EA049816B1 - T-CELL-MODULATING MULTIMERIC POLYPEPTIDES AND METHODS OF THEIR APPLICATION - Google Patents

Abstract

The present invention provides T-cell-modulating multimeric polypeptides that comprise an immunomodulatory polypeptide and a Wilms' tumor epitope-presenting peptide. The T-cell-modulating multimeric polypeptide is suitable for modulating T-cell activity and the immune response in an individual.

Description

Cross reference

This application claims priority to U.S. Provisional Patent Application No. 62/782,167, filed December 19, 2018, and U.S. Provisional Patent Application No. 62/814,684, filed March 6, 2019, which applications are incorporated herein by reference in their entirety.

Introduction

The adaptive immune response involves the interaction of the T cell receptor (TCR), present on the surface of T cells, with a small peptide antigen noncovalently displayed on the surface of the antigen-presenting cell (APC) by the major histocompatibility complex (MHC; also referred to in humans as the human leukocyte antigen (HLA) complex). This interaction represents the targeting mechanism of the immune system and is a prerequisite for the molecular interaction of T cell modulation (activation or inhibition) and effector function. Following epitope-specific cell targeting, target T cells are activated through interaction of costimulatory proteins located on APCs with similar costimulatory proteins on T cells. Both signals, epitope/TCR binding and interaction of APC costimulatory proteins with T cell costimulatory proteins, are required to direct T cell specificity and activation or inhibition. The TCR is specific for a given epitope; However, the costimulatory protein is not epitope-specific and is therefore typically expressed on all T cells or on large subsets of T cells.

Brief description of the invention

The present invention provides T-cell modulating multimeric polypeptides (TMMPs) comprising an immunomodulatory polypeptide and comprising a Wilms tumor epitope-presenting peptide. The T-cell modulating multimeric polypeptide is useful for modulating T-cell activity as well as for modulating an immune response in an individual.

Brief description of graphic materials

Fig. 1A-1F are schematic illustrations of various TMMPs according to the present invention.

Fig. 2A-2F are schematic representations of various disulfide-linked TMMPs of the present invention.

Fig. 3A-3E shows the amino acid sequence of WT-1 polypeptides. The sequences in Fig. 3A-3E are mapped to SEQ ID NOs: 399-403, respectively.

Fig. 4A-4E show the amino acid sequences of exemplary polypeptide chains of the TMMPs of the present invention. The sequences of the exemplary polypeptide chains in Fig. 4A-4E are depicted as SEQ ID NOs: 405-409, respectively. The epitope sequences are depicted as follows: Fig. 4D: CMTWNQMNL (SEQ IDNO:266); Fig. 4E: CYTWNQMNL (SEQ IDNO:267).

Figs. 5A-5G show the amino acid sequences of immunoglobulin Fc polypeptides. The sequences in Figs. 5A-5G are mapped to SEQ ID NOs: 410-421, respectively.

Figure 6 shows a multiple alignment of the amino acid sequences of the beta-2 microglobulin (P2M) precursors (i.e. including the leader sequence) from

Homo sapiens (NP 004039.1; SEQ ID NO: 19), Pan troglodytes (NP_001009066.1; SEQ ID NO: 19), Macaca mulatto (NP_001040602.1; SEQ ID

NO: 20), Bos taurus (NP_776318.1; SEQ ID NO: 21) and Mus musculus (NP_033865.2; SEQ ID

NO: 22).

Amino acids 1-20 constitute the signal peptide.

Fig. 7A-7C show the amino acid sequences of the full-length human HLA heavy chains from the alleles A*0101 (SEQ ID NO: 23), A*1101 (SEQ ID NO: 24), A*2402 (SEQ ID NO: 25), and A*3303 (SEQ ID NO: 26) (Fig. 7A); the full-length human HLA heavy chain from the allele B*0702 (SEQ ID NO: 27) (Fig. 7B), and the full-length HLA-C heavy chain (SEQ ID NO: 28) (Fig. 7C).

Fig. 8 shows an alignment of the amino acid sequences of eleven mature class I MHC heavy chains without their leader sequences, transmembrane domains, and intracellular domains. From top to bottom: SEQ ID NOs: 41-51.

Fig. 9A-9B show the alignment of the amino acid sequences of the HLA-A heavy chain (Fig. 9A; SEQ ID NOs: 198-206, respectively) and the consensus sequence (Fig. 9B; SEQ ID NO: 29).

Fig. 10A-10B show the alignment of the amino acid sequence of the HLA-B heavy chain (Fig. 10A; SEQ ID NO: 207-213, respectively) and the consensus sequence (Fig. 10B; SEQ ID NO: 30).

Fig. 11A-11B show the alignment of the amino acid sequences of the HLA-C heavy chain (Fig. 11A; SEQ ID NOs: 214-222, respectively) and the consensus sequence (Fig. 11B; SEQ ID NO: 31).

Figure 12 shows the consensus amino acid sequence for each heavy chain of HLA-E, -F, and -G (SEQ ID NOs: 32-34, respectively). Variable amino acid (aa) positions are shown as sequentially numbered X residues; amino acid positions 84, 139, and 236 are underlined with a double line.

Fig. 13 shows an alignment of the consensus amino acid sequences for HLA-A (SEQ ID NO: 35), -B (SEQ ID NO: 36), -C (SEQ ID NO: 37), -E (SEQ ID NO: 38), -F (SEQ ID NO: 39), and -G (SEQ ID NO: 40).

Fig. 14A-14I show the amino acid sequences of the polypeptide chains of the TMMPs of the present invention linked by two disulfide bonds. The sequences of the polypeptide chains in Fig. 14A-14I are depicted in SEQ ID NOs: 422-430, respectively. The epitope sequences are depicted as follows: Fig. 14B: VLDFAPPGA (SEQ id NO: 259); Fig. 14C

RMFPNAPYL (SEQ ID NO: 260);

RMFPNAPYL (SEQ ID NO: 260);

YMFPNAPYL (SEQ ID NO: 264).

fig. fig.

14F:

14H:

VLDFAPPGA (SEQ ID NO: 259);

YMFPNAPYL (SEQ ID NO: 264);

fig.

fig.

14G:

14I:

Fig. 15 shows expression and stability data for the WT1(37-45) epitope-containing gene of the present invention.

Fig. 16 shows expression and stability data for WT1(126-134) epitope-containing gene of the present invention.

Fig. 17A-17D are schematic representations of a doubly disulfide bonded TMMP of the present invention.

Fig. 18A-18C are schematic representations of exemplary configurations of disulfide-linked TMMPs of the present invention.

Fig. 19 shows schematic representations of examples of positions of immunomodulatory polypeptides in TMMPs of the present invention.

Figs. 20A-20R show the amino acid sequences of exemplary TMMP polypeptide chains of the present invention. The sequences of the exemplary polypeptide chains in Figs. 20A-20R are mapped as SEQ ID NOs: 431-448, respectively. The epitope sequences are mapped as follows: Fig. 20H: CYTWNQMNL (SEQ ID NO: 262); Fig. 20I CYTWNQMNL (SEQ ID NO: 262);

fig. 20J: CYTWNQMNL (SEQ ID NO: 262);

CYTWNQMNL (SEQ ID NO: 262);

NYMNLGATL (SEQ ID NO: 263);

NYMNLGATL (SEQ ID NO: 263);

NYMNLGATL (SEQ ID NO: 263).

fig. fig. fig.

fig. 20M: 20O: 20Q:

20K CYTWNQMNL (SEQ ID NO: 262); fig.

NYMNLGATL (SEQ ID NO: 263);

NYMNLGATL (SEQ ID NO: 263);

NYMNLGATL (SEQ ID NO: 263);

fig. fig. fig.

20L:

20N:

20P:

20R:

Fig. 21 shows the effect of TMMPs containing WT1 peptide epitopes and HLAA*02 heavy chains on the recruitment of antigen-specific CD8+ T cells.

Fig. 22 shows the effect of TMMPs containing WT1 peptide epitopes on the expansion of WT1-specific CD8+ T cells from total PBMCs during 8-day restimulation culture following 10-day culture-priming.

Fig. 23 shows TNF-α and IFN-γ production by expanded WT1-specific CD8+ T cells with WT1 37-45 TMMPs having either a G2C or R12C/G2C backbone.

Fig. 24 shows TNF-α and IFN-γ production by expanded WT1-specific CD8+ T cells with WT1 126-134 TMMPs bearing the R12C/G2C framework.

Fig. 25 shows the effect of disulfide bonds on IL-2-stimulated immune cell activation.

Fig. 26 shows the effect of the TMMP-containing IL-2 variant, as an immunomodulatory polypeptide, on CGLL-2 proliferation, compared with PROLEUKIN.

Fig. 27 shows the binding of 1715 + 2380 TMPPs to different Fc receptors.

Definitions

The terms polynucleotide and nucleic acid, as used interchangeably herein, refer to a polymeric form of nucleotides of any length - ribonucleotides or deoxyribonucleotides. Thus, the term includes, without limitation, single, double or multiple strands of DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids or a polymer containing purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural or derivatized nucleotide bases.

The terms peptide, polypeptide and protein are used interchangeably herein and refer to a polymeric form of amino acids of any length, which may include coding and non-coding amino acids, chemically or biochemically modified or derivatized amino acids and polypeptides having modified peptide backbones.

A polynucleotide or polypeptide has a certain percentage of sequence identity to another polynucleotide or polypeptide, meaning that when aligned, a certain percentage of the bases or amino acids are the same and in the same relative position when the two sequences are compared. Sequence identity can be determined in several different ways. To determine sequence identity, the sequence

- 2 049816 alignable sequences can be aligned using a variety of convenient methods and computer programs (e.g., BLAST, T-COFFEE, MUSCLE, MAFFT, etc.), available from Internet sites including ncbi.nlm.nili.gov/BLAST,ebi.ac.uk/Tools/msa/tcoffee/,ebi.ac.uk/Tools/msa/muscle/, mafftcbrc.jp/aligHMent/software/. See, e.g., Altschul et al. (1990), J. Mol. Bioi. 215:403–10.

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

The term immunological synapse or immune synapse, as used herein, generally refers to a 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, such as a lymphocyte, a T cell effector, a natural killer cell, and the like. The immunological synapse between an APC and a T cell is typically initiated by the interaction of a T cell antigen receptor and a major histocompatibility complex molecule, such as described in Bromley et al., Annu Rev Immunol. 2001;19:375-96; the contents of which are incorporated herein by reference in their entirety.

T cell includes all types of immune cells that express CD3, including helper T cells (CD4' cells), cytotoxic T cells (CD8' cells), T regulatory cells (Treg), and NK-T cells.

The term immunomodulatory polypeptide (also referred to as a costimulatory polypeptide) as used herein includes a polypeptide on an antigen presenting cell (APC) (e.g., a dendritic cell, a B cell, etc.) that specifically binds to a cognate co-immunomodulatory polypeptide on a T cell, thereby providing a signal that, in addition to the primary signal provided by, for example, binding of a TCR/CD3 complex to a peptide-loaded major histocompatibility complex (MHC) polypeptide, mediates a T cell response, including, but not limited to, proliferation, activation, differentiation, etc. The immunomodulatory polypeptide may include, but is not limited to, CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L, Fas ligand (FasL), inducible costimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM), CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, HVEM, lymphotoxin receptor beta, 3/TR6, ILT3, ILT4, HVEM, an agonist or an antibody that binds a Toll receptor ligand and a ligand that specifically binds to B7-H3.

As noted above, the immunomodulatory polypeptide (also referred to herein as MOD) specifically binds to the cognate co-immunomodulatory polypeptide on the T cell.

The immunomodulatory domain (MOD) of TMMP of the present invention binds a cognate coimmunomodulatory polypeptide that may be present on a target T cell.

Heterologous, as used herein, means a nucleotide or polypeptide that is not found in a native nucleic acid or protein, respectively.

The term recombinant, as used herein, 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 resulting in a construct having structural coding or non-coding sequences different from endogenous nucleic acids found in natural systems. DNA sequences encoding polypeptides may be assembled from cDNA fragments or from a series of synthetic oligonucleotides to provide a synthetic nucleic acid that is capable of expression from a recombinant transcriptional unit contained in a cell or in a cell-free transcription and translation system.

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

- 3 049816

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 is expressed as the dissociation constant (KD). The affinity can be at least 1-fold greater, at least 2-fold greater, at least 3-fold greater, at least 4-fold greater, at least 5-fold greater, at least 6-fold greater, at least 7-fold greater, at least 8-fold greater, at least 9-fold greater, at least 10-fold greater, at least 20-fold greater, at least 30-fold greater, at least 40-fold greater, at least 50-fold greater, at least 60-fold greater, at least 70-fold greater, at least 80-fold greater, at least 90-fold greater, at least 100-fold greater, or at least 1000-fold greater or more than the affinity of the antibody for unrelated amino acid sequences. The affinity of an antibody for a target protein can be, for example, from about 100 nM (nM) to about 0.1 nM, from about 100 nM to about 1 picomolar (pM), or from about 100 nM to about 1 femtomole (fM), or more. As used herein, the term avidity refers to the resistance of complexes of two or more agents to dissociate upon dilution. The terms immunoreactive and preferentially bind are used interchangeably herein to refer to antibodies and/or antigen-binding fragments thereof.

The term "binding" as used herein (e.g., in reference to the binding of a T cell modulating multimeric polypeptide (TMMP) to a polypeptide (e.g., a T cell receptor) on a T cell) refers to a non-covalent interaction between two molecules. Non-covalent binding refers to a direct association between two molecules, due to, for example, electrostatic, hydrophobic, ionic, and/or hydrogen bonding interactions, including interactions such as water bridges and salt bridges. Non-covalent binding interactions are typically characterized by a dissociation constant (KD) of less than ^10-6 M, less than ^10-7 M, less than ^10-8 M, less than ^10-9 M, less than ^10-10 M, less than ^10-11 M, less than ^10-12 M, less than ^10-13 M, less than ^10-14 M, or less than ^10-15 M. Affinity refers to the strength of non-covalent binding, with higher binding affinity correlating with a lower KD. Specific binding typically refers to binding with an affinity of at least about ^10-7 M or more, such as ^5x10-7 M, ^10-8 M, ^5x10-8 M, ^10-9 M, and more. Non-specific binding generally refers to binding (e.g., binding of a ligand to a molecule other than its designated binding site or receptor) with an affinity of less than about ^10-7 M (e.g., binding with an affinity of ^10-6 M, ^10-5 M, ^10-4 M). However, in some contexts, such as binding between a TCR and an MHC peptide/complex, specific binding may be in the range of 1 μM to 100 μM, or 100 μM to 1 mM. Covalent binding or covalent bond, as used herein, refers to the formation of one or more covalent chemical bonds between two different molecules.

The terms treatment, cure, etc. as used herein generally mean obtaining a desired pharmacological and/or physiological effect. The effect may be prophylactic in terms of preventing a disease or symptom in whole or in part, and/or may be therapeutic in terms of partially or completely curing the disease and/or adverse effects related to the disease. The term treatment as used herein encompasses any treatment of a disease or symptom in mammals, and includes: (a) preventing the disease or symptom from occurring in a subject that may be predisposed to acquiring the disease or symptom but has not yet been diagnosed as having it; (b) inhibiting the disease or symptom, i.e. stopping its development; and/or (c) alleviating the disease, i.e. causing the disease to regress. The therapeutic agent may be administered before, during, or after the onset of the disease or injury. Treatment of an ongoing disease, in which the treatment stabilizes or reduces the undesirable clinical symptoms of the patient, is of particular interest. It is desirable to carry out such treatment until complete loss of function in the affected tissues. It is desirable to administer the said treatment during the symptomatic stage of the disease, and in some cases after the symptomatic stage of the disease.

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

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

Where a range of values is provided, it shall be understood that each intermediate value, up to a tenth of the lower limit unless the context clearly states otherwise, between the upper and

- 4 049 816 the lower limit of this range and any other specified or intermediate value within the stated range are encompassed by the present invention. The upper and lower limits of these smaller ranges may be independently included in the smaller ranges and are also encompassed by the present invention, subject to any specifically excluded limit within the stated range. Where a stated range includes one or both limits, ranges excluding either or both of these included limits are also included in the invention.

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

The use of singular terms does not exclude plural terms unless the context clearly indicates otherwise. Thus, for example, a reference to a T-cell modulating multimeric polypeptide includes a plurality of such polypeptides, and a reference to an immunomodulatory polypeptide includes a reference to one or more immunomodulatory polypeptides and equivalents thereof known to those skilled in the art, and so forth. It should also be noted that the claims may be drafted to exclude any optional element. Thus, this statement is intended to serve as a precursor to the use of such exclusive terminology as solely, only, and the like in connection with the recitation of elements of the claims or the use of a negative limitation.

It should be understood that certain features of the invention that are described for clarity in the context of separate embodiments may also be presented in combination in a single embodiment. Conversely, various features of the invention that are described for brevity in the context of a single embodiment may also be presented separately or in any suitable subcombination. All combinations of embodiments related to this invention are specifically encompassed by this invention and are described herein as if each combination were individually and expressly disclosed. In addition, all subcombinations of various embodiments and elements thereof are also specifically encompassed by this invention and are described herein as if each such subcombination were individually and expressly disclosed herein.

The publications discussed herein are cited solely for their disclosure prior to the filing date of this application. Nothing herein shall be construed as an admission that the present invention is not entitled to precede such publication by virtue of prior invention. In addition, publication dates may differ from actual publication dates, which may require independent confirmation.

Detailed description

The present invention provides T-cell modulating multimeric polypeptides that comprise an immunomodulatory polypeptide and that comprise Wilms tumor epitope-presenting peptide-1 (WT-1). TMMP is useful for modulating T-cell activity as well as for modulating the immune response in an individual.

T cell modulating multimeric polypeptides.

The present invention provides a T cell modulating multimeric polypeptide (TMMP), comprising: a) a first polypeptide; and b) a second polypeptide, wherein the TMMP comprises an epitope; a first major histocompatibility complex (MHC) polypeptide; a second MHC polypeptide; one or more immunomodulatory polypeptides; and, optionally, an immunoglobulin (Ig) Fc polypeptide or a non-Ig framework. The present invention provides a TMMP, wherein the TMMP is a heterodimer comprising: a) a first polypeptide comprising a first MHC polypeptide; and b) a second polypeptide comprising a second MHC polypeptide, wherein the first polypeptide or the second polypeptide comprises an epitope; wherein the first polypeptide and/or the second polypeptide comprises one or more immunomodulatory polypeptides, which may be the same or different; and, optionally, an Ig Fc polypeptide or a non-Ig framework. The TMMP of the present invention is also referred to herein as the multimeric polypeptide of the present invention or synTac. In some cases, the peptide epitope present in the TMMP of the present invention is a WT-1 peptide.

The present invention provides a TMMP comprising a heterodimeric polypeptide comprising: a) a first polypeptide comprising: i) a peptide epitope; and ii) a first MHC polypeptide; b) a second polypeptide comprising a second MHC polypeptide; and c) at least one immunomodulatory polypeptide, wherein the first and/or second polypeptide comprises at least one (i.e., one or more) immunomodulatory polypeptide. Optionally, the first or second polypeptide comprises an Ig Fc polypeptide or a non-Ig scaffold. At least one of the one or more immunomodulatory

- 5,049,816 polypeptides is a variant of an immunomodulatory polypeptide that exhibits reduced affinity for a cognate co-immunomodulatory polypeptide compared to the affinity of the corresponding wild-type immunomodulatory polypeptide for the cognate co-immunomodulatory polypeptide. An epitope present in a TMMP of the invention binds to a T cell receptor (TCR) on a T cell with an affinity of at least 100 μM (e.g., at least 10 μM, at least 1 μM, at least 100 nM, at least 10 nM, or at least 1 nM). The TMMP of the invention binds to a first T cell with an affinity that is at least 25% higher than the affinity with which the TMMP binds to a second T cell, wherein the first T cell expresses on its surface a cognate coimmunomodulatory polypeptide and a TCR that binds the epitope with an affinity of at least 100 μM, and wherein the second T cell expresses on its surface a cognate coimmunomodulatory polypeptide but does not express on its surface a TCR that binds the epitope with an affinity of at least 100 μM (e.g., at least 10 μM, at least 1 μM, at least 100 nM, at least 10 nM, or at least 1 nM). In some cases, the peptide epitope present in the TMMP of the present invention is a WT-1 peptide.

The present invention provides a TMMR, wherein the TMMR is:

A) a heterodimer comprising: a) a first polypeptide comprising a first MHC polypeptide; and b) a second polypeptide comprising a second MHC polypeptide, wherein the first polypeptide or the second polypeptide comprises an epitope (e.g., a peptide that presents the epitope to a T cell); wherein the first polypeptide and/or the second polypeptide comprise one or more immunomodulatory polypeptides, which may be the same or different, and wherein at least one of the one or more immunomodulatory polypeptides may be a wild-type immunomodulatory polypeptide or a variant of a wild-type immunomodulatory polypeptide, wherein the variant immunomodulatory polypeptide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acid substitutions compared to the amino acid sequence of the corresponding wild-type immunomodulatory polypeptide; and wherein the first polypeptide or the second polypeptide optionally comprises an Ig Fc polypeptide or a non-Ig framework; or

B) a heterodimer comprising: a) a first polypeptide comprising a first MHC polypeptide; and b) a second polypeptide comprising a second MHC polypeptide, wherein the first polypeptide or the second polypeptide comprises an epitope; wherein the first polypeptide and/or the second polypeptide comprises one or more immunomodulatory polypeptides, which may be the same or different, wherein at least one of the one or more immunomodulatory polypeptides is a variant of a wild-type immunomodulatory polypeptide, wherein the variant immunomodulatory polypeptide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acid substitutions compared to the corresponding amino acid sequence of the wild-type immunomodulatory polypeptide, wherein at least one of the one or more immunomodulatory domains is a variant immunomodulatory polypeptide that exhibits reduced affinity for the cognate co-immunomodulatory polypeptide compared to the affinity of the corresponding wild-type immunomodulatory polypeptide for the cognate a co-immunomodulatory polypeptide, and wherein the epitope binds to a TCR on a T cell with an affinity of at least 10 ^-7 M such that: i) the TMMP polypeptide binds to a first T cell with an affinity that is at least 25% higher than the affinity with which the TMMP binds a second T cell, wherein the first T cell expresses on its surface a cognate co-immunomodulatory polypeptide and a TCR that binds the epitope with an affinity of at least 10 ^-7 M, and wherein the second T cell expresses on its surface a cognate co-immunomodulatory polypeptide but does not express on its surface a TCR that binds the epitope with an affinity of at least 10 ^-7 M; and/or ii) the ratio of the binding affinity of a control TMMP, where the control comprises a wild-type immunomodulatory polypeptide, to the cognate co-immunomodulatory polypeptide to the binding affinity of a TMMP comprising a variant wild-type immunomodulatory polypeptide to the cognate co-immunomodulatory polypeptide, as measured by biolayer interferometry, is in the range of 1.5:1 to 106:1; and wherein the variant immunomodulatory polypeptide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acid substitutions compared to the corresponding amino acid sequence of the wild-type immunomodulatory polypeptide; and wherein the first polypeptide or the second polypeptide optionally comprises an Ig Fc polypeptide or non-Ig scaffold; or

C) a heterodimer comprising: a) a first polypeptide comprising, in N-terminal to C-terminal direction: i) an epitope; ii) a first MHC polypeptide, and b) a second polypeptide comprising, in N-terminal to C-terminal direction: i) a second MHC polypeptide, and ii) optionally an immunoglobulin (Ig) Fc polypeptide or non-Ig framework, wherein the TMMP comprises one or more immunomodulatory domains that

- 6 049816 may be the same or different, wherein at least one of the one or more immunomodulatory domains is located: A) at the C-terminus of the first polypeptide; B) at the N-terminus of the second polypeptide; C) at the C-terminus of the second polypeptide, or D) at the C-terminus of the first polypeptide and at the N-terminus of the second polypeptide, and wherein at least one of the one or more immunomodulatory domains may be a wild-type immunomodulatory polypeptide or a variant of a wild-type immunomodulatory polypeptide, wherein the variant of the immunomodulatory polypeptide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acid substitutions compared to the amino acid sequence of the corresponding wild-type immunomodulatory polypeptide; and optionally wherein at least one of the one or more immunomodulatory domains is a variant of an immunomodulatory polypeptide that exhibits a reduced affinity for the cognate co-immunomodulatory polypeptide compared to the affinity of the corresponding wild-type immunomodulatory polypeptide for the cognate co-immunomodulatory polypeptide, and wherein the epitope binds to a TCR on a T cell with an affinity of at least 10 ^-7 M such that: i) the TMMP binds to a first T cell with an affinity that is at least 25% higher than the affinity with which the TMMP binds a second T cell, wherein the first T cell expresses on its surface a cognate co-immunomodulatory polypeptide and a TCR that binds the epitope with an affinity of at least 10 ^-7 M, and wherein the second T cell expresses on its surface a cognate co-immunomodulatory polypeptide, but does not express on its surface a TCR that binds the epitope with an affinity of at least ^10-7 M; and/or ii) the ratio of the binding affinity of a control TMMP, when the control comprises a wild-type immunomodulatory polypeptide, to the cognate co-immunomodulatory polypeptide to the binding affinity of a TMMP comprising a wild-type variant immunomodulatory polypeptide to the cognate co-immunomodulatory polypeptide, as measured by biolayer interferometry, is in the range of from 1.5:1 to ¹⁰⁶ :1; and wherein the immunomodulatory polypeptide variant comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acid substitutions compared to the corresponding amino acid sequence of the wild-type immunomodulatory polypeptide. In some cases, the peptide epitope present in the TMMP of the invention is a WT-1 peptide.

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

In some instances, an epitope present in a TMMP of the invention binds to the TCR on a T cell with an affinity of from about 10 ^-4 M to about 5 x 10 ^-4 M, from about 5 x 10 ^-4 M to about 10 ^-5 M, from about 10 ^-5 M to 5 x 10 ^-5 M, from about 5 x 10 ^-5 M to 10 v ⁶ M, from about 10 ^-6 M to about 5 x 10 ^-6 M, from about 5 x 10 ^-6 M to about 10 ^-7 M, from about 10 ^-7 M to about 5 x 10 ^-7 M, from about 5 x 10 ^-7 M to about 10 ^-8 M, or from about 10 ^-8 M to about 10 ^-9 M. Expressed differently, in some instances, an epitope present in a TMMP of the invention binds to the TCR on a T cell with an affinity of from about 1 nM to about 5 nM, from about 5 nM to about 10 nM, from about 10 nM to about 50 nM, from about 50 nM to about 100 nM, from about 0.1 μM to about 0.5 μM, from about 0.5 μM to about 1 μM, from about 1 μM to about 5 μM, from about 5 μM to about 10 μM, from about 10 μM to about 25 μM, from about 25 μM to about 50 μM, from about 50 μM to about 75 μM, from about 75 μM to about 100 μM.

- 7 049816

An immunomodulatory polypeptide present in a TMMP of the invention binds to a cognate co-immunomodulatory polypeptide with an affinity that is at least 10% less, at least 15% less, at least 20% less, at least 25% less, at least 30% less, at least 35% less, at least 40% less, at least 45% less, at least 50% less, at least 55% less, at least 60% less, at least 65% less, at least 70% less, at least 75% less, at least 80% less, at least 85% less, at least 90% less, at least 95% less, or more than 95% less than the affinity of the corresponding wild-type immunomodulatory polypeptide for the cognate co-immunomodulatory polypeptide.

In some cases, the immunomodulatory polypeptide variant present in the TMMP of the invention has a binding affinity for the cognate co-immunomodulatory polypeptide that is from 1 nM to 100 nM or from 100 nM to 100 μM. For example, in some cases, the immunomodulatory polypeptide variant present in the TMMP of the invention has a binding affinity for the cognate co-immunomodulatory polypeptide that is from about 100 nM to 150 nM, from about 150 nM to about 200 nM, from about 200 nM to about 250 nM, from about

250 nM to about 300 nM, from about 300 nM to about 350 nM, from about 350 nM to about 400 nM, from about

400 nM to about 500 nM, from about 500 nM to about 600 nM, from about 600 nM to about 700 nM, from about

700 nM to about 800 nM, about 800 nM to about 900 nM, about 900 nM to about 1 μM, about 1 μM to about 5 μM, about 5 μM to about 10 μM, about 10 μM to about 15 μM, about 15 μM to about 20 μM, about 20 μM to about 25 μM, about 25 μM to about 50 μM, about 50 μM to about 75 μM, or about 75 μM to about 100 μM. In some cases, the immunomodulatory polypeptide variant present in the TMMP of the present invention has a binding affinity for the cognate co-immunomodulatory polypeptide that is from about 1 nM to about 5 nM, from about 5 nM to about 10 nM, from about 10 nM to about 50 nM, from about 50 nM to about 100 nM.

The combination of the reduced affinity of the immunomodulatory polypeptide for its cognate co-immunomodulatory polypeptide and the affinity of the epitope for the TCR provides for improved selectivity of the TMMP of the invention. For example, the TMMP of the invention selectively binds to a first T cell that comprises on its surface both: i) a TCR specific for an epitope present in the TMMP and ii) a co-immunomodulatory polypeptide that binds to the immunomodulatory polypeptide present in the TMMP, compared to binding to a second T cell that comprises on its surface: i) a TCR specific for an epitope different from the epitope present in the TMMP and ii) a co-immunomodulatory polypeptide that binds to the immunomodulatory polypeptide present in the TMMP. For example, a TMMP of the present invention binds to a first T cell with an affinity that is at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 2-fold, at least 2.5-fold, at least 5-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 50-fold, at least 100-fold, or greater than 100-fold, greater than the affinity with which it binds a second T cell.

In some cases, a TMMP of the invention, when administered to an individual in need thereof, induces both an epitope-specific T cell response and an epitope-non-specific T cell response. In other words, in some cases, a TMMP of the invention, when administered to an individual in need thereof, induces an epitope-specific T cell response by modulating the activity of a first T cell that comprises on its surface both: i) a TCR specific for an epitope present on the TMMP; and ii) a co-immunomodulatory polypeptide that binds to the immunomodulatory polypeptide present on the TMMP; and induces an epitope-non-specific T cell response by modulating the activity of a second T cell that comprises on its surface both: i) a TCR specific for an epitope present on the TMMP; and ii) a co-immunomodulatory polypeptide that binds to an immunomodulatory polypeptide present in the TMMP. The ratio of epitope-specific T cell response to epitope-non-specific T cell response is at least 2:1, at least 5:1, at least 10:1, at least 15:1, at least 20:1, at least 25:1, at least 50:1, or at least 100:1. The ratio of epitope-specific T cell response to epitope-non-specific T cell response is from about 2:1 to about 5:1, from about 5:1 to about 10:1, from about 10:1 to about 15:1, from about 15:1 to about 20:1, from about 20:1 to about 25:1, from about 25:1 to about 50:1, or from about 50:1 to about 100:1, or greater than 100:1. Modulating T cell activity may involve one or more of: i) activating a cytotoxic T cell (e.g., CD8'); ii) inducing cytotoxic activity of a cytotoxic T cell (e.g., CD8 ⁺ ); iii) induction of production and release of cytotoxin (eg, perforin; granzyme; granulesin) by a cytotoxic T cell (eg, CD8 ⁺ ) iv) inhibition of autoreactive T cell activity; and the like.

- 8 049816

The combination of the reduced affinity of the immunomodulatory polypeptide for its cognate co-immunomodulatory polypeptide and the affinity of the epitope for the TCR provides for improved selectivity of the TMMP of the invention. Thus, for example, the TMMP of the invention binds with increased avidity to a first T cell that comprises on its surface both: i) a TCR specific for an epitope present in the TMMP and ii) a co-immunomodulatory polypeptide that binds to the immunomodulatory polypeptide present in the TMMP, compared to the avidity with which it binds to a second T cell that comprises on its surface: i) a TCR specific for an epitope different from the epitope present in the TMMP and ii) a co-immunomodulatory polypeptide that binds to the immunomodulatory polypeptide present in the TMMP.

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

The BLI assay can be performed using an Octet RED 96 instrument (Pal ForteBio) or a similar instrument as follows. A TMMP (e.g., a TMMP of the present invention; a control TMMP (wherein the control TMMP comprises a wild-type immunomodulatory polypeptide)) is immobilized on an insoluble support (biosensor). The immobilized TMMP is a target. The immobilization can be accomplished by immobilizing a capture antibody on an insoluble support, wherein the capture antibody immobilizes the TMMP. For example, the immobilization can be accomplished by immobilizing an anti-Fc antibody (e.g., an anti-human IgG Fc antibody) on an insoluble support, wherein the immobilizing anti-Fc antibodies bind to and immobilize the TMMP (wherein the TMMP comprises an Ig Fc polypeptide). The co-immunomodulatory polypeptide is applied at several different concentrations to the immobilized TMMP and the instrument response is recorded. The assays are performed in a liquid medium containing 25 mM HEPES pH 6.8, 5% polyethyleneglycol 6000, 50 mM KCl, 0.1% bovine serum albumin, and 0.02% Tween 20 nonionic detergent. Binding of the co-immunomodulatory polypeptide to the immobilized TMMP is carried out at 30°C. An anti-MHC class I monoclonal antibody can be used as a positive control for binding affinity. For example, anti-HLA class I monoclonal antibody W6/32 (American Type Culture Collection No. HB-95; Parham et al. (1979) J. Immunol. 123:342), which has a K _D of 7 nM, can be used. A standard curve can be prepared using serial dilutions of the anti-MHC class I monoclonal antibody. The co-immunomodulatory polypeptide, or mAb (monoclonal antibody) against class I MHC, is the analyte. BLI analyzes the interference pattern of white light reflected from two surfaces: i) the immobilized polypeptide (target); and ii) an internal reference layer. Changing the number of molecules (analyte; e.g., co-immunomodulatory polypeptide; anti-HLA antibody) bound to the biosensor tip causes a change in the interference pattern; this shift in the interference pattern can be measured in real time. Two kinetic terms that describe the affinity of the target-analyte interaction are the association constant ( _ka ) and the dissociation constant (kd). The ratio of these two variables (kd/ _a ) forms the affinity constant KD.

The BLI assay is performed in a multiwell plate. To perform the assay, the plate layout is defined, the assay steps are determined, and the biosensors are assigned in the Octet Data Acquisition software. The biosensor assembly is hydrated. The hydrated biosensor assembly and assay plate are equilibrated for 10 min on the Octet instrument. Once the data are acquired, the acquired data are loaded into the Octet data analysis software. The data are processed in the Processing window by specifying the reference subtraction method, Y-axis alignment, interstep correction, and Savitzky-Golay filtering. The data are analyzed in the Analysis window by specifying the assay steps (association and dissociation), selection of the curve fitting model (1:1), fitting method (global), and window of interest (in seconds). The quality of the fit is assessed. The KD values for each of the data (analyte concentration) can be averaged if they are within a 3-fold range. The KD error values should be within the same order of the affinity constant values; R ² values should be greater than 0.95. See, for example, Abdiche et al. (2008) J. Anal. Biochem. 377:209.

Unless otherwise stated, the affinity of a TMMP of the invention for a cognate co-immunomodulatory polypeptide, or the affinity of a control TMMP (wherein the control TMMP comprises a wild-type immunomodulatory polypeptide) for a cognate co-immunomodulatory polypeptide, is determined using BLI as described above.

In some cases, the ratio of: i) the binding affinity of the control TMMP (when the con

- 9 049816 the troll comprises a wild-type immunomodulatory polypeptide) for a cognate coimmunomodulatory polypeptide to ii) the binding affinity of the TMMP of the invention comprising the wild-type variant immunomodulatory polypeptide for the cognate coimmunomodulatory polypeptide, as measured by BLI (as described above), is at least 1.5:1, at least 2:1, at least 5:1, at least 10:1, at least 15:1, at least 20:1, at least 25:1, at least 50:1, at least 100:1, at least 500:1, at least 10 ² :1, at least 5x102:1, at least 10 ³ :1, at least 5x103:1, at least 10 ⁴ :1, at least 10 ⁵ :1, or at least 10 ⁶ :1. In some cases, the ratio of: i) the binding affinity of a control TMMP (where the control comprises a wild-type immunomodulatory polypeptide) to the cognate co-immunomodulatory polypeptide to ii) the binding affinity of a TMMP of the invention comprising a wild-type variant immunomodulatory polypeptide to the cognate co-immunomodulatory polypeptide, as measured by BLI (as described above), is in the range of from ^1.5 :1 to 10:1, from 10:1 to 50:1, from 50:1 to 10 ² :1, from 10 ² :1 to 10 ³ :1, from 10 ³ :1 to 10 ⁴ :1, from 10 ⁴ :1 to 10 ⁵ :1, or from 10 ⁵ :1 to 10 ⁶ :1.

As an example, when the control TMMP comprises a wild-type IL-2 polypeptide and when the TMMP of the invention comprises a variant IL-2 polypeptide (containing 1 to 10 amino acid substitutions compared to the amino acid sequence of the wild-type IL-2 polypeptide) as an immunomodulatory polypeptide, the ratio of: i) the binding affinity of the control TMMP to the IL-2 receptor (i.e., the cognate co-immunomodulatory polypeptide) to ii) the binding affinity of the TMMP of the invention to the IL-2 receptor, as measured by BLI, is at least 1.5:1, at least 2:1, at least 5:1, at least 10:1, at least 15:1, at least 20:1, at least 25:1, at least 50:1, at least 100:1, at least 500:1, at least 10 ² :1, at least 5x102:1, at least 10 ³ :1, at least 5x103:1, at least 10 ⁴ :1, at least 10 ⁵ :1 or at least 10 ⁶ :1. In some cases, when the control TMMP comprises a wild-type IL-2 polypeptide and when the TMMP of the invention comprises a variant IL-2 polypeptide (containing from 1 to 10 amino acid substitutions compared to the amino acid sequence of the wild-type IL-2 polypeptide) as an immunomodulatory polypeptide, the ratio of: i) the binding affinity of the control TMMP for the IL-2 receptor (i.e., the cognate co-immunomodulatory polypeptide) to ii) the binding affinity of the TMMP of the invention for the IL-2 receptor, as measured by BLI, is in the range of from 1.5:1 to 10 ⁶ :1, such as from 1.5:1 to 10:1, from 10:1 to 50:1, from 50:1 to 10 ² :1, from 10 ² :1 to 10 ³ :1, from 10 ³ :1 to ¹⁰⁴ :1, ¹⁰⁴ :1 to ¹⁰⁵ :1, or ¹⁰⁵ :1 to ¹⁰⁶ :1.

In another example, where the control TMMP comprises a wild-type PD-L1 polypeptide and where the TMMP of the invention comprises a variant PD-L1 polypeptide (comprising 1 to 10 amino acid substitutions compared to the amino acid sequence of the wild-type PD-L1 polypeptide) as an immunomodulatory polypeptide, the ratio of: i) the binding affinity of the control TMMP for the PD-1 polypeptide (i.e., the cognate co-immunomodulatory polypeptide) to ii) the binding affinity of the TMMP of the invention for the PD-1 polypeptide, as measured by BLI, is at least 1.5:1, at least 2:1, at least 5:1, at least 10:1, at least 15:1, at least 20:1, at least 25:1, at least 50:1, at least 100:1, at least 500:1, at least 10 ² :1, at least 5x102:1, at least 10 ³ :1, at least 5x103:1, at least 10 ⁴ :1, at least 10 ⁵ :1 or at least 10 ⁶ :1.

In another example, where the control TMMP comprises a wild-type CD80 polypeptide and where the TMMP of the invention comprises a variant CD80 polypeptide (comprising 1 to 10 amino acid substitutions compared to the amino acid sequence of the wild-type CD80 polypeptide) as an immunomodulatory polypeptide, the ratio of: i) the binding affinity of the control TMMP for the CTLA4 polypeptide (i.e., the cognate co-immunomodulatory polypeptide) to ii) the binding affinity of the TMMP of the invention for the CTLA4 polypeptide, as measured by BLI, is at least 1.5:1, at least 2:1, at least 5:1, at least 10:1, at least 15:1, at least 20:1, at least 25:1, at least 50:1, at least 100:1, at least 500:1, at least 10 ² :1, at least 5x102:1, at least 10 ³ :1, at least 5x103:1, at least 10 ⁴ :1, at least 10 ⁵ :1 or at least 10 ⁶ :1.

In another example, where the control TMMP comprises a wild-type CD80 polypeptide and where the TMMP of the invention comprises a variant CD80 polypeptide (containing from 1 to 10 amino acid substitutions compared to the amino acid sequence of the wild-type CD80 polypeptide) as an immunomodulatory polypeptide, the ratio of: i) the binding affinity of the control TMMP for the CD28 polypeptide (i.e., the cognate co-immunomodulatory polypeptide) to ii) the binding affinity of the TMMP of the invention for the CD28 polypeptide, as measured by BLI, is at least 1.5:1, at least 2:1, at least 5:1, at least 10:1,

- 10 049816 at least 15:1, at least 20:1, at least 25:1, at least 50:1, at least 100:1, at least 500:1, at least 10 ² :1, at least 5x102:1, at least 10 ³ :1, at least 5x103:1, at least 10 ⁴ :1, at least 10 ⁵ :1 or at least 10 ⁶ :1.

In another example, where the control TMMP comprises a wild-type 4-1BBL polypeptide and where the TMMP of the invention comprises a variant 4-1BBL polypeptide (containing 1 to 10 amino acid substitutions compared to the amino acid sequence of the wild-type 4-1BBL polypeptide) as an immunomodulatory polypeptide, the ratio of: i) the binding affinity of the control TMMP for the 4-1BB polypeptide (i.e., the cognate co-immunomodulatory polypeptide) to ii) the binding affinity of the TMMP of the invention for the 4-1BB polypeptide, as measured by BLI, is at least 1.5:1, at least 2:1, at least 5:1, at least 10:1, at least 15:1, at least 20:1, at least 25:1, at least 50:1, at least 100:1, at least 500:1, at least 10 ² :1, at least 5x102:1, at least 10 ³ :1, at least 5x103:1, at least 10 ⁴ :1, at least 10 ⁵ :1 or at least 10 ⁶ :1.

In another example, where the control TMMP comprises a wild-type CD86 polypeptide and where the TMMP of the invention comprises a variant CD86 polypeptide (comprising 1 to 10 amino acid substitutions compared to the amino acid sequence of the wild-type CD86 polypeptide) as an immunomodulatory polypeptide, the ratio of: i) the binding affinity of the control TMMP for the CD28 polypeptide (i.e., the cognate co-immunomodulatory polypeptide) to ii) the binding affinity of the TMMP of the invention for the CD28 polypeptide, as measured by BLI, is at least 1.5:1, at least 2:1, at least 5:1, at least 10:1, at least 15:1, at least 20:1, at least 25:1, at least 50:1, at least 100:1, at least 500:1, at least 10 ² :1, at least 5x102:1, at least 10 ³ :1, at least 5x103:1, at least 10 ⁴ :1, at least 10 ⁵ :1 or at least 10 ⁶ :1.

The binding affinity of a TMMP of the invention for a target T cell can be measured as follows: A) contacting a TMMP of the invention with a target T cell expressing on its surface: i) a cognate co-immunomodulatory polypeptide that binds a wild-type parent immunomodulatory polypeptide; and ii) a T cell receptor that binds to an epitope, wherein the TMMP comprises an epitope tag, such that the TMMP binds to the target T cell; B) contacting the TMMP bound to the target T cell with a fluorescently labeled binding agent (e.g., a fluorescently labeled antibody) that binds to the epitope tag, thereby creating a TMMP/target T cell/binding agent complex; C) measuring the mean fluorescence intensity (MFI) of the TMMP/target T cell/binding agent complex using flow cytometry. The epitope tag may be, for example, a FLAG tag, a hemagglutinin tag, a c-myc tag, a polyhistidine tag, etc. The MFI measured over a range of concentrations of the TMMP library member provides a measure of affinity. The MFI measured over a range of concentrations of the TMMP library member provides half the maximal effective concentration (EC ₅₀ ) of the TMMP. In some cases, the EC ₅₀ of the TMMP of the invention for a target T cell is in the nM (nanomolar) range; and the EC ₅₀ of the TMMP for a control T cell (wherein the control T cell expresses on its surface: i) a cognate co-immunomodulatory polypeptide that binds the parental wild-type immunomodulatory polypeptide; and ii) the T cell receptor (TCR) does not bind to an epitope present in TMMP) is in the μM (micromolar) range. In some cases, the ratio of the EC50 of the TMMP of the invention for a control T cell to the EC50 of the TMMP for a target T cell is at least 1.5:1, at least 2:1, at least 5:1, at least 10:1, at least 15:1, at least 20:1, at least 25:1, at least 50:1, at least 100:1, at least 500:1, at least 10 ² :1, at least 5x102:1, at least 10 ³ :1, at least 5x103:1, at least 10 ⁴ :1, at least 10 ⁵ :1, or at least 10 ⁶ :1. The ratio of the EC ₅₀ of the TMMP of the present invention for a control T cell to the EC ₅₀ of the TMMP for a target T cell is an expression of the selectivity of the TMMP.

In some cases, when measured as described in the preceding paragraph, the TMMP of the present invention exhibits selective binding to a target T cell, compared to binding of a TMMP library member to a control target T cell comprising: i) a cognate co-immunomodulatory polypeptide that binds a wild-type parental immunomodulatory polypeptide; and ii) a T cell receptor that binds to an epitope different from an epitope present in the TMMP library member.

Dimerized TMMPs.

The TMMP of the present invention may be dimerized; that is, the present invention relates to a multimeric polypeptide comprising a dimer of the TMMP of the present invention. Thus, in

- 11 049816 The present invention provides a TMMP comprising: A) a first heterodimer comprising: a) a first polypeptide comprising: i) a peptide epitope; and ii) a first major histocompatibility complex (MHC) polypeptide; and b) a second polypeptide i) a second MHC polypeptide, wherein the first heterodimer comprises one or more immunomodulatory polypeptides; and B) a second heterodimer comprising: a) a first polypeptide comprising: i) a peptide epitope; and ii) a first MHC polypeptide; and b) a second polypeptide i) a second MHC polypeptide, wherein the second heterodimer comprises one or more immunomodulatory polypeptides, and wherein the first heterodimer and the second heterodimer are covalently linked to each other. In some cases, the two TMMPs are identical to each other in amino acid sequence. In some cases, the first heterodimer and the second heterodimer are covalently linked to each other via a C-terminal region of a second polypeptide of the first heterodimer and a C-terminal region of a second polypeptide of the second heterodimer. In some cases, the first heterodimer and the second heterodimer are covalently linked to each other via a C-terminal amino acid of the second polypeptide of the first heterodimer and a C-terminal region of the second polypeptide of the second heterodimer; for example, in some cases, the C-terminal amino acid of the second polypeptide of the first heterodimer and the C-terminal region of the second polypeptide of the second heterodimer are linked to each other either directly or via a linker. The linker can be a peptide linker. The peptide linker may be from 1 amino acid to 200 amino acids in length (e.g., from 1 amino acid (aa) to 5 aa, from 5 aa to 10 aa, from 10 aa to 25 aa, from 25 aa to 50 aa, from 50 aa to 100 aa, from 100 aa to 150 aa, or from 150 aa to 200 aa). In some cases, the peptide epitope of the first heterodimer and the peptide epitope of the second heterodimer comprise the same amino acid sequence. In some cases, the first MHC polypeptide of the first and second heterodimer is MHC class I β2-microglobulin, and wherein the second MHC polypeptide of the first and second heterodimer is MHC class I heavy chain. In some cases, the immunomodulatory polypeptide of the first heterodimer and the immunomodulatory polypeptide of the second heterodimer comprise the same amino acid sequence. In some cases, the immunomodulatory polypeptide of the first heterodimer and the immunomodulatory polypeptide of the second heterodimer are variant immunomodulatory polypeptides that comprise from 1 to 10 amino acid substitutions compared to the corresponding parent wild-type immunomodulatory polypeptide, and wherein the from 1 to 10 amino acid substitutions result in a decrease in the affinity binding of the variant immunomodulatory polypeptide to the cognate co-immunomodulatory polypeptide. In some cases, the immunomodulatory polypeptide of the first heterodimer and the immunomodulatory polypeptide of the second heterodimer are each independently selected from the group consisting of IL-2, 4-1BBL, PD-L1, CD80, CD86, ICOS-L, OX-40L, FasL, JAG1 (CD339), TGFe, CD70 and ICAM. Examples of suitable MHC polypeptides, immunomodulatory polypeptides and peptide epitopes are described below.

MHC polypeptides.

As noted above, the TMMP of the present invention comprises MHC polypeptides. For the purposes of the present invention, the term major histocompatibility complex (MHC) polypeptides includes MHC polypeptides from 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 from other mammalian species (e.g., lagomorphs, non-human primates, canines, felines, ungulates (e.g., horses, bovines, sheep, goats, etc.), etc.). The term MHC polypeptide is intended to refer to class I MHC polypeptides (e.g., P-2 microglobulin and class I MHC heavy chain).

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

- 12 049816

In some cases, the MHC TMMP polypeptide is a human MHC polypeptide, and human MHC polypeptides are also referred to as human leukocyte antigen (HLA) polypeptides. In some cases, the MHC TMMP polypeptide is an HLA class I polypeptide, such as a β2-microglobulin polypeptide or an HLA class I heavy chain polypeptide. HLA class I 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.

Heavy chains of class I GKGS.

In some cases, the MHC class I heavy chain polypeptide present in the TMMP of the present invention comprises an amino acid sequence that has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to all or a portion (e.g., 50, 75, 100, 150, 200, or 250 consecutive amino acids) of the amino acid sequence of any of the human HLA heavy chain polypeptides depicted in Fig. 713. In some cases, the heavy chain of class I MHC has a length of 270 aa, 271 aa, 272 aa, 273 aa, 274 aa, 275 aa, 276 aa, 277 aa, 278 aa, 279 aa, 280 aa, 281 aa, 282 aa, 283 aa, 284 aa, 285 aa, 286 aa, 287 aa, 288 aa, 289 aa, or 290 aa. In some cases, the MHC class I heavy chain polypeptide present in the TMMP of the invention 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 identified as variable in the heavy chain consensus sequences) of any of the amino acid sequences depicted in Figs. 7-13. In some cases, the MHC class I heavy chain does not comprise transmembrane or cytoplasmic domains. As an example, an MHC class I TMMP heavy chain polypeptide of the present invention may comprise an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to amino acids 25-300 (without any, or partially without any, leader, transmembrane, or cytoplasmic sequences) or amino acids 25-365 (without leader) of the human HLA-A heavy chain polypeptides depicted in Figs. 7A, 7B, and 7C.

Figs. 7A, 7B, and 7C show the amino acid sequences of human leukocyte antigen (HLA) class I heavy chain polypeptides. The signal sequences, amino acids 1-24, are shown in bold and underlined. In Fig. 7A, element: 3A.1 is the heavy chain of HLA-A (HLA-A*O1:O1:O1:O1 or A*0101) (NCBI code NP_001229687.1), SEQ ID NO: 23; element 3A.2 is derived from HLA-A*1101 SEQ ID NO: 24; element 3A.3 is derived from HLA-A*2402 SEQ ID NO: 25; and element 3A.4 is derived from HLA-A*3303 SEQ ID NO: 26. In Fig. Figure 7B shows the sequence of HLA-B*07:02:01 (HLA-B*0702) GenBank NCBI accession code NP_005505.2 (see also GenBank code AUV50118.1). Figure 7C shows the sequence of HLA-C*O7O1 (GenBank code NP_001229971.1) (HLA-C*07:01:01:01 or HLA-Cw*070101, HLA-Cw*07 see GenBank code CAO78194.1).

Figure 8 shows a sequence alignment of eleven peptides of the mature class I MHC heavy chains without their leader sequences or transmembrane domains or intracellular domains. The aligned sequences represent human HLA-A, HLA-B, and HLA-C, the mouse H2K protein sequence, three HLA-A variants (var. 1, var. 2C, and var. 2CP), and three human HLA-A variants (HLA-A*1101; HLA-A*2402; and HLA-A*3303). The alignment indicates positions (84 and 139 of the mature proteins) at which cysteine residues can be inserted (e.g., by substitution) to form a disulfide bond to stabilize the MHC-H chain-β2M complex. The alignment also shows position 236 (of the mature polypeptide), which may be replaced by a cysteine residue that can form an intrachain disulfide bond with b2M (e.g., at aa 12). Each of these sites is marked with an arrow and the residues are shown in bold. The seventh HLA-A sequence shown in the alignment (alt. 2c) shows the sequence of variant 2 substituted with C residues at positions 84, 139 and 236. The boxes surrounding residues 84, 139 and 236 show groups of five amino acids on either side of these six sets of five residues, designated acc1 (for amino acid cluster 1), acc2 (for amino acid cluster 2), acc3 (for amino acid cluster 3), acc4 (for amino acid cluster 4), acc5 (for amino acid cluster 5) and acc6 (for amino acid cluster 6), which may be replaced by 1 to 5 amino acids independently selected from (i) any naturally occurring amino acid or (ii) any naturally occurring amino acid except proline or glycine.

According to Fig. 8, in some cases: i) acc1 (amino acid cluster 1) may be the amino acid sequence GTLRG (SEQ ID NO: 287) or this sequence with one or two amino acids deleted or substituted with other naturally occurring amino acids (e.g., L is substituted with I, V, A, or F); ii) acc2 (amino acid cluster 2) may be the amino acid sequence YNQSE (SEQ ID NO: 288) or this sequence with one or two amino acids deleted or substituted with other naturally occurring

- 13,049,816 amino acids (e.g. N is substituted with Q, Q is substituted with N, and/or E is substituted with D); iii) acc3 (amino acid cluster 3) may be the amino acid sequence TAADM (SEQ ID NO: 289) or this sequence with one or two amino acids deleted or substituted with other naturally occurring amino acids (e.g. T is substituted with S, A is substituted with G, D is substituted with E, and/or M is substituted with L, V, or I); iv) acc4 (amino acid cluster 4) may be the amino acid sequence AQTTK (SEQ ID NO: 290) or this sequence with one or two amino acids deleted or substituted with other naturally occurring amino acids (e.g. A is substituted with G, Q is substituted with N, or T is substituted with S, and/or K is substituted with R or Q); v) acc5 (amino acid cluster 5) may be the amino acid sequence VETRP (SEQ ID NO: 291) or this sequence with one or two amino acids deleted or substituted with other naturally occurring amino acids (e.g. V is substituted with I or L, E is substituted with D, T is substituted with S, and/or R is substituted with K); and/or vi) acc6 (amino acid cluster 6) may be the amino acid sequence GDGTF (SEQ ID NO: 292) or this sequence with one or two amino acids deleted or substituted with other naturally occurring amino acids (e.g. D is substituted with E, T is substituted with S, or F is substituted with L, W, or Y).

Figures 9 through 11 show the amino acid sequence alignments of the HLA class I heavy chains (without leader sequences or transmembrane domains or intracellular domains). The aligned amino acid sequences in Figure 9A represent the HLA-A class I heavy chains of the following alleles: A*0101, A*0201, A*0301, A*1101, A*2301, A*2402, A*2407, A*3303, and A*3401. The aligned amino acid sequences in Figure 10A represent the HLA-B class I heavy chains of the following alleles: B*0702, B*0801, B*1502, B*3802, B*4001, B*4601, and B*5301. The aligned amino acid sequences in Figure 11A represent the HLA-C class I heavy chains of the following alleles: C*0102, C*0303, C*0304, C*0401, C*0602, C*0701, C*0801, and C*1502. The alignment shows positions (84 and 139 of the mature proteins) at which cysteine residues can be inserted (e.g., by substitution) to form a disulfide bond to stabilize the H chain βΣΜ-βΣΜ complex. The alignment also shows position 236 (of the mature polypeptide) to be replaced by a cysteine residue that can form an intrachain disulfide bond with β2Μ (e.g., at aa 12). The boxes surrounding residues 84, 139, and 236 indicate groups of five amino acids on either side of these six sets of five residues, designated acc1 (for amino acid cluster 1), acc2 (for amino acid cluster 2), acc3 (for amino acid cluster 3), acc4 (for amino acid cluster 4), acc5 (for amino acid cluster 5), and acc6 (for amino acid cluster 6), which may be replaced by 1 to 5 amino acids independently selected from (i) any naturally occurring amino acid or (ii) any naturally occurring amino acid except proline or glycine.

Figs. 9A, 10A, and 11A are amino acid sequence alignments of the mature HLA-A, -B, and -C class I heavy chains, respectively. The sequences are for the intracellular portion of the mature protein (without leader sequences or transmembrane domains or intracellular domains). As described in Fig. 8, the aa positions of residues 84, 139, and 236 and their flanking residues (acc1 through acc6) may be substituted with from 1 to 5 amino acids independently selected from (i) any naturally occurring amino acid or (ii) any naturally occurring amino acid except proline or glycine, also shown. Figs. 9B, 10B, and 11B are consensus amino acid sequences for the HLA-A, -B, and -C sequences, respectively, shown in Figs. 9A, 10A, and 11A. The consensus sequences show variable amino acid positions as sequentially numbered X residues, and amino acid positions 84, 139, and 236 are double underlined.

According to Fig. 9A, in some cases: i) acc1 (amino acid cluster 1) may be the amino acid sequence GTLRG (SEQ ID NO: 287) or this sequence with one or two amino acids deleted or substituted with other naturally occurring amino acids (e.g., L is substituted with I, V, A, or F); ii) acc2 (amino acid cluster 2) may be the amino acid sequence YNQSE (SEQ ID NO: 288) or this sequence with one or two amino acids deleted or substituted with other naturally occurring amino acids (e.g., N is substituted with Q, Q is substituted with N, and/or E is substituted with D); iii) acc3 (amino acid cluster 3) may be the amino acid sequence TAADM (SEQ ID NO: 289) or that sequence with one or two amino acids deleted or substituted with other naturally occurring amino acids (e.g. T is substituted with S, A is substituted with G, D is substituted with E, and/or M is substituted with L, V, or I); iv) acc4 (amino acid cluster 4) may be the amino acid sequence AQTTK (SEQ ID NO: 290) or that sequence with one or two amino acids deleted or substituted with other naturally occurring amino acids (e.g. A is substituted with G, Q is substituted with N, or T is substituted with S, and or K is substituted with R or Q); v) acc5 (amino acid cluster 5) may be the amino acid sequence

- 14 049816 VETRP (SEQ ID NO: 291) or this sequence with one or two amino acids deleted or substituted with other naturally occurring amino acids (e.g. V is substituted with I or L, E is substituted with D, T is substituted with S, and/or R is substituted with K); and/or vi) acc6 (amino acid cluster 6) may be the amino acid sequence GDGTF (SEQ ID NO: 292) or this sequence with one or two amino acids deleted or substituted with other naturally occurring amino acids (e.g. D is substituted with E, T is substituted with S, or F is substituted with L, W or Y).

According to Fig. 10A, in some cases: i) acc1 (amino acid cluster 1) may be the amino acid sequence RNLRG (SEQ ID NO: 293) or this sequence with one or two amino acids deleted or substituted with other naturally occurring amino acids (e.g. N is substituted with T or I; and/or L is substituted with A; and/or the second R is substituted with L; and/or G is substituted with R); ii) acc2 (amino acid cluster 2) may be the amino acid sequence YNQSE (SEQ ID NO: 288) or this sequence with one or two amino acids deleted or substituted with other naturally occurring amino acids (e.g. N is substituted with Q, Q is substituted with N and/or E is substituted with D); iii) acc3 (amino acid cluster 3) may be the amino acid sequence TAADT (SEQ ID NO: 294) or this sequence with one or two amino acids deleted or substituted with other naturally occurring amino acids (e.g. the first T is substituted with S and/or A is substituted with G and/or D is substituted with E and/or the second T is substituted with S); iv) acc4 (amino acid cluster 4) may be the amino acid sequence AQITQ (SEQ ID NO: 295) or this sequence with one or two amino acids deleted or substituted with other naturally occurring amino acids (e.g. A is substituted with G and/or the first Q is substituted with N; and/or I is substituted with L or V; and/or T is substituted with S); v) acc5 (amino acid cluster 5) may be the amino acid sequence VETRP (SEQ ID NO: 291) or this sequence with one or two amino acids deleted or substituted with other naturally occurring amino acids (e.g. V is substituted with I or L, E is substituted with D, T is substituted with S, and/or R is substituted with K); and/or vi) acc6 (amino acid cluster 6) may be the amino acid sequence GDRTF (SEQ ID NO: 296) or this sequence with one or two amino acids deleted or substituted with other naturally occurring amino acids (e.g. D is substituted with E, and/or T is substituted with S; and/or R is substituted with K or H; and/or F is substituted with L, W or Y).

According to Fig. 11A, in some cases: i) acc1 (amino acid cluster 1) may be the amino acid sequence RNLRG (SEQ ID NO: 293) or this sequence with one or two amino acids deleted or substituted with other naturally occurring amino acids (e.g. N is substituted with K; and/or L is substituted with A or I; and/or the second R is substituted with H; and/or G is substituted with T or S); ii) acc2 (amino acid cluster 2) may be the amino acid sequence YNQSE (SEQ ID NO: 288) or this sequence with one or two amino acids deleted or substituted with other naturally occurring amino acids (e.g. N is substituted with Q, Q is substituted with N and/or E is substituted with D); iii) acc3 (amino acid cluster 3) may be the amino acid sequence TAADT (SEQ ID NO: 294) or this sequence with one or two amino acids deleted or substituted with other naturally occurring amino acids (e.g. the first T is substituted with S and/or A is substituted with G and/or D is substituted with E and/or the second T is substituted with S); iv) acc4 (amino acid cluster 4) may be the amino acid sequence AQITQ (SEQ ID NO: 295) or this sequence with one or two amino acids deleted or substituted with other naturally occurring amino acids (e.g. A is substituted with G and/or the first Q is substituted with N; and/or I is substituted with L; and/or the second Q is substituted with N or K); v) acc5 (amino acid cluster 5) may be the amino acid sequence VETRP (SEQ ID NO: 291) or this sequence with one or two amino acids deleted or substituted with other naturally occurring amino acids (e.g. V is substituted with I or L, E is substituted with D, T is substituted with S, and/or R is substituted with K or H); and/or vi) acc6 (amino acid cluster 6) may be the amino acid sequence GDGTF (SEQ ID NO: 292) or this sequence with one or two amino acids deleted or substituted with other naturally occurring amino acids (e.g. D is substituted with E; and/or T is substituted with S; and/or F is substituted with L, W or Y).

HLA-A.

In some cases, a TMMP of the present invention comprises an HLA-A heavy chain polypeptide. HLA-A heavy chain peptide sequences or portions thereof that may be included in a TMMP of the present invention include, but are not limited to, the alleles A*0101, A*0201, A*0301, A*1101, A*2301, A*2402, A*2407, A*3303, and A*3401, which are aligned without any, or substantially without, leader, transmembrane, and cytoplasmic sequences in Fig. 9A. Any of such alleles may comprise a mutation at one or more of positions 84, 139, and/or 236 (as shown in Fig. 9A) selected from: a tyrosine to alanine substitution at position 84 (Y84A); tyrosine to cysteine at position 84 (Y84C); alanine to cysteine at position 139 (A139C); and alanine to cysteine at

- 15 049816 position 236 (A236C). In addition, an HLA-A sequence having at least 75% (e.g. at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%) or 100% amino acid sequence identity with all or part (e.g. 50, 75, 100, 150, 200 or 250 consecutive amino acids) of the sequence of such HLA-A alleles (e.g. it may contain 1-25, 1-5, 510, 10-15, 15-20, 20-25 or 25-30 amino acid insertions, deletions and/or substitutions) may also be used.

In some cases, the TMMP of the present invention comprises an HLA-A heavy chain polypeptide comprising the following HLA-A consensus amino acid sequence:

GSHSMRYFX1TSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQX2MEPRAPWIE

OEGPEYWDX3X4TX5X6X7KAX8SQX9X10RX11X12LX13X14X15X16X17YYNOSEX18 GSHTX19QX20MX21GCDVGX22DX23RFLRGYX24QX25AYDGKDYIALX26EDLRSWT AADMAAOX27TX287X29KWEX30X31X32EAEQX33RX34YLX35GX36CVX37X38LRRY LENGKETLQRTDX39PKTHMTHHX40X41SDHEATLRCWALX42FYPAEITLTWQRDGE DOTODTELVETRPAGDGTFOKWAX43VVVPSGX44EORYTCHVOHEGLPKPLTLRWEX 45 (SEQ ID NO:29), in in which X1 is F, Y, S, or T; X2 is K or R; X3 is Q, G, E, or R; X4 is N or E; X5 is R or G; X6 is N or K; X7 is M or V; X8 is H or Q; X9 is T or I; X10 is D or H; X11 is A, V, or E; X12 is N or D; X13 is G or R; X14 is T or I; X15 is L or A; X16 is R or L; X17 is G or R; X18 is A or D; X19 is I, L, or V; X20 is I, R, or M; X21 is F or Y; X22 is S or P; X23 is W or G; X24 is R, H, or Q; X25 is D or Y; X26 is N or K; X27 is T or I; X28 is K or Q; X29 is R or H; X30 is A or T; X31 is A or V; X32 is H or R; X33 is R, L, Q or W; X34 is V or A; X35 is D or E; X36 is R or T; X37 is D or E; X38 is W or G; X39 is P or A; X40 is P or A; X41 is V or I; X42 is S or G; X43 is A or S; X44 is Q or E; and X45 is P or L.

As one example, an MHC class I TMMP heavy chain polypeptide may comprise an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following human HLA-A heavy chain amino acid sequence:

GSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEY

WDGETRKVKAHSQTHRVDLGTLRGYYNQSEAGSHTVQRMYGCDVGSDWRFLRGYHQ YAYDGKDYIALKEDLRSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGTCVEWLRRY LENGKETLQRTDAPKTHMTHHAVSDHEATLRCWALSFYPAEITLTWQRDGEDQTQDTE LVETRPAGDGTFQKWAAVVVPSGQEQRYTCHVQHEGLPKPLTLRWEP (SEQ ID

NO:297).

In some cases, an HLA-A heavy chain polypeptide suitable for inclusion in a TMMP of the present invention comprises the following amino acid sequence:

GSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEY

WDGETRKVKAHSQTHRVDLGTLRGYYNQSEAGSHTVQRMYGCDVGSDWRFLRGYHQ YAYDGKDYIALKEDLRSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGTCVEWLRRY LENGKETLQRTDAPKTHMTHHAVSDHEATLRCWALSFYPAEITLTWQRDGEDQTQDTE LVETRPAGDGTFQKWAAVVVPSGQEQRYTCHVQHEGLPKPLTLRWEP (SEQ ID

NO:297).

This HLA-A heavy chain polypeptide is also referred to as HLA-A*0201 or simply HLA-A02. In some cases, the C-terminal Pro is not included in the TMMP of the invention. For example, in some cases, an HLA-A02 polypeptide suitable for inclusion in the TMMP of the invention comprises the following amino acid sequence:

- 16 049816

GSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEY

WDGETRKVKAHSQTHRVDLGTLRGYYNQSEAGSHTVQRMYGCDVGSDWRFLRGYHQ

YAYDGKDYIALKEDLRSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGTCVEWLRRY

LENGKETLQRTDAPKTHMTHHAVSDHEATLRCWALSFYPAEITLTWQRDGEDQTQDTE

LVETRPAGDGTFQKWAAVVVPSGQEQRYTCHVQHEGLPKPLTLRWE (SEQ ID NO:449).

HLA-A (Y84A; A236C).

In some cases, the MHC class I heavy chain polypeptide comprises the Y84A and A236C substitutions. For example, in some cases, the MHC class I heavy chain polypeptide comprises an amino acid sequence that has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following human HLA-A heavy chain amino acid sequence (Y84A; A236C):

GSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEY

WDGETRKVKAHSQTHRVDLGTLRGAYNQSEAGSHTVQRMYGCDVGSDWRFLRGYHQ

YAYDGKDYIALKEDLRSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGTCVEWLRRY

LENGKETLQRTDAPKTHMTHHAVSDHEATLRCWALSFYPAEITLTWQRDGEDQTQDTE

LVETRPCGDGTFQKWAAVVVPSGQEQRYTCHVQHEGLPKPLTLRWEP (SEQ ID

NO:298), where amino acid 84 is Ala and amino acid 236 is Cys. In some cases, Cys-236 forms an interchain disulfide bond with Cys-12 of the B2M polypeptide variant that contains the R12C substitution.

In some cases, the HLA-A heavy chain polypeptide suitable for inclusion in the TMMP of the present invention is an HLA-A02 (Y84A; A236C) polypeptide comprising the following amino acid sequence:

GSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEY

WDGETRKVKAHSQTHRVDLGTLRGAYNQSEAGSHTVQRMYGCDVGSDWRFLRGYHQ

YAYDGKDYIALKEDLRSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGTCVEWLRRY

LENGKETLQRTDAPKTHMTHHAVSDHEATLRCWALSFYPAEITLTWQRDGEDQTQDTE LVETRPCGDGTFQKWAAVVVPSGQEQRYTCHVQHEGLPKPLTLRWEP (SEQ ID

NO:298).

In some cases, the HLA-A heavy chain polypeptide suitable for inclusion in the TMMP of the present invention is an HLA-A02 (Y84A; A236C) polypeptide comprising the following amino acid sequence:

GSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEY

WDGETRKVKAHSQTHRVDLGTLRGAYNQSEAGSHTVQRMYGCDVGSDWRFLRGYHQ

YAYDGKDYIALKEDLRSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGTCVEWLRRY

LENGKETLQRTDAPKTHMTHHAVSDHEATLRCWALSFYPAEITLTWQRDGEDQTQDTE

LVETRPCGDGTFQKWAAVVVPSGQEQRYTCHVQHEGLPKPLTLRWE (SEQ ID NO:450).

HLA-A (Y84C; A139C).

In some cases, the MHC class I heavy chain polypeptide comprises the Y84C and A139C substitutions. For example, in some cases, the MHC class I heavy chain polypeptide comprises an amino acid sequence that has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following human HLA-A heavy chain amino acid sequence (Y84C; A139C):

GSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEY

WDGETRKVKAHSQTHRVDLGTLRGCYNQSEAGSHTVQRMYGCDVGSDWRFLRGYHQ

YAYDGKDYIALKEDLRSWTAADMCAQTTKHKWEAAHVAEQLRAYLEGTCVEWLRRY

LENGKETLQRTDAPKTHMTHHAVSDHEATLRCWALSFYPAEITLTWQRDGEDQTQDTE

LVETRPAGDGTFQKWAAVVVPSGQEQRYTCHVQHEGLPKPLTLRWEP (SEQ ID

NO:299), where amino acid 84 is Cys and amino acid 139 is Cys. In some cases, Cys-84 forms an intrachain disulfide bond with Cys-139.

HLA-A11 (HLA-A*1101).

As one non-limiting example, the MHC class I heavy chain polypeptide TMMP

- 17 049816 may comprise an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% amino acid sequence identity with the following amino acid sequence of the human HLA-A11 heavy chain:

GSHSMRYFYTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEY

WDQETRNVKAQSQTDRVDLGTLRGYYNQSEDGSHTIQIMYGCDVGPDGRFLRGYRQD

AYDGKDYIALNEDLRSWTAADMAAQITKRKWEAAHAAEQQRAYLEGTCVEWLRRYL

ENGKETLQRTDPPKTHMTHHPISDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTEL

VETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLRWE (SEQ ID NO:300).

Such class I MHC heavy chain may be expressed in Asian populations, including populations of Asian descent.

HLA-A11 (Y84A; A236C).

As one non-limiting example, in some cases, the MHC class I heavy chain polypeptide is an HLA-A11 allele that comprises the Y84A and A236C substitutions. For example, in some cases, the MHC class I heavy chain polypeptide comprises an amino acid sequence that has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following human HLA-A A11 heavy chain amino acid sequence (Y84A; A236C):

GSHSMRYFYTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEY

WDQETRNVKAQSQTDRVDLGTLRGAYNQSEDGSHTIQIMYGCDVGPDGRFLRGYRQD

AYDGKDYIALNEDLRSWTAADMAAQITKRKWEAAHAAEQQRAYLEGTCVEWLRRYL

ENGKETLQRTDPPKTHMTHHPISDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTEL

VETRPCGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLRWE (SEQ ID NO:301), wherein amino acid 84 is Ala and amino acid 236 is Cys. In some instances, Cys-236 forms an interchain disulfide bond with Cys-12 of the B2M polypeptide variant that contains the R12C substitution.

HLA-A24 (HLA-A*2402).

In one non-limiting example, an MHC class I TMMP heavy chain polypeptide of the invention may comprise an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following human HLA-A24 heavy chain amino acid sequence:

GSHSMRYFSTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEY

WDEETGKVKAHSQTDRENLRIALRYYNQSEAGSHTLQMMFGCDVGSDGRFLRGYHQY

AYDGKDYIALKEDLRSWTAADMAAQITKRKWEAAHVAEQQRAYLEGTCVDGLRRYL

ENGKETLQRTDPPKTHMTHHPISDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTEL

VETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLRWEPSSQPTVPIVGIIA GLVLLGAVITGAVVAAVMWRRNSSDRKGGSYSQAASSDSAQGSDVSLTACKV (SEQ ID NO:302).

This class I MHC heavy chain may be expressed in Asian populations, including populations of Asian descent. In some cases, amino acid 84 is Ala. In some cases, amino acid 84 is Cys. In some cases, amino acid 236 is Cys. In some cases, amino acid 84 is Ala and amino acid 236 is Cys. In some cases, amino acid 84 is Cys and amino acid 236 is Cys.

HLA-A33 (HLA-A*3303).

In one non-limiting example, an MHC class I TMMP heavy chain polypeptide of the invention may comprise an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following human HLA-A33 heavy chain amino acid sequence:

- 18 049816

GSHSMRYFTTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEY WDRNTRNVKAHSQIDRVDLGTLRGYYNQSEAGSHTIQMMYGCDVGSDGRFLRGYQQD AYDGKDYIALNEDLRSWTAADMAAQITQRKWEAARVAEQLRAYLEGTCVEWLRRYL ENGKETLQRTDPPKTHMTHHAVSDHEATLRCWALSFYPAEITLTWQRDGEDQTQDTEL VETRPAGDGTFQKWASVVVPSGQEQRYTCHVQHEGLPKPLTLRWEPSSQPTIPIVGIIAG LVLFGAVFAGAVVAAVRWRRKSSDRKGGSYSQAASSDSAQGSDMSLTACKV (SEQ ID NO:303).

This class I MHC heavy chain may be expressed in Asian populations, including populations of Asian descent. In some cases, amino acid 84 is Ala. In some cases, amino acid 84 is Cys. In some cases, amino acid 236 is Cys. In some cases, amino acid 84 is Ala and amino acid 236 is Cys. In some cases, amino acid 84 is Cys and amino acid 236 is Cys.

HLA-B.

In some cases, a TMMP of the present invention comprises an HLA-B heavy chain polypeptide. HLA-B heavy chain peptide sequences or portions thereof that may be included in a TMMP of the present invention include, but are not limited to, the alleles B*0702, B*0801, B*1502, B*3802, B*4001, B*4601, and B*5301, which are aligned without any, or substantially without, leader, transmembrane, and cytoplasmic sequences in Fig. 10A. Any of such alleles may comprise a mutation at one or more of positions 84, 139, and/or 236 (as shown in Fig. 10A) selected from: a tyrosine to alanine substitution at position 84 (Y84A); a tyrosine to cysteine at position 84 (Y84C); alanine to cysteine at position 139 (A139C); and alanine to cysteine at position 236 (A236C). Furthermore, an HLA-B polypeptide comprising an amino acid sequence having at least 75% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%) or 100% amino acid sequence identity to all or a portion (e.g., 50, 75, 100, 150, 200, or 250 consecutive amino acids) of the sequence of such HLA-B alleles (e.g., it may comprise 1-25, 1-5, 5-10, 10-15, 15-20, 20-25, or 25-30 amino acid insertions, deletions, and/or substitutions) may also be used.

In some cases, the TMMP of the present invention comprises an HLA-B heavy chain polypeptide comprising the following HLA-B consensus amino acid sequence:

GSHSMRYFX1TX2X3SRPGRGEPRFIX4VGYVDDTX5FVRFDSDAX6SPRX7X8PR

APWIEOEGPEYWDRX9TQX10X11KTX12X13TQX14YX15X16NLX17X18X19X20YYNO

SEAGSHX21X22QX23MYGCDLGPDGRLLRGHDOSAYDGKDYIALNEDLX24SWTAADT

AAOIX25ORKX26EAARX27AEQX28RX29YLEGX30CVEWLRRYLENGKX31X32LX33R

ADPPKTHVTHHPX34SDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRPAGD

RTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLRWEP (SEQ ID NO:30), wherein X1 is H, Y or D; X2 is A or S; X3 is M or V; X4 is A, S or T; X5 is Q or L; X6 is A or T; X7 is E, M K or T; X8 is A or T; X9 is E or N; X10 is I or K; XII is Y, F, S or C; X12 is N or Q; X13 is A or T; X14 is D or Y; X15 is E or V; X16 is S or N; X17 is T, N or I; X18 is A or L; X19 is L or R; X20 is R or G; X21 is T or I; X22 is L or I; X23 is R or S; X24 is R or S; X25 is S or T; X26 is L or W; X27 is E or V; X28 is R, D, L or W; X29 is A or T; X30 is L, E or T; X31 is E or D; X32 is K or T; X33 is E or Q; and X34 is I or V.

In one non-limiting example, an MHC class I TMMP heavy chain polypeptide of the invention may comprise an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following human HLA-B heavy chain amino acid sequence:

- 19 049816 gshsmryfttsvsrpgrgeprfiavgyvddtqfvrfdsdaasqrmeprapwieqegpeywdrntrnvkahsqidrvdlgtlrgyynqs eagshtiqmmygcdvgsdgrflrgyqqdaydgkdyialnedlrswtaadmaaqitqrkweaarvaeqlraylegtcvewlrryleng ketlqrtdppkthmthhavsdheatlrcwalsfypaeitltwqrdgedqtqdtelvetrpagdgtfqkwasvvvpsgqeqrytchvqhe glpkpltlrwepssqptipivgiiaglvlfgavfagavvaavrwrrkssdrkggsysqaassdsaqgsdmsltackv (SEQ ID NO:304).

HLA-B (Y84A; A236C).

As one non-limiting example, in some cases, the MHC class I heavy chain polypeptide is an HLA-B polypeptide that comprises the Y84A and A236C substitutions. For example, in some cases, the MHC class I heavy chain polypeptide comprises an amino acid sequence that has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following human HLA-B heavy chain amino acid sequence (Y84A; A236C):

GSHSMRYFYTSVSRPGRGEPRFISVGYVDDTQFVRFDSDAASPREEPRAPWIEQEGPEY

WDRNTQIYKAQAQTDRESLRNLRGAYNQSEAGSHTLQSMYGCDVGPDGRLLRGHDQY AYDGKDYIALNEDLRSWTAADTAAQITQRKWEAAREAEQRRAYLEGECVEWLRRYLE NGKDKLERADPPKTHVTHHPISDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELV ETRPCGDRTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLRWEP (SEQ ID NO:305), wherein amino acid 84 is Ala and amino acid 236 is Cys. In some instances, Cys-236 forms an interchain disulfide bond with Cys-12 of the B2M polypeptide variant that contains the R12C substitution.

HLA-B (Y84C; A139C).

In some cases, the MHC class I heavy chain polypeptide comprises the Y84C and A139C substitutions. For example, in some cases, the MHC class I heavy chain polypeptide comprises an amino acid sequence that has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following human HLA-B heavy chain amino acid sequence (Y84C; A139C):

GSHSMRYFYTSVSRPGRGEPRFISVGYVDDTQFVRFDSDAASPREEPRAPWIEQEGPEY

WDRNTQIYKAQAQTDRESLRNLRGCYNQSEAGSHTLQSMYGCDVGPDGRLLRGHDQY AYDGKDYIALNEDLRSWTAADTCAQITQRKWEAAREAEQRRAYLEGECVEWLRRYLE NGKDKLERADPPKTHVTHHPISDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELV ETRPAGDRTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLRWEP (SEQ ID NO:306), wherein amino acid 84 is Cys and amino acid 139 is Cys. In some cases, Cys-84 forms an intrachain disulfide bond with Cys-139.

HLA-B*4702.

By way of example, in some cases, an MHC class I heavy chain polypeptide present in a TMMP of the invention comprises the amino acid sequence of HLA-B*0702 (SEQ ID NO: 207) in Fig. 10A, or a sequence having at least 75% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%) or 100% amino acid sequence identity to all or a portion (e.g., 50, 75, 100, 150, 200, or 250 consecutive amino acids) of such a sequence (e.g., it may contain 1-25, 1-5, 5-10, 10-15, 15-20, 20-25, or 25-30 amino acid insertions, deletions, and/or substitutions). In some cases, when the HLA-B TMMP heavy chain polypeptide of the invention has less than 100% identity to the sequence designated HLA-B in Fig. 8, or designated B*0702 in Fig. 10A, it may comprise a mutation at one or more positions 84, 139, and/or 236 selected from: a tyrosine to alanine substitution at position 84 (Y84A); a tyrosine to cysteine substitution at position 84 (Y84C); an alanine to cysteine at position 139 (A139C); and alanine to cysteine substitution at position 236 (A236C). In some cases, the HLA-B TMMP heavy chain polypeptide of the invention comprises the substitutions Y84A and A236C. In some cases, the HLA-B*0702 TMMP heavy chain polypeptide of the invention comprises the substitutions Y84C and A139C. In some cases, the HLA-B TMMP heavy chain polypeptide of the present invention comprises the substitutions Y84C, A139C and A236C.

HLA-C.

In some cases, the TMMP of the present invention comprises an HLA-C heavy chain polypeptide. The HLA-C heavy chain polypeptide or portions thereof that may be included in the TMMP of the present invention include, but are not limited to, the alleles C*0102, C*0303, C*0304, C*0401, C*0602, C*0701, C*0801, and C*1502 that are aligned without any, or substantially without any,

- 20 049 816 leader, transmembrane and cytoplasmic sequences of Fig. 11A. Any of such alleles may comprise a mutation at one or more positions 84, 139 and/or 236 (as shown in Fig. 11A) 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). In addition, an HLA-C polypeptide comprising an amino acid sequence having at least 75% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%) or 100% amino acid sequence identity with all or part (e.g., 50, 75, 100, 150, 200 or 250 consecutive amino acids) of the sequence of such HLA-C alleles (e.g., it may contain 1-25, 1-5, 5-10, 10-15, 15-20, 20-25 or 25-30 amino acid insertions, deletions and/or substitutions) can also be used.

In some cases, the TMMP of the present invention comprises an HLA-C heavy chain polypeptide comprising the following HLA-C consensus amino acid sequence:

X1SHSMX2YFX3TAVSX4PGRGEPX5FIX6VGYVDDTQFVX7FDSDAASPRGEPRX

8PWVEQEGPEYWDRETQX9YKRQAQX10DRVX11LRX12LRGYYNOSEX13X14SHX15X 16QX17MX18GCDX19GPDGRLLRGX20X21QX22AYDGKDYIALNEDLRSWTAADTAAQ ITORKX23EAARX24AEQX25RAYLEGX26CVEWLRRYLX27NGKX28TLORAEX29PKTH VTHHPX3QSDHEATLRCWALGFYPAEITLTWQX31DGEDOTODTELVETRPAGDGTFOK WAAVX32VPSGX33EORYTCHX34OHEGLX35EPLTLX36WX37P (SEQ ID NO:31),

Where X1 is C or G; X2 is R or K; X3 is F, Y, S or D; X4 is R or W; X5 is H or R; X6 is A or S; X7 is Q or R; X8 is A or E; X9 is N or K; X10 is T or A; X11 is S or N; X12 is N or K; X13 is A or D; X14 is G or R; X15 is T or I; X16 is L or I; X17 is W or R; X18 is C, Y, F or S; X19 is L or V; X20 is Y or H; X21 is D or N; X22 is Y, F, S or L; X23 is L or W; X24 is E, A or T; X25 is R, L or W; X26 is L or T; X27 is E or K; X28 is E or K; X29 is H or P; X30 is R or V; X31 is W or R; X32 is V or M; X33 is E or Q; X34 is M or V; X35 is P or Q; X36 is R or S; and X37 is P or G.

In one non-limiting example, an MHC class I TMMP heavy chain polypeptide of the invention may comprise an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following human HLA-C heavy chain amino acid sequence:

CSHSMRYFDTAVSRPGRGEPRFISVGYVDDTQFVRFDSDAASPRGEPRAPWVEQEGPEY

WDRETQNYKRQAQADRVSLRNLRGYYNQSEDGSHTLQRMYGCDLGPDGRLLRGYDQ SAYDGKDYIALNEDLRSWTAADTAAQITQRKLEAAARAAEQLRAYLEGTCVEWLRRYLE NGKETLQRAEPPKTHVTHHPLSDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELV ETRPAGDGTFQKWAAVVVPSGQEQRYTCHMQHEGLQEPLTLSWEP (SEQ ID NO:307).

HLA-C (Y84A; A236C).

As one non-limiting example, in some cases, the MHC class I heavy chain polypeptide is an HLA-C polypeptide that comprises the Y84A and A236C substitutions. For example, in some cases, the MHC class I heavy chain polypeptide comprises an amino acid sequence that has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following human HLA-C heavy chain amino acid sequence (Y84A; A236C):

CSHSMRYFDTAVSRPGRGEPRFISVGYVDDTQFVRFDSDAASPRGEPRAPWVEQEGPEY

WDRETQNYKRQAQADRVSLRNLRGAYNQSEDGSHTLQRMYGCDLGPDGRLLRGYDQ SAYDGKDYIALNEDLRSWTAADTAAQITQRKLEAARAAEQLRAYLEGTCVEWLRRYLE NGKETLQRAEPPKTHVTHHPLSDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELV ETRPCGDGTFQKWAAVVVPSGQEQRYTCHMQHEGLQEPLTLSWEP (SEQ ID NO:308), wherein amino acid 84 is Ala and amino acid 236 is Cys. In some instances, Cys-236 forms an interchain disulfide bond with Cys-12 of the B2M polypeptide variant that contains the R12C substitution.

- 21 049816

HLA-C (Y84C; A139C).

In some cases, the MHC class I heavy chain polypeptide comprises the Y84C and A139C substitutions. For example, in some cases, the MHC class I heavy chain polypeptide comprises an amino acid sequence that has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following human HLA-C heavy chain amino acid sequence (Y84C; A139C):

CSHSMRYFDTAVSRPGRGEPRFISVGYVDDTQFVRFDSDAASPRGEPRAPWVEQEGPEY

WDRETQNYKRQAQADRVSLRNLRGCYNQSEDGSHTLQRMYGCDLGPDGRLLRGYDQ SAYDGKDYIALNEDLRSWTAADTCAQITQRKLEAARAAEQLRAYLEGTCVEWLRRYLE NGKETLQRAEPPKTHVTHHPLSDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELV ETRPAGDGTFQKWAAVVVPSGQEQRYTCHMQHEGLQEPLTLSWEP (SEQ ID NO:397), wherein amino acid 84 is Cys and amino acid 139 is Cys. In some cases, Cys-84 forms an intrachain disulfide bond with Cys-139.

HLA-C*0701.

In some cases, the MHC class I TMMP heavy chain polypeptide of the invention comprises the amino acid sequence of HLA-C*0701 of Fig. 11A (labeled HLA-C in Fig. 8), or an amino acid sequence having at least 75% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%) or 100% amino acid sequence identity to all or a portion (e.g., 50, 75, 100, 150, 200, or 250 consecutive amino acids) of such a sequence (e.g., it may contain 1-25, 1-5, 5-10, 10-15, 15-20, 20-25, or 25-30 amino acid insertions, deletions, and/or substitutions). In some cases, when the HLA-C TMMP heavy chain polypeptide of the invention has less than 100% identity to the sequence designated HLA-C*0701 in Fig. 11A, it may comprise a mutation at one or more positions 84, 139, and/or 236 selected from: a tyrosine to alanine substitution at position 84 (Y84A); a tyrosine to cysteine substitution at position 84 (Y84C); an alanine to cysteine at position 139 (A139C); and alanine to cysteine substitution at position 236 (A236C). In some cases, the HLA-C TMMP heavy chain polypeptide of the invention comprises Y84A and A236C substitutions. In some cases, the HLA-C*0701 T cell-MMP heavy chain polypeptide or epitope conjugate thereof comprises Y84C and A139C substitutions. In some cases, the HLA-C TMMP heavy chain polypeptide of the present invention comprises the substitutions Y84C, A139C and A236C.

Non-classical heavy chains HLA-E, -F and -G of MHC class I.

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

Non-limiting examples of suitable HLA-E alleles include, but are not limited to,

HLA-E*0101 (HLA-E*01:01:01:01), HLA-E*01:03(HLAE*01:03:01:01), HLA-E*01:04, HLA-E*01:05, HLA-E*01:06, HLA-E*01:07, HLA-E*01:09 and HLA-E*01:10.

Non-limiting examples of suitable HLA-F alleles include, but are not limited to,

HLA-F*0101 (HLA-F*01:01:01:01), HLA-F*01:02, HLAF*01:03(HLA-F*01:03:01:01), HLA-F*01:04, HLA-F*01:05 and HLA-F*01:06.

Non-limiting examples of suitable HLA-G alleles include, but are not limited to,

HLA-G*0101 (HLA-G*01:01:01:01), HLA-G*01:02, HLAG*01:03(HLA-G*01:03:01:01), HLA-G*01:04 (HLA-G*01:04:01:01), HLA-G*01:06, HLAG*01:07, HLA-G*01:08, HLA-G*01:09: HLA-G*01:10, HLA-G*01:10, HLA-G*01:ll, HLAG*01:12, HLA-G*01:14, HLA-G*01:15, HLA-G*01:16, HLA-G*01:17, HLA-G*01:18: HLAG*01:19, HLA-G*01:20 and HLA-G*01:22.

The consensus sequences for the HLA alleles E, -F and -G are without any, or essentially without any, leader, transmembrane and cytoplasmic sequences.

- 22 049816 are plotted in Fig. 12 and aligned with the consensus sequences of the above-mentioned HLA-A, -B and -C alleles in Fig. 13.

Figure 12 shows the consensus sequence for each of HLA-E, -F, and -G with variable aa positions shown as sequentially numbered X residues and aa positions 84, 139, and 236 double underlined.

Figure 13 is an alignment of the consensus amino acid sequences for HLA-A, -B, -C, -E, -F, and -G, which are given in Figures 9-13. The variable residues in each sequence are listed as X with consecutive numbering removed. As indicated in Figure 8, aa positions 84, 139, and 236 are shown with their flanking clusters of five amino acids, which can be substituted with from 1 to 5 amino acids independently selected from (i) any naturally occurring amino acid or (ii) any naturally occurring amino acid except proline or glycine, also shown.

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

H2K mouse.

In some cases, an MHC class I heavy chain polypeptide present in a TMMP of the invention comprises the amino acid sequence of MOUSE H2K (SEQ ID NO: 45) in (MOUSE H2K in Fig. 8, or a sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to all or a portion (e.g., 50, 75, 100, 150, 200, or 250 consecutive amino acids) of such a sequence (e.g., it may comprise 1-25, 1-5, 5-10, 10-15, 15-20, 20-25, or 25-30 amino acid insertions, deletions, and/or substitutions). In some cases, where the MHC class I heavy chain polypeptide is present in a TMMP of the invention, ... the MOUSE H2K TMMP heavy chain polypeptide of the invention has less than 100% identity to the sequence designated MOUSE H2K in Fig. 8, it may comprise a mutation at one or more of positions 84, 139, and/or 236 selected from: a tyrosine to alanine substitution at position 84 (Y84A); a tyrosine to cysteine at position 84 (Y84C); an alanine to cysteine at position 139 (A139C); and an alanine to cysteine at position 236 (A236C). In some cases, the MOUSE H2K TMMP heavy chain polypeptide of the invention comprises Y84A and A236C substitutions. In some cases, the MOUSE H2K TMMP heavy chain polypeptide of the invention comprises Y84C and A139C substitutions. In some cases, the MOUSE H2K TMMP heavy chain polypeptide of the invention comprises Y84C, A139C substitutions and A236C.

- 23 049816

Examples of combinations

Table 1 below shows various combinations of MHC class I heavy chain sequence modifications that may be included in the TMMPs of the present invention.

______________________________Table 1______________________________

P/P number HLA Heavy Chain Sequence Range of sequence identities □ Specific substitutions in the ak position 84, 139 and/or 236 1 HLA-A Consensus (Fig. 9B) 75%-99.8%, 80%-99.8%, 85%-99.8%, 90%-99.8%, 95%-99.8%, 98%-99.8% or 99%-99.8%; or 1-25, 1-5, 5-10, 10-15, 15-20 or 20-25 aa insertions, deletions and/or substitutions (excluding variable residues) Not present; Y84C; Y84A; A139C; A236C; (Y84A and A236C); (Y84C and A139C); or (Y84C, A139C and A236C) 2 A*0101, A*0201, A*0301, A*1101, A*2402, A*2301, A*2402, A*2407, A*3303 or A*3401 (Fig. 9A) 75%-99.8%; 80%-99.8%; 85%-99.8%; 90%-99.8%; 95%-99.8%; 98%-99.8% or 99%-99.8%; or 1-25, 1-5, 5-10, 10-15, 15-20 or 20-25 aa insertions, deletions and/or substitutions Not present; Y84C; Y84A; A139C; A236C; (Y84A and A236C); (Y84C and A139C); or (Y84C, A139C and A236C) 3 HLA-B Consensus (Fig. 10B) 75%-99.8%, 80%-99.8%, 85%-99.8%, 90%-99.8%, 95%-99.8%, 98%-99.8% or 99%-99.8%; or 1-25, 1-5, 5-10, 10-15, 15-20 or 20-25 aa insertions, deletions and/or substitutions (excluding variable residues) Not present; Y84C; Y84A; A139C; A236C; (Y84A and A236C); (Y84C and A139C); or (Y84C, A139C and A236C) 4 B*0702, B*0801, B*1502, B*3802, B*4001, B*4601 or B*5301 (Fig. 10A) 75%-99.8%; 80%-99.8%; 85%-99.8%; 90%-99.8%; 95%-99.8%; 98%-99.8% or 99%-99.8%; or 1-25, 1-5, 5-10, 10-15, 15-20 or 20-25 aa insertions, deletions and/or substitutions Not present; Y84C; Y84A; A139C; A236C; (Y84A and A236C); (Y84C and A139C); or (Y84C, A139C and A236C) 5 HLA-C Consensus (Fig. 11B) 75%-99.8%, 80%-99.8%, 85%-99.8%, 90%-99.8%, 95%-99.8%, 98%-99.8% or 99%-99.8%; or 1-25, 1-5, 5-10, 10-15, 15-20 or 20-25 aa insertions, deletions and/or substitutions (excluding variable residues) Not present; Y84C; Y84A; A139C; A236C; (Y84A and A236C); (Y84C and A139C); or (Y84C, A139C and A236C) 6 C*0102, C*0303, C*0304, C*0401, C*0602, C*0701, C*0801 or C*1502 (Fig. PA) 75%-99.8%; 80%-99.8%; 85%-99.8%; 90%-99.8%; 95%-99.8%; 98%-99.8% or 99%-99.8%; or 1-25, 1-5, 5-10, 10-15, 15-20 or 20-25 aa insertions, deletions and/or substitutions Not present; Y84C; Y84A; A139C; A236C; (Y84A and A236C); (Y84C and A139C); or (Y84C, A139C and A236C) 7 HLA-E, F or G Consensus (Fig. 12) 75%-99.8%, 80%-99.8%, 85%-99.8%, 90%-99.8%, 95%-99.8%, 98%-99.8% or 99%-99.8%; or 1-25, 1-5, 5-10, 10-15, 15-20 or 20-25 aa insertions, deletions and/or substitutions (excluding variable residues) Not present; Y84C; Y84A; A139C; A236C; (Y84A and A236C); (Y84C and A139C); or (Y84C, A139C and A236C) 8 H2K mouse (Fig. 8) 75%-99.8%; 80%-99.8%; 85%-99.8%; 90%-99.8%; 95%-99.8%; 98%-99.8% or 99%-99.8%; or 1-25, 1-5, 5-10, 10-15, 15-20 or 20-25 aa insertions, deletions and/or substitutions Not present; Y84C; Y84A; A139C; A236C; (Y84A and A236C); (Y84C and A139C); or (Y84C, A139C and A236C)

□ The range of sequence identities represents the acceptable range of sequence identity of the MHC-H polypeptide sequence included in the TMMP relative to the corresponding portion of the sequences listed in Fig. 8-13, excluding variable residues in the consensus sequences.

Beta-2 microglobulin.

The TMMP b2-microglobulin (b2M) polypeptide of the present invention may be a human b2M polypeptide, a non-human primate b2M polypeptide, a mouse b2M polypeptide, and the like. In some cases, the b2M polypeptide comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the b2M amino acid sequence shown in Fig. 6. In some cases, the b2M polypeptide comprises an amino acid sequence that has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to amino acids 21-119 of the b2M amino acid sequence shown in Fig. 6.

In some cases, a suitable B2M polypeptide comprises the following amino acid sequence:

- 24 049816

IQRTPKIQVY SCHPAENGKS NFLNCYVSGF HPSDIEVDLLKNGERIEKVE HSDLSFSKDW SFYLLYYTEF TPTEKDEYAC RVNHVTLSQP KIVKWDRDM (SEQ ID NO:311);

and the HLA class I heavy chain polypeptide contains the following amino acid sequence:

GSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQE

GPEYWDGETRK VKAHSQTHRVDL(aa 1) { C } (aa2) AGSHT VQRM YGCD VGSD WRFLRGY HQ YAYDGKD YIALKEDLRS W(aa3) { C } (aa4))HKWE AAHVAEQLRAYLEGTC VEWLRRY LENGKETLQRTDAPKTHMTHHAVSDHEATLRCWALSFYPAEITLTWQRDGEDQTQDTE L(aa5)(C)(aa6)QKWAAVVVPSGQEQRYTCHVQHEGLPKPLTLRWEP (SEQ ID NO:309), where the cysteine residues designated as {C} form a disulfide bond between helices α1 and α2-1, and the residues (C) form a disulfide bond with the cysteine of the B2M polypeptide at position 12. In the above sequence, acc1 denotes amino acid cluster 1; acc2 denotes amino acid cluster 2; acc3 denotes amino acid cluster 3, acc4 denotes amino acid cluster 4, acc5 denotes amino acid cluster 5; and acc6 denotes amino acid cluster 6; see, for example, Fig. 10. Each occurrence of acc1, acc2, acc3, acc4, acc5, and acc6 is independently selected from 1-5 amino acid residues, wherein the amino acid residues are i) independently selected from any naturally occurring (e.g., encoded) amino acid or ii) any naturally occurring amino acid other than proline or glycine.

In some cases, the MHC polypeptide comprises a single amino acid substitution relative to a reference MHC polypeptide (wherein the reference MHC polypeptide may be a wild-type MHC polypeptide), wherein the single amino acid substitution replaces the amino acid with a cysteine (Cys) residue. Such cysteine residues, when present in the MHC polypeptide of the first TMMP polypeptide of the invention, may form a disulfide bond with a cysteine residue present in the second polypeptide chain of the TMMP of the invention.

In some cases, the first MHC polypeptide in the first TMMP polypeptide of the invention and/or the second MHC polypeptide in the second TMMP polypeptide of the invention comprise an amino acid substitution with an amino acid substitution of cysteine, wherein the substituted cysteine in the first MHC polypeptide forms a disulfide bond with a cysteine in the second MHC polypeptide, wherein the cysteine in the first MHC polypeptide forms a disulfide bond with the substituted cysteine in the second MHC polypeptide, or wherein the substituted cysteine located in the first MHC polypeptide forms a disulfide bond with the substituted cysteine in the second MHC polypeptide.

For example, in some cases, one of the following pairs of residues in HLA β2 microglobulin and HLA class I heavy chain is replaced by cysteines (where the residue numbers correspond to those in the mature polypeptide): 1) residue 12 of β2M, residue 236 of HLAI class heavy chain; 2) residue 12 of β2M, residue 237 of HLAI class heavy chain; 3) residue 8 of β2M, residue 234 of HLA class I heavy chain; 4) residue 10 of β2M, residue 235 of HLAI class heavy chain; 5) residue 24 of β2M, residue 236 of HLAI class heavy chain; 6) residue 28 of β2M, residue 232 of HLA class I heavy chain; 7) residue 98 of β2M, residue 192 of HLAI class heavy chain; 8) residue 99 of β2M, residue 234 of HLAI class heavy chain. 9) residue 3 B2M, residue 120 of the heavy chain of HLA class I; 10) residue 31 B2M, residue 96 of the heavy chain of HLA class I; 11) residue 53 B2M, residue 35 of the heavy chain of HLA class I; 12) residue 60 B2M, residue 96 of the heavy chain of HLA class I; 13) residue 60 B2M, residue 122 of the heavy chain of HLA class I; 14) residue 63 B2M, residue 27 of the heavy chain of HLA class I; 15) residue Arg3 e2M, residue Gly120 of the heavy chain of HLA class I; 16) residue His31 e2M, residue Gln96 of the heavy chain of HLA class I; 17) residue Asp53 e2M, residue Arg35 of the heavy chain of HLA class I; 18) residue Trp60 B2M, residue Gln96 of the heavy chain of HLA class I; 19) Trp60 residue B2M, Asp122 residue of HLA class I heavy chain; 20) Tyr63 residue B2M, Tyr27 residue of HLA class I heavy chain; 21) Lys6 residue E2M, Glu232 residue of HLA class I heavy chain; 22) Gln8 residue E2M, Arg234 residue of HLA class I heavy chain; 23) Tyr10 residue B2M, Pro235 residue of HLA class I heavy chain; 24) Ser11 residue E2M, Gln242 residue of HLA class I heavy chain; 25) Asn24 residue E2M, Ala236 residue of HLA class I heavy chain; 26) Ser28 residue E2M, Glu232 residue of HLA class I heavy chain; 27) residue Asp98 of e2M, residue His192 of the HLA class I heavy chain; and 28) residue Met99 of e2M, residue Arg234 of the HLA class I heavy chain. The amino acid numbering of the MHC/HLA class I heavy chain refers to the mature MHC/HLA class I heavy chain without the signal peptide. For example, in some cases, residue 236 of the mature amino acid sequence of HLA-A is replaced with Cys. In some cases, residue 236 of the mature amino acid sequence of HLA-B is replaced with Cys. In some cases, residue 236 of the mature amino acid sequence of HLA-C is replaced with Cys. In some cases, residue 32 (corresponding to Arg-12 of mature B2M) of the amino acid sequence shown in Fig. 6 is replaced with Cys.

- 25 049816

In some cases, the β2M polypeptide comprises the amino acid sequence: IQRTPKIQVY SRHPAENGKS NFLNCYVSGF HPSDIEVDLLKNGERIEKVE HSDLSFSKDW SFYLLYYTEF TPTEKDEYAC RVNHVTLSQP KIVKWDRDM (SEQ ID NO:310).

In some cases, the B2M polypeptide comprises the amino acid sequence: IQRTPKIQVY SCHPAENGKS NFLNCYVSGF HPSDIEVDLLKNGERIEKVE HSDLSFSKDW SFYLLYYTEF TPTEKDEYAC RVNHVTLSQP KIVKWDRDM (SEQ ID NO:311).

In some cases, the HLA class I heavy chain polypeptide contains the amino acid sequence:

GSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEY WDGETRKVKAHSQTHRVDLGTLRGYYNQSEAGSHTVQRMYGCDVGSDWRFLRGYHQ YAYDGKDYIALKEDLRSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGTCVEWLRRY LENGKETLQRTDAPKTHMTHHAVSDHEATLRCWALSFYPAEITLTWQRDGEDQTQDTE LVETRPAGDGTFQKWAAVVVPSGQEQRYTCHVQHEGLPKPLTLRWEP (SEQ ID NO:297).

In some cases, the HLA class I heavy chain polypeptide contains the amino acid sequence:

GSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEY WDGETRKVKAHSQTHRVDLGTLRGYYNQSEAGSHTVQRMYGCDVGSDWRFLRGYHQ YAYDGKDYIALKEDLRSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGTCVEWLRRY LENGKETLQRTDAPKTHMTHHAVSDHEATLRCWALSFYPAEITLTWQRDGEDQTQDTE LVETRPCGDGTFQKWAAVVVPSGQEQRYTCHVQHEGLPKPLTLRWEP (SEQ ID NO:312).

In some cases, the HLA class I heavy chain polypeptide contains the amino acid sequence:

GSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEY WDGETRKVKAHSQTHRVDLGTLRGAYNQSEAGSHTVQRMYGCDVGSDWRFLRGYHQ YAYDGKDYIALKEDLRSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGTCVEWLRRY LENGKETLQRTDAPKTHMTHHAVSDHEATLRCWALSFYPAEITLTWQRDGEDQTQDTE LVETRPCGDGTFQKWAAVVVPSGQEQRYTCHVQHEGLPKPLTLRWE (SEQ ID NO:313).

In some cases, the B2M polypeptide contains the following amino acid sequence: IQRTPKIQVY SCHPAENGKS NFLNCYVSGF HPSDIEVDLLKNGERIEKVE

HSDLSFSKDW SFYLLYYTEF TPTEKDEYAC RVNHVTLSQP KIVKWDRDM (SEQ ID NO:311);

and the HLA class I TMMP heavy chain polypeptide of the present invention comprises the following amino acid sequence:

GSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQE GPEYWDGETRKVKAHSQTHRVDLGTLRGYYNQSEAGSHTVQRMYGCDVGSDWRFLR GYHQYAYDGKDYIALKEDLRSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGTCVE WLRRYLENGKETLQRTDAPKTHMTHHAVSDHEATLRCWALSFYPAEITLTWQRDGED QTQDTELVETRPCGDGTFQKWAAVVVPSGQEQRYTCHVQHEGLPKPLTLRWEP (SEQ ID NO:312), where the Cys residues that are underlined and in bold form a disulfide bond with each other in TMMP.

In some cases, the B2M polypeptide comprises the amino acid sequence: IQRTPKIQVYSCHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDW SFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDM (SEQ ID NO:314).

In some cases, the first polypeptide and the second TMMP polypeptide of the invention are disulfide bonded to each other via: i) a Cys residue present in a linker connecting the peptide epitope and the β2M polypeptide in the first polypeptide chain; and ii) a Cys residue present in an MHC class I heavy chain in the second polypeptide chain. In some cases, the Cys residue present in the MHC class I heavy chain is a Cys introduced as a Y84C substitution. In some cases, the linker connecting the peptide epitope and the β2M polypeptide in

- 26 049816 of the first polypeptide chain, is GCGGS(G4S)n (SEQ ID NO: 315), where n is 1, 2, 3, 4, 5, 6, 7, 8 or 9. For example, in some cases, the linker comprises the amino acid sequence GCGGSGGGGSGGGGSGGGGS (SEQ ID NO: 316).

As another example, the linker comprises the amino acid sequence GCGGSGGGGSGGGGS (SEQ ID NO:317).

Examples of disulfide-linked first and second TMMP polypeptides of the present invention are schematically depicted in Figs. 2A-2F.

TMMPs linked by multiple disulfide bonds.

In some cases, the first polypeptide and the second TMMP polypeptide of the invention are linked to each other by at least two disulfide bonds (i.e., two interchain disulfide bonds). Examples of such multiple disulfide bonded TMMPs are shown schematically in Figs. 17A and 17B, and in Figs. 18A-18C. Furthermore, when the TMMP of the invention comprises an IgFc polypeptide, the heterodimeric TMMP can dimerize such that the disulfide bonds connect the IgFc polypeptides into two heterodimeric TMMPs. Such an arrangement is shown schematically in Figs. 17C and 17D, where the disulfide bonds are represented by dotted lines. Unless otherwise noted, the at least two disulfide bonds described for multiple disulfide-linked TMMPs in this section do not refer to the disulfide bonds linking IgFc polypeptides in dimerized TMMPs.

As noted above, in some cases, the first polypeptide and the second TMMP polypeptide of the invention are linked to each other by at least two disulfide bonds (i.e., two interchain disulfide bonds). For example, in some cases, the first polypeptide and the second TMMP polypeptide of the invention are linked to each other by 2 interchain disulfide bonds. As another example, in some cases, the first polypeptide and the second TMMP polypeptide of the invention are linked to each other by 3 interchain disulfide bonds. As another example, in some cases, the first polypeptide and the second TMMP polypeptide of the invention are linked to each other by 4 interchain disulfide bonds.

In some cases, when the peptide epitope in the first TMMP polypeptide of the invention is linked to the b2M polypeptide by a linker comprising Cys by at least one of two Cys disulfide bonds in the linker to a Cys in the class I MHC heavy chain in the second polypeptide. In some cases, when the peptide epitope in the first TMMP polypeptide of the invention is linked to the class I MHC heavy chain polypeptide by a linker by at least one of two Cys disulfide bonds in the linker to a Cys in the b2M polypeptide in the second polypeptide.

In some cases, a multiple disulfide-linked TMMP of the invention (e.g., a two-disulfide-linked TMMP) exhibits increased stability compared to a control TMMP comprising only one of the at least two disulfide bonds. In some cases, a multiple disulfide-linked TMMP of the invention (e.g., a two-disulfide-linked TMMP) of the invention exhibits increased stability in vitro compared to a control TMMP comprising only one of the at least two disulfide bonds. For example, in some cases, a multiple disulfide bonded TMMP of the present invention (e.g., a two disulfide bonded TMMP) exhibits at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 50%, at least 2-fold, at least 5-fold, or at least 10-fold greater stability in vitro, compared to a control TMMP comprising only one of the at least two disulfide bonds.

It can be determined whether a multiple disulfide-linked TMMP of the invention (e.g. a two-disulfide-linked TMMP) exhibits increased stability in vitro compared to a control TMMP comprising only one of the at least two disulfide bonds by measuring the amount of disulfide-linked heterodimeric TMMP of the invention present in a sample over a certain time and/or under a certain condition and/or during purification of the TMMP.

For example, in some cases, a multiple disulfide-linked TMMP (e.g., a di-disulfide-linked TMMP) of the invention exhibits at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 50%, at least 2-fold, at least 5-fold, or at least 10-fold greater in vitro stability than a control TMMP that includes only one of the at least two disulfide bonds when the TMMP is stored at 37°C for a certain period of time (e.g., for a period of time from about 1 week to about 2 weeks, from about 2 weeks to about 4 weeks, or from about 4 weeks to about 2 months). For example, in some cases, the amount of disulfide-linked heterodimeric TMMP remaining after storage of the multiple disulfide-linked TMMP (at least

- 27 049816 example of a two-disulfide-linked TMMP) according to the invention in vitro at 37°C for 28 days is at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 50%, at least 2 times, at least 5 times or at least 10 times greater than the amount of disulfide-linked heterodimeric TMMP remaining after storage of a control TMMP (a TMMP comprising only one of the at least two disulfide bonds present in a multiple disulfide-linked TMMP) in a test tube at 37°C for 28 days.

As an example, a double disulfide-linked TMMP comprising polypeptides 1715 and 2380, as depicted in Figs. 14A and 14B, exhibits greater in vitro stability than a TMMP comprising polypeptides 2405 and 2380, with polypeptide 2405 being depicted in Fig. 14D, wherein such TMMP comprises only one disulfide bond, wherein the one disulfide bond is formed between: i) the Cys from the G2C linker between the epitope and b2M; and ii) the Cys provided by the Y84C substitution in the MHC class I heavy chain. As another example, a double disulfide-linked TMMP comprising polypeptides 1715 and 2380, as depicted in Fig. 14A and 14B, exhibits higher stability in vitro, compared to TMMP containing polypeptides 1380 and 2380, wherein polypeptide 1380 is depicted in Fig. 14E, wherein such TMMP contains only one disulfide bond, wherein one disulfide bond is formed by: i) Cys provided by R12C substitution in polypeptide β2M; and ii) Cys provided by A236C substitution in MHC class I heavy chain.

In some cases, the multiple disulfide-linked TMMP of the present invention exhibits increased stability in vitro compared to a control TMMP comprising only one of the at least two disulfide bonds. For example, in some cases, the multiple disulfide-linked TMMP of the present invention exhibits at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 50%, at least 2-fold, at least 5-fold, or at least 10-fold greater stability in vivo compared to a control TMMP comprising only one of the at least two disulfide bonds.

In some cases, the presence of two disulfide bonds in a multiple disulfide-linked TMMP of the invention (e.g., a two-disulfide-linked TMMP) is designed to increase the production of the disulfide-linked heterodimeric TMMP, compared to the amount of disulfide-linked heterodimeric TMMP obtained when the TMMP is a control TMMP including only one of at least two disulfide bonds. For example, a multiple disulfide-linked TMMP of the invention (e.g., a two-disulfide-linked TMMP) can be produced in a mammalian cell in an in vitro cell culture, where the mammalian cell is cultured in a liquid cell culture medium. The TMMP can be secreted into the cell culture medium. The cells can be lysed, creating a cell lysate, and the TMMP can be present in the cell lysate. The TMMP may be purified from the cell culture medium and/or cell lysate. For example, when the TMMP comprises an IgG1 Fc polypeptide, the cell culture medium and/or cell lysate may be contacted with immobilized Protein A (e.g., the cell culture medium and/or cell lysate may be applied to a Protein A column wherein Protein A is immobilized on beads). TMMP present in the cell culture medium and/or cell lysate binds to the immobilized Protein A. After washing the column to remove unbound substances, the bound TMMP is eluted to obtain a Protein A eluate. The amount of disulfide-linked heterodimeric TMMP present in the Protein A eluate is at least 0.5%, at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, or at least 10% greater than the amount of disulfide-linked heterodimeric TMMP present in the Protein A eluate, wherein the TMMP is a control TMMP that contains only one of the at least two disulfide bonds present in the multiple disulfide-linked TMMP bonds (e.g., TMMP linked by two disulfide bonds). In some cases, the percentage of total TMMP protein in the eluate that is non-aggregated disulfide-linked heterodimeric TMMP is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%. The Protein A eluate may be subjected to size exclusion chromatography (SEC) and/or one or more other additional purification steps.

In some cases, the T cell modulating multimeric polypeptide of the invention comprises at least one heterodimer comprising: a) a first polypeptide comprising: i) a WT1 peptide epitope, wherein the WT1 peptide is at least 4 amino acids in length (e.g., from 4 amino acids to 25 amino acids; e.g., the WT1 peptide is 4, 5, 6, 7, 8, 9, 10-15, 15-20, or 20-25 amino acids in length); and ii) a first MHC polypeptide; b) a second polypeptide comprising a second MHC polypeptide, and c) at least one immunomodulatory polypeptide, wherein the first and/or second

- 28 049816 the polypeptide comprises an immunomodulatory polypeptide, and wherein the heterodimer comprises 2 disulfide bonds between the first polypeptide and the second polypeptide (i.e. the heterodimer consists of: i) a first disulfide bond linking the first polypeptide and the second polypeptide; and ii) a second disulfide bond linking the first polypeptide and the second polypeptide). In other words, the first polypeptide comprises a first Cys residue forming a disulfide bond (the first disulfide bond) with a first Cys residue in the second polypeptide; and the first polypeptide comprises a second Cys residue forming a disulfide bond (the second disulfide bond) with a second Cys residue in the second polypeptide.

In some cases, the TMMP of the invention comprises: a) a first polypeptide comprising, in an N-terminal to C-terminal direction: i) a peptide epitope; ii) a peptide linker; and iii) a β2M polypeptide; and b) a second polypeptide comprising an MHC class I heavy chain polypeptide, wherein one or both of the first and second polypeptides comprises at least one immunomodulatory polypeptide, wherein the TMMP comprises: a) a first disulfide bond between: i) a Cys present in the linker between the peptide epitope and the β2M polypeptide; and ii) a first Cys introduced into the MHC class I heavy chain polypeptide; and b) at least a second disulfide bond between the first polypeptide and the second polypeptide, wherein the at least second disulfide bond is between: i) a Cys in the first polypeptide that is C-terminal to the Cys present in the linker; and ii) a Cys in a second polypeptide that is C-terminal to the first Cys introduced into a class I MHC heavy chain polypeptide.

In some cases, the first and second disulfide bond-forming Cys residues in the first or second TMMP polypeptide of the present invention are spaced from about 10 amino acids to about 200 amino acids apart. For example, in some cases, the first and second disulfide bond-forming Cys residues in the first or second TMMP polypeptide of the invention are spaced from about 10 amino acids (aa) to about 15 aa, from about 15 aa to about 20 aa, from about 20 aa to about 25 aa, from about 25 aa to about 30 aa, from about 30 aa to about 40 aa, from about 40 aa to about 50 aa, from about 50 aa to about 60 aa, from about 60 aa to about 70 aa, from about 70 aa to about 80 aa, from about 80 aa to about 90 aa, from about 90 aa to about 100 aa, from about 100 aa to about 110 aa, from about 110 aa to about 120 aa, to about 120 ak to about 130 ak, from about 130 ak to about 140 ak, from about 140 ak to about 150 ak, from about 150 ak to about 160 ak, from about 160 ak to about 170 ak, from about 170 ak to about 180 ak, from about 180 ak to about 190 ak or from about 190 ak to about 200 ak.

By way of example, in some cases, the first and second disulfide bond-forming Cys residues in the first TMMP polypeptide of the present invention are spaced from about 10 amino acids to about 80 amino acid residues apart. For example, in some cases, the second disulfide bond-forming Cys residue in the first polypeptide is located at a distance of from about 10 amino acids to about 80 amino acids (e.g., from about 10 amino acids (aa) to about 15 aa, from about 15 aa to about 20 aa, from about 20 aa to about 25 aa, from about 25 aa to about 30 aa, from about 30 aa to about 40 aa, from about 40 aa to about 50 aa, from about 50 aa to about 60 aa, from about 60 aa to about 70 aa, or from about 70 aa to about 80 aa) toward the C-terminus of the first disulfide bond-forming Cys residue in the first polypeptide. In some cases, the second disulfide bond-forming Cys residue in the first polypeptide is 10 aa, 11 aa, 12 aa, 13 aa, 14 aa, 15 aa, 16 aa, 17 aa, 18 aa, 19 aa, 20 aa, 21 aa, 22 aa, 23 aa, 24 aa, or 25 aa C-terminal to the first disulfide bond-forming Cys residue in the first polypeptide. In some cases, the second disulfide bond-forming Cys residue in the first polypeptide is 15 aa C-terminal to the first disulfide bond-forming Cys residue in the first polypeptide. In some cases, the second disulfide bond-forming Cys residue in the first polypeptide is 20 aa C-terminal to the first disulfide bond-forming Cys residue in the first polypeptide. In some cases, the second disulfide bond-forming Cys residue in the first polypeptide is 25 aa C-terminal to the first disulfide bond-forming Cys residue in the first polypeptide.

In some cases, the first and second disulfide bond-forming Cys residues in the second TMMP polypeptide of the invention are located at a distance of from about 140 amino acids to about 160 amino acids from each other. For example, in some cases, the second disulfide bond-forming Cys residue in the second polypeptide is located at a distance of from about 140 amino acids to about 160 amino acids C-terminal to the first disulfide bond-forming Cys residue in the second polypeptide. In some cases, the second disulfide bond-forming Cys residue in the second polypeptide is amino acid (aa) 140, 141 aa, 142 aa, 143 aa, 144 aa, 145 aa, 146 aa, 147 aa, 148 aa, 149 aa, 150 aa, 151 aa, 152 aa, 153 aa, 154 aa, 155 aa, 156 aa, 157 aa, 158 aa, 159 aa, or 160 aa in a C-terminal direction to the first disulfide bond-forming Cys residue in the second polypeptide.

A multiple disulfide-linked TMMP of the present invention (e.g., a double disulfide-linked TMMP) may comprise: a) a first polypeptide comprising: i) a WT1 peptide (e.g., a WT1 peptide of from 4 amino acids to about 25 amino acids); and ii) a first

- 29 049816 MHC lipid, wherein the first polypeptide comprises a peptide linker between the WT1 peptide and the first MHC polypeptide, wherein the peptide linker comprises a Cys residue, and wherein the first MHC polypeptide is a B2M polypeptide comprising an amino acid substitution that introduces a Cys residue; b) and a second polypeptide comprising a second MHC polypeptide, wherein the second MHC polypeptide is a Class I heavy chain comprising a Y84C substitution and an A236C substitution, based on the amino acid numbering of HLA-A*O2O1 (depicted in Fig. 9A), or at corresponding positions of another Class I heavy chain allele, wherein the TMMP comprises a disulfide bond between a Cys residue in the peptide linker and a Cys residue at amino acid position 84 of the Class I heavy chain or the corresponding position of another Class I heavy chain allele, and wherein the TMMP comprises a disulfide bond between an introduced Cys residue in the B2M polypeptide and a Cys at amino acid position 236 of the Class I heavy chain or the corresponding position of another Class I heavy chain allele; and c) at least one immunomodulatory polypeptide, wherein the first and/or second polypeptide comprises at least one immunomodulatory polypeptide. Examples are schematically depicted in Fig. 17A and Fig. 17B.

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

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

The following examples are non-limiting examples of MHC class I heavy chain containing the Y84C substitution and the A236C substitution, based on the amino acid numbering of HLA-A*0201 (depicted in Fig. 9A), or at the corresponding positions in another class I heavy chain allele.

HLA-A.

In some cases, a multiple disulfide bonded TMMP of the present invention (e.g., a di-disulfide bonded TMMP) comprises: a) a first polypeptide comprising: i) a WT1 peptide (e.g., a WT1 peptide of from 4 amino acids to about 25 amino acids); and ii) a first MHC polypeptide, wherein the first polypeptide comprises a peptide linker between the WT1 peptide and the first MHC polypeptide, wherein the peptide linker comprises a Cys residue, and wherein the first MHC polypeptide is a B2M polypeptide comprising an amino acid substitution that introduces a Cys residue; b) and a second polypeptide comprising an HLA-A class I MHC heavy chain comprising an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least

- 30 049816 at least 99% or 100% identity of the amino acid sequence with the following amino acid sequence:

GSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEY WDGETRKVKAHSQTHRVDLGTLRGCYNQSEAGSHTVQRMYGCDVGSDWRFLRGYHQ YAYDGKDYIALKEDLRSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGTCVEWLRRY LENGKETLQRTDAPKTHMTHHAVSDHEATLRCWALSFYPAEITLTWQRDGEDQTQDTE LVETRPCGDGTFQKWAAVVVPSGQEQRYTCHVQHEGLPKPLTLRWEP (SEQ ID NO:341), wherein amino acid 84 is Cys and amino acid 236 is Cys; and c) at least one immunomodulatory polypeptide, wherein the first and/or second polypeptide comprises at least one immunomodulatory polypeptide. In some cases, the peptide linker comprises the amino acid sequence GCGGS (SEQ ID NO: 318). In some cases, the peptide linker comprises the amino acid sequence GCGGS(GGGGS)n (SEQ ID NO: 319), wherein n is an integer from 1 to 10. In some cases, the β2M polypeptide comprises an R12C substitution. For example, the β2M polypeptide may comprise an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence:

IQRTPKIQVYSCHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDW

SFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDM (SEQ ID NO:314), wherein amino acid 12 is Cys. The at least one immunomodulatory polypeptide can be a polypeptide that has an activating/stimulatory effect on a target T cell or a suppressive/inhibitory effect on a target T cell. For example, the at least one immunomodulatory polypeptide can be a cytokine (e.g., an IL2 polypeptide, an IL7 polypeptide, an IL12 polypeptide, an IL15 polypeptide, an IL17 polypeptide, an IL21 polypeptide, an IL27 polypeptide, an IL-23 polypeptide, a TGFe polypeptide, etc.; and including all family members, e.g., IL17A, IL-17B, IL-17C, IL-17D, IL-17E, IL-17F, IL-17E), 4-1BBL polypeptide, ICOS-L polypeptide, OX-40L polypeptide, CD80 polypeptide, CD86 polypeptide (CD80 and CD86 are also known as B7-1 and B7-2, respectively), CD40 polypeptide, CD70 polypeptide, JAG1 (CD339) polypeptide, ICAM (CD540) polypeptide, PD-L1 polypeptide, FasL polypeptide, PD-L2 polypeptide, PD-1H polypeptide (VISTA), ICOS-L polypeptide (CD275), GITRL polypeptide, HVEM polypeptide, CXCL10 polypeptide, CXCL9 polypeptide, CXCL11 polypeptide, CXCL13 polypeptide and CX3CL1 polypeptide, galectin-9 polypeptide, CD83 polypeptide, CD30L polypeptide, HLA-G, MICA polypeptide, MICB polypeptide, HVEM polypeptide (CD270), lymphotoxin receptor beta polypeptide, 3/TR6 polypeptide, ILT3 polypeptide, ILT4 polypeptide, CXCL10 polypeptide, CXCL9 polypeptide, CXCL11 polypeptide, CXCL13 polypeptide, or CX3CL1 polypeptide. These immunomodulatory polypeptides may be a wild-type polypeptide or a variant of a wild-type polypeptide. In some cases, the immunomodulatory polypeptide is an activating (stimulating) immunomodulatory polypeptide, for example, the immunomodulatory polypeptide can produce an activating/stimulating effect on a T cell. Examples of activating immunomodulatory polypeptides include, for example, CD80, CD86, 4-1BBL, OX40L, CD70, ICOS-L, CD40, ICAM (CD54), IL2, IL7, IL12, IL15, IL17, IL21, IL27, IL23, GITRL, TGFe, and lymphotoxin beta receptor. In some cases, the immunomodulatory polypeptide is an inhibitory (suppressive) immunomodulatory polypeptide, for example, the immunomodulatory polypeptide can produce a suppressive/inhibitory effect on a T cell. Examples of inhibitory immunomodulatory polypeptides include, for example, PD-1H, PD-L1, PD-L2, TGFe, FasL, HVEM, galectin-9, ILT3, and ILT4. Depending on the context, TGFe polypeptides can produce either an activating/stimulatory effect or a suppressive/inhibitory effect.

In some cases, at least one immunomodulatory polypeptide is a reduced affinity variant as described elsewhere herein. In some cases, the first or second polypeptide comprises an Fclg polypeptide.

In some cases, a multiple disulfide-linked TMMP of the invention (e.g., a two-disulfide-linked TMMP) comprises an HLA-A class I heavy chain polypeptide. In some cases, an HLA-A heavy chain polypeptide present in a multiple disulfide-linked TMMP of the invention (e.g., a two-disulfide-linked TMMP) comprises an amino acid sequence having at least 95%, at least 98%, or at least 99% amino acid sequence identity to an amino acid sequence

- 31 049816

HLAA*0101, HLA-A*0201, HLA-A*0202, HLA-A* 1101, HLA-A*2301, HLA-A*2402, HLAA*2407, HLA-A*3303, or HLA-A*3401, depicted in Fig. 9A, wherein the HLA-A heavy chain polypeptide comprises the Y84C and A236C substitutions. HLA-A*0101 (Y84C; A236C).

In some cases, the HLA-A heavy chain polypeptide present in a multiple disulfide-linked TMMP of the present invention (e.g., a two-disulfide-linked TMMP) comprises an amino acid sequence having at least 95%, at least 98%, or at least 99% or 100% amino acid sequence identity to the following amino acid sequence of HLA-A*0101 (Y84C; A236C): GSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQKMEPRAPWIEQ

EGPEYWDQETRNMKAHSQTDRANLGTLRGCYNQSEDGSHTIQIMYGCDVGPDGRFLR GYRQDAYDGKDYIALNEDLRSWTAADMAAQITKRKWEAVHAAEQRRVYLEGRCVDG LRRYLENGKETLQRTDPPKTHMTHHPISDHEATLRCWALGFYPAEITLTWQRDGEDQT QDTELVETRPCGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLRWE (SEQ ID NO:343), wherein amino acid 84 is Cys and amino acid 236 is Cys. HLA-A*0201 (Y84C; A236C).

In some cases, the HLA-A heavy chain polypeptide present in a multiple disulfide-linked TMMP of the present invention (e.g., a two-disulfide-linked TMMP) comprises an amino acid sequence having at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following HLA-A*0201 (Y84C; A236C) amino acid sequence: GSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEY WDGETRKVKAHSQTHRVDLGTLRGCYNQSEAGSHTVQRMYGCDVGSDWRFLRGYHQ YAYDGKDYIALKEDLRSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGTCVEWLRRY LENGKETLQRTDAPKTHMTHHAVSDHEATLRCWALSFYPAEITLTWQRDGEDQTQDTE LVETRPCGDGTFQKWAAVVVPSGQEQRYTCHVQHEGLPKPLTLRWEP (SEQ ID NO:341), wherein amino acid 84 is Cys and amino acid 236 is Cys. HLA-A*0202 (Y84C; A236C).

In some cases, the HLA-A heavy chain polypeptide present in a multiple disulfide-linked TMMP of the present invention (e.g., a two-disulfide-linked TMMP) comprises an amino acid sequence having at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following HLA-A*O2O2 (Y84C; A236C) amino acid sequence:

GSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEY WDGETRKVKAHSQTHRVDLGTLRGCYNQSEAGSHTVQRMYGCDVGSDWRFLRGYHQ YAYDGKDYIALKEDLRSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGTCVEWLRRY LENGKETLQRTDAPKTHMTHHAVSDHEATLRCWALSFYPAEITLTWQRDGEDQTQDTE LVETRPCGDGTFQKWAAVVVPSGQEQRYTCHVQHEGLPKPLTLRWE (SEQ ID NO:341), wherein amino acid 84 is Cys and amino acid 236 is Cys. HLA-A*1101 (Y84C; A236C).

In some cases, the HLA-A heavy chain polypeptide present in a multiple disulfide-linked TMMP of the present invention (e.g., a two-disulfide-linked TMMP) comprises an amino acid sequence having at least 95%, at least 98%, or at least 99% or 100% amino acid sequence identity to the following HLA-A*1101 (Y84C; A236C) amino acid sequence:

GSHSMRYFYTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEY WDQETRNVKAQSQTDRVDLGTLRGCYNQSEDGSHTIQIMYGCDVGPDGRFLRGYRQD AYDGKDYIALNEDLRSWTAADMAAQITKRKWEAAHAAEQQRAYLEGRCVEWLRRYL ENGKETLQRTDPPKTHMTHHPISDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTEL VETRPCGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLRWE (SEQ ID NO:344), wherein amino acid 84 is Cys and amino acid 236 is Cys. HLA-A*2301 (Y84C; A236C).

In some instances, the HLA-A heavy chain polypeptide present in a multiple disulfide-linked TMMP of the present invention (e.g., a two-disulfide-linked TMMP) comprises an amino acid sequence having at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following HLA-A*2301 (Y84C; A236C) amino acid sequence: GSHSMRYFSTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEY

WDEETGKVKAHSQTDRENLRIALRCYNQSEAGSHTLQMMFGCDVGSDGRFLRGYHQY AYDGKDYIALKEDLRSWTAADMAAQITQRKWEAARVAEQLRAYLEGTCVDGLRRYLE NGKETLQRTDPPKTHMTHHPISDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELV ETRPCGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLRWE (SEQ ID NO:345), wherein amino acid 84 is Cys and amino acid 236 is Cys. HLA-A*2402 (Y84C; A236C).

In some cases, the HLA-A heavy chain polypeptide present in a multiple disulfide-linked TMMP of the present invention (e.g., a two-disulfide-linked TMMP) comprises an amino acid sequence having at least 95%, at least 98%, or at least 99% or 100% amino acid sequence identity to the following amino acid sequence of HLA-A*2402 (Y84C; A236C):

GSHSMRYFSTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEY WDEETGKVKAHSQTDRENLRIALRCYNQSEAGSHTLQMMFGCDVGSDGRFLRGYHQY AYDGKDYIALKEDLRSWTAADMAAQITKRKWEAAHVAEQQRAYLEGTCVDGLRRYL ENGKETLQRTDPPKTHMTHHPISDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTEL VETRPCGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLRWE (SEQ ID NO:346), wherein amino acid 84 is Cys and amino acid 236 is Cys. HLA-A*2407 (Y84C; A236C).

In some cases, the HLA-A heavy chain polypeptide present in a multiple disulfide-linked TMMP of the present invention (e.g., a two-disulfide-linked TMMP) comprises an amino acid sequence having at least 95%, at least 98%, or at least 99% or 100% amino acid sequence identity to the following amino acid sequence of HLA-A*2407 (Y84C; A236C):

GSHSMRYFSTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEY WDEETGKVKAQSQTDRENLRIALRCYNQSEAGSHTLQMMFGCDVGSDGRFLRGYHQY AYDGKDYIALKEDLRSWTAADMAAQITKRKWEAAHVAEQQRAYLEGTCVDGLRRYL ENGKETLQRTDPPKTHMTHHPISDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTEL VETRPCGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLRWE (SEQ ID NO:347), wherein amino acid 84 is Cys and amino acid 236 is Cys.

HLA-A*3303 (Y84C; A236C).

In some cases, the HLA-A heavy chain polypeptide present in a multiple disulfide-linked TMMP of the present invention (e.g., a two-disulfide-linked TMMP) comprises an amino acid sequence having at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following HLA-A*3303 (Y84C; A236C) amino acid sequence:

GSHSMRYFTTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQ

EGPEYWDRNTRNVKAHSQIDRVDLGTLRGCYNQSEAGSHTIQMMYGCDVGSDGRFLR

GYQQDAYDGKDYIALNEDLRSWTAADMAAQITQRKWEAARVAEQLRAYLEGTCVEW

LRRYLENGKETLQRTDPPKTHMTHHAVSDHEATLRCWALSFYPAEITLTWQRDGEDQT

QDTELVETRPCGDGTFQKWASVVVPSGQEQRYTCHVQHEGLPKPLTLRWE (SEQ ID

NO:348), where amino acid 84 is Cys and amino acid 236 is Cys.

HLA-A*3401 (Y84C; A236C).

In some cases, the HLA-A heavy chain polypeptide present in a multiple disulfide-linked TMMP of the present invention (e.g., a two-disulfide-linked TMMP) comprises an amino acid sequence having at least 95%, at least 98%, or at least 99% or 100% amino acid sequence identity to the following amino acid sequence of HLA-A*3401 (Y84C; A236C):

- 33 049816

GSHSMRYFYTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEY WDRNTRKVKAQSQTDRVDLGTLRGCYNQSEDGSHTIQRMYGCDVGPDGRFLRGYQQ DAYDGKDYIALNEDLRSWTAADMAAQITQRKWETAHEAEQWRAYLEGTCVEWLRRY LENGKETLQRTDAPKTHMTHHAVSDHEATLRCWALSFYPAEITLTWQRDGEDQTQDTE LVETRPCGDGTFQKWASVVVPSGQEQRYTCHVQHEGLPKPLTLRWE (SEQ ID NO:349), wherein amino acid 84 is Cys and amino acid 236 is Cys. HLA-B.

In some cases, a multiple disulfide bonded TMMP of the present invention (e.g., a di-disulfide bonded TMMP) comprises: a) a first polypeptide comprising: i) a WT1 peptide (e.g., a WT1 peptide of from 4 amino acids to about 25 amino acids); and ii) a first MHC polypeptide, wherein the first polypeptide comprises a peptide linker between the WT1 peptide and the first MHC polypeptide, wherein the peptide linker comprises a Cys residue, and wherein the first MHC polypeptide is a B2M polypeptide comprising an amino acid substitution that introduces a Cys residue; b) and a second polypeptide comprising an HLA-B MHC class I heavy chain comprising an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence:

GSHSMRYFYTSVSRPGRGEPRFISVGYVDDTQFVRFDSDAASPREEPRAPWIEQEGPEY

WDRNTQIYKAQAQTDRESLRNLRGCYNQSEAGSHTLQSMYGCDVGPDGRLLRGHDQY AYDGKDYIALNEDLRSWTAADTAAQITQRKWEAAREAEQRRAYLEGECVEWLRRYLE NGKDKLERADPPKTHVTHHPISDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELV ETRPCGDRTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLRWEP (SEQ ID NO:350), wherein amino acid 84 is Cys and amino acid 236 is Cys; and c) at least one immunomodulatory polypeptide, wherein the first and/or second polypeptide comprises at least one immunomodulatory polypeptide. In some cases, the peptide linker comprises the amino acid sequence GCGGS (SEQ ID NO:318). In some cases, the peptide linker comprises the amino acid sequence GCGGS(GGGGS)n (SEQ ID NO: 342), wherein n is an integer from 1 to 10. In some cases, the B2M polypeptide comprises an R12C substitution. For example, the B2M polypeptide may comprise an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence:

IQRTPKIQVYSCHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDW

SFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDM (SEQ ID NO:314), wherein amino acid 12 is Cys. The at least one immunomodulatory polypeptide can be a polypeptide that has an activating/stimulatory effect on a target T cell or a suppressive/inhibitory effect on a target T cell. For example, the at least one immunomodulatory polypeptide can be a cytokine (e.g., an IL2 polypeptide, an IL7 polypeptide, an IL12 polypeptide, an IL15 polypeptide, an IL17 polypeptide, an IL21 polypeptide, an IL27 polypeptide, an IL-23 polypeptide, a TGFe polypeptide, etc.; and including all family members, e.g., IL17A, IL-17B, IL-17C, IL-17D, IL-17E, IL-17F, IL-17E), 4-1BBL polypeptide, ICOS-L polypeptide, OX-40L polypeptide, CD80 polypeptide, CD86 polypeptide (CD80 and CD86 are also known as B7-1 and B7-2, respectively), CD40 polypeptide, CD70 polypeptide, JAG1 (CD339) polypeptide, ICAM (CD540) polypeptide, PD-L1 polypeptide, FasL polypeptide, PD-L2 polypeptide, PD-1H polypeptide (VISTA), ICOS-L polypeptide (CD275), GITRL polypeptide, HVEM polypeptide, CXCL10 polypeptide, CXCL9 polypeptide, CXCL11 polypeptide, CXCL13 polypeptide and CX3CL1 polypeptide, galectin-9 polypeptide, CD83 polypeptide, CD30L polypeptide, HLA-G, MICA polypeptide, MICB polypeptide, HVEM polypeptide (CD270), lymphotoxin receptor beta polypeptide, 3/TR6 polypeptide, ILT3 polypeptide, ILT4 polypeptide, CXCL10 polypeptide, CXCL9 polypeptide, CXCL11 polypeptide, CXCL13 polypeptide, or CX3CL1 polypeptide. These immunomodulatory polypeptides may be a wild-type polypeptide or a variant of a wild-type polypeptide. In some cases, the immunomodulatory polypeptide is an activating (stimulatory) immunomodulatory polypeptide, for example, the immunomodulatory polypeptide can produce an activating/stimulatory effect on a T cell. Examples of activating immunomodulatory polypeptides include, for example, CD80, CD86, 4-1BBL, OX40L, CD70, ICOS-L, CD40, ICAM (CD54), IL2, IL7, IL12, IL15, IL17, IL21, IL27, IL23, GITRL, TGFe, and lymphotoxin beta receptor. In some cases, the immunomodulatory polypeptide is an inhibitory (suppressive) immunomodulatory polypeptide, for example, the immunomodulatory polypeptide can produce a suppressive/inhibitory effect on a T cell. Examples of inhibitory immunomodulatory polypeptides include, for

- 34 049816 example, PD-1H, PD-L1, PD-L2, TGFe, FasL, HVEM, galectin-9, ILT3 and ILT4. Depending on the context, TGFe polypeptides can produce either an activating/stimulatory effect or a suppressive/inhibitory effect.

In some cases, at least one immunomodulatory polypeptide is a reduced affinity variant as described elsewhere herein. In some cases, the first or second polypeptide comprises an FcIg polypeptide.

In some cases, a multiple disulfide-linked TMMP of the present invention (e.g., a di-disulfide-linked TMMP) comprises a class I HLA-B heavy chain polypeptide. In some cases, an HLA-B heavy chain polypeptide present in a multiple disulfide-linked TMMP of the present invention (e.g., a di-disulfide-linked TMMP) comprises an amino acid sequence that has at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence of HLA-B*O7O2, HLA-B*0801, HLA-B*1502, HLA-B*3802, HLA-B*4001, HLA-B*4601, or HLA-B*5301 depicted in Fig. 10A, wherein the HLA-B heavy chain polypeptide comprises the substitutions Y84C and A236C.

HLA-B*0702 (Y84C; A236C).

In some cases, the HLA-B heavy chain polypeptide present in a multiple disulfide-linked TMMP of the present invention (e.g., a two-disulfide-linked TMMP) comprises an amino acid sequence having at least 95%, at least 98%, or at least 99% or 100% amino acid sequence identity to the following amino acid sequence of HLA-B*0702 (Y84C; A236C):

GSHSMRYFYTSVSRPGRGEPRFISVGYVDDTQFVRFDSDAASPREEPRAPWIEQE

GPEYWDRNTQIYKAQAQTDRESLRNLRGCYNQSEAGSHTLQSMYGCDVGPDGRLLRG HDQYAYDGKDYIALNEDLRSWTAADTAAQITQRKWEAAREAEQRRAYLEGECVEWLR RYLENGKDKLERADPPKTHVTHHPISDHEATLRCWALGFYPAEITLTWQRDGEDQTQD TELVETRPCGDRTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLRWE (SEQ ID NO:350), wherein amino acid 84 is Cys and amino acid 236 is Cys.

HLA-B*0801 (Y84C; A236C).

In some cases, the HLA-B heavy chain polypeptide present in a multiple disulfide-linked TMMP of the present invention (e.g., a two-disulfide-linked TMMP) comprises an amino acid sequence having at least 95%, at least 98%, or at least 99% or 100% amino acid sequence identity to the following amino acid sequence of HLA-B*0801 (Y84C; A236C):

GSHSMRYFDTAMSRPGRGEPRFISVGYVDDTQFVRFDSDAASPREEPRAPWIEQE

GPEYWDRNTQIFKTNTQTDRESLRNLRGGCYNQSEAGSHTLQSMYGCDVGPDGRLLRGH NQYAYDGKDYIALNEDLRSWTAADTAAQITQRKWEAARVAEQDRAYLEGTCVEWLR RYLENGKDTLERADPPKTHVTHHPISDHEATLRCWALGFYPAEITLTWQRDGEDQTQD TELVETRPCGDRTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLRWE (SEQ ID

NO:351), where amino acid 84 is Cys and amino acid 236 is Cys.

HLA-B*1502 (Y84C; A236C).

In some cases, the HLA-B heavy chain polypeptide present in a multiple disulfide-linked TMMP of the present invention (e.g., a two-disulfide-linked TMMP) comprises an amino acid sequence having at least 95%, at least 98%, or at least 99% or 100% amino acid sequence identity to the following HLA-B*1502 (Y84C; A236C) amino acid sequence:

GSHSMRYFYTAMSRPGRGEPRFIAVGYVDDTQFVRFDSDAASPRMAPRAPWIEQ

EGPEYWDRNTQISKTNTQTYRESLRNLRGCYNQSEAGSHIIQRMYGCDVGPDGRLLRG YDQSAYDGKDYIALNEDLSSWTAADTAAQITQRKWEAAREAEQLRAYLEGLCVEWLR RYLENGKETLQRADPPKTHVTHHPISDHEATLRCWALGFYPAEITLTWQRDGEDQTQD TELVETRPCGDRTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLRWE (SEQ ID

NO:352), where amino acid 84 is Cys and amino acid 236 is Cys. HLA-B*3802 (Y84C; A236C).

In some cases, the HLA-B heavy chain polypeptide present in the multiple disulfide-linked TMMP of the present invention (e.g., a two-disulfide-linked

- 35 049816 TMMP protein bonds) comprises an amino acid sequence having at least 95%, at least 98%, or at least 99% or 100% amino acid sequence identity with the following amino acid sequence of HLA-B*3802 (Y84C; A236C):

GSHSMRYFYTSVSRPGRGEPRFISVGYVDDTQFVRFDSDAASPREEPRAPWIEQE GPEYWDRNTQICKTNTQTYRENLRTALRCYNQSEAGSHTLQRMYGCDVGPDGRLLRG HNQFAYDGKDYIALNEDLSSWTAADTAAQITQRKWEAARVAEQLRTYLEGTCVEWLR RYLENGKETLQRADPPKTHVTHHPISDHEATLRCWALGFYPAEITLTWQRDGEDQTQD TELVETRPCGDRTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLRWE (SEQ ID NO:353), wherein amino acid 84 is Cys and amino acid 236 is Cys. HLA-B*4001 (Y84C; A2346C).

In some cases, the HLA-B heavy chain polypeptide present in a multiple disulfide-linked TMMP of the present invention (e.g., a two-disulfide-linked TMMP) comprises an amino acid sequence having at least 95%, at least 98%, or at least 99% or 100% amino acid sequence identity to the following HLA-B*4001 (Y84C; A236C) amino acid sequence:

GSHSMRYFHTAMSRPGRGEPRFITVGYVDDTLFVRFDSDATSPRKEPRAPWIEQE GPEYWDRETQISKTNTQTYRESLRNLRGCYNQSEAGSHTLQRMYGCDVGPDGRLLRGH NQYAYDGKDYIALNEDLRSWTAADTAAQISQRKLEAARVAEQLRAYLEGECVEWLRR YLENGKDKLERADPPKTHVTHHPISDHEATLRCWALGFYPAEITLTWQRDGEDQTQDT ELVETRPCGDRTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLRWE (SEQ ID NO:354) wherein amino acid 84 is Cys and amino acid 236 is Cys. HLA-B*4601 (Y84C; A236C).

In some cases, the HLA-B heavy chain polypeptide present in a multiple disulfide-linked TMMP of the present invention (e.g., a two-disulfide-linked TMMP) comprises an amino acid sequence having at least 95%, at least 98%, or at least 99% or 100% amino acid sequence identity to the following amino acid sequence of HLA-B*4601 (Y84C; A236C):

GSHSMRYFYTAMSRPGRGEPRFIAVGYVDDTQFVRFDSDAASPRMAPRAPWIEQ EGPEYWDRETQKYKRQAQTDRVSLRNLRGCYNQSEAGSHTLQRMYGCDVGPDGRLLR GHDQSAYDGKDYIALNEDLSSWTAADTAAQITQRKWEAAREAEQWRAYLEGLCVEW LRRYLENGKETLQRADPPKTHVTHHPISDHEATLRCWALGFYPAEITLTWQRDGEDQTQ DTELVETRPCGDRTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLRWE (SEQ ID NO:355), wherein amino acid 84 is Cys and amino acid 236 is Cys. HLA-B*5301 (Y84C; A236C).

In some cases, the HLA-B heavy chain polypeptide present in a multiple disulfide-linked TMMP of the present invention (e.g., a two-disulfide-linked TMMP) comprises an amino acid sequence having at least 95%, at least 98%, or at least 99% or 100% amino acid sequence identity to the following amino acid sequence of HLA-B*5301 (Y84C; A236C):

GSHSMRYFYTAMSRPGRGEPRFIAVGYVDDTQFVRFDSDAASPRTEPRAPWIEQ EGPEYWDRNTQIFKTNTQTYRENLRIALRCYNQSEAGSHIIQRMYGCDLGPDGRLLRGH DQSAYDGKDYIALNEDLSSWTAADTAAQITQRKWEAARVAEQLRAYLEGLCVEWLRR YLENGKETLQRADPPKTHVTHHPVSDHEATLRCWALGFYPAEITLTWQRDGEDQTQDT ELVETRPCGDRTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLRWE (SEQ ID NO:356), wherein amino acid 84 is Cys and amino acid 236 is Cys. HLA-C.

In some cases, a multiple disulfide bonded TMMP of the present invention (e.g., a di-disulfide bonded TMMP) comprises: a) a first polypeptide comprising: i) 1 WT-1 peptide (e.g., a WT-1 peptide of from 4 amino acids to about 25 amino acids); and ii) a first MHC polypeptide, wherein the first polypeptide comprises a peptide linker between the WT-1 peptide and the first MHC polypeptide, wherein the peptide linker comprises a Cys residue, and wherein the first MHC polypeptide is a B2M polypeptide comprising an amino acid substitution,

- 36 049816 which introduces a Cys residue; b) and a second polypeptide comprising an HLA-C MHC class I heavy chain comprising an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99% or 100% amino acid sequence identity to the following amino acid sequence:

CSHSMRYFDTAVSRPGRGEPRFISVGYVDDTQFVRFDSDAASPRGEPRAPWVEQEGPEY WDRETQNYKRQAQADRVSLRNLRGCYNQSEDGSHTLQRMYGCDLGPDGRLLRGYDQ SAYDGKDYIALNEDLRSWTAADTAAQITQRKLEAARAAEQLRAYLEGTCVEWLRRYLE NGKETLQRAEPPKTHVTHHPLSDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELV ETRPCGDGTFQKWAAVVVPSGQEQRYTCHMQHEGLQEPLTLSWEP (SEQ ID NO:357), wherein amino acid 84 is Cys and amino acid 236 is Cys; and c) at least one immunomodulatory polypeptide, wherein the first and/or second polypeptide comprises at least one immunomodulatory polypeptide. In some cases, the peptide linker comprises the amino acid sequence GCGGS (SEQ ID NO: 318). In some cases, the peptide linker comprises the amino acid sequence GCGGS(GGGGS)n (SEQ ID NO: 342), wherein n is an integer from 1 to 10. In some cases, the β2M polypeptide comprises an R12C substitution. For example, the β2M polypeptide may comprise an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence:

TQRTPKTQVYSCHPAENGKSNFLNCYVSGFHPSDTEVDLLKNGERTEKVEHSDLSFSKDW

SFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDM (SEQ ID NO:314), wherein amino acid 12 is Cys. The at least one immunomodulatory polypeptide can be a polypeptide that has an activating/stimulatory effect on a target T cell or a suppressive/inhibitory effect on a target T cell. For example, the at least one immunomodulatory polypeptide can be a cytokine (e.g., an IL2 polypeptide, an IL7 polypeptide, an IL12 polypeptide, an IL15 polypeptide, an IL17 polypeptide, an IL21 polypeptide, an IL27 polypeptide, an IL-23 polypeptide, a TGFe polypeptide, etc.; and including all family members, e.g., IL17A, IL-17B, IL-17C, IL-17D, Il-Ite, IL-17F, IL-17E), 4-1BBL polypeptide, ICOS-L polypeptide, OX-40L polypeptide, CD80 polypeptide, CD86 polypeptide (CD80 and CD86 are also known as B7-1 and B7-2, respectively), CD40 polypeptide, CD70 polypeptide, JAG1 (CD339) polypeptide, ICAM (CD540) polypeptide, PD-L1 polypeptide, FasL polypeptide, PD-L2 polypeptide, PD-1H polypeptide (VISTA), ICOS-L polypeptide (CD275), GITRL polypeptide, HVEM polypeptide, CXCL10 polypeptide, CXCL9 polypeptide, CXCL11 polypeptide, CXCL13 polypeptide and CX3CL1 polypeptide, galectin-9 polypeptide, CD83 polypeptide, CD30L polypeptide, HLA-G, MICA polypeptide, MICB polypeptide, HVEM polypeptide (CD270), lymphotoxin receptor beta polypeptide, 3/TR6 polypeptide, ILT3 polypeptide, ILT4 polypeptide, CXCL10 polypeptide, CXCL9 polypeptide, CXCL11 polypeptide, CXCL13 polypeptide, or CX3CL1 polypeptide. These immunomodulatory polypeptides may be a wild-type polypeptide or a variant of a wild-type polypeptide. In some cases, the immunomodulatory polypeptide is an activating (stimulating) immunomodulatory polypeptide, for example, the immunomodulatory polypeptide can produce an activating/stimulating effect on a T cell. Examples of activating immunomodulatory polypeptides include, for example, CD80, CD86, 4-1BBL, OX40L, CD70, ICOS-L, CD40, ICAM (CD54), IL2, IL7, IL12, IL15, IL17, IL21, IL27, IL23, GITRL, TGFe, and lymphotoxin beta receptor. In some cases, the immunomodulatory polypeptide is an inhibitory (suppressive) immunomodulatory polypeptide, for example, the immunomodulatory polypeptide can produce a suppressive/inhibitory effect on a T cell. Examples of inhibitory immunomodulatory polypeptides include, for example, PD-1H, PD-L1, PD-L2, TGFe, FasL, HVEM, galectin-9, ILT3, and ILT4. Depending on the context, TGFe polypeptides can produce either an activating/stimulatory effect or a suppressive/inhibitory effect.

In some cases, at least one immunomodulatory polypeptide is a reduced affinity variant as described elsewhere herein. In some cases, the first or second polypeptide comprises an FcIg polypeptide.

In some cases, a multiple disulfide-linked TMMP of the present invention (e.g., a di-disulfide-linked TMMP) comprises an HLA-C class I heavy chain polypeptide. In some cases, an HLA-C heavy chain polypeptide present in a multiple disulfide-linked TMMP of the present invention (e.g., a di-disulfide-linked TMMP) comprises an amino acid sequence that has at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence of HLA-C*O1O2, HLA-C*0303, HLA-C*0304, HLA-C*0401, HLA-C*0602, HLA-C*0701, HLA-C*0702, HLA-C*0801, or HLA-C*1502 depicted in

- 37 049816 Fig. 11A, wherein the HLA-C heavy chain polypeptide contains the Y84C and A236C substitutions.

HLA-C*01:02 (Y84C; A236C).

In some cases, the HLA-C heavy chain polypeptide present in a multiple disulfide-linked TMMP of the present invention (e.g., a two-disulfide-linked TMMP) comprises an amino acid sequence having at least 95%, at least 98%, or at least 99% or 100% amino acid sequence identity to the following HLA-C*01:02 (Y84C; A236C) amino acid sequence:

CSHSMKYFFTSVSRPGRGEPRFISVGYVDDTQFVRFDSDAASPRGEPRAPWVEQE

GPEYWDRETQKYKRQAQTDRVSLRNLRGCYNQSEAGSHTLQWMCGCDLGPDGRLLR

GYDQYAYDGKDYIALNEDLRSWTAADTAAQITQRKWEAAREAEQRRAYLEGTCVEW

LRRYLENGKETLQRAEHPKTHVTHHPVSDHEATLRCWALGFYPAEITLTWQWDGEDQT

QDTELVETRPCGDGTFQKWAAVMVPSGEEQRYTCHVQHEGLPEPLTLRWEP (SEQ ID

NO:358), where amino acid 84 is Cys and amino acid 236 is Cys.

HLA-^0303 (Y84C; A236C).

In some cases, the HLA-C heavy chain polypeptide present in a multiple disulfide-linked TMMP of the present invention (e.g., a two-disulfide-linked TMMP) comprises an amino acid sequence having at least 95%, at least 98%, or at least 99% or 100% amino acid sequence identity to the following HLA-C*03:03 (Y84C; A236C) amino acid sequence:

GSHSMRYFYTAVSRPGRGEPHFIAVGYVDDTQFVRFDSDAASPRGEPRAPWVEQ

EGPEYWDRETQKYKRQAQTDRVSLRNLRGCYNQSEARSHIIQRMYGCDVGPDGRLLR

GYDQYAYDGKDYIALNEDLRSWTAADTAAQITQRKWEAAREAEQLRAYLEGLCVEWL

RRYLKNGKETLQRAEHPKTHVTHHPVSDHEATLRCWALGFYPAEITLTWQWDGEDQT

QDTELVETRPCGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLTLRWEP (SEQ ID

NO:359), where amino acid 84 is Cys and amino acid 236 is Cys.

HLA-C*0304 (Y84C; A236C).

In some cases, the HLA-C heavy chain polypeptide present in a multiple disulfide-linked TMMP of the present invention (e.g., a two-disulfide-linked TMMP) comprises an amino acid sequence having at least 95%, at least 98%, or at least 99% or 100% amino acid sequence identity to the following HLA-C*03:04 (Y84C; A236C) amino acid sequence:

GSHSMRYFYTAVSRPGRGEPHFIAVGYVDDTQFVRFDSDAASPRGEPRAPWVEQ

EGPEYWDRETQKYKRQAQTDRVSLRNLRGCYNQSEAGSHIIQRMYGCDVGPDGRLLR

GYDQYAYDGKDYIALNEDLRSWTAADTAAQITQRKWEAAREAEQLRAYLEGLCVEWL

RRYLKNGKETLQRAEHPKTHVTHHPVSDHEATLRCWALGFYPAEITLTWQWDGEDQT

QDTELVETRPCGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLTLRWEP (SEQ ID

NO :3 60), where amino acid 84 is Cys and amino acid 236 is Cys.

HLA-C*0401 (Y84C; A236C).

In some cases, the HLA-C heavy chain polypeptide present in a multiple disulfide-linked TMMP of the present invention (e.g., a two-disulfide-linked TMMP) comprises an amino acid sequence having at least 95%, at least 98%, or at least 99% or 100% amino acid sequence identity to the following HLA-C*04:01 (Y84C; A236C) amino acid sequence:

GSHSMRYFSTSVSWPGRGEPRFIAVGYVDDTQFVRFDSDAASPRGEPREPWVEQ

EGPEYWDRETQKYKRQAQADRVNLRKLRGCYNQSEDGSHTLQRMFGCDLGPDGRLLR

GYNQFAYDGKDYIALNEDLRSWTAADTAAQITQRKWEAAREAEQRRAYLEGTCVEWL

RRYLENGKETLQRAEHPKTHVTHHPVSDHEATLRCWALGFYPAEITLTWQWDGEDQT

QDTELVETRPCGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLTLRWKP (SEQ ID

NO:361), where amino acid 84 is Cys and amino acid 236 is Cys.

HLA-C*0602 (Y84C; A236C).

In some cases, the HLA-C heavy chain polypeptide present in the multiple disulfide-linked TMMP of the present invention (e.g., a two-disulfide-linked

- 38 049816 TMMP protein bonds) comprises an amino acid sequence having at least 95%, at least 98%, or at least 99% or 100% amino acid sequence identity with the following amino acid sequence of HLA-C*06:02 (Y84C; A236C):

CSHSMRYFDTAVSRPGRGEPRFISVGYVDDTQFVRFDSDAASPRGEPRAPWVEQ EGPEYWDRETQKYKRQAQADRVNLRKLRGCYNQSEDGSHTLQWMYGCDLGPDGRLL RGYDQSAYDGKDYIALNEDLRSWTAADTAAQITQRKWEAAREAEQWRAYLEGTCVE WLRRYLENGKETLQRAEHPKTHVTHHPVSDHEATLRCWALGFYPAEITLTWQRDGED QTQDTELVETRPCGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLTLRWEP (SEQ ID NO:362), wherein amino acid 84 is Cys and amino acid 236 is Cys. HLA-C*0701 (Y84C; A236C).

In some cases, the HLA-C heavy chain polypeptide present in a multiple disulfide-linked TMMP of the present invention (e.g., a two-disulfide-linked TMMP) comprises an amino acid sequence having at least 95%, at least 98%, or at least 99% or 100% amino acid sequence identity to the following HLA-C*07:01 (Y84C; A236C) amino acid sequence:

CSHSMRYFDTAVSRPGRGEPRFISVGYVDDTQFVRFDSDAASPRGEPRAPWVEQ EGPEYWDRETQNYKRQAQADRVSLRNLRGCYNQSEDGSHTLQRMYGCDLGPDGRLLR GYDQSAYDGKDYIALNEDLRSWTAADTAAQITQRKLEAARAAEQLRAYLEGTCVEWL RRYLENGKETLQRAEPPKTHVTHHPLSDHEATLRCWALGFYPAEITLTWQRDGEDQTQ DTELVETRPCGDGTFQKWAAVVVPSGQEQRYTCHMQHEGLQEPLTLSWEP (SEQ ID NO:357), wherein amino acid 84 is Cys and amino acid 236 is Cys. HLA-C*0702 (Y84C; A236C).

In some cases, the HLA-C heavy chain polypeptide present in a multiple disulfide-linked TMMP of the present invention (e.g., a two-disulfide-linked TMMP) comprises an amino acid sequence having at least 95%, at least 98%, or at least 99% or 100% amino acid sequence identity to the following amino acid sequence of HLA-C*07:02 (Y84C; A236C):

CSHSMRYFDTAVSRPGRGEPRFISVGYVDDTQFVRFDSDAASPRGEPRAPWVEQ EGPEYWDRETQKYKRQAQADRVSLRNLRGCYNQSEDGSHTLQRMSGCDLGPDGRLLR GYDQSAYDGKDYIALNEDLRSWTAADTAAQITQRKLEAARAAEQLRAYLEGTCVEWL RRYLENGKETLQRAEPPKTHVTHHPLSDHEATLRCWALGFYPAEITLTWQRDGEDQTQ DTELVETRPCGDGTFQKWAAVVVPSGQEQRYTCHMQHEGLQEPLTLSWEP (SEQ ID NO:404), wherein amino acid 84 is Cys and amino acid 236 is Cys. HLA-C*0801 (Y84C; A236C).

In some cases, the HLA-C heavy chain polypeptide present in a multiple disulfide-linked TMMP of the present invention (e.g., a two-disulfide-linked TMMP) comprises an amino acid sequence having at least 95%, at least 98%, or at least 99% or 100% amino acid sequence identity to the following HLA-C*08:01 (Y84C; A236C) amino acid sequence:

CSHSMRYFYTAVSRPGRGEPRFIAVGYVDDTQFVQFDSDAASPRGEPRAPWVEQ EGPEYWDRETQKYKRQAQTDRVSLRNLRGCYNQSEAGSHTLQRMYGCDLGPDGRLLR GYNQFAYDGKDYIALNEDLRSWTAADTAAQITQRKWEAARTAEQLRAYLEGTCVEWL RRYLENGKKTLQRAEHPKTHVTHHPVSDHEATLRCWALGFYPAEITLTWQRDGEDQTQ DTELVETRPCGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLTLRWGP (SEQ ID NO:363), wherein amino acid 84 is Cys and amino acid 236 is Cys. HLA-C*1502 (Y84C; A236C).

In some cases, the HLA-C heavy chain polypeptide present in a multiple disulfide-linked TMMP of the present invention (e.g., a two-disulfide-linked TMMP) comprises an amino acid sequence having at least 95%, at least 98%, or at least 99% or 100% amino acid sequence identity to the following HLA-C*15:02 (Y84C; A236C) amino acid sequence:

- 39 049816

CSHSMRYFYTAVSRPGRGEPHFIAVGYVDDTQFVRFDSDAASPRGEPRAPWVEQ EGPEYWDRETQNYKRQAQTDRVNLRKLRGCYNQSEAGSHIIQRMYGCDLGPDGRLLR GHDQLAYDGKDYIALNEDLRSWTAADTAAQITQRKWEAAREAEQLRAYLEGTCVEWL RRYLENGKETLQRAEHPKTHVTHHPVSDHEATLRCWALGFYPAEITLTWQRDGEDQTQ DTELVETRPCGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLTLRWEP (SEQ ID NO:364), wherein amino acid 84 is Cys and amino acid 236 is Cys.

Framework polypeptides.

The TMMP may comprise an Fc polypeptide or may comprise another suitable scaffold polypeptide.

Suitable scaffold polypeptides include antibody-based scaffold polypeptides and non-antibody-based scaffold polypeptides. 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, a pentapeptide repeat unit containing polypeptide (Val-Pro-Gly-X-Gly; SEQ ID NO: 59), wherein X is any amino acid other than proline), albumin-binding polypeptide, silk-like polypeptide (see, for example, Valluzzi et al. (2002) Philos Trans R Soc bond B Biol Sci. 357:165), silk-elastin-like polypeptide (SELP; see, for example, Megeed et al. (2002) Adv Drug Deliv Rev. 54:1075), and the like. Suitable XTEN polypeptides include, for example, those disclosed in WO 2009/023270, WO 2010/091122, WO 2007/103515, US 2010/0189682, and US 2009/0092582; see also Schellenberger et al. (2009) Nat Biotechnol. 27:1186). Suitable albumin polypeptides include, for example, human serum albumin.

Suitable scaffold polypeptides will in some cases be half-life enhancing polypeptides. Thus, in some cases, a suitable scaffold polypeptide will increase the in vivo half-life (e.g., serum half-life) of TMMP compared to a control TMMP lacking the scaffold polypeptide. For example, in some cases, the scaffold polypeptide increases the in vivo half-life (e.g., serum half-life) of the TMMP, compared to a control TMMP that lacks the scaffold polypeptide, by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 50%, at least about 2-fold, at least about 2.5-fold, at least about 5-fold, at least about 10-fold, at least about 25-fold, at least about 50-fold, at least about 100-fold, or more than 100-fold. As an example, in some cases, the Fc polypeptide increases the in vivo half-life (e.g., serum half-life) of the TMMP, compared to a control TMMP that lacks the Fc polypeptide, by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 50%, at least about 2-fold, at least about 2.5-fold, at least about 5-fold, at least about 10-fold, at least about 25-fold, at least about 50-fold, at least about 100-fold, or more than 100-fold.

Fc polypeptides.

In some cases, the first and/or second polypeptide chain of the TMMP of the present invention comprises an Fc polypeptide. The TMMP Fc polypeptide can be a human IgG1 Fc, a human IgG2 Fc, a human IgG3 Fc, a human IgG4 Fc, etc. In some cases, the Fc polypeptide comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to the amino acid sequence of the Fc region depicted in Figs. 5A-5G. In some cases, the Fc region comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to the human IgG1 Fc polypeptide shown in Fig. 5A. In some cases, the Fc region comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to the human IgG1 Fc polypeptide shown in Fig. 5A; and comprises an N77 substitution; for example, the Fc polypeptide comprises a N77A substitution. In some cases, the Fc polypeptide comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% identity

- 40 049816 amino acid sequence with the human IgG2 Fc polypeptide shown in Fig. 5A; for example, the Fc polypeptide comprises an amino acid sequence that has at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to amino acids 99-325 of the human IgG2 Fc polypeptide shown in Fig. 5A. In some cases, the Fc polypeptide comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to the human IgG3 Fc polypeptide shown in Fig. 5A; for example, the Fc polypeptide comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to amino acids 19-246 of the human IgG3 Fc polypeptide shown in Fig. 5A. In some cases, the Fc polypeptide comprises an amino acid sequence that has at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to the human IgM Fc polypeptide shown in Fig. 5B; for example, the Fc polypeptide comprises an amino acid sequence that has at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to amino acids 1-276 of the human IgM Fc polypeptide shown in Fig. 5B. In some cases, the Fc polypeptide comprises an amino acid sequence that has at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to the human IgA Fc polypeptide shown in Fig. 5C; for example, the Fc polypeptide comprises an amino acid sequence that has at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to amino acids 1-234 of the human IgA Fc polypeptide shown in Fig. 5C.

In some cases, the Fc polypeptide comprises an amino acid sequence that has at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to the human IgG4 Fc polypeptide shown in Fig. 5C. In some cases, the Fc polypeptide comprises an amino acid sequence that has at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to amino acids 100 to 327 of the human IgG4 Fc polypeptide shown in Fig. 5C.

In some cases, the IgG4 Fc polypeptide contains the following amino acid sequence:

PPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVE

VHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKG QPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:365).

In some cases, the Fc polypeptide present in a TMMP comprises the amino acid sequence depicted in Fig. 5A (human IgG1 Fc). In some cases, the Fc polypeptide present in a TMMP comprises the amino acid sequence depicted in Fig. 5A (human IgG1 Fc) except for a substitution of N297 (N77 of the amino acid sequence depicted in Fig. 5A) with an amino acid other than asparagine. In some cases, the Fc polypeptide present in a TMMP comprises the amino acid sequence depicted in Fig. 5C (human IgG1 Fc containing the N297A substitution), which is N77 of the amino acid sequence depicted in Fig. 5A). In some cases, the Fc polypeptide present in a TMMP comprises the amino acid sequence depicted in Fig. 5A (human IgG1 Fc), except for the substitution of L234 (L14 of the amino acid sequence depicted in Fig. 5A) with an amino acid other than leucine. In some cases, the Fc polypeptide present in the TMMP comprises the amino acid sequence depicted in Fig. 5A (human IgG1 Fc), except for the substitution of L234 (L14 of the amino acid sequence depicted in Fig. 5A) with an amino acid other than leucine.

- 41 049816

L235 (L15 of the amino acid sequence shown in Fig. 5A) is an amino acid other than leucine.

In some cases, the Fc polypeptide present in a TMMP comprises the amino acid sequence depicted in Fig. 5E. In some cases, the Fc polypeptide present in a TMMP comprises the amino acid sequence depicted in Fig. 5F. In some cases, the Fc polypeptide present in a TMMP comprises the amino acid sequence depicted in Fig. 5G (human IgG1 Fc comprising a L234A substitution and a L235A substitution corresponding to positions 14 and 15 of the amino acid sequence depicted in Fig. 5G). In some cases, the Fc polypeptide present in a TMMP comprises the amino acid sequence depicted in Fig. 5A (human IgG1 Fc) except for the substitution of P331 (P111 of the amino acid sequence depicted in Fig. 5A) with an amino acid other than proline; in some cases, the substitution is a P331S substitution. In some cases, the Fc polypeptide present in the TMMP comprises the amino acid sequence depicted in Fig. 5A (human IgG1 Fc), except for substitutions at L234 and L235 amino acids (L14 and L15 of the amino acid sequence depicted in Fig. 5A) with amino acids other than leucine. In some cases, the Fc polypeptide present in the TMMP comprises the amino acid sequence depicted in Fig. 5A (human IgG1 Fc), except for substitutions at L234 and L235 amino acids (L14 and L15 of the amino acid sequence depicted in Fig. 5A) with amino acids other than leucine and a substitution of P331 (P111 of the amino acid sequence depicted in Fig. 5A) with an amino acid other than proline. In some cases, the Fc polypeptide present in the TMMP comprises the amino acid sequence depicted in Fig. 5E (human IgG1 Fc containing L234F, L235E, and P331S substitutions (corresponding to amino acid positions 14, 15, and 111 of the amino acid sequence depicted in Fig. 5E). In some cases, the Fc polypeptide present in the TMMP is an IgG1 Fc polypeptide that contains L234A and L235A substitutions (L14 and L15 substitutions of the amino acid sequence depicted in Fig. 5A with Ala), as depicted in Fig. 5G.

Linkers.

The TMMP of the present invention may comprise one or more linkers, wherein the one or more linkers are between one or more of: i) an MHC class I polypeptide and an Ig Fc polypeptide, wherein such linker is referred to herein as L1; ii) an immunomodulatory polypeptide and an MHC class I polypeptide, wherein such linker is designated herein as L2; iii) a first immunomodulatory polypeptide and a second immunomodulatory polypeptide, wherein such linker is designated herein as L3; iv) a peptide antigen (epitope) and an MHC class I polypeptide; v) an MHC class I polypeptide and a dimerization polypeptide (e.g., a first or second member of a dimerization pair); and vi) a dimerization polypeptide (e.g., a first or second member of a dimerization pair) and an IgFc polypeptide.

Suitable linkers (also called spacers) can be easily selected and can be any of a number of suitable lengths, such as from 1 amino acid to 25 amino acids, from 3 amino acids to 20 amino acids, from 2 amino acids to 15 amino acids, from 3 to 12 amino acids, including from 4 to 10 amino acids, from 5 to 9 amino acids, from 6 to 8 amino acids, or from 7 to 8 amino acids. A suitable linker 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 is from 25 amino acids to 50 amino acids in length, such as 25 to 30, 30 to 35, 35 to 40, 40 to 45, or 45 to 50 amino acids.

Exemplary linkers include glycine polymers (G)n, glycine-serine polymers (including, for example, (GS)n, (GSGGS)n (SEQ ID NO: 366), and (GGGS)n (SEQ ID NO: 367), where n is an integer of at least one), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Polymers of glycine and glycine serine can be used; both Gly and Ser are relatively unstructured and thus can serve as a neutral linkage between the components. Polymers of glycine can be used; glycine has access to a significantly larger phi-psi space than even alanine and is much less restricted than residues with longer side chains (see Scheraga, Rev. Computational Chem. 11173-142 (1992)). Typical linkers may comprise amino acid sequences including, but not limited to, GGSG (SEQ ID NO: 368), GGSGG (SEQ ID NO: 369), GSGSG (SEQ ID NO: 370), GSGGG (SEQ ID NO: 371), GGGSG (SEQ ID NO: 372), GSSSG (SEQ ID NO: 373), and the like. Exemplary linkers may include, for example, Gly(Ser4) _n (SEQ ID NO: 374), wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some cases, the linker comprises the amino acid sequence (GSSSS) n (SEQ ID NO: 375), wherein n is 4. In some cases, the linker comprises the amino acid sequence (GSSSS) _n (SEQ ID NO: 376), wherein n is 5. In some cases, the linker comprises the amino acid sequence (GGGGS) _n (SEQ ID NO: 377), wherein n is 1. In some cases, the linker comprises the amino acid sequence (GGGGS) _n (SEQ ID NO: 378), wherein n is 2. In some cases, the linker comprises the amino acid sequence (GGGGS) _n (SEQ ID NO: 379), wherein n is 3. In some In some cases, the linker comprises the amino acid sequence (GGGGS) _n (SEQ ID NO: 380), where n is 4. In some cases, the linker comprises the amino acid sequence (GGGGS) _n

- 42 049816 (SEQ ID NO: 381), wherein n is 5. In some cases, the linker comprises the amino acid sequence (GGGGS) _n (SEQ ID NO: 382), wherein n is 6. In some cases, the linker comprises the amino acid sequence (GGGGS) _n (SEQ ID NO: 383), wherein n is 7. In some cases, the linker comprises the amino acid sequence (GGGGS) _n (SEQ ID NO: 384), wherein n is 8. In some cases, the linker comprises the amino acid sequence (GGGGS) _n (SEQ ID NO: 385), wherein n is 9. In some cases, the linker comprises the amino acid sequence (GGGGS) n (SEQ ID NO: 386), wherein n is 10. In some cases, the linker comprises the amino acid sequence AAAGG (SEQ ID NO: 387).

In some cases, a linker polypeptide present in a first TMMP polypeptide of the invention comprises a cysteine residue that can form a disulfide bond with a cysteine residue present in a second TMMP polypeptide of the invention. In some cases, for example, a suitable linker comprises the amino acid sequence GCGGSGGGGSGGGGS (SEQ ID NO:317).

As another example, a suitable linker may comprise the amino acid sequence GCGGS(G4S) _n (SEQ ID NO: 315), where n is 1, 2, 3, 4, 5, 6, 7, 8, or 9. For example, in some cases, the linker comprises the amino acid sequence GCGGSGGGGSGGGGSGGGGS (SEQ ID NO: 316).

As another example, the linker comprises the amino acid sequence GCGGSGGGGSGGGGS (SEQ IDNO:317).

Epitopes.

In some cases, the epitope (peptide presenting one or more epitopes) present in the TMMP of the present invention is a WT-1 peptide, for example, a WT-1 peptide that, together with MHC, presents an epitope for the TCR. The amino acid sequences of the WT-1 isoforms are shown in Fig. 3A-3E. A WT-1 peptide presenting one or more epitopes is referred to herein as a WT-1 peptide or a WT-1 epitope. In some cases, the WT-1 epitope present in the TMMP of the invention may be a peptide of 4 to 25 consecutive amino acids (e.g., 4 amino acids (aa), 5 aa, 6 aa, 7 aa, 8 aa, 9 aa, 10-15 aa, 15-20 aa, or 20-25 aa) of 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 WT-1 amino acid sequence depicted in any of Figs. 3A-3E. In some cases, the WT-1 epitope present in the TMMP of the invention may be a peptide of 4 to 25 consecutive amino acids (e.g., 4 amino acids (aa), 5 aa, 6 aa, 7 aa, 8 aa, 9 aa, 10-15 aa, 15-20 aa, or 20-25 aa) of 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 WT-1 amino acid sequence depicted in Fig. 3A. In some cases, the WT-1 epitope present in the TMMP of the invention may be a peptide of 4 to 25 consecutive amino acids (e.g., 4 amino acids (aa), 5 aa, 6 aa, 7 aa, 8 aa, 9 aa, 10-15 aa, 15-20 aa, or 20-25 aa) of 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 WT-1 amino acid sequence depicted in Fig. 3B. In some cases, the WT-1 epitope present in the TMMP of the invention may be a peptide of 4 to 25 consecutive amino acids (e.g., 4 amino acids (aa), 5 aa, 6 aa, 7 aa, 8 aa, 9 aa, 10-15 aa, 15-20 aa, or 20-25 aa) of 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 WT-1 amino acid sequence depicted in Fig. 3C. In some cases, the WT-1 epitope present in the TMMP of the invention may be a peptide of 4 to 25 consecutive amino acids (e.g., 4 amino acids (aa), 5 aa, 6 aa, 7 aa, 8 aa, 9 aa, 10-15 aa, 15-20 aa, or 20-25 aa) of 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 WT-1 amino acid sequence depicted in Fig. 3D. In some cases, the WT-1 epitope present in the TMMP of the invention may be a peptide of 4 to 25 consecutive amino acids (e.g., 4 amino acids (aa), 5 aa, 6 aa, 7 aa, 8 aa, 9 aa, 10-15 aa, 15-20 aa, or 20-25 aa) of 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 WT-1 amino acid sequence depicted in Fig. 3E. In some cases, the WT-1 epitope present in the TMMP of the invention is 6 amino acids in length. In some cases, the WT-1 epitope present in the TMMP of the invention is 7 amino acids in length. In some cases, the WT-1 epitope present in the TMMP of the invention is 8 amino acids in length. In some cases, the WT-1 epitope present in the TMMP of the invention is 9 amino acids in length. In some cases, the WT-1 epitope present in the TMMP of the invention is 10 amino acids in length. In some cases,

- 43 049816, the WT-1 epitope present in the TMMP of the invention is 11 amino acids in length. In some cases, the WT-1 epitope present in the TMMP of the invention is from 6 amino acids to 25 amino acids in length. In some cases, the WT-1 epitope present in the TMMP of the invention is from 6 amino acids to 20 amino acids in length. In some cases, the WT-1 epitope present in the TMMP of the invention is from 7 amino acids to 25 amino acids in length. In some cases, the WT-1 epitope present in the TMMP of the invention is from 7 amino acids to 20 amino acids in length. In some cases, the WT-1 epitope present in the TMMP of the invention is at least 4 amino acids in length, at least 6 amino acids in length, or at least 7 amino acids in length.

An epitope present in a TMMP of the present invention may be from about 4 amino acids to about 25 amino acids in length, for example, the epitope may be from 4 amino acids (aa) to 10 aa, from 10 aa to 15 aa, from 15 aa to 20 aa, or from 20 aa to 25 aa in length. For example, an epitope present in a TMMP of the present invention may be 4 amino acids (aa), 5 aa, 6 aa, 7 aa, 8 aa, 9 aa, 10 aa, 11 aa, 12 aa, 13 aa, 14 aa, 15 aa, 16 aa, 17 aa, 18 aa, 19 aa, 20 aa, 21 aa, 22 aa, 23 aa, 24 aa, or 25 aa in length. In some cases, the epitope present in the TMMP is 5 to 10 amino acids in length, such as 5 aa, 6 aa, 7 aa, 8 aa, 9 aa, or 10 aa.

The WT-1 epitope present in the TMMP of the present invention is a peptide specifically bound to a T cell, i.e., an epitope specifically bound to a WT-1 epitope-specific T cell. The epitope-specific T cell binds an epitope having a control amino acid sequence, but does not substantially bind an epitope that differs from the control amino acid sequence. For example, an epitope-specific T cell binds an epitope having a reference amino acid sequence and binds an epitope that differs from the reference amino acid sequence if, as a whole, it has an affinity that is less than 10 ^-6 M, less than 10 ^-5 M, or less than 10 ^-4 M. An epitope-specific T cell can bind the epitope for which it is specific with an affinity of at least 10 ^-7 M, at least 10 ^-8 M, at least 10 ^-9 M, or at least 10 ^-10 M.

Examples of WT-1 peptides suitable for inclusion in the TMMP of the present invention include, but are not limited to,

CMTWNQMNLGATLKG (SEQ ID NO:223), WNQMNLGATLKGVAA (SEQ ID NO:224), CMTWNYMNLGATLKG (SEQ ID NO:225), WNYMNLGATLKGVAA (SEQ ID NO:226), MTWNQMNLGATLKGV (SEQ ID NO:227), TWNQMNLGATLKGVA (SEQ ID NO:228), CMTWNLLMNLGATLKG (SEQ ID NO:229), MTWNLLMNLGATLKGV (SEQ ID NO:230), TWNLLMNLGATLKGVA (SEQ ID NO:231), WNLLMNLGATLKGVAA (SEQ ID NO:232), MNLGATLK (SEQ ID

NO:233), MTWNYMNLGATLKGV (SEQ ID NO:234), TWNYMNLGATLKGVA (SEQ ID

NO:235), CMTWNQMNLGATLKGVA (SEQ ID NO:236), CMTWNLMNLGATLKGVA (SEQ ID NO:237), CMTWNYMNLGATLKGVA (SEQ ID NO:238), GYLRNPTAC (SEQ ID NO:239), GALRNPTAL (SEQ ID NO:240), YALRNPTAC (SEQ ID NO:241), GLLRNPTAC (SEQ ID NO:242), RYRPHPGAL (SEQ ID NO:243), YQRPHPGAL (SEQ ID NO:244), RLRPHPGAL (SEQ ID NO:245), RIRPHPGAL (SEQ ID NO:246), QFPNHSFKHEDPMGQ (SEQ ID NO:247), HSFKHEDPY (SEQ ID NO:248), QFPNHSFKHEDPM (SEQ ID NO:249), QFPNHSFKHEDPY (SEQ ID NO:250), KRPFMCAYPGCNK (SEQ ID NO:251), KRPFMCAYPGCYK (SEQ ID NO:252), FMCAYPGCY (SEQ ID NO:253), FMCAYPGCK (SEQ ID NO:254), KRPFMCAYPGCNKRY (SEQ ID NO:255), SEKRPFMCAYPGCNK (SEQ ID NO:256), KRPFMCAYPGCYKRY (SEQ ID NO:257), NLMNLGATL (SEQ ID NO:258), VLDFAPPGA (SEQ ID NO:259); RMFPNAPYL (SEQ ID NO:260); CMTWNQMN (SEQ ID NO:261); CYTWNQMNL (SEQ ID NO:269); NYMNLGATL (SEQ ID NO:263);

YMFPNAPYL (SEQ ID NO:264); SLGEQQYSV (SEQ ID NO:265); CMTWNQMNL (SEQ ID NO:266) and NQMNLGATL (SEQ ID NO:267).

In some cases, the WT-1 peptide present in the TMMP of the invention is CMTWNQMN (SEQ ID NO: 261). In some cases, the WT-1 peptide present in the TMMP of the invention is CYTWNQMNL (SEQ ID NO: 269).

In some cases, the WT-1 peptide present in the TMMP of the present invention presents an HLA-A*2402-restricted epitope. WT-1 peptides that present an HLA-A*2402-restricted epitope include, for example,

- 44 049816

CMTWNQMN (SEQ ID NO:261); NYMNLGATL (SEQ ID NO:263) (WT-1 239-247; Q240Y);

CYTWNQMNL (SEQ ID NO:269) (WT-1 235-243); CMTWNQMNL (SEQ ID NO:266) (WT-1 235-243); NQMNLGATL (SEQ ID NO:267) (WT-1 239-247) and NLMNLGATL (SEQ ID NO:258) (WT-1 239-247; Q240L).

In some cases, the WT-1 peptide present in the TMMP of the present invention presents an HLA-A*0201-restricted epitope. WT-1 peptides that present an HLA-A*0201-restricted epitope include, for example,

VLDFAPPGA (SEQ ID NO:259) (WT-1 37-45); RMFPNAPYL (SEQ ID NO:260) (WT-1 126134); YMFPNAPYL (SEQ ID NO:264) (WT-1 126-134; R126Y); SLGEQQYSV (SEQ ID

NO:265) (WT-1 187-195) and NLMNLGATL (SEQ ID NO:258) (WT-1 239-247; Q240L).

HLA/peptide binding assays.

The question of whether a given peptide (e.g., WT-1 peptide) binds to HLA class I (containing the HLA heavy chain and the B2M polypeptide) and, when bound to the HLA complex, can effectively present an epitope to the TCR can be resolved by any of a number of well-established assays. Assays include binding assays and T-cell activation assays.

Cell-based binding assay.

As one example, a cell-based peptide-induced stabilization assay can be used to determine peptide-HLA class I binding. In this assay, the peptide of interest is allowed to bind to a TAP-deficient cell, i.e., a cell that contains a defective transporter associated with the antigen processing machinery (TAP) and, accordingly, rare class I surface molecules. Such cells include, for example, the human T2 cell line (T2(174xCEM.T2; American Type Culture Collection (ATCC) No. CRL-1992). Henderson et al. (1992) Science 255:1264. Without efficient TAP-mediated transport of cytosolic peptides into the endoplasmic reticulum, assembled class I complexes are structurally unstable and are only transiently retained on the cell surface. However, when T2 cells are incubated with an exogenous peptide capable of binding class I antigens, the surface peptide-HLA class I complexes are stabilized and can be detected by flow cytometry, for example, with a sorted (pan) monoclonal antibody to class I antigen. The stabilization and resulting increase in the lifetime of the peptide-HLA complexes on the cell surface due to the addition of the peptide confirms their identity. Analysis can be performed using flow cytometry, for example, in which the antibody pan-HLA class I contains a fluorescent label. Binding of the peptide to different allelic forms of the HLA H chains can be tested by genetically modifying T2 cells to express the HLA H allelic chain of interest.

The following is a non-limiting example of the use of the T2 assay to assess peptide binding to HLA A*0201. T2 cells are washed in cell culture medium and concentrated to ¹⁰⁶ cells/mL. Peptides of interest are prepared in cell culture medium and serially diluted to provide concentrations of 200 μM, 100 μM, 20 μM, and 2 μM. Cells are mixed 1:1 with each peptide dilution to provide a final volume of 200 μM and final peptide concentrations of 100 μM, 50 μM, 10 μM, and 1 μM. The HLA A*0201 binding peptide, GILGFVFTL (SEQ ID NO: 395), and the non-HLA A*0201-restricted peptide, HPVGEADYF (SEQ ID NO: 396) (HLA-B*35O1), were included in the assay as positive and negative controls, respectively. The cell/peptide mixtures were incubated at 37°C with 5% CO2 for ten minutes; then incubated at room temperature overnight. The cells were then incubated for 2 h at 37°C and stained with fluorescently labeled anti-human HLA antibody. The cells were washed twice with phosphate-buffered saline and analyzed by flow cytometry. The mean fluorescence intensity (MFI) of the anti-HLA antibody staining was used to measure the strength of binding.

Biochemical binding assay.

HLA polypeptides (HLA heavy chain polypeptide complexed with b2M polypeptide) can be tested for binding to a peptide of interest in a cell-free in vitro assay system. For example, a labeled reference peptide (e.g., fluorescently labeled) is allowed to bind to HLA polypeptides (HLA heavy chain polypeptide complexed with b2M polypeptide) to form an HLA-reference peptide complex. The ability of the peptide of interest to displace the labeled reference peptide from the HLA-reference peptide complex is tested. The relative binding affinity is calculated as the amount of test peptide required to displace the bound reference peptide. See, e.g., van der Burg et al. (1995) Human Immunol. 44:189.

As another example, a peptide of interest can be incubated with an HLA molecule (HLA heavy chain complexed with the B2M polypeptide) and the stabilization of the HLA/peptide complex can be measured in an enzyme immunoassay format. The ability of the peptide of interest to stabilize

- 45 049816 HLA molecule is compared with the ability of a control peptide to present a known T-cell epitope. Detection of stabilization is based on the presence or absence of the native conformation of the HLA/peptide complex, detected using an anti-HLA antibody. See, e.g., Westrop et al. (2009) J. Immunol. Methods 341:76; Steinitz et al. (2012) Blood 119:4073; and U.S. Patent No. 9,205,144.

T cell activation assays.

The question of whether a given HLA class I peptide (containing the HLA heavy chain and the B2M polypeptide) binds and, when bound to the HLA complex, can effectively present an epitope to the TCR can be resolved by assessing the T cell response to the peptide-HLA complex. T cell responses that can be measured include, for example, interferon gamma (IFNy) production, cytotoxic activity, etc.

ELISPOT analysis.

Suitable assays include, for example, enzyme-linked immunosorbent assay (ELISPOT). In this assay, IFNy production by CD8+ T cells is measured by adding an antigen-presenting cell (APC) that presents a peptide of interest complexed with HLA class I. Anti-IFNy antibody is immobilized on the wells of a multiwell plate. The APC is added to the wells and incubated for a period of time with the peptide of interest such that the peptide binds HLA class I on the surface of the APC. CD8' T cells specific for the peptide are added to the wells and the plate is incubated for about 24 h. The wells are then washed and any IFNy bound to the immobilized anti-IFNy antibody is detected using a detectably labeled anti-IFNy antibody. Any colorimetric assay can be used. For example, a detectably labeled IFNy antibody may be a biotin-labeled IFNy antibody, which may be detected using, for example, streptavidin conjugated to alkaline phosphatase. BCIP/NBT (5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium) solution is added for development of the assay. The presence of IFNy-secreting T cells is identified by the stained spots. Negative controls include APCs that have not been exposed to the peptide. APCs expressing different alleles of the HLA H chain may be used to determine whether the peptide of interest effectively binds to the HLAI class molecule consisting of a particular HLA H chain.

Cytotoxicity assays.

The question of whether a given peptide binds to a particular HLA class I H chain and, when bound to an HLA class I complex containing the H chain, can effectively present an epitope to the TCR can also be resolved using a cytotoxicity assay. The cytotoxicity assay involves incubating a target cell with a cytotoxic CD8+ T cell. The target cell displays on its surface a peptide/HLA class I complex containing the peptide of interest and an HLA class I molecule containing the HLA H chain to be tested. Target cells can be labeled with radioactive isotopes such as ⁵¹ Cr. Whether the target cell effectively presents an epitope to the TCR on the cytotoxic CD8+ T cell, thereby inducing cytotoxic activity from the CD8+ T cell toward the target cell, is determined by measuring the release of ⁵¹ Cr from the lysed target cell. Specific cytotoxicity can be calculated as the amount of cytotoxic activity in the presence of the peptide minus the amount of cytotoxic activity in the absence of the peptide.

Detection of antigen-specific T cells using peptide-HLA tetramers.

As another example, multimers (e.g., tetramers) of peptide-HLA complexes with fluorescent or heavy metal markers are generated. The multimers can then be used to identify and quantify specific T cells by flow cytometry (FACS) or mass cytometry (CyTOF). Detection of epitope-specific T cells provides direct evidence that the peptide-linked HLA molecule is capable of binding to a specific TCR on a subset of antigen-specific T cells. See, e.g., Klenerman et al. (2002) Nature Reviews Immunol. 2:263.

Immunomodulatory polypeptides.

In some cases, the immunomodulatory polypeptide present in the TMMP of the invention is a wild-type immunomodulatory polypeptide. In other cases, the immunomodulatory polypeptide present in the TMMP of the invention is a variant of an immunomodulatory polypeptide that exhibits reduced affinity for a co-immunomodulatory polypeptide compared to the affinity of the corresponding wild-type immunomodulatory polypeptide for a co-immunomodulatory polypeptide, suitable immunomodulatory domains that exhibit reduced affinity for a co-immunomodulatory domain may have from 1 amino acid (aa) to 20 aa differences from a wild-type immunomodulatory domain. For example, in some instances, a variant immunomodulatory polypeptide present in a TMMP of the invention differs in amino acid sequence by 1 aa, 2 aa, 3 aa, 4 aa, 5 aa, 6 aa, 7 aa, 8 aa, 9 aa, or 10 aa from the corresponding wild-type immunomodulatory polypeptide. As another example, in some instances, a variant immunomodulatory polypeptide

- 46 049 816 of the peptide present in the TMMP of the invention differs in amino acid sequence by 11 aa, 12 aa, 13 aa, 14 aa, 15 aa, 16 aa, 17 aa, 18 aa, 19 aa or 20 aa from the corresponding wild-type immunomodulatory polypeptide. As an example, in some cases, a variant immunomodulatory polypeptide present in the TMMP of the invention comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions compared to the corresponding reference (e.g., wild-type) immunomodulatory polypeptide. In some cases, a variant immunomodulatory polypeptide present in the TMMP of the invention comprises a single amino acid substitution compared to the corresponding reference (e.g., wild-type) immunomodulatory polypeptide. In some cases, the immunomodulatory polypeptide variant present in the TMMP of the invention comprises 2 amino acid substitutions (e.g., no more than 2 amino acid substitutions) compared to the corresponding reference (e.g., wild type) immunomodulatory polypeptide. In some cases, the immunomodulatory polypeptide variant present in the TMMP of the invention comprises 3 amino acid substitutions (e.g., no more than 3 amino acid substitutions) compared to the corresponding reference (e.g., wild type) immunomodulatory polypeptide. In some cases, the immunomodulatory polypeptide variant present in the TMMP of the invention comprises 4 amino acid substitutions (e.g., no more than 4 amino acid substitutions) compared to the corresponding reference (e.g., wild type) immunomodulatory polypeptide. In some cases, the immunomodulatory polypeptide variant present in the TMMP of the invention comprises 5 amino acid substitutions (e.g., no more than 5 amino acid substitutions) compared to the corresponding reference (e.g., wild-type) immunomodulatory polypeptide. In some cases, the immunomodulatory polypeptide variant present in the TMMP of the invention comprises 6 amino acid substitutions (e.g., no more than 6 amino acid substitutions) compared to the corresponding reference (e.g., wild-type) immunomodulatory polypeptide. In some cases, the immunomodulatory polypeptide variant present in the TMMP of the invention comprises 7 amino acid substitutions (e.g., no more than 7 amino acid substitutions) compared to the corresponding reference (e.g., wild-type) immunomodulatory polypeptide. In some cases, a variant immunomodulatory polypeptide present in a TMMP of the invention comprises 8 amino acid substitutions (e.g., no more than 8 amino acid substitutions) compared to the corresponding reference (e.g., wild-type) immunomodulatory polypeptide. In some cases, a variant immunomodulatory polypeptide present in a TMMP of the invention comprises 9 amino acid substitutions (e.g., no more than 9 amino acid substitutions) compared to the corresponding reference (e.g., wild-type) immunomodulatory polypeptide. In some cases, a variant immunomodulatory polypeptide present in a TMMP of the invention comprises 10 amino acid substitutions (e.g., no more than 10 amino acid substitutions) compared to the corresponding reference (e.g., wild-type) immunomodulatory polypeptide.

In some cases, a variant immunomodulatory polypeptide present in a TMMP of the invention comprises 11 amino acid substitutions (e.g., no more than 11 amino acid substitutions) compared to the corresponding reference (e.g., wild type) immunomodulatory polypeptide.

In some cases, a variant immunomodulatory polypeptide present in a TMMP of the invention comprises 12 amino acid substitutions (e.g., no more than 12 amino acid substitutions) compared to the corresponding reference (e.g., wild type) immunomodulatory polypeptide.

In some cases, a variant immunomodulatory polypeptide present in a TMMP of the invention comprises 13 amino acid substitutions (e.g., no more than 13 amino acid substitutions) compared to the corresponding reference (e.g., wild type) immunomodulatory polypeptide.

In some cases, a variant immunomodulatory polypeptide present in a TMMP of the invention comprises 14 amino acid substitutions (e.g., no more than 14 amino acid substitutions) compared to the corresponding reference (e.g., wild type) immunomodulatory polypeptide.

In some cases, a variant immunomodulatory polypeptide present in a TMMP of the invention comprises 15 amino acid substitutions (e.g., no more than 15 amino acid substitutions) compared to the corresponding reference (e.g., wild type) immunomodulatory polypeptide.

In some cases, a variant immunomodulatory polypeptide present in a TMMP of the invention comprises 16 amino acid substitutions (e.g., no more than 16 amino acid substitutions) compared to the corresponding reference (e.g., wild type) immunomodulatory polypeptide.

In some cases, the immunomodulatory polypeptide variant present in the TMMP of the present invention comprises 17 amino acid substitutions (e.g., no more than 17 amino acid substitutions

- 47 049816 me) compared to the corresponding reference (e.g. wild type) immunomodulatory polypeptide.

In some cases, a variant immunomodulatory polypeptide present in a TMMP of the invention comprises 18 amino acid substitutions (e.g., no more than 18 amino acid substitutions) compared to the corresponding reference (e.g., wild type) immunomodulatory polypeptide.

In some cases, a variant immunomodulatory polypeptide present in a TMMP of the invention comprises 19 amino acid substitutions (e.g., no more than 19 amino acid substitutions) compared to the corresponding reference (e.g., wild type) immunomodulatory polypeptide.

In some cases, a variant immunomodulatory polypeptide present in a TMMP of the invention comprises 20 amino acid substitutions (e.g., no more than 20 amino acid substitutions) compared to the corresponding reference (e.g., wild type) immunomodulatory polypeptide.

As discussed above, a variant immunomodulatory polypeptide suitable for inclusion in a TMMP of the present invention exhibits reduced affinity for a cognate co-immunomodulatory polypeptide compared to the affinity of the corresponding wild-type immunomodulatory polypeptide for a cognate co-immunomodulatory polypeptide.

Exemplary immunomodulatory polypeptide and cognate co-immunomodulatory polypeptide pairs include, but are not limited to:

a) 4-1BBL (immunomodulatory polypeptide) and 4-1BB (cognate co-immunomodulatory polypeptide);

b) PD-L1 (immunomodulatory polypeptide) and PD1 (cognate co-immunomodulatory polypeptide);

c) IL-2 (immunomodulatory polypeptide) and IL-2 receptor (cognate co-immunomodulatory polypeptide);

d) CD80 (immunomodulatory polypeptide) and CD86 (cognate co-immunomodulatory polypeptide);

e) CD86 (immunomodulatory polypeptide) and CD28 (cognate co-immunomodulatory polypeptide);

f) OX40L (CD252) (immunomodulatory polypeptide) and OX40 (CD134) (cognate coimmunomodulatory polypeptide);

g) Fas ligand (immunomodulatory polypeptide) and Fas (cognate co-immunomodulatory polypeptide);

h) ICOS-L (immunomodulatory polypeptide) and ICOS (cognate co-immunomodulatory polypeptide);

i) ICAM (immunomodulatory polypeptide) and LFA-1 (cognate co-immunomodulatory polypeptide);

j) CD30L (immunomodulatory polypeptide) and CD30 (cognate co-immunomodulatory polypeptide);

k) CD40 (immunomodulatory polypeptide) and CD40L (cognate co-immunomodulatory polypeptide);

l) CD83 (immunomodulatory polypeptide) and CD83L (cognate co-immunomodulatory polypeptide);

m) HVEM (CD270) (immunomodulatory polypeptide) and CD160 (cognate co-immunomodulatory polypeptide);

n) JAG1 (CD339) (immunomodulatory polypeptide) and Notch (cognate co-immunomodulatory polypeptide);

o) JAG1 (immunomodulatory polypeptide) and CD46 (cognate co-immunomodulatory polypeptide);

p) CD80 (immunomodulatory polypeptide) and CTLA4 (cognate co-immunomodulatory polypeptide);

q) CD86 (immunomodulatory polypeptide) and CTLA4 (cognate co-immunomodulatory polypeptide); and

r) CD70 (immunomodulatory polypeptide) and CD27 (cognate co-immunomodulatory polypeptide).

In some cases, the immunomodulatory polypeptide variant present in the TMMP of the invention has a binding affinity for the cognate co-immunomodulatory polypeptide that is from 100 nM to 100 μM. For example, in some cases, the immunomodulatory polypeptide variant present in the TMMP of the invention has a binding affinity for the cognate co-immunomodulatory polypeptide that is from about 100 nM to 150 nM, from about 150 nM to about 200 nM, from about 200 nM to about 250 nM, from about 250 nM to about

- 48 049816

300 nM, from about 300 nM to about 350 nM, from about 350 nM to about 400 nM, from about 400 nM to about 500 nM, from about 500 nM to about 600 nM, from about 600 nM to about 700 nM, from about 700 nM to about 800 nM, from about 800 nM to about 900 nM, from about 900 nM to about 1 μM, to about 1 μM to about 5 μM, from about 5 μM to about 10 μM, from about 10 μM to about 15 μM, from about 15 μM to about 20 μM, from about 20 μM to about 25 μM, from about 25 μM to about 50 μM, from about 50 μM to about 75 μM, or from about 75 μM to about 100 μM.

The immunomodulatory polypeptide variant present in the TMMP of the invention exhibits reduced affinity for the cognate co-immunomodulatory polypeptide. Similarly, the TMMP of the invention that comprises the immunomodulatory polypeptide variant exhibits reduced affinity for the cognate co-immunomodulatory polypeptide. Thus, for example, the TMMP of the invention that comprises the immunomodulatory polypeptide variant has a binding affinity for the cognate co-immunomodulatory polypeptide that is from 100 nM to 100 μM. For example, in some cases, a TMMP of the invention comprising a variant immunomodulatory polypeptide has a binding affinity for a cognate co-immunomodulatory polypeptide that is from about 100 nM to 150 nM, from about 150 nM to about 200 nM, from about 200 nM to about 250 nM, from about 250 nM to about 300 nM, from about 300 nM to about 350 nM, from about 350 nM to about 400 nM, from about 400 nM to about 500 nM, from about 500 nM to about 600 nM, from about 600 nM to about 700 nM, from about 700 nM to about 800 nM, from about 800 nM to about 900 nM, from about 900 nM to about 1 μM, to about 1 μM to about 5 μM, from about 5 μM to about 10 μM, from about 10 μM to about 15 μM, from about 15 μM to about 20 μM, from about 20 μM to about 25 μM, from about 25 μM to about 50 μM, from about 50 μM to about 75 μM, or from about 75 μM to about 100 μM.

As shown schematically in Fig. 19, the immunomodulatory polypeptide (i.e., one or more immunomodulatory polypeptides) can be present in the TMMP of the present invention at any of a variety of positions. Fig. 19 depicts the position of two copies of a variant IL-2 polypeptide; however, the immunomodulatory polypeptide can be any of a variety of immunomodulatory polypeptides described herein. As shown in Fig. 19, the immunomodulatory polypeptide can be: 1) N-terminal to the MHC class I heavy chain; 2) C-terminal to the MHC class I heavy chain and N-terminal to the Ig Fc polypeptide; in other words, located between the MHC class I heavy chain and the Ig Fc polypeptide; 3) C-terminal to the Ig Fc polypeptide; 4) N-terminal to a peptide epitope; or 5) C-terminal to the B2M polypeptide.

PD-L1 variants.

As one non-limiting example, in some cases, a variant immunomodulatory polypeptide present in a TMMP of the present invention is a variant PD-L1 polypeptide. Wild-type PD-L1 binds to PD1.

The wild-type human PD-L1 polypeptide may contain the following amino acid sequence:

MRIFAVFIFM TYWHLLNAFT VTVPKDLYVV

EYGSNMTIEC

YRQRARLLKD

NAPYNKINQR

KFPVEKQLDL QLSLGNAALQ ILVVDPVTSE

AALIVYWEME

ITDVKLQDAG

HELTCQAEGY

DKNIIQFVHG

VYRCMISYGG

PKAEVIWTSS

EEDLKVQHSS

ADYKRITVKV

DHQVLSGKTT

TTNSKREEKL FNVTSTLRIN TTTNEIFYCT FRRLDPEENH TAELVIPGNI LNVSIKICLT

LSPST(SEQIDNO:1).

The ectodomain of wild-type human PD-L1 may contain the following amino acid sequence:

FT VTVPKDLYVV EYGSNMTIEC KFPVEKQLDL

AALIVYWEME

ITDVKLQDAG

HELTCQAEGY

DKNIIQFVHG

VYRCMISYGG

PKAEVIWTSS

EEDLKVQHSS

ADYKRITVKV

DHQVLSGKTT

YRQRARLLKD

NAPYNKINQR

TTNSKREEKL

QLSLGNAALQ

ILVVDPVTSE

FNVTSTLRIN

TTTNEIFYCT FRRLDPEENH TAELVIPGNI LNVSIKI (SEQ ID NO:2).

A wild-type PD-1 polypeptide may contain the following amino acid sequence:

PGWFLDSPDR PWNPPTFSPA LLVVTEGDNA TFTCSFSNTS

ESFVLNWYRM VRARRNDSGT RPAGQFQTLV EDPSAVPVFS

SPSNQTDKLA

YLCGAISLAP

VGVVGGLLGS

VDYGELDFQW

AFPEDRSQPG KAQIKESLRA LVLLVWVLAV

REKTPEPPVP

QDCRFRVTQL

ELRVTERRAE

ICSRAARGTI

CVPEQTEYAT

PNGRDFHMSV

VPTAHPSPSP

GARRTGQPLK

IVFPSGMGTS

SPARRGSADG PRSAQPLRPE DGHCSWPL (SEQ ID NO:3).

In some cases, when the TMMP of the present invention comprises a variant PD-L1 polypeptide,

- 49 049816 cognate co-immunomodulatory polypeptide is a PD-1 polypeptide comprising the amino acid sequence of SEQ ID NO: 3.

In some cases, the PD-L1 polypeptide variant exhibits a reduced binding affinity for PD-1 (e.g., a PD-1 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 3) compared to the binding affinity of a PD-L1 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2. For example, in some cases, a PD-L1 polypeptide variant of the invention binds PD-1 (e.g., a PD-1 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 3) with a binding affinity that is at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55% less than 60% less, at least 65% less, at least 70% less, at least 75% less, at least 80% less, at least 85% less, at least 90% less, at least 95% less, or more than 95% less than the binding affinity of a PD-L1 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2.

In some cases, the PD-L1 polypeptide variant has a binding affinity for PD-1 that is from 1 nM to 1 mM. In some cases, the PD-L1 polypeptide variant present in the TMMP of the invention has a binding affinity for PD-1 that is from 100 nM to 100 μM. As another example, in some cases, a PD-L1 polypeptide variant has a binding affinity for PD1 (e.g., a PD1 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 3) that is from about 100 nM to 150 nM, from about 150 nM to about 200 nM, from about 200 nM to about 250 nM, from about 250 nM to about 300 nM, from about 300 nM to about 350 nM, from about 350 nM to about 400 nM, from about 400 nM to about 500 nM, from about 500 nM to about 600 nM, from about 600 nM to about 700 nM, from about 700 nM to about 800 nM, from about 800 nM to about 900 nM, from about 900 nM to about 1 μM, to about 1 μM to about 5 μM, from about 5 μM to about 10 μM, from about 10 μM to about 15 μM, from about 15 μM to about 20 μM, from about 20 μM to about 25 μM, from about 25 μM to about 50 μM, from about 50 μM to about 75 μM, or from about 75 μM to about 100 μM.

In some cases, the PD-L1 polypeptide variant has one amino acid substitution compared to the PD-L1 amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2. In some cases, the PD-L1 polypeptide variant has 2 to 10 amino acid substitutions compared to the PD-L1 amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2. In some cases, the PD-L1 polypeptide variant has 2 amino acid substitutions compared to the PD-L1 amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2. In some cases, the PD-L1 polypeptide variant has 3 amino acid substitutions compared to the PD-L1 amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2. In some cases, the PD-L1 polypeptide variant has 4 amino acid substitutions compared to the PD-L1 amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2. In some cases, the PD-L1 polypeptide variant has 5 amino acid substitutions compared to the PD-L1 amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2. In some cases, the PD-L1 polypeptide variant has 6 amino acid substitutions compared to the PDL1 amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2. In some cases, the PD-L1 polypeptide variant has 7 amino acid substitutions compared to the PD-L1 amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2. In some cases, the PD-L1 polypeptide variant has 8 amino acid substitutions compared to the PD-L1 amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2. In some cases, the PD-L1 polypeptide variant has 9 amino acid substitutions compared to the PD-L1 amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2. In some cases, the PD-L1 polypeptide variant has 10 amino acid substitutions compared to the PD-L1 amino acid sequence presented in SEQ ID NO: 1 or SEQ ID NO: 2.

A suitable PD-L1 variant comprises a polypeptide 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:

FT VTVPKXLYVV EYGSNMTIEC KFPVEKQLDL AALIVYWEME DKNIIQFVHG

EEDLKVQHSS YRQRARLLKD QLSLGNAALQ ITDVKLQDAG VYRCMISYGG

ADYKRITVKV NAPYNKINQR ILVVDPVTSE HELTCQAEGY PKAEVIWTSS DHQVLSGKTT TTNSKREEKL FNVTSTLRIN TTTNEIFYCT FRRLDPEENH TAELVIPGNI LNVSIKI (SEQ ID NO: 52), wherein X is any amino acid other than Asp. In some cases, X is Ala. In some cases, X is Arg.

- 50 049816

A suitable PD-L1 variant comprises a polypeptide 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:

FT VTVPKDLYVV EYGSNMTIEC KFPVEKQLDL AALXVYWEME DKNIIQFVHG EEDLKVQHSS YRQRARLLKD QLSLGNAALQ ITDVKLQDAG VYRCMISYGG ADYKRITVKV NAPYNKINQR ILVVDPVTSE HELTCQAEGY PKAEVIWTSS

DHQVLSGKTT TTNSKREEKL FNVTSTLRIN TTTNEIFYCT FRRLDPEENH TAELVIPGNI LNVSIKI (SEQ ID NO: 53), where X is any amino acid other than He. In some cases, X is Asp.

A suitable PD-L1 variant comprises a polypeptide 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:

FT VTVPKDLYVV EYGSNMTIEC KFPVEKQLDL AALIVYWEME DKNIIQFVHG EXDLKVQHSS YRQRARLLKD QLSLGNAALQ ITDVKLQDAG VYRCMISYGG ADYKRITVKV NAPYNKINQR ILVVDPVTSE HELTCQAEGY PKAEVIWTSS

DHQVLSGKTT TTNSKREEKL FNVTSTLRIN TTTNEIFYCT FRRLDPEENH TAELVIPGNI LNVSIKI (SEQ ID NO:54), where X is any amino acid other than Glu. In some cases, X is Arg.

CD80 variants.

In some cases, the variant immunomodulatory polypeptide present in the TMMP of the present invention is a variant CD80 polypeptide. Wild-type CD80 binds to CD28. Wild-type CD80 also binds to CD86.

The amino acid sequence of the wild-type human CD80 ectodomain may be as follows:

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:4).

The amino acid sequence of wild-type CD28 may be as follows: MLRLLLALNL FPSIQVTGNK ILVKQSPMLV AYDNAVNLSC KYSYNLFSRE

FRASLHKGLD SAVEVCVVYG NYSQQLQVYS KTGFNCDGKL GNESVTFYLQ

NLYVNQTDIY FCKIEVMYPP PYLDNEKSNG TIIHVKGKHL CPSPLFPGPS

KPFWVLVVVG GVLACYSLLV TVAFIIFWVR SKRSRLLHSD YMNMTPRRPG PTRKHYQPYA PPRDFAAYRS (SEQ ID NO:5).

In some cases, when the TMMP of the present invention comprises a variant CD80 polypeptide, the cognate co-immunomodulatory polypeptide is a CD28 polypeptide comprising the amino acid sequence of SEQ ID NO: 5.

The amino acid sequence of wild-type CD28 may be as follows:

MLRLLLALNL FPSIQVTGNK ILVKQSPMLV AYDNAVNLSW KHLCPSPLFP

GPSKPFWVLV VVGGVLACYS LLVTVAFIIF WVRSKRSRLL HSDYMNMTPR

RPGPTRKHYQ PYAPPRDFAA YRS (SEQ ID NO:6)

The amino acid sequence of wild-type CD28 may be as follows: MLRLLLALNL FPSIQVTGKH LCPSPLFPGP SKPFWVLVVV GGVLACYSLL VTVAFIIFWV RSKRSRLLHS DYMNMTPRRP GPTRKHYQPY APPRDFAAYR S (SEQ ID NO:7).

In some instances, the CD80 variant polypeptide exhibits a reduced binding affinity for the CD28 polypeptide compared to the binding affinity of a CD80 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 4 for CD28. For example, in some instances, the CD80 variant polypeptide binds CD28 with a binding affinity that is at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%,

- 51 049816 at least 55% less, at least 60% less, at least 65% less, at least 70% less, at least 75% less, at least 80% less, at least 85%, at least 90%, at least 95% less, or more than 95% less than the binding affinity of a CD80 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 4 to CD28 (e.g., a CD28 polypeptide comprising the amino acid sequence set forth in one of SEQ ID NO: 5, 6, or 7).

In some cases, the CD80 polypeptide variant has a binding affinity for CD28 that ranges from 100 nM to 100 μM. As another example, in some cases, a CD80 polypeptide variant of the invention has a binding affinity for CD28 (e.g., a CD28 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7) that is from about 100 nM to 150 nM, from about 150 nM to about 200 nM, from about 200 nM to about 250 nM, from about 250 nM to about 300 nM, from about 300 nM to about 350 nM, from about 350 nM to about 400 nM, from about 400 nM to about 500 nM, from about 500 nM to about 600 nM, from about 600 nM to about 700 nM, from about 700 nM to about 800 nM, from about 800 nM to about 900 nM, from about 900 nM to about 1 μM, to about 1 μM to about 5 μM, from about 5 μM to about 10 μM, from about 10 μM to about 15 μM, from about 15 μM to about 20 μM, from about 20 μM to about 25 μM, from about 25 μM to about 50 μM, from about 50 μM to about 75 μM, or from about 75 μM to about 100 μM.

In some cases, the CD80 polypeptide variant has one amino acid substitution compared to the CD80 amino acid sequence set forth in SEQ ID NO: 4. In some cases, the CD80 polypeptide variant has 2 to 10 amino acid substitutions compared to the CD80 amino acid sequence set forth in SEQ ID NO: 4. In some cases, the CD80 polypeptide variant has 2 amino acid substitutions compared to the CD80 amino acid sequence set forth in SEQ ID NO: 4. In some cases, the CD80 polypeptide variant has 3 amino acid substitutions compared to the CD80 amino acid sequence set forth in SEQ ID NO: 4. In some cases, the CD80 polypeptide variant has 4 amino acid substitutions compared to the CD80 amino acid sequence set forth in SEQ ID NO: 4. In some cases, the CD80 polypeptide variant has 5 amino acid substitutions compared to the CD80 amino acid sequence set forth in SEQ ID NO: 4. In some cases, the CD80 polypeptide variant CD80 has 6 amino acid substitutions compared to the amino acid sequence of CD80 set forth in SEQ ID NO: 4. In some cases, the CD80 polypeptide variant has 7 amino acid substitutions compared to the amino acid sequence of CD80 set forth in SEQ ID NO: 4. In some cases, the CD80 polypeptide variant has 8 amino acid substitutions compared to the amino acid sequence of CD80 set forth in SEQ ID NO: 4. In some cases, the CD80 polypeptide variant has 9 amino acid substitutions compared to the amino acid sequence of CD80 set forth in SEQ ID NO: 4. In some cases, the CD80 polypeptide variant has 10 amino acid substitutions compared to the amino acid sequence of CD80 set forth in SEQ ID NO: 4.

Suitable CD80 variants include a polypeptide 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 one of the following amino acid sequences:

VIHVTK EVKEVATLSC GHXVSVEELA QTRIYWQKEK KMVLTMMSGD

MNIWPEYKNR TIFDITNNLS IVILALRPSD EGTYECVVLK YEKDAFKREH LAEVTLSVKA DFPTPSISDF EIPTSNIRRI ICSTSGGFPE PHLSWLENGE ELNAINTTVS QDPETELYAV SSKLDFNMTT NHSFMCLIKY GHLRVNQTFN WNTTKQEHFP DN (SEQ ID NO: 55), wherein X is any amino acid other than Asn. In some cases, X is Ala;

VIHVTK EVKEVATLSC GHNVSVEELA QTRIYWQKEK KMVLTMMSGD

MNIWPEYKNR TIFDITXNLS IVILALRPSD EGTYECVVLK YEKDAFKREH LAEVTLSVKA DFPTPSISDF EIPTSNIRRI ICSTSGGFPE PHLSWLENGE ELNAINTTVS QDPETELYAV SSKLDFNMTT NHSFMCLIKY GHLRVNQTFN WNTTKQEHFP DN (SEQ ID NO: 56), wherein X is any amino acid other than Asn. In some cases, X is Ala;

- 52 049816

VIHVTK EVKEVATLSC GHNVSVEELA QTRIYWQKEK KMVLTMMSGD MNIWPEYKNR TIFDITNNLS XVILALRPSD EGTYECVVLK YEKDAFKREH LAEVTLSVKA DFPTPSISDF EIPTSNIRRI ICSTSGGFPE PHLSWLENGE ELNAINTTVS QDPETELYAV SSKLDFNMTT NHSFMCLIKY GHLRVNQTFN WNTTKQEHFP DN (SEQ ID NO:57), wherein X is any amino acid other than Ile. In some cases, X is Ala;

VIHVTK EVKEVATLSC GHNVSVEELA QTRIYWQKEK KMVLTMMSGD MNIWPEYKNR TIFDITNNLS IVILALRPSD EGTYECVVLX YEKDAFKREH

LAEVTLSVKA DFPTPSISDF EIPTSNIRRI ICSTSGGFPE PHLSWLENGE ELNAINTTVS QDPETELYAV SSKLDFNMTT NHSFMCLIKY GHLRVNQTFN WNTTKQEHFP DN (SEQ ID NO:5-8), wherein X is any amino acid other than Lys. In some cases, X is Ala;

VIHVTK EVKEVATLSC GHNVSVEELA QTRIYWQKEK KMVLTMMSGD MNIWPEYKNR TIFDITNNLS IVILALRPSD EGTYECVVLK YEKDAFKREH

LAEVTLSVKA DFPTPSISDF EIPTSNIRRI ICSTSGGFPE PHLSWLENGE ELNAINTTVS XDPETELYAV SSKLDFNMTT NHSFMCLIKY GHLRVNQTFN WNTTKQEHFP DN (SEQ ID NO:60), wherein X is any amino acid other than Gln. In some cases, X is Ala;

VIHVTK EVKEVATLSC GHNVSVEELA QTRIYWQKEK KMVLTMMSGD MNIWPEYKNR TIFDITNNLS IVILALRPSD EGTYECVVLK YEKDAFKREH LAEVTLSVKA DFPTPSISDF EIPTSNIRRI ICSTSGGFPE PHLSWLENGE ELNAINTTVS QXPETELYAV SSKLDFNMTT NHSFMCLIKY GHLRVNQTFN WNTTKQEHFP DN (SEQ ID NO:61), wherein X is any amino acid other than Asp. In some cases, X is Ala;

VIHVTK EVKEVATLSC GHNVSVEEXA QTRIYWQKEK KMVLTMMSGD MNIWPEYKNR TIFDITNNLS IVILALRPSD EGTYECVVLK YEKDAFKREH LAEVTLSVKA DFPTPSISDF EIPTSNIRRI ICSTSGGFPE PHLSWLENGE ELNAINTTVS QDPETELYAV SSKLDFNMTT NHSFMCLIKY GHLRVNQTFN WNTTKQEHFP DN (SEQ ID NO:62), wherein X is any amino acid other than Leu. In some cases, X is Ala;

VIHVTK EVKEVATLSC GHNVSVEELA QTRIXWQKEK KMVLTMMSGD MNIWPEYKNR TIFDITNNLS IVILALRPSD EGTYECVVLK YEKDAFKREH LAEVTLSVKA DFPTPSISDF EIPTSNIRRI ICSTSGGFPE PHLSWLENGE ELNAINTTVS QDPETELYAV SSKLDFNMTT NHSFMCLIKY GHLRVNQTFN WNTTKQEHFP DN (SEQ ID NO:63), wherein X is any amino acid other than Tyr. In some cases, X is Ala;

VIHVTK EVKEVATLSC GHNVSVEELA QTRIYWXKEK KMVLTMMSGD MNIWPEYKNR TIFDITNNLS IVILALRPSD EGTYECVVLK YEKDAFKREH LAEVTLSVKA DFPTPSISDF EIPTSNIRRI ICSTSGGFPE PHLSWLENGE ELNAINTTVS QDPETELYAV SSKLDFNMTT NHSFMCLIKY GHLRVNQTFN WNTTKQEHFP DN (SEQ ID NO:64), wherein X is any amino acid other than Gln. In some cases, X is Ala;

- 53 049816

VIHVTK EVKEVATLSC GHNVSVEELA QTRIYWQKEK KXVLTMMSGD MNIWPEYKNR TIFDITNNLS IVILALRPSD EGTYECVVLK YEKDAFKREH LAEVTLSVKA DFPTPSISDF EIPTSNIRRI ICSTSGGFPE PHLSWLENGE ELNAINTTVS QDPETELYAV SSKLDFNMTT NHSFMCLIKY GHLRVNQTFN WNTTKQEHFP DN (SEQ ID NO:65), wherein X is any amino acid other than Met. In some cases, X is Ala;

VIHVTK EVKEVATLSC GHNVSVEELA QTRIYWQKEK KMXLTMMSGD MNIWPEYKNR TIFDITNNLS IVILALRPSD EGTYECVVLK YEKDAFKREH LAEVTLSVKA DFPTPSISDF EIPTSNIRRI ICSTSGGFPE PHLSWLENGE ELNAINTTVS QDPETELYAV SSKLDFNMTT NHSFMCLIKY GHLRVNQTFN WNTTKQEHFP DN (SEQ ID NO:66), wherein X is any amino acid other than Val. In some cases, X is Ala;

VIHVTK EVKEVATLSC GHNVSVEELA QTRIYWQKEK KMVLTMMSGD MNXWPEYKNR TIFDITNNLS IVILALRPSD EGTYECVVLK YEKDAFKREH LAEVTLSVKA DFPTPSISDF EIPTSNIRRI ICSTSGGFPE PHLSWLENGE ELNAINTTVS QDPETELYAV SSKLDFNMTT NHSFMCLIKY GHLRVNQTFN WNTTKQEHFP DN (SEQ ID NO:67), wherein X is any amino acid other than Ile. In some cases, X is Ala;

VIHVTK EVKEVATLSC GHNVSVEELA QTRIYWQKEK KMVLTMMSGD MNIWPEXKNR TIFDITNNLS IVILALRPSD EGTYECVVLK YEKDAFKREH LAEVTLSVKA DFPTPSISDF EIPTSNIRRI ICSTSGGFPE PHLSWLENGE ELNAINTTVS QDPETELYAV SSKLDFNMTT NHSFMCLIKY GHLRVNQTFN WNTTKQEHFP DN (SEQ ID NO:68), wherein X is any amino acid other than Tyr. In some cases, X is Ala;

VIHVTK EVKEVATLSC GHNVSVEELA QTRIYWQKEK KMVLTMMSGD MNIWPEYKNR TIFXITNNLS IVILALRPSD EGTYECVVLK YEKDAFKREH LAEVTLSVKA DFPTPSISDF EIPTSNIRRI ICSTSGGFPE PHLSWLENGE ELNAINTTVS QDPETELYAV SSKLDFNMTT NHSFMCLIKY GHLRVNQTFN WNTTKQEHFP DN (SEQ ID NO:69), wherein X is any amino acid other than Asp. In some cases, X is Ala;

VIHVTK EVKEVATLSC GHNVSVEELA QTRIYWQKEK KMVLTMMSGD MNIWPEYKNR TIFDITNNLS IVILALRPSD EGTYECVVLK YEKDAFKREH LAEVTLSVKA DXPTPSISDF EIPTSNIRRI ICSTSGGFPE PHLSWLENGE ELNAINTTVS QDPETELYAV SSKLDFNMTT NHSFMCLIKY GHLRVNQTFN WNTTKQEHFP DN (SEQ ID NO:70), wherein X is any amino acid other than Phe. In some cases, X is Ala;

VIHVTK EVKEVATLSC GHNVSVEELA QTRIYWQKEK KMVLTMMSGD MNIWPEYKNR TIFDITNNLS IVILALRPSD EGTYECVVLK YEKDAFKREH LAEVTLSVKA DFPTPSISDF EIPTSNIRRI ICSTSGGFPE PHLSWLENGE ELNAINTTVX QDPETELYAV SSKLDFNMTT NHSFMCLIKY GHLRVNQTFN WNTTKQEHFP DN (SEQ IDNO:71), wherein X is any amino acid other than Ser. In some cases, X is Ala; and

- 54 049816

VIHVTK EVKEVATLSC GHNVSVEELA QTRIYWQKEK KMVLTMMSGD

MNIWPEYKNR TIFDITNNLS IVILALRPSD EGTYECVVLK YEKDAFKREH

LAEVTLSVKA DFPTXSISDF EIPTSNIRRI ICSTSGGFPE PHLSWLENGE ELNAINTTVS QDPETELYAV SSKLDFNMTT NHSFMCLIKY GHLRVNQTFN WNTTKQEHFP DN (SEQ ID NO:72), where X is any amino acid other than Pro. In some cases, X is Ala.

CD86 variants.

In some cases, the variant immunomodulatory polypeptide present in the TMMP of the invention is a variant CD86 polypeptide. Wild-type CD86 binds to CD28. In some cases, when the TMMP of the invention comprises a variant CD86 polypeptide, the cognate co-immunomodulatory polypeptide is a CD28 polypeptide comprising the amino acid sequence of SEQ ID NO: 5.

The amino acid sequence of the complete ectodomain of wild-type human CD86 may be as follows:

APLKIQAYFNETADLPCQFANSQNQSLSELVVFWQDQENLVLNEVYLGKEKFDS

VHSKYMNRTSFDSDSWTLRLHNLQIKDKGLYQCIIHHKKPTGMIRIHQMNSELSVLANF SQPEIVPISNITENVYINLTCSSIHGYPEPKKMSVLLRTKNSTIEYDGIMQKSQDNVTELYD VSISLSVSFPDVTSNMTIFCILETDKTRLLSSSPFSIELEDPQPPPDHIP (SEQ ID NO:8).

The amino acid sequence of the wild-type human IgV CD86 domain may be as follows:

APLKIQAYFNETADLPCQFANSQNQSLSELVVFWQDQENLVLNEVYLGKEKFDS

VHSKYMNRTSFDSDSWTLRLHNLQIKDKGLYQCIIHHKKPTGMIRIHQMNSELSVL (SEQ ID NO:9).

In some cases, the CD86 polypeptide variant exhibits reduced binding affinity for the CD28 polypeptide compared to the binding affinity of a CD86 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 8 or SEQ ID NO: 9 for CD28. For example, in some cases, the CD86 polypeptide variant binds CD28 with a binding affinity that is at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55% less, at least 60% less, at least 65% less, at least 70% less, at least 75% less, at least 80% less, at least 85%, at least 90%, at least 95% less, or more than 95% less than the binding affinity of a CD86 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 8 or SEQ ID NO: 9 to CD28 (e.g., a CD28 polypeptide comprising the amino acid sequence presented in one of SEQ ID NO: 5, 6 or 7).

In some cases, the CD86 polypeptide variant has a binding affinity for CD28 that ranges from 100 nM to 100 μM. As another example, in some cases, a CD86 polypeptide variant of the invention has a binding affinity for CD28 (e.g., a CD28 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 5, 6, or 7) that is from about 100 nM to 150 nM, from about 150 nM to about 200 nM, from about 200 nM to about 250 nM, from about 250 nM to about 300 nM, from about 300 nM to about 350 nM, from about 350 nM to about 400 nM, from about 400 nM to about 500 nM, from about 500 nM to about 600 nM, from about 600 nM to about 700 nM, from about 700 nM to about 800 nM, from about 800 nM to about 900 nM, from about 900 nM to about 1 μM, to about 1 μM to about 5 μM, from about 5 μM to about 10 μM, from about 10 μM to about 15 μM, from about 15 μM to about 20 μM, from about 20 μM to about 25 μM, from about 25 μM to about 50 μM, from about 50 μM to about 75 μM, or from about 75 μM to about 100 μM.

In some cases, the CD86 polypeptide variant has one amino acid substitution compared to the CD86 amino acid sequence set forth in SEQ ID NO: 8. In some cases, the CD86 polypeptide variant has 2 to 10 amino acid substitutions compared to the CD86 amino acid sequence set forth in SEQ ID NO: 8. In some cases, the CD86 polypeptide variant has 2 amino acid substitutions compared to the CD86 amino acid sequence set forth in SEQ ID NO: 8. In some cases, the CD86 polypeptide variant has 3 amino acid substitutions compared to the CD86 amino acid sequence set forth in SEQ ID NO: 8. In some cases, the CD86 polypeptide variant has 4 amino acid substitutions compared to the CD86 amino acid sequence set forth in SEQ ID NO: 8. In some cases, the CD86 polypeptide variant has 5 amino acid substitutions compared to the CD86 amino acid sequence set forth in SEQ ID NO: 8. In some cases, the CD86 polypeptide variant The CD86 polypeptide has 6 amino acid substitutions compared to the amino acid sequence of CD86, represented

- 55 049816 as set forth in SEQ ID NO: 8. In some cases, the CD86 polypeptide variant has 7 amino acid substitutions compared to the CD86 amino acid sequence presented in SEQ ID NO: 8. In some cases, the CD86 polypeptide variant has 8 amino acid substitutions compared to the CD86 amino acid sequence presented in SEQ ID NO: 8. In some cases, the CD86 polypeptide variant has 9 amino acid substitutions compared to the CD86 amino acid sequence presented in SEQ ID NO: 8. In some cases, the CD86 polypeptide variant has 10 amino acid substitutions compared to the CD86 amino acid sequence presented in SEQ ID NO: 8.

In some cases, the CD86 polypeptide variant has one amino acid substitution compared to the CD86 amino acid sequence set forth in SEQ ID NO: 9. In some cases, the CD86 polypeptide variant has 2 to 10 amino acid substitutions compared to the CD86 amino acid sequence set forth in SEQ ID NO: 9. In some cases, the CD86 polypeptide variant has 2 amino acid substitutions compared to the CD86 amino acid sequence set forth in SEQ ID NO: 9. In some cases, the CD86 polypeptide variant has 3 amino acid substitutions compared to the CD86 amino acid sequence set forth in SEQ ID NO: 9. In some cases, the CD86 polypeptide variant has 4 amino acid substitutions compared to the CD86 amino acid sequence set forth in SEQ ID NO: 9. In some cases, the CD86 polypeptide variant has 5 amino acid substitutions compared to the CD86 amino acid sequence set forth in SEQ ID NO: 9. In some cases, the CD86 polypeptide variant The CD86 polypeptide has 6 amino acid substitutions compared to the amino acid sequence of CD86 presented in SEQ ID NO: 9. In some cases, the CD86 polypeptide variant has 7 amino acid substitutions compared to the amino acid sequence of CD86 presented in SEQ ID NO: 9. In some cases, the CD86 polypeptide variant has 8 amino acid substitutions compared to the amino acid sequence of CD86 presented in SEQ ID NO: 9. In some cases, the CD86 polypeptide variant has 9 amino acid substitutions compared to the amino acid sequence of CD86 presented in SEQ ID NO: 9. In some cases, the CD86 polypeptide variant has 10 amino acid substitutions compared to the amino acid sequence of CD86 presented in SEQ ID NO: 9.

Suitable CD86 variants include a polypeptide 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 one of the following amino acid sequences:

APLKIQAYFNETADLPCQFANSQNQSLSELVVFWQDQENLVLNEVYLGKEKFDS

VHSKYMXRTSFDSDSWTLRLHNLQIKDKGLYQCIIHHKKPTGMIRIHQMNSELSVLANF

SQPEIVPISNITENVYINLTCSSIHGYPEPKKMSVLLRTKNSTIEYDGIMQKSQDNVTELYD

VSISLSVSFPDVTSNMTIFCILETDKTRLLSSPFSIELEDPQPPPDHIP (SEQ ID NO:73), wherein X is any amino acid other than Asn. In some cases, X is Ala;

APLKIQAYFNETADLPCQFANSQNQSLSELVVFWQDQENLVLNEVYLGKEKFDS

VHSKYMNRTSFXSDSWTLRLHNLQIKDKGLYQCIIHHKKPTGMIRIHQMNSELSVLANF SQPEIVPISNITENVYINLTCSSIHGYPEPKKMSVLLRTKNSTIEYDGIMQKSQDNVTELYD VSISLSVSFPDVTSNMTIFCILETDKTRLLSSPFSIELEDPQPPPDHIP (SEQ ID NO:74), wherein X is any amino acid other than Asp. In some cases, X is Ala;

APLKIQAYFNETADLPCQFANSQNQSLSELVVFWQDQENLVLNEVYLGKEKFDS

VHSKYMNRTSFDSDSXTLRLHNLQIKDKGLYQCIIHHKKPTGMIRIHQMNSELSVLANFS QPEIVPISNITENVYINLTCSSIHGYPEPKKMSVLLRTKNSTIEYDGIMQKSQDNVTELYD VSISLSVSFPDVTSNMTIFCILETDKTRLLSSPFSIELEDPQPPPDHIP (SEQ ID NO:75), wherein X is any amino acid other than Trp. In some cases, X is Ala;

APLKIQAYFNETADLPCQFANSQNQSLSELVVFWQDQENLVLNEVYLGKEKFDS

VHSKYMNRTSFDSDSWTLRLHNLQIKDKGLYQCIIHXKKPTGMIRIHQMNSELSVLANF SQPEIVPISNITENVYINLTCSSIHGYPEPKKMSVLLRTKNSTIEYDGIMQKSQDNVTELYD VSISLSVSFPDVTSNMTIFCILETDKTRLLSSPFSIELEDPQPPPDHIP (SEQ ID NO:76), wherein X is any amino acid other than His. In some cases, X is Ala;

- 56 049816

APLKIQAYFNETADLPCQFANSQNQSLSELVVFWQDQENLVLNEVYLGKEKFDS

VHSKYMXRTSFDSDSWTLRLHNLQIKDKGLYQCIIHHKKPTGMIRIHQMNSELSVL (SEQ ID NO:77), wherein X is any amino acid other than Asn. In some cases, X is Ala;

APLKIQAYFNETADLPCQFANSQNQSLSELVVFWQDQENLVLNEVYLGKEKFDS

VHSKYMNRTSFXSDSWTLRLHNLQIKDKGLYQCIIHHKKPTGMIRIHQMNSELSVL (SEQ ID NO:78), wherein X is any amino acid other than Asp. In some cases, X is Ala;

APLKIQAYFNETADLPCQFANSQNQSLSELVVFWQDQENLVLNEVYLGKEKFDS VHSKYMNRTSFDSDSXTLRLHNLQIKDKGLYQCIIHHKKPTGMIRIHQMNSELSVL (SEQ ID NO:79), wherein X is any amino acid other than Trp. In some cases, X is Ala;

APLKIQAYFNETADLPCQFANSQNQSLSELVVFWQDQENLVLNEVYLGKEKFDS VHSKYMNRTSFDSDSWTLRLHNLQIKDKGLYQCIIHXKKPTGMIRIHQMNSELSVL (SEQ ID NO:80), wherein X is any amino acid other than His. In some cases, X is Ala;

APLKIQAYFNETADLPCQFANSQNQSLSELVVFWQDQENLXLNEVYLGKEKFDS VHSKYMNRTSFDSDSWTLRLHNLQIKDKGLYQCIIHHKKPTGMIRIHQMNSELSVLANF SQPEIVPISNITENVYINLTCSSIHGYPEPKKMSVLLRTKNSTIEYDGIMQKSQDNVTELYD VSISLSVSFPDVTSNMTIFCILETDKTRLLSSPFSIELEDPQPPPDHIP (SEQ ID NO:81), wherein X is any amino acid other than Val. In some cases, X is Ala;

APLKIQAYFNETADLPCQFANSQNQSLSELVVFWQDQENLXLNEVYLGKEKFDS VHSKYMNRTSFDSDSWTLRLHNLQIKDKGLYQCIIHHKKPTGMIRIHQMNSELSVL (SEQ ID NO:82), wherein X is any amino acid other than Val. In some cases, X is Ala;

APLKIQAYFNETADLPCQFANSQNQSLSELVVFWXDQENLVLNEVYLGKEKFDS VHSKYMNRTSFDSDSWTLRLHNLQIKDKGLYQCIIHHKKPTGMIRIHQMNSELSVLANF SQPEIVPISNITENVYINLTCSSIHGYPEPKKMSVLLRTKNSTIEYDGIMQKSQDNVTELYD VSISLSVSFPDVTSNMTIFCILETDKTRLLSSPFSIELEDPQPPPDHIP (SEQ ID NO:83), wherein X is any amino acid other than Gln. In some cases, X is Ala;

APLKIQAYFNETADLPCQFANSQNQSLSELVVFWXDQENLVLNEVYLGKEKFDS VHSKYMNRTSFDSDSWTLRLHNLQIKDKGLYQCIIHHKKPTGMIRIHQMNSELSVL (SEQ ID NO:84), wherein X is any amino acid other than Gln. In some cases, X is Ala;

APLKIQAYFNETADLPCQFANSQNQSLSELVVXWQDQENLVLNEVYLGKEKFDS VHSKYMNRTSFDSDSWTLRLHNLQIKDKGLYQCIIHHKKPTGMIRIHQMNSELSVLANF SQPEIVPISNITENVYINLTCSSIHGYPEPKKMSVLLRTKNSTIEYDGIMQKSQDNVTELYD VSISLSVSFPDVTSNMTIFCILETDKTRLLSSPFSIELEDPQPPPDHIP (SEQ ID NO:85), wherein X is any amino acid other than Phe. In some cases, X is Ala;

APLKIQAYFNETADLPCQFANSQNQSLSELVVXWQDQENLVLNEVYLGKEKFDS VHSKYMNRTSFDSDSWTLRLHNLQIKDKGLYQCIIHHKKPTGMIRIHQMNSELSVL (SEQ ID NO:86), wherein X is any amino acid other than Phe. In some cases, X is Ala;

APLKIQAYFNETADLPCQFANSQNQSLSELVVFWQDQENLVLNEVYLGKEKFDS VHSKYMNRTSFDSDSWTXRLHNLQIKDKGLYQCIIHHKKPTGMIRIHQMNSELSVLANF

- 57 049816

SQPEIVPISNITENVYINLTCSSIHGYPEPKKMSVLLRTKNSTIEYDGIMQKSQDNVTELYD

VSISLSVSFPDVTSNMTIFCILETDKTRLLSSPFSIELEDPQPPPDHIP (SEQ ID NO:87), wherein X is any amino acid other than Leu. In some cases, X is Ala;

APLKIQAYFNETADLPCQFANSQNQSLSELVVFWQDQENLVLNEVYLGKEKFDS

VHSKYMNRTSFDSDSWTXRLHNLQIKDKGLYQCIIHHKKPTGMIRIHQMNSELSVL (SEQ ID NO:88), wherein X is any amino acid other than Leu. In some cases, X is Ala;

APLKIQAYFNETADLPCQFANSQNQSLSELVVFWQDQENLVLNEVYLGKEKFDS VHSKXMNRTSFDSDSWTLRLHNLQIKDKGLYQCIIHHKKPTGMIRIHQMNSELSVLANF SQPEIVPISNITENVYINLTCSSIHGYPEPKKMSVLLRTKNSTIEYDGIMQKSQDNVTELYD VSISLSVSFPDVTSNMTIFCILETDKTRLLSSPFSIELEDPQPPPDHIP (SEQ ID NO:89), wherein X is any amino acid except Tyr. In some cases, X is Ala;

APLKIQAYFNETADLPCQFANSQNQSLSELVVFWQDQENLVLNEVYLGKEKFDS

VHSKXMNRTSFDSDSWTLRLHNLQIKDKGLYQCIIHHKKPTGMIRIHQMNSELSVL (SEQ ID NO:90), wherein X is any amino acid other than Tyr. In some cases, X is Ala;

APLKIQAYFNETADLPCQFANSQNQSLSELVVFWQDQENLVLNEVYLGKEKFDS VHSKYMXRTSFDSDSWTLRLHNLQIKDKGLYQCIIHXKKPTGMIRIHQMNSELSVLANF SQPEIVPISNITENVYINLTCSSIHGYPEPKKMSVLLRTKNSTIEYDGIMQKSQDNVTELYD VSISLSVSFPDVTShMTIFCILETDKTRLLSSPFSIELEDPQPPPDHIP (SEQ ID NO:91), wherein the first X is any amino acid other than Asn and the second X is any amino acid other than His. In some cases, the first and second X are both Ala;

APLKIQAYFNETADLPCQFANSQNQSLSELVVFWQDQENLVLNEVYLGKEKFDS VHSKYMXRTSFDSDSWTLRLHNLQIKDKGLYQCIIHXKKPTGMIRIHQMNSELSVL (SEQ ID NO:92), wherein the first X is any amino acid other than Asn and the second X is any amino acid other than His. In some cases, the first and second X are both Ala;

APLKIQAYFNETADLPCQFANSQNQSLSELVVFWQDQENLVLNEVYLGKEKFDS VHSKYMNRTSFX1SDSWTLRLHNLQIKDKGLYQCIIHX2KKPTGMIRIHQMNSELSVLAN FSQPEIVPISNITENVYINLTCSSIHGYPEPKKMSVLLRTKNSTIEYDGIMQKSQDNVTELY DVSISLSVSFPDVTSNMTIFCILETDKTRLLSSPFSIELEDPQPPPDHIP (SEQ ID NO:93), wherein X1 is any amino acid other than Asp and X2 is any amino acid other than His. In some cases, X1 is Ala and _X2 is Ala;

APLKIQAYFNETADLPCQFANSQNQSLSELVVFWQDQENLVLNEVYLGKEKFDS VHSKYMNRTSFX1SDSWTLRLHNLQIKDKGLYQCIIHX2KKPTGMIRIHQMNSELSVL (SEQ ID NO:94), wherein the first X is any amino acid other than Asn and the second X is any amino acid other than His. In some cases, the first and second X are both Ala;

APLKIQAYFNETADLPCQFANSQNQSLSELVVFWQDQENLVLNEVYLGKEKFDS VHSKYMX1RTSFX2SDSWTLRLHNLQIKDKGLYQCIIHX3KKPTGMIRIHQMNSELSVLA NFSQPEIVPISNITENVYINLTCSSIHGYPEPKKMSVLLRTKNSTIEYDGIMQKSQDNVTEL YDVSISLSVSFPDVTSNMTIFCILETDKTRLLSSPFSIELEDPQPPPDHIP (SEQ ID NO:95), wherein X1 is any amino acid other than Asn, X2 is any amino acid other than Asp, and _X3 is any amino acid other than His. In some cases, X1 is Ala, X2 is Ala, and X3 is Ala; and

- 58 049816

APLKIQAYFNETADLPCQFANSQNQSLSELVVFWQDQENLVLNEVYLGKEKFDS VHSKYMX1RTSFX2SDSWTLRLHNLQIKDKGLYQCIIHX3KKPTGMIRIHQMNSELSVL (SEQ ID NO:96), wherein X1 is any amino acid other than Asn, X2 is any amino acid other than Asp, and X3 is any amino acid other than His. In some cases, X1 is Ala, _X2 is Ala, and X3 is Ala.

Options 4-1BBL.

In some cases, the immunomodulatory polypeptide variant present in the TMMP of the present invention is a 4-1BBL polypeptide variant. Wild-type 4-1BBL binds to 4-1BB (CD137).

The amino acid sequence of wild-type 4-1BBL could be as follows MEYASDASLD PEAPWPPAPR ARACRVLPWA LVAGLLLLLL LAAACAVFLA CPWAVSGARA SPGSAASPRL REGPELSPDD PAGLLDLRQG MFAQLVAQNV LLIDGPLSWY SDPGLAGVSL TGGLSYKEDT KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNS AFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 10).

In some cases, the 4-1BBL polypeptide variant is a human tumor necrosis factor (TNF) 4-1BBL homology domain (THD) variant.

The wild-type amino acid sequence of human THD 4-1BBL may be, for example, one of SEQ ID NOs: 11-13, i.e. the following: PAGLLDLRQG MFAQLVAQNV LLIDGPLSWY SDPGLAGVSL TGGLSYKEDT KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNS AFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 11).

D PAGLLDLRQG MFAQLVAQNV LLIDGPLSWY SDPGLAGVSL TGGLSYKEDT KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNS AFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 12).

D PAGLLDLRQG MFAQLVAQNV LLIDGPLSWY SDPGLAGVSL TGGLSYKEDT KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNS AFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPA (SEQ ID NO: 13).

The amino acid sequence of wild-type 4-1BB may be as follows: MGNSCYNIVA TLLLVLNFER TRSLQDPCSN CPAGTFCDNN RNQICSPCPP

NSFSSAGGQR TCDICRQCKG VFRTRKECSS TSNAECDCTP GFHCLGAGCS

MCEQDCKQGQ ELTKKGCKDC CFGTFNDQKR GICRPWTNCS LDGKSVLVNG

TKERDVVCGP SPADLSPGAS SVTPPAPARE PGHSPQIISF FLALTSTALL FLLFFLTLRF SVVKRGRKKL LYIFKQPFMR PVQTTQEEDG CSCRFPEEEE GGCEL (SEQ ID NO: 14).

In some cases, when the TMMP of the present invention comprises a variant of the 41BBL polypeptide, the cognate co-immunomodulatory polypeptide is a 4-1BB polypeptide comprising the amino acid sequence of SEQ ID NO: 14.

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

In some cases, a 4-1BBL polypeptide variant has a binding affinity for 4-1BB that

- 59 049816 is from 100 nM to 100 µM. As another example, in some cases, a 4-1BBL polypeptide variant has a binding affinity for 4-1BB (e.g., a 4-1BB polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 14) that is from about 100 nM to 150 nM, from about 150 nM to about 200 nM, from about 200 nM to about 250 nM, from about 250 nM to about 300 nM, from about 300 nM to about 350 nM, from about 350 nM to about 400 nM, from about 400 nM to about 500 nM, from about 500 nM to about 600 nM, from about 600 nM to about 700 nM, from about 700 nM to about 800 nM, from about 800 nM to about 900 nM, from about 900 nM to about 1 μM, to about 1 μM to about 5 μM, from about 5 μM to about 10 μM, from about 10 μM to about 15 μM, from about 15 μM to about 20 μM, from about 20 μM to about 25 μM, from about 25 μM to about 50 μM, from about 50 μM to about 75 μM, or from about 75 μM to about 100 μM.

In some cases, the 4-1BBL polypeptide variant has one amino acid substitution compared to the 4-1BBL amino acid sequence presented in one of SEQ ID NOs: 10-13. In some cases, the 4-1BBL polypeptide variant has 2 to 10 amino acid substitutions compared to the 4-1BBL amino acid sequence presented in one of SEQ ID NOs: 10-13. In some cases, the 4-1BBL polypeptide variant has 2 amino acid substitutions compared to the 4-1BBL amino acid sequence presented in one of SEQ ID NOs: 10-13. In some cases, the 4-1BBL polypeptide variant has 3 amino acid substitutions compared to the 4-1BBL amino acid sequence presented in one of SEQ ID NOs: 10-13. In some cases, the 4-1BBL polypeptide variant has 4 amino acid substitutions compared to the 4-1BBL amino acid sequence presented in one of SEQ ID NOs: 10-13. In some cases, the 4-1BBL polypeptide variant has 5 amino acid substitutions compared to the 4-1BBL amino acid sequence presented in one of SEQ ID NOs: 10-13. In some cases, the 4-1BBL polypeptide variant has 6 amino acid substitutions compared to the 4-1BBL amino acid sequence presented in one of SEQ ID NOs: 10-13. In some cases, the 4-1BBL polypeptide variant has 7 amino acid substitutions compared to the 4-1BBL amino acid sequence presented in one of SEQ ID NOs: 10-13. In some cases, the 4-1BBL polypeptide variant has 8 amino acid substitutions compared to the 4-1BBL amino acid sequence presented in one of SEQ ID NOs: 1013. In some cases, the 4-1BBL polypeptide variant has 9 amino acid substitutions compared to the 4-1BBL amino acid sequence presented in one of SEQ ID NOs: 10-13. In some cases, the 4-1BBL polypeptide variant has 10 amino acid substitutions compared to the 4-1BBL amino acid sequence presented in one of SEQ ID NOs: 10-13.

Suitable 4-1BBL variants include a polypeptide 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 one of the following amino acid sequences:

PAGLLDLRQG MFAQLVAQNV LLIDGPLSWY SDPGLAGVSL TGGLSYXEDT

KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA

LTVDLPPASS EARNS AFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ

GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 97), wherein X is any amino acid other than Lys. In some cases, X is Ala;

PAGLLDLRQG MFAQLVAQNV LLIDGPLSWY SDPGLAGVSL TGGLSYKEDT

KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNS AFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWXLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 98), wherein X is any amino acid other than Gln. In some cases, X is Ala;

PAGLLDLRQG XFAQLVAQNV LLIDGPLSWY SDPGLAGVSL TGGLSYKEDT

KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNS AFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 99), where X is any amino acid other than Met. In some cases, X is Ala;

PAGLLDLRQG MXAQLVAQNV LLIDGPLSWY SDPGLAGVSL TGGLSYKEDT

KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNS AFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 100), wherein X is any amino acid other than Phe. In some cases, X is Ala;

PAGLLDLRQG MFAXLVAQNV LLIDGPLSWY SDPGLAGVSL TGGLSYKEDT KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNS AFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 101), wherein X is any amino acid other than Gln. In some cases, X is Ala;

PAGLLDLRQG MFAQXVAQNV LLIDGPLSWY SDPGLAGVSL TGGLSYKEDT KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNS AFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 102), wherein X is any amino acid other than Leu. In some cases, X is Ala;

PAGLLDLRQG MFAQLXAQNV LLIDGPLSWY SDPGLAGVSL TGGLSYKEDT KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNS AFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 103), wherein X is any amino acid other than Val. In some cases, X is Ala;

PAGLLDLRQG MFAQLVAXNV LLIDGPLSWY SDPGLAGVSL TGGLSYKEDT KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNS AFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 104), wherein X is any amino acid other than Gln. In some cases, X is Ala;

PAGLLDLRQG MFAQLVAXNV LLIDGPLSWY SDPGLAGVSL TGGLSYKEDT KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNS AFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 105), wherein X is any amino acid other than Asn. In some cases, X is Ala;

PAGLLDLRQG MFAQLVAQNX LLIDGPLSWY SDPGLAGVSL TGGLSYKEDT KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNS AFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 106), wherein X is any amino acid other than Val. In some cases, X is Ala;

PAGLLDLRQG MFAQLVAQNV XLIDGPLSWY SDPGLAGVSL TGGLSYKEDT KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNS AFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 107), wherein X is any amino acid other than Leu. In some cases, X is Ala;

PAGLLDLRQG MFAQLVAQNV LXIDGPLSWY SDPGLAGVSL TGGLSYKEDT KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNS AFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 108), wherein X is any amino acid other than Leu. In some cases, X is Ala;

PAGLLDLRQG MFAQLVAQNV LLXDGPLSWY SDPGLAGVSL TGGLSYKEDT KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNS AFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 109), wherein X is any amino acid other than Ile. In some cases, X is Ala;

PAGLLDLRQG MFAQLVAQNV LLIXGPLSWY SDPGLAGVSL TGGLSYKEDT KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNS AFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 110), wherein X is any amino acid other than Asp. In some cases, X is Ala;

PAGLLDLRQG MFAQLVAQNV LLIDXPLSWY SDPGLAGVSL TGGLSYKEDT KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNS AFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO:111), wherein X is any amino acid other than Gly. In some cases, X is Ala;

PAGLLDLRQG MFAQLVAQNV LLIGGXLSWY SDPGLAGVSL TGGLSYKEDT KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNS AFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 112), wherein X is any amino acid other than Pro. In some cases, X is Ala;

PAGLLDLRQG MFAQLVAQNV LLIGGPXSWY SDPGLAGVSL TGGLSYKEDT KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNS AFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 113), wherein X is any amino acid other than Leu. In some cases, X is Ala;

PAGLLDLRQG MFAQLVAQNV LLIGGPLXWY SDPGLAGVSL TGGLSYKEDT KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNS AFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 114), wherein X is any amino acid other than Ser. In some cases, X is Ala;

PAGLLDLRQG MFAQLVAQNV LLIGGPLSXY SDPGLAGVSL TGGLSYKEDT KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNS AFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 115), wherein X is any amino acid other than Trp. In some cases, X is Ala;

PAGLLDLRQG MFAQLVAQNV LLIGGPLSWX SDPGLAGVSL TGGLSYKEDT KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNS AFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 116), wherein X is any amino acid other than Tyr. In some cases, X is Ala;

PAGLLDLRQG MFAQLVAQNV LLIGGPLSWY X DPGLAGVSL TGGLSYKEDT KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNS AFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 117), wherein X is any amino acid other than Ser. In some cases, X is Ala;

PAGLLDLRQG MFAQLVAQNV LLIGGPLSWY SX PGLAGVSL TGGLSYKEDT KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNS AFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 118), wherein X is any amino acid other than Asp. In some cases, X is Ala;

PAGLLDLRQG MFAQLVAQNV LLIGGPLSWY SDXGLAGVSL TGGLSYKEDT KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNS AFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 119), wherein X is any amino acid other than Pro. In some cases, X is Ala;

PAGLLDLRQG MFAQLVAQNV LLIGGPLSWY SDPXLAGVSL TGGLSYKEDT KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNS AFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 120), wherein X is any amino acid other than Gly. In some cases, X is Ala;

PAGLLDLRQG MFAQLVAQNV LLIGGPLSWY SDPGXAGVSL TGGLSYKEDT KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNS AFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 121), wherein X is any amino acid other than Leu. In some cases, X is Ala;

PAGLLDLRQG MFAQLVAQNV LLIGGPLSWY SDPGLAXVSL TGGLSYKEDT KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNS AFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 122), wherein X is any amino acid other than Gly. In some cases, X is Ala;

PAGLLDLRQG MFAQLVAQNV LLIGGPLSWY SDPGLAGXSL TGGLSYKEDT KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNS AFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 123), wherein X is any amino acid other than Val. In some cases, X is Ala;

PAGLLDLRQG MFAQLVAQNV LLIGGPLSWY SDPGLAGVXL TGGLSYKEDT KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNS AFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 124), wherein X is any amino acid other than Ser. In some cases, X is Ala;

PAGLLDLRQG MFAQLVAQNV LLIGGPLSWY SDPGLAGVSX TGGLSYKEDT KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNS AFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 125), wherein X is any amino acid other than Leu. In some cases, X is Ala;

PAGLLDLRQG MFAQLVAQNV LLIGGPLSWY SDPGLAGVSL XGGLSYKEDT KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNS AFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 126), wherein X is any amino acid other than Thr. In some cases, X is Ala;

PAGLLDLRQG MFAQLVAQNV LLIGGPLSWY SDPGLAGVSL TXGLSYKEDT KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNS AFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 127), wherein X is any amino acid other than Gly. In some cases, X is Ala;

PAGLLDLRQG MFAQLVAQNV LLIGGPLSWY SDPGLAGVSL TGXLSYKEDT KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNS AFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 128), wherein X is any amino acid other than Gly. In some cases, X is Ala;

PAGLLDLRQG MFAQLVAQNV LLIGGPLSWY SDPGLAGVSL TGGXSYKEDT KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNS AFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 129), wherein X is any amino acid other than Leu. In some cases, X is Ala;

PAGLLDLRQG MFAQLVAQNV LLIGGPLSWY SDPGLAGVSL TGGLXYKEDT KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNS AFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 130), wherein X is any amino acid other than Ser. In some cases, X is Ala;

PAGLLDLRQG MFAQLVAQNV LLIGGPLSWY SDPGLAGVSL TGGLSXKEDT KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNS AFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 131), wherein X is any amino acid other than Tyr. In some cases, X is Ala;

PAGLLDLRQG MFAQLVAQNV LLIGGPLSWY SDPGLAGVSL TGGLSYKXDT KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNS AFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 132), wherein X is any amino acid other than Glu. In some cases, X is Ala;

PAGLLDLRQG MFAQLVAQNV LLIGGPLSWY SDPGLAGVSL TGGLSYKEXT KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNS AFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 133), wherein X is any amino acid other than Asp. In some cases, X is Ala;

PAGLLDLRQG MFAQLVAQNV LLIGGPLSWY SDPGLAGVSL TGGLSYKEDX KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNS AFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 134), wherein X is any amino acid other than Thr. In some cases, X is Ala;

PAGLLDLRQG MFAQLVAQNV LLIGGPLSWY SDPGLAGVSL TGGLSYKEDT XELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNS AFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 135), wherein X is any amino acid other than Lys. In some cases, X is Ala;

PAGLLDLRQG MFAQLVAQNV LLIGGPLSWY SDPGLAGVSL TGGLSYKEDT KXLVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNS AFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 136), wherein X is any amino acid other than Glu. In some cases, X is Ala;

PAGLLDLRQG MFAQLVAQNV LLIGGPLSWY SDPGLAGVSL TGGLSYKEDT KELVVAKAGV YYVXFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNS AFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 137), wherein X is any amino acid other than Phe. In some cases, X is Ala;

PAGLLDLRQG MFAQLVAQNV LLIGGPLSWY SDPGLAGVSL TGGLSYKEDT KELVVAKAGV YYVFXQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNS AFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 138), wherein X is any amino acid other than Phe. In some cases, X is Ala;

PAGLLDLRQG MFAQLVAQNV LLIGGPLSWY SDPGLAGVSL TGGLSYKEDT KELVVAKAGV YYVFFXLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNS AFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 139), wherein X is any amino acid other than Gln. In some cases, X is Ala;

PAGLLDLRQG MFAQLVAQNV LLIGGPLSWY SDPGLAGVSL TGGLSYKEDT KELVVAKAGV YYVFFQXELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNS AFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 140), wherein X is any amino acid other than Leu. In some cases, X is Ala;

PAGLLDLRQG MFAQLVAQNV LLIGGPLSWY SDPGLAGVSL TGGLSYKEDT KELVVAKAGV YYVFFQLXLR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNS AFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 141), wherein X is any amino acid other than Glu. In some cases, X is Ala;

PAGLLDLRQG MFAQLVAQNV LLIGGPLSWY SDPGLAGVSL TGGLSYKEDT KELVVAKAGV YYVFFQLEXR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNS AFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 142), wherein X is any amino acid other than Leu. In some cases, X is Ala;

PAGLLDLRQG MFAQLVAQNV LLIGGPLSWY SDPGLAGVSL TGGLSYKEDT KELVVAKAGV YYVFFQLELX RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNS AFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 143), wherein X is any amino acid other than Arg. In some cases, X is Ala;

PAGLLDLRQG MFAQLVAQNV LLIGGPLSWY SDPGLAGVSL TGGLSYKEDT KELVVAKAGV YYVFFQLELR XVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNS AFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 144), wherein X is any amino acid other than Arg. In some cases, X is Ala;

PAGLLDLRQG MFAQLVAQNV LLIGGPLSWY SDPGLAGVSL TGGLSYKEDT KELVVAKAGV YYVFFQLELR RXVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNS AFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 145), wherein X is any amino acid other than Val. In some cases, X is Ala;

PAGLLDLRQG MFAQLVAQNV LLIGGPLSWY SDPGLAGVSL TGGLSYKEDT KELVVAKAGV YYVFFQLELR RVXAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNS AFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 146), wherein X is any amino acid other than Val. In some cases, X is Ala;

PAGLLDLRQG MFAQLVAQNV LLIGGPLSWY SDPGLAGVSL TGGLSYKEDT KELVVAKAGV YYVFFQLELR RVVAXEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNS AFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 147), wherein X is any amino acid other than Gly. In some cases, X is Ala;

PAGLLDLRQG MFAQLVAQNV LLIGGPLSWY SDPGLAGVSL TGGLSYKEDT KELVVAKAGV YYVFFQLELR RVVAGXGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNS AFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 148), wherein X is any amino acid other than Glu. In some cases, X is Ala;

PAGLLDLRQG MFAQLVAQNV LLIGGPLSWY SDPGLAGVSL TGGLSYKEDT KELVVAKAGV YYVFFQLELR RVVAGEXSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNS AFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 149), wherein X is any amino acid other than Gly. In some cases, X is Ala;

PAGLLDLRQG MFAQLVAQNV LLIGGPLSWY SDPGLAGVSL TGGLSYKEDT KELVVAKAGV YYVFFQLELR RVVAGEGXGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNS AFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 150), wherein X is any amino acid other than Ser. In some cases, X is Ala;

PAGLLDLRQG MFAQLVAQNV LLIGGPLSWY SDPGLAGVSL TGGLSYKEDT KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVXLPPASS EARNS AFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 151), wherein X is any amino acid other than Asp. In some cases, X is Ala;

PAGLLDLRQG MFAQLVAQNV LLIGGPLSWY SDPGLAGVSL TGGLSYKEDT KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDXPPASS EARNS AFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 152), wherein X is any amino acid other than Leu. In some cases, X is Ala;

PAGLLDLRQG MFAQLVAQNV LLIGGPLSWY SDPGLAGVSL TGGLSYKEDT KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLXPASS EARNS AFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 153), wherein X is any amino acid other than Pro. In some cases, X is Ala;

PAGLLDLRQG MFAQLVAQNV LLIGGPLSWY SDPGLAGVSL TGGLSYKEDT KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPAXS EARNSAFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 154), wherein X is any amino acid other than Ser. In some cases, X is Ala;

PAGLLDLRQG MFAQLVAQNV LLIGGPLSWY SDPGLAGVSL TGGLSYKEDT KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASX EARNSAFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 155), wherein X is any amino acid other than Ser. In some cases, X is Ala;

PAGLLDLRQG MFAQLVAQNV LLIGGPLSWY SDPGLAGVSL TGGLSYKEDT KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS XARNSAFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 156), wherein X is any amino acid other than Glu. In some cases, X is Ala;

PAGLLDLRQG MFAQLVAQNV LLIGGPLSWY SDPGLAGVSL TGGLSYKEDT KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EAXNSAFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 157), wherein X is any amino acid other than Arg. In some cases, X is Ala;

PAGLLDLRQG MFAQLVAQNV LLIGGPLSWY SDPGLAGVSL TGGLSYKEDT KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARXSAFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 158), wherein X is any amino acid other than Asn. In some cases, X is Ala;

PAGLLDLRQG MFAQLVAQNV LLIGGPLSWY SDPGLAGVSL TGGLSYKEDT KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNXAFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 159), wherein X is any amino acid other than Ser. In some cases, X is Ala;

PAGLLDLRQG MFAQLVAQNV LLIGGPLSWY SDPGLAGVSL TGGLSYKEDT KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNS AXGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 160), wherein X is any amino acid other than Phe. In some cases, X is Ala;

PAGLLDLRQG MFAQLVAQNV LLIGGPLSWY SDPGLAGVSL TGGLSYKEDT KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ GRLLHLSAGX RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 161), wherein X is any amino acid other than Gln. In some cases, X is Ala;

PAGLLDLRQG MFAQLVAQNV LLIGGPLSWY SDPGLAGVSL TGGLSYKEDT KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ GRLLHLSAGQ XLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 162), wherein X is any amino acid other than Arg. In some cases, X is Ala;

PAGLLDLRQG MFAQLVAQNV LLIGGPLSWY SDPGLAGVSL TGGLSYKEDT KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ GRLLHLSAGQ RXGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 163), wherein X is any amino acid other than Leu. In some cases, X is Ala;

PAGLLDLRQG MFAQLVAQNV LLIGGPLSWY SDPGLAGVSL TGGLSYKEDT KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNS AFGFQ GRLLHLSAGQ RLXVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 164), wherein X is any amino acid other than Gly. In some cases, X is Ala;

PAGLLDLRQG MFAQLVAQNV LLIGGPLSWY SDPGLAGVSL TGGLSYKEDT KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNS AFGFQ GRLLHLSAGQ RLGXHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 165), wherein X is any amino acid other than Val. In some cases, X is Ala;

PAGLLDLRQG MFAQLVAQNV LLIGGPLSWY SDPGLAGVSL TGGLSYKEDT KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNS AFGFQ GRLLHLSAGQ RLGVXLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 166), wherein X is any amino acid other than His. In some cases, X is Ala;

PAGLLDLRQG MFAQLVAQNV LLIGGPLSWY SDPGLAGVSL TGGLSYKEDT KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNS AFGFQ GRLLHLSAGQ RLGVHXHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 167), wherein X is any amino acid other than Leu. In some cases, X is Ala;

PAGLLDLRQG MFAQLVAQNV LLIGGPLSWY SDPGLAGVSL TGGLSYKEDT KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNS AFGFQ GRLLHLSAGQ RLGVHLXTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 168), wherein X is any amino acid other than His. In some cases, X is Ala;

PAGLLDLRQG MFAQLVAQNV LLIGGPLSWY SDPGLAGVSL TGGLSYKEDT KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNS AFGFQ GRLLHLSAGQ RLGVHLHXEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 169), wherein X is any amino acid other than Thr. In some cases, X is Ala;

PAGLLDLRQG MFAQLVAQNV LLIGGPLSWY SDPGLAGVSL TGGLSYKEDT KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNS AFGFQ GRLLHLSAGQ RLGVHLHTXA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 170), wherein X is any amino acid other than Glu. In some cases, X is Ala;

PAGLLDLRQG MFAQLVAQNV LLIGGPLSWY SDPGLAGVSL TGGLSYKEDT KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNS AFGFQ GRLLHLSAGQ RLGVHLHTEA XARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 171), wherein X is any amino acid other than Arg. In some cases, X is Ala;

PAGLLDLRQG MFAQLVAQNV LLIGGPLSWY SDPGLAGVSL TGGLSYKEDT KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNS AFGFQ GRLLHLSAGQ RLGVHLHTEA RAXHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 172), wherein X is any amino acid other than Arg. In some cases, X is Ala;

PAGLLDLRQG MFAQLVAQNV LLIGGPLSWY SDPGLAGVSL TGGLSYKEDT KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNS AFGFQ GRLLHLSAGQ RLGVHLHTEA RARXAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 173), wherein X is any amino acid other than His. In some cases, X is Ala;

PAGLLDLRQG MFAQLVAQNV LLIGGPLSWY SDPGLAGVSL TGGLSYKEDT KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNS AFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAXQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 174), wherein X is any amino acid other than Trp. In some cases, X is Ala;

PAGLLDLRQG MFAQLVAQNV LLIGGPLSWY SDPGLAGVSL TGGLSYKEDT KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNS AFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQXTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 175), wherein X is any amino acid other than Leu. In some cases, X is Ala;

PAGLLDLRQG MFAQLVAQNV LLIGGPLSWY SDPGLAGVSL TGGLSYKEDT KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNS AFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLXQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 176), wherein X is any amino acid other than Thr. In some cases, X is Ala;

PAGLLDLRQG MFAQLVAQNV LLIGGPLSWY SDPGLAGVSL TGGLSYKEDT KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNS AFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTX GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 177), wherein X is any amino acid other than Gln. In some cases, X is Ala;

PAGLLDLRQG MFAQLVAQNV LLIGGPLSWY SDPGLAGVSL TGGLSYKEDT KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNS AFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ XATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 178), wherein X is any amino acid other than Gly. In some cases, X is Ala;

PAGLLDLRQG MFAQLVAQNV LLIGGPLSWY SDPGLAGVSL TGGLSYKEDT KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNS AFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GAXVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 179), wherein X is any amino acid other than Thr. In some cases, X is Ala; and

PAGLLDLRQG MFAQLVAQNV LLIGGPLSWY SDPGLAGVSL TGGLSYKEDT KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNS AFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATXLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 180), where X is any amino acid other than Val. In some cases, X is Ala.

IL-2 variants.

In some cases, the immunomodulatory polypeptide variant present in the TMMP of the invention is an IL-2 polypeptide variant. Wild-type IL-2 binds to the IL-2 receptor (IL-2R), which is a heterotrimeric polypeptide comprising IL-2Ra, IL-2Re, and IL-2Ry.

The amino acid sequence of wild-type IL-2 may be as follows:

- 69 049816

APTSSSTKKT QLQLEHLLLD LQMILNGINN YKNPKLTRML TFKFYMPKKA TELKHLQCLEEELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE

TTFMCEYADE TATIVEFLNRWITFCQSIIS TLT (SEQ ID NO: 15).

Wild-type IL2 binds to the IL2 receptor (IL2R) on the cell surface. The IL2 receptor is in some cases a heterotrimeric polypeptide containing an alpha chain (IL-2Ra, also called CD25), a beta chain (IL-2Re, also called CD122), and a gamma chain (IL-2RY, also called CD132). The amino acid sequences of human IL-2Ra, IL2RP, and IL-2Ry may be as follows.

Human IL-2Ra:

ELCDDDPPE IPHATFKAMA YKEGTMLNCE CKRGFRRIKS

GSLYMLCTGN

SPMQPVDQAS

RGPAESVCKM

SSHSSWDNQC QCTSSATRNT TKQVTPQPEE

LPGHCREPPP WENEATERIY HFVVGQMVYY

TSCLVTTTDF

THGKTRWTQP

QIQTEMAATM

QLICTGEMET

ETSIFTTEYQ

SQFPGEEKPQ

VAVAGVFLLL

QKERKTTEMQ QCVQGYRALH ASPEGRPESE

ISVLLLSGLT

WQRRQRKSRR TI (SEQ ID NO: 16).

Human IL-2Re:

VHAWPDRRRW

REGVRWRVMA

FERHLEFEAR KPLQGEFTTW VYLLINCRNT

VNG

NQTCELLPVS

IQDFKPFENL

TLSPGHTWEE

SPWSQPLAFR

GPWLKKVLKC

TSQFTCFYNS

QASWACNLIL RLMAPISLQV APLLTLKQKQ TKPAALGKDT

NTPDPSKFFS

RANISCVWSQ

GAPDSQKLTT

VHVETHRCNI

EWICLETLTP

IPWLGHLLVG

QLSSEHGGDV

DGALQDTSCQ

VDIVTLRVLC

SWEISQASHY

DTQYEFQVRV LSGAFGFIIL

QKWLSSPFPS

SSFSPGGLAP EISPLEVLER DKVTQLLLQQ DKVPEPASLS SNHSLTSCFT NQGYFFFHLP

DALEIEACQV

FPSRDDLLLF

YFTYDPYSEE

DPDEGVAGAP

SPSLLGGPSP

PSTAPGGSGA

PLGPPTPGVP

DLVDFQPPPE

LVLREAGEEV

TGSSPQPLQP GEERMPPSLQ PDAGPREGVS

LSGEDDAYCT

ERVPRDWDPQ

FPWSRPPGQG

EFRALNARLP LNTDAYLSLQ ELQGQDPTHL V (SEQ ID NO: 17).

Human IL-2RY:

LNTTILTP NGNEDTTADF FLTTMPTDSL

SVSTLPLPEV

QCFVFNVEYM ITSGCQLQKK TLHKLSESQL PSVDGQKRYT EAVVISVGSM

NCTWNSSSEP

EIHLYQTFVV

ELNWNNRFLN

FRVRSRFNPL

GLIISLLCVY

QPTNLTLHYW QLQDPREPRR HCLEHLVQYR CGSAQHWSEW

FWLERTMPRI

YKNSDNDKVQ QATQMLKLQN TDWDHSWTEQ

SHPIHWGSNT

PTLKNLEDLV

KCSHYLFSEE

Lvipwapenl

SVDYRHKFSL

SKENPFLFAL

TEYHGNFSAW

SGVSKGLAES

LQPDYSERLC

LVSEIPPKGG

ALGEGPGASP

CNQHSPYWAP

PCYTLKPET (SEQ ID NO: 18).

In some cases, when the TMMP of the present invention comprises a variant IL-2 polypeptide, the cognate co-immunomodulatory polypeptide is an IL-2R polypeptide comprising a polypeptide comprising the amino acid sequence of SEQ ID NOs: 16, 17, and 18.

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

In some cases, the IL-2 polypeptide variant has a binding affinity for IL-2R that is between 100 nM and 100 μM. As another example, in some cases, the IL-2 polypeptide variant has a binding affinity for IL-2R (e.g., an IL-2R polypeptide comprising a polypeptide

- 70 049816 comprising the amino acid sequence presented in SEQ ID NO: 16-18), which is from about 100 nM to 150 nM, from about 150 nM to about 200 nM, from about 200 nM to about 250 nM, from about 250 nM to about 300 nM, from about 300 nM to about 350 nM, from about 350 nM to about 400 nM, from about 400 nM to about 500 nM, from about 500 nM to about 600 nM, from about 600 nM to about 700 nM, from about 700 nM to about 800 nM, from about 800 nM to about 900 nM, from about 900 nM to about 1 μM, to about 1 μM to about 5 μM, from about 5 μM to about 10 μM, from about 10 μM to about 15 μM, from about 15 μM to about 20 μM, from about 20 μM to about 25 μM, from about 25 μM to about 50 μM, from about 50 μM to about 75 μM, or from about 75 μM to about 100 μM.

In some cases, the IL-2 polypeptide variant has one amino acid substitution compared to the IL-2 amino acid sequence set forth in SEQ ID NO: 15. In some cases, the IL-2 polypeptide variant has 2 to 10 amino acid substitutions compared to the IL-2 amino acid sequence set forth in SEQ ID NO: 15. In some cases, the IL-2 polypeptide variant has 2 amino acid substitutions compared to the IL-2 amino acid sequence set forth in SEQ ID NO: 15. In some cases, the IL-2 polypeptide variant has 3 amino acid substitutions compared to the IL-2 amino acid sequence set forth in SEQ ID NO: 15. In some cases, the IL-2 polypeptide variant has 4 amino acid substitutions compared to the IL-2 amino acid sequence set forth in SEQ ID NO: 15. In some cases, the IL-2 polypeptide variant has 5 amino acid substitutions compared to the IL-2 amino acid sequence set forth in provided in SEQ ID NO: 15. In some cases, the IL-2 polypeptide variant has 6 amino acid substitutions compared to the IL-2 amino acid sequence provided in SEQ ID NO: 15. In some cases, the IL-2 polypeptide variant has 7 amino acid substitutions compared to the IL-2 amino acid sequence provided in SEQ ID NO: 15. In some cases, the IL-2 polypeptide variant has 8 amino acid substitutions compared to the IL-2 amino acid sequence provided in SEQ ID NO: 15. In some cases, the IL-2 polypeptide variant has 9 amino acid substitutions compared to the IL-2 amino acid sequence provided in SEQ ID NO: 15. In some cases, the IL-2 polypeptide variant has 10 amino acid substitutions compared to the IL-2 amino acid sequence provided in SEQ ID NO: 15.

Suitable IL-2 variants include a polypeptide 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 one of the following amino acid sequences:

APTSSSTKKT QLQLEHLLLD LQMILNGINN YKNPKLTRML TXI<FYMPI<I<A

TELKHLQCLE EELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNR WITFCQSIIS TLT (SEQ ID NO: 181), wherein X is any amino acid other than Phe. In some cases, X is Ala. In some cases, X is Met. In some cases, X is Pro. In some cases, X is Ser. In some cases, X is Thr. In some cases, X is Trp. In some cases, X is Tyr. In some cases, X is Val. In some cases, X is His;

APTSSSTKKT QLQLEHLLLX LQMILNGINN YKNPKLTRML TFKFYMPKKA

TELKHLQCLE EELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNR WITFCQSIIS TLT (SEQ ID NO: 182), wherein X is any amino acid other than Asp. In some cases, X is Ala;

APTSSSTKKT QLQLXHLLLD LQMILNGINN YKNPKLTRML TFKFYMPKKA

TELKHLQCLE EELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNR WITFCQSIIS TLT (SEQ ID NO: 183), where X is any amino acid other than Glu. In some cases, X is Ala.

APTSSSTKKT QLQLEXLLLD LQMILNGINN YKNPKLTRML TFKFYMPKKA

TELKHLQCLE EELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNR WITFCQSIIS TLT (SEQ ID NO: 184), wherein X is any amino acid other than His. In some cases, X is Ala. In some cases, X is Thr. In some cases, X is Asn. In some cases, X is Cys. In some cases, X is Gln. In some cases, X is Met. In some cases, X is Val. In some cases, X is Trp;

- 71 049816

APTSSSTKKT QLQLEXLLLD LQMILNGINN YKNPKLTRML TFKFYMPKKA

TELKHLQCLE EELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE

TTFMCEYADE TATIVEFLNR WITFCQSIIS TLT (SEQ ID NO: 185), wherein X is any amino acid other than His. In some cases, X is Ala. In some cases, X is Arg. In some cases, X is Asn. In some cases, X is Asp. In some cases, X is Cys. In some cases, X is Glu. In some cases, X is Gln. In some cases, X is Gly. In some cases, X is Ile. In some cases, X is Lys. In some cases, X is Leu. In some cases, X is Met. In some cases, X is Phe. In some cases, X is Pro. In some cases, X is Ser. In some cases, X is Thr. In some cases, X is Tyr. In some cases, X is Trp. In some cases, X is Val;

APTSSSTKKT QLQLEHLLLD LQMILNGINN YKNPKLTRML TFKFXMPKKA

TELKHLQCLE EELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE

TTFMCEYADE TATIVEFLNR WITFCQSIIS TLT (SEQ ID NO: 186), wherein X is any amino acid other than Tyr. In some cases, X is Ala;

APTSSSTKKT QLQLEHLLLD LQMILNGINN YKNPKLTRML TFKFYMPKKA

TELKHLQCLE EELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE

TTFMCEYADE TATIVEFLNR WITFCXSIIS TLT (SEQ ID NO: 187), wherein X is any amino acid other than Gln. In some cases, X is Ala;

APTSSSTKKT QLQLEX1LLLD LQMILNGINN YKNPKLTRML TFKFYMPKKA

TELKHLQCLE EELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE

TTFMCEYADE TATIVEFLNR WITFCQSIIS TLT (SEQ ID NO: 188), wherein X1 is any amino acid other than His, and wherein X2 is any amino acid other than Phe. In some instances, _X1 is Ala. In some instances, X2 is Ala. In some instances, X1 is Ala and X2 is Ala. In some instances, X1 is Thr and X2 is Ala;

APTSSSTKKT QLQLEHLLLX1 LQMILNGINN YKNPKLTRML TFKFYMPKKA

TELKHLQCLE EELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE

TTFMCEYADE TATIVEFLNR WITFCQSIIS TLT (SEQ ID NO: 189), wherein X1 is any amino acid other than Asp; and wherein X2 is any amino acid other than Phe. In some instances, _X1 is Ala. In some instances, X2 is Ala. In some instances, X1 is Ala and X2 is Ala;

APTSSSTKKT QLQLFHLLLF LQMILNGINN YKNPKLTRML TFKFYMPKKA

TELKHLQCLE EELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE

TTFMCEYADE TATIVEFLNR WITFCQSIIS TLT (SEQ ID NO: 190), wherein X1 is any amino acid other than Glu; wherein X2 is any amino acid other than Asp; and wherein X3 is any amino acid other than Phe. In some instances, X1 is Ala. In some instances, X2 is Ala. In some instances, X3 is Ala. In some instances, X1 is Ala, X2 is Ala, and X3 is Ala;

APTSSSTKKT QLQLEFLLLF LQMILNGINN YKNPKLTRML TFKFYMPKKA

TELKHLQCLE EELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE

TTFMCEYADE TATIVEFLNR WITFCQSIIS TLT (SEQ ID NO: 191), wherein X ₁ is any amino acid other than His; wherein X ₂ is any amino acid other than Asp; and wherein X ₃ is any amino acid other than Phe. In some instances, X1 is Ala. In some instances, X2 is Ala. In some instances, X3 is Ala. In some instances, X1 is Ala, X2 is Ala, and X3 is Ala;

APTSSSTKKT QLQLEHLLLF LQMILNGINN YKNPKLTRML TFKFYMPKKA

TELKHLQCLE EELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE

TTFMCEYADE TATIVEFLNR WITFCFSIIS TLT (SEQ ID NO: 192), wherein X1 is any amino acid other than Asp; wherein X2 is any

- 72 049816 any amino acid other than Phe; and wherein X3 is any amino acid other than Gln. In some cases, X1 is Ala. In some cases, X2 is Ala. In some cases, X3 is Ala. In some cases, X1 is Ala, X2 is Ala, and X3 is Ala;

APTSSSTKKT QLQLEHLLLX1 LQMILNGINN YKNPKLTRML TX2KFX3MPKKA

TELKHLQCLE EELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE

TTFMCEYADE TATIVEFLNR WITFCQSIIS TLT (SEQ ID NO: 193), wherein X1 is any amino acid other than Asp; wherein X2 is any amino acid other than Phe; and wherein X3 is any amino acid other than Tyr. In some instances, X1 is Ala. In some instances, X ₂ is Ala. In some instances, X3 is Ala. In some instances, X1 is Ala, X2 is Ala, and X3 is Ala;

APTSSSTKKT QLQLEX1LLLX2 LQMILNGINN YKNPKLTRML TX3KFX4MPKKA

TELKHLQCLE EELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE

TTFMCEYADE TATIVEFLNR WITFCQSIIS TLT (SEQ ID NO: 194), wherein X1 is any amino acid other than His; wherein X2 is any amino acid other than Asp; wherein X3 is any amino acid other than Phe; and wherein _X4 is any amino acid other than Tyr. In some instances, X1 is Ala. In some instances, _X2 is Ala. In some instances, _X3 is Ala. In some instances, X4 is Ala. In some instances, X1 is Ala, X2 is Ala, X3 is Ala, and X4 is Ala;

APTSSSTKKT QLQLEHLLLX1 LQMILNGINN YKNPKLTRML TX2KFX3MPKKA

TELKHLQCLE EELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE

TTFMCEYADE TATIVEFLNR WITFCX4SIIS TLT (SEQ ID NO: 195), wherein X1 is any amino acid other than Asp; wherein _X2 is any amino acid other than Phe; wherein X3 is any amino acid other than Tyr; and wherein X4 is any amino acid other than Gln. In some instances, X1 is Ala. In some instances, _X2 is Ala. In some instances, _X3 is Ala. In some instances, X4 is Ala. In some instances, X1 is Ala, X2 is Ala, _X3 is Ala, and _X4 is Ala;

APTSSSTKKT QLQLEX1LLLX2 LQMILNGINN YKNPKLTRML TX3KFX4MPKKA

TELKHLQCLE EELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE

TTFMCEYADE TATIVEFLNR WITFCX5SIIS TLT (SEQ ID NO: 196), wherein X1 is any amino acid other than His; wherein X2 is any amino acid other than Asp; wherein _{X3 is any amino acid other than Phe; wherein X4} is any amino acid other than Tyr; and wherein _X5 is any amino acid other than Gln. In some instances, X1 is Ala. In some instances, X2 is Ala. In some instances, X3 is Ala. In some instances, X4 is Ala. In some instances, X5 is Ala. In some instances, X1 is Ala, _X2 is Ala, _X3 is Ala, _X4 is Ala, and X5 is Ala; and

APTSSSTKKT QLQLEX1LLLD LQMILNGINN YKNPKLTRML TX2KFYMPKKA

TELKHLQCLE EELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE

TTFMCEYADE TATIVEFLNR WITFCX3SIIS TLT (SEQ ID NO: 197), wherein X1 is any amino acid other than His; wherein X2 is any amino acid other than Phe; and wherein X3 is any amino acid other than Gln. In some instances, _X1 is Ala. In some instances, _X2 is Ala. In some instances, X3 is Ala. In some instances, X1 is Ala, X2 is Ala, and X3 is Ala.

Additional polypeptides.

The TMMP polypeptide chain of the present invention may comprise one or more polypeptides in addition to those described above. Suitable additional polypeptides include epitope tags and affinity domains. The one or more additional polypeptides may be included at the N-terminus of the TMMP polypeptide chain, at the C-terminus of the TMMP polypeptide chain, or within the TMMP polypeptide chain.

Epitope tag.

Suitable epitope tags include, but are not limited to, hemagglutinin (HA; e.g., YPYDVPDYA (SEQ ID NO:271); FLAG (e.g., DYKDDDDK (SEQ ID NO:272); c-myc (e.g.,

- 73 049816

EQKLISEEDL; SEQ ID NO:273) _{and the} like.

Affinity domain.

Affinity domains include peptide sequences that can interact with a binding partner, such as one immobilized on a solid support, useful for identification or purification. DNA sequences encoding several consecutive individual amino acids, such as histidine, when fused to an expressed protein can be used for one-step purification of a recombinant protein by high-affinity binding to a resin column such as nickel-sepharose. Typical affinity domains include

His5 (ННННН) (SEQ ID NO:271), HisX6 (НННННН) (SEQ ID NO:275),

C-myc (EQKLISEEDL) (SEQ ID NO:276), Flag (DYKDDDDK) (SEQ ID NO:277), StrepTag (WSHPQFEK) (SEQ ID NO:278), hemagglutinin, e.g., ^{HA Tag} ( ^{SE Q} ID NO:279), glutathione-S-transferase (GST), thioredoxin reductase, cellulose binding domain, RYIRS (SEQ ID NO:280), Phe-His-His-Thr (SEQ ID NO:281), chitin binding domain, S peptide, T7 peptide, SH2 domain, C-terminal RNA tag, WEAAAREACCRECCARA (SEQ ID NO:282), metal binding domains, e.g., zinc binding domains or calcium binding domains domains such as those from calcium-binding proteins such as calmodulin, troponin C, calcineurin B, myosin light chain, recoverin, S-modulin, visinin, VILIP, neurocalcin, hippocalcin, frequinin, caltracin, calpain large subunit, S100 proteins, parvalbumin, calbindin D9K, calbindin D28K and calretinin, inteins, biotin, streptavidin, MyoD, Id, leucine zipper sequences and maltose binding protein.

Drug conjugates.

The TMMP polypeptide chain of the present invention may comprise a small molecule drug associated (e.g., covalently attached) with the polypeptide chain. For example, when the TMMP of the present invention comprises an Fc polypeptide, the Fc polypeptide may comprise a covalently associated small molecule drug. In some cases, the small molecule drug is a chemotherapeutic agent for cancer, such as a cytotoxic agent. The TMMP polypeptide chain of the present invention may comprise a cytotoxic agent associated (e.g., covalently attached) with the polypeptide chain. For example, when the TMMP of the present invention comprises an Fc polypeptide, the Fc polypeptide may comprise a covalently associated cytotoxic agent. Cytotoxic agents include prodrugs.

A drug (e.g., a chemotherapeutic anticancer agent) can be linked directly or indirectly to a polypeptide chain of a TMMP of the present invention. For example, when a TMMP of the present invention comprises an Fc polypeptide, a drug (e.g., a chemotherapeutic anticancer agent) can be linked directly or indirectly to the Fc polypeptide. A direct linkage can include a linkage directly to an amino acid side chain. An indirect linkage can be linked through a linker. A drug (e.g., a chemotherapeutic anticancer agent) can be linked to a polypeptide chain (e.g., an Fc polypeptide) of a TMMP of the present invention through a thioether bond, an amide bond, a carbamate bond, a disulfide bond, or an ether bond.

Linkers include cleavable linkers and non-cleavable linkers. In some cases, the linker is a protease cleavable linker. Suitable linkers include, for example, peptides (e.g., 2 to 10 amino acids in length; e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids in length), alkyl chains, poly(ethylene glycol), disulfide groups, thioether groups, acid labile groups, photolabile groups, peptidase labile groups, and esterase labile groups. Non-limiting examples of suitable linkers include: i) X-succinimides-(CHmaleimidepropionamide)-tetraethyleneglycol]ester (NHS-PEG4-maleimidine); ii) N-succinimidyl 4-(2-pyridyldithio)butanoate (SPDB); N-succinimidyl 4-(2-pyridyldithio)2-sulfbutanoate (sulf-SPDB); N-succinimidyl 4-(2-pyridyldithio)pentanoate (SPP); N-succinimidyl 4-(N-maleimidomethyl)-cyclohexane-1-carboxy-(6-amidocaproate) (LC-SMCC); k-maleimidundecanodic acid N-succinimidyl ester (KMUA); γ-maleimide-butyric acid N-succinimidyl ester (GMBS); ε-maleimidocaproic acid N-hydroxysuccinimide ester (EMCS); m-maleimide benzoyl-X-hydroxysuccinimide ester (MBS); N-(α-maleimidacetoxy)succinimide ester (AMAS); succinimidyl 6-(p-maleimidopropionamide)hexanoate (SMPH); N-succinimidyl 4-(p-maleimidophenyl)butyrate (SMPB); N-(p-maleimidophenyl)isocyanate (PMPI); N-succinimidyl 4(2-pyridylthio)pentanoate (SPP); N-succinimidyl (4-iodoacetyl)aminobenzoate (SIAB); 6-maleimidocaproyl (MC); maleimidopropanoyl (MP); p-aminobenzyloxtocarbonyl (PAB); N-succinimidyl 4-(maleimidomethyl)cyclohexanecarboxylate (SMCC); N-succinimidyl 4-(N-maleimidomethyl)-cyclohexane-1-carboxy-(6-amidecaproate), long chain analog of SMCC (LC-SMCC); N-succinimidyl ester of 3-maleimidopropanoic acid (BMPS); N-succinimidyl iodoacetate (SIA); N-succinimidyl bromoacetate (SBA) and N-succinimidyl 3

- 74 049816 (bromoacetamide) propionate (SBAP).

A polypeptide (e.g., an Fc polypeptide) can be modified with crosslinking reagents such as succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), sulfo-SMCC, maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), sulfo-MBS, or succinimidyl iodoacetate, as described in the literature, to introduce 1-10 reactive groups. The modified Fc polypeptide is then reacted with a thiol-containing cytotoxic agent to form a conjugate.

For example, when a TMMP of the present invention comprises an Fc polypeptide, wherein the polypeptide chain comprising the Fc polypeptide may have the formula (A)-(L)-(C), wherein (A) is a polypeptide chain comprising the Fc polypeptide; wherein (L), if present, is a linker; and wherein (C) is a cytotoxic agent. (L), if present, links (A) to (C). In some cases, the polypeptide chain comprising the Fc polypeptide may comprise more than one cytotoxic agent (e.g., 2, 3, 4, or 5, or more than 5 cytotoxic agents).

Suitable drugs include, for example, rapamycin.

Suitable drugs include, for example, retinoids such as all-trans retinoic acid (ATRA); vitamin D3; a vitamin D3 analog; and the like. As noted above, in some cases the drug is a cytotoxic agent. Cytotoxic agents are known in the art. A suitable cytotoxic agent may be any compound that results in or causes cell death or in any way reduces cell viability and includes, for example, maytansinoids and maytansinoid analogues, benzodiazepines, taxoids, CC-1065 and CC-1065 analogues, duocarmycins and duocarmycin analogues, enediynes such as calicheamicins, dolastatin and dolastatin analogues including auristatins, tomaimycin derivatives, leptomycin derivatives, methotrexate, cisplatin, carboplatin, daunorubicin, mitochloroacetic alcohol, zuclofin sulfine, morincillin, morincillin, molibucin, rifin sulfine, cycocylphosphine, morincillin, morincholuxin, morinfincil, albumin, morinfucin, chlorgin, chlorgin, rifin, chlorgin, rifin, chlorgincin, molibuxin, ciloxin, chlorgincin, alpha-quinsulfin doxorubicin.

For example, in some cases, the cytotoxic agent is a compound that inhibits microtubule formation in eukaryotic cells. Such agents include, for example, a maytansinoid, a benzodiazepine, a taxoid, CC-1065, duocarmycin, a duocarmycin analog, calicheamicin, dolastatin, a dolastatin analog, auristatin, tomaimycin, and leptomycin, or a prodrug of any of the foregoing. Maytansinoid compounds include, for example, N(2')-deacetyl-N(2')-(3-mercapto-1-oxopropyl)maytansine (DM1); N(2')-deacetyl-N(2')-(4-mercapto-1-oxopentyl)-maytansine (DM3) and N(2')-deacetyl-N2-(4-mercapto-4-methyl-1-oxopentyl)-maytansine (DM4). Benzodiazepines include, for example, indolinobenzodiazepines and oxazolidinobenzodiazepines.

Cytotoxic agents include taxol; cytochalasin B; gramicidin D; ethidium bromide; emetine; mitomycin; etoposide; tenopside; vincristine; vinblastine; colchicine; doxorubicin; daunorubicin; dihydroxy anthracindione; maytansine or its analogue or derivative; auristatin or functional peptide analogue or derivative; dolastatin 10 or 15, or its analogue; irinotecan or its analogue; mitoxantrone; mithramycin; actinomycin D; 1-dehydrotestosterone; glucocorticoid; procaine; tetracaine; lidocaine; propranolol; puromycin; calicheamicin or its analogue or derivative; antimetabolite; 6-mercaptopurine; 6-thioguanine; cytarabine; fludarabine; 5-fluorouracil; decarbazine; hydroxyurea; asparaginase; gemcitabine; cladribine; alkylating agent; platinum derivative; duocarmycin A; duocarmycin SA; rachelmycin (CC-1065) or its analogue or derivative; antibiotic; pyrrole[2,1-c][1,4]-benzodiazepines (PDB); diphtheria toxin; ricin toxin; cholera toxin; Shiga-like toxin; LT toxin; C3 toxin; Shiga toxin; pertussis toxin; tetanus toxin; soybean protease inhibitor Bowman-Birk; Pseudomonas exotoxin; alorin; saporin; modeccin; gelanin; abrin A chain; modeccin A chain; alpha-sarcin; Aleurites fordii proteins; dianthin proteins; Phytolacca americana proteins; Momordica charanstica inhibitor; Curcin; Crotin; Saponaria officinalis inhibitors; Gelonin; Mitogellin; Restrictocin; Fenomycin; Enomycin toxins; Ribonucleases (RNases); DNase I; Staphylococcal enterotoxin A; Pokeweed antiviral protein; Diphtheria toxin and Pseudomonas endotoxin.

Examples of TMMR.

The TMMP of the invention comprises at least one heterodimeric polypeptide comprising: a) a first polypeptide comprising: i) a WT-1 peptide epitope; and ii) a first MHC polypeptide; b) a second polypeptide comprising a second MHC polypeptide; and c) at least one immunomodulatory polypeptide, wherein the first and/or second polypeptide comprises an immunomodulatory polypeptide. Thus, in some cases, the TMMP of the invention comprises at least one heterodimer comprising: a) a first polypeptide comprising: i) a WT-1 peptide epitope; ii) a first MHC polypeptide; and iii) at least one immunomodulatory polypeptide; and b) a second polypeptide comprising a second MHC polypeptide. In some examples, the TMMP of the invention comprises at least one heterodimeric polypeptide comprising: a) a first polypeptide comprising: i) a WT-1 peptide epitope; and ii) a first MHC polypeptide; and b) the second polyp

- 75 049 816 a tid comprising a second MHC polypeptide; and ii) at least one immunomodulatory polypeptide. In some cases, a TMMP of the present invention comprises at least one heterodimer comprising: a) a first polypeptide comprising: i) a WT-1 peptide epitope; ii) a first MHC polypeptide; and iii) at least one immunomodulatory polypeptide; and b) a second polypeptide comprising: i) a second MHC polypeptide, and ii) at least one immunomodulatory polypeptide. In some cases, the at least one immunomodulatory polypeptide is a wild-type immunomodulatory polypeptide. In other cases, the at least one immunomodulatory polypeptide is a variant of an immunomodulatory polypeptide that exhibits reduced affinity for a co-immunomodulatory polypeptide compared to the affinity of the corresponding wild-type immunomodulatory polypeptide for a co-immunomodulatory polypeptide. In some cases, the TMMP of the present invention comprises two immunomodulatory polypeptides, wherein the two immunomodulatory polypeptides have the same amino acid sequence.

In some cases, the TMMP of the invention comprises: a) a first polypeptide comprising, in N-terminal to C-terminal direction: i) a WT-1 peptide epitope; ii) a first MHC polypeptide; and iii) at least one immunomodulatory polypeptide; and b) a second polypeptide comprising, in N-terminal to C-terminal direction: i) a second MHC polypeptide; and ii) an Ig Fc polypeptide. In some cases, the first MHC polypeptide is a B2M polypeptide; and the second MHC polypeptide is an HLA heavy chain polypeptide. In some cases, the HLA heavy chain polypeptide is an HLA-A24 polypeptide. In some cases, the HLA heavy chain polypeptide is HLA-A24 with the A236C substitution. In some cases, the first polypeptide comprises, from N-terminal to C-terminal: i) a WT-1 peptide epitope; ii) a first MHC polypeptide; and iii) two immunomodulatory polypeptides, wherein the two immunomodulatory polypeptides have the same amino acid sequence. In some cases, the Ig Fc polypeptide is a human IgG1 Fc polypeptide. In some cases, the Ig Fc polypeptide is an IgG1 Fc polypeptide comprising the substitutions L234A and L235A. In some cases, the first and second polypeptides are linked to each other by a disulfide bond. In some cases, the immunomodulatory polypeptide is a variant IL-2 polypeptide comprising the substitutions H16A and F42A. In some cases, the immunomodulatory polypeptide is a variant IL-2 polypeptide comprising the substitutions H16T and F42A. In some cases, the peptide linker is between one or more of: i) a second MHC polypeptide and the Ig Fc polypeptide; ii) an epitope and the first MHC polypeptide; iii) a first MHC polypeptide and the immunomodulatory polypeptide; and (when the TMMP comprises two immunomodulatory polypeptides on the first polypeptide chain) iv) between the two immunomodulatory polypeptides. In some cases, the peptide linker comprises the amino acid sequence AAAGG (SEQ ID NO: 283). In some cases, the peptide linker comprises the amino acid sequence (GGGGS) _n (SEQ ID NO: 284), where n is an integer from 1 to 10 (e.g., where n is 2, 3, or 4). In some cases, the WT-1 peptide epitope is CMTWNQMN (SEQ ID NO: 261). In some cases, the WT-1 peptide epitope is CYTWNQMNL (SEQ ID NO: 286).

In some cases, the TMMP of the invention comprises: a) a first polypeptide comprising, in N-terminal to C-terminal direction: i) a WT-1 peptide epitope; and ii) a first MHC polypeptide; and b) a second polypeptide comprising, in N-terminal to C-terminal direction: i) at least an immunomodulatory polypeptide; ii) a second MHC polypeptide; and iii) an IgFc polypeptide. In some cases, the first MHC polypeptide is a B2M polypeptide; and the second MHC polypeptide is an HLA heavy chain polypeptide. In some cases, the HLA heavy chain polypeptide is an HLA-A24 polypeptide. In some cases, the HLA heavy chain polypeptide is HLA-A24 with the A236C substitution. In some cases, the second polypeptide comprises, in N-terminal to C-terminal direction: i) two immunomodulatory polypeptides, wherein the two immunomodulatory polypeptides have the same amino acid sequence; ii) a second MHC polypeptide; and iii) an Ig Fc polypeptide. In some cases, the Ig Fc polypeptide is a human IgG1 Fc polypeptide. In some cases, the Ig Fc polypeptide is an IgG1 Fc polypeptide comprising the substitutions L234A and L235A. In some cases, the first and second polypeptides are linked to each other by a disulfide bond. In some cases, the immunomodulatory polypeptide is a variant IL-2 polypeptide comprising the substitutions H16A and F42A. In some cases, the immunomodulatory polypeptide is a variant IL-2 polypeptide comprising the substitutions H16T and F42A. In some cases, the peptide linker is between one or more of: i) the second MHC polypeptide and the Ig Fc polypeptide; ii) the epitope and the first MHC polypeptide; iii) the first MHC polypeptide and the immunomodulatory polypeptide; and (when the TMMP comprises two immunomodulatory polypeptides on the second polypeptide chain) iv) between the two immunomodulatory polypeptides. In some cases, the peptide linker comprises the amino acid sequence AAAGG (SEQ ID NO: 283). In some cases, the peptide linker comprises the amino acid sequence (GGGGS) _n (SEQ ID NO: 284), where n is an integer from 1 to 10 (e.g., where n is 2, 3, or 4). In some cases,

- 76 049816 the WT-1 peptide epitope is CMTWNQMN (SEQ ID NO: 261). In some cases, the WT-1 peptide epitope is CYTWNQMNL (SEQ ID NO: 286).

In some cases, the TMMP of the invention comprises: a) a first polypeptide comprising, in N-terminal to C-terminal direction: i) a WT-1 peptide epitope; and ii) a first MHC polypeptide; and b) a second polypeptide comprising, in N-terminal to C-terminal direction: i) a second MHC polypeptide; ii) an IgFc polypeptide; and iii) at least one immunomodulatory polypeptide. In some cases, the first MHC polypeptide is a B2M polypeptide; and the second MHC polypeptide is an HLA heavy chain polypeptide. In some cases, the HLA heavy chain polypeptide is an HLA-A24 polypeptide. In some cases, the HLA heavy chain polypeptide is HLA-A24 with the A236C substitution. In some cases, the second polypeptide comprises, in N-terminal to C-terminal direction: i) a second MHC polypeptide; ii) an IgFc polypeptide; and iii) two immunomodulatory polypeptides, wherein the two immunomodulatory polypeptides have the same amino acid sequence. In some cases, the Ig Fc polypeptide is a human IgG1 Fc polypeptide. In some cases, the Ig Fc polypeptide is an IgG1 Fc polypeptide comprising the substitutions L234A and L235A. In some cases, the first and second polypeptides are linked to each other by a disulfide bond. In some cases, the immunomodulatory polypeptide is a variant IL-2 polypeptide comprising the substitutions H16A and F42A. In some cases, the immunomodulatory polypeptide is a variant IL-2 polypeptide comprising the substitutions H16T and F42A. In some cases, the peptide linker is between one or more of: i) a second MHC polypeptide and the Ig Fc polypeptide; ii) an epitope and the first MHC polypeptide; iii) an Ig Fc polypeptide and the immunomodulatory polypeptide; and (when the TMMP comprises two immunomodulatory polypeptides on the second polypeptide chain) iv) between the two immunomodulatory polypeptides. In some cases, the peptide linker comprises the amino acid sequence AAAGG (SEQ ID NO: 283). In some cases, the peptide linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO: 284), wherein n is an integer from 1 to 10 (e.g., wherein n is 2, 3, or 4). In some cases, the WT-1 peptide epitope is CMTWNQMN (SEQ ID NO: 261). In some cases, the WT-1 peptide epitope is CYTWNQMNL (SEQ ID NO: 286).

In some cases, the TMMP of the present invention comprises: a) a first polypeptide comprising, in N-terminal to C-terminal direction: i) a WT-1 peptide epitope; and ii) a first MHC polypeptide; and b) a second polypeptide comprising, in N-terminal to C-terminal direction: i) at least an immunomodulatory polypeptide; ii) a second MHC polypeptide; and iii) an IgFc polypeptide. In some cases, the first MHC polypeptide is a B2M polypeptide; and the second MHC polypeptide is an HLA heavy chain polypeptide. In some cases, the HLA heavy chain polypeptide is an HLA-A24 polypeptide. In some cases, the HLA heavy chain polypeptide is HLA-A24 with an A236C substitution. In some cases, the IgFc polypeptide is a human IgG1 Fc polypeptide. In some cases, the Ig Fc polypeptide is an IgG1 Fc polypeptide comprising the substitutions L234A and L235A. In some cases, the first and second polypeptides are linked to each other by a disulfide bond. In some cases, the immunomodulatory polypeptide is an IL-2 polypeptide variant comprising the substitutions H16A and F42A. In some cases, the immunomodulatory polypeptide is an IL-2 polypeptide variant comprising the substitutions H16T and F42A. In some cases, the WT-1 peptide epitope is CMTWNQMN (SEQ ID NO: 261).

In some cases, the WT-1 peptide epitope is CYTWNQMNL (SEQ ID NO: 286).

In some cases, the TMMP of the invention comprises: a) a first polypeptide comprising, in N-terminal to C-terminal direction: i) at least one immunomodulatory polypeptide; ii) a WT-1 peptide epitope; and iii) a first MHC polypeptide; and b) a second polypeptide comprising, in N-terminal to C-terminal direction: i) a second MHC polypeptide; and ii) an IgFc polypeptide. In some cases, the first MHC polypeptide is a B2M polypeptide; and the second MHC polypeptide is an HLA heavy chain polypeptide. In some cases, the HLA heavy chain polypeptide is an HLA-A24 polypeptide. In some cases, the HLA heavy chain polypeptide is HLA-A24 with the A236C substitution. In some cases, the first polypeptide comprises, in N-terminal to C-terminal direction: i) two immunomodulatory polypeptides, wherein the two immunomodulatory polypeptides have the same amino acid sequence; ii) a WT-1 peptide epitope; and iii) a first MHC polypeptide. In some cases, the Ig Fc polypeptide is a human IgG1 Fc polypeptide. In some cases, the Ig Fc polypeptide is an IgG1 Fc polypeptide comprising the substitutions L234A and L235A. In some cases, the first and second polypeptides are linked to each other by a disulfide bond. In some cases, the immunomodulatory polypeptide is a variant IL-2 polypeptide comprising the substitutions H16A and F42A. In some cases, the immunomodulatory polypeptide is a variant IL-2 polypeptide comprising the substitutions H16T and F42A. In some cases, the peptide linker is between one or more of: i) the second MHC polypeptide and the Ig Fc polypeptide; ii) the epitope and the first MHC polypeptide; iii) the immunomodulatory polypeptide and the epitope; and (when TMMP contains two immunomodulatory polypeptides on

- 77 049816 of the first polypeptide chain) iv) between two immunomodulatory polypeptides. In some cases, the peptide linker comprises the amino acid sequence ^ ^Α θθ (SEQ ID NO: 283).

In some cases, the peptide linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO: 284), where n is an integer from 1 to 10 (e.g., where n is 2, 3, or 4). In some cases, the WT-1 peptide epitope is CMTWNQMN (SEQ id NO: 261).

In some cases, the WT-1 peptide epitope is CYTWNQMNL (SEQ ID NO: 286).

In some cases, the TMMP of the invention comprises: a) a first polypeptide comprising, in N-terminal to C-terminal direction: i) a WT-1 peptide epitope; and ii) a first MHC polypeptide; and b) a second polypeptide comprising, in N-terminal to C-terminal direction: i) a second MHC polypeptide; ii) at least one immunomodulatory polypeptide; and iii) at least an IgFc polypeptide. In some cases, the first MHC polypeptide is a B2M polypeptide; and the second MHC polypeptide is an HLA heavy chain polypeptide. In some cases, the HLA heavy chain polypeptide is an HLA-A24 polypeptide. In some cases, the HLA heavy chain polypeptide is HLA-A24 with the A236C substitution. In some cases, the second polypeptide comprises, in N-terminal to C-terminal direction: i) a second MHC polypeptide; ii) two immunomodulatory polypeptides, wherein the two immunomodulatory polypeptides have the same amino acid sequence; and iii) an Ig Fc polypeptide. In some cases, the Ig Fc polypeptide is a human IgG1 Fc polypeptide. In some cases, the Ig Fc polypeptide is an IgG1 Fc polypeptide comprising the substitutions L234A and L235A. In some cases, the first and second polypeptides are linked to each other by a disulfide bond. In some cases, the immunomodulatory polypeptide is an IL-2 polypeptide variant comprising the substitutions H16A and F42A. In some cases, the immunomodulatory polypeptide is an IL-2 polypeptide variant comprising the substitutions H16T and F42A. In some cases, the peptide linker is between one or more of: i) a second MHC polypeptide and the immunomodulatory polypeptide; ii) an immunomodulatory polypeptide and the Ig Fc polypeptide; iii) an epitope and a first MHC polypeptide; iii) a first MHC polypeptide and an immunomodulatory polypeptide; and (when the TMMP comprises two immunomodulatory polypeptides on a second polypeptide chain) iv) between the two immunomodulatory polypeptides. In some cases, the peptide linker comprises the amino acid sequence AAAGG (SEQ ID NO: 283).

In some cases, the peptide linker comprises the amino acid sequence (GGGGS) _n (SEQ ID NO: 284), where n is an integer from 1 to 10 (e.g., where n is 2, 3, or 4). In some cases, the WT-1 peptide epitope is CMTWNQMN (SEQ id NO: 261).

In some cases, the WT-1 peptide epitope is CYTWNQMNL (SEQ ID NO: 286).

As noted above, and as schematically depicted in Fig. 19, the immunomodulatory polypeptide (i.e., one or more immunomodulatory polypeptides) can be present in the TMMPs of the invention at any of a variety of positions. Fig. 19 depicts the position of two copies of a variant IL-2 polypeptide; however, the immunomodulatory polypeptide can be any of a variety of immunomodulatory polypeptides described herein. As depicted in Fig. 19, the immunomodulatory polypeptide can be: 1) N-terminal to the MHC class I heavy chain (position 1); 2) C-terminal to the MHC class I heavy chain and N-terminal to the Ig Fc polypeptide; in other words, located between the MHC class I heavy chain and the Ig Fc polypeptide (position 2); 3) C-terminal to the Ig Fc polypeptide (position 3); 4) N-terminal to a peptide epitope (position 4); or 5) C-terminal to a β2M polypeptide (position 5). Position 1 refers to the position of the immunomodulatory polypeptide on the same polypeptide chain as the MHC class I heavy chain and is N-terminal to the MHC class I heavy chain; for example, when the TMMP comprises: a) a first polypeptide comprising, in N-terminal to C-terminal direction: i) a peptide epitope (e.g., WT-1 peptide); and ii) a β2M polypeptide; and b) a second polypeptide comprising, in N-terminal to C-terminal direction: i) one or more immunomodulatory polypeptides; and ii) an MHC class I heavy chain polypeptide. Position 2 refers to a position of the immunomodulatory polypeptide on the same polypeptide chain as the MHC class I heavy chain and is at the C-terminus of the MHC class I heavy chain; but not at the C-terminus of the polypeptide chain; for example, when a TMMP comprises: a) a first polypeptide comprising, in N-terminal to C-terminal direction: i) a peptide epitope (e.g., a WT-1 peptide); and ii) a B2M polypeptide; and b) a second polypeptide comprising, in N-terminal to C-terminal direction: i) an MHC class I heavy chain polypeptide; ii) one or more immunomodulatory polypeptides; and iii) an Ig Fc polypeptide. Position 3 refers to a position of the immunomodulatory polypeptide on the same polypeptide chain as the MHC class I heavy chain and is at the C-terminus of the polypeptide chain; for example, when the TMMP comprises: a) a first polypeptide comprising, in N-terminal to C-terminal direction: i) a peptide epitope (e.g., a WT-1 peptide); and ii) a B2M polypeptide; and b) a second polypeptide comprising, in N-terminal to C-terminal direction: i) an MHC class I heavy chain polypeptide; ii) an Ig Fc polypeptide; and iii) one or more immunomodulatory polypeptides. Position 4 refers to the position of the immunomodulatory polypeptide on the same polypeptide chain as the B2M polypeptide and is N-terminal to the peptide epitope and the B2M polypeptide; for example, when the TMMP comprises: a) a first polypeptide comprising

- 78 049816 comprising, in N-terminal to C-terminal direction: i) one or more immunomodulatory polypeptides; ii) a peptide epitope (e.g., a WT-1 peptide); and iii) a β2M polypeptide; and b) a second polypeptide comprising an MHC class I heavy chain polypeptide (e.g., a second polypeptide comprising, in N-terminal to C-terminal direction: i) an MHC class I heavy chain polypeptide; and ii) an Ig Fc polypeptide. Position 5 refers to the position of the immunomodulatory polypeptide on the same polypeptide chain as the β2M polypeptide and is C-terminal to the β2M polypeptide (e.g., located at the C-terminus of the polypeptide chain); for example, when a TMMP comprises: a) a first polypeptide comprising, in N-terminal to C-terminal direction: i) a peptide epitope (e.g., a WT-1 peptide); and ii) a β2M polypeptide; iii) one or more immunomodulatory polypeptides; and b) a second polypeptide comprising an MHC class I heavy chain polypeptide (e.g., a second polypeptide comprising, in N-terminal to C-terminal direction: i) an MHC class I heavy chain polypeptide; and ii) an Ig Fc polypeptide.

Furthermore, as discussed above and as schematically depicted in Fig. 18A-18C, the first polypeptide chain and the second polypeptide chain of the TMMP of the present invention can be linked by one or more disulfide bonds. For example, the TMMP of the present invention can comprise: a) a first polypeptide chain comprising a B2M polypeptide with an R12C substitution; and b) a second polypeptide chain comprising an MHC class I heavy chain polypeptide with an A236C substitution; such that the disulfide bond is formed between the Cys at position 12 of the B2M polypeptide in the first polypeptide chain and the Cys at position 236 of the MHC class I heavy chain polypeptide in the second polypeptide chain. As another example, the TMMP of the present invention can comprise: a) a first polypeptide comprising, in N-terminal to C-terminal direction: i) a peptide epitope; ii) a peptide linker comprising the sequence GCGG(G4S)n (SEQ ID NO: 393), wherein n is 1, 2, or 3; and iii) a p2M polypeptide; and b) a second polypeptide comprising an MHC class I heavy chain polypeptide with a Y84C substitution such that the disulfide bond is between the Cys in the peptide linker in the first polypeptide chain and the Cys at position 84 of the MHC class I polypeptide in the second polypeptide chain. In other examples, a TMMP of the invention may comprise: a) a first polypeptide comprising, in N-terminal to C-terminal direction: i) a peptide epitope; ii) a peptide linker comprising the sequence GCGG(G4S) _n (SEQ ID NO: 393), wherein n is 1, 2, or 3; and iii) a p2M polypeptide with an R12C substitution; and b) a second polypeptide comprising an MHC class I heavy chain polypeptide with a Y84C substitution and an A236C substitution; such that: i) a first disulfide bond is formed between a Cys in the first linker in the first polypeptide chain and a Cys at position 84 of the MHC class I heavy chain polypeptide in the second polypeptide chain; and ii) a second disulfide bond between a Cys at position 12 of the B2M polypeptide in the first polypeptide chain and a Cys at position 236 of the MHC class I heavy chain polypeptide in the second polypeptide chain. For simplicity, the first disulfide bond is referred to as G2C/Y84C; and the second disulfide bond is referred to as R12C/A236C. A TMMP of the present invention may comprise: a) a G2C/Y84C disulfide bond but not an R12C/A236C disulfide bond; b) the R12C/A236C disulfide bond but not the G2C/Y84C disulfide bond; or c) the G2C/Y84C disulfide bond and the R12C/A236C disulfide bond.

The TMMP of the invention may comprise: a) a G2C/Y84C disulfide bond but not an R12C/A236C disulfide bond; and b) at least one immunomodulatory polypeptide at position 1. The TMMP of the invention may comprise: a) a G2C/Y84C disulfide bond but not an R12C/A236C disulfide bond; and b) at least one immunomodulatory polypeptide at position 2. The TMMP of the invention may comprise: a) a G2C/Y84C disulfide bond but not an R12C/A236C disulfide bond; and b) at least one immunomodulatory polypeptide at position 3. The TMMP of the invention may comprise: a) a G2C/Y84C disulfide bond but not an R12C/A236C disulfide bond; and b) at least one immunomodulatory polypeptide at position 4. The TMMP of the present invention may comprise: a) a G2C/Y84C disulfide bond, but not a R12C/A236C disulfide bond; and b) at least one immunomodulatory polypeptide at position 5.

The TMMP of the present invention may comprise: a) an R12C/A236C disulfide bond, but not a G2C/Y84C disulfide bond; and at least one immunomodulatory polypeptide at position 1. The TMMP of the present invention may comprise: a) an R12C/A236C disulfide bond, but not a G2C/Y84C disulfide bond; and at least one immunomodulatory polypeptide at position 2. The TMMP of the present invention may comprise: a) an R12C/A236C disulfide bond, but not a G2C/Y84C disulfide bond; and at least one immunomodulatory polypeptide at position 3. The TMMP of the present invention may comprise: a) an R12C/A236C disulfide bond, but not a G2C/Y84C disulfide bond; and at least one immunomodulatory polypeptide at position 4. The TMMP of the present invention may comprise: a) an R12C/A236C disulfide bond, but not a G2C/Y84C disulfide bond; and at least one immunomodulatory polypeptide at position 5.

The TMMP of the invention may comprise: a) a G2C/Y84C disulfide bond and an R12C/A236C disulfide bond; and b) and at least one immunomodulatory polypeptide at position 1. The TMMP of the invention may comprise: a) a G2C/Y84C disulfide bond and an R12C/A236C disulfide bond; and b) and at least one immunomodulatory polypeptide at position 2. The TMMP of the invention may comprise: a) a G2C/Y84C disulfide bond and an R12C/A236C disulfide bond

- 79 049816 R12C/A236C bond; and b) and at least one immunomodulatory polypeptide at position 3. The TMMP of the present invention may comprise: a) a G2C/Y84C disulfide bond and an R12C/A236C disulfide bond; and b) and at least one immunomodulatory polypeptide at position 4. The TMMP of the present invention may comprise: a) a G2C/Y84C disulfide bond and an R12C/A236C disulfide bond; and b) and at least one immunomodulatory polypeptide at position 5.

Non-limiting examples of amino acid sequences of the first and second polypeptide chains of the TMMP of the present invention are shown in Figs. 4A-4K and Figs. 20A-20R.

In some cases, the TMMP of the present invention comprises: a) a first polypeptide chain comprising an amino acid sequence designated 2752, as depicted in Fig. 4D; and b) a second polypeptide chain comprising an amino acid sequence designated 3159, as depicted in Fig. 4C.

In some cases, a TMMP of the present invention comprises: a) a first polypeptide chain comprising an amino acid sequence designated 2753, as depicted in Fig. 4E; and b) a second polypeptide chain comprising an amino acid sequence designated 3159, as depicted in Fig. 4C. Such a TMMP comprises: a) an immunomodulatory polypeptide at position 3, as depicted in Fig. 19; and b) an R12C/A236C disulfide bond (but not a G2C/Y84C disulfide bond).

In some cases, the TMMP of the present invention comprises: a) a first polypeptide chain comprising an amino acid sequence designated 2752, as depicted in Fig. 4D; and b) a second polypeptide chain comprising an amino acid sequence designated 2750, as depicted in Fig. 4B.

In some cases, a TMMP of the present invention comprises: a) a first polypeptide chain comprising an amino acid sequence designated 2753, as depicted in Fig. 4E; and b) a second polypeptide chain comprising an amino acid sequence designated 2750, as depicted in Fig. 4B. Such a TMMP comprises: a) an immunomodulatory polypeptide at position 1, as depicted in Fig. 19; and b) an R12C/A236C disulfide bond (but not a G2C/Y84C disulfide bond).

In some cases, the TMMP of the present invention comprises: a) a first polypeptide chain comprising an amino acid sequence designated 2752, as depicted in Fig. 4D; and b) a second polypeptide chain comprising an amino acid sequence designated 3158, as depicted in Fig. 4A.

In some cases, the TMMP of the present invention comprises: a) a first polypeptide chain comprising an amino acid sequence designated 2753, as depicted in Fig. 4E; and b) a second polypeptide chain comprising an amino acid sequence designated 3158, as depicted in Fig. 4A.

In some cases, the TMMP of the present invention comprises: a) a first polypeptide chain comprising an amino acid sequence designated 2380, as depicted in Fig. 14B; and b) a second polypeptide chain comprising an amino acid sequence designated 1715, as depicted in Fig. 14A.

In some cases, the TMMP of the present invention comprises: a) a first polypeptide chain comprising an amino acid sequence designated 2381, as depicted in Fig. 14B; and b) a second polypeptide chain comprising an amino acid sequence designated 1715, as depicted in Fig. 14A.

In some cases, the TMMP of the present invention comprises: a) a first polypeptide chain comprising an amino acid sequence designated 2380, as depicted in Fig. 14B; and b) a second polypeptide chain comprising an amino acid sequence designated 2405, as depicted in Fig. 14D.

In some cases, a TMMP of the present invention comprises: a) a first polypeptide chain comprising an amino acid sequence designated 2381, as depicted in Fig. 14B; and b) a second polypeptide chain comprising an amino acid sequence designated 2405, as depicted in Fig. 14D. In some cases, a TMMP of the present invention comprises: a) a first polypeptide chain comprising an amino acid sequence designated 2762, as depicted in Fig. 14F; and b) a second polypeptide chain comprising an amino acid sequence designated 2405, as depicted in Fig. 14D.

In some cases, the TMMP of the present invention comprises: a) a first polypeptide chain comprising an amino acid sequence designated 2380, as depicted in Fig. 14B; and b) a second polypeptide chain comprising an amino acid sequence designated 1380, as depicted in Fig. 14E.

In some cases, the TMMP of the present invention comprises: a) a first polypeptide chain comprising an amino acid sequence designated 2381, as depicted in Fig. 14B; and b) a second polypeptide chain comprising an amino acid sequence designated 1380, as depicted in Fig. 14E.

- 80 049816

In some cases, a TMMP of the present invention comprises: a) a first polypeptide chain comprising an amino acid sequence designated 3592, as depicted in Fig. 20A; and b) a second polypeptide chain comprising an amino acid sequence designated 3188, as depicted in Fig. 20H. Such a TMMP comprises: a) an immunomodulatory polypeptide at position 1, as depicted in Fig. 19; and b) both a G2C/Y84C disulfide bond and an R12C/A236C disulfide bond.

In some cases, a TMMP of the present invention comprises: a) a first polypeptide chain comprising an amino acid sequence designated 3425, as depicted in Fig. 20B; and b) a second polypeptide chain comprising an amino acid sequence designated 3188, as depicted in Fig. 20H. Such a TMMP comprises: a) an immunomodulatory polypeptide at position 3, as depicted in Fig. 19; and b) both a G2C/Y84C disulfide bond and an R12C/A236C disulfide bond.

In some cases, the TMMP of the present invention comprises: a) a first polypeptide chain comprising an amino acid sequence designated 3196, as depicted in Fig. 20C; and b) a second polypeptide chain comprising an amino acid sequence designated 3604, as depicted in Fig. 201. Such a TMMP comprises: a) an immunomodulatory polypeptide at position 5, as depicted in Fig. 19; and b) both a G2C/Y84C disulfide bond and an R12C/A236C disulfide bond.

In some cases, a TMMP of the present invention comprises: a) a first polypeptide chain comprising an amino acid sequence designated 2764, as depicted in Fig. 20D; and b) a second polypeptide chain comprising an amino acid sequence designated 3603, as depicted in Fig. 20J. Such a TMMP comprises: a) an immunomodulatory polypeptide at position 5, as depicted in Fig. 19; and b) an R12C/A236C disulfide bond (but not a G2C/Y84C disulfide bond).

In some cases, a TMMP of the present invention comprises: a) a first polypeptide chain comprising an amino acid sequence designated 3593, as depicted in Fig. 20E; and b) a second polypeptide chain comprising an amino acid sequence designated 3192, as depicted in Fig. 20K. Such a TMMP comprises: a) an immunomodulatory polypeptide at position 1, as depicted in Fig. 19; and b) a G2C/Y84C disulfide bond (but not an R12C/A236C disulfide bond).

In some cases, a TMMP of the present invention comprises: a) a first polypeptide chain comprising an amino acid sequence designated 3426, as depicted in Fig. 20F; and b) a second polypeptide chain comprising an amino acid sequence designated 3192, as depicted in Fig. 20K. Such a TMMP comprises: a) an immunomodulatory polypeptide at position 3, as depicted in Fig. 19; and b) a G2C/Y84C disulfide bond (but not an R12C/A236C disulfide bond).

In some cases, a TMMP of the present invention comprises: a) a first polypeptide chain comprising an amino acid sequence designated 3197, as depicted in Fig. 20G; and b) a second polypeptide chain comprising an amino acid sequence designated 3605, as depicted in Fig. 20L. Such a TMMP comprises: a) an immunomodulatory polypeptide at position 5, as depicted in Fig. 19; and b) a G2C/Y84C disulfide bond (but not an R12C/A236C disulfide bond).

In some cases, a TMMP of the present invention comprises: a) a first polypeptide chain comprising an amino acid sequence designated 3592, as depicted in Fig. 20A; and b) a second polypeptide chain comprising an amino acid sequence designated 3529, as depicted in Fig. 20M. Such a TMMP comprises: a) an immunomodulatory polypeptide at position 1, as depicted in Fig. 19; and b) both a G2C/Y84C disulfide bond and an R12C/A236C disulfide bond.

In some cases, a TMMP of the present invention comprises: a) a first polypeptide chain comprising an amino acid sequence designated 3425, as depicted in Fig. 20B; and b) a second polypeptide chain comprising an amino acid sequence designated 3529, as depicted in Fig. 20M. Such a TMMP comprises: a) an immunomodulatory polypeptide at position 3, as depicted in Fig. 19; and b) both a G2C/Y84C disulfide bond and an R12C/A236C disulfide bond; and also comprises a WT1 epitope 239-247 (Q240Y).

In some cases, a TMMP of the present invention comprises: a) a first polypeptide chain comprising an amino acid sequence designated 3196, as depicted in Fig. 20C; and b) a second polypeptide chain comprising an amino acid sequence designated 3709, as depicted in Fig. 20N. Such a TMMP comprises: a) an immunomodulatory polypeptide at position 3, as depicted in Fig. 19; and b) both a G2C/Y84C disulfide bond and an R12C/A236C disulfide bond.

In some cases, the TMMP of the present invention comprises: a) a first polypeptide chain comprising an amino acid sequence designated 2750, as depicted in Fig. 4B; and

- 81 049816

b) a second polypeptide chain comprising the amino acid sequence designated 3528, as depicted in Fig. 20O. Such a TMMP comprises: a) an immunomodulatory polypeptide at position 1, as depicted in Fig. 19; and b) an R12C/A236C disulfide bond (but not a G2C/Y84C disulfide bond).

In some cases, a TMMP of the present invention comprises: a) a first polypeptide chain comprising an amino acid sequence designated 3159, as depicted in Fig. 4C; and b) a second polypeptide chain comprising an amino acid sequence designated 3528, as depicted in Fig. 20O. Such a TMMP comprises: a) an immunomodulatory polypeptide at position 3, as depicted in Fig. 19; and b) an R12C/A236C disulfide bond (but not a G2C/Y84C disulfide bond).

In some cases, a TMMP of the present invention comprises: a) a first polypeptide chain comprising an amino acid sequence designated 2764, as depicted in Fig. 20D; and b) a second polypeptide chain comprising an amino acid sequence designated 3708, as depicted in Fig. 20P. Such a TMMP comprises: a) an immunomodulatory polypeptide at position 5, as depicted in Fig. 19; and b) an R12C/A236C disulfide bond (but not a G2C/Y84C disulfide bond).

In some cases, a TMMP of the present invention comprises: a) a first polypeptide chain comprising an amino acid sequence designated 3593, as depicted in Fig. 20E; and b) a second polypeptide chain comprising an amino acid sequence designated 3530, as depicted in Fig. 20Q. Such a TMMP comprises: a) an immunomodulatory polypeptide at position 1, as depicted in Fig. 19; and b) a G2C/Y84C disulfide bond (but not an R12C/A236C disulfide bond).

In some cases, a TMMP of the present invention comprises: a) a first polypeptide chain comprising an amino acid sequence designated 3426, as depicted in Fig. 20F; and b) a second polypeptide chain comprising an amino acid sequence designated 3530, as depicted in Fig. 20Q. Such a TMMP comprises: a) an immunomodulatory polypeptide at position 3, as depicted in Fig. 19; and b) a G2C/Y84C disulfide bond (but not an R12C/A236C disulfide bond).

In some cases, a TMMP of the present invention comprises: a) a first polypeptide chain comprising an amino acid sequence designated 3197, as depicted in Fig. 20G; and b) a second polypeptide chain comprising an amino acid sequence designated 3710, as depicted in Fig. 20R. Such a TMMP comprises: a) an immunomodulatory polypeptide at position 5, as depicted in Fig. 19; and b) a G2C/Y84C disulfide bond (but not an R12C/A236C disulfide bond).

In some cases, a TMMP of the present invention comprises: a) a first polypeptide chain comprising an amino acid sequence designated 3426, as depicted in Fig. 20F; and b) a second polypeptide chain comprising an amino acid sequence designated 3529, as depicted in Fig. 20M. Such a TMMP comprises: a) an immunomodulatory polypeptide at position 3, as depicted in Fig. 19; and b) a G2C/Y84C disulfide bond (but not an R12C/A236C disulfide bond), and also includes the WT1 epitope 239-247 (Q240Y).

In some cases, a TMMP of the present invention comprises: a) a first polypeptide chain comprising an amino acid sequence designated 3425, as depicted in Fig. 20B; and b) a second polypeptide chain comprising an amino acid sequence designated 3528, as depicted in Fig. 20O. Such a TMMP comprises: a) an immunomodulatory polypeptide at position 3, as depicted in Fig. 19; and b) an R12C/A236C disulfide bond (but not a G2C/Y84C disulfide bond) and also includes the WT1 epitope 239-247 (Q240Y).

In some cases, a TMMP of the present invention comprises: a) a first polypeptide chain comprising an amino acid sequence designated 3425, as depicted in Fig. 20B; and b) a second polypeptide chain comprising an amino acid sequence designated 3530, as depicted in Fig. 20Q. Such a TMMP comprises: a) an immunomodulatory polypeptide at position 3, as depicted in Fig. 19; and b) a G2C/Y84C disulfide bond (but not an R12C/A236C disulfide bond), and also includes the WT1 epitope 239-247 (Q240Y).

In some cases, a TMMP of the present invention comprises: a) a first polypeptide chain comprising an amino acid sequence designated 3159, as depicted in Fig. 4C; and b) a second polypeptide chain comprising an amino acid sequence designated 3188, as depicted in Fig. 20H. Such a TMMP comprises: a) an immunomodulatory polypeptide at position 3, as depicted in Fig. 19; and b) an R12C/A236C disulfide bond (but not a G2C/Y84C disulfide bond), and also includes the WT1 epitope 235-243 (M236Y).

Methods for producing a multimeric T-cell modulating polypeptide. The present invention provides a method for producing a TMMP comprising one or more variant immunomodulatory polypeptides that exhibit reduced affinity for a cognate co-immunomodulatory polypeptide compared to the affinity of the corresponding parent immunomodulatory polypeptide.

- 82 049 816 a wild-type polypeptide to a co-immunomodulatory polypeptide, comprising: A) creating a TMMP library comprising a plurality of members, wherein each member comprises: a) a first polypeptide comprising: i) an epitope and ii) a first major MHC polypeptide, and b) a second polypeptide comprising: i) a second MHC polypeptide; and ii) optionally an Ig Fc polypeptide or non-Ig framework, wherein each member comprises a different variant of the immunomodulatory polypeptide on the first polypeptide, the second polypeptide, or both the first and second polypeptides; B) determining the affinity of each library member for a cognate co-immunomodulatory polypeptide; and C) selecting a member that exhibits reduced affinity for the cognate co-immunomodulatory polypeptide. In some cases, affinity is determined by biolayer interferometry (BLI) using purified TMMP library members and a cognate co-immunomodulatory polypeptide. BLI methods are well known to those skilled in the art. The BLI assay is described above. See, e.g., Lad et al. (2015) J. Biomol. Screen. 20(4): 498–507 and Shah and Duncan (2014) J. Vis. Exp. 18:e51383.

The present invention provides a method for producing a TMMP that exhibits selective binding to a T cell, comprising: A) generating a TMMP library comprising a plurality of members, wherein each member comprises: a) a first polypeptide comprising: i) an epitope; and ii) a first MHC polypeptide; and b) a second polypeptide comprising: i) a second MHC polypeptide; and ii) optionally an immunoglobulin (Ig) Fc polypeptide or non-Ig framework, wherein each member comprises a different variant immunomodulatory polypeptide on the first polypeptide, the second polypeptide, or both the first and second polypeptides, wherein the variant immunomodulatory polypeptide differs in amino acid sequence by 1-10 amino acids from the parent wild-type immunomodulatory polypeptide; B) contacting a TMMP library member with a target T cell expressing on its surface: i) a cognate co-immunomodulatory polypeptide that binds a wild-type parental immunomodulatory polypeptide; and ii) a T cell receptor that binds to an epitope, wherein the TMMP library member comprises an epitope tag, such that the TMMP library member binds to the target T cell; C) contacting the TMMP library member that binds to the target T cell with a fluorescently labeled binding agent that binds to the epitope tag, creating a TMMP library member/target T cell/binding agent complex; D) measuring the mean fluorescence intensity (MFI) of the TMMP library member/target T cell/binding agent complex using flow cytometry, wherein the MFI measured over a range of concentrations of the TMMP library member provides a measure of affinity and apparent avidity; and E) selecting a TMMP library member that selectively binds the target T cell as compared to binding of the TMMP library member to a control T cell that comprises: i) a cognate co-immunomodulatory polypeptide that binds the parent wild-type immunomodulatory polypeptide; and ii) a T cell receptor that binds to an epitope different from an epitope present on the TMMP library member. In some cases, the TMMP library member that is identified as selectively binding to the target T cell is isolated from the library.

In some cases, the parental pairs of a wild-type immunomodulatory polypeptide and a cognate immunomodulatory polypeptide are selected from:

IL-2 and IL-2 receptor;

4-1BBL and 4-1BB;

PD-L1 and PD-1;

CD70 and CD27;

TGFe and TGFe receptor;

CD80 and CD28;

CD86 and CD28;

OX40L and OX40;

FasL and Fas;

ICOS-L and ICOS;

ICAM and LFA-1;

JAG1 and Notch;

JAG1 and CD46;

CD80 and CTLA4; and

CD86 and CTLA4.

The present invention provides a method for producing a TMMP comprising one or more variant immunomodulatory polypeptides that exhibit reduced affinity for a cognate co-immunomodulatory polypeptide compared to the affinity of a corresponding wild-type parent immunomodulatory polypeptide for the co-immunomodulatory polypeptide, comprising selecting from a TMMP library comprising a plurality of members a member that exhibits reduced affinity for the cognate co-immunomodulatory polypeptide, wherein the plurality of members comprises: a) a first polypeptide comprising: i) an epitope; and ii) a first MHC polypeptide; and b) a second polypeptide comprising: i) a second MHC polypeptide; and ii) optionally an Ig Fc polypeptide or framework

- 83 049816 not from Ig, wherein the library members comprise a plurality of variants of an immunomodulatory polypeptide present in the first polypeptide, in the second polypeptide, or in both the first and second polypeptides. In some cases, the selection step includes determining the affinity using biolayer binding interferometry between the TMMP library members and the cognate coimmunomodulatory polypeptide. In some cases, the TMMP is as described above.

In some cases, the method further comprises: a) contacting the selected TMMP library member with a target T cell expressing on its surface: i) a cognate coimmunomodulatory polypeptide that binds a wild-type parent immunomodulatory polypeptide; and ii) a T cell receptor that binds to an epitope, wherein the TMMP library member comprises an epitope tag, such that the TMMP library member binds to the target T cell; b) contacting the selected TMMP library member with the target T cell with a fluorescently labeled binding agent that binds to the epitope tag, creating a selected TMMP library member/target T cell/binding agent complex; and c) measuring the mean fluorescence intensity (MFI) of the selected TMMP library member/target T cell/binding agent complex using flow cytometry, wherein the MFI measured over a range of concentrations of the selected TMMP library member provides a measure of affinity and apparent avidity. A selected TMMP library member that specifically binds to a target T cell, as compared to the binding of a TMMP library member to a control target T cell comprising: i) a cognate co-immunomodulatory polypeptide that binds a wild-type parental immunomodulatory polypeptide; and ii) a T cell receptor that binds to an epitope different from the epitope present on the TMMP library member, is identified as selectively binding to the target T cell. In some cases, the binding agent is an antibody specific for an epitope tag. In some cases, the immunomodulatory polypeptide variant comprises 1 to 20 amino acid substitutions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid substitutions) compared to the corresponding parent wild-type immunomodulatory polypeptide. In some cases, the TMMP comprises two immunomodulatory polypeptide variants. In some cases, the two immunomodulatory polypeptide variants comprise the same amino acid sequence. In some cases, the first polypeptide comprises one of the two immunomodulatory polypeptide variants, and wherein the second polypeptide comprises the second of the two immunomodulatory polypeptide variants. In some cases, the two immunomodulatory polypeptide variants are in the same polypeptide chain of the TMMP. In some cases, the two immunomodulatory polypeptide variants are in the first polypeptide chain of the TMMP. In some cases, two variants of immunomodulatory polypeptides are found in the second polypeptide chain of TMMP.

In some cases, the method further comprises isolating the selected TMMP library member from the library. In some cases, the method further comprises providing a nucleic acid comprising a nucleotide sequence encoding the selected TMMP library member. In some cases, the nucleic acid is present in a recombinant expression vector. In some cases, the nucleotide sequence is operably linked to a transcriptional control element that functions in a eukaryotic cell. In some cases, the method further comprises introducing the nucleic acid into a eukaryotic host cell and culturing the cell in a liquid medium to synthesize the encoded selected TMMP library member in the cell. In some cases, the method further comprises isolating the synthesized selected TMMP library member from the cell or from a liquid culture medium containing the cell. In some cases, the selected TMMP library member comprises an IgFc polypeptide. In some cases, the method further comprises conjugating a drug to the IgFc polypeptide. In some cases, the drug is a cytotoxic agent selected from a maytansinoid, a benzodiazepine, a taxoid, CC-1065, duocarmycin, a duocarmycin analog, calicheamicin, dolastatin, a dolastatin analog, auristatin, tomaimycin, and leptomycin, or a prodrug of any of the foregoing. In some cases, the drug is a retinoid. In some cases, the parent wild-type immunomodulatory polypeptide and cognate immunomodulatory polypeptides are selected from: IL-2 and the IL-2 receptor; 4-1BBL and 4-1BB; PD-L1 and PD-1; CD70 and CD27; TGFe and the TGFe receptor; CD80 and CD28; CD86 and CD28; OX40L and OX40; FasL and Fas; ICOS-L and ICOS; ICAM and LFA-1; JAG1 and Notch; JAG1 and CD46; CD80 and CTLA4, and CD86 and CTLA4.

The present invention provides a method for producing a TMMP comprising one or more variant immunomodulatory polypeptides that exhibit reduced affinity for a cognate co-immunomodulatory polypeptide compared to the affinity of a corresponding wild-type parent immunomodulatory polypeptide for the co-immunomodulatory polypeptide, comprising: A) providing a library of TMMPs comprising a plurality of members, wherein the plurality of members comprises: a) a first polypeptide comprising: i) an epitope and ii) a first major MHC polypeptide, and b) a second polypeptide comprising: i) a second MHC polypeptide; and ii) optionally an Ig Fc polypeptide or

- 84 049816 a non-Ig framework, wherein the library members comprise different variants of an immunomodulatory polypeptide present in the first polypeptide, the second polypeptide, or both the first and second polypeptides; and B) selecting from the library a member that exhibits reduced affinity for the cognate co-immunomodulatory polypeptide. In some cases, the selection step comprises determining the affinity using biolayer binding interferometry between the TMMP library members and the cognate co-immunomodulatory polypeptide. In some cases, the TMMP is as described above.

In some cases, the method further comprises: a) contacting the selected TMMP library member with a target T cell expressing on its surface: i) a cognate coimmunomodulatory polypeptide that binds a wild-type parent immunomodulatory polypeptide; and ii) a T cell receptor that binds to an epitope, wherein the TMMP library member comprises an epitope tag, such that the TMMP library member binds to the target T cell; b) contacting the selected TMMP library member with the target T cell with a fluorescently labeled binding agent that binds to the epitope tag, creating a selected TMMP library member/target T cell/binding agent complex; and c) measuring the mean fluorescence intensity (MFI) of the selected TMMP library member/target T cell/binding agent complex using flow cytometry, wherein the MFI measured over a range of concentrations of the selected TMMP library member provides a measure of affinity and apparent avidity. A selected TMMP library member that specifically binds to a target T cell, as compared to the binding of a TMMP library member to a control target T cell comprising: i) a cognate co-immunomodulatory polypeptide that binds a wild-type parental immunomodulatory polypeptide; and ii) a T cell receptor that binds to an epitope different from the epitope present on the TMMP library member, is identified as selectively binding to the target T cell. In some cases, the binding agent is an antibody specific for an epitope tag. In some cases, the immunomodulatory polypeptide variant comprises 1 to 20 amino acid substitutions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid substitutions) compared to the corresponding parent wild-type immunomodulatory polypeptide. In some cases, the TMMP comprises two immunomodulatory polypeptide variants. In some cases, the two immunomodulatory polypeptide variants comprise the same amino acid sequence. In some cases, the first polypeptide comprises one of the two immunomodulatory polypeptide variants, and wherein the second polypeptide comprises the second of the two immunomodulatory polypeptide variants. In some cases, the two immunomodulatory polypeptide variants are in the same polypeptide chain of the TMMP. In some cases, the two immunomodulatory polypeptide variants are in the first polypeptide chain of the TMMP. In some cases, two variants of immunomodulatory polypeptides are found in the second polypeptide chain of TMMP.

In some cases, the method further comprises isolating the selected TMMP library member from the library. In some cases, the method further comprises providing a nucleic acid comprising a nucleotide sequence encoding the selected TMMP library member. In some cases, the nucleic acid is present in a recombinant expression vector. In some cases, the nucleotide sequence is operably linked to a transcriptional control element that functions in a eukaryotic cell. In some cases, the method further comprises introducing the nucleic acid into a eukaryotic host cell and culturing the cell in a liquid medium to synthesize the encoded selected TMMP library member in the cell. In some cases, the method further comprises isolating the synthesized selected TMMP library member from the cell or from a liquid culture medium containing the cell. In some cases, the selected TMMP library member comprises an IgFc polypeptide. In some cases, the method further comprises conjugating a drug to the IgFc polypeptide. In some cases, the drug is a cytotoxic agent selected from a maytansinoid, a benzodiazepine, a taxoid, CC-1065, duocarmycin, a duocarmycin analog, calicheamicin, dolastatin, a dolastatin analog, auristatin, tomaimycin, and leptomycin, or a prodrug of any of the foregoing. In some cases, the drug is a retinoid. In some cases, the parent wild-type immunomodulatory polypeptide and cognate immunomodulatory polypeptides are selected from IL-2 and the IL-2 receptor; 4-1BBL and 4-1BB; PD-L1 and PD-1; TGFe and the TGFe receptor; CD80 and CD28; CD86 and CD28; OX40L and OX40; FasL and Fas; ICOS-L and ICOS; CD70 and CD27; ICAM and LFA-1; JAG1 and Notch; JAG1 and CD46; CD80 and CTLA4, and CD86 and CTLA4.

Nucleic acids.

The present invention relates to a nucleic acid comprising a nucleotide sequence encoding a TMMP according to the present invention. The present invention relates to a nucleic acid comprising a nucleotide sequence encoding a TMMP according to the present invention.

The present invention relates to nucleic acids containing nucleotide sequences

- 85 049816 entities encoding the TMMPs of the invention. In some cases, the individual polypeptide chains of the TMMPs of the invention are encoded by separate nucleic acids. In some cases, all of the polypeptide chains of the TMMPs of the invention are encoded by a single nucleic acid. In some cases, the first nucleic acid comprises a nucleotide sequence encoding a first TMMP polypeptide of the invention, and the second nucleic acid comprises a nucleotide sequence encoding a second TMMP polypeptide of the invention. In some cases, a single nucleic acid comprises a nucleotide sequence encoding a first TMMP polypeptide of the invention and a second TMMP polypeptide of the invention.

Individual nucleic acids encoding individual polypeptide chains of a multimeric polypeptide.

The present invention relates to nucleic acids comprising nucleotide sequences encoding TMMPs of the present invention. As indicated above, in some cases, individual polypeptide chains of the TMMPs of the present invention are encoded by separate nucleic acids. In some cases, nucleotide sequences encoding individual polypeptide chains of the TMMPs of the present invention are operably linked to transcriptional control elements, for example promoters, such as promoters that function in a eukaryotic cell, wherein the promoter can be a constitutive promoter or an inducible promoter.

The present invention provides a first nucleic acid and a second nucleic acid, wherein the first nucleic acid comprises a nucleotide sequence encoding a first TMMP polypeptide of the present invention, wherein the first polypeptide comprises, in an N-terminal to C-terminal direction: a) an epitope (e.g., a T-cell epitope); b) a first MHC polypeptide; and c) an immunomodulatory polypeptide (e.g., a reduced affinity variant as described above); and wherein the second nucleic acid comprises a nucleotide sequence encoding a second TMMP polypeptide of the present invention, wherein the second polypeptide comprises, in an N-terminal to C-terminal direction: a) a second MHC polypeptide; and b) an IgFc polypeptide. Suitable T-cell epitopes, MHC polypeptides, immunomodulatory polypeptides, and IgFc polypeptides are described above. In some cases, the nucleotide sequences encoding the first and second polypeptides are operably linked to transcriptional control elements. In some cases, the transcriptional control element is a promoter that functions in a eukaryotic cell. In some cases, the nucleic acids are present in separate expression vectors.

The present invention provides a first nucleic acid and a second nucleic acid, wherein the first nucleic acid comprises a nucleotide sequence encoding a first TMMP polypeptide of the present invention, wherein the first polypeptide comprises, in an N-terminal to C-terminal direction: a) an epitope (e.g., a T-cell epitope); b) a first MHC polypeptide; and wherein the second nucleic acid comprises a nucleotide sequence encoding a second TMMP polypeptide of the present invention, wherein the second polypeptide comprises, in an N-terminal to C-terminal direction: a) an immunomodulatory polypeptide (e.g., a reduced affinity variant as described above); b) a second MHC polypeptide; and c) an IgFc polypeptide. Suitable T-cell epitopes, MHC polypeptides, immunomodulatory polypeptides, and IgFc polypeptides are described above. In some cases, the nucleotide sequences encoding the first and second polypeptides are operably linked to transcriptional control elements. In some cases, the transcriptional control element is a promoter that functions in a eukaryotic cell. In some cases, the nucleic acids are present in separate expression vectors.

A nucleic acid encoding two or more polypeptides present in a multimeric polypeptide.

The present invention relates to a nucleic acid comprising nucleotide sequences encoding at least a first polypeptide and a second polypeptide of a TMMP of the invention. In some cases, when a TMMP of the invention comprises a first, second and third polypeptide, the nucleic acid comprises a nucleotide sequence encoding the first, second and third polypeptides. In some cases, the nucleotide sequences encoding the first polypeptide and the second TMMP polypeptide of the invention comprise a proteolytically cleavable linker inserted between the nucleotide sequence encoding the first polypeptide and the nucleotide sequence encoding the second polypeptide. In some cases, the nucleotide sequences encoding the first polypeptide and the second TMMP polypeptide of the invention comprise an internal ribosome entry site (IRES) inserted between the nucleotide sequence encoding the first polypeptide and the nucleotide sequence encoding the second polypeptide. In some cases, the nucleotide sequences encoding the first polypeptide and the second TMMP polypeptide of the present invention include a ribosome skipping signal (or cis-acting hydrolase element, CHYSEL (SEQ ID NO: 394)) inserted between the nucleotide sequence encoding the first polypeptide and the nucleotide sequence encoding the second polypeptide. Exemplary nucleic acids are described below.

- 86 049816, wherein a proteolytically cleavable linker is provided between the nucleotide sequences encoding the first polypeptide and the second TMMP polypeptide of the invention; in any of these embodiments, instead of the nucleotide sequence encoding the proteolytically cleavable linker, an IRES or ribosome skipping signal can be used.

In some cases, the first nucleic acid (e.g., a recombinant expression vector, mRNA, viral RNA, etc.) comprises a nucleotide sequence encoding a first polypeptide chain of the TMMP of the present invention, and the second nucleic acid (e.g., a recombinant expression vector, mRNA, viral RNA, etc.) comprises a nucleotide sequence encoding a second polypeptide chain of the multimeric polypeptide of the present invention. In some cases, the nucleotide sequence encoding the first polypeptide and the second nucleotide sequence encoding the second polypeptide are each operably linked to transcriptional control elements, such as promoters, such as promoters that function in a eukaryotic cell, wherein the promoter can be a constitutive promoter or an inducible promoter.

The present invention relates to a nucleic acid comprising a nucleotide sequence encoding a recombinant polypeptide, wherein the recombinant polypeptide comprises, in N-terminal to C-terminal direction: a) an epitope (e.g., a T-cell epitope); b) a first MHC polypeptide; c) an immunomodulatory polypeptide (e.g., a reduced affinity variant as described above); d) a proteolytically cleavable linker; e) a second MHC polypeptide; and f) an immunoglobulin (Ig) Fc polypeptide. The present invention relates to a nucleic acid comprising a nucleotide sequence encoding a recombinant polypeptide, wherein the recombinant polypeptide comprises, in N-terminal to C-terminal direction: a) a first leader peptide; b) an epitope; c) a first MHC polypeptide; d) an immunomodulatory polypeptide (e.g., a reduced affinity variant as described above); e) a proteolytically cleavable linker; f) a second leader peptide; g) a second MHC polypeptide; and h) an IgFc polypeptide. The invention relates to a nucleic acid comprising a nucleotide sequence encoding a recombinant polypeptide, wherein the recombinant polypeptide comprises, in N-terminal to C-terminal direction: a) an epitope; b) a first MHC polypeptide; c) a proteolytically cleavable linker; d) an immunomodulatory polypeptide (e.g., a reduced affinity variant as described above); e) a second MHC polypeptide; and f) an IgFc polypeptide. In some cases, the first leader peptide and the second leader peptide are a β2-M leader peptide. In some cases, the nucleotide sequence is operably linked to a transcriptional control element. In some cases, the transcriptional control element is a promoter that functions in a eukaryotic cell.

Suitable MHC polypeptides are described above. In some cases, the first MHC polypeptide is a β2-microglobulin polypeptide; and wherein the second MHC polypeptide is a class I MHC heavy chain polypeptide. In some cases, the β2-microglobulin polypeptide comprises an amino acid sequence having at least 85% amino acid sequence identity to the β2M amino acid sequence depicted in Fig. 6. In some cases, the class I MHC heavy chain polypeptide is a heavy chain of HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, HLA-G, HLA-K, or HLA-L. In some cases, the class I MHC heavy chain polypeptide comprises an amino acid sequence having at least 85% amino acid sequence identity to the amino acid sequence depicted in any of Figs. 3A-3C.

Suitable Fc polypeptides are described above. In some cases, the Ig Fc polypeptide is an IgG1 Fc polypeptide, an IgG2 Fc polypeptide, an IgG3 Fc polypeptide, an IgG4 Fc polypeptide, an IgA Fc polypeptide, or an IgM Fc polypeptide. In some cases, the Ig Fc polypeptide comprises an amino acid sequence having at least 85% amino acid sequence identity to the amino acid sequence depicted in Figs. 5A-5G.

Suitable immunomodulatory polypeptides are described above.

Suitable proteolytically cleavable linkers are described above. In some cases, the proteolytically cleavable linker comprises an amino acid sequence selected from:

a) LEVLFQGP (SEQ ID NO:388); b) ENLYTQS (SEQ

ID NO:389); c) DDDDK (SEQ ID NO:390); d) LVPR (SEQ ID NO:391); and e) GSGATNFSLLKQAGDVEENPGP (SEQ ID NO:392).

In some cases, the linker between the epitope and the first MHC polypeptide comprises a first Cys residue, and the second MHC polypeptide comprises an amino acid substitution to provide a second Cys residue, such that the first and second Cys residues provide a disulfide bond between the linker and the second MHC polypeptide. In some cases, the first MHC polypeptide comprises an amino acid substitution to provide a first Cys residue, and the second MHC polypeptide comprises an amino acid substitution to provide a second Cys residue, such that the first Cys residue and the second Cys residue provide a disulfide bond between the first MHC polypeptide and the second MHC polypeptide.

- 87 049816

Recombinant expression vectors.

The present invention provides recombinant expression vectors comprising the nucleic acids of the present invention. In some cases, the recombinant expression vector is a vector that is not derived from a virus. 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 adenoviral construct, a recombinant lentiviral construct, a recombinant retroviral construct, a non-integrating viral vector, etc.

Suitable expression vectors include, but are not limited to, viral vectors (e.g., vaccinia virus; poliovirus; adenovirus (see, e.g., Li et al., Invest Opthalmol Vis Sci 35:2543 2549, 1994; Borras et al., Gene Ther 6:515 524, 1999; Li and Davidson, PNAS 92:7700 7704, 1995; Sakamoto et al., H Gene Ther 5:1088 1097, 1999; WO 94/12649, WO 93/03769; WO 93/19191; WO 94/28938; WO 95/11984; and WO 95/00655); adeno-associated virus (see, for example, AN et al., Hum Gene Ther 9:81 86, 1998, Flannery et al., PNAS 94:6916 6921, 1997; Bennett et al., Invest Opthalmol Vis Sci 38:2857 2863, 1997; Jomary et al., Gene Ther 4:683 690, 1997, Rolling et al., Hum Gene Ther 10:641 648, 1999; Hum Mol Genet 5:591 594, 1996; et al., Virol. (1988) 166:154-165; and Flotte et al., PNAS (1993) 90:10613-10617); SV40; herpes simplex virus; human immunodeficiency virus (see, e.g., Miyoshi et al., PNAS 94:10319 23, 1997; Takahashi et al., J Virol 73:7812 7816, 1999); retroviral vector (e.g., murine leukemia virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous sarcoma virus, Harvey sarcoma virus, avian leukemia virus, lentivirus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus); and the like.

Numerous suitable expression vectors are known to those skilled in the art, and many are commercially available. The following vectors are provided as examples; for eukaryotic host cells: pXT1, pSG5 (Stratagene), pSVK3, pBPV, pMSG, and pSVLSV40 (Pharmacia). However, any other vector may be used as long as it is compatible with the host cell.

Depending on the host/vector system used, any of the appropriate transcriptional and translational control elements, including constitutive and inducible promoters, transcriptional enhancer elements, transcriptional terminators, etc., can be used in the expression vector (see, e.g., Bitter et al. (1987) Methods in Enzymology, 153:516-544).

In some cases, the nucleotide sequence encoding the DNA-targeting RNA and/or the site-directed modifying polypeptide is operably linked to a control element, such as a transcriptional control element such as a promoter. The transcriptional control element may be functional in any eukaryotic cell, such as a mammalian cell; or a prokaryotic cell (e.g., a bacterial cell or an archaeal cell). In some cases, the nucleotide sequence encoding the DNA-targeting RNA and/or the site-directed modifying polypeptide is operably linked to multiple control elements that provide for expression of the nucleotide sequence encoding the DNA-targeting RNA and/or the site-directed modifying polypeptide in both prokaryotic and eukaryotic cells.

Non-limiting examples of suitable eukaryotic promoters (promoters that function in a eukaryotic cell) include the cytomegalovirus (CMV) early-stage promoters, herpes simplex virus (HSV) thymidine kinase, SV40 early and late, retrovirus long terminal repeats (LTRs), and mouse metallothionein-I. The selection of a suitable vector and promoter is within the ordinary level of skill in the art. The expression vector may also contain a ribosome binding site for translation initiation and a transcription terminator. The expression vector may also contain appropriate sequences for enhancing expression.

Genetically modified host cells.

The present invention provides a genetically modified host cell, wherein the host cell is genetically modified with a nucleic acid of the present invention.

Suitable host cells include eukaryotic cells such as yeast cells, insect cells, and mammalian cells. In some cases, the host cell is a cell 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, but are not limited to, HeLa cells (e.g., American Type Culture Collection (ATCC) #CCL-2), CHO cells (e.g., ATCC #CRL9618, CCL61, CRL9096), 293 cells (e.g., ATCC #CRL-1573), Vero cells, NIH 3T3 cells (e.g., ATCC #CRL-1658), Huh-7 cells, BHK cells (e.g., ATCC #CCL10), PC12 cells (ATCC #CRL1721), COS cells, COS-7 cells (ATCC #CRL1651), RAT1 cells, mouse L cells (ATCC #CCLI.3), human embryonic kidney (HEK) cells (ATCC #CRL1573), HLHepG2, and the like.

- 88 049816

In some cases, the host cell is a mammalian cell that has been genetically modified so that it does not synthesize endogenous MHC b2-M.

In some cases, the host cell is a mammalian cell that has been genetically modified so that it does not synthesize endogenous class I MHC heavy chain. In some cases, the host cell is a mammalian cell that has been genetically modified so that it does not synthesize endogenous β2-M MHC and so that it does not synthesize endogenous class I MHC heavy chain.

Compositions.

The present invention relates to compositions, including pharmaceutical compositions, comprising a TMMP (synTac) of the present invention. The present invention relates to compositions, including pharmaceutical compositions, comprising a TMMP of the present invention. The present invention relates to compositions, including pharmaceutical compositions, comprising a nucleic acid or a recombinant expression vector of the present invention.

Compositions containing a multimeric polypeptide.

The composition of the present invention may contain, in addition to the TMMP of the present invention, one or more of the following: a salt, such as NaCl, MgCl ₂ , KCl, MgSO ₄ , etc.; a buffering agent, such as Tris buffer, N-(2-hydroxyethyl) piperazine-N-(2-ethanesulfonic acid) (HEPES), 2-(N-morpholino)ethanesulfonic acid (MES), sodium salt of 2-(N-morpholino)ethanesulfonic acid (MES), 3-(N-morpholino)propanesulfonic acid (MOPS), N-tris[hydroxymethyl]methyl-3-aminopropanesulfonic acid (TAPS), etc.; a solubilizing agent; a detergent, such as a nonionic detergent such as Tween-20, etc.; a protease inhibitor; glycerol; and the like.

The composition may comprise a pharmaceutically acceptable excipient, a variety of which are known in the art and need not be discussed in detail herein. Pharmaceutically acceptable excipients are described in detail in various publications including, for example, Remington: The Science and Practice of Pharmacy, ^19th Ed. (1995), or latest edition, Mack Publishing Co; A. Gennaro (2000) Remington: The Science and Practice of Pharmacy, ^20th edition, Lippincott, Williams, &Wilkins; Pharmaceutical Dosage Forms and Drug Delivery Systems (1999) HC Ansel et al., eds ^7th ed., Lippincott, Williams, &Wilkins; and Handbook of Pharmaceutical Excipients (2000) AH Kibbe et al., eds., ^3rd ed. Amer. Pharmaceutical Assoc.

The pharmaceutical composition may comprise the TMMP of the invention and a pharmaceutically acceptable excipient. In some cases, the pharmaceutical composition in question will be suitable for administration to a subject, such as being sterile. For example, in some cases, the pharmaceutical composition in question will be suitable for administration to a human subject, such as when the composition is sterile and does not contain detectable pyrogens and/or other toxins.

The protein compositions may contain other components such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose, magnesium, carbonate, and the like. The compositions may contain pharmaceutically acceptable auxiliary substances necessary to approximate physiological conditions, such as pH adjusting and buffering agents, toxicity adjusting agents, and the like, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, hydrochloride, sulfate salt, solvates (e.g., mixed ionic salts, water, organics), hydrates (e.g., water), and the like.

For example, the compositions may include an aqueous solution, a powder form, granules, tablets, pills, suppositories, capsules, suspensions, sprays, and the like. The composition may be formulated according to the various routes of administration described below.

When the TMMP of the present invention is administered by injection (e.g., subcutaneously, intraperitoneally, intramuscularly, and/or intravenously) directly into tissue, the formulation may be provided as a ready-to-use dosage form or as an aqueous form (e.g., a reconstitutable, shelf-stable powder) or an aqueous form such as a liquid comprised of pharmaceutically acceptable carriers and excipients. Protein-containing preparations may also be provided to increase the serum half-life of the TMMP following administration. For example, the TMMP may be provided as a liposome composition prepared as a colloid or by other conventional methods for increasing the serum half-life. There are numerous methods for preparing liposomes, as described, for example, in Szoka et al., 1980 Ann. Rev. Biophys. Bioeng. 9:467, U.S. Patents 4,235,871, 4,501,728, and 4,837,028. The formulations may also be provided in controlled-release or sustained-release forms.

Other examples of formulations suitable for parenteral administration include isotonic sterile injection solutions, antioxidants, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient, suspending agents, solubilizers, thickeners, stabilizers and preservatives. For example, the pharmaceutical under consideration

- 89 049816 the composition may be present in a container, for example, a sterile container such as a syringe. The compositions may be presented in sealed single- or multiple-dose containers such as ampoules and vials, and may be stored under frozen (lyophilized) conditions requiring only the addition of a sterile liquid excipient, for example, water for injection, immediately before use. Injectable solutions and suspensions for immediate administration may be prepared from sterile powders, granules and tablets.

The concentration of the TMMP of the present invention in the composition can vary widely (e.g., from less than about 0.1%, typically from or from at least about 2% up to 20% to 50% or more by weight) and will typically be selected based primarily on fluid volumes, viscosity, and patient-related factors in accordance with the particular route of administration selected and the needs of the patient.

The present invention provides a container containing a composition of the present invention, for example a liquid composition. The container may be, for example, a syringe, an ampoule, and the like. In some cases, the container is sterile. In some cases, both the container and the composition are sterile.

The present invention relates to compositions, including pharmaceutical compositions, comprising a TMMP of the present invention. The composition may comprise: a) a TMMP of the present invention; and b) an excipient as described above. In some cases, the excipient is a pharmaceutically acceptable excipient.

In some cases, the TMMP of the invention is present in a liquid composition. Accordingly, the invention relates to compositions (e.g., liquid compositions, including pharmaceutical compositions) comprising the TMMP of the invention. In some cases, the composition of the invention comprises: a) the TMMP of the invention; and b) a saline solution (e.g., 0.9% NaCl). In some cases, the composition is sterile. In some cases, the composition is suitable for administration to a human subject, for example, where the composition is sterile and free of detectable pyrogens and/or other toxins. Accordingly, the invention relates to a composition comprising: a) the TMMP of the invention; and b) a saline solution (e.g., 0.9% NaCl), wherein the composition is sterile and free of detectable pyrogens and/or other toxins.

Compositions containing a nucleic acid or a recombinant expression vector

The present invention relates to compositions, e.g., pharmaceutical compositions, comprising a nucleic acid or a recombinant expression vector of the present invention. A wide variety of pharmaceutically acceptable excipients are known in the art and need not be discussed in detail herein. Pharmaceutically acceptable excipients are described in detail in various publications including, for example, A. Gennaro (2000) Remington: The Science and Practice of Pharmacy, ^20th edition, Lippincott, Williams, &Wilkins; Pharmaceutical Dosage Forms and Drug Delivery Systems (1999) H. C. Ansel et al, eds ^7th ed., Lippincott, Williams, &Wilkins; and Handbook of Pharmaceutical Excipients (2000) A. H. Kibbe et al., eds., ^3rd ed. Amer. Pharmaceutical Assoc.

The composition of the present invention may comprise: a) one or more nucleic acids or one or more recombinant expression vectors containing nucleotide sequences encoding TMMP; and b) one or more of: a buffer, a surfactant, an antioxidant, a hydrophilic polymer, a dextrin, a chelating agent, a suspending agent, a solubilizer, a thickener, a stabilizer, a bacteriostatic agent, a wetting agent, and a preservative. Suitable buffers include, but are not limited to, (such as N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES), bis(2-hydroxyethyl)amino-tris(hydroxymethyl)methane (BIS-Tris), N-(2-hydroxyethyl)piperazine-N-3-propanesulfonic acid (EPPS or HEPPS), glycylglycine, N-2hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES), 3-(N-morpholino)propanesulfonic acid (MOPS), piperazine-N-bis(2-ethanesulfonic acid) (PIPES), sodium bicarbonate, 3-(N-tris(hydroxymethyl)-methyl-amino-)-2-hydroxy-propanesulfonic acid) TAPSO, (N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES), (tris(hydroxymethyl)methylglycine (Tricine), tris(hydroxymethyl)-aminomethane (Tris), etc.). Suitable salts include, for example, NaCl, MgCl ₂ , KCl, MgSO ₄ , etc.

The pharmaceutical formulation of the invention may comprise a nucleic acid or recombinant expression vector of the invention in an amount of from about 0.001% to about 90% (w/w). In the description of the formulations below, the nucleic acid or recombinant expression vector in question should be understood to include the nucleic acid or recombinant expression vector of the invention. For example, in some cases, the formulation of the invention comprises a nucleic acid or recombinant expression vector of the invention.

The target nucleic acid or recombinant expression vector may be mixed, encapsulated, conjugated or otherwise associated with other compounds or mixtures of compounds; such compounds may include, for example, liposomes or receptor-targeting molecules. The target nucleic acid or recombinant expression vector may be combined in

- 90 049816 with one or more components that promote absorption, distribution and/or absorption.

The composition of the subject nucleic acid or recombinant expression vector may be presented in any of a variety of possible dosage forms, such as, but not limited to, tablets, capsules, gelcaps, liquid syrups, soft gels, suppositories and enemas. The target composition of the nucleic acid or recombinant expression vector may also be prepared as a suspension in an aqueous, non-aqueous or mixed medium. Aqueous suspensions may further contain substances that increase the viscosity of the suspension, including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension may also contain stabilizers.

The composition containing the nucleic acid or recombinant expression vector may be a liposomal composition. As used herein, the term liposome means a vesicle consisting of amphiphilic lipids arranged in a spherical bilayer or bilayers. Liposomes are unilamellar or multilamellar vesicles that have a membrane formed of lipophilic material and an aqueous interior that contains the composition to be delivered. Cationic liposomes are positively charged liposomes that can interact with negatively charged DNA molecules to form a stable complex. Liposomes that are pH sensitive or negatively charged are believed to entrap DNA rather than form a complex with it. Both cationic and non-cationic liposomes can be used to deliver the target nucleic acid or recombinant expression vector.

Liposomes also include sterically stabilized liposomes, a term which, as used herein, refers to liposomes containing one or more specialized lipids that, when incorporated into the liposomes, result in an increased circulation duration compared to liposomes lacking such specialized lipids. Examples of sterically stabilized liposomes are those in which a portion of the vesicle-forming lipid portion of the liposome contains one or more glycolipids or is derivatized with one or more hydrophilic polymers, such as a polyethylene glycol (PEG) moiety. Liposomes and their use are further described in U.S. Patent No. 6,287,860, which is incorporated herein by reference in its entirety.

The compositions and formulations of this disclosure may also include surfactants. The use of surfactants in drug products, formulations, and emulsions is well known in the art. Surfactants and their use are further described in U.S. Patent No. 6,287,860.

In one embodiment, various penetration enhancers are included for efficient delivery of nucleic acids. In addition to facilitating diffusion of non-lipophilic drugs across cell membranes, penetration enhancers also increase the permeability of lipophilic drugs. Penetration enhancers can be classified as belonging to one of five broad categories, i.e., surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants. Penetration enhancers and their use are further described in U.S. Patent No. 6,287,860, which is incorporated herein by reference in its entirety.

Compositions and formulations for oral administration include powders or granules, microparticles, nanoparticles, suspensions or solutions in water or non-aqueous media, capsules, gelcaps, sachets, tablets or mini-tablets. Thickening agents, flavoring agents, diluents, emulsifiers, dispersing agents or binders may be desirable. Suitable oral preparations include those in which the antisense nucleic acid is administered in combination with one or more penetration enhancers, surfactants and chelators. Suitable surfactants include, but are not limited to, fatty acids and/or esters or salts thereof, bile acids and/or salts thereof. Suitable bile acids/salts and fatty acids and their use are further described in U.S. Patent No. 6,287,860. Also suitable are combinations of penetration enhancers, such as fatty acids/salts in combination with bile acids/salts. An example of a suitable combination is sodium lauric acid, capric acid and UDCA (ursodeoxycholic acid). Other penetration enhancers include, but are not limited to, polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether. Suitable penetration enhancers also include propylene glycol, dimethyl sulfoxide, triethanolamine, N,N-dimethylacetamide, KH-dimethylformamide. 2-pyrrolidone and its derivatives, tetrahydrofurfuryl alcohol and AZONE™.

Methods of modulating T cell activity.

The present invention provides a method for selectively modulating the activity of an epitope-specific T cell comprising contacting the T cell with a TMMP of the present invention, wherein contacting the T cell with a TMMP of the present invention selectively modulates the activity of the epitope-specific T cell. In some cases, the contacting occurs in vitro. In some

- 91 049816 In some cases, contact occurs in vivo. In some cases, contact occurs ex vivo.

In some cases, such as when the target T cell is a CD8+ T cell, the TMMP contains MHC class I polypeptides (e.g., β2-microglobulin and MHC class I heavy chain).

When the TMMP of the present invention comprises an immunomodulatory polypeptide that is an activating polypeptide, contact of a T cell with the TMMP activates an epitope-specific T cell. In some cases, the epitope-specific T cell is a T cell that is specific for an epitope present on a cancer cell, and contact of the epitope-specific T cell with the TMMP increases the cytotoxic activity of the T cell with respect to the cancer cell. In some cases, the epitope-specific T cell is a T cell that is specific for an epitope present on a cancer cell, and contact of the epitope-specific T cell with the TMMP increases the number of epitope-specific T cells

In some cases, the epitope-specific T cell is a T cell that is specific for an epitope present on a cell infected with a virus, and contact of the epitope-specific T cell with TMMP increases the cytotoxic activity of the T cell toward the cell infected with the virus. In some cases, the epitope-specific T cell is a T cell that is specific for an epitope present on a cell infected with a virus, and contact of the epitope-specific T cell with TMMP increases the number of epitope-specific T cells.

When the TMMP of the present invention comprises an immunomodulatory polypeptide that is an inhibitory polypeptide, contact of a T cell with the TMMP inhibits the epitope-specific T cell. In some cases, the epitope-specific T cell is an autoreactive T cell that is specific for an epitope present in an autoantigen, and contacting reduces the number of autoreactive T cells.

The present invention provides a method of modulating an immune response in an individual, the method comprising administering to the individual an effective amount of a TMMP of the present invention. Administration of the TMMP induces an epitope-specific T cell response (e.g., a WT-1 epitope-specific T cell response) and an epitope-non-specific T cell response, wherein the ratio of the epitope-specific T cell response to the epitope-non-specific T cell response is at least 2:1. In some cases, the ratio of the epitope-specific T cell response to the epitope-non-specific T cell response is at least 5:1. In some cases, the ratio of the epitope-specific T cell response to the epitope-non-specific T cell response is at least 10:1. In some cases, the ratio of epitope-specific T cell response to epitope-non-specific T cell response is at least 25:1. In some cases, the ratio of epitope-specific T cell response to epitope-non-specific T cell response is at least 50:1. In some cases, the ratio of epitope-specific T cell response to epitope-non-specific T cell response is at least 100:1. In some cases, the individual is a human. In some cases, the modulation increases the cytotoxic T cell response to a cancer cell, such as a WT-1-expressing cancer cell. In some cases, administration is intravenous, subcutaneous, intramuscular, systemic, intralymphatic, distal to the treatment site, local, or at or near the treatment site.

The present invention provides a method of delivering a costimulatory (i.e., immunomodulatory) polypeptide selective for target T cells, comprising contacting a mixed population of T cells with a TMMP of the present invention, wherein the mixed population of T cells comprises target T cells and non-target T cells, wherein the target T cell is specific for an epitope present in the TMMP (e.g., wherein the target T cell is specific for a WT-1 epitope present in the TMMP), and wherein said contacting step delivers one or more costimulatory polypeptides (immunomodulatory polypeptides) present in the TMMP to the target T cell. In some cases, the population of T cells is an in vitro population. In some cases, the population of T cells is an in vivo population in an individual. In some cases, the method comprises administering to the individual a TMMP. In some cases, the T cell is a cytotoxic T cell. In some cases, the mixed population of T cells is an in vitro population of mixed T cells obtained from the individual, and the contacting step results in activation and/or proliferation of the target T cell, resulting in a population of activated and/or proliferated target T cells; in some of these examples, the method further comprises administering the population of activated and/or proliferated target T cells to the individual.

The present invention provides a method for detecting in a mixed population of T cells obtained from an individual the presence of a target T cell that binds an epitope of interest (e.g., a WT-1 epitope), the method comprising: a) contacting in vitro the mixed population of T cells with a TMMP of the present invention, wherein the TMMP comprises the epitope of interest (e.g., a WT1 epitope); and b) detecting activation and/or proliferation of the T cells in response to said contacting.

- 92 049816 cycle, with activated and/or proliferated T cells indicating the presence of target T cells.

Treatment methods.

The present invention provides a method of treating an individual comprising administering to the individual an amount of a TMMP of the present invention or one or more nucleic acids encoding a TMMP effective to treat the individual. Also provided is a TMMP of the present invention for use in a method of treating a human or animal organism. In some cases, the method of treating according to the present invention comprises administering to an individual in need thereof one or more recombinant expression vectors comprising nucleotide sequences encoding a TMMP of the present invention. In some cases, the method of treating according to the present invention comprises administering to an individual in need thereof one or more mRNA molecules comprising nucleotide sequences encoding a TMMP of the present invention. In some cases, the method of treating according to the present invention comprises administering to an individual in need thereof a TMMP of the present invention. Conditions that can be treated include, for example, cancer and autoimmune disorders, as described below.

In some cases, a TMMP of the invention, when administered to an individual in need thereof, induces both an epitope-specific T cell response and an epitope-non-specific T cell response. In other words, in some cases, a TMMP of the invention, when administered to an individual in need thereof, induces an epitope-specific T cell response by modulating the activity of a first T cell that comprises on its surface both: i) a TCR specific for an epitope present on the TMMP; and ii) a co-immunomodulatory polypeptide that binds to the immunomodulatory polypeptide present on the TMMP; and induces an epitope-non-specific T cell response by modulating the activity of a second T cell that comprises on its surface both: i) a TCR specific for an epitope present on the TMMP; and ii) a co-immunomodulatory polypeptide that binds to an immunomodulatory polypeptide present in the TMMP. The ratio of epitope-specific T cell response to epitope-non-specific T cell response is at least 2:1, at least 5:1, at least 10:1, at least 15:1, at least 20:1, at least 25:1, at least 50:1, or at least 100:1. The ratio of epitope-specific T cell response to epitope-non-specific T cell response is from about 2:1 to about 5:1, from about 5:1 to about 10:1, from about 10:1 to about 15:1, from about 15:1 to about 20:1, from about 20:1 to about 25:1, from about 25:1 to about 50:1, or from about 50:1 to about 100:1, or greater than 100:1. Modulating T cell activity may involve one or more of: i) activating a cytotoxic T cell (e.g., CD8'); ii) inducing cytotoxic activity of a cytotoxic T cell (e.g., CD8 ⁺ ); iii) induction of production and release of cytotoxin (eg, perforin; granzyme; granulesin) by a cytotoxic T cell (eg, CD8 ⁺ ) iv) inhibition of autoreactive T cell activity; and the like.

The combination of the reduced affinity of the immunomodulatory polypeptide for its cognate co-immunomodulatory polypeptide and the affinity of the epitope for the TCR provides for improved selectivity of the TMMP of the invention. Thus, for example, the TMMP of the invention binds with increased avidity to a first T cell that comprises on its surface both: i) a TCR specific for an epitope present in the TMMP and ii) a co-immunomodulatory polypeptide that binds to the immunomodulatory polypeptide present in the TMMP, compared to the avidity with which it binds to a second T cell that comprises on its surface: i) a TCR specific for an epitope different from the epitope present in the TMMP and ii) a co-immunomodulatory polypeptide that binds to the immunomodulatory polypeptide present in the TMMP.

The present invention relates to a method for selectively modulating the activity of an epitope-specific T cell in an individual, comprising administering to the individual an effective amount of a multimeric polypeptide of the present invention or one or more nucleic acids (e.g., expression vectors; mRNA, etc.) comprising nucleotide sequences encoding TMMPs, wherein the TMMP selectively modulates the activity of an epitope-specific T cell in the individual. Selectively modulating the activity of an epitope-specific T cell can treat a disease or disorder in the individual. Thus, the present invention provides a method of treatment comprising administering to an individual in need thereof an effective amount of a TMMP of the present invention.

In some cases, the immunomodulatory polypeptide (MOD) is an activating polypeptide and the TMMP activates an epitope-specific T cell. In some cases, the epitope is a cancer-associated epitope and the TMMP increases the activity of a T cell specific for the cancer-associated epitope. In some cases, the MOD is an activating polypeptide and the TMMP activates a WT-1 epitope-specific T cell. In some cases, the T cell is a T helper cell (CD4+ cell), a cytotoxic T cell (CD8+ cell), or a NK T cell. In some cases, the epitope is a WT-1 epitope and the TMMP increases

- 93 049816 activity of T cells specific for a cancer cell expressing a WT-1 epitope (e.g., T helper cells (CD4' cells), cytotoxic T cells (CD8' cells) and/or NK T cells). Activation of CD4+ T cells may include an increase in the proliferation of CD4+ T cells and/or the activation or enhancement of the release of cytotoxins by CD4+ T cells. Activation of NK-T cells and/or CD8+ cells may include: an increase in the proliferation of NK-T cells and/or CD8+ cells; and/or the activation of the release of cytokines such as interferon by NK-T cells and/or CD8+ cells. In some cases, the TMMP of the present invention reduces the proliferation and/or activity of regulatory T cells (Treg). Tregs are FoxP3 ⁺ , CD4 ⁺ T cells. In some cases, such as when the TMMP of the present invention comprises an inhibitory immunomodulatory polypeptide (e.g., PDL1, FasL, etc.), the TMMP reduces the proliferation and/or activity of Tregs.

In some cases, the immunomodulatory polypeptide is an activating polypeptide and the TMMP activates an epitope-specific T cell. In some cases, the epitope is a cancer-associated epitope and the TMMP increases the activity of a T cell specific for the cancer-associated epitope.

When the TMMP of the present invention comprises a WT-1 peptide epitope, the TMMP can be administered to an individual with a WT-1-expressing cancer. WT1-expressing cancers include: leukemia, desmoplastic small round cell tumor, gastric cancer, colon cancer, lung cancer, breast cancer, germ cell tumor, ovarian cancer, uterine cancer, thyroid cancer, liver cancer, kidney cancer, Kaposi's sarcoma, sarcoma, hepatocellular carcinoma, Wilms' tumor, acute myelogenous leukemia (AML), myelodysplastic syndrome (MDS), non-small cell lung cancer (NSCLC), myeloma, pancreatic cancer, colorectal cancer, mesothelioma, soft tissue sarcoma, neuroblastoma, and nephroblastoma.

When the TMMP of the present invention comprises a WT-1 peptide epitope, the TMMP can be administered to a subject in need thereof for treating acute myeloid leukemia (AML) in the subject. When the TMMP of the present invention comprises a WT-1 peptide epitope, the TMMP can be administered to a subject in need thereof for treating myeloma in the subject. When the TMMP of the present invention comprises a WT-1 peptide epitope, the TMMP can be administered to a subject in need thereof for treating ovarian cancer in the subject. When the TMMP of the present invention comprises a WT-1 peptide epitope, the TMMP can be administered to a subject in need thereof for treating pancreatic cancer in the subject. When the TMMP of the present invention comprises a WT-1 peptide epitope, the TMMP can be administered to a subject in need thereof for treating non-small cell lung cancer (NSCLC) in the subject. When the TMMP of the present invention comprises a WT-1 peptide epitope, the TMMP can be administered to a subject in need thereof for treating colorectal cancer (CRC) in the subject. When the TMMP of the present invention comprises a WT-1 peptide epitope, the TMMP can be administered to a subject in need thereof for treating breast cancer in the subject. When the TMMP of the present invention comprises a WT-1 peptide epitope, the TMMP can be administered to a subject in need thereof for treating Wilms tumor in the subject. When the TMMP of the present invention comprises a WT-1 peptide epitope, the TMMP can be administered to a subject in need thereof for treating mesothelioma in the subject. When the TMMP of the present invention comprises a WT-1 peptide epitope, the TMMP can be administered to a subject in need thereof for treating soft tissue sarcoma in the subject. When the TMMP of the present invention comprises a WT-1 peptide epitope, the TMMP can be administered to a subject in need thereof to treat neuroblastoma in the subject. When the TMMP of the present invention comprises a WT-1 peptide epitope, the TMMP can be administered to a subject in need thereof to treat nephroblastoma in the subject.

The present invention provides a method of treating cancer in an individual, the method comprising administering to the individual an effective amount of a TMMP of the present invention or one or more nucleic acids (e.g., expression vectors; mRNA, etc.) comprising nucleotide sequences encoding the TMMP, wherein the TMMP comprises a T-cell epitope that is a malignant epitope, and wherein the TMMP comprises a stimulatory immunomodulatory polypeptide. In some cases, an effective amount of a TMMP of the present invention 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 TMMP of the present invention 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 administration of the TMMP or in the absence of administration of the TMMP. In some cases, an effective amount of a TMMP of the present invention 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.

In some cases, the effective amount of TMMP of the present invention is

- 94 049816 an amount that, when administered in one or more doses to an individual in need thereof, reduces the tumor mass in the individual. For example, in some cases, an effective amount of TMMP according to the present invention is an amount that, when administered in one or more doses to an individual in need thereof (an individual having a tumor), reduces the tumor mass 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 tumor mass in the individual before the administration of TMMP or in the absence of the administration of TMMP. In some cases, an effective amount of TMMP of the present invention is an amount that, when administered in one or more doses to an individual in need thereof (an individual having a tumor), reduces the volume of the tumor in the individual. For example, in some cases, an effective amount of TMMP of the present invention is an amount that, when administered in one or more doses to an individual in need thereof (an individual having a tumor), reduces the volume of the tumor 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 volume of the tumor in the individual before the administration of TMMP or in the absence of administration of TMMP. In some cases, an effective amount of a TMMP of the invention is an amount that, when administered in one or more doses to an individual in need thereof, increases the survival time of the individual. For example, in some cases, an effective amount of a TMMP of the invention is an amount that, when administered in one or more doses to an individual in need thereof, increases 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 administration of the multimeric polypeptide.

In some cases, the epitope-specific T cell is a T cell that is specific for an epitope present on a cell infected with a virus, and contact of the epitope-specific T cell with TMMP increases the cytotoxic activity of the T cell toward the cell infected with the virus. In some cases, the epitope-specific T cell is a T cell that is specific for an epitope present on a cell infected with a virus, and contact of the epitope-specific T cell with TMMP increases the number of epitope-specific T cells.

Thus, the present invention provides a method of treating a viral infection in an individual, wherein the method comprises administering to the individual an effective amount of a TMMP according to the present invention or one or more nucleic acids comprising nucleotide sequences encoding a TMMP, wherein the TMMP comprises a T-cell epitope that is a viral epitope, and wherein the TMMP comprises a stimulatory immunomodulatory polypeptide. In some cases, an effective amount of a TMMP is an amount that, when administered in one or more doses to an individual in need thereof, reduces the number of virus-infected cells in the individual. For example, in some cases, an effective amount of a TMMP of the present invention is an amount that, when administered in one or more doses to an individual in need thereof, reduces the number of virus-infected 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 virus-infected cells in the individual before administration of the TMMP or in the absence of administration of the TMMP. In some cases, an effective amount of a TMMP of the present invention is an amount that, when administered in one or more doses to an individual in need thereof, reduces the number of virus-infected cells in the individual to undetectable levels.

Thus, the present invention provides a method of treating an infection in an individual, wherein the method comprises administering to the individual an effective amount of a TMMP of the present invention or one or more nucleic acids comprising nucleotide sequences encoding a TMMP, wherein the TMMP comprises a T-cell epitope that is a pathogen-associated epitope, and wherein the TMMP comprises a stimulatory immunomodulatory polypeptide. In some cases, an effective amount of a TMMP of the present invention is an amount that, when administered in one or more doses to an individual in need thereof, reduces the number of pathogens in the individual. For example, in some cases, an effective amount of a TMMP of the present invention is an amount that, when administered in one or more doses to an individual in need thereof, reduces the amount of pathogens 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 amount of pathogens in the individual.

- 95 049816 before administration of TMMP or in the absence of administration of TMMP. In some cases, an effective amount of TMMP according to the invention is an amount that, when administered in one or more doses to an individual in need thereof, reduces the number of pathogens in the individual to undetectable levels. Pathogens include viruses, bacteria, protozoa, and the like.

In some cases, the immunomodulatory polypeptide is an inhibitory polypeptide and the TMMP inhibits the activity of an epitope-specific T cell. In some cases, the epitope is an autoepitope and the TMMP selectively inhibits the activity of T cells specific for that epitope.

The present invention provides a method for treating an autoimmune disorder in an individual, the method comprising administering to the individual an effective amount of a TMMP of the present invention or one or more nucleic acids comprising nucleotide sequences encoding a TMMP, wherein the TMMP comprises a T-cell epitope that is an autoepitope, and wherein the TMMP comprises an inhibitory immunomodulatory polypeptide. In some cases, an effective amount of a TMMP of the present invention is an amount that, when administered in one or more doses to an individual in need thereof, reduces the number of autoreactive T 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 autoreactive T cells in the individual before administration of the TMMP or in the absence of administration of the TMMP. In some cases, an effective amount of TMMP is an amount that, when administered in one or more doses to an individual in need thereof, reduces the production of Th2 cytokines in the individual. In some cases, an effective amount of a TMMP of the present invention is an amount that, when administered in one or more doses to an individual in need thereof, alleviates one or more symptoms associated with an autoimmune disease in the individual.

As noted above, in some instances, when performing a method of treating a subject, the TMMP of the invention is administered to the individual in need thereof as a TMMP per se. In other instances, when performing a method of treating a subject, one or more nucleic acids comprising nucleotide sequences encoding the TMMP of the invention are administered to the individual in need thereof. Thus, in other instances, one or more nucleic acids of the invention, such as one or more recombinant expression vectors of the invention, are administered to the individual in need thereof.

Compositions.

Suitable formulations are described above, wherein suitable formulations comprise a pharmaceutically acceptable excipient. In some cases, a suitable composition comprises: a) a TMMP of the invention; and b) a pharmaceutically acceptable excipient. In some cases, a suitable composition comprises: a) a nucleic acid comprising a nucleotide sequence encoding a TMMP of the invention; and b) a pharmaceutically acceptable excipient; in some cases, the nucleic acid is mRNA. In some cases, a suitable composition comprises: a) a first nucleic acid comprising a nucleotide sequence encoding a first TMMP polypeptide of the invention; b) a second nucleic acid comprising a nucleotide sequence encoding a second TMMP polypeptide of the invention; and c) a pharmaceutically acceptable excipient. In some cases, a suitable composition comprises: a) a nucleic acid comprising a nucleotide sequence encoding a multimeric polypeptide of the invention; and b) a pharmaceutically acceptable excipient; in some cases, the nucleic acid is mRNA. In some cases, a suitable composition comprises: a) a first recombinant expression vector comprising a nucleotide sequence encoding a first TMMP polypeptide of the invention;

b) a second recombinant expression vector comprising a nucleotide sequence encoding a second TMMP polypeptide of the invention; and

c) a pharmaceutically acceptable excipient.

Suitable pharmaceutically acceptable excipients are described above.

Doses.

The appropriate dosage can be determined by the attending physician or other qualified medical personnel based on various clinical factors. As is well known in the medical art, dosages for any patient depend on many factors, including the patient's size, body surface area, age, the particular polypeptide or nucleic acid to be administered, the patient's sex, the time and route of administration, general health, and other drugs administered concurrently. The TMMP of the present invention can be administered in amounts from 1 ng/kg body weight to 20 mg/kg body weight per dose, such as from 0.1 mg/kg body weight to 10 mg/kg body weight, such as from 0.5 mg/kg body weight to 5 mg/kg body weight; however, dosages below or above this approximate range are contemplated, especially taking into account the above factors. If the regimen is a continuous infusion, it can also be from 1 mcg to 10 mg per kilogram of body weight per minute. The TMMP of the present invention can be administered in an amount of from about 1 mg/kg body weight to 50 mg/kg body weight, such as from about 1 mg/kg body weight to about 5 mg/kg body weight, from about 5 mg/kg body weight to about 10 mg/kg body weight, from about 10 mg/kg body weight to about 15 mg/kg body weight, from about 15 mg/kg body weight to about 20 mg/kg body weight, from about 20 mg/kg body weight to about 25 mg/kg body weight, from about 25 mg/kg body weight to about 30 mg/kg body weight, from about 30 mg/kg body weight to about 35 mg/kg body weight, from about 35 mg/kg body weight to about 40 mg/kg body weight, or from about 40 mg/kg body weight to about 50 mg/kg body weight.

In some cases, a suitable dose of TMMP of the present invention is from 0.01 μg to 100 g per kg of body weight, from 0.1 μg to 10 g per kg of body weight, from 1 μg to 1 g per kg of body weight, from 10 μg to 100 mg per kg of body weight, from 100 μg to 10 mg per kg of body weight, or from 100 μg to 1 mg per kg of body weight. Those skilled in the art can readily estimate the repetition frequency for dosing based on measured residence times and concentrations of the administered agent in body fluids or tissues. Following successful treatment, it may be desirable for the patient to undergo maintenance therapy to prevent recurrence of the disease state, in which case the TMMP of the present invention is administered in maintenance doses ranging from 0.01 μg to 100 g per kg body weight, from 0.1 μg to 10 g per kg body weight, from 1 μg to 1 g per kg body weight, from 10 μg to 100 mg per kg body weight, from 100 μg to 10 mg per kg body weight, or from 100 μg to 1 mg per kg body weight.

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

In some cases, multiple doses of the TMMP of the invention, the nucleic acid of the invention, or the recombinant expression vector of the invention are administered. The frequency of administration of the TMMP of the invention, the nucleic acid of the invention, or the recombinant expression vector of the invention may vary depending on any of a variety of factors, such as the severity of the symptoms, etc. For example, in some cases, the TMMP of the invention, the nucleic acid of the invention, or the recombinant expression vector of the invention is administered once per month, twice per month, three times per month, every other week (qow), once per week (qw), twice per week (biw), three times per week (tiw), four times per week, five times per week, six times per week, every other day (qod), daily (qd), twice per day (qid), or three times per day (tid).

The duration of administration of the TMMP of the invention, the nucleic acid of the invention, or the recombinant expression vector of the invention, e.g., the period of time during which the TMMP of the invention, the nucleic acid of the invention, or the recombinant expression vector of the invention is administered, can vary depending on any of a variety of factors, such as the response of the patient, etc. For example, the TMMP of the invention, the nucleic acid of the invention, or the recombinant expression vector of the invention can be administered for a period of time of from about one day to about one week, from about two weeks to four weeks, from one month to two months, from two months to four months, from four months to six months, from six months to eight months, from eight months to 1 year, from about 1 year to about 2 years, or from about 2 years to about 4 years, or more.

Routes of administration.

The active agent (TMMP of the invention, nucleic acid of the invention, or recombinant expression vector of the invention) is administered to the individual using any available method and route suitable for drug delivery, including in vivo and ex vivo methods, as well as systemic and localized routes of administration.

Conventional and pharmaceutically acceptable routes of administration include intratumoral, peritumoral, intramuscular, intralymphatic, intratracheal, intracranial, subcutaneous, intradermal, topical, intravenous, intraarterial, rectal, nasal, oral and other enteral and parenteral routes of administration. Routes of administration may be combined if desired or adjusted depending on the TMMP and/or the desired effect. The TMMP of the invention or the nucleic acid or recombinant expression vector of the invention may be administered in single or multiple doses.

In some cases, a TMMP of the invention, a nucleic acid of the invention, or a recombinant expression vector of the invention is administered intravenously. In some cases, a TMMP of the invention, a nucleic acid of the invention, or a recombinant expression vector of the invention is administered intramuscularly. In some cases, a TMMP of the invention, a nucleic acid of the invention, or a recombinant expression vector of the invention is administered intralymphatically. In some cases, a TMMP of the invention, a nucleic acid of the invention, or a recombinant expression vector of the invention is administered intravenously.

- 97 049816 the expression vectors of the invention are administered locally. In some cases, the TMMPs of the invention, the nucleic acid of the invention, or the recombinant expression vector of the invention are administered through the tumor. In some cases, the TMMPs of the invention, the nucleic acid of the invention, or the recombinant expression vector of the invention are administered intratumorally. In some cases, the TMMPs of the invention, the nucleic acid of the invention, or the recombinant expression vector of the invention are administered intracranially. In some cases, the TMMPs of the invention, the nucleic acid of the invention, or the recombinant expression vector of the invention are administered subcutaneously.

In some cases, the TMMP of the invention is administered intravenously. In some cases, the TMMP of the invention is administered intramuscularly. In some cases, the TMMP of the invention is administered locally. In some cases, the TMMP of the invention is administered through a tumor. In some cases, the TMMP of the invention is administered intratumorally. In some cases, the TMMP of the invention is administered intracranially. In some cases, the TMMP of the invention is administered subcutaneously. In some cases, the TMMP of the invention is administered intralymphatically.

The TMMP of the invention, the nucleic acid of the invention, or the recombinant expression vector of the invention may be administered to the host using any available conventional methods and routes suitable for the delivery of conventional drugs, including systemic or local routes. In general, routes of administration contemplated for use in the method of the invention include, but are not limited to, enteral, parenteral, and inhalation routes.

Parenteral routes of administration other than inhalation administration include, but are not limited to, topical, transdermal, subcutaneous, intramuscular, intraorbital, intracapsular, intraspinal, intramural, intratumoral, intralymphatic, peritumoral and intravenous routes, i.e., any route of administration other than via the alimentary canal. Parenteral administration may be performed to effect systemic or local delivery of a TMMP of the invention, a nucleic acid of the invention, or a recombinant expression vector of the invention. Where systemic delivery is desired, administration typically includes invasive or systemically absorbed topical or mucosal administration of pharmaceuticals.

Combination therapy.

In some cases, the method of the present invention for treating cancer in an individual comprises: a) administering a TMMP of the present invention; and b) administering at least one therapeutic agent or therapeutic intervention. Suitable additional therapeutic agents include, but are not limited to, a small molecule chemotherapeutic anticancer agent and an immune checkpoint inhibitor. Suitable additional therapeutic interventions include, for example, radiation, surgery (e.g., surgical resection of a tumor), and the like.

The method of treatment according to the present invention may comprise co-administering the TMMP of the present invention and at least one additional therapeutic agent. By co-administering is meant that both the TMMP of the present invention and the at least one additional therapeutic agent are administered to the individual, although not necessarily at the same time, to achieve a therapeutic effect that results from the administration of both the TMMP and the at least one additional therapeutic agent. The administration of the TMMP and the at least one additional therapeutic agent may be substantially simultaneous, for example, the TMMP may be administered to the 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 TMMP of the present invention is administered to an individual undergoing or having undergone treatment with at least one additional therapeutic agent. The administration of the TMMP may occur at different times and/or with different frequencies.

As an example, a method of treatment according to the present invention may comprise co-administering a TMMP of the present invention and an immune checkpoint inhibitor, such as an antibody specific for an immune checkpoint. By co-administering is meant that both a TMMP of the present invention and an immune checkpoint inhibitor (e.g., an antibody specific for an immune checkpoint polypeptide) are administered to an individual, although not necessarily simultaneously, in order to achieve a therapeutic effect that results from the administration of both the TMMP and the immune checkpoint inhibitor (e.g., an antibody specific for an immune checkpoint polypeptide). The administration of the TMMP and the immune checkpoint inhibitor (e.g., an antibody specific for an immune checkpoint polypeptide) can be substantially simultaneous, such as the TMMP can be administered to the individual over a period of from about 1 minute to about 24 hours (e.g., over about 1 minute, over about 5 minutes, over about 15

- 98 049 816 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 administration of the immune checkpoint inhibitor (e.g., an antibody specific for an immune checkpoint polypeptide). In some cases, the TMMPs of the invention are administered to an individual who is being treated or has been treated with an immune checkpoint inhibitor (e.g., an antibody specific for an immune checkpoint polypeptide). The administration of the TMMP and the immune checkpoint inhibitor (e.g., an antibody specific for an immune checkpoint polypeptide) may occur at different times and/or with different frequencies.

Exemplary immune checkpoint inhibitors include inhibitors that target an immune checkpoint polypeptide 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 a checkpoint inhibitory molecule selected from A2AR, B7-H3, B7-H4, BTLA, CTLA-4, IDO, KIR, LAG3, PD-1, TIM3, CD96, TIGIT, and VISTA.

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

Suitable 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), arelumab (Merck Serono), ipilimumab (YERVOY, Bristol-Myers Squibb), tremelimumab (Pfizer), pidilizumab (CureTech, Ltd.), IMP321 (Immutep SA), MGA271 (Macrogenics), BMS986016 (Bristol-Meyers Squibb), lirilumab (Bristol-Myers Squibb), urelumab (Bristol-Meyers Squibb), PF05082566 (Pfizer), IPH2101 (Innate Pharma/Bristol-Myers Squibb), MEDI-6469 (Medlmmune/AZ), CP-870, 893 (Genentech), mogamulizumab (Kyowa Hakko Kirin), varlilumab (CellDex 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 U.S. Patent Publication No. 2017/0044259. For pidilizumab, see, for example, Rosenblatt et al. (2011) J. Immunother. 34:409-18. In some cases, the immune checkpoint inhibitor is an anti-CTLA-4 monoclonal 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 monoclonal antibody. In some cases, the anti-PD-L1 monoclonal antibody is BMS-935559, MEDI4736, MPDL3280A (also known as RG7446), KN035, or MSB0010718C. In some cases, the anti-PD-L1 monoclonal antibody is MPDL3280A (atezolizumab) or MEDI4736 (durvalumab). For durvalumab, see, e.g., WO 2011/066389. For atezolizumab, see, e.g., U.S. Patent No. 8,217,149.

Subjects suitable for treatment.

Subjects suitable for treatment by the method of the present invention include individuals who have cancer, including individuals who have been diagnosed with cancer, individuals who have been treated for cancer but have failed to respond to the treatment, and individuals who have been treated for cancer and who initially responded but have subsequently become refractory to the treatment. Subjects suitable for treatment by the method of the present invention include individuals who have an infection (e.g., an infection with a pathogen such as a bacterium, virus, protozoa, etc.), including individuals who have been diagnosed with an infection and individuals who have been treated for the infection but have failed to respond to the treatment. Subjects suitable for treatment by the method of the present invention include individuals who have an infection (e.g., an infection with a pathogen such as a bacterium, virus, protozoa, etc.), including individuals who have been diagnosed with an infection and individuals who have been treated for the infection but have failed to respond to the treatment.

- 99 049816 the invention include individuals who have a bacterial infection, including individuals who have been diagnosed with a bacterial infection, and individuals who have been treated for a bacterial infection but who have failed to respond to the treatment. Subjects suitable for treatment by the method of the invention include individuals who have a viral infection, including individuals who have been diagnosed with a viral infection, and individuals who have been treated for a viral infection but who have failed to respond to the treatment. Subjects suitable for treatment by the method of the invention include individuals who have an autoimmune disease, including individuals who have been diagnosed with an autoimmune disease, and individuals who have been treated for an autoimmune disease but who have failed to respond to the treatment.

Examples of non-limiting aspects of the invention.

Aspects of set A.

The aspects, including embodiments, of the present invention 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 invention, numbered 1-95, are set forth below. As will be understood by those skilled in the art after reading this description, each of the individually numbered aspects may be used or combined with any of the preceding or following individually numbered aspects. This is intended to support all such combinations of aspects and is not limited to the combinations of aspects expressly provided below:

Aspect 1. A T-cell modulating multimeric polypeptide comprising at least one heterodimeric polypeptide comprising: a) a first polypeptide comprising: i) a Wilms tumor-1 (WT-1) peptide epitope; and ii) a first major histocompatibility complex (MHC) polypeptide; b) a second polypeptide comprising a second MHC polypeptide; and c) at least one immunomodulatory polypeptide, wherein the first and/or second polypeptide comprises an immunomodulatory polypeptide.

Aspect 2. The T-cell modulating multimeric polypeptide of aspect 1, wherein at least one of the one or more immunomodulatory domains is a variant of an immunomodulatory polypeptide that exhibits a reduced affinity for the cognate co-immunomodulatory polypeptide compared to the affinity of the corresponding wild-type immunomodulatory polypeptide for the cognate co-immunomodulatory polypeptide, and wherein the epitope binds to a T-cell receptor (TCR) on a T-cell with an affinity of at least 10 ^-7 M such that: i) the T-cell modulating multimeric polypeptide binds to a first T-cell with an affinity that is at least 25% higher than the affinity with which the T-cell modulating multimeric polypeptide binds to a second T-cell, wherein the first T-cell expresses on its surface the cognate a co-immunomodulatory polypeptide and a TCR that binds the epitope with an affinity of at least 10 ^-7 M, and wherein the second T cell expresses on its surface a cognate co-immunomodulatory polypeptide but does not express on its surface a TCR that binds the epitope with an affinity of at least 10 ^-7 M; and/or ii) the ratio of the binding affinity of a control T cell modulating multimeric polypeptide, where the control comprises a wild-type immunomodulatory polypeptide, for the cognate co-immunomodulatory polypeptide to the binding affinity of a T cell modulating multimeric polypeptide comprising a variant wild-type immunomodulatory polypeptide for the cognate co-immunomodulatory polypeptide, as measured by biolayer interferometry, is in the range of from 1.5:1 to 10 ⁶ :1.

Aspect 3. A T-cell modulating multimeric polypeptide according to aspect 2, wherein a) the T-cell modulating multimeric polypeptide binds to a first T-cell with an affinity that is at least 50%, at least 2-fold, at least 5-fold or at least 10-fold greater than the affinity with which it binds to a second T-cell; and/or b) the immunomodulatory polypeptide variant binds the co-immunomodulatory polypeptide with an affinity of from about 10 ^-4 M to about 10 ^-7 M, from about 10 ^-4 M to about 10 ^-6 M, from about 10 ^-4 M to about 10 ^-5 M; and/or c) wherein the ratio of the binding affinity of the control T-cell modulating multimeric polypeptide, where the control comprises an immunomodulatory polypeptide, to the cognate co-immunomodulatory polypeptide to the binding affinity of the T-cell modulating multimeric polypeptide comprising a wild-type variant immunomodulatory polypeptide to the cognate co-immunomodulatory polypeptide, as measured by bilayer interferometry, is at least 10:1, at least 50:1, at least ¹⁰² :1, or at least ¹⁰³ :1.

Aspect 4. A T-cell modulating multimeric polypeptide according to any of aspects 1-3, wherein the first or second polypeptide comprises an immunoglobulin (Ig) Fc polypeptide.

Aspect 5. The T cell modulating multimeric polypeptide of aspect 4, wherein the Ig Fc polypeptide is an IgG1 Fc polypeptide.

Aspect 6. The T cell modulating multimeric polypeptide of aspect 5, wherein the IgG1 Fc comprises one or more amino acid substitutions selected from N297A, L234A, L235A, L234F, L235E and

- 100 049816

P331S.

Aspect 7. A T cell modulating multimeric polypeptide according to any of aspects 1-6, wherein: a1) the first polypeptide comprises, in N-terminal to C-terminal direction: i) a WT-1 peptide epitope; ii) a first MHC polypeptide; and iii) at least one immunomodulatory polypeptide; and b2) the second polypeptide comprises, in N-terminal to C-terminal direction: i) a second MHC polypeptide; and ii) an immunoglobulin (Ig) Fc polypeptide; or a2) the first polypeptide comprises, in N-terminal to C-terminal direction: i) a WT-1 peptide epitope; and ii) a first MHC polypeptide; and b2) the second polypeptide comprises, in N-terminal to C-terminal direction: i) at least one immunomodulatory polypeptide; ii) a second MHC polypeptide; and iii) an Ig Fc polypeptide; or a3) the first polypeptide comprises, in N-terminal to C-terminal direction: i) a WT-1 peptide epitope; and ii) a first MHC polypeptide; and b3) the second polypeptide comprises, in N-terminal to C-terminal direction: i) a second MHC polypeptide; and ii) an Ig Fc polypeptide; and iii) at least one immunomodulatory polypeptide; or a4) the first polypeptide comprises, in N-terminal to C-terminal direction: i) a WT-1 peptide epitope; and ii) a first MHC polypeptide; and b4) the second polypeptide comprises, in N-terminal to C-terminal direction: i) a second MHC polypeptide; and ii) at least one immunomodulatory polypeptide; or a5) the first polypeptide comprises, in N-terminal to C-terminal direction: i) a WT-1 peptide epitope; and ii) a first MHC polypeptide; and b5) the second polypeptide comprises, in an N-terminal to C-terminal direction: i) at least one immunomodulatory polypeptide; and ii) a second MHC polypeptide; or a6) the first polypeptide comprises, in an N-terminal to C-terminal direction: i) a WT-1 peptide epitope; ii) a first MHC polypeptide; and iii) at least one immunomodulatory polypeptide; and b6) the second polypeptide comprises: i) a second MHC polypeptide.

Aspect 8. A T-cell modulating multimeric polypeptide according to any of aspects 1-7, wherein the first polypeptide comprises a peptide linker between the WT-1 epitope and the first MHC polypeptide and/or wherein the second polypeptide comprises a peptide linker between the immunomodulatory polypeptide and the second MHC polypeptide.

Aspect 9. A T-cell modulating multimeric polypeptide according to aspect 8, wherein the peptide linker comprises the amino acid sequence (GGGGS) _n (SEQ ID NO: 284), where n is an integer from 1 to 10.

Aspect 10. A T-cell modulating multimeric polypeptide according to any of aspects 1-9, wherein the first MHC polypeptide is a β2-microglobulin polypeptide; and wherein the second MHC polypeptide is a class I MHC heavy chain polypeptide.

Aspect 11. The T cell modulating multimeric polypeptide of any one of aspects 1-10, wherein the at least one immunomodulatory polypeptide is selected from the group consisting of a cytokine (e.g. IL2 polypeptide, IL7 polypeptide, IL12 polypeptide, IL15 polypeptide, IL17 polypeptide, IL21 polypeptide, IL27 polypeptide, IL-23 polypeptide, TGFe polypeptide, etc.; and including all family members, e.g. IL17A, IL-17B, IL-17C, IL-17D, IL-17E, IL-17F, IL-17E), 41BBL polypeptide, ICOS-L polypeptide, OX-40L polypeptide, CD80 polypeptide, CD86 polypeptide, (CD80 and CD86 are also known as B7-1 and B7-2, respectively), CD40 polypeptide, CD70 polypeptide, JAG1 (CD339) polypeptide, ICAM (CD540) polypeptide, PD-L1 polypeptide, FasL polypeptide, PD-L2 polypeptide, PD-1H (VISTA) polypeptide, ICOS-L (CD275) polypeptide, GITRL polypeptide, HVEM polypeptide, CXCL10 polypeptide, CXCL9 polypeptide, CXCL11 polypeptide, CXCL13 polypeptide and CX3CL1 polypeptide, galectin-9 polypeptide, CD83 polypeptide, CD30L polypeptide, HLA-G polypeptide, MICA polypeptide, MICB polypeptide, HVEM (CD270) polypeptide, lymphotoxin beta receptor polypeptide, 3/TR6 polypeptide, ILT3 polypeptide, ILT4 polypeptide, CXCL10, CXCL9 polypeptide, CXCL11 polypeptide, CXCL13 polypeptide and CX3CL1 polypeptide, and combinations thereof.

Aspect 12. A T-cell modulating multimeric polypeptide according to any of aspects 1-11, wherein at least one immunomodulatory polypeptide is an IL-2 polypeptide.

Aspect 13. A T-cell modulating multimeric polypeptide according to any of aspects 1-12, wherein the multimeric polypeptide comprises at least two immunomodulatory polypeptides, and wherein at least two of the immunomodulatory polypeptides are the same.

Aspect 14. A T-cell modulating multimeric polypeptide according to aspect 13, wherein 2 or more immunomodulatory polypeptides are in tandem.

Aspect 15. A T-cell modulating multimeric polypeptide according to any of aspects 1-14, wherein the first polypeptide and the second polypeptide are covalently linked to each other.

Aspect 16. A T-cell modulating multimeric polypeptide according to aspect 15, wherein the covalent bond is via a disulfide bond.

Aspect 17. The T-cell modulating multimeric polypeptide of any one of aspects 1-16, wherein the first MHC polypeptide or the linker between the epitope and the first MHC polypeptide comprises an amino acid substitution to provide a first Cys residue, wherein the second MHC polypeptide comprises an amino acid substitution to provide a second Cys residue, and wherein the disulfide bond

- 101 049816 is located between the first and second Cys residues.

Aspect 18. A T cell modulating multimeric polypeptide according to any of aspects 1-17, wherein the WT-1 peptide epitope has a length of from about 4 to about 25 amino acids.

Aspect 19. A T cell modulating multimeric polypeptide according to any of aspects 1-18, wherein the WT-1 peptide epitope comprises an amino acid sequence from the group consisting of:

NLMNLGATL (SEQ ID NO:258), NYMNLGATL (SEQ ID NO:263), CMTWNQMNLGATLKG (SEQ ID NO:223), WNQMNLGATLKGVAA (SEQ ID NO:224), CMTWNYMNLGATLKG (SEQ ID NO:225), WNYMNLGATLKGVAA (SEQ ID NO:226), MTWNQMNLGATLKGV (SEQ ID NO:227), TWNQMNLGATLKGVA (SEQ ID NO:228), CMTWNLMNLGATLKG (SEQ ID NO:229), MTWNLLMNLGATLKGV (SEQ ID NO:230), TWNLLMNLGATLKGVA (SEQ ID NO:231), WNLLMNLGATLKGVAA (SEQ ID NO:232), MNLGATLK (SEQ ID NO:233), MTWNYMNLGATLKGV SEQ ID NO:234), TWNYMNLGATLKGVA (SEQ ID NO:235), CMTWNQMNLGATLKGVA (SEQ ID NO:236), CMTWNLMNLGATLKGVA (SEQ ID NO:237), CMTWNYMNLGATLKGVA (SEQ ID NO:238), GYLRNPTAC (SEQ ID NO:239), GALRNPTAL (SEQ ID NO:240), YALRNPTAC (SEQ ID NO:241), GLLRNPTAC (SEQ ID NO:242), RYRPHPGAL (SEQ ID NO:243), YQRPHPGAL (SEQ ID NO:244), RLRPHPGAL (SEQ ID NO:245), RIRPHPGAL (SEQ ID NO:246), QFPNHSFKHEDPMGQ (SEQ ID NO:247), HSFKHEDPY (SEQ ID NO:248), QFPNHSFKHEDPM (SEQ ID NO:249), QFPNHSFKHEDPY (SEQ ID NO:250), KRPFMCAYPGCNK (SEQ ID NO:251), KRPFMCAYPGCYK (SEQ ID NO:252), FMCAYPGCY (SEQ ID NO:253), FMCAYPGCK (SEQ ID NO:254), KRPFMCAYPGCNKRY (SEQ ID NO:255), SEKRPFMCAYPGCNK (SEQ ID NO:256), KRPFMCAYPGCYKRY (SEQ ID NO:257), NLMNLGATL (SEQ ID NO:258), VLDFAPPGA (SEQ ID NO:259), RMFPNAPYL (SEQ ID NO:260), CMTWNQMN (SEQ ID NO:261), CYTWNQMNL (SEQ ID NO:269), NYMNLGATL (SEQ ID NO:263), YMFPNAPYL (SEQ ID NO:264), SLGEQQYSV (SEQ ID NO:265), CMTWNQMNL (SEQ ID NO:266) and NQMNLGATL (SEQ ID NO:267).

Aspect 20. A T cell modulating multimeric polypeptide according to any of aspects 1-18, wherein the WT-1 peptide comprises the amino acid sequence CMTWNQMNL (SEQ ID NO:266) or CYTWNQMNL (SEQ ID NO:269).

Aspect 21. A T-cell modulating multimeric polypeptide according to any of aspects 1-20, wherein the first or second MHC polypeptide comprises: a) an amino acid sequence having at least 95% amino acid sequence identity to an amino acid sequence

HLAA*0101, HLA-A*0201, HLA-A*0201, HLA-A*1101, HLA-A*2301, HLA-A*2402, HLAA*2407, HLA-A*3303 or HLA-A*3401 depicted in Fig. 9A; or b) an amino acid sequence having at least 95% amino acid sequence identity to the amino acid sequence

HLAВ*0702, HLA-B*0801, HLA-B*1502, HLA-B*3802, HLA-B*4001, HLA-B*4601 or HLAB*5301 depicted in Fig. 10A; or c) an amino acid sequence having at least 95% amino acid sequence identity with

HLA-C*0102, HLA-C*0303, HLA-C*0304, HLA-C*0401, HLA-C*0602, HLA-C*0701, HLAC*0702, HLA-C*0801 or HLA-C* 1502 shown in FIG. 11A.

Aspect 22. A T-cell modulating multimeric polypeptide according to any of aspects 1-20, wherein the first MHC polypeptide is a B2M polypeptide; and wherein the second MHC polypeptide comprises an amino acid sequence having at least 95% amino acid sequence identity to an HLA-A*2402 polypeptide.

Aspect 23. The T-cell modulating multimeric polypeptide of any of aspects 1-20, wherein the first MHC polypeptide is a B2M polypeptide; and wherein the second MHC polypeptide is an HLA-A*1101 polypeptide.

- 102 049816

Aspect 24. A T-cell modulating multimeric polypeptide according to any of aspects 1-20, wherein the first MHC polypeptide is a B2M polypeptide; and wherein the second MHC polypeptide comprises an amino acid sequence having at least 95% amino acid sequence identity to an HLA-A*3303 polypeptide.

Aspect 25. A T cell modulating multimeric polypeptide according to any of aspects 1-20, wherein the first MHC polypeptide is a B2M polypeptide; and wherein the second MHC polypeptide comprises an amino acid sequence having at least 95% amino acid sequence identity to the HL A-A*0201 polypeptide.

Aspect 26. The T cell modulating multimeric polypeptide of any of aspects 21-25, wherein the second MHC heavy chain polypeptide comprises Cys at position 236.

Aspect 27. A T cell modulating multimeric polypeptide according to any of aspects 21-26, wherein the B2M polypeptide comprises Cys at position 12.

Aspect 28. A T cell modulating multimeric polypeptide according to any of aspects 1-27, wherein the immunomodulatory polypeptide is a variant of an IL-2 polypeptide comprising: i) an H16A substitution and an F42A substitution; or ii) an H16T substitution and an F42A substitution.

Aspect 29. A T cell modulating multimeric polypeptide according to any of aspects 4-28, wherein the multimeric polypeptide comprises first and second heterodimers, and wherein the first and second heterodimers are covalently linked by one or more disulfide bonds between the Ig Fc polypeptides of the first and second heterodimers.

Aspect 30. A nucleic acid comprising a nucleic sequence encoding a first or second polypeptide according to any of aspects 1-28, wherein the first or second polypeptide comprises at least one immunomodulatory domain.

Aspect 31. An expression vector comprising the nucleic acid of aspect 30.

Aspect 32. A method for selectively modulating the activity of a Wilms tumor epitope-1 (WT-1)-specific T cell, comprising contacting the T cell with a T cell modulating multimeric polypeptide according to any of aspects 1-29, wherein said contacting selectively modulates the activity of the WT-1 epitope-specific T cell.

Aspect 22. A method for treating a patient with a cancerous disease, comprising administering to the patient an effective amount of a pharmaceutical composition comprising a T-cell modulating multimeric polypeptide according to any of aspects 1-29,

Aspect 34. The method according to aspect 33, wherein the cancer is hepatocellular carcinoma, pancreatic cancer, gastric cancer, colorectal cancer, hepatoblastoma or corpus luteum tumor of the ovary.

Aspect 35. The method according to aspect 33 or aspect 34, wherein said administration is carried out intramuscularly.

Aspect 36. The method according to aspect 33 or aspect 34, wherein said administration is carried out intravenously.

Aspect 37. A method for modulating an immune response in an individual, comprising administering to the individual an effective amount of a T cell modulating multimeric polypeptide (TMMP) according to any of aspects 1-29, wherein said administration induces an epitope-specific T cell response (e.g. a T cell response specific for a WT-1 epitope present in the TMMP) and an epitope-non-specific T cell response, wherein the ratio of epitope-specific T cell response to epitope-non-specific T cell response is at least 2:1.

Aspect 38. The method of aspect 37, wherein the ratio of epitope-specific T cell response to epitope-non-specific T cell response is at least 5:1.

Aspect 39. The method of aspect 37, wherein the ratio of epitope-specific T cell response to epitope-non-specific T cell response is at least 10:1.

Aspect 40. The method of aspect 37, wherein the ratio of epitope-specific T cell response to epitope-non-specific T cell response is at least 25:1.

Aspect 41. The method of aspect 37, wherein the ratio of epitope-specific T cell response to epitope-non-specific T cell response is at least 50:1.

Aspect 42. The method of aspect 37, wherein the ratio of epitope-specific T cell response to epitope-non-specific T cell response is at least 100:1.

Aspect 43. The method according to any of aspects 37-42, wherein the individual is a human.

Aspect 44. The method according to any of aspects 37-43, wherein said modulating comprises enhancing a cytotoxic T cell response to a cancer cell (e.g. a WT-1 expressing cancer cell).

Aspect 45. The method according to any of aspects 37-44, wherein said administration is intravenous, subcutaneous, intramuscular, systemic, intralymphatic, distal to the treatment site, local or at or near the treatment site.

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Aspect 46. The method according to any of aspects 37-45, wherein the epitope-non-specific T cell response is less than the epitope-non-specific T cell response that is induced by a control T cell modulating multimeric polypeptide comprising a corresponding wild-type immunomodulatory polypeptide.

Aspect 47. A method for delivering a costimulatory (i.e. immunomodulatory) polypeptide selective for target T cells, comprising contacting a mixed population of T cells with a multimeric polypeptide (TMMP) according to any of aspects 1-29, wherein the mixed population of T cells comprises target T cells and non-target T cells, wherein the target T cell is specific for an epitope present in the TMMP (e.g. the target T cell is specific for a WT-1 epitope present in the TMMP), and wherein said contacting delivers one or more costimulatory polypeptides present in the TMMP to the target T cell.

Aspect 48. The method according to aspect 47, wherein the population of T cells is used in vitro.

Aspect 49. The method of aspect 47, wherein the population of T cells is applied to an individual in vivo.

Aspect 50. The method of aspect 49, comprising administering the multimeric polypeptide to the individual.

Aspect 51. The method according to any of aspects 47-50, wherein the target T cell is a cytotoxic T cell.

Aspect 52. The method of aspect 47, wherein the mixed population of T cells is a population of in vitro mixed T cells obtained from an individual, and wherein said contacting results in activation and/or proliferation of the target T cell, creating a population of activated and/or proliferated target T cells.

Aspect 53. The method of aspect 52, further comprising administering the population of activated and/or proliferated target T cells to the individual.

Aspect 54. A method for detecting in a mixed population of T cells obtained from an individual the presence of a target T cell that binds a WT-1 epitope of interest, comprising: a) contacting in vitro a mixed population of T cells with a T cell modulating multimeric polypeptide (TMMP) according to any of aspects 1-29, wherein the TMMP comprises a WT-1 epitope of interest; and b) detecting activation and/or proliferation of T cells in response to said contact, wherein activated and/or proliferated T cells indicate the presence of a target T cell.

Aspect 55. A T-cell modulating multimeric polypeptide comprising: at least one heterodimer comprising: a) a first polypeptide comprising: i) a Wilms tumor-1 (WT-1) peptide epitope, wherein the WT-1 peptide has a length of from about 4 amino acids to about 25 amino acids; and ii) a first major histocompatibility complex (MHC) class I polypeptide; b) a second polypeptide comprising a second MHC class I polypeptide, and c) at least one immunomodulatory polypeptide, wherein the first and/or second polypeptide comprises an immunomodulatory polypeptide, and wherein the first and second polypeptides are covalently linked to each other via at least 2 disulfide bonds.

Aspect 56. The T cell modulating multimeric polypeptide of aspect 55, wherein at least one of the at least one immunomodulatory polypeptide is a variant immunomodulatory polypeptide that exhibits a reduced affinity for the cognate co-immunomodulatory polypeptide compared to the affinity of the corresponding wild-type immunomodulatory polypeptide for the cognate co-immunomodulatory polypeptide, and wherein the epitope binds to a T cell receptor (TCR) on a T cell with an affinity of at least 10 ^-7 M such that: i) the T cell modulating multimeric polypeptide binds to a first T cell with an affinity that is at least 25% higher than the affinity with which the T cell modulating multimeric polypeptide binds to a second T cell, wherein the first T cell expresses on its surface the cognate a co-immunomodulatory polypeptide and a TCR that binds the epitope with an affinity of at least 10 ^-7 M, and wherein the second T cell expresses on its surface a cognate co-immunomodulatory polypeptide but does not express on its surface a TCR that binds the epitope with an affinity of at least 10 ^-7 M; and/or ii) the ratio of the binding affinity of a control T cell modulating multimeric polypeptide, where the control comprises a wild-type immunomodulatory polypeptide, for the cognate co-immunomodulatory polypeptide to the binding affinity of a T cell modulating multimeric polypeptide comprising a variant wild-type immunomodulatory polypeptide for the cognate co-immunomodulatory polypeptide, as measured by biolayer interferometry, is in the range of from 1.5:1 to 10 ⁶ :1.

Aspect 57. A T cell modulating multimeric polypeptide according to aspect 56, wherein a) the T cell modulating multimeric polypeptide binds to a first T cell with an affinity that is at least 50%, at least 2-fold, at least 5-fold, or at least 10-fold greater than the affinity with which it binds to a second T cell; and/or b) an immunomodulatory variant

- 104 049816 the duplicating polypeptide binds the co-immunomodulatory polypeptide with an affinity of from about 10 ^-4 M to about 10 ^-7 M, from about 10 ^-4 M to about 10 ^-6 M, from about 10 ^-4 M to about 10 ^-5 M; and/or c) wherein the ratio of the binding affinity of the control T-cell modulating multimeric polypeptide, when the control comprises an immunomodulatory polypeptide, to the cognate co-immunomodulatory polypeptide to the binding affinity of the T-cell modulating multimeric polypeptide comprising the wild-type immunomodulatory polypeptide variant to the cognate co-immunomodulatory polypeptide, as measured by bilayer interferometry, is at least 10:1, at least 50:1, at least 10 ² :1, or at least 10 ³ :1.

Aspect 58. A T cell modulating multimeric polypeptide according to any of aspects 55-57, wherein the first or second polypeptide comprises an immunoglobulin (Ig) Fc polypeptide.

Aspect 59. The T cell modulating multimeric polypeptide of aspect 58, wherein the Ig Fc polypeptide is an IgG1 Fc polypeptide.

Aspect 60. A T cell modulating multimeric polypeptide according to aspect 58, wherein the Ig Fc polypeptide is an IgG4 Fc polypeptide.

Aspect 61. A T cell modulating multimeric polypeptide according to aspect 59, wherein the IgG1 Fc comprises one or more amino acid substitutions selected from N297A, L234A, L235A, L234F, L235E and P331S.

Aspect 62. A T cell modulating multimeric polypeptide according to any of aspects 55-61, wherein a1) the first polypeptide comprises, in the direction from N-terminus to C-terminus:

i) WT-1 peptide epitope;

ii) a first MHC class I polypeptide; and iii) at least one immunomodulatory polypeptide; and b1) the second polypeptide comprises, in an N-terminal to C-terminal direction:

i) a second MHC class I polypeptide; and ii) an immunoglobulin (Ig) Fc polypeptide; or a2) the first polypeptide comprises, in N-terminal to C-terminal direction:

i) a WT-1 peptide epitope; and ii) a first MHC class I polypeptide; and b2) the second polypeptide comprises, in N-terminal to C-terminal direction:

i) at least one immunomodulatory polypeptide;

ii) a second MHC class I polypeptide; and iii) an Ig Fc polypeptide; or a3) the first polypeptide comprises, in N-terminal to C-terminal direction:

i) a WT-1 peptide epitope; and ii) a first MHC class I polypeptide; and b3) the second polypeptide comprises, in N-terminal to C-terminal direction:

i) a second MHC class I polypeptide; and ii) an Ig Fc polypeptide; and iii) at least one immunomodulatory polypeptide; or a4) the first polypeptide comprises, in N-terminal to C-terminal direction:

i) a WT-1 peptide epitope; and ii) a first MHC class I polypeptide; and b4) the second polypeptide comprises, in N-terminal to C-terminal direction:

i) a second MHC class I polypeptide; and ii) at least one immunomodulatory polypeptide; or a5) the first polypeptide comprises, in N-terminal to C-terminal direction:

i) a WT-1 peptide epitope; and ii) a first MHC class I polypeptide; and b5) the second polypeptide comprises, in N-terminal to C-terminal direction:

i) at least one immunomodulatory polypeptide; and ii) a second MHC class I polypeptide; or a6) the first polypeptide comprises, in N-terminal to C-terminal direction:

i) WT-1 peptide epitope;

ii) a first MHC class I polypeptide; and iii) at least one immunomodulatory polypeptide; and b6) the second polypeptide comprises:

i) the second polypeptide of class I MHC.

Aspect 63. A T-cell modulating multimeric polypeptide according to any of aspects 55-62, wherein the first MHC polypeptide is a β2-microglobulin polypeptide; and wherein the second MHC polypeptide is a class I MHC heavy chain polypeptide.

Aspect 64. A T cell modulating multimeric polypeptide according to any of aspects 55-63, in

- 105 049816 wherein at least one immunomodulatory polypeptide is selected from the group consisting of a cytokine (e.g., an IL2 polypeptide, an IL7 polypeptide, an IL12 polypeptide, an IL15 polypeptide, an IL17 polypeptide, an IL21 polypeptide, an IL27 polypeptide, an IL-23 polypeptide, a TGFe polypeptide, etc.; and including all family members, e.g., IL17A, IL-17B, IL-17C, IL-17D, IL-17E, IL-17F, IL-17E), a 41BBL polypeptide, an ICOS-L polypeptide, an OX-40L polypeptide, a CD80 polypeptide, a CD86 polypeptide (CD80 and CD86 are also known as B7-1 and B7-2, respectively), a CD40 polypeptide, a CD70, JAG1 (CD339) polypeptide, ICAM (CD540) polypeptide, PD-L1 polypeptide, FasL polypeptide, PD-L2 polypeptide, PD-1H (VISTA) polypeptide, ICOS-L (CD275) polypeptide, GITRL polypeptide, HVEM polypeptide, CXCL10 polypeptide, CXCL9 polypeptide, CXCL11 polypeptide, CXCL13 polypeptide and CX3CL1 polypeptide, galectin-9 polypeptide, CD83 polypeptide, CD30L polypeptide, HLA-G polypeptide, MICA polypeptide, MICB polypeptide, HVEM (CD270) polypeptide, lymphotoxin beta receptor polypeptide, 3/TR6 polypeptide, ILT3 polypeptide, ILT4 polypeptide, CXCL10 polypeptide, CXCL9 polypeptide, CXCL11 polypeptide, CXCL13 polypeptide and CX3CL1 polypeptide, and combinations thereof.

Aspect 65. A T cell modulating multimeric polypeptide according to any of aspects 55-63, wherein at least one immunomodulatory polypeptide is an IL2 polypeptide.

Aspect 66. A T-cell modulating multimeric polypeptide according to any of aspects 55-65, wherein the multimeric polypeptide comprises at least two immunomodulatory polypeptides, and wherein at least two of the immunomodulatory polypeptides are the same.

Aspect 67. A T-cell modulating multimeric polypeptide according to aspect 66, wherein 2 or more immunomodulatory polypeptides are in tandem.

Aspect 68. A T cell modulating multimeric polypeptide according to any of aspects 55-67, wherein: a) the first disulfide bond is between: i) a Cys present in a linker between a WT-1 peptide epitope and a first MHC class I polypeptide, wherein the first MHC class I polypeptide is a β2M polypeptide; and ii) a Cys residue introduced by a Y84C substitution in a second MHC class I polypeptide, wherein the second MHC class I polypeptide is a MHC class I heavy chain polypeptide; and b) the second disulfide bond is between: i) a Cys residue introduced in the β2M polypeptide by a R12C substitution; and ii) a Cys residue introduced in the MHC class I heavy chain polypeptide by a A236C substitution.

Aspect 69. A T cell modulating multimeric polypeptide according to aspect 68, wherein the linker comprises the amino acid sequence GCGGS (SEQ ID NO: 318).

Aspect 70. A T cell modulating multimeric polypeptide according to aspect 69, wherein the linker comprises the amino acid sequence GCGGS(GGGGS) _n (SEQ ID NO: 342), where n is an integer from 1 to 10.

Aspect 71. A T cell modulating multimeric polypeptide according to any of aspects 55-70, wherein the WT-1 peptide epitope is from about 4 to about 15 amino acids in length.

Aspect 72. The T cell modulating multimeric polypeptide of any of aspects 55-71, wherein the WT-1 peptide epitope comprises an amino acid sequence from the group consisting of:

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NLMNLGATL (SEQ ID NO:258),

NYMNLGATL (SEQ ID NO:263), CMTWNQMNLGATLKG (SEQ ID NO:223), WNQMNLGATLKGVAA (SEQ ID NO:224), CMTWNYMNLGATLKG (SEQ ID NO:225), WNYMNLGATLKGVAA (SEQ ID NO:226), MTWNQMNLGATLKGV (SEQ ID NO:227), TWNQMNLGATLKGVA (SEQ ID NO:228), CMTWNLMNLGATLKG (SEQ ID NO:229), MTWNLMNLGATLKGV (SEQ ID NO:230), TWNLMNLGATLKGVA (SEQ ID NO:231), WNLLMNLGATLKGVAA (SEQ ID NO:232), MNLGATLK (SEQ ID NO:233), MTWNYMNLGATLKGV SEQ ID NO:234), TWNYMNLGATLKGVA (SEQ ID NO:235), CMTWNQMNLGATLKGVA (SEQ ID NO:236), CMTWNYMNLGATLKGVA (SEQ ID NO:237), CMTWNYMNLGATLKGVA (SEQ ID NO:238), GYLRNPTAC (SEQ ID NO:239), GALRNPTAL (SEQ ID NO:240), YALRNPTAC (SEQ ID NO:241), GLLRNPTAC (SEQ ID NO:242), RYRPHPGAL (SEQ ID NO:243), YQRPHPGAL (SEQ ID NO:244), RLRPHPGAL (SEQ ID NO:245), RIRPHPGAL (SEQ ID NO:246), QFPNHSFKHEDPMGQ (SEQ ID NO:247), HSFKHEDPY (SEQ ID NO:248), QFPNHSFKHEDPM (SEQ ID NO:249), QFPNHSFKHEDPY (SEQ ID NO:250), KRPFMCAYPGCNK (SEQ ID NO:251), KRPFMCAYPGCYK (SEQ ID NO:252), FMCAYPGCY (SEQ ID NO:253), FMCAYPGCK (SEQ ID NO:254), KRPFMCAYPGCNKRY (SEQ ID NO:255), SEKRPFMCAYPGCNK (SEQ ID NO:256), KRPFMCAYPGCYKRY (SEQ ID NO:257), NLMNLGATL (SEQ ID NO:258), VLDFAPPGA (SEQ ID NO:259), RMFPNAPYL (SEQ ID NO:260), CMTWNQMN (SEQ ID NO:261), CYTWNQMNL (SEQ ID NO:269), NYMNLGATL (SEQ ID NO:263), YMFPNAPYL (SEQ ID NO:264), SLGEQQYSV (SEQ ID NO:265), CMTWNQMNL (SEQ ID NO:266) and NQMNLGATL (SEQ ID NO:267).

Aspect 73. A T cell modulating multimeric polypeptide according to any of aspects 55-71, wherein the WT-1 peptide comprises the amino acid sequence VLDFAPPGA (SEQ ID NO:259) or RMFPNAPYL (SEQ ID NO:260).

Aspect 74. A T cell modulating multimeric polypeptide according to any of aspects 55-73, wherein the first or second MHC class I polypeptide comprises: a) an amino acid sequence having at least 95% amino acid sequence identity to an amino acid sequence

HLAA*0101, HLA-A*0201, HLA-A*0201, HLA-A*1101, HLA-A*2301, HLA-A*2402, HLAA*2407, HLA-A*3303, or HLA-A*3401 depicted in Fig. 9A; or b) an amino acid sequence having at least 95% amino acid sequence identity to the amino acid sequence ^of HLA-B*0702, HLA-B*0801, HLA-B*1502, HLA-B*3802, HLA-B*4001, HLA-B*4601, or HLAB*5301 depicted in Fig. 10A; or c) an amino acid sequence having at least 95% amino acid sequence identity to

HLA-C*0102, HLA-C*0303, HLA-C*0304, HLA-C*0401, HLA-C*0602, HLA-C*0701, HLAC*0702, HLA-C*0801 or HLA-C* 1502 shown in FIG. 11A.

Aspect 75. A T cell modulating multimeric polypeptide according to any of aspects 55-74, wherein the first MHC polypeptide is a B2M polypeptide; and wherein the second MHC polypeptide comprises an amino acid sequence having at least 95% amino acid sequence identity to an HLA-A*2402 polypeptide.

Aspect 76. A T-cell modulating multimeric polypeptide according to any of aspects 55-74, wherein the first MHC polypeptide is a B2M polypeptide; and wherein the second MHC polypeptide is an HLA-A*1101 polypeptide.

Aspect 77. A T cell modulating multimeric polypeptide according to any of aspects 55-74, wherein the first MHC polypeptide is a B2M polypeptide; and wherein the second MHC polypeptide comprises an amino acid sequence having at least 95% amino acid sequence identity to an HLA-A*3303 polypeptide.

Aspect 78. A T cell modulating multimeric polypeptide according to any of aspects 55-74, wherein the first MHC polypeptide is a b2M polypeptide; and wherein the second polypeptide

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MHC comprises an amino acid sequence that has at least 95% amino acid sequence identity with the HLA-A*O2O 1 polypeptide.

Aspect 79. A T cell modulating multimeric polypeptide according to any of aspects 55-78, wherein at least one immunomodulatory polypeptide is a variant of an IL-2 polypeptide comprising: i) an H16A substitution and an F42A substitution; or ii) an H16T substitution and an F42A substitution.

Aspect 80. A T cell modulating multimeric polypeptide according to any of aspects 55-79, wherein the multimeric polypeptide comprises a first and a second heterodimer.

Aspect 81. A nucleic acid comprising a nucleic sequence encoding a first or second polypeptide according to any of aspects 55-80, wherein the first or second polypeptide comprises at least one immunomodulatory domain.

Aspect 82. An expression vector comprising the nucleic acid of aspect 81.

Aspect 83. A method for selectively modulating the activity of a Wilms tumor epitope-1 (WT-1)-specific T cell, comprising contacting the T cell with a T cell modulating multimeric polypeptide according to any of aspects 55-80, wherein said contacting selectively modulates the activity of the WT-1 epitope-specific T cell.

Aspect 84. A method of treating a patient with a cancerous disease, comprising administering to the patient an effective amount of a pharmaceutical composition comprising a T-cell modulating multimeric polypeptide according to any of aspects 55-80,

Aspect 85. The method of aspect 84, wherein the cancer expresses the WT-1 protein.

Aspect 86. The method according to aspect 84 or aspect 85, wherein the cancer is acute myeloid leukemia, myeloma, ovarian cancer, pancreatic cancer, non-small cell lung cancer, colorectal cancer, breast cancer, Wilms tumor, mesothelioma, soft tissue sarcoma, neuroblastoma or nephroblastoma.

Aspect 87. The method according to any of aspects 84-86, wherein said administration is carried out intramuscularly.

Aspect 88. The method according to any of aspects 84-86, wherein said administration is carried out intravenously.

Aspect 89. The method according to any of aspects 84-88, further comprising administering to the individual one or more checkpoint inhibitors.

Aspect 90. The method according to aspect 89, wherein the checkpoint inhibitor is an antibody that binds to a polypeptide selected from the group consisting of CD27, CD28, CD40, CD122, CD96, CD73, CD47, OX40, GITR, CSF1R, JAK, PI3K delta, PI3K gamma, TAM, arginase, CD137, ICOS, A2AR, B7-H3, B7-H4, BTLA, CTLA-4, LAG3, TIM3, VISTA, CD96, TIGIT, CD122, PD-1, PD-L1 and PD-L2.

Aspect 91. The method of aspect 90, wherein the checkpoint inhibitor is an antibody specific for PD-1, PD-L1 or CTLA4.

Aspect 92. The method of aspect 89, wherein the one or more checkpoint inhibitors are selected from the group consisting of nivolumab, pembrolizumab, pidilizumab, AMP-224, MPDL3280A, MDX-1105, MEDI-4736, arelumab, ipilimumab, tremelimumab, pidilizumab, IMP321, MGA271, BMS-986016, lirilumab, urelumab, PF-05082566, IPH2101, MEDI-6469, CP-870,893, mogamulizumab, varlilumab, avelumab, galiximab, AMP-514, AUNP 12, indoximod, NLG-919, INCB024360, KN035, and their combinations.

Aspect 93. A method for modulating an immune response in an individual, comprising administering to the individual an effective amount of a T-cell modulating multimeric polypeptide according to any of aspects 55-80, wherein said administration induces an epitope-specific T-cell response and an epitope-non-specific T-cell response, wherein the ratio of epitope-specific T-cell response to epitope-non-specific T-cell response is at least 2:1.

Aspect 94. A method for delivering an immunomodulatory polypeptide selective for a target T cell, comprising contacting a mixed population of T cells with a T cell modulating multimeric polypeptide according to any of aspects 55-80, wherein the mixed population of T cells comprises target T cells and non-target T cells, wherein the target T cells are specific for a WT-1 epitope present in the T cell modulating multimeric polypeptide, and wherein said contacting delivers one or more immunomodulatory polypeptides present in the T cell modulating multimeric polypeptide to the target T cell.

Aspect 95. A method for detecting in a mixed population of T cells obtained from an individual the presence of a target T cell that binds a Wilms tumor-1 (WT-1) epitope, comprising: a) contacting in vitro the mixed population of T cells with a T cell modulating multimeric polypeptide according to any of aspects 55-80, wherein the T cell modulating multimeric polypeptide comprises a WT-1 epitope; and b) detecting activation and/or proliferation of T cells in response to said contact, wherein the activated and/or proliferated T cells indicate the presence of the target T cell.

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Aspects of recruitment B.

The aspects, including embodiments, of the present invention 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 invention, numbered 1-36, are set forth below. As will be understood by those skilled in the art after reading this description, each of the individually numbered aspects may be used or combined with any of the preceding or following individually numbered aspects. This is intended to support all such combinations of aspects and is not limited to the combinations of aspects expressly provided below:

Aspect 1. A T cell modulating multimeric polypeptide comprising: at least one heterodimer comprising: a) a first polypeptide comprising: i) a Wilms tumor-1 (WT-1) peptide epitope, wherein the WT-1 peptide has a length of at least 4 amino acids; and ii) a first major histocompatibility complex (MHC) class I polypeptide; b) a second polypeptide comprising a second MHC class I polypeptide, and c) at least one activating immunomodulatory polypeptide, wherein the first and/or second polypeptide comprises an immunomodulatory polypeptide, and wherein the WT-1 peptide epitope comprises an amino acid sequence selected from the group consisting of:

NLMNLGATL (SEQ ID NO:258), NYMNLGATL (SEQ ID NO:263), CMTWNQMNLGATLKG (SEQ ID NO:223), WNQMNLGATLKGVAA (SEQ ID NO:224), CMTWNYMNLGATLKG (SEQ ID NO:225), WNYMNLGATLKGVAA (SEQ ID NO:226), MTWNQMNLGATLKGV (SEQ ID NO:227), TWNQMNLGATLKGVA (SEQ ID NO:228), CMTWNLMNLGATLKG (SEQ ID NO:229), MTWNLLMNLGATLKGV (SEQ ID NO:230), TWNLLMNLGATLKGVA (SEQ ID NO:231), WNLLMNLGATLKGVAA (SEQ ID NO:232), MNLGATLK (SEQ ID NO:233), MTWNYMNLGATLKGV SEQ ID NO:234), TWNYMNLGATLKGVA (SEQ ID NO:23 5), CMTWNQMNLGATLKGVA (SEQ ID NO:236), CMTWNLMNLGATLKGVA (SEQ ID NO:237), CMTWNYMNLGATLKGVA (SEQ ID NO:238), GYLRNPTAC (SEQ ID NO:239), GALRNPTAL (SEQ ID NO:240), YALRNPTAC (SEQ ID NO:241), GLLRNPTAC (SEQ ID NO:242), RYRPHPGAL (SEQ ID NO:243), YQRPHPGAL (SEQ ID NO:244), RLRPHPGAL (SEQ ID NO:245), RIRPHPGAL (SEQ ID NO:246), QFPNHSFKHEDPMGQ (SEQ ID NO:247), HSFKHEDPY (SEQ ID NO:248), QFPNHSFKHEDPM (SEQ ID NO:249), QFPNHSFKHEDPY (SEQ ID NO:250), KRPFMCAYPGCNK (SEQ ID NO:251), KRPFMCAYPGCYK (SEQ ID NO:252), FMCAYPGCY (SEQ ID NO:253), FMCAYPGCK (SEQ ID NO:254), KRPFMCAYPGCNKRY (SEQ ID NO:255), SEKRPFMCAYPGCNK (SEQ ID NO:256), KRPFMCAYPGCYKRY (SEQ ID NO:257), NLMNLGATL (SEQ ID NO:258), VLDFAPPGA (SEQ ID NO:259), RMFPNAPYL (SEQ ID NO:260), CMTWNQMN (SEQ ID NO:261), CYTWNQMNL (SEQ ID NO:269), NYMNLGATL (SEQ ID NO:263), YMFPNAPYL (SEQ ID NO:264), SLGEQQYSV (SEQ ID NO:265), CMTWNQMNL (SEQ ID NO:266) and NQMNLGATL (SEQ ID NO:267), wherein optionally the first or second polypeptide comprises an immunoglobulin (Ig) Fc polypeptide.

Aspect 2. The T-cell modulating multimeric polypeptide of aspect 1, wherein at least one of the one or more immunomodulatory domains is a variant of the immunomodulatory polypeptide that exhibits a reduced affinity for the cognate co-immunomodulatory polypeptide compared to the affinity of the corresponding wild-type immunomodulatory polypeptide for the cognate co-immunomodulatory polypeptide, and wherein the epitope binds to the T-cell receptor (TCR) on a T-cell with an affinity of at least 10 ^-7 M, such that:

i) the T cell modulating multimeric polypeptide binds to a first T cell with an affinity that is at least 25% higher than the affinity with which the T cell modulating multimeric polypeptide binds to a second T cell, wherein the first T cell expresses on its surface a cognate co-immunomodulatory polypeptide and a TCR that binds the epitope with an affinity of at least 10 ^-7 M, and wherein the second T cell expresses on its surface a cognate co-immunomodulatory polypeptide but does not express on its surface a TCR that binds the epitope with an affinity of at least 10 ^-7 M; and/or ii) a ratio of the binding affinity of a control T cell modulating multimeric polypeptide, wherein the control comprises a wild-type immunomodulatory polypeptide, to the cognate co-immunomodulatory polypeptide.

- 109 049816 immunomodulatory polypeptide to the binding affinity of the T-cell modulating multimeric polypeptide comprising a wild-type immunomodulatory polypeptide variant to a cognate co-immunomodulatory polypeptide, as measured by biolayer interferometry, is in the range of 1.5:1 to 10 ⁶ :1.

Aspect 3. A T cell modulating multimeric polypeptide according to aspect 2, wherein:

a) the T cell modulating multimeric polypeptide binds to a first T cell with an affinity that is at least 50%, at least 2-fold, at least 5-fold, or at least 10-fold higher than the affinity with which it binds to a second T cell; and/or

b) the immunomodulatory polypeptide variant binds to the co-immunomodulatory polypeptide with an affinity of from about 10 ^-4 M to about 10 ^-7 M, from about 10 ^-4 M to about 10 ^-6 M, from about 10 ^-4 M to about 10 ^-5 M; and/or

c) wherein the ratio of the binding affinity of the control T-cell modulating multimeric polypeptide, when the control comprises a wild-type immunomodulatory polypeptide, to the cognate co-immunomodulatory polypeptide to the binding affinity of the T-cell modulating multimeric polypeptide comprising a variant wild-type immunomodulatory polypeptide to the cognate co-immunomodulatory polypeptide, as measured by biolayer interferometry, is at least 10:1, at least 50:1, at least ¹⁰² :1, or at least ¹⁰³ :1.

Aspect 4. A T-cell modulating multimeric polypeptide according to any of aspects 1-3, wherein a1) the first polypeptide comprises, in the direction from the N-terminus to the C-terminus:

i) WT-1 peptide epitope;

ii) a first MHC polypeptide; and iii) at least one immunomodulatory polypeptide; and b1) the second polypeptide comprises, in an N-terminal to C-terminal direction:

i) a second MHC polypeptide; and ii) an immunoglobulin (Ig) Fc polypeptide; or a2) the first polypeptide comprises, in N-terminal to C-terminal direction:

i) a WT-1 peptide epitope; and ii) a first MHC polypeptide; and b2) the second polypeptide comprises, in N-terminal to C-terminal direction:

i) at least one immunomodulatory polypeptide;

ii) a second MHC polypeptide; and iii) an IgFc polypeptide; or a3) a first polypeptide comprises, in N-terminal to C-terminal direction:

i) a WT-1 peptide epitope; and ii) a first MHC polypeptide; and b3) the second polypeptide comprises, in N-terminal to C-terminal direction:

i) a second MHC polypeptide; and ii) an IgFc polypeptide; and iii) at least one immunomodulatory polypeptide; or a4) the first polypeptide comprises, in N-terminal to C-terminal direction:

i) a WT-1 peptide epitope; and ii) a first MHC polypeptide; and b4) the second polypeptide comprises, in N-terminal to C-terminal direction:

i) a second MHC polypeptide; and ii) at least one immunomodulatory polypeptide; or a5) the first polypeptide comprises, in N-terminal to C-terminal direction:

i) a WT-1 peptide epitope; and ii) a first MHC polypeptide; and b5) the second polypeptide comprises, in N-terminal to C-terminal direction:

i) at least one immunomodulatory polypeptide; and ii) a second MHC polypeptide; or a6) the first polypeptide comprises, in N-terminal to C-terminal direction:

i) WT-1 peptide epitope;

ii) a first MHC polypeptide; and iii) at least one immunomodulatory polypeptide; and b6) the second polypeptide comprises: i) a second MHC polypeptide.

Aspect 5. A T cell modulating multimeric polypeptide according to any of aspects 1-4, wherein:

(a) the first MHC polypeptide is a β2-microglobulin polypeptide; and the second MHC polypeptide is a class I MHC heavy chain polypeptide; or

- 110 049816

b) the first MHC polypeptide is a class I MHC heavy chain polypeptide; and the second MHC polypeptide is a β2-microglobulin polypeptide.

Aspect 6. A T cell modulating multimeric polypeptide according to aspect 5, wherein:

a) the first polypeptide contains, in the direction from the N-terminus to the C-terminus:

i) WT-1 peptide epitope; and ii) β2-microglobulin polypeptide; And

b) the second polypeptide contains, in the direction from the N-terminus to the C-terminus:

i) at least one immunomodulatory polypeptide;

ii) MHC class I heavy chain polypeptide; and iii) Ig Fc polypeptide.

Aspect 7. A T cell modulating multimeric polypeptide according to aspect 5, wherein:

a) the first polypeptide contains, in the direction from the N-terminus to the C-terminus:

i) WT-1 peptide epitope; and ii) β2-microglobulin polypeptide; And

b) the second polypeptide contains, in the direction from the N-terminus to the C-terminus:

i) a class I MHC heavy chain polypeptide; and ii) an Ig Fc polypeptide; and iii) at least one immunomodulatory polypeptide.

Aspect 8. A T cell modulating multimeric polypeptide according to any of aspects 1-7, wherein at least one immunomodulatory polypeptide is selected from the group consisting of a cytokine, a 4-1BBL polypeptide, an ICOS-L polypeptide, an OX-40L polypeptide, a CD80 polypeptide, a CD86 polypeptide, a CD40 polypeptide, a CD70 polypeptide and combinations thereof.

Aspect 9. A T-cell modulating multimeric polypeptide according to any of aspects 1-8, wherein at least one immunomodulatory polypeptide is an IL-2 polypeptide.

Aspect 10. A T-cell modulating multimeric polypeptide according to any of aspects 1-9, wherein the multimeric polypeptide comprises at least two immunomodulatory polypeptides, and wherein at least two of the immunomodulatory polypeptides are the same, optionally wherein 2 or more immunomodulatory polypeptides are in tandem.

Aspect 11. A T cell modulating multimeric polypeptide according to any of aspects 1-10, wherein the immunomodulatory polypeptide is a variant of an IL-2 polypeptide that exhibits reduced affinity for the IL-2 receptor compared to the affinity of a wild-type IL-2 polypeptide for the IL-2 receptor.

Aspect 12. A T-cell modulating multimeric polypeptide according to aspect 11, wherein the IL-2 polypeptide variant comprises: i) an H16A substitution and an F42A substitution; or ii) an H16T substitution and an F42A substitution.

Aspect 13. A T-cell modulating multimeric polypeptide according to any of aspects 1-12, wherein the first polypeptide and the second polypeptide are covalently linked to each other, optionally wherein the covalent linkage is via a disulfide bond.

Aspect 14. A T-cell modulating multimeric polypeptide according to any of aspects 1-13, wherein the first MHC polypeptide or the linker between the epitope and the first MHC polypeptide comprises an amino acid substitution to provide a first Cys residue, wherein the second MHC polypeptide comprises an amino acid substitution to provide a second Cys residue, and wherein the disulfide bond is between the first and second Cys residues.

Aspect 15. A T cell modulating multimeric polypeptide according to any of aspects 1-14, wherein: a) the polypeptide comprises a disulfide bond between: i) a Cys present in a linker between a WT-1 peptide epitope and a first MHC class I polypeptide, wherein the first MHC class I polypeptide is a B2M polypeptide; and ii) a Cys residue introduced by a Y84C substitution into a second MHC class I polypeptide, wherein the second MHC class I polypeptide is a MHC class I heavy chain polypeptide.

Aspect 16. A T cell modulating multimeric polypeptide according to any of aspects 1-14, wherein the polypeptide comprises a disulfide bond between i) a Cys residue introduced into a first MHC class I polypeptide by substitution R12C, wherein the first MHC class I polypeptide is a β2M polypeptide; and ii) a Cys residue introduced into a second MHC class I polypeptide by substitution A236C, wherein the second MHC class I polypeptide is a MHC class I heavy chain polypeptide.

Aspect 17. A T cell modulating multimeric polypeptide according to any one of aspects 1-14, wherein the polypeptide comprises a first disulfide bond between: i) a Cys present in a linker between a WT-1 peptide epitope and a first MHC class I polypeptide, wherein the first MHC class I polypeptide is a β2M polypeptide; and ii) a Cys residue introduced by a Y84C substitution into a second MHC class I polypeptide, wherein the second MHC class I polypeptide is a MHC class I heavy chain polypeptide; and a second disulfide bond between: i) a Cys residue introduced into a β2M polypeptide by a R12C substitution; and ii) a Cys residue introduced into a heavy chain polypeptide

- 111 049816

GKGS I class by replacing A236C.

Aspect 18. A T cell modulating multimeric polypeptide according to aspect 15 or aspect 17, wherein the linker between the WT-1 peptide epitope and the first MHC is GCGGS(G4S)n (SEQ ID NO: 315), wherein n is 1, 2, 3, 4, 5, 6, 7, 8 or 9.

Aspect 19. A T cell modulating multimeric polypeptide according to any of aspects 1-18, wherein the WT-1 peptide epitope has a length of from about 4 to about 25 amino acids.

Aspect 20. A T cell modulating multimeric polypeptide according to any of aspects 1-19, wherein the WT-1 peptide comprises an amino acid sequence

CMTWNQMNL (SEQ ID NO: 266), CYTWNQMNL (SEQ ID NO:269), NYMNLGATL (SEQ

ID NO:263), VLDFAPPGA (SEQ ID NO:259), YMFPNAPYL (SEQ ID NO:264), SLGEQQYSV (SEQ ID NO:265), RMFPNAPYL (SEQ ID NO:260) and NLMNLGATL (SEQ ID NO:258).

Aspect 21. A T cell modulating multimeric polypeptide according to any of aspects 1-20, wherein the first or second MHC polypeptide comprises:

a) an amino acid sequence having at least 95% amino acid sequence identity with the amino acid sequence

HLA-A*0101, HLA-A*0201, HLA-A*0201, HLA-A*1101, HLAA*2301, HLA-A*2402, HLA-A*2407, HLA-A*3303 or HLA-A*3401 shown in FIG. 9A; or

b) an amino acid sequence having at least 95% amino acid sequence identity with the amino acid sequence

HLA-B*0702, HLA-B*0801, HLA-B*1502, HLA-B*3802, HLAB*4001, HLA-B*4601, or HLA-B*5301 shown in FIG. 10A; or

c) an amino acid sequence having at least 95% amino acid sequence identity with

HLA-C*0102, HLA-C*0303, HLAC*0304, HLA-C*0401, HLA-C*0602, HLA-C*0701, HLA-C*0702, HLA-C*0801 or HLAC*1502, shown in FIG. 11A.

Aspect 22. A T cell modulating multimeric polypeptide according to any of aspects 1-21, wherein the first MHC polypeptide is a B2M polypeptide; and wherein the second MHC polypeptide comprises an amino acid sequence having at least 95% amino acid sequence identity to an HLA-A*2402 polypeptide, and wherein the epitope is selected from the group consisting of:

RMFPNAPYL (SEQ ID NO:260), CYTWNQMNL (SEQ ID NO:269) and NYMNLGATL (SEQ

ID NO:263).

Aspect 23. A T cell modulating multimeric polypeptide according to any of aspects 1-21, wherein the first MHC polypeptide is a B2M polypeptide; and wherein the second MHC polypeptide comprises an amino acid sequence having at least 95% amino acid sequence identity to an HLA-A*O2O1 polypeptide, and wherein the epitope is selected from the group consisting of:

VLDFAPPGA (SEQ ID NO:259), RMFPNAPYL (SEQ ID NO:260) and YMFPNAPYL (SEQ ID

NO:264).

Aspect 24. A T cell modulating multimeric polypeptide according to any of aspects 1-23, wherein the multimeric polypeptide comprises a first and a second heterodimer, and wherein the first and second heterodimers are covalently linked by one or more disulfide bonds between the Ig Fc polypeptides of the first and second heterodimers.

Aspect 25. A nucleic acid comprising a nucleic sequence encoding the first or second polypeptide according to any of aspects 1-24.

Aspect 26. An expression vector comprising the nucleic acid of aspect 25.

Aspect 27. A method for selectively modulating the activity of a Wilms tumor epitope-1 (WT-1)-specific T cell, comprising contacting the T cell with a T cell modulating multimeric polypeptide according to any of aspects 1-24, wherein said contacting selectively modulates the activity of the WT-1 epitope-specific T cell.

Aspect 28. A method for treating a patient with a cancerous disease, comprising administering to the patient an effective amount of a pharmaceutical composition comprising a T-cell modulating multimeric polypeptide according to any of aspects 1-24,

Aspect 29. The method of aspect 28, wherein the cancer is an acute myocardial infarction.

- 112 049816 hematopoietic leukemia, myeloma, ovarian cancer, pancreatic cancer, non-small cell lung cancer, colorectal cancer, breast cancer, Wilms tumor, mesothelioma, soft tissue sarcoma, neuroblastoma or nephroblastoma.

Aspect 30. The method of aspect 28 or aspect 29, further comprising administering to the individual one or more checkpoint inhibitors.

Aspect 31. The method of aspect 30, wherein the checkpoint inhibitor is an antibody that binds to a polypeptide selected from the group consisting of CD27, CD28, CD40, CD122, CD96, CD73, CD47, OX40, GITR, CSF1R, JAK, PI3K delta, PI3K gamma, TAM, arginase, CD137, ICOS, A2AR, B7-H3, B7-H4, BTLA, CTLA-4, LAG3, TIM3, VISTA, CD96, TIGIT, CD122, PD-1, PD-L1 and PD-L2.

Aspect 32. The method of aspect 31, wherein the checkpoint inhibitor is an antibody specific for PD-1, PD-L1 or CTLA4.

Aspect 33. The method of aspect 30, wherein the one or more checkpoint inhibitors are selected from the group consisting of nivolumab, pembrolizumab, pidilizumab, AMP-224, MPDL3280A, MDX-1105, MEDI-4736, arelumab, ipilimumab, tremelimumab, pidilizumab, IMP321, MGA271, BMS-986016, lirilumab, urelumab, PF-05082566, IPH2101, MEDI-6469, CP-870,893, mogamulizumab, varlilumab, avelumab, galiximab, AMP-514, AUNP 12, indoximod, NLG-919, INCB024360, KN035, and their combinations.

Aspect 34. A method for modulating an immune response in an individual, comprising administering to the individual an effective amount of a T cell modulating multimeric polypeptide according to any of aspects 1-24, wherein said administration induces an epitope-specific T cell response and an epitope-non-specific T cell response, and wherein the ratio of epitope-specific T cell response to epitope-non-specific T cell response is at least 2:1.

Aspect 35. A method for delivering an immunomodulatory polypeptide selective for a target T cell, comprising contacting a mixed population of T cells with a T cell modulating multimeric polypeptide according to any of aspects 1-24, wherein the mixed population of T cells comprises target T cells and non-target T cells, wherein the target T cells are specific for a WT-1 epitope present in the T cell modulating multimeric polypeptide, and wherein said contacting delivers one or more immunomodulatory polypeptides present in the T cell modulating multimeric polypeptide to the target T cell.

Aspect 36. A method for detecting in a mixed population of T cells obtained from an individual the presence of a target T cell that binds a WT-1 epitope, comprising: a) contacting in vitro a mixed population of T cells with a T cell modulating multimeric polypeptide according to any of aspects 1-24, wherein the T cell modulating multimeric polypeptide comprises a WT-1 epitope; and b) detecting activation and/or proliferation of T cells in response to said contact, wherein activated and/or proliferated T cells indicate the presence of the target T cell.

Examples

The following examples are provided to provide those skilled in the art with a complete disclosure and description of how to make and use the present invention, but they are not intended to limit the scope of what the inventors consider to be their invention, nor to indicate that the experiments reported below represent all or the only experiments performed. Care has been taken to maintain accuracy with respect to the numbers used (e.g., amounts, temperatures, etc.), but some experimental errors and variations should be allowed for. Unless otherwise indicated, parts refer to parts by weight, molecular weight refers to average molecular weight, temperature is in degrees Celsius, and pressure represents atmospheric or near-atmospheric pressure. Standard abbreviations may be used, such as bp, base pair(s); kbp, kilobase pair(s); pL, picoliter(s); s or sec, second(s); min, minute(s); hr, hour(s); ac, amino acid(s); thousand(-s) nucleotide pairs; b. o., base pair(s); nt, nucleotide(s); i/m, intramuscular(s); i/p, intraperitoneal(s); s/c, subcutaneous(s), etc.

Example 1.

The effect of linking two polypeptide chains of the TMMP heterodimer via two disulfide bonds on stability and production was investigated.

The following TMMPs were generated: a) a TMMP containing polypeptides 1715 + 2380; and b) a TMMP containing polypeptides 1715 and 2381. The amino acid sequences of the polypeptide chains are shown in Figs. 14A-14C. As shown in Fig. 15 and Fig. 16, the TMMPs include: i) HLA-A class I heavy chain polypeptides from the A02:01 allele; and ii) two copies of IL2 (H16A; F42A) immunomodulatory (MOD) polypeptides.

Polypeptide 2380 contains the WT1 peptide WT1(37-45), while polypeptide 2381 contains the WT1 peptide WT1(126-134). TMMP 1715-2380 is a homodimer of a heterodimer containing

- 113 049816 polypeptide 1715 and polypeptide 2380. Similarly, TMMPs 1715-2381 are a homodimer of a heterodimer comprising polypeptide 1715 and polypeptide 2381. Thus, TMMPs comprise: i) 2 copies of the 1715 + 2380 heterodimer linked by 2 disulfide bonds between the IgFc polypeptide present in the 1715 polypeptides; or ii) 2 copies of the 1715 + 2381 heterodimer linked by 2 disulfide bonds between the IgFc polypeptide present in the 1715 polypeptides. This arrangement is schematically depicted in Fig. 17C.

TMMP 1715 + 2380 is a heterodimer with two disulfide bonds: a) the first disulfide bond is between: i) the Cys present in the linker between the WT1 peptide and the B2M chain in the 2380 polypeptide; and ii) the Cys introduced by the Y84C substitution in the class I heavy chain present in the 1715 polypeptide; and b) the second disulfide bond is between: i) the Cys introduced by the R12C substitution in the B2M polypeptide present in the 2380 polypeptide; and ii) the Cys introduced by the A236C substitution in the class I heavy chain present in the 1715 polypeptide.

TMMP 1715 + 2381 is a heterodimer with two disulfide bonds: a) the first disulfide bond is between: i) the Cys present in the linker between the WT1 peptide and the B2M chain in the 2381 polypeptide; and ii) the Cys introduced by the Y84C substitution in the class I heavy chain present in the 1715 polypeptide; and b) the second disulfide bond is between: i) the Cys introduced by the R12C substitution in the B2M polypeptide present in the 2381 polypeptide; and ii) the Cys introduced by the A236C substitution in the class I heavy chain present in the 1715 polypeptide.

TMMPs were produced in ExpiCHO cells (adapted from Chinese hamster ovary (CHO) cells; ThermoFisher; see, e.g., Jain et al. (2017) Protein Expr. Purif. 134:38) and purified from the cell culture medium in which the cells were grown. Two purification steps were performed. In the first step, the cell culture medium was clarified, and the clarified cell culture medium was purified on a Protein A purification column. In the second purification step, the eluate from the protein column was subjected to size exclusion chromatography.

The stability of purified TMMPs was investigated. The amount of heterodimeric TMMPs present was determined after storing purified TMMPs in liquid solution (phosphate-buffered saline (PBS) containing 365 mM NaCl, pH 7.4) for 28 days at 37°C or for 28 days at 42°C.

In addition, purified TMMPs were subjected to 3 freeze/thaw cycles.

The results are shown in Fig. 15 and Fig. 16.

As shown in Fig. 15, the homodimer of the 1715-2380 heterodimer (called monomer in Fig. 15) represents 80% of the eluate from the Protein A column. As shown in Fig. 16, the homodimer of the 1715-2381 heterodimer (called monomer in Fig. 16) represents 79% of the eluate from the Protein A column.

Homodimers of heterodimers 1715-2380 and 1715-2381 were found to be stable to 3 freeze/thaw cycles.

The unfolding temperatures of the peptide/HLA, IL-2, and Fc domains of different TMMPs, expressed as T _m (°C), are shown in Fig. 15 and Fig. 16. In addition, the temperature at which aggregation occurs (T _m °C) is shown in Fig. 15 and Fig. 16.

Example 2. Biochemical characterization of TMMPs containing WT1 epitopes, HLA-A*02 heavy chains, either one or two disulfide chains between the two polypeptide chains of the heterodimer, and IL-2 variant immunomodulatory polypeptides at position 1.

The characteristics of the constructs used in this study are summarized in Table 2.

Table 2

Constructions Epitope S-S connection(s) IL-2 position 2405 + 2762 WT-1 (37-45) G2C 1 1715 +2380 WT-1 (37-45) G2C + R12C 1 2405 + 2763 WT-1 (126-134) G2C 1 1715 +2381 WT-1 (126-134) G2C + R12C 1 2405 + 3626 WT-1 (126-134 (R126Y)) G2C 1 1715 + 3625 WT-1 (126-134 (R126Y)) G2C + R12C 1

The amino acid sequences of the polypeptide chains of the constructs are shown in Fig. 14A-14I.

G2C indicates that the TMMP comprises a disulfide bond between: i) a Cys in the peptide linker between the peptide epitope and the B2M polypeptide; and ii) a Cys at position 84 of the MHC class I heavy chain, wherein the MHC class I heavy chain contains a Y84C substitution.

R12C indicates that the TMMP comprises a disulfide bond between: i) Cys at position 12 in the B2M polypeptide, wherein the B2M polypeptide comprises an R12C substitution; and ii) Cys at position 236 of the class I MHC heavy chain, wherein the class I MHC heavy chain comprises an A236C substitution.

G2C + R12C indicates that the TMMP contains both a G2C disulfide bond and an R12C disulfide bond.

WT-1 (37-45) is VLDFAPPGA (SEQ IDNO:259).

WT-1 (126-134) is RMFPNAPYL (SEQ ID NO:260).

WT-1 (126-134 (R126Y)) is YMFPNAPYL (SEQ ID NO:264) and is also called mi

- 114 049816 motop 126-134.

The position of IL-2 is shown schematically in Fig. 19.

Results obtained.

The ability of TMMPs to stimulate antigen-specific proliferation of CD8+ T cells was investigated. TMMPs included, as an epitope: i) WT1 37-45; ii) WT1 126-134; or iii) WT1 126-134 (R126Y). All TMMPs included the heavy chains of MHC class I of the A02 allele.

Peripheral blood mononuclear cells (PBMCs) obtained from human donors were incubated in vitro with TMMP at different concentrations (0 nM, 10 nM, 100 nM, 300 nM, or 1000 nM) for 10 days. After a 10-day incubation period, the number of cells specific for a given epitope was determined. The data from PBMCs obtained from two donors (Leukopaque 7 and Leukopaque 12) are shown in Fig. 21.

The data presented in Fig. 21 demonstrate for two donors that WT1-specific TMMPs can induce an expansion of WT1-specific CD8+ T cells from total PBMCs during a 10-day culture stimulation. This expansion was achieved from PBMCs that contained little or no detectable WT1-specific T cell precursors, indicating that TMMPs were able to induce antigen-specific responses in donors from an unprimed or naive repertoire. The data demonstrate that TMMPs specific for any of the three selected WT1 peptides (37-45, 126-134, and 126-134 R126Y) and on any of the two disulfide scaffolds examined (G2C and R12C/G2C) induce the expansion of WT1-specific CD8+ T cells from total PBMCs.

PBMCs from different human donors (L7, L10, and L12) were stimulated for 10 days in vitro with the indicated WT1 peptides in the presence of recombinant human IL-2 and then restimulated for 8 days with TMMPs containing the same peptides and containing either a G2C disulfide bond or both R12C and G2C disulfide bonds. The data are shown in Fig. 22.

The data presented in Fig. 22 demonstrate in PBMCs from three donors that WT1-containing TMMPs can expand WT1-specific CD8+ T cells from total PBMCs during an 8-day restimulation culture period following a 10-day priming culture. This expansion occurred from cells that contained detectable amounts of WT1-specific T cell precursors, indicating that TMMPs were able to lead to the expansion of antigen-specific T cells in donors with a primed/pre-existing WT-1-specific T cell repertoire. The data demonstrate that TMMPs specific for three WT1 peptides (37-45, 126-134, and 126-134 R126Y) and on either of the two disulfide scaffolds examined (G2C and R12C/G2C) induce such buildup.

The ability of CD8' T cells expanded by exposure to TMMPs containing the WT1 37-45 peptide and containing either a G2C disulfide bond or both R12C and G2C disulfide bonds was studied to produce TNF-α and IFN-γ in response to target cells (T2 cells) presenting the WT1-37-45 peptide or an irrelevant peptide (SL9). Phorbol 12-myristate 13-acetate (PMA) and ionomycin (iono) were used as positive controls. CD8+ T cells treated with medium were used as negative controls. TMMP-expanded CD8+ T cells were incubated with target cells (T2 cells) loaded with either the WT1 peptide or an irrelevant peptide (SL9 peptide from HIV). The response demonstrated by IFN-γ and TNFα production by CD8+ T cells relative to T2 cells injected with the WT 37-45 peptide was compared with that of T2 cells injected with the SL9 peptide. The data are shown in Fig. 23.

The data shown in Fig. 23 demonstrate the selective polyfunctionality of WT1 37-45-specific CD8+ T cells expanded with WT1 37-45 TMMPs having either a G2C or R12C/G2C backbone. Measurable responses (TNF-α and IFN-γ production) were observed only upon recognition of target cells presenting the WT1 37-45 peptide but not the SL9 peptide. Positive and negative control wells show that in the absence of stimulation there is no basal activity in CD8+ T cells (as shown in the medium-only wells) and that both antigen-specific and non-antigen-specific cells are able to demonstrate functional responses to strong antigen-non-specific stimulation (PMA + ionomycin).

Using the same assay, the ability of CD8 T cells expanded with TMMPs containing the WT1 126-134 peptide (WT1 126) and containing R12C and G2C disulfide bonds to produce TNF-α and IFN-γ in response to target cells (T2 cells) presenting the WT1-126134 peptide, the WT1-126-134 R126Y peptide, or an irrelevant peptide (SL9) was examined. The data are shown in Fig. 24.

The data presented in Fig. 24 demonstrate the selective polyfunctionality of WT1 126-134-specific CD8 ⁺ T cells expanded with WT1 126-134-specific immunoSTATs on a R12C/G2C scaffold.

A measurable response (TNF-α and IFN-γ production) was observed only upon recognition of target cells presenting the WT1 126-134 peptide or the WT1 126-134 R126Y peptide, but not the SL9 peptide.

- 115 049816

Positive and negative control wells show that in the absence of stimulation there is no basal activity in CD8' T cells (as shown in medium-only wells) and that both antigen-specific and non-antigen-specific cells are able to demonstrate functional responses to strong antigen-non-specific stimulation (PMA + ionomycin).

The effect of disulfide bonds on IL-2-stimulated immune cell activation was investigated. CD69 is an early activation marker on most lymphocytes and some other immune cells. Cells upregulate CD69 under various types of stimulatory conditions, including IL-2 stimulation. CD69 upregulation was assessed on NK cells, CD4 ⁺ T cells, and CD8 ⁺ T cells. CD69 upregulation demonstrates that the IL-2 polypeptides present in TMMPs are active and their function is attenuated compared to the control (recombinant human IL-2). PBMCs from various human donors were incubated with the various ISTs outlined in Table . ISTs containing a CMV epitope or a MART-1 epitope were included as controls. Data from one human donor (Leukopaque 6) are shown. Data are shown in Fig. 25. Figure 25 shows the upregulation of CD69 on NK cells as a relevant and representative example of a cell that readily upregulates CD69 in response to IL-2. Similar data were observed on CD8 ⁺ T cell gates and CD4 ⁺ T cell gates.

The data presented in Fig. 25 demonstrate that the IL-2 immunomodulatory polypeptide engineered at position 1 of HLA-A02-specific immuno-STATs built on different disulfide scaffolds (R12C, G2C, and R12C/G2C) is functional (as observed by induction of CD69 on the surface of the corresponding immune cell) and attenuated compared to recombinant human wild-type IL-2.

To assess the activity of IL-2 immunomodulatory polypeptide variants present in TMMPs, a CTLL-2 proliferation assay was performed. CTLL-2 cell growth is IL-2 dependent; therefore, CTLL-2 proliferation is indicative of the amount and/or activity of IL-2 present in the culture medium (e.g., when IL-2 is produced by T cells contacting TMMPs). Gillis et al. (1978) J. Immunol. 120:2027.

The data are shown in Fig. 26. The data shown in Fig. 26 demonstrate that the IL-2 immunomodulatory polypeptide engineered at position 1 of HLA-A02-specific immunoSTATs (TMMPs) built on different disulfide scaffolds (R12C, G2C, and R12C/G2C) is functional (as observed by induction of CTLL-2 proliferation) and attenuated compared to PROLEUKIN.

The ability of the TMMPs used in these experiments to bind to FcRn was examined. TMMPs contain the Fc region of Ig that can bind to FcRn. Binding to FcRn is indicative of prolonged half-life in vivo. Souders et al. (2015) MAbs 7:912. Data for TMPP 1715 + 2380 are shown in Fig. 27.

The ability of TMPP 1715+2380 to bind to other Fc receptors was examined. TMPP 1715+2380 contains an Ig Fc region with LALA substitutions that reduce Ig Fc binding to FcRI, RIIA, IIB, IIIA-F, and IIIB, thereby reducing Ig Fc-mediated effector functions. The data are shown in Fig. 27.

Materials and methods.

Fig. 21.

Leukopacks (leukocyte-rich blood products) from two healthy donors were obtained using apheresis equipment. Leukopacks were diluted with an equal volume of phosphate-buffered saline (PBS) at room temperature. PBMCs were isolated from the diluted Leukopacks by density gradient centrifugation as follows: 30 ml of the diluted Leukopack was added to a layer of 13 ml Ficoll-Paque in a 50 ml conical tube and centrifuged at 400 g for 30 min at room temperature in a swing-out rotor without brake. The cell layer (lymphocytes, monocytes, and platelets) was collected from the plasma-Ficoll interface, transferred to a new 50 ml conical tube, and washed with a 3-fold excess of PBS by centrifugation at 300 g for 10 min at room temperature. After careful removal of the supernatant, the cells were resuspended and washed with 50 ml PBS by centrifugation at 200 g for 10 min at room temperature to remove platelets. After washing and platelet removal, the resulting PBMCs were pooled from 50 ml tubes, resuspended in PBS, counted, pelleted by centrifugation at 300 g for 10 min, and resuspended to a final concentration of 50 x 10 ⁶ cells ml in cell freezing medium.

PBMCs from healthy human donors were prepared from two leukopaques as described above. On the day of the experiment, the cells were thawed in a 37°C water bath and washed in ImmunoCult™-XF Warm Cell Expansion Medium (Stemcell Technologies) by centrifugation at 350 x g for 6 min. The supernatant was removed and the cells were resuspended in ImmunoCult™ medium. Viable cell numbers were assessed using a Countess automated cell counter (Invitrogen, California). The medium volume was adjusted to obtain a cell concentration of 5 x 10 ⁶ cells/mL, and 2 mL of cells (equivalent to 10 6 cells/mL) were seeded.

- 116 049816 (10x10 ⁶ cells) per 6-well plate. PBMCs were stimulated with the indicated amounts of immuno-STAT or medium alone in a total medium volume of 4 ml. Cells were stimulated for 10 days at 37°C, 5% CO2, with medium replaced on days 5 and 7 by aspirating 2 ml of culture supernatant from the wells and replenishing with 2 ml of fresh medium.

After culturing, the cells were collected and pelleted by centrifugation at 350 xg for 5 min, the number of viable cells was determined using a Countess automated cell counter (Invitrogen, California), and the cells were analyzed by flow cytometry staining with: viability dye, appropriate WT1 peptide-specific tetramers HLA-A*0201 (MBL International) and antibodies against CD69, CD3, CD14, CD19, CD127, CD56, CD4 (Biolegend), CD8, CD25 (BD Biosciences). Stained cells were washed and analyzed by flow cytometry.

Data collection was performed using an Attune NxT flow cytometry instrument (Invitrogen). Collected data were exported as fcs files and analyzed using Flowjo software (Tree Star, Oregon).

Based on the frequency of antigen-specific T cells, the volumes and events analyzed by flow cytometry, and the number of cells collected at the end of culture, the number of antigen-specific T cells was counted and plotted on the graphs shown.

Fig. 22.

PBMCs from two donors were expanded for 10 days with 10 micrograms/ml of the indicated peptides and 50 U/ml of recombinant human IL-2. Expansion was performed in 6-well plates with a total of 10 million cells in 4 ml of Immunocult medium per well. Cells were stimulated for 10 days at 37°C, 5% CO2, with the medium replaced on days 5 and 7 by aspirating 2 ml of culture supernatant from the wells and replacing with 2 ml of fresh medium containing 50 U/ml of recombinant human IL-2. After culturing, cells were harvested and pelleted by centrifugation at 350 g for 5 min, and the number of viable cells was determined using a Countess automated cell counter (Invitrogen, California). Each stimulation condition was performed in at least 3 wells of a six-well plate. PBMCs from a single well were used to assess the frequency/quantity of WT1-specific CD8' T cells in culture by flow cytometry staining with viability dye, appropriate WT1 peptide-specific tetramers HLA-A*0201 (MBL International) and antibodies against CD69, CD3, CD14, CD19, CD127, CD56, CD4 (Biolegend), CD8, CD25 (BD Biosciences). Stained cells were washed and analyzed by flow cytometry. At least two wells were used to enrich for CD8+ T cells using the CD8+ T cell negative selection kit from Stem Cell Technologies. Purified CD8+ T cells were restimulated for 8 days with the indicated TMMPs in the presence of autologous mitomycin C-pretreated PBMCs at a 1:2 ratio, to a final cell count of 5-10 million cells in 4 ml Immunocult medium per well. The TMMPs used were at concentrations previously established as optimal for the combination of donor and specific TMMP.

After culturing, the cells were collected and pelleted by centrifugation at 350 g for 5 min, the number of viable cells was determined using a Countess automated cell counter (Invitrogen, California), and they were analyzed by flow cytometry staining with: viability dye, appropriate WT1 peptide-specific tetramers HLA-A*0201 (MBL International) and antibodies against CD69, CD3, CD14, CD19, CD127, CD56, CD4 (Biolegend), CD8, CD25 (BD Biosciences). Stained cells were washed and analyzed by flow cytometry.

Data collection was performed using an Attune NxT flow cytometry instrument (Invitrogen). Collected data were exported as fcs files and analyzed using Flowjo software (Tree Star, Oregon).

Based on the frequency of antigen-specific T cells, the volumes and events analyzed by flow cytometry, and the number of cells collected at the end of culture, the number of antigen-specific T cells was counted and plotted on the graphs shown.

Fig. 23.

PBMCs from two donors were expanded for 10 days with 10 micrograms/ml WT1 37-45 peptide and 50 U/ml recombinant human IL-2. Expansion was performed in 6-well plates with a total of 10 million cells in 4 ml Immunocult medium per well. Cells were stimulated for 10 days at 37°C, 5% CO ₂ with medium replaced on days 5 and 7 by aspirating 2 ml of culture supernatant from wells and replacing with 2 ml of fresh medium containing 50 U/ml recombinant human IL-2. After culturing, cells were collected and pelleted by centrifugation at 350 g for 5 min, and the number of living cells was determined using a Countess automated cell counter (Invitrogen, California). Each stimulation was performed in at least 3 wells of a six-well plate. PBMC from one well were used to assess the frequency/number of WT1 37-45 peptide-specific CD8+ T cells in the culture by flow cytometry staining with: viability dye, appropriate WT1 37-45 peptide-specific HLA-A*O2O1 tetramers (MBL International) and antibodies against CD69, CD3, CD14, CD19, CD127, CD56, CD4 (Biolegend), CD8, CD25 (BD Biosci

- 117 049816 ences). Stained cells were washed and analyzed by flow cytometry. Data were collected using an Attune NxT flow cytometry instrument (Invitrogen). Collected data were exported as fcs files and analyzed using Flowjo software (Tree Star, Oregon).

At least two wells were used to enrich for CD8+ T cells using the CD8+ T cell negative selection kit from Stem Cell Technologies. Purified CD8+ T cells were restimulated for 8 days with the indicated WT1 37-45-specific immuno-STATs on either a G2C or R12C/G2C scaffold in the presence of autologous mitomycin C-pretreated PBMCs at a 1:2 ratio, to a final content of 5-10 million cells in 4 ml of Immunocult medium per well. WT1 37-45-specific immuno-STAT on either a G2C or R12C/G2C scaffold was used at the concentration previously established as optimal for that donor.

After culture, the cells were collected and pelleted by centrifugation at 350 g for 5 min, the number of living cells was determined using a Countess automated cell counter (Invitrogen, California), and CD8' T cells were enriched using a Cd8 ⁺ T cell negative selection kit from Stem Cell Technologies.

Target cells, T2 cells (ATCC), were treated with 5 μg/ml of WT1 37-45 peptide or human immunodeficiency virus-1 (HIV-1) SL9 Gag77-85 peptide for 2 h at 37°C, 5% CO ₂ . After peptide loading, T2 cells were washed twice and resuspended in ImmunoCult™-XF cell growth medium (Stemcell Technologies).

Enriched CD8 ⁺ T cells and peptide-loaded T2 cells were mixed at a 1:1 ratio (1 x ¹⁰⁶ cells for each) in a final volume of 200 μl per well in 96-well plates. Control wells were supplemented with medium and phorbol 12-myristate; phorbol 13-acetate (PMA)/ionomycin as negative and positive controls, respectively. Anti-CD107a antibody was added directly to the cells 0.5 to 1 h after stimulation. Cells were stimulated for 5 h, washed with PBS, and stained for viability using FVS780 for 10 min on ice. Cells were washed, stained with WT1 peptide-specific 37-45 tetramers (APC and PE-labeled) for 15 min at room temperature. Subsequently, the cells were washed and stained with antibodies against CD3 and CD8 for 30 min on ice. The stained cells were washed twice and resuspended in intracellular (IC) buffer overnight at 4°C. The next day, the cells were washed and resuspended in permeabilization buffer and incubated for 5 min at room temperature. Permeabilized cells were washed and stained with antibodies against interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), resuspended in permeabilization buffer for 30 min at room temperature. The stained cells were washed, resuspended in 2 ml FACS buffer and transfected in a 96-well deep-well plate. Data acquisition was performed using an Attune NxT flow cytometry instrument (Thermofisher Scientific, MA). The collected data were exported as fcs files and analyzed using Flowjo software (Tree Star, Oregon).

Fig. 24.

MHC from two donors were expanded for 10 days with 10 micrograms/ml WT1 peptide 126134 and 50 U/ml recombinant human IL-2. Expansion was performed in 6-well plates with a total of 10 million cells in 4 ml Immunocult medium per well. Cells were stimulated for 10 days at 37°C, 5% CO2 with medium replaced on days 5 and 7 by aspirating 2 ml of culture supernatant from wells and replacing with 2 ml of fresh medium containing 50 U/ml recombinant human IL-2. After cultivation, cells were collected and pelleted by centrifugation at 350 g for 5 min, and the number of living cells was determined using a Countess automated cell counter (Invitrogen, California). Each stimulation was performed in at least 3 wells of a six-well plate. PBMC from one well were used to assess the frequency/number of WT1 126-134 peptide-specific CD8 ⁺ T cells in the culture by flow cytometry staining with viability dye, appropriate WT1 126-134 peptide-specific HLA-A*0201 tetramers (MBL International) and antibodies against CD69, CD3, CD14, CD19, CD127, CD56, CD4 (Biolegend), CD8, CD25 (BD Biosciences). Stained cells were washed and analyzed by flow cytometry. Data were collected using an Attune NxT flow cytometry instrument (Invitrogen). Collected data were exported as fcs files and analyzed using Flowjo software (Tree Star, OR).

At least two wells were used to enrich for CD8 ⁺ T cells using the CD8 ⁺ T cell negative selection kit from Stem Cell Technologies. Purified CD8 ⁺ T cells were restimulated for 8 days with WT1 126-134-specific immuno-STAT on an R12C/G2C scaffold in the presence of autologous mitomycin C-pretreated PBMCs at a 1:2 ratio to yield 5-10 million cells in 4 ml of Immunocult medium per well. The concentration of WT1 126-134-specific immuno-STAT on an R12C/G2C scaffold previously established as optimal for the donor was used.

- 118 049816

After culture, the cells were collected and pelleted by centrifugation at 350 g for 5 min, the number of living cells was determined using a Countess automated cell counter (Invitrogen, California), and CD8' T cells were enriched using a Cd8 ⁺ T cell negative selection kit from Stem Cell Technologies.

Target cells, T2 cells (ATCC), were treated with 5 μg/ml WT1 126-134 peptide, WT1 126134 R126Y peptide, or human immunodeficiency virus-1 (HIV-1) SL9 Gag77-85 peptide for 2 h at 37°C, 5% CO ₂ . After peptide loading, T2 cells were washed twice and resuspended in ImmunoCult™-XF Cell Growth Medium (Stemcell Technologies).

Enriched CD8 ⁺ T cells and peptide-loaded T2 cells were mixed at a 1:1 ratio (1 x ¹⁰⁶ cells for each) in a final volume of 200 μl per well in 96-well plates. Control wells were supplemented with medium and phorbol 12-myristate; phorbol 13-acetate (PMA)/ionomycin as negative and positive controls, respectively. Anti-CD107a antibody was added directly to the cells 0.5 to 1 h after stimulation. Cells were stimulated for 5 h, washed with PBS, and stained for viability using FVS780 for 10 min on ice. Cells were washed, stained with WT1 peptide-specific 126-134 tetramers (APC and PE-labeled) for 15 min at room temperature. Subsequently, the cells were washed and stained with antibodies against CD3 and CD8 for 30 min on ice. The stained cells were washed twice and resuspended in intracellular (IC) buffer overnight at 4°C. The next day, the cells were washed and resuspended in permeabilization buffer and incubated for 5 min at room temperature. Permeabilized cells were washed and stained with antibodies against interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), resuspended in permeabilization buffer for 30 min at room temperature. The stained cells were washed, resuspended in 2 ml FACS buffer and transfected in a 96-well deep-well plate. Data acquisition was performed using an Attune NxT flow cytometry instrument (Thermofisher Scientific, MA). The collected data were exported as fcs files and analyzed using Flowjo software (Tree Star, Oregon).

Fig. 25.

PBMCs from healthy human donors were prepared from Leucopacks obtained from Hemacare (Northridge, CA) and stored cryopreserved at -150°C until the day of the experiment.

On the day of the experiment, cells were thawed in a water bath for 1 min, washed with 10 ml of warm ImmunoCult™-XF cell growth medium (Stemcell Technologies, Vancouver, Canada), pelleted by centrifugation (350 g, 5 min), and resuspended in 10 ml of medium. Cells were counted using a Countess automated cell counter (Invitrogen, California), the medium volume was adjusted to a cell concentration of 3.8 x 10 ⁶ cells/ml, and 237.5 μl of cell suspensions were added to 96-well round-bottom plates.

In Immunocult medium, 20x dilution series of the indicated immuno-STAT and rh-IL-2 were prepared. For stimulation of PBMC, 12.5 μl of 20x dilution series were added to wells containing cells and mixed to obtain final concentrations of the analyzed drugs. PBMC were incubated at 37°C, 5% CO2 for 20 to 24 h.

Following stimulation, cells were pelleted by centrifugation at 350 g for 5 min. Supernatants were collected, frozen, and stored at -20°C until further analysis. Pellets of PBMC were washed twice with PBS and stained for 10 min at 4°C in 50 μl of FVS780 live/dead stain. Staining was quenched with 200 μl of staining buffer, and cells were pelleted by centrifugation (350 x g, 5 min). Cells were stained for 30 min at 4°C with 50 μl of antibodies against CD3, CD4, CD8, CD14, CD19, CD56, and CD69. For staining, cells were washed with staining buffer, pelleted by centrifugation (350xg, 5 min), resuspended in 130 µl staining buffer and analyzed by flow cytometry using an Attune® flow cytometer. Collected data were exported as fcs files and analyzed using Flowjo software (Tree Star, OR).

CD69 upregulation was assessed on different cell subsets that are sensitive to CD69 upregulation upon IL-2 stimulation. Based on the expression levels of surface markers used in the staining, gates were created to identify NK cells, CD8 ⁺ T cells, and CD4 ⁺ T cells.

Fig. 26.

One day before the assay, CTLL-2 cells were washed with medium and cultured at 1 x ¹⁰⁵ cells/mL in a 75-T flask for 24 h at 37°C, 5% CO2 to deplete IL-2. After 24 h of depletion culture, cells were seeded at 5000 cells/well in a volume of 100 microliters/well in a 96-well flat-bottomed plate with a cell culture cluster lid (Costar corning, cat. no. 3599). Viability and cell numbers were checked before stimulation using a Vi cell viability assay (Beckman-Coulter).

A series of dilutions (10 points; dilutions in 3-fold steps) of the indicated immuno-STATs were prepared.

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Claims (24)

(TMMP) or Proleukin (Prometheus Therapeutics) in complete RPMI medium supplemented with 10% HI FBS as 2x stock solutions for the final concentrations analyzed. 100 μl of these 2x dilution series were added to pre-seeded cells in 96-well plates and mixed to obtain the final concentrations of the drugs analyzed. Each concentration was tested in triplicate. Cells were incubated for 3 days at 37°C, 5% CO ₃ . After three days of culture, 100 µl of cells from each well were transferred to flat-bottomed white 96-well plates treated for tissue culture attachment. 100 µl of CellTiter-Glo® reagent was prepared using the CellTiter-Glo Luminescent Cell Viability Assay Kit (Promega cat# G7571) following the manufacturer’s instructions and added to the cells. Cells and CellTiter-Glo® reagent were mixed by placing the plates on an orbital shaker for 2 min to induce cell lysis. The plates were then incubated at room temperature for 10 min to stabilize the luminescent signal. Luminescence was measured on a Biotek synergy neo2 multi-mode plate reader, Gen5 3.04 software. Fig. 27. All experiments were performed on an Octet HTX system (ForteBio). Anti-Penta-His (HIS1K) biosensors for kinetic measurements (ForteBio, #18-5122) were hydrated in assay buffer and pre-adapted in glycine at pH 1.7. Assay buffer was used for all assays except the FcRn assay. The buffer used for FcRn contained PBS, 0.1% bovine serum albumin (BSA), 0.02% Tween-20, pH 7.2. The assay buffer used for FcRn reagents contained PBS, 0.1% BSA, 0.02% Tween-20, pH 6. Each His-tagged receptor was immobilized on HIS1K biosensors at a concentration of 5 μg/mL (FcRI exclusion: 10 μg/mL) for 120 s. Antigen-loaded HIS1K biosensors were then loaded into a 7-point 1:3 dilution series of each individual antibody starting at a concentration of 300 nM. A well containing assay buffer alone was used to test for non-specific binding between the buffer and loaded biosensors. Association was observed for 60 s, followed by dissociation in 60 s. A short baseline period (60 s) was created using dissociation buffer after HIS1K loading. Although the present invention has been described with reference to specific embodiments thereof, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. Furthermore, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps to the purpose, spirit and scope of the present invention. All such modifications are intended to be within the scope of the appended claims. CLAUSE OF THE INVENTION 1. A T cell modulating multimeric polypeptide (TMMP) comprising at least one heterodimer comprising: a) a first polypeptide comprising: i) Wilms tumor epitope-1 (WT-1) peptide, wherein the WT-1 peptide is from 4 to 25 amino acids in length; and ii) a first major histocompatibility complex (MHC) class I polypeptide, wherein the first MHC class I polypeptide is a β2-microglobulin (β2M) polypeptide; b) a second polypeptide comprising: i) a second MHC class I polypeptide, wherein the second MHC class I polypeptide is an MHC class I heavy chain polypeptide; ii) one or more immunomodulatory polypeptides, wherein the one or more immunomodulatory polypeptides are one or more variant IL-2 polypeptides that exhibit reduced affinity for the IL-2 receptor and that comprise an H16 substitution or an H16 substitution and an F42 substitution according to the amino acid numbering of SEQ ID NO: 15; and iii) an immunoglobulin (Ig) Fc polypeptide, wherein the first polypeptide and the second polypeptide are covalently linked to each other via at least first and second disulfide bonds, wherein the first disulfide bond is formed between a Cys residue in a Cys-containing linker between a WT-1 epitope peptide and a β2M polypeptide of said first polypeptide and a Cys residue in an MHC class I heavy chain polypeptide of said second polypeptide, and wherein the second disulfide bond is formed between a Cys residue in a β2M polypeptide of said first polypeptide and a Cys residue in an MHC class I heavy chain polypeptide of said second polypeptide. 2. TMMR according to item 1, characterized in that a) the first polypeptide includes, in order from N-terminus to C-terminus: i) WT-1 peptide epitope, - 120 049816 ii) containing a Cys linker, and iii) a β2M polypeptide, and b) the second polypeptide comprises, in order from N-terminus to C-terminus: i) one or more variant IL-2 polypeptides, ii) an optional linker, iii) an MHC class I heavy chain polypeptide, iv) an optional linker, and v) an Ig Fc polypeptide, wherein when the TMMP comprises more than one IL-2 variant polypeptide, the TMMP may comprise one or more linkers between said IL-2 variant polypeptides. 3. TMMR according to item 1, characterized in that a) the first polypeptide comprises, in order from N-terminus to C-terminus: i) a WT-1 peptide epitope, ii) a Cys linker, and iii) a B2M polypeptide, and b) the second polypeptide comprises, in order from N-terminus to C-terminus: i) an MHC class I heavy chain polypeptide, ii) an optional linker, iii) an Ig Fc polypeptide, iv) an optional linker, and v) one or more variant IL-2 polypeptides, wherein when the TMMP comprises more than one variant IL-2 polypeptide, the TMMP may comprise one or more linkers between said variant IL-2 polypeptides. 4. The TMMP according to any one of claims 1-3, wherein the variant IL-2 polypeptide comprises: i) replacement of H16A and replacement of F42A or ii) replacement of H16T and replacement of F42A. 5. The TMMP according to any one of claims 1 to 4, characterized in that the MHC class I heavy chain polypeptide comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 199, SEQ ID NO: 201 or SEQ ID NO: 203. 6. The TMMP of claim 2, wherein the TMMP comprises two variant IL-2 polypeptides that are in tandem and optionally connected by a linker, and wherein each of the IL-2 variants comprises an amino acid sequence that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 188, wherein X1 is Ala and _X2 is Ala, or X1 is Thr and _X2 is Ala, wherein the first disulfide bond is formed between (i) a Cys residue in the linker between the WT-1 peptide epitope and the B2M polypeptide, and (ii) a Cys residue at position 84 in an MHC class I heavy chain polypeptide, and wherein the second disulfide bond is formed between a Cys residue at position 12 of the B2M polypeptide, based on the amino acid numbering of SEQ ID NO: 311, and a Cys residue at position 236 MHC class I heavy chain polypeptide according to the amino acid numbering of SEQ ID NO: 199, wherein the MHC class I heavy chain polypeptide comprises an amino acid sequence that is at least 95% identical to the amino acids presented in SEQ ID NO: 199, wherein the amino acid at position 84 is Cys, and the amino acid at position 236 is Cys, and wherein the Ig Fc polypeptide has at least 95% amino acid sequence identity to the amino acid sequence presented in any of SEQ ID NOs: 418-421. 7. The TMMP of claim 6, wherein the variant IL-2 polypeptides are connected by a linker, and each variant IL-2 polypeptide has an amino acid sequence as set forth in SEQ ID NO: 188, wherein X1 is Ala and _X2 is Ala. 8. The TMMP of claim 6 or 7, wherein the MHC class I heavy chain polypeptide comprises an amino acid sequence having at least 95% amino acid sequence identity with SEQ ID NO: 199, and wherein the epitope is VLDFAPPGA (SEQ ID NO: 259). 9. The TMMP of claim 3, which comprises two variant IL-2 polypeptides in tandem and optionally connected by a linker, and wherein each of the variant IL-2 polypeptides comprises an amino acid sequence having at least 95% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 188, wherein X1 is Ala and _X2 is Ala, or wherein X1 is Thr and _X2 is Ala, wherein the first disulfide bond is formed between (i) a Cys residue in the linker between the WT-1 peptide epitope and the B2M polypeptide and (ii) a Cys residue at position 84 of the MHC class I heavy chain polypeptide, and wherein the second disulfide bond is formed between a Cys residue at position 12 of the B2M polypeptide, based on the amino acid numbering of SEQ ID NO: 311, and a Cys residue at position 236 MHC class I heavy chain polypeptide based on the amino acid numbering of SEQ ID NO: 199, wherein the MHC class I heavy chain polypeptide comprises an amino acid sequence having - 121 049816 having at least 95% amino acid sequence identity to the amino acids set forth in any of SEQ ID NOs: 41-43 and 198-206, wherein amino acid 84 is Cys and amino acid 236 is Cys, and wherein the Ig Fc polypeptide has at least 95% amino acid sequence identity to the amino acid sequence set forth in any of SEQ ID NOs: 418-421. 10. The TMMP of claim 9, wherein the MHC class I heavy chain polypeptide comprises an amino acid sequence having at least 95% amino acid sequence identity to SEQ ID NO: 201. 11. The TMMP of claim 9, wherein the MHC class I heavy chain polypeptide comprises an amino acid sequence having at least 95% amino acid sequence identity to SEQ ID NO: 203. 12. The TMMP of any one of claims 9-11, wherein the variant IL-2 polypeptides are connected by a linker, and each of the variant IL-2 polypeptides has an amino acid sequence as set forth in SEQ ID NO: 188, wherein X1 is Ala, and X2 is Ala. 13. The TMMP according to any one of claims 1 to 12, wherein the linker between the WT-1 peptide epitope and the B2M polypeptide comprises the amino acid sequence presented in SEQ ID NO: 316 or SEQ ID NO: 317. 14. The TMMP of claim 7, wherein the first polypeptide comprises an amino acid sequence as set forth in SEQ ID NO: 423, the MHC class I heavy chain polypeptide comprises an amino acid sequence having at least 98% amino acid sequence identity to the amino acid sequence GSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEY WDGETRKVKAHSQTHRVDLGTLRGCYNQSEAGSHTVQRMYGCDVGSDWRFLRGYHQ YAYDGKDYIALKEDLRSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGTCVEWLRRY LENGKETLQRTDAPKTHMTHHAVSDHEATLRCWALSFYPAEITLTWQRDGEDQTQDTE LVETRPCGDGTFQKWAAVVVPSGQEQRYTCHVQHEGLPKPLTLRWEh the Ig Fc polypeptide comprises an amino acid sequence having at least 98% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 421. 15. The TMMP of claim 14, wherein the MHC class I heavy chain polypeptide comprises an amino acid sequence having an amino acid sequence GSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEY WDGETRKVKAHSQTHRVDLGTLRGCYNQSEAGSHTVQRMYGCDVGSDWRFLRGYHQ YAYDGKDYIALKEDLRSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGTCVEWLRRY LENGKETLQRTDAPKTHMTHHAVSDHEATLRCWALSFYPAEITLTWQRDGEDQTQDTE LVETRPCGDGTFQKWAAVVVPSGQEQRYTCHVQHEGLPKPLTLRWE. 16. A TMMP comprising a first and a second heterodimer according to any one of claims 1 to 14, wherein the first and second heterodimers are identical in amino acid sequence and are covalently linked to each other by one or more disulfide bonds between the Ig Fc polypeptides of these first and second heterodimers. 17. A TMMP comprising a first and second heterodimer according to claim 15, wherein the first and second heterodimers are identical in amino acid sequence and are covalently linked to each other by one or more disulfide bonds between the Ig Fc polypeptides of the first and second heterodimers. 18. The TMMP of claim 17, wherein two disulfide bonds link the Ig Fc polypeptide of one heterodimer to the Ig Fc polypeptide of another heterodimer. 19. A pharmaceutical composition containing TMMP according to claim 17. 20. A pharmaceutical composition containing TMMP according to claim 18. 21. A pharmaceutical composition comprising one or more nucleic acids containing nucleotide sequences encoding the first and second TMMP polypeptides according to any one of claims 1-18. 22. A pharmaceutical composition comprising at least one expression vector, wherein said at least one expression vector comprises nucleotide sequences encoding the first and second TMMP polypeptides according to any one of claims 1-18. 23. A method for producing TMMP according to any one of claims 1 to 18, comprising culturing a host cell genetically modified with one or more expression vectors containing nucleotide sequences encoding the first and second polypeptides of the multimeric polypeptide, under conditions under which the genetically modified host cell produces TMMP. 24. An in vitro composition of genetically modified host cells, wherein the host cells - 122 -