases, the β2M 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 β2M amino acid sequence shown in FIG. 1B, and amino acid 12 is Cys. In some cases, the HLA-A * The 0201 polypeptide is HLA-A shown in FIG. *2B, wherein amino acid 14 is Ala and amino acid 15 is Ala. In some cases, the Ig Fc 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 shown in FIG. 2B, wherein amino acid 84 is Cys and amino acid 236 is Cys. In some cases, the TTP is an anti-CD19 scFv. In some cases, the Ig Fc 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 shown in FIG. 2B, wherein amino acid 14 is Ala and amino acid 15 is Ala. In some cases, the first and second variant IL-2 polypeptides comprise an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: APTSSSTKKTQLQLEALLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 878), in which amino acid 16 is Ala and amino acid 42 is Ala. In some cases, the anti-CD19 scFv 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 following amino acid sequence: DIQLTQSPASLAVSLGQRATISCKASQSVDYDGDSYLNWYQQIPGQPPKLLIYDASNLVSGIPPRFSGSGSGTDFTLNIHPVEKVDAATYHCQQSTEDPWTFGGGTKLEIKGGGGSGGGGSGGGGSQVQLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIWPGDGDTNYNGKFKGKATLTADESSSTAYMQLSSLASEDSAVYFCARRETTTVGRYYYAMDYWGQGTTVTVSS (SEQ ID NO: 872).In some cases, the TEP 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 depicted in Figure 23 A. In some cases, the TEP 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 depicted in Figure 23B.

[0291] TEPs without immunomodulatory polypeptides As discussed above, the present disclosure provides a TEP that does not include at least one MOD. For example, the TEP may include a single polypeptide chain (or a dimer of two single polypeptide chains, as described above) that includes (a) a peptide epitope, which is a peptide having a length of about 4 amino acids to about 25 amino acids, (b) a β2M polypeptide, (c) an MHC class I heavy chain polypeptide, (d) a scaffold component, such as an Ig Fc polypeptide or a non-Ig scaffold polypeptide, and (e) a TTP, and the TEP may include one or more independently selected linkers interposed between any two of components (a)-(e), and the TEP does not include a MOD (e.g., does not include a MOD listed in Table 2, e.g., does not include an IL-2 polypeptide, a 4-1BBL polypeptide). Such a TEP may also be referred to herein as a "MOD-free TEP." Components (a)-(e) of a MOD-free TEP may be arranged in various configurations, as shown generally in Figures 22A-22B. The components (other than the MOD) are as described above.

[0292] Nucleic Acids, Recombinant Expression Vectors, and Engineered Host Cells The present disclosure provides a nucleic acid comprising a nucleotide sequence encoding a TEP of the present disclosure. In some cases, the nucleotide sequence encoding the TEP is operably 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 a recombinant expression vector.

[0293] The present disclosure provides a recombinant expression vector comprising the nucleic acid encoding TEP.In some cases, the recombinant expression vector is a non-viral vector.In some cases, the recombinant expression vector is a viral construct, such as a recombinant adeno-associated viral construct (see, for example, U.S. Patent No. 7,078,387), a recombinant adenovirus construct, a recombinant lentivirus construct, a recombinant retrovirus construct, a non-integral viral vector, etc.

[0294] Suitable expression vectors are well known to those skilled in the art.Suitable expression vectors are disclosed in published PCT applications WO2020132138A1 and WO2019 / 051091, the disclosures of which are expressly incorporated herein by reference as they relate to such expression vectors, including, in particular, paragraphs

[0515] to

[0520] of WO2020132138A1 and paragraphs

[0401] to

[0406] of WO2019 / 051091.

[0295] The disclosure further provides genetically modified host cells, where the host cells are genetically modified with a nucleic acid, or expression vector, described herein.

[0296] Suitable host cells include eukaryotic cells such as yeast, insect cells, and mammalian cells. In some cases, the host cells are cells of a mammalian cell line. Suitable mammalian cell lines include human cell lines, non-human primate cell lines, rodent (e.g., mouse, rat) cell lines, and the like. Suitable mammalian cell lines include HeLa cells (e.g., American Type Culture Collection (ATCC) No. CCL-2), CHO cells (e.g., ATCC Nos. CRL9618, CCL61, CRL9096), 293 cells (e.g., ATCC No. CRL-1573), Vero cells, NIH 3T3 cells (e.g., ATCC No. CRL-1658), Huh-7 cells, BHK cells (e.g., ATCC No. CCL10), PC12 cells (ATCC No. CRL1721), COS cells, COS-7 cells (ATCC No. CRL1651), RAT1 cells, mouse L cells (ATCC No. CCLI.3), human embryonic kidney (HEK) cells (ATCC No. CRL1573), HLHepG2 cells, and the like.

[0297] In some cases, the host cell is a mammalian cell that has been genetically modified so that it does not synthesize endogenous MHC β2M.

[0298] In some cases, the host cell is a mammalian cell that has been genetically modified to not synthesize endogenous MHC class I heavy chains. In some cases, the host cell is a mammalian cell that has been genetically modified to not synthesize endogenous MHC β2M and to not synthesize endogenous MHC class I heavy chains.

[0299] Methods for Producing T Cell Engaging Polypeptides The TEP of the present disclosure can be produced by culturing the genetically modified host cells of the present disclosure in a suitable culture medium in vitro, and such culturing results in the production of the TEP. For example, a mammalian host cell (e.g., a CHO cell) can be genetically modified with a recombinant expression vector comprising a nucleotide sequence encoding the TEP of the present disclosure, and the genetically modified mammalian host cell can be cultured in vitro in a suitable culture medium such that the genetically modified mammalian host cell produces the TEP. The TEP can be isolated, for example, from the culture medium in which the genetically modified mammalian host cell is cultured and / or from a cell lysate of the genetically modified mammalian host cell. The TEP can be isolated using any of a variety of well-established methods. If the TEP comprises an Ig Fc polypeptide at its C-terminus, intracellular processing can remove the C-terminal Lys residue from the C-terminus of the Ig Fc polypeptide. See, e.g., van den Bremer et al. (2015) mAbs 7:4, and Sissolak et al. (2019) J. Industrial Microbiol. & Biotechnol. 46:1167. As described above, two TEPs, each comprising an Ig Fc polypeptide (e.g., IgG1 Fc), may spontaneously form a homodimer of two TEPS, with the individual TEPs linked by one or more disulfide bonds between their respective Ig Fc portions.

[0300] composition The present disclosure provides compositions, including pharmaceutical compositions, that include the TEPs or dimerized TEPs disclosed herein.The present disclosure provides compositions, including pharmaceutical compositions, that include the nucleic acids or recombinant expression vectors.

[0301] Compositions containing TEP or dimerized TEP In addition to the TEP or dimerized TEP, the composition can include one or more pharma- ceutically acceptable excipients, such as carriers, diluents, buffers, salts, solubilizers, surfactants, stabilizers, or other additives, which may, for example, aid in the manufacturing process, protect, support, or enhance stability, bioavailability, and / or patient acceptability. Pharmaceutically acceptable excipients are well known to those of skill in the art.

[0302] When the TEP or dimerized TEP is administered directly into a tissue as an injection (e.g., subcutaneously, intraperitoneally, intramuscularly, and / or intravenously), the formulation may be provided in a ready-to-use form that can be directly injected or infused into a patient, or mixed with a saline solution for injection, optionally in a non-aqueous form (e.g., a shelf-stable powder that can be reconstituted) or in an aqueous form (e.g., a liquid consisting of a pharma- ceutically acceptable carrier and excipients). The formulation may also be provided to extend the serum half-life of the TEP after administration. For example, the TEP or dimerized TEP may be provided as a liposomal formulation prepared into a colloid, or using other conventional techniques to extend serum half-life. The formulation may also be provided in a controlled release form or a sustained release form.

[0303] The concentration of TEP or dimerized TEP in the liquid composition formulation can vary widely (e.g., less than about 0.1% by weight, typically or at least about 2% to 20% to 50% by weight, or more). Within this range are concentrations of about 5 to about 15 mg / mL, about 8 to about 12 mg / mL, and about 9 to about 11 mg / mL, including about 5 mg / mL, about 6 mg / mL, about 7 mg / mL, about 8 mg / mL, about 9 mg / mL, about 10 mg / mL, about 11 mg / mL, about 12 mg / mL, about 13 mg / mL, about 14 mg / mL, and about 15 mg / mL. The concentration can depend on a number of factors, including the stability of the TEP in the liquid composition.

[0304] In some cases, the TEP or dimerized TEP is present in a liquid composition. In some cases, the composition comprises a) the TEP or dimerized TEP, and b) saline (e.g., 0.9% NaCl). In some cases, the composition is sterile and suitable for administration to a human subject.

[0305] Compositions Comprising Nucleic Acids or Recombinant Expression Vectors The present disclosure provides pharmaceutical compositions comprising a nucleic acid (e.g., DNA, RNA, or mRNA) or a recombinant expression vector encoding a TEP. Published PCT applications WO2020132138A1 and WO2019 / 051091 disclose methods for preparing such compositions. See, for example, paragraphs

[0537] to

[0546] of WO2020132138A1 and paragraphs

[0423] to

[0432] of WO2019 / 051091, the disclosures of which are expressly incorporated herein by reference.

[0306] Methods for modulating T cell activity The disclosure provides methods of selectively modulating activity of epitope-specific T cells (e.g., T cells comprising a TCR specific for a virus (e.g., a viral peptide in complex with an MHC polypeptide), e.g., a T cell comprising a TCR specific for SARS-CoV-2 (e.g., a SARS-CoV-2 peptide in complex with an MHC polypeptide), and / or a T cell specific for a cancer-associated antigen), the method comprising contacting the T cell with a TEP, wherein contacting the T cell with the TEP comprises at least one MOD selectively modulating activity of the epitope-specific T cell. In some cases, the contacting occurs in vitro. In some cases, the contacting occurs in vivo. In some cases, the contacting occurs ex vivo.

[0307] When the TEP comprises an activating polypeptide, MOD, contacting the T cells with the TEP activates the epitope-specific T cells.

[0308] In some cases, the TEP comprises i) an activating polypeptide, MOD, ii) a CMV peptide, and iii) a TTP that targets cancer cells, and the TEP contacts T cells specific for the CMV peptide present in the TEP. In these cases, contacting the CMV-specific T cells with the TEP activates the CMV-specific T cells and / or increases the proliferation of the CMV-specific T cells. In some cases, contacting the CMV-specific T cells with the TEP increases the number and / or cytotoxic activity of the T cells against cancer cells targeted by the TTP present in the TEP. As one non-limiting example, the TEP comprises i) an activating polypeptide, MOD (e.g., an IL-2 polypeptide), ii) a CMV peptide as a peptide epitope, and iii) a TTP that is an scFv that binds to Her2, and contacting the TEP with cytotoxic T cells that bind the CMV peptide activates the T cells and increases their cytotoxic activity against Her2-expressing cancer cells.

[0309] In some cases, the TEP includes i) an activating polypeptide, MOD, ii) a SARS-CoV-2 peptide, and iii) a TTP that targets cancer cells, and the TEP is contacted with T cells specific for the SARS-CoV-2 peptide present in the TEP. In these cases, contacting the SARS-CoV-2-specific T cells with the TEP activates the SARS-CoV-2-specific T cells and / or increases the proliferation of the SARS-CoV-2-specific T cells. In some cases, contacting the SARS-CoV-2-specific T cells with the TEP increases the number and / or cytotoxic activity of the T cells against cancer cells targeted by the TTP present in the TEP. As one non-limiting example, the TEP comprises i) a MOD, which is an activating polypeptide (e.g., an IL-2 polypeptide), ii) a SARS-CoV-2 peptide as a peptide epitope, and iii) a TTP, which is an scFv that binds to Her2, and by contacting the TEP with a cytotoxic T cell that binds to the SARS-CoV-2 peptide, the T cell is activated and its cytotoxic activity against Her2-expressing cancer cells is increased.

[0310] The present disclosure provides a method for modulating an immune response in an individual, the method comprising administering an effective amount of a TEP to the individual. The administration of the TEP induces epitope-specific T cell responses (e.g., CMV-specific responses, SARS-CoV-2-specific responses, etc.) and epitope-nonspecific T cell responses, and the ratio of epitope-specific to epitope-nonspecific T cell responses is at least 2:1 or more, e.g., at least 5:1, 10:1, at least 25:1 or more. In some cases, the modulation increases the cytotoxic T cell response against cancer cells, e.g., cancer cells expressing an antigen targeted by the TTP present in the TEP.

[0311] "Regulating the activity" of T cells includes, for example, i) cytotoxicity (e.g., CD8 + ) activating T cells; ii) cytotoxicity (e.g., CD8 + ) inducing T cell cytotoxic activity, and / or iii) cytotoxic (e.g., CD8 + ) inducing the production and release of cytotoxins (e.g., perforin, granzymes, granulysin) by T cells.

[0312] The present disclosure provides a method of selectively delivering an immunomodulatory polypeptide (MOD) to a target T cell, the method comprising contacting a mixed population of T cells with a TEP, the mixed population of T cells comprising target T cells and non-target T cells, the target T cells being specific for an epitope present in the TEP (e.g., the target T cells are specific for an epitope present in the TEP), and the contacting step delivers one or more MODs present in the TEP to the target T cells. In some cases, the population of T cells is in vitro. In some cases, the population of T cells is in vivo within an individual. In some cases, the method comprises administering the TEP to the individual. In some cases, the T cells are cytotoxic T cells. In some cases, the mixed population of T cells is an in vitro population of mixed T cells obtained from an individual, and the contacting step results in activation and / or proliferation of the target T cells to generate a population of activated and / or expanded target T cells, and in some of these cases, the method further comprises administering the population of activated and / or expanded target T cells to the individual.

[0313] The present disclosure provides a method for detecting the presence of target T cells that bind an epitope of interest (e.g., a SARS-CoV-2 epitope) in a mixed population of T cells obtained from an individual, the method comprising: a) contacting the mixed population of T cells in vitro with a TEP, the TEP comprising the epitope of interest (e.g., a SARS-CoV-2 epitope); and b) detecting activation and / or proliferation of the T cells in response to said contact, wherein activated and / or proliferated T cells indicate the presence of the target T cells.

[0314] Treatment method The present disclosure provides a method of treating an individual, the method comprising administering to the individual a TEP (e.g., a homodimeric TEP), or one or more nucleic acids encoding the TEP, in an amount effective to treat the individual. A TEP for use in a method of treating the human or animal body is also provided. In some cases, the method of treatment comprises administering to an individual in need thereof one or more recombinant expression vectors comprising a nucleotide sequence encoding the TEP. In some cases, the method of treatment comprises administering to an individual in need thereof one or more mRNA molecules comprising a nucleotide sequence encoding the TEP. In some cases, the method of treatment comprises administering to an individual in need thereof a TEP. Treatable conditions include, for example, cancer, as described below, and autoimmune diseases.

[0315] In some cases, the method of the present disclosure includes administering two different TEPs, for example, when the two TEPs are different from each other in amino acid sequence.For example, in some cases, the method of the present disclosure includes administering a first TEP and a second TEP, where the first TEP includes a first peptide epitope and the second TEP includes a second peptide epitope that is different from the first peptide epitope in amino acid sequence.The first and second TEPs can include the same or different MHC class I heavy chain polypeptides.The first and second TEPs can include the same or different TTPs.The first and second TEPs can be administered substantially simultaneously or at different times.

[0316] A TEP comprising at least one MOD can both 1) modulate the activity of epitope-specific T cells (e.g., T cells specific for an epitope present in the TEP) and 2) target the TEP to a target cell. For example, the TTP can bind the TEP to a cancer cell, and the TEP engages and activates epitope-specific T cells, e.g., increasing the cytotoxic activity of the T cells, which then leads to the killing of the cancer cell. For example, in some cases, the TEP 1) targets the TEP to a cancer cell and 2) induces a cytotoxic T cell response against a viral epitope (e.g., a SARS-CoV-2 peptide, a CMV peptide, or other viral peptide) present in the TEP.

[0317] As discussed above, depending on the affinity of the wild-type MOD for its co-MOD, the combination of reduced affinity of the MOD for its co-MOD and affinity of the epitope for the TCR provides enhanced selectivity of the TEP. Thus, for example, the TEP binds with higher affinity to a first T cell presenting both i) a TCR specific for an epitope present in the TEP, and ii) a co-immunomodulatory polypeptide that binds to an immunomodulatory polypeptide present in the TEP, compared to the affinity of the TEP binding to a second T cell presenting i) a TCR specific for an epitope other than the epitope present in the TEP, and ii) a co-immunomodulatory polypeptide that binds to an immunomodulatory polypeptide present in the TEP. A variant MOD may be advantageous in multiple situations, for example, where the wild-type MOD has a relatively high affinity for the co-MOD (e.g., IL-2 for IL-2R) and / or may have multiple effects upon binding (e.g., IL-2, which can both activate epitope-specific T cells and upregulate the production of Tregs), or where the MOD can bind to multiple co-MODs (e.g., CD80, which can bind both CD28 and CTLA4). In such cases, reducing affinity for the co-MOD may be advantageous by reducing off-target binding of the TEP, increasing desired activation of the TEP, and / or reducing or substantially eliminating undesired activation of the TEP.

[0318] The present disclosure provides a method for selectively regulating the activity of epitope-specific T cells in an individual, the method comprising administering to the individual an effective amount of a TEP or one or more nucleic acids (e.g., expression vectors, mRNA, etc.) comprising a nucleotide sequence encoding a TEP, the TEP selectively regulating the activation of epitope-specific T cells in the individual. By selectively regulating the activity of epitope-specific T cells, it is possible to treat a disease or disorder in the individual. Thus, the present disclosure provides a method of treatment comprising administering an effective amount of a TEP to an individual in need thereof.

[0319] The cancer that can be treated by the method includes any cancer that can be targeted by TTP.The cancer that can be treated by the method includes carcinoma, sarcoma, melanoma, leukemia, lymphoma and multiple myeloma.The cancer that can be treated by the method of the present disclosure includes solid tumor.The cancer that can be treated by the method includes metastatic cancer.

[0320] Cancers that can be treated by the methods disclosed herein include, but are not limited to, esophageal cancer, hepatocellular carcinoma, basal cell carcinoma (a form of skin cancer), squamous cell carcinoma (various tissues), bladder cancer, including transitional cell carcinoma (malignant neoplasm of the bladder), bronchogenic carcinoma, colon cancer, colorectal cancer, gastric cancer, lung cancer, including small cell and non-small cell carcinoma of the lung, adrenal cortical carcinoma, thyroid cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, renal cell carcinoma, ductal carcinoma in situ or bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, uterine cancer, testicular cancer, osteogenic carcinoma, epithelial carcinoma, and nasopharyngeal carcinoma.

[0321] Sarcomas that can be treated by the methods disclosed herein include, but are not limited to, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, chordoma, osteogenic sarcoma, osteosarcoma, angiosarcoma, endothelial sarcoma, lymphangiosarcoma, lymphangioendothelial sarcoma, synovium, mesothelioma, Ewing's sarcoma, leiomyosarcoma, rhabdomyosarcoma, and other soft tissue sarcomas.

[0322] Other solid tumors that can be treated by the methods disclosed herein include, but are not limited to, glioma, astrocytoma, medulloblastoma, craniopharyngioma, epithelioma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, and retinoblastoma.

[0323] Leukemias that may be suitable for therapy with the methods disclosed herein include, but are not limited to, a) chronic myeloproliferative syndromes (neoplastic disorders of pluripotent hematopoietic stem cells), b) acute myeloid leukemia (neoplastic transformation of pluripotent hematopoietic stem cells or hematopoietic cells with restricted lineage potential), c) chronic lymphocytic leukemia (CLL; clonal proliferation of immunologically immature and dysfunctional small lymphocytes), including B-cell CLL, T-cell CLL prolymphocytic leukemia, and hairy cell leukemia, and d) acute lymphoblastic leukemia (characterized by accumulation of lymphoblasts). Lymphomas that can be treated using the present methods include, but are not limited to, B-cell lymphomas (e.g., Burkitt's lymphoma), Hodgkin's lymphoma, non-Hodgkin's lymphoma, and the like.

[0324] Other cancers that can be treated by the methods disclosed herein include atypical meningioma, islet cell carcinoma, medullary thyroid carcinoma, mesenchymoma, hepatocellular carcinoma, hepatoblastoma, renal clear cell carcinoma, and mediastinal neurofibroma.

[0325] When the TEP comprises i) a peptide epitope that presents a viral epitope when in the MHC / peptide complex of the TEP, ii) a TTP that targets a cancer-associated antigen, the TEP can be administered to an individual in need thereof to treat cancer in the individual, where i) the TEP activates T cells specific for the viral epitope (e.g., a SARS-CoV-2 epitope, a CMV epitope, etc.), and ii) the cancer expresses the cancer epitope bound by the TTP. The present disclosure provides a method for treating cancer in an individual, the method comprising administering to the individual an effective amount of a TEP, the TEP comprising i) a peptide epitope that presents a viral epitope (e.g., a SARS-CoV-2 epitope, a CMV epitope, etc.) when in the MHC / peptide complex of the TEP, ii) a TTP that targets a cancer-associated antigen, and iii) a stimulatory immune-modulating polypeptide (e.g., an IL-2 polypeptide, a 4-1BBL polypeptide, etc.).

[0326] In some cases, an "effective amount" of a TEP is an amount that, when administered in one or more doses to an individual in need thereof, reduces the number of cancer cells in the individual. For example, in some cases, an "effective amount" of a TEP is an amount that, when administered in one or more doses to an individual in need thereof, reduces the number of cancer cells in the individual by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% compared to the number of cancer cells in the individual before or in the absence of administration of the TEP. In some cases, an "effective amount" of a TEP is an amount that, when administered in one or more doses to an individual in need thereof, reduces the number of cancer cells in the individual to undetectable levels.

[0327] In some cases, an "effective amount" of a TEP is an amount that, when administered in one or more doses to an individual in need thereof, reduces tumor mass / tumor volume in the individual. In some cases, an "effective amount" of a TEP is an amount that, when administered in one or more doses to an individual in need thereof, extends the survival time of the individual. For example, in some cases, an "effective amount" of a TEP is an amount that, when administered in one or more doses to an individual in need thereof, extends the survival time of the individual by at least 1 month, at least 2 months, at least 3 months, 3 months to 6 months, 6 months to 1 year, 1 year to 2 years, 2 years to 5 years, 5 years to 10 years, or more than 10 years, compared to the expected survival time of the individual in the absence of the TEP.

[0328] In some cases, an "effective amount" of a TEP is an amount that, when administered in one or more doses to an individual in need thereof, either as a monotherapy or as part of a combination therapy, reduces the overall tumor burden in the individual, i.e., the amount of cancer in the body, or alternatively keeps the total tumor burden in the patient relatively stable for a period of time sufficient for the patient to have confirmed "stable disease" as determined by standard RECIST criteria. See, e.g., Aykan and Ozatli (2020) World J. Clin. Oncol. 11:53.

[0329] In some cases, an effective amount of a TEP is an amount that, when administered in one or more doses to an individual in need thereof, either as a monotherapy or as part of a combination therapy, reduces tumor size by a sufficient amount, and for a sufficient duration, for the patient to have a confirmed "partial response" as determined by standard RECIST criteria.

[0330] In some cases, an effective amount of a TEP is an amount that, when administered in one or more doses to an individual in need thereof (e.g., an individual having a tumor), either as a monotherapy or as part of a combination therapy, reduces tumor size by a sufficient amount, and for a sufficient duration, for the patient to have a "confirmed complete response" as determined by standard RECIST criteria.

[0331] As mentioned above, in some cases, in carrying out the present treatment method, the TEP is administered to an individual in need thereof as the TEP itself. In other examples, in carrying out the present treatment method, one or more nucleic acids comprising a nucleotide sequence encoding the TEP are administered to an individual in need thereof. Thus, in other examples, one or more nucleic acids of the present disclosure, for example, one or more recombinant expression vectors of the present disclosure, are administered to an individual in need thereof.

[0332] In some cases, SARS-CoV-2 vaccines (e.g., Moderna, Pfizer, or J&J vaccines) have been shown to stimulate CD8 T cells in patients that recognize SARS CoV-2 peptides in the TEP. + To increase the proportion of T cells, the TEP is administered to the patient several days (e.g., 3–14 days, or 7–10 days) before administration of the TEP (which contains a SARS-CoV-2 peptide).

[0333] dose The appropriate dose can be determined by the attending physician or other qualified medical personnel based on various clinical factors. As is well known in the medical arts, the dose for any one patient depends on many factors, including the size, body surface area, age of the patient, the particular polypeptide or nucleic acid being administered, the patient's sex, the duration and route of administration, general health, and other drugs being administered concomitantly. The TEP of the present disclosure may be administered in amounts ranging from 1 ng / kg body weight to 20 mg / kg body weight or more per dose, e.g., 0.1 mg / kg body weight to 10 mg / kg body weight, e.g., 1 mg / kg body weight to 5 mg / kg body weight, or 5 mg / kg body weight to 10 mg / kg body weight, 10-15 mg / kg body weight or more, although doses below or above this exemplary range are contemplated, particularly considering the factors listed above. If the regimen is a continuous infusion, it may range from 1 μg to 10 mg per kilogram of body weight per minute. Generally, the TEPs of the present disclosure can be administered in an amount of about 1 mg / kg body weight to about 20 mg / kg body weight, such as about 1 mg / kg body weight to about 5 mg / kg body weight, about 5 mg / kg body weight to about 10 mg / kg body weight, about 10 mg / kg body weight to about 15 mg / kg body weight, or about 15 mg / kg body weight to about 20 mg / kg body weight. Typical ranges can be 1 mg / kg body weight to 5 mg / kg body weight, or 5 mg / kg body weight to about 10 mg / kg body weight, such as 1, 2, 4, 5, 6, 7, or 8 mg / kg body weight.

[0334] Following successful treatment, it may be desirable to have the patient undergo maintenance therapy in which TEP is administered at a maintenance dose within the ranges described above to prevent recurrence of the condition.

[0335] Those of skill in the art will readily appreciate that dosage levels may vary depending on the particular TEP, the severity of the symptoms, and the subject's susceptibility to side effects. Preferred dosages for a given compound can be readily determined by those of skill in the art using a variety of methods.

[0336] The frequency of administration of TEP can vary depending on any of a variety of factors, but is generally administered once a week, once every two weeks, once every three weeks, once every four weeks, once a month, or less than once a month, such as once every five weeks, once every six weeks, once every two months, once every three months, etc., but can also be administered more frequently than once a week, such as twice a week (biw), three times a week (tiw), four times a week, five times a week, six times a week, every other day (qod), or every day (qd). In some cases, TEP is administered once every three weeks. Administration should generally be discontinued upon disease progression or unacceptable toxicity.

[0337] The duration of administration of TEP can vary depending on any of a variety of factors, such as, for example, the response of the patient.For example, TEP can be administered for a period ranging from 1 month to about 2 months, from about 2 months to about 4 months, from about 4 months to about 6 months, from about 6 months to about 8 months, from about 8 months to about 1 year, from about 1 year to about 2 years, or from about 2 years to about 4 years or more.Typically, TEP continues to be administered at least as long as the patient continues to receive clinically determined benefits, which is likely to be at least several months to multiple years.

[0338] Combination therapy In some cases, the method of the present disclosure for treating cancer in an individual includes a) administering a TEP, and b) administering at least one additional therapeutic agent or treatment. Suitable additional therapeutic agents include, but are not limited to, small molecule cancer chemotherapeutic agents and immune checkpoint inhibitors. Suitable additional treatments include, for example, radiation, surgery (e.g., surgical removal of tumor), and the like.

[0339] The therapeutic methods of the present disclosure may include co-administration of a TEP and at least one additional therapeutic agent. By "co-administration" it is meant that both the TEP and the at least one additional therapeutic agent are administered to an individual, not necessarily at the same time, to achieve a therapeutic effect that is the result of administering both the TEP and the at least one additional therapeutic agent. The administration of the TEP and the at least one additional therapeutic agent may be substantially simultaneous, for example, the TEP may be administered to an individual within about 1 minute to about 24 hours (e.g., within about 1 minute, within about 5 minutes, within about 15 minutes, within about 30 minutes, within about 1 hour, within about 4 hours, within about 8 hours, within about 12 hours, or within about 24 hours) of the administration of the at least one additional therapeutic agent. In some cases, the TEP of the present disclosure is administered to an individual who is undergoing treatment with at least one additional therapeutic agent or has been treated with at least one additional therapeutic agent. The administration of the TEP may occur at different times and / or at different frequencies.

[0340] By way of example, the therapeutic methods of the present disclosure may include co-administration of a TEP and an immune checkpoint inhibitor, such as an antibody specific for an immune checkpoint. By "co-administration" it is meant that both the TEP and the immune checkpoint inhibitor (e.g., an antibody specific for an immune checkpoint polypeptide) are administered to an individual, not necessarily at the same time, to achieve a therapeutic effect that is the result of administering both the TEP and the immune checkpoint inhibitor (e.g., an antibody specific for an immune checkpoint polypeptide). The administration of the TEP and the immune checkpoint inhibitor (e.g., an antibody specific for an immune checkpoint polypeptide) may be substantially simultaneous, for example, the TEP can be administered to an individual within about 1 minute to about 24 hours (e.g., within about 1 minute, within about 5 minutes, within about 15 minutes, within about 30 minutes, within about 1 hour, within about 4 hours, within about 8 hours, within about 12 hours, within about 24 hours, within 1 week, within 3 weeks, 3 weeks, 4 weeks, 1 month) after administration of the immune checkpoint inhibitor (e.g., an antibody specific for an immune checkpoint polypeptide). In some cases, the TEP of the present disclosure is administered to an individual who is undergoing or has been treated with an immune checkpoint inhibitor (e.g., an antibody specific for an immune checkpoint polypeptide). The administration of the TEP and the immune checkpoint inhibitor (e.g., an antibody specific for an immune checkpoint polypeptide) may occur at different times and / or at different frequencies. If there is an established dosing interval for the checkpoint inhibitor, it may be possible to administer the TEP on the same day as the checkpoint inhibitor, depending on the interval. For example, in some cases where the dosing schedule for pembrolizumab is once every three weeks, a pharmaceutical composition comprising the TEP may be administered on the same day.

[0341] Exemplary immune checkpoint inhibitors include inhibitors that target immune checkpoint polypeptides, such as CD27, CD28, CD40, CD122, CD96, CD73, CD47, OX40, GITR, CSF1R, JAK, PI3K delta, PI3K gamma, TAM, arginase, CD137 (also known as 4-1BB), ICOS, A2AR, B7-H3, B7-H4, BTLA, CTLA-4, LAG3, TIM3, VISTA, CD96, TIGIT, CD122, PD-1, PD-L1, and PD-L2. In some cases, the immune checkpoint polypeptide is a stimulatory checkpoint molecule selected from CD27, CD28, CD40, ICOS, OX40, GITR, CD122, and CD137. In some cases, the immune checkpoint polypeptide is an inhibitory checkpoint molecule selected from A2AR, B7-H3, B7-H4, BTLA, CTLA-4, IDO, KIR, LAG3, PD-1, TIM3, CD96, TIGIT, and VISTA.

[0342] In some cases, the immune checkpoint inhibitor is an antibody specific for an immune checkpoint polypeptide. In some cases, the anti-immune checkpoint antibody is a monoclonal antibody. In some cases, the anti-immune checkpoint antibody is humanized or deimmunized such that the antibody does not substantially elicit an immune response in humans. In some cases, the anti-immune checkpoint antibody is a humanized monoclonal antibody. In some cases, the anti-immune checkpoint antibody is a deimmunized monoclonal antibody. In some cases, the anti-immune checkpoint antibody is a fully human monoclonal antibody. In some cases, the anti-immune checkpoint antibody inhibits binding of an immune checkpoint polypeptide to a ligand of the immune checkpoint polypeptide. In some cases, the anti-immune checkpoint antibody inhibits binding of an immune checkpoint polypeptide to a receptor of the immune checkpoint polypeptide.

[0343] Suitable anti-immune checkpoint antibodies include, but are not limited to, nivolumab (Bristol-Myers Squibb), pembrolizumab (Merck), pidilizumab (Curetech), AMP-224 (GlaxoSmithKline / Amplimmune), MPDL3280A (Roche), MDX-1105 (Medarex, Inc. / Bristol Myer Squibb), MEDI-4736 (Medimmune / AstraZeneca), allerumab (Merck Serono), ipilimumab (YERVOY, (Bristol-Myers Squibb), tremelimumab (Pfizer), pidilizumab (CureTech, Ltd.), IMP321 (Immutep SA), MGA271 (Macrogenics), BMS-986016 (Bristol-Meyers Squibb), lirilumab (Bristol-Myers Squibb), urelumab (Bristol-Myers Squibb), PF-05082566 (Pfizer), IPH2101 (Innate Pharma / Bristol-Myers Squibb), MEDI-6469 (MedImmune / AZ), CP-870, 893 (Genentech), mogamulizumab (Kyowa Hakko Kirin), valilumab (CelIDex Therapeutics), avelumab (EMD Serono), galiximab (Biogen Idec), AMP-514 (Amplimmune / AZ), AUNP 12 (Aurigene and Pierre Fabre), indoximod (NewLink Genetics), NLG-919 (NewLink Genetics), INCB024360 (Incyte), KN035, and combinations thereof. For example, in some cases, the immune checkpoint inhibitor is an anti-PD-1 antibody. Suitable anti-PD-1 antibodies include, for example, nivolumab, pembrolizumab (also known as MK-3475), pidilizumab, SHR-1210, PDR001, and AMP-224. In some cases, the anti-PD-1 monoclonal antibody is nivolumab, pembrolizumab, or PDR001.Suitable anti-PD1 antibodies are described in US Patent Publication No. 2017 / 0044259. For pidilizumab, see, e.g., Rosenblatt et al. (2011) J. Immunother. 34:409-18. In some cases, the immune checkpoint inhibitor is an anti-CTLA-4 antibody. In some cases, the anti-CTLA-4 antibody is ipilimumab or tremelimumab. For tremelimumab, see, e.g., Ribas et al. (2013) J. Clin. Oncol. 31:616-22. In some cases, the immune checkpoint inhibitor is an anti-PD-L1 antibody. In some cases, the anti-PD-L1 monoclonal antibody is BMS-935559, MEDI4736, MPDL3280A (also known as RG7446), KN035, or MSB0010718C. In some embodiments, the anti-PD-L1 monoclonal antibody is MPDL3280A (atezolizumab), or MEDI4736 (durvalumab). For durvalumab, see, e.g., International Publication No. WO 2011 / 066389. For atezolizumab, see, e.g., U.S. Patent No. 8,217,149. In some cases, the immune checkpoint inhibitor is an anti-TIGIT antibody that binds to T cell immunoreceptor with immunoglobulin and ITIM domains (TIGIT). In some cases, the anti-TIGIT antibody is BMS-986207 (Bristol-Myers Squibb). In some cases, the anti-TIGIT antibody is tiragumab. In some cases, the anti-TIGIT antibody is EOS88448 (EOS-448). See, e.g., U.S. Pat. Nos. 11,008,390 and 10,189,902, U.S. Patent Application Publication No. 2017 / 0088613, and WO 2019 / 137541.

[0344] Among such checkpoint inhibitors, antibodies against PD-1, PD-L1, and CTLA-4 are the most common, with at least nivolumab, tremelimumab, pembrolizumab, ipilimumab, cemiplimab, atezolizumab, avelumab, tisleizumab, and durvalumab approved by the FDA and / or regulatory agencies outside the U.S. The TEPs of the present disclosure may also be co-administered with a combination of checkpoint inhibitors, such as a combination of (i) an antibody against PD-1, or PD-L1, and (ii) an antibody against CTLA-4.

[0345] Suitable subjects for treatment Suitable subjects for treatment using the methods include individuals with cancer, including individuals who have been diagnosed with cancer, individuals who have been treated for cancer but have not responded to the treatment, and individuals who have been treated for cancer and initially responded but have subsequently become non-responsive to treatment and / or have experienced disease progression during previous treatment.

[0346] In some cases, the subject is an individual undergoing treatment with an immune checkpoint inhibitor. In some cases, the subject is an individual undergoing treatment with an immune checkpoint inhibitor, but whose disease has progressed despite such treatment. In some cases, the subject is an individual undergoing treatment with a cancer chemotherapeutic agent or has been treated with a cancer chemotherapeutic agent. In some cases, the subject is an individual preparing to undergo treatment with an immune checkpoint inhibitor, undergoing treatment with an immune checkpoint inhibitor, or an individual who has been treated with an immune checkpoint inhibitor. In some cases, the subject is an individual preparing to undergo, undergoing, or has undergone treatment with a cancer chemotherapeutic agent, radiation therapy, surgery, and / or another therapeutic agent. In some cases, the pharmaceutical composition comprising TEP is administered in an adjuvant or neoadjuvant setting.

[0347] Examples of Non-Limiting Aspects of the Disclosure The aspects including the embodiments of the subject matter described above may be useful alone or in combination with one or more other aspects or embodiments. Without limiting the above description, certain non-limiting aspects of the disclosure are provided below. As will be apparent to those skilled in the art upon reading this disclosure, each of the individually numbered aspects may be used or combined with any of the preceding or subsequent individually numbered aspects. This is intended to provide support for all such combinations of aspects, and is not limited to the combinations of aspects explicitly provided below.

[0348] Embodiment 1. A single chain T-cell engaging polypeptide (TEP) comprising: (a) a peptide epitope, the peptide having a length of about 4 amino acids to about 25 amino acids or about 8 amino acids to about 12 amino acids; (b) a β2 microglobulin (β2M) polypeptide; (c) a major histocompatibility complex (MHC) class I heavy chain polypeptide; (d) an immunoglobulin (Ig) Fc polypeptide or a non-Ig scaffold polypeptide; and (e) a tumor targeting polypeptide (TTP); and, optionally, one or more immune modulating polypeptides, and may include one or more independently selected linkers interposed between any two of components (a)-(e).

[0349] Aspect 2. In order from the N-terminus to the C-terminus, (a1) the peptide; (b1) the β2M polypeptide; (c1) the MHC class I heavy chain polypeptide; (d1) the TTP; (e1) an Ig Fc polypeptide; and (f1) one or more immune modulating polypeptides; or (a2) the peptide; (b2) the β2M polypeptide; (c2) the MHC class I heavy chain polypeptide; (d2) one or more immune modulating polypeptides; (e2) an Ig Fc polypeptide; and (f2) the TTP; or (a3) the peptide; (b3) the β2M polypeptide; (c3) the MHC class I heavy chain polypeptide; (d3) one or more immune modulating polypeptides; (e3) the TTP; and (f3) an Ig Fc polypeptide; or 2. The TEP of embodiment 1, comprising: (a4) the peptide; (b4) the β2M polypeptide; (c4) the MHC class I heavy chain polypeptide; (d4) the TTP; (e4) one or more immune modulating polypeptides; and (f4) an Ig Fc polypeptide.

[0350] Aspect 3. The TEP of aspect 1 or 2, wherein said TEP comprises one or more immunomodulatory polypeptides, and at least one of said one or more immunomodulatory polypeptides is a variant immunomodulatory polypeptide that exhibits reduced affinity for said cognate coimmunomodulatory polypeptide compared to the affinity of a corresponding wild-type immunomodulatory polypeptide for said cognate coimmunomodulatory polypeptide.

[0351] Embodiment 4. The TEP of embodiment 3, wherein at least one of said one or more immune modulating polypeptides is a variant IL-2 polypeptide comprising a) an H16A substitution and an F42A substitution, or b) an H16T substitution and an F42A substitution, and optionally said variant IL-2 polypeptide comprises an amino acid sequence having at least 95%, at least 98%, or at least 99% amino acid sequence identity to the IL-2 amino acid sequence depicted in FIG. 16A.

[0352] Embodiment 5. The TEP of any one of embodiments 1-4, wherein said Ig Fc polypeptide is an IgG1 Fc polypeptide, optionally wherein said IgG1 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), optionally wherein said Ig Fc polypeptide comprises an amino acid sequence having at least 95%, at least 98%, or at least 99% amino acid sequence identity to the IgG1 Fc amino acid sequence depicted in FIG. 2A, and optionally wherein said Ig Fc polypeptide does not comprise the C-terminal Lys that is present in a wild-type Ig Fc polypeptide.

[0353] Embodiment 6 The TEP according to embodiment 5, wherein the IgG1 Fc polypeptide comprises a L234A and a L235A substitution.

[0354] Embodiment 7. The TEP of any one of embodiments 1-6, wherein said TEP comprises at least two immunomodulatory polypeptides, and at least two of said immunomodulatory polypeptides have an identical amino acid sequence.

[0355] Embodiment 8. The TEP of embodiment 7, wherein said two or more immunomodulatory polypeptides are in tandem, and optionally, said two or more immunomodulatory polypeptides are separated by a peptide linker.

[0356] ...

Claims

1. (a) a peptide epitope, the peptide having a length of about 4 amino acids to about 25 amino acids; (b) a β2 microglobulin (β2M) polypeptide; and (c) a major histocompatibility complex (MHC) class I heavy chain polypeptide; and (d) an immunoglobulin (Ig) Fc polypeptide or a non-Ig scaffold polypeptide; (e) a tumor targeting polypeptide (TTP); and Optionally, one or more immunomodulatory polypeptides. Including, may include one or more independently selected linkers interposed between any two of components (a)-(e); Single chain T cell engaging polypeptide (TEP).

2. From the N-terminus to the C-terminus, (a1) the peptide; (b1) the β2M polypeptide; and (c1) the MHC class I heavy chain polypeptide; and (d1) the TTP; (e1) an Ig Fc polypeptide; and (f1) one or more immunomodulatory polypeptides; Contains or (a2) the peptide; and (b2) the β2M polypeptide; and (c2) the MHC class I heavy chain polypeptide; and (d2) one or more immunomodulatory polypeptides; (e2) an Ig Fc polypeptide; and (f2) the TTP; Contains or (a3) the peptide; (b3) the β2M polypeptide; and (c3) the MHC class I heavy chain polypeptide; and (d3) one or more immunomodulatory polypeptides; (e3) the TTP; and (f3) Ig Fc polypeptide; Contains or (a4) the peptide; (b4) the β2M polypeptide; (c4) the MHC class I heavy chain polypeptide; and (d4) the TTP; (e4) one or more immunomodulatory polypeptides; (f4) Ig Fc polypeptide; 2. The TEP of claim 1 .

3. The TEP of claim 1 or claim 2, wherein the TEP comprises one or more immunomodulatory polypeptides, and at least one of the one or more immunomodulatory polypeptides is a variant immunomodulatory polypeptide that exhibits a lower affinity for the cognate coimmunomodulatory polypeptide compared to the affinity of the corresponding wild-type immunomodulatory polypeptide for the cognate coimmunomodulatory polypeptide.

4. 4. The TEP of claim 3, wherein at least one of the one or more immune modulating polypeptides is a variant IL-2 polypeptide comprising (a) an H16A and an F42A substitution, or (b) an H16T and an F42A substitution.

5. The TEP of any one of claims 1 to 4, wherein the Ig Fc polypeptide is an IgG1 Fc polypeptide, and optionally, the IgG1 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).

6. The TEP of claim 5 , wherein the IgG1 Fc polypeptide comprises a L234A and a L235A substitution.

7. The TEP of any one of claims 1 to 6, wherein the TEP comprises at least two immunomodulatory polypeptides, and at least two of the immunomodulatory polypeptides have an identical amino acid sequence.

8. 8. The TEP of claim 7, wherein the two or more immunomodulatory polypeptides are in tandem, and optionally the two or more immunomodulatory polypeptides are separated by a peptide linker.

9. The TEP according to any one of claims 1 to 8, wherein the peptide epitope is a peptide of a viral antigen.

10. 10. The TEP of claim 9, wherein the viral antigen is a cytomegalovirus (CMV) polypeptide.

11. The TEP of claim 10 , wherein the CMV polypeptide is a CMV pp65 polypeptide.

12. 12. The TEP of claim 11, wherein the peptide has the amino acid sequence NLVPMVATV (SEQ ID NO: 913) and has a length of 9 amino acids.

13. 10. The TEP of claim 9, wherein the viral antigen is a SARS-CoV-2 polypeptide.

14. 14. The TEP of claim 13, wherein the SARS-CoV-2 peptide comprises 4 to 25 consecutive amino acids of a SARS-CoV-2 surface glycoprotein.

15. The SARS-CoV-2 peptide is a surface glycoprotein peptide shown in FIG. 13J, and optionally the SARS-CoV-2 peptide is selected from the group consisting of STQDLFLPFF (SEQ ID NO:845), FCNDPFLGVYY (SEQ ID NO:846), SSANNCTFEY (SEQ ID NO:847), STECSNLLLQY (SEQ ID NO:848), YTNSFTRGVY (SEQ ID NO:849), CVADYSVLY (SEQ ID NO:850), LTDEMIAQY (SEQ ID NO:851), KIADYNYKL (SEQ ID NO:219), KLPDDFTGCV (SEQ ID NO:852). , SFELLHAPATV (SEQ ID NO: 853), LLFNKVTLA (SEQ ID NO: 221), VLNDILSRL (SEQ ID NO: 222), RLDKVEAEV (SEQ ID NO: 223), RLQSLQTYV (SEQ ID NO: 224), HLMSFPQSA (SEQ ID NO: 225), RLNEVAKNL (SEQ ID NO: 854), NLNESLIDL (SEQ ID NO: 855), FIAGLIAIV (SEQ ID NO: 226), VVFLHVTYV (SEQ ID NO: 856), YLQPRTFLL (SEQ ID NO: 218), SIIAYTMSL (SEQ ID NO: 220), TLDSKTQSL (SEQ ID NO: 53 4), KCYGVSPTK (SEQ ID NO: 857), TVYDPLQPELDSFK (SEQ ID NO: 333), GVYFASTEK (SEQ ID NO: 289), GTHWFVTQR (SEQ ID NO: 332), YYVGYLQPRTF (SEQ ID NO: 392), NYNYLYRLF (SEQ ID NO: 394), YFPLQSYGF (SEQ ID NO: 395), VYDPLQPELDSF (SEQ ID NO: 858), YEQYIKWPWYI (SEQ ID NO: 859), KWPWYIWLGF (SEQ ID NO: 860), GTITSGWTF (SEQ ID NO: 861), QYIKWPWYI (SEQ ID NO: 400), RFD NPVLPF (SEQ ID NO: 389), EILDITPCSF (SEQ ID NO: 862), FVFKNIDGY (SEQ ID NO: 592), SVASQSIIAY (SEQ ID NO: 863), WTAGAAAYY (SEQ ID NO: 145), VASQSIIAY (SEQ ID NO: 597), TPINLVRDL (SEQ ID NO: 476), SPRRARSVA (SEQ ID NO: 481), APHGVVFLHV (SEQ ID NO: 864), LPQGFSAL (SEQ ID NO: 477), QPYRVVVL (SEQ ID NO: 479), MIAQYTSAL (SEQ ID NO: 865), FPQSAPHGVVF (SEQ ID NO: 600),15. The TEP of claim 14, selected from the group consisting of SEPVLKGVKL (SEQ ID NO: 484), TEKSNIIRGW (SEQ ID NO: 711), TECSNLLLQY (SEQ ID NO: 866), and REGVFVSNGTHW (SEQ ID NO: 715).

16. 16. The TEP of claim 15, wherein the SARS-CoV-2 peptide has an amino acid sequence selected from YLQPRTFLL (SEQ ID NO:218), YLQPRTLFL (SEQ ID NO:887), YLQPRTLIL (SEQ ID NO:1201), and YLQPRTLVL (SEQ ID NO:1202), and the SARS-CoV-2 peptide has a length of 9 amino acids.

17. 14. The TEP of claim 13, wherein the SARS-CoV-2 polypeptide is a nonstructural polypeptide, optionally wherein the nonstructural polypeptide is NSP13.

18. 18. The TEP of claim 17, wherein the SARS-CoV-2 peptide has the amino acid sequence VMPLSAPTL and has a length of 9 amino acids.

19. The TEP of any one of claims 1 to 18, wherein the TEP is a single chain Fv (scFv) antibody or a nanobody.

20. The TEP of any one of claims 1 to 19, wherein the TEP binds to an antigen selected from mesothelin, TROP-2, Her2, CD19, WT1, MUC1, BCMA, PSMA, B7-H3, CEACAM, CD20, CD22, CD30, CD38, CD138, mesothelin, and claudin polypeptides.

21. The TEP of claim 20, wherein the TEP binds to a CD19 antigen.

22. 20. The TEP of claim 19, wherein the TEP comprises an amino acid sequence set forth in any one of Figures 17A-17L.

23. 23. The TEP of any one of claims 1 to 22, wherein the β2M polypeptide and the MHC heavy chain polypeptide are linked by an intrachain disulfide bond linking a Cys residue in the β2M polypeptide and a Cys residue in the MHC heavy chain polypeptide.

24. 24. The TEP of claim 23, wherein the Cys at amino acid residue 12 of the β2M polypeptide is disulfide bonded to the Cys at amino acid residue 236 of the MHC heavy chain polypeptide.

25. 25. The TEP of any one of claims 1 to 24, wherein the TEP comprises a linker between the peptide epitope and the β2M polypeptide, and an intrachain disulfide bond links a Cys present in the linker to a Cys at position 84 of the MHC heavy chain polypeptide.

26. The TEP of any one of claims 1 to 25, wherein the MHC class I heavy chain is an HLA-E allele heavy chain polypeptide.

27. The TEP of any one of claims 1 and 5-26, which does not contain an immunomodulatory polypeptide.

28. (i) a homodimer comprising two single-chain TEPs having identical amino acid sequences, wherein the two single-chain TEPs comprise an Ig Fc polypeptide, and the two single-chain TEPs are covalently linked by one or more disulfide bonds between the Ig Fc polypeptides; or (ii) A heterodimer comprising two single-chain TEPs having different amino acid sequences, the two single-chain TEPs comprising an Ig Fc polypeptide having an interspecies binding sequence. A dimer comprising two of the single-chain TEPs according to any one of claims 1 to 27,

29. A nucleic acid comprising a nucleotide sequence encoding the TEP of any one of claims 1 to 27.

30. 30. A recombinant expression vector comprising the nucleic acid of claim 29.

31. 28. A method for producing a T-cell engaging polypeptide (TEP), the method comprising culturing a host cell in vitro, the host cell being genetically modified with a recombinant expression vector comprising a nucleotide sequence encoding a TEP according to any one of claims 1 to 27, the culturing being carried out under conditions providing for the production of the TEP by the genetically modified host cell.

32. 29. A method of selectively modulating the activity of epitope-specific T cells, the method comprising contacting said T cells with a T cell engaging polypeptide of any one of claims 1 to 27 or a dimer of claim 28, wherein said contacting selectively modulates the activity of said epitope-specific T cells.

33. 29. A method of treating a patient with cancer comprising administering to the patient an effective amount of a pharmaceutical composition comprising a T cell polypeptide according to any one of claims 1 to 27 or a dimer according to claim 28.

34. 34. The method of claim 33, wherein the cancer is a carcinoma, sarcoma, melanoma, leukemia, or lymphoma.

35. 35. The method of claim 33 or 34, wherein the administration is intramuscular, intravenous, peritumoral, or intratumoral.

36. 36. The method of any one of claims 33 to 35, comprising administering to the patient a SARS-CoV-2 vaccine prior to administering to the patient an effective amount of a pharmaceutical composition comprising a T cell engaging polypeptide.

37. The method of any one of claims 33 to 35, wherein the patient is a patient who has received a SARS-CoV-2 vaccine.

38. 38. The method of any one of claims 33-37, comprising co-administering an immune checkpoint inhibitor to the patient, optionally wherein the immune checkpoint inhibitor is an antibody specific for PD-L1, PD-1, TIGIT, LAG3, or CTLA4.

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