EA046369B1 - MULTIMERIC POLYPEPTIDES MODULATING T-CELLS AND METHODS OF THEIR APPLICATION - Google Patents

Description

Cross reference

This application claims benefit from U.S. Provisional Patent Application No. 62/438,272, filed Dec. 22, 2016, U.S. Provisional Patent Application No. 62/470,774, filed March 13, 2017, U.S. Provisional Patent Application No. 62/555,435, filed September 7, 2017, and U.S. Provisional Patent Application No. 62/582,132, filed November 6, 2017, each of which is incorporated herein by reference in its entirety.

Incorporation by reference of a sequence listing provided as a text file

The Sequence List is provided in the form of a text file CUEB107WO_SEQ_LISTING_171133_ST25.txt, created on November 14, 2017 and having a size of 153 KB. The contents of the text file 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 presented non-covalently on the surface of the antigen-presenting cell (APC) by the major histocompatibility complex. (MHC; also called human leukocyte antigen complex (HLA) in humans). This interaction represents the targeting mechanism of the immune system and is a necessary molecular interaction for the modulation (activation or inhibition) and effector function of T cells. Following epitope-specific targeting of the target cells, the targeted T cells are activated through the interaction of costimulatory proteins present on APCs with costimulatory partner proteins on the T cells. Both signals—epitope/TCR binding and interaction of APC costimulatory proteins with T cell costimulatory proteins—are required to control T cell specificity and activation or inhibition. TCR is specific for a given epitope; however, costimulatory protein is not epitope specific and is instead typically expressed on all T cells or large subsets of T cells.

Brief description of the invention

This invention relates to variant immunomodulatory polypeptides and hybrid polypeptides containing variant immunomodulatory peptides. This invention relates to multimeric T cell modulating polypeptides and compositions containing them, wherein said multimeric T cell modulating polypeptides comprise a variant immunomodulatory polypeptide of the present invention. This invention relates to nucleic acids containing nucleotide sequences encoding multimeric polypeptides that modulate T cells, and to host cells containing these nucleic acids. The present invention provides methods for modulating the activity of a T cell, said methods comprising contacting the T cell with a multimeric T cell modulating polypeptide of the present invention.

Brief description of graphic materials

Fig. 1A-1D schematically show various embodiments of the multimeric T cell modulating polypeptide of the present invention. In these embodiments, disulfide bonds are formed between MHC polypeptides (eg, HLA) present in the individual polypeptides.

Fig. 2A-2Q show the amino acid sequence of wild-type human IL-2 (Fig. 2A); and amino acid sequences of variant IL-2 polypeptides (FIGS. 2B-2Q).

Fig. 3A-3C show the amino acid sequences of the alpha chain (FIG. 3A), beta chain (FIG. 3B), and gamma chain (FIG. 3C) of the IL-2 receptor.

Fig. 4A-4C show the amino acid sequences of immunoglobulin Fc polypeptides.

Fig. 5A-5C show the amino acid sequences of class I human leukocyte antigen (HLA) heavy chain polypeptides. Signal sequences are underlined.

Fig. 6 shows a multiple amino acid sequence alignment of beta-2-microglobulin (b2M) precursors (i.e., including the leader sequence) from Homo sapiens (NP_004039.1; SEQ ID NO: 95), Pan troglodytes (NP_001009066.1; SEQ ID NO: 96), Macaca mulatto (NP_001040602.1; SEQ ID NO: 97), Bos Taurus (NP_776318.1; SEQ ID NO: 98) and Mus musculus (NP_033865.2; SEQ ID NO: 99). Amino acids 1-20 constitute the signal peptide.

Fig. 7A, 7B demonstrate the production of IL-2/synTac (Cue-IL-2-a and Cue-Kn-2-b) of the present invention after transient transfection. Fig. 7A shows the crude product; fig. 7B shows the purified product.

Fig. 8A, 8B demonstrate IL-2/synTac production of the present invention, wherein the IL-2 polypeptide is present in the light chain (light chain polypeptide chain (e.g., B2M) of the MHC class I molecule) or the heavy chain (heavy chain polypeptide chain MHC class I molecules).

In fig. Figure 9 demonstrates the expression level of IL-2/syn-Tacs, in which IL-2 is a di

- 1 046369 coma type (dt) or contains various combinations of F42A, D20K, Q126A, E15A, Y45A and H16A.

Fig. 10 shows the expression of IL-2/synTac according to the present invention, in which IL-2 is present in one copy (1X), two copies (2X) or three copies (3X) in synTac.

Fig. 11 demonstrates in vitro stimulation of antigen-specific CD8+ T cells and non-specific CD8' T cells by IL-2/synTac of the present invention, wherein the variant IL-2 containing the F42A and H16A substitutions is present in two copies in synTac.

Fig. 12 demonstrates the binding of IL-2/synTac to specific (lymphocytic choriomeningitis virus; LCMV) or nonspecific (OT1; ovalbumin-recognizing) CD8' T cells.

Fig. 13 demonstrates IL-2/synTac-mediated signaling in antigen-specific (LCMV) or nonspecific (BL6) CD8+ T cells.

Fig. 14A-14F demonstrate the percentage of cells positive for phospho-signal transducer and activator of transcription 5 (pSTAT5 - phospho-signal transducer and activator of transcription 5) after stimulation of CD8 ⁺ antigen-specific (LCMV) or non-specific (BL6) cells with IL-2/synTac according to this invention, at different concentrations of IL-2/synTac.

Fig. 15 demonstrates the in vivo activity of IL-2/synTac according to the present invention. The left panel demonstrates the fold change in the number of antigen-specific CD8 ⁺ T cells after administration of phosphate-buffered saline (PSB), recombinant IL-2 (rIL-2) or IL-2/synTac according to the present invention. The right panel shows antigen-specific and non-antigen-specific responses after administration of PBS, rIL-2 or IL-2/synTac according to this invention.

Fig. 16A, 16B show the effects of increasing dose (FIG. 16A) and route of administration (FIG. 16B).

Fig. 17A, 17B demonstrate the effect of IL-2 copy number on in vivo antitumor efficacy.

Fig. 18 shows the serum half-life of IL-2/synTac of the present invention following intraperitoneal administration of 10 mg/kg IL-2/synTac.

Fig. 19 demonstrates the stability of the IL-2/synTac of the present invention 2 hours after intraperitoneal administration of 10 mg/kg IL-2/synTac.

Fig. 20 shows size exclusion chromatography data of IL-2/synTac of the present invention after incubating IL-2/synTac at 4°C or 37°C for 5 days.

Fig. 21 shows the amino acid sequence of the IL-2/synTac heavy chain of the present invention with a leader peptide, wherein the IL-2/synTac heavy chain contains an IgG1 Fc with an N297A substitution.

Fig. 22 shows the amino acid sequence of the IL-2/synTac heavy chain of the present invention without the leader peptide, wherein the IL-2/synTac heavy chain contains an IgG1 Fc with an N297A substitution.

Fig. 23A, 23B show the nucleotide sequence (FIG. 23A) encoding the IL-2/synTac heavy chain shown in FIG. 21; and a key (FIG. 23B) for the sequence.

Fig. 24 shows the amino acid sequence of the IL-2/synTac heavy chain of the present invention with a leader peptide, wherein the IL-2/synTac heavy chain contains an IgG1 Fc with substitutions L234A and L235A.

Fig. 25 shows the amino acid sequence of the IL-2/synTac heavy chain of the present invention without the leader peptide, wherein the IL-2/synTac heavy chain contains an IgG1 Fc with substitutions L234A and L235A.

Fig. 26A, 26B show the nucleotide sequence (FIG. 26A) encoding the IL-2/synTac heavy chain depicted in FIG. 24; and a key (Fig. 26B) for the sequence.

Fig. 27 shows the amino acid sequence of the IL-2/synTac heavy chain of the present invention with a leader peptide, wherein the IL-2/synTac heavy chain contains an IgG1 Fc with substitutions L234F, L235E and P331S.

Fig. 28 shows the amino acid sequence of the IL-2/synTac heavy chain of the present invention without the leader peptide, wherein the IL-2/synTac heavy chain contains an IgG1 Fc with substitutions L234F, L235E and P331S.

Fig. 29A, 29B show the nucleotide sequence (FIG. 29A) encoding the IL-2/synTac heavy chain depicted in FIG. 27; and a key (Fig. 29B) for the sequence.

Fig. 30 shows the amino acid sequence of the IL-2/synTac light chain of the present invention with a leader peptide, wherein the IL-2/synTac light chain contains the E7 epitope of human papilloma virus (HPV).

Fig. 31 shows the amino acid sequence of the IL-2/synTac light chain of the present invention without the leader peptide, wherein the IL-2/synTac light chain contains the HPV E7 epitope.

Fig. 32 shows the nucleotide sequence encoding the IL-2/synTac light chain depicted in FIG. thirty.

Fig. 33A-33D show the amino acid sequences of wild-type human IgG1 Fc (FIG. 33A), IgG1 Fc with L234F, L235E, and P331S substitutions (FIG. 33B), IgG1 Fc with N297A substitution (FIG. 33C), and

- 2 046369

Fc IgG1 with substitutions L234A and L235A (Fig. 33D).

Fig. 34A-34C show the amino acid sequence of β2-microglobulin polypeptide (R12C) (FIG. 34A), variant IL-2 polypeptide (H16A; F42A) (FIG. 34B), and MHC class I A0201 H chain (Y84A); A236C) (Fig. 34C).

Fig. 35 demonstrates IL-2/synTac-mediated expansion of human CMV(CMV)-specific CD8+ T cells.

Fig. 36 shows expression data and receptor binding data for synTac with variant IL-2 polypeptides.

Fig. 37 demonstrates the binding of IL-2/synTac to primary human E7 HPV16(11-20)specific CD8+ T cells as detected by flow cytometry.

Fig. 38 demonstrates the effect of variant IL-2/synTac binding to primary HPV16 E7(11-20)-specific human CD8+ T cells on SLP76 phosphorylation.

Fig. 39 demonstrates the effect of variant IL-2/synTac binding to primary human HPV16 E7(11-20)-specific T cells on the production of CD25, granzyme B and CD107a.

Fig. 40 demonstrates the effect of variant IL-2/synTac binding to primary HPV16 E7(11-20)-specific human CD8+ T cells on IFN-γ production.

Definitions

The terms polynucleotide and nucleic acid, used interchangeably herein, refer to the polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, the term includes, but is not limited to, single-, double- or multi-stranded DNA or RNA, genomic DNA, cDNA DNA-RNA hybrids or 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 the polymeric form of amino acids of any length, which may include coded and non-coded 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 with another polynucleotide or polypeptide, meaning that when aligned, that percentage of 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 various ways. To determine sequence identity, sequences can be aligned using a variety of convenient methods and computer programs (eg, BLAST, T-COFFEE, MUSCLE, MAFFT, etc.) available on the World Wide Web at sites including ncbi.nlm.nih. gov/BLAST, ebi.ac.uk/Tools/msa/tcoffee/, ebi.ac.uk/Tools/msa/muscle/, mafft.cbrc.jp/alignment/software/. See, for example, Altschul et al. (1990), J. Mol. Bioi., 215:403-10.

The term conservative amino acid substitution refers to the interchangeability of amino acid residues having similar side chains in proteins. For example, the group of amino acids having aliphatic side chains consists of glycine, alanine, valine, leucine and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains consists of serine and threonine; a group of amino acids having amide-containing side chains consists of asparagine and glutamine; a group of amino acids having aromatic side chains consists of phenylalanine, tyrosine and tryptophan; a group of amino acids having main side chains consists of lysine, arginine and histidine; a 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. Examples of conservative groups of amino acid substitutions are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine-glycine and asparagine-glutamine.

As used herein, the term binding (eg, in relation to the binding of a multimeric T cell modulating polypeptide of the present invention to a polypeptide (eg, a T cell receptor on a T cell) refers to a non-covalent interaction between them. Binding interactions are typically characterized by a constant dissociation (KD) 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 binding strength, increased binding affinity correlating with lower KD.

As used herein, the term immunological synapse or immune synapse generally refers to the natural interface between two interacting immune cells of the adaptive immune response, including, for example, the interface between an antigen presenting cell (APC) or target cell and an effector cell, such as a lymphocyte, an effector T cell. cell, natural killer cell, etc. The immunological synapse between APC and T cell is usually initiated by the interaction of the T cell antigen receptor and major histocompatibility complex molecules, for example, as described in Bromley et al., Annu. Rev. Immunol., 2001,

- 3 046369

19:375-96, the disclosure of which is incorporated herein by reference in its entirety.

The T cell includes all types of immune cells that express CD3, including T helper cells (CD4+ cells), cytotoxic T cells (CD8+ cells), regulatory T cells (Tregs), and NK-T cells.

A costimulatory polypeptide as used herein includes a polypeptide on an antigen presenting cell (APC) (e.g., a dendritic cell, B cell, etc.) that specifically binds a cognate costimulatory polypeptide on a T cell, thereby providing a signal that, in addition to the primary signal provided by, for example, the binding of the TKP/CD3 complex to a major histocompatibility complex (MHC) polypeptide containing the peptide, mediates the T cell response, including, but not limited to, proliferation, activation , differentiation, etc. The costimulatory ligand 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, agonist or antibody that binds the Toll ligand receptor, and a ligand that specifically binds to B7-P3. A costimulatory ligand also includes, but is not limited to, an antibody that specifically binds to a costimulatory molecule present on a T cell, such as, but not limited to, CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS , lymphocyte function-associated antigen-1, CD2, LIGHT, NKG2C, B7-H3 and a ligand that specifically binds CD83.

The modulator domain (MOD) of the multimeric T cell modulating polypeptide of the present invention includes a costimulatory polypeptide, for example an IL-2 polypeptide, such as a variant IL-2 polypeptide of the present invention.

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

Recombinant, as used herein, means that a particular nucleic acid (DNA or RNA) is the product of various combinations of cloning, restriction digestion, polymerase chain reaction (PCR) and/or ligation steps resulting in a construct having a structural coding or non-coding sequence, different from endogenous nucleic acids present in natural systems. DNA sequences encoding polypeptides can be assembled from cDNA fragments or from a series of synthetic oligonucleotides to produce a synthetic nucleic acid that is capable of being expressed from a recombinant transcription unit contained in a cell or cell-free transcription and translation system.

The terms recombinant expression vector or DNA construct are used interchangeably herein to refer to a DNA molecule containing a vector and a single insert. Recombinant expression vectors are typically generated 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 DNA regulatory sequences.

The cell has been genetically modified, or transformed, or transfected with exogenous DNA, such as a recombinant expression vector, when such DNA has been introduced into the cell. The presence of exogenous DNA results in permanent or temporary genetic changes. Transforming DNA may or may not be integrated (covalently linked) into the cell's genome. For example, in prokaryotes, yeast and mammalian cells, transforming DNA may be supported on an episomal element such as a plasmid. As for eukaryotic cells, a stably transformed cell is a cell in which the transforming DNA is integrated into the chromosome so that it is inherited by daughter cells through chromosome replication.

As used herein, host cell means a eukaryotic cell in vivo or in vitro, or a cell of a multicellular organism (e.g., a cell line) cultured as a single-cell entity, wherein the eukaryotic cells can be or have been used as recipients for a nucleic acid (e.g., an expression vector, which contains a nucleotide sequence encoding a multimeric polypeptide of this invention), and include the progeny of the parent cell that has been genetically modified with the nucleic acid. It is understood that the progeny of an individual cell may not necessarily be completely identical in morphology or genomic or complete DNA complement to the original parent due to natural, accidental or intentional mutation. A recombinant host cell (also called a genetically modified host cell) is a host cell into which a heterologous nucleic acid, such as an expression vector, has been introduced. For example, a genetically modified eukaryotic host cell is genetically modified by introducing into a suitable eukaryotic host cell a heterologous nucleic acid, e.g.

- 4 046369 nucleic acid that is foreign to the eukaryotic host cell, or a recombinant nucleic acid that is not normally found in the eukaryotic host cell.

Terms treatment, treat, treatment, process, etc. are used herein to denote the achievement of a desired pharmacological and/or physiological effect. The effect may be prophylactic in terms of complete or partial prevention of a disease or symptom thereof and/or may be therapeutic in terms of partial or complete cure of a disease and/or an adverse effect associated with the disease. As used herein, treatment/treatment encompasses any treatment/treatment of a disease or symptom in a mammal and includes (a) preventing the occurrence of the disease or symptom in a subject who may be predisposed to acquiring the disease or symptom but has not yet been diagnosed as having it;

(b) suppression of a disease or symptom, e.g. cessation of their development; or (c) alleviation of the disease, i.e. causing regression of the disease.

The therapeutic agent may be administered before, during, or after the onset of the disease or injury. Treatment of ongoing disease, in which treatment stabilizes or reduces the patient's adverse clinical symptoms, is of particular interest. It is advisable to carry out such treatment before complete loss of function in the affected tissues occurs. The therapy is preferably administered to the subject 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 mammalian subject for which diagnosis, treatment or therapy is required. Mammals include, for example, humans, non-human primates, rodents (e.g., rats, mice), lagomorphs (e.g., rabbits), ungulates (e.g., cows, sheep, pigs, horses, goats, etc.), etc. .d.

Before the present invention is further described, it should be understood that the present invention is not limited to the specific embodiments described, as they may, of course, vary. It should also be understood that the terminology used herein is intended to describe specific embodiments only and is not intended to be limiting, as the scope of this invention will be limited only by the appended claims.

When a range of values is provided, every intermediate value, to the tenth of a lower limit, unless the context clearly requires otherwise, between the upper and lower limits of that range and any other specified or intermediate value within the specified range is intended to be within the scope of the invention. The upper and lower limits of these smaller ranges may be independently included in the smaller ranges and are also covered by the invention, subject to any specifically excluded limit within said range. If the specified range includes one or both of the limits, ranges excluding one or both of these included limits are also included in this invention.

Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one skilled in the art to which this invention relates. Although any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of this invention, the preferred methods and materials are described herein. All publications referred to herein are incorporated herein by reference for the purpose of disclosure and description of the methods and/or materials in connection with which such publications are cited.

It should be noted that as used herein and in the accompanying claims, the singular number includes the plural unless the context clearly dictates otherwise. Thus, for example, a reference to a variant IL-2 polypeptide includes a plurality of such polypeptides, and a reference to a class I HLA heavy chain polypeptide includes a reference to one or more class I HLA heavy chain polypeptides and equivalents thereof known to those skilled in the art and so on. Further. In addition, it should be noted that the claims may be drafted in such a way as to exclude any optional element. As such, this statement is intended to serve as a precursor to the use of exclusive terminology such as sole, only, etc. in connection with the enumeration of claim elements or the use of a negative limitation.

It will be understood that certain features of the invention, which for clarity are described in the context of individual embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which for brevity are described in the context of a single embodiment, may also be provided separately or in any suitable subcombination. All combinations of embodiments related to this invention are specifically covered by this invention and are disclosed herein as if

- 5 046369 each combination was disclosed individually and explicitly. In addition, all subcombinations of the various embodiments and elements thereof are also specifically embraced by this invention and are disclosed herein in the same manner as if each such combination were individually and explicitly disclosed herein.

The publications discussed herein are intended solely for their disclosure prior to the filing date of this application. Nothing herein should be construed as an admission that this invention is not entitled to precede such publication by virtue of a prior invention. In addition, publication dates provided may differ from actual publication dates, which may need to be independently verified.

Detailed description of the invention

This invention relates to variant immunomodulatory polypeptides and hybrid polypeptides containing variant immunomodulatory peptides. This invention relates to multimeric T cell modulating polypeptides and compositions containing them, wherein said multimeric T cell modulating polypeptides comprise a variant immunomodulatory polypeptide of the present invention. This invention relates to nucleic acids containing nucleotide sequences encoding multimeric polypeptides that modulate T cells, and to host cells containing these nucleic acids. The present invention provides methods for modulating T cell activity; these methods involve contacting a T cell with a multimeric T cell modulating polypeptide of the present invention.

In embodiments described herein, the multimeric polypeptide functions as a surrogate APC and mimics the adaptive immune response. The multimeric polypeptide does this by interacting the TCR present on the surface of the T cell with an epitope presenting peptide in complex with the MHC present in the multimeric polypeptide. This interaction provides the multimeric polypeptide with the ability to achieve epitope-specific targeting to cells. In embodiments described herein, the multimeric polypeptide also has at least one immunomodulatory protein (also referred to herein as a modulatory domain or MOD) that interacts with a co-stimulatory protein partner (also referred to herein as an immunomodulatory polypeptide, cognate immunomodulatory polypeptide or cognate costimulatory protein, etc.) on the T cell. Both signals—epitope/MHC binding to the TCR and immunomodulatory polypeptide binding to the cognate costimulatory polypeptide—then drive both the desired T cell specificity and inhibition or activation/proliferation. As further described herein, the at least one immunomodulatory protein may be a variant of a naturally occurring immunomodulatory protein (e.g., naturally occurring IL-2), wherein the variant exhibits reduced affinity for its costimulatory protein partner on a T cell ( e.g., IL-2R) versus the affinity of a natural immunomodulatory protein for a costimulatory protein partner.

The multimeric T cell modulating polypeptide of this invention is also referred to as synTac polypeptide. The synTac polypeptide of the present invention contains a variant modulator domain, wherein the variant modulator domain exhibits a reduced binding affinity for an immunomodulatory polypeptide (a cognate costimulatory polypeptide, e.g., a cognate costimulatory polypeptide on the surface of a T cell) compared to the affinity of the wild-type modulator domain for the immunomodulatory polypeptide . The synTac polypeptide of the present invention can modulate the activity of a target T cell. The synTac polypeptide of the present invention provides increased specificity for target cells.

Variant immunomodulatory polypeptides.

The present invention provides IL-2 variant modulating polypeptides. The amino acid sequence of wild type human IL-2 is shown in FIG. 2A. The amino acid sequence of wild type human IL-2 polypeptide may be as follows:

APTSSSTKKT OLOLEHLLLD

LQMILNGINN YKNPKLTRML TFKFYMPKKA TELKHLQCLEEELKPLEEVL

NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNRWITFCQSIIS

TLT (SEQIDNO:1).

Wild-type IL-2 binds to the IL-2 receptor (IL-2R) on the cell surface. The IL-2 receptor is, in some cases, a heterotrimeric polypeptide containing an alpha chain (IL-2Ra; also called CD25), a beta chain (IL-2Rb; also called CD122), and a gamma chain (IL-2Py, also called CD132 ). The amino acid sequences of human IL-2Ra, IL-2Rβ, and ΠΛ-2Ργ are shown in FIG. 3A-3C.

In some cases, the variant IL-2 polypeptide of the present invention exhibits reduced binding affinity to IL-2R compared to the binding affinity of an IL-2 polypeptide containing the amino acid sequence depicted in FIG. 2A, with IL-2R. For example, in some cases, a variant IL-2 polypeptide of the present invention binds IL-2R with a binding affinity that is less than the binding affinity of an IL-2 polypeptide containing the amino acid sequence depicted in FIG. 2A, with an IL-2R polypeptide containing alpha, beta and gamma chains containing the amino acid sequences shown in FIG. 3A-3C. For example, in some cases, a variant IL-2 polypeptide of the present invention binds IL-2R with a binding affinity that is at least 5%, at least 10%, at least 15%, at least 20%, by at least 25%, by at least 30%, by at least 35%, by at least 40%, by at least 45%, by at least 50%, by at least 55%, by at least 60%, at least 65%, at least 70% less, at least 75% less, at least 80% less, at least 85% less, at least 90 % less, at least 95% less, or greater than 95% less than the IL-2 binding affinity of a polypeptide containing the amino acid sequence depicted in FIG. 2A, with IL-2R (eg, IL-2R containing alpha, beta and gamma chains containing the amino acid sequences (mature forms) shown in Fig. 3A-3C). In such cases, binding affinity is determined using the procedure described below.

In some cases, the variant IL-2 polypeptide of the present invention has a binding affinity for IL-2R that ranges from 100 nM to 100 μM. As another example, in some cases, a variant IL-2 polypeptide of the present invention has binding affinity for IL-2R (e.g., IL-2R containing alpha, beta, and gamma chains containing the amino acid sequences depicted in FIG. 3A -3C), which is from about 100 nM to about 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 such cases, binding affinity is determined using the procedure described below.

The variant IL-2 polypeptide of the present invention may have one amino acid substitution relative to a wild-type IL-2 polypeptide (eg, an IL-2 polypeptide containing the amino acid sequence shown in FIG. 2A or as set forth in SEQ ID NO: 1). In some cases, the variant IL-2 polypeptide of the present invention has 2 to 10 amino acid substitutions relative to a wild-type IL-2 polypeptide (e.g., an IL-2 polypeptide containing the amino acid sequence depicted in FIG. 2A or as set forth in SEQ ID NO: 1). In some cases, the variant IL-2 polypeptide of the present invention has 2 amino acid substitutions relative to a wild-type IL-2 polypeptide (e.g., an IL-2 polypeptide containing the amino acid sequence depicted in FIG. 2A, or as set forth in SEQ ID NO: 1) . In some cases, a variant IL-2 polypeptide of the present invention has 3 amino acid substitutions relative to a wild-type IL-2 polypeptide (e.g., an IL-2 polypeptide containing the amino acid sequence depicted in FIG. 2A or as set forth in SEQ ID NO: 1) . In some cases, the variant IL-2 polypeptide of the present invention has 4 amino acid substitutions relative to a wild-type IL-2 polypeptide (e.g., an IL-2 polypeptide containing the amino acid sequence depicted in FIG. 2A, or as set forth in SEQ ID NO: 1) . In some cases, the variant IL-2 polypeptide of the present invention has 5 amino acid substitutions relative to a wild-type IL-2 polypeptide (e.g., an IL-2 polypeptide containing the amino acid sequence depicted in FIG. 2A or as set forth in SEQ ID NO: 1) . In some cases, the variant IL-2 polypeptide of the present invention has 6 amino acid substitutions relative to a wild-type IL-2 polypeptide (e.g., an IL-2 polypeptide containing the amino acid sequence depicted in FIG. 2A or as set forth in SEQ ID NO: 1) . In some cases, the variant IL-2 polypeptide of the present invention has 7 amino acid substitutions relative to a wild-type IL-2 polypeptide (e.g., an IL-2 polypeptide containing the amino acid sequence depicted in FIG. 2A or as set forth in SEQ ID NO: 1) . In some cases, the variant IL-2 polypeptide of the present invention has 8 amino acid substitutions relative to a wild-type IL-2 polypeptide (e.g., an IL-2 polypeptide containing the amino acid sequence depicted in FIG. 2A or as set forth in SEQ ID NO: 1) . In some cases, the variant IL-2 polypeptide of the present invention has 9 amino acid substitutions relative to a wild-type IL-2 polypeptide (e.g., an IL-2 polypeptide containing the amino acid sequence depicted in FIG. 2A or as set forth in SEQ ID NO: 1) . In some cases, the variant IL-2 polypeptide of the present invention has 10 amino acid substitutions relative to a wild-type IL-2 polypeptide (e.g., an IL-2 polypeptide containing the amino acid sequence depicted in FIG. 2A or as set forth in SEQ ID NO: 1) .

- 7 046369

The IL-2 variant polypeptide of the present invention may be 120 to 140 amino acids in length, such as 120 to 125 amino acids, 125 to 130 amino acids, 130 to 135 amino acids, or 135 to 140 amino acids. In some cases, the variant IL-2 polypeptide of this invention is 133 amino acids in length.

Replacement E15.

In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to that depicted in FIG. 2D, wherein amino acid 15 is an amino acid other than glutamic acid, for example wherein amino acid 15 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn , Gln, Lys, Arg, His or Asp. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to that depicted in FIG. 2D, wherein amino acid 15 is Ala, Gly, Val, Leu or Ile. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to that depicted in FIG. 2D, with amino acid 15 being Ala. In some cases, the variant IL-2 polypeptide of the present invention comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the amino acid sequence depicted in FIG. . 2D, with amino acid 15 being Gly. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to that depicted in FIG. 2D, with amino acid 15 being Val. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to that depicted in FIG. 2D, with amino acid 15 being Leu. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to that depicted in FIG. 2D, with amino acid 15 being Ile. In some cases, the E15 variant IL-2 polypeptides described above have a binding affinity for IL-2R that is from about 100 nM to about 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, from about 1 μM to about 5 μM, from about 5 μM to about 10 μM, from 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, such variant IL-2 polypeptides bind IL-2R with a binding affinity that is at least 5% less, 10% less, at least 15% less, at least 20% less, at least 25% less, at least 30% less, at least 35% less, at least 40% less, at least 45% less, at least 50% less, at least 55 % less, at least 60% less, at least 65% less, at least 70% less, at least 75% less, at least 80% less, at least 85% less, at least 90% less, at least 95% less, or greater than 95% less than the IL-2 binding affinity of a polypeptide containing the amino acid sequence depicted in FIG. 2A, with IL-2R (eg, IL-2R containing alpha, beta and gamma chains containing the amino acid sequences (mature forms) shown in Fig. 3A-3C). In some cases, such a variant IL-2 polypeptide is 133 amino acids in length.

Replacement H16.

In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to that depicted in FIG. 2E, wherein amino acid 16 is an amino acid other than histidine, for example, wherein amino acid 16 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, Asp or Glu. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to that depicted in FIG. 2E, with amino acid 16 being Ala, Gly, Val, Leu

- 8 046369 or Ile. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to that depicted in FIG. 2E, wherein amino acid 16 is Ala. In some cases, the variant IL-2 polypeptide of the present invention comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the amino acid sequence depicted in FIG. . 2E, wherein amino acid 16 is Gly. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to that depicted in FIG. 2E, wherein amino acid 16 is Val. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to that depicted in FIG. 2E, wherein amino acid 16 is Leu. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to that depicted in FIG. 2E, with amino acid 16 being Ile. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to that depicted in FIG. 2E, wherein amino acid 16 is Asn. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to that depicted in FIG. 2E, wherein amino acid 16 is Asp. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to that depicted in FIG. 2E, wherein amino acid 16 is Cys. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to that depicted in FIG. 2E, wherein amino acid 16 is Gln. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to that depicted in FIG. 2E, wherein amino acid 16 is Glu. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to that depicted in FIG. 2E, with amino acid 16 being Met. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to that depicted in FIG. 2E, wherein amino acid 16 is Phe. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to that depicted in FIG. 2E, with amino acid 16 being Ser. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to that depicted in FIG. 2E, with amino acid 16 being Thr. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to that depicted in FIG. 2E, wherein amino acid 16 is Trp. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to that depicted in FIG. 2E, wherein amino acid 16 is Tyr. In some cases, the H16 variant IL-2 polypeptides described above have a binding affinity for IL-2R that is from about 100 nM to about 150 nM, from about 150 nM to about 200 nM, from about 200 nM to about 250 nM, from about 250 to about 300 nM, from about 300 to about 350 nM, from about 350 to about 400 nM, from about 400 to about 500 nM, from about 500 to about 600 nM, from about 600 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, from about 1 µM to about 5 µM, from

- 9 046369 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, such variant IL-2 polypeptides bind IL-2R with a binding affinity that is at least 5% less, 10% less, at least 15% less, at least 20% less, at least 25% less, at least 30% less, at least 35% less, at least 40% less, at least 45% less, at least 50% less, at least 55 % less, at least 60% less, at least 65% less, at least 70% less, at least 75% less, at least 80% less, at least 85% less, at least 90% less, at least 95% less, or greater than 95% less than the IL-2 binding affinity of a polypeptide containing the amino acid sequence depicted in FIG. 2A with IL-2R (eg, IL-2R containing alpha, beta and gamma chains containing the amino acid sequences (mature forms) depicted in Fig. 3A-3C). In some cases, such a variant IL-2 polypeptide is 133 amino acids in length.

Replacement D20.

In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to that depicted in FIG. 2C, wherein amino acid 20 is an amino acid other than aspartic acid, for example, wherein amino acid 20 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His or Glu. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to that depicted in FIG. 2C, wherein amino acid 20 is Ala, Gly, Val, Leu or Ile. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to that depicted in FIG. 2C, wherein amino acid 20 is Ala. In some cases, the variant IL-2 polypeptide of the present invention comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the amino acid sequence depicted in FIG. . 2C, wherein amino acid 20 is Gly. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to that depicted in FIG. 2C, wherein amino acid 20 is Val. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to that depicted in FIG. 2C, wherein amino acid 20 is Leu. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to that depicted in FIG. 2C, wherein amino acid 20 is Ile. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to that depicted in FIG. 2C, wherein amino acid 20 is Asn, Gln, Lys, Arg or His. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to that depicted in FIG. 2C, wherein amino acid 20 is Lys. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to that depicted in FIG. 2C, wherein amino acid 20 is Asn. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to that depicted in FIG. 2C, wherein amino acid 20 is Gln. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to that depicted in FIG. 2C, wherein amino acid 20 is His. In some cases, the D20 variant IL-2 polypeptides described above have binding affinities for IL-2R that range from about 100 nM to about 150 nM, from about 150 nM to about 200 nM, from about

- 10 046369

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, about 1 μM to about 5 μM, about 5 μM to about 10 μM, about μM to about 15 μM, about 15 μM to about 20 μM, 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, such variant IL-2 polypeptides bind IL-2R with a binding affinity that is at least 5% less, 10% less, at least 15% less, at least 20% less, at least 25% less, at least 30% less, at least 35% less, at least 40% less, at least 45% less, at least 50% less, at least 55 % less, at least 60% less, at least 65% less, at least 70% less, at least 75% less, at least

80% less, at least 85% less, at least 90% less, at least 95% less, or greater than 95% less than the IL-2 binding affinity of a polypeptide containing the amino acid sequence depicted in FIG. . 2A with IL-2R (eg, IL-2R containing alpha, beta and gamma chains containing the amino acid sequences (mature forms) shown in Fig. 3A-3C). In some cases, such a variant IL-2 polypeptide is 133 amino acids in length.

Replacement F42.

In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to that depicted in FIG. 2B, wherein amino acid 42 is an amino acid other than phenylalanine, for example wherein amino acid 42 is Gly, Ala, Val, Leu, Ile, Pro, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to that depicted in FIG. 2B, wherein amino acid 42 is Ala, Gly, Val, Leu or Ile. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to that depicted in FIG. 2B, wherein amino acid 42 is Ala. In some cases, the variant IL-2 polypeptide of the present invention comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the amino acid sequence depicted in FIG. . 2B, wherein amino acid 42 is Gly. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to that depicted in FIG. 2B, wherein amino acid 42 is Val. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to that depicted in FIG. 2B, wherein amino acid 42 is Leu. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to that depicted in FIG. 2B, wherein amino acid 42 is Ile. In some cases, the F42 variant IL-2 polypeptides described above have binding affinities for IL-2R that range from about 100 nM to about 150 nM, from about 150 nM to about 200 nM, from about 200 nM to about 250 nM , from about 250 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, from about 1 μM to about 5 μM, from about 5 μM to about 10 μM, from 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, such variant IL-2 polypeptides bind IL-2R with a binding affinity that is at least 5% less, 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 binding affinity of the IL-2 polypeptide

- 11 046369 yes, containing the amino acid sequence shown in FIG. 2A with IL-2R (eg, IL-2R comprising alpha, beta and gamma chains containing the amino acid sequences (mature forms) shown in FIGS. 3A-3C). In some cases, such a variant IL-2 polypeptide is 133 amino acids in length.

Replacing Y45.

In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to that depicted in FIG. 2F, wherein amino acid 45 is an amino acid other than tyrosine, for example, wherein amino acid 45 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to that depicted in FIG. 2F, wherein amino acid 45 is Ala, Gly, Val, Leu or Ile. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to that depicted in FIG. 2F, wherein amino acid 45 is Ala. In some cases, the variant IL-2 polypeptide of the present invention comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the amino acid sequence depicted in FIG. . 2F, with amino acid 45 being Gly. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to that depicted in FIG. 2F, wherein amino acid 45 is Val. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to that depicted in FIG. 2F, with amino acid 45 being Leu. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to that depicted in FIG. 2F, with amino acid 45 being Ile. In some cases, the Y45 variant IL-2 polypeptides described above have a binding affinity for IL-2R that is from about 100 nM to about 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 about 700 to about 800 nM, about 800 to about 900 nM, about 900 to about 1 µM, about 1 to about 5 µM, about 5 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, such variant IL-2 polypeptides bind IL-2R with a binding affinity of at least 5%, 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% 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 binding affinity of IL-2 polypeptide containing the amino acid sequence depicted in FIG. 2A with IL-2R (eg, IL-2R comprising alpha, beta and gamma chains containing the amino acid sequences (mature forms) shown in FIGS. 3A-3C). In some cases, such a variant IL-2 polypeptide is 133 amino acids in length.

Replacement Q126.

In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to that depicted in FIG. 2G, wherein amino acid 126 is an amino acid other than glutamine, for example, wherein amino acid 126 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Lys, Arg, His, Asp or Glu. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to that depicted in FIG. 2G, wherein amino acid 126 is Ala, Gly, Val, Leu or Ile. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to that shown in FIG. 2G, with amino acid 126 being Ala. In some cases, the variant IL-2 polypeptide of the present invention includes an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the amino acid sequence depicted in FIG. . 2G, with amino acid 126 being Gly. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to that depicted in FIG. 2G, wherein amino acid 126 is Val. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to that depicted in FIG. 2G, with amino acid 126 being Leu. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to that depicted in FIG. 2G, with amino acid 126 being Ile. In some cases, the Q126 variant IL-2 polypeptides described above have binding affinities for IL-2R that range from about 100 nM to about 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, from about 1 μM to about 5 μM, from about 5 μM to about 10 μM, from 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, such variant IL-2 polypeptides bind IL-2R with a binding affinity that is at least 5% less, 10% less, at least 15% less, at least 20% 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 80% less, at least 85% less, at least 90% less, at least 95% less or greater than 95% less than the binding affinity of an IL-2 polypeptide containing the amino acid sequence depicted in FIG. 2A with IL-2R (eg, IL-2R comprising alpha, beta and gamma chains containing the amino acid sequences (mature forms) shown in FIGS. 3A-3C). In some cases, such a variant IL-2 polypeptide is 133 amino acids in length.

Replacements F42 and H16.

In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to that depicted in FIG. 2H, wherein amino acid 42 is an amino acid other than phenylalanine, for example, wherein amino acid 42 is Gly, Ala, Val, Leu, Ile, Pro, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu; and wherein amino acid 16 is an amino acid other than histidine, for example wherein amino acid 16 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln , Lys, Arg, Asp or Glu. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to that depicted in FIG. 2H, wherein amino acid 42 is Ala, Gly, Val, Leu or Ile; and wherein amino acid 16 is Ala, Gly, Val, Leu or Ile. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to that depicted in FIG. 2H, wherein amino acid 42 is Ala and amino acid 16 is Ala. In some cases, the variant IL-2 polypeptide of the present invention comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the amino acid sequence depicted in FIG. . 2H, wherein amino acid 42 is Ala and amino acid 16 is Gly. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to that depicted in FIG. 2H, with amino acid 42 being Val and amino acid 16

- 13 046369 represents Ala. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the amino acid sequence depicted in FIG. 2H, wherein amino acid 42 is Leu and amino acid 16 is Ala. In some cases, the variant IL-2 polypeptide of the present invention comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the amino acid sequence depicted in FIG. . 2H, wherein amino acid 42 is Ile and amino acid 16 is Ala. In some cases, the variant IL-2 F42/H16 polypeptides described above have a binding affinity for IL-2R that is from about 100 nM to about 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, 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, such variant IL-2 polypeptides bind IL-2R with a binding affinity that is at least 5% less, 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%, at least 50% less, at least 55% less, at least 60% less, at least 65% less, at least 70% less, at least 75% less, at least 80% less, at least 85 % less, at least 90% less, at least 95% less, or greater than 95% less than the IL-2 binding affinity of a polypeptide containing the amino acid sequence depicted in FIG. 2A with IL-2R (eg, IL-2R comprising alpha, beta and gamma chains containing the amino acid sequences (mature forms) shown in FIGS. 3A-3C). In some cases, such a variant IL-2 polypeptide is 133 amino acids in length.

Replacements F42 and D20.

In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to that depicted in FIG. 2I, wherein amino acid 42 is an amino acid other than phenylalanine, for example, wherein amino acid 42 is Gly, Ala, Val, Leu, Ile, Pro, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu; and wherein amino acid 20 is an amino acid other than aspartic acid, for example wherein amino acid 20 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His or Glu. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to that depicted in FIG. 2I, wherein amino acid 42 is Ala, Gly, Val, Leu or Ile; and wherein amino acid 20 is Ala, Gly, Val, Leu or Ile. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to that depicted in FIG. 2I, wherein amino acid 42 is Ala, Gly, Val, Leu or Ile; and wherein amino acid 20 is Asn, Gln, Lys, Arg or His. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to that depicted in FIG. 2I, wherein amino acid 42 is Ala and amino acid 20 is Ala. In some cases, the variant IL-2 polypeptide of the present invention comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the amino acid sequence depicted in FIG. . 2I, wherein amino acid 42 is Ala and amino acid 20 is Gly. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to that depicted in FIG. 2I, wherein amino acid 42 is Val and amino acid 20 is Ala. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the amino acid sequence , shown in Fig. 2I, wherein amino acid 42 is Leu and amino acid 20 is Ala. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the amino acid sequence depicted in FIG. 2I, wherein amino acid 42 is Ile and amino acid 20 is Ala. In some cases, the variant IL-2 polypeptide of the present invention comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the amino acid sequence depicted in FIG. . 2I, wherein amino acid 42 is Ala and amino acid 20 is Asn. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to that depicted in FIG. 2I, wherein amino acid 42 is Ala and amino acid 20 is Gln. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to that depicted in FIG. 2I, wherein amino acid 42 is Ala and amino acid 20 is Lys. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to that depicted in FIG. 2I, wherein amino acid 42 is Ala and amino acid 20 is Arg. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to that depicted in FIG. 2I, wherein amino acid 42 is Ala and amino acid 20 is His. In some cases, the variant IL-2 polypeptides with F42/D20 substitutions described above have binding affinities for IL-2R that range from about 100 nM to about 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, 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, 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, such variant IL-2 polypeptides bind IL-2R with a binding affinity that is at least 5% less, at least 10% less, at least 15% less, at least 20% 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 at least 60% less, at least 65% less, at least 70% less, at least 80% less, at least 85% less, at least 90% less, at least 95% less than or greater than 95% less than the IL-2 binding affinity of a polypeptide containing the amino acid sequence depicted in FIG. 2A with IL-2R (eg, IL-2R comprising alpha, beta and gamma chains containing the amino acid sequences (mature forms) shown in FIGS. 3A-3C). In some cases, such a variant IL-2 polypeptide is 133 amino acids in length.

Replacements F42, D20 and E15.

In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to that depicted in FIG. 2J, wherein amino acid 42 is an amino acid other than phenylalanine, for example wherein amino acid 42 is Gly, Ala, Val, Leu, Ile, Pro, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu; wherein amino acid 20 is an amino acid other than aspartic acid, for example wherein amino acid 20 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln Lys, Arg, His or Glu; and wherein amino acid 15 is an amino acid other than glutamic acid, for example wherein amino acid 15 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His or Asp. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to that depicted in FIG. 2J, wherein amino acid 42 is Ala, Gly, Val, Leu or Ile; wherein amino acid 20 is Ala, Gly, Val, Leu or Ile; and wherein amino acid 15 is Ala, Gly, Val, Leu or Ile. In some cases, a variant IL-2 polypeptide for a given

- 15 046369 of the invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to that shown in FIG. 2J, wherein amino acid 42 is Ala, Gly, Val, Leu or Ile; wherein amino acid 20 is Asn, Gln, Lys, Arg or His; and wherein amino acid 15 is Ala, Gly, Val, Leu or Ile. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to that depicted in FIG. 2J, wherein amino acid 42 is Ala, amino acid 20 is Ala, and amino acid 15 is Ala. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the amino acid sequence depicted in FIG. 2J, wherein amino acid 42 is Ala, amino acid 20 is Gly, and amino acid 15 is Gly. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to that depicted in FIG. 2J, wherein amino acid 42 is Val, amino acid 20 is Ala, and amino acid 15 is Gly. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to that depicted in FIG. 2J, wherein amino acid 42 is Leu, amino acid 20 is Ala, and amino acid 15 is Gly. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to that depicted in FIG. 2J, wherein amino acid 42 is Ile, amino acid 20 is Ala, and amino acid 15 is Ala. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the amino acid sequence depicted in FIG. 2J, wherein amino acid 42 is Ala, amino acid 20 is Asn, and amino acid 15 is Ala. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the amino acid sequence depicted in FIG. 2I, wherein amino acid 42 is Ala, amino acid 20 is Gln, and amino acid 15 is Ala. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the amino acid sequence depicted in FIG. 2J, wherein amino acid 42 is Ala, amino acid 20 is Lys, and amino acid 15 is Ala. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the amino acid sequence depicted in FIG. 2J, wherein amino acid 42 is Ala, amino acid 20 is Arg, and amino acid 15 is Ala. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the amino acid sequence depicted in FIG. 2I, wherein amino acid 42 is Ala, amino acid 20 is His, and amino acid 15 is Ala. In some cases, the variant IL-2 polypeptides F42/D20/E15 described above have binding affinities for IL-2R that range from about 100 nM to about 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, from about 1 μM to about 5 μM, from about 5 μM to about 10 μM, from 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, such variant IL-2 polypeptides bind IL-2R with a binding affinity that is at least 5% less, 10% less, at least 15% less, at least 20% less, at least 30% less, at least 35% less, at least 40% less, at least 45% less, by

- 16 046369 at least 50% less, at least 55% less, at least 60% less, at least 65% less, at least 70% less, at least 75% less, at least 80% less, at least 85% less, at least 90% less, at least 95% less, or greater than 95% less than the binding affinity of the IL-2 polypeptide comprising the amino acid sequence , shown in Fig. 2A with IL-2R (eg, IL-2R comprising alpha, beta and gamma chains containing the amino acid sequences (mature forms) shown in FIGS. 3A-3C). In some cases, such a variant IL-2 polypeptide is 133 amino acids in length.

Replacements F42, D20 and H16.

In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to that depicted in FIG. 2K, wherein amino acid 42 is an amino acid other than phenylalanine, for example, wherein amino acid 42 is Gly, Ala, Val, Leu, Ile, Pro, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu; wherein amino acid 20 is an amino acid other than aspartic acid, for example wherein amino acid 20 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln Lys, Arg, His or Glu; and wherein amino acid 16 is an amino acid other than histidine, for example wherein amino acid 16 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln , Lys, Arg, Asp or Glu. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to that depicted in FIG. 2K, wherein amino acid 42 is Ala, Gly, Val, Leu or Ile; wherein amino acid 20 is Ala, Gly, Val, Leu or Ile; and wherein amino acid 16 is Ala, Gly, Val, Leu or Ile. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to that depicted in FIG. 2K, wherein amino acid 42 is Ala, Gly, Val, Leu or Ile; wherein amino acid 20 is Asn, Gln, Lys, Arg or His; and wherein amino acid 16 is Ala, Gly, Val, Leu or Ile. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to that depicted in FIG. 2K, wherein amino acid 42 is Ala, amino acid 20 is Ala and amino acid 16 is Ala. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the amino acid sequence depicted in FIG. 2K, wherein amino acid 42 is Ala, amino acid 20 is Gly, and amino acid 16 is Gly. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to that depicted in FIG. 2K, wherein amino acid 42 is Val, amino acid 20 is Ala, and amino acid 16 is Gly. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to that depicted in FIG. 2K, wherein amino acid 42 is Leu, amino acid 20 is Ala, and amino acid 16 is Gly. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to that depicted in FIG. 2K, wherein amino acid 42 is Ile, amino acid 20 is Ala, and amino acid 16 is Ala. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the amino acid sequence depicted in FIG. 2K, wherein amino acid 42 is Ala, amino acid 20 is Asn, and amino acid 16 is Ala. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the amino acid sequence depicted in FIG. 2K, wherein amino acid 42 is Ala, amino acid 20 is Gln, and amino acid 16 is Ala. In some cases, the variant IL-2 polypeptide of the present invention comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with an amino acid sequence shown in Fig. 2K, wherein amino acid 42 is Ala, amino acid 20 is Lys, and amino acid 16 is Ala. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the amino acid sequence depicted in FIG. 2K, wherein amino acid 42 is Ala, amino acid 20 is Arg, and amino acid 16 is Ala. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the amino acid sequence depicted in FIG. 2K, wherein amino acid 42 is Ala, amino acid 20 is His, and amino acid 16 is Ala. In some cases, the variant IL-2 polypeptides F42/D20/H16 described above have a binding affinity for IL-2R that is from about 100 nM to about 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, from about 1 μM to about 5 μM, from about 5 μM to about 10 μM, from 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, such variant IL-2 polypeptides bind IL-2R with a binding affinity that is at least 5% less, at least 10% less, at least 15% less, at least 20% less, at least 30% less, at least 35% less, at least 40% less, at least 45%, at least 50%, at least 55% less, at least 60%, at least 65%, at least 70%, at least 75% less, at least 80% less, at least 85% less, at least 90% less, according to at least 95% less or greater than 95% less than the binding affinity of an IL-2 polypeptide containing the amino acid sequence depicted in FIG. 2A with IL-2R (eg, IL-2R comprising alpha, beta and gamma chains containing the amino acid sequences (mature forms) shown in FIGS. 3A-3C). In some cases, such a variant IL-2 polypeptide is 133 amino acids in length.

Replacements F42, D20 and Q126.

In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to that depicted in FIG. 2L, wherein amino acid 42 is an amino acid other than phenylalanine, for example wherein amino acid 42 is Gly, Ala, Val, Leu, Ile, Pro, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu; wherein amino acid 20 is an amino acid other than aspartic acid, for example wherein amino acid 20 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln Lys, Arg, His or Glu; and wherein amino acid 126 is an amino acid other than glutamine, for example wherein amino acid 126 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Lys, Arg, His, Asp or Glu. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to that depicted in FIG. 2L, wherein amino acid 42 is Ala, Gly, Val, Leu or Ile; wherein amino acid 20 is Ala, Gly, Val, Leu or Ile; and wherein amino acid 126 is Ala, Gly, Val, Leu or Ile. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to that depicted in FIG. 2L, wherein amino acid 42 is Ala, Gly, Val, Leu or Ile; wherein amino acid 20 is Asn, Gln, Lys, Arg or His; and wherein amino acid 126 is Ala, Gly, Val, Leu or Ile. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to that depicted in FIG. 2L, wherein amino acid 42 is Ala, amino acid 20 is Ala, and amino acid 126 is Ala. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the amino acid sequence depicted in FIG. 2L, with amino

- 18 046369 acid 42 is Ala, amino acid 20 is Gly, and amino acid 126 is Gly. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to that depicted in FIG. 2L, wherein amino acid 42 is Val, amino acid 20 is Ala, and amino acid 126 is Gly. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to that depicted in FIG. 2L, wherein amino acid 42 is Leu, amino acid 20 is Ala, and amino acid 126 is Gly. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to that depicted in FIG. 2L, wherein amino acid 42 is Ile, amino acid 20 is Ala, and amino acid 126 is Ala. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the amino acid sequence depicted in FIG. 2L, wherein amino acid 42 is Ala, amino acid 20 is Asn, and amino acid 126 is Ala. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the amino acid sequence depicted in FIG. 2L, wherein amino acid 42 is Ala, amino acid 20 is Gln, and amino acid 126 is Ala. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the amino acid sequence depicted in FIG. 2L, wherein amino acid 42 is Ala, amino acid 20 is Lys, and amino acid 126 is Ala. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the amino acid sequence depicted in FIG. 2L, wherein amino acid 42 is Ala, amino acid 20 is Arg, and amino acid 126 is Ala. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the amino acid sequence depicted in FIG. 2L, wherein amino acid 42 is Ala, amino acid 20 is His, and amino acid 126 is Ala. In some cases, the variant IL-2 polypeptides F42/D20/Q126 described above have binding affinities for IL-2R that range from about 100 nM to about 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, 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 , 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 up to about 100 µM. In some cases, such variant IL-2 polypeptides bind IL-2R with a binding affinity that is at least 5% less, 10% less, at least 15% less, at least 20% less, at least 30% less, at least by

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 80% less, at least

85% less, at least 90% less, at least 95% less, or greater than 95% less than the IL-2 binding affinity of a polypeptide containing the amino acid sequence depicted in FIG. 2A, with IL-2R (eg, IL-2R comprising alpha, beta and gamma chains containing the amino acid sequences (mature forms) depicted in FIGS. 3A-3C). In some cases, this variant IL-2 polypeptide is 133 amino acids long.

Replacements F42, D20 and Y45.

In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to that depicted in FIG. 2M, wherein amino acid 42 is an amino acid other than phenylalanine, at

- 19 046369 example, wherein amino acid 42 is Gly, Ala, Val, Leu, Ile, Pro, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu; wherein amino acid 20 is an amino acid other than aspartic acid, for example wherein amino acid 20 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln Lys, Arg, His or Glu; and wherein amino acid 45 is an amino acid other than tyrosine, for example wherein amino acid 45 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys , Arg, His, Asp or Glu. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to that depicted in FIG. 2M, wherein amino acid 42 is Ala, Gly, Val, Leu or Ile; wherein amino acid 20 is Ala, Gly, Val, Leu or Ile; and wherein amino acid 45 is Ala, Gly, Val, Leu or Ile. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to that depicted in FIG. 2M, wherein amino acid 42 is Ala, Gly, Val, Leu or Ile; wherein amino acid 20 is Asn, Gln, Lys, Arg or His; and wherein amino acid 45 is Ala, Gly, Val, Leu or Ile. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to that depicted in FIG. 2M, wherein amino acid 42 is Ala, amino acid 20 is Ala and amino acid 45 is Ala. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the amino acid sequence depicted in FIG. 2M, wherein amino acid 42 is Ala, amino acid 20 is Gly, and amino acid 45 is Gly. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to that depicted in FIG. 2M, wherein amino acid 42 is Val, amino acid 20 is Ala, and amino acid 45 is Gly. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to that depicted in FIG. 2M, wherein amino acid 42 is Leu, amino acid 20 is Ala, and amino acid 45 is Gly. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to that depicted in FIG. 2M, wherein amino acid 42 is Ile, amino acid 20 is Ala, and amino acid 45 is Ala. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the amino acid sequence depicted in FIG. 2M, wherein amino acid 42 is Ala, amino acid 20 is Asn, and amino acid 45 is Ala. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the amino acid sequence depicted in FIG. 2M, wherein amino acid 42 is Ala, amino acid 20 is Gln, and amino acid 45 is Ala. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the amino acid sequence depicted in FIG. 2M, wherein amino acid 42 is Ala, amino acid 20 is Lys, and amino acid 45 is Ala. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the amino acid sequence depicted in FIG. 2M, wherein amino acid 42 is Ala, amino acid 20 is Arg, and amino acid 45 is Ala. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the amino acid sequence depicted in FIG. 2M, wherein amino acid 42 is Ala, amino acid 20 is His, and amino acid 45 is

- 20 046369

Ala. In some cases, the variant IL-2 polypeptides F42/D20/Y45 described above have a binding affinity for IL-2R that is from about 100 nM to about 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, from about 1 μM to about 5 μM, from about 5 μM to about 10 μM, from 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, such variant IL-2 polypeptides bind IL-2R with a binding affinity that is at least 5% less, at least 10% less, at least

15% less, at least 20% 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 80% less, at least

85% less, at least 90% less, at least 95% less, or greater than 95% less than the IL-2 binding affinity of a polypeptide containing the amino acid sequence depicted in FIG. 2A, with IL-2R (eg, IL-2R comprising alpha, beta and gamma chains containing the amino acid sequences (mature forms) shown in FIGS. 3A-3C). In some cases, this variant IL-2 polypeptide is 133 amino acids long.

Replacements F42, D20, Y45 and H16.

In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to that depicted in FIG. 2N, wherein amino acid 42 is an amino acid other than phenylalanine, for example wherein amino acid 42 is Gly, Ala, Val, Leu, Ile, Pro, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu; wherein amino acid 20 is an amino acid other than aspartic acid, for example wherein amino acid 20 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln Lys, Arg, His or Glu; wherein amino acid 45 is an amino acid other than tyrosine, for example wherein amino acid 45 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu; and wherein amino acid 16 is an amino acid other than histidine, for example wherein amino acid 16 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln , Lys, Arg, Asp or Glu. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to that depicted in FIG. 2N, wherein amino acid 42 is Ala, Gly, Val, Leu or Ile; wherein amino acid 20 is Ala, Gly, Val, Leu or Ile; wherein amino acid 45 is Ala, Gly, Val, Leu or Ile; and wherein amino acid 16 is Ala, Gly, Val, Leu or Ile. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to that depicted in FIG. 2N, wherein amino acid 42 is Ala, Gly, Val, Leu or Ile; wherein amino acid 20 is Asn, Gln, Lys, Arg or His; wherein amino acid 45 is Ala, Gly, Val, Leu or Ile; and wherein amino acid 16 is Ala, Gly, Val, Leu or Ile. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to that depicted in FIG. 2N, wherein amino acid 42 is Ala, amino acid 20 is Ala, amino acid 45 is Ala and amino acid 16 is Ala. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the amino acid sequence depicted in FIG. 2N, wherein amino acid 42 is Ala, amino acid 20 is Gly, amino acid 45 is Gly and amino acid 16 is Ala. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the amino acid sequence depicted in FIG. 2N, wherein amino acid 42 is Val, amino acid 20 is Ala, amino acid 45 is Gly and amino acid 16 is Ala. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the amino acid sequence shown in Fig. 2N, wherein amino acid 42 is Leu, amino acid 20 is Ala, amino acid 45 is Gly and amino acid 16 is Val. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to that depicted in FIG. 2N, wherein amino acid 42 is Ile, amino acid 20 is Ala, amino acid 45 is Ala and amino acid 16 is Gly. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to that depicted in FIG. 2N, wherein amino acid 42 is Ala, amino acid 20 is Asn, amino acid 45 is Ala and amino acid 16 is Ala. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the amino acid sequence depicted in FIG. 2N, wherein amino acid 42 is Ala, amino acid 20 is Gln, amino acid 45 is Ala and amino acid 16 is Ala. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the amino acid sequence depicted in FIG. 2N, wherein amino acid 42 is Ala, amino acid 20 is Lys, amino acid 45 is Ala and amino acid 16 is Ala. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the amino acid sequence depicted in FIG. 2N, wherein amino acid 42 is Ala, amino acid 20 is Arg, amino acid 45 is Ala and amino acid 16 is Ala. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the amino acid sequence depicted in FIG. 2N, wherein amino acid 42 is Ala, amino acid 20 is His, amino acid 45 is Ala and amino acid 16 is Ala. In some cases, the variant IL-2 polypeptides F42/D20/Y45/H16 described above have binding affinities for IL-2R that range from about 100 nM to about 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, such variant IL-2 polypeptides bind IL-2R with a binding affinity that is at least 5% less, 10% less, at least 15% less, at least 20% 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 80% less, at least 85% less, at least 90% less, at least 95% less or greater than 95% less than the binding affinity of an IL-2 polypeptide containing the amino acid sequence depicted in FIG. 2A with IL-2R (eg, IL-2R comprising alpha, beta and gamma chains containing the amino acid sequences (mature forms) shown in FIGS. 3A-3C). In some cases, such a variant IL-2 polypeptide is 133 amino acids in length.

Replacements F42, D20, Y45 and Q126.

In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to that depicted in FIG. 2O, wherein amino acid 42 is an amino acid other than phenylalanine, for example, wherein amino acid 42 is Gly, Ala, Val, Leu, Ile, Pro, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu; wherein amino acid 20 is an amino acid other than aspartic acid, for example wherein amino acid 20 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln Lys, Arg, His or Glu; wherein amino acid 45 is an amino acid other than tyrosine, for example wherein amino acid 45 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or

- 22 046369

Glu; and wherein amino acid 126 is an amino acid other than glutamine, for example wherein amino acid 126 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Lys , Arg, His, Asp or Glu. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to that depicted in FIG. 2O, wherein amino acid 42 is Ala, Gly, Val, Leu or Ile; wherein amino acid 20 is Ala, Gly, Val, Leu or Ile; wherein amino acid 45 is Ala, Gly, Val, Leu or Ile; and wherein amino acid 126 is Ala, Gly, Val, Leu or Ile. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to that depicted in FIG. 2O, wherein amino acid 42 is Ala, Gly, Val, Leu or Ile; wherein amino acid 20 is Asn, Gln, Lys, Arg or His; wherein amino acid 45 is Ala, Gly, Val, Leu or Ile; and wherein amino acid 126 is Ala, Gly, Val, Leu or Ile. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to that depicted in FIG. 2O, wherein amino acid 42 is Ala, amino acid 20 is Ala, amino acid 45 is Ala and amino acid 126 is Ala. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the amino acid sequence depicted in FIG. 2O, wherein amino acid 42 is Ala, amino acid 20 is Gly, amino acid 45 is Gly and amino acid 126 is Ala. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the amino acid sequence depicted in FIG. 2O, wherein amino acid 42 is Val, amino acid 20 is Ala, amino acid 45 is Gly and amino acid 126 is Ala. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the amino acid sequence depicted in FIG. 2O, wherein amino acid 42 is Leu, amino acid 20 is Ala, amino acid 45 is Gly and amino acid 126 is Val. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to that depicted in FIG. 2O, wherein amino acid 42 is Ile, amino acid 20 is Ala, amino acid 45 is Ala and amino acid 126 is Gly. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to that depicted in FIG. 2O, wherein amino acid 42 is Ala, amino acid 20 is Asn, amino acid 45 is Ala and amino acid 126 is Ala. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the amino acid sequence depicted in FIG. 2O, wherein amino acid 42 is Ala, amino acid 20 is Gln, amino acid 45 is Ala and amino acid 126 is Ala. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the amino acid sequence depicted in FIG. 2O, wherein amino acid 42 is Ala, amino acid 20 is Lys, amino acid 45 is Ala and amino acid 126 is Ala. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the amino acid sequence depicted in FIG. 2O, wherein amino acid 42 is Ala, amino acid 20 is Arg, amino acid 45 is Ala and amino acid 126 is Ala. In some cases, a variant IL-2 polypeptide of the present invention comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with an amino acid sequence shown in Fig. 2O, wherein amino acid 42 is Ala, amino acid 20 is His, amino acid 45 is Ala and amino acid 126 is Ala. In some cases, the variant IL-2 polypeptides F42/D20/Y45/Q126 described above have binding affinities for IL-2R that range from about 100 nM to about 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, 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, such variant IL-2 polypeptides bind IL-2R with a binding affinity that is at least 5% less, at least 10% less, at least 15% less, at least 20% less, at least 30% less, at least 35% less, at least by

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 80% less, at least 85% less, at least

90% less, at least 95% less, or greater than 95% less than the IL-2 binding affinity of a polypeptide containing the amino acid sequence depicted in FIG. 2A with IL-2R (eg, IL-2R comprising alpha, beta and gamma chains containing the amino acid sequences (mature forms) shown in FIGS. 3A-3C). In some cases, such a variant IL-2 polypeptide is 133 amino acids in length.

Replacements F42, D20, Y45, H16 and Q126.

In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to that depicted in FIG. 2P, wherein amino acid 42 is an amino acid other than phenylalanine, for example wherein amino acid 42 is Gly, Ala, Val, Leu, Ile, Pro, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu; wherein amino acid 20 is an amino acid other than aspartic acid, for example wherein amino acid 20 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln Lys, Arg, His or Glu; wherein amino acid 45 is an amino acid other than tyrosine, for example wherein amino acid 45 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu; wherein amino acid 126 is an amino acid other than glutamine, for example wherein amino acid 126 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Lys, Arg, His, Asp or Glu; and wherein amino acid 16 is an amino acid other than histidine, for example wherein amino acid 16 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln , Lys, Arg, Asp or Glu. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to that depicted in FIG. 2P, wherein amino acid 42 is Ala, Gly, Val, Leu or Ile; wherein amino acid 20 is Ala, Gly, Val, Leu or Ile; wherein amino acid 45 is Ala, Gly, Val, Leu or Ile; wherein amino acid 126 is Ala, Gly, Val, Leu or Ile; and wherein amino acid 16 is Ala, Gly, Val, Leu or Ile. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to that depicted in FIG. 2P, wherein amino acid 42 is Ala, Gly, Val, Leu or Ile; wherein amino acid 20 is Asn, Gln, Lys, Arg or His; wherein amino acid 45 is Ala, Gly, Val, Leu or Ile; wherein amino acid 126 is Ala, Gly, Val, Leu or Ile; and wherein amino acid 16 is Ala, Gly, Val, Leu or Ile. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to that depicted in FIG. 2P, wherein amino acid 42 is Ala, amino acid 20 is Ala, amino acid 45 is Ala, amino acid 126 is Ala, and amino acid 16 is Ala. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the amino acid sequence depicted in FIG. 2P, wherein amino acid 42 is Ala, amino acid 20 is Gly, amino acid 45 is Gly, amino acid 126 is Ala and amino acid 16 is Ala. In some cases, an option

- 24 046369

The IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the amino acid sequence depicted in FIG. 2P, wherein amino acid 42 is Val, amino acid 20 is Ala, amino acid 45 is Gly, amino acid 126 is Ala and amino acid 16 is Ala. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the amino acid sequence depicted in FIG. 2P, wherein amino acid 42 is Leu, amino acid 20 is Ala, amino acid 45 is Gly, amino acid 126 is Val and amino acid 16 is Ala. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the amino acid sequence depicted in FIG. 2P, wherein amino acid 42 is Ile, amino acid 20 is Ala, amino acid 45 is Ala, amino acid 126 is Gly and amino acid 16 is Ala. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the amino acid sequence depicted in FIG. 2P, wherein amino acid 42 is Ala, amino acid 20 is Asn, amino acid 45 is Ala, amino acid 126 is Ala and amino acid 16 is Ala. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the amino acid sequence depicted in FIG. 2P, wherein amino acid 42 is Ala, amino acid 20 is Gln, amino acid 45 is Ala, amino acid 126 is Ala and amino acid 16 is Ala. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the amino acid sequence depicted in FIG. 2P, wherein amino acid 42 is Ala, amino acid 20 is Lys, amino acid 45 is Ala, amino acid 126 is Ala and amino acid 16 is Ala. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the amino acid sequence depicted in FIG. 2P, wherein amino acid 42 is Ala, amino acid 20 is Arg, amino acid 45 is Ala, amino acid 126 is Ala and amino acid 16 is Ala. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the amino acid sequence depicted in FIG. 2P, wherein amino acid 42 is Ala, amino acid 20 is His, amino acid 45 is Ala, amino acid 126 is Ala and amino acid 16 is Ala. In some cases, the F42/D20/Y45/H16/Q126 substitution variant IL-2 polypeptide described above has a binding affinity for IL-2R that is from about 100 nM to about 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 5 µM to about 5 µM, about 5 µM to about 10 µM, 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, such variant IL-2 polypeptides bind IL-2R with a binding affinity that is at least 5% less, 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%, at least 50% less, at least 55% less, at least 60% less, at least 65% less, at least 70% less, at least 75%, at least 80% less, at least 85% less than, at least 90% less than, at least 95% less than, or greater than 95% less than the IL-2 binding affinity of a polypeptide containing the amino acid sequence depicted in FIG. 2A with IL-2R (for example, IL-2R, including alpha, beta and gamma chains,

- 25 046369 containing the amino acid sequences (mature forms) shown in FIG. 3A-3C). In some cases, such a variant IL-2 polypeptide is 133 amino acids in length.

Replacements F42, Q126 and H16.

In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to that depicted in FIG. 2Q, wherein amino acid 42 is an amino acid other than phenylalanine, for example, wherein amino acid 42 is Gly, Ala, Val, Leu, Ile, Pro, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu; wherein amino acid 126 is an amino acid other than glutamine, for example wherein amino acid 126 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Lys, Arg, His, Asp or Glu; and wherein amino acid 16 is an amino acid other than histidine, for example wherein amino acid 16 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln , Lys, Arg, Asp or Glu. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to that depicted in FIG. 2Q, wherein amino acid 42 is Ala, Gly, Val, Leu or Ile; wherein amino acid 126 is Ala, Gly, Val, Leu or Ile; and wherein amino acid 16 is Ala, Gly, Val, Leu or Ile. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to that depicted in FIG. 2Q, wherein amino acid 42 is Ala, Gly, Val, Leu or Ile; wherein amino acid 126 is Asn, Gln, Lys, Arg or His; and wherein amino acid 16 is Ala, Gly, Val, Leu or Ile. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to that depicted in FIG. 2Q, wherein amino acid 42 is Ala, amino acid 126 is Ala, and amino acid 16 is Ala. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the amino acid sequence depicted in FIG. 2Q, wherein amino acid 42 is Ala, amino acid 126 is Gly, and amino acid 16 is Gly. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to that depicted in FIG. 2Q, wherein amino acid 42 is Val, amino acid 126 is Ala, and amino acid 16 is Gly. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to that depicted in FIG. 2Q, wherein amino acid 42 is Leu, amino acid 126 is Ala, and amino acid 16 is Gly. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to that depicted in FIG. 2Q, wherein amino acid 42 is Ile, amino acid 126 is Ala, and amino acid 16 is Ala. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the amino acid sequence depicted in FIG. 2Q, wherein amino acid 42 is Ala, amino acid 126 is Asn, and amino acid 16 is Ala. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the amino acid sequence depicted in FIG. 2Q, wherein amino acid 42 is Ala, amino acid 126 is Ala, and amino acid 16 is Ala. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the amino acid sequence depicted in FIG. 2Q, wherein amino acid 42 is Ala, amino acid 126 is Lys, and amino acid 16 is Ala. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or

- 26 046369 at least 99% amino acid sequence identity with the amino acid sequence shown in FIG. 2Q, wherein amino acid 42 is Ala, amino acid 126 is Arg, and amino acid 16 is Ala. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the amino acid sequence depicted in FIG. 2Q, wherein amino acid 42 is Ala, amino acid 126 is His, and amino acid 16 is Ala. In some cases, the IL-2 variant F42/Q126/H16 polypeptides have binding affinities for IL-2R that are about 100 nM to about 150 nM, about 150 nM to about 200 nM, about 200 nM to about 250 nM, from about 250 nM to about 300 nM, about 300 nM to about 350 nM, about 350 nM to about 400 nM, about 400 nM to about 500 nM, about 500 nM to about 600 nM, about 600 nM to about 700 nM, from about 700 nM to about 800 nM, from about 800 nM to about 900 nM, from about 900 nM to about 1 µM, from about 1 µM to about 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, such variant IL-2 polypeptides bind IL-2R with a binding affinity that is at least 5% less, 10% less, at least 15% less, at least 20% less, at least on

30% less, at least 35% less, at least 40% less, at least

45% less, at least 50% less, at least 55% less, at least

60% less, at least 65% less, at least 70% less, at least

75% less, at least 80% less, at least 85% less, at least

90% less, at least 95% less, or greater than 95% less than the binding affinity of an IL-2 polypeptide containing the amino acid sequence depicted in FIG. 2A with IL-2R (eg, IL-2R comprising alpha, beta and gamma chains containing the amino acid sequences (mature forms) shown in FIGS. 3A-3C). In some cases, such a variant IL-2 polypeptide is 133 amino acids in length. Fusion polypeptides.

The present invention provides IL-2 fusion polypeptides. The fusion polypeptide of this invention contains

a) a variant IL-2 polypeptide according to this invention; And

b) heterologous merger partner.

In some cases, the heterologous fusion partner is fused to the N-terminus of the variant IL-2 polypeptide. In some cases, the heterologous fusion partner is fused to the C-terminus of the variant IL-2 polypeptide. In some cases, the IL-2 fusion polypeptide of the present invention comprises a first heterologous fusion partner fused to the N-terminus of the variant IL-2 polypeptide and a second heterologous fusion partner fused to the C-terminus of the variant IL-2 polypeptide.

The total length of the IL-2 fusion polypeptide of this invention can vary from 135 to 2000 amino acids. For example, the IL-2 fusion polypeptide of the present invention may range from 135 to 150 amino acids, 150 to 175 amino acids, 175 to 200 amino acids, 200 to 225 amino acids, 225 to 250 amino acids, 250 to 275 amino acids, from 275 to 300 amino acids, from 300 to 350 amino acids, from 350 to 400 amino acids, from 400 to 450 amino acids, from 450 to 500 amino acids, from 500 to 600 amino acids, from 600 to 700 amino acids, from 700 to 800 amino acids, from 800 up to 900 amino acids, 900 to 1000 amino acids, 1000 to 1250 amino acids, 1250 to 1500 amino acids, 1500 to 1750 amino acids, or 1750 to 2000 amino acids.

Suitable fusion partners include, but are not limited to, the transmembrane domain; Antibody Fc region; antigen-binding region of an antibody; cytokine (except IL-2); immunomodulatory domain; intracellular signaling domain, etc.

Multimeric polypeptides modulating T cells.

The present invention provides multimeric (eg, heterodimeric, heterotrimeric) polypeptides. These multimeric polypeptides are T cell modulating polypeptides and are also referred to herein as multimeric T cell modulating polypeptides or synTac (for immunological synapse for T cell activation). Fig. 1A-1D show schematic representations of multimeric T cell modulating polypeptides of the present invention. The multimeric T cell modulating polypeptide of this invention is also referred to as IL-2/synTac, synTac polypeptide or multimeric polypeptide.

In some cases, the synTac polypeptide of this invention contains a wild-type IL-2 polypeptide. In some cases, the synTac polypeptide of this invention contains one copy of the wild-type IL-2 polypeptide. In some cases, the synTac polypeptide of this invention contains two copies of the wild-type IL-2 polypeptide. In some cases, the synTac polypeptide of this invention contains three copies of the wild-type IL-2 polypeptide. In some cases, the wild-type IL-2 polypeptide contains an amino acid sequence that is at least 90%

- 27 046369 at least 95%, at least 98% or at least 99% amino acid sequence identity with the amino acid sequence depicted in FIG. 2A.

In some cases, the synTac polypeptide of the present invention contains a variant IL-2 polypeptide of the present invention. As noted above, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention exhibits reduced binding affinity to IL-2R compared to the binding affinity of wild-type IL-2 to IL-2R. A multimeric polypeptide of the present invention that contains a variant IL-2 polypeptide of the present invention also exhibits reduced binding affinity for IL-2R compared to a control multimeric polypeptide containing wild-type IL-2 with IL-2R (e.g., IL-2R, containing alpha, beta and gamma polypeptides containing the amino acid sequences (mature form) shown in Fig. 3A-3C).

In some cases, the synTac polypeptide of the present invention exhibits reduced binding affinity for IL-2R compared to the IL-2 binding affinity of a polypeptide containing the amino acid sequence depicted in FIG. 2A with IL-2R. For example, in some cases, the synTac polypeptide of the present invention binds IL-2R with a binding affinity that is less than the binding affinity of a control synTac polypeptide containing an IL-2 polypeptide containing the amino acid sequence depicted in FIG. 2A with IL-2R, which contains alpha, beta and gamma polypeptides containing the amino acid sequences (mature form) shown in FIG. 3A-3C. For example, in some cases, the synTac polypeptide of the present invention 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% 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 binding affinity of a synTac control polypeptide containing an IL-2 polypeptide containing the amino acid sequence depicted in FIG. 2A with IL-2R (eg, IL-2R containing alpha, beta and gamma polypeptides containing the amino acid sequences (mature form) shown in Fig. 3A-3C).

In some cases, the synTac polypeptide of the present invention has a binding affinity for IL-2R that ranges from 100 nM to about 100 μM. In some cases, the synTac polypeptide of the present invention has a binding affinity for IL-2R that is about 100 to 500 nM. For example, in some cases, the synTac polypeptide of the present invention has binding affinity for IL-2R (e.g., IL-2R containing alpha, beta and gamma polypeptides containing the amino acid sequences (mature form) depicted in FIGS. 3A-3C ), which ranges from about 100 nM to about 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, about 400 nM to about 450 nM, or about 450 nM to about 500 nM. In some cases, the synTac polypeptide of the present invention has a binding affinity for IL-2R (e.g., IL-2R containing alpha, beta and gamma polypeptides containing the amino acid sequences (mature form) depicted in FIGS. 3A-3C) from about 500 nM to 1 µM. For example, in some cases, the synTac polypeptide of the present invention has binding affinity for IL-2R (e.g., IL-2R containing alpha, beta and gamma polypeptides containing the amino acid sequences (mature form) depicted in FIGS. 3A-3C ), which is 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, or from about 900 nM to about 1 μM. In some cases, the synTac polypeptide of the present invention has binding affinity for IL-2R (e.g., IL-2R containing alpha, beta and gamma polypeptides containing the amino acid sequences (mature form) depicted in FIGS. 3A-3C.) from about 1 to 10 µM. For example, in some cases, the synTac polypeptide of the present invention has binding affinity for IL-2R (e.g., IL-2R containing alpha, beta and gamma polypeptides containing the amino acid sequences (mature form) depicted in FIGS. 3A-3C ), which is from about 1 to 2 μM, from about 2 μM to about 3 μM, from about 3 μM to about 4 μM, from about 4 μM to about 5 μM, from about 5 μM to about 6 μM, from about 6 µM to about 7 µM, from about 7 µM to about 8 µM, from about 8 µM to about 9 µM, or from about 9 µM to about 10 µM. In some cases, the synTac polypeptide of the present invention has a binding affinity for IL-2R (e.g., IL-2R containing alpha, beta, and gamma polypeptides containing the amino acid sequences (mature form) depicted in FIGS. 3A-3C) of about 10 up to 100 µM. For example, in some cases, the synTac polypeptide of the present invention has binding affinity for IL-2R (e.g., IL-2R containing alpha, beta and gamma polypeptides containing the amino acid sequences (mature form) depicted in FIGS. 3A-3C ), which is from about 10 μM to about 20 μM, from about 20 μM to about 30 μM, from about 30 μM to about 40 μM, from about 40 μM to about 50 μM, from about 50 μM to about 60 μM,

- 28 046369 from about 60 μM to about 70 μM, from about 70 μM to about 80 μM, from about 80 μM to about 90 μM or from about 90 to about 100 μM.

Determination of binding affinity.

The binding affinity between an immunomodulatory polypeptide and its cognate coimmunomodulatory polypeptide can be determined by biolayer interferometry (BLI bio-layer interferometry) using the purified immunomodulatory polypeptide and the purified cognate coimmunomodulatory polypeptide. The binding affinity between the synTac of the present invention and its cognate coimmunomodulatory polypeptide can also be determined by BLI using purified synTac and the cognate coimmunomodulatory 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 specific and relative binding affinities described in this invention between an immunomodulatory polypeptide and its cognate coimmunomodulatory polypeptide or between synTac and its cognate immunomodulatory polypeptide can be determined using the following procedures.

To determine the binding affinity between the synTac of the present invention and its cognate coimmunomodulatory polypeptide, a BLI assay can be performed using an Octet RED 96 instrument (Pal ForteBio) or similar instrument as indicated below. To determine the binding affinity of a multimeric T cell modulating polypeptide (e.g., synTac of the present invention or a control multimeric T cell modulating polypeptide (wherein the control multimeric T cell modulating polypeptide contains a wild type immunomodulatory polypeptide)), multimeric the T cell modulating polypeptide is immobilized on an insoluble carrier (biosensor). The immobilized, multimeric T cell modulating polypeptide is the target. Immobilization can be accomplished by immobilizing a capture antibody on an insoluble support, wherein the capture antibody immobilizes a multimeric T cell modulating polypeptide. For example, immobilization can be accomplished by immobilizing anti-Fc antibodies (e.g., anti-human Fc IgG) onto an insoluble support, wherein the immobilized anti-Fc antibodies bind to and immobilize the multimeric T cell modulating polypeptide (wherein the multimeric T cell modulating polypeptide contains IgFc polypeptide). The coimmunomodulatory polypeptide is applied at several different concentrations to the immobilized, multimeric T cell modulating polypeptide and the response of the device is recorded. Assays are carried out in a liquid medium containing 25 mM HEPES, pH 6.8, 5% poly(ethylene glycol) 6000, 50 mM KCl, 0.1% bovine serum albumin and 0.02% nonionic detergent Tween 20. Binding of coimmunomodulatory polypeptide to immobilized T cell modulation of multimeric polypeptide is carried out at 30°C. As a positive control for binding affinity, an anti-MHC class I monoclonal antibody can be used. For example, the anti-HSV class I monoclonal antibody W6/32 can be used (American Type Culture Collection, No. HB-95; Parham et al. (1979), J. Immunol ., 123:342) with K _D 7 nM. A standard curve can be prepared using serial dilutions of an anti-MHC class I monoclonal antibody. A coimmunomodulatory polypeptide, or anti-MHC class I mAb, is the analyte. BLI analyzes the interference pattern of white light reflected from two surfaces

i) from an immobilized polypeptide (target/target); and ii) an internal reference layer.

Changing the number of molecules (analyte; e.g., coimmunomodulatory polypeptide; anti-NAS 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 target/target and analyte interactions are association constant (k _a ) and dissociation constant (k d ). The ratio of these two terms (k _d /k _a ) gives the affinity constant K _D .

As noted above, determination of the binding affinity between an immunomodulatory polypeptide (eg, IL-2 or an IL-2 variant) and its cognate coimmunomodulatory polypeptide (eg, IL-2R) can also be determined using BLI. The analysis is similar to that described above for the synTac multimeric polypeptide. BLI analysis can be performed using an Octet RED 96 (Pal ForteBio) or similar instrument as follows. A synTac component immunomodulatory polypeptide of the present invention (eg, a variant IL-2 polypeptide of the present invention); and a control immunomodulatory polypeptide (wherein the control immunomodulatory polypeptide comprises a wild-type immunomodulatory polypeptide, e.g., wild-type IL-2)) is immobilized on an insoluble carrier (biosensor). The immunomodulatory polypeptide is the target/target. Immobilization can be accomplished by immobilizing a capture antibody on an insoluble support, wherein the capture antibody immobilizes the immunomodulatory polypeptide. For example, if the target/target is fused with an immunoaffinity tag (e.g. FLAG, human IgG Fc), immobilization can be achieved by immobilization

- 29 046369 tion using an appropriate antibody to an immunoaffinity tag (for example, anti-human Fc IgG) on an insoluble carrier, wherein the immobilized antibodies bind and immobilize the immunomodulatory polypeptide (wherein the immunomodulatory polypeptide contains an Ig Fc polypeptide). The coimmunomodulatory polypeptide (or polypeptides) are applied at several different concentrations to the immobilized immunomodulatory polypeptide and the response of the device is recorded. Alternatively, the coimmunomodulatory polypeptide(s) is immobilized in a biosensor (eg, for an IL-2 receptor heterotrimer, as a monomeric subunit, a heterodimeric subcomplex, or a complete heterotrimer) and the immunomodulatory polypeptide is applied at several different concentrations to the immobilized coimmunomodulatory polypeptide(s), and the instrument responds registered. Assays are carried out in a liquid medium containing 25 mM HEPES, pH 6.8, 5% poly(ethylene glycol) 6000, 50 mM KCl, 0.1% bovine serum albumin and 0.02% nonionic detergent Tween 20. Binding of coimmunomodulatory polypeptide to immobilized immunomodulatory polypeptide 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, the anti-HSV class I monoclonal antibody W6/32 can be used (American Type Culture Collection, No. HB-95; Parham et al. (1979), J. Immunol ., 123: 342) with a KD of 7 nM. A standard curve can be prepared using serial dilutions of an anti-MHC class I monoclonal antibody. A coimmunomodulatory polypeptide, or anti-MHC class I mAb, is the analyte. BLI analyzes the interference pattern of white light reflected from two surfaces

i) from an immobilized polypeptide (target/target); and ii) an internal reference layer.

Changing the number of molecules (analyte; e.g., coimmunomodulatory polypeptide; anti-NAS 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 target/target and analyte interactions are association constant (ka) and dissociation constant (kd). The ratio of these two terms (kd/ka) gives the affinity constant _KD . Determination of the binding affinity of both a wild-type immunomodulatory polypeptide (eg, IL-2) to its receptor (eg, IL-2R) and a variant immunomodulatory polypeptide (eg, variant IL-2 as described herein) to its cognate coimmunomodulatory polypeptide (eg, its receptor) (eg, IL-2R) thus allows the relative binding affinity of a variant coimmunomodulatory polypeptide compared with a wild-type coimmunomodulatory polypeptide to a cognate coimmunomodulatory polypeptide to be determined. Those. it can be determined whether the binding affinity of a variant immunomodulatory polypeptide to its receptor (its cognate immunomodulatory polypeptide) is reduced compared to the binding affinity of a wild-type immunomodulatory polypeptide to the same cognate immunomodulatory polypeptide and, if so, what the percentage reduction is compared to the binding affinity of a coimmunomodulatory polypeptide wild type.

The BLI assay is performed in a multiwell plate. To perform the analysis, a plate layout is created, analysis steps are planned, and biosensors are assigned in Octet Data Acquisition software. The biosensor assembly is hydrated. The hydrated biosensor assembly and assay plate are adapted within 10 min on an Octet instrument. Immediately after receiving, the resulting data is loaded into the Octet data analysis program. The data is processed in the processing window by specifying the method of reference subtraction, Y-axis alignment, inter-step correction and Savitzky-Golay filtering. Data are analyzed in the analysis window by specifying the steps for analysis (association and dissociation), selecting a 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 data curve (analyte concentration) can be averaged if they are within the 3-fold range. The KD error values must be within the same order of magnitude 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.

In some cases, the ratio of i) the binding affinity of a control, multimeric T cell modulatory polypeptide (wherein the control includes a wild-type immunomodulatory polypeptide, e.g., wild-type IL-2) to a cognate coimmunomodulatory polypeptide (e.g., IL-2R) to ii) the affinity binding of a multimeric T cell modulating polypeptide of the present invention comprising a wild-type variant immunomodulatory polypeptide (e.g., variant IL-2) to a cognate immunomodulatory polypeptide (e.g., IL-2R) when measured by BLI (as described above) is at least 1.5:1, at least 2:1, at least 5:1, at least 10:1, at least 15:1, at least 20:1, at least 25:1 , at least 50:1, at least 100:1, at least 500:1, at least 10 ² :1, at least 5x102:1, at least 10 ³ :1, at least 5x10 ³ :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, multimeric T cell modulating polypeptide (wherein the control includes a wild-type immunomodulatory polypeptide) to a cognate coimmunomodulatory polypeptide to ii) the affinity

- 30 046369 The binding ratio of a T cell immunomodulatory multimeric polypeptide of the present invention comprising a wild-type variant immunomodulatory polypeptide to a cognate coimmunomodulatory polypeptide, as measured by BLI, is in the range of 1.5:1 to ¹⁰⁶ :1, for example 1.5: 1 to 10:1, 10:1 to 50:1, 50:1 to 102:1, ¹⁰² :1 to 103:1, ¹⁰³ :1 to ¹⁰⁴ :1, ¹⁰⁴ :1 up to 10 ⁵ :1 or from 10 ⁵ :1 to 10 ⁶ :1.

In some cases, the ratio of i) the binding affinity of a control immunomodulatory polypeptide (wherein the control includes a wild-type immunomodulatory polypeptide, e.g., wild-type IL2) to a cognate coimmunomodulatory polypeptide (e.g., IL-2R) to ii) the binding affinity of an immunomodulatory polypeptide of the invention containing the variant wild-type immunomodulatory polypeptide (e.g., variant IL-2) with a cognate immunomodulatory polypeptide (e.g., IL-2R), when measured by BLI (as described above), is at least 1.5:1, at least 2:1, at least 5:1, at least 10:1, at least 15:1, at least 20:1, at least 25:1, at least 50:1, at least 100:1, according to at least 500:1, at least 102:1, at least 5x102:1, at least ¹⁰³ :1, at least 5x103:1, at least ¹⁰⁴ :1, at least ¹⁰⁵ :1 or at least 10 ⁶ :1. In some cases, the ratio of i) the binding affinity of a control immunomodulatory polypeptide (wherein the control includes a wild-type immunomodulatory polypeptide) to a cognate coimmunomodulatory polypeptide to ii) the binding affinity of an immunomodulatory polypeptide of the present invention containing a wild-type variant immunomodulatory polypeptide to a cognate coimmunomodulatory polypeptide, as measured by BLI , is in the range from 1.5:1 to 10 ⁶ :1, for example from 1.5:1 to 10:1, from 10:1 to 50:1, from 50:1 to 102:1, from 102:1 up 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.

The variant IL-2 polypeptide present in the synTac polypeptide of the present invention may have one amino acid substitution relative to a wild-type IL-2 polypeptide (e.g., an IL-2 polypeptide containing the amino acid sequence depicted in FIG. 2A or as set forth in SEQ ID NO : 1). In some cases, the variant IL-2 polypeptide present in the synTac polypeptide of the present invention has from 2 to 10 amino acid substitutions relative to the wild-type IL-2 polypeptide (e.g., an IL-2 polypeptide containing the amino acid sequence depicted in Fig. 2A or as indicated in SEQ ID NO: 1). In some cases, the variant IL-2 polypeptide present in the synTac polypeptide of the present invention has 2 amino acid substitutions relative to the wild-type IL-2 polypeptide (e.g., an IL-2 polypeptide containing the amino acid sequence depicted in FIG. 2A or as set forth in SEQ ID NO: 1). In some cases, the variant IL-2 polypeptide present in the synTac polypeptide of the present invention has 3 amino acid substitutions relative to the wild-type IL-2 polypeptide (e.g., an IL-2 polypeptide containing the amino acid sequence depicted in FIG. 2A or as set forth in SEQ ID NO: 1). In some cases, the variant IL-2 polypeptide present in the synTac polypeptide of the present invention has 4 amino acid substitutions relative to the wild-type IL-2 polypeptide (e.g., an IL-2 polypeptide containing the amino acid sequence depicted in FIG. 2A or as set forth in SEQ ID NO: 1). In some cases, the variant IL-2 polypeptide present in the synTac polypeptide of the present invention has 5 amino acid substitutions relative to the wild-type IL-2 polypeptide (e.g., an IL-2 polypeptide containing the amino acid sequence depicted in FIG. 2A or as set forth in SEQ ID NO: 1). In some cases, the variant IL-2 polypeptide present in the synTac polypeptide of the present invention has 6 amino acid substitutions relative to the wild-type IL-2 polypeptide (e.g., an IL-2 polypeptide containing the amino acid sequence depicted in FIG. 2A or as set forth in SEQ ID NO: 1). In some cases, the variant IL-2 polypeptide present in the synTac polypeptide of the present invention has 7 amino acid substitutions relative to the wild-type IL-2 polypeptide (e.g., an IL-2 polypeptide containing the amino acid sequence depicted in FIG. 2A or as set forth in SEQ ID NO: 1). In some cases, the variant IL-2 polypeptide present in the synTac polypeptide of the present invention has 8 amino acid substitutions relative to the wild-type IL-2 polypeptide (e.g., an IL-2 polypeptide containing the amino acid sequence depicted in FIG. 2A or as set forth in SEQ ID NO: 1). In some cases, the variant IL-2 polypeptide present in the synTac polypeptide of the present invention has 9 amino acid substitutions relative to the wild-type IL-2 polypeptide (e.g., an IL-2 polypeptide containing the amino acid sequence depicted in FIG. 2A or as set forth in SEQ ID NO: 1). In some cases, the variant IL-2 polypeptide present in the synTac polypeptide of the present invention has 10 amino acid substitutions relative to the wild-type IL-2 polypeptide (e.g., an IL-2 polypeptide containing the amino acid sequence depicted in FIG. 2A or as set forth in SEQ ID NO: 1).

In some cases, the multimeric polypeptide of the present invention comprises a first polypeptide and a second polypeptide, wherein the first polypeptide comprises an amino terminus (N-terminus) to a carboxyl terminus

- 31 046369 strong end (C-end)

a) epitope (for example, T-cell epitope);

b) the first polypeptide of the major histocompatibility complex (MHC); And

c) an immunomodulatory polypeptide (eg, a variant IL-2 polypeptide of the present invention); and wherein the second polypeptide contains, in order from N-terminus to C-terminus

a) a second MHC polypeptide; And

b) Immunoglobulin Fc polypeptide (Ig).

In other cases, the multimeric polypeptide of the present invention comprises a first polypeptide and a second polypeptide, wherein the first polypeptide comprises N-terminus to C-terminus

a) epitope (for example, T-cell epitope); And

b) the first MHC polypeptide; and wherein said second polypeptide comprises, in order from N-terminus to C-terminus

a) an immunomodulatory polypeptide (for example, a variant IL-2 polypeptide of this invention);

b) a second MHC polypeptide; And

c) Fc polypeptide Ig.

In some cases, the first and second MHC polypeptides are class I MHC polypeptides; for example, in some cases, the first MHC polypeptide is a class I MHC b2-microglobulin (B2M or b2M) polypeptide and the second MHC polypeptide is a class I MHC heavy chain (H chain); or the first MHC polypeptide is a class I MHC H chain, and the second MHC polypeptide is a class I MHC polypeptide B2M). In other cases, the first and second MHC polypeptides are class II MHC polypeptides; for example, in some cases, the first MHC polypeptide is a class II MHC α chain polypeptide and the second MHC polypeptide is a class II MHC β chain polypeptide. In other cases, the first polypeptide is a class II MHC β-chain polypeptide and the second MHC polypeptide is a class II MHC α-chain polypeptide. In some cases, the multimeric polypeptide comprises two or more immunomodulatory polypeptides, wherein at least one of the immunomodulatory polypeptides is a variant IL-2 immunomodulatory polypeptide of the present invention. When the multimeric polypeptide of the present invention includes two or more immunomodulatory polypeptides, in some cases the two or more immunomodulatory polypeptides are present on the same polypeptide chain and may be in tandem. When the multimeric polypeptide of the present invention includes two or more immunomodulatory polypeptides, in some cases two or more immunomodulatory polypeptides are present in separate polypeptides. In some cases, the multimeric polypeptide of this invention is a heterodimer. In some cases, the multimeric polypeptide of this invention is a trimeric polypeptide.

In some cases, the multimeric polypeptide of this invention contains

a) the first polypeptide containing in order from N-terminus to C-terminus

i) an epitope, and ii) a first MHC polypeptide; And

b) a second polypeptide containing in order from N-terminus to C-terminus

i) a second MHC polypeptide, and ii) an Ig Fc polypeptide, and iii) an immunomodulatory domain (eg, a variant IL-2 polypeptide of the present invention).

In some cases, the multimeric polypeptide of this invention contains

a) the first polypeptide containing in order from N-terminus to C-terminus

i) an epitope, and ii) a first MHC polypeptide; And

b) a second polypeptide containing in order from N-terminus to C-terminus

i) a second MHC polypeptide, and ii) an immunomodulatory domain (eg, a variant IL-2 polypeptide of the present invention).

In some cases, the multimeric polypeptide of this invention contains

a) the first polypeptide containing in order from N-terminus to C-terminus

i) an epitope, and ii) a first MHC polypeptide; And

b) a second polypeptide containing in order from N-terminus to C-terminus

i) an immunomodulatory domain (eg, a variant IL-2 polypeptide of the present invention), and ii) a second MHC polypeptide.

In some cases, the multimeric polypeptide of this invention contains

a) the first polypeptide containing in order from N-terminus to C-terminus

- 32 046369

i) an epitope, ii) a first MHC polypeptide, and iii) an immunomodulatory domain (eg, a variant IL-2 polypeptide of the present invention); And

b) a second polypeptide containing in order from N-terminus to C-terminus

i) a second MHC polypeptide.

In some cases, when the multimeric polypeptide of the present invention contains a non-β scaffold, the non-β scaffold is an XTEN peptide, a transferrin polypeptide, an Fc receptor polypeptide, an elastin-like polypeptide, a silk-like polypeptide, or a silk-like elastin-like polypeptide.

In some cases, the multimeric polypeptide of this invention is monovalent. In some cases, the multimeric polypeptide of this invention is multivalent. In some cases, the multivalent multimeric polypeptide of the present invention comprises an immunoglobulin Fc polypeptide on one of the first or second polypeptide. For example, depending on the Fc polypeptide present in the multimeric polypeptide of the present invention, the multimeric polypeptide may be a homodimer, wherein two molecules of the multimeric polypeptide are present in the homodimer, and wherein the two molecules of the multimeric polypeptide may be disulfide linked to each other, for example, through Fc- a polypeptide present in two molecules. As another example, the multimeric polypeptide of the present invention may comprise three, four or five multimeric polypeptide molecules, wherein the multimeric polypeptide molecules may be disulfide linked to each other, for example, through an Fc polypeptide present in the molecules.

In some cases, the multimeric polypeptide of this invention contains

a) the first polypeptide containing in order from N-terminus to C-terminus

i) an epitope, ii) a B2M polypeptide, and iii) a variant IL-2 polypeptide of the present invention; And

b) a second polypeptide containing in order from N-terminus to C-terminus

i) MHC class I heavy chain, and ii) Fc polypeptide.

In some cases, the multimeric polypeptide of this invention contains

a) the first polypeptide containing in order from N-terminus to C-terminus

i) an epitope, and ii) a B2M polypeptide; And

b) a second polypeptide containing in order from N-terminus to C-terminus

i) a variant IL-2 polypeptide of the present invention, ii) a MHC class I heavy chain, and iii) an Fc polypeptide.

In some cases, the multimeric polypeptide of this invention contains

a) the first polypeptide containing in order from N-terminus to C-terminus

i) an epitope, ii) a B2M polypeptide, iii) a first variant IL-2 polypeptide of the present invention, iv) a second variant IL-2 polypeptide of the present invention, and

v) a third variant IL-2 polypeptide of the present invention; And

b) a second polypeptide containing in order from N-terminus to C-terminus

i) MHC class I heavy chain, and ii) Fc polypeptide.

In some cases, the first, second and third variant IL-2 polypeptides have the same amino acid sequence. In some cases, the first, second and third variant IL-2 polypeptides differ from each other in amino acid sequence. In some cases, the multimeric polypeptide of this invention contains

a) the first polypeptide containing in order from N-terminus to C-terminus

i) an epitope, and ii) a B2M polypeptide; And

b) a second polypeptide containing in order from N-terminus to C-terminus

i) a first variant IL-2 polypeptide of the present invention, ii) a second variant IL-2 polypeptide of the present invention, and iii) a third variant IL-2 polypeptide of the present invention, iv) MHC class I heavy chain, and

v) Fc polypeptide.

- 33 046369

In some cases, the first, second and third variant IL-2 polypeptides have the same amino acid sequence. In some cases, the first, second and third variant IL-2 polypeptides differ from each other in amino acid sequence.

Linkers.

The multimeric polypeptide of the present invention may include linker peptides located between, for example, an epitope and an MHC polypeptide; between MHC polypeptide and immunomodulatory polypeptide; between the MHC polypeptide and the Ig Fc polypeptide; between the first variant IL-2 polypeptide and the second variant IL-2 polypeptide; or between a second variant IL-2 polypeptide and a third variant IL-2 polypeptide.

Suitable linkers (also called spacers) can be easily selected and can be of any suitable length, such as 1 to 25 amino acids, 3 to 20 amino acids, 2 to 15 amino acids, 3 to 12 amino acids, including 4 up to 10 amino acids, 5 to 9 amino acids, 6 to 8 amino acids, or 7 to 8 amino acids. A suitable linker may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 amino acids in length.

Exemplary linkers include (G)n glycine polymers, glycine-serine polymers (including, for example, (GS)n, (GSGGS)n (SEQ ID NO: 89) and (GGGS)n (SEQ ID NO: 86), wherein n is an integer of at least 1), glycine-alanine polymers, alanine-serine polymers and other flexible linkers known in the art. Glycine and glycineserine polymers can be used; both Gly and Ser are relatively unstructured and can therefore serve as neutral linkers between components. Glycine polymers can be used; glycine accesses significantly more 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)). Exemplary linkers may contain amino acid sequences including, but not limited to, GGSG (SEQ ID NO: 2), GGSGG (SEQ ID NO: 3), GSGSG (SEQ ID NO: 4), GSGGG (SEQ ID NO: 5), GGGSG (SEQ ID NO: 6), GSSSG (SEQ ID NO: 7), etc. Exemplary linkers may include, for example, Gly(Ser ₄ ) _n , where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some cases, the linker contains the amino acid sequence (GSSSS)n (SEQ ID NO: 93), where n is 4. In some cases, the linker contains the amino acid sequence (GSSSS)n (SEQ ID NO: 94), where n is 5. In some cases, the linker contains the amino acid sequence (GGGGS)n (SEQ ID NO : 9), where n is 1. In some cases, the linker contains the amino acid sequence (GGGGS) _n (SEQ ID NO: 92), and n is 2. In some cases, the linker contains the amino acid sequence (GGGGS)n (SEQ ID NO: 89 ), wherein n is 3. In some cases, the linker contains the amino acid sequence (GGGGS) _n (SEQ ID NO: 90), and n is 4. In some cases, the linker contains the amino acid sequence (GGGGS) _n (SEQ ID NO: 91), where n is equal to 5.

In some cases, the linker polypeptide present in the first polypeptide of the multimeric polypeptide of the present invention includes a cysteine residue that can form a disulfide bond with a cysteine residue present in the second polypeptide of the multimeric polypeptide of the present invention. In some cases, for example, a suitable linker contains the amino acid sequence GCGASGGGGSGGGGS (SEQ ID NO: 10).

Epitopes.

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

The epitope present in the multimeric polypeptide of the present invention is specifically associated with a T cell, i.e. the epitope is specifically bound to an epitope-specific T cell. An epitope-specific T cell binds an epitope having a reference amino acid sequence, but does not substantially bind an epitope that differs from the reference amino acid sequence. For example, an epitope-specific T cell binds an epitope having a control amino acid sequence, and binds an epitope that differs from the control amino acid sequence, if at all, with an affinity of less than 10-6 M, less than 10-5 M, or less than 10 -4 M. Epitope specific T cells can bind to an epitope for which they are 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.

Suitable epitopes include, but are not limited to, epitopes present in a cancer-associated antigen. Cancer-associated antigens include, but are not limited to, α-folate receptor; carbonic anhydrase IX (CAIX); CD19; CD20; CD22; CD30; CD33; CD44v7/8; carcinoembryonic

- 34 046369 antigen (CEA - carcinoembryonic antigen); epithelial glycoprotein-2 (EGP-2 - epithelial glycoprotein-2); epithelial glycoprotein-40 (EGP-40 - epithelial glycoprotein-40); folate binding protein (FBP folate binding protein); fetal acetylcholine receptor; ganglioside antigen GD2; HER2/Neu; IL-13R-a2; kappa light chain; LeY; cell adhesion molecule L1; melanoma-associated antigen (MAGE - melanoma-associated antigen); MAGE-A1; mesothelin; MUC1; NKG2D ligands; oncofetal antigen (h5T4); prostate stem cell antigen (PSCA - prostate stem cell antigen); prostate-specific membrane antigen (PSMA - prostate-specific membrane antigen); tumor-associated glycoprotein-72 (TAG-72 - tumor-associate glycoprotein-72); and vascular endothelial growth factor receptor-2 (VEGF-R2). See, for example, Vigneron et al. (2013), Cancer Immunity, 13:15; and Vigneron (2015), BioMed Res. Int'l Article ID 948501. In some cases, the epitope is an epitope of the human papillomavirus E7 antigen; see, for example, Ramos et al. (2013), J. Immunother., 36:66.

In some cases, the epitope is HPV16E7/82-90 (LLMGTLGIV; SEQ ID NO: 11). In some cases, the epitope is HPV16E7/86-93 (TLGIVCPI; SEQ ID NO: 12). In some cases, the epitope is HPV16E7/11-20 (YMLDLQPETT; SEQ ID NO: 13). In some cases, the epitope is HPV16E7/11-19 (YMLDLQPET; SEQ ID NO: 87). See, for example, Ressing et al. ((1995) J. Immunol., 154:5934) for additional suitable HPV (human papilloma virus) epitopes.

MHC polypeptides.

As noted above, the multimeric polypeptide of this invention includes MHC polypeptides. For purposes of this invention, the term major histocompatibility complex (MHC) polypeptides includes MHC polypeptides of various species, including human MHC polypeptides (also called human leukocyte antigens (HLAs)), rodent MHC polypeptides (e.g., mouse, rat, etc.). ) and MHC polypeptides from other mammalian species (eg, lagomorphs, non-human primates, canines, felines, ungulates (eg, horses, bovines, sheep, captilians, etc.), etc. The term MHC polypeptide is intended to be includes class I MHC polypeptides (eg, β-2-microglobulin and class I MHC heavy chain) and class II MHC polypeptides (eg, class IIa MHC polypeptide and class II MHC β polypeptide).

As noted above, in some embodiments of the multimeric polypeptide of this invention, the first and second MHC polypeptides are class I MHC polypeptides; for example, in some cases, the first MHC polypeptide is a class I MHC b2-microglobulin (b2M) polypeptide and the second MHC polypeptide is a class I MHC heavy chain (H chain). In other cases, the first and second MHC polypeptides are class II MHC polypeptides; for example, in some cases, the first MHC polypeptide is a class II MHC α chain polypeptide and the second MHC polypeptide is a class II MHC β chain polypeptide. In other cases, the first polypeptide is a class II MHC β-chain polypeptide and the second MHC polypeptide is a class II MHC α-chain polypeptide.

In some cases, the MHC polypeptide of the multimeric polypeptide of the present invention is a human MHC polypeptide, and human MHC polypeptides are also referred to as human leukocyte antigen (HLA) polypeptides. In some cases, the MHC polypeptide of the multimeric polypeptide of the present invention is a class I HLA polypeptide, such as a β2-microglobulin polypeptide or a class I HLA heavy chain polypeptide. Class I HLA heavy chain polypeptides include HLA heavy chain polypeptides, HLA heavy chain polypeptides, HLA heavy chain polypeptides , HLA-C heavy chain polypeptides, HLA-E heavy chain polypeptides, HLA-F heavy chain polypeptides and HLA-G heavy chain polypeptides. In some cases, the MHC polypeptide of the multimeric polypeptide of the present invention is a class II HLA polypeptide, for example, a class II HLA α-chain or a class II HLA β-chain. Class II MHC polypeptides include DPa and β polypeptides, DMa and β polypeptides, DOAa and β polypeptides, DOBa and β polypeptides, DQa and β polypeptides, and DRa and β MHC class II polypeptides.

As an example, the MHC class I heavy chain polypeptide of the multimeric 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 with amino acids 25-365 of the amino acid sequence of the human HLA-A heavy chain polypeptide shown in FIG. 5A.

As an example, the MHC class I heavy chain polypeptide of the multimeric 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 with amino acids 25-365 in the amino acid sequence of the following HLA-A heavy chain amino acid sequence:

- 35 046369

GSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEY WDGETRKVKAHSQTHRVDLGTLRGYYNQSEAGSHTVQRMYGCDVGSDWRFLRGYHQ YAYDGKDYIALKEDLRSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGTCVEWLRRY LENGKETLQRTDAPKTHMTH HAVSDHEATLRCWALSFYPAEITLTWQRDGEDQTQDTE LVETRPAGDGTFQKWAAVVVPSGQEQRYTCHVQHEGLPKPLTLRWEP (SEQ ID NO: 14).

As another example, the MHC class I heavy chain polypeptide of a multimeric 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 with amino acids 25-362 of the amino acid sequence of the human HLA-B heavy chain polypeptide shown in FIG. 5V.

As another example, the MHC class I heavy chain polypeptide of the multimeric 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 with amino acids 25-362 of the amino acid sequence of the human HLA-C heavy chain polypeptide shown in FIG. 5C.

As another example, the MHC class I heavy chain polypeptide of the multimeric 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 with the following amino acid sequence:

GPHSLRYFVTAVSRPGLGEPRFIAVGYVDDTQFVRFDSDADNPRFEPRAPWMEQEGPEY

WEEQTQRAKSDEQWFRVSLRTAQRYYNQSKGGSHTFQRMFGCDVGSDWRLLRGYQQ FAYDGRDYIALNEDLKTWTAADTAALITRRKWEQAGDAEYYRAYLEGECVEWLRRYL ELGNETLLRTDSPKAHVTYHPRSQVDVTLRCWALGFYPADITLTWQLNGEDLTQDMEL VETRPAGDGTFQKWAAVVVPLGKE QNYTCHVHHKGLPEPLTLRW (SEQ ID NO:15).

The multimeric b2-microglobulin (b2M) polypeptide of the present invention may be a human b2M polypeptide, a non-human primate b2M polypeptide, a murine b2M polypeptide, and the like. In some cases, the B2M polypeptide contains an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99 or 100%, amino acid sequence identity with the B2M amino acid sequence shown in FIG. 6. In some cases, the B2M polypeptide contains an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least At least 99 or 100% amino acid sequence identity between amino acids 21 and 119 to the B2M amino acid sequence shown in FIG. 6.

In some cases, the MHC polypeptide contains 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 an amino acid with a cysteine (Cys) residue. Such cysteine residues, when present in the MHC polypeptide of the first polypeptide of the multimeric polypeptide of the present invention, can form a disulfide bond with a cysteine residue present in the second polypeptide chain of the multimeric polypeptide of the present invention.

In some cases, the first MHC polypeptide in the first polypeptide of the multimeric polypeptide of the present invention and/or the second MHC polypeptide in the second polypeptide of the multimeric polypeptide of the present invention includes an amino acid substitution to replace the amino acid with a cysteine, wherein the substituted cysteine in the first MHC polypeptide forms a disulfide bond with the cysteine at a second MHC polypeptide, wherein the cysteine in the first MHC polypeptide forms a disulfide bond with a substituted cysteine in the second MHC polypeptide, or wherein the substituted cysteine in the first MHC polypeptide forms a disulfide bond with a substituted cysteine in the second MHC polypeptide.

For example, in some cases, one of the following pairs of residues in HLA β2-microglobulin and class I HLA heavy chain is replaced by cysteines (with the residue numbers corresponding to the residue numbers of the mature polypeptide):

1) residue B2M 12, class I HLA heavy chain residue 236;

- 36 046369

2) β2Μ residue 12, class I HLA heavy chain residue 237;

3) residue B2M 8, class I HLA heavy chain residue 234;

4) residue B2M 10, class I HLA heavy chain residue 235;

5) residue B2M 24, class I HLA heavy chain residue 236;

6) residue B2M 28, class I HLA heavy chain residue 232;

7) residue B2M 98, class I HLA heavy chain residue 192;

8) residue B2M 99, class I HLA heavy chain residue 234;

9) residue B2M 3, class I HLA heavy chain residue 120;

10) residue B2M 31, class I HLA heavy chain residue 96;

11) residue B2M 53, class I HLA heavy chain residue 35;

12) residue B2M 60, class I HLA heavy chain residue 96;

13) residue B2M 60, class I HLA heavy chain residue 122;

14) residue B2M 63, class I HLA heavy chain residue 27;

15) residue 2M Arg3, class I HLA heavy chain residue Gly120;

16) residue 2M His31, class I HLA heavy chain residue Gln96;

17) residue 2M Asp53, class I HLA heavy chain residue Arg35;

18) residue 2M Trp60, heavy chain residue of class I HLA Gln96;

19) residue B2M Trp60, class I HLA heavy chain residue Asp122;

20) B2M residue Trp63, class I HLA heavy chain residue Tyr27;

21) residue 2M Lys6, class I HLA heavy chain residue Glu232;

22) residue 2M Gln8, class I HLA heavy chain residue Arg234;

23) residue B2M Tyr10, heavy chain residue of HLA class I Pro235;

24) residue 2M Ser11, class I HLA heavy chain residue Gln242;

25) residue 2M Asn24, class I HLA heavy chain residue Ala236;

26) residue 2M Ser28, class I HLA heavy chain residue Glu232;

27) residue 2M Asp98, class I HLA heavy chain residue His192; And

28) residue B2M Met99, class I HLA heavy chain residue Arg234.

The amino acid numbering of the class I MHC/HSLA heavy chain refers to the mature MHC/HSLA class I heavy chain without the signal peptide. For example, in the amino acid sequence depicted in FIG. 5A, which includes the signal peptide Gly120 is Gly144; Gln96 is Gln120, etc. In some cases, the B2M polypeptide contains the R12C substitution, and the class I HLA heavy chain contains the A236C substitution; in such cases, a disulfide bond is formed between Cys-12 of the B2M polypeptide and Cys-236 of the class I HLA heavy chain. For example, in some cases, residue 236 of the mature HLA-A amino acid sequence (i.e., residue 260 of the amino acid sequence shown in FIG. 5A) is replaced by Cys. In some cases, residue 236 of the mature HLA-B amino acid sequence (ie, residue 260 of the amino acid sequence shown in FIG. 5B) is replaced with Cys. In some cases, residue 236 of the mature HLA-C amino acid sequence (ie, residue 260 of the amino acid sequence shown in Fig. 5C) is replaced by Cys. In some cases, residue 32 (corresponding to mature B2M Arg-12) of the amino acid sequence depicted in FIG. 6 is replaced by Cys.

In some cases, the B2M polypeptide includes the amino acid sequence IQRTPKIQVY SRHPAENGKS NFLNCYVSGF

HPSDIEVDLLKNGERIEKVE HSDLSFSKDW SFYLLYYTEF TPTEKDEYAC

RVNHVTLSQP KIVKWDRDM (SEQ ID NO: 16).

In some cases, the B2M polypeptide includes the amino acid sequence IQRTPKIQVY SCHPAENGKS

NFLNCYVSGF HPSDIEVDLLKNGERIEKVE HSDLSFSKDW SFYLLYYTEF

TPTEKDEYAC RVNHVTLSQP KIVKWDRDM (SEQ ID NO: 17).

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

GSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEY

WDGETRKVKAHSQTHRVDLGTLRGYYNQSEAGSHTVQRMYGCDVGSDWRFLRGYHQ YAYDGKDYIALKEDLRSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGTCVEWLRRY LENGKETLQRTDAPKTHMTHHAVSDHEATLRCWALSFYPAEITLTWQRDGEDQTQDTE LVETRPAGDGTFQKWAAV VVPSGQEQRYTCHVQHEGLPKPLTLRWEP (SEQ ID NO: 14).

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

GSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEY

WDGETRKVKAHSQTHRVDLGTLRGYYNQSEAGSHTVQRMYGCDVGSDWRFLRGYHQ YAYDGI<DYIALI<EDLRSWTAADMAAQTTKHI<WEAAHVAEQLRAYLEGTCVEWLRRY LENGKETLQRTDAPKTHMTHHAVSDHEATLRCWALSFYPAEITLTWQRDGEDQTQDTE LVETRPCGDGTFQKWA AVVVPSGQEQRYTCHVQHEGLPKPLTLRWEP (SEQ ID

NO: 18).

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

GSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEY WDGETRKVKAHSQTHRVDLGTLRGAYNQSEAGSHTVQRMYGCDVGSDWRFLRGYHQ YAYDGI<DYIALI<EDLRSWTAADMAAQTTI<HI<WEAAHVAEQLRAYLEGTCVEWLRRY LENGKETLQRTDAPKTHM THHAVSDHEATLRCWALSFYPAEITLTWQRDGEDQTQDTE LVETRPCGDGTFQKWAAVVVPSGQEQRYTCHVQHEGLPKPLTLRWE (SEQ ID NO: 19).

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

HSDLSFSKDW SFYLLYYTEF TPTEKDEYAC RVNHVTLSQP KIVKWDRDM (SEQ ID NO: 17);

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

GSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEY WDGETRKVKAHSQTHRVDLGTLRGYYNQSEAGSHTVQRMYGCDVGSDWRFLRGYHQ YAYDGI<DYIALI<EDLRSWTAADMAAQTTI<HI<WEAAHVAEQLRAYLEGTCVEWLRRY LENGKETLQRTDAPKTH MTHHAVSDHEATLRCWALSFYPAEITLTWQRDGEDQTQDTE LVETRPCGDGTFQKWAAVVVPSGQEQRYTCHVQHEGLPKPLTLRWEP (SEQ ID NO: 18), wherein the Cys residues that are underlined and in bold form a disulfide bond with each other in the multimeric polypeptide.

In some cases, the B2M polypeptide contains the amino acid sequence IQRTPKIQVYSCHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDW

SFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDM (SEQ ID NO: 17)

Immunomodulatory polypeptides.

The multimeric polypeptide of the present invention contains a variant IL-2 polypeptide of the present invention, as described above, which is a variant of a naturally occurring costimulatory protein, wherein the variant exhibits reduced affinity for its cognate costimulatory protein partner on a T cell (eg , IL-2R) compared with the affinity of the naturally occurring IL-2 polypeptide for its partner costimulatory protein (IL-2R). Thus, the multimeric polypeptide of the present invention contains the variant IL2 polypeptide present in the multimeric polypeptide of the present invention.

In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the sequence shown in Fig. 2E, wherein amino acid 16 is an amino acid other than histidine, for example, wherein amino acid 16 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, Asp or Glu. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the sequence shown in Fig. 2E, wherein amino acid 16 is Ala, Gly, Val, Leu or Ile. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the sequence shown in Fig. 2E, wherein amino acid 16 is Ala. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with amino acid sequence shown

- 38 046369 in Fig. 2E, wherein amino acid 16 is Gly. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the sequence shown in Fig. 2E, wherein amino acid 16 is Val. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the sequence shown in Fig. 2E, wherein amino acid 16 is Leu. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the sequence shown in Fig. 2E, with amino acid 16 being Ile. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the sequence shown in Fig. 2E, wherein amino acid 16 is Asn. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the sequence shown in Fig. 2E, wherein amino acid 16 is Asp. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the sequence shown in Fig. 2E, wherein amino acid 16 is Cys. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the sequence shown in Fig. 2E, wherein amino acid 16 is Gln. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the sequence shown in Fig. 2E, wherein amino acid 16 is Glu. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the sequence shown in Fig. 2E, with amino acid 16 being Met. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the sequence shown in Fig. 2E, wherein amino acid 16 is Phe. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the sequence shown in Fig. 2E, with amino acid 16 being Ser. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the sequence shown in Fig. 2E, with amino acid 16 being Thr. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the sequence shown in Fig. 2E, wherein amino acid 16 is Trp. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the sequence shown in Fig. 2E, wherein amino acid 16 is Tyr. Somehow

- 39 046369 In some cases, the variant IL-2 polypeptide has a binding affinity for IL-2R that is from about 100 nM to about 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 to about 800 nM, from about 800 to about 900 nM, from about 900 to about 1 μM, from about 1 to about 5 μM, from about 5 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 variant IL-2 polypeptide is 133 amino acids long.

Replacement F42.

In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the sequence shown in Fig. 2B, wherein amino acid 42 is an amino acid other than phenylalanine, for example wherein amino acid 42 is Gly, Ala, Val, Leu, Ile, Pro, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the sequence shown in Fig. 2B, wherein amino acid 42 is Ala, Gly, Val, Leu or Ile. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the sequence shown in Fig. 2B, with amino acid 42 being Ala. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with amino acid sequence shown in FIG. 2B, wherein amino acid 42 is Gly. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the sequence shown in Fig. 2B, wherein amino acid 42 is Val. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the sequence shown in Fig. 2B, wherein amino acid 42 is Leu. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the sequence shown in Fig. 2B, wherein amino acid 42 is Ile. In some cases, a single copy of the variant IL-2 polypeptide is present in the multimeric polypeptide of the present invention. In some cases, the multimeric polypeptide of the present invention contains two copies of a variant IL-2 polypeptide, for example, when the two copies are in tandem without a linker between the two copies or in tandem and separated by a linker peptide. In some cases, the multimeric polypeptide of the present invention contains three copies of a variant IL-2 polypeptide, for example, when the three copies are in tandem without a linker between the three copies or in tandem and separated by a linker peptide. In some cases, when the IL-2/synTac of the present invention contains a class I HPA heavy chain and B2M, the IL-2 polypeptide(s) is/are on a polypeptide chain containing a class I HPA heavy chain. In some cases, when the IL-2 polypeptide(s) The 2/synTac of the present invention contains a class I and B2M HLA heavy chain, the IL-2 polypeptide(s) is/are on a polypeptide chain containing the B2M polypeptide. In some cases, the variant IL-2 polypeptide or synTac containing it has a binding affinity for IL-2R that is from about 100 nM to about 150 nM, from about 150 nM to about 200 nM, from about 200 nM to about 250 nM , from about 250 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 to 700 nM, 700 to 800 nM, 800 to 900 nM, 900 nM to 1 µM, 1 to 5 µM, 5 to 10 µM, about 10 µM to about 15 µM, 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 to 100 μM. In some cases, variant IL-2

- 40 046369 the lipeptide present in the multimeric polypeptide of the present invention has a length of 133 amino acids.

Replacing Y45.

In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the sequence shown in Fig. 2F, wherein amino acid 45 is an amino acid other than tyrosine, for example, wherein amino acid 45 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the sequence shown in Fig. 2F, wherein amino acid 45 is Ala, Gly, Val, Leu or Ile. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the sequence shown in Fig. 2F, wherein amino acid 45 is Ala. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with amino acid sequence shown in FIG. 2F, with amino acid 45 being Gly. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the sequence shown in Fig. 2F, wherein amino acid 45 is Val. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the sequence shown in Fig. 2F, with amino acid 45 being Leu. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the sequence shown in Fig. 2F, with amino acid 45 being Ile. In some cases, a single copy of the variant IL-2 polypeptide is present in the multimeric polypeptide of the present invention. In some cases, the multimeric polypeptide of the present invention contains two copies of a variant IL-2 polypeptide, for example, when the two copies are in tandem without a linker between the two copies or in tandem and separated by a linker peptide. In some cases, the multimeric polypeptide of the present invention contains three copies of a variant IL-2 polypeptide, for example, when the three copies are in tandem without a linker between the three copies or in tandem and separated by a linker peptide. In some cases, when the IL-2/synTac of the present invention contains a class I HPA heavy chain and B2M, the IL-2 polypeptide(s) is/are on a polypeptide chain containing a class I HPA heavy chain. In some cases, when the IL-2 polypeptide(s) The 2/synTac of the present invention contains a class I and B2M HLA heavy chain, the IL-2 polypeptide(s) is/are on a polypeptide chain containing the B2M polypeptide. In some cases, the variant IL-2 polypeptide or synTac containing it has a binding affinity for IL-2R that is from about 100 nM to about 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 to 700 nM, 700 to 800 nM, 800 to 900 nM, 900 nM to 1 µM, 1 to 5 µM, 5 to 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 to 100 µM. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention is 133 amino acids in length.

Replacement Q126.

In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the sequence shown in Fig. 2G, at

- 41 046369 wherein amino acid 126 is an amino acid other than glutamine, for example amino acid 126 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Lys, Arg, His, Asp or Glu. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the sequence shown in Fig. 2G, wherein amino acid 126 is Ala, Gly, Val, Leu or Ile. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the sequence shown in Fig. 2G, with amino acid 126 being Ala. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with amino acid sequence shown in FIG. 2G, with amino acid 126 being Gly. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the sequence shown in Fig. 2G, wherein amino acid 126 is Val. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the sequence shown in Fig. 2G, with amino acid 126 being Leu. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the sequence shown in Fig. 2G, with amino acid 126 being Ile. In some cases, a single copy of the variant IL-2 polypeptide is present in the multimeric polypeptide of the present invention. In some cases, the multimeric polypeptide of the present invention contains two copies of a variant IL-2 polypeptide, for example, when the two copies are in tandem without a linker between the two copies or in tandem and separated by a linker peptide. In some cases, the multimeric polypeptide of the present invention contains three copies of a variant IL-2 polypeptide, for example, when the three copies are in tandem without a linker between the three copies or in tandem and separated by a linker peptide. In some cases, when the IL-2/synTac of the present invention contains a class I HPA heavy chain and B2M, the IL-2 polypeptide(s) is/are on a polypeptide chain containing a class I HPA heavy chain. In some cases, when the IL-2 polypeptide(s) The 2/synTac of the present invention contains a class I and B2M HLA heavy chain, the IL-2 polypeptide(s) is/are on a polypeptide chain containing the B2M polypeptide. In some cases, the variant IL-2 polypeptide or synTac containing it has a binding affinity for IL-2R that is from about 100 nM to about 150 nM, from about 150 nM to about 200 nM, from about 200 nM to about 250 nM , from about 250 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 to 700 nM, from 700 to 800 nM, from 800 to 900 nM, from 900 to 1 µM, from 1 to 5 µM, from 5 to 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 to 100 μM. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention is 133 amino acids in length.

Replacements F42 and H16.

In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the sequence shown in Fig. 2H, wherein amino acid 42 is an amino acid other than phenylalanine, for example, wherein amino acid 42 is Gly, Ala, Val, Leu, Ile, Pro, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu; and wherein amino acid 16 is an amino acid other than histidine, for example wherein amino acid 16 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln , Lys, Arg, Asp or Glu. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having less amino acid sequence identity

- 42 046369 at least 90%, at least 95%, at least 98% or at least 99% with the amino acid sequence shown in FIG. 2H, wherein amino acid 42 is Ala, Gly, Val, Leu or Ile; and wherein amino acid 16 is Ala, Gly, Val, Leu or Ile. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the sequence shown in Fig. 2H, wherein amino acid 42 is Ala and amino acid 16 is Ala. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the amino acid the sequence shown in Fig. 2H, wherein amino acid 42 is Ala and amino acid 16 is Gly. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the sequence shown in Fig. 2H, wherein amino acid 42 is Val and amino acid 16 is Ala. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the amino acid the sequence shown in Fig. 2H, wherein amino acid 42 is Leu and amino acid 16 is Ala. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the sequence shown in Fig. 2H, wherein amino acid 42 is Ile and amino acid 16 is Ala. In some cases, a single copy of the variant IL-2 polypeptide is present in the multimeric polypeptide of the present invention. In some cases, the multimeric polypeptide of the present invention contains two copies of a variant IL-2 polypeptide, for example, when the two copies are in tandem without a linker between the two copies or in tandem and separated by a linker peptide. In some cases, the multimeric polypeptide of the present invention contains three copies of a variant IL-2 polypeptide, for example, when the three copies are in tandem without a linker between the three copies or in tandem and separated by a linker peptide. In some cases, when the IL-2/synTac of the present invention contains a class I HPA heavy chain and B2M, the IL-2 polypeptide(s) is/are on a polypeptide chain containing a class I HPA heavy chain. In some cases, when the IL-2 polypeptide(s) The 2/synTac of the present invention contains a class I and B2M HLA heavy chain, the IL-2 polypeptide(s) is/are on a polypeptide chain containing the B2M polypeptide. In some cases, the multimeric polypeptide of the present invention contains 2 copies of a variant IL-2 containing the F42A and H16A substitutions, wherein the multimeric polypeptide contains class I and B2M HLA heavy chain polypeptides, and wherein 2 copies of IL-2 (F42A, H16A) are located on polypeptide chain containing the heavy chain of class I HLA. In some cases, the variant IL-2 polypeptide or synTac containing it has a binding affinity for IL-2R that is from about 100 nM to about 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 to 700 nM, from 700 to 800 nM, from 800 to 900 nM, from 900 nM to 1 µM, from 1 to 5 µM, from 5 to 10 µM, from 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 100 μM. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention is 133 amino acids in length. In some cases, the variant IL-2 polypeptide contains the amino acid sequence depicted in FIG. 34B (containing replacements H16A and F42A).

Replacements F42 and D20.

In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the sequence shown in Fig. 2I, wherein amino acid 42 is an amino acid other than phenylalanine, for example, wherein amino acid 42 is Gly, Ala, Val, Leu, Ile, Pro, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu; and wherein amino acid 20 is an amino acid other than aspartic acid, for example wherein amino acid 20 is Gly, Ala, Val,

- 43 046369

Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His or Glu. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the sequence shown in Fig. 2I, wherein amino acid 42 is Ala, Gly, Val, Leu or Ile; and wherein amino acid 20 is Ala, Gly, Val, Leu or Ile. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the sequence shown in Fig. 2I, wherein amino acid 42 is Ala, Gly, Val, Leu or Ile; and wherein amino acid 20 is Asn, Gln, Lys, Arg or His. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the sequence shown in Fig. 2I, wherein amino acid 42 is Ala and amino acid 20 is Ala. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the amino acid the sequence shown in Fig. 2I, wherein amino acid 42 is Ala and amino acid 20 is Gly. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the sequence shown in Fig. 2I, wherein amino acid 42 is Val and amino acid 20 is Ala. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the amino acid the sequence shown in Fig. 2I, wherein amino acid 42 is Leu and amino acid 20 is Ala. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the sequence shown in Fig. 2I, wherein amino acid 42 is Ile and amino acid 20 is Ala. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the amino acid the sequence shown in Fig. 2I, wherein amino acid 42 is Ala and amino acid 20 is Asn. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the sequence shown in Fig. 2I, wherein amino acid 42 is Ala and amino acid 20 is Gln. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the sequence shown in Fig. 2I, wherein amino acid 42 is Ala and amino acid 20 is Lys. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the sequence shown in Fig. 2I, wherein amino acid 42 is Ala and amino acid 20 is Arg. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the sequence shown in Fig. 2I, wherein amino acid 42 is Ala and amino acid 20 is His. In some cases, a single copy of the variant IL-2 polypeptide is present in the multimeric polypeptide of the present invention. In some cases, the multimeric polypeptide of this invention contains two copies of a variant IL-2 polypeptide

- 44 046369 yes, for example, when two copies are in tandem without a linker between the two copies or in tandem and separated by a linker peptide. In some cases, the multimeric polypeptide of the present invention contains three copies of a variant IL-2 polypeptide, for example, when the three copies are in tandem without a linker between the three copies or in tandem and separated by a linker peptide. In some cases, when the IL-2/synTac of the present invention contains a class I HPA heavy chain and B2M, the IL-2 polypeptide(s) is/are on a polypeptide chain containing a class I HPA heavy chain. In some cases, when the IL-2 polypeptide(s) The 2/synTac of the present invention contains a class I and B2M HLA heavy chain, the IL-2 polypeptide(s) is/are on a polypeptide chain containing the B2M polypeptide. In some cases, the variant IL-2 polypeptide or synTac containing it has a binding affinity for IL-2R that is from about 100 nM to about 150 nM, from about 150 nM to about 200 nM, from about 200 nM to about 250 nM , from about 250 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 to 700 nM, 700 to 800 nM, 800 to 900 nM, 900 nM to 1 µM, 1 to 5 µM, 5 to 10 µM, about 10 µM to about 15 µM, 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 to 100 μM. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention is 133 amino acids in length.

Replacements F42, D20 and E15.

In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the sequence shown in Fig. 2J, wherein amino acid 42 is an amino acid other than phenylalanine, for example wherein amino acid 42 is Gly, Ala, Val, Leu, Ile, Pro, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu; wherein amino acid 20 is an amino acid other than aspartic acid, for example wherein amino acid 20 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln , Lys, Arg, His or Glu; and wherein amino acid 15 is an amino acid other than glutamic acid, for example wherein amino acid 15 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His or Asp. In some cases, the variant IL-2 polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to that depicted in FIG. 2J, wherein amino acid 42 is Ala, Gly, Val, Leu or Ile; wherein amino acid 20 is Ala, Gly, Val, Leu or Ile; and wherein amino acid 15 is Ala, Gly, Val, Leu or Ile. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the sequence shown in Fig. 2J, wherein amino acid 42 is Ala, Gly, Val, Leu or Ile; wherein amino acid 20 is Asn, Gln, Lys, Arg or His; and wherein amino acid 15 is Ala, Gly, Val, Leu or Ile. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the sequence shown in Fig. 2J, wherein amino acid 42 is Ala, amino acid 20 is Ala, and amino acid 15 is Ala. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the amino acid the sequence shown in Fig. 2J, wherein amino acid 42 is Ala, amino acid 20 is Gly, and amino acid 15 is Gly. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the sequence shown in Fig. 2J, wherein amino acid 42 is Val, amino acid 20 is Ala, and amino acid 15 is Gly. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the sequence shown in Fig. 2J, wherein amino acid 42 is Leu, amino acid 20 is Ala, and amino acid 15 is Gly.

- 45 046369

In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the sequence shown in Fig. 2J, wherein amino acid 42 is Ile, amino acid 20 is Ala, and amino acid 15 is Ala. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the amino acid the sequence shown in Fig. 2J, wherein amino acid 42 is Ala, amino acid 20 is Asn, and amino acid 15 is Ala. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the amino acid the sequence shown in Fig. 2I, wherein amino acid 42 is Ala, amino acid 20 is Gln, and amino acid 15 is Ala. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the amino acid the sequence shown in Fig. 2J, wherein amino acid 42 is Ala, amino acid 20 is Lys, and amino acid 15 is Ala. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the amino acid the sequence shown in Fig. 2J, wherein amino acid 42 is Ala, amino acid 20 is Arg, and amino acid 15 is Ala. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the amino acid the sequence shown in Fig. 2I, wherein amino acid 42 is Ala, amino acid 20 is His, and amino acid 15 is Ala. In some cases, a single copy of the variant IL-2 polypeptide is present in the multimeric polypeptide of the present invention. In some cases, the multimeric polypeptide of the present invention contains two copies of a variant IL-2 polypeptide, for example, when the two copies are in tandem without a linker between the two copies or in tandem and separated by a linker peptide. In some cases, when the IL-2/synTac of the present invention contains a class I HLA heavy chain and B2M, the IL-2 polypeptide(s) is on a polypeptide chain containing a class I HLA heavy chain. In some cases, when the IL-2/synTac The synTac of the present invention contains a class I HLA heavy chain and a B2M IL-2 polypeptide(s) located on a polypeptide chain containing the B2M polypeptide. In some cases, the multimeric polypeptide of the present invention contains three copies of a variant IL-2 polypeptide, for example, when the three copies are in tandem without a linker between the three copies or in tandem and separated by a linker peptide. In some cases, the variant IL-2 polypeptide or synTac containing it has a binding affinity for IL-2R that is from about 100 nM to about 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 to 700 nM, 700 to 800 nM, 800 to 900 nM, 900 nM to 1 µM, 1 to 5 µM, 5 to 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 to 100 µM. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention is 133 amino acids in length.

Replacements F42, D20 and H16.

In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the sequence shown in Fig. 2K, wherein amino acid 42 is an amino acid other than phenylalanine, for example, wherein amino acid 42 is Gly, Ala, Val, Leu, Ile, Pro, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu; wherein amino acid 20 is an amino acid other than aspartic acid, for example wherein amino acid 20 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln , Lys, Arg, His or Glu; and at the same time amino acid 16

- 46 046369 is an amino acid other than histidine, for example, amino acid 16 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys , Arg, Asp or Glu. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the sequence shown in Fig. 2K, wherein amino acid 42 is Ala, Gly, Val, Leu or Ile; wherein amino acid 20 is Ala, Gly, Val, Leu or Ile; and wherein amino acid 16 is Ala, Gly, Val, Leu or Ile. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the sequence shown in Fig. 2K, wherein amino acid 42 is Ala, Gly, Val, Leu or Ile; wherein amino acid 20 is Asn, Gln, Lys, Arg or His; and wherein amino acid 16 is Ala, Gly, Val, Leu or Ile. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the sequence shown in Fig. 2K, wherein amino acid 42 is Ala, amino acid 20 is Ala, and amino acid 16 is Ala. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the amino acid the sequence shown in Fig. 2K, wherein amino acid 42 is Ala, amino acid 20 is Gly, and amino acid 16 is Gly. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the sequence shown in Fig. 2K, wherein amino acid 42 is Val, amino acid 20 is Ala, and amino acid 16 is Gly. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the sequence shown in Fig. 2K, wherein amino acid 42 is Leu, amino acid 20 is Ala, and amino acid 16 is Gly. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the sequence shown in Fig. 2K, wherein amino acid 42 is Ile, amino acid 20 is Ala, and amino acid 16 is Ala. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the amino acid the sequence shown in Fig. 2K, wherein amino acid 42 is Ala, amino acid 20 is Asn, and amino acid 16 is Ala. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the amino acid the sequence shown in Fig. 2K, wherein amino acid 42 is Ala, amino acid 20 is Gln, and amino acid 16 is Ala. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the amino acid the sequence shown in Fig. 2K, wherein amino acid 42 is Ala, amino acid 20 is Lys, and amino acid 16 is Ala. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the amino acid the sequence shown in Fig. 2K, wherein amino acid 42 is Ala, amino acid 20 is Arg, and amino acid 16 is Ala. In some cases, a variant IL-2 polypeptide present in the multimer

- 47 046369 novel polypeptide of this invention, contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the amino acid sequence depicted in FIG. 2K, wherein amino acid 42 is Ala, amino acid 20 is His, and amino acid 16 is Ala. In some cases, a single copy of the variant IL-2 polypeptide is present in the multimeric polypeptide of the present invention. In some cases, the multimeric polypeptide of the present invention contains two copies of a variant IL-2 polypeptide, for example, when the two copies are in tandem without a linker between the two copies or in tandem and separated by a linker peptide. In some cases, the multimeric polypeptide of the present invention contains three copies of a variant IL-2 polypeptide, for example, when the three copies are in tandem without a linker between the three copies or in tandem and separated by a linker peptide. In some cases, when the IL-2/synTac of the present invention contains a class I HPA heavy chain and B2M, the IL-2 polypeptide(s) is/are on a polypeptide chain containing a class I HPA heavy chain. In some cases, when the IL-2 polypeptide(s) The 2/synTac of the present invention contains a class I and B2M HLA heavy chain, the IL-2 polypeptide(s) is/are on a polypeptide chain containing the B2M polypeptide. In some cases, the variant IL-2 polypeptide or synTac containing it has a binding affinity for IL-2R that is from about 100 nM to about 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 to 700 nM, 700 to 800 nM, 800 to 900 nM, 900 nM to 1 µM, 1 to 5 µM, 5 to 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 to 100 µM. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention is 133 amino acids in length.

Replacements F42, D20 and Q126.

In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the sequence shown in Fig. 2L, wherein amino acid 42 is an amino acid other than phenylalanine, for example, wherein amino acid 42 is Gly, Ala, Val, Leu, Ile, Pro, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu; wherein amino acid 20 is an amino acid other than aspartic acid, for example wherein amino acid 20 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln Lys, Arg, His or Glu; and wherein amino acid 126 is an amino acid other than glutamine, for example wherein amino acid 126 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Lys , Arg, His, Asp or Glu. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the sequence shown in Fig. 2L, wherein amino acid 42 is Ala, Gly, Val, Leu or Ile; wherein amino acid 20 is Ala, Gly, Val, Leu or Ile; and wherein amino acid 126 is Ala, Gly, Val, Leu or Ile. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the sequence shown in Fig. 2L, wherein amino acid 42 is Ala, Gly, Val, Leu or Ile; wherein amino acid 20 is Asn, Gln, Lys, Arg or His; and wherein amino acid 126 is Ala, Gly, Val, Leu or Ile. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the sequence shown in Fig. 2L, wherein amino acid 42 is Ala, amino acid 20 is Ala, and amino acid 126 is Ala. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the amino acid the sequence shown in Fig. 2L, wherein amino acid 42 is Ala, amino acid 20 is Gly, and amino acid 126 is Gly. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of this invention contains an amino acid sequence having at least 90% amino acid sequence identity, at least

- 48 046369 at least 95%, at least 98% or at least 99% with the amino acid sequence shown in FIG. 2L, wherein amino acid 42 is Val, amino acid 20 is Ala, and amino acid 126 is Gly. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the sequence shown in Fig. 2L, wherein amino acid 42 is Leu, amino acid 20 is Ala, and amino acid 126 is Gly. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the sequence shown in Fig. 2L, wherein amino acid 42 is Ile, amino acid 20 is Ala, and amino acid 126 is Ala. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the amino acid the sequence shown in Fig. 2L, wherein amino acid 42 is Ala, amino acid 20 is Asn, and amino acid 126 is Ala. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the amino acid the sequence shown in Fig. 2L, wherein amino acid 42 is Ala, amino acid 20 is Gln, and amino acid 126 is Ala. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the amino acid the sequence shown in Fig. 2L, wherein amino acid 42 is Ala, amino acid 20 is Lys, and amino acid 126 is Ala. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the amino acid the sequence shown in Fig. 2L, wherein amino acid 42 is Ala, amino acid 20 is Arg, and amino acid 126 is Ala. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the amino acid the sequence shown in Fig. 2L, wherein amino acid 42 is Ala, amino acid 20 is His, and amino acid 126 is Ala. In some cases, a single copy of the variant IL-2 polypeptide is present in the multimeric polypeptide of the present invention. In some cases, the multimeric polypeptide of the present invention contains two copies of a variant IL-2 polypeptide, for example, when the two copies are in tandem without a linker between the two copies or in tandem and separated by a linker peptide. In some cases, the multimeric polypeptide of the present invention contains three copies of a variant IL-2 polypeptide, for example, when the three copies are in tandem without a linker between the three copies or in tandem and separated by a linker peptide. In some cases, when the IL-2/synTac of the present invention contains a class I HPA heavy chain and B2M, the IL-2 polypeptide(s) is/are on a polypeptide chain containing a class I HPA heavy chain. In some cases, when the IL-2 polypeptide(s) The 2/synTac of the present invention contains a class I and B2M HLA heavy chain, the IL-2 polypeptide(s) is/are on a polypeptide chain containing the B2M polypeptide. In some cases, the variant IL-2 polypeptide or synTac containing it has a binding affinity for IL-2R that is from about 100 nM to about 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 to 700 nM, 700 to 800 nM, 800 to 900 nM, 900 nM to 1 µM, 1 to 5 µM, 5 to 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 to 100 µM. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention is 133 amino acids in length.

Replacements F42, D20 and Y45.

In some cases, a variant IL-2 polypeptide present in a multimeric polypeptide

- 49 046369 de according to this invention, contains an amino acid sequence having at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity with the amino acid sequence shown in FIG. 2M, wherein amino acid 42 is an amino acid other than phenylalanine, for example, wherein amino acid 42 is Gly, Ala, Val, Leu, Ile, Pro, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu; wherein amino acid 20 is an amino acid other than aspartic acid, for example wherein amino acid 20 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln , Lys, Arg, His or Glu; and wherein amino acid 45 is an amino acid other than tyrosine, for example wherein amino acid 45 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys , Arg, His, Asp or Glu. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the sequence shown in Fig. 2M, wherein amino acid 42 is Ala, Gly, Val, Leu or Ile; wherein amino acid 20 is Ala, Gly, Val, Leu or Ile; and wherein amino acid 45 is Ala, Gly, Val, Leu or Ile. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the sequence shown in Fig. 2M, wherein amino acid 42 is Ala, Gly, Val, Leu or Ile; wherein amino acid 20 is Asn, Gln, Lys, Arg or His; and wherein amino acid 45 is Ala, Gly, Val, Leu or Ile. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the sequence shown in Fig. 2M, wherein amino acid 42 is Ala, amino acid 20 is Ala, and amino acid 45 is Ala. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the amino acid the sequence shown in Fig. 2M, wherein amino acid 42 is Ala, amino acid 20 is Gly, and amino acid 45 is Gly. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the sequence shown in Fig. 2M, wherein amino acid 42 is Val, amino acid 20 is Ala, and amino acid 45 is Gly. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the sequence shown in Fig. 2M, wherein amino acid 42 is Leu, amino acid 20 is Ala, and amino acid 45 is Gly. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the sequence shown in Fig. 2M, wherein amino acid 42 is Ile, amino acid 20 is Ala, and amino acid 45 is Ala. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the amino acid the sequence shown in Fig. 2M, wherein amino acid 42 is Ala, amino acid 20 is Asn, and amino acid 45 is Ala. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the amino acid the sequence shown in Fig. 2M, wherein amino acid 42 is Ala, amino acid 20 is Gln, and amino acid 45 is Ala. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with

- 50 046369 amino acid sequence shown in FIG. 2M, wherein amino acid 42 is Ala, amino acid 20 is Lys, and amino acid 45 is Ala. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the amino acid the sequence shown in Fig. 2M, wherein amino acid 42 is Ala, amino acid 20 is Arg, and amino acid 45 is Ala. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the amino acid the sequence shown in Fig. 2M, wherein amino acid 42 is Ala, amino acid 20 is His, and amino acid 45 is Ala. In some cases, a single copy of a variant IL-2 polypeptide is present in the multimeric polypeptide of the invention. In some cases, the multimeric polypeptide of the present invention contains two copies of a variant IL-2 polypeptide, for example, when the two copies are in tandem without a linker between the two copies or in tandem and separated by a linker peptide. In some cases, the multimeric polypeptide of the present invention contains three copies of a variant IL-2 polypeptide, for example, when the three copies are in tandem without a linker between the three copies or in tandem and separated by a linker peptide. In some cases, when the IL-2/synTac of the present invention contains a class I HPA heavy chain and B2M, the IL-2 polypeptide(s) is/are on a polypeptide chain containing a class I HPA heavy chain. In some cases, when the IL-2 polypeptide(s) The 2/synTac of the present invention contains a class I and B2M HLA heavy chain, the IL-2 polypeptide(s) is/are on a polypeptide chain containing the B2M polypeptide. In some cases, the variant IL-2 polypeptide or synTac containing it has a binding affinity for IL-2R that is from about 100 nM to about 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 to 700 nM, 700 to 800 nM, 800 to 900 nM, 900 nM to 1 µM, 1 to 5 µM, 5 to 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 to 100 µM. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention is 133 amino acids in length.

Replacements F42, D20, Y45 and H16.

In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the sequence shown in Fig. 2N, wherein amino acid 42 is an amino acid other than phenylalanine, for example wherein amino acid 42 is Gly, Ala, Val, Leu, Ile, Pro, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu; wherein amino acid 20 is an amino acid other than aspartic acid, for example wherein amino acid 20 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln , Lys, Arg, His or Glu; wherein amino acid 45 is an amino acid other than tyrosine, for example wherein amino acid 45 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu; and wherein amino acid 16 is an amino acid other than histidine, for example wherein amino acid 16 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln , Lys, Arg, Asp or Glu. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the sequence shown in Fig. 2N, wherein amino acid 42 is Ala, Gly, Val, Leu or Ile; wherein amino acid 20 is Ala, Gly, Val, Leu or Ile; wherein amino acid 45 is Ala, Gly, Val, Leu or Ile; and wherein amino acid 16 is Ala, Gly, Val, Leu or Ile. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the sequence shown in Fig. 2N, wherein amino acid 42 is Ala, Gly, Val, Leu or Ile; wherein amino acid 20 is Asn, Gln, Lys, Arg or His; wherein amino acid 45 is Ala, Gly, Val, Leu or Ile; and wherein amino acid 16 is Ala, Gly, Val, Leu or Ile. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains

- 51 046369 an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the amino acid sequence shown in FIG. 2N, wherein amino acid 42 is Ala, amino acid 20 is Ala, amino acid 45 is Ala and amino acid 16 is Ala. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity. relative to the amino acid sequence shown in FIG. 2N, wherein amino acid 42 is Ala, amino acid 20 is Gly, amino acid 45 is Gly and amino acid 16 is Ala. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity. relative to the amino acid sequence shown in FIG. 2N, wherein amino acid 42 is Val, amino acid 20 is Ala, amino acid 45 is Gly and amino acid 16 is Ala. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity. relative to the amino acid sequence shown in FIG. 2N, wherein amino acid 42 is Leu, amino acid 20 is Ala, amino acid 45 is Gly and amino acid 16 is Val. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the sequence shown in Fig. 2N, wherein amino acid 42 is Ile, amino acid 20 is Ala, amino acid 45 is Ala and amino acid 16 is Gly. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the sequence shown in Fig. 2N, wherein amino acid 42 is Ala, amino acid 20 is Asn, amino acid 45 is Ala and amino acid 16 is Ala. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity. relative to the amino acid sequence shown in FIG. 2N, wherein amino acid 42 is Ala, amino acid 20 is Gln, amino acid 45 is Ala and amino acid 16 is Ala. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity. relative to the amino acid sequence shown in FIG. 2N, wherein amino acid 42 is Ala, amino acid 20 is Lys, amino acid 45 is Ala and amino acid 16 is Ala. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity. relative to the amino acid sequence shown in FIG. 2N, wherein amino acid 42 is Ala, amino acid 20 is Arg, amino acid 45 is Ala and amino acid 16 is Ala. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity. relative to the amino acid sequence shown in FIG. 2N, wherein amino acid 42 is Ala, amino acid 20 is His, amino acid 45 is Ala and amino acid 16 is Ala. In some cases, one copy of the variant IL-2 polypeptide is present in the multimeric polypeptide of the present invention. In some cases, the multimeric polypeptide of the present invention contains two copies of a variant IL-2 polypeptide, for example, when the two copies are in tandem without a linker between the two copies or in tandem and separated by a linker peptide. In some cases, the multimeric polypeptide of the present invention contains three copies of the variant IL-2 polypeptide, for example, when the three copies are in tandem without a linker between the three copies or in tandem and separated by a linker

- 52 046369 peptide. In some cases, when the IL-2/synTac of the present invention contains a class I HPA heavy chain and B2M, the IL-2 polypeptide(s) is/are on a polypeptide chain containing a class I HPA heavy chain. In some cases, when the IL-2 polypeptide(s) The 2/synTac of the present invention contains a class I and B2M HLA heavy chain, the IL-2 polypeptide(s) is/are on a polypeptide chain containing the B2M polypeptide. In some cases, the variant IL-2 polypeptide or synTac containing it has a binding affinity for IL-2R that is from about 100 nM to about 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 to 700 nM, 700 to 800 nM, 800 to 900 nM, 900 nM to 1 µM, 1 to 5 µM, 5 to 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 to 100 µM. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention is 133 amino acids in length.

Replacements F42, D20, Y45 and Q126.

In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the sequence shown in Fig. 2O, wherein amino acid 42 is an amino acid other than phenylalanine, for example, wherein amino acid 42 is Gly, Ala, Val, Leu, Ile, Pro, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu; wherein amino acid 20 is an amino acid other than aspartic acid, for example wherein amino acid 20 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln , Lys, Arg, His or Glu; wherein amino acid 45 is an amino acid other than tyrosine, for example wherein amino acid 45 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu; and wherein amino acid 126 is an amino acid other than glutamine, for example wherein amino acid 126 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Lys , Arg, His, Asp or Glu. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the sequence shown in Fig. 2O, wherein amino acid 42 is Ala, Gly, Val, Leu or Ile; wherein amino acid 20 is Ala, Gly, Val, Leu or Ile; wherein amino acid 45 is Ala, Gly, Val, Leu or Ile; and wherein amino acid 126 is Ala, Gly, Val, Leu or Ile. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the sequence shown in Fig. 2O, wherein amino acid 42 is Ala, Gly, Val, Leu or Ile; wherein amino acid 20 is Asn, Gln, Lys, Arg or His; wherein amino acid 45 is Ala, Gly, Val, Leu or Ile; and wherein amino acid 126 is Ala, Gly, Val, Leu or Ile. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the sequence shown in Fig. 2O, wherein amino acid 42 is Ala, amino acid 20 is Ala, amino acid 45 is Ala and amino acid 126 is Ala. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity. relative to the amino acid sequence shown in FIG. 2O, wherein amino acid 42 is Ala, amino acid 20 is Gly, amino acid 45 is Gly and amino acid 126 is Ala. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity. relative to the amino acid sequence shown in FIG. 2O, wherein amino acid 42 is Val, amino acid 20 is Ala, amino acid 45 is Gly and amino acid 126 is Ala. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having an amino acid sequence identity of at least 90%, at least 95%, at least 98%, or at least

- 53 046369 at least 99% with respect to the amino acid sequence shown in FIG. 2O, wherein amino acid 42 is Leu, amino acid 20 is Ala, amino acid 45 is Gly and amino acid 126 is Val. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the sequence shown in Fig. 2O, wherein amino acid 42 is Ile, amino acid 20 is Ala, amino acid 45 is Ala and amino acid 126 is Gly. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the sequence shown in Fig. 2O, wherein amino acid 42 is Ala, amino acid 20 is Asn, amino acid 45 is Ala and amino acid 126 is Ala. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity. relative to the amino acid sequence shown in FIG. 2O, wherein amino acid 42 is Ala, amino acid 20 is Gln, amino acid 45 is Ala and amino acid 126 is Ala. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity. relative to the amino acid sequence shown in FIG. 2O, wherein amino acid 42 is Ala, amino acid 20 is Lys, amino acid 45 is Ala and amino acid 126 is Ala. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity. relative to the amino acid sequence shown in FIG. 2O, wherein amino acid 42 is Ala, amino acid 20 is Arg, amino acid 45 is Ala and amino acid 126 is Ala. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity. relative to the amino acid sequence shown in FIG. 2O, wherein amino acid 42 is Ala, amino acid 20 is His, amino acid 45 is Ala and amino acid 126 is Ala. In some cases, one copy of the variant IL-2 polypeptide is present in the multimeric polypeptide of the present invention. In some cases, the multimeric polypeptide of the present invention contains two copies of a variant IL-2 polypeptide, for example, when the two copies are in tandem without a linker between the two copies or in tandem and separated by a linker peptide. In some cases, the multimeric polypeptide of the present invention contains three copies of a variant IL-2 polypeptide, for example, when the three copies are in tandem without a linker between the three copies or in tandem and separated by a linker peptide. In some cases, when the IL-2/synTac of the present invention contains a class I HPA heavy chain and B2M, the IL-2 polypeptide(s) is/are on a polypeptide chain containing a class I HPA heavy chain. In some cases, when the IL-2 polypeptide(s) The 2/synTac of the present invention contains a class I and B2M HLA heavy chain, the IL-2 polypeptide(s) is/are on a polypeptide chain containing the B2M polypeptide. In some cases, the variant IL-2 polypeptide or synTac containing it has a binding affinity for IL-2R that is from about 100 nM to about 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 to 700 nM, 700 to 800 nM, 800 to 900 nM, 900 nM to 1 µM, 1 to 5 µM, 5 to 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 to 100 µM. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention is 133 amino acids in length.

Replacements F42, D20, Y45, H16 and Q126.

In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90% amino acid sequence identity, at least 95%, at least

- 54 046369

98% or at least 99% with the amino acid sequence shown in FIG. 2P, wherein amino acid 42 is an amino acid other than phenylalanine, for example wherein amino acid 42 is Gly, Ala, Val, Leu, Ile, Pro, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu; wherein amino acid 20 is an amino acid other than aspartic acid, for example wherein amino acid 20 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln , Lys, Arg, His or Glu; wherein amino acid 45 is an amino acid other than tyrosine, for example wherein amino acid 45 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu; wherein amino acid 126 is an amino acid other than glutamine, for example wherein amino acid 126 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Lys, Arg, His, Asp or Glu; and wherein amino acid 16 is an amino acid other than histidine, for example wherein amino acid 16 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln , Lys, Arg, Asp or Glu. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the sequence shown in Fig. 2P, wherein amino acid 42 is Ala, Gly, Val, Leu or Ile; wherein amino acid 20 is Ala, Gly, Val, Leu or Ile; wherein amino acid 45 is Ala, Gly, Val, Leu or Ile; wherein amino acid 126 is Ala, Gly, Val, Leu or Ile; and wherein amino acid 16 is Ala, Gly, Val, Leu or Ile. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the sequence shown in Fig. 2P, wherein amino acid 42 is Ala, Gly, Val, Leu or Ile; wherein amino acid 20 is Asn, Gln, Lys, Arg or His; wherein amino acid 45 is Ala, Gly, Val, Leu or Ile; wherein amino acid 126 is Ala, Gly, Val, Leu or Ile; and wherein amino acid 16 is Ala, Gly, Val, Leu or Ile. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the sequence shown in Fig. 2P, wherein amino acid 42 is Ala, amino acid 20 is Ala, amino acid 45 is Ala, amino acid 126 is Ala, and amino acid 16 is Ala. In some cases, a variant IL-2 polypeptide is present in a multimeric polypeptide, the invention includes an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to relative to the amino acid sequence shown in FIG. 2P, wherein amino acid 42 is Ala, amino acid 20 is Gly, amino acid 45 is Gly, amino acid 126 is Ala and amino acid 16 is Ala. In some cases, a variant IL-2 polypeptide is present in a multimeric polypeptide, the invention includes an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to relative to the amino acid sequence shown in FIG. 2P, wherein amino acid 42 is Val, amino acid 20 is Ala, amino acid 45 is Gly, amino acid 126 is Ala and amino acid 16 is Ala. In some cases, a variant IL-2 polypeptide is present in a multimeric polypeptide, the invention includes an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to relative to the amino acid sequence shown in FIG. 2P, wherein amino acid 42 is Leu, amino acid 20 is Ala, amino acid 45 is Gly, amino acid 126 is Val and amino acid 16 is Ala. In some cases, a variant IL-2 polypeptide is present in a multimeric polypeptide. The present invention includes an amino acid sequence having at least 99% amino acid sequence identity with the amino acid sequence shown in FIG. 2P, wherein amino acid 42 is Ile, amino acid 20 is Ala, amino acid 45 is Ala, amino acid 126 is Gly and amino acid 16 is Ala. In some cases, a variant IL-2 polypeptide is present in a multimeric polypeptide, the invention includes an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to relative to the amino acid sequence shown in FIG. 2P, wherein amino acid 42 is Ala, amino acid

- 55 046369 is Asn, amino acid 45 is Ala, amino acid 126 is Ala and amino acid 16 is Ala. In some cases, a variant IL-2 polypeptide is present in a multimeric polypeptide, the invention includes an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to relative to the amino acid sequence shown in FIG. 2P, wherein amino acid 42 is Ala, amino acid 20 is Gln, amino acid 45 is Ala, amino acid 126 is Ala and amino acid 16 is Ala. In some cases, a variant IL-2 polypeptide is present in a multimeric polypeptide; the present invention includes an amino acid sequence having amino acid sequence identity with the amino acid sequence shown in FIG. 2P, wherein amino acid 42 is Ala, amino acid 20 is Lys, amino acid 45 is Ala, amino acid 126 is Ala and amino acid 16 is Ala. In some cases, a variant IL-2 polypeptide is present in a multimeric polypeptide; the present invention includes an amino acid sequence having amino acid sequence identity with the amino acid sequence shown in FIG. 2P, wherein amino acid 42 is Ala, amino acid 20 is Arg, amino acid 45 is Ala, amino acid 126 is Ala and amino acid 16 is Ala. In some cases, a variant IL-2 polypeptide is present in a multimeric polypeptide, the invention includes an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to relative to the amino acid sequence shown in FIG. 2P, wherein amino acid 42 is Ala, amino acid 20 is His, amino acid 45 is Ala, amino acid 126 is Ala and amino acid 16 is Ala. In some cases, one copy of the variant IL-2 polypeptide is present in the multimeric polypeptide of the present invention. In some cases, the multimeric polypeptide of the present invention contains two copies of a variant IL-2 polypeptide, for example, when the two copies are in tandem without a linker between the two copies or in tandem and separated by a linker peptide. In some cases, the multimeric polypeptide of the present invention contains three copies of a variant IL-2 polypeptide, for example, when the three copies are in tandem without a linker between the three copies or in tandem and separated by a linker peptide. In some cases, when the IL-2/synTac of the present invention contains a class I HPA heavy chain and B2M, the IL-2 polypeptide(s) is/are on a polypeptide chain containing a class I HPA heavy chain. In some cases, when the IL-2 polypeptide(s) The 2/synTac of the present invention contains a class I and B2M HLA heavy chain, the IL-2 polypeptide(s) is/are on a polypeptide chain containing the B2M polypeptide. In some cases, the variant IL-2 polypeptide or synTac containing it has a binding affinity for IL-2R that is from about 100 nM to about 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 to 700 nM, 700 to 800 nM, 800 to 900 nM, 900 nM to 1 µM, 1 to 5 µM, 5 to 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 to 100 µM. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention is 133 amino acids in length.

Replacements F42, Q126 and H16.

In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the sequence shown in FIG. 2Q, wherein amino acid 42 is an amino acid other than phenylalanine, for example, wherein amino acid 42 is Gly, Ala, Val, Leu, Ile, Pro, Tyr, Tip, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, His, Asp or Glu; wherein amino acid 126 is an amino acid other than glutamine, for example wherein amino acid 126 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Lys, Arg, His, Asp or Glu; and wherein amino acid 16 is an amino acid other than histidine, for example wherein amino acid 16 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln , Lys, Arg, Asp or Glu. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the sequence shown in Fig. 2Q, wherein amino acid 42 is Ala, Gly, Val, Leu or Ile; wherein amino acid 126 represents Ala, Gly,

- 56 046369

Val, Leu or Ile; and wherein amino acid 16 is Ala, Gly, Val, Leu or Ile. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the sequence shown in Fig. 2Q, wherein amino acid 42 is Ala, Gly, Val, Leu or Ile; wherein amino acid 126 is Asn, Gln, Lys, Arg or His; and wherein amino acid 16 is Ala, Gly, Val, Leu or Ile. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the sequence shown in Fig. 2Q, wherein amino acid 42 is Ala, amino acid 126 is Ala, and amino acid 16 is Ala. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the amino acid the sequence shown in Fig. 2Q, wherein amino acid 42 is Ala, amino acid 126 is Gly, and amino acid 16 is Gly. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the sequence shown in Fig. 2Q, wherein amino acid 42 is Val, amino acid 126 is Ala, and amino acid 16 is Gly. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the sequence shown in Fig. 2Q, wherein amino acid 42 is Leu, amino acid 126 is Ala, and amino acid 16 is Gly. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the sequence shown in Fig. 2Q, wherein amino acid 42 is Ile, amino acid 126 is Ala, and amino acid 16 is Ala. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the amino acid the sequence shown in Fig. 2Q, wherein amino acid 42 is Ala, amino acid 126 is Asn, and amino acid 16 is Ala. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the amino acid the sequence shown in Fig. 2Q, wherein amino acid 42 is Ala, amino acid 126 is Ala, and amino acid 16 is Ala. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the amino acid the sequence shown in Fig. 2Q, wherein amino acid 42 is Ala, amino acid 126 is Lys, and amino acid 16 is Ala. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the amino acid the sequence shown in Fig. 2Q, wherein amino acid 42 is Ala, amino acid 126 is Arg, and amino acid 16 is Ala. In some cases, the variant IL-2 polypeptide present in the multimeric polypeptide of the present invention contains an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with the amino acid the sequence shown in Fig. 2Q, wherein amino acid 42 is Ala, amino acid 126 is His, and amino acid 16 is Ala. In some cases, a single copy of a variant IL-2 polypeptide is present in the multimeric polypeptide of the invention. In some cases, the multimeric polypeptide of this invention contains two copies of a variant IL-2 polypeptide, for example,

- 57 046369 when two copies are in tandem without a linker between the two copies or in tandem and separated by a linker peptide. In some cases, the multimeric polypeptide of the present invention contains three copies of a variant IL-2 polypeptide, for example, when the three copies are in tandem without a linker between the three copies or in tandem and separated by a linker peptide. In some cases, when the IL-2/synTac of the present invention contains a class I HPA heavy chain and B2M, the IL-2 polypeptide(s) is/are on a polypeptide chain containing a class I HPA heavy chain. In some cases, when the IL-2 polypeptide(s) The 2/synTac of the present invention contains a class I and B2M HLA heavy chain, the IL-2 polypeptide(s) is/are on a polypeptide chain containing the B2M polypeptide. In some cases, the variant IL-2 polypeptide or synTac comprising the variant IL-2 polypeptide has a binding affinity for IL-2R that is from about 100 nM to about 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, from about 1 μM to about 5 μM, from 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 7 5 µM to about 100 µM. In some cases, the variant IL-2 polypeptide is 133 amino acids long.

Multiple immunomodulatory domains.

As noted above, in some cases, the multimeric polypeptide of the present invention comprises two or more immunomodulatory polypeptides, wherein at least one of the two or more immunomodulatory polypeptides is a variant IL-2 polypeptide of the present invention.

In some cases, the multimeric polypeptide of the present invention contains two or more copies of a variant IL-2 polypeptide of the present invention. In some cases, two or more variant IL-2 polypeptides are on the same polypeptide chain of a multimeric polypeptide of the present invention. In some cases, two or more variant IL-2 polypeptides are on separate polypeptide chains of the multimeric polypeptide of the present invention.

In some cases, the multimeric polypeptide of the present invention comprises a first immunomodulatory polypeptide and at least a second immunomodulatory polypeptide, wherein the first immunomodulatory polypeptide is a variant IL-2 polypeptide of the present invention and the second immunomodulatory polypeptide is not an IL-2 polypeptide. For example, in some cases, the second immunomodulatory polypeptide is a member of the tumor necrosis factor (TNF) superfamily; for example, FasL polypeptide, 4-1BBL polypeptide, CD40 polypeptide, OX40L polypeptide, CD30L polypeptide, CD70 polypeptide, etc. In some cases, the second immunomodulatory polypeptide of the multimeric polypeptide of this invention is a T cell costimulatory polypeptide and is a member of the immunoglobulin (Ig) superfamily; for example, CD7 polypeptide, CD86 polypeptide, ICAM polypeptide, etc. In some cases, the second immunomodulatory polypeptide is 4-1BBL, OX40L, ICOS-L, ICAM, PD-L1, CD86, FasL and PD-L2. Suitable multimeric polypeptide immunomodulatory polypeptides of the present invention include, for example, CD7, CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, HVEM, lymphotoxin beta receptor, 3/TR6, ILT3, ILT4 or HVEM. In some cases, the second immunomodulatory polypeptide is a variant (eg, a variant of naturally occurring 4-1BBL) that exhibits an affinity (determined as described above) for its partner costimulatory protein found in the T cell that is reduced relative to the affinity of the naturally occurring costimulatory protein (immunomodulatory polypeptide) to its partner (cognate) costimulatory protein. In some cases, the multimeric polypeptide of the present invention comprises a first immunomodulatory polypeptide and at least a second immunomodulatory polypeptide, neither of which is a variant IL-2 polypeptide. It should be understood that this invention generally relates to the use of immunomodulatory polypeptides that are variants of naturally occurring immunomodulatory polypeptides, which variants exhibit an affinity (defined as described above) for co-stimulatory protein analogues that is reduced compared to the affinity of naturally occurring costimulatory protein (immunomodulatory polypeptide) for partner (cognate) costimulatory protein.

Additional T cell modulatory domains (MODs) that may be included in the multimeric polypeptide of the present invention include naturally occurring or synthetic human gene products (protein), affinity reagents (e.g., antibody, antibody fragment, single chain Fv, aptamers, nanobodies) targeting the human gene product, including, but not limited to, all secreted proteins resulting from classical and non-classical (e.g. FGF2, IL1, S100A4) secretion mechanisms, and the ecto-domains of all cell surface proteins, anchored by natural genetically encoded protein segments

- 58 046369 (single or multiple membrane segments) or post-translational modifications such as GPI links). Any natural or synthetic affinity reagent (eg, antibody, antibody fragment, single chain Fv, aptamer, nanobody, lectin, etc.) targeting a cell surface glycan or other post-translational modification (eg, sulfation). Examples include, but are not limited to, members of the TNF/TNF family (OX40L, ICOSL, FASL, LTA, LTB TRAIL, CD153, TNF8P9, RANKL, TWEAK, TNF13, TNF13b, TNFSF14, TNFSF15, TNFSF18, CD40LG, CD70) or affinity reagents , aimed at members of the TNF/TNOR family; members of the immunoglobulin superfamily (VISTA, PD1, PD-L1, PD-L2, B71, B72, CTLA4, CD28, TIM3, CD4, CD8, CD19), T-cell receptor chains, ICOS, ICOS ligand, NPLA2, butyrophilins, BTLA, B7-H3, B7-H4, CD3, CD79a, CD79b, IgSF CAMS (including CD2, CD58, CD48, CD150, CD229, CD244, ICAM-1), leukocyte immunoglobulin-like receptors (LILR - Leukocyte immunoglobulin like receptor), receptors like killer cell immunoglobulin like receptor (KIR) members of the lectin superfamily, selectins, cytokines/chemokines and cytokine/chemokine receptors, growth factors and growth factor receptors), adhesion molecules (integrins, fibronectins, cadherins) or multispan integral ectodomains membrane protein or affinity reagents targeting the immunoglobulin superfamily and listed gene products. In addition, active homologs/orthologues of these gene products, including, but not limited to, viral sequences (e.g., CMV, EBV), bacterial sequences, fungal sequences, eukaryotic pathogens (e.g., Schistosoma, Plasmodium, Babesia, Eimeria, Theileria, Toxoplasma ), Entamoeba, Leishmania and Trypanosoma) and mammalian-derived coding regions. Besides. The MOD may contain a small molecule drug targeting a human gene product.

Framework polypeptides.

The multimeric T cell modulating polypeptide of the present invention comprises an Fc polypeptide or other suitable framework polypeptide.

Suitable framework polypeptides include antibody-based framework polypeptides and non-antibody-based frameworks. Non-antibody based scaffolds include, for example, albumin, XTEN (extended recombinant) polypeptide, transferrin, Fc receptor polypeptide, elastin-like polypeptide (see, e.g., Hassouneh et al. (2012), Methods Enzymol., 502:215; e.g. polypeptide containing a pentapeptide repeat unit (Val-Pro-Gly-X-Gly; SEQ ID NO: 100), 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. Lond. B. Biol. Sci., 357:165) silk-elastin-like polypeptide (SELP; see, for example, Megeed et al. (2002), Adv. Drug. Deliv. Rev., 54:1075), etc. 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 framework polypeptides are, in some cases, half-life enhancing polypeptides. Thus, in some cases, a suitable framework polypeptide increases the in vivo half-life (eg, serum half-life) of the multimeric polypeptide compared to a control multimeric polypeptide lacking the framework polypeptide. For example, in some cases, the framework polypeptide increases the in vivo half-life (e.g., serum half-life) of the multimeric polypeptide relative to a control multimeric polypeptide lacking the framework polypeptide by at least about 10%, with at least about 15% %, at least about 20%, at least about 25%, at least about 50%, at least about 2 times, at least about 2.5 times, at least about 5 times, at least about 10 times, at least about 25 times, at least about 50 times, at least about 100 times, or more than 100 times. For example, in some cases, the Fc polypeptide increases the in vivo half-life (e.g., serum half-life) of the multimeric polypeptide compared to a control multimeric polypeptide lacking the Fc polypeptide by at least about 10%, by at least about 10%. 15%, at least about 20%, at least about 25%, at least about 50%, at least about 2 times, at least about 2.5 times, at least about 5 times, at least about 10 times, at least about 25 times, at least about 50 times, at least about 100 times, or more than 100 times.

Fc polypeptides.

In some cases, the first and/or second polypeptide chains of the multimeric polypeptide of this invention comprise an Fc polypeptide. The Fc polypeptide of the multimeric polypeptide of the present invention may 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 with

- 59 046369 amino acid sequence of the Fc region shown in FIG. 4A-4C. 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% , with at least about 98%, at least about 99, or 100% amino acid sequence identity with the human IgG1 Fc polypeptide shown in FIG. 4A. 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%, with at least about 98%, at least about 99 or 100% amino acid sequence identity with the human IgG1 Fc polypeptide shown in FIG. 4A; and contains a replacement for N77; for example, the Fc polypeptide contains the 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%, with at least about 98%, at least about 99 or 100% amino acid sequence identity with the human IgG2 Fc polypeptide shown in FIG. 4A; for example, an Fc polypeptide contains 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 with amino acids 99-325 of the human IgG2 Fc polypeptide shown in FIG. 4A. 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%, with at least about 98%, at least about 99 or 100% amino acid sequence identity with the human IgG3 Fc polypeptide shown in FIG. 4A; for example, an Fc polypeptide contains 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 with amino acids 19-246 of the human IgG3 Fc polypeptide shown in FIG. 4A. 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%, with at least about 98%, at least about 99 or 100% amino acid sequence identity with the human IgM Fc polypeptide shown in FIG. 4B; for example, an Fc polypeptide contains 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 with amino acids 1-276 of the human IgM Fc polypeptide shown in FIG. 4B. 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%, with at least about 98%, at least about 99 or 100% amino acid sequence identity with the human IgA Fc polypeptide shown in FIG. 4C; for example, an Fc polypeptide contains 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 with amino acids 1-234 of the human IgA Fc polypeptide shown in FIG. 4C.

In some cases, the Fc polypeptide present in the multimeric polypeptide of the present invention contains the amino acid sequence depicted in FIG. 33A (human IgG1 Fc). In some cases, the Fc polypeptide present in the multimeric polypeptide of the present invention contains the amino acid sequence depicted in FIG. 33A (human IgG1 Fc), except for the substitution of N297 with an amino acid other than asparagine. In some cases, the Fc polypeptide present in the multimeric polypeptide of the present invention contains the amino acid sequence depicted in FIG. 33C (human IgG1 Fc containing the N297A substitution). In some cases, the Fc polypeptide present in the multimeric polypeptide of the present invention contains the amino acid sequence depicted in FIG. 33A (human IgG1 Fc), except for the replacement of L234 with an amino acid other than leucine. In some cases, the Fc polypeptide present in the multimeric polypeptide of the present invention contains the amino acid sequence depicted in FIG. 33A (human IgG1 Fc), except for the replacement of L235 with an amino acid other than leucine. In some cases, the Fc polypeptide present in the multimeric polypeptide of the present invention contains the amino acid sequence depicted in FIG. 33D (Human IgG1 Fc containing L234A substitution and L235A substitution). In some

- 60 046369 cases, the Fc polypeptide present in the multimeric polypeptide of the present invention contains the amino acid sequence shown in FIG. 33A (human IgG1 Fc), except for the replacement of P331 with an amino acid other than proline; in some cases the replacement is a P331S replacement. In some cases, the Fc polypeptide present in the multimeric polypeptide of the present invention contains the amino acid sequence depicted in FIG. 33A (human IgG1 Fc), except for substitutions in L234 and L235 with amino acids other than leucine. In some cases, the Fc polypeptide present in the multimeric polypeptide of the present invention contains the amino acid sequence depicted in FIG. 33A (human IgG1 Fc), except for substitutions in L234 and L235 with amino acids other than leucine, and substitution of P331 with an amino acid other than proline. In some cases, the Fc polypeptide present in the multimeric polypeptide of the present invention contains the amino acid sequence depicted in FIG. 33B (human IgG1 Fc containing substitutions L234F, L235E and P331S).

Additional polypeptides.

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

Epitope tag.

Suitable epitope tags include, but are not limited to, hemagglutinin (HA; e.g., YPYDVPDYA (SEQ ID NO: 20); FLAG (e.g., DYKDDDDK (SEQ ID NO: 21); c-myc (e.g., EQKLISEEDL; SEQ ID NO) 22), etc.

Affine 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 the recombinant protein by high affinity binding to a resin column such as nickel-Sepharose. Exemplary affinity domains include His5 (HHHHH) (SEQ ID NO: 23), HisX6 (HHHHHH) (SEQ ID NO: 24), C-myc (EQKLISEEDL) (SEQ ID NO: 22), FLAG (DYKDDDDK) (SEQ ID NO : 21), StrepTag (WSHPQFEK) (SEQ ID NO: 25), hemagglutinin, e.g. HA tag (YPYDVPDYA) (SEQ ID NO: 20), glutathione^-transferase (GST), thioredoxin, cellulose binding domain, RYIRS (SEQ ID NO : 26), Phe-His-His-Thr (SEQ ID NO: 88), chitin-binding domain, S-peptide, T7 peptide, SH2 domain, C-terminal RNA tag, WEAAAREACCRECCARA (SEQ ID NO: 27) , metal-binding domains, e.g. zinc-binding domains or calcium-binding domains, such as domains from calcium-binding proteins, e.g. calmodulin, troponin C, calcineurin B, myosin light chain, recurein, S-modulin, visinin, VILIP , neurocalcin, hippocalcin, friquentin, caltractin, calpain large subunit, S100 proteins, parvalbumin, calbindin D9K, calbindin D28K and calretinin, inteins, biotin, streptavidin, MyoD, Id, leucine zipper and maltose binding protein sequences.

Examples of multimeric polypeptides of the present invention.

The following are non-limiting embodiments of the multimeric IL-2 polypeptide/synTac of the present invention.

In some cases, the multimeric IL-2 polypeptide/synTac of the present invention comprises a) a first polypeptide containing, in order from N-terminus to C-terminus

i) an epitope, ii) a β2-microglobulin (β2M) polypeptide containing the amino acid sequence shown in FIG. 34A; And

b) a second polypeptide containing in order from N-terminus to C-terminus

i) a variant IL-2 polypeptide of the present invention, ii) a major histocompatibility complex (MHC) heavy chain polypeptide containing the amino acid sequence shown in FIG. 34C, and iii) an IgG1 Fc polypeptide containing one or more amino acid substitutions selected from N297A, L234A, L235A, L234F, L235E and P331S.

In some cases, the variant IL-2 polypeptide contains the substitution H16A and F42A. In some cases, the IgG1 Fc polypeptide contains the N297A substitution. In some cases, the IgG1 Fc polypeptide contains an L234A substitution and an L235A substitution. In some cases, the IgG1 Fc polypeptide contains the L234F substitution and the L235E substitution. In some cases, the IgG1 Fc polypeptide contains the L234F substitution, the L235E substitution, and the P331S substitution. In some cases, the second polypeptide contains two copies of the variant IL-2 polypeptide. In some cases, the first polypeptide contains a peptide linker between the epitope and the B2M polypeptide. In neko

- 61 046369 in which cases the second polypeptide contains a peptide linker between one or more of

a) a first copy of a variant IL-2 polypeptide and a second copy of a variant IL-2 polypeptide;

b) variant IL-2 polypeptide and MHC heavy chain polypeptide; And

c) between the MHC heavy chain polypeptide and the IgG1 Fc polypeptide.

In some cases, the peptide linker is selected from (GGGGS) ₃ (SEQ ID NO: 89), (GGGGS) ₄ (SEQ ID NO: 90) and AAAGG (SEQ ID NO: 28). In some cases, the IgG1 Fc polypeptide contains the amino acid sequence shown in FIG. 33 In some cases, the IgG1 Fc polypeptide contains the amino acid sequence shown in FIG. 33C. In some cases, the IgG1 Fc polypeptide contains the amino acid sequence shown in FIG. 33D.

In some cases, the multimeric polypeptide of this invention contains

a) the first polypeptide containing in order from N-terminus to C-terminus

i) an epitope, ii) a β2-microglobulin polypeptide containing the amino acid sequence shown in FIG. 34A; And

b) a second polypeptide containing in order from N-terminus to C-terminus

i) a variant IL-2 polypeptide containing the amino acid sequence shown in FIG. 34B, ii) a major histocompatibility complex (MHC) heavy chain polypeptide containing the amino acid sequence shown in FIG. 34C, and iii) an IgG1 Fc polypeptide containing one or more amino acid substitutions selected from N297A, L234A, L235A, L234F, L235E and P331S.

In some cases, the IgG1 Fc polypeptide contains the N297A substitution. In some cases, the IgG1 Fc polypeptide contains an L234A substitution and an L235A substitution. In some cases, the IgG1 Fc polypeptide contains the L234F substitution and the L235E substitution. In some cases, the IgG1 Fc polypeptide contains the L234F substitution, the L235E substitution, and the P331S substitution. In some cases, the IgG1 Fc polypeptide contains the amino acid sequence shown in FIG. 33 In some cases, the IgG1 Fc polypeptide contains the amino acid sequence shown in FIG. 33C. In some cases, the IgG1 Fc polypeptide contains the amino acid sequence shown in FIG. 33D. In some cases, the second polypeptide includes two copies of the variant IL-2 polypeptide. In some cases, the first polypeptide contains a peptide linker between the epitope and the B2M polypeptide. In some cases, the second polypeptide contains a peptide linker between one or more of

a) a first copy of a variant IL-2 polypeptide and a second copy of a variant IL-2 polypeptide;

b) variant IL-2 polypeptide and MHC heavy chain polypeptide; And

c) between the MHC heavy chain polypeptide and the IgG1 Fc polypeptide.

In some cases, the peptide linker is selected from (GGGGS) ₃ (SEQ ID NO: 89), (GGGGS) ₄ (SEQ ID NO: 90) and AAAGG (SEQ ID NO: 28).

In some cases, the multimeric polypeptide of this invention contains

a) the first polypeptide containing in order from N-terminus to C-terminus

i) an epitope containing the amino acid sequence YMLDLQPETT (SEQ ID NO: 13), ii) a β2-microglobulin polypeptide containing the amino acid sequence shown in FIG. 34A; And

b) a second polypeptide containing in order from N-terminus to C-terminus

i) a variant IL-2 polypeptide containing the amino acid sequence shown in FIG. 34B, ii) a major histocompatibility complex (MHC) heavy chain polypeptide containing the amino acid sequence shown in FIG. 34C, and iii) an IgG1 Fc polypeptide containing the amino acid sequence shown in FIG. 33A, 33B, 33C or 33D.

In some cases, the IgG1 Fc polypeptide contains the amino acid sequence shown in FIG. 33 In some cases, the IgG1 Fc polypeptide contains the amino acid sequence shown in FIG. 33C. In some cases, the IgG1 Fc polypeptide contains the amino acid sequence shown in FIG. 33D. In some cases, the second polypeptide contains two copies of the variant IL-2 polypeptide. In some cases, the first polypeptide contains a peptide linker between the epitope and the B2M polypeptide. In some cases, the second polypeptide contains a peptide linker between one or more of

a) a first copy of a variant IL-2 polypeptide and a second copy of a variant IL-2 polypeptide;

b) variant IL-2 polypeptide and MHC heavy chain polypeptide; And

c) between the MHC heavy chain polypeptide and the IgG1 Fc polypeptide.

In some cases, the peptide linker is selected from (GGGGS) ₃ (SEQ ID NO: 89), (GGGGS) ₄ (SEQ ID NO: 90) and AAAGG (SEQ ID NO: 28). In some cases, the IgG1 Fc polypeptide contains the amino acid sequence shown in FIG. 33In some cases, the Fc polypeptide IgG1 with

- 62 046369 contains the amino acid sequence shown in FIG. 33C. In some cases, the IgG1 Fcpolypeptide contains the amino acid sequence depicted in FIG. 33D.

In some cases, the multimeric polypeptide of this invention contains

a) a first polypeptide containing the amino acid sequence shown in FIG. 31; And

b) a second polypeptide containing the amino acid equivalent of that shown in FIG. 22.

In some cases, the multimeric polypeptide of this invention contains

a) a first polypeptide containing the amino acid sequence shown in FIG. 31; And

b) a second polypeptide containing the amino acid equivalent of that shown in FIG. 25.

In some cases, the multimeric polypeptide of this invention contains

a) a first polypeptide containing the amino acid sequence shown in FIG. 31; And

b) a second polypeptide containing the amino acid equivalent shown in FIG. 28.

Nucleic acids.

This invention relates to a nucleic acid containing a nucleotide sequence encoding a variant IL-2 polypeptide of this invention. This invention relates to a nucleic acid containing a nucleotide sequence encoding an IL-2 fusion polypeptide of the present invention.

This invention relates to nucleic acids containing nucleotide sequences encoding a multimeric polypeptide according to this invention. In some cases, individual polypeptide chains of a multimeric polypeptide of this invention are encoded by separate nucleic acids. In some cases, all polypeptide chains of a multimeric polypeptide of the present invention are encoded by a single nucleic acid. In some cases, the first nucleic acid comprises a nucleotide sequence encoding a first polypeptide of a multimeric polypeptide of the present invention; and the second nucleic acid contains a nucleotide sequence encoding a second polypeptide of the multimeric polypeptide of the present invention. In some cases, the single nucleic acid contains a nucleotide sequence encoding a first polypeptide of a multimeric polypeptide of the invention and a second polypeptide of a multimeric polypeptide of the invention.

Non-limiting examples of nucleic acids of the present invention are depicted in FIG. 23A, 26A, 29A and 32.

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

This invention relates to nucleic acids containing nucleotide sequences encoding a multimeric polypeptide according to this invention. As noted above, in some cases, individual polypeptide chains of a multimeric polypeptide of this invention are encoded in separate nucleic acids. In some cases, the nucleotide sequences encoding individual polypeptide chains of a multimeric polypeptide of the present invention are operably linked to transcription control elements, such as promoters, such as promoters that function in a eukaryotic cell, wherein the promoter may be a constitutive promoter or an inducible promoter.

This invention provides a first nucleic acid and a second nucleic acid, wherein the first nucleic acid comprises a nucleotide sequence encoding a first polypeptide of a multimeric polypeptide of the invention, wherein the first polypeptide is contained in order from N-terminus to C-terminus

a) epitope (for example, T-cell epitope);

b) the first MHC polypeptide; And

c) an immunomodulatory polypeptide (eg, a variant IL-2 polypeptide of the present invention); and wherein the second nucleic acid comprises a nucleotide sequence encoding a second polypeptide of the multimeric polypeptide of the present invention, wherein the second polypeptide comprises, in order from N-terminus to C-terminus

a) a second MHC polypeptide; And

b) Fc polypeptide Ig.

Suitable T cell epitopes, MHC polypeptides, immunomodulatory polypeptides and Ig Fc polypeptides are described above. In some cases, the nucleotide sequences encoding the first and second polypeptides are operably linked to transcription control elements. In some cases, the transcription control element is a promoter that functions in a eukaryotic cell. In some cases, the nucleic acids are present in separate expression vectors.

This invention relates to a first nucleic acid and a second nucleic acid, with

- 63 046369 than the first nucleic acid contains a nucleotide sequence encoding a first polypeptide of a multimeric polypeptide of the present invention, the first polypeptide being contained in order from N-terminus to C-terminus

a) epitope (for example, T-cell epitope); And

b) the first MHC polypeptide; and wherein the second nucleic acid comprises a nucleotide sequence encoding a second polypeptide of the multimeric polypeptide of the present invention, wherein the second polypeptide comprises in order from N-terminus to C-terminus

a) an immunomodulatory polypeptide (for example, a variant IL-2 polypeptide of this invention);

b) a second MHC polypeptide; And

c) Fc polypeptide Ig.

Suitable T cell epitopes, MHC polypeptides, immunomodulatory polypeptides and Ig Fc polypeptides are described above. In some cases, the nucleotide sequences encoding the first and second polypeptides are operably linked to transcription control elements. In some cases, the transcription 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 multimeric polypeptide of the present invention. In some cases, when the multimeric polypeptide of this invention includes a first, second and third polypeptide, the nucleic acid includes a nucleotide sequence encoding the first, second and third polypeptides. In some cases, the nucleotide sequences encoding the first polypeptide and the second polypeptide of the multimeric polypeptide of the present invention include 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 polypeptide of the multimeric polypeptide of the present invention include an internal ribosome entry site (IRES) located 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 polypeptide of the multimeric polypeptide of the present invention include a ribosome skipping signal (or a cis-acting hydrolase element) located between the nucleotide sequence encoding the first polypeptide and the nucleotide sequence sequence encoding the second polypeptide. Examples of nucleic acids are described below, wherein a proteolytically cleavable linker is provided between the nucleotide sequences encoding the first polypeptide and the second polypeptide of the multimeric polypeptide of the present invention; in any of these embodiments, an IRES or ribosome skipping signal may be used instead of a nucleotide sequence encoding a proteolytically cleavable linker.

In some cases, the first nucleic acid (eg, recombinant expression vector, mRNA, viral RNA, etc.) contains a nucleotide sequence encoding the first polypeptide chain of a multimeric polypeptide of the present invention; and the second nucleic acid (eg, recombinant expression vector, mRNA, viral RNA, etc.) contains 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 transcription control elements, such as promoters, such as promoters that function in a eukaryotic cell, wherein the promoter may be a constitutive promoter or an inducible promoter promoter

This invention relates to a nucleic acid containing a nucleotide sequence encoding a recombinant polypeptide, wherein the recombinant polypeptide contains in the order from N-terminus to C-terminus

a) epitope (for example, T-cell epitope);

b) the first MHC polypeptide;

c) an immunomodulatory polypeptide (eg, a variant IL-2 polypeptide of the present invention);

d) a proteolytically cleavable linker;

e) a second MHC polypeptide; And

f) Immunoglobulin Fc polypeptide (Ig).

- 64 046369

This invention relates to a nucleic acid containing a nucleotide sequence encoding a recombinant polypeptide, wherein the recombinant polypeptide contains in the order from N-terminus to C-terminus

a) the first leader peptide;

b) epitope;

c) the first MHC polypeptide;

d) an immunomodulatory polypeptide (eg, a variant IL-2 polypeptide of the present invention);

e) a proteolytically cleavable linker;

f) a second leader peptide;

g) a second MHC polypeptide; And

h) Fc polypeptide Ig.

This invention relates to a nucleic acid containing a nucleotide sequence encoding a recombinant polypeptide, wherein the recombinant polypeptide contains in the order from N-terminus to C-terminus

a) epitope;

b) the first MHC polypeptide;

c) a proteolytically cleavable linker;

d) an immunomodulatory polypeptide (eg, a variant IL-2 polypeptide of the present invention);

e) a second MHC polypeptide; And

f) Ig Fc polypeptide.

In some cases, the first leader peptide and the second leader peptide are a B2-M leader peptide. In some cases, the nucleotide sequence is operably linked to a transcription control element. In some cases, the transcription 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 contains an amino acid sequence having at least 85% amino acid sequence identity with one of the amino acid sequences presented in FIG. 6. In some cases, the MHC class I heavy chain polypeptide is the heavy chain MHA-A, MLA-B, MLA-C, MLA-E, MHA-F, MLA-G, MLA-K, or MLA-L. In some cases, the MHC class I heavy chain polypeptide contains an amino acid sequence having at least 85% amino acid sequence identity with the amino acid sequence shown in one of FIG. 5A-5C. In some cases, the first MHC polypeptide is a class II MHC alpha chain polypeptide; and wherein the second MHC polypeptide is a class II MHC beta chain polypeptide.

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 contains an amino acid sequence having at least 85% amino acid sequence identity with the amino acid sequence depicted in FIG. 4A-4C.

Suitable immunomodulatory polypeptides are described above.

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

a) LEVLFQGP (SEQ ID NO: 29);

b) ENLYTQS (SEQ ID NO: 30);

c) DDDDK (SEQ ID NO: 31);

d) LVPR (SEQ ID NO: 32); And

f) GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 33).

In some cases, the linker between the epitope and the first MHC polypeptide contains a first Cys residue, and the second MHC polypeptide contains an amino acid substitution to produce 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 contains an amino acid substitution to produce a first Cys residue, and the second MHC polypeptide contains an amino acid substitution to produce 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.

Recombinant expression vectors.

The present invention provides recombinant expression vectors containing the nucleic acids of the present invention. In some cases, the recombinant expression vector is

- 65 046369 non-viral vector. In some embodiments, the recombinant expression vector is a viral construct, such as a recombinant adeno-associated viral construct (see, e.g., U.S. Pat. No. 7,078,387), a recombinant adenoviral construct, a recombinant lentiviral construct, a recombinant retroviral construct, a non-integrating viral vector, and the like.

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, Ali 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; Ali et al., Hum. Mol. Genet., 5:591-594, 1996 ; Srivastava in WO 93/09239, Samulski et al., J. Vir. (1989), 63:3822-3828; Mendelson et al., Virol. (1988), 166:154-165; and Flotte et al., PNAS (1993), 90:10613-10617); SV40; herpes simplex virus; human immunodeficiency virus (see, for example, Miyoshi et al., PNAS, 94:10319-23, 1997; Takahashi et al., J. Virol., 73:7812-7816, 1999); retroviral vectors (for example, 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), etc. .P.

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

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

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

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

Genetically modified host cells.

The present invention provides a genetically modified host cell, wherein said 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 (eg, mouse, rat) cell lines, and the like. Suitable mammalian cell lines include, but are not limited to, HeLa cells (eg, American Type Culture Collection (ATCC) No. CCL-2), CHO cells (eg, ATCC No. CRL9618, CCL61, CRL9096), 293 cells (eg, ATCC No. CRL-1573), Vero cells, NIH 3T3 cells (e.g. ATCC No. CRL-1658), Huh-7 cells, BHK cells (e.g. ATCC No. CCL10), PC12 cells (ATCC No. CRL1721) COS cells, COS-7 cells (ATCC No. CRL1651), RAT1 cells, mouse L cells (ATCC No. CCLI. 3), human embryonic kidney (HEK) cells (ATCC No. CRL1573), HLHepG2 cells, etc.

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.

Methods for producing a multimeric polypeptide.

- 66 046369

The present invention provides methods for producing the multimeric polypeptide of the present invention. These methods typically involve culturing in a host cell culture medium that is genetically modified with a recombinant expression vector containing a nucleotide sequence encoding a multimeric polypeptide; and isolating the multimeric polypeptide from the genetically modified host cell and/or culture medium. A host cell that is genetically modified with a recombinant expression vector containing a nucleotide sequence encoding a multimeric polypeptide is also called an expression host. As noted above, in some cases, individual polypeptide chains of a multimeric polypeptide of this invention are encoded in separate recombinant expression vectors. In some cases, all polypeptide chains of a multimeric polypeptide of the present invention are encoded in a single recombinant expression vector.

Isolation of a multimeric polypeptide from an expression host cell (eg, from a lysate of an expression host cell) and/or the culture medium in which the host cell is cultured can be accomplished using standard protein purification methods.

For example, the lysate can be obtained from an expression host and the lysate purified using high performance liquid chromatography (HPLC), size exclusion chromatography, gel electrophoresis, affinity chromatography, or other purification method. Alternatively, when the multimeric polypeptide is secreted from the host expression cell into the culture medium, the multimeric polypeptide can be purified from the culture medium using HPLC, size exclusion chromatography, gel electrophoresis, affinity chromatography, or other purification technique. In some cases, the compositions that are used will comprise at least 80 wt%, at least about 85 wt%, at least about 95 wt%, or at least about 99.5 wt% of the desired product, relative to to contamination associated with the method of preparing the product and its cleaning. Percentages may be based on total protein content.

In some cases, for example, when a multimeric polypeptide contains an affinity tag, the multimeric polypeptide can be purified using an immobilized binding partner of the affinity tag.

Compositions.

The present invention provides compositions, including pharmaceutical compositions, containing a variant IL-2 polypeptide of the present invention. This invention relates to compositions, including pharmaceutical compositions, containing a multimeric polypeptide of this invention. The present invention provides compositions, including pharmaceutical compositions, containing a nucleic acid or recombinant expression vector of the present invention.

Compositions containing a multimeric polypeptide.

The composition of this invention may contain, in addition to the multimeric polypeptide of this invention, one or more of the following components: a salt, for example, NaCl, MgCl ₂ , KCl, MgSO ₄ , etc.; buffering agent, e.g. Tris buffer, N(2-hydroxyethyl)piperazine-H'-(2-ethanesulfonic acid) (HEPES), 2-(I-morpholino)ethanesulfonic acid (MES), 2-(N-morpholino)ethanesulfonic acid salt acids (MES), 3-(N-morpholino)propanesulfonic acid (MOPS), N-tris[hydroxymethyl]methyl-3-aminopropanesulfonic acid (TAPS), etc.; solubilizing agent; a detergent, for example a non-ionic detergent such as Tween-20, etc.; protease inhibitor; glycerin, etc.

The composition may contain a pharmaceutically acceptable excipient, a variety of which are known in the art and do not need to be discussed in detail herein. Pharmaceutically acceptable excipients have been 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), 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 pharmaceutical composition may contain a multimeric polypeptide of this invention and a pharmaceutically acceptable excipient. In some cases, the pharmaceutical composition in question will be suitable for administration to a subject, for example, will be sterile. For example, in some embodiments, the pharmaceutical composition in question will be suitable for administration to a human, for example, if the composition is sterile and free of 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 excipients 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 salts, solvates (e.g. mixed ionic salts, water, organics), hydrates (e.g. water), etc.

- 67 046369

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

When the multimeric polypeptide of the present invention is administered by injection (e.g., subcutaneously, intraperitoneally, intramuscularly, and/or intravenously) directly into tissue, the drug may be provided as a ready-to-use dosage form or as a non-aqueous form (e.g., reconstituted, stable at stored powder) or aqueous form, such as a liquid, consisting of pharmaceutically acceptable carriers and excipients. Protein-containing drugs may also be provided to increase the serum half-life of the target protein after administration. For example, the protein may be provided as a liposomal composition, formulated as a colloid, or by other conventional means to increase serum half-life. Many methods are available for the preparation of liposomes, as described, for example, in Szoka et al., 1980, Ann. Rev. Biophys. Bioeng., 9:467; US Pat. Nos. 4,235,871, 4,501,728, and 4,837,028. The formulations may also be provided in controlled-release or slow-release forms.

Other examples of formulations suitable for parenteral administration include isotonic sterile injectable solutions, antioxidants, bacteriostats and solutes that render the formulation isotonic with the blood of the intended recipient, suspending agents, solubilizers, thickeners, stabilizers and preservatives. For example, the pharmaceutical composition in question may be present in a container, such as a sterile container such as a syringe. The compositions may be provided in sealed unit or multiple dose containers, such as ampoules and vials, and may be stored in a freeze-dried (lyophilized) state, requiring only the addition of a sterile liquid excipient, such as water for injection, immediately prior to use. Injectable solutions and suspensions for immediate administration can be prepared from sterile powders, granules and tablets.

The concentration of the multimeric polypeptide of the present invention in the composition can vary widely (e.g., from less than about 0.1%, typically from or at least about 2% to from about 20 to 50% or more by weight) and will typically be selected according to based primarily on fluid volumes, viscosities, and patient-related factors according to the specific route of administration selected and the patient's needs.

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, ampoule, or the like. In some cases the container is sterile. In some cases, both the container and the composition are sterile.

The present invention provides compositions, including pharmaceutical compositions, containing a variant IL-2 polypeptide of the present invention. The composition may contain

a) a variant IL-2 polypeptide according to this invention; And

b) excipient as described above for multimeric polypeptides.

In some cases, the excipient is a pharmaceutically acceptable excipient. Compositions containing a nucleic acid or recombinant expression vector.

The present invention provides compositions, for example pharmaceutical compositions, containing a nucleic acid or 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: he 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 this invention may include

a) said nucleic acid or recombinant expression vector; 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 Nbis(2-hydroxyethyl)-2aminoethanesulfonic acid (BES), bis(2-hydroxyethyl)amino-tris(hydroxymethyl)methane (BIS-Tris), N(2-hydroxyethyl) ) piperazine-H'3-propanesulfonic acid (EPPS or HEPPS), glycylglycine, N-2hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), 3-(N-morpholino)propane sulfonic acid (MOPS), piperazine-N'-2-ethanesulfonic acid -bis(2-ethanesulfonic acid) (PIPES), sodium bicarbonate, 3-(Ntris(hydroxymethyl)methylamino)-2-hydroxypropanesulfonic acid) TAPSO, (Ntris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES), Ntris(hydroxymethyl) methylglycine (tricine), tris (hydroxymethyl) aminomethane (Tris), etc.). Suitable salts include, for example, NaCl, MgCl ₂ , KCl, MgSO ₄ , etc.

The pharmaceutical composition of this invention may include a nucleic acid

- 68 046369 or a recombinant expression vector according to this invention in an amount of from about 0.001% to about 90% (w/w). In the description of the compositions below, the subject nucleic acid or recombinant expression vector will be understood to include the nucleic acid or recombinant expression vector of the present invention. For example, in some embodiments, a composition of this invention comprises a nucleic acid or recombinant expression vector of this 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-targeted molecules. The target nucleic acid or recombinant expression vector may be combined in a formulation with one or more components that facilitate uptake, distribution and/or absorption.

The target nucleic acid or recombinant expression vector composition can be formulated in any of a variety of possible dosage forms, such as, but not limited to, tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories and enemas. The target nucleic acid or recombinant expression vector composition may also be formulated 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 composed of amphiphilic lipids arranged in a spherical bilayer or bilayers. Liposomes are single- or multi-layered vesicles that have a membrane formed from a lipophilic material and an aqueous interior that contains the delivery composition. 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 thought to capture DNA rather than form complexes with it. Both cationic and non-cationic liposomes can be used to deliver the nucleic acid or recombinant expression vector of interest.

Liposomes also include sterically stabilized liposomes, a term which as used herein refers to liposomes containing one or more specialized lipids which, when incorporated into the liposomes, result in increased circulation time compared to liposomes lacking such specialized lipids. Examples of sterically stabilized liposomes are those in which 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 US Pat. No. 6,287,860, which is incorporated herein by reference in its entirety.

The preparations and compositions of this invention 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 US Pat. No. 6,287,860.

In one embodiment, various penetration enhancers are included for efficient delivery of nucleic acids. In addition to promoting the 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 US Pat. No. 6,287,860, which is incorporated herein by reference in its entirety.

Compositions and preparations for oral administration include powders or granules, microparticles, nanoparticles, suspensions or solutions in water or non-aqueous media, capsules, gel capsules, sachets, tablets or mini-tablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersants or binders may be desirable. Suitable oral formulations 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 uses are further described in US Pat. No. 6287860. Also suitable are combinations of penetration enhancers, for example fatty acids/salts in combination with bile acids/salts. An example of a suitable combination is the sodium salt of lauric acid, capric acid and UDCA (ursodeoxycholic acid). Other penetration enhancers include, but are not limited to, polyoxyethylene-9-lauryl ether and floor

- 69 046369 noxyethylene-20-cetyl ether. Suitable penetration enhancers also include propylene glycol, dimethyl sulfoxide, triethanoamine, N.N-dimethylacetamide. N.N-dimethylformamide. 2-pyrrolidone and its derivatives, tetrahydrofurfuryl alcohol and AZONE™.

Methods for modulating T cell activity.

The present invention provides a method of selectively modulating the activity of an epitope-specific T cell, comprising contacting the T cell with a multimeric polypeptide of the present invention, wherein contact of the T cell with the multimeric polypeptide of the present invention selectively modulates the activity of the epitope-specific T cell. In some cases, contact occurs in vitro. 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 multimeric polypeptide contains class I MHC polypeptides (eg, β2-microglobulin and class I MHC heavy chain). In some cases, for example, when the target T cell is a CD4+ T cell, the multimeric polypeptide comprises MHC class II polypeptides (eg, MHC class II α chain; MHC class II β chain).

When the multimeric polypeptide of the present invention includes an immunomodulatory polypeptide that is an activating polypeptide, contact of a T cell with the multimeric polypeptide activates the epitope-specific T cell. In some cases, an epitope-specific T cell is a T cell that is specific for an epitope present in a cancer cell, and contact of the epitope-specific T cell with a multimeric polypeptide increases the cytotoxic activity of the T cell toward the cancer cell. In some cases, an 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 a multimeric polypeptide increases the number of epitope-specific T cells.

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

When the multimeric polypeptide of the present invention includes an immunomodulatory polypeptide that is an inhibitory polypeptide, contact of a T cell with the multimer 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 a self-antigen, and the contact reduces the number of autoreactive T cells.

Methods for selective delivery of a costimulatory polypeptide (eg, IL-2).

The present invention provides a method of delivering a costimulatory polypeptide, such as IL-2, or a reduced-affinity variant of a naturally occurring costimulatory polypeptide, such as a variant IL-2, disclosed herein to a selected T cell or a selected population of T cells . for example, such that the TCR is specific for a given epitope. The present invention provides a method of delivering a costimulatory polypeptide, such as IL-2, or a reduced-affinity variant of a naturally occurring costimulatory polypeptide, such as a variant IL-2 disclosed herein, selectively to a target T cell bearing a TCR specific for IL-2. an epitope present in the multimeric polypeptide of the present invention. The method involves contacting a population of T cells with a multimeric polypeptide of the present invention. The T cell population may be a mixed population that includes

i) a target T cell, and ii) non-target T cells that are not epitope specific (e.g., T cells that are specific for an epitope(s) other than the epitope to which the epitope specific T cell binds) .

The epitope-specific T cell is specific for the epitope-presenting peptide present in the multimeric polypeptide and binds to the HLA peptide complex or the MHC peptide complex provided by the multimeric polypeptide. Contacting a population of T cells with a multimeric polypeptide delivers a costimulatory polypeptide (eg, IL-2 or reduced-affinity variant IL-2) present in the multimeric polypeptide selectively to T cells that are specific for the epitope present in the multimeric polypeptide.

Thus, the present invention provides a method for delivering a costimulatory polypeptide, such as IL-2, or a reduced affinity variant of a naturally occurring costimulatory polypeptide, such as a variant IL-2 disclosed herein, or a combination thereof, from

- 70 046369 selectively to the target T cell, the method comprising contacting a mixed population of T cells with a multimeric polypeptide of this invention. The mixed T cell population includes targeted T cells and non-targeted T cells. the target T cell is specific for an epitope present in the multimeric polypeptide. Contacting a mixed population of T cells with a multimeric polypeptide of the present invention delivers the costimulatory polypeptide(s) present in the multimeric polypeptide to the target T cell.

For example, the multimeric polypeptide of the present invention is contacted with a population of T cells comprising

i) target T cell(s) that are specific for an epitope present in the multimeric polypeptide, and ii) non-target T cell(s), such as T cell(s) that are specific for the second epitope(s) that are not epitope present in the multimeric polypeptide.

Contact with said population results in selective delivery of the costimulatory polypeptide(s) (e.g., a naturally occurring costimulatory polypeptide (e.g., naturally occurring IL-2) or a reduced-affinity variant of a naturally occurring costimulatory polypeptide (e.g., a variant IL-2), disclosed herein)), which is present in the multimeric polypeptide, to the target T cell. Thus, for example, less than 50%, less than 40%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, or less than 4, 3, 2, or 1% non-targeted T cells bind the multimeric polypeptide, and as a result, the costimulatory polypeptide (eg, IL-2 or variant IL-2) is not delivered to the non-target T cell.

In some cases, the population of T cells is contained in a test tube. In some cases, the T cell population is in vitro, and the biological response (eg, T cell activation and/or expansion and/or phenotypic differentiation) of the target T cell population to the multimeric polypeptide of the invention is induced in the context of an in vitro culture. For example, a mixed population of T cells can be obtained from an individual and can be contacted with a multimeric polypeptide in vitro. Such exposure may involve single or multiple exposure of a population of T cells to a specific dose(s) and/or exposure schedule(s). In some cases, said contact results in selective binding/activation and/or expansion of target T cells within a population of T cells and results in the formation of a population of activated and/or expanded target T cells. As an example, the mixed population of T cells may be peripheral blood mononuclear cells (PBMCs). For example, PBMCs from a patient can be obtained by standard methods of drawing blood and enriching PBMCs before exposure to 0.1-1000 nM of the multimeric polypeptide of this invention under standard lymphocyte culture conditions. At time points before, during, and after exposure to a mixed population of T cells at a specific dose and schedule, the abundance of target T cells in in vitro culture can be monitored using specific peptide-MHC multimers and/or phenotypic markers and/or functional activity ( eg ELISpot cytokine assays). In some cases, after achieving the optimal number and/or phenotype of antigen-specific cells in vitro, all or part of the population of activated and/or expanded target T cells is administered to the individual (the individual from whom the mixed population of T cells was obtained).

In some cases, the population of T cells is contained in a test tube. For example, a mixed population of T cells is obtained from an individual and contacted with a multimeric polypeptide of the invention in vitro. Such exposure, which may involve single or multiple exposure of T cells to a specific dose(s) and/or exposure schedule(s) in the context of an in vitro cell culture, can be used to determine whether the mixed population of T cells includes T cells which are specific to the epitope presented by the multimeric polypeptide. The presence of T cells that are specific for an epitope of a multimeric polypeptide can be determined by analyzing a sample containing a mixed population of T cells, which T cell population contains T cells that are not specific for the epitope (non-target T cells) and may contain epitope-specific T cells (target T cells). Known assays can be used to detect activation and/or proliferation of target T cells, thereby providing an ex vivo assay that can determine whether a particular multimeric polypeptide (synTac) has an epitope that binds to T cells present in an individual, and therefore, whether the multimeric polypeptide would have potential use as a therapeutic composition for that individual. Suitable known assays for detecting activation and/or proliferation of target T cells include, for example, flow cytometric characterization of T cell phenotype and/or antigen specificity and/or proliferation. Such an assay to detect the presence of epitope-specific T cells, such as a companion diagnostic, may further include additional assays (eg, ELISpot assays for effector cytokines) and/or appropriate controls (eg, antigen-specific and antigen-nonspecific multimeric peptide-ASH staining reagents) to detect whether said multimeric polypeptide selectively binds/activates and/or causes proliferation of target T cells. Ta

- 71 046369 Thus, for example, 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, the method comprising:

a) contacting in vitro a mixed population of T cells with a multimeric polypeptide of this invention, wherein the multimeric polypeptide contains an 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 the target T cell.

Alternatively and/or additionally, if activation and/or expansion (proliferation) of the desired T cell population is achieved using a multimeric polypeptide, then all or a portion of the T cell population including the activated/expanded T cells can be administered back to the individual as therapy.

In some cases, a population of T cells resides in vivo in an individual. In such cases, the method of this invention for selectively delivering a costimulatory polypeptide (eg, IL-2 or reduced-affinity IL-2) to an epitope-specific T cell involves administering the individual multimeric polypeptide.

An epitope-specific T cell to which a costimulatory polypeptide (eg, IL-2 or reduced affinity IL-2) is selectively delivered is also referred to herein as a targeted T cell. In some cases, the target T cell is a regulatory T cell (Treg). In some cases, Treg inhibits or suppresses the activity of an autoreactive T cell.

In some cases, the target T cell is a cytotoxic T cell. For example, the target T cell may be a cytotoxic T cell specific for a cancer epitope (eg, an epitope presented by a cancer cell).

Methods of treatment.

The present invention provides a method of selectively modulating the activity of an epitope-specific T cell in an individual, the method comprising administering to the individual an amount of a multimeric polypeptide of the invention or one or more nucleic acids encoding a multimeric polypeptide effective to selectively modulate the activity of an epitope-specific T cell in the individual. In some cases, the method of treatment of this invention includes administering to an individual in need thereof one or more recombinant expression vectors containing nucleotide sequences encoding a multimeric polypeptide of this invention. In some cases, the method of treatment of this invention includes administering to an individual in need thereof one or more mRNA molecules containing nucleotide sequences encoding a multimeric polypeptide of this invention. In some cases, the method of treatment of this invention includes administering to an individual in need thereof a multimeric polypeptide of this invention.

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

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

The present invention provides a method of treating cancer in an individual, the method comprising administering to the individual an effective amount of a multimeric polypeptide of the invention or one or more nucleic acids (e.g., expression vectors; mRNA, etc.) containing nucleotide sequences encoding the multimeric polypeptide, at wherein the multimeric polypeptide contains a T cell epitope that is a cancer epitope, and wherein the multimeric polypeptide contains one or more stimulatory immunomodulatory polypeptides as described herein. In some cases, an effective amount of a multimeric polypeptide 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 multimeric polypeptide of this 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

- 72 046369 by 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95% compared with the number of cancer cells in the individual before administration of the multimeric polypeptide or in the absence of administration of the multimeric polypeptide. In some cases, an effective amount of a multimeric polypeptide of this 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, an effective amount of a multimeric polypeptide of this invention is an amount that, when administered in one or more doses to an individual in need thereof, reduces tumor burden in the individual. For example, in some cases, an effective amount of a multimeric polypeptide of this invention is an amount that, when administered in one or more doses to an individual in need thereof, reduces the individual's tumor burden by at least 10%, by at least 15%, by at least 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 burden in the individual before administration of the multimeric polypeptide or in the absence of administration of the multimeric polypeptide. In some cases, an effective amount of a multimeric polypeptide of this invention is an amount that, when administered in one or more doses to an individual in need thereof, increases the individual's survival time. For example, in some cases, an effective amount of a multimeric polypeptide of the present invention is an amount that, when administered in one or more doses to an individual in need thereof, increases the individual's survival time by at least 1 month, by at least 2 months, by at least 3 months, from 3 to 6 months, for a period from 6 months to 1 year, for a period from 1 year to 2 ac, for a period from 2 to 5 ac, for a period from 5 to 10 ac or for a period of more than 10 ac compared with the expected survival time of an individual in the absence of administration of the multimeric polypeptide.

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

Thus, the present invention provides a method of treating a viral infection 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 containing nucleotide sequences encoding the multimeric polypeptide, wherein the multimeric polypeptide contains a T cell epitope that represents is a viral epitope, and wherein the multimeric polypeptide contains one or more stimulatory immunomodulatory polypeptides, as described herein. In some cases, an effective amount of a multimeric polypeptide 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 multimeric polypeptide of this 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%, by 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 or in the absence of administration of the multimeric polypeptide. In some cases, an effective amount of a multimeric polypeptide of this 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, comprising administering to the individual an effective amount of a multimeric polypeptide of the present invention or one or more nucleic acids containing nucleotide sequences encoding a multimeric polypeptide, wherein the multimeric polypeptide contains a T cell epitope that is associated with pathogen epitope, and wherein the multimeric polypeptide contains one or more stimulatory immunomodulatory polypeptides, as described herein. In some cases, an effective amount of a multimeric polypeptide 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 multimeric polypeptide of this invention is

- 73 046369 an amount which, when administered in one or more doses to an individual in need thereof, reduces the number 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 number of pathogens in the individual before administration of the multimeric polypeptide or in the absence of administration of the multimeric polypeptide. In some cases, an effective amount of a multimeric polypeptide of this invention is an amount that, when administered in one or more doses to an individual in need thereof, reduces the pathogen burden in the individual to undetectable levels. Pathogens include viruses, bacteria, protozoa, etc.

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

This invention provides a method of treating an autoimmune disorder in an individual, the method comprising administering to the individual an effective amount of a multimeric polypeptide of the invention or one or more nucleic acids containing nucleotide sequences encoding the multimeric polypeptide, wherein the multimeric polypeptide contains a T cell epitope that is self epitope, and wherein the multimeric polypeptide contains an inhibitory immunomodulatory polypeptide. In some cases, an effective amount of a multimeric polypeptide is an amount that, when administered in one or more doses to an individual in need thereof, reduces the number of autoreactive T cells 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%, according to at least 90% or at least 95% compared to the number of autoreactive T cells in the individual before or in the absence of administration of the multimeric polypeptide. In some cases, an effective amount of a multimeric polypeptide 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 multimeric polypeptide 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 cases, when implementing a method of treating a subject, the multimeric polypeptide of this invention is administered to the individual in need thereof as a polypeptide by itself. In other cases, in carrying out a method of treating a subject, one or more nucleic acids containing nucleotide sequences encoding a multimeric polypeptide of the present invention are administered to an individual in need thereof. Thus, in other cases, one or more nucleic acids of the present invention, for example, one or more recombinant expression vectors of the present invention, are administered to an individual in need thereof.

Drugs.

Suitable preparations are described above, and suitable preparations include a pharmaceutically acceptable excipient. In some cases, a suitable composition contains

a) a multimeric polypeptide according to this invention; And

b) a pharmaceutically acceptable excipient.

In some cases, a suitable composition contains

a) a nucleic acid containing a nucleotide sequence encoding a multimeric polypeptide according to this invention; And

b) a pharmaceutically acceptable excipient; in some cases the nucleic acid is mRNA.

In some cases, a suitable composition contains

a) a first nucleic acid containing a nucleotide sequence encoding a first polypeptide of the multimeric polypeptide of the present invention;

b) a second nucleic acid containing a nucleotide sequence encoding a second polypeptide of the multimeric polypeptide of the present invention; And

c) a pharmaceutically acceptable excipient.

In some cases, a suitable composition contains

a) a recombinant expression vector containing a nucleotide sequence encoding a multimeric polypeptide according to this invention; And

b) a pharmaceutically acceptable excipient.

In some cases, a suitable composition contains

a) the first recombinant expression vector containing the nucleotide sequence that

- 74 046369 directing the first polypeptide of the multimeric polypeptide according to this invention;

b) a second recombinant expression vector containing a nucleotide sequence encoding a second polypeptide of the multimeric polypeptide of the present invention; And

c) a pharmaceutically acceptable excipient.

Suitable pharmaceutically acceptable excipients are described above.

Doses.

The appropriate dosage may be determined by the attending physician or other qualified health care personnel based on various clinical factors. As is well known in the medical field, dosages for any one patient depend on many factors, including the patient's size, body surface area, age, the particular polypeptide or nucleic acid to be administered, the sex of the patient, time and route of administration, general health, and others. medications administered together with the dose. The multimeric polypeptide of the present invention may be administered in amounts ranging from 1 ng/kg body weight to 20 mg/kg body weight per dose, such as 0.1 to 10 mg/kg body weight, such as 0.5 to 5 mg/kg body weight bodies; 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 range from 1 mcg to 10 mg per kilogram of body weight per minute. The multimeric polypeptide of this invention can be administered in an amount of from about 1 to 50 mg/kg body weight, for example 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 the multimeric polypeptide of this invention is 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 mcg to 100 mg per kg body weight, 100 mcg to 10 mg per kg body weight, or 100 mcg to 1 mg per kg body weight. Those skilled in the art can easily estimate the repetition frequency for dosing based on the measured residence times and concentrations of the administered agent in body fluids or tissues. After successful treatment, it may be desirable for the patient to undergo maintenance therapy to prevent recurrence of the disease state, in which the multimeric polypeptide of this 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, 1 mcg to 1 g per kg body weight, 10 mcg to 100 mg per kg body weight, 100 mcg to 10 mg per kg body weight, or 100 mcg to 1 mg per kg body weight bodies.

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

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

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

Routes of administration.

Active agent (multimeric polypeptide according to this invention, nucleic acid according to

- 75 046369 of this invention or a recombinant expression vector of this invention) is administered to an 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.

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

In some embodiments, a multimeric polypeptide of the invention, a nucleic acid of the invention, or a recombinant expression vector of the invention is administered intravenously. In some embodiments, a multimeric polypeptide of the invention, a nucleic acid of the invention, or a recombinant expression vector of the invention is administered intramuscularly. In some embodiments, a multimeric polypeptide of the invention, a nucleic acid of the invention, or a recombinant expression vector of the invention is administered locally. In some embodiments, a multimeric polypeptide of the invention, a nucleic acid of the invention, or a recombinant expression vector of the invention is administered intratumorally. In some embodiments, a multimeric polypeptide of the invention, a nucleic acid of the invention, or a recombinant expression vector of the invention is administered peritumorally. In some embodiments, a multimeric polypeptide of the invention, a nucleic acid of the invention, or a recombinant expression vector of the invention is administered intracranially. In some embodiments, a multimeric polypeptide of the invention, a nucleic acid of the invention, or a recombinant expression vector of the invention is administered subcutaneously.

In some embodiments, the multimeric polypeptide of this invention is administered intravenously. In some embodiments, the multimeric polypeptide of this invention is administered intramuscularly. In some embodiments, the multimeric polypeptide of this invention is administered locally. In some embodiments, the multimeric polypeptide of this invention is administered intratumorally. In some embodiments, the multimeric polypeptide of this invention is administered peritumorally. In some embodiments, the multimeric polypeptide of this invention is administered intracranially. In some embodiments, the multimeric polypeptide is administered subcutaneously.

A multimeric polypeptide of the present invention, a nucleic acid of the present invention, or a recombinant expression vector of the present invention can be administered to a host using any available conventional methods and routes suitable for the delivery of conventional drugs, including systemic or localized routes. In general, routes of administration contemplated for use in the method of this invention include, but are not limited to, enteral, parenteral and inhalation routes.

Parenteral routes of administration other than inhalation include, but are not limited to, topical, transdermal, subcutaneous, intramuscular, intraorbital, intracapsular, intraspinal, intrasternal, intratumoral, peritumoral, and intravenous, i.e. any route of administration other than the alimentary canal. Parenteral administration can be carried out to effect systemic or local delivery of a multimeric polypeptide of the present invention, a nucleic acid of the present invention, or a recombinant expression vector of the present invention. When systemic delivery is desired, administration typically involves invasive or systemically absorbed topical or mucosal administration of pharmaceuticals.

Subjects eligible for treatment.

Subjects suitable for treatment by the method of this invention include individuals who have cancer, including individuals who have been diagnosed with cancer, individuals who have been treated for cancer but who have not responded to treatment, and individuals who have been treated for cancer and who initially responded but subsequently became refractory to treatment. Subjects suitable for treatment by the method of this invention include individuals who have an infection (eg, infection with a pathogen such as a bacterium, virus, protozoan, etc.). Includes individuals who have been diagnosed with an infection and individuals who have been treated for an infection but have failed to respond to treatment. Subjects suitable for treatment by the method of this 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 have failed to respond to treatment. Subjects suitable for treatment by the method of this invention include individuals who have a viral infection, including individuals

- 76,046,369 individuals who were diagnosed with a viral infection and individuals who were treated for a viral infection but who failed to respond to treatment. Subjects suitable for treatment by the method of this 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 have failed to respond to treatment.

In some cases, for example, when the epitope is an HPV epitope, the subject suitable for treatment by the method of this invention is an individual who has been diagnosed with HPV-associated cancer or HPV cancer. HPV-associated and HPV-mediated cancers include, for example, head and neck cancer; cervical cancer; and cancer of the genitourinary system.

Examples of non-limiting aspects of the invention.

Aspects, including embodiments of this subject matter described above, may be useful alone or in combination with one or more other aspects or embodiments. Without limiting the foregoing description, certain non-limiting aspects of the invention, numbered 1-132, are demonstrated below. As those skilled in the art will appreciate upon reading this invention, each of the separately numbered aspects may be used or combined with any of the preceding or following separately numbered aspects. This is intended to provide support for all such aspect combinations and is not limited to the aspect combinations expressly provided below.

Aspect 1. A variant IL-2 polypeptide comprising an amino acid sequence having at least 85% identity to the amino acid sequence set forth in SEQ ID NO: 1, wherein said variant IL-2 polypeptide has one or more amino acid substitutions relative to the sequence set forth in SEQ ID NO: 1, wherein said variant IL-2 polypeptide exhibits reduced binding affinity to the IL-2 receptor (IL-2R) containing alpha, beta and gamma polypeptides having the amino acid sequences depicted in FIG. 3A-3C, compared with the binding affinity of the amino acid sequence of IL-2 indicated in one of SEQ ID NO: 1 with IL-2R.

Aspect 2. The variant IL-2 polypeptide of aspect 1, wherein the variant includes substitution of one or more of E15, H16, D20, F42, Y45 and Q126.

Aspect 3. The variant IL-2 polypeptide of aspect 1 or aspect 2, wherein said variant immunomodulatory polypeptide exhibits less than 10% to less than 50% of the binding affinity exhibited by the IL-2 amino acid sequence set forth in SEQ ID NO: 1c IL-2R.

Aspect 4. A variant IL-2 polypeptide according to any one of aspects 1-3, wherein said variant includes substitutions of F42 for Ala, Gly, Val, Ile or Leu.

Aspect 5. A variant IL-2 polypeptide according to any one of aspects 1-3, wherein said variant includes the F42 and D20 substitutions or the F42 and H16 substitutions.

Aspect 6. A variant IL-2 polypeptide according to any one of aspects 1-3, wherein said variant includes substitutions F42, D20 and Y45; or wherein said variant includes the replacements F42, H16 and Q126.

Aspect 7. Multimeric polypeptide containing

a) the first polypeptide containing in order from N-terminus to C-terminus

i) an epitope, ii) the first polypeptide of the major histocompatibility complex (MHC); and

b) a second polypeptide containing in order from N-terminus to C-terminus

i) a second MHC polypeptide; and ii) optionally an immunoglobulin (Ig) Fc polypeptide or non-Ig framework, wherein the multimeric polypeptide contains one or more immunomodulatory domains, wherein the one or more immunomodulatory domains are located on

A) C-terminus of the first polypeptide;

B) N-terminus of the second polypeptide;

C) 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, wherein at least one of the immunomodulatory domains contains a variant of a naturally occurring costimulatory protein, and wherein said variant exhibits reduced affinity for its partner costimulatory protein compared to the affinity of a naturally occurring costimulatory protein. the nature of the costimulatory protein to the partner costimulatory protein.

Aspect 8. Multimeric polypeptide containing

a) the first polypeptide containing in order from N-terminus to C-terminus

i) an epitope, ii) the first polypeptide of the major histocompatibility complex (MHC); and

b) a second polypeptide containing in order from N-terminus to C-terminus

i) a second MHC polypeptide, and ii) optionally an immunoglobulin (Ig) Fc polypeptide or non-Ig framework,

- 77 046369 wherein the multimeric polypeptide contains one or more immunomodulatory domains, wherein one or more immunomodulatory domains are located on

A) C-terminus of the first polypeptide;

B) N-terminus of the second polypeptide;

C) C-terminus of the second polypeptide; or

D) the C-terminus of the first polypeptide and the N-terminus of the second polypeptide, wherein at least one of the one or more immunomodulatory domains is a variant IL-2 polypeptide according to any one of aspects 1-6, and wherein the multimeric polypeptide exhibits reduced binding affinity with the IL-2 receptor (IL-2R) containing alpha, beta and gamma polypeptides having the amino acid sequences shown in FIG. 3A-3C, compared with the binding affinity of a control multimeric polypeptide containing the amino acid sequence of IL-2 given in SEQ ID NO: 1 for the IL-2R polypeptide.

Aspect 9. The multimeric polypeptide of aspect 8, in which

a) the first polypeptide contains in the direction from the N-terminus to the C-terminus

i) epitope, ii) first MHC polypeptide; and iii) a variant IL-2 polypeptide; and

b) the second polypeptide contains in the direction from the N-terminus to the C-terminus

i) a second MHC polypeptide; and ii) an Ig Fc polypeptide.

Aspect 10. The multimeric polypeptide of aspect 8, in which

a) the first polypeptide contains in the direction from the N-terminus to the C-terminus

i) an epitope, and ii) a first MHC polypeptide; and

b) the second polypeptide contains in the direction from the N-terminus to the C-terminus

i) a variant IL-2 polypeptide, ii) a second MHC polypeptide, and iii) an Ig Fc polypeptide.

Aspect 11. The multimeric polypeptide of aspect 8, in which

a) the first polypeptide contains in the direction from the N-terminus to the C-terminus

i) an epitope, and ii) a first MHC polypeptide; and

b) the second polypeptide contains in the direction from the N-terminus to the C-terminus

i) a second MHC polypeptide; and ii) a variant IL-2 polypeptide.

Aspect 12. The multimeric polypeptide of aspect 8, in which

a) the first polypeptide contains in the direction from the N-terminus to the C-terminus

i) an epitope, and ii) a first MHC polypeptide; and

b) a second polypeptide containing, in order from N-terminus to C-terminus

i) a variant IL-2 polypeptide; and ii) a second MHC polypeptide.

Aspect 13. The multimeric polypeptide of aspect 8, in which

a) the first polypeptide contains in the direction from the N-terminus to the C-terminus

i) an epitope, ii) a first MHC polypeptide, and iii) a variant IL-2 polypeptide; and

b) the second polypeptide contains a second MHC polypeptide.

Aspect 14. The multimeric polypeptide of aspect 7 or 8, wherein the non-Ig framework is an XTEN polypeptide, a transferrin polypeptide, an elastin-like polypeptide, a silk-like polypeptide, or a silk-elastin-like polypeptide.

Aspect 15. The multimeric polypeptide of any one of aspects 7-14, 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 16. The multimeric polypeptide of aspect 15, wherein the β2-microglobulin polypeptide comprises an amino acid sequence having at least 85% amino acid sequence identity with one of the amino acid sequences shown in FIG. 6.

Aspect 17. The multimeric polypeptide of aspect 15, wherein the MHC class I heavy chain polypeptide is an HLA-A, HLA-B, or HLA-C heavy chain.

Aspect 18. The multimeric polypeptide of aspect 15, wherein the MHC heavy chain polypeptide

- 78 046369 class I contains an amino acid sequence having at least 85% amino acid sequence identity with the amino acid sequence presented in one of FIGS. 5A-5C.

Aspect 19. The multimeric polypeptide of any one of aspects 7-14, wherein the first MHC polypeptide is a class II MHC alpha chain polypeptide; and wherein the second MHC polypeptide is a class II MHC beta chain polypeptide.

Aspect 20. The multimeric polypeptide of any one of aspects 7-19, wherein the epitope is a T cell epitope.

Aspect 21. The multimeric polypeptide of any one of aspects 7-13 and 15-20, wherein the multimeric polypeptide comprises an Fc polypeptide, and wherein the Ig Fc polypeptide is an IgG1 Fc polypeptide, an IgG2 Fc polypeptide, an IgG3 Fc polypeptide, an Fc - IgG4 polypeptide - IgA Fc polypeptide or IgM Fc polypeptide.

Aspect 22. The multimeric polypeptide of aspect 21, wherein the Ig Fc polypeptide comprises an amino acid sequence having at least 85% amino acid sequence identity with the amino acid sequence depicted in FIG. 4A-4C.

Aspect 23. A multimeric polypeptide according to any one of aspects 7-22, wherein the first polypeptide and the second polypeptide are non-covalently linked.

Aspect 24. A multimeric polypeptide according to any one of aspects 7-22, wherein the first polypeptide and the second polypeptide are covalently linked to each other.

Aspect 25. The multimeric polypeptide of aspect 24, wherein the covalent bond is via a disulfide bond.

Aspect 26. The multimeric polypeptide of aspect 25, wherein the first MHC polypeptide or a linker between the epitope and the first MHC polypeptide contains an amino acid substitution to provide a first Cys residue, and the second MHC polypeptide contains an amino acid substitution to provide a second Cys residue, and wherein a disulfide bond is between the first and the second Cys residues.

Aspect 27. The multimeric polypeptide of any one of aspects 7-26, comprising a linker inserted between the epitope and the first MHC polypeptide.

Aspect 28. The multimeric polypeptide of any one of aspects 7-26, comprising a linker inserted between the MHC polypeptide and the immunomodulatory polypeptide.

Aspect 29. A multimeric polypeptide according to any one of aspects 7-28, comprising 2 variant IL-2 polypeptides.

Aspect 30. A multimeric polypeptide according to any one of aspects 8-28, comprising 3 variant IL-2 polypeptides.

Aspect 31. The multimeric polypeptide of aspect 29 or aspect 30, wherein 2 or 3 variant IL-2 polypeptides are in tandem, wherein the multimeric polypeptide contains a linker between said variant IL-2 polypeptides.

Aspect 32. The multimeric polypeptide of any one of aspects 8-31, wherein said variant IL-2 comprises a substitution of one or more of E15, H16, D20, F42, Y45, and Q126.

Aspect 33. A multimeric polypeptide according to any one of aspects 8-32, wherein said variant IL-2 comprises a substitution of F42 for Ala, Gly, Val, Ile or Leu.

Aspect 34. The multimeric polypeptide of aspect 33, wherein said variant IL-2 contains substitutions F42 and D20 or substitutions F42 and H16.

Aspect 35. The multimeric polypeptide of aspect 33, wherein said variant IL-2 contains substitutions F42, D20 and Y45; or wherein said variant IL-2 contains substitutions F42, H16 and Q126.

Aspect 36. Nucleic acid containing a nucleotide sequence encoding a recombinant polypeptide,

i) wherein the recombinant polypeptide contains, in order from N-terminus to C-terminus

a) epitope,

b) the first polypeptide of the major histocompatibility complex (MHC),

c) immunomodulatory polypeptide,

d) a proteolytically cleavable linker or ribosome passage signal,

e) a second MHC polypeptide, and

f) an immunoglobulin (Ig) Fc polypeptide, wherein the immunomodulatory polypeptide is a variant of a naturally occurring costimulatory protein, and wherein the variant exhibits reduced affinity for its costimulatory protein partner compared to the affinity of the naturally occurring costimulatory protein for its costimulatory protein partner; or ii) wherein the recombinant polypeptide contains, in order from N-terminus to C-terminus

a) epitope,

b) the first MHC polypeptide,

c) a proteolytically cleavable linker or ribosome passage signal,

d) immunomodulatory polypeptide,

- 79 046369

e) a second MHC polypeptide, and

f) an Ig Fc polypeptide, wherein the immunomodulatory polypeptide is a variant of a naturally occurring costimulatory protein, and wherein the variant exhibits reduced affinity for its partner costimulatory protein compared to the affinity of the naturally occurring costimulatory protein for its partner costimulatory protein.

Aspect 37. Nucleic acid containing a nucleotide sequence encoding a recombinant polypeptide,

i) wherein the recombinant polypeptide contains, in order from N-terminus to C-terminus

a) epitope,

b) the first polypeptide of the major histocompatibility complex (MHC),

c) immunomodulatory polypeptide,

d) a proteolytically cleavable linker or ribosome passage signal,

e) a second MHC polypeptide, and

f) an immunoglobulin (Ig) Fc polypeptide, wherein the immunomodulatory polypeptide is a variant of the immunomodulatory polypeptide of any one of aspects 1-6; or ii) wherein the recombinant polypeptide contains, in order from N-terminus to C-terminus

a) epitope,

b) the first MHC polypeptide,

c) a proteolytically cleavable linker or ribosome passage signal,

d) immunomodulatory polypeptide,

e) a second MHC polypeptide, and

f) an Ig Fc polypeptide, wherein the immunomodulatory polypeptide is a variant of the immunomodulatory polypeptide of any one of aspects 1-6.

Aspect 38. The nucleic acid of aspect 36 or 37, 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 39. The nucleic acid of aspect 38, wherein the β2-microglobulin polypeptide comprises an amino acid sequence having at least 85% amino acid sequence identity with one of the amino acid sequences shown in FIG. 6.

Aspect 40. The nucleic acid of aspect 38, wherein the class I MHC heavy chain polypeptide is an HLA-A, HLA-B, or HLA-C heavy chain.

Aspect 41. The nucleic acid of aspect 40, wherein the MHC class I heavy chain polypeptide comprises an amino acid sequence having at least 85% amino acid sequence identity with the amino acid sequence shown in any of FIG. 5A-5C.

Aspect 42. The nucleic acid of aspect 36 or 37, wherein the first MHC polypeptide is a class II MHC alpha chain polypeptide and wherein the second MHC polypeptide is a class II MHC beta chain polypeptide.

Aspect 43. The nucleic acid of any one of aspects 36-42, wherein the epitope is a T cell epitope.

Aspect 44. The nucleic acid of any one of aspects 36-43, wherein 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.

Aspect 45. The nucleic acid of aspect 44, wherein the Ig Fc polypeptide comprises an amino acid sequence having at least 85% amino acid sequence identity with the amino acid sequence depicted in FIG. 4A-4C.

Aspect 46. The nucleic acid of any one of aspects 37-45, wherein said variant immunomodulatory IL-2 polypeptide comprises a substitution of one or more of E15, H16, D20, F42, Y45, and Q126.

Aspect 47. The nucleic acid of any one of aspects 36-46, wherein the multimeric polypeptide comprises a second immunomodulatory polypeptide selected from CD7, CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, HVEM, lymphotoxin beta receptor 3/ TR6, ILT3, ILT4 and HVEM.

Aspect 48. The nucleic acid of any one of aspects 36-47, wherein the proteolytically cleavable linker or ribosome skip signal comprises an amino acid sequence selected from

a) LEVLFQGP (SEQ ID NO: 29);

b) ENLYTQS (SEQ ID NO: 30);

c) furin cleavage site; d) LVPR (SEQ ID NO:32);

- 80 046369

e) GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 33);

f) GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 34);

g) GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO: 35); and

h) GSGVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 36).

Aspect 49. The nucleic acid of aspect 36-48, wherein the recombinant polypeptide is contained in order from N-terminus to C-terminus

a) the first leader peptide;

b) epitope;

c) the first MHC polypeptide;

d) immunomodulatory polypeptide;

e) a proteolytically cleavable linker or ribosome passage signal;

f) a second leader peptide;

g) a second MHC polypeptide; and

h) Immunoglobulin Fc polypeptide (Ig).

Aspect 50. The nucleic acid of aspect 49, wherein the first leader peptide and the second leader peptide are a B2-M leader peptide.

Aspect 51. A nucleic acid as defined in any one of aspects 36-50, wherein the nucleotide sequence is operably linked to a transcription control element.

Aspect 52. The nucleic acid of aspect 51, wherein the transcription control element is a promoter that functions in a eukaryotic cell.

Aspect 53. The nucleic acid of any one of aspects 36-52, wherein the first MHC polypeptide or a linker between the epitope and the first MHC polypeptide contains an amino acid substitution to produce a first Cys residue, and the second MHC polypeptide contains an amino acid substitution to produce a second Cys residue, and wherein the first and a second Cys residue provides a disulfide bond between the first MHC polypeptide and the second MHC polypeptide.

Aspect 54. A recombinant expression vector comprising a nucleic acid according to any one of aspects 36-52, and wherein the vector is optionally a viral vector or a non-viral vector.

Aspect 55. A host cell genetically modified with the recombinant expression vector of aspect 54.

Aspect 56. The host cell of aspect 55, wherein the host cell is in vitro and wherein the host cell is optionally genetically modified such that the cell does not produce endogenous MHC β2-microglobulin polypeptide.

Aspect 57. Composition comprising

a) a first nucleic acid containing a nucleotide sequence encoding a first polypeptide, in order from N-terminus to C-terminus

i) an epitope, ii) a first MHC polypeptide, and iii) an immunomodulatory domain, wherein the immunomodulatory polypeptide is a variant of a naturally occurring costimulatory protein, and wherein the variant exhibits reduced affinity for its partner costimulatory protein compared to the affinity of a naturally occurring costimulatory protein protein to partner costimulatory protein; and

b) a first nucleic acid containing a nucleotide sequence encoding a second polypeptide containing, in order from N-terminus to C-terminus

i) a second MHC polypeptide; and ii) an Ig Fc polypeptide.

Aspect 58. Composition comprising

a) a first nucleic acid containing a nucleotide sequence encoding a first polypeptide, in order from N-terminus to C-terminus

i) an epitope, and ii) a first MHC polypeptide; and

b) a first nucleic acid containing a nucleotide sequence encoding a second polypeptide, in order from N-terminus to C-terminus

i) an immunomodulatory domain, wherein the immunomodulatory domain is a variant of a naturally occurring costimulatory protein, and wherein said variant exhibits a reduced affinity for its partner costimulatory protein compared to the affinity of the naturally occurring costimulatory protein for a partner costimulatory protein, ii) a second MHC polypeptide, and iii) Ig Fc polypeptide.

Aspect 59. Composition comprising

- 81 046369

a) a first nucleic acid containing a nucleotide sequence encoding a first polypeptide containing, in order from N-terminus to C-terminus

i) an epitope, ii) a first MHC polypeptide, and iii) an immunomodulatory domain, wherein the immunomodulatory domain is a variant IL-2 polypeptide as defined in any one of aspects 1-6; and

b) a first nucleic acid containing a nucleotide sequence encoding a second polypeptide, in order from N-terminus to C-terminus

i) a second MHC polypeptide; and ii) an Ig Fc polypeptide.

Aspect 60. Composition comprising

a) a first nucleic acid containing a nucleotide sequence encoding a first polypeptide, in order from N-terminus to C-terminus

i) an epitope, and ii) a first MHC polypeptide; and

b) a first nucleic acid containing a nucleotide sequence encoding a second polypeptide, in order from N-terminus to C-terminus

i) an immunomodulatory domain, wherein the immunomodulatory domain is a variant IL-2 polypeptide according to any one of aspects 1-6, ii) a second MHC polypeptide, and iii) an Ig Fc polypeptide.

Aspect 61. The composition of any one of aspects 57-60, wherein the first and/or second nucleic acid is present in the recombinant expression vector.

Aspect 62. A host cell genetically modified by a composition according to any one of aspects 57-61.

Aspect 63. A method for producing a multimeric polypeptide according to any one of aspects 7-36, comprising

a) culturing the host cell according to any one of aspects 55, 56 and 62 in vitro in a culture medium under conditions under which the host cell synthesizes the multimeric polypeptide; and

b) isolating the multimeric polypeptide from the host cell and/or from the culture medium.

Aspect 64. The method of aspect 63, wherein the second polypeptide contains an affinity tag, and wherein said release comprises contacting the multimeric polypeptide produced by the cell with a binding partner for the affinity tag, wherein the binding partner is immobilized, thereby immobilizing the multimeric polypeptide.

Aspect 65. The method of aspect 64, comprising eluting the immobilized multimeric polypeptide.

Aspect 66. A method of selectively activating an epitope-specific T cell, comprising contacting the T cell with a multimeric polypeptide according to any one of aspects 7-35, wherein said contact selectively activates the epitope-specific T cell.

Aspect 67. The method of aspect 66, wherein said contacting occurs in vitro.

Aspect 68. The method of aspect 66, wherein said contacting occurs in vivo.

Aspect 69. The method of aspect 66, wherein the epitope is a cancer-associated epitope, and wherein said administration selectively increases the activity of a T cell specific for the cancer-associated epitope.

Aspect 70. A method of treating cancer in an individual, comprising administering to the individual an effective amount

a) a multimeric polypeptide according to any one of aspects 7-35; or

b) one or more recombinant expression vectors containing nucleotide sequences encoding a multimeric polypeptide according to any one of aspects 7-35; or

c) one or more mRNAs containing nucleotide sequences encoding a multimeric polypeptide according to any one of aspects 7-35, wherein said epitope is a cancer-associated epitope, and wherein said administration is effective to selectively activate an epitope-specific cancer T cell in an individual.

Aspect 71. The method of aspect 70, wherein said administration is subcutaneous.

Aspect 72. The method of aspect 70, wherein said administration is intravenous.

Aspect 73. The method of aspect 70, wherein said administration is peritumoral.

Aspect 74. The method of aspect 70, wherein said administration is systemic.

Aspect 75. The method of aspect 70, wherein said administration is distal to the site of treatment.

Aspect 76. The method of aspect 70, wherein said administration is local.

Aspect 77. The method of aspect 70, wherein said administration occurs at or near the site of treatment.

- 82 046369

Aspect 78. Composition comprising

a) a multimeric polypeptide according to any one of aspects 7-35; and

b) a pharmaceutically acceptable excipient.

Aspect 79. Composition comprising

a) a nucleic acid according to any one of aspects 36-53 or a recombinant expression vector according to aspect 54; and

b) a pharmaceutically acceptable excipient.

Aspect 80. Multimeric polypeptide containing

a) the first polypeptide containing in order from N-terminus to C-terminus

i) epitope, ii) β2-microglobulin polypeptide f2M). containing the amino acid sequence shown in FIG. 34A; and

b) a second polypeptide containing in order from N-terminus to C-terminus

i) a variant of a naturally occurring costimulatory protein, wherein said variant exhibits a reduced affinity for its partner costimulatory protein compared to the affinity of the naturally occurring costimulatory protein for a partner costimulatory protein, which may optionally be a variant IL-2 polypeptide in any one of aspects 1 -6, ii) a major histocompatibility complex (MHC) heavy chain polypeptide containing the amino acid sequence shown in FIG. 34C, and iii) an IgG1 Fc polypeptide containing one or more amino acid substitutions selected from N297A, L234A, L235A, L234F, L235E and P331S (N77A, L14A, L15A, L14F, L15E and P111S, respectively, based on amino acid numbering shown in Fig. 33A).

Aspect 81. The multimeric polypeptide of aspect 80, wherein the IgG1 Fc polypeptide contains the substitution N297A (N77A based on the amino acid numbering depicted in FIG. 33A).

Aspect 82. The multimeric polypeptide of aspect 80, wherein the IgG1 Fc polypeptide contains an L234A substitution and an L235A substitution (L14A and L15A based on the amino acid numbering depicted in FIG. 33A).

Aspect 83. The multimeric polypeptide of aspect 80, wherein the IgG1 Fc polypeptide contains the L234F substitution and the L235E substitution (L14F and L15E based on the amino acid numbering depicted in FIG. 33A).

Aspect 84. The multimeric polypeptide of aspect 80, wherein the IgG1 Fc polypeptide contains the L234F substitution, the L235E substitution, and the P331S substitutions (L14F, L15E, and P111S substitutions based on the amino acid numbering depicted in FIG. 33A).

Aspect 85. The multimeric polypeptide of any one of aspects 80-84, wherein the second polypeptide contains two copies of the variant IL-2 polypeptide.

Aspect 86. The multimeric polypeptide of any one of aspects 80-85, wherein the first polypeptide comprises a peptide linker between the epitope and the B2M polypeptide.

Aspect 87. The multimeric polypeptide of any one of aspects 80-86, wherein the second polypeptide comprises a peptide linker between one or more of

a) a first copy of a variant IL-2 polypeptide and a second copy of a variant IL-2 polypeptide;

b) variant IL-2 polypeptide and MHC heavy chain polypeptide; and

c) between the MHC heavy chain polypeptide and the IgG1 Fc polypeptide.

Aspect 88. The multimeric polypeptide of aspect 86 or 87, wherein the peptide linker is selected from (GGGGS)3, (GGGGS) ₄ and AAAGG.

Aspect 89. Multimeric polypeptide containing

a) the first polypeptide containing in order from N-terminus to C-terminus

i) an epitope, ii) a β2-microglobulin polypeptide containing the amino acid sequence shown in FIG. 34A; and

b) a second polypeptide containing in order from N-terminus to C-terminus

i) a variant IL-2 polypeptide containing the amino acid sequence shown in FIG. 34B, ii) a major histocompatibility complex (MHC) heavy chain polypeptide containing the amino acid sequence shown in FIG. 34C, and iii) an IgG1 Fc polypeptide containing one or more amino acid substitutions selected from N297A, L234A, L235A, L234F, L235E and P331S (N77A, L14A, L15A, L14F, L15E and P111S, respectively, based on amino acid numbering shown in Fig. 33A).

Aspect 90. The multimeric polypeptide of aspect 89, wherein the IgG1 Fc polypeptide contains the substitution N297A (N77A based on the amino acid numbering depicted in FIG. 33A).

Aspect 91. The multimeric polypeptide of aspect 89, wherein the IgG1 Fc polypeptide contains an L234A substitution and an L235A substitution (L14A and L15A based on the amino acid numbering depicted in FIG. 33A).

- 83 046369

Aspect 92. The multimeric polypeptide of aspect 89, wherein the IgG1 Fc polypeptide contains the L234F substitution and the L235E substitution (L14F and L15E based on the amino acid numbering depicted in FIG. 33A).

Aspect 93. The multimeric polypeptide of aspect 89, wherein the IgG1 Fc polypeptide contains the L234F substitution, the L235E substitution, and the P331S substitution (L14F, L15E, and P111S based on the amino acid numbering depicted in FIG. 33A).

Aspect 94. The multimeric polypeptide of any one of aspects 89-93, wherein the second polypeptide contains two copies of the variant IL-2 polypeptide.

Aspect 95. The multimeric polypeptide of any one of aspects 89-94, wherein the first polypeptide comprises a peptide linker between the epitope and the B2M polypeptide.

Aspect 96. The multimeric polypeptide of any one of aspects 89-95, wherein the second polypeptide comprises a peptide linker between one or more of

a) a first copy of a variant IL-2 polypeptide and a second copy of a variant IL-2 polypeptide;

b) variant IL-2 polypeptide and MHC heavy chain polypeptide; and

c) MHC heavy chain polypeptide and IgG1 Fc polypeptide.

Aspect 97. The multimeric polypeptide of aspect 95 or 96, wherein the peptide linker is selected from (GGGGS) ₃ , (GGGGS)4 and AAAGG.

Aspect 98. Multimeric polypeptide containing

a) the first polypeptide containing, in order from N-terminus to C-terminus:

i) an epitope containing the amino acid sequence YMLDLQPETT (SEQ ID NO: 13), ii) a β2-microglobulin polypeptide containing the amino acid sequence shown in FIG. 34A; and

b) a second polypeptide containing in order from N-terminus to C-terminus

i) a variant IL-2 polypeptide containing the amino acid sequence shown in FIG. 34B, ii) a major histocompatibility complex (MHC) heavy chain polypeptide containing the amino acid sequence shown in FIG. 34C, and iii) an IgG1 Fc polypeptide containing the amino acid sequence shown in FIG. 33A, 33B, 33C or 33D.

Aspect 99. The multimeric polypeptide of aspect 98, wherein the IgG1 Fc polypeptide comprises the amino acid sequence depicted in FIG. 33B.

Aspect 100. The multimeric polypeptide of aspect 98, wherein the IgG1 Fc polypeptide contains the amino acid sequence depicted in FIG. 33C.

Aspect 101. The multimeric polypeptide of aspect 98, wherein the IgG1 Fc polypeptide contains the amino acid sequence depicted in FIG. 33D.

Aspect 102. A multimeric polypeptide according to any one of aspects 98-101, wherein the second polypeptide contains two copies of the variant IL-2 polypeptide.

Aspect 103. The multimeric polypeptide of any one of aspects 98-102, wherein the first polypeptide comprises a peptide linker between the epitope and the B2M polypeptide.

Aspect 104. The multimeric polypeptide of any one of aspects 98-103, wherein the second polypeptide comprises a peptide linker between one or more of

a) a first copy of a variant IL-2 polypeptide and a second copy of a variant IL-2 polypeptide;

b) variant IL-2 polypeptide and MHC heavy chain polypeptide; and

c) MHC heavy chain polypeptide and IgG1 Fc polypeptide.

Aspect 105. The multimeric polypeptide of aspect 103 or aspect 104, wherein the peptide linker is selected from (GGGGS) ₃ , (GGGGS) ₄ and AAAGG. Aspect 106. Multimeric polypeptide containing

a) a first polypeptide containing the amino acid sequence shown in FIG. 31;

b) a second polypeptide containing the amino acid sequence shown in FIG. 22.

Aspect 107. Multimeric polypeptide containing

a) a first polypeptide containing the amino acid sequence shown in FIG. 31;

b) a second polypeptide containing the amino acid sequence shown in FIG. 25.

Aspect 108. Multimeric polypeptide containing

a) a first polypeptide containing the amino acid sequence shown in FIG. 31;

b) a second polypeptide containing the amino acid sequence shown in FIG. 28.

Aspect 109. Pharmaceutical composition containing:

a) a multimeric polypeptide according to any one of aspects 80-108; and

b) a pharmaceutically acceptable excipient.

Aspect 110. One or more nucleic acids comprising nucleotide sequences encoding a first and/or second polypeptide of a multimeric polypeptide, according to any one of aspects 80-108.

Aspect 111. One or more nucleic acids of aspect 110, wherein the nucleic acid(s) is/are present in the recombinant expression vectors.

- 84 046369

Aspect 112. A method of selectively activating an epitope-specific T cell, comprising contacting the T cell with a multimeric polypeptide according to any one of aspects 80-108, wherein said contact selectively activates the epitope-specific T cell.

Aspect 113. The method of aspect 112, wherein said contacting occurs in vitro.

Aspect 114. The method of aspect 112, wherein said contacting occurs in vivo.

Aspect 115. A method comprising administering to a subject an effective amount

a) a multimeric polypeptide according to any one of aspects 80-108; or

b) one or more recombinant expression vectors containing nucleotide sequences encoding a multimeric polypeptide according to any one of aspects 80-108; or

c) one or more mRNAs containing nucleotide sequences encoding a multimeric polypeptide according to any one of aspects 80-108, wherein said administration induces a T cell response to the epitope in an individual.

Aspect 116. The method of aspect 115, wherein said administration is subcutaneous.

Aspect 117. The method of aspect 115, wherein said administration is intravenous.

Aspect 118. The method of aspect 115, wherein said administration is systemic.

Aspect 119. The method of aspect 115, wherein said administration is intramuscular.

Aspect 120. The method of aspect 115, wherein said administration is distal to the site of treatment.

Aspect 121. The method of aspect 115, wherein said administration is local.

Aspect 122. The method of aspect 115, wherein said administration is at or near the site of treatment.

Aspect 123. A method of delivering a costimulatory polypeptide selectively to a target T cell, comprising contacting a mixed population of T cells with the multimeric polypeptide of any one of aspects 7-35 and 80-108, wherein the mixed population of T cells comprises target T cells and non-target T cells -cells, wherein the target T cell is specific for an epitope present in the multimeric polypeptide, and wherein said contact delivers a costimulatory polypeptide present in the multimeric polypeptide to the target T cell.

Aspect 124. A method of delivering IL-2 or variant IL-2 selectively to a target T cell, comprising contacting a mixed population of T cells with a multimeric polypeptide according to any one of aspects 835 and 80-108, wherein the mixed population of T cells includes the target T cells. cells and non-target T cells, wherein the target T cells are specific for an epitope present in the multimeric polypeptide, and wherein said contact delivers the variant IL-2 or IL-2 present in the multimeric polypeptide to the target T cell.

Aspect 125. The method of aspect 123 or 124, wherein the T cell population is in vitro.

Aspect 126. The method of aspect 123 or 124, wherein the T cell population is located in vivo in the individual.

Aspect 127. The method of aspect 126, comprising administering the multimeric polypeptide to an individual.

Aspect 128. The method of any one of aspects 123-127, wherein the target T cell is a regulatory T cell.

Aspect 129. The method of any one of aspects 123-127, wherein the target T cell is a cytotoxic T cell.

Aspect 130. The method of aspect 123 or 124, wherein the mixed population of T cells is a mixed population of T cells obtained from an individual in vitro, and wherein said contact results in activation and/or proliferation of the target T cells, generating a population of activated and /or proliferated target T cells.

Aspect 131. The method of claim 130, further comprising administering a population of activated and/or proliferated target T cells to an individual.

Aspect 132. A method of 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, the method comprising a) contacting the mixed population of T cells in vitro with a multimeric polypeptide according to any one of claims. 7-35 and 80-108, wherein the multimeric polypeptide contains an 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 the target T cell.

Examples

The following examples are provided to provide those skilled in the art with complete disclosure and description of how to make and use this invention, and are not intended to limit the scope of what the inventors consider their invention to be, nor are they intended to provide that that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy in terms of numbers used (e.g. quantity, temperature, etc.), but some considerations should be taken into account

- 85 046369 experimental errors and deviations. Unless otherwise noted, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric pressure. Standard abbreviations can be used, for example, by - base pair(s);

kb - kilobase(s);

pl - picolitre(s);

s - second(s);

min - minute(s);

h - hour(s);

ak - amino acid(s);

kb - kilobase(s);

nt - nucleotide(s);

kDa - kilodalton(s);

IM - intramuscular;

i.p. - intraperitoneal;

s/c - subcutaneously, etc.

Example 1. Production of IL-2/synTac.

The production of IL-2/synTac by transiently transfected mammalian cells was analyzed. As shown in FIG. 7A, production levels (in mg/L culture medium) of two different IL-2/synTac 6-7 days after transient transfection of cells were above 90 mg/L.

IL-2/synTac produced by mammalian cells was purified and subjected to reducing and non-reducing polyacrylamide gel electrophoresis. The results are shown in FIG. 7B. Dimensions are given in kDa.

IL-2/synTac was prepared in which the IL-2 polypeptide was in the light chain (i.e., a polypeptide containing the MHC class I light chain; e.g., B2M) or in the heavy chain (i.e., a polypeptide containing the heavy MHC chain class I). Expression levels and stability of IL-2/synTac were analyzed.

SynTac was produced in mammalian cells. As shown in FIG. 8A, IL-2/synTac containing IL-2 on the heavy chain was produced at levels approximately 25 times higher than that of IL-2/synTac containing IL-2 on the light chain.

IL-2/synTac produced by mammalian cells was subjected to reducing and non-reducing polyacrylamide gel electrophoresis; and the gels were stained with Coomassie blue. As shown in FIG. 8B, IL-2/synTac containing IL-2 on the heavy chain was more stable than IL-2/synTac containing IL-2 on the light chain. Dimensions are given in kDa.

Expression levels of IL-2/synTac, which includes variant IL-2, were assessed. In fig. 9 shows the expression level of IL-2/syn-Tacs, in which IL-2 is wild type (wt) or contains various combinations of F42A, D20K, Q126A, E15A, Y45A and H16A. Expression levels are expressed as percentage change relative to synTac expression levels with wild-type IL-2.

The effect of IL-2 copy number in IL-2/synTac on expression levels was assessed. IL-2/synTac contained one copy (1X), two copies (2X), or three copies (3X) in synTac. Various IL-2/synTac were produced in mammalian cells and expression levels were analyzed. These data are shown in Fig. 10. IL-2/synTac with one or two copies of IL-2 show similar expression levels, whereas IL-2/synTac with three copies of IL-2 show lower expression levels. Expression levels are expressed as fold change relative to the expression level of IL-2/synTac with one copy of IL-2.

Example 2. IL-2/synTac activity in vitro.

To achieve maximum specificity of targeting through the T-cell receptor, the affinity of the costimulatory polypeptide for its ligand should be lower than the affinity of MHC for TCR. The affinity of peptide/MHC for TCR can be about 10 μM.

IL-2/synTac was created containing two copies of a variant IL-2 containing the F42A and H16A substitutions. IL-2/synTac-induced costimulatory signaling was tested in antigen-specific CD8+ T cells and nonspecific CD8+ T cells. Antigen-specific CD8+ T cells and non-specific CD8+ T cells were contacted with different concentrations of IL-2/synTac.

As shown in FIG. 11, IL-2/synTac induced costimulatory signaling in antigen-specific CD8+ T cells at significantly lower concentrations than in nonspecific CD8+ T cells.

The selectivity of IL-2/synTac binding was tested. CD8+ T cells were isolated from the spleens of LCMV or OT1 mice. CD8+ T cells were incubated with various concentrations of IL-2/synTac and allowed to bind for 20 min. IL-2/synTac contain IgG2a Fc. Binding of IL-2/synTac to CD8+ T cells was detected using phycoerythrin (PE)-labeled anti-IgG2a antibody. PE fluorescence was determined using flow cytometry to determine the percentage of cells associated with IL-2/synTac.

- 86 046369

As shown in FIG. 12, IL-2/synTac binds in an antigen-specific manner to LCMV CD8+ T cells but does not exhibit significant binding to OT1 CD8+ T cells. Thus, IL-2/synTac selectively binds to CD8+ T cells specific for an epitope present in IL-2/synTac.

It was determined whether IL-2/synTac selectively activates targeted T cells. CD8+ T cells were isolated from the spleens of LCMV or OT1 mice. The IL-2/synTac used contained either the single amino acid substitution F42A or the substitutions F42A and H16A. CD8+ T cells were stimulated with different concentrations of IL-2/synTac for 20 min. Cells were then stained with PE-labeled anti-phospho-STAT5 antibody. PE fluorescence was determined using flow cytometry to determine the percentage of cells that are phospho-STAT5 positive, with (|)oc(|io-STAT5 being an activation marker.

As shown in FIG. 13, IL-2/synTac induced CD8+ stimulation (as indicated by % phospho^TAT5-positive cells) in antigen-specific (LCMV) CD8+ T cells at significantly lower concentrations than in nonspecific (BL6) CD8' T cells.

The specific activities of different IL-2/synTacs were analyzed. IL-2/synTac containing one copy of IL-2, two copies of IL-2 or three copies of IL-2, with IL-2 containing various combinations of F42A, D20K, Q126A, E15A, H16A and Y45A substrates were tested at various concentrations on stimulation of CD8+ antigen-specific (LCMV) or non-specific (BL6) cells. The percentage of cells positive for phospho-signal transducer and activator of transcription 5 (pSTAT5 - phospho-signal transducer and activator of transcription 5) was determined. The data is shown in Fig. 14A-14F.

Example 3 IL-2/synTac activity in vivo.

The in vivo activity of IL-2/synTac was tested. The in vivo fold change in antigen-specific CD8+ T cells was tested following administration of phosphate buffered saline (PBS), recombinant IL-2 (rIL-2) or IL-2/synTac of the present invention. The data is shown in FIG. 15, left panel. These data show that IL-2/synTac is 10 times more effective than rIL-2.

The in vivo specificity of IL-2/synTac was tested. After administration of PBS, rIL-2, or IL-2/synTac, antigen-specific and antigen-nonspecific responses were assessed. Data are expressed as the percentage of lymph node cells that were antigen-specific or antigen-nonspecific after administration of PBS, rIL-2, or IL-2/synTac. As shown in FIG. 15, right panel, IL-2/synTac induced an antigen-specific response (expressed as % maximum dilution of carboxyfluorescein succinimidyl ester (CFSE), an index of T cell proliferation). In contrast, the response induced by rIL-2 was not antigen-specific.

A dose-response analysis was performed. IL-2/synTac (F42A, H16A) was administered intraperitoneally at concentrations of 4, 8 and 16 mg/kg. The results are shown in FIG. 16A. As shown in FIG. 16A, IL-2/synTac administered at 4 or 8 mg/kg gave similar results; IL-2/synTac administered at a dose of 16 mg/kg caused the most potent immunostimulatory activity.

The effect of the IL-2/synTac route was tested. IL-2/synTac (F42A, H16A) was administered at a dose of 4 mg/kg either subcutaneously (SubQ - subcutaneously) or intraperitoneally (IP - intraperitoneally). As shown in FIG. 16B, subcutaneous administration resulted in greater immunostimulatory activity than IP administration.

The effect of IL-2 copy number on efficacy has been established. IL-2/synTac containing one copy of IL-2 (F42A, H16A) or two copies of IL-2 (F42A, H16A) was injected into mice bearing tumors carrying the HPV E7 epitope. The epitope included in IL-2/synTac was the HPV E7 epitope. As shown in FIG. 17A and 17B, IL-2/synTac containing two copies of IL-2 (F42A, H16A) were more effective in reducing tumor size than IL-2/synTac containing only one copy of IL-2 (F42A, H16A).

Example 4 PK/PD and stability studies of IL-2/synTac.

A pharmacokinetic (PK) analysis of IL-2/synTac was performed. IL-2/synTac (F42A, D20K, H16A) was administered IP at a dose of 10 mg/kg. At various time points after administration, serum samples were obtained and IL-2/synTac levels were measured in the serum samples. As shown in FIG. 18, the serum half-life of IL-2/synTac was approximately 4 h.

IL-2/synTac was administered intraperitoneally to C57BL/6 mice at a dose of 10 mg/kg, and serum was collected 2 h after injection. IL-2/synTac included a His6 tag. 100 ng of input protein or the equivalent of 40 μl of serum was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and probed with anti-(His) ₆ antibody or anti-β-2Μ antibody. The results shown in FIG. 19 show that IL-2/synTac remains stable and intact for at least 2 hours in vivo.

IL-2/synTac was stored at 4°C or 37°C for 5 days. 0.5 mg of each sample (at 10 mg/ml) was analyzed by size exclusion chromatography. As shown in FIG. 20, IL-2/synTac is stable and intact for at least 5 days at 4 or 37°C.

- 87 046369

Example 5: IL-2L>upTac-mediated expansion of human CMV-specific CD8+ T cells.

Peripheral blood mononuclear cells (PBMCs) from human donors were screened for reactivity to a pool of cytomegalovirus (CMV) peptides using an enzyme-linked immunosorbent spot (ELISPOT) assay for IFN-gamma. PBMCs were classified according to the spot forming count (SFC) as high, intermediate, low, or non-CMV precursor groups. PBMCs from each group were stimulated with doses of IL-2/synTac (CUE: IL-2; synTac, including 2 copies of the variant IL-2 MOD containing the H16A and F42A substitutions) ranging from 30 to 2 nM. Fifty percent of the conditioned medium was replaced with fresh medium on day 5. On day 7, samples were stained with a panel of antibodies and analyzed by flow cytometry. Pentamer staining targeting the CMV peptide NLVPMVATV (SEQ ID NO: 37) was used to determine the frequency of antigen-specific CD8+ cells. Data are shown in FIG. 35. The _EC50 of IL-2/synTac has been determined to range from about 1 nM to about 5 nM. Fig. 35 demonstrates the fold increase in antigen-specific CD8+ cells compared to untreated controls. The numerical values on the x-axis represent the number of SFCs of each donor PBMC. Error bars represent the mean +/- SD values from technical copies of each data point.

The data shown in FIG. 35 indicate that IL-2/synTac is effective in expanding the number of epitope-specific CD8 ⁺ T cells when there is a measurable (eg, pentamer or SFC staining) precursor population of such epitope-specific CD8 ⁺ T cells.

Example 6. IL-2/synTac with amino acid substitutions in H16.

IL-2/synTac variants were generated with substitutions in H16. Expression levels and affinity for IL-2R were determined. Affinity for IL-2R was determined using BL. The data is shown in FIG. 36.

Example 7: Effects of IL-2/synTac on Primary Human Antigen-Specific CD8 ⁺ T Cells.

The IL-2/synTac variant bound to primary human CD8 ⁺ T cells. The IL-2/synTac variant includes

i) HPV16 E7 (11-20) (YMLDLQPETT; SEQ ID NO: 13) as an epitope presenting peptide; and ii) 2 copies of variant IL-2 MOD containing substitutions H16A and F42A). The binding of variant IL-2/synTac to HPV16 E7-specific CD8 ⁺ T cells (11–20) or to bulk CD8 ⁺ T cells was assessed. The data is shown in FIG. 37.

Fig. 37 demonstrates binding of the IL-2/synTac variant of the present invention to primary human E7 HPV16(11-20)-specific CD8 ⁺ T cells as detected by flow cytometry. The _EC50 for binding to HPV16(11-20) E7-specific CD8 ⁺ T cells was 2.6 nM. Thus, the variant IL-2/synTac exhibited high-affinity interaction with tumor antigen-specific primary human T cells. Binding was highly selective for antigen-specific T cells compared to binding to nontarget (general population) CD8 ⁺ T cells.

The effect of variant IL-2/synTac binding to primary E7 HPV16(11-20)-specific human CD8 ⁺ T cells on phosphorylation of the T cell receptor proximal marker (TCR) SLP76 was assessed. The data is shown in FIG. 38.

Fig. 38 demonstrates the effect of variant IL-2/synTac binding to primary E7 HPV16(11-20)-specific human CD8 ⁺ T cells on SLP76 phosphorylation. Binding of variant IL-2/synTac to primary E7 HPV16(11-20)-specific human CD8 ⁺ T cells resulted in a rapid increase in SLP76 phosphorylation. The effect was strong (EC ₅₀ =65 nM). The effect was also selective, as the control IL-2/synTac, which contains a CMV peptide instead of HPV16(11-20) E7, resulted in only low levels of SLP76 phosphorylation.

Key markers of T cell activation and cytolytic activity were assessed. Primary E7 HPV16(11-20)-specific human T cells were incubated for 2 days with 0 or 100 nM variant IL-2/synTac. The IL-2/synTac variant includes

i) HPV16 E7 (11-20) as epitope-presenting peptide; and ii) 2 copies of the variant IL-2 MOD containing the H16A and F42A substitutions.

Evaluated products

i) CD25, a marker of CD8 ⁺ T cell activation;

ii) granzyme B, a key mediator of target cell death through the granule-mediated pathway; and iii) CD107a, a marker of degranulation on CD8 ⁺ T cells.

The data is shown in FIG. 39.

Fig. 39 demonstrates the effect of variant IL-2/synTac binding to primary E7 HPV16(11-20)-specific human T cells on the production of CD25, granzyme B and CD107a. Data show that binding of variant IL-2/synTac to primary E7 HPV16(11-20)-specific human T cells induces differentiation of T cells into cytolytic effector cells, o

-

Claims (44)

as evidenced by increased expression of CD25, granzyme B, and CD107a. The effect of variant IL-2/synTac binding to primary E7 HPV16(11-20)-specific human CD8+ T cells on IFN-γ production was assessed. An ELISpot assay was used to detect IFN-γ production. The data is shown in FIG. 40. Fig. 40 demonstrates the effect of variant IL-2/synTac binding to primary E7 HPV16(11-20)-specific human CD8+ T cells on IFN-γ production. Data show that binding of variant IL-2/synTac to primary E7 HPV16(11-20)-specific human CD8+ T cells resulted in dose-dependent secretion of IFN-γ. No IFN-γ production was observed with the control IL-2/synTac, which contains a CMV peptide in place of HPV16 E7 ( 11 – 20 ). Although the present invention has been described with reference to specific embodiments thereof, those skilled in the art will appreciate that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, 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 this invention. All such modifications are intended to come within the scope of the appended claims. CLAIM 1. Multimeric polypeptide containing a heterodimer containing a) the first polypeptide containing i) an epitope of human papillomavirus, and ii) a first major histocompatibility complex (MHC) class I polypeptide, wherein the first MHC peptide is a b2-microglobulin (b2M) polypeptide; b) a second polypeptide containing i) a second MHC class I polypeptide, wherein the second MHC class I polypeptide is an HLA-A heavy chain polypeptide, ii) at least one immunomodulatory polypeptide, wherein the at least one immunomodulatory polypeptide is a variant IL-2 polypeptide that contains an amino acid a sequence having at least 98% amino acid sequence identity with the amino acid sequence set forth in SEQ ID NO: 44, wherein amino acid 16 is Ala and amino acid 42 is Ala, where the percentage identity is determined by sequence alignment performed by BLAST, and iii) Fc- immunoglobulin polypeptide (Ig). 2. Multimeric polypeptide according to claim 1, where the specified HPV epitope is represented by the HPV E7 epitope. 3. The multimeric polypeptide according to claim 1 or 2, wherein the b2M polypeptide contains an amino acid sequence having at least 95% amino acid sequence identity with amino acids 21-119 in SEQ ID NO: 95. 4. Multimeric polypeptide according to any one of claims 1 to 3, wherein the B2M polypeptide contains the amino acid sequence presented in SEQ ID NO: 17. 5. The multimeric polypeptide according to any one of claims 1 to 4, wherein the HLA-A heavy chain polypeptide contains an amino acid sequence having at least 95% amino acid sequence identity with the amino acid sequence presented in SEQ ID NO: 19, where the percentage of identity is determined by alignment sequences by BLAST. 6. The multimeric polypeptide of claim 5, wherein the HLA-A heavy chain polypeptide contains a Cys at residue 236 of the HLA-A heavy chain polypeptide based on the numbering set forth in SEQ ID NO: 19. 7. A multimeric polypeptide as claimed in any one of claims 1 to 6, wherein the Ig Fc polypeptide comprises an amino acid sequence having at least about 95% amino acid sequence identity to SEQ ID NO: 57, wherein the percentage identity is determined by a sequence alignment performed by BLAST. 8. The multimeric polypeptide of claim 7, wherein the Ig Fc polypeptide contains an L14A substitution and an L15A substitution based on the amino acid numbering set forth in SEQ ID NO: 57. 9. The multimeric polypeptide according to any one of claims 1 to 8, wherein the multimeric polypeptide contains two copies of the variant IL-2 polypeptide, wherein each copy contains the amino acid sequence of SEQ ID NO: 84. 10. The multimeric polypeptide of claim 9, wherein said first polypeptide comprises a peptide linker between the epitope and a β2-microglobulin polypeptide, and said second polypeptide comprises an independently selected peptide linker between one or more of a) a first copy of a variant IL-2 polypeptide and a second copy of a variant IL-2 polypeptide; b) one or two copies of the specified IL-2 polypeptide and the specified severe polypeptide - 89 046369 GKGS chain class I; and c) said MHC class I heavy chain polypeptide and Ig Fc polypeptide. 11. The multimeric polypeptide of claim 9, wherein the first polypeptide and the second polypeptide are covalently linked to each other via a disulfide bond, wherein the disulfide bond links the Cys at amino acid position 12 of the B2M polypeptide to the Cys at amino acid position 236 of the MHC class I heavy chain polypeptide. 12. Multimeric polypeptide according to claim 1, containing a) the first polypeptide containing in order from N-terminus to C-terminus i) an HPV epitope, ii) a peptide linker, iii) a B2M polypeptide, wherein the B2M polypeptide contains an amino acid sequence having at least 95% amino acid sequence identity with amino acids 21-119 in SEQ ID NO: 95; b) a second polypeptide containing in order from N-terminus to C-terminus i) variant IL-2 polypeptide, ii) peptide linker, iii) variant IL-2 polypeptide, iv) optional peptide linker, v) an HLA-A heavy chain polypeptide, wherein the HLA-A heavy chain polypeptide contains an amino acid sequence having at least 95% amino acid sequence identity with the amino acid sequence set forth in SEQ ID NO: 19, vi) an optional peptide linker, and vii ) an immunoglobulin Ig Fc polypeptide, wherein the Ig Fc polypeptide is an IgG1 Fc polypeptide that contains an amino acid sequence having at least about 95% amino acid sequence identity to SEQ ID NO: 57, wherein the peptide liqueurs are independently selected and where the percentage identity is determined by sequence alignment , carried out by BLAST. 13. The multimeric polypeptide of claim 12, wherein the first polypeptide and the second polypeptide are covalently linked to each other via a disulfide bond between the Cys residue at amino acid position 12 of the B2M polypeptide and the Cys residue at amino acid position 236 of the MHC class I heavy chain polypeptide. 14. The multimeric polypeptide according to claim 12, wherein said HPV epitope is an HPV E7 epitope. 15. Multimeric polypeptide according to claim 1, where the heterodimeric polypeptide contains a) the first polypeptide containing i) a human papillomavirus epitope, wherein the epitope comprises the amino acid sequence YMLDLQPETT (SEQ ID NO: 13), and ii) a first major histocompatibility complex (MHC) polypeptide, wherein the first MHC peptide is a b2-microglobulin (b2M) polypeptide containing the amino acid sequence given in SEQ ID NO: 17; And b) a second polypeptide containing i) two copies of an IL-2 polypeptide, wherein each copy contains the amino acid sequence set forth in SEQ ID NO: 84, ii) a second MHC class I polypeptide, wherein the second MHC polypeptide is a class I MHC heavy chain polypeptide containing the amino acid sequence set forth in SEQ ID NO: 19, and iii) an immunoglobulin Fc polypeptide (Ig). 16. The multimeric polypeptide of claim 15, wherein the Ig Fc polypeptide comprises an amino acid sequence having at least about 95% amino acid sequence identity to SEQ ID NO: 57, wherein the percentage identity is determined by a sequence alignment performed by BLAST, and wherein the Ig Fc polypeptide contains an L14A substitution and an L15A substitution based on the amino acid sequence numbering set forth in SEQ ID NO: 57, wherein the multimeric polypeptide comprises a covalent bond between a first polypeptide and a second polypeptide, and wherein the covalent bond comprises a disulfide bond between a Cys residue at amino acid position 12 of the B2M polypeptide and the residue Cys at amino acid position 236 of a class I MHC heavy chain polypeptide, where the first polypeptide contains a peptide linker between the epitope and a B2M polypeptide, and where the second polypeptide contains a peptide linker between one or more of a) a first copy of an IL-2 polypeptide and a second copy of an IL-2 polypeptide; b) one of two copies of the IL-2 polypeptide and the MHC class I heavy chain polypeptide; And c) MHC class I heavy chain polypeptide and Ig Fc polypeptide. - 90 046369 17. A homodimer containing two heterodimers according to any one of claims 1 to 16. 18. Pharmaceutical composition containing a homodimer according to claim 17. 19. Use of the pharmaceutical composition according to claim 18 for the production of a medicament for the treatment of HPV-associated cancer or HPV-related cancer in an individual. 20. Nucleic acid containing a) the first nucleotide sequence encoding the first polypeptide containing i) a human papillomavirus epitope, wherein the epitope comprises the amino acid sequence YMLDLQPETT (SEQ ID NO: 13), and ii) a first major histocompatibility complex (MHC) class I polypeptide, wherein the first MHC peptide is a b2-microglobulin (b2M) polypeptide containing the amino acid sequence given in SEQ ID NO: 17; And b) a second nucleotide sequence encoding a second polypeptide containing i) two copies of an IL-2 polypeptide, wherein each copy contains the amino acid sequence set forth in SEQ ID NO: 84, ii) a second MHC polypeptide, wherein the second MHC polypeptide is a class I MHC heavy chain polypeptide containing the amino acid sequence set forth in SEQ ID NO: 19, and iii) immunoglobulin Fc polypeptide (Ig). 21. The nucleic acid of claim 20, wherein the Ig Fc polypeptide comprises an amino acid sequence having at least about 95% amino acid sequence identity to SEQ ID NO: 57, wherein the percentage identity is determined by a sequence alignment performed by BLAST, and wherein The Ig Fc polypeptide contains an L14A substitution and a L15A substitution based on the amino acid sequence numbering set forth in SEQ ID NO: 57, where the first polypeptide contains a peptide linker between an epitope and a B2M polypeptide, and where the second polypeptide contains a peptide linker between one or more of a) a first copy of an IL-2 polypeptide and a second copy of an IL-2 polypeptide; b) one of two copies of the IL-2 polypeptide and the MHC class I heavy chain polypeptide; And c) MHC class I heavy chain polypeptide and Ig Fc polypeptide. 22. An expression vector containing the nucleic acid according to claim 20 or 21. 23. An expression host cell that is genetically modified with the expression vector of claim 22, wherein the host cell is in vitro. 24. An expression host cell that is genetically modified with the nucleic acid of claim 20 or 21, wherein the host cell is in vitro. 25. A method for producing the first and second polypeptides encoded by the nucleic acid of claim 20 or 21, where the method includes cultivating the genetically modified expression host cell of claim 23 or 24 in a culture medium under conditions under which said cell synthesizes the first and second polypeptides . 26. Composition of the first and second nucleic acids to obtain a heterodimer, where a) the first nucleic acid contains a first nucleotide sequence encoding a first polypeptide containing i) a human papillomavirus epitope, wherein the epitope comprises the amino acid sequence YMLDLQPETT (SEQ ID NO: 13), and ii) a first major histocompatibility complex (MHC) polypeptide, wherein the first MHC peptide is a b2-microglobulin (b2M) polypeptide containing the amino acid sequence set forth in SEQ ID NO: 17; and b) the second nucleic acid contains a second nucleotide sequence encoding a second polypeptide containing i) two copies of an IL-2 polypeptide, each copy containing the amino acid sequence set forth in SEQ ID NO: 84, ii) a second MHC polypeptide, wherein the second MHC polypeptide is a class I MHC heavy chain polypeptide containing the amino acid sequence set forth in SEQ ID NO: 19, and iii) immunoglobulin Fc polypeptide (Ig). 27. The nucleic acid composition of claim 26, wherein the Ig Fc polypeptide comprises an amino acid sequence having at least about 95% amino acid sequence identity to SEQ ID NO: 57, wherein the percentage identity is determined by a sequence alignment performed by BLAST, and wherein the Ig Fc polypeptide contains an L14A substitution and an L15A substitution based on the amino acid sequence numbering set forth in SEQ ID NO: 57, where the first polypeptide contains a peptide linker between an epitope and a B2M polypeptide, and wherein the second polypeptide contains a peptide linker between one or more of a) a first copy of an IL-2 polypeptide and a second copy of an IL-2 polypeptide; - 91 046369 b) one of two copies of the IL-2 polypeptide and the MHC class I heavy chain polypeptide; And c) MHC class I heavy chain polypeptide and Ig Fc polypeptide. 28. A composition for producing a heterodimer, where the composition contains one or more expression vectors containing nucleic acid molecules according to claim 26 or 27. 29. An expression host cell that is genetically modified with one or more expression vectors according to claim 28, wherein the host cell is in vitro. 30. An expression host cell that is genetically modified with the nucleic acids of claim 26 or 27, wherein the host cell is in vitro. 31. A method for producing a heterodimer encoded by a nucleic acid according to claim 26 or 27, where the method includes culturing the expressing host cell according to claim 29 or 30 in a culture medium under conditions under which said cell synthesizes the first and second polypeptides. 32. Heterodimer containing a) the first polypeptide containing i) a cancer-associated epitope other than that of human papillomavirus, ii) a first major histocompatibility complex (MHC) class I polypeptide, wherein the first MHC peptide is a b2-microglobulin (b2M) polypeptide containing an amino acid sequence having at least 95 % amino acid sequence identity to amino acids 21-119 in SEQ ID NO: 95, where the percentage identity is determined by sequence alignment performed by BLAST; b) a second polypeptide containing i) two copies of an IL-2 polypeptide, wherein each copy contains the amino acid sequence set forth in SEQ ID NO: 84, wherein the two IL-2 polypeptides are linked by a peptide linker, ii) a second MHC polypeptide, wherein the second MHC polypeptide is a MHC heavy chain polypeptide class I, and iii) immunoglobulin Fc polypeptide (Ig). 33. The heterodimer of claim 32, wherein the Ig Fc polypeptide is an IgG1 Fc polypeptide comprising an amino acid sequence having at least about 95% amino acid sequence identity to SEQ ID NO: 57, wherein the percent identity is determined by a sequence alignment performed by BLAST, and wherein the Ig Fc polypeptide contains an L14A substitution and an L15A substitution based on the amino acid sequence numbering set forth in SEQ ID NO: 57, and the second polypeptide contains a linker between one or more of a) a first copy of an IL-2 polypeptide and a second copy of an IL-2 polypeptide; b) one of two copies of the IL-2 polypeptide and the MHC heavy chain polypeptide; And c) MHC heavy chain polypeptide and IgG1 Fc polypeptide. 34. The heterodimer of claim 32 or 33, wherein the multimeric polypeptide comprises a covalent bond between the first polypeptide and the second polypeptide and wherein the covalent bond comprises a disulfide bond that connects (i) a Cys residue at amino acid position 12 of the polypeptide in 2M and (ii) a Cys residue in amino acid position 236 of the MHC class I heavy chain polypeptide based on the numbering shown in SEQ ID NO: 19. 35. A homodimer comprising two heterodimers according to any one of claims 32 to 34, wherein the two heterodimers are linked to each other by one or more disulfide bonds that connect the Ig Fc polypeptide of one heterodimer to the Ig Fc polypeptide of the other heterodimer. 36. Pharmaceutical composition containing a heterodimer according to any one of claims 32-35. 37. Use of the pharmaceutical composition according to claim 36 for the production of a medicament for the treatment of cancer in an individual. 38. Use according to claim 37, wherein the homodimer is administered in an amount of from about 1 mg/kg to about 5 mg/kg body weight. 39. Use according to claim 37, wherein the homodimer is administered in an amount of from about 5 mg/kg to about 10 mg/kg body weight. 40. Nucleic acid containing a) a first nucleotide sequence encoding the first polypeptide according to any one of claims 32-34; And b) a second nucleotide sequence encoding a second polypeptide according to any one of claims 32-34. 41. An expression vector containing the nucleic acid according to claim 40. 42. An expression host cell that is genetically modified with the expression vector of claim 41, wherein the host cell is in vitro. 43. A method for producing the first and second polypeptides encoded by the nucleic acid of claim 40, where the method includes cultivating the expression host cell of claim 42 in a culture medium under conditions under which said cell synthesizes the first and second polypeptides. 44. A composition containing first and second nucleic acids, where -