Interleukin-2 Polypeptides, Fusion Polypeptides, and Methods of Use Thereof - Patent application

Abstract

Disclosed are IL-2 variant polypeptides with reduced affinity for IL-2Rα and IL-2Rβ, and compositions comprising one or more IL-2 variant polypeptides, e.g., fusion polypeptides, and methods for their use in treatments involving, e.g., cancer vaccines, TCR-T cell therapy, and CAR-T cell therapy. Such polypeptides do not systemically activate multiple immune cell subsets as native IL-2 delivered at high doses does, but rather primarily activate only T cells whose T cell receptors (TCRs) engage peptide-MHC complexes (pMHC) presented by antigen-presenting cells, thus providing a useful therapeutic index for pharmaceutical compositions comprising such polypeptides.

Description

[Technical Field]

[0001] cross reference This application claims the benefit of U.S. Provisional Patent Application No. 63 / 405,751, filed September 12, 2022, which is incorporated herein by reference in its entirety.

[0002] Incorporation by Reference of Electronically Submitted Materials The Sequence Listing is provided herein as Sequence Listing XML "CUEB-144WO_SEQ_LIST", created on September 11, 2023, and having a size of 272,239 bytes. The contents of the Sequence Listing XML are incorporated herein by reference in their entirety. [Background technology]

[0003] preface Interleukin-2 (IL-2) is a cytokine produced by helper T cells to amplify immune responses. IL-2 has many immunostimulatory and immunoregulatory functions, including T cell proliferation, activation of the cytotoxic functions of natural killer (NK) cells, T lymphocytes, and monocytes, and activation of regulatory T cells (Tregs).

[0004] The known ability of IL-2 to activate T cells led to the development of aldesleukin, a recombinant IL-2 product marketed under the trade name Proleukin®. Aldesleukin has been approved for use in the treatment of metastatic melanoma and renal cell carcinoma, with the hope that IL-2 will activate sufficient numbers of cancer-specific T cells in patients that can reduce or kill the cancer. Unfortunately, aldesleukin is a potent stimulator of T cells and is associated with potentially severe side effects, including coma or even death. Therefore, it is recommended that aldesleukin be administered only in hospital settings where cardiopulmonary or intensive care medicine specialists are readily available.

[0005] Over the years, numerous variants of IL-2 have been investigated to obtain the beneficial activities of IL-2 while reducing its known side effects. Because the IL-2 receptor (IL-2R) on T cells contains three polypeptide chains, i.e., IL-2Rα (also known as CD25), IL-2Rβ (also known as CD122), and IL-2Rγ (also known as CD132), such variants typically increase the affinity of the IL-2 polypeptide for one or more of the IL-2R receptor chains and / or decrease the affinity of the IL-2 polypeptide for one or more of the IL-2R receptor chains. For example, binding of IL-2 to IL-2Rα is known to upregulate the activity of Tregs. Therefore, numerous "non-alpha" IL-2 variants, with substantially reduced ability to bind to IL-2Rα, have been developed for applications in which Treg activation is undesirable.

[0006] IL-2 variants that reduce the affinity of IL-2 to bind to both IL-2Rα and IL-2Rβ have been described. See, for example, WO2018 / 119114 (Patent Document 1) (Cue Biopharma, Inc.) and Quayle et al., Clin Cancer Res 2020;26:1953-64 (Non-Patent Document 1), which disclose IL-2 variants containing mutations that substantially eliminate binding to IL-2Rα and also significantly reduce binding affinity to IL-2Rβ.

[0007] IL-2 variant polypeptides, in which the IL-2 variant polypeptides have such reduced affinity for IL-2Rα and IL-2Rβ, and compositions comprising one or more IL-2 variant polypeptides, e.g., fusion polypeptides, have been found to have unexpected properties that can provide benefits in many different medical applications. More specifically, it has been found that IL-2 variant polypeptides and compositions comprising one or more IL-2 variant polypeptides, e.g., fusion polypeptides, do not systemically activate multiple immune cell subsets as does native IL-2 delivered in high doses, but rather predominantly activate only T cells whose T cell receptors (TCRs) engage peptide-MHC complexes (pMHC) presented by antigen-presenting cells, thus providing a useful therapeutic index for pharmaceutical compositions comprising such IL-2 variant polypeptides or compositions comprising one or more IL-2 variant polypeptides, e.g., fusion polypeptides. Furthermore, it has been found that such IL-2 variant polypeptides, and compositions comprising one or more IL-2 variant polypeptides, e.g., fusion polypeptides, when engaging with IL-2 receptors on chimeric antigen receptor-T cells (CAR-T cells), TCR-T cells, and other cytotoxic cells (e.g., macrophages and NK cells) bearing exogenous activating receptors such as chimeric antigen receptors (CARs) and TCRs, can provide homeostatic signals that can prolong the survival of such cells and / or provide activation signals to cells that cause the cells to proliferate and retain their cytotoxic function. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] WO2018 / 119114 [Non-patent literature]

[0009] [Non-Patent Document 1] Quayle et al., Clin Cancer Res 2020;26:1953-64 Summary of the Invention

[0010] overview Disclosed herein are methods of using IL-2 variants, and fusion polypeptides comprising such variants, which variants have reduced binding to IL-2Rα and IL-2Rβ. [Brief explanation of the drawings]

[0011] [Figure 1] 1A-1C are schematic diagrams of higher order forms of IL-2 variant polypeptides of the present disclosure. [Figure 2] 2A-2C are schematic diagrams of fusion polypeptides containing a variant IL-2 polypeptide at the N-terminal position. [Figure 3] 3A-3C are schematic diagrams of fusion polypeptides containing a variant IL-2 polypeptide at the C-terminal position. [Figure 4] 4A-4C are schematic diagrams of fusion polypeptides containing variant IL-2 polypeptides at the N-terminal and C-terminal positions. [Figure 5] 5A-5B are schematic diagrams of fusion polypeptide homodimers containing a variant IL-2 polypeptide at the N-terminal position. [Figure 6] 6A-6B are schematic diagrams of fusion polypeptide homodimers containing a variant IL-2 polypeptide at the C-terminal position. [Figure 7] 7A-7B are schematic diagrams of fusion polypeptide homodimers containing variant IL-2 polypeptides at the N-terminal and C-terminal positions. [Figure 8A] FIG. 1 is a schematic diagram of a fusion polypeptide heterodimer comprising a variant IL-2 polypeptide at the N-terminal and C-terminal positions. [Figure 8B] FIG. 1 is a schematic diagram of a fusion polypeptide heterodimer comprising a variant IL-2 polypeptide at the N-terminal and C-terminal positions. [Figure 8C] 1 is a schematic diagram of a fusion polypeptide heterodimer comprising a variant IL-2 polypeptide at the N-terminal and C-terminal positions. [Figure 8D] FIG. 1 is a schematic diagram of a fusion polypeptide heterodimer comprising a variant IL-2 polypeptide at the N-terminal and C-terminal positions. [Figure 8E] FIG. 1 is a schematic diagram of a fusion polypeptide heterodimer comprising a variant IL-2 polypeptide at the N-terminal and C-terminal positions. [Figure 8F] FIG. 1 is a schematic diagram of a fusion polypeptide heterodimer comprising a variant IL-2 polypeptide at the N-terminal and C-terminal positions. [Figure 9A] The amino acid sequences of the immunoglobulin Fc polypeptides are provided (SEQ ID NOS: 23-35, respectively). [Figure 9B] The amino acid sequences of the immunoglobulin Fc polypeptides are provided (SEQ ID NOS: 23-35, respectively). [Figure 9C] The amino acid sequences of the immunoglobulin Fc polypeptides are provided (SEQ ID NOS: 23-35, respectively). [Figure 9D] The amino acid sequences of the immunoglobulin Fc polypeptides are provided (SEQ ID NOS: 23-35, respectively). [Figure 9E] The amino acid sequences of the immunoglobulin Fc polypeptides are provided (SEQ ID NOS: 23-35, respectively). [Figure 9F] The amino acid sequences of the immunoglobulin Fc polypeptides are provided (SEQ ID NOS: 23-35, respectively). [Figure 9G] The amino acid sequences of the immunoglobulin Fc polypeptides are provided (SEQ ID NOS: 23-35, respectively). [Figure 9H] The amino acid sequences of the immunoglobulin Fc polypeptides are provided (SEQ ID NOS: 23-35, respectively). [Figure 9I] The amino acid sequences of the immunoglobulin Fc polypeptides are provided (SEQ ID NOS: 23-35, respectively). [Figure 9J]The amino acid sequences of the immunoglobulin Fc polypeptides are provided (SEQ ID NOS: 23-35, respectively). [Figure 9K] The amino acid sequences of the immunoglobulin Fc polypeptides are provided (SEQ ID NOS: 23-35, respectively). [Figure 9L] The amino acid sequences of the immunoglobulin Fc polypeptides are provided (SEQ ID NOS: 23-35, respectively). [Figure 9M] The amino acid sequences of the immunoglobulin Fc polypeptides are provided (SEQ ID NOS: 23-35, respectively). [Figure 10] 10A-10C are schematic diagrams of fusion polypeptides comprising a variant IL-2 polypeptide at the N-terminal position and one or more additional proteins (designated "A") at the C-terminal position. [Figure 11] 11A-11C are schematic diagrams of fusion polypeptides comprising a variant IL-2 polypeptide at the C-terminal position and one or more additional proteins (designated "A") at the N-terminal position. [Figure 12] 12A-12B are schematic diagrams of fusion polypeptide homodimers comprising a variant IL-2 polypeptide at the N-terminal position and one or more additional proteins (designated "A") at the C-terminal position. [Figure 13] 13A-13B are schematic diagrams of fusion polypeptide homodimers comprising a variant IL-2 polypeptide at the C-terminal position and one or more additional proteins (designated "A") at the N-terminal position. [Figure 14A] FIG. 1 is a schematic diagram of a fusion polypeptide heterodimer comprising a variant IL-2 polypeptide at the N- and / or C-terminal position and one or more additional proteins (designated "A") at the N- and / or C-terminal position. [Figure 14B] FIG. 1 is a schematic diagram of a fusion polypeptide heterodimer comprising a variant IL-2 polypeptide at the N- and / or C-terminal position and one or more additional proteins (designated "A") at the N- and / or C-terminal position. [Figure 14C]FIG. 1 is a schematic diagram of a fusion polypeptide heterodimer comprising a variant IL-2 polypeptide at the N- and / or C-terminal position and one or more additional proteins (designated "A") at the N- and / or C-terminal position. [Figure 14D] FIG. 1 is a schematic diagram of a fusion polypeptide heterodimer comprising a variant IL-2 polypeptide at the N- and / or C-terminal position and one or more additional proteins (designated "A") at the N- and / or C-terminal position. [Figure 14E] FIG. 1 is a schematic diagram of a fusion polypeptide heterodimer comprising a variant IL-2 polypeptide at the N- and / or C-terminal position and one or more additional proteins (designated "A") at the N- and / or C-terminal position. [Figure 14F] FIG. 1 is a schematic diagram of a fusion polypeptide heterodimer comprising a variant IL-2 polypeptide at the N- and / or C-terminal position and one or more additional proteins (designated "A") at the N- and / or C-terminal position. [Figure 14G] FIG. 1 is a schematic diagram of a fusion polypeptide heterodimer comprising a variant IL-2 polypeptide at the N- and / or C-terminal position and one or more additional proteins (designated "A") at the N- and / or C-terminal position. [Figure 14H] FIG. 1 is a schematic diagram of a fusion polypeptide heterodimer comprising a variant IL-2 polypeptide at the N- and / or C-terminal position and one or more additional proteins (designated "A") at the N- and / or C-terminal position. [Figure 14I] FIG. 1 is a schematic diagram of a fusion polypeptide heterodimer comprising a variant IL-2 polypeptide at the N- and / or C-terminal position and one or more additional proteins (designated "A") at the N- and / or C-terminal position. [Figure 14J]FIG. 1 is a schematic diagram of a fusion polypeptide heterodimer comprising a variant IL-2 polypeptide at the N- and / or C-terminal position and one or more additional proteins (designated "A") at the N- and / or C-terminal position. [Figure 14K] FIG. 1 is a schematic diagram of a fusion polypeptide heterodimer comprising a variant IL-2 polypeptide at the N- and / or C-terminal position and one or more additional proteins (designated "A") at the N- and / or C-terminal position. [Figure 14L] FIG. 1 is a schematic diagram of a fusion polypeptide heterodimer comprising a variant IL-2 polypeptide at the N- and / or C-terminal position and one or more additional proteins (designated "A") at the N- and / or C-terminal position. [Figure 14M] FIG. 1 is a schematic diagram of a fusion polypeptide heterodimer comprising a variant IL-2 polypeptide at the N- and / or C-terminal position and one or more additional proteins (designated "A") at the N- and / or C-terminal position. [Figure 14N] FIG. 1 is a schematic diagram of a fusion polypeptide heterodimer comprising a variant IL-2 polypeptide at the N- and / or C-terminal position and one or more additional proteins (designated "A") at the N- and / or C-terminal position. [Figure 15A] 1 provides a comparison of the binding signals of CUE-1646 versus CUE-1647 on human IL-2Rα and IL-2Rβ (see Example 1). [Figure 15B] 1 provides a comparison of the binding signals of CUE-1646 versus CUE-1647 on human IL-2Rα and IL-2Rβ (see Example 1). [Figure 15C] 1 provides a comparison of the binding signals of CUE-1646 versus CUE-1647 on human IL-2Rα and IL-2Rβ (see Example 1). [Figure 15D] , provides a comparison of the binding signals of CUE-1646 versus CUE-1647 on human IL-2Rα and IL-2Rβ (see Example 1). [Figure 16A]Representative sensorgrams and corresponding fits for CUE-1646 and CUE-1647 binding to human IL-2Rα and IL-2Rβ are provided (see Example 1). [Figure 16B] Representative sensorgrams and corresponding fits for CUE-1646 and CUE-1647 binding to human IL-2Rα and IL-2Rβ are provided (see Example 1). [Figure 16C] Representative sensorgrams and corresponding fits for CUE-1646 and CUE-1647 binding to human IL-2Rα and IL-2Rβ are provided (see Example 1). [Figure 16D] Representative sensorgrams and corresponding fits for CUE-1646 and CUE-1647 binding to human IL-2Rα and IL-2Rβ are provided (see Example 1). [Figure 17] Table 4 is provided showing the mean affinity and kinetic measurements of CUE-1646 and CUE-1647 binding to human IL-2Rα and IL-2Rβ (see Example 1). [Figure 18A] The amino acid sequences of exemplary anti-mesothelin scFvs (SEQ ID NOS: 88-95, respectively) are provided. [Figure 18B] The amino acid sequences of exemplary anti-mesothelin scFvs (SEQ ID NOS: 88-95, respectively) are provided. [Figure 18C] The amino acid sequences of exemplary anti-mesothelin scFvs (SEQ ID NOS: 88-95, respectively) are provided. [Figure 18D] The amino acid sequences of exemplary anti-mesothelin scFvs (SEQ ID NOS: 88-95, respectively) are provided. [Figure 18E] The amino acid sequences of exemplary anti-mesothelin scFvs (SEQ ID NOS: 88-95, respectively) are provided. [Figure 18F] The amino acid sequences of exemplary anti-mesothelin scFvs (SEQ ID NOS: 88-95, respectively) are provided. [Figure 18G] The amino acid sequences of exemplary anti-mesothelin scFvs (SEQ ID NOS: 88-95, respectively) are provided. [Figure 18H] The amino acid sequences of exemplary anti-mesothelin scFvs (SEQ ID NOS: 88-95, respectively) are provided. [Figure 19A] The amino acid sequences of exemplary anti-TROP-2 scFvs (SEQ ID NOs: 262-265, respectively) are provided. [Figure 19B] The amino acid sequences of exemplary anti-TROP-2 scFvs (SEQ ID NOs: 262-265, respectively) are provided. [Figure 19C] The amino acid sequences of exemplary anti-TROP-2 scFvs (SEQ ID NOs: 262-265, respectively) are provided. [Figure 19D] The amino acid sequences of exemplary anti-TROP-2 scFvs (SEQ ID NOs: 262-265, respectively) are provided. [Figure 20] The amino acid sequence of an exemplary variant IL-2 fusion polypeptide (SEQ ID NO:266) is provided. [Figure 21] The amino acid sequence of the polypeptide designated "1646" (SEQ ID NO: 255) is provided. [Figure 22] The amino acid sequence of the polypeptide designated "1647" (SEQ ID NO: 256) is provided. [Figure 23] Figures 23A-23B provide the amino acid sequences of a variant IL-2 / Fc fusion polypeptide designated "2657" or "Rgt-2657" (Figure 23A, SEQ ID NO: 257) and a variant IL-2 / Fc fusion polypeptide designated "2657Δ" (Figure 23B, SEQ ID NO: 267), which lacks the C-terminal Lys. [Figure 24] 1 provides the amino acid sequence of the IL-2 / Fc fusion polypeptide designated "3151" or "Rgt-3151" (SEQ ID NO: 258). [Figure 25] Figures 25A-25B provide the amino acid sequences of a variant IL-2 / Fc fusion polypeptide designated "2656" or "Rgt-2656" (Figure 25A, SEQ ID NO: 259) and a variant IL-2 / Fc fusion polypeptide designated "2656Δ" (Figure 25B, SEQ ID NO: 268), which lacks the C-terminal Lys. [Figure 26]Figures 26A-26B provide the amino acid sequences of the polypeptides designated "4123" (Figure 26A, SEQ ID NO: 260) and "4124" (Figure 26B, SEQ ID NO: 261). [Figure 27] 1 shows the effect of the IL-2 / Fc fusion polypeptide Rgt-2657 on the proliferation of NK cells. [Figure 28] 28A-28B show the effect of IL-2 / Fc fusion polypeptide Rgt-2657 on antigen-specific CD8+ T cells in vivo. [Figure 29] 1 shows the effect of IL-2 valency on the proliferation of CTLL-2 cells. [Figure 30A] FIG. 1 is a schematic diagram of a heterodimeric fusion polypeptide comprising a variant IL-2 polypeptide and, in some cases, one or more additional proteins (designated "A"). [Figure 30B] FIG. 1 is a schematic diagram of a heterodimeric fusion polypeptide comprising a variant IL-2 polypeptide and, in some cases, one or more additional proteins (designated "A"). [Figure 30C] FIG. 1 is a schematic diagram of a heterodimeric fusion polypeptide comprising a variant IL-2 polypeptide and, in some cases, one or more additional proteins (designated "A"). [Figure 30D] FIG. 1 is a schematic diagram of a heterodimeric fusion polypeptide comprising a variant IL-2 polypeptide and, in some cases, one or more additional proteins (designated "A"). [Figure 30E] FIG. 1 is a schematic diagram of a heterodimeric fusion polypeptide comprising a variant IL-2 polypeptide and, in some cases, one or more additional proteins (designated "A"). [Figure 30F] FIG. 1 is a schematic diagram of a heterodimeric fusion polypeptide comprising a variant IL-2 polypeptide and, in some cases, one or more additional proteins (designated "A"). [Figure 30G]FIG. 1 is a schematic diagram of a heterodimeric fusion polypeptide comprising a variant IL-2 polypeptide and, in some cases, one or more additional proteins (designated "A"). [Figure 30H] FIG. 1 is a schematic diagram of a heterodimeric fusion polypeptide comprising a variant IL-2 polypeptide and, in some cases, one or more additional proteins (designated "A"). DETAILED DESCRIPTION OF THE INVENTION

[0012] definition The terms "polynucleotide" and "nucleic acid" are used interchangeably herein to refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, the term includes, but is not limited to, single-, double-, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers containing purine and pyrimidine bases, or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases.

[0013] The terms "peptide," "polypeptide," and "protein" are used interchangeably herein to refer to polymeric forms of amino acids of any length, and may include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides with modified peptide backbones.

[0014] A polynucleotide or polypeptide has a certain percentage of "sequence identity" to another polynucleotide or polypeptide, meaning that when two sequences are compared and aligned, that percentage of bases or amino acids are the same and in the same relative positions. Sequence identity can be determined in many different ways; for example, sequences can be aligned using a variety of convenient methods and computer programs, such as BLAST (Basic Local Alignment Search Tool), which is available on the World Wide Web at sites including https: / / blast.ncbi.nlm.nih.gov / Blast.cgi. Unless otherwise specified, "sequence identity" referred to herein is determined by BLAST with default settings selected.

[0015] The term "conservative amino acid substitution" refers to the interchangeability of amino acid residues in proteins with similar side chains. For example, the group of amino acids with aliphatic side chains consists of glycine, alanine, valine, leucine, and isoleucine; the group of amino acids with aliphatic hydroxyl side chains consists of serine and threonine; the group of amino acids with amide-containing side chains consists of asparagine and glutamine; the group of amino acids with aromatic side chains consists of phenylalanine, tyrosine, and tryptophan; the group of amino acids with basic side chains consists of lysine, arginine, and histidine; the group of amino acids with acidic side chains consists of glutamic acid and aspartic acid; and the group of amino acids with sulfur-containing side chains consists of cysteine ​​and methionine. Exemplary conservative amino acid substitution groups are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine-glycine, and asparagine-glutamine.

[0016] "T cells" are helper T cells (CD4 + cells), cytotoxic T cells (CD8 +These include all types of immune cells that express CD3, including T cells, regulatory T cells (Tregs), and NK-T cells.

[0017] As used herein, "recombinant" means that a particular nucleic acid (DNA or RNA) is the product of various combinations of cloning, restriction, polymerase chain reaction (PCR), and / or ligation steps that result in a construct having structural coding or non-coding sequences distinguishable from the endogenous nucleic acid present in a natural system. A DNA sequence encoding a polypeptide may be assembled from cDNA fragments or a series of synthetic oligonucleotides to provide a synthetic nucleic acid that can be expressed from a recombinant transcription unit contained within a cellular or cell-free transcription and translation system.

[0018] The term "recombinant expression vector" is used interchangeably herein to refer to a DNA molecule comprising a vector and at least one insert. Recombinant expression vectors are typically constructed for the purpose of expressing and / or propagating an insert or for the construction of other recombinant nucleotide sequences. The insert may or may not be operably linked to a promoter sequence and may or may not be operably linked to DNA regulatory sequences.

[0019] The term "nanobody" (Nb) as used herein refers to the smallest antigen-binding fragment or single variable domain (V) derived from a naturally occurring heavy chain antibody. HH), and are known to those skilled in the art. They are derived from heavy chain-only antibodies found in camelids (see Hamers-Casterman et al. (1993) Nature 363:446; Desmyter et al. (1996) Nature Structural Biol. 3:803; and Desmyter et al. (2015) Curr. Opin. Struct. Biol. 32:1). In the "camelid" family, immunoglobulins that lack light polypeptide chains are found. "Camelids" includes Old World camelids (Bactrian camels (Camelus bactrianus) and dromedaries (Camelus dromedarius)) and New World camelids (e.g., alpacas (Llama paccos), Llama glama (Llama glama), guanacos (Llama guanicoe), and Llama vicugna (Llama vicugna)). Single variable domain heavy chain antibodies are referred to herein as nanobodies or V HH They are called antibodies.

[0020] "Single-chain Fv" or "sFv" or "scFv" antibody fragments are fragments of the V of an antibody. H and V L domains, and these domains are present in a single polypeptide chain. In some embodiments, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains that enables the sFv to form the desired structure for antigen binding. For a review of sFvs, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0021] As used herein, the term "CDR" or "complementarity-determining region" is intended to mean the non-contiguous antigen-binding sites found within the variable regions of both heavy and light chain polypeptides. CDRs are described by Kabat et al. (1977) J. Biol. Chem. 252:6609, Kabat et al., US Dept. of Health and Human Services, "Sequences of proteins of immunological interest" (1991) (also referred to herein as Kabat 1991), Chothia et al. (1987) J. Mol. Biol. 196:901 (also referred to herein as Chothia 1987), and MacCallum et al. (1996) J. Mol. Biol. 262:732 (also referred to herein as MacCallum 1996), and the definitions include overlapping or subsets of amino acid residues when compared with each other. Nevertheless, application of either definition to refer to the CDRs of an antibody or grafted antibody or variant thereof is intended to be within the scope of the term as defined and used herein. The amino acid residues that encompass the CDRs as defined by each of the above cited references are set forth in Table 1 below for comparison.

[0022] (Table 1) CDR definitions TIFF2025532538000002.tif33132 1 Residue numbering follows the nomenclature of Kabat et al., 1991 (supra). 2 Residue numbering follows the nomenclature of Chothia et al. (1987), supra. 3 Residue numbering follows the nomenclature of MacCallum et al. (1996) (supra).

[0023] As used herein, the terms "CDR-H1," "CDR-H2," and "CDR-H3" refer to the first, second, and third CDRs, respectively, in a heavy chain variable region. The terms "CDR-H1," "CDR-H2," and "CDR-H3" can be used interchangeably with "VH CDR1," "VH CDR2," and "VH CDR3," respectively. As used herein, the terms "CDR-1," "CDR-2," and "CDR-3" refer to the first, second, and third CDRs, respectively, of the variable region of either chain.

[0024] As used herein, the term "affinity" refers to the equilibrium constant for the reversible binding of two agents (e.g., an antibody and an antigen), and is also referred to as the dissociation constant (K D )

[0025] As used herein, the term "binding" refers to a non-covalent interaction between two molecules. Non-covalent binding refers to a direct association between two molecules through electrostatic, hydrophobic, ionic, and / or hydrogen-bonding interactions, including, for example, interactions such as salt bridges and water bridges. As used herein, "covalent binding" or "covalent bond" refers to the formation of one or more covalent chemical bonds between two different molecules.

[0026] As used herein, the terms "treatment," "treating," and the like generally refer to achieving a desired pharmacological and / or physiological effect. The effect may be prophylactic, in terms of completely or partially preventing a disease or its symptoms, and / or therapeutic, in terms of partially or completely curing a disease and / or the adverse effects caused by the disease. As used herein, "treatment" encompasses any treatment of a disease or condition in a mammal, including (a) preventing the onset of the disease or condition in a subject susceptible to, but not yet diagnosed with, the disease or condition; (b) inhibiting, i.e., eliminating or reducing, the disease or one or more symptoms; and / or (c) alleviating the disease, i.e., regressing or substantially eliminating the disease. Therapeutic agents may be administered before, during, or after the onset of disease or injury. Treatment of ongoing disease, which stabilizes or reduces undesirable clinical symptoms in a patient, is of particular interest. Such treatment is desirably performed before complete loss of function of affected tissues. The therapy will desirably be administered during, and in some cases after, the symptomatic stage of the disease.

[0027] The terms "individual," "subject," "host," and "patient" are used interchangeably herein to refer to any mammalian subject for whom diagnosis, treatment, or therapy is desired. Mammals include, for example, humans, non-human primates, rodents (e.g., rats, mice), lagomorphs (e.g., rabbits), ungulates (e.g., cows, sheep, pigs, horses, goats, etc.), etc.

[0028] Unless otherwise indicated, the term "substantially" is intended to encompass both "entirely" and "largely, but not entirely." For example, an Ig Fc that "substantially does not induce ADCC" means an Ig Fc that does not induce ADCC at all or that does not induce ADCC for the most part.

[0029] As used herein, the term "about" when used in connection with a quantity indicates that the quantity may vary by 10% of the stated amount. For example, "about 100" means an amount of 90 to 110. When "about" is used in the context of a range, "about" when used in connection with the lower end of a range means that the lower amount includes an amount 10% lower than the lower end of the range, and "about" when used in connection with the upper end of a range means that the upper amount includes an amount 10% higher than the upper end of the range. For example, about 100 to about 1000 means that the range extends from 90 to 1100.

[0030] Before the present disclosure is further described, it is to be understood that this disclosure is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting, as the scope of the present disclosure will be limited only by the appended claims.

[0031] Where a range of numerical values ​​is provided, it is understood that each intervening value between the upper and lower limits of that range, to one-tenth of the unit of the lower limit unless the context clearly dictates otherwise, and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these narrower ranges may independently be included in the narrower range and are also encompassed within the disclosure, subject to any specifically excluded value in the stated range. Where an stated range includes one or both of those upper and lower limits, ranges excluding either or both of those included limits are also encompassed within the disclosure.

[0032] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of this disclosure, the preferred methods and materials are described below. All publications mentioned herein are incorporated by reference to disclose and describe the methods and / or materials in connection with the content in which the publications are cited.

[0033] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a "variant IL-2 polypeptide" includes a plurality of such polypeptides. It should be further noted that the claims may be drafted to exclude any element. Accordingly, this statement is intended to serve as a predicate for use of exclusive terminology, such as "solely," "only," or a "negative" limitation in connection with the recitation of claim elements.

[0034] It is to be understood that certain features of the present disclosure that are, for clarity, described in the context of individual embodiments, may also be provided in combination within a single embodiment. Conversely, various features of the present disclosure that are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination. All combinations of the embodiments belonging to the present disclosure are expressly embraced by the present disclosure and are disclosed herein as if all combinations were individually and specifically disclosed. In addition, all subcombinations of the various embodiments and elements thereof are also expressly embraced by the present disclosure and are disclosed herein as if all such subcombinations were individually and specifically disclosed herein.

[0035] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present disclosure. Nothing herein should be construed as an admission that the present disclosure is not entitled to antedate such publication. Further, the dates of publication provided may be different from the actual publication dates, which may need to be independently confirmed.

[0036] Detailed Description The present disclosure provides IL-2 variant polypeptides and compositions comprising IL-2 variant polypeptides, e.g., fusion polypeptides, where the variants have reduced binding to IL-2Rα and IL-2Rβ, as well as methods for the use of such IL-2 variant polypeptides and compositions comprising IL-2 variant polypeptides.

[0037] In some cases, the method includes administering a composition including an immunomodulatory protein, such as an IL-2 variant polypeptide, and one or more IL-2 variant polypeptides, e.g., fusion polypeptides, to an individual who is also undergoing a therapy involving administering one of the following: (a) therapy with modified or unmodified T cells, e.g., TCR-T therapy or tumor-infiltrating lymphocytes (TILs); (b) a product (e.g., a cancer vaccine) that can associate with the TCR of a T cell or be processed by the immune system to be presented by a pMHC complex to the TCR of a T cell; (c) a precursor of a product (e.g., a nucleic acid vaccine, such as an mRNA vaccine) that can associate with the TCR of a T cell or that can be processed by the immune system to be presented by MHC to the TCR of a T cell; (d) CAR-T cells, or (e) A modified cell comprising one or more exogenous activated receptors capable of interacting with a target cell.

[0038] In some cases, the method includes administering a composition including an immunomodulatory protein, such as an IL-2 variant polypeptide, and one or more IL-2 variant polypeptides, e.g., fusion polypeptides, to an individual who is also undergoing a therapy involving administering one of the following: (a) TCR-bearing modified or unmodified T cells, e.g., TCR-T therapy or TIL therapy, or (b) a modified cell comprising a chimeric antigen receptor (CAR), wherein the CAR binds to a target antigen and the modified cell comprises an intracellular signaling domain that is activated by interaction of the modified cell with IL-2; or (c) a modified cell containing one or more exogenously activated receptors; or (d) a product (e.g., a vaccine) that can be processed by the immune system into one or more antigens that can associate with the TCR of a T cell or be presented by the major histocompatibility complex (MHC) to the TCR of a T cell, optionally wherein the one or more antigens are cancer-associated antigens; or (e) one or more nucleic acids (e.g., nucleic acid vaccines such as mRNA vaccines) encoding one or more polypeptides that can be processed by the immune system into one or more antigens that can associate with the TCR of a T cell or be presented by the major histocompatibility complex (MHC) to the TCR of a T cell, optionally wherein the one or more antigens are cancer-associated antigens; or (f) at least one immune checkpoint inhibitor (CPI). When an individual is administered a first composition comprising (a), (b), (c), (d) or (e), the individual may also be administered a CPI.

[0039] As described above, IL-2 variant polypeptides, in which the IL-2 variant polypeptides have such reduced affinity for IL-2Rα and IL-2Rβ, and compositions comprising one or more IL-2 variant polypeptides, e.g., fusion polypeptides, have been found to have unexpected properties that may provide benefits in many different medical applications. More specifically, it has been found that IL-2 variant polypeptides and compositions comprising one or more IL-2 variant polypeptides, e.g., fusion polypeptides, do not systemically activate multiple immune cell subsets as native IL-2 does, but rather predominantly (i.e., the majority) or preferentially (i.e., more frequently) activate T cells whose T cell receptor (TCR) has engaged a peptide-MHC complex (pMHC) presented by an antigen-presenting cell, as opposed to activating T cells whose T cell receptor (TCR) has not engaged a peptide-MHC complex (pMHC) presented by an antigen-presenting cell. For example, a variant IL-2 polypeptide of the present disclosure, or a fusion polypeptide comprising a variant IL-2 polypeptide, can activate a T cell whose TCR is engaged with pMHC to an extent at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 75%, or at least 100% (or twice as much) as a T cell whose TCR is not engaged with pMHC, more than the extent to which the TCR is activated by the same variant IL-2 polypeptide. Accordingly, this property can provide a useful therapeutic index for pharmaceutical compositions comprising such IL-2 variant polypeptides or compositions comprising one or more IL-2 variant polypeptides, e.g., fusion polypeptides, comprising one or more IL-2 variant polypeptides. That is, pharmaceutical compositions comprising such IL-2 variant polypeptides or compositions comprising one or more IL-2 variant polypeptides, e.g., fusion polypeptides, can be administered within a dosage range in which such compositions are therapeutically effective without unacceptable adverse events or toxicity.Furthermore, it has been found that such IL-2 variant polypeptides, and compositions comprising one or more IL-2 variant polypeptides, e.g., fusion polypeptides, when engaging with IL-2 receptors on CAR-T cells, TCR-T cells, and other cytotoxic cells bearing exogenous activating receptors such as CAR and TCR (e.g., macrophages and NK cells), can provide a homeostatic signal that can prolong the survival of such cells and / or provide an activating signal to the cells that causes the cells to proliferate and retain their cytotoxic function. The term "exogenous activating receptor" includes receptors that are not normally present in the cell but are instead introduced into the cell to provide a desired functionality, for example, by introducing into the cell a nucleic acid comprising a nucleotide sequence encoding the activating receptor.

[0040] Variant IL-2 polypeptides, fusion proteins and methods are described below.

[0041] Variant IL-2 Polypeptides As discussed above, the IL-2 variants contain mutations that substantially eliminate binding to IL-2Rα and also reduce, but do not substantially eliminate, binding affinity to IL-2Rβ. By substantially eliminating binding to IL-2Rα, the IL-2 variants do not significantly upregulate the production of Tregs, which is undesirable in applications where T cell cytotoxic activity is desired. The reduced binding affinity to IL-2Rβ, combined with the substantially eliminated binding to IL-2Rα, greatly reduces the ability of such IL-2 variants to systemically activate T cells.

[0042] The wild-type IL-2 amino acid sequence can be as follows: APTSSSTKKT QLQLEHLLLD LQMILNGINN YKNPKLTRML TFKFYMPKKA TELKHLQCLEEELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNRWITFCQSIIS TLT (SEQ ID NO: 1).

[0043] As mentioned above, the IL-2 receptor comprises the IL-2Rα, IL-2Rβ, and IL-2Rγ chains. The amino acid sequences of these three chains are as follows:

[0044] Human IL-2Rα: ELCDDDPPE IPHATFKAMA YKEGTMLNCE CKRGFRRIKS GSLYMLCTGN SSHSSWDNQC QCTSSATRNT TKQVTPQPEE QKERKTTEMQ SPMQPVDQAS LPGHCREPPP WENEATERIY HFVVGQMVYY QCVQGYRALH RGPAESVCKM THGKTRWTQP QLICTGEMET SQFPGEEKPQ ASPEGRPESE TSCLVTTTDF QIQTEMAATM ETSIFTTEYQ VAVAGCVFLL ISVLLLSGLT WQRRQRKSRR TI (SEQ ID NO: 2).

[0045] Human IL-2Rβ:VNG TSQFTCFYNS RANISCVWSQ DGALQDTSCQ VHAWPDRRRW NQTCELLPVS QASWACNLIL GAPDSQKLTT VDIVTLRVLC REGVRWRVMA IQDFKPFENL RLMAPISLQV VHVETHRCNI SWEISQASHY FERHLEFEAR TLSPGHTWEE APLLTLKQKQ EWICLETLTP DTQYEFQVRV KPLQGEFTTW SPWSQPLAFR TKPAALGKDT IPWLGHLLVG LSGAFGFIIL VYLLINCRNT GPWLKKVLKC NTPDPSKFFS QLSSEHGGDV QKWLSSPFPS SSFSPGGLAP EISPLEVLER DKVTQLLLQQ DKVPEPASLS SNHSLTSCFT NQGYFFFHLP DALEIEACQV YFTYDPYSEE DPDEGVAGAP TGSSPQPLQP LSGEDDAYCT FPSRDDLLLF SPSLLGGPSP PSTAPGGSGA GEERMPPSLQ ERVPRDWDPQ PLGPPTPGVP DLVDFQPPPE LVLREAGEEV PDAGPREGVS FPWSRPPGQG EFRALNARLP LNTDAYLSLQ ELQGQDPTHL V (SEQ ID NO: 3).

[0046] Human IL-2Rγ: LNTTILTP NGNEDTTADF FLTTMPTDSL SVSTLPLPEV QCFVFNVEYM NCTWNSSSEP QPTNLTLHYW YKNSDNDKVQ KCSHYLFSEE ITSGCQLQKK EIHLYQTFVV QLQDPREPRR QATQMLKLQN LVIPWAPENL TLHKLSESQL ELNWNNRFLN HCLEHLVQYR TDWDHSWTEQ SVDYRHKFSL PSVDGQKRYT FRVRSRFNPL CGSAQHWSEW SHPIHWGSNT SKENPFLFAL EAVVISVGSM GLIISLLCVY FWLERTMPRI PTLKNLEDLV TEYHGNFSAW SGVSKGLAES LQPDYSERLC LVSEIPPKGG ALGEGPGASP CNQHSPYWAP PCYTLKPET (SEQ ID NO: 4).

[0047] Mutations that can reduce the binding of IL-2 to IL-2Rα include, for example, substitutions, including (but not limited to) conservative amino acid substitutions, at one or more of the following amino acids: R38, F42, K43, Y45, E62, P65, E68, V69, and L72. For example, an IL-2 variant can include substitutions at one, two, three, four, five, or more of the foregoing amino acids. Exemplary substitutions include the following from Table 2:

[0048] (Table 2) TIFF2025532538000003.tif67160

[0049] In some cases, the variant IL-2 polypeptide exhibits reduced binding affinity to IL-2Rα compared to the binding affinity to IL-2Rα of an IL-2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1. For example, in some cases, the variant IL-2 polypeptide binds to IL-2Rα with a binding affinity that is at least 10% lower, at least 15% lower, at least 20% lower, at least 25%, at least 30% lower, at least 35% lower, at least 40% lower, at least 45% lower, at least 50% lower, at least 55% lower, at least 60% lower, at least 65% lower, at least 70% lower, at least 75% lower, at least 80% lower, at least 85% lower, at least 90% lower, at least 95% lower, at least 98% lower, or at least 99% lower than the binding affinity of an IL-2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 for IL-2Rα. In some cases, the binding affinity is at least 75% lower. In some cases, the binding affinity is at least 80% lower. In some cases, the binding affinity is at least 85% lower. In some cases, the binding affinity is at least 90% lower. In some cases, the binding affinity is at least 95% lower. In some cases, the binding affinity is at least 98% lower. In some cases, the binding affinity is at least 99% lower. In some cases, the binding affinity is reduced by at least 50-fold. In some cases, the binding affinity is reduced by at least 75-fold. In some cases, the binding affinity is reduced by at least 100-fold. In some cases, the binding affinity is reduced by about 110-fold.

[0050] In some cases, the variant IL-2 polypeptide retains at least some minimal binding to IL-2Rα. For example, in some cases, the variant IL-2 polypeptide exhibits 2% to 50% of the binding of wild-type IL-2 (e.g., an IL-2 polypeptide comprising the amino acid sequence of SEQ ID NO: 1) to an IL-2Rα polypeptide. For example, in some cases, the variant IL-2 polypeptide exhibits 2% to 5%, 5% to 10%, 10% to 15%, 15% to 20%, 20% to 25%, 25% to 30%, 30% to 35%, 35% to 40%, 40% to 45%, or 45% to 50% of the binding of wild-type IL-2 to an IL-2Rα polypeptide.

[0051] As mentioned above, substitution of F42 with an amino acid other than Phe, e.g., Ala, has been shown to substantially reduce binding of IL-2 variants to IL-2Rα. See Quayle et al., Clin Cancer Res 2020;26:1953-64, which reported that the F42A substitution caused a 110-fold decrease in binding to IL-2Rα. Thus, binding to IL-2Rα is virtually eliminated by this mutation.

[0052] Mutations that can reduce binding of IL-2 to IL-2Rβ include, for example, substitutions, including (but not limited to) conservative amino acid substitutions, at one or more of the following amino acids: E15, H16, L19, D20, D84, S87, N88, V91, and I92. For example, an IL-2 variant can include substitutions at one, two, three, four, five, or more of the foregoing amino acids. Exemplary substitutions include the following from Table 3:

[0053] (Table 3) TIFF2025532538000004.tif67160

[0054] In some cases, the variant IL-2 polypeptide exhibits reduced binding affinity to IL-2Rβ compared to the binding affinity to IL-2Rβ of an IL-2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1. For example, in some cases, the variant IL-2 polypeptide binds to IL-2Rβ with a binding affinity that is at least 10% lower, at least 15% lower, at least 20% lower, at least 25%, at least 30% lower, at least 35% lower, at least 40% lower, at least 45% lower, at least 50% lower, at least 55% lower, at least 60% lower, at least 65% lower, at least 70% lower, at least 75% lower, at least 80% lower, at least 85% lower, at least 90% lower, at least 95% lower, at least 98% lower, or at least 99% lower than the binding affinity of an IL-2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 for IL-2Rβ. In some cases, the binding affinity is at least 25% lower. In some cases, the binding affinity is at least 30% lower. In some cases, the binding affinity is at least 35% lower. In some cases, the binding affinity is at least 40% lower. In some cases, the binding affinity is at least 45% lower. In some cases, the binding affinity is at least 50% lower. In some cases, the binding affinity is reduced by about 2-fold. In some cases, the binding affinity is reduced by about 3-fold.

[0055] In some cases, the variant IL-2 polypeptide retains binding to IL-2Rβ. For example, in some cases, the variant IL-2 polypeptide exhibits 2% to 50% of the binding of wild-type IL-2 (e.g., an IL-2 polypeptide comprising the amino acid sequence of SEQ ID NO: 1) to an IL-2Rβ polypeptide. For example, in some cases, the variant IL-2 polypeptide exhibits 2% to 5%, 5% to 10%, 10% to 15%, 15% to 20%, 20% to 25%, 25% to 30%, 30% to 35%, 35% to 40%, 40% to 45%, or 45% to 50% of the binding of wild-type IL-2 to an IL-2Rβ polypeptide.

[0056] As mentioned above, substitution of F42 with an amino acid other than His, e.g., Ala, has been shown to reduce binding of IL-2 variants to IL-2Rβ. See Quayle et al., Clin Cancer Res 2020;26:1953-64, who reported that an H16A substitution caused a three-fold decrease in binding to IL-2Rβ.

[0057] Some exemplary combinations of mutations that reduce binding of an IL-2 variant polypeptide to IL-2Rα and IL-2Rβ include the following from Table 4:

[0058] (Table 4) TIFF2025532538000005.tif196158TIFF2025532538000006.tif214158TIFF2025532538000007.tif23115 8TIFF2025532538000008.tif245158TIFF2025532538000009.tif190158TIFF2025532538000010.tif40158

[0059] Exemplary IL-2 variant amino acid sequences include amino acid sequences having at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity to any one of the following IL-2 variant amino acid sequences (I) to (XVIII):

[0060] (I) In the TIFF2025532538000011.tif17150 sequence, X1 is any amino acid other than Arg, for example, Ala, Asp, or Glu; the amino acid sequence comprises one or more additional mutations that reduce binding of the IL-2 variant to IL-2Rβ by at least 25%, at least 50%, or at least 75%, at least about two-fold, or at least about three-fold (compared to the binding affinity of wild-type IL-2 to IL-2Rβ); the IL-2 variant exhibits reduced binding affinity to IL-2Rα by at least 50-fold, at least 75-fold, at least 100-fold, or about 110-fold (compared to the binding affinity of wild-type IL-2 to IL-2Rα); and the IL-2 variant may comprise one or more mutations that reduce binding of the IL-2 variant to IL-2Rα.

[0061] (II) In the TIFF2025532538000012.tif17152 sequence, X1 is any amino acid other than Phe, for example, Ala or Lys, and the amino acid sequence comprises one or more additional mutations that reduce binding of the IL-2 variant to IL-2Rβ by at least 25%, at least 50%, or at least 75%, at least about two-fold, or at least about three-fold (compared to the binding affinity of wild-type IL-2 to IL-2Rβ), and the IL-2 variant exhibits reduced binding affinity to IL-2Rα by at least 50-fold, at least 75-fold, at least 100-fold, or about 110-fold (compared to the binding affinity of wild-type IL-2 to IL-2Rα), and the IL-2 variant may comprise one or more mutations that reduce binding of the IL-2 variant to IL-2Rα. In some cases, X1 may alternatively be Gly, Val, Leu, Ile, Arg, Asn, Cys, Glu, Gln, Ile, Lys, Met, Phe, Pro, Ser, Tyr, Trp, or Val.

[0062] (III) In the TIFF2025532538000013.tif17151 sequence, X1 is any amino acid other than Lys, for example, Glu; the amino acid sequence comprises one or more additional mutations that reduce binding of the IL-2 variant to IL-2Rβ by at least 25%, at least 50%, or at least 75%, at least about two-fold, or at least about three-fold (compared to the binding affinity of wild-type IL-2 to IL-2Rβ); the IL-2 variant exhibits reduced binding affinity to IL-2Rα by at least 50-fold, at least 75-fold, at least 100-fold, or about 110-fold (compared to the binding affinity of wild-type IL-2 to IL-2Rα); and the IL-2 variant may comprise one or more mutations that reduce binding of the IL-2 variant to IL-2Rα.

[0063] (IV) In the TIFF2025532538000014.tif17151 sequence, X1 is any amino acid other than Tyr, the amino acid sequence comprises one or more additional mutations that reduce binding of the IL-2 variant to IL-2Rβ by at least 25%, at least 50%, or at least 75%, at least about two-fold, or at least about three-fold (compared to the binding affinity of wild-type IL-2 to IL-2Rβ), the IL-2 variant exhibits reduced binding affinity to IL-2Rα by at least 50-fold, at least 75-fold, at least 100-fold, or about 110-fold (compared to the binding affinity of wild-type IL-2 to IL-2Rα), and the IL-2 variant may comprise one or more mutations that reduce binding of the IL-2 variant to IL-2Rα.

[0064] (V) In the TIFF2025532538000015.tif17151 sequence, X1 is any amino acid other than Glu, for example, Gln; the amino acid sequence comprises one or more additional mutations that reduce binding of the IL-2 variant to IL-2Rβ by at least 25%, at least 50%, or at least 75%, at least about two-fold, or at least about three-fold (compared to the binding affinity of wild-type IL-2 to IL-2Rβ); the IL-2 variant exhibits reduced binding affinity to IL-2Rα by at least 50-fold, at least 75-fold, at least 100-fold, or about 110-fold (compared to the binding affinity of wild-type IL-2 to IL-2Rα); and the IL-2 variant may comprise one or more mutations that reduce binding of the IL-2 variant to IL-2Rα.

[0065] (VI) In the TIFF2025532538000016.tif17152 sequence, X1 is any amino acid other than Pro, the amino acid sequence comprises one or more additional mutations that reduce binding of the IL-2 variant to IL-2Rβ by at least 25%, at least 50%, or at least 75%, at least about two-fold, or at least about three-fold (compared to the binding affinity of wild-type IL-2 to IL-2Rβ), the IL-2 variant exhibits reduced binding affinity to IL-2Rα by at least 50-fold, at least 75-fold, at least 100-fold, or about 110-fold (compared to the binding affinity of wild-type IL-2 to IL-2Rα), and the IL-2 variant may comprise one or more mutations that reduce binding of the IL-2 variant to IL-2Rα.

[0066] (VII) In the TIFF2025532538000017.tif17151 sequence, X1 is any amino acid other than Glu, the amino acid sequence comprises one or more additional mutations that reduce binding of the IL-2 variant to IL-2Rβ by at least 25%, at least 50%, or at least 75%, at least about two-fold, or at least about three-fold (compared to the binding affinity of wild-type IL-2 to IL-2Rβ), the IL-2 variant exhibits reduced binding affinity to IL-2Rα by at least 50-fold, at least 75-fold, at least 100-fold, or about 110-fold (compared to the binding affinity of wild-type IL-2 to IL-2Rα), and the IL-2 variant may comprise one or more mutations that reduce binding of the IL-2 variant to IL-2Rα.

[0067] (VIII) In the TIFF2025532538000018.tif17151 sequence, X1 is any amino acid other than Val, the amino acid sequence comprises one or more additional mutations that reduce binding of the IL-2 variant to IL-2Rβ by at least 25%, at least 50%, or at least 75%, at least about two-fold, or at least about three-fold (compared to the binding affinity of wild-type IL-2 to IL-2Rβ), the IL-2 variant exhibits reduced binding affinity to IL-2Rα by at least 50-fold, at least 75-fold, at least 100-fold, or about 110-fold (compared to the binding affinity of wild-type IL-2 to IL-2Rα), and the IL-2 variant may comprise one or more mutations that reduce binding of the IL-2 variant to IL-2Rα.

[0068] (IX) In the TIFF2025532538000019.tif17151 sequence, X1 is any amino acid other than Leu, the amino acid sequence comprises one or more additional mutations that reduce binding of the IL-2 variant to IL-2Rβ by at least 25%, at least 50%, or at least 75%, at least about two-fold, or at least about three-fold (compared to the binding affinity of wild-type IL-2 to IL-2Rβ), the IL-2 variant exhibits reduced binding affinity to IL-2Rα by at least 50-fold, at least 75-fold, at least 100-fold, or about 110-fold (compared to the binding affinity of wild-type IL-2 to IL-2Rα), and the IL-2 variant may comprise one or more mutations that reduce binding of the IL-2 variant to IL-2Rα.

[0069] (X) In the TIFF2025532538000020.tif17150 sequence, X1 is any amino acid other than Glu, for example, Ala, and the binding of the IL-2 variant to IL-2Rβ is reduced by at least two-fold or at least three-fold compared to the binding affinity of wild-type IL-2 to IL-2Rβ (compared to the binding affinity of wild-type IL-2 to IL-2Rβ), the IL-2 includes one or more mutations that reduce the binding affinity of the IL-2 variant to IL-2Rα (compared to the binding affinity of wild-type IL-2 to IL-2Rα) by at least 50-fold, at least 75-fold, at least 100-fold, or about 110-fold, and the IL-2 variant may have one or more additional mutations that reduce binding of the IL-2 variant to IL-2Rβ.

[0070] (XI) In the TIFF2025532538000021.tif17150 sequence, X1 is any amino acid other than His, for example, Ala, Glu, Thr, or Asp; binding of the IL-2 variant to IL-2Rβ (compared to the binding affinity of wild-type IL-2 to IL-2Rβ) is reduced by at least two-fold or at least three-fold compared to the binding affinity of wild-type IL-2 to IL-2Rβ; the IL-2 comprises one or more mutations that reduce the binding affinity of the IL-2 variant to IL-2Rα (compared to the binding affinity of wild-type IL-2 to IL-2Rα) by at least 50-fold, at least 75-fold, at least 100-fold, or about 110-fold; and the IL-2 variant may have one or more additional mutations that reduce binding of the IL-2 variant to IL-2Rβ. In some cases, X1 is Gly, Val, Leu, Ile, Arg, Asn, Cys, Glu, Gln, Ile, Lys, Met, Phe, Pro, Ser, Tyr, Trp, or Val.

[0071] (XII) In the TIFF2025532538000022.tif17150 sequence, X1 is any amino acid other than Leu, the binding of the IL-2 variant to IL-2Rβ is reduced by at least two-fold or at least three-fold compared to the binding affinity of wild-type IL-2 to IL-2Rβ (compared to the binding affinity of wild-type IL-2 to IL-2Rβ), the IL-2 comprises one or more mutations that reduce the binding affinity of the IL-2 variant to IL-2Rα (compared to the binding affinity of wild-type IL-2 to IL-2Rα) by at least 50-fold, at least 75-fold, at least 100-fold, or about 110-fold, and the IL-2 variant may have one or more additional mutations that reduce binding of the IL-2 variant to IL-2Rβ.

[0072] (XIII) In the TIFF2025532538000023.tif17150 sequence, X1 is any amino acid other than Asp, for example, Asn; the binding of the IL-2 variant to IL-2Rβ (compared to the binding affinity of wild-type IL-2 to IL-2Rβ) is reduced by at least two-fold or at least three-fold compared to the binding affinity of wild-type IL-2 to IL-2Rβ; the IL-2 includes one or more mutations that reduce the binding affinity of the IL-2 variant to IL-2Rα (compared to the binding affinity of wild-type IL-2 to IL-2Rα) by at least 50-fold, at least 75-fold, at least 100-fold, or about 110-fold; and the IL-2 variant may have one or more additional mutations that reduce binding of the IL-2 variant to IL-2Rβ.

[0073] (XIV) In the TIFF2025532538000024.tif17151 sequence, X1 is any amino acid other than Asp, for example, His, Lys, or Arg; the binding of the IL-2 variant to IL-2Rβ (compared to the binding affinity of wild-type IL-2 to IL-2Rβ) is reduced by at least two-fold or at least three-fold compared to the binding affinity of wild-type IL-2 to IL-2Rβ; the IL-2 comprises one or more mutations that reduce the binding affinity of the IL-2 variant to IL-2Rα (compared to the binding affinity of wild-type IL-2 to IL-2Rα) by at least 50-fold, at least 75-fold, at least 100-fold, or about 110-fold; and the IL-2 variant may have one or more additional mutations that reduce binding of the IL-2 variant to IL-2Rβ.

[0074] (XV) In the TIFF2025532538000025.tif17152 sequence, X1 is any amino acid other than Ser, the binding of the IL-2 variant to IL-2Rβ is reduced by at least two-fold or at least three-fold compared to the binding affinity of wild-type IL-2 to IL-2Rβ (compared to the binding affinity of wild-type IL-2 to IL-2Rβ), the IL-2 comprises one or more mutations that reduce the binding affinity of the IL-2 variant to IL-2Rα (compared to the binding affinity of wild-type IL-2 to IL-2Rα) by at least 50-fold, at least 75-fold, at least 100-fold or about 110-fold, and the IL-2 variant may have one or more additional mutations that reduce binding of the IL-2 variant to IL-2Rβ.

[0075] (XVI) In the TIFF2025532538000026.tif17151 sequence, X1 is any amino acid other than Asn, for example, Ser, Ala, Gly, Arg, Thr, or Asp; the binding of the IL-2 variant to IL-2Rβ (compared to the binding affinity of wild-type IL-2 to IL-2Rβ) is reduced by at least two-fold or at least three-fold compared to the binding of wild-type IL-2 to IL-2Rβ; the IL-2 comprises one or more mutations that reduce the binding affinity of the IL-2 variant to IL-2Rα (compared to the binding affinity of wild-type IL-2 to IL-2Rα) by at least 50-fold; and the IL-2 variant may comprise one or more additional mutations that reduce the binding of the IL-2 variant to IL-2Rβ by at least 75-fold, at least 100-fold, or about 110-fold.

[0076] (XVII) In the TIFF2025532538000027.tif17152 sequence, X1 is any amino acid other than Val, for example, Glu, Ala, or Thr; the binding of the IL-2 variant to IL-2Rβ (compared to the binding affinity of wild-type IL-2 to IL-2Rβ) is reduced by at least two-fold or at least three-fold compared to the binding affinity of wild-type IL-2 to IL-2Rβ; the IL-2 comprises one or more mutations that reduce the binding affinity of the IL-2 variant to IL-2Rα (compared to the binding affinity of wild-type IL-2 to IL-2Rα) by at least 50-fold, at least 75-fold, at least 100-fold, or about 110-fold; and the IL-2 variant may have one or more additional mutations that reduce binding of the IL-2 variant to IL-2Rβ.

[0077] (XVIII) In the TIFF2025532538000028.tif17152 sequence, X1 is any amino acid other than Ile, for example, Ala, and the binding of the IL-2 variant to IL-2Rβ is reduced by at least two-fold or at least three-fold compared to the binding affinity of wild-type IL-2 to IL-2Rβ (compared to the binding affinity of wild-type IL-2 to IL-2Rβ), the IL-2 comprises one or more mutations that reduce the binding affinity of the IL-2 variant to IL-2Rα (compared to the binding affinity of wild-type IL-2 to IL-2Rα) by at least 50-fold, at least 75-fold, at least 100-fold, or about 110-fold, and the IL-2 variant may have one or more additional mutations that reduce binding of the IL-2 variant to IL-2Rβ.

[0078] Determination of the binding affinity of IL-2 variants to IL-2Rα and IL-2Rβ is performed as described in Example 1 below.

[0079] Higher Forms of IL-2 Variant Polypeptides 1A-1C, IL-2 variant polypeptides of the present disclosure can be linked together to form higher order chains, e.g., dimers, trimers, tetramers, etc. IL-2 variant polypeptides can be joined by one or more independently selected linkers (discussed below). Linkers in such higher order forms can include linkers, e.g., rigid linkers or short flexible linkers, that enhance the avidity of the IL-2 variant polypeptides for their target cells and / or improve the yield of such higher order IL-2 variant polypeptides.

[0080] Compositions Comprising IL-2 Variant Polypeptides Compositions comprising one or more IL-2 variant polypeptides include fusion polypeptides on Ig Fc or other scaffold polypeptides, as well as compositions in which one or more IL-2 variant polypeptides, or fusion polypeptides comprising one or more IL-2 variant polypeptides, are associated with a carrier.

[0081] Fusion Polypeptides As noted above, the present disclosure also provides compositions comprising IL-2 variant polypeptides, eg, fusion polypeptides.

[0082] The heterologous fusion partner may include one or more additional heterologous polypeptides, such as an Ig Fc or other scaffold polypeptide, and a functional protein as described below. Incorporation of an Ig Fc polypeptide or other scaffold polypeptide into the fusion polypeptide can increase the stability, manufacturability, and / or in vivo half-life of the resulting fusion polypeptide. Typically, the fusion polypeptide does not include an MHC class I polypeptide or an MHC class II polypeptide. For example, the fusion polypeptide typically does not include an MHC class I heavy chain polypeptide, a β2-microglobulin (β2M) polypeptide, an MHC class II alpha chain polypeptide, or an MHC class II beta chain polypeptide. In some cases, the fusion polypeptide includes (a) at least one IL-2 variant polypeptide and (b) an immunoglobulin (Ig) Fc polypeptide, but does not include any other heterologous polypeptides. In some cases, the fusion polypeptide includes (a) at least one IL-2 variant polypeptide, (b) a peptide linker, and (c) an Ig Fc polypeptide, but does not include any other heterologous polypeptides. In some cases, the fusion polypeptide comprises (a) at least one IL-2 variant polypeptide, (b) optionally a peptide linker, (c) an Ig Fc polypeptide, and at least one other heterologous polypeptide, such as a functional protein described below.

[0083] Suitable scaffold polypeptides include antibody-based scaffold polypeptides and non-antibody-based scaffolds. Non-antibody-based scaffolds include, for example, fibronectin-based scaffold proteins, albumin, XTEN (extended recombinant) polypeptides, transferrin, Fc receptor polypeptides, elastin-like polypeptides (see, for example, Hassouneh et al. (2012) Methods Enzymol. 502:215, for example, polypeptides comprising pentapeptide repeating units (Val-Pro-Gly-X-Gly, SEQ ID NO: 36), where X is an amino acid other than proline), albumin-binding polypeptides, silk-like polypeptides (see, for example, Valluzzi et al. (2002) Philos Trans R Soc Lond B Biol Sci. 357:165), silk-elastin-like polypeptides (SELPs, see, for example, Megeed et al. (2002) Adv Drug Deliv Rev. 54:1075) and the like. Suitable XTEN polypeptides include, for example, WO 2009 / 023270, WO 2010 / 091122, WO 2007 / 103515, U.S. Patent Application Publication No. 2010 / 0189682, U.S. Patent Application No. 2009 / 0092582, and Schellenberger et al. (2009) Nat Biotechnol. 27:1186. Suitable albumin polypeptides include, for example, human serum albumin.

[0084] In some cases, the fusion polypeptide comprises at least one IL-2 variant polypeptide fused to the N-terminus of an Ig Fc fusion partner (see, e.g., Figures 2A-2C). In some cases, the fusion polypeptide comprises at least one IL-2 variant polypeptide fused to the C-terminus of an Ig Fc fusion partner (see, e.g., Figures 3A-3C). In some cases, the fusion polypeptide comprises at least one IL-2 variant polypeptide fused to the N-terminus of an Ig Fc fusion partner and at least one IL-2 variant polypeptide fused to the C-terminus of an Ig Fc fusion partner (see, e.g., Figures 4A-4C). In Figures 2A-2C, 3A-3C, and 4A-4C, the Ig Fc polypeptide of the fusion polypeptide has been modified using known methods to prevent dimer formation with another fusion protein comprising Ig Fc. For example, Wang et al.,Front.Immunol.8:1545(Nov.2017), Ying et al.,mAbs 6:5,1201-1210;September / October 2014, and Ying et al.,J.Biol.Chem.,vol.287,no.23,pp.19399-19408,June See 1,2012.

[0085] In some cases, the fusion polypeptides can form dimers or higher-order fusion polypeptides. In some cases, the dimers are homodimers in which each fusion polypeptide contains the same amino acid sequence (see, e.g., Figures 5A-5B, 6A-6B, 7A, and 7B). When the fusion partner is an Ig Fc, e.g., an IgG1 Fc, disulfide bonds (typically two) can spontaneously form to link the two fusion polypeptides, thereby forming a homodimer.

[0086] In some instances, it may be desirable to form heterodimeric fusion polypeptides in which each fusion polypeptide comprises a different amino acid sequence (see, e.g., Figures 8A-8F and 30A-30G). In such instances, it may be desirable to utilize an Ig Fc polypeptide comprising an interspecies binding sequence.

[0087] Interspecies dimerization sequences, such as "knobs-in-hole" sequences, allow the selective dimerization of two different fusion polypeptides that differ from each other in amino acid sequence. Interspecies binding sequences favor the formation of heterodimers with their cognate polypeptide sequences (i.e., the interspecies sequence and its counterpart interspecies sequence), particularly those based on Ig Fc sequence variants. Such interspecies polypeptide sequences include knobs-in-hole sequences and knobs-in-hole sequences that promote the formation of one or more disulfide bonds. For example, one interspecies binding pair includes the T366Y and Y407T mutant pair at the CH3 domain interface of IgG1 or corresponding residues in other immunoglobulins. See Ridgway et al., Protein Engineering 9:7, 617-621 (1996). A second interspecies binding pair involves a knob formed by a T366W substitution and a hole formed by a triple substitution of T366S, L368A, and Y407V on a complementary Ig Fc sequence. See Xu et al. mAbs 7:1, 231-242 (2015). Another interspecies binding pair has a first Fc polypeptide with Y349C, T366S, L368A, and Y407V substitutions and a second Ig Fc polypeptide with S354C and T366W substitutions (a disulfide bond can form between Y349C and S354C). See, e.g., Brinkmann and Konthermann, mAbs 9:2, 182-212 (2015). Ig Fc polypeptide sequences, with or without knobs-in-holes modifications, can be stabilized by the formation of disulfide bonds (e.g., hinge region disulfide bonds) between the Ig Fc polypeptides. Thus, in some cases, a dimerization fusion polypeptide may be a heterodimeric fusion polypeptide comprising two fusion polypeptides that are not identical in amino acid sequence; such a dimerization fusion polypeptide may be referred to as a "heterodimeric fusion polypeptide."

[0088] Interspecies dimerization sequences can also be used to allow a fusion polypeptide to be linked to a molecule that does not contain an IL-2 variant. For example, a fusion polypeptide containing an IL-2 variant can be linked to a molecule containing a polypeptide that binds to a cancer-associated antigen (e.g., an antibody or a binding fragment thereof, such as a single-chain Fc polypeptide (scFvs) or nanobody), thereby localizing the fusion polypeptide containing the IL-2 variant to tissues that contain the cancer-associated antigen. As discussed below, a fusion polypeptide containing at least one IL-2 variant can be linked to a different fusion polypeptide containing a protein with a different activity.

[0089] Carrier containing IL-2 variant polypeptide In some examples, it may be desirable to associate one or more IL-2 variant polypeptides with a carrier, such as a lipid vesicle (e.g., liposome) or micelle, a nanoparticle, a PEGylated protein (including site-specific PEGylation, i.e., containing one or more poly(ethylene glycol) (PEG) moieties), or an artificial antigen-presenting cell, such as engineered red blood cells and enucleated cells (see, e.g., US 2019 / 0290686). Such carriers and methods for their production are well known in the art and may provide desirable characteristics, such as increased stability, manufacturability, in vivo half-life, and / or targeting of the IL-2 variant(s) to target tissues in the body.

[0090] Ig Fc polypeptide As described above, a fusion polypeptide comprising one or more IL-2 variants can include an Ig Fc polypeptide. The Ig Fc polypeptide can be, for example, human IgG1 Fc, human IgG2 Fc, human IgG3 Fc, human IgG4 Fc, or a variant of a wild-type Ig Fc polypeptide. Variants include naturally occurring variants, non-naturally occurring variants, and combinations thereof. For example, the Ig Fc can be a variant of an Fc polypeptide, such as human IgG1 Fc, which has a substantially reduced ability to affect complement-dependent cytotoxicity (CDC) or antibody-dependent cellular cytotoxicity (ADCC). See, for example, the variant human IgG1 Fc polypeptides in Figures 9B and 9D.

[0091] In some cases, the Ig Fc polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity to the Ig Fc amino acid sequence shown in any one of Figures 9A-9M. The C-terminal Lys in any of the Ig Fc polypeptides shown in Figures 9A-9I and 9L-9M may be deleted.

[0092] In some cases, the Ig Fc polypeptide is an IgG1 Fc polypeptide or a variant of an IgG1 Fc polypeptide. For example, in some cases, the Ig Fc polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity to the human IgG1 Fc polypeptide shown in Figure 9A, with or without a C-terminal Lys. As another example, in some cases, the Ig Fc polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity to the Ig Fc polypeptide shown in Figure 9B, with or without a C-terminal Lys, and the Ig Fc polypeptide comprises an Ala at position 14 and an Ala at position 15, both of which are known as "LALA" mutations. In any of the above embodiments, the Ig Fc polypeptide can have an N77 substitution, i.e., the Ig Fc polypeptide can have an amino acid other than Asn at position 77, and in some cases, the Ig Fc polypeptide has Ala at position 77. In some cases, the Ig Fc polypeptide comprises the amino acid sequence shown in Figure 9A, with or without a C-terminal Lys. In some cases, the Ig Fc polypeptide comprises the amino acid sequence shown in Figure 9B, with or without a C-terminal Lys.

[0093] In some cases, the Ig Fc polypeptide is an IgG1 Fc polypeptide or a variant of an IgG1 Fc polypeptide, where the variant includes naturally occurring variants, non-naturally occurring variants, and combinations thereof. For example, in some cases, the Ig Fc polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity to the human IgG1 Fc polypeptide shown in Figure 9C, with or without a C-terminal Lys, and the Ig Fc polypeptide comprises Glu at position 136 and Met at position 138. As another example, in some cases, the Ig Fc polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity to the human IgG1 Fc polypeptide shown in Figure 9D, with or without a C-terminal Lys, wherein the Ig Fc polypeptide comprises Ala at positions 14 and 15, and the Ig Fc polypeptide comprises Glu at position 136 and Met at position 138. In any of the above embodiments, the Ig Fc polypeptide can have an N77 substitution, i.e., the Ig Fc polypeptide can have an amino acid other than Asn at position 77, and in some cases, the Ig Fc polypeptide has Ala at position 77. In some cases, the Ig Fc polypeptide comprises the amino acid sequence shown in Figure 9C, with or without a C-terminal Lys. In some cases, the Ig Fc polypeptide comprises the amino acid sequence shown in Figure 9D, with or without a C-terminal Lys.

[0094] In some cases, the Ig Fc polypeptide comprises the amino acid sequence shown in FIG. 9E with or without a C-terminal Lys (human IgG1 Fc comprises an L234F substitution, an L235E substitution, and a P331S substitution, where L234 corresponds to amino acid 14 of the amino acid sequence shown in FIG. 9A, L235 corresponds to amino acid 15 of the amino acid sequence shown in FIG. 9E, and P331 corresponds to amino acid 111 of the amino acid sequence shown in FIG. 9E). In some cases, the Ig Fc polypeptide comprises the amino acid sequence shown in FIG. 9F with or without a C-terminal Lys, including an N279A substitution (amino acid sequence of N77A shown in FIG. 9F). The substitution at N297 leads to the removal of a carbohydrate modification, resulting in an antibody sequence with reduced complement component 1q ("C1q") binding compared to the wild-type protein, and therefore reduced complement-dependent cytotoxicity (CDC). In some cases, the Ig Fc polypeptide comprises a substitution at K322. The K322 (e.g., K322A) substitution exhibits a substantial decrease in FcγR binding affinity and a substantial decrease in antibody-dependent cell-mediated cytotoxicity (ADCC), and C1q binding and CDC function are substantially or completely eliminated.

[0095] In some cases, the Ig Fc polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity to a human IgG2 Fc polypeptide shown in Figure 9G, with or without a C-terminal Lys, for example, the Ig Fc polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity to amino acids 99-325 of the human IgG2 Fc polypeptide shown in Figure 9G (e.g., the Ig Fc polypeptide has a length of approximately 227 amino acids). In some cases, the Ig Fc polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity to the human IgG3 Fc polypeptide shown in Figure 9H, with or without a C-terminal Lys, for example, the Ig Fc polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity to amino acids 19-246 of the human IgG3 Fc polypeptide shown in Figure 9H (e.g., the Ig Fc polypeptide has a length of approximately 228 amino acids). In some cases, the Ig Fc polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity to the human IgM Fc polypeptide shown in Figure 9J, for example, the Ig Fc polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity to amino acids 1-276 of the human IgM Fc polypeptide shown in Figure 9J.In some cases, the Ig Fc polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity to the human IgA Fc polypeptide shown in Figure 9K, for example, the Ig Fc polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity to amino acids 1-234 of the human IgA Fc polypeptide shown in Figure 9K.

[0096] In some cases, the Ig Fc polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity to the human IgE Fc polypeptide shown in Figure 9L, with or without a C-terminal Lys. In some cases, the Ig Fc polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity to the human IgE Fc polypeptide shown in Figure 9L.

[0097] In some cases, the Ig Fc polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity to the human IgG4 Fc polypeptide shown in Figure 9M, with or without the C-terminal Lys. In some cases, the Ig Fc polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity to amino acids 100-327 of the human IgG4 Fc polypeptide shown in Figure 9M (e.g., the Ig Fc polypeptide is approximately 228 amino acids in length, or 227 amino acids in length if the C-terminal Lys is removed).

[0098] In some instances, the IgG4 Fc polypeptide comprises the following amino acid sequence: PPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKCTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 37).

[0099] As mentioned above, disulfide bonds (typically two) can spontaneously form between two Ig Fc polypeptides, for example, between the Ig Fc heavy chain CH2 and CH3 domains, to form a dimer comprising two fusion polypeptides.

[0100] Fusion polypeptide comprising an IL-2 variant polypeptide and a functional protein In some cases, it may be desirable for a fusion polypeptide comprising one or more IL-2 variant polypeptides to also include one or more proteins that are not IL-2 variant polypeptides ("functional proteins"). Such functional proteins can include, for example, immunomodulatory polypeptides, scFvs or nanobodies that target cancer-associated antigens, T cell receptors (TCRs), TCR-like antibody fragments, proteins that target NK cells, e.g., anti-NK antibodies, and receptor trap proteins, e.g., TGF-β trap proteins.

[0101] Fusion polypeptides comprising one or more IL-2 variant polypeptides and one or more functional proteins can be arranged in a variety of configurations. Figures 10A-10C, 11A-11C, and 30A-30G illustrate fusion polypeptides comprising one or more IL-2 variant polypeptides and a heterologous fusion partner comprising both an Ig Fc polypeptide and a functional protein. Figures 12A-12B and 13A-13B illustrate homodimers comprising individual fusion polypeptides, each comprising one or more IL-2 variant polypeptides and a heterologous fusion partner comprising both an Ig Fc polypeptide and a functional protein. Figures 30A-30G illustrate heterodimers comprising one or more variant IL-2 polypeptides, an Ig Fc polypeptide, and, in some cases, a functional protein. As illustrated in Figures 30A-30G, in some cases, the heterodimer comprises a first Ig Fc polypeptide and a second Ig Fc polypeptide, where the first Ig Fc polypeptide is not fused to a variant IL-2 polypeptide and the second Ig Fc polypeptide is fused to a variant IL-2 polypeptide. The first and / or second polypeptides may also be fused to one or more functional proteins (referred to as "A"). When the first and second polypeptides of the heterodimer each comprise a functional protein, the functional proteins may be the same or different (i.e., the functional proteins may have the same amino acid sequence or may differ in amino acid sequence).

[0102] Figures 14A-14N illustrate heterodimeric polypeptides comprising individual fusion polypeptides, each of which comprises one or more IL-2 variant polypeptides and one or more functional proteins. Such proteins can be prepared using the interspecies linkage sequences discussed above. When a heterodimeric polypeptide comprises two or more functional proteins, the functional proteins can be the same or different. See, for example, Figures 14C, 14G, 14I, 14K, 14L, 14M, and 14N, where functional proteins A and A' can be the same or different.

[0103] As mentioned above, non-antibody polypeptide scaffolds may be used in place of the Ig Fc polypeptide.

[0104] Also as noted above, the IL-2 variant polypeptide may alternatively be provided on a carrier such as a lipid vesicle (e.g., liposome) or micelle, a nanoparticle, a PEGylated protein (including site-specific PEGylation), a fibronectin-based scaffold protein, or an artificial antigen-presenting cell such as an engineered red blood cell or enucleated cell (see, e.g., US 2019 / 0290686). In such instances, the functional protein is similarly associated with the carrier via means known in the art.

[0105] Some exemplary functional proteins are described below.

[0106] Cancer targeting polypeptide (CTP) The functional protein can include a cancer targeting polypeptide (CTP) that is specific for a cancer-associated epitope. A "cancer-associated" epitope is an epitope present in a cancer-associated antigen. In some cases, the CTP is an antibody. In some cases, the CTP is a single-chain T-cell receptor (scTCR). In some cases, the target of the CTP is a peptide / HLA (pHLA) complex on the surface of a cancer cell, and the peptide can be a cancer-associated peptide (e.g., a peptide fragment of a cancer-associated antigen).

[0107] Cancer-associated antigens that can be targeted by CTP Cancer-associated antigens that can be targeted by cancer targeting polypeptides include, for example, NY-ESO (New York esophageal squamous cell carcinoma 1), MART-1 (melanoma antigen recognized by T cells 1, also known as Melan-A), HPV (human papillomavirus) E6, BCMA (B-cell maturation antigen), CD123, CD133, CD171, CD19, CD20, CD22, CD30, CD33, CEA (carcinoembryonic antigen), EGFR (epidermal growth factor receptor), EGFRvIII (epidermal growth factor receptor variant III), and EpCAM (epithelial cell adhesion molecule). , EphA2 (ephrin type A receptor 2), disialoganglioside GD2, GPC3 (glypican-3), HER2, IL13Ralpha2 (interleukin-13 receptor subunit alpha-2), LeY (difucosylated type 2 blood-associated antigen), MAGE-A3 (melanoma-associated antigen 3), melanoma glycoprotein, mesothelin, MUC1 (mucin 1), MUC16 (mucin-16), myelin, NKG2D (natural killer group 2D), ligand, PSMA (prostate-specific membrane antigen), and ROR1 (type I receptor tyrosine kinase-like orphan receptor).

[0108] Cancer-associated antigens that can be targeted by CTP include 17-1A antigen, alpha-fetoprotein (AFP), alpha-actin-4, A3, antigen specific for the A33 antibody, ART-4, B7, Ba733, BAGE, bcl-2, bcl-6, BCMA, BrE3 antigen, CA125, CAMEL, CAP-1, carbonic anhydrase IX (CAIX), CASP-8 / m, CCL19, CCL21, CD1, CD1a, CD2, CD3, CD4, CD5, CD8, CD11A, CD 14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD29, CD30, CD32b, CD33, CD37, CD38, CD40, CD40L, CD44, CD45, CD46, CD52, CD54, CD55, CD59, CD64, CD66a-e, CD67, CD70, CD70L, CD74, CD79a, CD79b, CD80, CD83, CD95, CD123, CD126, CD132, CD133, CD138, CD14 7, CD154, CD171, CDC27, CDK-4 / m, CDKN2A, CEA, CEACAM5, CEACAM6, claudins (e.g., claudin-1, claudin-10, claudin-18 (e.g., claudin-18, isoform 2)), complement factors (e.g., C3, C3a, C3b, C5a, and C5), colon-specific antigen p (CSAp), c-Met, CTLA-4, CXCR4, CXCR7, CXCL12, DAM, Dickkopf-related protein (DKK) ), ED-B fibronectin, epidermal growth factor receptor (EGFR), EGFRvIII, EGP-1 (TROP-2), EGP-2, ELF2-M, Ep-CAM, EphA2, EphA3, fibroblast activation protein (FAP), fibroblast growth factor (FGF), Flt-1, Flt-3, folate binding protein, folate receptor, G250 antigen, gangliosides (e.g., GC2, GD3, and GM2), GAGE, GD2, gp100, GPC3, GRO-13, HLA-DR, and HM1.24, human chorionic gonadotropin (HCG) and its subunits, HER2, HER3, HMGB-1, hypoxia-inducible factor (HIF-1), HIF-1a, HSP70-2M, HST-2, Ia, IFN-gamma, IFN-alpha, IFN-beta, IFN-X, IL-4R, IL-6R, IL-13R, IL13Ralpha2, IL-15R, IL-17R, IL-18 R, IL-2, IL-6, IL-8, IL-12, IL-15, IL-17, IL-18, IL-23, IL-25, ILGF, ILGF-1R, insulin-like growth factor 1 (IGF-1), IGF-1R, integrin αVβ3, integrin α5β1, KC4 antigen, killer cell immunoglobulin-like receptor (KIR), Kras, KS-1 antigen, KS1-4, LDR / FUT, Le. gamma , macrophage migration inhibitory factor (MIF), MAGE, MAGE-3, MART-1, MART-2, mCRP, MCP-1, melanoma glycoprotein, mesothelin, MIP-1A, MIP-1B, MIF, mucins (e.g., MUC1, MUC2, MUC3, MUC4, MUC5ac, MUC13, MUC16, MUM-1 / 2, and MUM-3), NCA66, NCA95, NCA90, nectin-4, NY-ESO-1, PAM4 antigen, pancreatic cancer mucins, PD-1, PD-L1, PD-1 receptor, placental proliferation These include, but are not limited to, factors, p53, PLAGL2, prostatic acid phosphatase, PSA, PRAME, PSMA, P1GF, RSS, RANTES, SAGE, 5100, survivin, survivin-2B, T101, TAC, TAG-72, tenascin, Thomson-Friedreich antigen, Tn antigen, TNF-alpha, tumor necrosis antigen, TRAG-3, TRAIL receptor, vascular endothelial growth factor (VEGF), VEGF receptor (VEGFR), WT-1, and COL6A3 exon 6.

[0109] In some cases, the cancer-associated antigen is an antigen associated with a blood cancer. Examples of such antigens include, but are not limited to, BCMA, C5, CD19, CD20, CD22, CD25, CD30, CD33, CD38, CD40, CD45, CD52, CD56, CD66, CD74, CD79a, CD79b, CD80, CD138, CTLA-4, CXCR4, DKK, EphA3, GM2, HLA-DR beta, integrin αVβ3, IGF-R1, IL6, KIR, PD-1, PD-L1, TRAILR1, TRAILR2, transferrin receptor, and VEGF. In some cases, the cancer-associated antigen is an antigen expressed by malignant B cells, such as CD19, CD20, CD22, CD25, CD38, CD40, CD45, CD74, CD80, CTLA-4, IGF-R1, IL6, PD-1, TRAILR2, or VEGF.

[0110] In some cases, the cancer-associated antigen is an antigen associated with a solid tumor. Examples of such antigens include CAIX, cadherin, CEA, c-MET, CTLA-4, EGFR family members, EpCAM, EphA3, FAP, folate binding protein, FR-alpha, gangliosides (such as GC2, GD3, and GM2), HER2, HER3, IGF-1R, integrin αVβ3, integrin α5β1, Le gamma , Liv1, mesothelin, mucin, NaPi2b, PD-1, PD-L1, PD-1 receptor, pgA33, PSMA, RANKL, ROR1, TAG-72, tenascin, TRAILR1, TRAILR2, VEGF, VEGFR, and others listed above.

[0111] CTP targeting peptide / HLA complexes In some cases, the target of the CTP is a peptide-HLA (pHLA) complex on the surface of a cancer cell, and the peptide may be a cancer-associated peptide (e.g., a peptide fragment of a cancer-associated antigen). In such cases, the functional protein may be a T cell receptor or other polypeptide that binds to a pHLA complex on the surface of a cancer cell. Such pHLA complexes are known in the art. Cancer-associated peptides are known in the art. In some cases, the cancer-associated peptide is a peptide fragment of an HLA-A * It binds to the HLA complex, which contains the 0201 heavy chain and the β2M polypeptide.

[0112] In some cases, epitopes present on pHLA on the surface of cancer cells are associated with HLA-A * 0101, A * 0201, A * 0301, A * 1101, A * 2301, A * 2402, A * 2407, A * 3303, and / or A * 3401. In some cases, epitopes present on pHLA on the surface of cancer cells bind to HLA-B complexes containing HLA heavy chains such as HLA-B. * 0702, B * 0801, B * 1502, B * 3802, B * 4001, B * 4601, and / or B * 5301. In some cases, epitopes present on pHLA on the surface of cancer cells are associated with C * 0102, C * 0303, C * 0304, C * 0401, C * 0602, C * 0701, C * 702, C * 0801, and / or C * It binds to HLA complexes containing HLA heavy chains such as 1502.

[0113] In some cases, the epitope is a cancer-associated epitope of any one of the following cancer-associated antigens: a MUC1 polypeptide, an LMP2 polypeptide, an epidermal growth factor receptor (EGFR) vIII polypeptide, a HER-2 / neu polypeptide, or a melanoma antigen family A, 3 (MAGE) polypeptide. A3) polypeptide, p53 polypeptide, mutant p53 polypeptide, NY-ESO-1 polypeptide, folate hydrolase (prostate-specific membrane antigen; PSMA) polypeptide, carcinoembryonic antigen (CEA) polypeptide, claudin polypeptide (e.g., claudin-1, claudin-10, claudin-18 (e.g., claudin-18, isoform 2)), nectin-4 polypeptide, melanoma antigen recognized by T cells (melanA / MART1) polypeptide, Ras polypeptide, gp100 polypeptide, proteinase 3 (PR1) polypeptide, bcr-abl polypeptide, tyrosinase polypeptide, survivin polypeptide, prostate-specific antigen (PSA) polypeptide, hTERT polypeptide, sarcoma metastasis breakpoint polypeptide, synovial sarcoma X (SSX) breakpoint polypeptide, EphA2 polypeptide, acid phosphatase, prostate (PAP) polypeptide, melanoma inhibitor of apoptosis (ML-IAP), epithelial cell adhesion molecule (EpCAM) polypeptide, ERG (TMPRSS2) ETS fusion) polypeptide, NA17 polypeptide, paired box-3 (PAX3) polypeptide, anaplastic lymphoma kinase (ALK) polypeptide, androgen receptor polypeptide, cyclin B1 polypeptide, N-myc proto-oncogene (MYCN) polypeptide, Ras homolog gene family member C (RhoC) polypeptide, tyrosinase-related protein-2 (TRP-2) polypeptide, mesothelin polypeptide, prostate stem cell antigen (PSCA) polypeptide, melanoma-associated antigen-1 (MAGE A1) polypeptide, cytochrome P450 1B1 (CYP1B1) polypeptide, placenta-specific protein 1 (PLAC1) polypeptide, BORIS polypeptide (also known as CCCTC-binding factor or CTCF), ETV6-AML polypeptide, breast cancer antigen NY-BR-1 polypeptide (also known as ankyrin repeat domain-containing protein 30A), regulator of G-protein signaling (RGS5) polypeptide, squamous cell carcinoma cell antigen recognized by T cells (SART3) polypeptide, carbonic anhydrase IX polypeptide, paired box-5 (PAX5) polypeptide, OY-TES1 (testis antigen, also known as acrosin-binding protein) polypeptide, sperm protein 17 polypeptide, lymphoid cell-specific protein-tyrosine kinase (LCK) polypeptide, high-molecular-weight melanoma-associated antigen (HMW-MAA), A-kinase anchor protein-4 (AKAP-4), synovial sarcoma X breakpoint 2 (SSX 2) polypeptides, X antigen family member 1 (XAGE1) polypeptide, B7 homolog 3 (B7H3, also known as CD276) polypeptide, legumain polypeptide (LGMN1, also known as asparaginyl endopeptidase), tyrosine kinase-2 with Ig and EGF homology domains (Tie-2, also known as angiopoietin-1 receptor) polypeptide, P antigen family member 4 (PAGE4) polypeptide, vascular endothelial growth factor receptor 2 (VEGF2) polypeptide, MAD-CT-1 polypeptide, fibroblast activation protein (FAP) polypeptide, platelet-derived growth factor receptor beta (PDGFβ) polypeptide, MAD-CT-2 polypeptide, Fos-related antigen-1 (FOSL) polypeptide, human papilloma virus (HPV) antigen, alpha-fetoprotein (AFP) antigen, and Wilms' tumor-1 (WT1) antigen.

[0114] CTP, an antibody As described above, in some cases, the CTP is an antibody specific for a cancer-associated antigen. In some cases, the CTP is an antibody specific for a peptide / HLA complex on the surface of a cancer cell, and the peptide can be a cancer-associated peptide (e.g., a peptide of a cancer-associated antigen).

[0115] Non-limiting examples of cancer-associated antigen targeting antibodies include abituzumab (anti-CD51), LL1 (anti-CD74), LL2 or RFB4 (anti-CD22), veltuzumab (hA20, anti-CD20), rituximab (anti-CD20), obinutuzumab (GA101, anti-CD20), daratuzumab (anti-CD38), lambrolizumab (anti-PD-1 receptor), nivolumab (anti-PD-1 receptor), ipilimumab (anti-CTLA-4), RS7 (anti-TROP-2), PAM4 or KC4 (both, anti-mucins), MN-14 (anti-CEA), MN-15 or MN-3 (anti-CEACAM6), Mu-9 (anti-colon specific antigen-p), Immunoglobulin G (IMG) and IL-11 (anti-IL-11). 31 (anti-alpha-fetoprotein), R1 (anti-IGF-1R), A19 (anti-CD19), TAG-72 (e.g., CC49), Tn, J591 or HuJ591 (anti-PSMA), AB-PG1-XG1-026 (anti-PSMA dimer), D2 / B (anti-PSMA), G250 (anti-carbonic anhydrase IX), L243 (anti-HLA-DR), alemtuzumab (anti-CD52), oportuzumab (anti-EpCAM), bevacizumab (anti VEGF), cetuximab (anti-EGFR), gemtuzumab (anti-CD33), ibritumomab tiuxetan (anti-CD20), panitumumab (anti-EGFR), tositumomab (anti-CD20), PAM4 (also known as clivatuzumab, anti-mucin), trastuzumab (anti-HER2), pertuzumab (anti-HER2), polatuzumab (anti-CD79b), and anetuzumab (anti-mesothelin).

[0116] In some cases, the cancer targeting polypeptide is an antibody. In some cases, the cancer targeting polypeptide is a single-chain antibody. In some cases, the cancer targeting polypeptide is an scFv. In some cases, the cancer targeting polypeptide is a nanobody (also referred to as a single-domain antibody (sdAb)). In some cases, the cancer targeting polypeptide is a heavy-chain nanobody. In some cases, the cancer targeting polypeptide is a light-chain nanobody.

[0117] The VH and VL amino acid sequences of various tumor antigen-binding antibodies are known in the art, as they are the light chain and heavy chain CDRs of such antibodies.See, for example, Ling et al. (2018) Frontiers Immunol.9:469, International Publication No. 2005 / 012493, US Patent Application Publication No. 2019 / 0119375, US Patent Application Publication No. 2013 / 0066055.The following are non-limiting examples of tumor antigen-binding antibodies: Exemplary antibodies with known light and heavy chain sequences include anti-Her2 antibodies, anti-CD19 antibodies, anti-mesothelin antibodies, antibodies against trophoblast cell surface antigen 2 (Trop-2) (also known as epithelial glycoprotein 1, gastrointestinal tumor-associated antigen GA733-1, membrane component chromosome 1 surface marker 1, and tumor-associated calcium signal transducer 2), anti-BCMA (B-cell maturation antigen) antibodies, anti-MUC16 (also known as CA125), and anti-claudin-18 isoform 2 ("claudin-18.2").

[0118] In some cases, the antibody targets human serum albumin (HSA). By binding to HSA, such an anti-HSA antibody can tether a fusion protein containing an IL-2 variant polypeptide(s) to HSA, thereby potentially extending the serum half-life of the fusion protein. Alternatively, as discussed below, one or more IL-2 variant polypeptides may be fused directly to the heavy or light chain of an anti-HSA antibody.

[0119] The following are non-limiting examples of cancer-associated antigen-binding antibodies that can be the CTPs in the IL-2 variant fusion polypeptides of the present disclosure. Any of the CTPs described below can constitute "A" or "A'" in any of Figures 10A-10C, 11A-11C, 12A-12B, and 14A-14N.

[0120] In some cases, an IL-2 variant fusion polypeptide includes a first CTP (e.g., "A") and a second CTP (e.g., "A'"), where the first CTP is specific for a first cancer-associated antigen and the second CTP is specific for a second cancer-associated antigen that is different from the first cancer-associated antigen. For example, in some cases, an IL-2 variant fusion polypeptide includes (i) a first CTP (e.g., "A"), where the first CTP is an anti-CD19 antibody, and (ii) a second CTP (e.g., "A'"), where the second CTP is an anti-CD20 antibody. As another example, in some cases, an IL-2 variant fusion polypeptide includes (i) a first CTP (e.g., "A"), where the first CTP is an anti-Her2 antibody, and (ii) a second CTP (e.g., "A'"), where the second CTP is an anti-EGFR antibody.

[0121] In some cases, an IL-2 variant fusion polypeptide comprises a first CTP (e.g., "A") and a second CTP (e.g., "A'"), where the first CTP is specific for a first epitope on a cancer-associated antigen and the second CTP is specific for a second epitope on the same cancer-associated antigen. In some cases, the first epitope and the second epitope are non-identical and non-overlapping epitopes, such that the cancer-associated antigen can be bound simultaneously by the first CTP and the second CTP.

[0122] anti-Her2 In some cases, the CTP present in the IL-2 variant fusion polypeptide of the present disclosure comprises an anti-Her2 antibody. In some cases, the anti-Her2 antibody is an scFv polypeptide. In some cases, the anti-Her2 antibody is a nanobody.

[0123] In some instances, the anti-Her2 antibody has (a) the following amino acid sequence: DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYE and (b) a light chain comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to KHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 38), and the following amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYY. CSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNV NHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD and a heavy chain comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 39).

[0124] In some cases, the anti-Her2 antibody comprises a light chain variable region (VL) present in the light chain amino acid sequence provided above and a heavy chain variable region (VH) present in the heavy chain amino acid sequence provided above. For example, the anti-Her2 antibody may comprise (a) a VL comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence: DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO: 40); and b) It may include a VH comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS (SEQ ID NO: 41). In some cases, the anti-Her2 antibody has, from N-terminus to C-terminus, (a) at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS (SEQ ID NO: 41). (b) a linker; and (c) a VL comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence: DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO: 40).Suitable linkers are described elsewhere herein and include, for example, (GGGGS)n (SEQ ID NO: 42), where n is an integer between 1 and 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10).

[0125] In some cases, the anti-Her2 antibody comprises a VL CDR1, a VL CDR2, and a VL CDR3 present in the light chain amino acid sequence provided above, and a VH CDR1, a CDR2, and a CDR3 present in the heavy chain amino acid sequence provided above. H and V L CDRs are as defined by Kabat (see, e.g., Table 1, above, and Kabat 1991). H and V L CDRs are as defined by Chothia (see, e.g., Table 1, above, and Chothia 1987). H and V L CDRs are as defined by MacCallum (see, eg, Table 1 above, and MacCallum 1996).

[0126] For example, an anti-Her2 antibody may comprise a VL CDR1 having the amino acid sequence RASQDVNTAVA (SEQ ID NO: 43), a VL CDR2 having the amino acid sequence SASFLY (SEQ ID NO: 44), a VL CDR3 having the amino acid sequence QQHYTTPP (SEQ ID NO: 45), a VH CDR1 having the amino acid sequence GFNIKDTY (SEQ ID NO: 46), a VH CDR2 having the amino acid sequence IYPTNGYT (SEQ ID NO: 47), and a VH CDR3 having the amino acid sequence SRWGGDGFYAMDY (SEQ ID NO: 48).

[0127] In some cases, the anti-Her2 antibody is an scFv antibody. For example, the anti-Her2 scFv can comprise an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO: 49).

[0128] As another example, in some instances, the anti-Her2 antibody may have (a) the following amino acid sequence: DIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYIYPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADY (b) a light chain variable region (VL) comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to EKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 50); and (b) the following amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVRQAPGKGLEWVADVNPNSGGSIYNQRFKGRFTLSVDRSKNTLYLQMNSLRAEDT AVYYCARNLGPSFYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN VNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWL and a heavy chain variable region (VH) comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 51).

[0129] In some instances, the anti-Her2 antibody comprises a VL present in the light chain amino acid sequence provided above and a VH present in the heavy chain amino acid sequence provided above. For example, the anti-Her2 antibody may comprise: (a) a VL comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence: DIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYIYPYTFGQGTKVEIK (SEQ ID NO: 52); (b) A VH comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVRQAPGKGLEWVADVNPNSGGSIYNQRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQGTLVTVSS (SEQ ID NO: 53).

[0130] In some cases, the anti-Her2 antibody comprises a VL CDR1, a VL CDR2, and a VL CDR3 present in the light chain amino acid sequence provided above, and a VH CDR1, a CDR2, and a CDR3 present in the heavy chain amino acid sequence provided above. H and V L CDRs are as defined by Kabat (see, e.g., Table 1, above, and Kabat 1991). H and V L CDRs are as defined by Chothia (see, e.g., Table 1, above, and Chothia 1987). H and V L CDRs are as defined by MacCallum (see, eg, Table 1 above, and MacCallum 1996).

[0131] For example, an anti-HER2 antibody may comprise a VL CDR1 having the amino acid sequence KASQDVSIGVA (SEQ ID NO: 54), a VL CDR2 having the amino acid sequence SASYRY (SEQ ID NO: 55), a VL CDR3 having the amino acid sequence QQYYIYPY (SEQ ID NO: 56), a VH CDR1 having the amino acid sequence GFTFTDYTMD (SEQ ID NO: 57), a VH CDR2 having the amino acid sequence ADVNPNSGGSIYNQRFKG (SEQ ID NO: 58), and a VH CDR3 having the amino acid sequence ARNLGPSFYFDY (SEQ ID NO: 59).

[0132] In some cases, the anti-Her2 antibody is an scFv. For example, in some cases, the anti-Her2 scFv comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO: 49).

[0133] anti-CD19 In some cases, the CTP present in the IL-2 variant fusion polypeptide of the present disclosure includes an anti-CD19 antibody. In some cases, the anti-CD19 antibody is an scFv polypeptide. In some cases, the anti-CD19 antibody is a nanobody. Anti-CD19 antibodies are known in the art, and the VH and VL or VH and VL CDRs of any anti-CD19 antibody can be used in the CTP. See, for example, International Publication No. WO 2005 / 012493.

[0134] In some instances, the anti-CD19 antibody comprises a VL CDR1 comprising the amino acid sequence KASQSVDYDGDSYLN (SEQ ID NO: 60), a VL CDR2 comprising the amino acid sequence DASNLVS (SEQ ID NO: 61), and a VL CDR3 comprising the amino acid sequence QQSTEDPWT (SEQ ID NO: 62). In some instances, the anti-CD19 antibody comprises a VH CDR1 comprising the amino acid sequence SYWMN (SEQ ID NO: 63), a VH CDR2 comprising the amino acid sequence QIWPGDGDTNYNGKFKG (SEQ ID NO: 64), and a VH CDR3 comprising the amino acid sequence RETTTVGRYYYAMDY (SEQ ID NO: 65). In some instances, the anti-CD19 antibody comprises a VL CDR1 comprising the amino acid sequence KASQSVDYDGDSYLN (SEQ ID NO: 60), a VL CDR2 comprising the amino acid sequence DASNLVS (SEQ ID NO: 61), a VL CDR3 comprising the amino acid sequence QQSTEDPWT (SEQ ID NO: 62), a VH CDR1 comprising the amino acid sequence SYWMN (SEQ ID NO: 63), a VH CDR2 comprising the amino acid sequence QIWPGDGDTNYNGKFKG (SEQ ID NO: 64), and a VH CDR3 comprising the amino acid sequence RETTTVGRYYYAMDY (SEQ ID NO: 65).

[0135] In some cases, the anti-CD19 antibody is an scFv. For example, in some cases, the anti-CD19 scFv comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: DIQLTQSPASLAVSLGQRATISCKASQSVDYDGDSYLNWYQQIPGQPPKLLIYDASNLVSGIPPRFSGSGSGTDFTLNIHPVEKVDAATYHCQQSTEDPWTFGGGTKLEIKGGGGSGGGGSGGGGSQVQLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIWPGDGDTNYNGKFKGKATLTADESSSTAYMQLSSLASEDSAVYFCARRETTTVGRYYYAMDYWGQGTTVTVS (SEQ ID NO: 66).

[0136] In some instances, the anti-CD19 antibody is an scFv polypeptide comprising the following amino acid sequence: DIQLTQSPASLAVSLGQRATISCKASQSVDYDGDSYLNWYQQIPGQPPKLLIYDASNLVSGIPPRFSGSGSGTDFTLNIHPVEKVDAATYHCQQSTEDPWTFGGGTKLEIKGGGGSGGGGSGGGGSQVQLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIWPGDGDTNYNGKFKGKATLTADESSSTAYMQLSSLASEDSAVYFCARRETTTVGRYYYAMDYWGQGTTVTVSS (SEQ ID NO: 67).

[0137] Antimesothelin In some cases, the CTP present in the IL-2 variant fusion polypeptide of the present disclosure comprises an anti-mesothelin antibody. In some cases, the anti-mesothelin antibody is an scFv polypeptide. In some cases, the anti-mesothelin antibody is a nanobody. Anti-mesothelin antibodies are known in the art, and the VH and VL or VH and VL CDRs of any anti-mesothelin antibody can be used as the CTP in the IL-2 variant fusion polypeptide. See U.S. Patent Application Publication No. 2019 / 0000944, WO 2009 / 045957, WO 2014 / 031476, U.S. Patent No. 8,460,660, U.S. Patent Application Publication No. 2013 / 0066055, and WO 2009 / 068204. In some cases, the CTP is an anti-mesothelin scFv or anti-mesothelin nanobody that includes the VH and VL CDRs present in any one of the amino acid sequences set forth in Figures 18A-18H. In some cases, the CTP is an anti-mesothelin scFv that includes the amino acid sequence set forth in any one of Figures 18A-18H.

[0138] In some instances, the anti-mesothelin antibody has (a) the following amino acid sequence: DIALTQPASVSGSPGQSITISCTGTSSDIGGYNSVSWYQQHPGKAPKLMIYGVNNRPSGVSNRFSGSSKSGNTASLTISGLQAEDEADYYCSSYDIESATPVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKGDSSPVKAGVETTTPSKQSNNKYAASSYLSLTP and (b) a light chain comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to EQWKSHRSYSCQVTHEGSTVEKTVAPTESS (SEQ ID NO: 68), and the following amino acid sequence: QVELVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQAPGKGLEWMGIIDPGDSRTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAM. YYCARGQLYGGTYMDGWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYIC NVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 69).

[0139] In some instances, the anti-mesothelin antibody comprises a VL present in the light chain amino acid sequence provided above and a VH present in the heavy chain amino acid sequence provided above. For example, an anti-mesothelin antibody can include (a) a VL comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence: DIALTQPASVSGSPGQSITISCTGTSSDIGGYNSVSWYQQHPGKAPKLMIYGVNNRPSGVSNRFSGSSKSGNTASLTISGLQAEDEADYYCSSYDIESATPVFGGGTK (SEQ ID NO: 70); and (b) a VH comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence: QVELVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQAPGKGLEWMGIIDPGDSRTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARGQLYGGTYMDGWGQGTLVTVSS (SEQ ID NO: 71).

[0140] In some cases, the anti-mesothelin antibody comprises a VL CDR1, a VL CDR2, and a VL CDR3 present in the light chain amino acid sequence provided above, and a VH CDR1, a CDR2, and a CDR3 present in the heavy chain amino acid sequence provided above. H and V L CDRs are as defined by Kabat (see, e.g., Table 1, above, and Kabat 1991). H and V L CDRs are as defined by Chothia (see, e.g., Table 1, above, and Chothia 1987). H and V L CDRs are as defined by MacCallum (see, eg, Table 1 above, and MacCallum 1996).

[0141] For example, an anti-mesothelin antibody may comprise a VL CDR1 having the amino acid sequence TGTSSDIGGYNSVS (SEQ ID NO: 72), a VL CDR2 having the amino acid sequence LMIYGVNNRPS (SEQ ID NO: 73), a VL CDR3 having the amino acid sequence SSYDIESATP (SEQ ID NO: 74), a VH CDR1 having the amino acid sequence GYSFTSYWIG (SEQ ID NO: 75), a VH CDR2 having the amino acid sequence WMGIIDPGDSRTRYSP (SEQ ID NO: 76), and a VH CDR3 having the amino acid sequence GQLYGGTYMDG (SEQ ID NO: 77).

[0142] The anti-mesothelin antibody can be an scFv. In one non-limiting example, the anti-mesothelin scFv has the following amino acid sequence: TIFF2025532538000029.tif23163, where VH CDR1, CDR2, and CDR3 are underlined and VL CDR1, CDR2, and CDR3 are bold and underlined.

[0143] As one non-limiting example, an anti-mesothelin scFv may have the following amino acid sequence: TIFF2025532538000030.tif30160, where VH CDR1, CDR2, and CDR3 are underlined and VL CDR1, CDR2, and CDR3 are bold and underlined.

[0144] In some instances, the CTP is a suitable anti-mesothelin antibody comprising (a) a VL CDR1, VL CDR2, and VL CDR3 present in a light chain variable region (VL) comprising the following amino acid sequence: EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPIFTFGPGTKVDIK (SEQ ID NO: 80), and (b) a VH CDR1, CDR2, and CDR3 present in a heavy chain variable region (VH) comprising the following amino acid sequence: QMQLVESGGGVVQPGRSLRLSCTASGFTFSNNGMHWVRQAPGKGLEWVAVIWFDGMNKFYVDSVKGRFTISRDNSKNTLYLEMNSLRAEDTAIYYCAREGDGSGIYYYYGMDVWGQGTTVTVSS (SEQ ID NO: 81). In some cases, V H and V L CDRs are as defined by Kabat (see, e.g., Table 1, above, and Kabat 1991). H and V L CDRs are as defined by Chothia (see, e.g., Table 1, above, and Chothia 1987). H and V L CDRs are as defined by MacCallum (see, eg, Table 1 above, and MacCallum 1996).

[0145] In some instances, the CTP comprises (a) a VL region comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPIFTFGPGTKVDIK (SEQ ID NO: 80); and (b) An anti-mesothelin antibody comprising a VH region having an amino acid sequence that has at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence: QMQLVESGGGVVQPGRSLRLSCTASGFTFSNNGMHWVRQAPGKGLEWVAVIWFDGMNKFYVDSVKGRFTISRDNSKNTLYLEMNSLRAEDTAIYYCAREGDGSGIYYYYGMDVWGQGTTVTVSS (SEQ ID NO: 81).

[0146] In some cases, the CTP comprises, from N-terminus to C-terminus, (a) an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: QMQLVESGGGVVQPGRSLRLSCTASGFTFSNNGMHWVRQAPGKGLEWVAVIWFDGMNKFYVDSVKGRFTISRDNSKNTLYLEMNSLRAEDTAIYYCAREGDGSGIYYYYGMDVWGQGTTVTVSS (SEQ ID NO: 81). (b) a VH region, (b) a peptide linker, and (c) a VL region comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPIFTFGPGTKVDIK (SEQ ID NO: 80). In some cases, the peptide linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO: 42), where n is an integer between 1 and 10 (e.g., n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). In some cases, the peptide linker comprises the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 82) and is 15 amino acids in length.

[0147] In some instances, the CTP comprises, in order from N-terminus to C-terminus, (a) a VL region comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPIFTFGPGTKVDIK (SEQ ID NO: 80); (b) a peptidylphosphorylamine (PMP) domain comprising a peptide fragment (PMP) and a peptide fragment (PMP) that is fused to the peptide fragment. and (c) an anti-mesothelin scFv comprising a VH region comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: QMQLVESGGGVVQPGRSLRLSCTASGFTFSNNGMHWVRQAPGKGLEWVAVIWFDGMNKFYVDSVKGRFTISRDNSKNTLYLEMNSLRAEDTAIYYCAREGDGSGIYYYYGMDVWGQGTTVTVSS (SEQ ID NO: 81). In some cases, the peptide linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO: 42), where n is an integer between 1 and 10 (e.g., n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). In some cases, the peptide linker comprises the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 82) and is 15 amino acids in length.

[0148] In some instances, the CTP is an anti-mesothelin antibody comprising (a) a VL CDR1, VL CDR2, and VL CDR3 present in a light chain variable region (VL) comprising the following amino acid sequence: DIELTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPGRFSGSGSGNSYSLTISSVEAEDDATYYCQQWSKHPLTFGSGTKVEIK (SEQ ID NO: 83), and (b) a VH CDR1, CDR2, and CDR3 present in a heavy chain variable region (VH) comprising the following amino acid sequence: QVQLQQSGPELEKPGASVKISCKASGYSFTGYTMNWVKQSHGKSLEWIGLITPYNGASSYNQKFRGKATLTVDKSSSTAYMDLLSLTSEDSAVYFCARGGYDGRGFDYWGSGTPVTVSS (SEQ ID NO: 84). H and V L CDRs are as defined by Kabat (see, e.g., Table 1, above, and Kabat 1991). H and V L CDRs are as defined by Chothia (see, e.g., Table 1, above, and Chothia 1987). H and V L The CDRs are as defined by MacCallum (see, e.g., Table 1 above, and MacCallum 1996). See, e.g., amatuximab.

[0149] In some instances, the CTP comprises (a) a VL region comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: DIELTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPGRFSGSGSGNSYSLTISSVEAEDDATYYCQQWSKHPLTFGSGTKVEIK (SEQ ID NO: 83); and An anti-mesothelin antibody comprising a VH region having an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence below: QVQLQQSGPELEKPGASVKISCKASGYSFTGYTMNWVKQSHGKSLEWIGLITPYNGASSYNQKFRGKATLTVDKSSSTAYMDLLSLTSEDSAVYFCARGGYDGRGFDYWGSGTPVTVSS (SEQ ID NO: 84).

[0150] In some instances, the CTP comprises, in order from N-terminus to C-terminus, (a) a VL region comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: DIELTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPGRFSGSGSGNSYSLTISSVEAEDDATYYCQQWSKHPLTFGSGTKVEIK (SEQ ID NO: 83); and (b) a peptide linker. and (c) an anti-mesothelin scFv comprising a VH region comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: QVQLQQSGPELEKPGASVKISCKASGYSFTGYTMNWVKQSHGKSLEWIGLITPYNGASSYNQKFRGKATLTVDKSSSTAYMDLLSLTSEDSAVYFCARGGYDGRGFDYWGSGTPVTVSS (SEQ ID NO: 84). In some cases, the peptide linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO: 42), where n is an integer between 1 and 10 (e.g., n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). In some cases, the peptide linker comprises the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 82) and is 15 amino acids in length.

[0151] In some cases, the CTP comprises, from N-terminus to C-terminus, (a) an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: QVQLQQSGPELEKPGASVKISCKASGYSFTGYTMNWVKQSHGKSLEWIGLITPYNGASSYNQKFRGKATLTVDKSSSTAYMDLLSLTSEDSAVYFCARGGYDGRGFDYWGSGTPVTVSS (SEQ ID NO: 84); An anti-mesothelin scFv comprising: (a) a VH region, (b) a peptide linker, and (c) a VL region comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: DIELTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPGRFSGSGSGNSYSLTISSVEAEDDATYYCQQWSKHPLTFGSGTKVEIK (SEQ ID NO: 83). In some cases, the peptide linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO: 42), where n is an integer between 1 and 10 (e.g., n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). In some cases, the peptide linker comprises the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 82) and is 15 amino acids in length.

[0152] In some instances, the CTP is an anti-mesothelin antibody comprising (a) a VL CDR1, VL CDR2, and VL CDR3 present in a light chain variable region (VL) comprising the following amino acid sequence: DIALTQPASVSGSPGQSITISCTGTSSDIGGYNSVSWYQQHPGKAPKLMIYGVNNRPSGVSNRFSGSSKSGNTASLTISGLQAEDEADYYCSSYDIESATPVFGGGTKLTVLG (SEQ ID NO: 85), and (b) a VH CDR1, CDR2, and CDR3 present in a heavy chain variable region (VH) comprising the following amino acid sequence: QVELVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQAPGKGLEWMGIIDPGDSRTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARGQLYGGTYMDGWGQGTLVTVSS (SEQ ID NO: 71). H and V L CDRs are as defined by Kabat (see, e.g., Table 1, above, and Kabat 1991). H and V L CDRs are as defined by Chothia (see, e.g., Table 1, above, and Chothia 1987). H and V L CDRs are as defined by MacCallum (see, eg, Table 1 above, and MacCallum 1996).

[0153] In some instances, the CTP comprises (a) a VL region comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: DIALTQPASVSGSPGQSITISCTGTSSDIGGYNSVSWYQQHPGKAPKLMIYGVNNRPSGVSNRFSGSSKSGNTASLTISGLQAEDEADYYCSSYDIESATPVFGGGTKLTVLG (SEQ ID NO: 85); and ) A VH region comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: QVELVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQAPGKGLEWMGIIDPGDSRTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARGQLYGGTYMDGWGQGTLVTVSS (SEQ ID NO: 71).

[0154] In some instances, the CTP comprises, in order from N-terminus to C-terminus, (a) a VL region comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: DIALTQPASVSGSPGQSITISCTGTSSDIGGYNSVSWYQQHPGKAPKLMIYGVNNRPSGVSNRFSGSSKSGNTASLTISGLQAEDEADYYCSSYDIESATPVFGGGTKLTVLG (SEQ ID NO: 85); and (b) a peptide and (c) an anti-mesothelin scFv comprising a VH region comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: QVELVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQAPGKGLEWMGIIDPGDSRTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARGQLYGGTYMDGWGQGTLVTVSS (SEQ ID NO: 71). In some cases, the peptide linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO: 42), where n is an integer between 1 and 10 (e.g., n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). In some cases, the peptide linker comprises the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 82) and is 15 amino acids in length.

[0155] In some instances, the CTP comprises, in order from N-terminus to C-terminus, (a) a VH region comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: QVELVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQAPGKGLEWMGIIDPGDSRTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARGQLYGGTYMDGWGQGTLVTVSS (SEQ ID NO: 71). an anti-mesothelin scFv comprising a VL region comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: DIALTQPASVSGSPGQSITISCTGTSSDIGGYNSVSWYQQHPGKAPKLMIYGVNNRPSGVSNRFSGSSKSGNTASLTISGLQAEDEADYYCSSYDIESATPVFGGGTKLTVLG (SEQ ID NO: 85). In some cases, the peptide linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO: 42), where n is an integer between 1 and 10 (e.g., n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). In some cases, the peptide linker comprises the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 82) and is 15 amino acids in length.

[0156] In some instances, the CTP is an anti-mesothelin antibody comprising (a) a VL CDR1, VL CDR2, and VL CDR3 present in a light chain variable region (VL) comprising the following amino acid sequence: DIQMTQSPSSLSASVGDRVTITCSASSSVSYMHWYQQKSGKAPKLLIYDTSKLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSKHPLTFGQGTKLEIK (SEQ ID NO: 86), and (b) a VH CDR1, CDR2, and CDR3 present in a heavy chain variable region (VH) comprising the following amino acid sequence: QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYTMNWVRQAPGQGLEWMGLITPYNGASSYNQKFRGKATMTVDTSTSTVYMELSSLRSEDTAVYYCARGGYDGRGFDYWGQGTLVTVSS (SEQ ID NO: 87). H and V L CDRs are as defined by Kabat (see, e.g., Table 1, above, and Kabat 1991). H and V L CDRs are as defined by Chothia (see, e.g., Table 1, above, and Chothia 1987). H and V L CDRs are as defined by MacCallum (see, e.g., Table 1 above, and MacCallum 1996). See, e.g., RG7787.

[0157] In some instances, the CTP comprises (a) a VL region comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: DIQMTQSPSSLSASVGDRVTITCSASSSVSYMHWYQQKSGKAPKLLIYDTSKLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSKHPLTFGQGTKLEIK (SEQ ID NO: 86); and An anti-mesothelin antibody comprising a VH region having an amino acid sequence that has at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence below: QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYTMNWVRQAPGQGLEWMGLITPYNGASSYNQKFRGKATMTVDTSTSTVYMELSSLRSEDTAVYYCARGGYDGRGFDYWGQGTLVTVSS (SEQ ID NO: 87).

[0158] In some instances, the CTP comprises, in order from N-terminus to C-terminus, (a) a VL region comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: DIQMTQSPSSLSASVGDRVTITCSASSSVSYMHWYQQKSGKAPKLLIYDTSKLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSKHPLTFGQGTKLEIK (SEQ ID NO: 86); and (b) a peptide linker. and (c) an anti-mesothelin scFv comprising a VH region comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYTMNWVRQAPGQGLEWMGLITPYNGASSYNQKFRGKATMTVDTSTSTVYMELSSLRSEDTAVYYCARGGYDGRGFDYWGQGTLVTVSS (SEQ ID NO: 87). In some cases, the peptide linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO: 42), where n is an integer between 1 and 10 (e.g., n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). In some cases, the peptide linker comprises the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 82) and is 15 amino acids in length.

[0159] In some cases, the CTP comprises, from N-terminus to C-terminus, (a) an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYTMNWVRQAPGQGLEWMGLITPYNGASSYNQKFRGKATMTVDTSTSTVYMELSSLRSEDTAVYYCARGGYDGRGFDYWGQGTLVTVSS (SEQ ID NO: 87); An anti-mesothelin scFv comprising (b) a VH region, (b) a peptide linker, and (c) a VL region comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: DIQMTQSPSSLSASVGDRVTITCSASSSVSYMHWYQQKSGKAPKLLIYDTSKLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSKHPLTFGQGTKLEIK (SEQ ID NO: 86). In some cases, the peptide linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO: 42), where n is an integer between 1 and 10 (e.g., n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). In some cases, the peptide linker comprises the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 82) and is 15 amino acids in length.

[0160] In some instances, the anti-mesothelin scFv has the following amino acid sequence: TIFF2025532538000031.tif23160, in which the VH sequence is in italics, the (GGGGS)3 (SEQ ID NO: 82) linker is in bold and underlined, and the VL sequence is underlined.

[0161] In some instances, the anti-mesothelin scFv has the following amino acid sequence: TIFF2025532538000032.tif23159, in which the VL sequence is underlined, the (GGGGS)3 (SEQ ID NO: 82) linker is bold and underlined, and the VL sequence is italicized.

[0162] In some instances, the anti-mesothelin scFv has the following amino acid sequence: TIFF2025532538000033.tif23160, in which the VH sequence is in italics, the (GGGGS)3 (SEQ ID NO: 82) linker is in bold and underlined, and the VL sequence is underlined.

[0163] In some instances, the anti-mesothelin scFv has the following amino acid sequence: TIFF2025532538000034.tif23158, in which the VL sequence is underlined, the (GGGGS)3 (SEQ ID NO: 82) linker is bold and underlined, and the VL sequence is italicized.

[0164] In some instances, the anti-mesothelin scFv has the following amino acid sequence: TIFF2025532538000035.tif23160, in which the VH sequence is in italics, the (GGGGS)3 (SEQ ID NO: 82) linker is in bold and underlined, and the VL sequence is underlined.

[0165] In some instances, the anti-mesothelin scFv has the following amino acid sequence: TIFF2025532538000036.tif23159, in which the VL sequence is underlined, the (GGGGS)3 (SEQ ID NO: 82) linker is bold and underlined, and the VL sequence is italicized.

[0166] In some instances, the anti-mesothelin scFv has the following amino acid sequence: TIFF2025532538000037.tif23159, in which the VH sequence is in italics, the (GGGGS)3 (SEQ ID NO: 82) linker is in bold and underlined, and the VL sequence is underlined.

[0167] In some instances, the anti-mesothelin scFv has the following amino acid sequence: TIFF2025532538000038.tif23159, in which the VL sequence is underlined, the (GGGGS)3 (SEQ ID NO: 82) linker is bold and underlined, and the VL sequence is italicized.

[0168] anti-PSMA Prostate-specific membrane antigen (PSMA) (also known as folate hydrolase 1 (FOLH1), membrane glutamate carboxypeptidase, and N-acetylated alpha-linked acidic dipeptidase 1) is upregulated in prostate cancer cells and is used as a diagnostic and prognostic indicator for prostate cancer. In some cases, the CTP present in the IL-2 variant fusion polypeptide of the present disclosure comprises an anti-PSMA antibody. In some cases, the anti-PSMA antibody is an scFv polypeptide. In some cases, the anti-PSMA antibody is a nanobody. Anti-PSMA antibodies are known in the art, and the VH and VL or VH and VL CDRs of any anti-PSMA antibody can be used as the CTP. See, e.g., U.S. Patent No. 10,179,819 and U.S. Patent Application Publication No. 2021 / 0277141.

[0169] anti-CD22 CD22 (also known as B lymphocyte cell adhesion molecule, sialic acid-binding Ig-like lectin 2, or SIGLEC2) is a sialic acid-binding adhesion molecule that is primarily restricted to the B cell lineage and expressed on most B-lineage malignancies. In some cases, the CTP present in an IL-2 variant fusion polypeptide of the present disclosure comprises an anti-CD22 antibody. In some cases, the anti-CD22 antibody is an scFv polypeptide. In some cases, the anti-CD22 antibody is a nanobody.

[0170] Anti-CD22 antibodies are known in the art, and the VH and VL or VH and VL CDRs of any anti-CD22 antibody can be used as CTPs. See, for example, Xiao et al. (2009) Mabs 1:297 (which describes the fully human anti-CD22 m971 scFv) and U.S. Patent Application Publication No. 2020 / 0147134. Examples of anti-CD22 antibodies include epratuzumab and inotuzumab. See, for example, Lenoard et al. (2007) Oncogene 26:3704 and U.S. Patent No. 5,789,554 (which describes epratuzumab), and DiJoseph et al. (2007) Leukemia 21:2240 (which describes inotuzumab).

[0171] For example, an anti-CD22 antibody may comprise: (i) a heavy chain variable region (VH) CDR1 having the amino acid sequence: GDSVSSNSAA (SEQ ID NO: 96); (ii) a VH CDR2 having the amino acid sequence: TYYRSKWYN (SEQ ID NO: 97); (iii) a VH CDR3 having the amino acid sequence: AREVTGDLEDAFDI (SEQ ID NO: 98); (iv) a light chain variable region (VL) CDR1 having the amino acid sequence: QTIWSY (SEQ ID NO: 99); v) a VL CDR2 having the amino acid sequence: AAS (Ala-Ala-Ser); and (vi) a VL CDR3 having the amino acid sequence: QQSYSIPQT (SEQ ID NO: 100).

[0172] anti-TROP-2 Trophoblast cell surface antigen 2 (Trop-2) (also known as epithelial glycoprotein-1, gastrointestinal tumor-associated antigen GA733-1, membrane component staining 1 surface marker-1, and tumor-associated calcium signal transducer-2) is a transmembrane glycoprotein and the protein product of the TACSTD2 gene that is upregulated in many cancer types. In some cases, the CTP present in the IL-2 variant fusion polypeptide of the present disclosure comprises an anti-TROP-2 antibody. In some cases, the anti-TROP-2 antibody is an scFv polypeptide. In some cases, the anti-TROP-2 antibody is a nanobody.

[0173] In some cases, the CTP is an anti-TROP-2 scFv or anti-TROP-2 nanobody comprising the VH and VL CDRs present in any one of the amino acid sequences set forth in Figures 19A-19D. In some cases, the CTP is an anti-TROP-2 scFv comprising the amino acid sequence set forth in any one of Figures 19A-19D.

[0174] Anti-TROP-2 antibodies are known in the art, and the VH and VL, or VH and VL CDRs, of any anti-TROP-2 antibody can be used as a CTP. (See, e.g., U.S. Patent No. 7,238,785.) In some cases, the anti-TROP-2 antibody comprises (i) the light chain CDR sequences CDR1 (KASQDVSIAVA; SEQ ID NO: 101), CDR2 (SASYRYT; SEQ ID NO: 102), and CDR3 (QQHYITPLT; SEQ ID NO: 103), and (ii) the heavy chain CDR sequences CDR1 (NYGMN; SEQ ID NO: 104), CDR2 (WINTYTGEPTYTDDFKG; SEQ ID NO: 105), and CDR3 (GGFGSSYWYFDV; SEQ ID NO: 106).

[0175] In some instances, the anti-TROP-2 antibody comprises (i) the heavy chain CDR sequences CDR1 (TAGMQ; SEQ ID NO: 107), CDR2 (WINTHSGVPKYAEDFKG; SEQ ID NO: 108), and CDR3 (SGFGSSYWYFDV; SEQ ID NO: 109), and (ii) the light chain CDR sequences CDR1 (KASQDVSTAVA; SEQ ID NO: 110), CDR2 (SASYRYT; SEQ ID NO: 102), and CDR3 (QQHYITPLT; SEQ ID NO: 103).

[0176] In some instances, the CTP is an anti-TROP2 antibody comprising (a) a VL CDR1, VL CDR2, and VL CDR3 present in a light chain variable region (VL) comprising the following amino acid sequence: DIQLTQSPSSLSASVGDRVSITCKASQDVSIAVAWYQQKPGKAPKLLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDFAVYYCQQHYITPLTFGAGTKVEIK (SEQ ID NO: 111), and (b) a VH CDR1, CDR2, and CDR3 present in a heavy chain variable region (VH) comprising the following amino acid sequence: QVQLQQSGSELKKPGASVKVSCKASGYTFTNYGMNWVKQAPGQGLKWMGWINTYTGEPTYTDDFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCARGGFGSSYWYFDVWGQGSLVTVSS (SEQ ID NO: 112). H and V L CDRs are as defined by Kabat (see, e.g., Table 1, above, and Kabat 1991). H and V L CDRs are as defined by Chothia (see, e.g., Table 1, above, and Chothia 1987). H and V L CDRs are as defined by MacCallum (see, eg, Table 1 above, and MacCallum 1996).

[0177] In some instances, the CTP comprises (a) a VL region comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: DIQLTQSPSSLSASVGDRVSITCKASQDVSIAVAWYQQKPGKAPKLLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDFAVYYCQQHYITPLTFGAGTKVEIK (SEQ ID NO: 111); and an anti-TROP-2 antibody comprising a VH region having an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence: QVQLQQSGSELKKPGASVKVSCKASGYTFTNYGMNWVKQAPGQGLKWMGWINTYTGEPTYTDDFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCARGGFGSSYWYFDVWGQGSLVTVSS (SEQ ID NO: 112).

[0178] In some instances, the CTP comprises, in order from N-terminus to C-terminus, (a) a VL region comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: DIQLTQSPSSLSASVGDRVSITCKASQDVSIAVAWYQQKPGKAPKLLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDFAVYYCQQHYITPLTFGAGTKVEIK (SEQ ID NO: 111); (b) a peptide and (c) an anti-TROP-2 scFv comprising a VH region comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: QVQLQQSGSELKKPGASVKVSCKASGYTFTNYGMNWVKQAPGQGLKWMGWINTYTGEPTYTDDFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCARGGFGSSYWYFDVWGQGSLVTVSS (SEQ ID NO: 112). In some cases, the peptide linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO: 42), where n is an integer between 1 and 10 (e.g., n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). In some cases, the peptide linker comprises the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 82) and is 15 amino acids in length.

[0179] In some cases, the CTP comprises, from N-terminus to C-terminus, (a) an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: QVQLQQSGSELKKPGASVKVSCKASGYTFTNYGMNWVKQAPGQGLKWMGWINTYTGEPTYTDDFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCARGGFGSSYWYFDVWGQGSLVTVSS (SEQ ID NO: 112). an anti-TROP-2 scFv comprising (a) a VH region comprising: (b) a peptide linker; and (c) a VL region comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: DIQLTQSPSSLSASVGDRVSITCKASQDVSIAVAWYQQKPGKAPKLLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDFAVYYCQQHYITPLTFGAGTKVEIK (SEQ ID NO: 111). In some cases, the peptide linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO: 42), where n is an integer between 1 and 10 (e.g., n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). In some cases, the peptide linker comprises the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 82) and is 15 amino acids in length.

[0180] In some instances, the CTP is an anti-TROP2 antibody comprising (a) a VL CDR1, VL CDR2, and VL CDR3 present in a light chain variable region (VL) comprising the following amino acid sequence: DIQMTQSPSSLSASVGDRVTITCKASQDVSTAVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFAVYYCQQHYITPLTFGQGTKLEIK (SEQ ID NO: 113), and (b) a VH CDR1, CDR2, and CDR3 present in a heavy chain variable region (VH) comprising the following amino acid sequence: QVQLVQSGAEVKKPGASVKVSCKASGYTFTTAGMQWVRQAPGQGLEWMGWINTHSGVPKYAEDFKGRVTISADTSTSTAYLQLSSLKSEDTAVYYCARSGFGSSYWYFDVWGQGTLVTVSS (SEQ ID NO: 114). H and V L CDRs are as defined by Kabat (see, e.g., Table 1, above, and Kabat 1991). H and V L CDRs are as defined by Chothia (see, e.g., Table 1, above, and Chothia 1987). H and V L CDRs are as defined by MacCallum (see, eg, Table 1 above, and MacCallum 1996).

[0181] In some instances, the CTP comprises (a) a VL region comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: DIQMTQSPSSLSASVGDRVTITCKASQDVSTAVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFAVYYCQQHYITPLTFGQGTKLEIK (SEQ ID NO: 113); and an anti-TROP-2 antibody comprising a VH region having an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence: QVQLVQSGAEVKKPGASVKVSCKASGYTFTTAGMQWVRQAPGQGLEWMGWINTHSGVPKYAEDFKGRVTISADTSTSTAYLQLSSLKSEDTAVYYCARSGFGSSYWYFDVWGQGTLVTVSS (SEQ ID NO: 114).

[0182] In some instances, the CTP comprises, in order from N-terminus to C-terminus, (a) a VL region comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: DIQMTQSPSSLSASVGDRVTITCKASQDVSTAVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFAVYYCQQHYITPLTFGQGTKLEIK (SEQ ID NO: 113); and (c) an anti-TROP-2 scFv comprising a VH region comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: QVQLVQSGAEVKKPGASVKVSCKASGYTFTTAGMQWVRQAPGQGLEWMGWINTHSGVPKYAEDFKGRVTISADTSTSTAYLQLSSLKSEDTAVYYCARSGFGSSYWYFDVWGQGTLVTVSS (SEQ ID NO: 114). In some cases, the peptide linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO: 42), where n is an integer between 1 and 10 (e.g., n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). In some cases, the peptide linker comprises the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 82) and is 15 amino acids in length.

[0183] In some instances, the CTP comprises, from N-terminus to C-terminus, (a) an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: QVQLVQSGAEVKKPGASVKVSCKASGYTFTTAGMQWVRQAPGQGLEWMGWINTHSGVPKYAEDFKGRVTISADTSTSTAYLQLSSLKSEDTAVYYCARSGFGSSYWYFDVWGQGTLVTVSS (SEQ ID NO: 114). an anti-TROP-2 scFv comprising (a) a VH region comprising: (b) a peptide linker; and (c) a VL region comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: DIQMTQSPSSLSASVGDRVTITCKASQDVSTAVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFAVYYCQQHYITPLTFGQGTKLEIK (SEQ ID NO: 113). In some cases, the peptide linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO: 42), where n is an integer between 1 and 10 (e.g., n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). In some cases, the peptide linker comprises the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 82) and is 15 amino acids in length.

[0184] anti-BCMA In some cases, the CTP present in an IL-2 variant fusion polypeptide of the present disclosure comprises an anti-BCMA antibody. In some cases, the anti-BCMA antibody is an scFv polypeptide. In some cases, the anti-BCMA antibody is a nanobody. Anti-BCMA (B cell maturation antigen) antibodies are known in the art, and the VH and VL or VH and VL CDRs of any anti-BCMA antibody can be used as the CTP. See, e.g., WO 2014 / 089335, U.S. Patent No. 2019 / 0153061, and WO 2017 / 093942.

[0185] In some instances, the anti-BCMA antibody has (a) the following amino acid sequence: QSVLTQPPSASGTPGQRVTISCSGSSSNIGSNTVNWYQQLPGTAPKLLIFNYHQRPSGVPDRFSGSKSGSSASLAISGLQSEDEADYYCAAWDDSLNGWVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPDSKQSNNKYAASSYLSLTPEQW a light chain comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to KSHRSYSCQVTHEGSTVEKTVAPTECS (SEQ ID NO: 115); and (b) the following amino acid sequence: EVQLVESGGGLVKPGGSLRLSCAASGFTFGDYALSWFRQAPGKGLEWVGVSRSKAYGGTTDYAASVKGRFTISRDDSKSTAYLQMNSLKTEDTAV YYCASSGYSSGWTPFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYIC NVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 116).

[0186] In some instances, the anti-BCMA antibody comprises a VL present in the light chain amino acid sequence provided above and a VH present in the heavy chain amino acid sequence provided above. For example, the anti-BCMA antibody may comprise (a) a VL comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: QSVLTQPPSASGTPGQRVTISCSGSSSNIGSNTVNWYQQLPGTAPKLLIFNYHQRPSGVPDRFSGSKSGSSASLAISGLQSEDEADYYCAAWDDSLNGWVFGGGTKLTVLG (SEQ ID NO: 117); and (b) The VH may comprise an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence: EVQLVESGGGLVKPGGSLRLSCAASGFTFGDYALSWFRQAPGKGLEWVGVSRSKAYGGTTDYAASVKGRFTISRDDSKSTAYLQMNSLKTEDTAVYYCASSGYSSGWTPFDYWGQGTLVTVSSASTKGPSV (SEQ ID NO: 118).

[0187] In some cases, the anti-BCMA antibody comprises a VL CDR1, a VL CDR2, and a VL CDR3 present in the light chain amino acid sequence provided above, and a VH CDR1, a CDR2, and a CDR3 present in the heavy chain amino acid sequence provided above. H and V L CDRs are as defined by Kabat (see, e.g., Table 1, above, and Kabat 1991). H and V L CDRs are as defined by Chothia (see, e.g., Table 1, above, and Chothia 1987). H and V L CDRs are as defined by MacCallum (see, eg, Table 1 above, and MacCallum 1996).

[0188] For example, an anti-BCMA antibody may comprise a VL CDR1 having the amino acid sequence SSNIGSNT (SEQ ID NO: 119), a VL CDR2 having the amino acid sequence NYH, a VL CDR3 having the amino acid sequence AAWDDSLNGWV (SEQ ID NO: 120), a VH CDR1 having the amino acid sequence GFTFGDYA (SEQ ID NO: 121), a VH CDR2 having the amino acid sequence SRSKAYGGTT (SEQ ID NO: 122), and a VH CDR3 having the amino acid sequence ASSGYSSGWTPFDY (SEQ ID NO: 123).

[0189] The anti-BCMA antibody may be an scFv. As one non-limiting example, the anti-BCMA scFv may comprise the following amino acid sequence: QVQLVQSGAEVKKPGSSVKVSCKASGGTFSNYWMHWVRQAPGQGLEWMGATYRGHSDTYYNQKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARGAIYNGYDVLDNWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKLLIYYTSNLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYRKLPWTFGQGTKLEIKR (SEQ ID NO: 124).

[0190] As another example, the anti-BCMA scFv may comprise the following amino acid sequence: DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKLLIYYTSNLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYRKLPWTFGQGTKLEIKRGGGGSGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGGTFSNYWMHWVRQAPGQGLEWMGATYRGHSDTYYNQKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARGAIYNGYDVLDNWGQGTLVTVSS (SEQ ID NO: 125).

[0191] In some cases, the anti-BCMA antibody may comprise a VL CDR1 having the amino acid sequence SASQDISNYLN (SEQ ID NO: 126), a VL CDR2 having the amino acid sequence YTSNLHS (SEQ ID NO: 127), a VL CDR3 having the amino acid sequence QQYRKLPWT (SEQ ID NO: 128), a VH CDR1 having the amino acid sequence NYWMH (SEQ ID NO: 129), a VH CDR2 having the amino acid sequence ATYRGHSDTYYNQKFKG (SEQ ID NO: 130), and a VH CDR3 having the amino acid sequence GAIYNGYDVLDN (SEQ ID NO: 131).

[0192] In some instances, the anti-BCMA antibody comprises (a) a light chain comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKLLIYYTSNLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYRKLPWTFGQGTKLEIKR (SEQ ID NO: 132).

[0193] In some instances, the anti-BCMA antibody comprises (a) a heavy chain comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: QVQLVQSGAEVKKPGSSVKVSCKASGGTFSNYWMHWVRQAPGQGLEWMGATYRGHSDTYYNQKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARGAIYDGYDVLDNWGQGTLVTVSS (SEQ ID NO: 133).

[0194] In some instances, the anti-BCMA antibody (e.g., the antibody referred to in the literature as belantamab) comprises a light chain comprising the amino acid sequence: DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKLLIYYTSNLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYRKLPWTFGQGTKLEIKR (SEQ ID NO: 132), and a heavy chain comprising the amino acid sequence: QVQLVQSGAEVKKPGSSVKVSCKASGGTFSNYWMHWVRQAPGQGLEWMGATYRGHSDTYYNQKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARGAIYDGYDVLDNWGQGTLVTVSS (SEQ ID NO: 133).

[0195] In some cases, the anti-BCMA antibody has a cancer chemotherapeutic agent linked to the antibody. For example, in some cases, the anti-BCMA antibody is GSK2857916 (belantamab-mafodotin) and monomethyl auristatin F (MMAF) is linked to the anti-BCMA antibody belantamab via a maleimidocaproyl linker.

[0196] anti-MUC1 In some cases, the CTP present in an IL-2 variant fusion polypeptide of the present disclosure comprises an anti-MUC1 antibody. In some cases, the anti-MUC1 antibody is an scFv polypeptide. In some cases, the anti-MUC1 antibody is a nanobody. For example, the CTP can be specific for a MUC1 polypeptide present on cancer cells. In some cases, the CTP is specific for a truncated form of MUC1, see, e.g., Fessler et al. (2009) Breast Cancer Res. Treat. 118:113. In some cases, the CTP is an antibody specific for a glycosylated MUC1 peptide, see, e.g., Naito et al. (2017) ACS Omega 2:7493 and U.S. Patent No. 10,017,580.

[0197] As one non-limiting example, the CTP can be a single-chain Fv specific for MUC1. See, e.g., Singh et al. (2007) Mol. Cancer Ther. 6:562, Thie et al. (2011) PloSOne 6:e15921, Imai et al. (2004) Leukemia 18:676, Posey et al. (2016) Immunity 44:1444, EP 3130607, EP 3164418, WO 2002 / 044217, and U.S. Patent Application Publication No. 2018 / 0112007. In some cases, the CTP is an scFv specific for the MUC1 peptide VTSAPDTRPAPGSTAPPAHG (SEQ ID NO: 134). In some cases, the CTP is an scFv specific for the MUC1 peptide SNIKFRPGSVVVQLTLAFREGTINVHDVETQFNQYKTEAASRY (SEQ ID NO: 135). In some cases, the CTP is an scFv specific for the MUC1 peptide SVVVQLTLAFREGTINVHDVETQFNQYKTEAASRY (SEQ ID NO: 136). In some cases, the CTP is an scFv specific for the MUC1 peptide LAFREGTINVHDVETQFNQY (SEQ ID NO: 137). In some cases, the CTP is an scFv specific for the MUC1 peptide SNIKFRPGSVVVQLTLAAFREGTIN (SEQ ID NO: 138).

[0198] As an example, an anti-MUC1 antibody can include a VH CDR1 having the amino acid sequence RYGMS (SEQ ID NO: 139), a VH CDR2 having the amino acid sequence TISGGGTYIYYPDSVKG (SEQ ID NO: 140), a VH CDR3 having the amino acid sequence DNYGRNYDYGMDY (SEQ ID NO: 141), a VL CDR1 having the amino acid sequence SATSSVSYIH (SEQ ID NO: 142), a VL CDR2 having the amino acid sequence STSNLAS (SEQ ID NO: 143), and a VL CDR3 having the amino acid sequence QQRSSSPFT (SEQ ID NO: 144). See, e.g., U.S. Patent Application Publication No. 2018 / 0112007.

[0199] As another example, an anti-MUC1 antibody can comprise a VH CDR1 having the amino acid sequence GYAMS (SEQ ID NO: 145), a VH CDR2 having the amino acid sequence TISSGGTYIYYPDSVKG (SEQ ID NO: 146), a VH CDR3 having the amino acid sequence LGGDNYYEYFDV (SEQ ID NO: 147), a VL CDR1 having the amino acid sequence RASKSVSTSGYSYMH (SEQ ID NO: 148), a VL CDR2 having the amino acid sequence LASNLES (SEQ ID NO: 149), and a VL CDR3 having the amino acid sequence QHSRELPFT (SEQ ID NO: 150). See, e.g., U.S. Patent Application Publication No. 2018 / 0112007.

[0200] As another example, an anti-MUC1 antibody can comprise a VH CDR1 having the amino acid sequence DYAMN (SEQ ID NO: 151), a VH CDR2 having the amino acid sequence VISTFSGNINFNQKFKG (SEQ ID NO: 152), a VH CDR3 having the amino acid sequence SDYYGPYFDY (SEQ ID NO: 153), a VL CDR1 having the amino acid sequence RSSQTIVHSNGNTYLE (SEQ ID NO: 154), a VL CDR2 having the amino acid sequence KVSNRFS (SEQ ID NO: 155), and a VL CDR3 having the amino acid sequence (FQGSHVPFT (SEQ ID NO: 156). See, e.g., U.S. Patent Application Publication No. 2018 / 0112007.

[0201] As another example, an anti-MUC1 antibody can comprise a VH CDR1 having the amino acid sequence GYAMS (SEQ ID NO: 145), a VH CDR2 having the amino acid sequence TISSGGTYIYYPDSVKG (SEQ ID NO: 146), a VH CDR3 having the amino acid sequence LGGDNYYEY (SEQ ID NO: 157), a VL CDR1 having the amino acid sequence TASKSVSTSGYSYMH (SEQ ID NO: 158), a VL CDR2 having the amino acid sequence LVSNLES (SEQ ID NO: 159), and a VL CDR3 having the amino acid sequence QHIRELTRSE (SEQ ID NO: 160). See, e.g., U.S. Patent Application Publication No. 2018 / 0112007.

[0202] anti-MUC16 In some cases, the CTP present in the IL-2 variant fusion polypeptide of the present disclosure comprises an anti-MUC16 antibody. MUC16 is also known as CA125. In some cases, the anti-MUC16 antibody is an scFv polypeptide. In some cases, the anti-MUC16 antibody is a nanobody. See, e.g., Yin et al. (2002) Int. J. Cancer 98:737. For example, the CTP can be specific for a MUC16 polypeptide present on cancer cells. See, e.g., U.S. Patent Application Publication No. 2018 / 0118848 and U.S. Patent Application Publication No. 2018 / 0112008.

[0203] As an example, an anti-MUC16 antibody may comprise a VH CDR1 having the amino acid sequence GFTFSNYY (SEQ ID NO: 161), a VH CDR2 having the amino acid sequence ISGRGSTI (SEQ ID NO: 162), a VH CDR3 having the amino acid sequence VKDRGGYSPY (SEQ ID NO: 163), a VL CDR1 having the amino acid sequence QSISTY (SEQ ID NO: 164), a VL CDR2 having the amino acid sequence TAS, and a VL CDR3 having the amino acid sequence QQSYSTPPIT (SEQ ID NO: 165). See, e.g., U.S. Patent Application Publication No. 2018 / 0118848.

[0204] Anti-claudin-18.2 In some cases, the CTP present in an IL-2 variant fusion polypeptide of the present disclosure comprises an anti-claudin-18 isoform 2 ("claudin-18.2") antibody. In some cases, the anti-claudin-18.2 antibody is an scFv polypeptide. In some cases, the anti-claudin-18.2 antibody is a nanobody. See, e.g., WO 2013 / 167259. In some cases, the CTP is an antibody specific for TEDEVQSYPSKHDYV (SEQ ID NO: 166) or EVQSYPSKHDYV (SEQ ID NO: 167).

[0205] As an example, an anti-claudin-18.2 antibody may include a VH CDR1 having the amino acid sequence GYTFTDYS (SEQ ID NO: 168), a VH CDR2 having the amino acid sequence INTETGVP (SEQ ID NO: 169), a VH CDR3 having the amino acid sequence ARRTGFDY (SEQ ID NO: 170), a VL CDR1 having the amino acid sequence KNLLHSDGITY (SEQ ID NO: 171), a VL CDR2 having the amino acid sequence RVS, and a VL CDR3 having the amino acid sequence VQVLELPFT (SEQ ID NO: 172).

[0206] As another example, an anti-claudin-18.2 antibody may comprise a VH CDR1 having the amino acid sequence GFTFSSYA (SEQ ID NO: 173), a VH CDR2 having the amino acid sequence ISDGGSYS (SEQ ID NO: 174), a VH CDR3 having the amino acid sequence ARDSYYDNSYVRDY (SEQ ID NO: 175), a VL CDR1 having the amino acid sequence QDINTF (SEQ ID NO: 176), a VL CDR2 having the amino acid sequence RTN, and a VL CDR3 having the amino acid sequence LQYDEFPLT (SEQ ID NO: 177).

[0207] anti-EGFR In some cases, the CTP present in the IL-2 variant fusion polypeptide of the present disclosure is an antibody specific for epidermal growth factor receptor (EGFR), see, e.g., U.S. Patent No. 6,217,866 and US2015 / 0141620. In some cases, the EGFR-specific CTP is an scFv. In some cases, the EGFR-specific CTP is a nanobody. In some cases, the EGFR-specific CTP comprises the VL and VH CDRs present in cetuximab.

[0208] In some cases, a suitable anti-EGFR antibody comprises a VL CDR present in the following amino acid sequence: DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGA (SEQ ID NO: 178). See, e.g., WO 2020 / 225552.

[0209] In some instances, a suitable anti-EGFR antibody has the following amino acid sequence: QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRV EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 179). See, e.g., WO 2020 / 225552.

[0210] In some instances, the anti-EGFR antibody has (a) the following amino acid sequence: QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV VTPSSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 179), and (b) a heavy chain comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence: DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVE. and a light chain comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to SEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGA (SEQ ID NO: 178).

[0211] As one example, an anti-EGFR antibody may comprise a VH CDR1 having the amino acid sequence NYGVH (SEQ ID NO: 180), a VH CDR2 having the amino acid sequence VIWSGGNTDYNTPFTS (SEQ ID NO: 181), a VH CDR3 having the amino acid sequence ALTYYDYEFAY (SEQ ID NO: 182), a VL CDR1 having the amino acid sequence RASQSIGTNIH (SEQ ID NO: 183), a VL CDR2 having the amino acid sequence YASESIS (SEQ ID NO: 184), and a VL CDR3 having the amino acid sequence QQNNNQPTT (SEQ ID NO: 185). See, e.g., WO 2020 / 225552.

[0212] As another example, an anti-EGFR antibody can comprise a VH CDR1 having the amino acid sequence SYWIE (SEQ ID NO: 186), a VH CDR2 having the amino acid sequence EILPGSKKTNYNEKFKG (SEQ ID NO: 187), a VH CDR3 having the amino acid sequence YYYRNDDYGMDT (SEQ ID NO: 188), a VL CDR1 having the amino acid sequence SASQDIRNYLN (SEQ ID NO: 189), a VL CDR2 having the amino acid sequence YTSTLHS (SEQ ID NO: 190), and a VL CDR3 having the amino acid sequence QQYSKIPYT (SEQ ID NO: 191) (see, e.g., U.S. Patent No. 6,217,866).

[0213] As another example, an anti-EGFR antibody can comprise a VH CDR1 having the amino acid sequence NYDMS (SEQ ID NO: 192), a VH CDR2 having the amino acid sequence YIGNGGNTYSPDTVKG (SEQ ID NO: 193), a VH CDR3 having the amino acid sequence HYGYDGRF (SEQ ID NO: 194), a VL CDR1 having the amino acid sequence RSSQSLEHSNGDTYLH (SEQ ID NO: 195), a VL CDR2 having the amino acid sequence KVSNRFS (SEQ ID NO: 196), and a VL CDR3 having the amino acid sequence CQSTHVPWT (SEQ ID NO: 197). See, e.g., U.S. Patent No. 6,217,866.

[0214] anti-CD20 In some cases, the CTP present in the IL-2 variant fusion polypeptide of the present disclosure comprises an anti-CD20 antibody. Anti-CD20 antibodies are known in the art; see, for example, US2015 / 0141620. In some cases, the CD20-specific CTP comprises the VL and VH CDRs present in rituximab. In some cases, the CD20-specific CTP comprises the VL and VH CDRs present in rituximab. In some cases, the anti-CD20 antibody is an scFv polypeptide. In some cases, the anti-CD20 antibody is a nanobody.

[0215] In some instances, a suitable anti-CD20 antibody has the following amino acid sequence: QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVASASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKK It contains the VH CDRs present in AEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 198).

[0216] In some instances, a suitable anti-CD20 antibody comprises a VL CDR present in the following amino acid sequence: QIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 199).

[0217] In some instances, a suitable anti-CD20 antibody may have (a) the following amino acid sequence: QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVASASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLS SVVTVPSSSLGTQTYICNVNHKPSNTKVDKKAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 198), and (b) the following amino acid sequence: QIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAE. DAATYYCQQWTSNPPTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 199), comprising a VL region comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to DAATYYCQQWTSNPPTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 199).

[0218] mAb fusion polypeptide CTP In embodiments, one or more IL-2 variant polypeptides can be part of a mAb fusion polypeptide (not shown). The mAb fusion polypeptide can comprise a heterodimer, where the first polypeptide of the heterodimer comprises an antibody heavy chain polypeptide and the second polypeptide of the heterodimer comprises an antibody light chain polypeptide, with either the first or second polypeptide comprising one or more variant IL-2 polypeptides, for example, at the N-terminus of the antibody light chain polypeptide, the C-terminus of the antibody light chain polypeptide, or the C-terminus of the antibody heavy chain polypeptide. Such fusion polypeptides are referred to herein as "monoclonal antibody fusions" or "mAb fusions." The VH and VL polypeptides are on separate polypeptides of the heterodimer, and together, the VH and VL polypeptides bind to a cancer epitope and thus together constitute a CTP. Such mAbs can be directed against any of the cancer targets listed above or against HSA.

[0219] T cell receptor (TCR) As mentioned above, in some cases, the functional protein is a TCR, such as a single-chain T cell receptor or "scTCR." The functional protein is an scTCR specific for a peptide / HLA complex on the surface of a cancer cell, and the peptide can be a cancer-associated peptide (e.g., a peptide of a cancer-associated antigen). The amino acid sequences of scTCRs specific for cancer-associated peptides bound to HLA complexes are known in the art. See, for example, U.S. Patent Application Publication No. 2019 / 0135914, U.S. Patent Application Publication No. 2019 / 0062398, and U.S. Patent Application Publication No. 2018 / 0371049.

[0220] An scTCR comprises an alpha chain variable region (Vα) and a beta chain variable region (Vβ) covalently linked via a suitable peptide linker sequence. For example, Vα can be covalently linked to Vβ via a suitable peptide linker (L) sequence fused to the C-terminus of Vα and the N-terminus of Vβ. An scTCR can have a Vα-L-Vβ structure. An scTCR can also comprise a constant domain (also referred to as a constant region). In some cases, an scTCR comprises, from N-terminus to C-terminus, (i) a TCR α chain variable domain polypeptide, (ii) a peptide linker, (iii) a TCR β chain variable domain polypeptide, and (iv) a TCR β chain constant region extracellular domain polypeptide. In some cases, the scTCR comprises, from N-terminus to C-terminus, (i) a TCR β chain variable domain polypeptide, (ii) a peptide linker, (iii) a TCR α chain variable domain polypeptide, and (iv) a TCR α chain constant region extracellular domain polypeptide.

[0221] The amino acid sequences of scTCRs specific for peptide / HLA complexes, where the peptide is a cancer-associated peptide, are known in the art (see, e.g., U.S. Patent Application Publication No. 2019 / 0135914, U.S. Patent Application Publication No. 2019 / 0062398, U.S. Patent Application Publication No. 2018 / 0371049, U.S. Patent Application Publication No. 2019 / 0144563, and U.S. Patent Application Publication No. 2019 / 0119350).

[0222] Alternatively, similar to the mAb fusion polypeptides described above, one or more IL-2 variant polypeptides can be part of a TCR fusion polypeptide (also not shown). Such a TCR fusion polypeptide can comprise a heterodimer, in which a first polypeptide of the heterodimer comprises at least the variable region (and optionally the constant region) of the TCR α chain, and a second polypeptide of the heterodimer comprises at least the variable region (and optionally the constant region) of the TCR β chain, with either the first or second polypeptide comprising one or more variant IL-2 polypeptides, e.g., at the C-terminus of the variable region of the polypeptide comprising the α chain polypeptide(s) and / or at the C-terminus of the variable region of the polypeptide comprising the β chain polypeptide(s). Such fusion polypeptides are referred to herein as "TCR fusion polypeptides." The α chain polypeptide(s) and β chain polypeptide(s) are on separate polypeptides of the heterodimer and together bind to the pHLA complex on the surface of cancer cells, thus constituting the CTP.

[0223] Immunomodulatory Polypeptides Functional proteins can also include wild-type or variant immunomodulatory polypeptides, e.g., wild-type or variant immunostimulatory polypeptides, e.g., the B7 family of costimulatory receptors, e.g., CD80, CD86, cytokines, e.g., IL-7, IL-12, IL-15, or IL-21, TNF superfamily members, e.g., CD-40, 4-1BBL, and OX40, or chemokines, e.g., CCL19, CCL21, CXCL9 / 10 / 11, or CXCL12.

[0224] Linker The IL-2 variant polypeptides or fusion polypeptides of the present disclosure may comprise one or more independently selected linkers, i.e., contiguous stretches of two or more amino acids that link one or more IL-2 variant polypeptides together in a higher order IL-2 variant polypeptide, such as a dimer, trimer, or that link one or more components of a fusion polypeptide described herein.

[0225] Suitable linkers (also called "spacers") can be readily selected and can be any of a number of suitable lengths, such as from 1 to 25 amino acids, from 25 to 50 amino acids, or greater than 50 amino acids in length.

[0226] Generally, a linker can be a flexible linker or a rigid linker. A linker can also contain a cysteine ​​residue if it is desired to attach the linker to another part of the polypeptide via a disulfide bond. Such linkers are typically not cleavable linkers, i.e., they are not designed to be cleaved in vivo.

[0227] Flexible Peptide Linkers Exemplary flexible linkers include glycine polymers (G) n , glycine-serine polymers (e.g., (GS) n , (GSGGS) n (SEQ ID NO: 200), (GGGGS) n (SEQ ID NO: 201), and (GGGS) n(SEQ ID NO: 202), where n is an integer of at least one, glycine-alanine polymers, alanine-serine polymers, and other flexible peptide linkers known in the art. Glycine and glycine-serine polymers can be used; both Gly and Ser are relatively unstructured and can therefore function as neutral tethers between components. Glycine polymers can be used; glycine has access to significantly more phi-psi space than alanine and is significantly less restricted than residues with longer side chains (see Scheraga, Rev. Computational Chem. 11173-142 (1992)). Exemplary linkers can include amino acid sequences, including, but not limited to, GGSG (SEQ ID NO: 203), GGSGG (SEQ ID NO: 204), GSGSG (SEQ ID NO: 205), GSGGG (SEQ ID NO: 206), GGGSG (SEQ ID NO: 207), GSSSG (SEQ ID NO: 208), and the like. An exemplary linker can include, for example, Gly(Ser4)n (SEQ ID NO: 209), where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some cases, the linker includes the amino acid sequence (GSSSS)n (SEQ ID NO: 210), where n is 4. In some cases, the linker includes the amino acid sequence (GSSSS)n (SEQ ID NO: 211), where n is 5. An exemplary linker can include, for example, (GGGGS) n (SEQ ID NO:42), where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some cases, the linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO:212), where n is 2. In some cases, the linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO:82), where n is 3. In some cases, the linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO:213), where n is 4. In some cases, the linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO:214), where n is 5.

[0228] Exemplary flexible peptide linkers include, for example, (GGGGS)n (SEQ ID NO:42), also referred to as a "G4S" linker, where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some cases, the linker comprises the amino acid sequence AAAGG (SEQ ID NO:215). Linkers having the amino acid sequence AAAGG (SEQ ID NO:215) are also suitable.

[0229] As used in this disclosure, a "short flexible peptide linker" refers to a flexible peptide linker containing fewer than 15 amino acids, i.e., 2-14 amino acids. For example, a short flexible peptide linker can contain 2-4, 2-5, or 3-6 amino acids (e.g., a GGS linker), or 4-8, 5-10, or 10-14 amino acids. Included within this range are flexible peptide linkers containing 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 amino acids.

[0230] Rigid Peptide Linkers In some cases, the peptide linker is a rigid peptide linker. As used herein, the term "rigid peptide linker" refers to a linker comprising a contiguous stretch of two or more amino acids that effectively separates protein domains by maintaining a substantially fixed distance / spatial separation between the domains, thereby reducing or substantially eliminating unfavorable interactions between such domains. Rigid peptide linkers are known in the art and generally adopt a relatively well-defined conformation when in solution. Rigid peptide linkers include those that have a specific secondary and / or tertiary structure in solution, and are typically long enough to impart a secondary or tertiary structure to the linker. Rigid peptide linkers include proline-rich peptide linkers and peptide linkers with an inflexible helical structure, such as an α-helical structure. Rigid peptide linkers are described, for example, in Chen et al. (2013) Adv. Drug Deliv. Rev. 65:1357, and Klein et al. (2014) Protein Engineering, Design & Selection 27:325.

[0231] Examples of rigid peptide linkers include, for example, (EAAAK)n (SEQ ID NO:216), A(EAAAK)n (SEQ ID NO:217), A(EAAAK)nA (SEQ ID NO:218), A(EAAAK)nALEA(EAAAK)nA (SEQ ID NO:219), (Lys-Pro)n (SEQ ID NO:220), (Glu-Pro)n (SEQ ID NO:221), (Thr-Pro-Arg)n (SEQ ID NO:222), and (Ala-Pro)n (SEQ ID NO:223), where n is an integer between 1 and 20 (e.g., n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20). Non-limiting examples of suitable rigid peptide linkers comprising EAAAK (SEQ ID NO:224) include EAAAK (SEQ ID NO:224), (EAAAK)2 (SEQ ID NO:225), (EAAAK)3 (SEQ ID NO:226), A(EAAAK)4ALEA(EAAAK)4A (SEQ ID NO:227), and AEAAAKEAAAKA (SEQ ID NO:228). Non-limiting examples of suitable rigid peptide linkers comprising (AP)n include PAPAP (SEQ ID NO:229) (also referred to herein as "(AP)2"), APAPAPAP (SEQ ID NO:230) (also referred to herein as "(AP)4"), APAPAPAPAPAPAP (SEQ ID NO:231) (also referred to herein as "(AP)6"), APAPAPAPAPAPAPAPAP (SEQ ID NO:232) (also referred to herein as "(AP)8"), and APAPAPAPAPAPAPAPAPAPAP (SEQ ID NO:233) (also referred to herein as "(AP)10"). Non-limiting examples of suitable rigid peptide linkers comprising (KP)n include KPKP (SEQ ID NO:234) (also referred to herein as "(KP)2"), KPKPKPKP (SEQ ID NO:235) (also referred to herein as "(KP)4"), KPKPKPKPKPKP (SEQ ID NO:236) (also referred to herein as "(KP)6"), KPKPKPKPKPKPKPKP (SEQ ID NO:237) (also referred to herein as "(KP)8"), and KPKPKPKPKPKPKPKPKPKPKP (SEQ ID NO:238) (also referred to herein as "(KP)10").Non-limiting examples of suitable rigid peptide linkers comprising (EP)n include EPEP (SEQ ID NO:239) (also referred to herein as "(EP)2"), EPEPEPEPEP (SEQ ID NO:240) (also referred to herein as "(EP)4"), EPEPEPEPEPEPEP (SEQ ID NO:241) (also referred to herein as "(EP)6"), EPEPEPEPEPEPEPEPEP (SEQ ID NO:242) (also referred to herein as "(EP)8"), and EPEPEPEPEPEPEPEPEPEPEP (SEQ ID NO:243) (also referred to herein as "(EP)10").

[0232] Generally, when a higher order IL-2 variant polypeptide or fusion polypeptide comprises a rigid peptide linker and / or a short flexible peptide linker, the rigid peptide linker and / or the short flexible peptide linker can be positioned between any two of the components. In some cases, the use of a rigid peptide linker or a short flexible peptide linker can increase the thermal stability of the resulting polypeptide.

[0233] Cysteine-containing linkers As noted above, in some cases, the linker peptide contains a cysteine ​​residue that can form an interchain disulfide bond with a cysteine ​​residue present elsewhere in the polypeptide chain. Exemplary cysteine-containing linkers include GCGGS (SEQ ID NO:244), CGGGS (SEQ ID NO:245), CGGGS(GGGGS)n (SEQ ID NO:246), where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, e.g., 1, 2, or 3; GGCGS (SEQ ID NO:247), GGCGS(GGGGS)n (SEQ ID NO:248), where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, e.g., 1, 2, or 3; , 1, 2, or 3; GGGCS (SEQ ID NO: 249), GGGCS(GGGGS)n (SEQ ID NO: 250), where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, e.g., 1, 2, or 3; GGGGC (SEQ ID NO: 251), and GGGGC(GGGGS)n (SEQ ID NO: 252), where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, e.g., 1, 2, or 3.

[0234] Exemplary Polypeptides Exemplary polypeptides are shown in the figures. Exemplary IL-2 variant polypeptides include those in which an F42 substitution (e.g., with Ala or Lys) is made to substantially reduce binding of the IL-2 variant to IL-2Rα, e.g., by at least about 100-fold, e.g., F42A or F42K. As discussed above, IL-2 variant polypeptides having an F42A or F42K substitution also have one or more substitutions that reduce binding of the variant to IL-2Rβ, e.g., substitutions at E15, H16, or N88. Of these, an H16 substitution has been shown to reduce binding to IL-2Rβ by about 3-fold. H16 substitutions include, e.g., Ala, Glu, Thr, or Asp. Thus, exemplary variants can include any one of the following pairs of substitutions: H16A, F42A; H16T, F42A; H16E, F42A; H16D, F42A; H16A, F42K; H16T, F42K; H16E, F42K; or H16D, F42K.

[0235] Thus, for example, the higher order forms of IL-2 variant polypeptides illustrated in Figures 1A-1C can comprise IL-2 variants comprising any one of the substitution combinations set forth in Table 3, including, for example, any one of the following pairs of substitutions: H16A, F42A; H16T, F42A; H16E, F42A; H16D, F42A; H16A, F42K; H16T, F42K; H16E, F42K; or H16D, F42K. The IL-2 variant polypeptides may have one or more additional substitutions and may be connected by linkers as described above.

[0236] Similarly, the fusion polypeptides illustrated in Figures 2A-2C, 3A-3C, 4A-4C, 5A-5B, 6A-6B, 7A-7B, 8A-8F, 10A-10C, 11A-11C, 12A-12B, 13A-13B, 14A-14N, and 30A-30G can also include variant IL-2 polypeptides comprising any one of the substitution combinations listed in Table 3, including, for example, any one of the following pairs of substitutions: H16A, F42A; H16T, F42A; H16E, F42A; H16D, F42A; H16A, F42K; H16T, F42K; H16E, F42K; or H16D, F42K. The IL-2 variant polypeptide may have one or more additional substitutions, and one or more of the components of the fusion polypeptide may be joined by a linker as described above. Exemplary fusion proteins may utilize any one of the Ig Fc polypeptides in Figures 9A-9M, such as a human IgG1 Fc variant polypeptide having a substantially reduced ability to effect complement dependent cytotoxicity (CDC) or antibody dependent cellular cytotoxicity (ADCC), e.g., a variant polypeptide having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the human variant human IgG1 Fc polypeptide of Figure 9B (SEQ ID NO: 24) and Figure 9D (SEQ ID NO: 26).

[0237] As one non-limiting example, a fusion polypeptide (e.g., as depicted in Figures 11B and 13B) may comprise, from N-terminus to C-terminus: (i) an anti-CD19 scFv comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: DIQLTQSPASLAVSLGQRATISCKASQSVDYDGDSYLNWYQQIPGQPPKLLIYDASNLVSGIPPRFSGSGSGTDFTLNIHPVEKVDAATYHCQQSTEDPWTFGGGTKLEIKGGGGSGGGGSGGGGSQVQLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIWPGDGDTNYNGKFKGKATLTADESSSTAYMQLSSLASEDSAVYFCARRETTTVGRYYYAMDYWGQGTTVTVSS (SEQ ID NO: 67); (ii) a peptide linker, (iii) the amino acid sequence: an Ig Fc polypeptide comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF2025532538000039.tif23159, wherein amino acid 14 is Ala and amino acid 15 is Ala; (iv) a peptide linker, (v) a first copy of a variant IL-2 polypeptide, wherein the variant IL-2 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: APTSSSTKKTQLQLEALLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 254), and wherein amino acid 16 is other than His and amino acid 42 is other than Phe; (vi) a peptide linker, and (vii) a second copy of a variant IL-2 polypeptide, wherein the variant IL-2 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following amino acid sequence: APTSSSTKKTQLQLEALLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 254), wherein amino acid 16 is other than His and amino acid 42 is other than Phe. may include:

[0238] In some cases, the fusion polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence shown in Figure 20. In some cases, the fusion polypeptide comprises the amino acid sequence shown in Figure 20.

[0239] In some cases, the fusion polypeptide comprises, consists essentially of, or consists of an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity to any one of the amino acid sequences shown in Figures 21-26. In some cases, the fusion polypeptide comprises, consists essentially of, or consists of a homodimer having two copies of an immunomodulatory protein, each copy having an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity to the "2657" amino acid sequence shown in Figure 23A (and set forth in SEQ ID NO:257). Polypeptide 2657 spontaneously forms a homodimer with a second copy of 2657, the two copies being linked by two disulfide bonds linking the Ig Fc polypeptide in each copy of 2657. Figure 5B shows the structure of a homodimer comprising two polypeptides such as 2657. In some cases, the fusion polypeptide comprises, consists essentially of, or consists of a homodimer having two copies of an immunomodulatory protein, each copy having an amino acid sequence with at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity to the "2657Δ" amino acid sequence shown in Figure 23B (and set forth in SEQ ID NO:267). Polypeptide 2657Δ spontaneously forms homodimers with a second copy of 2657Δ, the two copies being linked by two disulfide bonds linking the Ig Fc polypeptides in each copy of 2657Δ. The fusion polypeptide designated 2657Δ and shown in Figure 23B lacks the C-terminal Lys present in 2657.

[0240] In some cases, the fusion polypeptide comprises, consists essentially of, or consists of a homodimer having two copies of an immunomodulatory protein, each copy having an amino acid sequence with at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity to the "2656" amino acid sequence shown in Figure 25A (and set forth in SEQ ID NO: 259). Polypeptide 2656 also spontaneously forms homodimers with a second copy of 2656, the two copies being linked by two disulfide bonds linking the Ig Fc polypeptides in each copy of 2656. Figure 5A shows the structure of a homodimer comprising two polypeptides such as 2656. In some cases, the fusion polypeptide comprises, consists essentially of, or consists of a homodimer having two copies of an immunomodulatory protein, each copy having an amino acid sequence with at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity to the "2656Δ" amino acid sequence shown in Figure 25B (and set forth in SEQ ID NO:268). Polypeptide 2656Δ also spontaneously forms homodimers with a second copy of 2656Δ, the two copies being linked by two disulfide bonds linking the Ig Fc polypeptides in each copy of 2656Δ. The fusion polypeptide designated 2656Δ and shown in Figure 25B lacks the C-terminal Lys present in 2656.

[0241] In some cases, the heterodimeric fusion polypeptide comprises, consists essentially of, or consists of (a) a first polypeptide comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity to the "4123" amino acid sequence shown in Figure 26A (and set forth in SEQ ID NO: 260), and (b) a second polypeptide comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity to the "4124" amino acid sequence shown in Figure 26B (and set forth in SEQ ID NO: 261). Figure 30A shows the structure of a heterodimer comprising two polypeptides, such as 4123 and 4124.

[0242] Methods for producing IL-2 variant or fusion polypeptides The present disclosure provides methods for obtaining the IL-2 variant polypeptides or fusion polypeptides described herein.

[0243] nucleic acid The present disclosure provides nucleic acids comprising a nucleotide sequence encoding an IL-2 variant polypeptide or fusion polypeptide described herein. When an IL-2 variant polypeptide or fusion polypeptide comprises a single chain, or when a fusion polypeptide comprises a homodimer of two single-chain polypeptides, the single-chain polypeptide can be encoded in a single nucleic acid.

[0244] When the fusion polypeptide is a heterodimer, the fusion polypeptide can be encoded by a single nucleic acid or two separate nucleic acids. That is, in some cases, the individual polypeptide chains of the heterodimeric fusion polypeptide described herein can be encoded by separate nucleic acids and operably linked to a transcriptional control element, for example, a promoter, such as a promoter functional in eukaryotic cells, which promoter can be a constitutive promoter or an inducible promoter. When the individual polypeptide chains of the heterodimeric fusion polypeptide are encoded by a single nucleic acid, the nucleic acid can include, for example, a proteolytically cleavable linker interposed between the nucleotide sequences encoding the first polypeptide, an internal ribosome entry site (IRES) interposed between the nucleotide sequences encoding the first polypeptide and the nucleotide sequences encoding the second polypeptide, or a ribosome skipping signal (or cis-acting hydrolase element, CHYSEL) interposed between the nucleotide sequences encoding the first polypeptide and the nucleotide sequences encoding the second polypeptide. In some cases, the nucleotide sequence encoding the first polypeptide and the second nucleotide sequence encoding the second polypeptide are each operably linked to a transcriptional control element such as a promoter, e.g., a promoter that is functional in a eukaryotic cell, and the promoter may be a constitutive promoter or an inducible promoter.

[0245] Recombinant Expression Vectors The present disclosure provides a recombinant expression vector comprising the nucleic acid of the present disclosure.In some cases, the recombinant expression vector is a non-viral vector.In some cases, the recombinant expression vector is a viral construct, such as a recombinant adeno-associated virus construct (see, for example, U.S. Patent No. 7,078,387), a recombinant adenovirus construct, a recombinant lentivirus construct, a recombinant retrovirus construct, a non-integral viral vector, etc.

[0246] Suitable expression vectors are known in the art and include 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; WO94 / 12649, WO93 / 03769, WO93 / 19191, WO94 / 28938, WO 95 / 11984 and WO95 / 00655), adeno-associated virus (see, e.g., 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 WO93 / 09239,Samulski et al.,J.Vir.(1989)63:3822-3828;Mendelson et al.,Virol.(1988)166:154-165;and Flotte et al. al., PNAS (1993) 90:10613-10617), SV40, herpes simplex virus, human immunodeficiency virus (e.g., Miyoshi et al., PNAS 94:10319 23, 1997; Takahashi et al., J Virol 73:7812 7816, 1999), retroviral vectors (e.g., murine leukemia virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous sarcoma virus, Harvey sarcoma virus, avian leukosis virus, lentivirus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus).

[0247] Numerous suitable expression vectors are known to those of skill in the art, many of which are commercially available. For eukaryotic host cells, the following vectors are provided as examples: pXT1, pSG5 (Stratagene), pSVK3, pBPV, pMSG, and pSVLSV40 (Pharmacia). However, any other vector may be used as long as it is compatible with the host cell.

[0248] Depending on the host / vector system utilized, any of a number of suitable transcriptional and translational control elements, including constitutive and inducible promoters, transcriptional enhancer elements, transcriptional terminators, etc., may be used in the expression vector (see, e.g., Bitter et al (1987) Methods in Enzymology, 153:516-544).

[0249] In some cases, the nucleotide sequence encoding the IL-2 variant polypeptide or fusion polypeptide is operably linked to a control element, e.g., a transcription control element such as a promoter. The transcription control element may be functional in either eukaryotic cells (e.g., mammalian cells) or prokaryotic cells (e.g., bacterial or archaeal cells). In some cases, the nucleotide sequence encoding the IL-2 variant polypeptide or fusion polypeptide is operably linked to multiple control elements that allow expression of the nucleotide sequence encoding the IL-2 variant polypeptide or fusion polypeptide in both prokaryotic and eukaryotic cells.

[0250] Non-limiting examples of suitable eukaryotic promoters (promoters functional in eukaryotic cells) include those from cytomegalovirus (CMV) immediate early, herpes simplex virus (HSV) thymidine kinase, early and late SV40, long terminal repeats (LTRs) from retroviruses, and mouse metallothionein-I. Selection of appropriate vectors and promoters is well within the level of ordinary skill in the art. Expression vectors may also contain a ribosome binding site for translation initiation and a transcription terminator. Expression vectors may also include appropriate sequences for amplifying expression.

[0251] Genetically modified host cells The present disclosure provides genetically modified host cells, which host cells are genetically modified with one or more nucleic acids of the present disclosure.

[0252] Suitable host cells include eukaryotic cells such as yeast, insect cells, and mammalian cells. In some cases, the host cells are cells of a mammalian cell line. Suitable mammalian cell lines include human cell lines, non-human primate cell lines, rodent (e.g., mouse, rat) cell lines, and the like. Suitable mammalian cell lines include HeLa cells (e.g., American Type Culture Collection (ATCC) No. CCL-2), CHO cells (e.g., ATCC Nos. CRL9618, CCL61, CRL9096), 293 cells (e.g., ATCC No. CRL-1573), Vero cells, NIH 3T3 cells (e.g., ATCC No. CRL-1658), Huh-7 cells, BHK cells (e.g., ATCC No. CCL10), PC12 cells (ATCC No. CRL1721), COS cells, COS-7 cells (ATCC No. CRL1651), RAT1 cells, mouse L cells (ATCC No. CCLI.3), human fetal liver (HEK) cells (ATCC No. CRL1573), HLHepG2 cells, and the like.

[0253] In some cases, the host cell is a mammalian cell that has been genetically modified so that it does not synthesize endogenous MHC β2-M.

[0254] In some cases, the host cell is a mammalian cell that has been genetically modified not to synthesize endogenous MHC class I heavy chains. In some cases, the host cell is a mammalian cell that has been genetically modified not to synthesize endogenous MHC β2-M and not to synthesize endogenous MHC class I heavy chains.

[0255] The host cells are then cultured under conditions such that the host cells produce the desired IL-2 variant polypeptide or fusion polypeptide, after which the product is recovered and purified.

[0256] Pharmaceutical Composition The present disclosure provides pharmaceutical compositions comprising IL-2 variant polypeptides, or compositions comprising one or more IL-2 variant polypeptides, e.g., fusion polypeptides. Such pharmaceutical compositions may contain one or more pharmaceutically acceptable excipients, the types of which are well known in the art and need not be discussed in detail herein. See, e.g., Sheskey et al., "Handbook of Pharmaceutical Excipients" (2020), 9th Edition (latest edition), and / or "Remington: The Science and Practice of Pharmacy," 23rd Edition (2020), 23rd Edition (latest edition).

[0257] In some cases, the pharmaceutical composition is suitable for administration to a subject, e.g., sterile. For example, in some cases, the pharmaceutical composition is suitable for administration to a human subject, e.g., if the composition is sterile and substantially free of detectable pyrogens and / or other toxins, or if such detectable pyrogens and / or other toxins are present at levels within acceptable limits set by applicable regulatory authorities, e.g., USF&DA.

[0258] For example, compositions may include aqueous solutions, powder forms, granules, tablets, pills, suppositories, capsules, suspensions, sprays, etc. Compositions may be formulated for various routes of administration, as described below.

[0259] When the pharmaceutical composition is administered directly into a tissue as an injection (e.g., subcutaneously, intraperitoneally, intramuscularly, and / or intravenously), the formulation may be provided in a ready-to-use dosage form, i.e., a non-aqueous form (e.g., a shelf-stable powder that can be reconstituted) or an aqueous form (e.g., a liquid composed of pharmaceutically acceptable carriers and excipients). Protein-containing formulations may also be provided to extend the serum half-life of the IL-2 variant polypeptide or fusion protein. For example, the IL-2 variant polypeptide or fusion protein may be provided as a liposomal formulation prepared into a colloid, or may be provided using other conventional techniques for extending serum half-life. The formulation may also be provided in a controlled-release or sustained-release form.

[0260] In some cases, the pharmaceutical composition is a liquid composition comprising saline (e.g., 0.9% NaCl).

[0261] How to use As described above, compositions comprising an IL-2 variant and one or more IL-2 variant polypeptides, e.g., fusion polypeptides, described herein do not systemically activate multiple immune cell subsets as does native IL-2 delivered at high doses, but rather predominantly or preferentially activate only T cells whose T cell receptors (TCRs) engage peptide-MHC complexes (pMHC) presented by antigen-presenting cells. Thus, pharmaceutical compositions comprising such proteins can have a useful therapeutic index, i.e., the blood concentration of the drug required for efficacy is significantly lower than the concentration at which the drug produces unacceptable adverse events or becomes toxic, such that the drug can be effective and reasonably well tolerated by patients. Furthermore, it has been found that compositions comprising such IL-2 variants and one or more IL-2 variant polypeptides, e.g., fusion polypeptides, when engaging with IL-2 receptors on CAR-T cells and other cytotoxic cells (e.g., macrophages and NK cells) bearing exogenous activating receptors such as CARs and TCRs, can provide homeostatic signals that can prolong the survival of such cells and / or provide activation signals to cells that cause the cells to proliferate and retain their cytotoxic function. Thus, compositions comprising IL-2 variants and one or more IL-2 variant polypeptides, e.g., fusion polypeptides, are suitable for use in combination with certain medical treatments, such as cancer vaccines, cell therapies, and immune checkpoint inhibitors.

[0262] One such method of treatment involves administering a therapeutically effective amount of a pharmaceutical composition comprising an IL-2 variant polypeptide or a composition comprising one or more IL-2 variant polypeptides, e.g., a fusion polypeptide, to a patient who has received, is receiving, or will receive modified or unmodified T cells having a T cell receptor (TCR), e.g., the TCR is specific for the patient's cancer. Such therapies include, for example, (i) TCR-T cell therapy, in which the patient's T cells are depleted and modified to express a TCR specific for a cancer antigen expressed on a particular human leukocyte antigen (HLA), and (ii) tumor-infiltrating lymphocytes (TILs) therapy, in which TILs are depleted from the patient, grown in large numbers in vitro, and then administered to the patient. When co-administered (at the same or different times), an IL-2 variant polypeptide or a composition comprising one or more IL-2 variant polypeptides, e.g., a fusion polypeptide, does not systemically activate multiple immune cell subsets as native IL-2 delivered in high doses does, but rather can associate with IL-2 receptors on such modified or unmodified T cells to provide an activation signal that can prolong the survival of such cells and / or to provide homeostatic signals to the cells that allow them to proliferate and retain their cytotoxic function.

[0263] Another method of treatment involves administering a therapeutically effective amount of a pharmaceutical composition comprising an IL-2 variant polypeptide or a composition comprising one or more IL-2 variant polypeptides, e.g., a fusion polypeptide, to a patient who has received, is receiving, or will receive a product (e.g., a vaccine such as a polypeptide cancer vaccine) that can associate with the TCR of a T cell or can be processed by the immune system to be presented by the TCR of a T cell via an antigen-presenting cell (APC) via a major histocompatibility complex (MHC), or a precursor of a product that can associate with the TCR of a T cell or can be processed by the immune system to be presented by the TCR of a T cell via an MHC, e.g., an mRNA cancer vaccine that produces a protein that can be processed by the immune system to be presented by the TCR of a T cell via an antigen-presenting cell (APC) via an MHC. Such cancer vaccines are known to generate T cells in the patient, but such T cells may not be activated to destroy cancer cells. When co-administered (at the same or different times), an IL-2 variant polypeptide or a composition comprising one or more IL-2 variant polypeptides, e.g., a fusion polypeptide, does not substantially activate multiple immune cell subsets systemically, as does native IL-2 delivered at high doses. Rather, it predominantly or preferentially activates only T cells whose TCRs associate with pMHC presented by APCs. Thus, when co-administered with a cancer vaccine, an IL-2 variant polypeptide or a composition comprising one or more IL-2 variant polypeptides, e.g., a fusion polypeptide, can deliver an IL-2 activation signal to T cells associated with APCs presenting the antigen of the cancer vaccine. As a result, T cells can become cytotoxic against cancer cells bearing pMHC presenting the antigen of the cancer vaccine. Many types of vaccines are known for eliciting T cell responses against antigens (e.g., cancer-associated antigens), which can be enhanced when the vaccines are used in combination with the fusion polypeptides described herein.Examples of such vaccines include, but are not limited to, inactivated vaccines that use killed versions of disease-causing pathogens, live attenuated vaccines that use weakened forms of disease-causing pathogens, messenger RNA (mRNA) vaccines that produce proteins to induce immune responses, subunit, recombinant, polysaccharide, and conjugate vaccines that use specific fragments of antigens, such as their proteins, sugars, or capsids (the casing surrounding germ cells), as well as toxoid and vector vaccines.As referred to herein, the term "cancer vaccine" refers to any vaccine that can generate T cells and induce an immune response that can then attack cancer cells.Such cancer vaccines can be either preventive vaccines that can reduce the likelihood that individuals will develop cancers associated with the proteins present in the cancer vaccine, or that can reduce the likelihood that individuals will develop cancers associated with the proteins encoded by the nucleic acids present in the cancer vaccine, or therapeutic vaccines that can induce T cells that can attack and kill cancer cells.

[0264] Another method of treatment involves administering a therapeutically effective amount of one or more IL-2 variant polypeptides or a composition comprising one or more IL-2 variant polypeptides, e.g., fusion polypeptides, to a patient who has received, is receiving, or will receive modified cells (e.g., T cells, macrophages, or NK cells) comprising a chimeric antigen receptor (CAR), where the CAR binds to a target antigen and the modified cells comprise an intracellular signaling domain that is activated by interaction of the modified cells with IL-2. When co-administered (at the same or different times), the IL-2 variant polypeptides or a composition comprising one or more IL-2 variant polypeptides, e.g., fusion polypeptides, do not substantially activate multiple immune cell subsets systemically as native IL-2 delivered at high doses does, but rather can associate with IL-2 receptors on such cells to provide a homeostatic signal that can prolong the survival of such cells and / or to provide an activation signal to cells that causes the cells to proliferate and retain their cytotoxic function.

[0265] Another method of treatment involves administering a therapeutically effective amount of one or more IL-2 variant polypeptides or a composition comprising one or more IL-2 variant polypeptides, e.g., fusion polypeptides, to a patient who has received, is receiving, or will receive cells (e.g., macrophages or NK cells) that have been modified to express one or more exogenous activating receptors (e.g., CARs or TCRs). When co-administered (at the same or different times), the IL-2 variant polypeptides or a composition comprising one or more IL-2 variant polypeptides, e.g., fusion polypeptides, do not substantially activate multiple immune cell subsets systemically as native IL-2 delivered in high doses does, but rather can associate with and activate the modified cells and / or provide homeostatic signals that can prolong the survival of such cells and / or provide activation signals to cells that allow them to proliferate and retain their cytotoxic function.

[0266] Another treatment method involves administering to a cancer patient a therapeutically effective amount of one or more IL-2 variant polypeptides or a composition comprising one or more IL-2 variant polypeptides, e.g., fusion polypeptides, as monotherapy or in combination with another therapy, such as an immune checkpoint inhibitor (CPI). For example, if a CPI demonstrates therapeutic benefit in the treatment of a particular cancer (either alone or in combination with another agent), such therapeutic benefit is likely due, at least in part, to the CPI's ability to enhance the patient's T cell response to that cancer. In such cases, administering a composition comprising one or more IL-2 variant polypeptides, e.g., fusion polypeptides, may enhance the effectiveness of the treatment by enhancing the activation and / or proliferation of the patient's cancer-specific T cells. Administering a composition comprising one or more IL-2 variant polypeptides, e.g., fusion polypeptides, may also enhance the effectiveness of CPI treatment by enhancing the activation and / or proliferation of other immune cells, such as NK cells.

[0267] As described above, IL-2 variant polypeptide(s) or compositions comprising one or more IL-2 variant polypeptides, e.g., fusion polypeptides, do not substantially activate multiple immune cell subsets systemically, as does native IL-2 delivered at high doses; rather, they predominantly or preferentially activate only T cells whose TCRs engage peptide-MHC complexes (pMHC) presented by APCs. For example, if such T cells are specific for an antigen expressed on the surface of cancer cells, the IL-2 polypeptide(s) activate these T cells for potential killing of the cancer cells. In this way, such polypeptides can provide immune stimulation in a manner that provides a therapeutic index that allows for the administration of active amounts of IL-2 variant polypeptides without the toxicity observed with native IL-2 or other IL-2 variants.

[0268] Cancers that can be treated with the methods of the present disclosure include carcinomas, sarcomas, melanomas, leukemias, and lymphomas.Cancers that can be treated with the methods of the present disclosure include solid tumors.Cancers that can be treated with the methods of the present disclosure include metastatic cancers.

[0269] Cancers that can be treated by the methods disclosed herein include, but are not limited to, esophageal cancer, hepatocellular carcinoma, basal cell carcinoma (a form of skin cancer), squamous cell carcinoma (various tissues), bladder cancer including transitional cell carcinoma (a malignant neoplasm of the bladder), bronchogenic carcinoma, colon cancer, colorectal cancer, gastric cancer, lung cancer including small cell carcinoma and non-small cell carcinoma of the lung, adrenocortical carcinoma, thyroid cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, renal cell carcinoma, ductal carcinoma in situ or bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, uterine cancer, testicular cancer, osteogenic carcinoma, epithelial carcinoma, and nasopharyngeal carcinoma.

[0270] Sarcomas that can be treated by the methods disclosed herein include, but are not limited to, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, chordoma, osteogenic sarcoma, osteosarcoma, angiosarcoma, endothelial sarcoma, lymphangiosarcoma, lymphangioendothelial sarcoma, synovioma, mesothelioma, Ewing's sarcoma, leiomyosarcoma, rhabdomyosarcoma, and other soft tissue sarcomas.

[0271] Other solid tumors that can be treated by the methods disclosed herein include, but are not limited to, glioma, astrocytoma, medulloblastoma, craniopharyngioma, epithelioma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, and retinoblastoma.

[0272] Leukemias that may be suitable for therapy using the methods disclosed herein include, but are not limited to, (a) chronic myeloproliferative syndromes (neoplastic disorders of pluripotent hematopoietic stem cells), (b) acute myeloid leukemia (neoplastic transformation of pluripotent hematopoietic stem cells or hematopoietic cells with restricted lineage potential), (c) chronic lymphocytic leukemia (CLL; clonal proliferation of immunologically immature and dysfunctional small lymphocytes), including B-cell CLL, T-cell CLL prolymphocytic leukemia, and hairy cell leukemia, and (d) acute lymphoblastic leukemia (characterized by the accumulation of lymphoblasts). Lymphomas that can be treated using this method include, but are not limited to, B-cell lymphomas (e.g., Burkitt's lymphoma), Hodgkin's lymphoma, non-Hodgkin's lymphoma, etc.

[0273] Other cancers that can be treated by the methods disclosed herein include atypical meningioma, islet cell carcinoma, medullary thyroid carcinoma, mesenchymoma, hepatocellular carcinoma, hepatoblastoma, renal clear cell carcinoma, and mediastinal neurofibroma.

[0274] dose Appropriate dosages can be determined by the attending physician or other qualified medical personnel based on various clinical factors. As is well known in the medical arts, the dosage for any single patient depends on many factors, including the patient's size, body surface area, age, the amount of IL-2 variant polypeptide administered (e.g., a fusion polypeptide containing one, two, four, or more variant IL-2 polypeptides), the patient's gender, the duration and route of administration, general health, and other concomitant medications. Depending on these factors, an IL-2 variant polypeptide or a composition containing one or more IL-2 variant polypeptides, e.g., a fusion polypeptide, may be administered at a dosage of 0.1 mg / kg to 20 mg / kg body weight per dose, e.g., 0.1 mg / kg to 0.5 mg / kg body weight, 0.5 mg / kg to 1 mg / kg body weight, 1 mg / kg to 5 mg / kg body weight, 5 mg / kg to 10 mg / kg body weight, 10 mg / kg to 15 mg / kg body weight, and 15 mg / kg to 20 mg / kg body weight.

[0275] Those skilled in the art can readily estimate repetition rates for administration based on the measured residence time and concentration of the administered agent in bodily fluids or tissues. After successful treatment, it may be desirable to have the patient undergo maintenance therapy in which the pharmaceutical composition of the present disclosure is administered at a maintenance dose within the above ranges to prevent recurrence of the condition.

[0276] Those skilled in the art will readily appreciate that dosage levels may vary depending on the function of the particular IL-2 variant polypeptide or fusion protein, the severity of the symptoms, and the subject's susceptibility to side effects. Preferred dosages for a given compound can be readily determined by those skilled in the art using a variety of methods.

[0277] In some cases, administration of multiple doses of one or more IL-2 variant polypeptides or compositions comprising one or more IL-2 variant polypeptides, e.g., fusion polypeptides, can vary depending on any of a variety of factors, e.g., the severity of symptoms, etc. For example, in some cases, administration is once a month, approximately every three weeks, twice a month, three times a month, every other week (qow), once a week (qw), or more frequently than once a week.

[0278] The duration of administration of one or more IL-2 variant polypeptides or compositions comprising one or more IL-2 variant polypeptides, e.g., fusion polypeptides, can vary depending on any of a variety of factors, e.g., patient response, etc. For example, administration can be a one-time or can include multiple administrations occurring over a period ranging from less than one month, e.g., one day to one week, two weeks to four weeks, or more than one month, e.g., from about one month to about two months, from about two months to about four months, from about four months to about six months, from about six months to about eight months, from about eight months to about one year, from about one year to about two years, or from about two years to about four years, or more.

[0279] Administration route Pharmaceutical compositions comprising IL-2 variant polypeptides or compositions comprising one or more IL-2 variant polypeptides, e.g., fusion polypeptides, are administered to an individual using any available method and route suitable for drug delivery, including in vivo and ex vivo methods, and systemic and local administration routes.

[0280] Common and pharmaceutically acceptable routes of administration include intratumoral, peritumoral, intramuscular, intralymphatic, intratracheal, intracranial, subcutaneous, intradermal, topical application, intravenous, intraarterial, rectal, nasal, oral, and other enteral and parenteral routes of administration. Routes of administration may be combined as needed or adjusted depending on the IL-2 variant polypeptide or fusion protein and / or the desired effect.

[0281] In some cases, the pharmaceutical composition is administered intravenously, intramuscularly, or subcutaneously. In some cases, the pharmaceutical composition is administered locally. In some cases, the pharmaceutical composition is administered intratumorally. In some cases, the pharmaceutical composition is administered peritumorally. In some cases, the pharmaceutical composition is administered intracranially or intralymphatically.

[0282] Combination therapy As described above, pharmaceutical compositions comprising IL-2 variant polypeptides or compositions comprising one or more IL-2 variant polypeptides, e.g., fusion polypeptides, can be co-administered with modified or unmodified T cells (e.g., in TCR-T or TIL therapy), vaccines, including cancer vaccines, modified cells (e.g., T cells, macrophages, or NK cells) comprising a chimeric antigen receptor (CAR), or modified cells (e.g., macrophages and NK cells) comprising one or more exogenous activating receptors. Such pharmaceutical compositions can also be co-administered to an individual in need thereof in combination with one or more additional therapeutic agents or treatments, including, for example, immune checkpoint inhibitors.

[0283] "Co-administration" means that both an IL-2 variant polypeptide or fusion polypeptide of the present disclosure and at least one additional therapeutic agent are administered to an individual, although not necessarily simultaneously, to achieve a therapeutic effect resulting from the administration of both a pharmaceutical composition comprising an IL-2 variant polypeptide or a composition comprising one or more IL-2 variant polypeptides, e.g., a fusion polypeptide, and at least one additional therapeutic agent. The administration of the pharmaceutical composition and the additional therapeutic agent(s) may be substantially simultaneous; for example, they may be administered to an individual within about 1 minute to about 24 hours (e.g., within about 1 minute, within about 5 minutes, within about 15 minutes, within about 30 minutes, within about 1 hour, within about 4 hours, within about 8 hours, within about 12 hours, or within about 24 hours) of the administration of the at least one additional therapeutic agent. In some cases, a pharmaceutical composition comprising an IL-2 variant polypeptide or a composition comprising one or more IL-2 variant polypeptides, e.g., a fusion polypeptide, is administered to an individual who is undergoing treatment with, is undergoing treatment with, or has been treated with, at least one additional therapeutic agent. Administration of a pharmaceutical composition comprising an IL-2 variant polypeptide or a composition comprising one or more IL-2 variant polypeptides, eg, a fusion polypeptide, can occur at different times and / or with different frequencies.

[0284] As another example, a therapeutic method of the present disclosure may involve co-administration of a pharmaceutical composition comprising an IL-2 variant polypeptide or a composition comprising one or more IL-2 variant polypeptides, e.g., a fusion polypeptide, and an immune checkpoint inhibitor, such as an antibody specific for an immune checkpoint. By "co-administration," it is meant that the pharmaceutical composition and the immune checkpoint-specific antibody therapy are administered to an individual, although not necessarily simultaneously, to achieve a therapeutic effect that is the result of administering both the pharmaceutical composition comprising an IL-2 variant polypeptide or a composition comprising one or more IL-2 variant polypeptides, e.g., a fusion polypeptide, and the immune checkpoint inhibitor. The administration of a pharmaceutical composition comprising an IL-2 variant polypeptide or a composition comprising one or more IL-2 variant polypeptides, e.g., a fusion polypeptide, and an immune checkpoint-specific antibody may be substantially simultaneous, e.g., within about 1 minute to about 24 hours (e.g., within about 1 minute, within about 5 minutes, within about 15 minutes, within about 30 minutes, within about 1 hour, within about 2 hours, within about 4 hours, within about 8 hours, within about 12 hours, or within about 24 hours) of the administration of the immune checkpoint-specific antibody. In some cases, a pharmaceutical composition comprising an IL-2 variant polypeptide or a composition comprising one or more IL-2 variant polypeptides, e.g., a fusion polypeptide, is administered to an individual undergoing or who has previously been treated with an immune checkpoint-specific antibody. The administration of a pharmaceutical composition comprising an IL-2 variant polypeptide or a composition comprising one or more IL-2 variant polypeptides, e.g., a fusion polypeptide, and an immune checkpoint-specific antibody may occur at different times and / or at different frequencies.

[0285] Exemplary immune checkpoint inhibitors include inhibitors that target immune checkpoint polypeptides such as CD27, CD28, CD40, CD122, CD96, CD73, CD47, OX40, GITR, CSF1R, JAK, PI3K delta, PI3K gamma, TAM, arginase, CD137 (also known as 4-1BB), ICOS, A2AR, B7-H3, B7-H4, BTLA, CTLA-4, LAG3, TIM3, VISTA, CD96, TIGIT, CD122, PD-1, PD-L1, and PD-L2. In some cases, the immune checkpoint polypeptide is a stimulatory checkpoint molecule selected from CD27, CD28, CD40, ICOS, OX40, GITR, CD122, and CD137. In some cases, the immune checkpoint polypeptide is an inhibitory checkpoint molecule selected from A2AR, B7-H3, B7-H4, BTLA, CTLA-4, IDO, KIR, LAG3, PD-1, TIM3, CD96, TIGIT, and VISTA.

[0286] In some cases, the immune checkpoint inhibitor is an antibody specific for an immune checkpoint. Suitable anti-immune checkpoint antibodies include, but are not limited to, nivolumab (Bristol-Myers Squibb), pembrolizumab (Merck), pidilizumab (Curetech), AMP-224 (GlaxoSmithKline / Amplimmune), MPDL3280A (Roche), MDX-1105 (Medarex, Inc. / Bristol Myer Squibb), MEDI-4736 (Medimmune / AstraZeneca), arelumab (Merck Serono), ipilimumab (YERVOY, (Bristol-Myers Squibb), tremelimumab (Pfizer), pidilizumab (CureTech, Ltd.), IMP321 (Immutep SA), MGA271 (Macrogenics), BMS-986016 (Bristol-Myers Squibb), lirilumab (Bristol-Myers Squibb), urelumab (Bristol-Myers Squibb), PF-05082566 (Pfizer), IPH2101 (Innate Pharma / Bristol-Myers Squibb), MEDI-6469 (MedImmune / AZ), CP-870,893 (Genentech), mogamulizumab (Kyowa Hakko Kirin), varlilumab (CelIDex Therapeutics), avelumab (EMD Serono), galiximab (Biogen Idec), AMP-514 (Amplimmune / AZ), AUNP 12 (Aurigene and Pierre Fabre), Indoximod (NewLink Genetics), NLG-919 (NewLink Genetics), INCB024360 (Incyte), KN035, and combinations thereof. For example, in some cases, the immune checkpoint inhibitor is an anti-PD-1 antibody.Suitable anti-PD-1 antibodies include, for example, nivolumab, cemiplimab, pembrolizumab (also known as MK-3475), pidilizumab, SHR-1210, PDR001, and AMP-224. In some cases, the anti-PD-1 monoclonal antibody is nivolumab, cemiplimab, pembrolizumab, or PDR001. Suitable anti-PD1 antibodies are described in U.S. Patent Publication No. 2017 / 0044259. For pidilizumab, see, for example, Rosenblatt et al. (2011) J. Immunother. 34:409-18. In some cases, the immune checkpoint inhibitor is an anti-CTLA-4 antibody. In some cases, the anti-CTLA-4 antibody is ipilimumab or tremelimumab. For tremelimumab, see, e.g., Ribas et al. (2013) J. Clin. Oncol. 31:616-22. In some cases, the immune checkpoint inhibitor is an anti-PD-L1 antibody. In some cases, the anti-PD-L1 monoclonal antibody is BMS-935559, MEDI4736, MPDL3280A (also known as RG7446), KN035, or MSB0010718C. In some embodiments, the anti-PD-L1 monoclonal antibody is MPDL3280A (atezolizumab) or MEDI4736 (durvalumab). For durvalumab, see, e.g., WO 2011 / 066389. For atezolizumab, see, e.g., U.S. Patent No. 8,217,149. In some cases, the anti-TIGIT antibody is tiraglumab (RG6058; MTIG7192A) (see U.S. Patent No. 2018 / 0186875). In some cases, the anti-TIGIT antibody is vibostolimab (MK-7684) (see U.S. Patent No. 2018 / 0066055). In some cases, the anti-TIGIT antibody is etigilimab (OMP-313M32).

[0287] Suitable subjects for treatment Suitable subjects for treatment using the methods of the present disclosure include individuals with cancer (either a first-time cancer or a recurrent cancer), who may develop cancer, including individuals who have been diagnosed with cancer, individuals who have been treated for cancer but have not responded to the treatment, and individuals who have been treated for cancer and initially responded but have subsequently become unresponsive to the treatment.

[0288] Examples of Non-Limiting Embodiments of the Disclosure Mode Set A Aspects, including embodiments of the present subject matter described above, may be useful alone or in combination with one or more other aspects or embodiments. Without limiting the foregoing, certain non-limiting aspects of the disclosure are provided below. As will be apparent to one of ordinary skill in the art upon reading this disclosure, each individually numbered aspect may be used or combined with any preceding or subsequent individually numbered aspect. This is intended to provide support for all such combinations of aspects, and is not limited to the combinations of aspects explicitly provided below.

[0289] 1. To the individual: (i) a first composition, (a) a modified or unmodified T cell having a T cell receptor (TCR), a product capable of associating with the TCR of the T cell or being processed by the immune system to be presented by a major histocompatibility complex (MHC) to the TCR of the T cell, or a precursor of a product capable of associating with the TCR of the T cell or being processed by the immune system to be presented by an MHC to the TCR of the T cell; or (b) a modified cell comprising a chimeric antigen receptor (CAR), wherein the CAR binds to a target antigen and the modified cell comprises an intracellular signaling domain that is activated by interaction of the modified cell with IL-2; or (c) modified cells containing one or more exogenously activated receptors; a first composition comprising: (ii) a second composition comprising an immunomodulatory protein 1. A method comprising administering the immunomodulatory protein comprises one or more variant IL-2 polypeptides having at least two amino acid substitutions relative to that set forth in SEQ ID NO:1; one or more variant IL-2 polypeptides bind to the IL-2R alpha chain (IL-2Rα) and have a binding affinity for IL-2Rα that is lower than the affinity of a wild-type IL-2 polypeptide for IL-2Rα when assayed under the same conditions, and the IL-2Rα has the amino acid sequence set forth in SEQ ID NO:2; one or more variant IL-2 polypeptides bind to the IL-2R beta chain (IL-2Rβ) and have a binding affinity for IL-2Rβ that is lower than the affinity of a wild-type IL-2 polypeptide for IL-2Rβ when assayed under the same conditions, the IL-2Rβ having the amino acid sequence set forth in SEQ ID NO:3, the second composition preferentially activates T cells whose TCR has engaged with an antigen presented by MHC compared to T cells whose TCR has not engaged with an antigen presented by MHC, and the first composition and the second composition are administered simultaneously or at different times. method.

[0290] 2. 2. The method of embodiment 1, wherein the at least one variant IL-2 polypeptide exhibits at least a two-fold decrease in binding affinity to IL-2Rβ compared to the binding affinity of the wild-type IL-2 polypeptide to IL-2Rβ, and at least a 50-fold decrease in binding affinity to IL-2Rα compared to the binding affinity of the wild-type IL-2 polypeptide to IL-2Rα.

[0291] 3. 3. The method of embodiment 1 or 2, wherein the one or more variant IL-2 polypeptides comprise at least one substitution that reduces the affinity of the variant IL-2 polypeptide for IL-2Rα, and optionally the at least one substitution is selected from a substitution at R38, F42, K43, Y45, E62, P65, E68, V69, L72, and a combination thereof.

[0292] 4. 4. The method of any one of aspects 1 to 3, wherein at least one variant IL-2 polypeptide comprises a substitution of the phenylalanine amino acid F42, optionally wherein the phenylalanine is substituted with Ala, GIy, Val, Ile, or Leu.

[0293] 5. 5. The method of any one of aspects 1 to 4, wherein the one or more variant IL-2 polypeptides comprise at least one substitution that reduces the affinity of the variant IL-2 polypeptide for IL-2Rβ, and optionally the at least one substitution is selected from a substitution at E15, H16, L19, D20, D84, S87, N88, V91, I92, and a combination thereof.

[0294] 6. 6. The method of any one of aspects 1 to 5, wherein at least one variant IL-2 polypeptide comprises a substitution of the histidine amino acid H16, optionally wherein the histidine is substituted with Ala, Gly, Val, Leu, Thr, Ile, Asp, or Glu.

[0295] 7. 7. The method of any one of aspects 1 to 6, wherein at least one variant IL-2 polypeptide comprises a substitution of asparagine amino acid N88, optionally wherein the asparagine is substituted with Gly, Ala, Ser, Thr, Arg, or Asp.

[0296] 8. 8. The method of any one of aspects 1 to 7, wherein at least one variant IL-2 polypeptide comprises a substitution of amino acids F42 and H16, optionally with phenylalanine substituted with Ala and histidine substituted with Ala, Thr, Asp, or Glu.

[0297] 9. 9. The method of any one of aspects 1 to 8, wherein at least one variant IL-2 polypeptide comprises (i) an H16A substitution and an F42A substitution, (ii) an H16T substitution and an F42A substitution, (iii) an H16E substitution and an F42A substitution, and (iv) an H16D substitution and an F42A substitution.

[0298] 10. 10. The method of any one of embodiments 1 to 9, wherein at least one variant IL-2 polypeptide comprises substitutions at F42, H16, and N88.

[0299] 11. 11. The method of embodiment 10, wherein the asparagine amino acid N88 is substituted with Gly, Ala, Ser, Thr, Arg, or Asp.

[0300] 12. 12. The method of any one of aspects 1 to 11, wherein the immunomodulatory protein comprises two or more variant IL-2 polypeptides, each variant polypeptide comprising the same amino acid sequence.

[0301] 13. 13. The method of embodiment 12, wherein the immunomodulatory protein comprises two variant IL-2 polypeptides, wherein the two variant IL-2 polypeptides are in tandem and connected by independently selected linkers, optionally wherein the linkers comprise glycine and serine.

[0302] 14. 14. The method of any one of aspects 1 to 13, wherein the immunomodulatory protein further comprises a carrier.

[0303] 15. The method of embodiment 14, wherein the carrier is a lipid vesicle (e.g., a liposome) or micelle, a nanoparticle, a PEGylated protein, a fibronectin-based scaffold protein, or an artificial antigen-presenting cell such as an engineered red blood cell or an enucleated cell (e.g., a platelet).

[0304] 16. 14. The method of any of aspects 1-13, wherein the immunomodulatory protein further comprises an immunoglobulin (Ig) scaffold polypeptide or a non-Ig scaffold polypeptide.

[0305] 17. 17. The method of embodiment 16, wherein the immunomodulatory protein comprises a non-Ig scaffold selected from an XTEN polypeptide, a transferrin polypeptide, an elastin-like polypeptide, a silk-like polypeptide, or a silk-elastin-like polypeptide.

[0306] 18. 17. The method of embodiment 16, wherein the immunomodulatory protein comprises an Fc polypeptide, and 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.

[0307] 19. 20. The method of embodiment 18, wherein the Ig Fc polypeptide is a variant having a substantially reduced effector function, e.g., a substantially reduced ability to effect complement-dependent cytotoxicity (CDC) and / or antibody-dependent cellular cytotoxicity (ADCC).

[0308] 20. 20. The method of embodiment 18 or 19, wherein the Ig Fc polypeptide comprises one or more amino acid substitutions selected from N297A, L234A, L235A, L234F, L235E, G237A, and P331S, wherein N297, L234, L235, G237, and P331 correspond to N77, L14, L15, G17, and P111, respectively, of the amino acid sequence depicted in Figure 9A; and optionally, the Ig Fc polypeptide comprises amino acid substitutions L234A and L235A.

[0309] twenty one. The method according to any one of aspects 18 to 20, wherein the Ig Fc polypeptide is an IgG1 Fc polypeptide comprising an amino acid sequence having at least 95% amino acid sequence identity to the amino acid sequence shown in any one of Figures 9A to 9M.

[0310] twenty two. 22. The method of any one of aspects 18 to 21, wherein the immunomodulatory protein comprises a homodimer of two immunomodulatory proteins, each of which comprises an Ig Fc polypeptide, and wherein the Ig Fc of one immunomodulatory protein is linked to the Ig Fc of the other immunomodulatory protein by one or more disulfide bonds.

[0311] twenty three. 23. The method of embodiment 22, wherein each homodimeric immunomodulatory protein comprises two variant IL-2 polypeptides in tandem, wherein the variant IL-2 polypeptides are joined by independently selected linkers.

[0312] twenty four. Each homodimeric immunomodulatory protein is composed of, from the N-terminus to the C-terminus: (i) a variant IL-2 polypeptide; (ii) an independently selected linker; (iii) a variant IL-2 polypeptide; (iv) an independently selected linker, and (v) Ig Fc polypeptide 23. The method of embodiment 22, comprising:

[0313] twenty five. Each homodimeric immunomodulatory protein is composed of, from the N-terminus to the C-terminus: (i) a variant IL-2 polypeptide; (ii) an independently selected linker; (iii) a variant IL-2 polypeptide; (iv) an independently selected linker; (v) an Ig Fc polypeptide; (vi) independently selected linkers; (vii) a variant IL-2 polypeptide; (viii) an independently selected linker, and (ix) a variant IL-2 polypeptide 23. The method of embodiment 22, comprising:

[0314] 26. Each homodimeric immunomodulatory protein is composed of, from the N-terminus to the C-terminus: (i) a variant IL-2 polypeptide; (ii) an independently selected linker; (iii) an Ig Fc polypeptide; (iv) an independently selected linker, and (v) a variant IL-2 polypeptide 23. The method of embodiment 22, comprising:

[0315] 27. Each homodimeric immunomodulatory protein is composed of, from the N-terminus to the C-terminus: (i) an Ig Fc polypeptide; (ii) an independently selected linker; (iii) a variant IL-2 polypeptide; (iv) an independently selected linker, and (v) a variant IL-2 polypeptide 23. The method of embodiment 22, comprising:

[0316] 28. 22. The method of any one of aspects 18 to 21, wherein the immunomodulatory protein comprises a heterodimer of two immunomodulatory proteins, one of which comprises an Ig Fc polypeptide comprising an interspecies dimerization sequence, and the other immunomodulatory protein comprises an Ig Fc polypeptide comprising a counterpart interspecies sequence.

[0317] 29. One of the immunomodulatory proteins in the heterodimer is, in the N-terminal to C-terminal direction, (i) a variant IL-2 polypeptide; (ii) an independently selected linker; (iii) a variant IL-2 polypeptide; (iv) an independently selected linker, and (v) an Ig Fc polypeptide comprising an interspecies binding sequence; Including, The other immunomodulatory protein in the heterodimer is arranged in the N-terminal to C-terminal direction as follows: (i) an Ig Fc polypeptide comprising a counterpart interspecies binding sequence; (ii) an independently selected linker; (iii) a variant IL-2 polypeptide; (iv) an independently selected linker, and (v) a variant IL-2 polypeptide Including, 29. The method of embodiment 28.

[0318] 30. One of the immunomodulatory proteins in the heterodimer is, in the N-terminal to C-terminal direction, (i) a variant IL-2 polypeptide; (ii) an independently selected linker, and (iii) an Ig Fc polypeptide comprising an interspecies binding sequence; Including, The other immunomodulatory protein in the heterodimer is arranged in the N-terminal to C-terminal direction as follows: (i) an Ig Fc polypeptide comprising a counterpart interspecies binding sequence; (ii) an independently selected linker, and (iii) a variant IL-2 polypeptide Including, 29. The method of embodiment 28.

[0319] 31. 31. The method of any one of aspects 1-30, wherein the first composition comprises one or more products, and the one or more products are capable of being processed by the immune system into one or more antigens that can engage with the TCR of a T cell or be presented by a major histocompatibility complex (MHC) to the TCR of a T cell, and optionally the one or more antigens are cancer-associated antigens.

[0320] 32. 31. The method of any one of aspects 1-30, wherein the first composition comprises one or more nucleic acids encoding one or more polypeptides, wherein the one or more polypeptides are capable of engaging a TCR of a T cell or being processed by the immune system into one or more antigens that can be presented by a major histocompatibility complex (MHC) to the TCR of a T cell, and optionally the one or more antigens are cancer-associated antigens.

[0321] 33. 33. The method of embodiment 31 or 32, wherein the first composition comprises an anti-cancer vaccine.

[0322] 34. 31. The method of any one of aspects 1 to 30, wherein the first composition comprises a TCR-T cell comprising an exogenous TCR, and optionally, the TCR binds to a cancer-associated antigen when the cancer-associated antigen is presented to the TCR by an MHC.

[0323] 35. 31. The method of any one of aspects 1-30, wherein the first composition comprises tumor infiltrating lymphocytes (TILS), and optionally, the TILS have been modified to reduce the sensitivity of the TILS to T cell inhibitory signals.

[0324] 36. Cancer-associated antigens include alpha-fetoprotein, Wilms' tumor 1 (WT-1), mutant KRAS, e.g., mutant KRAS containing a G12C or G12D mutation, melanoma antigen recognized by T cells 1 (MART-I), melanoma-associated antigen (MAGE), MAGE-A1, MAGE-A3, MAGE-A4, MAGEA4 / A8, human papillomavirus (HPV) antigen E6, HPV antigen E7, New York esophageal squamous cell carcinoma 1 (NY-ESO-1), MUC-1 (mucin-1), cytoplasmic leukocyte antigen (CLAMP), and leukocyte antigens. 36. The method of any one of aspects 31 to 35, wherein the antigen is selected from: prostate cell surface associated), mesothelin, survivin, prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), prostate-specific antigen (PSA), mutant p53 polypeptide, Ras polypeptide, nuclear factor erythroid 2-related factor 2 (NFE2L2), beta-catenin, PIK3CA (phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha), and BRAF.

[0325] 37. the first composition comprises a CAR having an antigen-binding domain specific for a cancer-associated antigen, and optionally the cell is a T cell, a macrophage, or an NK cell, and optionally (i) when the CAR is not bound to the cancer-associated antigen, the second composition provides the cell with a homeostatic signal for survival; and / or (ii) when the CAR is not bound to the cancer-associated antigen, the second composition provides an activating signal to the cell that causes the cell to proliferate and retain its cytotoxic function; A method according to any one of aspects 1 to 30.

[0326] 38. 38. The method of embodiment 37, wherein the antigen-binding domain is a single chain Fv polypeptide or a nanobody.

[0327] 39. Cancer-associated antigens include AFP, BCMA, CD10, CD117, CD123, CD133, CD128, CD171, CD19, CD20, CD22, CD30, CD33, CD34, CD38, CD5, CD56, CD7, CD70, CD80, CD86, CEA, CLD18, CLL-1, cMet, EGFR, EGFRvIII, EpCAM, EphA2, GD-2, glypican-3, GPC3, HER-2, kappa immunoglobulin, LeY, and L The method of aspect 37 or 38, wherein the first composition is selected from MP1, mesothelin, MG7, MUC1, an NKG2D ligand, PD-L1, PSCA, PSMA, ROR1, ROR1R, TACI, and VEGFR2, e.g., BCMA and CD19, and optionally wherein the first composition comprises TECARTUS®, KYMRIAH®, ABECMA®, BREYANZI®, or YESCARTA®.

[0328] 40. 40. The method according to any one of aspects 1 to 39, for the treatment of cancer in an individual.

[0329] 41. 41. The method of embodiment 40, further comprising administering to the individual at least one immune checkpoint inhibitor (CPI), wherein the CPI, the first composition, and the second composition are administered simultaneously or at different times.

[0330] 42. 42. The method of embodiment 41, wherein the at least one immune checkpoint inhibitor comprises an antibody specific for the immune checkpoint inhibitor.

[0331] 43. 43. The method of embodiment 42, wherein the antibody is specific for an immune checkpoint inhibitor selected from CD27, CD28, CD40, CD122, CD96, CD73, CD47, OX40, GITR, CSF1R, JAK, PI3K delta, PI3K gamma, TAM, arginase, CD137 (also known as 4-1BB), ICOS, A2AR, B7-H3, B7-H4, BTLA, CTLA-4, LAG3, TIM3, VISTA, CD96, TIGIT, CD122, PD-1, PD-L1, and PD-L2, and optionally the immune checkpoint inhibitor is a PD-1, PD-L1, CTLA-4, TIGIT, or LAG3-specific antibody.

[0332] 44. 37. The method according to any one of aspects 1 to 36, for the prevention of cancer in an individual.

[0333] 45. 37. The method according to any one of aspects 1 to 36, for the treatment of cancer in an individual.

[0334] 46. The method of any one of embodiments 1-30, wherein the individual is administered a CAR-T therapy product, a TCR-T therapy product, or a CAR-NK therapy product.

[0335] 47. 47. The method of embodiment 46, wherein the individual is administered a CAR-T therapy product, wherein the CAR-T cell therapy product comprises a population of modified autologous T cells comprising a CAR or allogeneic T cells comprising a CAR, wherein the CAR comprises an antigen-binding domain specific for a cancer-associated antigen.

[0336] 48. 48. The method of embodiment 47, wherein the antigen-binding domain is a single chain Fv polypeptide or a nanobody.

[0337] 49. 49. The method of embodiment 47 or 48, wherein the cancer-associated antigen is selected from AFP, BCMA, CD10, CD117, CD123, CD133, CD128, CD171, CD19, CD20, CD22, CD30, CD33, CD34, CD38, CD5, CD56, CD7, CD70, CD80, CD86, CEA, CLD18, CLL-1, cMet, EGFR, EGFRvIII, EpCAM, EphA2, GD-2, glypican-3, GPC3, HER-2, kappa immunoglobulin, LeY, LMP1, mesothelin, MG7, MUC1, NKG2D ligand, PD-L1, PSCA, PSMA, ROR1, ROR1R, TACI, and VEGFR2.

[0338] 50. 50. The method of any one of aspects 1-49, further comprising administering to the individual an immune checkpoint inhibitor.

[0339] 51. 51. The method of embodiment 50, wherein the immune checkpoint inhibitor is an antibody specific for an immune checkpoint inhibitor selected from CD27, CD28, CD40, CD122, CD96, CD73, CD47, OX40, GITR, CSF1R, JAK, PI3K delta, PI3K gamma, TAM, arginase, CD137 (also known as 4-1BB), ICOS, A2AR, B7-H3, B7-H4, BTLA, CTLA-4, LAG3, TIM3, VISTA, CD96, TIGIT, CD122, PD-1, PD-L1, and PD-L2.

[0340] 52. 31. The method of any one of aspects 1 to 30, wherein the first composition comprises one or more products that are a vaccine, and the one or more products are capable of being processed by the immune system into one or more antigens that can engage with the TCR of a T cell or be presented by a major histocompatibility complex (MHC) to the TCR of a T cell.

[0341] 53. 31. The method of any one of aspects 1 to 30, wherein the first composition is a vaccine comprising a nucleic acid comprising a nucleotide sequence encoding a polypeptide capable of being processed by the immune system into one or more antigens that can be presented by a major histocompatibility complex (MHC) to a TCR of a T cell.

[0342] 54. 54. The method of embodiment 52 or 53, wherein the one or more antigens are cancer-associated antigens.

[0343] 55. 53. The method of embodiment 52, wherein the vaccine comprises one or more products that can be processed by the immune system into two or more cancer-associated antigens that can associate with the TCR of a T cell or be presented by a major histocompatibility complex (MHC) to the TCR of a T cell.

[0344] 56. 53. The method of embodiment 52, wherein the vaccine comprises one or more nucleic acids comprising one or more nucleotide sequences encoding a plurality of polypeptides, wherein the polypeptides are capable of being processed by the immune system into two or more cancer-associated antigens that can associate with a TCR of a T cell or be presented by a major histocompatibility complex (MHC) to the TCR of a T cell.

[0345] 57. 2A-C, 3A-C, 4A-C, 5A-B, 6A-B, 7A-B, 8A-F, 10A-C, 11A-C, 12A-B, 13A-B, and 14A-N, wherein the immunomodulatory protein comprises one or more variant IL-2 polypeptides having at least two amino acid substitutions relative to that shown in SEQ ID NO: 1, one or more variant IL-2 polypeptides bind to the IL-2R alpha chain (IL-2Rα) and have a binding affinity for IL-2Rα that is lower than the affinity of a wild-type IL-2 polypeptide for IL-2Rα when assayed under the same conditions, and the IL-2Rα has the amino acid sequence set forth in SEQ ID NO:2; one or more variant IL-2 polypeptides bind to the IL-2R beta chain (IL-2Rβ) and have a binding affinity for IL-2Rβ that is lower than the affinity of a wild-type IL-2 polypeptide for IL-2Rβ when assayed under the same conditions, and the IL-2Rβ has the amino acid sequence set forth in SEQ ID NO:3; Fusion polypeptides.

[0346] 58. 58. The fusion polypeptide of embodiment 57, wherein the fusion protein comprises one or more mutations capable of reducing binding of IL-2 to IL-2Rα, and the one or more mutations are selected from substitutions at one or more of the amino acids R38, F42, K43, Y45, E62, P65, E68, V69, and L72.

[0347] 59. 59. The fusion polypeptide of embodiment 58, wherein the fusion protein comprises a substitution of amino acid F42, optionally wherein Phe is substituted with Ala or Lys.

[0348] 60. 60. The fusion polypeptide of any one of aspects 57 to 59, wherein the fusion protein comprises one or more mutations capable of reducing binding of IL-2 to IL-2Rβ, wherein the one or more mutations are selected from substitutions at one or more of the following amino acids: E15, H16, L19, D20, D84, S87, N88, V91, I92.

[0349] 61. 61. The fusion polypeptide of embodiment 60, wherein the fusion protein comprises a substitution of amino acid H16, optionally wherein His is substituted with Ala, Glu, Thr, or Asp.

[0350] 62. 58. The fusion polypeptide of embodiment 57, wherein the fusion protein comprises a substitution selected from E15A with R38A, R38D, or R38E.

[0351] 63. 58. The fusion polypeptide of embodiment 57, wherein the fusion protein comprises substitutions selected from: H16A with R38A, R38D, or R38E; H16T with R38A, R38D, or R38E; H16E with R38A, R38D, or R38E; and H16D with R38A, R38D, or R38E.

[0352] 64. 58. The fusion polypeptide of embodiment 57, wherein the fusion protein comprises substitutions selected from: D84H with R38A, R38D, or R38E; D84K with R38A, R38D, or R38E; and D84R with R38A, R38D, or R38E.

[0353] 65. 58. The fusion polypeptide of embodiment 57, wherein the fusion protein comprises substitutions selected from: R38A with N88S, N88A, N88G, N88R, N88T, or N88D; R38D with N88S, N88A, N88G, N88R, N88T, or N88D; and R38E with N88S, N88A, N88G, N88R, N88T, or N88D.

[0354] 66. 58. The fusion polypeptide of embodiment 57, wherein the fusion protein comprises substitutions selected from: R38A with V91E, V91A, or V91T; R38D with V91E, V91A, or V91T; and R38E with V91E, V91A, or V91T.

[0355] 67. 58. The fusion polypeptide of embodiment 57, wherein the fusion protein comprises a substitution selected from R38A, I92A, R38D, I92A and R38E, I92A.

[0356] 68. 58. The fusion polypeptide of embodiment 57, wherein the fusion protein comprises a substitution selected from E15A, F42A and E15A, F42K.

[0357] 69. 58. The fusion polypeptide of embodiment 57, wherein the fusion protein comprises substitutions selected from H16A, F42A; H16T, F42A; H16E, F42A; H16D, F42A; H16A, F42K; H16T, F42K; and H16E, F42K; H16D, F42K.

[0358] 70. 58. The fusion polypeptide of embodiment 57, wherein the fusion protein comprises substitutions selected from: F42A with N88S, N88A, N88G, N88R, N88T, or N88D; and F42K with N88S, N88A, N88G, N88R, N88T, or N88D.

[0359] 71. 58. The fusion polypeptide of embodiment 57, wherein the fusion protein comprises substitutions selected from: F42A with V91E, V91A, or V91T; and F42K with V91E, V91A, or V91T.

[0360] 72. 58. The fusion polypeptide of embodiment 57, wherein the fusion protein comprises substitutions selected from: F42A with I92A; and F42K with I92A.

[0361] 73. 58. The fusion polypeptide of embodiment 57, wherein the fusion protein comprises E15A and K43E substitutions.

[0362] 74. 58. The fusion polypeptide of embodiment 57, wherein the fusion protein comprises substitutions selected from H16A, K43E; H16T, K43E; H16E, K43E; and H16D, K43E.

[0363] 75. 58. The fusion polypeptide of embodiment 57, wherein the fusion protein comprises a substitution selected from K43E with D84H, D84K, or D84R.

[0364] 76. 58. The fusion polypeptide of embodiment 57, wherein the fusion protein comprises a substitution selected from K43E with N88S, N88A, N88G, N88R, N88T, or N88D.

[0365] 77. 58. The fusion polypeptide of embodiment 57, wherein the fusion protein comprises a substitution selected from K43E with V91E, V91A, or V91T.

[0366] 78. 58. The fusion polypeptide of embodiment 57, wherein the fusion protein comprises K43E and I92A or E15A, E62Q substitutions.

[0367] 79. 58. The fusion polypeptide of embodiment 57, wherein the fusion protein comprises substitutions selected from H16A, E62Q; H16T, E62Q; H16E, E62Q; and H16D, E62Q.

[0368] 80. 58. The fusion polypeptide of embodiment 57, wherein the fusion protein comprises a substitution selected from E62Q with D84H, D84K, or D84R.

[0369] 81. 58. The fusion polypeptide of embodiment 57, wherein the fusion protein comprises a substitution selected from E62Q with N88S, N88A, N88G, N88R, N88T, or N88D.

[0370] 82. 58. The fusion polypeptide of embodiment 57, wherein the fusion protein comprises a substitution selected from E62Q with V91E, V91A, or V91T.

[0371] 83. 58. The fusion polypeptide of embodiment 57, wherein the fusion protein comprises a substitution selected from E62Q and I92A.

[0372] 84. 58. The fusion polypeptide of embodiment 57, wherein the fusion protein comprises a substitution selected from E62Q with V91E, V91A, or V91T.

[0373] 85. 69. The fusion polypeptide of embodiment 68, wherein the fusion protein comprises substitutions at F42, E15 and N88.

[0374] 86. 69. The fusion polypeptide of embodiment 68, wherein the fusion protein comprises substitutions at F42, E15 and V91.

[0375] 87. 70. The fusion polypeptide of embodiment 69, wherein the fusion protein comprises substitutions at F42, H16 and D84.

[0376] 88. 70. The fusion polypeptide of embodiment 69, wherein the fusion protein comprises substitutions at F42, H16 and N88.

[0377] 89. 70. The fusion polypeptide of embodiment 69, wherein the fusion protein comprises substitutions at F42, H16 and V91.

[0378] 90. 70. The fusion polypeptide of embodiment 69, wherein the fusion protein comprises substitutions at F42, H16 and I92.

[0379] 91. A fusion polypeptide according to any one of aspects 57 to 90, wherein the fusion protein is as shown in any one of Figures 10A-C, 11A-C, 12A-B and 13A-B, and 14A-N, and comprises a functional protein that is a cancer targeting polypeptide (CTP).

[0380] 92. 92. The fusion polypeptide of embodiment 91, wherein the target of the cancer targeting polypeptide is a peptide-HLA complex on the surface of a cancer cell.

[0381] 93. 92. The fusion polypeptide of embodiment 91, wherein the target of the CTP is a cancer-associated epitope.

[0382] 94. 92. The fusion polypeptide of embodiment 91, wherein the CTP is an antibody specific for a cancer-associated antigen.

[0383] 95. 92. The fusion polypeptide of embodiment 91, wherein the CTP is an antibody specific for a peptide / HLA complex on the surface of a cancer cell, and the peptide can be a cancer-associated peptide (e.g., a peptide of a cancer-associated antigen).

[0384] 96. 92. The fusion polypeptide of embodiment 91, wherein the functional protein is a TCR, such as an "scTCR," a single-chain T-cell receptor specific for a peptide / HLA complex on the surface of a cancer cell, and the peptide can be a cancer-associated peptide (e.g., a peptide of a cancer-associated antigen).

[0385] 97. 92. The fusion polypeptide of embodiment 91, wherein the functional protein comprises a wild-type or variant immunomodulatory polypeptide, such as a wild-type or variant immunostimulatory polypeptide, such as a member of the B7 family of costimulatory receptors, e.g., CD80, CD86, a cytokine, such as IL-7, IL-12, IL-15 or IL-21, a TNF superfamily member, such as CD-40, 4-1BBL and OX40, or a chemokine, such as CCL19, CCL21, CXCL9 / 10 / 11, or CXCL12.

[0386] 98. 98. The fusion polypeptide of any one of embodiments 57 to 97, comprising one or more independently selected linkers.

[0387] 99. 98. The fusion polypeptide of any one of aspects 57 to 97, comprising a variant Ig Fc polypeptide having a substantially reduced effector function, such as a substantially reduced ability to effect complement dependent cytotoxicity (CDC) and / or antibody dependent cellular cytotoxicity (ADCC).

[0388] 100. 90. The fusion polypeptide of embodiment 99, wherein the Ig Fc polypeptide comprises one or more amino acid substitutions selected from N297A, L234A, L235A, L234F, L235E, G237A and P331S, wherein N297, L234, L235, G237 and P331 correspond to N77, L14, L15, G17 and P111, respectively, of the amino acid sequence shown in Figure 9A.

[0389] 101. 101. The fusion polypeptide of embodiment 99 or 100, wherein the Ig Fc polypeptide is an IgG1 Fc polypeptide comprising an amino acid sequence having at least 95% amino acid sequence identity to the amino acid sequence shown in any one of Figures 9A to 9M.

[0390] 102. 102. The fusion polypeptide of any one of aspects 57 to 101, comprising a homodimer of two fusion polypeptides, each of which comprises an Ig Fc polypeptide, wherein the Ig Fc of one immunomodulatory protein is linked to the Ig Fc of the other immunomodulatory protein by one or more disulfide bonds.

[0391] 103. 103. The fusion polypeptide of embodiment 102, wherein each homodimeric immunomodulatory protein comprises two variant IL-2 polypeptides in tandem, wherein the variant IL-2 polypeptides are linked by independently selected linkers.

[0392] 104. 104. A method of treating cancer, comprising administering to a patient a pharmaceutical composition comprising an effective amount of the fusion polypeptide of any one of aspects 57 to 103, wherein the pharmaceutical composition is co-administered with a second pharmaceutical composition that is a cancer vaccine.

[0393] 105. 104. A method of treating cancer, comprising administering to a patient a pharmaceutical composition comprising an effective amount of the fusion polypeptide of any one of aspects 57 to 103, wherein the pharmaceutical composition is co-administered with a second pharmaceutical composition that is a TCR-T cell therapy.

[0394] 106. 104. A method of treating cancer, comprising administering to a patient a pharmaceutical composition comprising an effective amount of the fusion polypeptide of any one of aspects 57 to 103, wherein the pharmaceutical composition is co-administered with a second pharmaceutical composition that is a CAR-T cell therapy.

[0395] 107. A method of treating cancer, comprising administering to a patient a pharmaceutical composition comprising an effective amount of the fusion polypeptide of any one of embodiments 57 to 103, wherein the pharmaceutical composition is co-administered with a second pharmaceutical composition that is an engineered cell (e.g., a macrophage or NK cell) comprising one or more exogenous activating receptors (e.g., a CAR or a TCR).

[0396] 108. A method of treating cancer, comprising administering to a patient a pharmaceutical composition comprising an effective amount of the fusion polypeptide of any one of aspects 57 to 103, wherein the pharmaceutical composition is co-administered with a second pharmaceutical composition that is an immune checkpoint inhibitor (e.g., an anti-PD1 antibody).

[0397] 109. 109. The method of treating cancer according to any of embodiments 104 to 108, wherein the fusion polypeptide is administered before, simultaneously with, or after administration of the second pharmaceutical composition.

[0398] 110. Use of a fusion polypeptide according to any of aspects 57 to 103 for the preparation of a medicament for treating cancer, to be administered before, simultaneously with, or after administration of a cancer vaccine.

[0399] 111. Use of the fusion polypeptide according to any of aspects 57 to 103 for the preparation of a medicament for treating cancer, which is to be administered before, simultaneously with, or after administration of a pharmaceutical composition that is a TCR-T cell therapy.

[0400] 112. 104. Use of the fusion polypeptide according to any of aspects 57 to 103 for the preparation of a medicament for treating cancer, to be administered before, simultaneously with, or after administration of a pharmaceutical composition that is a CAR-T cell therapy.

[0401] 113. Use of a fusion polypeptide according to any of aspects 57 to 103 for the preparation of a medicament for treating cancer, to be administered before, simultaneously with, or after administration of a pharmaceutical composition that is a modified cell other than a T cell (e.g., a macrophage or an NK cell) that comprises one or more exogenous activating receptors (e.g., a CAR or a TCR).

[0402] 114. Use of a fusion polypeptide according to any of aspects 57 to 103 for the preparation of a medicament for treating cancer, the medicament being to be administered before, simultaneously with, or after administration of a pharmaceutical composition that is an immune checkpoint inhibitor (e.g., an anti-PD1 antibody).

[0403] 115. 104. The fusion polypeptide of any one of aspects 94 to 103, wherein the CTP is an antibody specific for a cancer-associated antigen selected from the group consisting of epidermal growth factor receptor (EGFR), Her2, CD19, mesothelin, prostate-specific membrane antigen (PSMA), CD22, TROP-2, B-cell maturation antigen (BCMA), mucin-1 (MUC1), claudin 18.2, and CD20.

[0404] 116. 116. The fusion polypeptide of any one of aspects 94 to 103 and 115, wherein the fusion polypeptide comprises a first CTP and a second CTP, wherein the first CTP is a first antibody specific to a first cancer-associated antigen, and the second CTP is an antibody specific to a second cancer-associated antigen.

[0405] 117. 117. The fusion polypeptide of embodiment 116, wherein the first antibody is specific for CD19 and the second antibody is specific for CD20.

[0406] 118. 117. The fusion polypeptide of embodiment 116, wherein the first antibody is specific for Her2 and the second antibody is specific for EGFR.

[0407] 119. 119. A method of treating cancer, comprising administering to a patient a pharmaceutical composition comprising an effective amount of the fusion polypeptide of any one of aspects 115 to 118, wherein the pharmaceutical composition is co-administered with a second pharmaceutical composition that is a cancer vaccine.

[0408] 120. 119. A method of treating cancer, comprising administering to a patient a pharmaceutical composition comprising an effective amount of the fusion polypeptide of any one of aspects 115 to 118, wherein the pharmaceutical composition is co-administered with a second pharmaceutical composition that is a TCR-T cell therapy.

[0409] 121. 119. A method of treating cancer, comprising administering to a patient a pharmaceutical composition comprising an effective amount of the fusion polypeptide of any one of aspects 115 to 118, wherein the pharmaceutical composition is co-administered with a second pharmaceutical composition that is a CAR-T cell therapy.

[0410] 122. 119. A method of treating cancer, comprising administering to a patient a pharmaceutical composition comprising an effective amount of the fusion polypeptide of any one of embodiments 115-118, wherein the pharmaceutical composition is co-administered with a second pharmaceutical composition that is an engineered cell (e.g., a macrophage or NK cell) comprising one or more exogenous activating receptors (e.g., a CAR or a TCR).

[0411] one two three. 119. A method of treating cancer, comprising administering to a patient a pharmaceutical composition comprising an effective amount of the fusion polypeptide of any one of aspects 115 to 118, wherein the pharmaceutical composition is co-administered with a second pharmaceutical composition that is an immune checkpoint inhibitor (e.g., an anti-PD1 antibody).

[0412] 124. 125. The method of treating cancer according to any of embodiments 119 to 124, wherein the fusion polypeptide is administered before, simultaneously with, or after administration of the second pharmaceutical composition.

[0413] Mode Set B Aspects, including embodiments of the present subject matter described above, may be useful alone or in combination with one or more other aspects or embodiments. Without limiting the foregoing, certain non-limiting aspects of the disclosure are provided below. As will be apparent to one of ordinary skill in the art upon reading this disclosure, each individually numbered aspect may be used or combined with any preceding or subsequent individually numbered aspect. This is intended to provide support for all such combinations of aspects, and is not limited to the combinations of aspects explicitly provided below.

[0414] Aspect 1. To the individual: (i) a first composition, (a) a modified or unmodified T cell having a T cell receptor (TCR), or (b) a modified cell comprising a chimeric antigen receptor (CAR), wherein the CAR binds to a target antigen and the modified cell comprises an intracellular signaling domain that is activated by interaction of the modified cell with IL-2; or (c) a modified cell containing one or more exogenously activated receptors; or (d) a product that can be processed by the immune system into one or more antigens that can associate with the TCR of a T cell or be presented by the major histocompatibility complex (MHC) to the TCR of a T cell, optionally wherein the one or more antigens are cancer-associated antigens; or (e) one or more nucleic acids encoding one or more polypeptides that can be processed by the immune system into one or more antigens that can associate with the TCR of a T cell or be presented by a major histocompatibility complex (MHC) to the TCR of a T cell, optionally wherein the one or more antigens are cancer-associated antigens; or (f) at least one immune checkpoint inhibitor (CPI) a first composition comprising: (ii) a second composition comprising an immunomodulatory protein 1. A method comprising administering The immunomodulatory protein comprises one or more variant IL-2 polypeptides having at least two amino acid substitutions relative to that set forth in SEQ ID NO: 1, wherein the one or more variant IL-2 polypeptides bind to the IL-2R alpha chain (IL-2Rα) with a binding affinity to IL-2Rα that is lower than the affinity of a wild-type IL-2 polypeptide for IL-2Rα when assayed under the same conditions, the IL-2Rα having the amino acid sequence set forth in SEQ ID NO: 2, and the one or more variant IL-2 polypeptides bind to the IL-2R beta chain (IL-2Rβ) with a binding affinity to IL-2Rβ that is lower than the affinity of a wild-type IL-2 polypeptide for IL-2Rα when assayed under the same conditions. the binding affinity of the first composition to the TCR is lower than the affinity of a wild-type IL-2 polypeptide for IL-2Rβ when assayed under the same conditions, the IL-2Rβ has the amino acid sequence set forth in SEQ ID NO:3, the second composition preferentially activates T cells whose TCR is engaged with an antigen presented by an MHC compared to T cells whose TCR is not engaged with an antigen presented by an MHC, the first composition and the second composition are administered at the same time or at different times, and the individual may also be administered a CPI when administered the first composition comprising (a), (b), (c), (d) or (e). method.

[0415] Aspect 2. 2. The method of embodiment 1, wherein at least one of the one or more variant IL-2 polypeptides exhibits at least a two-fold decrease in binding affinity to IL-2Rβ compared to the binding affinity of the wild-type IL-2 polypeptide for IL-2Rβ, and at least a 50-fold decrease in binding affinity to IL-2Rα compared to the binding affinity of the wild-type IL-2 polypeptide for IL-2Rα.

[0416] Aspect 3. 3. The method of embodiment 1 or 2, wherein at least one of the one or more variant IL-2 polypeptides comprises at least one substitution that reduces the affinity of the variant IL-2 polypeptide for IL-2Rα, and optionally the at least one substitution is selected from a substitution at R38, F42, K43, Y45, E62, P65, E68, V69, L72, and a combination thereof.

[0417] Aspect 4. 4. The method of any one of aspects 1 to 3, wherein at least one of the one or more variant IL-2 polypeptides comprises a substitution of the phenylalanine amino acid F42, optionally wherein the phenylalanine is substituted with Ala, GIy, Val, Ile, or Leu.

[0418] Aspect 5. 5. The method of any one of aspects 1 to 4, wherein at least one of the one or more variant IL-2 polypeptides comprises at least one substitution that reduces the affinity of the variant IL-2 polypeptide for IL-2Rβ, and optionally the at least one substitution is selected from a substitution at E15, H16, L19, D20, D84, S87, N88, V91, I92, and a combination thereof.

[0419] Aspect 6. 6. The method of any one of aspects 1 to 5, wherein at least one of the one or more variant IL-2 polypeptides comprises a substitution of the histidine amino acid H16, optionally wherein the histidine is substituted with Ala, Gly, Val, Leu, Thr, Ile, Asp, or Glu.

[0420] Aspect 7. 7. The method of any one of aspects 1-6, wherein at least one of the one or more variant IL-2 polypeptides comprises a substitution of asparagine amino acid N88, optionally wherein the asparagine is substituted with Gly, Ala, Ser, Thr, Arg, or Asp.

[0421] Aspect 8. 8. The method of any one of aspects 1 to 7, wherein at least one of the one or more variant IL-2 polypeptides comprises a substitution of amino acids F42 and H16, optionally with phenylalanine substituted with Ala and histidine substituted with Ala, Thr, Asp, or Glu.

[0422] Aspect 9. 9. The method of any one of aspects 1-8, wherein at least one of the one or more variant IL-2 polypeptides comprises (i) an H16A substitution and an F42A substitution, (ii) an H16T substitution and an F42A substitution, (iii) an H16E substitution and an F42A substitution, and (iv) an H16D substitution and an F42A substitution.

[0423] Aspect 10. 10. The method of any one of embodiments 1 to 9, wherein at least one of the one or more variant IL-2 polypeptides comprises substitutions at F42, H16, and N88.

[0424] Aspect 11. 11. The method of embodiment 10, wherein the asparagine amino acid N88 is substituted with Gly, Ala, Ser, Thr, Arg, or Asp.

[0425] Aspect 12. 12. The method of any one of aspects 1 to 11, wherein the immunomodulatory protein comprises two or more variant IL-2 polypeptides, each variant IL-2 polypeptide comprising the same amino acid sequence.

[0426] Aspect 13. 13. The method of embodiment 12, wherein the immunomodulatory protein comprises two variant IL-2 polypeptides, wherein the two variant IL-2 polypeptides are in tandem and connected by independently selected linkers, optionally wherein the linkers comprise glycine and serine.

[0427] Aspect 14. 14. The method of any one of aspects 1 to 13, wherein the immunomodulatory protein further comprises a carrier.

[0428] Aspect 15. The method of embodiment 14, wherein the carrier is a lipid vesicle (e.g., a liposome) or micelle, a nanoparticle, a PEGylated protein, or an artificial antigen-presenting cell, such as an engineered red blood cell or an enucleated cell (e.g., a platelet).

[0429] Aspect 16. 14. The method of any of aspects 1-13, wherein the immunomodulatory protein further comprises an immunoglobulin (Ig) scaffold polypeptide or a non-Ig scaffold polypeptide.

[0430] Aspect 17. 17. The method of embodiment 16, wherein the immunomodulatory protein comprises a non-Ig scaffold selected from an XTEN polypeptide, a transferrin polypeptide, an elastin-like polypeptide, a silk-like polypeptide, a fibronectin-based scaffold protein, or a silk-elastin-like polypeptide.

[0431] Aspect 18. 17. The method of embodiment 16, wherein the immunomodulatory protein is a fusion polypeptide comprising (a) one or more variant IL-2 polypeptides and (b) an Fc polypeptide, and 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.

[0432] Aspect 19. 20. The method of embodiment 18, wherein the Ig Fc polypeptide is a variant having a substantially reduced effector function, e.g., a substantially reduced ability to effect complement-dependent cytotoxicity (CDC) and / or antibody-dependent cellular cytotoxicity (ADCC).

[0433] Aspect 20. 20. The method of embodiment 18 or 19, wherein the Ig Fc polypeptide comprises one or more amino acid substitutions selected from N297A, L234A, L235A, L234F, L235E, G237A, and P331S, wherein N297, L234, L235, G237, and P331 correspond to N77, L14, L15, G17, and P111, respectively, of the amino acid sequence depicted in Figure 9A.

[0434] Aspect 21. The method according to any one of aspects 18 to 20, wherein the Ig Fc polypeptide is an IgG1 Fc polypeptide comprising an amino acid sequence having at least 95% amino acid sequence identity to the amino acid sequence shown in any one of Figures 9A to 9M.

[0435] Aspect 22. 22. The method of any one of aspects 18 to 21, wherein the immunomodulatory protein comprises a homodimer of two immunomodulatory proteins, each of which comprises an Ig Fc polypeptide, and wherein the Ig Fc of one immunomodulatory protein is linked to the Ig Fc of the other immunomodulatory protein by one or more disulfide bonds.

[0436] Aspect 23. 23. The method of embodiment 22, wherein each homodimeric immunomodulatory protein comprises two variant IL-2 polypeptides in tandem, wherein the variant IL-2 polypeptides are joined by independently selected linkers.

[0437] Aspect 24. Each homodimeric immunomodulatory protein is composed of, from the N-terminus to the C-terminus: (i) a variant IL-2 polypeptide; (ii) an independently selected linker; (iii) a variant IL-2 polypeptide; (iv) an independently selected linker, and (v) Ig Fc polypeptide 23. The method of embodiment 22, comprising:

[0438] Aspect 25. Each homodimeric immunomodulatory protein is composed of, from the N-terminus to the C-terminus: (i) a variant IL-2 polypeptide; (ii) an independently selected linker; (iii) a variant IL-2 polypeptide; (iv) an independently selected linker; (v) an Ig Fc polypeptide; (vi) independently selected linkers; (vii) a variant IL-2 polypeptide; (viii) an independently selected linker, and (ix) a variant IL-2 polypeptide 23. The method of embodiment 22, comprising:

[0439] Aspect 26. Each homodimeric immunomodulatory protein is composed of, from the N-terminus to the C-terminus: (i) a variant IL-2 polypeptide; (ii) an independently selected linker; (iii) an Ig Fc polypeptide; (iv) an independently selected linker, and (v) a variant IL-2 polypeptide 23. The method of embodiment 22, comprising:

[0440] Aspect 27. Each homodimeric immunomodulatory protein is composed of, from the N-terminus to the C-terminus: (i) an Ig Fc polypeptide; (ii) an independently selected linker; (iii) a variant IL-2 polypeptide; (iv) an independently selected linker, and (v) a variant IL-2 polypeptide 23. The method of embodiment 22, comprising:

[0441] Aspect 28. 22. The method of any one of aspects 18 to 21, wherein the immunomodulatory protein comprises a heterodimer of two immunomodulatory proteins, one of which comprises an Ig Fc polypeptide comprising an interspecies dimerization sequence and the other of which comprises an Ig Fc polypeptide comprising a counterpart interspecies sequence; or wherein the immunomodulatory protein comprises a heterodimer comprising a first polypeptide and a second polypeptide, wherein the first polypeptide comprises an Ig Fc polypeptide comprising an interspecies dimerization sequence and the second polypeptide comprises an Ig Fc polypeptide comprising a counterpart interspecies sequence, and wherein either the first polypeptide or the second polypeptide comprises one or more variant IL-2 polypeptides.

[0442] Aspect 29. (A) One of the immunomodulatory proteins in the heterodimer is arranged in the N-terminal to C-terminal direction as follows: (i) a variant IL-2 polypeptide; (ii) an independently selected linker; (iii) a variant IL-2 polypeptide; (iv) an independently selected linker, and (v) an Ig Fc polypeptide comprising an interspecies binding sequence; Including, The other immunomodulatory protein in the heterodimer is arranged in the N-terminal to C-terminal direction as follows: (i) an Ig Fc polypeptide comprising a counterpart interspecies binding sequence; (ii) an independently selected linker; (iii) a variant IL-2 polypeptide; (iv) an independently selected linker, and (v) a variant IL-2 polypeptide Contains, or (B) The first polypeptide in the heterodimer is arranged in the N-terminal to C-terminal direction as follows: (i) a variant IL-2 polypeptide; (ii) an independently selected linker; (iii) a variant IL-2 polypeptide; (iv) an independently selected linker, and (v) an Ig Fc polypeptide comprising an interspecies binding sequence; Including, the second polypeptide in the heterodimer comprises an Ig Fc polypeptide containing a counterpart interspecies binding sequence but does not comprise a variant IL-2 polypeptide; or (C) The first polypeptide in the heterodimer is arranged in the N-terminal to C-terminal direction as follows: (i) an Ig Fc polypeptide comprising an interspecies binding sequence; (ii) an independently selected linker; (iii) a variant IL-2 polypeptide; (iv) an independently selected linker; (v) a variant IL-2 polypeptide Including, the second polypeptide in the heterodimer comprises an Ig Fc polypeptide comprising a counterpart interspecies binding sequence, but does not comprise a variant IL-2 polypeptide; 29. The method of embodiment 28.

[0443] Aspect 30. One of the immunomodulatory proteins in the heterodimer is, in the N-terminal to C-terminal direction, (i) a variant IL-2 polypeptide; (ii) an independently selected linker, and (iii) an Ig Fc polypeptide comprising an interspecies binding sequence; Including, The other immunomodulatory protein in the heterodimer is arranged in the N-terminal to C-terminal direction as follows: (i) an Ig Fc polypeptide comprising a counterpart interspecies binding sequence; (ii) an independently selected linker, and (iii) a variant IL-2 polypeptide Including, 29. The method of embodiment 28.

[0444] Aspect 31. (A) The first polypeptide in the heterodimer is arranged in the N-terminal to C-terminal direction as follows: (i) a variant IL-2 polypeptide; (ii) an independently selected linker, and (iii) an Ig Fc polypeptide comprising an interspecies binding sequence; Including, the second polypeptide in the heterodimer comprises an Ig Fc polypeptide containing a counterpart interspecies binding sequence but does not comprise a variant IL-2 polypeptide; or (B) The first polypeptide in the heterodimer is arranged in the N-terminal to C-terminal direction as follows: (i) an Ig Fc polypeptide comprising an interspecies binding sequence; (ii) an independently selected linker, and (iii) a variant IL-2 polypeptide Including, the second polypeptide in the heterodimer comprises an Ig Fc polypeptide comprising a counterpart interspecies binding sequence, but does not comprise a variant IL-2 polypeptide; 29. The method of embodiment 28.

[0445] Aspect 32. 32. The method of any one of aspects 1-31, wherein the first composition comprises one or more products, and the one or more products are capable of being processed by the immune system into one or more antigens that can engage with the TCR of a T cell or be presented by a major histocompatibility complex (MHC) to the TCR of a T cell, and optionally the one or more antigens are cancer-associated antigens.

[0446] Aspect 33. 32. The method of any one of aspects 1-31, wherein the first composition comprises one or more nucleic acids encoding one or more polypeptides, wherein the one or more polypeptides are capable of engaging a TCR of a T cell or being processed by the immune system into one or more antigens that can be presented by a major histocompatibility complex (MHC) to the TCR of a T cell, and optionally the one or more antigens are cancer-associated antigens.

[0447] Aspect 34. 32. The method of any one of aspects 1 to 31, wherein the first composition comprises a TCR-T cell comprising an exogenous TCR, and optionally, the TCR binds to a cancer-associated antigen when the cancer-associated antigen is presented to the TCR by an MHC.

[0448] Aspect 35. 32. The method of any one of aspects 1-31, wherein the first composition comprises tumor infiltrating lymphocytes (TILS), and optionally, the TILS have been modified to reduce the sensitivity of the TILS to T cell inhibitory signals.

[0449] Aspect 36. Cancer-associated antigens include alpha-fetoprotein, Wilms' tumor 1 (WT-1), mutant KRAS, e.g., mutant KRAS containing a G12C or G12D mutation, melanoma antigen recognized by T cells 1 (MART-1), melanoma-associated antigen (MAGE), MAGE-A1, MAGE-A3, MAGE-A4, human papillomavirus (HPV) antigen E6, HPV antigen E7, New York esophageal squamous cell carcinoma 1 (NY-ESO-1), MUC-1 (mucin-1, cell surface-associated antigen), and the like. ), mesothelin, survivin, prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), prostate-specific antigen (PSA), mutant p53 polypeptide, Ras polypeptide, nuclear factor erythroid 2-related factor 2 (NFE2L2), beta-catenin, PIK3CA (phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha), and BRAF.

[0450] Aspect 37. 32. The method of any one of embodiments 1-31, wherein a first composition comprises a CAR having an antigen-binding domain specific for a cancer-associated antigen, and optionally the cell is a T cell, a macrophage, or an NK cell, and optionally, (i) when the CAR is not bound to the cancer-associated antigen, the second composition provides a homeostatic signal to the cell for survival, and / or (ii) when the CAR is not bound to the cancer-associated antigen, the second composition provides an activation signal to the cell that causes the cell to proliferate and retain its cytotoxic function.

[0451] Aspect 38. 38. The method of embodiment 37, wherein the antigen-binding domain is a single chain Fv polypeptide or a nanobody.

[0452] Aspect 39. 39. The method of embodiment 37 or 38, wherein the cancer-associated antigen is selected from AFP, BCMA, CD10, CD117, CD123, CD133, CD128, CD171, CD19, CD20, CD22, CD30, CD33, CD34, CD38, CD5, CD56, CD7, CD70, CD80, CD86, CEA, CLD18, CLL-1, cMet, EGFR, EGFRvIII, EpCAM, EphA2, GD-2, glypican-3, GPC3, HER-2, kappa immunoglobulin, LeY, LMP1, mesothelin, MG7, MUC1, NKG2D ligand, PD-L1, PSCA, PSMA, ROR1, ROR1R, TACI, and VEGFR2.

[0453] Aspect 40. 40. The method according to any one of aspects 1 to 39, for the treatment of cancer in an individual.

[0454] Aspect 41. 41. The method of embodiment 40, wherein the first composition comprises (a), (b), (c), (d), or (e), and the method further comprises administering to the individual at least one immune checkpoint inhibitor (CPI), wherein the CPI, the first composition, and the second composition are administered simultaneously or at different times.

[0455] Aspect 42. 42. The method of embodiment 41, wherein the at least one immune checkpoint inhibitor comprises an antibody specific for an immune checkpoint.

[0456] Aspect 43. 43. The method of embodiment 42, wherein the antibody is specific for an immune checkpoint selected from CD27, CD28, CD40, CD122, CD96, CD73, CD47, OX40, GITR, CSF1R, JAK, PI3K delta, PI3K gamma, TAM, arginase, CD137 (also known as 4-1BB), ICOS, A2AR, B7-H3, B7-H4, BTLA, CTLA-4, LAG3, TIM3, VISTA, CD96, TIGIT, CD122, PD-1, PD-L1, and PD-L2; and optionally, the immune checkpoint inhibitor is a PD-1, PD-L1, CTLA-4, TIGIT, and LAG3-specific antibody.

[0457] Aspect 44. 37. The method according to any one of aspects 1 to 36, for the prevention of cancer in an individual.

[0458] Aspect 45. 37. The method according to any one of aspects 1 to 36, for the treatment of cancer in an individual.

[0459] Aspect 46. The method of any one of embodiments 1-30, wherein the individual is administered a CAR-T therapy product, a TCR-T therapy product, or a CAR-NK therapy product.

[0460] Aspect 47. 47. The method of embodiment 46, wherein the individual is administered a CAR-T therapy product, wherein the CAR-T cell therapy product comprises a population of modified autologous T cells comprising a CAR or allogeneic T cells comprising a CAR, wherein the CAR comprises an antigen-binding domain specific for a cancer-associated antigen.

[0461] Aspect 48. 48. The method of embodiment 47, wherein the antigen-binding domain is a single chain Fv polypeptide or a nanobody.

[0462] Aspect 49. 49. The method of embodiment 47 or 48, wherein the cancer-associated antigen is selected from AFP, BCMA, CD10, CD117, CD123, CD133, CD128, CD171, CD19, CD20, CD22, CD30, CD33, CD34, CD38, CD5, CD56, CD7, CD70, CD80, CD86, CEA, CLD18, CLL-1, cMet, EGFR, EGFRvIII, EpCAM, EphA2, GD-2, glypican-3, GPC3, HER-2, kappa immunoglobulin, LeY, LMP1, mesothelin, MG7, MUC1, NKG2D ligand, PD-L1, PSCA, PSMA, ROR1, ROR1R, TACI, and VEGFR2.

[0463] Aspect 50. 50. The method of any one of aspects 44-49, further comprising administering to the individual an immune checkpoint inhibitor.

[0464] Aspect 51. 51. The method of embodiment 50, wherein the immune checkpoint inhibitor is an antibody specific for an immune checkpoint selected from CD27, CD28, CD40, CD122, CD96, CD73, CD47, OX40, GITR, CSF1R, JAK, PI3K delta, PI3K gamma, TAM, arginase, CD137 (also known as 4-1BB), ICOS, A2AR, B7-H3, B7-H4, BTLA, CTLA-4, LAG3, TIM3, VISTA, CD96, TIGIT, CD122, PD-1, PD-L1, and PD-L2; and optionally, the CPI is an antibody specific for an immune checkpoint selected from CTLA-4, TIGIT, PD-1 and PD-L1, or PD-1 or PD-L1.

[0465] Aspect 52. 32. The method of any one of aspects 1-31, wherein the first composition is a vaccine comprising one or more products, wherein the one or more products are capable of engaging with a TCR of a T cell or being processed by the immune system into one or more antigens that can be presented by a major histocompatibility complex (MHC) to the TCR of a T cell, and optionally the one or more antigens are cancer-associated antigens.

[0466] Aspect 53. 32. The method of any one of aspects 1 to 31, wherein the first composition is a vaccine comprising a nucleic acid, the nucleic acid comprising a nucleotide sequence encoding a polypeptide capable of being processed by the immune system into one or more antigens that can be presented by a major histocompatibility complex (MHC) to a TCR of a T cell, and optionally, the one or more antigens are cancer-associated antigens.

[0467] Aspect 54. 53. The method of embodiment 52, wherein the vaccine comprises one or more products that can be processed by the immune system into two or more cancer-associated antigens that can associate with the TCR of a T cell or be presented by a major histocompatibility complex (MHC) to the TCR of a T cell.

[0468] Aspect 55. 54. The method of embodiment 53, wherein the vaccine comprises one or more nucleic acids comprising one or more nucleotide sequences encoding a plurality of polypeptides, wherein the polypeptides are capable of being processed by the immune system into two or more cancer-associated antigens that can associate with a TCR of a T cell or be presented by a major histocompatibility complex (MHC) to the TCR of a T cell.

[0469] Aspect 56. The immunomodulatory protein is selected from the group consisting of: (i) an immunomodulatory protein comprising, consisting essentially of, or consisting of a homodimer of the 2657 protein shown in Figure 23A, wherein two copies of the 2657 protein are linked by two disulfide bonds linking the Ig Fc polypeptide in each copy of 2657; (ii) an immunomodulatory protein comprising, consisting essentially of, or consisting of a homodimer of the 2656 protein shown in Figure 25A, wherein two copies of the 2656 protein are linked by two disulfide bonds linking the Ig Fc polypeptide in each copy of 2656; and (iii) an immunomodulatory protein comprising, consisting essentially of, or consisting of a homodimer of the 2657Δ protein shown in Figure 23B, wherein two copies of the 2657Δ protein are linked by two disulfide bonds linking the Ig Fc polypeptide in each copy of 2657Δ. 2656Δprotein as shown in FIG. 25A, wherein the 2656Δ protein is linked by two disulfide bonds linking an Ig Fc polypeptide; (iv) an immunomodulatory protein comprising, consisting essentially of, or consisting of a homodimer of the 2656Δ protein as shown in FIG. 25B, wherein two copies of the 2656Δ protein are linked by two disulfide bonds linking an Ig Fc polypeptide in each copy of 2656Δ; and (v) a heterodimer comprising the 4123 protein as shown in FIG. 26A and the 4124 protein as shown in FIG. 26B.1. A fusion polypeptide comprising one or more variant IL-2 polypeptides having at least two amino acid substitutions relative to that set forth in SEQ ID NO: 1, as set forth in any one of Figures 2A-2C, 3A-3C, 4A-4C, 5A-5B, 6A-6B, 7A-7B, 8A-8F, 10A-10C, 11A-11C, 12A-12B, 13A-14B, 14A-14N, and 30A-30G, wherein the one or more variant IL-2 polypeptides bind to the IL-2R alpha chain (IL-2Rα); A fusion polypeptide having a binding affinity to IL-2Rα that is lower than the affinity of a wild-type IL-2 polypeptide for IL-2Rα when assayed under the same conditions, wherein IL-2Rα has the amino acid sequence set forth in SEQ ID NO:2, and one or more variant IL-2 polypeptides bind to the IL-2R beta chain (IL-2Rβ) and have a binding affinity to IL-2Rβ that is lower than the affinity of a wild-type IL-2 polypeptide for IL-2Rβ when assayed under the same conditions, wherein IL-2Rβ has the amino acid sequence set forth in SEQ ID NO:3.

[0470] Aspect 57. 1. A fusion polypeptide comprising one or more variant IL-2 polypeptides as shown in any one of Figures 2A-2C, 3A-3C, 4A-4C, 5A-5B, 6A-6B, 7A-7B, 8A-8F, 10A-10C, 11A-11C, 12A-12B, 13A-14B, 14A-14N, and 30A-30G, and having at least two amino acid substitutions relative to that shown in SEQ ID NO: 1, one or more variant IL-2 polypeptides bind to the IL-2R alpha chain (IL-2Rα) and have a binding affinity for IL-2Rα that is lower than the affinity of a wild-type IL-2 polypeptide for IL-2Rα when assayed under the same conditions, and the IL-2Rα has the amino acid sequence set forth in SEQ ID NO:2; one or more variant IL-2 polypeptides bind to the IL-2R beta chain (IL-2Rβ) and have a binding affinity for IL-2Rβ that is lower than the affinity of a wild-type IL-2 polypeptide for IL-2Rβ when assayed under the same conditions, and the IL-2Rβ has the amino acid sequence set forth in SEQ ID NO:3; Fusion polypeptides.

[0471] Aspect 58. 58. The fusion polypeptide of embodiment 57, wherein at least one of the one or more variant IL-2 polypeptides comprises one or more mutations capable of reducing binding of IL-2 to IL-2Rα, and the one or more mutations are selected from substitutions at one or more of the amino acids R38, F42, K43, Y45, E62, P65, E68, V69, and L72.

[0472] Aspect 59. 59. The fusion polypeptide of embodiment 58, wherein at least one of the one or more variant IL-2 polypeptides comprises a substitution of amino acid F42, optionally with Phe substituted with Ala or Lys.

[0473] Aspect 60. 60. The fusion polypeptide of any one of aspects 57 to 59, wherein at least one of the one or more variant IL-2 polypeptides comprises one or more mutations capable of reducing binding of IL-2 to IL-2Rβ, wherein the one or more mutations are selected from substitutions at one or more of the following amino acids: E15, H16, L19, D20, D84, S87, N88, V91, I92.

[0474] Aspect 61. 61. The fusion polypeptide of embodiment 60, wherein at least one of the one or more variant IL-2 polypeptides comprises a substitution of amino acid H16, optionally wherein His is substituted with Ala, Glu, Thr, or Asp.

[0475] Aspect 62. 58. The fusion polypeptide of embodiment 57, wherein at least one of the one or more variant IL-2 polypeptides comprises a substitution selected from E15A with R38A, R38D, or R38E.

[0476] Aspect 63. 58. The fusion polypeptide of embodiment 57, wherein at least one of the one or more variant IL-2 polypeptides comprises a substitution selected from: H16A with R38A, R38D, or R38E; H16T with R38A, R38D, or R38E; H16E with R38A, R38D, or R38E; and H16D with R38A, R38D, or R38E.

[0477] Aspect 64. 58. The fusion polypeptide of embodiment 57, wherein at least one of the one or more variant IL-2 polypeptides comprises a substitution selected from: D84H with R38A, R38D, or R38E; D84K with R38A, R38D, or R38E; and D84R with R38A, R38D, or R38E.

[0478] Aspect 65. 58. The fusion polypeptide of embodiment 57, wherein at least one of the one or more variant IL-2 polypeptides comprises a substitution selected from: R38A with N88S, N88A, N88G, N88R, N88T, or N88D; R38D with N88S, N88A, N88G, N88R, N88T, or N88D; and R38E with N88S, N88A, N88G, N88R, N88T, or N88D.

[0479] Aspect 66. 58. The fusion polypeptide of embodiment 57, wherein at least one of the one or more variant IL-2 polypeptides comprises a substitution selected from: R38A with V91E, V91A, or V91T; R38D with V91E, V91A, or V91T; and R38E with V91E, V91A, or V91T.

[0480] Aspect 67. 58. The fusion polypeptide of embodiment 57, wherein at least one of the one or more variant IL-2 polypeptides comprises a substitution selected from R38A, I92A, R38D, I92A and R38E, I92A.

[0481] Aspect 68. 58. The fusion polypeptide of embodiment 57, wherein at least one of the one or more variant IL-2 polypeptides comprises a substitution selected from E15A, F42A and E15A, F42K.

[0482] Aspect 69. 58. The fusion polypeptide of embodiment 57, wherein at least one of the one or more variant IL-2 polypeptides comprises a substitution selected from H16A, F42A; H16T, F42A; H16E, F42A; H16D, F42A; H16A, F42K; H16T, F42K; and H16E, F42K; H16D, F42K.

[0483] Aspect 70. 58. The fusion polypeptide of embodiment 57, wherein at least one of the one or more variant IL-2 polypeptides comprises a substitution selected from: F42A with N88S, N88A, N88G, N88R, N88T, or N88D; and F42K with N88S, N88A, N88G, N88R, N88T, or N88D.

[0484] Aspect 71. 58. The fusion polypeptide of embodiment 57, wherein at least one of the one or more variant IL-2 polypeptides comprises a substitution selected from: F42A with V91E, V91A, or V91T; and F42K with V91E, V91A, or V91T.

[0485] Aspect 72. 58. The fusion polypeptide of embodiment 57, wherein at least one of the one or more variant IL-2 polypeptides comprises a substitution selected from: F42A with I92A; and F42K with I92A.

[0486] Aspect 73. 58. The fusion polypeptide of embodiment 57, wherein at least one of the one or more variant IL-2 polypeptides comprises an E15A and a K43E substitution.

[0487] Aspect 74. 58. The fusion polypeptide of embodiment 57, wherein at least one of the one or more variant IL-2 polypeptides comprises a substitution selected from H16A,K43E; H16T,K43E; H16E,K43E; and H16D,K43E.

[0488] Aspect 75. 58. The fusion polypeptide of embodiment 57, wherein at least one of the one or more variant IL-2 polypeptides comprises a substitution selected from K43E with D84H, D84K, or D84R.

[0489] Aspect 76. 58. The fusion polypeptide of embodiment 57, wherein at least one of the one or more variant IL-2 polypeptides comprises a substitution selected from K43E with N88S, N88A, N88G, N88R, N88T, or N88D.

[0490] Aspect 77. 58. The fusion polypeptide of embodiment 57, wherein at least one of the one or more variant IL-2 polypeptides comprises a substitution selected from K43E with V91E, V91A, or V91T.

[0491] Aspect 78. 58. The fusion polypeptide of embodiment 57, wherein at least one of the one or more variant IL-2 polypeptides comprises the following substitutions: K43E and I92A, or E15A, E62Q.

[0492] Aspect 79. 58. The fusion polypeptide of embodiment 57, wherein at least one of the one or more variant IL-2 polypeptides comprises a substitution selected from H16A, E62Q; H16T, E62Q; H16E, E62Q; and H16D, E62Q.

[0493] Aspect 80. 58. The fusion polypeptide of embodiment 57, wherein at least one of the one or more variant IL-2 polypeptides comprises a substitution selected from E62Q with D84H, D84K, or D84R.

[0494] Aspect 81. 58. The fusion polypeptide of embodiment 57, wherein at least one of the one or more variant IL-2 polypeptides comprises a substitution selected from E62Q with N88S, N88A, N88G, N88R, N88T, or N88D.

[0495] Aspect 82. 58. The fusion polypeptide of embodiment 57, wherein at least one of the one or more variant IL-2 polypeptides comprises a substitution selected from E62Q with V91E, V91A, or V91T.

[0496] Aspect 83. 58. The fusion polypeptide of embodiment 57, wherein at least one of the one or more variant IL-2 polypeptides comprises a substitution selected from E62Q and I92A.

[0497] Aspect 84. 58. The fusion polypeptide of embodiment 57, wherein at least one of the one or more variant IL-2 polypeptides comprises a substitution selected from E62Q with V91E, V91A, or V91T.

[0498] Aspect 85. 69. The fusion polypeptide of embodiment 68, wherein at least one of the one or more variant IL-2 polypeptides comprises substitutions at F42, E15 and N88.

[0499] Aspect 86. 69. The fusion polypeptide of embodiment 68, wherein at least one of the one or more variant IL-2 polypeptides comprises substitutions at F42, E15 and V91.

[0500] Aspect 87. 70. The fusion polypeptide of embodiment 69, wherein at least one of the one or more variant IL-2 polypeptides comprises substitutions at F42, H16 and D84.

[0501] Aspect 88. 70. The fusion polypeptide of embodiment 69, wherein at least one of the one or more variant IL-2 polypeptides comprises substitutions at F42, H16 and N88.

[0502] Aspect 89. 70. The fusion polypeptide of embodiment 69, wherein at least one of the one or more variant IL-2 polypeptides comprises substitutions at F42, H16 and V91.

[0503] Aspect 90. 70. The fusion polypeptide of embodiment 69, wherein at least one of the one or more variant IL-2 polypeptides comprises substitutions at F42, H16 and I92.

[0504] Aspect 91. 10A-10C, 11A-11C, 12A-12B, 13A-13B, 14A-14N, and 30A-30G, and wherein the fusion polypeptide comprises a functional protein that is a cancer targeting polypeptide (CTP).

[0505] Aspect 92. 92. The fusion polypeptide of embodiment 91, wherein the target of the cancer targeting polypeptide is a peptide-HLA complex on the surface of a cancer cell.

[0506] Aspect 93. 92. The fusion polypeptide of embodiment 91, wherein the target of the CTP is a cancer-associated epitope.

[0507] Aspect 94. 92. The fusion polypeptide of embodiment 91, wherein the CTP is an antibody specific for a cancer-associated antigen.

[0508] Aspect 95. 92. The fusion polypeptide of embodiment 91, wherein the CTP is an antibody specific for a peptide / HLA complex on the surface of a cancer cell, and the peptide can be a cancer-associated peptide (e.g., a peptide of a cancer-associated antigen).

[0509] Aspect 96. 92. The fusion polypeptide of embodiment 91, wherein the functional protein is a TCR, such as an "scTCR," a single-chain T-cell receptor specific for a peptide / HLA complex on the surface of a cancer cell, and the peptide can be a cancer-associated peptide (e.g., a peptide of a cancer-associated antigen).

[0510] Aspect 97. 92. The fusion polypeptide of embodiment 91, wherein the functional protein comprises a wild-type or variant immunomodulatory polypeptide, such as a wild-type or variant immunostimulatory polypeptide, such as a member of the B7 family of costimulatory receptors, e.g., CD80, CD86, a cytokine, such as IL-7, IL-12, IL-15 or IL-21, a TNF superfamily member, such as CD-40, 4-1BBL and OX40, or a chemokine, such as CCL19, CCL21, CXCL9 / 10 / 11, or CXCL12.

[0511] Aspect 98. 98. The fusion polypeptide of any one of embodiments 57 to 97, comprising one or more independently selected linkers.

[0512] Aspect 99. To the individual: (i) a first composition, (a) a modified or unmodified T cell having a T cell receptor (TCR), or (b) a modified cell comprising a chimeric antigen receptor (CAR), wherein the CAR binds to a target antigen and the modified cell comprises an intracellular signaling domain that is activated by interaction of the modified cell with IL-2; or (c) a modified cell containing one or more exogenously activated receptors; or (d) one or more products that can be processed by the immune system into one or more antigens that can associate with the TCR of a T cell or be presented by a major histocompatibility complex (MHC) to the TCR of a T cell, optionally wherein the one or more antigens are cancer-associated antigens; or (e) one or more nucleic acids encoding one or more polypeptides that can be processed by the immune system into one or more antigens that can associate with the TCR of a T cell or be presented by a major histocompatibility complex (MHC) to the TCR of a T cell, optionally wherein the one or more antigens are cancer-associated antigens; or (f) at least one immune checkpoint inhibitor (CPI) a first composition comprising: (ii) a second composition comprising the fusion polypeptide according to any one of aspects 57 to 98. 1. A method comprising administering when the individual is administered a first composition comprising (a), (b), (c), (d), or (e), the individual may also be administered a CPI; The first composition and the second composition are administered at the same time or at different times; method.

[0513] Aspect 100. 100. The method of embodiment 99, wherein the second composition preferentially activates T cells whose TCR is engaged with an MHC-presented antigen compared to T cells whose TCR is not engaged with an MHC-presented antigen.

[0514] Aspect 101. 101. The method of embodiment 99 or 100, wherein the first composition comprises modified or unmodified T cells having a T cell receptor (TCR).

[0515] Aspect 102. 101. The method of embodiment 99 or 100, wherein the first composition comprises a modified cell comprising a chimeric antigen receptor (CAR), wherein the CAR binds to a target antigen, and wherein the modified cell comprises an intracellular signaling domain that is activated by interaction of the modified cell with IL-2.

[0516] Aspect 103. 101. The method of embodiment 99 or 100, wherein the first composition comprises modified cells comprising one or more exogenously activated receptors.

[0517] Aspect 104. 101. The method of embodiment 99 or 100, wherein the first composition comprises one or more products, and the one or more products are capable of being processed by the immune system into one or more antigens that can associate with a TCR of a T cell or be presented by a major histocompatibility complex (MHC) to the TCR of a T cell, and optionally the one or more antigens are cancer-associated antigens.

[0518] Aspect 105. 101. The method of embodiment 99 or 100, wherein the first composition comprises one or more nucleic acids encoding one or more polypeptides, wherein the one or more polypeptides are capable of associating with a TCR of a T cell or being processed by the immune system into one or more antigens that can be presented by a major histocompatibility complex (MHC) to the TCR of a T cell, and optionally the one or more antigens are cancer-associated antigens.

[0519] Aspect 106. 101. The method of embodiment 99 or 100, wherein the first composition comprises a CPI.

[0520] Aspect 107. The method of any of embodiments 101-105, further comprising administering a CPI.

[0521] Aspect 108. 108. The method of any one of aspects 99 to 107, wherein the CPI is an antibody specific for an immune checkpoint selected from CD27, CD28, CD40, CD122, CD96, CD73, CD47, OX40, GITR, CSF1R, JAK, PI3K delta, PI3K gamma, TAM, arginase, CD137 (also known as 4-1BB), ICOS, A2AR, B7-H3, B7-H4, BTLA, CTLA-4, LAG3, TIM3, VISTA, CD96, TIGIT, CD122, PD-1, PD-L1, and PD-L2.

[0522] Aspect 109. 108. The method of any one of aspects 99 to 107, wherein the CPI is an antibody specific for an immune checkpoint selected from CTLA-4, TIGIT, PD-1, and PD-L1.

[0523] Aspect 110. 108. The method of any one of aspects 99 to 107, wherein the CPI is an antibody specific for an immune checkpoint selected from PD-1 and PD-L1. [Example]

[0524] The following examples are presented so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of the invention as envisioned by the inventors, nor are they intended to represent that the following experiments are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should be accounted for.

[0525] Example 1 - Determination of binding affinity to IL-2Rα and IL-2Rβ Kinetic measurements were performed using biolayer interferometry on a ForteBio Octet Red 96e plate using an anti-human IgG Fc capture (AHC) against human IL-2R. All experiments were performed at 30°C with an orbital shaking speed of 1000 rpm. Samples were diluted in assay buffer (25 mM HEPES, pH 6.8, 50 mM KCl, 5% polyethylene glycol (PEG) 6000, 0.1% bovine serum albumin (BSA), 0.02% Tween 20) in a 96-well non-binding black plate (Greiner 655900).

[0526] All proteins were diluted in assay buffer containing 25 mM HEPES, pH 6.8, 50 mM KCl, 5% PEG 6000, 0.1% BSA, and 0.02% Tween 20. Receptor ligands were prepared to a final concentration of 20 nM for IL-2Rα-Fc and 25 nM for IL-2Rβ-Fc and dispensed into columns of a 96-well non-binding plate in a volume of 200 μl / well. Analyte solutions were serially diluted two-fold to seven concentrations, starting from 1000 nM for CUE-1646 (WT-IL-2-FLAG-) or 3000 nM for CUE-1647 (H16A, F42A mutant IL-2-FLAG) against human IL-2R. Analytes were added to the 96-well plate in a volume of 200 μl / well. Kinetic measurements were performed over an appropriate concentration range depending on the robustness of the signal and fit.

[0527] AHC or HIS1K biosensors were prehydrated for 10 min and normalized for 60 s (s) in assay buffer. For each run, a column of eight biosensors was simultaneously immersed in IL-2R ligand solution for 300 s. Ligand immobilization on the biosensors ranged from 1.6–1.4 nM for human IL-2Rα and 2.0–1.7 nM for human IL-2Rβ. A 120 s baseline step in assay buffer alone washed away any unbound ligand and allowed the signal to stabilize before analyte association. The interaction kinetics were measured by immersing eight loaded biosensors in analyte solutions of varying concentrations of either CUE-1646 (amino acid sequence shown in Figure 21) or CUE-1647 (amino acid sequence shown in Figure 22) for 300 s. A reference well with buffer alone was included in each run to compensate for any baseline drift of the immobilized IL-2R. This was followed by a 300-second dissociation period into wells containing assay buffer only, and dissociation of the analyte from the immobilized receptor ligand was monitored. Blank biosensors were also subjected to analyte dilution to check for nonspecific binding on the sensor. A new biosensor was used for each binding experiment. Binding sensorgrams were collected using standard kinetic acquisition rates.

[0528] Raw data were processed and analyzed using ForteBio's Data Analysis 11.0 software. For each run, various analyte concentrations were subtracted from the corresponding reference wells to correct for any background drift. The sensorgram Y-axis was aligned to the baseline for a common reference point, and an inter-step correction was aligned to the dissociation to correct for any misalignment between the association and dissociation steps. Sensorgrams were processed using Savitzky-Golay filtering to remove high-frequency noise from the curves. The processed curves were globally fit to a 1:1 bin...

Claims

1. To the individual: (i) a first composition, (a) a modified or unmodified T cell having a T cell receptor (TCR); or (b) a modified cell comprising a chimeric antigen receptor (CAR), wherein the CAR binds to a target antigen, and wherein the modified cell comprises an intracellular signaling domain that is activated by interaction of the modified cell with IL-2; or (c) a modified cell containing one or more exogenously activated receptors; or (d) a product that can be processed by the immune system into one or more antigens that can associate with the TCR of a T cell or be presented by the major histocompatibility complex (MHC) to the TCR of a T cell, optionally wherein the one or more antigens are cancer-associated antigens; or (e) one or more nucleic acids encoding one or more polypeptides capable of associating with the TCR of a T cell or being processed by the immune system into one or more antigens that can be presented by a major histocompatibility complex (MHC) to the TCR of a T cell, optionally wherein the one or more antigens are cancer-associated antigens; or (f) at least one immune checkpoint inhibitor (CPI); the first composition comprising: (ii) a second composition comprising an immunomodulatory protein; 1. A method comprising administering the immunomodulating protein comprises one or more variant IL-2 polypeptides having at least two amino acid substitutions relative to that set forth in SEQ ID NO:1; the one or more variant IL-2 polypeptides bind to the IL-2R alpha chain (IL-2Rα) and have a binding affinity for IL-2Rα that is lower than the affinity of a wild-type IL-2 polypeptide for IL-2Rα when assayed under the same conditions, wherein the IL-2Rα has the amino acid sequence set forth in SEQ ID NO:2; and the one or more variant IL-2 polypeptides bind to the IL-2R beta chain (IL-2Rβ) and have a binding affinity for IL-2Rβ that is lower than the affinity of a wild-type IL-2 polypeptide for IL-2Rβ when assayed under the same conditions, and the IL-2Rβ has the amino acid sequence set forth in SEQ ID NO:3; the second composition preferentially activates T cells in which the TCR is engaged with an antigen presented by an MHC compared to T cells in which the TCR is not engaged with an antigen presented by an MHC; the first composition and the second composition are administered at the same time or at different times; When the individual is administered a first composition comprising (a), (b), (c), (d), or (e), the individual may also be administered a CPI; The method.

2. 2. The method of claim 1, wherein at least one of the one or more variant IL-2 polypeptides exhibits at least a two-fold decrease in binding affinity to IL-2Rβ compared to the binding affinity of the wild-type IL-2 polypeptide for IL-2Rβ and at least a fifty-fold decrease in binding affinity to IL-2Rα compared to the binding affinity of the wild-type IL-2 polypeptide for IL-2Rα.

3. 3. The method of claim 1 or 2, wherein at least one of the one or more variant IL-2 polypeptides comprises at least one substitution that reduces the affinity of the variant IL-2 polypeptide for IL-2Rα, and optionally the at least one substitution is selected from a substitution at R38, F42, K43, Y45, E62, P65, E68, V69, L72, and a combination thereof.

4. 4. The method of any one of claims 1-3, wherein at least one of the one or more variant IL-2 polypeptides comprises a substitution of the phenylalanine amino acid F42, optionally wherein the phenylalanine is substituted with Ala, Gly, Val, Ile, or Leu.

5. 5. The method of any one of claims 1-4, wherein at least one of the one or more variant IL-2 polypeptides comprises at least one substitution that reduces the affinity of the variant IL-2 polypeptide for IL-2Rβ, and optionally the at least one substitution is selected from a substitution at E15, H16, L19, D20, D84, S87, N88, V91, I92, and a combination thereof.

6. 6. The method of any one of claims 1-5, wherein at least one of the one or more variant IL-2 polypeptides comprises a substitution of histidine amino acid H16, optionally wherein the histidine is substituted with Ala, Gly, Val, Leu, Thr, Ile, Asp, or Glu.

7. 7. The method of any one of claims 1-6, wherein at least one of the one or more variant IL-2 polypeptides comprises a substitution of asparagine amino acid N88, optionally wherein the asparagine is substituted with Gly, Ala, Ser, Thr, Arg, or Asp.

8. 8. The method of any one of claims 1-7, wherein at least one of the one or more variant IL-2 polypeptides comprises a substitution of amino acids F42 and H16, and optionally, the phenylalanine is substituted with Ala and the histidine is substituted with Ala, Thr, Asp, or Glu.

9. 9. The method of any one of claims 1-8, wherein at least one of the one or more variant IL-2 polypeptides comprises (i) an H16A substitution and an F42A substitution, (ii) an H16T substitution and an F42A substitution, (iii) an H16E substitution and an F42A substitution, and (iv) an H16D substitution and an F42A substitution.

10. 10. The method of any one of claims 1 to 9, wherein at least one of the one or more variant IL-2 polypeptides comprises substitutions at F42, H16, and N88.

11. 11. The method of claim 10, wherein the asparagine amino acid N88 is substituted with Gly, Ala, Ser, Thr, Arg, or Asp.

12. The method of any one of claims 1 to 11, wherein the immunomodulatory protein comprises two or more variant IL-2 polypeptides, each variant IL-2 polypeptide comprising the same amino acid sequence.

13. 13. The method of claim 12, wherein the immunomodulatory protein comprises two variant IL-2 polypeptides, the two variant IL-2 polypeptides being in tandem and linked by independently selected linkers, optionally wherein the linkers comprise glycine and serine.

14. The method of any one of claims 1 to 13, wherein the immunomodulatory protein further comprises a carrier.

15. 15. The method of claim 14, wherein the carrier is a lipid vesicle (e.g., a liposome) or micelle, a nanoparticle, a PEGylated protein, or an artificial antigen-presenting cell such as an engineered red blood cell or an enucleated cell (e.g., a platelet).

16. The method of any one of claims 1 to 13, wherein the immunomodulatory protein further comprises an immunoglobulin (Ig) scaffold polypeptide or a non-Ig scaffold polypeptide.

17. 17. The method of claim 16, wherein the immunomodulatory protein comprises a non-Ig scaffold selected from an XTEN polypeptide, a transferrin polypeptide, an elastin-like polypeptide, a silk-like polypeptide, a fibronectin-based scaffold protein, or a silk-elastin-like polypeptide.

18. 17. The method of claim 16, wherein the immunomodulatory protein is a fusion polypeptide comprising (a) the one or more variant IL-2 polypeptides and (b) an Fc polypeptide, 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.

19. 19. The method of claim 18, wherein the Ig Fc polypeptide is a variant having a substantially reduced effector function, e.g., a substantially reduced ability to confer complement-dependent cytotoxicity (CDC) and / or antibody-dependent cellular cytotoxicity (ADCC).

20. 20. The method of claim 18 or 19, wherein the Ig Fc polypeptide comprises one or more amino acid substitutions selected from N297A, L234A, L235A, L234F, L235E, G237A and P331S, wherein N297, L234, L235, G237 and P331 correspond to N77, L14, L15, G17 and P111, respectively, of the amino acid sequence shown in Figure 9A.

21. 21. The method of any one of claims 18-20, wherein the Ig Fc polypeptide is an IgG1 Fc polypeptide comprising an amino acid sequence having at least 95% amino acid sequence identity to the amino acid sequence shown in any one of Figures 9A-9M.

22. 22. The method of any one of claims 18 to 21, wherein the immunomodulatory protein comprises a homodimer of two immunomodulatory proteins, each of which comprises an Ig Fc polypeptide, and wherein the Ig Fc of one immunomodulatory protein is linked to the Ig Fc of the other immunomodulatory protein by one or more disulfide bonds.

23. 23. The method of claim 22, wherein each of the immunomodulatory proteins of the homodimer comprises two variant IL-2 polypeptides in tandem, the variant IL-2 polypeptides being linked by independently selected linkers.

24. Each of the immunomodulating proteins of the homodimer comprises, in N-terminal to C-terminal direction: (i) a variant IL-2 polypeptide; (ii) an independently selected linker; (iii) a variant IL-2 polypeptide; (iv) an independently selected linker, and (v) Ig Fc polypeptide 23. The method of claim 22, comprising:

25. Each of the immunomodulating proteins of the homodimer comprises, in N-terminal to C-terminal direction: (i) a variant IL-2 polypeptide; (ii) an independently selected linker; (iii) a variant IL-2 polypeptide; (iv) an independently selected linker; (v) an Ig Fc polypeptide; (vi) an independently selected linker; (vii) a variant IL-2 polypeptide; (viii) an independently selected linker, and (ix) variant IL-2 polypeptide 23. The method of claim 22, comprising:

26. Each of the immunomodulating proteins of the homodimer comprises, in N-terminal to C-terminal direction: (i) a variant IL-2 polypeptide; (ii) an independently selected linker; (iii) an Ig Fc polypeptide; (iv) an independently selected linker, and (v) variant IL-2 polypeptides 23. The method of claim 22, comprising:

27. Each of the immunomodulating proteins of the homodimer comprises, in N-terminal to C-terminal direction: (i) an Ig Fc polypeptide; (ii) an independently selected linker; (iii) a variant IL-2 polypeptide; (iv) an independently selected linker, and (v) variant IL-2 polypeptides 23. The method of claim 22, comprising:

28. the immunomodulating protein comprises a heterodimer of two immunomodulating proteins, one of which comprises an Ig Fc polypeptide comprising an interspecies dimerization sequence and the other immunomodulating protein comprises an Ig Fc polypeptide comprising a counterpart interspecies sequence; or the immunomodulating protein comprises a heterodimer comprising a first polypeptide and a second polypeptide, the first polypeptide comprising an Ig Fc polypeptide comprising an interspecies dimerization sequence; the second polypeptide comprises an Ig Fc polypeptide comprising a counterpart interspecies sequence; and wherein either the first polypeptide or the second polypeptide comprises the one or more variant IL-2 polypeptides; The method according to any one of claims 18 to 21.

29. (A) one of the immunomodulatory proteins of the heterodimer is, in the N-terminal to C-terminal direction, (i) a variant IL-2 polypeptide; (ii) an independently selected linker; (iii) a variant IL-2 polypeptide; (iv) an independently selected linker, and (v) an Ig Fc polypeptide comprising an interspecies binding sequence; Including, The other immunomodulatory protein of the heterodimer is, in the N-terminal to C-terminal direction, (i) an Ig Fc polypeptide comprising a counterpart interspecies binding sequence; (ii) an independently selected linker; (iii) a variant IL-2 polypeptide; (iv) an independently selected linker, and (v) variant IL-2 polypeptides Contains, or (B) the first polypeptide in the heterodimer is arranged, in an N-terminal to C-terminal direction, as follows: (i) a variant IL-2 polypeptide; (ii) an independently selected linker; (iii) a variant IL-2 polypeptide; (iv) an independently selected linker, and (v) an Ig Fc polypeptide comprising an interspecies binding sequence; Including, the second polypeptide in the heterodimer comprises an Ig Fc polypeptide comprising a counterpart interspecies binding sequence but does not comprise a variant IL-2 polypeptide; or (C) the first polypeptide in the heterodimer is, in an N-terminal to C-terminal direction, (i) an Ig Fc polypeptide comprising an interspecies binding sequence; (ii) an independently selected linker; (iii) a variant IL-2 polypeptide; (iv) an independently selected linker; (v) variant IL-2 polypeptides Including, the second polypeptide in the heterodimer comprises an Ig Fc polypeptide comprising a counterpart interspecies binding sequence, but does not comprise a variant IL-2 polypeptide; 29. The method of claim 28.

30. One of the immunomodulatory proteins of the heterodimer comprises, in the N-terminal to C-terminal direction: (i) a variant IL-2 polypeptide; (ii) an independently selected linker, and (iii) an Ig Fc polypeptide comprising an interspecies binding sequence; Including, The other immunomodulatory protein of the heterodimer has, in the N-terminal to C-terminal direction: (i) an Ig Fc polypeptide comprising a counterpart interspecies binding sequence; (ii) an independently selected linker, and (iii) a variant IL-2 polypeptide Including, 29. The method of claim 28.

31. (A) the first polypeptide in the heterodimer is arranged, in an N-terminal to C-terminal direction, as follows: (i) a variant IL-2 polypeptide; (ii) an independently selected linker, and (iii) an Ig Fc polypeptide comprising an interspecies binding sequence; Including, the second polypeptide in the heterodimer comprises an Ig Fc polypeptide comprising a counterpart interspecies binding sequence but does not comprise a variant IL-2 polypeptide; or (B) the first polypeptide in the heterodimer is arranged, in an N-terminal to C-terminal direction, as follows: (i) an Ig Fc polypeptide comprising an interspecies binding sequence; (ii) an independently selected linker, and (iii) a variant IL-2 polypeptide Including, the second polypeptide in the heterodimer comprises an Ig Fc polypeptide comprising a counterpart interspecies binding sequence, but does not comprise a variant IL-2 polypeptide; 29. The method of claim 28.

32. 31. The method of any one of claims 1 to 30, wherein the first composition comprises one or more products that can be processed by the immune system into one or more antigens that can associate with the TCR of a T cell or be presented by a major histocompatibility complex (MHC) to the TCR of a T cell, and optionally the one or more antigens are cancer-associated antigens.

33. 31. The method of any one of claims 1 to 30, wherein the first composition comprises one or more nucleic acids encoding one or more polypeptides, wherein the one or more polypeptides are capable of associating with the TCR of a T cell or being processed by the immune system into one or more antigens that can be presented by a major histocompatibility complex (MHC) to the TCR of a T cell, and optionally the one or more antigens are cancer-associated antigens.

34. 31. The method of any one of claims 1 to 30, wherein the first composition comprises TCR-T cells comprising an exogenous TCR, and optionally, wherein the TCR binds to a cancer-associated antigen when the cancer-associated antigen is presented to the TCR by MHC.

35. 31. The method of any one of claims 1 to 30, wherein the first composition comprises tumor infiltrating lymphocytes (TILS), optionally modified to reduce the sensitivity of the TILS to T cell inhibitory signals.

36. The cancer-associated antigen is selected from the group consisting of alpha-fetoprotein, Wilms' tumor 1 (WT-1), mutant KRAS, e.g., mutant KRAS containing a G12C or G12D mutation, melanoma antigen recognized by T cells 1 (MART-1), melanoma-associated antigen (MAGE), MAGE-A1, MAGE-A3, MAGE-A4, human papillomavirus (HPV) antigen E6, HPV antigen E7, New York esophageal squamous cell carcinoma 1 (NY-ESO-1), MUC-1 (mucin-1), cell surface associated antigen (CEA-1), and the like.

36. The method of any one of claims 31 to 35, wherein the antigen is selected from the group consisting of prostate cancer cells, mesothelin, survivin, prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), prostate-specific antigen (PSA), mutant p53 polypeptide, Ras polypeptide, nuclear factor erythroid 2-related factor 2 (NFE2L2), beta-catenin, PIK3CA (phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha), and BRAF.

37. the first composition comprises a CAR having an antigen-binding domain specific for a cancer-associated antigen, and optionally the cell is a T cell, a macrophage, or an NK cell, and optionally (i) when the CAR is not bound to a cancer-associated antigen, the second composition provides a homeostatic signal to the cell for survival, and / or (ii) when the CAR is not bound to a cancer-associated antigen, the second composition provides an activation signal to the cell that causes the cell to proliferate and retains its cytotoxic function; The method according to any one of claims 1 to 30.

38. 38. The method of claim 37, wherein the antigen-binding domain is a single-chain Fv polypeptide or a nanobody.

39. 39. The method of claim 37 or 38, wherein the cancer-associated antigen is selected from AFP, BCMA, CD10, CD117, CD123, CD133, CD128, CD171, CD19, CD20, CD22, CD30, CD33, CD34, CD38, CD5, CD56, CD7, CD70, CD80, CD86, CEA, CLD18, CLL-1, cMet, EGFR, EGFRvIII, EpCAM, EphA2, GD-2, glypican-3, GPC3, HER-2, kappa immunoglobulin, LeY, LMP1, mesothelin, MG7, MUC1, NKG2D ligand, PD-L1, PSCA, PSMA, ROR1, ROR1R, TACI, and VEGFR2.

40. 40. The method of any one of claims 1 to 39, for the treatment of cancer in said individual.

41. 41. The method of claim 40, wherein the first composition comprises (a), (b), (c), (d), or (e), and the method further comprises administering to the individual at least one immune checkpoint inhibitor (CPI), wherein the CPI, first composition, and second composition are administered simultaneously or at different times.

42. 42. The method of claim 41, wherein the at least one immune checkpoint inhibitor comprises an antibody specific for the immune checkpoint.

43. 43. The method of claim 42, wherein the antibody is specific for an immune checkpoint selected from CD27, CD28, CD40, CD122, CD96, CD73, CD47, OX40, GITR, CSF1R, JAK, PI3K delta, PI3K gamma, TAM, arginase, CD137 (also known as 4-1BB), ICOS, A2AR, B7-H3, B7-H4, BTLA, CTLA-4, LAG3, TIM3, VISTA, CD96, TIGIT, CD122, PD-1, PD-L1, and PD-L2, and optionally, the immune checkpoint inhibitor is an antibody specific for PD-1, PD-L1, CTLA-4, TIGIT, and LAG3.

44. The method of any one of claims 1 to 36, for the prevention of cancer in said individual.

45. The method of any one of claims 1 to 36, for the treatment of cancer in said individual.

46. 31. The method of any one of claims 1 to 30, wherein the individual is administered a CAR-T, TCR-T, or CAR-NK therapy product.

47. 47. The method of claim 46, wherein the individual is administered a CAR-T therapy product, wherein the CAR-T cell therapy product comprises a population of modified autologous T cells comprising a CAR or allogeneic T cells comprising a CAR, wherein the CAR comprises an antigen-binding domain specific for a cancer-associated antigen.

48. 48. The method of claim 47, wherein the antigen-binding domain is a single-chain Fv polypeptide or a nanobody.

49. 49. The method of claim 47 or 48, wherein the cancer-associated antigen is selected from AFP, BCMA, CD10, CD117, CD123, CD133, CD128, CD171, CD19, CD20, CD22, CD30, CD33, CD34, CD38, CD5, CD56, CD7, CD70, CD80, CD86, CEA, CLD18, CLL-1, cMet, EGFR, EGFRvIII, EpCAM, EphA2, GD-2, glypican-3, GPC3, HER-2, kappa immunoglobulin, LeY, LMP1, mesothelin, MG7, MUC1, NKG2D ligand, PD-L1, PSCA, PSMA, ROR1, ROR1R, TACI, and VEGFR2.

50. 50. The method of any one of claims 44 to 49, further comprising administering to the individual an immune checkpoint inhibitor.

51. 51. The method of claim 50, wherein the immune checkpoint inhibitor is an antibody specific for an immune checkpoint selected from CD27, CD28, CD40, CD122, CD96, CD73, CD47, OX40, GITR, CSF1R, JAK, PI3K delta, PI3K gamma, TAM, arginase, CD137 (also known as 4-1BB), ICOS, A2AR, B7-H3, B7-H4, BTLA, CTLA-4, LAG3, TIM3, VISTA, CD96, TIGIT, CD122, PD-1, PD-L1 and PD-L2, and optionally the CPI is an antibody specific for an immune checkpoint selected from CTLA-4, TIGIT, PD-1 and PD-L1, or PD-1 or PD-L1.

52. 31. The method of any one of claims 1 to 30, wherein the first composition is a vaccine comprising one or more products that can be processed by the immune system into one or more antigens that can associate with the TCR of a T cell or be presented by a major histocompatibility complex (MHC) to the TCR of a T cell, and optionally the one or more antigens are cancer-associated antigens.

53. 31. The method of any one of claims 1 to 30, wherein the first composition is a vaccine comprising a nucleic acid comprising a nucleotide sequence encoding a polypeptide capable of being processed by the immune system into one or more antigens that can be presented by the major histocompatibility complex (MHC) to the TCR of a T cell, and optionally the one or more antigens are cancer-associated antigens.

54. 53. The method of claim 52, wherein the vaccine comprises one or more products that can be processed by the immune system into two or more cancer-associated antigens that can associate with the TCR of a T cell or be presented by the major histocompatibility complex (MHC) to the TCR of a T cell.

55. 54. The method of claim 53, wherein the vaccine comprises one or more nucleic acids comprising one or more nucleotide sequences encoding multiple polypeptides, wherein the polypeptides can be processed by the immune system into two or more cancer-associated antigens that can associate with the TCR of a T cell or be presented by the major histocompatibility complex (MHC) to the TCR of a T cell.

56. The immunomodulatory protein is (i) an immunomodulatory protein comprising, consisting essentially of, or consisting of a homodimer of the 2657 protein shown in FIG. 23A, wherein two copies of the 2657 protein are linked by two disulfide bonds linking the Ig Fc polypeptide in each copy of 2657; (ii) an immunomodulatory protein comprising, consisting essentially of, or consisting of a homodimer of the 2656 protein shown in FIG. 25A, wherein two copies of the 2656 protein are linked by two disulfide bonds linking the Ig Fc polypeptide in each copy of 2656; or (iii) an immunomodulatory protein comprising, consisting essentially of, or consisting of a homodimer of the 2657Δ protein shown in FIG. 23B, wherein two copies of the 2657Δ protein are linked by two disulfide bonds linking the Ig Fc polypeptide in each copy of 2657Δ. 26A and 26B.

56. The method of any one of claims 1 to 55, wherein the immunomodulatory protein is selected from the group consisting of: (i) an immunomodulatory protein comprising, consisting essentially of, or consisting of a homodimer of the 2656Δ protein shown in Figure 25B, wherein two copies of the 2656Δ protein are linked by two disulfide bonds linking the Ig Fc polypeptides in each copy of 2656Δ; and (ii) a heterodimer comprising the 4123 protein shown in Figure 26A and the 4124 protein shown in Figure 26B.

57. 1. A fusion polypeptide comprising one or more variant IL-2 polypeptides as shown in any one of Figures 2A-2C, 3A-3C, 4A-4C, 5A-5B, 6A-6B, 7A-7B, 8A-8F, 10A-10C, 11A-11C, 12A-12B, 13A-14B, 14A-14N, and 30A-30G, and having at least two amino acid substitutions relative to that shown in SEQ ID NO:1, the one or more variant IL-2 polypeptides bind to the IL-2R alpha chain (IL-2Rα) and have a binding affinity for IL-2Rα that is lower than the affinity of a wild-type IL-2 polypeptide for IL-2Rα when assayed under the same conditions, and the IL-2Rα has the amino acid sequence set forth in SEQ ID NO:2; the one or more variant IL-2 polypeptides bind to the IL-2R beta chain (IL-2Rβ) and have a binding affinity for IL-2Rβ that is lower than the affinity of a wild-type IL-2 polypeptide for IL-2Rβ when assayed under the same conditions, and the IL-2Rβ has the amino acid sequence set forth in SEQ ID NO:

3. The fusion polypeptide.

58. 58. The fusion polypeptide of claim 57, wherein at least one of the one or more variant IL-2 polypeptides comprises one or more mutations capable of reducing binding of IL-2 to IL-2Rα, wherein the one or more mutations are selected from substitutions at one or more of amino acids R38, F42, K43, Y45, E62, P65, E68, V69, and L72.

59. 59. The fusion polypeptide of claim 58, wherein at least one of the one or more variant IL-2 polypeptides comprises a substitution of amino acid F42, optionally with Phe substituted with Ala or Lys.

60. 60. The fusion polypeptide of any one of claims 57 to 59, wherein at least one of the one or more variant IL-2 polypeptides comprises one or more mutations capable of reducing binding of IL-2 to IL-2Rβ, wherein the one or more mutations are selected from substitutions at one or more of the following amino acids: E15, H16, L19, D20, D84, S87, N88, V91, I92.

61. 61. The fusion polypeptide of claim 60, wherein at least one of the one or more variant IL-2 polypeptides comprises a substitution of amino acid H16, optionally with Ala, Glu, Thr, or Asp.

62. 58. The fusion polypeptide of claim 57, wherein at least one of the one or more variant IL-2 polypeptides comprises a substitution selected from E15A with R38A, R38D, or R38E.

63. 58. The fusion polypeptide of claim 57, wherein at least one of the one or more variant IL-2 polypeptides comprises a substitution selected from H16A with R38A, R38D, or R38E; H16T with R38A, R38D, or R38E; H16E with R38A, R38D, or R38E; and H16D with R38A, R38D, or R38E.

64. 58. The fusion polypeptide of claim 57, wherein at least one of the one or more variant IL-2 polypeptides comprises a substitution selected from: D84H with R38A, R38D, or R38E; D84K with R38A, R38D, or R38E; and D84R with R38A, R38D, or R38E.

65. 58. The fusion polypeptide of claim 57, wherein at least one of the one or more variant IL-2 polypeptides comprises a substitution selected from: R38A with N88S, N88A, N88G, N88R, N88T, or N88D; R38D with N88S, N88A, N88G, N88R, N88T, or N88D; and R38E with N88S, N88A, N88G, N88R, N88T, or N88D.

66. 58. The fusion polypeptide of claim 57, wherein at least one of the one or more variant IL-2 polypeptides comprises a substitution selected from: R38A with V91E, V91A, or V91T; R38D with V91E, V91A, or V91T; and R38E with V91E, V91A, or V91T.

67. 58. The fusion polypeptide of claim 57, wherein at least one of the one or more variant IL-2 polypeptides comprises a substitution selected from R38A, I92A, R38D, I92A and R38E, I92A.

68. 58. The fusion polypeptide of claim 57, wherein at least one of the one or more variant IL-2 polypeptides comprises a substitution selected from E15A, F42A and E15A, F42K.

69. 58. The fusion polypeptide of claim 57, wherein at least one of the one or more variant IL-2 polypeptides comprises a substitution selected from H16A, F42A; H16T, F42A; H16E, F42A; H16D, F42A; H16A, F42K; H16T, F42K; and H16E, F42K; H16D, F42K.

70. 58. The fusion polypeptide of claim 57, wherein at least one of the one or more variant IL-2 polypeptides comprises a substitution selected from: F42A with N88S, N88A, N88G, N88R, N88T, or N88D; and F42K with N88S, N88A, N88G, N88R, N88T, or N88D.

71. 58. The fusion polypeptide of claim 57, wherein at least one of the one or more variant IL-2 polypeptides comprises a substitution selected from: F42A with V91E, V91A, or V91T; and F42K with V91E, V91A, or V91T.

72. 58. The fusion polypeptide of claim 57, wherein at least one of the one or more variant IL-2 polypeptides comprises a substitution selected from: F42A with I92A; and F42K with I92A.

73. 58. The fusion polypeptide of claim 57, wherein at least one of the one or more variant IL-2 polypeptides comprises the substitutions E15A and K43E.

74. 58. The fusion polypeptide of claim 57, wherein at least one of the one or more variant IL-2 polypeptides comprises a substitution selected from H16A, K43E; H16T, K43E; H16E, K43E; and H16D, K43E.

75. 58. The fusion polypeptide of claim 57, wherein at least one of the one or more variant IL-2 polypeptides comprises a substitution selected from K43E with D84H, D84K, or D84R.

76. 58. The fusion polypeptide of claim 57, wherein at least one of the one or more variant IL-2 polypeptides comprises a substitution selected from K43E with N88S, N88A, N88G, N88R, N88T, or N88D.

77. 58. The fusion polypeptide of claim 57, wherein at least one of the one or more variant IL-2 polypeptides comprises a substitution selected from K43E with V91E, V91A, or V91T.

78. 58. The fusion polypeptide of claim 57, wherein at least one of the one or more variant IL-2 polypeptides comprises the substitutions K43E and I92A or E15A, E62Q.

79. 58. The fusion polypeptide of claim 57, wherein at least one of the one or more variant IL-2 polypeptides comprises a substitution selected from H16A, E62Q; H16T, E62Q; H16E, E62Q; and H16D, E62Q.

80. 58. The fusion polypeptide of claim 57, wherein at least one of the one or more variant IL-2 polypeptides comprises a substitution selected from E62Q with D84H, D84K, or D84R.

81. 58. The fusion polypeptide of claim 57, wherein at least one of the one or more variant IL-2 polypeptides comprises a substitution selected from E62Q with N88S, N88A, N88G, N88R, N88T, or N88D.

82. 58. The fusion polypeptide of claim 57, wherein at least one of the one or more variant IL-2 polypeptides comprises a substitution selected from E62Q with V91E, V91A, or V91T.

83. 58. The fusion polypeptide of claim 57, wherein at least one of the one or more variant IL-2 polypeptides comprises a substitution selected from E62Q and I92A.

84. 58. The fusion polypeptide of claim 57, wherein at least one of the one or more variant IL-2 polypeptides comprises a substitution selected from E62Q with V91E, V91A, or V91T.

85. 69. The fusion polypeptide of claim 68, wherein at least one of the one or more variant IL-2 polypeptides comprises substitutions at F42, E15, and N88.

86. 69. The fusion polypeptide of claim 68, wherein at least one of the one or more variant IL-2 polypeptides comprises substitutions at F42, E15, and V91.

87. 70. The fusion polypeptide of claim 69, wherein at least one of the one or more variant IL-2 polypeptides comprises substitutions at F42, H16, and D84.

88. 70. The fusion polypeptide of claim 69, wherein at least one of the one or more variant IL-2 polypeptides comprises substitutions at F42, H16, and N88.

89. 70. The fusion polypeptide of claim 69, wherein at least one of the one or more variant IL-2 polypeptides comprises substitutions at F42, H16, and V91.

90. 70. The fusion polypeptide of claim 69, wherein at least one of the one or more variant IL-2 polypeptides comprises substitutions at F42, H16, and I92.

91. 10A-10C, 11A-11C, 12A-12B and 13A-13B, 14A-14N, and 30A-30G, and wherein the fusion polypeptide comprises a functional protein that is a cancer targeting polypeptide (CTP).

92. 92. The fusion polypeptide of claim 91, wherein the target of the cancer targeting polypeptide is a peptide-HLA complex on the surface of a cancer cell.

93. 92. The fusion polypeptide of claim 91, wherein the target of the CTP is a cancer-associated epitope.

94. 92. The fusion polypeptide of claim 91, wherein the CTP is an antibody specific for a cancer-associated antigen.

95. The fusion polypeptide of claim 91, wherein the CTP is an antibody specific to a peptide / HLA complex on the surface of a cancer cell, and the peptide may be a cancer-associated peptide (e.g., a peptide of a cancer-associated antigen).

96. The fusion polypeptide of claim 91, wherein the functional protein is a TCR, such as an "scTCR," a single-chain T-cell receptor specific for a peptide / HLA complex on the surface of a cancer cell, and the peptide can be a cancer-associated peptide (e.g., a peptide of a cancer-associated antigen).

97. 92. The fusion polypeptide of claim 91, wherein the functional protein comprises a wild-type or variant immunomodulatory polypeptide, e.g., a wild-type or variant immunostimulatory polypeptide, e.g., the B7 family of costimulatory receptors, e.g., CD80, CD86, a cytokine, e.g., IL-7, IL-12, IL-15, or IL-21, a TNF superfamily member, e.g., CD-40, 4-1BBL, and OX40, or a chemokine, e.g., CCL19, CCL21, CXCL9 / 10 / 11, or CXCL12.

98. 98. The fusion polypeptide of any one of claims 57 to 97, comprising one or more independently selected linkers.

99. To the individual: (i) a first composition, (a) a modified or unmodified T cell having a T cell receptor (TCR); or (b) a modified cell comprising a chimeric antigen receptor (CAR), wherein the CAR binds to a target antigen, and wherein the modified cell comprises an intracellular signaling domain that is activated by interaction of the modified cell with IL-2; or (c) a modified cell containing one or more exogenously activated receptors; or (d) one or more products that can be processed by the immune system into one or more antigens that can associate with the TCR of a T cell or be presented by a major histocompatibility complex (MHC) to the TCR of a T cell, optionally wherein the one or more antigens are cancer-associated antigens; or (e) one or more nucleic acids encoding one or more polypeptides capable of associating with the TCR of a T cell or being processed by the immune system into one or more antigens that can be presented by a major histocompatibility complex (MHC) to the TCR of a T cell, optionally wherein the one or more antigens are cancer-associated antigens; or (f) at least one immune checkpoint inhibitor (CPI); the first composition comprising: (ii) a second composition comprising a fusion polypeptide according to any one of claims 57 to 98.

1. A method comprising administering when the individual is administered a first composition comprising (a), (b), (c), (d), or (e), the individual may also be administered a CPI; The first composition and the second composition are administered at the same time or at different times. The method.

100. 100. The method of claim 99, wherein the second composition preferentially activates T cells in which the TCR is engaged with an MHC-presented antigen compared to T cells in which the TCR is not engaged with an MHC-presented antigen.

101. 101. The method of claim 99 or 100, wherein the first composition comprises modified or unmodified T cells having a T cell receptor (TCR).

102. 101. The method of claim 99 or 100, wherein the first composition comprises a modified cell comprising a chimeric antigen receptor (CAR), wherein the CAR binds to a target antigen, and the modified cell comprises an intracellular signaling domain that is activated by interaction of the modified cell with IL-2.

103. 101. The method of claim 99 or 100, wherein the first composition comprises modified cells comprising one or more exogenously activated receptors.

104. 101. The method of claim 99 or 100, wherein the first composition comprises one or more products, which can be processed by the immune system into one or more antigens that can associate with the TCR of a T cell or be presented by a major histocompatibility complex (MHC) to the TCR of a T cell, and optionally the one or more antigens are cancer-associated antigens.

105. 101. The method of claim 99 or 100, wherein the first composition comprises one or more nucleic acids encoding one or more polypeptides, wherein the one or more polypeptides are capable of associating with the TCR of a T cell or being processed by the immune system into one or more antigens that can be presented by a major histocompatibility complex (MHC) to the TCR of a T cell, and optionally the one or more antigens are cancer-associated antigens.

106. 101. The method of claim 99 or 100, wherein the first composition comprises a CPI.

107. 106. The method of any one of claims 101 to 105, further comprising administering a CPI.

108. 108. The method of any one of claims 99 to 107, wherein the CPI is an antibody specific for an immune checkpoint selected from CD27, CD28, CD40, CD122, CD96, CD73, CD47, OX40, GITR, CSF1R, JAK, PI3K delta, PI3K gamma, TAM, arginase, CD137 (also known as 4-1BB), ICOS, A2AR, B7-H3, B7-H4, BTLA, CTLA-4, LAG3, TIM3, VISTA, CD96, TIGIT, CD122, PD-1, PD-L1, and PD-L2.

109. The method of any one of claims 99 to 107, wherein the CPI is an antibody specific for an immune checkpoint selected from CTLA-4, TIGIT, PD-1, and PD-L1.

110. The method of any one of claims 99 to 107, wherein the CPI is an antibody specific for an immune checkpoint selected from PD-1 and PD-L1.

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