Engineered cytokines and targeted cytokine delivery

Abstract

The present disclosure provides engineered IL-15 muteins and fusion proteins comprising the same, and uses thereof. The present disclosure also provides compositions and methods for delivering cytokines to particular cells, as well as fusion proteins that specifically bind to CD8+KIR+ T regulatory cells and further comprise a cytokine such as a IL-15. Also provided are treatments for diseases or disorders, such as an inflammatory disease, an autoimmune disease, cancer, or an infectious disease.

Description

[0001] ENGINEERED CYTOKINES AND TARGETED CYTOKINE DELIVERY REFERENCE TO AN ELECTRONIC SEQUENCE LISTING This application contains a Sequence Listing which has been submitted electronically in xml format and is hereby incorporated by reference in its entirety. Said xml copy, created on April 14, 2025, is named 368576_41102_SL.xml and is 698,948 bytes in size. BACKGROUND Cytokines are a class of small immunomodulatory proteins. Because of their immunostimulatory or immunosuppressive effects, cytokines, cytokine receptor agonists, and cytokine receptor antagonists have been used in therapies for treating diseases. In some approaches, cytokines have been conjugated to other molecules, particularly antibodies or antibody-like molecules, so that they can be delivered to particular tissues or cell types. See, e.g., Spangler et al., J Immunol (2018) 201(7): 2094–2106. Interleukin-15 (IL-15) is a cytokine with pleiotropic effects on the immune system, and has been used to increase immune cell proliferation and activity. The adaptive immune system has a number of cell subtypes, including T cells subsets and B cell subsets. T cell subsets include a variety of types of T cells, including naïve T lymphocytes and effector T lymphocytes, such as cytotoxic T cells and helper T cells, and regulatory T cells (also known as "Tregs"). The activity of these T cell types is achieved by a balance between the activity of effector T cells and regulation by regulatory T cells. While effector T cells promote inflammation, regulatory T cells are generally thought to control it. Therefore, regulatory T cells play an important role in autoimmune pathogenesis by maintaining self-tolerance, limiting autoimmunity, and controlling expansion and activation of autoreactive CD4+ T effector cells. CD8+ regulatory T cells ("CD8+ Tregs") have been identified as playing a role in autoimmune disorders. See, e.g., Li et al., Science (2022) 376:eabi9591. Modulation of the activity of, presence of, and / or abundance of CD8+ Tregs, and thereby activating

[0002] 1 170886321.1 or deactivating this immune response network, offers a therapeutic approach for treating a variety of diseases. See, e.g., WO2022 / 169825. BRIEF SUMMARY In some aspects, the present disclosure provides engineered IL-15 mutants and uses thereof. For example, the present disclosure provides, in some embodiments, IL-15 mutants comprising the amino acid sequences according to SEQ ID NOs:10-94. In some embodiments, the IL-15 mutant has reduced or no binding to IL15Ra, IL2Rb, and / or IL2Rg. In some further aspects, IL-15 fusion proteins are provided. For example, IL-15 mutants may be expressed as a fusion protein with one or more antibody binding domains. In some aspects, pharmaceutical compositions comprising a IL-15 mutant or fusion protein as disclosed herein are provided. In some additional aspects, the present disclosure provides methods of treatment using a IL-15 mutant or fusion protein thereof, or a pharmaceutical composition as disclosed herein. In some aspects, the present disclosure provides methods of increasing CD8+ Treg activity comprising administering to a subject in need thereof a molecule that comprises (1) a binding domain that binds to one or more proteins expressed on the surface of a CD8+ Treg cell and (2) a cytokine. In some aspects, the present disclosure provides methods of increasing CD8+ Treg proliferation comprising administering to a subject in need thereof a molecule that comprises (1) a binding domain that binds to one or more proteins expressed on the surface of a CD8+ Treg cell and (2) a cytokine. In some aspects, the present disclosure provides methods of treating an autoimmune disease, comprising administering to a subject in need thereof a molecule that comprises (1) a binding domain that binds to one or more proteins expressed on the surface of a CD8+ Treg cell, and (2) a cytokine.

[0003] 2 170886321.1 In some aspects, the present disclosure provides methods of suppressing an immune response mediated by pathogenic immune cells, comprising administering to a subject in need thereof a molecule that comprises (1) a binding domain that binds to one or more proteins expressed on the surface of a CD8+ Treg cell, and (2) a cytokine, whereby the number or activity of pathogenic immune cells is decreased. In some of the aforementioned embodiments, the molecule comprises a fusion protein comprising (1) an antibody or antibody fragment that binds to one or more proteins expressed on the surface of a CD8+ Treg cell, and (2) a cytokine. Also provided in the present disclosure are fusion proteins comprising: (a) a binding agent comprising: (i) a first binding domain that specifically binds to a KIR protein, and (ii) a second binding domain that specifically binds to a protein expressed on CD8+KIR+ T regulatory cells (Tregs) other than a KIR protein; and (b) a cytokine. Methods of using the fusion proteins are also provided by the disclosure. BRIEF DESCRIPTION OF THE DRAWINGS FIG.1 shows an exemplary anti-CD8α-IL-15 fusion protein. FIG.2 shows an exemplary anti-KIR-IL-15 fusion protein. FIGS.3A to 3C relate to activation of CD8+ Tregs by a dual-targeting anti- CD8 / anti-KIR binding protein. FIG.3A shows a bi-specific antibody having a CD8- binding domain and a binding domain that targets KIR2DL1 / 2 / 3 (anti-CD8 scFv / anti- KIR FAB-Fc). FIG.3B shows degranulation and FIG.3C shows granzyme B levels following administration of anti-CD8 scFv / anti-KIR FAB-Fc (at doses of 10 μg / mL, 1 μg / mL, or 0.1 μg / mL) or a monoclonal KIR blockade (at 20 μg). FIG.4 shows dose-dependent reductions in proinflammatory cytokines following administration of anti-CD8 scFv / anti-KIR FAB-Fc (at doses of 10 μg / mL or 1 μg / mL). FIG.5 shows CD4+ T cell death following administration of anti-CD8 scFv / anti-KIR FAB-Fc (at doses of 10 μg / mL, 1 μg / mL, or 0.1 μg / mL) or a monoclonal KIR blockade (at 20 μg).

[0004] 3 170886321.1 FIG.6 shows preferential binding of the bi-specific blocker anti-CD8 scFv / anti- KIR FAB-Fc (anti-KIR2DL1 / 2 / 3 and anti-CD8) to CD8+ T cells relative to NK cells. FIG.7 shows preferential binding of the bi-specific blocker anti-CD8 scFv / anti- KIR FAB-Fc (anti-KIR2DL1 / 2 / 3 and anti-CD8) to CD8+ T cells relative to NK cells and CD4+ T cells. FIG.8 shows a bi-specific antibody (Fab-scFv-Fc) having a domain that binds to CD8 and a domain that binds KIR, with a cytokine (e.g., IL-15) attached to the Fc domain of the anti-KIR heavy chain. FIGS.9A to 9B show higher cytokine potency for an IL-15 variant-antibody fusion protein compared to the wild-type IL-15 alone. FIG.9A shows signaling in different cell types following exposure to anti-CD8 scFv / anti-KIR FAB-Fc-IL-15 fusion protein. FIG.9B shows signaling in different cell types following exposure to wild-type IL-15. FIGS.10A to 10B show preferential signaling in CD8+KIR2D+ Tregs for an IL-15 variant-antibody fusion protein compared to the wild-type IL-15 alone. FIG.10A shows signaling in different cell types following exposure to anti-CD8 scFv / anti-KIR FAB-Fc-IL-15 fusion protein. FIG.10B shows signaling in different cell types following exposure to wild-type IL-15. FIG.11 shows low off-target proliferation of CD4-CD8-KIR2D- cells on Day 9, for wild-type IL-15 and IL-15 variant-CD8xKIR. FIGS.12A to 12C show proliferation of cell subsets after incubation with wild- type cytokines or variant-antibody fusion proteins at different concentrations (0.1 nM, FIG.12A; 1nM, FIG.12B; 10nM, FIG.12C). FIGS.13A to 13D show selective signaling on targeted CD8+ cells (relative to non-targeted CD4+ cells) following exposure to CD8-targeted IL-15 wild-type (FIG. 13A) or variants v9 (FIG.13B), v67 (FIG.13C), and v84 (FIG.13D). FIGS.14A to 14I show signaling by KIR+ CD8+ cells following exposure to anti-KIR-IL-15 fusion proteins.

[0005] 4 170886321.1 FIG.15 shows signaling potency differential between KIR+ and KIR- cells exposed to anti-KIR-IL-15 fusion proteins. FIG.16 shows maximal signaling between KIR+ and KIR- cells exposed to anti-KIR-IL-15 fusion proteins. FIGS.17A to 17J show cell proliferation (as measured by Ki67) for KIR+ cells and KIR- cells, following exposure to IL-15 or anti-KIR-IL-15 fusion proteins. FIGS.18A to 18E show potency of IL-15 or anti-KIR-IL-15 fusion proteins for CD8+KIR+ cells and CD8+KIR- cells. FIGS.19A to 19B show induction of gene expression following culture of KIR+CD8+ cells from healthy subjects (FIG.19A) or celiac subjects (FIG.19B) with anti-KIR-IL-15 fusion proteins. FIGS.20A to 20B show induction of gene expression following culture of KIR+CD8+ cells from healthy subjects (FIG.20A) or celiac subjects (FIG.20B) with anti-KIR-IL-15 fusion proteins. FIG.21 shows KIR+ CD8+ Treg cells isolated from NSG mice engrafted with PBMC from a healthy human donor and administered saline, an untargeted Fc IL-15v9 mutein, or an anti-KIR-IL-15v9 fusion protein. FIG.22 shows fluorescence over time of GFP-labeled SKW expressing an autoimmune TCR in co-culture with CD8+ Treg that had been cultured in anti-CD8-IL- 15 WT or anti-CD8-IL-15 v9. FIG.23 shows survival of NSG mice engrafted with CD4+ and CD8+ T cells, and treated with anti-KIR-IL15 v67 or saline. Mice that received anti-KIR-IL15 v67 tended towards increased survival relative to those that received saline control. FIGS.24A to 24B show KIR+ CD8+ Treg levels (FIG.24A) and Granzyme B activity (FIG.24B) in NSG mice that received treatment with anti-KIR-IL15 v67 or saline. FIGS.25A to 25B show expansion (FIG.25A) and activation (FIG.25B) of CD8+ Tregs in vitro by wild-type cytokines.

[0006] 5 170886321.1 FIGS.26A-26C show flow cytometry characterization of CD8+ Treg from different patient populations following incubation with wild-type IL-15, including expression levels of T-bet (FIG.26A), CD69 (FIG.26B, and (ICOS FIG.26C). FIGS.27A-27C show evaluation of a targeted IL-15 mutein in a type-1 diabetes (T1D) organoid model. PBMCs from a T1D patient were thawed and cultured + / - T1D antigenic peptides and + / - targeted IL-15 mutein for 48 hours and then were added to the culture media of pancreatic organoids from a healthy donor and incubated for 72 hours (FIG.27A). The targeted IL-15 mutein resulted in greater expansion of CD8+ Tregs in the T1D patient PBMCs, with or without T1D antigenic peptides (FIG.27B). Addition of the targeted IL-15 mutein resulted in fewer apoptotic Beta cells in the pancreatic organoid (FIG.27C). DETAILED DESCRIPTION The present disclosure provides, in some embodiments, engineered IL-15 mutants, or "IL-15 muteins". The present disclosure also provides, in some embodiments, IL-15 muteins attached to other molecules, such as in antibody-mutein fusion proteins and antibody-mutein conjugates, and related methods for delivering the IL-15 muteins to cells and / or tissues. The present disclosure also provides therapeutic uses of the IL-15 muteins, antibody-mutein fusion proteins, and antibody-mutein conjugates described herein. The present disclosure also provides methods of delivering cytokines to particular cells and / or tissues. In some embodiments, the methods also involve modulating the activity, presence, and / or abundance of T cells. Also provided in further embodiments are binding agents, including fusion proteins comprising binding domains comprising antibodies or antibody fragments (e.g., antigen-binding fragments of antibodies) that target KIR and further comprising a cytokine, and related pharmaceutical compositions, methods of activating CD8+ regulatory T cells (CD8+ Treg cells), and methods of treating or preventing disease (e.g.¸ an autoimmune disease). The present disclosure provides methods of delivering cytokines to particular cells and / or tissues. In some embodiments, the methods also involve modulating the

[0007] 6 170886321.1 activity, presence, and / or abundance of T cells. Also provided in further embodiments are binding agents, including fusion proteins comprising binding domains comprising antibodies or antibody fragments (e.g., antigen-binding fragments of antibodies) that target KIR and further comprising a cytokine, and related pharmaceutical compositions, methods of activating CD8+ regulatory T cells (CD8+ Treg cells), and methods of treating or preventing disease (e.g.¸ an autoimmune disease). I. Glossary Prior to setting forth this disclosure in more detail, definitions of certain terms to be used herein are provided. Additional definitions are set forth throughout this disclosure. Unless the context requires otherwise, throughout the present specification and claims, the word "comprise" and variations thereof, such as "comprises" and "comprising", are to be construed in an open, inclusive sense, that is, as "including, but not limited to". "Consisting of" shall mean excluding more than trace elements of other ingredients and substantial method steps disclosed herein, and in the case of an amino acid or nucleic acid sequence, excluding additional amino acids or nucleotides, respectively. The term "consisting essentially of" limits the scope of a claim to the specified materials or steps, or to those that do not materially affect the basic characteristics of a claimed invention. For example, a composition consisting essentially of the elements as defined herein would not exclude trace contaminants from an isolation and purification method and would not exclude pharmaceutically acceptable carriers, such as phosphate buffered saline, preservatives, and the like. Similarly, a protein consists essentially of a particular amino acid sequence when the protein includes additional amino acids that contribute to at most 20% of the length of the protein and do not substantially affect the activity of the protein (e.g., alters the activity of the protein by no more than 50%). Embodiments defined by each of these transitional terms are within the scope of this invention. In the present description, the term "about" means + 20% of the indicated range, value, or structure, unless otherwise indicated.

[0008] 7 170886321.1 It should be understood that the terms "a" and "an" as used herein includes "one" or "one or more" of the enumerated components unless stated otherwise. The use of the alternative (e.g., "or") should be understood to mean either one, both, or any combination thereof of the alternatives, and may be used synonymously with "and / or". As used herein, the terms "include" and "have" are used synonymously, which terms and variants thereof (e.g., "including", "having") are intended to be construed as non-limiting. The word "substantially" does not exclude "completely"; e.g., a composition which is "substantially free" from Y may be completely free from Y. Where necessary, the word "substantially" may be omitted from definitions provided herein. "Optional" or "optionally" means that the subsequently described element, component, event, or circumstance may or may not occur, and that the description includes instances in which the element, component, event, or circumstance occurs and instances in which they do not. As used herein, "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an α-carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refer to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that function in a manner similar to a naturally occurring amino acid.

[0009] 8 170886321.1 As used herein, the terms "peptide," "polypeptide," and "protein," and variations of these terms, refer to a molecule that comprises at least two amino acids joined to each other by a (normal or modified) peptide bond. For example, a peptide, polypeptide, or protein may comprise or be composed of a plurality of amino acids selected from the 20 amino acids defined by the genetic code or an amino acid analog or mimetic, each being linked to at least one other by a peptide bond. A peptide, polypeptide, or protein can comprise or be composed of L-amino acids and / or D-amino acids (or analogs or mimetics thereof). The terms "peptide", "polypeptide," "protein" also include "peptidomimetics" which are defined as peptide analogs containing non- peptidic structural elements, which peptides are capable of mimicking or antagonizing the biological action(s) of a natural parent peptide. In certain embodiments, a peptidomimetic lacks characteristics such as enzymatically scissile peptide bonds. A peptide, polypeptide, or protein may comprise amino acids other than the 20 amino acids defined by the genetic code in addition to these amino acids, or it can be composed of amino acids other than the 20 amino acids defined by the genetic code. In certain embodiments, a peptide, polypeptide, or protein in the context of the present disclosure can comprise amino acids that are modified by natural processes, such as post-translational maturation processes, or by chemical processes (e.g., synthetic processes), which are known in the art and include those described herein. Such modifications can appear anywhere in the polypeptide; e.g., in the peptide skeleton; in the amino acid chain; or at the carboxy- or amino-terminal ends. A peptide or polypeptide can be branched, such as following an ubiquitination, or may be cyclic, with or without branching. The terms "peptide", "polypeptide", and "protein" also include modified peptides, polypeptides, and proteins. For example, peptide, polypeptide, or protein modifications can include acetylation, acylation, ADP- ribosylation, amidation, covalent fixation of a nucleotide or of a nucleotide derivative, covalent fixation of a lipid or of a lipidic derivative, the covalent fixation of a phosphatidylinositol, covalent or non-covalent cross-linking, cyclization, disulfide bond formation, demethylation, glycosylation including pegylation, hydroxylation,

[0010] 9 170886321.1 iodization, methylation, myristoylation, oxidation, proteolytic processes, phosphorylation, prenylation, racemization, seneloylation, sulfatation, or amino acid addition such as arginylation or ubiquitination. Such modifications have been described in the literature (see Proteins Structure and Molecular Properties (1993) 2nd Ed., T. E. Creighton, New York; Post-translational Covalent Modifications of Proteins (1983) B. C. Johnson, Ed., Academic Press, New York; Seifter et al. (1990) Analysis for protein modifications and nonprotein cofactors, Meth. Enzymol.182: 626-646 and Rattan et al., (1992) Protein Synthesis: Post-translational Modifications and Aging, Ann NY Acad Sci, 663: 48-62). Accordingly, the terms "peptide", "polypeptide", and "protein" can include, for example, lipopeptides, lipoproteins, glycopeptides, glycoproteins, and the like. Variants of proteins, peptides, and polypeptides of this disclosure are also contemplated. In certain embodiments, variant proteins, peptides, and polypeptides comprise or consist of an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identical to an amino acid sequence of a defined or reference amino acid sequence as described herein. "Protein" and "polypeptide" are often used in reference to relatively large polypeptides, whereas the term "peptide" is often used in reference to small polypeptides, but usage of these terms in the art overlaps. The terms "protein" and "(poly)peptide" are used interchangeably herein when referring to an encoded gene product and fragments thereof. Additionally, as used herein, "(poly)peptide" and "protein" may be used interchangeably in reference to a polymer of amino acid residues, such as a plurality of amino acid monomers linked by peptide bonds. "Nucleic acid molecule" or "polynucleotide" or "nucleic acid" refers to a polymeric compound including covalently linked nucleotides, which can be made up of natural subunits (e.g., purine or pyrimidine bases) or non-natural subunits (e.g., morpholine ring). Purine bases include adenine, guanine, hypoxanthine, and xanthine, and pyrimidine bases include uracil, thymine, and cytosine. Nucleic acid monomers can be linked by phosphodiester bonds or analogs of such linkages. Analogs of phosphodiester linkages include phosphorothioate, phosphorodithioate,

[0011] 10 170886321.1 phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoranilidate, phosphoramidate, and the like. Nucleic acid molecules include polyribonucleic acid (RNA), polydeoxyribonucleic acid (DNA), which includes cDNA, genomic DNA, and synthetic DNA, any of which may be single or double-stranded. If single-stranded, the nucleic acid molecule may be the coding strand or non-coding (anti-sense strand). Polynucleotides (including oligonucleotides), and fragments thereof may be generated, for example, by polymerase chain reaction (PCR) or by in vitro translation, or generated by any of ligation, scission, endonuclease action, or exonuclease action. A nucleic acid molecule encoding an amino acid sequence includes all nucleotide sequences that encode the same amino acid sequence. Some versions of the nucleotide sequences may also include intron(s) to the extent that the intron(s) may be removed through co- or post-transcriptional mechanisms. Different nucleotide sequences may encode the same amino acid sequence as the result of the redundancy or degeneracy of the genetic code, or by splicing, or both. Variants of nucleic acid molecules of this disclosure are also contemplated. Variant nucleic acid molecules are at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.9% identical a nucleic acid molecule of a defined or reference polynucleotide as described herein, or that hybridize to a polynucleotide under stringent hybridization conditions of 0.015M sodium chloride, 0.0015M sodium citrate at about 65-68ºC or 0.015M sodium chloride, 0.0015M sodium citrate, and 50% formamide at about 42ºC. Nucleic acid molecule variants retain the capacity to encode a binding domain having a functionality described herein, such as specifically binding a target molecule. As used herein, the term "sequence variant" refers to any sequence having one or more alterations in comparison to a reference sequence, whereby a reference sequence is any published sequence and / or any of the sequences disclosed herein, i.e., SEQ ID NO:1 to SEQ ID NO:466. Thus, the term "sequence variant" includes nucleotide sequence variants and amino acid sequence variants. In certain

[0012] 11 170886321.1 embodiments, a sequence variant in the context of a nucleotide sequence, the reference sequence is also a nucleotide sequence, whereas in certain embodiments for a sequence variant in the context of an amino acid sequence, the reference sequence is also an amino acid sequence. A "sequence variant" as used herein can be, for example, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the reference sequence. "Percent sequence identity" refers to a relationship between two or more sequences, as determined by comparing the sequences. Methods to determine sequence identity can be designed to give the best match between the sequences being compared. For example, the sequences may be aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment). Further, non-homologous sequences may be disregarded for comparison purposes. The percent sequence identity referenced herein is calculated over the length of the reference sequence, unless indicated otherwise. Methods to determine sequence identity and similarity can be found in publicly available computer programs. Sequence alignments and percent identity calculations may be performed using a BLAST program (e.g., BLAST 2.0, BLASTP, BLASTN, or BLASTX). The mathematical algorithm used in the BLAST programs can be found in Altschul et al., Nucleic Acids Res. (1997) 25:3389-3402. Within the context of this disclosure, it will be understood that where sequence analysis software is used for analysis, the results of the analysis are based on the "default values" of the program referenced. "Default values" mean any set of values or parameters that originally load with the software when first initialized. A "sequence variant" in the context of a nucleic acid (nucleotide) sequence has an altered sequence in which one or more of the nucleotides in the reference sequence is deleted or substituted, or one or more nucleotides are inserted into the sequence of the reference nucleotide sequence. Nucleotides are referred to herein by the standard one- letter designation (A, C, G, or T). Due to the degeneracy of the genetic code, a

[0013] 12 170886321.1 "sequence variant" of a nucleotide sequence can either result in a change in the respective reference amino acid sequence, i.e., in an amino acid "sequence variant" or not. In certain embodiments, a nucleotide sequence variant does not result in an amino acid sequence variant (e.g., a silent mutation). In some embodiments, a nucleotide sequence variant that results in one or more "non-silent" mutation is contemplated. In some embodiments, a nucleotide sequence variant of the present disclosure encodes an amino acid sequence that is at least 80%, at least 85 %, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a reference amino acid sequence. Nucleotide and amino sequences as disclosed herein refer also to codon- optimized versions of a reference or wild-type nucleotide or amino acid sequence. In any of the embodiments described herein, a polynucleotide of the present disclosure may be codon-optimized for a host cell containing the polynucleotide (see, e.g., Scholten et al., Clin. Immunol. (2006) 119:135-145). Codon optimization can be performed using known techniques and tools, e.g., using the GenScript® OptimumGeneTM tool, or the GeneArt Gene Synthesis Tool (Thermo Fisher Scientific). Codon-optimized sequences include sequences that are partially codon- optimized (i.e., at least one codon is optimized for expression in the host cell) and those that are fully codon-optimized. A "sequence variant" in the context of an amino acid sequence has an altered sequence in which one or more of the amino acids is deleted, substituted, or inserted in comparison to a reference amino acid sequence. As a result of the alterations, such a sequence variant has an amino acid sequence which is at least 80%, at least 85 %, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the reference amino acid sequence. For example, per 100 amino acids of the reference sequence a variant sequence that has no more than 10 alterations, i.e., any combination of deletions, insertions, or substitutions, is "at least 90% identical" to the reference sequence.

[0014] 13 170886321.1 A "conservative substitution" refers to amino acid substitutions that do not significantly affect or alter binding characteristics of a particular protein. Generally, conservative substitutions are ones in which a substituted amino acid residue is replaced with an amino acid residue having a similar side chain. Conservative substitutions include a substitution found in one of the following groups: Group 1: Alanine (Ala or A), Glycine (Gly or G), Serine (Ser or S), Threonine (Thr or T); Group 2: Aspartic acid (Asp or D), Glutamic acid (Glu or Z); Group 3: Asparagine (Asn or N), Glutamine (Gln or Q); Group 4: Arginine (Arg or R), Lysine (Lys or K), Histidine (His or H); Group 5: Isoleucine (Ile or I), Leucine (Leu or L), Methionine (Met or M), Valine (Val or V); and Group 6: Phenylalanine (Phe or F), Tyrosine (Tyr or Y), Tryptophan (Trp or W). Additionally or alternatively, amino acids can be grouped into conservative substitution groups by similar function, chemical structure, or composition (e.g., acidic, basic, aliphatic, aromatic, or sulfur-containing). For example, an aliphatic grouping may include, for purposes of substitution, Gly, Ala, Val, Leu, and Ile. Other conservative substitutions groups include: sulfur-containing: Met and Cysteine (Cys or C); acidic: Asp, Glu, Asn, and Gln; small aliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr, Pro, and Gly; polar, negatively charged residues and their amides: Asp, Asn, Glu, and Gln; polar, positively charged residues: His, Arg, and Lys; large aliphatic, nonpolar residues: Met, Leu, Ile, Val, and Cys; and large aromatic residues: Phe, Tyr, and Trp. Additional information can be found in Creighton (1984) Proteins, W.H. Freeman and Company. Amino acid sequence insertions can include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include the fusion to the N- or C-terminus of an amino acid sequence to a reporter molecule or an enzyme. In general, alterations in the sequence variants do not abolish or significantly reduce a desired functionality of the respective reference sequence. For example, it is preferred that a variant sequence of the present disclosure does not significantly reduce

[0015] 14 170886321.1 or completely abrogate the functionality of a sequence of an antibody, or antigen- binding fragment thereof, to bind to the same epitope as compared to antibody or antigen-binding fragment having (or encoded by) the reference sequence. Guidance in determining which nucleotides and amino acid residues, respectively, may be substituted, inserted, or deleted without abolishing a desired structure or functionality can be found by using, e.g., known computer programs. As used herein, a nucleic acid sequence or an amino acid sequence "derived from" a designated nucleic acid, peptide, polypeptide, or protein refers to the origin of the nucleic acid, peptide, polypeptide, or protein. A nucleic acid sequence or amino acid sequence that is derived from a particular sequence may have an amino acid sequence that is essentially identical to that sequence or a portion thereof, from which it is derived, whereby "essentially identical" includes sequence variants as defined above. A nucleic acid sequence or amino acid sequence that is derived from a particular peptide or protein, may be derived from the corresponding domain in the particular peptide or protein. In this context, "corresponding" refers to possession of a same functionality or characteristic of interest. For example, an "extracellular domain" corresponds to another "extracellular domain" (of another protein), or a "transmembrane domain" corresponds to another “transmembrane domain” (of another protein). "Corresponding" parts of peptides, proteins, and nucleic acids are thus easily identifiable to one of ordinary skill in the art. Likewise, a sequence "derived from" another (e.g., "source") sequence can be identified by one of ordinary skill in the art as having its origin in the source sequence. A nucleic acid sequence or an amino acid sequence derived from another nucleic acid, peptide, polypeptide, or protein may be identical to the starting nucleic acid, peptide, polypeptide, or protein (from which it is derived). However, a nucleic acid sequence or an amino acid sequence derived from another nucleic acid, peptide, polypeptide, or protein may also have one or more mutations relative to the starting nucleic acid, peptide, polypeptide, or protein (from which it is derived), in particular a nucleic acid sequence or an amino acid sequence derived from another nucleic acid, peptide, polypeptide, or protein may be a functional sequence variant as described

[0016] 15 170886321.1 above of the starting nucleic acid, peptide, polypeptide, or protein (from which it is derived). For example, in a peptide / protein, one or more amino acid residues may be substituted with other amino acid residues, or one or more amino acid residue insertions or deletions may occur. As used herein, the term "mutation" relates to a change in a nucleic acid sequence and / or in an amino acid sequence in comparison to a reference sequence, e.g., a corresponding genomic, wild-type, or reference sequence. The resulting "mutated" nucleic acid sequence and / or in an amino acid may be referred to as a "mutant". A mutation, e.g., in comparison to a reference genomic sequence, may be, for example, a (naturally occurring) somatic mutation, a spontaneous mutation, an induced mutation, e.g., induced by enzymes, chemicals, or radiation, or a mutation obtained by site- directed mutagenesis (molecular biology methods for making specific and intentional changes in the nucleic acid sequence and / or in the amino acid sequence) Thus, the terms "mutation" or "mutating" shall be understood to also include physically making or inducing a mutation, e.g., in a nucleic acid sequence or in an amino acid sequence. A mutation includes substitution, deletion, and insertion of one or more nucleotides or amino acids as well as inversion of several successive nucleotides or amino acids. To achieve a mutation in an amino acid sequence, a mutation may be introduced into the nucleotide sequence encoding said amino acid sequence in order to express a (recombinant) mutated polypeptide. A mutation may be achieved, for example, by altering (e.g., by site-directed mutagenesis) a codon (e.g., by altering one, two, or three nucleotide bases therein) of a nucleic acid molecule encoding one amino acid to provide a codon that encodes a different amino acid, or that encodes a same amino acid, or by synthesizing a sequence variant. The term "introduced" in the context of inserting a nucleic acid molecule into a cell, means "transfection", or "transformation", or "transduction" and includes reference to the incorporation of a nucleic acid molecule into a eukaryotic or prokaryotic cell wherein the nucleic acid molecule may be incorporated into the genome of a cell (e.g.,

[0017] 16 170886321.1 chromosome, plasmid, plastid, or mitochondrial DNA), converted into an autonomous replicon, or transiently expressed (e.g., transfected mRNA). The term "recombinant", as used herein (e.g., a recombinant antibody, a recombinant protein, a recombinant nucleic acid, or the like, refers to any molecule (antibody, protein, nucleic acid, or the like) which is prepared, expressed, created, or isolated by recombinant means, and which is not naturally occurring. "Recombinant" can be used synonymously with "engineered" or "non-natural" and can refer to an organism, microorganism, cell, nucleic acid molecule, or vector that includes at least one genetic alteration or has been modified by introduction of an exogenous nucleic acid molecule, wherein such alterations or modifications are introduced by genetic engineering (i.e., human intervention). Genetic alterations include, for example, modifications introducing expressible nucleic acid molecules encoding proteins, fusion proteins, or enzymes, or other nucleic acid molecule additions, deletions, or substitutions or other functional disruption of a cell's genetic material. Additional modifications include, for example, non-coding regulatory regions in which the modifications alter expression of a polynucleotide, gene, or operon. As used herein, "heterologous" or "non-endogenous" or "exogenous" refers to any gene, protein, compound, nucleic acid molecule, or activity that is not native to a host cell or a subject, or any gene, protein, compound, nucleic acid molecule, or activity native to a host cell or a subject that has been altered. Heterologous, non-endogenous, or exogenous includes genes, proteins, compounds, or nucleic acid molecules that have been mutated or otherwise altered such that the structure, activity, or both is different as between the native and altered genes, proteins, compounds, or nucleic acid molecules. In certain embodiments, heterologous, non-endogenous, or exogenous genes, proteins, or nucleic acid molecules may not be endogenous to a host cell or a subject, but instead nucleic acids encoding such genes, proteins, or nucleic acid molecules may have been added to a host cell by conjugation, transformation, transfection, electroporation, or the like, wherein the added nucleic acid molecule may integrate into a host cell genome or can exist as extra-chromosomal genetic material (e.g., as a plasmid or other self-

[0018] 17 170886321.1 replicating vector). The term "homologous" or "homolog" refers to a gene, protein, compound, nucleic acid molecule, or activity found in or derived from a host cell, species, or strain. For example, a heterologous or exogenous polynucleotide or gene encoding a polypeptide may be homologous to a native polynucleotide or gene and encode a homologous polypeptide or activity, but the polynucleotide or polypeptide may have an altered structure, sequence, expression level, or any combination thereof. A non-endogenous polynucleotide or gene, as well as the encoded polypeptide or activity, may be from the same species, a different species, or a combination thereof. As used herein, the term "endogenous" or "native" refers to a polynucleotide, gene, protein, compound, molecule, or activity that is normally present in a host cell or a subject. As used herein, the terms "cell", "cell line", " and "cell culture" are used interchangeably and all such designations include progeny. Thus, the words "transformants" and "transformed cells" include the primary subject cell and cultures derived therefrom without regard for the number of transfers. It is also understood that all progeny may not be precisely identical in DNA content, due to deliberate or inadvertent mutations. Variant progeny that have the same or substantially the same function, phenotype, or biological activity as screened for in the originally transformed cell are included. Where distinct designations are intended, it will be clear from the context. The terms "isolated" or "partially purified" as used herein refer in the case of a nucleic acid, polypeptide, or protein, to a nucleic acid, polypeptide, or protein separated from at least one other component (e.g., nucleic acid or polypeptide or protein) that is present with the nucleic acid, polypeptide, or protein as found in its natural source and / or that would be present with the nucleic acid, polypeptide, or protein when expressed by a cell, or secreted in the case of secreted polypeptides and proteins. A chemically synthesized nucleic acid, polypeptide, or protein, or one synthesized using in vitro transcription / translation, is considered "isolated." The terms "purified" or "substantially purified" refer to an isolated nucleic acid, polypeptide, or protein that is at

[0019] 18 170886321.1 least 95% by weight the subject nucleic acid, polypeptide, or protein, including, for example, at least 96%, at least 97%, at least 98%, at least 99%, or more. II. IL-15 Mutants Cytokines are a class of small immunomodulatory proteins that can have immunostimulatory or immunosuppressive effects. Cytokines signal by bringing their respective receptor chains in proximity, and the affinity of cytokines to receptors determines their potency. Cytokines have different interfaces that bind to different receptor chains, which in turn cross-phosphorylate signaling proteins, such as the JAK / STAT, AKT, and MAPK pathways. The STAT5 pathway is commonly used to measure cell activation. Interleukin-15 (IL-15) is a cytokine with pleiotropic effects on the immune system. The wild-type human form of IL-15 is translated as a 162-amino acid precursor polypeptide. This precursor is processed post-translationally to form a 114-amino acid polypeptide consisting of SEQ ID NO:95. The wild-type human IL-15 precursor sequence can also be found under NCBI Reference Sequence No. NP_000576.1, which sequence is incorporated herein by reference. The nucleic acid molecule encoding wild- type human IL-15 can be found under NCBI Reference Sequence NG_029605.2, which sequence is incorporated herein by reference. Mutant forms of IL-15 can be generated that have reduced binding to receptor subunits, often to resolve difficulties associated with toxicity and / or off-target effects. For example, mutant forms of IL-15 with abolished or reduced affinity to the IL-15 receptor alpha and / or beta / gamma are described in WO2019 / 166946A1. As used herein, "IL-15" includes any form of IL-15 and variants thereof, including human IL-15 and all mammalian species of IL-15, and includes wild-type and variant forms. In some embodiments, the present disclosure provides an isolated IL-15 peptide comprising or consisting of the amino acid sequence according to any one of SEQ ID NOs:10-94.

[0020] 19 170886321.1 In some embodiments, the present disclosure provides an isolated IL-15 peptide having one or more amino acid substitutions or deletions relative to SEQ ID NO:95, wherein the one or more amino acid substitutions or deletions comprises or consists of: a. an N-terminal truncation in which the first three amino acids (NWV) are deleted; b. an N-terminal truncation in which the first five amino acids (NWVNV) are deleted; c. V49Y; d. E46K; e. E53A; f. E53K; g. Y26A; h. Y26K; i. E89K; j. D8K; k. K10V; l. K11A; m. D61K; n. E64G; o. N65G; p. L69W; q. D30K; r. H105A; s. M109A; t. V49G; u. E53S; v. E53R; w. T24K; x. D8A; y. K10Q; z. D61A; aa. N65W; bb. Q108A; cc. V49A; dd. Y26A and E46K; ee. Y26A and V49G; ff. Y26A and V49Y; gg. Y26A and E53A; hh. Y26A and E53K; ii. Y26A and E53R; jj. Y26A and E89K; kk. Y26K and E53A; ll. E46K and V49Y; mm. E46K and E53A; nn. E46K and E53K; oo. E46K and E53R; pp. E46K and E89K; qq. V49G and E53K; rr. V49G and E89K; ss. V49Y and E53A; tt. V49Y and E53K; uu. V49Y and E53R; vv. V49Y and E89K; ww. E53A and E89K; xx. E53K and E89K; yy. E53R and E89K; zz. E53S and E89K; aaa. K10Q and D61A; bbb. K10Q and E64G; ccc. K10V and D61A; ddd. K10V and E64G; eee. D61A and E64G; fff. K10Q, Y26A, and V49G; ggg. K10Q, Y26A, V49G, and E64G; hhh. Y26A, V49G, and E64G; iii. K10Q, V49G, and E53K; jjj. K10Q, V49G, E53K, and E64G; kkk. V49G, E53K, and E64G; lll. K10Q, V49Y, and E53A; mmm. K10Q, V49Y, E53A, and E64G; nnn. V49Y, E53A, and E64G; ooo. K10Q, V49Y, and E53R; ppp. K10Q, V49Y, E53R, and E64G; qqq. V49Y, E53R, and E64G; rrr. K10Q, V49Y, and E89K; sss. K10Q, V49Y, E64G, and E89K; ttt. V49Y, E64G, and E89K; uuu. K10Q, E53A, and E89K; vvv. K10Q, E53A, E64G, and E89K; www. E53A, E64G, and E89K; xxx. K10Q, E53S, and E89K; yyy. K10Q, E53S, E64G, and E89K; zzz. E53S, E64G, and E89K; aaaa. V49Y, E53A, and M109A; bbbb. K10Q, V49Y, E53A, and M109A; cccc. V49Y, E53R, and M109A; dddd. V49Y, E89K, and M109A; eeee. K10Q, V49Y, E89K, and M109A; ffff. E53A, E89K, and M109A; or gggg. K10Q, E53A, E89K, and M109A.

[0021] 20 170886321.1 In some embodiments, the present disclosure provides an isolated IL-15 peptide comprising or consisting of the amino acid sequence according to any one of SEQ ID NOs:10-94, or has one or more of the mutations shown in Table 1 and Table 2. In some embodiments, the present disclosure provides an isolated IL-15 peptide encoded by the nucleic acid sequence according to any one of SEQ ID NOs:321-405. III. IL-15 Fusion Proteins and Conjugates In some embodiments, the present disclosure provides a molecule wherein an IL-15 protein is conjugated to another molecule or expressed as part of a fusion protein. For example, the IL-15 may be conjugated to, or expressed as part of a fusion protein with, a binding agent such as a binding agent derived from an antibody or from a non- antibody format. In some embodiments, the binding agent comprises an antibody, or a binding domain derived from an antibody, or antigen binding portions thereof (i.e., an antibody fragment). In some embodiments, the antibody fragment is a Fab, Fab', F(ab')2, Fv, scFv, or a single domain antibody (also referred to as a VHH, VNAR, sdAb, or nanobody). In one aspect, the present disclosure provides an IL-15 mutein that is conjugated to, or expressed as a fusion protein with an antibody, or an antibody fragment (e.g., an antigen-binding antibody fragment), that binds an antigen. In some embodiments, the IL-15 mutant comprises or consists of the amino acid sequence according to any one of SEQ ID NOs:10-94, or has one or more of the mutations shown in Table 1 and Table 2. In some embodiments, the antibody, or an antibody fragment (e.g., an antigen- binding antibody fragment), is attached to the IL-15 mutein by a linker. In some embodiments, the linker is a peptide, e.g., about 2 to 20 amino acids. In some embodiments, the linker is a glycine-serine (GS)-linker, such as a (G4S)n, (SG4)n, or G4(SG4)n linker peptide, wherein n is generally a number between 1 and 10, typically between 2 and 4. In some embodiments, the linker comprises a FLAG tag, a 6His tag, a 8His tag, or an AVI tag. In some embodiments, the linker comprises SEQ ID NO:107. In some embodiments, the linker consists of SEQ ID NO:107. In some embodiments, the linker comprises SEQ ID NO:108. In some embodiments, the linker consists of SEQ

[0022] 21 170886321.1 ID NO:108. In some embodiments, the linker comprises SEQ ID NO:437. In some embodiments, the linker comprises SEQ ID NO:438. Antibodies generally are comprised of a heavy chain and a light chain. Each heavy chain is composed of a variable region (abbreviated as VH) and a constant region. The heavy chain constant region may include three domains CH1, CH2, and CH3 and optionally a fourth domain, CH4. Each of these domains is referred to as an "Fc domain". As used herein, when a binding agent includes an Fc domain, it can include one or more Fc domains, or an entire Fc region, unless otherwise specified by context. Each light chain is composed of a variable region (abbreviated as VL) and a constant region or constant domain. The light chain constant region is a CL domain. The VH and VL regions may be further divided into hypervariable regions referred to as complementarity-determining regions (CDRs) and interspersed with conserved regions referred to as framework regions (FR). Each VH and VL region thus consists of three CDRs and four FRs that are arranged from the N terminus to the C terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. This structure is well known to those skilled in the art. As used herein, and unless the context clearly indicates otherwise, "antibody" refers to an intact antibody comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds (though it will be understood that heavy chain antibodies, which lack light chains, are still encompassed by the term "antibody"), as well as any antigen-binding portion or fragment of an intact antibody that has or retains the ability to bind to the antigen target molecule recognized by the intact antibody, such as, for example, a scFv, Fab, or F(ab')2fragment. Thus, the term "antibody" herein is used in the broadest sense and includes polyclonal and monoclonal antibodies, including intact antibodies and functional (antigen-binding) antibody fragments thereof, including fragment antigen-binding (Fab) fragments, F(ab')2 fragments, Fab' fragments, Fv fragments, recombinant IgG (rIgG) fragments, single chain antibody fragments, including single chain variable fragments (scFv), and single domain antibodies (e.g., sdAb, sdFv, nanobody) fragments. The term encompasses genetically engineered and / or

[0023] 22 170886321.1 otherwise modified forms of immunoglobulins, such as intrabodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies, and heteroconjugate antibodies, multispecific, e.g., bispecific, antibodies, diabodies, triabodies, and tetrabodies, tandem di-scFv, tandem tri-scFv. Unless otherwise stated, the term "antibody" should be understood to encompass functional antibody fragments thereof. The term also encompasses intact or full-length antibodies, including antibodies of any class or sub-class thereof, including IgG and sub-classes thereof, IgM, IgE, IgA, and IgD. As used herein, the terms "antigen binding fragment", "fragment", and "antibody fragment" are used interchangeably to refer to any fragment of an antibody of the disclosure that retains the antigen-binding activity of the antibody. Examples of antibody fragments include, but are not limited to, a single chain antibody, Fab, Fab’, F(ab')2, Fv, and scFv. Human antibodies are known (e.g., van Dijk and van de Winkel, Curr. Opin. Chem. Biol. (2001) 5:368-374). Human antibodies can be produced in transgenic animals (e.g., mice) that are capable, upon immunization, of producing a full repertoire or a selection of human antibodies in the absence of endogenous immunoglobulin production. Transfer of the human germ-line immunoglobulin gene array in such germ- line mutant mice will result in the production of human antibodies upon antigen challenge (see, e.g., Jakobovits, Proc. Natl. Acad. Sci. USA (1993) 90:2551-2555; Jakobovits, Nature (1993) 362:255-258; Bruggemann, Year Immunol. (1993) 7:3340). Human antibodies can also be produced in phage display libraries (Hoogenboom and Winter, J. Mol. Biol. (1992) 227:381-388; Marks et al., J. Mol. Biol. (1991) 222:581- 597). Additional techniques for the preparation of human monoclonal antibodies are known in the art (Cole, Monoclonal Antibodies and Cancer Therapy (1985), Alan R. Liss, p.77; and Boerner et al., J. Immunol. (1991) 147:86-95). Human monoclonal antibodies may be prepared by using improved EBV-B cell immortalization (Traggiai et al., Nat Med. (2004) 10(8):871-5). The term "human antibody" as used herein also

[0024] 23 170886321.1 comprises such antibodies which are modified, e.g., in the variable region, to generate properties according to the antibodies and antibody fragments of the present disclosure. Antibodies according to the present disclosure can be of any isotype (e.g., IgA, IgG, IgM, IgE, IgD; i.e., comprising a α, γ, µ, ɛ, or δ heavy chain). Within the IgG isotype, for example, antibodies may be IgG1, IgG2, IgG3, or IgG4 subclass. In specific embodiments, an antibody of the present disclosure is an IgG1 antibody. Antibodies or antigen binding fragments provided herein may include a κ or a λ light chain. As used herein, the term "variable region" (variable region of a light chain (VL), variable region of a heavy chain (VH)) denotes each variable region polypeptide of the pair of light and heavy chains, which, in most instances, is involved directly in binding the antibody to the antigen. The terms "VL" and "VH" refer to the variable binding region from an antibody light and heavy chain, respectively. The variable binding regions are made up of discrete, well-defined sub-regions known as "complementarity- determining regions" (CDRs) and "framework regions" (FRs). The terms "complementarity-determining region" and "CDR" are synonymous with "hypervariable region" or "HVR," and are known in the art to refer to sequences of amino acids within TCR or antibody variable regions, which confer antigen specificity and / or binding affinity and are separated by framework sequence. In general, there are three CDRs in each variable region of an immunoglobulin binding protein; e.g., for antibodies, the VH and VL regions comprise six CDRs: HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3 (also referred to herein as CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3, respectively). As used herein, a "variant" of a CDR refers to a functional variant of a CDR sequence having up to 1-3 amino acid substitutions, deletions, or combinations thereof. It will be understood that in certain embodiments, an antibody or antigen binding fragment of the present disclosure can comprise all or part of a heavy chain (HC), a light chain (LC), or both. For example, a full-length intact IgG antibody monomer typically includes a VH, a CH1, a CH2, a CH3, a VL, and a CL. Fc components are described further herein. In certain embodiments, an antibody or

[0025] 24 170886321.1 antigen binding fragment of the present disclosure comprises a CDRH1, a CDRH2, a CDRH3, a CDRL1, a CDRL2, and a CDRL3 according to any one of the presently disclosed VH and VL sequences, respectively. Fragments of the antibodies described herein can be obtained from the antibodies by methods that include digestion with enzymes, such as pepsin or papain, and / or by cleavage of disulfide bonds by chemical reduction. Alternatively, fragments of the antibodies can be obtained by cloning and expression of part of the sequences of the heavy or light chains. The present disclosure encompasses single-chain Fv fragments (scFv) derived from the heavy and light chains of an antibody as described herein, including, for example, an scFv comprising the CDRs from an antibody according to the present description, heavy or light chain monomers and dimers, single domain heavy chain antibodies, single domain light chain antibodies, as well as single chain antibodies, in which the heavy and light chain variable domains are joined by a peptide linker. In certain embodiments, an antibody according to the present disclosure, or an antigen binding fragment thereof, comprises a purified antibody, a monoclonal antibody, a single chain antibody, Fab, Fab′, F(ab')2, Fv, or scFv. Throughout this disclosure, antibodies, antigen binding fragments thereof, and fusion proteins may individually or collectively (e.g., in any combination) be referred to as "binding proteins" or "binding agents". Proteins according to the present disclosure may be provided in purified form. For example, an IL-15 or antibody may be present in a composition that is substantially free of other polypeptides, e.g., where less than 90% (by weight), usually less than 60% and more usually less than 50% of the composition is made up of other polypeptides. Binding proteins according to the present disclosure may be immunogenic in human and / or in non-human (or heterologous) hosts; e.g., in mice. For example, an antibody may have an idiotope that is immunogenic in non-human hosts, but not in a human host. Antibodies of the disclosure for human use include those that are not typically isolated from hosts such as mice, goats, rabbits, rats, non-primate mammals,

[0026] 25 170886321.1 or the like, and in some instances are not obtained by humanization or from xeno-mice. Also contemplated herein are variant forms of the disclosed antibodies, which are engineered so as to reduce known or potential immunogenicity and / or other potential liabilities, or to confer a desired structure and / or functionality of the antibody in a non- human animal, such as a mouse (e.g., a "murinized " antibody wherein one or more human amino acid residue, sequence, or motif is replaced by a residue, sequence, or motif that has reduced or abrogated immunogenicity or other liability, or has a desired structure and / or function, in a mouse; e.g., for model studies using a mouse). As used herein, "fusion protein" refers to a protein that, in a single chain, has at least two distinct domains or motifs, wherein the domains or motifs are not naturally found together, or in the given arrangement, in a protein. A polynucleotide encoding a fusion protein may be constructed using PCR, recombinantly engineered, or the like, or such fusion proteins can be synthesized. For example, IL-15 muteins, antibodies, or antigen-binding fragments of antibodies such as those described herein, including but not limited to scFv, may, in certain embodiments, be comprised in a fusion protein that is capable of specifically binding to an antigen as described herein. Immunoglobulin sequences can be aligned to a numbering scheme (e.g., Kabat, Chothia, EU, International Immunogenetics Information System (IMGT), and AHo), which can allow equivalent residue positions to be annotated and for different molecules to be compared using Antigen receptor Numbering And Receptor Classification (ANARCI) software tool (Bioinformatics (2016) 15:298-300). See also Dondelinger et al., Front. Immunol. (2018) 9:2278. As used herein, "specifically binds" or "specific for" refers to an association or union of a binding protein (e.g., an antibody or antigen binding fragment thereof) or a binding domain to a target molecule with an affinity or Ka (i.e., an equilibrium association constant of a particular binding interaction with units of 1 / M) equal to or greater than 105M-1(which equals the ratio of the on-rate [Kon] to the off rate [Koff] for this association reaction), while not significantly associating or uniting with any other molecules or components in a sample. Binding proteins or binding domains may be

[0027] 26 170886321.1 classified as "high-affinity" binding proteins or binding domains or as "low-affinity" binding proteins or binding domains. "High-affinity" binding proteins or binding domains refer to those binding proteins or binding domains having a Ka of at least 107M-1, at least 108M-1, at least 109M-1, at least 1010M-1, at least 1011M-1, at least 1012M-1, or at least 1013M-1. "Low-affinity" binding proteins or binding domains refer to those binding proteins or binding domains having a Ka of up to 107M-1, up to 106M-1, or up to 105M-1. Alternatively, affinity may be defined as an equilibrium dissociation constant (Kd) of a particular binding interaction with units of M (e.g., 10-5M to 10-13M). The terms "binding" and "specifically binding" and similar references do not encompass non-specific sticking. Binding of a binding protein can be determined or assessed using an appropriate assay, such as, for example, Surface Plasmon Resonance (SPR) methods, e.g., a Biacore™ system; kinetic exclusion assays such as KinExA®; and BioLayer interferometry (e.g., using the ForteBio® Octet platform); isothermal titration calorimetry (ITC), or the like, an antigen-binding ELISA (e.g., direct or indirect) with imaging by, e.g., optical density at 450nm, or by flow cytometry, or the like. The term "epitope" or "antigenic epitope" includes any molecule, structure, amino acid sequence, or protein determinant that is recognized and specifically bound by a cognate binding molecule, such as an immunoglobulin or other binding molecule, domain, or protein. Epitopic determinants generally contain chemically active surface groupings of molecules, such as amino acids or sugar side chains, and can have specific three-dimensional structural characteristics, as well as specific charge characteristics. An epitope to which binding protein binds may be linear (continuous) or conformational (discontinuous). A linear or a sequential epitope is an epitope that is recognized by an antibody according to its linear sequence of amino acids, or primary structure. A conformational epitope may be recognized according to a three- dimensional shape and protein structure. In the case of a conformational epitope (3D structure), the amino acid sequence typically forms a 3D structure as epitope and, thus, the amino acids forming the epitope may be or may be not located in adjacent positions

[0028] 27 170886321.1 of the primary structure (i.e., maybe or may be not consecutive amino acids in the amino acid sequence). a. Multispecific Antibodies and Binding Proteins Antibodies and antibody fragments of the present disclosure may, in embodiments, be multispecific (e.g., bispecific, trispecific, tetraspecific, or the like), and may be provided in any multispecific format, as disclosed herein. Multispecific antibodies are monoclonal antibodies that have binding specificities for at least two different sites or antigens. In certain embodiments, an antibody or antigen binding fragment of the present disclosure is a multispecific antibody, such as a bispecific or trispecific antibody. Formats for bispecific antibodies are disclosed in, for example, Spiess et al., Mol. Immunol.67(2):95 (2015), and in Brinkmann and Kontermann, mAbs 9(2):182-212 (2017), which bispecific formats and methods of making the same are incorporated herein by reference and include, for example, Bispecific T cell Engagers (BiTEs), DARTs, Knobs-Into-Holes (KIH) assemblies, scFv-CH3-KIH assemblies, KIH Common Light-Chain antibodies, TandAbs, Triple Bodies, TriBi Minibodies, Fab-scFv, scFv-CH-CL-scFv, F(ab')2-scFv2, tetravalent HCabs, Intrabodies, CrossMabs, Dual Action Fabs (DAFs) (two-in-one or four-in-one), DutaMabs, DT-IgG, Charge Pairs, Fab-arm Exchange, SEEDbodies, Triomabs, LUZ-Y assemblies, Fcabs, κλ-bodies, orthogonal Fabs, DVD-IgGs, IgG(H)-scFv, scFv-(H)IgG, IgG(L)-scFv, scFv-(L)IgG, IgG(L,H)-Fv, IgG(H)-V, V(H)-IgG, IgG(L)-V, V(L)-IgG, KIH IgG-scFab, 2scFv-IgG, IgG-2scFv, scFv4-Ig, Zybody, and DVI-IgG (four-in-one). Bispecific and multi-specific antibodies include the following: an scFv1-ScFv2, an ScFv12-Fc-scFv22, an IgG-scFv, a DVD-Ig, a triomab / quadroma, a two-in-one IgG, a scFv2-Fc, a TandAb, an scFv-HSA-scFv, an scFv-VHH, a Fab-scFv-Fc, a Fab-VHH- Fc, a dAb-IgG, an IgG-VHH, a Tandem scFv-Fc, a (scFv1)2-Fc-(VHH)2, a scFv-Fc, a one-armed tandem scFv-Fc, and a DART-Fc. An IgG-scFv may be an IgG(H)-scFv, scFv-(H)IgG, IgG(L)-scFv, svFc-(L)IgG, 2scFV-IgG, or IgG-2scFv. Techniques for making multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs

[0029] 28 170886321.1 having different specificities (see Milstein and Cuello, Nature (1983) 305:537); WO 93 / 08829; and Traunecker et al., EMBO J. (1991) 10:3655), and "knob-in-hole" engineering (see, e.g., U.S. Pat. No.5,731,168). Multi-specific antibodies may also be made by engineering electrostatic steering effects for making antibody Fc- heterodimeric molecules (WO 2009 / 089004A1); cross-linking of two or more antibodies or fragments (see, e.g., U.S. Pat. No.4,676,980, and Brennan et al., Science (1985) 229:81); using leucine zippers to produce bi-specific antibodies (see, e.g., Kostelny et al., J. Immunol. (1992) 148(5):1547-1553); using "diabody" technology for making bispecific antibody fragments (see, e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA (1993) 90:6444-6448); using single-chain Fv (scFv) dimers (see, e.g., Gruber et al., J. Immunol. (1994) 152:5368); and preparing trispecific antibodies (e.g., Tutt et al. J. Immunol. (1991) 147:60). Engineered antibodies with three or more functional antigen binding sites, including "Octopus antibodies", are also included herein (see, e.g., US 2006 / 0025576A1). Antibodies or antigen binding fragments disclosed herein also include a "Dual Acting Fab" or "DAF" comprising an antigen binding site that binds to two different antigens (see, e.g., US 2008 / 0069820 and Bostrom et al., Science (2009) 323:1610-14). "CrossMab" antibodies are also included herein (see, e.g., WO 2009 / 080251, WO 2009 / 080252, WO2009 / 080253, WO2009 / 080254, and WO2013 / 026833). In some embodiments, the antibodies or antigen binding fragments disclosed herein comprise different antigen-binding sites, fused to one or the other of the two subunits of the Fc domain; thus, the two subunits of the Fc domain may be comprised in two non-identical polypeptide chains. Recombinant co-expression of these polypeptides and subsequent dimerization leads to several possible combinations of the two polypeptides. To improve the yield and purity of the bispecific molecules in recombinant production, it is advantageous to introduce in the Fc domain of the binding agent a modification promoting the association of the desired polypeptides.

[0030] 29 170886321.1 Accordingly, in particular aspects relates to a binding agent (e.g., an antibody or antigen binding fragment thereof) comprising (a) at least a first binding domain, (b) a second binding domain, and (c) a Fc domain composed of a first and a second subunit capable of stable association, wherein the Fc domain comprises a modification promoting the association of the first and second subunit of the Fc domain. The site of most extensive protein-protein interaction between the two subunits of a human IgG Fc domain is in the CH3 domain of the Fc domain. Thus, in one aspect said modification is in the CH3 domain of the Fc domain. In a specific aspect, the Fc modification is a so-called "knob-into-hole" modification, comprising a "knob" modification in one of the two subunits of the Fc domain and a "hole" modification in the other one of the two subunits of the Fc domain. In a particular aspect, the first subunit of the Fc domain comprises the amino acid substitutions S354C and T366W (EU numbering) and the second subunit of the Fc domain comprises the amino acid substitutions Y349C, T366S, and Y407V (numbering according to Kabat EU index). The knob-into-hole technology is known in the art (e.g., U.S. Pat. Nos.5,731,168 and 7,695,936; Ridgway et al., Prot Eng (1996) 9:617-621; Carter, J Immunol Meth (2001) 248:7-15). Generally, the method involves introducing a protuberance ("knob") at the interface of a first polypeptide and a corresponding cavity ("hole") in the interface of a second polypeptide, such that the protuberance can be positioned in the cavity so as to promote heterodimer formation and hinder homodimer formation. Protuberances are constructed by replacing small amino acid side chains from the interface of the first polypeptide with larger side chains (e.g., tyrosine or tryptophan). Compensatory cavities of identical or similar size to the protuberances are created in the interface of the second polypeptide by replacing large amino acid side chains with smaller ones (e.g., alanine or threonine). Accordingly, in some embodiments, in a CH3 domain of an Fc domain an amino acid residue is replaced with an amino acid residue having a larger side chain volume, thereby generating a protuberance within the CH3 domain which is positionable in a cavity within a CH3 domain of a second Fc domain, and in the CH3

[0031] 30 170886321.1 domain of the second Fc domain an amino acid residue is replaced with an amino acid residue having a smaller side chain volume, thereby generating a cavity within the CH3 domain of the second Fc domain within which the protuberance within the CH3 domain of the first Fc domain is positionable. The protuberance and cavity can be made by altering the nucleic acid encoding the polypeptides, e.g., by site-specific mutagenesis, or by peptide synthesis. In a specific embodiment, in the CH3 domain of the first Fc domain the threonine residue at position 366 is replaced with a tryptophan residue (T366W), and in the CH3 domain of the second Fc domain the tyrosine residue at position 407 is replaced with a valine residue (Y407V). In another embodiment, in the second Fc domain additionally the threonine residue at position 366 is replaced with a serine residue (T366S) and the leucine residue at position 368 is replaced with an alanine residue (L368A). In some embodiments, in the first Fc domain additionally the serine residue at position 354 is replaced with a cysteine residue (S354C), and in the second Fc domain additionally the tyrosine residue at position 349 is replaced by a cysteine residue (Y349C). Introduction of these two cysteine residues results in the formation of a disulfide bridge between the two Fc domains that further stabilizes the dimer (Carter (2001), J Immunol Methods 248, 7-15). In some embodiments, the first Fc domain comprises the amino acid substitutions S354C and T366W (EU numbering) and the second Fc domain comprises the amino acid substitutions Y349C, T366S, and Y407V (numbering according to Kabat EU index). In some embodiments, a modification promoting association of the first and the second Fc domains comprises a modification mediating electrostatic steering effects, for example, as described in WO2009 / 089004. Generally, this method involves replacement of one or more amino acid residues at the interface of the two Fc domains by charged amino acid residues so that homodimer formation becomes electrostatically unfavorable but heterodimerization electrostatically favorable. In some embodiments, a binding agent (e.g., an antibody or antigen binding fragment thereof) comprises one or more scFvs or "single-chain variable fragments".

[0032] 31 170886321.1 An scFv is a fusion protein of the variable regions of the heavy (VH) and light chain (VL) variable regions of an antibody, connected with a short linker peptide of ten to about 25 amino acids. The linker is usually rich in glycine for flexibility, as well as serine or threonine for solubility, and can either connect the N-terminus of the VH with the C-terminus of the VL, or vice versa. This protein retains the specificity of the original antibody, despite removal of the constant regions and the introduction of the linker. scFv antibodies are, described in, e.g., Houston, Methods in Enzymol. (1991) 203:46-96. Methods for making scFv molecules and designing suitable peptide linkers are described in, for example, U.S. Pat. No.4,704,692; U.S. Pat. No.4,946,778; Raag and Whitlow, FASEB (1995) 9:73-80; Bird and Walker, TIBTECH (1991) 9:132-137). Binding agents (e.g., an antibody or antigen binding fragment thereof) that are scFv-Fcs have been described by Sokolowska-Wedzina et al., Mol. Cancer Res. (2017) 15(8):1040-1050. In some embodiments, a binding agent (e.g., an antibody or antigen binding fragment thereof) is a "bispecific T cell engager" or BiTE (see, e.g., WO2004 / 106381; WO2005 / 061547; WO2007 / 042261; WO2008 / 119567). This approach utilizes two antibody variable domains arranged on a single polypeptide. For example, a single polypeptide chain can include two single chain Fv (scFv) fragments, each having a variable heavy chain (VH) and a variable light chain (VL) domain separated by a polypeptide linker of a length sufficient to allow intramolecular association between the two domains. This single polypeptide further includes a polypeptide spacer sequence between the two scFv fragments. Each scFv recognizes a different epitope, and these epitopes may be specific for different proteins, such that both proteins are bound by the BiTE. As it is a single polypeptide, the bispecific T cell engager may be expressed using any prokaryotic or eukaryotic cell expression system known in the art, e.g., a CHO cell line. However, specific purification techniques (see, e.g., EP1691833) may be necessary to separate monomeric bispecific T cell engagers from other multimeric species, which may have biological activities other than the intended activity of the monomer. In one exemplary purification scheme, a solution containing secreted

[0033] 32 170886321.1 polypeptides is first subjected to a metal affinity chromatography, and polypeptides are eluted with a gradient of imidazole concentrations. This eluate is further purified using anion exchange chromatography, and polypeptides are eluted using with a gradient of sodium chloride concentrations. Finally, this eluate is subjected to size exclusion chromatography to separate monomers from multimeric species. In some embodiments, a binding agent that is a bispecific antibody is composed of a single polypeptide chain comprising two single chain FV fragments (scFV) fused to each other by a peptide linker. A single-domain antibody is an antibody fragment consisting of a single monomeric variable antibody domain. Single-domain antibodies can be derived from the variable domain of the antibody heavy chain from camelids (e.g., nanobodies or VHH fragments). Furthermore, the term single-domain antibody includes an autonomous human heavy chain variable domain (aVH) or VNAR fragments derived from sharks (see, e.g., Hasler et al., Mol. Immunol. (2016) 75:28-37). Techniques for producing single-domain antibodies (DABs or VHH) are known in the art (e.g., Cossins et al., Prot Express Purif (2006) 51:253-259; Li et al., Immunol. Lett. (2017) 188:89- 95). Single-domain antibodies may be obtained, for example, from camels, alpacas, or llamas by standard immunization techniques (see, e.g., Muyldermans et al., TIBS (2001) 26:230-235; Yau et al., J Immunol Methods (2003) 281:161-75; and Maass et al., J Immunol Methods (2007) 324:13-25). A VHH may have potent antigen-binding capacity and can interact with novel epitopes that are inacessible to conventional VH- VL pairs (see, e.g., Muyldermans et al., TIBS (2001) 26:230-235). Alpaca serum IgG contains about 50% camelid heavy chain only IgG antibodies (HCAbs) (see, e.g., Maass et al., J Immunol Methods (2007) 324:13-25). Alpacas may be immunized with antigens and VHHs can be isolated that bind to and neutralize the target antigen (see, e.g., Maass et al., J Immunol Methods (2007) 324:13-25). PCR primers that amplify alpaca VHH coding sequences have been identified and may be used to construct alpaca VHH phage display libraries, which can be used for antibody fragment isolation by

[0034] 33 170886321.1 standard biopanning techniques well known in the art (see, e.g., Maass et al., J Immunol Methods (2007) 324:13-25). In some embodiments, a binding agent (e.g., an antibody or antigen binding fragment thereof) is a IgG-scFV. IgG-scFv formats include IgG(H)-scFv, scFv-(H)IgG, IgG(L)-scFv, svFc-(L)IgG, 2scFV-IgG, and IgG-2scFv. These and other bispecific antibody formats and methods of making them have been described (e.g., Brinkmann and Kontermann, MAbs (2017) 9(2):182-212; Wang et al., Antibodies (2019) 8:43; Dong et al., MAbs (2011) 3:273-88; Natsume et al., J. Biochem. (2006) 140(3):359- 368; Cheal et al., Mol. Cancer Ther. (2014) 13(7):1803-1812; Bates and Power, Antibodies (2019) 8:28). Igg-like dual-variable domain antibodies (DVD-Ig) have been described (e.g., Wu et al., Nat Biotechnol (2007) 25:1290-97; Hasler et al., Mol. Immunol. (2016) 75:28-37; WO08 / 024188; WO07 / 024715). Triomabs have been described (Chelius et al., Mabs (2010) 2(3):309-319).2-in- 1-IgGs have also been described (Kontermann et al., Drug Discovery Today (2015) 20(7):838-847). Tandem antibody or TandAb have been described (e.g., Kontermann et al., Drug Discovery Today (2015) 20(7):838-847). ScFv-HSA-scFv antibodies have also been described (e.g., Kontermann et al., Drug Discovery Today (2015) 20(7):838-847). In some embodiments, the binding agent (e.g., an antibody or antigen binding fragment thereof) is a scaffold antigen binding protein, such as for example, fibronectin and designed ankyrin repeat proteins (DARPins), which have been used as alternative scaffolds for antigen-binding domains (see, e.g., Gebauer and Skerra, Curr Opin Chem Biol (2009) 13:245-255; Stumpp et al., Drug Discovery Today (2008) 13: 695-701). In some embodiments, a scaffold antigen binding protein is selected from the group consisting of Lipocalins (Anticalin), a Protein A-derived molecule such as Z-domain of Protein A (Affibody), an A-domain (Avimer / Maxibody), a serum transferrin (trans- body); a designed ankyrin repeat protein (DARPin), a fibronectin (AdNectin), a C-type

[0035] 34 170886321.1 lectin domain (Tetranectin); a variable domain of a new antigen receptor beta-lactamase (VNAR fragments), a human gamma-crystallin or ubiquitin (Affilin molecules); a kunitz type domain of human protease inhibitors; and microbodies such as the proteins from the knottin family, peptide aptamers, and fibronectin (adnectin). Lipocalins are a family of extracellular proteins that transport small hydrophobic molecules such as steroids, bilins, retinoids, and lipids. They have a rigid beta-sheet secondary structure with a number of loops at the open end of the conical structure which can be engineered to bind to different target antigens. Anticalins are between 160-180 amino acids in size, and are derived from lipocalins. For further details, see Skerra, Biochim Biophys Acta (2000) 1482: 337-350; U.S. Pat. No.7,250,297 B1; and US2007 / 0224633. Designed Ankyrin Repeat Proteins (DARPins) are derived from Ankyrin which is a family of proteins that mediate attachment of integral membrane proteins to the cytoskeleton. A single ankyrin repeat is a 33-residue motif consisting of two alpha- helices and a beta-turn. They can be engineered to bind different target antigens by randomizing residues in the first alpha-helix and a beta-turn of each repeat. Their binding interface can be increased by increasing the number of modules (a method of affinity maturation). For further details, see Binz et al., J. Mol. Biol. (2003) 332:489- 503; Kohl et al., PNAS (2003) 100(4):1700-1705; Zahnd et al., J. Mol. Biol. (2007) 369(4):1015-1028; and US2004 / 0132028A1. b. Fc Domain Modifications In some embodiments, a binding protein (e.g., antibody or an antibody fragment (e.g., an antigen-binding fragment)) of the present disclosure comprises an Fc moiety. In certain embodiments, the Fc moiety may be derived from human origin, e.g., from human IgG1, IgG2, IgG3, and / or IgG4, or from another Ig class or isotype. In specific embodiments, an antibody or antigen binding fragments can comprise an Fc moiety derived from human IgG1. As used herein, the term "Fc moiety" refers to a sequence comprising, consisting, consisting essentially of, or derived from a portion of an immunoglobulin heavy chain beginning in the hinge region just upstream of the papain cleavage site

[0036] 35 170886321.1 (e.g., residue 216 in native IgG, taking the first residue of heavy chain constant region to be 114) and ending at the C-terminus of the immunoglobulin heavy chain. Accordingly, an Fc moiety may be a complete Fc moiety or a portion (e.g., a domain) thereof. In certain embodiments, a complete Fc moiety comprises a hinge domain, a CH2 domain, and a CH3 domain (e.g., EU amino acid positions 216-446). An additional lysine residue (K) is sometimes present at the extreme C-terminus of the Fc moiety, but is often cleaved from a mature antibody. Amino acid positions within an Fc moiety can be numbered according to the EU numbering system of Kabat (see, e.g., Kabat et al., "Sequences of Proteins of Immunological Interest", U.S. Dept. Health and Human Services, 1983 and 1987). Amino acid positions of an Fc moiety can also be numbered according to the IMGT numbering system (including unique numbering for the C-domain and exon numbering) and the Kabat numbering system. In some embodiments, an Fc moiety comprises at least one of: a hinge (e.g., upper, middle, and / or lower hinge region) domain, a CH2 domain, a CH3 domain, or a variant, portion, or fragment thereof. In some embodiments, an Fc moiety comprises at least a hinge domain, a CH2 domain or a CH3 domain. In further embodiments, the Fc moiety is a complete Fc moiety. The Fc moiety may also comprise one or more amino acid insertions, deletions, or substitutions relative to a naturally occurring Fc moiety. For example, at least one of a hinge domain, CH2 domain, or CH3 domain, or a portion thereof, may be deleted. For example, an Fc moiety may comprise or consist of: (i) hinge domain (or a portion thereof) fused to a CH2 domain (or a portion thereof), (ii) a hinge domain (or a portion thereof) fused to a CH3 domain (or a portion thereof), (iii) a CH2 domain (or a portion thereof) fused to a CH3 domain (or a portion thereof), (iv) a hinge domain (or a portion thereof), (v) a CH2 domain (or a portion thereof), or (vi) a CH3 domain or a portion thereof. An Fc moiety of the present disclosure may be modified such that it varies in amino acid sequence from the complete Fc moiety of a naturally occurring immunoglobulin molecule, while retaining or enhancing at least one desirable function conferred by the naturally occurring Fc moiety, and / or reducing an undesired function

[0037] 36 170886321.1 of a naturally occurring Fc moiety. Such functions include, for example, Fc receptor (FcR) binding, antibody half-life modulation (e.g., by binding to FcRn), ADCC function, protein A binding, protein G binding, and complement binding. Portions of naturally occurring Fc moieties which are involved with such functions have been described in the art. In some embodiments, an Fc region or Fc domain has substantially no binding to at least one Fc receptor selected from FcyRI (CD64), FcyRIIA (CD32a), FcyRIIB (CD32b), FcyRIIIA (CD16a), and FcyRIIIB (CD16b). In some embodiments, an Fc region or domain exhibits substantially no binding to any of the Fc receptors selected from FcyRI (CD64), FcyRIIA (CD32a), FcyRIIB (CD32b), FcyRIIIA (CD16a), and FcyRIIIB (CD16b). As used herein, "substantially no binding" refers to weak to no binding to a selected Fcgamma receptor or receptors. In some embodiments, "substantially no binding" refers to a reduction in binding affinity (e.g., increase in Kd) to a Fc gamma receptor of at least 1000-fold. In some embodiments, an Fc domain or region is an Fc null. As used herein, an "Fc null" refers to an Fc region or Fc domain that exhibits weak to no binding to any of the Fcgamma receptors. In some embodiments, an Fc null domain or region exhibits a reduction in binding affinity (e.g., increase in Kd) to Fc gamma receptors of at least 1000-fold. In some embodiments, an Fc domain has reduced or substantially no effector function activity. As used herein, "effector function activity" refers to antibody dependent cellular cytotoxicity (ADCC), antibody dependent cellular phagocytosis (ADCP), and / or complement dependent cytotoxicity (CDC). In some embodiments, an Fc domain exhibits reduced ADCC, ADCP, or CDC activity, as compared to a wild- type Fc domain. In some embodiments, an Fc domain exhibits a reduction in ADCC, ADCP, and CDC, as compared to a wild-type Fc domain. In some embodiments, an Fc domain exhibits substantially no effector function (i.e., the ability to stimulate ADCC, ADCP, or CDC). As used herein, "substantially no effector function" refers to a reduction in effector function activity of at least 1000-fold, as compared to a wild-type Fc domain.

[0038] 37 170886321.1 In some embodiments, an Fc domain has reduced or no ADCC activity. As used herein reduced or no ADCC activity refers to a decrease in ADCC activity of an Fc domain by of a factor of at least 10, at least 20, at least 30, at least 50, at least 100, or at least 500. In some embodiments, an Fc domain has reduced or no CDC activity. As used herein reduced or no CDC activity refers to a decrease in CDC activity of an Fc domain by of a factor of at least 10, at least 20, at least 30, at least 50, at least 100, or at least 500. In vitro and / or in vivo cytotoxicity assays can be conducted to confirm the reduction / depletion of ADCC and / or CDC activity. For example, Fc receptor (FcR) binding assays can be conducted to ensure that the antibody lacks Fcgamma receptor (hence likely lacking ADCC activity). The primary cells for mediating ADCC, NK cells, express FcgammaRIII only, whereas monocytes express FcgammaRI, FcgammaRII, and FcgammaRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol.9:457-492 (1991). Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest are described in U.S. Pat. No.5,500,362 (see, e.g., Hellstrom et al. Proc. Nat’l Acad. Sci. USA (1986) 83:7059-7063; Hellstrom et al., Proc. Nat’l Acad. Sci. USA (1985) 82:1499-1502); U.S. Pat. No.5,821,337 (see Bruggemann et al., J. Exp. Med. (1987) 166:1351-1361). Alternatively, non-radioactive assays methods may be employed (see, e.g., ACTITM non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, Calif.; and CytoTox 96TM non-radioactive cytotoxicity assay (Promega, Madison, Wis.). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al., Proc. Nat’l Acad. Sci. USA (1998) 95:652-656. C1q binding assays may also be carried out to confirm that an antibody or Fc domain or region is unable to bind C1q and hence lacks CDC activity or has reduced

[0039] 38 170886321.1 CDC activity. See, e.g., C1q and C3c binding ELISA in WO2006 / 029879 and WO2005 / 100402. To assess complement activation, a CDC assay may be performed (see, e.g., Gazzano-Santoro et al., J. Immunol. Methods (1996) 202:163; Cragg et al., Blood (2003) 101:1045-1052; and Cragg and Glennie, Blood (2004) 103:2738-2743). In some embodiments, an Fc domain has reduced or no ADCP activity. As used herein reduced or no ADCP activity refers to a decrease in ADCP activity of an Fc domain by of a factor of at least 10, at least 20, at least 30, at least 50, at least 100, or at least 500. ADCP binding assays may also be carried out to confirm that an antibody or Fc domain or region lacks ADCP activity or has reduced ADCP activity. See, e.g., US2019 / 0079077 and US2019 / 0048078 and the references disclosed therein. Antibodies with reduced effector function activity include those with substitution of one or more of Fc region residues 238, 265, 269, 270, 297, 327, and 329 (see U.S. Pat. No.6,737,056). Such Fc mutants include Fc mutants with substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327, including the so- called “DANA” Fc mutant with substitution of residues 265 and 297 to alanine (see U.S. Pat. No.7,332,581). Certain antibody variants with diminished binding to FcRs are also known (see, e.g., U.S. Pat. No.6,737,056; WO2004 / 056312; Shields et al., J. Biol. Chem. (2001) 9(2):6591-6604). In certain embodiments, a binding agent comprises an Fc domain or region with one or more amino acid substitutions which diminish FcgammaR binding, e.g., substitutions at positions 234 and 235 of the Fc region (EU numbering of residues). In some embodiments, the substitutions are L234A and L235A (LALA). In some embodiments, the Fc domain further comprises D265A and / or P329G in an Fc region derived from a human IgG1 Fc region. In some embodiments, the substitutions are L234A, L235A, and P329G (LALA-PG) in an Fc region derived from a human IgG1 Fc region. (See, e.g., WO 2012 / 130831). In some embodiments, the substitutions are L234A, L235A, and D265A (LALA-DA) in an Fc region derived from a human IgG1 Fc region.

[0040] 39 170886321.1 In some embodiments, alterations are made in the Fc region that result in altered (i.e., either diminished) C1q binding and / or Complement Dependent Cytotoxicity (CDC) (see, e.g., U.S. Pat. No.6,194,551; WO99 / 51642; Idusogie et al., J. Immunol. (2000) 164:4178-4184). c. Anti-CD8 Antibodies and Antigen-Binding Fragments Thereof CD8alpha (CD8α) is a protein that is expressed on T cells. CD8alpha polypeptides include, but are not limited to, those having the amino acid sequences set forth in NP_001759.3, NP001139345.1, NP_741969.1, NP_001369627.1, NP_757362.1, NP_001171571.1, NP_742100.1, NP_742099.1, and NP_004922; these amino acid sequences are incorporated by reference herein. In some embodiments, an IL-15 mutein is conjugated to or expressed as a fusion protein comprising an anti-CD8α antibody or antigen-binding fragment thereof. For example, in some embodiments, the antibody or fragment thereof comprises: a light chain variable region (VL) having CDRL1, CDRL2, and CDRL3 amino acid sequences according to SEQ ID NO:97, SEQ ID NO:98, and SEQ ID NO:99, respectively; and a heavy chain variable region (VH) having CDRH1, CDRH2, and CDRH3 amino acid sequences according to SEQ ID NO:101, SEQ ID NO:102, and SEQ ID NO:103, respectively. In some embodiments, the antibody or fragment thereof comprises: a light chain variable region (VL) comprising or consisting of the amino acid sequence according to SEQ ID NO:96; and a heavy chain variable region (VH) comprising or consisting of the amino acid sequence according to SEQ ID NO:100. In some embodiments, the antibody or fragment thereof comprises: a first chain, a second chain, and a third chain, wherein: (i) the first chain is a light chain comprising or consisting of the amino acid sequence according to SEQ ID NO:104; (ii) the second chain is a heavy chain comprising or consisting of the amino acid sequence according to SEQ ID NO:106; and (iii) the third chain is a heavy chain comprising or consisting of the amino acid sequence according to SEQ ID NO:105 covalently connected to the IL-15 peptide by a linker. In some embodiments, the linker comprises or consists of the amino acid

[0041] 40 170886321.1 sequence according to SEQ ID NO:107 or 108. In some embodiments, the IL-15 peptide / linker is covalently connected at the C-terminal end of the third chain. In some embodiments, the present disclosure provides an isolated antibody-IL- 15 fusion protein comprising: a first peptide chain, a second peptide chain, and a third peptide chain, wherein: (i) the first peptide chain comprises or consists of the amino acid sequence according to SEQ ID NO:104; (ii) the second peptide chain comprises or consists of the amino acid sequence according to SEQ ID NO:106; and (iii) the third peptide chain comprises or consists of the amino acid sequence according to any one of SEQ ID NOs:119-204. In some embodiments, the present disclosure provides a fusion protein comprising: a first chain, a second chain, and a third chain, wherein: (i) the first chain is a light chain comprising or consisting of the amino acid sequence according to SEQ ID NO:104; (ii) the second chain is a heavy chain comprising or consisting of the amino acid sequence according to SEQ ID NO:106; and (iii) the third chain is a heavy chain comprising or consisting of the amino acid sequence according to SEQ ID NO:105; wherein a IL-15 peptide is covalently connected to the C-terminal end of the second chain or the third chain, e.g., by a linker. In some embodiments, the IL-15 peptide is covalently connected to the C-terminal end of the second chain by a linker. In some embodiments, the IL-15 peptide is covalently connected to the C-terminal end of the third chain by a linker. In some embodiments, the linker comprises or consists of the amino acid sequence according to SEQ ID NO:107 or 108. d. Anti-KIR Antibodies and Antigen-Binding Fragments Thereof KIR2DL1 is a protein expressed on NK cells and on some T cells. It is also known as CD158A, KIR-K64, KIR221, KIR2DL3, NKAT, NKAT-1, NKAT1, and p58.1. KIR2DL1 polypeptides include, but are not limited to, those having the amino acid sequence set forth in NP_055033.2; this amino acid sequence is incorporated by reference herein. KIR2DL2 is a protein expressed on NK cells and on some T cells. It is also known as CD158B1, CD158b, NKAT-6, NKAT6, and p58.2. KIR2DL2 polypeptides

[0042] 41 170886321.1 include, but are not limited to, those having the amino acid sequence set forth in NP_055034.2; this amino acid sequence is incorporated by reference herein. KIR2DL3 is a protein expressed on NK cells and on some T cells. It is also known as CD158B2, CD158b, GL183, KIR-023GB, KIR-K7b, KIR-K7c, KIR2DL, KIR2DS5, KIRCL23, NKAT, NKAT2, NKAT2A, NKAT2B, and p58. KIR2DL3 polypeptides include, but are not limited to, those having the amino acid sequence set forth in NP_056952.2; this amino acid sequence is incorporated by reference herein. KIR3DL1 is a protein expressed on NK cells and on some T cells. It is also known as CD158E1, KIR, KIR2DL5B, KIR3DL1 / S1, NKAT-3, NKAT3, NKB1, and NKB1B. KIR3DL1 polypeptides include, but are not limited to, those having the amino acid sequences set forth in NP_037421.2 and NP_001309097.1; these amino acid sequences are incorporated by reference herein. KIR3DL2 is a protein expressed on NK cells and on some T cells. It is also known as 3DL2, CD158K, KIR-3DL2, NKAT-4, NKAT4, NKAT4B, and p140. KIR3DL2 polypeptides include, but are not limited to, those having the amino acid sequences set forth in NP_006728.2 and NP_001229796.1; these amino acid sequences are incorporated by reference herein. In some embodiments, an IL-15 mutein is conjugated to or expressed as a fusion protein comprising an anti-KIR antibody or antigen-binding fragment thereof. For example, in some embodiments, the antibody or fragment thereof comprises: a light chain variable region (VL) having CDRL1, CDRL2, and CDRL3 amino acid sequences according to SEQ ID NO:407, SEQ ID NO:408, and SEQ ID NO:409, respectively; and a heavy chain variable region (VH) having CDRH1, CDRH2, and CDRH3 amino acid sequences according to SEQ ID NO:411, SEQ ID NO:412, and SEQ ID NO:413, respectively. In some embodiments, the antibody or fragment thereof comprises: a light chain variable region (VL) comprising or consisting of the amino acid sequence according to SEQ ID NO:406; and a heavy chain variable region (VH) comprising or consisting of the amino acid sequence according to SEQ ID NO:410. In some embodiments, the antibody or fragment thereof comprises: a first chain, a second chain,

[0043] 42 170886321.1 and a third chain, wherein: (i) the first chain is a light chain comprising or consisting of the amino acid sequence according to SEQ ID NO:414; (ii) the second chain is a heavy chain comprising or consisting of the amino acid sequence according to SEQ ID NO:416; and (iii) the third chain is a heavy chain comprising or consisting of the amino acid sequence according to SEQ ID NO:415 covalently connected to the IL-15 peptide by a linker. In some embodiments, the linker comprises or consists of the amino acid sequence according to SEQ ID NO:107, 108, 437, or 438. In some embodiments, the present disclosure provides an isolated antibody-IL- 15 fusion protein comprising: a first peptide chain, a second peptide chain, and a third peptide chain, wherein: (i) the first peptide chain comprises or consists of the amino acid sequence according to SEQ ID NO:414; (ii) the second peptide chain comprises or consists of the amino acid sequence according to SEQ ID NO:416; and (iii) the third peptide chain comprises or consists of the amino acid sequence according to any one of SEQ ID NOs:417-425. e. Anti-KIR / Anti-CD8 Bispecific Antibodies and Antigen-Binding Fragments Thereof In some embodiments, an IL-15 mutein is conjugated to or expressed as a fusion protein comprising a bispecific anti-KIR / anti-CD8 antibody or antigen-binding fragment thereof. For example, in some embodiments, the antibody or fragment thereof comprises an anti-KIR-targeting portion comprising: a light chain variable region (VL) having CDRL1, CDRL2, and CDRL3 amino acid sequences according to SEQ ID NO:407, SEQ ID NO:408, and SEQ ID NO:409, respectively; and a heavy chain variable region (VH) having CDRH1, CDRH2, and CDRH3 amino acid sequences according to SEQ ID NO:411, SEQ ID NO:412, and SEQ ID NO:413, respectively. In some embodiments, the anti-KIR-targeting portion comprises: a light chain variable region (VL) comprising or consisting of the amino acid sequence according to SEQ ID NO:406; and a heavy chain variable region (VH) comprising or consisting of the amino acid sequence according to SEQ ID NO:410. In some embodiments, the antibody or fragment thereof comprises an anti-CD8-targeting portion comprising: a light chain

[0044] 43 170886321.1 variable region (VL) having CDRL1, CDRL2, and CDRL3 amino acid sequences according to SEQ ID NO:97, SEQ ID NO:98, and SEQ ID NO:99, respectively; and a heavy chain variable region (VH) having CDRH1, CDRH2, and CDRH3 amino acid sequences according to SEQ ID NO:101, SEQ ID NO:102, and SEQ ID NO:103, respectively. In some embodiments, the anti-CD8-targeting portion comprises: a light chain variable region (VL) comprising or consisting of the amino acid sequence according to SEQ ID NO:96; and a heavy chain variable region (VH) comprising or consisting of the amino acid sequence according to SEQ ID NO:100. In some embodiments, the present disclosure provides an IL-15 (e.g., a wild- type IL-15 or an IL-15 mutein) conjugated to or expressed as a fusion protein comprising a bispecific anti-KIR / anti-CD8 antibody or antigen-binding fragment thereof, wherein the antibody or fragment thereof comprises: (1) a first light chain variable region (VL) having CDRL1, CDRL2, and CDRL3 amino acid sequences according to SEQ ID NO:448, SEQ ID NO:449, and SEQ ID NO:450, respectively; and a first heavy chain variable region (VH) having CDRH1, CDRH2, and CDRH3 amino acid sequences according to SEQ ID NO:451, SEQ ID NO:452, and SEQ ID NO:453, respectively; and (2) a second light chain variable region (VL) having CDRL1, CDRL2, and CDRL3 amino acid sequences according to SEQ ID NO:440, SEQ ID NO:441, and SEQ ID NO:442, respectively; and a second heavy chain variable region (VH) having CDRH1, CDRH2, and CDRH3 amino acid sequences according to SEQ ID NO:443, SEQ ID NO:444, and SEQ ID NO:445, respectively. In some embodiments, the present disclosure provides an IL-15 (e.g., a wild- type IL-15 or an IL-15 mutein) conjugated to or expressed as a fusion protein comprising a bispecific anti-KIR / anti-CD8 antibody or antigen-binding fragment thereof, wherein the antibody or fragment thereof comprises: (1) a first light chain variable region (VL) comprising or consisting of the amino acid sequence according to SEQ ID NO:454; and a first heavy chain variable region (VH) comprising or consisting of the amino acid sequence according to SEQ ID NO:455; and (2) a second light chain variable region (VL) comprising or consisting of the amino acid sequence according to

[0045] 44 170886321.1 SEQ ID NO:446; and a second heavy chain variable region (VH) comprising or consisting of the amino acid sequence according to SEQ ID NO:447. In some embodiments, the present disclosure provides an IL-15 (e.g., a wild- type IL-15 or an IL-15 mutein) conjugated to or expressed as a fusion protein comprising a bispecific anti-KIR / anti-CD8 antibody or antigen-binding fragment thereof, wherein the antibody or fragment thereof comprises: (i) a light chain comprising or consisting of the amino acid sequence according to SEQ ID NO:456; (ii) a heavy chain comprising or consisting of the amino acid sequence according to SEQ ID NO:457; and (iii) a scFv-heavy chain comprising or consisting of the amino acid sequence according to SEQ ID NO:458. IV. Modulation of CD8+KIR+ Regulatory T cells Certain regulatory T cells (or "Tregs") are characterized in human peripheral blood mononuclear cells (PBMC) by the phenotype of being CD8+ KIR+, and are typically MHC Class I restricted. CD8 Tregs regulate immune balance by eliminating self-reactive and potentially pathogenic CD4+ T cells. Control of pathogenic CD4+ T cells appears impaired in patients with rheumatoid arthritis and systemic lupus erythematosus (SLE), in part due to reduced prevalence and function of CD8+ Tregs. Animal models of rheumatologic diseases suggest that increasing the prevalence of CD8+ Treg may correct immune system imbalance and reduce autoimmune disease pathology and progression. IL-15 is a pleiotropic cytokine that results in activation and proliferation of various lymphocyte populations. IL-15 expands CD8+ Treg effectively in vitro, but also expands conventional CD8+ and NK cells. To limit the effects of IL-15 to CD8+ Treg, IL-15 can be delivered as an antibody-tethered IL-15 mutein. Point mutations at IL-15 residues that interact with the IL-15 receptor chains can reduce binding affinity between the IL-15 mutein and the IL-15 receptor complex, resulting in a mutein that has greatly reduced potency across all cell subsets. Fusion of the IL-15 mutein to a cell-specific targeting antibody can restore IL-15 mutein functions in only select immune cell subsets.

[0046] 45 170886321.1 In some embodiments, the KIR proteins expressed by the cells can include one or more of the inhibitory KIR proteins, e.g., KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL5, KIR3DL1, and KIR3DL2; and may specifically include one or more of KIR2DL2, KIR2DL3, and KIR3DL1. In some embodiments, the CD8+KIR+ Tregs are not HLA E (Qa-1b) restricted. (See, e.g., Lohwasser et al., International Immunology (2001) 13:321-327; Sarantopoulos et al., J. Clin. Invest. (2004) 114(9):1218-1221, for a general explanation of murine Qa-1b and human HLA E restriction.) In some embodiments, the CD8+KIR+ Tregs can also be characterized as being CD44+, CD122+, and are not HLA E (Qa-1b) restricted. In some embodiments, the CD8+KIR+ Tregs can also be characterized as being CD28-. In some embodiments, the CD8+KIR+ Tregs can also be characterized as being CD28-, CD44+, and CD122+. In some embodiments, the CD8+KIR+ Tregs can also be characterized as being CD28-, CD44+, and CD122+, and are not HLA E (Qa-1b) restricted. In some embodiments, the CD8+KIR+ Tregs express the following antigens: CD3, CD8, PD-1, CD16, CD122, CD39, CXCR3, ICOS, CD103, and inhibitory KIR proteins. In some embodiments, CD8+KIR+ Tregs express one or more of the following antigens: CD3, CD27, CD38, CD39, CD40L, CD45RA, CD45RB, CD45RO, CD73, CD103 (ITGAE), CD122, CD166, CD177, CCR7, CXCR3, CXCR5, HLA-DR, ICOS, LAG-3 / CD223, OX-40, PD-1, S1000A8 / 9, TIM-3, TLT-2, 2B4, and 41BB. In some embodiments, CD8+KIR+ Tregs express one or more of the following antigens: CD3, CD5, CD16, CD27, CD38, CD39, CD40L, CD45RA, CD45RB, CD45RO, CD73, CD103 (ITGAE), CD122, CD166, CD177, CCR7, CXCR3, CXCR5, HLA-DR, ICOS, KLRB1, KLRG1, LAG-3 / CD223, NKG2C, NKG2D, OX-40, PD-1, S1000A8 / 9, TIM- 3, TLT-2, 2B4, and 41BB. In some embodiments, CD8+KIR+ Tregs express one or more of the following antigens: CD39, KLRB1, KLRG1, NKG2C, NKG2D, CXCR3, and CD122. Some embodiments provided herein are methods or compositions relating to binding agents comprising binding domains that specifically bind to antigens expressed

[0047] 46 170886321.1 on CD8+KIR+ regulatory T cells (Tregs). In some embodiments, the CD8+KIR+ Tregs are MHC class I restricted. In some embodiments, the CD8+KIR+ Tregs are not MHC Qa-1 (HLA-E) restricted. Also provided in further embodiments are methods of using the binding agents for the treatment of autoimmune disease, infectious disease, or cancer. V. Production of Proteins IL-15 proteins described herein can be prepared by methods known in the art, including synthetic and recombinant methods. The proteins may be made by chemical synthesis. The proteins may be made by generating a polynucleotide encoding them and expressing the polynucleotide in a cell using various methods known in the art. In various embodiments, proteins described herein can be produced in human, murine, or other animal-derived cells lines. Recombinant DNA expression can be used to produce IL-15 muteins as well as fusion proteins thereof. This allows the production of antibodies as well as a spectrum of antigen binding portions and other binding agents (including fusion proteins) in a host species of choice. The production of antibodies, antigen binding portions thereof, and other binding agents in bacteria, yeast, transgenic animals, and chicken eggs are also alternatives for cell-based production systems. Nucleic acid molecules encoding the amino acid sequence of a protein described herein can be prepared by a variety of methods known in the art. These methods include, but are not limited to, preparation of synthetic nucleotide sequences encoding the protein. In addition, oligonucleotide-mediated (or site-directed) mutagenesis, PCR- mediated mutagenesis, and cassette mutagenesis can be used to prepare nucleotide sequences encoding the protein. A nucleic acid sequence encoding at least a protein described herein can be recombined with vector DNA in accordance with conventional techniques, such as, for example, blunt-ended or staggered-ended termini for ligation, restriction enzyme digestion to provide appropriate termini, filling in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and ligation with appropriate ligases. Techniques for such manipulations are known in the art (see, e.g., Maniatis et al., Molecular Cloning, Lab. Manual (Cold Spring Harbor Lab. Press,

[0048] 47 170886321.1 NY, 1982 and 1989); Ausubel et al., Current Protocols in Molecular Biology (John Wiley & Sons), 1987-1993), and can be used to construct nucleic acid sequences and vectors that encode the protein. Where a binding agent as described herein comprises antibodies or antigen binding portions thereof, in some embodiments, a VH polypeptide is encoded by a first nucleic acid. In some embodiments, a VL polypeptide is encoded by a second nucleic acid. In some embodiments, the VH and VL polypeptides are encoded by one nucleic acid. A nucleic acid molecule, such as DNA, is said to be "capable of expressing" a polypeptide if it contains nucleotide sequences that contain transcriptional and translational regulatory information and such sequences are "operably linked" to nucleotide sequences that encode the polypeptide. An operable linkage is a linkage in which the regulatory DNA sequences and the DNA sequence sought to be expressed (e.g., an antibody or antigen binding portion thereof) are connected in such a way as to permit gene expression of a polypeptide(s) or antigen binding portions in recoverable amounts. The precise nature of the regulatory regions needed for gene expression may vary from organism to organism, as is well known in the analogous art. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd ed.) (1989) Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press; Ausubel et al., Current Protocols in Molecular Biology (John Wiley & Sons) (1987-1993). Accordingly, the expression of a protein as described herein can occur in either prokaryotic or eukaryotic cells. Suitable hosts include bacterial or eukaryotic hosts, including yeast, insects, fungi, bird, and mammalian cells either in vivo or in situ, or host cells of mammalian, insect, bird, or yeast origin. The mammalian cell or tissue can be of human, primate, hamster, rabbit, rodent, cow, pig, sheep, horse, goat, dog, or cat origin, but any other mammalian cell may be used. Further, by use of, for example, the yeast ubiquitin hydrolase system, in vivo synthesis of ubiquitin-transmembrane polypeptide fusion proteins can be accomplished. The fusion proteins so produced can be processed in vivo or purified and processed in vitro, allowing synthesis of an antibody or antigen binding portion thereof as described herein with a specified amino

[0049] 48 170886321.1 terminus sequence. Moreover, problems associated with retention of initiation codon- derived methionine residues in direct yeast (or bacterial) expression may be avoided. (See, e.g., Sabin et al., Bio / Technol. (1989) 7:705-709; Miller et al., Bio / Technol. (1989) 7:698-704.) Any of a series of yeast gene expression systems incorporating promoter and termination elements from the actively expressed genes coding for glycolytic enzymes produced in large quantities when yeast are grown in medium rich in glucose can be utilized to obtain recombinant antibodies or antigen-binding portions thereof or other binding agents. Known glycolytic genes can also provide very efficient transcriptional control signals. For example, the promoter and terminator signals of the phosphoglycerate kinase gene can be utilized. Production of antibodies or antigen-binding portions thereof and other binding agents in insects can be achieved, for example, by infecting an insect host with a baculovirus engineered to express a polypeptide by methods known to those of ordinary skill in the art. See Ausubel et al., Current Protocols in Molecular Biology (John Wiley & Sons) (1987-1993). In some embodiments, an introduced nucleic acid sequence encoding a protein is incorporated into a plasmid or viral vector capable of autonomous replication in a recipient host cell. Any of a wide variety of vectors can be employed for this purpose and are known and available to those of ordinary skill in the art. See, e.g., Ausubel et al., Current Protocols in Molecular Biology (John Wiley & Sons) (1987-1993). Factors of importance in selecting a particular plasmid or viral vector include: the ease with which recipient cells that contain the vector may be recognized and selected from those recipient cells which do not contain the vector; the number of copies of the vector which are desired in a particular host; and whether it is desirable to be able to "shuttle" the vector between host cells of different species. Exemplary prokaryotic vectors known in the art include plasmids such as those capable of replication in E. coli. Other gene expression elements useful for the expression of DNA encoding antibodies or antigen-binding portions thereof and other binding agents include, but are not limited to, (a) viral transcription promoters and their

[0050] 49 170886321.1 enhancer elements, such as the SV40 early promoter (Okayama et al., Mol. Cell. Biol. (1983) 3:280), Rous sarcoma virus LTR (Gorman et al., PNAS (1982) 79:6777), and Moloney murine leukemia virus LTR (Grosschedl et al., Cell (1985) 41:885); (b) splice regions and polyadenylation sites such as those derived from the SV40 late region (Okayarea et al., Mol. Cell. Biol. (1983) 3:280), and (c) polyadenylation sites such as in SV40 (Okayama et al., Mol. Cell. Biol. (1983) 3:280). Immunoglobulin-encoding DNA genes can be expressed as described by Weidle et al., 51 Gene (1987)51:21, using as expression elements the SV40 early promoter and its enhancer, the mouse immunoglobulin H chain promoter enhancers, SV40 late region mRNA splicing, rabbit S-globin intervening sequence, immunoglobulin and rabbit S-globin polyadenylation sites, and SV40 polyadenylation elements. For immunoglobulin-encoding nucleotide sequences, the transcriptional promoter can be, for example, human cytomegalovirus, the promoter enhancers can be cytomegalovirus and mouse / human immunoglobulin. In some embodiments, for expression of DNA coding regions in rodent cells, the transcriptional promoter can be a viral LTR sequence, the transcriptional promoter enhancers can be either or both the mouse immunoglobulin heavy chain enhancer and the viral LTR enhancer, and the polyadenylation and transcription termination regions. In other embodiments, DNA sequences encoding other proteins are combined with the above-recited expression elements to achieve expression of the proteins in mammalian cells. For expression of an antibody or antigen-binding portion thereof, each coding region or gene fusion may be assembled in, or inserted into, an expression vector. Recipient cells capable of expressing the variable region(s) or antigen binding portions thereof are then transfected singly with nucleotides encoding an antibody or an antibody polypeptide or antigen-binding portion thereof, or are co-transfected with a polynucleotide(s) encoding VH and a VL chain coding regions. The transfected recipient cells are cultured under conditions that permit expression of the incorporated

[0051] 50 170886321.1 coding regions and the expressed antibody chains or intact antibodies or antigen binding portions are recovered from the culture. In some embodiments, nucleic acids containing the coding regions encoding an antibody or antigen-binding portion thereof are assembled in separate expression vectors that are then used to co-transfect a recipient host cell. Each vector can contain one or more selectable genes. For example, in some embodiments, two selectable genes are used, a first selectable gene designed for selection in a bacterial system and a second selectable gene designed for selection in a eukaryotic system, wherein each vector has a set of coding regions. This strategy results in vectors which first direct the production, and permit amplification, of the nucleotide sequences in a bacterial system. The DNA vectors so produced and amplified in a bacterial host are subsequently used to co- transfect a eukaryotic cell, and allow selection of a co-transfected cell carrying the desired transfected nucleic acids (e.g., encoding antibody heavy and light chains). Non- limiting examples of selectable genes for use in a bacterial system are the gene that confers resistance to ampicillin and the gene that confers resistance to chloramphenicol. Selectable genes for use in eukaryotic transfectants include the xanthine guanine phosphoribosyl transferase gene (designated gpt) and the phosphotransferase gene from Tn5 (designated neo). Alternatively, the fused nucleotide sequences encoding VH and VL chains can be assembled on the same expression vector. For transfection of the expression vectors and production of the antibodies or antigen binding portions thereof or other binding agents, the recipient cell line can be a Chinese Hamster ovary cell line (e.g., DG44) or a myeloma cell. Myeloma cells can synthesize, assemble, and secrete immunoglobulins encoded by transfected immunoglobulin genes and possess the mechanism for glycosylation of the immunoglobulin. For example, in some embodiments, the recipient cell is the recombinant Ig-producing myeloma cell SP2 / 0. SP2 / 0 cells only produce immunoglobulins encoded by the transfected genes. Myeloma cells can be grown in culture or in the peritoneal cavity of a mouse, where secreted immunoglobulin can be obtained from ascites fluid.

[0052] 51 170886321.1 An expression vector encoding an antibody or antigen-binding portion thereof or other binding agent can be introduced into an appropriate host cell by any of a variety of suitable means, including such biochemical means as transformation, transfection, protoplast fusion, calcium phosphate-precipitation, and application with polycations such as diethylaminoethyl (DEAE) dextran, and such mechanical means as electroporation, direct microinjection, and microprojectile bombardment, as known to one of ordinary skill in the art (see Johnston et al., Science (1988) 240:1538). Yeast provides certain advantages over bacteria for the production of immunoglobulin heavy and light chains. Yeasts carry out post-translational peptide modifications including glycosylation. A number of recombinant DNA strategies exist that utilize strong promoter sequences and high copy number plasmids which can be used for production of the desired proteins in yeast. Yeast recognizes leader sequences of cloned mammalian gene products and secretes polypeptides bearing leader sequences (i.e., pre-polypeptides). See, e.g., Hitzman et al., 11th Intl. Conf. Yeast, Genetics & Molec. Biol. (Montpelier, France, 1982). Yeast gene expression systems can be routinely evaluated for the levels of production, secretion, and the stability of antibodies, and assembled antibodies and antigen binding portions thereof. Various yeast gene expression systems incorporating promoter and termination elements from the actively expressed genes coding for glycolytic enzymes produced in large quantities when yeasts are grown in media rich in glucose can be utilized. Known glycolytic genes can also provide very efficient transcription control signals. For example, the promoter and terminator signals of the phosphoglycerate kinase (PGK) gene can be utilized. Another example is the translational elongation factor 1alpha promoter. A number of approaches can be taken for evaluating optimal expression plasmids for the expression of immunoglobulins in yeast. See II DNA Cloning 45, (Glover, ed., IRL Press, 1985) and, e.g., U.S. Publication No. US 2006 / 0270045 A1. Bacterial strains can also be utilized as hosts for the production of the antibody molecules or antigen binding portions thereof or other binding agents described herein.

[0053] 52 170886321.1 E. coli K12 strains such as E. coli W3110, Bacillus species, enterobacteria such as Salmonella typhimurium or Serratia marcescens, and various Pseudomonas species can be used. Plasmid vectors containing replicon and control sequences which are derived from species compatible with a host cell are used in connection with these bacterial hosts. The vector carries a replication site, as well as specific genes which are capable of providing phenotypic selection in transformed cells. A number of approaches can be taken for evaluating the expression plasmids for the production of antibodies and antigen binding portions thereof in bacteria (see II DNA Cloning 45, Glover, ed., IRL Press, 1985; Ausubel et al., Current Protocols in Molecular Biology (John Wiley & Sons), 1987-1993; Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd ed.) (1989) Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press; Coligan et al. (eds), Current Protocols in Immunology, Greene Publishing Assoc and Wiley Interscience, NY (1992- 1996)). Host mammalian cells can be grown in vitro or in vivo. Mammalian cells provide post-translational modifications to immunoglobulin molecules including leader peptide removal, folding and assembly of VH and VL chains, glycosylation of the antibody molecules, and secretion of functional antibody and / or antigen binding portions thereof. Mammalian cells that can be useful as hosts for the production of antibody proteins, in addition to the cells of lymphoid origin described above, include cells of fibroblast origin, such as Vero or CHO-K1 cells. Exemplary eukaryotic cells that can be used to express immunoglobulin polypeptides include, but are not limited to, COS cells, including COS 7 cells; 293 cells, including 293-6E cells; CHO cells, including CHO-S, CHO-K1, and DG44 cells; PERC6TMcells (Crucell); and NSO cells. In some embodiments, a particular eukaryotic host cell is selected based on its ability to make desired post-translational modifications to the heavy chains and / or light chains. For example, in some embodiments, CHO cells produce polypeptides that have a higher level of sialylation than the same polypeptide produced in 293 cells.

[0054] 53 170886321.1 In some embodiments, one or more proteins disclosed herein can be produced in vivo in an animal that has been engineered or transfected with one or more nucleic acid molecules encoding the polypeptides, according to any suitable method. In some embodiments, an antibody or antigen-binding portion thereof is produced in a cell-free system. Non-limiting exemplary cell-free systems are described in, e.g., Sitaraman et al., Methods Mol. Biol. (2009) 498:229-44; Spirin, Trends Biotechnol. (2004) 22:538-45; Endo et al., Biotechnol. Adv. (2003) 21:695-713. Many vector systems are available for the expression of the VH and VL chains in mammalian cells (see II DNA Cloning 45, Glover, ed., IRL Press, 1985). Various approaches can be followed to obtain intact antibodies. As discussed above, it is possible to co-express VH and VL chains and optionally the associated constant regions in the same cells to achieve intracellular association and linkage of VH and VL chains into complete tetrameric H2L2 antibodies or antigen-binding portions thereof. The co- expression can occur by using either the same or different plasmids in the same host. Nucleic acids encoding the VH and VL chains or antigen binding portions thereof can be placed into the same plasmid, which is then transfected into cells, thereby selecting directly for cells that express both chains. Alternatively, cells can be transfected first with a plasmid encoding one chain, for example the VL chain, followed by transfection of the resulting cell line with a VH chain plasmid containing a second selectable marker. Cell lines producing antibodies, antigen-binding portions thereof or other binding agents via either route could be transfected with plasmids encoding additional copies of peptides, VH, VL, or VH plus VL chains in conjunction with additional selectable markers to generate cell lines with enhanced properties, such as higher production of assembled antibodies or antigen binding portions thereof or enhanced stability of the transfected cell lines. Antibodies or antigen binding portions can be expressed in plant cell culture, or plants grown conventionally. The expression in plants may be systemic, limited to sub- cellular plastids, or limited to seeds (endosperms). See, e.g., U.S. Patent Pub. No. 2003 / 0167531; U.S. Pat. No.6,080,560; U.S. Pat. No.6,512,162; and WO / 0129242.

[0055] 54 170886321.1 For intact antibodies, the variable regions (VH and VL) of the antibodies are typically linked to at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. Human constant region DNA sequences can be isolated in accordance with well-known procedures from a variety of human cells, such as immortalized B-cells (see, e.g., WO87 / 02671; which is incorporated by reference herein in its entirety). An antibody can contain both light chain and heavy chain constant regions. The heavy chain constant region can include CH1, hinge, CH2, CH3, and, sometimes, CH4 regions. In some embodiments, the CH2 domain can be deleted or omitted. Alternatively, techniques described for the production of single chain antibodies (see, e.g., U.S. Pat. No.4,946,778; Bird, Science (1988) 242:423-42; Huston et al., Proc. Natl. Acad. Sci. USA (1988) 85:5879-5883; Ward et al., Nature (1989) 334:544- 54; which are incorporated by reference herein in their entireties) can be adapted to produce single chain antibodies that specifically bind to the desired antigen. Single chain antibodies are formed by linking the heavy and light chain variable regions of the Fv region via an amino acid bridge, resulting in a single chain polypeptide. Techniques for the assembly of functional Fv fragments in E. coli can also be used (see, e.g., Skerra et al., Science (1988) 242:1038-1041; which is incorporated by reference herein in its entirety). Intact (e.g., whole) antibodies, their dimers, individual light and heavy chains, or antigen binding portions thereof can be recovered and purified by known techniques, e.g., immunoadsorption or immunoaffinity chromatography, chromatographic methods such as HPLC (high performance liquid chromatography), ammonium sulfate precipitation, gel electrophoresis, or any combination of these. See generally, Scopes, Protein Purification (Springer-Verlag, N.Y., 1982). Substantially pure antibodies or antigen binding portions thereof of at least about 90% to 95% homogeneity are advantageous, as are those with 98% to 99% or more homogeneity, particularly for pharmaceutical uses. Once purified, partially or to homogeneity as desired, an intact antibody or antigen binding portions thereof can then be used therapeutically or in

[0056] 55 170886321.1 developing and performing assay procedures, immunofluorescent staining, and the like. See generally, Vols. I & II Immunol. Meth. (Lefkovits & Pernis, eds., Acad. Press, NY, 1979 and 1981). VI. Pharmaceutical Formulations In some aspects, the present disclosure relates to compositions comprising active ingredients (e.g., including a fusion protein or other binding molecule as described herein or a nucleic acid encoding the fusion protein or other binding molecule as described herein). In some embodiments, the composition is a pharmaceutical composition. As used herein, the term "pharmaceutical composition" refers to the active agent in combination with a pharmaceutically acceptable carrier or excipient accepted for use in the pharmaceutical industry. The phrase "pharmaceutically acceptable" is employed herein to refer to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio. The preparation of a pharmacological composition that contains active ingredients dissolved or dispersed therein is well understood in the art and need not be limited based on any particular formulation. Typically such compositions are prepared as injectable either as liquid solutions or suspensions; however, solid forms suitable for rehydration, or suspensions, in liquid prior to use can also be prepared. A preparation can also be emulsified or presented as a liposome composition. An antibody or antigen binding portion thereof or other binding agent can be mixed with excipients that are pharmaceutically acceptable and compatible with the active ingredient and in amounts suitable for use in the therapeutic methods described herein. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol or the like and combinations thereof. In addition, if desired, a pharmaceutical composition can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents and the like which enhance or maintain the effectiveness of the active ingredient. The

[0057] 56 170886321.1 pharmaceutical compositions as described herein can include pharmaceutically acceptable salts of the components therein. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of a polypeptide) that are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like. Physiologically tolerable carriers are well known in the art. Exemplary liquid carriers are sterile aqueous solutions that contain the active ingredients and water, and may contain a buffer such as sodium phosphate at physiological pH value, physiological saline or both, such as phosphate-buffered saline. Still further, aqueous carriers can contain more than one buffer salt, as well as salts such as sodium and potassium chlorides, dextrose, polyethylene glycol and other solutes. Liquid compositions can also contain liquid phases in addition to and to the exclusion of water. Exemplary of such additional liquid phases are glycerin, vegetable oils such as cottonseed oil, and water-oil emulsions. The amount of an active agent that will be effective in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques. In some embodiments, a pharmaceutical composition comprising an antibody or antigen-binding portion thereof or other binding agent or a nucleic acid encoding an antibody or antigen-binding portion thereof or other binding agent as described herein can be a lyophilisate. In some embodiments, a syringe comprising a therapeutically effective amount of a fusion protein or a pharmaceutical composition described herein is provided. VII. Methods of Treatment and Related Uses In some aspects, the IL-15 muteins, or conjugates and fusion proteins comprising the IL-15 muteins, as described herein, can be used in therapeutic methods. For example, the IL-15 mutein, an antibody-IL-15 mutein conjugate, or an antibody-IL-

[0058] 57 170886321.1 15 mutein fusion protein may be administered to a subject, e.g., a human, to prevent or to treat a disease. In some embodiments, an IL-15 mutein, an antibody-IL-15 mutein conjugate, or an antibody-IL-15 mutein fusion protein may be administered to a subject having a tumor or cancer. In some embodiments, an IL-15 mutein, an antibody-IL-15 mutein conjugate, or an antibody-IL-15 mutein fusion protein may be administered to a subject having an inflammatory disease. As used herein, a "subject" refers to a human or animal. Usually the animal is a vertebrate such as a primate, rodent, domestic animal, or game animal. Primates include chimpanzees, cynomolgus monkeys, spider monkeys, and macaques, e.g., Rhesus. Rodents include mice, rats, woodchucks, ferrets, rabbits, and hamsters. Domestic and game animals include cows, horses, pigs, deer, bison, buffalo, feline species, e.g., domestic cat, canine species, e.g., dog, fox, wolf, avian species, e.g., chicken, emu, ostrich, and fish, e.g., trout, catfish, and salmon. In certain embodiments, the subject is a mammal, e.g., a primate, e.g., a human. The terms, "patient", "individual", and "subject" are used interchangeably herein. Preferably, the subject is a mammal. The mammal can be a human, non-human primate, mouse, rat, dog, cat, horse, or cow, but is not limited to these examples. Mammals other than humans can be advantageously used, for example, as subjects that represent animal models of, for example, various cancers. In addition, the methods described herein can be used to treat domesticated animals and / or pets. A subject can be any sex, e.g., male or female. In certain embodiments, the subject is a human. A "subject in need" of treatment for a particular disease or condition can be a subject having that disease or condition or diagnosed as having that disease or condition. In other embodiments, a subject "at risk of developing" a disease or condition refers to a subject diagnosed as being at risk for developing the disease or condition. As used herein, the terms "treat", "treatment", "treating", or "amelioration" when used in reference to a disease, disorder, or medical condition, refer to therapeutic treatments for a condition, wherein the object is to reverse, alleviate, ameliorate, inhibit,

[0059] 58 170886321.1 slow down, or stop the progression or severity of a symptom or condition. The term "treating" includes reducing or alleviating at least one adverse effect or symptom of a condition. Treatment is generally "effective" if one or more symptoms or clinical markers are reduced. Alternatively, treatment is "effective" if the progression of a condition is reduced or halted. That is, "treatment" includes not just the improvement of symptoms or markers, but also a cessation or at least slowing of progress or worsening of symptoms that would be expected in the absence of treatment. Beneficial or desired clinical results may include, but are not limited to, alleviation of one or more symptom(s), diminishment of extent of the deficit, stabilized (i.e., not worsening) state of disease or symptom, and an increased lifespan as compared to that expected in the absence of treatment. As used herein, the term "administering" refers to applying a method or route to a molecule or pharmaceutical composition described herein that results in delivery of the molecule or pharmaceutical composition to a target cell or tissue in the body. In some embodiments, an IL-15 mutein or a pharmaceutical composition comprising the same, or a nucleic acid encoding the IL-15 mutein or a pharmaceutical composition comprising the same, can be administered by any appropriate route which results in an effective treatment in the subject. The dosage ranges for an IL-15 mutein, an antibody-IL-15 mutein conjugate, or an antibody-IL-15 mutein fusion protein depend upon the potency, and encompass amounts large enough to produce the desired effect. The dosage should not be so large as to cause unacceptable adverse side effects. Generally, the dosage will vary with the age, condition, and sex of the subject and can be determined by one of skill in the art. The dosage can also be adjusted by the individual physician in the event of any complication. Administration of the doses recited above can be repeated. In a preferred embodiment, the doses recited above are administered weekly, biweekly, every three weeks, or monthly for several weeks or months. The duration of treatment depends upon the subject’s clinical progress and responsiveness to treatment.

[0060] 59 170886321.1 In some embodiments, a dose can be administered intravenously. In some embodiments, an intravenous administration can be an infusion occurring over a period of from about 10 minutes to about 4 hours. In some embodiments, an intravenous administration can be an infusion occurring over a period of from about 30 minutes to about 90 minutes. In some embodiments, a dose can be administered subcutaneously. Pharmaceutical compositions containing a binding agent can be administered in a unit dose. The term "unit dose" when used in reference to a pharmaceutical composition refers to physically discrete units suitable as unitary dosage for the subject, each unit containing a predetermined quantity of active material (e.g., IL-15), calculated to produce the desired therapeutic effect in association with the required physiologically acceptable diluent, i.e., carrier, or vehicle. In some aspects, the IL-15 muteins, antibody-IL-15 mutein conjugates, antibody-IL-15 mutein fusion proteins disclosed herein are for use in any of the aforementioned methods. In some aspects, the IL-15 muteins, antibody-IL-15 mutein conjugates, antibody-IL-15 mutein fusion proteins disclosed herein are used in manufacture of a medicament for use in any of the aforementioned methods. In some aspects, the cytokine-fusion proteins (e.g., antibodies and antigen binding fragments thereof, binding proteins) as described herein can be used in a method(s) comprising administering the fusion protein, or pharmaceutical compositions as described herein, to a subject having an inflammatory disease. In some aspects, the cytokine-fusion proteins (e.g., antibodies and antigen binding fragments thereof, binding proteins) as described herein can be used in a method comprising administering the fusion protein, or pharmaceutical compositions as described herein, to a subject having an autoimmune disease. In some embodiments, the autoimmune disease is a rheumatological disorder, fibrotic disorder, gastrointestinal disorder, endocrinological disorder, neurological disorder, or skin disorder. In some embodiments, the autoimmune disease is autoimmune-induced hepatitis, Addison's Disease, Alopecia Areata, Alport's Syndrome, Ankylosing Spondylitis, Anti-

[0061] 60 170886321.1 phospholipid Syndrome, Arthritis, Ascariasis, Aspergillosis Atopic Allergy, Atopic Dermatitis, Atopic Rhinitis, Autoimmune Hemolytic Anemia, Autoimmune Hepatitis, Autoimmune Myositis, Behcet's Disease, Bird-Fancier's Lung, Bronchial Asthma, Caplan's Syndrome, Cardiomyopathy, Celiac Disease, Chagas' Disease, Chronic Glomerulonephritis, Chronic Graft versus Host Disease, Cogan's Syndrome, Cold Agglutinin Disease, CREST Syndrome, Crohn’s Disease, Cryoglobulinemia, Cushing’s Syndrome, Dermatomyositis, Discoid Lupus, Dressier's Syndrome, Eaton-Lambert Syndrome, Encephalomyelitis, Endocrine ophthalmopathy, Erythematosus, Evan's Syndrome, Felty's Syndrome, Fibromyalgia, Fuch’s Cyclitis, Gastric Atrophy, Gastrointestinal Allergy, Giant Cell Arteritis, Glomerulonephritis, Goodpasture's Syndrome, Graft v. Host Disease, Graves' Disease, Guillain-Barre Disease (Syndrome), Hashimoto’s Thyroiditis, Hemolytic Anemia, Henoch-Schonlein Purpura, Hyperviscosity Syndrome, Idiopathic Adrenal Atrophy, Idiopathic Pulmonary Fibrosis, Idiopathic Thrombocytopenic Purpura, IgA Nephropathy, Inflammatory Bowel Disease (Syndrome), Insulin-Dependent Diabetes Mellitus (IDDM or Type I), Juvenile Arthritis, Juvenile Idiopathic Arthritis, Juvenile Diabetes Mellitus (Type I), Lambert-Eaton Syndrome Laminitis, Lichen Planus, Lupoid Hepatitis, Lupus, Lupus Nephritis, Lymphopenia, Macroglobulinemia, Meniere’s Disease, Mixed Connective Tissue Disease, Monoclonal Gammopathy of Undermined Origin, Multiple Sclerosis, Myasthenia Gravis, Myocarditis, Pemphigus / Pemphigoid, Pernicious Anemia, POEMS syndrome, Polyglandular Syndromes, Polyarteritis Nodosa, Polymyositis, Presenile Dementia, Primary Agammaglobulinemia, Primary Biliary Cirrhosis / Cholangitis, Psoriasis, Psoriatic Arthritis, Raynauds Phenomenon, Reiter's Syndrome, Rheumatic Fever, Rheumatoid Arthritis, Sampter's Syndrome, Schmidt's Syndrome, Scleroderma / Systemic Sclerosis, Shulman's Syndrome, Sjörgen's Syndrome, Stiff-Man Syndrome, Sympathetic Ophthalmia, Systemic Lupus Erythematosus, Takayasu's Arteritis, Temporal Arteritis, Thyroiditis, Thrombocytopenia, Thyrotoxicosis, Toxic Epidermal Necrolysis, Type B Insulin Resistance, Type I Diabetes Mellitus, Ulcerative Colitis, Uveitis, Vitiligo, Waldenstrom’s Macroglobulinemia, and / or Wegener's

[0062] 61 170886321.1 Granulomatosis. In some embodiments the autoimmune disease is autoimmune hepatitis, celiac disease, Crohn's disease, juvenile idiopathic arthritis, inflammatory bowel disease (IBD), insulin-dependent diabetes mellitus (IDDM or type 1 diabetes), lupus nephritis, myasthenia gravis, myocarditis, multiple sclerosis (MS), pemphigus / pemphigoid, primary biliary cirrhosis / cholangitis, rheumatoid arthritis (RA), scleroderma / systemic sclerosis, Sjögren's syndrome (SS), systemic lupus erythematosus (SLE), or ulcerative colitis. In some embodiments, the autoimmune disease is selected from autoimmune hepatitis, celiac disease, Crohn’s disease, inflammatory bowel disease (IBD), insulin-dependent diabetes mellitus (IDDM or type 1 diabetes), multiple sclerosis (MS), rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), or ulcerative colitis. In some aspects, the cytokine-fusion proteins (e.g., antibodies and antigen binding fragments thereof, binding proteins) as described herein can be used in a method of treating complications of a transplant associated with graft versus host disease (GVHD), comprising administering the fusion protein, or pharmaceutical compositions as described herein, to a subject. In some aspects, the fusion proteins (e.g., antibodies and antigen binding fragments thereof, binding proteins) as described herein can be used in a method comprising administering the fusion protein, or pharmaceutical compositions as described herein, to a subject to modulate an immune response to a virus in a subject. In some embodiments, the fusion protein or pharmaceutical composition is administered to suppress, reduce, or prevent an immune response to a virus. In some embodiments, the immune response that is suppresed, reduced, or prevented is an immune response to a virus, or antigenic portions thereof. In some embodiments, the virus is a viral vector, and the administration of the fusion protein suppresses, reduces, or prevents the induction of undesired immune responses associated with vector-mediated delivery of genetic material. The use of viral vectors, such as adeno-associated virus (AAV) vectors, to deliver genes of interest is currently an important tool for therapeutic approaches involving gene replacement, gene silencing, gene addition, and gene

[0063] 62 170886321.1 editing. However, host immune responses can limit the effectiveness of these approaches (Wang et al., Nat Rev Drug Discov (2019) 18, 358–378). For example, the host may produce neutralizing antibodies against the vector capsids based on exposure to the wild-type virus, blocking gene delivery. The host may also produce neutralizing antibodies against the vector capsid that limit the effectiveness of re-administration of the vector in therapies requiring repeated dosing. Additionally, hosts can mount a cytotoxic T lymphocyte (CTL)-mediated cytotoxicity that clears transduced cells. In a subset of 'reactive patients', AAV mediated gene delivery can be associated with inflammatory side effects and toxicities mediated by pathogenic CD4+ T cells. Accordingly, the fusion proteins (e.g., antibodies and antigen binding fragments thereof, binding proteins) as described herein can be used in a method comprising administering the fusion protein, or pharmaceutical compositions as described herein, to a subject to suppress, reduce, or prevent an immune response to a viral vector. As used herein, "an immune response to a virus" or "an immune response to a viral vector" may refer to any immune response to a virus, a viral vector, or antigenic portions thereof, e.g., viral proteins or fragments thereof. In some embodiments, the immune response may be activation or proliferation of CD4+ T cells. The immune response may be characterized by, for example, pro-inflammatory cytokine (e.g., IFN-ɣ) production by CD4+ T cells. In some embodiments, the viral vector has been, is, or will be administered to the subject. In some embodiments, the immune response to the viral vector is induced by administration of a viral vector to the subject. In some embodiments, the virus or viral vector is a retrovirus, adenovirus, parvovirus, coronavirus, ortho-myxovirus, rhabdovirus, paramyxovirus, picornavirus, alphavirus, herpesvirus, poxvirus, Norwalk virus, togavirus, flavivirus, reoviruses, papovavirus, hepadnavirus, or hepatitis virus. In some embodiments, the virus or viral vector is an adeno-associated virus (AAV) vector, e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh.8, AAVrh.10, AAVrh.43, AAVrh.74, or AAVhu.37, or a variant thereof. In some embodiments, the virus or viral vector is an adenovirus vector. In some embodiments, the virus or viral vector is a lentivirus vector.

[0064] 63 170886321.1 As used herein, to "activate or stimulate" CD8+ Tregs, or activated CD8+ Tregs, refers to an increase of the regulatory T cell functions of such cells, such as the ability to suppress an immune response. Activation or stimulation of CD8+ Tregs may include removal of a suppressive effect on such cells, so as to restore the CD8+ Tregs (e.g., restore balance to the immune system or restore balanced immune activity in the subject prior to receiving a viral vector). Activation or stimulation of CD8+ Tregs may also include results of such activation or stimulation, including removal of a CD4+ cells, B cells, or other cells mediating an immune response, such as by elimination, for example, cytolysis, of such cells. In some embodiments, the CD8+ Tregs are contacted with a binding agent (e.g., cytokine-antibody fusion proteins or conjugates) in vivo. In some embodiments, the CD8+ Tregs are contacted with a binding agent (e.g., cytokine-antibody fusion proteins or conjugates) ex vivo. The activated CD8+ Tregs can then be administered in an effective amount to a subject in need thereof. In some embodiments, the activated CD8+ Tregs exert a suppressive effect on other immune cells, such as CD4+ T cells, antibody producing B cells, antigen presenting dendritic cells, or antigen presenting cells. In some embodiments, the activated CD8+ Tregs exert a suppressive effect on other immune cells, such as CD4+ T cells, antibody producing B cells, and antigen presenting dendritic cells. In some embodiments, the activated CD8+ Tregs deplete other immune cells, such as CD4+ T cells, antibody producing B cells, and antigen presenting dendritic cells. In some embodiments, the activated CD8+ Tregs modulate the activity of undesired immune cells and decrease the titer of antibodies in the subject. In some embodiments, the activated CD8+ Tregs decrease the titer of antibodies in the subject. In some embodiments, the CD8+ Tregs are CD39+ and KIR+. In some embodiments, a cytokine, cytokine-fusion protein, or pharmaceutical composition comprising the same, as described herein, is administered with an immunosuppressive agent, such as a corticosteroid. In some embodiments, the immunosuppressive agent is one or more of: a calcineurin inhibitor, e.g., a cyclosporin

[0065] 64 170886321.1 or an ascomycin, e.g., cyclosporin A (NEORAL®), FK506 (tacrolimus), pimecrolimus, an mTOR inhibitor, e.g., rapamycin or a derivative thereof, e.g., sirolimus (RAPAMUNE®), everolimus (Certican®), temsirolimus, zotarolimus, biolimus-7, biolimus-9, a rapalog, e.g., ridaforolimus, azathioprine, campath 1H, a S1P receptor modulator, e.g., fingolimod or an analogue thereof, an anti-IL-8 antibody, mycophenolic acid or a salt thereof, e.g., sodium salt, e.g., mycophenolate mofetil (CELLCEPT®), OKT3 (ORTHOCLONE OKT3®), Prednisone, ATGAM®, THYMOGLOBULIN®, Brequinar Sodium, OKT4, T10B9.A-3A, 33B3.1, 15- deoxyspergualine, tresperimus, leflunomide ARAVA®, CTLAI-Ig, anti-CD25, anti- IL2R , basiliximab (SIMULECT®), Daclizumab (ZENAPAX®), mizorbine, methotrexate, dexamethasone, ISAtx-247, SDZ ASM 981 (pimec rolimus, Elidel®), CTLA4lg (Abatacept), belatacept, LFA3lg, etanercept (sold as Enbrel® by Immunex), adalimumab (Humira®), infliximab (Remicade®), an anti-LFA-1 antibody, natalizumab (Antegren®), Enlimomab, gavilimomab, antithymocyte immunoglobulin, siplizumab, alefacept efalizumab, pentase, mesalazine, asacol, codeine phosphate, benorylate, fenbufen, naprosin, diclofenac, etodolac, and indomethacin, tocilizumab (Actemra), siltuximab (Sylvant), secukibumab (Cosentyx), ustekinumab (Stelara), risankizumab, sifalimumab, aspirin, ibuprofen, imlifidase, a proteasome inhibitor, arsenic trioxide, and rabbit anti-thymocyte globulin. See, e.g., Chu et al., Frontiers in Immunology (2021) 12:658038. In some embodiments, a cytokine, cytokine-fusion protein, or pharmaceutical composition comprising the same, as described herein, is administered with an anti- inflammatory agent, such as a corticosteroid. In some embodiments, the anti- inflammatory agent is one or more of: methotrexate, dexamethasone, dexamethasone alcohol, dexamethasone sodium phosphate, fluromethalone acetate, fluromethalone alcohol, lotoprendol etabonate, medrisone, prednisolone acetate, prednisolone sodium phosphate, difluprednate, rimexolone, hydrocortisone, hydrocortisone, lodoxamide tromethamine, aspirin, ibuprofen, suprofen, piroxicam, meloxicam, flubiprofen, naproxan, ketoprofen, tenoxicam, diclofenac sodium, ketotifen fumarate, diclofenac

[0066] 65 170886321.1 sodium, nepafenac, bromfenac, flurbiprofen sodium, suprofen, celecoxib, naproxen, rofecoxib, glucocorticoids, diclofenac, and any combination thereof. In some embodiments, the anti-inflammatory agent is one or more nonsteroidal anti- inflammatory drugs (NSAIDs), such as naproxen sodium (Anaprox), celecoxib (Celebrex), sulindac (Clinoril), oxaprozin (Daypro), salsalate (Disalcid), diflunisal (Dolobid), piroxicam (Feldene), indomethacin (Indocin), etodolac (Lodine), meloxicam (Mobic), naproxen (Naprosyn), nabumetone (Relafen), ketorolac tromethamine (Toradol), naproxen / esomeprazole (Vimovo), and diclofenac (Voltaren), and combinations thereof. In some embodiments of the aforementioned methods, cytokine-fusion protein as described herein is administered in a dose of from about 0.01 mg / kg to about 20 mg / kg (i.e., about 0.01 mg to about 20 mg of fusion protein per kg of body weight). In some embodiments, the fusion protein is administered in a dose of from about 0.5 mg / kg to about 15 mg / kg. In some embodiments, the fusion protein is administered in a dose of from about 0.5 mg / kg to about 5 mg / kg. In some embodiments, the fusion protein is administered in a dose of from about 0.01 mg / kg to about 10 mg / kg. In some embodiments, the fusion protein is administered in a dose of from about 0.1 mg / kg to about 10 mg / kg. In some embodiments, the fusion protein is administered in a dose of from about 0.5 mg / kg to about 10 mg / kg. In some embodiments, the fusion protein is administered in a dose of from about 0.05 mg / kg to about 0.1 mg / kg. In some embodiments, the fusion protein is administered in a dose of from about 0.5 mg / kg to about 1.0 mg / kg. In some embodiments, the fusion protein is administered in a dose of up to about 10 mg / kg. In some embodiments, the fusion protein is administered in a dose of about 10 mg / kg. In some embodiments, the fusion protein is administered in a dose of from 0.01 mg / kg to 20 mg / kg. In some embodiments, the fusion protein is administered in a dose of from 0.5 mg / kg to 15 mg / kg. In some embodiments, the fusion protein is administered in a dose of from 0.5 mg / kg to 5 mg / kg. In some embodiments, the fusion protein is administered in a dose of from 0.01 mg / kg to 10 mg / kg. In some embodiments, the fusion protein is administered in a dose of from 0.1

[0067] 66 170886321.1 mg / kg to 10 mg / kg. In some embodiments, the fusion protein is administered in a dose of from 0.5 mg / kg to 10 mg / kg. In some embodiments, the fusion protein is administered in a dose of from 0.05 mg / kg to 0.1 mg / kg. In some embodiments, the fusion protein is administered in a dose of from 0.5 mg / kg to 1.0 mg / kg. In some 5 embodiments, the fusion protein is administered in a dose of up to 10 mg / kg. In some embodiments, the fusion protein is administered in a dose of 10 mg / kg. In some embodiments, the fusion protein is administered in a dose of about 50 mg / kg. In some embodiments, the fusion protein is administered in a dose of 50 mg / kg. In some embodiments, the dose range can be titrated to maintain serum levels between 0.10 ug / mL and 1000 ug / mL. In some aspects, fusion proteins or binding agents (e.g., antibodies and antigen binding fragments thereof, binding proteins) disclosed herein are for use in the aforementioned methods, or are used in the manufacture of a medicament for use in the aforementioned methods. 5 VIII. Example Embodiments Embodiments of the present disclosure include, but are not limited to, the following examples: 1. An isolated IL-15 peptide comprising the amino acid sequence according to any one of SEQ ID NOs:67, 51, 44, 84, 92, 43, 45, 10-43, 46-50, 52-83, 85-91, and0 93-94. 2. An isolated IL-15 peptide consisting of the amino acid sequence according to any one of SEQ ID NOs:67, 51, 44, 84, 92, 43, 45, 10-43, 46-50, 52-83, 85-91, and 93-94. 3. An isolated IL-15 peptide having one or more amino acid substitutions5 or deletions relative to SEQ ID NO:95, wherein the one or more amino acid substitutions or deletions comprises or consists of: a. K10Q, Y26A, and V49G; b. E46K and E89K; c. Y26A and E53R;

[0068] 67 170886321.1 d. E53A, E64G, and E89K; e. K10Q, V49Y, E89K, and M109A; f. Y26A and E53K; g. Y26A and E89K; h. an N-terminal truncation in which the first three amino acids (NWV) are deleted; i. an N-terminal truncation in which the first five amino acids (NWVNV) are deleted; j. V49Y; k. E46K; l. E53A; m. E53K; n. Y26A; o. Y26K; p. E89K; q. D8K; r. K10V; s. K11A; t. D61K; u. E64G; v. N65G; w. L69W; x. D30K; y. H105A; z. M109A; aa. V49G; bb. E53S; cc. E53R; dd. T24K;

[0069] 68 170886321.1 ee. D8A; ff. K10Q; gg. D61A; hh. N65W; ii. Q108A; jj. V49A; kk. Y26A and E46K; ll. Y26A and V49G; mm. Y26A and V49Y; nn. Y26A and E53A; oo. Y26K and E53A; pp. E46K and V49Y; qq. E46K and E53A; rr. E46K and E53K; ss. E46K and E53R; tt. V49G and E53K; uu. V49G and E89K; vv. V49Y and E53A; ww. V49Y and E53K; xx. V49Y and E53R; yy. V49Y and E89K; zz. E53A and E89K; aaa. E53K and E89K; bbb. E53R and E89K; ccc. E53S and E89K; ddd. K10Q and D61A; eee. K10Q and E64G; fff. K10V and D61A; ggg. K10V and E64G;

[0070] 69 170886321.1 hhh. D61A and E64G; iii. K10Q, Y26A, V49G, and E64G; jjj. Y26A, V49G, and E64G; kkk. K10Q, V49G, and E53K; lll. K10Q, V49G, E53K, and E64G; mmm. V49G, E53K, and E64G; nnn. K10Q, V49Y, and E53A; ooo. K10Q, V49Y, E53A, and E64G; ppp. V49Y, E53A, and E64G; qqq. K10Q, V49Y, and E53R; rrr. K10Q, V49Y, E53R, and E64G; sss. V49Y, E53R, and E64G; ttt. K10Q, V49Y, and E89K; uuu. K10Q, V49Y, E64G, and E89K; vvv. V49Y, E64G, and E89K; www. K10Q, E53A, and E89K; xxx. K10Q, E53A, E64G, and E89K; yyy. K10Q, E53S, and E89K; zzz. K10Q, E53S, E64G, and E89K; aaaa. E53S, E64G, and E89K; bbbb. V49Y, E53A, and M109A; cccc. K10Q, V49Y, E53A, and M109A; dddd. V49Y, E53R, and M109A; eeee. V49Y, E89K, and M109A; ffff. E53A, E89K, and M109A; or gggg. K10Q, E53A, E89K, and M109A. 4. The isolated IL-15 peptide of any one of embodiments 1-3, wherein the peptide comprises SEQ ID NO:67.

[0071] 70 170886321.1 5. The isolated IL-15 peptide of any one of embodiments 1-3, wherein the one or more amino acid substitutions or deletions relative to SEQ ID NO:95 are K10Q, Y26A, and V49G. 6. The isolated IL-15 peptide of any one of embodiments 1-3, wherein the peptide comprises SEQ ID NO:51. 7. The isolated IL-15 peptide of any one of embodiments 1-3, wherein the one or more amino acid substitutions or deletions relative to SEQ ID NO:95 are E46K and E89K. 8. The isolated IL-15 peptide of any one of embodiments 1-3, wherein the peptide comprises SEQ ID NO:44. 9. The isolated IL-15 peptide of any one of embodiments 1-3, wherein the one or more amino acid substitutions or deletions relative to SEQ ID NO:95 are Y26A and E53R. 10. The isolated IL-15 peptide of any one of embodiments 1-3, wherein the peptide comprises SEQ ID NO:84. 11. The isolated IL-15 peptide of any one of embodiments 1-3, wherein the one or more amino acid substitutions or deletions relative to SEQ ID NO:95 are E53A, E64G, and E89K. 12. The isolated IL-15 peptide of any one of embodiments 1-3, wherein the peptide comprises SEQ ID NO:92. 13. The isolated IL-15 peptide of any one of embodiments 1-3, wherein the one or more amino acid substitutions or deletions relative to SEQ ID NO:95 are K10Q, V49Y, E89K, and M109A. 14. The isolated IL-15 peptide of any one of embodiments 1-3, wherein the peptide comprises SEQ ID NO:43. 15. The isolated IL-15 peptide of any one of embodiments 1-3, wherein the one or more amino acid substitutions or deletions relative to SEQ ID NO:95 are Y26A and E53K.

[0072] 71 170886321.1 16. The isolated IL-15 peptide of any one of embodiments 1-3, wherein the peptide comprises SEQ ID NO:45. 17. The isolated IL-15 peptide of any one of embodiments 1-3, wherein the one or more amino acid substitutions or deletions relative to SEQ ID NO:95 are Y26A and E89K. 18. An isolated fusion protein comprising (i) a IL-15 peptide comprising or consisting of the amino acid sequence as set forth in any one of embodiments 1-17 and (ii) an antibody or fragment thereof. 19. The isolated fusion protein of embodiment 18, wherein the IL-15 peptide is covalently connected to the antibody or fragment thereof by a linker. 20. The isolated fusion protein of embodiment 19, wherein the linker comprises or consists of SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:437, or SEQ ID NO:438. 21. The isolated fusion protein of embodiment 18, wherein the antibody or fragment thereof comprises: a light chain variable region (VL) having CDRL1, CDRL2, and CDRL3 amino acid sequences according to SEQ ID NO:97, SEQ ID NO:98, and SEQ ID NO:99, respectively; and a heavy chain variable region (VH) having CDRH1, CDRH2, and CDRH3 amino acid sequences according to SEQ ID NO:101, SEQ ID NO:102, and SEQ ID NO:103, respectively. 22. The isolated fusion protein of embodiment 18 or embodiment 21, wherein the antibody or fragment thereof comprises: a light chain variable region (VL) comprising or consisting of the amino acid sequence according to SEQ ID NO:96; and a heavy chain variable region (VH) comprising or consisting of the amino acid sequence according to SEQ ID NO:100. 23. The isolated fusion protein of any one of embodiments 18, 21, and 22, wherein the antibody or fragment thereof comprises: a first chain, a second chain, and a third chain, wherein: (i) the first chain is a light chain comprising or consisting of the amino acid sequence according to SEQ ID NO:104;

[0073] 72 170886321.1 (ii) the second chain is a heavy chain comprising or consisting of the amino acid sequence according to SEQ ID NO:106; and (iii) the third chain is a heavy chain comprising or consisting of the amino acid sequence according to SEQ ID NO:105 covalently connected to the IL-15 peptide by a linker. 24. The isolated fusion protein of embodiment 23, wherein the linker comprises or consists of the amino acid sequence according to SEQ ID NO:107 or 108. 25. The isolated fusion protein of embodiment 23, wherein the linker comprises or consists of the amino acid sequence according to SEQ ID NO:437 or SEQ ID NO:438. 26. An isolated antibody-IL-15 fusion protein comprising: a first peptide chain, a second peptide chain, and a third peptide chain, wherein: (i) the first peptide chain comprises or consists of the amino acid sequence according to SEQ ID NO:104; (ii) the second peptide chain comprises or consists of the amino acid sequence according to SEQ ID NO:106; and (iii) the third peptide chain comprises or consists of the amino acid sequence according to any one of SEQ ID NOs:109-204. 27. The fusion protein of embodiment 26, wherein the third peptide chain comprises or consists of the amino acid sequence according to SEQ ID NO:153. 28. The fusion protein of embodiment 26, wherein the third peptide chain comprises or consists of the amino acid sequence according to SEQ ID NO:176. 29. The fusion protein of embodiment 26, wherein the third peptide chain comprises or consists of the amino acid sequence according to SEQ ID NO:193. 30. The fusion protein of embodiment 26, wherein the third peptide chain comprises or consists of the amino acid sequence according to SEQ ID NO:201. 31. The fusion protein of embodiment 26, wherein the third peptide chain comprises or consists of the amino acid sequence according to SEQ ID NO:160.

[0074] 73 170886321.1 32. The fusion protein of embodiment 26, wherein the third peptide chain comprises or consists of the amino acid sequence according to SEQ ID NO:152. 33. The fusion protein of embodiment 26, wherein the third peptide chain comprises or consists of the amino acid sequence according to SEQ ID NO:154. 34. The isolated fusion protein of embodiment 18, wherein the antibody or fragment thereof comprises: a light chain variable region (VL) having CDRL1, CDRL2, and CDRL3 amino acid sequences according to SEQ ID NO:407, SEQ ID NO:408, and SEQ ID NO:409, respectively; and a heavy chain variable region (VH) having CDRH1, CDRH2, and CDRH3 amino acid sequences according to SEQ ID NO:411, SEQ ID NO:142, and SEQ ID NO:413, respectively. 35. The isolated fusion protein of embodiment 18 or embodiment 34, wherein the antibody or fragment thereof comprises: a light chain variable region (VL) comprising or consisting of the amino acid sequence according to SEQ ID NO:406; and a heavy chain variable region (VH) comprising or consisting of the amino acid sequence according to SEQ ID NO:410. 36. The isolated fusion protein of any one of embodiments 18, 34, and 36, wherein the antibody or fragment thereof comprises: a first chain, a second chain, and a third chain, wherein: (i) the first chain is a light chain comprising or consisting of the amino acid sequence according to SEQ ID NO:414; (ii) the second chain is a heavy chain comprising or consisting of the amino acid sequence according to SEQ ID NO:416; and (iii) the third chain is a heavy chain comprising or consisting of the amino acid sequence according to SEQ ID NO:415 covalently connected to the IL-15 peptide by a linker. 37. The isolated fusion protein of embodiment 36, wherein the linker comprises or consists of the amino acid sequence according to SEQ ID NO:107, 108, 437, or 438.

[0075] 74 170886321.1 38. An isolated antibody-IL-15 fusion protein comprising: a first peptide chain, a second peptide chain, and a third peptide chain, wherein: (i) the first peptide chain comprises or consists of the amino acid sequence according to SEQ ID NO:414; (ii) the second peptide chain comprises or consists of the amino acid sequence according to SEQ ID NO:416; and (iii) the third peptide chain comprises or consists of the amino acid sequence according to any one of SEQ ID NOs:417-425. 39. The fusion protein of embodiment 38, wherein the third peptide chain comprises or consists of the amino acid sequence according to SEQ ID NO:419. 40. The fusion protein of embodiment 38, wherein the third peptide chain comprises or consists of the amino acid sequence according to SEQ ID NO:420. 41. The fusion protein of embodiment 38, wherein the third peptide chain comprises or consists of the amino acid sequence according to SEQ ID NO:421. 42. The fusion protein of embodiment 38, wherein the third peptide chain comprises or consists of the amino acid sequence according to SEQ ID NO:422. 43. The fusion protein of embodiment 38, wherein the third peptide chain comprises or consists of the amino acid sequence according to SEQ ID NO:423. 44. The fusion protein of embodiment 38, wherein the third peptide chain comprises or consists of the amino acid sequence according to SEQ ID NO:424. 45. The fusion protein of embodiment 38, wherein the third peptide chain comprises or consists of the amino acid sequence according to SEQ ID NO:425. 46. The isolated fusion protein of embodiment 18, wherein the antibody or fragment thereof is bispecific and comprises: (1) a first light chain variable region (VL) having CDRL1, CDRL2, and CDRL3 amino acid sequences according to SEQ ID NO:407, SEQ ID NO:408, and SEQ ID NO:409, respectively; and a first heavy chain variable region (VH) having CDRH1, CDRH2, and CDRH3 amino acid sequences according to SEQ ID NO:411, SEQ ID NO:142, and SEQ ID NO:413, respectively; and (2) a second light chain variable region (VL) having CDRL1, CDRL2, and CDRL3

[0076] 75 170886321.1 amino acid sequences according to SEQ ID NO:97, SEQ ID NO:98, and SEQ ID NO:99, respectively; and a second heavy chain variable region (VH) having CDRH1, CDRH2, and CDRH3 amino acid sequences according to SEQ ID NO:101, SEQ ID NO:102, and SEQ ID NO:103, respectively. 47. The isolated fusion protein of embodiment 18 or embodiment 46, wherein the antibody or fragment thereof is bispecific and comprises: (1) a first light chain variable region (VL) comprising or consisting of the amino acid sequence according to SEQ ID NO:406; and a first heavy chain variable region (VH) comprising or consisting of the amino acid sequence according to SEQ ID NO:410; and (2) a second light chain variable region (VL) comprising or consisting of the amino acid sequence according to SEQ ID NO:96; and a second heavy chain variable region (VH) comprising or consisting of the amino acid sequence according to SEQ ID NO:100. 48. The isolated fusion protein of any one of embodiments 46 or 47, further comprising a linker connecting the IL-15 to the antibody or fragment thereof. 49. The isolated fusion protein of embodiment 48, wherein the linker comprises or consists of the amino acid sequence according to SEQ ID NO:107, 108, 437, or 438. 50. The isolated fusion protein of embodiment 18, wherein the antibody or fragment thereof is bispecific and comprises: (1) a first light chain variable region (VL) having CDRL1, CDRL2, and CDRL3 amino acid sequences according to SEQ ID NO:448, SEQ ID NO:449, and SEQ ID NO:450, respectively; and a first heavy chain variable region (VH) having CDRH1, CDRH2, and CDRH3 amino acid sequences according to SEQ ID NO:451, SEQ ID NO:452, and SEQ ID NO:453, respectively; and (2) a second light chain variable region (VL) having CDRL1, CDRL2, and CDRL3 amino acid sequences according to SEQ ID NO:440, SEQ ID NO:441, and SEQ ID NO:442, respectively; and a second heavy chain variable region (VH) having CDRH1, CDRH2, and CDRH3 amino acid sequences according to SEQ ID NO:443, SEQ ID NO:444, and SEQ ID NO:445, respectively.

[0077] 76 170886321.1 51. The isolated fusion protein of embodiment 18, wherein the antibody or fragment thereof is bispecific and comprises: (1) a first light chain variable region (VL) comprising or consisting of the amino acid sequence according to SEQ ID NO:454; and a first heavy chain variable region (VH) comprising or consisting of the amino acid sequence according to SEQ ID NO:455; and (2) a second light chain variable region (VL) comprising or consisting of the amino acid sequence according to SEQ ID NO:446; and a second heavy chain variable region (VH) comprising or consisting of the amino acid sequence according to SEQ ID NO:447. 52. The isolated fusion protein of embodiment 18, wherein the antibody or fragment thereof is bispecific and comprises: (i) a light chain comprising or consisting of the amino acid sequence according to SEQ ID NO:456; (ii) a heavy chain comprising or consisting of the amino acid sequence according to SEQ ID NO:457; and (iii) a heavy chain comprising or consisting of the amino acid sequence according to SEQ ID NO:458. 53. The isolated fusion protein of any one of embodiments 50-52, wherein the heavy chain comprising or consisting of the amino acid sequence according to SEQ ID NO:458 is covalently connected to the IL-15 peptide by a linker. 54. The isolated fusion protein of embodiment 53, wherein the linker comprises or consists of the amino acid sequence according to SEQ ID NO:107, 108, 437, or 438. 55. An isolated nucleic acid molecule comprising a polynucleotide sequence that encodes the peptide or fusion protein of any one of embodiments 1-49. 56. The isolated nucleic acid molecule of embodiment 55, wherein the polynucleotide sequence comprises or consists of any one of SEQ ID NOs:207-405 and 426-436. 57. An isolated nucleic acid molecule comprising a polynucleotide sequence that encodes the peptide or fusion protein of any one of embodiments 1-54.

[0078] 77 170886321.1 58. The isolated nucleic acid molecule of embodiment 57, wherein the polynucleotide sequence comprises or consists of any one of SEQ ID NOs:207-405, 426-436, and 460-463. 59. The isolated nucleic acid molecule of embodiment 55 or embodiment 56, further comprising a polynucleotide sequence that encodes a signal peptide. 60. The isolated nucleic acid molecule of embodiment 59, wherein the signal peptide comprises or consists of the amino acid sequence of SEQ ID NO:205. 61. The isolated nucleic acid molecule of embodiment 59, wherein the polynucleotide sequence that encodes a signal peptide comprises or consists of SEQ ID NO:206. 62. The isolated nucleic acid molecule of embodiment 57 or embodiment 58, further comprising a polynucleotide sequence that encodes a signal peptide. 63. The isolated nucleic acid molecule of embodiment 62, wherein the signal peptide comprises or consists of the amino acid sequence of SEQ ID NO:205. 64. The isolated nucleic acid molecule of embodiment 62, wherein the polynucleotide sequence that encodes a signal peptide comprises or consists of SEQ ID NO:206. 65. A recombinant expression vector comprising the nucleic acid molecule according to any one of embodiments 55-64. 66. A pharmaceutical composition comprising the peptide according to any one of embodiments 1-17 and a pharmaceutically acceptable carrier. 67. A pharmaceutical composition comprising the fusion protein according to any one of embodiments 18-49 and a pharmaceutically acceptable carrier. 68. A pharmaceutical composition comprising the nucleic acid molecule or vector according to any one of embodiments 55-61 and 65 and a pharmaceutically acceptable carrier. 69. A pharmaceutical composition comprising the fusion protein according to any one of embodiments 18-54 and a pharmaceutically acceptable carrier.

[0079] 78 170886321.1 70. A pharmaceutical composition comprising the nucleic acid molecule or vector according to any one of embodiments 55-65 and a pharmaceutically acceptable carrier. 71. A method for treating a disease in a subject in need thereof, comprising administering the peptide according to any one of embodiments 1-17, or the pharmaceutical composition according to embodiment 66, to the subject. 72. A method for treating a disease in a subject in need thereof, comprising administering the fusion protein according to any one of embodiments 18-49, or the pharmaceutical composition according to embodiment 67, to the subject. 73. A method for treating a disease in a subject in need thereof, comprising administering the nucleic acid molecule or vector according to any one of embodiments 55-61 and 65, or the pharmaceutical composition according to embodiment 68, to the subject. 74. A method for treating a disease in a subject in need thereof, comprising administering the nucleic acid molecule or vector according to any one of embodiments 55-65, or the pharmaceutical composition according to embodiment 70, to the subject. 75. Use of the peptide, fusion protein, nucleic acid molecule, vector, or pharmaceutical composition of any one of embodiments 1-70 in a method for treating a disease in a subject in need thereof. 76. The peptide, fusion protein, nucleic acid molecule, vector, or pharmaceutical composition of any one of embodiments 1-70 for use in a method for treating a disease in a subject in need thereof. 77. Use of the peptide, fusion protein, nucleic acid molecule, vector, or pharmaceutical composition of any one of embodiments 1-70 in the manufacture of a medicament for treating a disease in a subject in need thereof. 78. The method, use, or composition for use of any one of embodiments 71- 77, wherein the disease is cancer. 79. The method, use, or composition for use of any one of embodiments 71- 77, wherein the disease is an inflammatory disease.

[0080] 79 170886321.1 80. The method, use, or composition for use of any one of embodiments 71- 77, wherein the disease is an autoimmune disease. 81. The method, use, or composition for use of any one of embodiments 71- 77, wherein the disease is celiac disease. 82. The method, use, or composition for use of any one of embodiments 71- 77, wherein the disease is Crohn's disese. 83. The method, use, or composition for use of any one of embodiments 71- 77, wherein the disease is rheumatoid arthritis. 84. The method, use, or composition for use of any one of embodiments 71- 77, wherein the disease is diabetes. 85. The method, use, or composition for use of any one of embodiments 71- 77, wherein the disease is Sjögren's syndrome (SS). 86. The method, use, or composition for use of any one of embodiments 71- 77, wherein the disease is lupus. 87. The method, use, or composition for use of any one of embodiments 71- 77, wherein the disease is systemic lupus erythematosus (SLE). 88. The method, use, or composition for use of any one of embodiments 71- 77, wherein the disease is graft versus host disease (GVHD). 89. A host cell comprising the nucleic acid molecule according to any one of embodiments 55-64. 90. A fusion protein comprising: (a) a binding agent comprising: (i) a first binding domain that specifically binds to a KIR protein, and (ii) a second binding domain that specifically binds to a protein expressed on CD8+KIR+ T regulatory cells (Tregs) other than a KIR protein; and (b) a cytokine. 91. The fusion protein of embodiment 90, wherein the binding agent binds to CD8+KIR+ Tregs.

[0081] 80 170886321.1 92. The fusion protein of embodiment 90 or embodiment 91, wherein the cytokine is a IL-15. 93. The fusion protein of embodiment 92, wherein the IL-15 comprises or consists of an amino acid sequence according to SEQ ID NO:67. 94. The fusion protein of embodiment 92, wherein the IL-15 comprises or consists of an amino acid sequence according to SEQ ID NO:51. 95. The fusion protein of embodiment 92, wherein the IL-15 comprises or consists of an amino acid sequence according to any one of SEQ ID NO:1-94. 96. The fusion protein of embodiment 92, wherein the IL-15 comprises or consists of an amino acid sequence according to SEQ ID NO:44. 97. The fusion protein of embodiment 92, wherein the IL-15 comprises or consists of an amino acid sequence according to SEQ ID NO:84. 98. The fusion protein of embodiment 92, wherein the IL-15 comprises or consists of an amino acid sequence according to SEQ ID NO:92. 99. The fusion protein of embodiment 92, wherein the IL-15 comprises or consists of an amino acid sequence according to SEQ ID NO:43. 100. The fusion protein of embodiment 92, wherein the IL-15 comprises or consists of an amino acid sequence according to SEQ ID NO:45. 101. A method of increasing CD8+ Treg activity comprising administering to a subject in need thereof a molecule that comprises (1) a binding domain that binds to one or more proteins expressed on the surface of a CD8+ Treg cell and (2) a cytokine. 102. A method of increasing CD8+ Treg proliferation comprising administering to a subject in need thereof a molecule that comprises (1) a binding domain that binds to one or more proteins expressed on the surface of a CD8+ Treg cell and (2) a cytokine. 103. A method of treating an autoimmune disease, comprising administering to a subject in need thereof a molecule that comprises (1) a binding domain that binds to one or more proteins expressed on the surface of a CD8+ Treg cell, and (2) a cytokine.

[0082] 81 170886321.1 104. A method of suppressing an immune response mediated by pathogenic immune cells, comprising administering to a subject in need thereof a molecule that comprises (1) a binding domain that binds to one or more proteins expressed on the surface of a CD8+ Treg cell, and (2) a cytokine, whereby the number or activity of pathogenic immune cells is decreased. 105. The method of any one of embodiments 101-104, wherein the molecule comprises a fusion protein comprising (1) an antibody or antibody fragment that binds to one or more proteins expressed on the surface of a CD8+ Treg cell, and (2) a cytokine. 106. The method of embodiment 105, wherein the fusion protein comprises the fusion protein according to any one of embodiments 18-54. IX. Examples a. EXAMPLE 1: Engineered IL-15 Muteins A panel of IL-15 mutants, or "muteins", was designed to selectively impact binding to IL15Ra, IL2Rb, and / or IL2Rg. The panel contained single, double, or multiple amino acid substitutions that were designed to influence IL-15 binding to one or more of the IL15R subunits. The IL-15 single mutation muteins generated and tested in Examples 2 through 5 are listed in Table 1. The IL-15 combination mutation muteins generated and tested in Examples 2 through 5 are listed in Table 2. Table 1. List of IL-15 single mutation muteins tested Variant ID IL-15 Mutation(s) Amino Acid DNA SEQ ID SEQ ID NO: NO: 170886321.1 v9 N1G D30N E46G 9 319 V49R E64Q Nt i l t ti Table 2. List of IL-15 combination mutation muteins tested Variant IL-1 Amino Acid DNA SEQ ID ID 5 Mutation(s) SEQ ID NO: NO: 170886321.1 v45 Y26A E89K 45 356 v46 Y26K E53A 46 357 47 E4K V4Y 47 170886321.1 v89 K10Q V49Y E53A M109A 89 400 v90 V49Y E53R M109A 90 401 1 V4 Y E K M1 A 1 4 2 . : o ecu e es gn an asm ons ruc on of Anti- CD8α-IL-15 Fusion Proteins Anti-CD8α-IL-15 fusion proteins having an anti-CD8α antibody portion and an IL-15 portion (as in FIG.1) were generated. The antibody portion was a monoclonal antibody (mAb) having anti-CD8a Mb1b binding domains, and included knobs-into-holes (KiH) mutations in the human IgG1 (hIgG1) Fc portion to enable heterodimeric heavy chain assembly. The C- terminus of the anti-CD8α Mb1b knob heavy chain was selected for adjoining the IL-15 portion. The anti-CD8α Mb1b variable heavy (VH) binding domain was designed to directly adjoin to the human IgG1 CH1-CH2-CH3 domains to form an intact IgG1 heavy chain. The anti-CD8α Mb1b variable light (VL) binding domain likewise adjoined to the human kappa constant region, thereby forming an intact kappa light chain. The VL binding domain had the amino acid sequence of SEQ ID NO:96, with light CDR1, CDR2, and CDR3 sequences according to Chothia notation as set forth in SEQ ID NOs:97, 98, and 99, respectively. The VH binding domain had the amino acid sequence of SEQ ID NO:100, with heavy CDR1, CDR2, and CDR3 sequences according to Chothia notation as set forth in SEQ ID NOs:101, 102, and 103, respectively. The knobs-into-holes mutations were in the hIgG1 CH3 domain. The anti-CD8α Mb1b heavy chain knob mutations corresponded to the following amino acid substitutions noted in EU index positions: S354C and T366W. The anti-CD8α Mb1b heavy chain hole mutations corresponded to the following amino acid substitutions noted in EU index positions: Y349C, T366S, L368A, and Y407V.

[0083] 85 170886321.1 The hIgG1 Fc contained a subset of mutations in the CH2 domain to reduce effector function. The mutations corresponded to the following amino acid substitutions noted in EU index positions: L234A, L235E, G237A, A330S, and P331S. The anti-CD8α Mb1b light chain had the amino acid sequence of SEQ ID NO:104. The anti-CD8α Mb1b knob heavy chain had the amino acid sequence of SEQ ID NO:105. The anti-CD8α Mb1b hole heavy chain had the amino acid sequence of SEQ ID NO:106. The anti-CD8α-IL-15 fusion proteins included a IL-15 portion selected from wild type (SEQ ID NO:95) and variants v1-v94 (SEQ ID NOs:1-94) described above in Example 1. Each IL-15 portion was tethered to the antibody by the introduction of a Gly-Ser linker of SEQ ID NO:107 or an (EAAAK)3 linker of SEQ ID NO:108, at the C- terminus of the anti-CD8a Mb1b knob heavy chain. Thus, the anti-CD8α-IL-15 fusion proteins had knob heavy chains corresponding to one of SEQ ID NOs:109-204. Plasmid construction and sequencing for each anti-CD8a-IL-15 fusion protein was performed. Synthesized inserts were subsequently subcloned by ligation into the commercially available transient mammalian expression vector pcDNA3.4 (Invitrogen). The ligation mixture was transformed into E.coli. Resulting clones were screened by restriction digestion to confirm presence of an appropriate size insert. Positive transformants underwent DNA sequencing analysis to confirm the sequence of each insert. DNA with the correct sequence was subsequently used for expression. c. EXAMPLE 3: Expression of Anti-CD8α-IL-15 Fusion Proteins Lyophilized plasmid DNA generated from Example 2 was reconstituted in sterile water to a concentration of 1 mg / mL. After reconstitution, each plasmid was transformed into E.coli DH10B (Thermo Scientific Cat #1829001) via electroporation using a Bio-Rad Gene Pulser II instrument set to 1.75 kV, 100 Ohms and 25mF and allowed to undergo overnight selection on LB Amp plates (Teknova Cat #L1004) at 37C. A swab of transformants from each transformation was used to inoculate separate

[0084] 86 170886321.1 35 mL Plasmid+ media (HTS Labs Cat #446300) containing 100 mg / ml Ampicillin (Teknova Cat #A9626) cultures for generating plasmid DNA. The cultures were allowed to grow overnight in an Innova42 incubator (New Brunswick Scientific) set to 37C, 300 rpm in vented 125 mL flasks (HTS Labs Cat #931147). Following overnight incubation, each culture was pelleted via centrifugation in a Sorvall Lynx 4000 centrifuge set to 7,000 X g at 4C for 15 minutes and the supernatant discarded. Plasmid DNA from the resulting bacterial pellets was processed using a Macherey Nagel plasmid DNA purification kit (NucleoBond Xtra Midi, Cat #740410) as per manufacturer instructions. DNA was resuspended in sterile water to approximately 1.5 mg / mL using the NanoDrop (Fisher Scientific, Cat #13-400-519) instrument to quantitate. Assembly of each intact anti-CD8α-IL-15 fusion protein results from expression of three simultaneously co-transfected plasmids: one plasmid encoding the anti-CD8α light chain (nucleic acid sequence of anti-CD8α light chain as set forth in SEQ ID NO:207), a second plasmid encoding the anti-CD8α knob heavy chain linked to the IL- 15 portion (nucleic acid sequence of anti-CD8α knob heavy chain as set forth in one of SEQ ID NOs:208-247 and 249-309, for each IL-15 sequence), and a third plasmid encoding the anti-CD8α hole heavy chain (nucleic acid sequence of anti-CD8α hole heavy chain as set forth in SEQ ID NO:248). The signal sequences used in the plasmid constructs corresponded to SEQ ID NOs:205 (amino acid) and 206 (nucleic acid). For each anti-CD8α-IL-15 fusion protein transfection, 50mL cultures of Expi293F cells (Thermo Scientific Cat #A14527) were cultured to a density of 1.5E6 cells / mL in 125mL flasks (HTS Labs Cat #931110) in preparation for transient expression. The cells were allowed to grow overnight at 37C, 8% CO2, 150 rpm in Expi293 Expression Medium (Thermo Scientific Cat #A1435101). The following day the cells were transfected using PEI (Polyethylenimine, Polysciences Cat #23966-1) as the complexing reagent using a DNA concentration of 1 mg / L. DNA / PEI mixtures were allowed to incubate for 30 minutes before adding to the Expi293F cells. The transfected cultures were returned to the incubator set to the conditions listed above. One day post-

[0085] 87 170886321.1 transfection, each culture was given a bolus feed of 2.5 mL CHO Efficient Feed B Nutrient Supplement (Thermo Scientific Cat #A1024001) and 400 mL 500 mM Valproic acid (Fisher Cat #501786601). The cultures were allowed to incubate in a Kuhner Climo-Shaker ISF1-XC incubator at 37C, 8% CO2, 150 rpm for another 5 days. At 6 days post-transfection, the cultures were sampled for cell density and viability, and harvested. A sample was taken for Protein A titer analysis on the Octet Red 384 using Protein A sensors (Sartorius Cat #18-5010). The measured value was run against a standard curve of purified protein to determine culture titers. For harvest, cultures were clarified via centrifugation in a Sorvall Lynx 4000 centrifuge set to 7,000 x g, 4C for 20 minutes. The supernatants were decanted into a fresh tube and the pellets discarded. d. EXAMPLE 4: Purification of Anti-CD8α-IL-15 Fusion Proteins An increased throughput purification was developed to accommodate the large number of anti-CD8α-IL-15 fusion proteins. Specifically, a medium-throughput 1-step purification process was implemented for generating approximately 1 mg of each IL-15. Following cell culture harvest, the supernatants containing the anti-CD8α-IL-15 fusion proteins underwent enrichment via protein A affinity interaction. The resin used for protein A enrichment was AmMag Protein A Magnetic Beads (GenScript, Cat #L00695), which allows for the enrichment of Fc-containing molecules from crude feed streams like cell culture supernatants described in previous section. Before the harvested cell supernatant was applied to the protein A beads, a sanitization step was performed with 0.1 N sodium hydroxide (MilliporeSigma, Cat #221465) to remove any adventitious agents or endotoxin that may be present on the beads from previous batches or storage. Following sanitization, the beads were collected using a magnetic separation rack (New England Biolabs, Cat #S1507S) and the buffer was discarded. The beads were then equilibrated with 20 mM sodium phosphate (MilliporeSigma, Cat #S9763), 150 mM sodium chloride (MilliporeSigma, Cat #S9888), pH 7.2 in order to match the pH and conductivity of the load material. As before, the beads were collected and the buffer discarded. Once the beads were fully

[0086] 88 170886321.1 equilibrated, they were adjusted to a 25% slurry in equilibration buffer. The beads were then applied to the harvested cell supernatant at a volume of 0.2 mL per 50 mL of harvested cell supernatant. The beads were incubated with each of the harvested anti- CD8α-IL-15 fusion protein cell supernatants overnight with rocking at room temperature. The following day, the loaded beads were collected and transferred to a 24-well plate (Agilent, Cat #204023) and washed with 20 mM sodium phosphate, 150 mM sodium chloride, pH 7.2. The beads were collected using a magnetic 24 well plate block (Alpaqua Cat #A000440) and the buffer was discarded. Following the wash, a transition was performed with 50 mM sodium acetate (MilliporeSigma, Cat #241245), pH 5.0 to remove the salt. The beads were collected as before and the buffer discarded. Elution followed with 5 mL of 40 mM sodium acetate (MilliporeSigma, Cat #241245), pH 3.5. The beads were magnetically isolated and the elution was collected. A summary of the operating parameters for the protein A enrichment is presented below in Table 3. Anti-CD8α-IL-15 fusion proteins were eluted from the protein A mag beads at low pH. To prevent the low pH from inducing protein degradation or aggregation, the proteins were immediately neutralized with 1.0 M Tris-HCl (MilliporeSigma, Cat #108315), pH 8.5 to a target of pH 5.0. Following neutralization, the anti-CD8α-IL-15 fusion proteins were filtered into a 15 mL conical vial (MilliporeSigma, Cat #T1943) with a 0.2 micron syringe filter (MilliporeSigma, Cat #SLGPR33RB) to remove any precipitates that may be present and control bioburden. Table 3. Operating parameters for protein A chromatography Step Buffer Parameter N A N A R i i t A M P t i A

[0087] 89 170886321.1 20 mM sodium Buffer Volume (mL): 10 Equilibration phosphate, 150 mM Cycles: 2 di m hl rid H 72 S rn t nt Di rd d e. EXAMPLE 5: Characterization of anti-CD8α-IL-15 fusion proteins Following purification of the anti-CD8α-IL-15 fusion proteins using the procedure described in Example 4, performance and quality of the anti-CD8α-IL-15 mutein fusion proteins were assessed using analytical assays and process attributes. The assays included concentration, size exclusion chromatography-high performance liquid chromatography (SEC-HPLC), reduced and non-reduced CE-SDS analysis by LabChip, and endotoxin to confirm purity. The purity of the anti-CD8α-IL-15 fusion proteins was evaluated by SEC-HPLC using an Agilent 1100 series HPLC. SEC-HPLC identifies any size variants such as high molecular weight species (HMW) and low molecular weight species (LMW). Operating parameters for the SEC-HPLC assay are presented in Table 4. Table 4. Operating parameters for SEC-HPLC Parameter Value n 170886321.1 Mobile phase 0.1 M sodium phosphate, 0.1 M sodium sulfate, pH 6.5 Injection l 5 µL a Perkin Elmer GXII Touch Protein Analyzer (LabChip). Samples were analyzed with the LabChip Protein Express assay under both reducing and non-reducing conditions. An internal molecular weight ladder standard (PerkinElmer, Cat #CLS0008) was used to determine calibrated molecular weights for the samples. The assay was operated according to the manufacturer’s recommendation. The anti-CD8α-IL-15 fusion proteins were assayed for endotoxin contamination using a Charles River nexgen-pts with limulus amebocyte lysate (LAL) assay cartridges (Charles River, Cat #PTS201). The assay was operated according to the manufacturer’s recommendation. The concentration of the purified anti-CD8α-IL-15 fusion proteins was analyzed by UV absorbance at 280 nm using a NanoDrop (Fisher Scientific, Cat #13-400-519). Binding affinities of the anti-CD8α-IL-15 fusion proteins to IL15R-alpha (R&D Systems Cat #AVI7194), IL2R-beta (Acro Biosystems Cat #ILB-H82E3), and IL2R- beta / gamma heterodimer (Acro Biosystems Cat #ILG-H82F3) were determined by Bio- Layer Interferometry (BLI) using an Octet Red 384 instrument (Sartorius). Biotinylated receptors were captured onto a streptavidin coated biosensor (Sartorius Cat #18-5019). A dilution series of each anti-CD8α-IL-15 fusion protein was prepared from 400 nM to 6.25 nM and allowed to bind to the receptor. Binding was performed at 25C in Octet Kinetics Buffer (Sartorius Cat #18-1105). Ten minutes of association followed by 10 minutes of dissociation were utilized for these measurements. Buffer reference subtraction was applied to the binding curves. Association and dissociation rate constants were globally fit to a 1:1 Langmuir binding model, and KD binding affinity was calculated.

[0088] 91 170886321.1 Tables 5, 6, and 7 detail the results for the anti-CD8α-IL-15 fusion proteins that included wild type IL-15 or IL-15 variants with single mutations binding to IL15R- alpha (Table 5), IL2R-beta (Table 6), and IL2R-beta / gamma heterodimer (Table 7). Tables 8, 9, and 10 detail the results for the anti-CD8α-IL-15 fusion proteins for IL-15 variants with combination mutations binding to IL15R-alpha (Table 8), IL2R-beta (Table 9), and IL2R-beta / gamma heterodimer (Table 10). Table 5. Binding affinity and kinetic rate constants for samples of the anti-CD8α- IL-15 fusion proteins that included wild type IL-15 or IL-15 variants with single mutations binding to IL15R-alpha IL-1 ka(1 / Ms) kd(1 / s) Variant ID 5 Mutation(s) Association Dissociation KD(M) 9 170886321.1 v35 D61A 2.6E+06 1.9E-04 7.2E-11 v22 D61K N / D Table 6: Binding affinity and kinetic rate constants for samples of the anti-CD8α- IL-15 fusion proteins that included wild type IL-15 or IL-15 variants with single mutations binding to IL2R-beta IL ka(1 / Ms) kd(1 / s) Variant ID -15 Association Dissociation KD(M) 170886321.1 v29 V49G 9.9E+04 1.1E-02 1.1E-07 v4 V49R 2.5E+05 4.4E-03 1.8E-08 Table 7. Binding affinity and kinetic rate constants for samples of the anti-CD8α- IL-15 fusion proteins that included wild type IL-15 or IL-15 variants with single mutations binding to IL2R-beta / gamma heterodimer IL-15 ka(1 / Ms) kd(1 / s) Variant ID Association Dissociation KD(M) 170886321.1 v32 T24K 4.6E+05 5.0E-03 1.1E-08 v16 Y26A 2.1E+05 2.1E-03 1.0E-08 Table 8. Binding affinity and kinetic rate constants for samples of anti-CD8α-IL- 15 fusion proteins that include IL-15 variants with combination mutations binding to IL15R-alpha Variant ka(1 / Ms) kd(1 / s) IL1 M tti A iti Di iti K M 170886321.1 Variant ka(1 / Ms) kd(1 / s) ID IL-15 Mutations Association Dissociation KD(M) rt ntnt rt ntnt d. d. d. 170886321.1 Variant ka(1 / Ms) kd(1 / s) ID IL-15 Mutations Association Dissociation KD(M) rt ntnt rt ntnt d. d. d. d. d. Table 9. Binding affinity and kinetic rate constants for samples of anti-CD8α-IL- 15 fusion proteins that include IL-15 variants with combination mutations binding to IL2R-beta Variant ka(1 / Ms) kd(1 / s) IL-15 Mutations Association Dissociation K (M) 97 170886321.1 Variant ka(1 / Ms) kd(1 / s) ID IL-15 Mutations Association Dissociation KD(M) rt ntnt rt ntnt 170886321.1 Variant ka(1 / Ms) kd(1 / s) ID IL-15 Mutations Association Dissociation KD(M) r t n t nt r t n t nt Table 10. Binding affinity and kinetic rate constants for samples of anti-CD8α-IL- 15 fusion proteins that include IL-15 variants with combination mutations binding to IL2R-beta / gamma heterodimer Variant ka(1 / Ms) kd(1 / s) IL-15 Mutations Association Dissociation KD(M) 170886321.1 Variant ka(1 / Ms) kd(1 / s) ID IL-15 Mutations Association Dissociation KD(M) rt ntnt rt ntnt 170886321.1 Variant ka(1 / Ms) kd(1 / s) ID IL-15 Mutations Association Dissociation KD(M) r t n t nt r t n t nt The anti-CD8α-IL-15 fusion proteins were then assessed in a pSTAT5 assay to determine activity on primary T cells. Signaling downstream of CD122 / CD132 was measured by detection of phosphorylated STAT5 (pSTAT5) using flow cytometry and the A647-anti-pSTAT5 antibody clone 47 / STAT5 (BD Biosciences #612599). PBMCs were resuspended in 50uL X-VIVO 15 media and plated at 100-200k cells / well in a 96- well round bottom plate. Test articles were added to a final concentration between 100nM and 0.01pM and incubated for 30 minutes at 37°C. Cells were then fixed with paraformaldehyde (PFA) at a final concentration of 2% for 15 minutes at room temperature. Cells were centrifuged and washed with FACS buffer (PBS with 2% FBS and 1mM EDTA), then permeabilized with ice cold methanol-based buffer (True-Phos Perm Buffer Biolegend #425401) at -20°C for 1 hour. Cells were centrifuged and washed with FACS buffer twice, then stained with A647-anti-pSTAT5 and the antibody panel in Table 11 in FACS buffer for 30 minutes at room temperature. Cells were washed with FACS buffer and fixed with 2% PFA for 15 minutes at room temperature. After fixing, cells were washed one more time with FACS buffer and then resuspended in 100uL of FACS buffer for flow analysis. Table 11. Antibody panel used in pSTAT5 assay Target Label Clone Vendor Catalog # 170886321.1 Helios AF488 D8W4X Cell Signaling 56424S KIR2D (pan) PE NKVFS1 Miltenyi 130-123-710 Activity of these muteins on CD4+ and CD8+ cells is shown in Table 12. The proportion of CD4+ or CD8+ T cells which had detectable pSTAT5 was determined by gating in FlowJo (Treestar, Ashland, OR) and plotted against the concentration of the CD8-IL15 protein using GraphPad Prism (GraphPad, Boston, MA). The EC50 was then calculated for both CD4+ and CD8+ cells. Where an EC50 could not be calculated, due to the response not reaching 50% of the wild-type IL-15 control, the sample was assigned a value of 100nM, the highest concentration in the assay; those results should be taken to represent a value that is at least 100nM, The EC50 on CD4+ cells was then divided by the EC50 on CD8+ cells as a metric for the selective activity of the mutein (Table 12). Table 12. Activity of CD8-targeted IL-15 muteins on CD4+ and CD8+ T cells in a pSTAT5 assay Variant Mutations CD4 EC50 CD8 EC50 CD4 / CD8 ratio 170886321.1 v53 V49G E89K 4094.0 1.6 2566.8 v54 V49Y E53A 498.0 1.2 427.5 103 170886321.1 v87 E53S E64G E89K 37151.0 10.3 3617.4 v88 V49Y E53A 1673.0 3.7 456.9 A number of the muteins showed a greater than 1000-fold difference in signaling between CD8+ and CD4+ T cells, indicating that these muteins successfully achieved differential signaling between a targeted cell and a non-targeted cell. f. EXAMPLE 6: Molecule Design and Plasmid Construction of Anti-KIR- IL-15 Fusion Proteins Anti-KIR-IL-15 fusion proteins having an anti-KIR2DL antibody portion and an IL-15 portion (as in FIG.2) were generated. The mAb portion consisted of anti- KIR2DL IPH2102 binding domains, and utilized knobs-into-holes (KiH) mutations in the human IgG1 (hIgG1) Fc to enable heterodimeric heavy chain assembly. The C- terminus of the anti-KIR2DL IPH2102 knob heavy chain was selected for adjoining each IL15 mutein. The anti-KIR2DL IPH2102 variable heavy (VH) binding domain was designed to directly adjoin to the human IgG1 CH1-CH2-CH3 domains to form an intact IgG1 heavy chain. The anti-KIR2DL IPH2102 variable light (VL) binding domain likewise adjoined to the human kappa constant region, thereby forming an intact kappa light chain. The VL binding domain had the amino acid sequence of SEQ ID NO:406, with light CDR1, CDR2, and CDR3 sequences according to Chothia notation as set forth in SEQ ID NOs:407, 408, and 409, respectively. The VH binding domain had the amino

[0089] 104 170886321.1 acid sequence of SEQ ID NO:410, with heavy CDR1, CDR2, and CDR3 sequences according to Chothia notation as set forth in SEQ ID NOs:411, 412, and 413, respectively. Additional refinements were introduced into the anti-KIR2DL mAb to facilitate heavy chain:light chain assembly. Two mutations were introduced into the constant regions of the molecule: one of these amino acid substitutions is in the anti-KIR2DL IPH2102 light chain kappa constant (Ck) domain at EU index position Q124 (Q124K). The second substitution is in the anti-KIR2DL IPH2102 heavy chain constant 1 (CH1) domain at EU index position K147 (K147E). The introduction of charged residue exchanges in the Ck and CH1 domains may improve heavy chain:light chain assembly, thereby resulting in a more homogeneous product. The hIgG1 Fc contained a subset of mutations in the CH2 domain to reduce effector function. The mutations corresponded to the amino acid substitutions at the following EU index positions: L234A, L235E, G237A, A330S, and P331S. The knobs-into-holes mutations were in the hIgG1 CH3 domain. The anti- KIR2DL IPH2102 heavy chain knob mutations corresponded to amino acid substitutions at EU index positions S354C and T366W. The anti-KIR2DL IPH2102 heavy chain hole mutations corresponded to amino acid substitutions at EU index positions Y349C, T366S, L368A, and Y407V. The wild-type IL-15 and the following IL-15 muteins from Example 1 were used in the fusion proteins: v9, v43, v44, v45, v51, v67, v84, and v92 (see Tables 1 and 2). Each IL-15 mutein was tethered to the antibody by introduction of a Gly-Ser linker at the C-terminus of the anti-KIR2DL IPH2102 knob heavy chain. Synthesized inserts were subsequently subcloned by ligation into the commercially available transient mammalian expression vector pcDNA3.4 (Invitrogen). The ligation mixture was transformed into E.coli. Resulting clones were screened by restriction digestion to confirm presence of an appropriate size insert. Positive transformants underwent DNA sequencing analysis to confirm the sequence of each insert. DNA with the correct sequence was subsequently used for expression.

[0090] 105 170886321.1 g. EXAMPLE 7: Expression of Anti-KIR-IL-15 Fusion Proteins Lyophilized plasmid DNA generated from Example 6 was reconstituted in sterile water to a concentration of 1 mg / mL. After reconstitution, each plasmid was transformed into E.coli DH10B (Thermo Scientific Cat #1829001) via electroporation using a Bio-Rad Gene Pulser II instrument set to 1.75 kV, 100 Ohms, and 25mF and allowed to undergo overnight selection on LB Amp plates (Teknova Cat #L1004) at 37C. A swab of transformants from each transformation was used to inoculate separate 35 mL Plasmid+ media (HTS Labs Cat #446300) containing 100 mg / mL Ampicillin (Teknova Cat #A9626) cultures for generating plasmid DNA. The cultures were allowed to grow overnight in an Innova42 incubator (New Brunswick Scientific) set to 37C, 300 rpm in vented 125 mL flasks (HTS Labs Cat #931147). Following overnight incubation, each culture was pelleted via centrifugation in a Sorvall Lynx 4000 centrifuge set to 7,000 X g at 4C for 15 minutes and the supernatant discarded. Plasmid DNA from the resulting bacterial pellets was processed using a Macherey Nagel plasmid DNA purification kit (NucleoBond Xtra Midi, Cat #740410) as per manufacturer instructions. DNA was resuspended in sterile water to approximately 1.5 mg / mL using the NanoDrop (Fisher Scientific, Cat #13-400-519) instrument to quantitate. Assembly of each intact anti-KIR2DL-IL-15 fusion protein molecule results from expression of three simultaneously co-transfected plasmids: one plasmid encoding the anti-KIR2DL light chain (nucleic acid sequence of anti-KIR2DL as set forth in SEQ ID NO:426), the second plasmid encoding the anti-KIR2DL heavy chain knob linked to an IL-15 portion (nucleic acid sequence of anti-KIR2DL knob heavy chain as set forth in one of SEQ ID NOs:428-436, for each IL-15 sequence), and the third plasmid encoding the anti-KIR2DL hole heavy chain (nucleic acid sequence of anti-KIR2DL hole heavy chain as set forth in SEQ ID NO:427). The signal sequences used in the plasmid constructs corresponded to SEQ ID NOs:205 (amino acid) and 206 (nucleic acid). These plasmids were transfected in a ratio of 1:1:0.5, respectively.

[0091] 106 170886321.1 For each anti-KIR2DL-IL-15 fusion protein transfection, 450mL cultures of Expi293F cells (Thermo Scientific Cat #A14527) were cultured to a density of 1.5E6 cells / mL in 500mL flasks (HTS Labs Cat #931112) in preparation for transient expression. The cells were allowed to grow overnight at 37C, 8% CO2, 150 rpm in Expi293 Expression Medium (Thermo Scientific Cat #A1435101). The following day the cells were transfected using PEI (Polyethylenimine, Polysciences Cat #23966-1) as the complexing reagent using a DNA concentration of 0.83 mg / L (0.33mg / L light chain, 0.33mg / L heavy chain knob, and 0.16mg / L heavy chain hole). DNA / PEI mixtures were allowed to incubate for 30 minutes before adding to the Expi293F cells. The transfected cultures were returned to the incubator set to the conditions listed above. One day post-transfection, each culture was given a bolus feed of 22.5 mL CHO Efficient Feed B Nutrient Supplement (Thermo Scientific Cat #A1024001) and 3.6 mL 500 mM Valproic acid (Fisher Cat #501786601). The cultures were allowed to incubate in a Kuhner Climo-Shaker ISF1-XC incubator at 37C, 8% CO2, 150 rpm for another 5 days. At 6 days post-transfection, the cultures were sampled for cell density and viability, and harvested. A sample was taken for Protein A titer analysis on the Octet Red 384 using Protein A sensors (Sartorius Cat #18-5010). The measured value was run against a standard curve of purified protein to determine culture titers. For harvest, cultures were clarified via addition of 0.1% PDADMAC (Sigma Aldrich Cat #409014) followed by centrifugation in a Sorvall Lynx 4000 centrifuge set to 7,000 x g, 4C for 20 minutes. The supernatants were 0.2um filtered (Thermo Scientific Cat # 5660010) and the pellets discarded. h. EXAMPLE 8: Purification of Anti-KIR-IL-15 Fusion Proteins Anti-KIR2DL-IL-15 fusion proteins produced in Example 6 and Example 7 and corresponding control proteins were purified with a two-step chromatography process to achieve desirable product quality characteristics. The proteins were first captured with protein A affinity chromatography to remove process related impurities. The proteins were subsequently purified with anion exchange polishing chromatography to

[0092] 107 170886321.1 remove product related impurities and misassembled molecules. Each step is described in further detail below. Following cell culture harvest, the supernatants containing the anti-KIR2DL-IL- 15 fusion proteins underwent capture via protein A affinity interaction. They were captured using HiTrap columns prepacked with MabSelect PrismA resin (Cytiva, Cat #17549852). This allows for the enrichment of Fc-containing molecules from crude feed streams like cell culture supernatants described in previous section. A summary of the operating parameters for protein A capture is presented below in Table 13. Protein A chromatography began with a sanitization step which was performed with 1.0 N sodium hydroxide (MilliporeSigma, Cat #221465) to remove any adventitious agents or endotoxin that may be present on the column from previous batches or storage. Following sanitization, the column was equilibrated with 20 mM sodium phosphate (MilliporeSigma, Cat #S9763), 150 mM sodium chloride (MilliporeSigma, Cat #S9888), pH 7.2 to match the pH and conductivity of the load material. Once the column was equilibrated, the harvested cell supernatant was applied to the column. It was then washed with 20 mM sodium phosphate, 150 mM sodium chloride, pH 7.2 to remove any unbound components from the resin. A second wash was performed with 50 mM sodium carbonate (MilliporeSigma, Cat #137014), 0.5 M sodium chloride (MilliporeSigma, Cat #S9888), pH 10.0 to remove non-specifically bound components. Following the wash, a transition was performed with 50 mM Tris- HCl (MilliporeSigma, Cat #108315), pH 7.5 to remove the salt. Elution followed with 40 mM sodium acetate (MilliporeSigma, Cat #241245), pH 3.5. Protein A elution occurs at low pH. To prevent the low pH from inducing protein degradation or aggregation, the proteins were immediately neutralized with 1.0 M Tris-HCl (MilliporeSigma, Cat #108315), pH 8.5 to a target of pH 7.5. Following neutralization, proteins were filtered into a 15 mL conical vial (MilliporeSigma, Cat #T1943) with a 0.2 micron syringe filter (MilliporeSigma, Cat #SLGPR33RB) to remove any precipitates that may be present and to control bioburden.

[0093] 108 170886321.1 Table 13. Operating parameters for protein A chromatography Step Buffer Parameter N / A N / A Resin: Cytiva MabSelect PrismA After protein A capture, the proteins were further purified with anion exchange chromatography (AEX) to remove product-related impurities and misassembled molecules. The anion exchange chromatography step was performed using HiTrap columns prepacked with Capto Q ImpRes resin (Cytiva, Cat #29400462). A summary of the operating parameters for anion exchange chromatography is presented below in Table 14. AEX began with a sanitization step which was performed with 1.0 N sodium hydroxide to remove any adventitious agents or endotoxin that may be present on the column from previous batches or storage. Following sanitization, the column was charged with 50 mM Tris-HCl, 1.0 M sodium chloride, pH 7.5. The column was then equilibrated 50 mM Tris-HCl, pH 7.5 to match the pH and conductivity of the load material. Once the column was equilibrated, the diluted and filtered protein A eluate was applied to the column. It is then washed with 50 mM Tris-HCl, pH 7.5 to remove

[0094] 109 170886321.1 any unbound components from the resin. The protein was eluted from the column with a gradient of 50 mM Tris-HCl, pH 7.5 to 50 mM Tris-HCl, 1.0 M sodium chloride, pH 7.5 over a length of 20 CVs. Fractions were collected and pooled following analysis of the resulting chromatogram. Table 14. Operating Parameters for Anion Exchange Chromatography Step Buffer Parameter N / A N / A Resin: Cytiva Capto Q ImpRes Following AEX, the collected fractions were pooled based on a strategy to minimize product-related contaminants. The pooled proteins were then buffer exchanged to 50 mM Tris-HCl, pH 7.5 and concentrated to a target of 1 g / L. The proteins were then aliquoted and inventoried in preparation for further downstream analytics and functional testing. i. EXAMPLE 9: Characterization of Anti-KIR-IL-15 Fusion Proteins Post-purification, a panel of analytical assays was compiled to validate the performance and quality of the anti-KIR2DL-IL-15 fusion proteins. The assays included size exclusion chromatography-high performance liquid chromatography

[0095] 110 170886321.1 (SEC-HPLC), liquid chromatography mass spectrometry (LC-MS) to confirm identity, reduced and non-reduced CE-SDS analysis by LabChip, endotoxin to confirm purity, and concentration. The analytical assays are summarized in the subsequent sections. SEC-HPLC The purity of anti-KIR2DL-IL-15 fusion proteins was evaluated by SEC-HPLC using an Agilent 1100 series HPLC. SEC-HPLC identifies any size variants such as high molecular weight species (HMW) and low molecular weight species (LMW). Operating parameters for the SEC-HPLC assay are presented in Table 15. Table 15. Operating parameters for SEC-HPLC Parameter Value Tosoh TSKgel G2000SWxl-G4000SWxl Guard Column A summary of SEC-HPLC results is presented in Table 16. Table 16. Summary of SEC-HPLC results Variant SEC- SEC- SEC- Mutation HPLC HPLC HPLC ) 170886321.1 LC-MS The identity of anti-KIR2DL-IL-15 fusion proteins was confirmed by LC-MS using a Q-TOF Sciex 5600 instrument. Operating parameters for the LC-MS assay are presented in Table 17. Anti-KIR2DL-IL-15 fusion proteins were first treated with PNGase F (New England Biolabs, Cat #P0710S) following the manufacturer recommended procedure to remove the glycans from the heavy chains of the molecule. Following deglycosylation, the samples were analyzed by LC / MS at the University of Washington Mass Spectrometry center. The samples were first separated on a polymeric reversed-phase (PLRP) protein column (Agilent, Cat #PL1912-1502), which isolates the protein from the aqueous mobile phase. The protein is eluted from the PLRP column via an acetonitrile gradient and injected directly into an AB Sciex 5600 mass spectrometer. Table 17. Operating parameters for LC-MS LC Operating Parameters PtValue 170886321.19.10.3 90 10MS Operating Parameters A summary of LC-MS results is presented in Table 18. Proposed modifications were determined based on mass difference from theoretical molecular weight and modifications that are known to routinely be present in antibody-like molecules. The identity of a molecule was confirmed if the observed mass has a small delta compared to the expected mass with the expected post-translational modifications. Table 18. Summary of LC-MS results Delt Observ a m N) 170886321.1 19x S-S E46K 2x PyroQ 51 159061 159256 1 L 159061 0 Y CE-SDS Anti-KIR2DL-IL-15 fusion proteins were also evaluated for CE-SDS purity with a Perkin Elmer GXII Touch Protein Analyzer (a.k.a LabChip). Samples were analyzed with the LabChip Protein Express assay under both reducing (R) and non- reducing (NR) conditions. An internal molecular weight ladder standard (PerkinElmer, Cat #CLS0008) was used to determine calibrated molecular weights for the samples. The assay was operated according to the manufacturer’s recommendation. A summary of Labchip results is presented in Table 19. Table 19. Summary of Labchip results NR CE- NR CE- V i t R E DS ) 170886321.1 v51 E46K E89K 0.0 > 99.9 96.6 67 K10 Y26A V49G 00 999 999 Endotoxin Anti-KIR2DL-IL-15 fusion proteins were assayed for endotoxin contamination using a Charles River nexgen-pts with limulus amebocyte lysate (LAL) assay cartridges (Charles River, Cat #PTS201). The assay was operated according to the manufacturer’s recommendation. A summary of endotoxin results is presented in Table 20. Table 20. Summary of endotoxin results Variant Mutat Endotoxin ID ion (EU / mg) Concentration The concentration of the purified anti-KIR2DL-IL-15 fusion proteins was analyzed by UV absorbance at 280 nm using a NanoDrop (Fisher Scientific, Cat #13- 400-519). A summary of concentration results is presented in Table 21.

[0096] 115 170886321.1 Table 21. Summary of concentration results Variant Mu Concentration ID tation (g / L) N A il t IL1 114 Binding Affinity Binding affinities of the anti-KIR2DL-IL-15 fusion proteins to IL15R-alpha (R&D Systems, P / N AVI7194), IL2R-beta (Acro Biosystems, P / N ILB-H82E3), and IL2R-beta / gamma heterodimer (Acro Biosystems, P / N ILG-H82F3) were determined by BLI (Bio-Layer Interferometry). Utilizing an Octet Red 384 instrument (Sartorius), biotinylated receptors were captured onto a streptavidin coated biosensor (Sartorius, P / N 18-5019). A dilution series of each anti-KIR2DL-IL-15 fusion protein was prepared and allowed to bind to the receptor. For interactions with IL15R-alpha, a dilution series from 800 – 12.5 nM was utilized for the IL15 mutein samples, while a dilution series from 100 – 1.56 nM was prepared for the wild-type control. For interactions with IL2R-beta and IL2R-beta / gamma, a dilution series from 400 – 6.25 nM was prepared for all samples. Binding was performed at 25ºC in Octet Kinetics Buffer (Sartorius, P / N 18-1105). Ten minutes of association followed by 10 minutes of dissociation were utilized for these measurements. Buffer reference subtraction was applied to the binding curves. Association and dissociation rate constants were globally fit to a 1:1 Langmuir binding model, and KD binding affinity was calculated. Table 22A, Table 22B, and Table 23 detail the results for IL15R-alpha, IL2R- beta, and IL2R-beta / gamma heterodimer, respectively.

[0097] 116 170886321.1 Table 22A. Binding affinity and kinetic rate constants for anti-KIR2DL-IL-15 fusion proteins samples binding to IL15R-alpha Variant ka (1 / Ms) kd (1 / s) ID Mutation Association Dissociation KD (M) 1 6 6 fusion proteins samples binding to IL2R-beta ka (1 / Ms) kd (1 / s) Variant M t ti A i ti Di i ti K M) 8 7 8 8 8 7 7 7 7 Table 23. Binding affinity and kinetic rate constants for anti-KIR2DL-IL-15 fusion proteins samples binding to IL2R-beta / gamma heterodimer

[0098] 117 170886321.1 Variant ka(1 / Ms) kd(1 / s) ID Mutation Association Dissociation KD(M) r t n t nt r t n t nt 9 9 9 9 9 8 8 9 9 Melting Temperature (Tm) The anti-KIR2DL-IL-15 fusion proteins were evaluated for thermal transitions. Utilizing an UNcle instrument (Unchained Labs), melting temperature (Tm) and aggregation temperature (Tagg) of the molecules were determined. The proteins were analyzed in a pH 7.5, Tris-HCl buffer. Thermal scans were performed in replicate (N=3), and intrinsic fluorescence was monitored from 25ºC to 95ºC with a ramp rate of 0.5ºC / minute. Data analysis was performed with UNcle software version 6.0. Thermal transitions were assigned with the barycentric mean (BCM) analysis method. Aggregation temperature was determined with the 266 nm laser. Three distinct thermal transitions were identified for these proteins. Table 24 details the results of this analysis. Table 24. Thermal transitions for anti-KIR2DL-IL-15 fusion protein samples Variant ID Mutation Tm1 Tm2 Tm3 Tagg 266 (ºC) (ºC) (ºC) (ºC)

[0099] 118 170886321.1 v45 Y26A E89K 51.4 61.2 79.5 64.6 c Binding Agent Bispecific Binding Agent Targeting KIR and CD8 A CrossMab was prepared using a Fab that binds to KIR2L1 / 2 / 3 (prepared from IPH2102 IgG1r mAb (parental antibody VH and VL sequences, SEQ ID NOs:410 and 406, respectively)) and an scFv that binds to CD8alpha (prepared from Mb1b IgG1r mAb (parental antibody VH and VL sequences, SEQ ID NOs:100 and 96)) (FIG.3A). The Fab and scFv were attached to an IgG1 hinge-CH2-CH3 in which the CH3 domain was engineered to contain the "knobs-into-holes" mutations to enforce correct association of the two heterodimeric heavy chains. The "knob" heavy chain included mutations S354C and T366W. The "hole" heavy chain included mutations Y349C, T366S, L368A, and Y407V. The KIR2L1 / 2 / 3 - CD8alpha CrossMAb was tested for co-binding to KIR2DL1 or KIR2DL3 and CD8alpha by biolayer interferometry using an Octet instrument. For the co-binding studies, the CrossMAb was captured to AHC (anti-human Fc) biosensors using 2-fold dilutions ranging from 0.3125 ug / ml to 20 ug / ml. The analytes (KIR2DL1, KIR2DL3, and CD8alpha) were kept constant at 100 nM. Analyte co-binding following capture was analyzed in two ways: first the association of KIR2DL1 or KIR2DL3 followed directly by the association of CD8alpha, or the association of CD8alpha followed by the direct association of KIRDL1 or KIR2DL3. KIR2DL1, KIR2DL3, and CD8alpha were tagged with a hexahistidine peptide. The CrossMab was able to co-bind targets KIR2DL1 or KIR2DL3 and CD8alpha.

[0100] 119 170886321.1 CrossMAb affinity for KIR2DL1, KIR2DL3, and CD8alpha ligands was measured and compared to the anti-CD8alpha and anti-KIR2DL1 / L2 / L3 parental antibodies using the Octet instrument (Table 25). For kinetic analysis, the CrossMAb was captured to AHC (anti-human Fc) biosensors using a load concentration of 1.25 ug / ml. Each analyte (KIR2DL1, KIR2DL3, and CD8alpha) concentration ranged from 6.25 nM to 200 nM. Analyte binding following capture was analyzed first for the association of KIR2DL1, KIR2DL3, or CD8alpha followed by the dissociation of each analyte independently. This ensured that ka (on rate), kd (off rate), and KD values could be obtained and directly compared to the parental antibodies. The kinetic analysis revealed that the CrossMab retained affinity for targets KIR2DL1, KIR2DL3, and CD8alpha. The affinities of the parental antibodies anti-KIR2DL1 / L2 / L3 IPH2102 IgG1r mAb and anti-CD8alpha Mb1b IgG1r mAb were also analyzed. For kinetic analysis, the parental antibodies were separately captured to AHC (anti-human Fc) biosensors using a load concentration of 1.25 ug / ml. For IPH2102 IgG1r mAb, the KIR2DL1, or KIR2DL3 analytes ranged from 6.25 nM to 200 nM. Analyte binding following capture was analyzed first for association of KIR2DL1 or KIR2DL3 followed by the dissociation of each analyte independently. Likewise, for the Mb1b IgG1r mAb, the CD8alpha analyte ranged from 6.25 nM to 200 nM. Analyte binding following capture was analyzed first for association followed by the dissociation of CD8alpha. Table 25. Comparison of affinities between the parental antibodies and the CrossMAb Protein Target Affinity KD (nM) IPH2102 t l tib d KIR2DL1 L2L3 KIR2DL1 138 M

[0101] 120 170886321.1 The anti-KIR / anti-CD8 bispecific molecule was shown to preferentially bind to CD8+ Tregs and was associated with increased CD8+ Treg activity and decreases in CD4+ T cells. Briefly, CD8+ Treg were enriched from Celiac patient PBMCs and cultured with autologous CD4+ T cells and antigen presenting cells pulsed with a gliadin peptide cocktail and analyzed using flow cytometry (FIG.3B and FIG.3C), bioplex assay detecting 34 analytes (ProCarta Plex; FIG.4), and longitudinal imaging using incucyte, followed by flow cytometry (FIG.5). Gliadin peptide restimulation of celiac patient-derived PBMCs in the presence of a bi- and mono-specific KIR blockade indicated that the bi-specific blockade (anti-KIR2DL1 / 2 / 3 and anti-CD8) resulted in greater effects on CD8+ Treg cell activity than blocking KIR alone (FIG.3B and FIG. 3C). These effects were also dose-dependent (FIG.3B and FIG.3C). Dose-dependent reductions were also observed for an extensive panel of proinflammatory cytokines (FIG.4) and chemokines. Bi-specific KIR-CD8 blockade was also associated with greater and dose-dependent effects on CD4+ T cell survival (FIG.5). Preferential binding of the bi-specific blocker (anti-KIR2DL1 / 2 / 3 and anti-CD8) to CD8+ T cells was demonstrated, despite 10-fold fewer CD8+ T cells expressing KIR compared to NK cells (FIG.6 and FIG.7). Delivery of IL-15 Untargeted cytokines (e.g., common-ɣ cytokines such as IL-15 and IL-7) can expand CD8+ Tregs in vitro. However, they may be too toxic for use in patients; the maximum tolerated dose for IL-2, IL-7, IL-15, and IL-21 is below 100ug / kg, with minimal or no therapeutic index. Untargeted cytokines may also have poor pharmacokinetic properties, e.g., a short half-life (< 6hrs) driven by consumption of homeostatic cytokines. Targeting cytokines to CD8+ Treg cells may allow selective activation and expansion of CD8+ Treg cells, allowing a reduction of potency. Cytokine potency may also be decreased by introducing mutations into cytokine polypeptides at specific sites to avoid off-target activity.

[0102] 121 170886321.1 Cytokines signal by bringing their respective receptor chains in proximity. Cytokines have different interfaces that bind to different receptor chains, which in turn cross-phosphorylate signaling proteins, such as the JAK / STAT, AKT, and MAPK pathways. The affinity of cytokines to receptors determines their potency. Phosphorylation of STAT5 is involved in signal transduction and antibody detection of phosphorylated STAT5 ("pSTAT5") can be used to measure cell activation (Table 26). Table 26. pSTAT5 signaling assay protocol 1Isolate PBMCs from fresh whole blood, rest overnight in X-VIVO 15 mediaor As part of the pSTAT5 assay, cells are permeabilized with 90-100% methanol buffer on ice for staining of phospho-transcription factors. First, it was determined that BioLegend True-Phos buffer indicates higher %pSTAT5+ and pSTAT5 MFI compared to 100% methanol (based on titration of wild- type IL-15 on frozen human PBMCs, and comparing 100% methanol permeabilization on ice to BioLegend True-Phos permeabilization buffer at -20C). That is, the use of

[0103] 122 170886321.1 BioLegend True-Phos buffer for permeabilization was associated with higher detection of phosphorylated STAT5 compared to assays using methanol. Second, an antibody panel was identified that allowed for detection by flow cytometry even after permeabilization. Preliminary tests of antibodies identified some clones that retain binding after permeabilization, including CD3 (SK7), CD4 (SK3), CD8 (SK1), and pSTAT5 (47 / STAT5). Antibodies to KIR2D, CD3, CD4, CD8, NKp46, and Helios were tested (see Table 27). Table 27. Antibody panel for CD8+ Treg pSTAT5 signaling assays Target Fluor Clone Isotype Vendor Dilution Zombie BV510 / 480 --- --- BioLegend 1000 The antibody panel enabled CD8+KIR2D+ Treg demarcation after methanol permeabilization. The antibodies showed low NKp46+ staining. Additionally, virtually all CD8+KIR2D+ cells were Helios+, but not all CD8+Helios+ cells were KIR2D+. Finally, pSTAT5 titrations looked very similar between fresh and frozen cells. The KIR2L1 / 2 / 3 - CD8alpha CrossMAb was expressed as a fusion protein including either wild-type or a variant / mutant form of IL-15 ("TM-15-CD8xKIR" or "IL-15 variant KIR x CD8 fusion") (FIG.8). The IL-15 variant had the amino acid sequence of SEQ ID NO:9 (v9 in Table 1).

[0104] 123 170886321.1 Administration of TM-15-CD8xKIR to cells in vitro resulted in reduced EC50 (i.e., higher potency) on CD8+KIR2D+ cells (24x lower EC50 on CD3+KIR2D+ cells vs CD3+KIR2D- cells;14x lower EC50 on CD8+KIR2D+ cells vs CD8+KIR2D- cells; 2.5x lower EC50 on CD8+KIR2D- cells vs CD3+KIR2D- cells), while administration of wild-type IL-15 alone was associated with similar EC50 and maxiumum sigaling across cell subsets (FIG.9A, FIG.9B). The changes in EC50 suggest KIR2D binding is driving this selectivity. TM-15-CD8xKIR also showed preferential signaling in CD8+KIR2D+ Tregs (FIG.10A, FIG.10B). A proliferation assay was developed for evaluating a targeted mutein's ability to selectively expand CD8+ Tregs. While pSTAT5 titrations provide an easy method to evaluate CD8+ Treg selectivity of targeted muteins, they do not demonstrate downstream effects of signaling, and low or transient signaling may not result in downstream effects such as proliferation or activation. Thus, a proliferation assay protocol was developed for determining downstream effects of targeted cytokines. This assay allows assessment of selective expansion of different cell subsets. New panels can be implemented to characterize activation or other phenotypic changes. The use of total PBMCs allows evaluation of off target effects on NK cells and CD4+ cells. Starting with total PBMCs or PBMCs depleted of CD14+ cells, the cells were stained with CellTrace Violet proliferation dye, plated at 100k cells / well, and incubated in 50uL plain X-VIVO 15 or X-VIVO 15 + 2ng / mL PMA (for low level stimulation). Short titration curves of targeted muteins or wild-type cytokines were generated, with test articles diluted to 20nM, 2nM, and 0.2nM concentration (1 plate with plain media, 1 plate with 4ng / mL PMA) and added at 50uL of test articles to cells. Test articles were replenished on Day5 / 6 and Day9 / 10, by removing 50uL media and added 50uL fresh media with diluted test articles. Four replicate plates were generated for Day3, Day6, Day9, and Day13 or Day14 timepoints and stained. Based on data from the first two timepoints, very little proliferation appears to have occurred at Day3, and some cell subsets were proliferating at Day6.

[0105] 124 170886321.1 Viability comparisons, based on analysis of average cell viability of cells treated with IL-15 variant-CD8xKIR or IL-15 using total PBMCs and plain media, showed highest number of viable cells on Day 9. Gating on proliferation dye peaks of total viable cells receiving 1nM IL-15 favored Day 9 timepoint. Day 3 and Day 6 showed low proliferation; Day 13 showed proliferation of the majority of viable cells. Viability and proliferation data indicated that Day ~9 provides the most useful assessment. Selective proliferation of CD8+KIR2D+ cells was observed on Day 9 with IL-15 variant-CD8xKIR. IL-15 variant-CD8xKIR showed higher proliferation of CD8+KIR2D+ cells than IL-15. Proliferation of CD4-CD8-KIR2D- cells gives insight into off-target effects of test articles.Low off-target proliferation of CD4-CD8-KIR2D- cells was observed on Day9 (FIG.11). Cells undergoing 4+ divisions indicate high proliferation. At 0.1nM of the test article, IL-15 variant-CD8xKIR showed higher proliferation of KIR2D+ cells (FIG. 12A). At 1nM of the test article, higher proliferation of CD8+KIR2D+ cells was observed, but also higher off-target cell proliferation (FIG.12B). At 10nM of the test article, IL-15v-CD8xKIRs still showed CD8+KIR2D+ selectivity, but high off-target proliferation (FIG.12C). k. EXAMPLE 11: in vivo Expansion of CD8+ Tregs with Targeted Muteins in a C57BL / 6 mouse In this example, the ability of targeted muteins to selectively expand CD8+ Tregs was tested in the C57BL / 6 mouse model. Two constructs were generated with paratopes binding either mouse CD8 or Ly49 and the IL-15 v9 mutein to examine the ability of targeted IL-15 to expand CD8+ or Ly49+ cells in vivo. Ly49 was chosen as the functional homolog of KIR in mice (Schenkel et al., Front. Immuol.2013 Apr 16, doi:10.3389 / fimmu.2013.00090). Mice were intravenously dosed with targeted mutein constructs at three different dose levels.75uL peripheral blood draws were performed at 4, 7, and 15 days after dosing, as well as 4 days before dosing. Peripheral blood samples were lysed in 1mL ACK buffer for 5 minutes at room temperature. Lysed samples were centrifuged and

[0106] 125 170886321.1 supernatants aspirated. The resulting cell pellets were transferred to a 96-well round bottom plate, where they were first stained with Fixable Viability Dye eFluor780 (eBioscience #65-0865-18) and TruStain FcX (Biolegend #101320) diluted in PBS for 15 minutes at room temperature. Cells were washed with FACS buffer and then stained with the antibody panel described in Table 28 for 30 minutes at room temperature. Cells were then washed once and resuspended in 100uL FACS buffer for flow analysis. Table 28. Antibody panel used in Example 11 Target Label Clone Vendor Catalog # CD3 BV785 17A2 BioLegend 100232 2 Untargeted mFc-IL-15 v9 showed selective expansion of NK and CD8+Ly49+ cells 5-7 days after injection of the test article. CD8-targeted IL-15 showed selective expansion of the entire CD8+ T cell population, over other CD45+ lymphocytes. CD8- IL-15v9 also expanded NK1.1+ cells at the highest dose. Ly49-targeted IL-15 showed selective expansion of CD8+Ly49+ Treg cells in C57BL / 6 mice over the conventional CD8+ population, suggesting that targeting IL-15 to this population can expand CD8+ Treg. Ly49-IL-15 v9 also expanded NK1.1+ NK cells, which also express Ly49. In summary, these data suggest that in vivo, targeting of IL-15 via antibody to CD8+ Treg cells results in rapid expansion of this population.

[0107] 126 170886321.1 l. EXAMPLE 12: Selectivity of CD8-Targeted IL-15 Muteins In this example, the ability of CD8-targeted cytokine muteins to selectively signal in CD8+ Tregs was measured in primary human PBMCs. Anti-CD8α-IL-15 fusion proteins having an anti-CD8α antibody portion and an IL-15 portion were generated as in Example 2. CD122 / CD132 signaling was measured by detection of phosphorylated STAT5 (pSTAT5) using flow cytometry and the A647-anti-pSTAT5 antibody clone 47 / STAT5 (BD Biosciences #612599). PBMCs were resuspended in 50uL X-VIVO 15 media and plated at 100-200k cells / well in a 96-well round bottom plate. Test articles were added to a final concentration between 100nM and 0.01pM and incubated for 30 minutes at 37°C. Cells were then fixed with paraformaldehyde (PFA) at a final concentration of 2% for 15 minutes at room temperature. Cells were centrifuged and washed with FACS buffer (PBS with 2% FBS and 1mM EDTA), then permeabilized with ice cold methanol-based buffer (True-Phos Perm Buffer Biolegend #425401) at -20°C for 1 hour. Cells were centrifuged and washed with FACS buffer twice, then stained with A647-anti-pSTAT5 and the antibody panel in Table 11 in FACS buffer for 30 minutes at room temperature. Cells were washed with FACS buffer and fixed with 2% PFA for 15 minutes at room temperature. After fixing, cells were washed one more time with FACS buffer and then resuspended in 100uL of FACS buffer for flow analysis. CD8-targeted wild-type IL-15 shows a small degree of selectivity between targeted CD8+ T cells and non-targeted CD4+ cells. Reduced-potency IL-15 variants v9, v67, and v84 show selective signaling on targeted CD8+ cells over non-targeted CD4+ cells (FIGS.13A, 13B, 13C, 13D). m. EXAMPLE 13: Selectivity signaling of KIR-targeted IL-15 Muteins In this example, the ability of KIR-targeted cytokine muteins to selectively signal in KIR+CD8+ Tregs was measured in primary human PBMCs. Anti-KIR-IL-15 fusion proteins having an anti-KIR antibody portion and an IL-15 portion were generated as in Example 6. Similar to Example 12, signaling downstream of CD122 / CD132 was measured by detection of phosphorylated STAT5 (pSTAT5) using

[0108] 127 170886321.1 flow cytometry and the A647-anti-pSTAT5 antibody clone 47 / STAT5 (BD Biosciences #612599). PBMCs were resuspended in 50uL X-VIVO 15 media and plated at 100-200k cells / well in a 96-well round bottom plate. Test articles were added to a final concentration between 100nM and 0.01pM and incubated for 30 minutes at 37°C. Cells were then fixed with paraformaldehyde (PFA) at a final concentration of 2% for 15 minutes at room temperature. Cells were centrifuged and washed with FACS buffer (PBS with 2% FBS and 1mM EDTA), then permeabilized with ice cold methanol-based buffer (True-Phos Perm Buffer Biolegend #425401) at -20°C for 1 hour. Cells were centrifuged and washed with FACS buffer twice, then stained with A647-anti-pSTAT5 and the antibody panel in Table 11 in FACS buffer for 30 minutes at room temperature. Cells were washed with FACS buffer and fixed with 2% PFA for 15 minutes at room temperature. After fixing, cells were washed one more time with FACS buffer and then resuspended in 100uL of FACS buffer for flow analysis. Similar to previous experiments, targeted wild-type IL-15 showed a small difference between KIR+ targeted cells and KIR- non-targeted cells (FIGS.14A-14I). The difference between KIR+ CD8+ cells and KIR- CD8+ cells is then quantified by comparison of EC50 generated from curve fits from FIGS.14A-14I. An anti-KIR antibody with WT IL-15, shows similar signaling potency on KIR+ and KIR- cells (FIGS.14A-14I and Table 29). Table 29. Potency and differential signaling on KIR+ and KIR- cells CD8+KIR2D- CD8+KIR2D+ Fold EC50 EC50 KIR+ / KIR- .6 .8 .8 .7 .9 .2 .9 128 170886321.1 v84 5204 2.863 1817.7 v92 9954 9.607 1036.1 - 5 mu e ns genera ed s owed a grea er marg n o d eren a n s gnaling potency between KIR+ and KIR- cells compared to WT IL-15 (FIG.15 and Table 29). Of the IL-15 muteins tested, v9, v51, v67, and v84 showed the greatest differential KIR+ and KIR- cells. Maximal signaling for WT IL-15 and all cytokine muteins was also assessed, based on CD8+KIR+ curves shown in FIGS.14A-14I. All muteins assessed had similar maximal signaling to WT IL-15, suggesting that despite the lower potency, muteins stimulated all cells able to respond to IL-15 in the culture (FIG.16). n. EXAMPLE 14: in vitro Proliferation Assay of Targeted Cytokine Mutein Molecules Using Human PBMCs In this example, the downstream functional effect of targeted muteins was assessed in primary human PBMCs by characterizing the proliferation of immune cell subsets. Anti-KIR-IL-15 fusion proteins having an anti-KIR antibody portion and an IL- 15 portion were generated as in Example 6. Flow cytometry was used to identify cell subsets and Ki67 to identify cells which were in cell cycle at the time of the experiment. PBMCs were washed with PBS and plated at 100k cells / well in a 96-well round bottom plate. Test articles were then added to cells for a final volume of 100uL and final concentration between 10nM and 0.1pM, then incubated at 37°C for 5 days. On Day 5, 50uL of culture media was removed and replaced with fresh media with diluted test articles. On Day 8, cells were washed once with PBS and then stained with Zombie NIR viability dye (Biolegend #423106) and Human TruStain Fcx (Biolegend # 422302) diluted in PBS for 15 minutes at room temperature. Cells were washed with FACS buffer, then fixed and permeabilized with diluted Fixation buffer from the True-Nuclear Transcription Factor Buffer Set (Biolegend #424401) for 1 hour at room temperature. Cells were centrifuged and washed twice with diluted Permeabilization Buffer and stained with the antibody panel in Table 30A in Permeabilization Buffer for 30 minutes

[0109] 129 170886321.1 at room temperature. Cells were then washed once and resuspended in 100uL FACS buffer for flow analysis. Table 30A. Antibody panel used in proliferation assay of Example 14 Target Label Clone Vendor Catalog # Helios AF488 D8W4X Cell Signaling 56424S p g g, y g induced greater difference in potency of proliferation (as measured by Ki67) between KIR+ cells and KIR- cells than KIR antibody conjugated to wild-type IL-15 (FIGS. 17A-17J). Muteins v9, v43, v44, v45, v51, v67, and v84 showed similar potency on KIR+ cells to WT IL-15, suggesting that these muteins fully signal to generate functional proliferative response (FIGS.17A-17J). v92 did not show a similar maximal response compared to the other muteins and WT IL-15, suggesting that it does not fully stimulate T or NK cells to proliferate. o. EXAMPLE 15: Activity of KIR-targeted IL-15 muteins in cells from an SLE donor In this example, the ability of KIR-targeted cytokine muteins to selectively signal in KIR+CD8+ Tregs was measured in PBMC from a donor with SLE. Anti-KIR- IL-15 fusion proteins having an anti-KIR antibody portion and an IL-15 portion were generated as in Example 6. Similar to Example 12, signaling downstream of CD122 / CD132 was measured by detection of phosphorylated STAT5 (pSTAT5) using flow cytometry and the A647-anti-pSTAT5 antibody clone 47 / STAT5 (BD Biosciences #612599).

[0110] 130 170886321.1 PBMCs were resuspended in 50uL X-VIVO 15 media and plated at 100-200k cells / well in a 96-well round bottom plate. Test articles were added to a final concentration between 100nM and 0.01pM and incubated for 30 minutes at 37°C. Cells were then fixed with paraformaldehyde (PFA) at a final concentration of 2% for 15 minutes at room temperature. Cells were centrifuged and washed with FACS buffer (PBS with 2% FBS and 1mM EDTA), then permeabilized with ice cold methanol-based buffer (True-Phos Perm Buffer Biolegend #425401) at -20°C for 1 hour. Cells were centrifuged and washed with FACS buffer twice, then stained with A647-anti-pSTAT5 and the antibody panel in Table 11 in FACS buffer for 30 minutes at room temperature. Cells were washed with FACS buffer and fixed with 2% PFA for 15 minutes at room temperature. After fixing, cells were washed one more time with FACS buffer and then resuspended in 100uL of FACS buffer for flow analysis. Similar to previous experiments, targeted wild-type IL-15 showed a small difference in potency between CD8+KIR+ targeted cells and CD8+KIR- non-targeted cells (FIGS.18A, 18B, 18C, 189D, 18E). KIR-targeted WT IL-15 showed small differences between targeted (CD8+KIR2D+) and non-targeted (CD4+) populations. Muteins v9, v51, and v67 showed large differences in potency between targeted (CD8+KIR2D+) and non-targeted (CD4+ and CD8+KIR2D-) populations, suggesting that the differences seen in healthy donors also holds in autoimmune. p. EXAMPLE 16: Analysis of Clonal Expansion of CD8+ Treg in culture with targeted wild-type and mutein IL-15 Anti-KIR-IL-15 fusion proteins having an anti-KIR antibody portion and an IL- 15 portion were generated as in Example 6. PBMC from a single healthy and a single celiac donor were thawed and plated in huTCM (X-Vivo 15 (Lonza) containing 5% human AB serum (Gemini Bio), 1x penicillin / streptomycin (Gibco), and 1x GlutaMax (Gibco)) with either 0.8 nM of CD8-IL-15 WT or CD8-IL-15 v9. After four days, half the media was replaced with media containing 1.6nM concentration of either CD8-IL- 15 WT or CD8-IL-15 v9. Cells were then cultured in IL-15 test articles for a further

[0111] 131 170886321.1 four days. Cells that had been cultured in IL-15 were then stained with the antibodies in Table 30B. Freshly thawed cells from the same donor were also stained, as pre- expansion samples. The cell samples were sorted on a BD Fusion, and KIR2D+KIR3DL1+CD8+CD4-CD3+ cells collected. Table 30B. Antibodies used to stain cells in Example 16 Target Label Clone Vendor KIR3DL1 PE DX9 Biolegend nalyzed in a human TCRB assay. Comparison of the CD8+ Treg clones expanded by CD8-targeted wild-type IL- 15 and IL-15 v9 suggests that similar clones are expanded by both wild-type and mutein IL-15, as the majority of the clones lie on the identity line. Analysis of TCRB gene usage using Morisita Index suggests that similar turnover was seen with treatment with targeted IL-15 wild-type or IL-15 v9. Despite the mutations introduced, the v9 IL-15 mutein performed similarly to unmodified IL-15. q. EXAMPLE 17: RNAseq of KIR+CD8+ cells Expanded with IL-15 PBMCs from healthy and celiac donors were thawed and plated at 2e6 c / ml in a 12-well plate (2e6 cells total / well). Wild-type IL-15 ("WT IL-15", ), CD8-targeted WT IL-15 (anti-CD8α-IL-15, SEQ ID NOs:104, 106, and 204), and CD8-targeted IL-15 v9 mutein (v9, SEQ ID NO:9; anti-CD8α-IL-15v9, SEQ ID NOs:104, 106, and 118) were each added to cells at 2 different concentrations (0.78 and 3.125 nM). The antibody portion of the CD8-targeted muteins comprised an anti-CD8α Mb1b light chain having the amino acid sequence of SEQ ID NO:104; an anti-CD8α Mb1b knob heavy chain having the amino acid sequence of SEQ ID NO:105; and an anti-CD8α Mb1b hole heavy chain having the amino acid sequence of SEQ ID NO:106, with the IL-15

[0112] 132 170886321.1 tethered to the antibody via a linker at the C-terminus of the anti-CD8α Mb1b knob heavy chain, as in Example 2 above. Cells were cultured at 37°C for 8 days and cytokines were replaced on day 4. On day 8, cells were stained with PE anti-KIR3DL1 (clone DX9), BV421 anti-CD3 (clone OKT3), AF488 anti-CD8a (clone SK1), and BV786 anti-CD4 (clone OKT4) (all from Biolegend), and PE anti-pan KIR2D (clone NKVSF1; Miltenyi). Cells were washed and incubated with anti-PE MicroBeads (Miltenyi) and PE-labeled KIR+ cells were isolated using an MS Column (Miltenyi) following kit directions. Subsequent isolation of CD3+CD8+KIR+ T cells was performed using a BD FACSAria Fusion cytometer. Sorted cells were pelleted, flash frozen in a dry ice / isopropanol slurry, and frozen at -80°C. RNA was purified from frozen cells by using Trizol (Thermofisher). Library generation was then performed using a SMART-Seq® v4 Ultra® Low Input RNA Kit (Clontech) on polyadenylated RNA, followed by second-strand synthesis. Libraries were then generated and run on an Illumina NextSeq 2500. The resulting sequences were aligned to the human genome and resulting genes with expression differences identified. IL-15 induced a large set of genes in both healthy and celiac patients, including genes associated with proliferation (MKI67), function (KIR2DL1, KIR2DL3, ICOS), and cytotoxicity (GZMA, GZMB) (FIGS.19A, 19B). Minimal differences in gene expression were seen between targeted WT IL-15 and IL-15 v9, suggesting that both WT and IL-15 mutein induce similar gene expression programs (FIGS.20A, 20B). r. EXAMPLE 18: Expansion of KIR+CD8+ Treg in a PBMC-engrafted mouse model To test the ability of KIR-targeted therapeutics in vivo, NSG mice were engrafted with PBMC from a healthy human donor and administered either an untargeted Fc IL-15v9 mutein or an anti-KIR-IL-15v9 fusion protein having an anti- KIR antibody portion and an IL-15v9 portion were generated as in Example 6.1 x 107 cells were injected i.v. on study day 0, followed by intraperitoneal injection of either

[0113] 133 170886321.1 0.03mg / kg untargeted Fc-IL-15v9, 0.074mg / kg targeted KIR-IL-15v9 or saline on days 1, 8, 15, 22, 29, and 36 after PBMC transfer. At day 38, mice were bled for flow cytometry. Blood from mice was taken, placed into wells of a 96 deep-well plate, spun down, and subjected to RBC lysis by adding ACK lysis buffer (Gibco) for 5 minutes at room temperature. The lysis was stopped by adding 1ml PBS to each well and the plate washed twice in PBS. The cells were then stained with Zombie NIR live / dead stain and Fc block (Biolegend) for 20 mins at room temperature in the dark. Cells were then washed once in PBS, and once in AnnexinV buffer (Biolegend), followed by addition of antibodies staining extracellular targets (Table 31; all except Helios and Granzyme B), and left at room temperature for 30 minutes in the dark. The cells were then washed again in Annexin V buffer and placed into Fix / Perm buffer (eBioscience Foxp3 / Transcription factor Fix / Perm kit) for 30 minutes at room temperature, followed by washing in 1x Perm buffer (eBioscience) The antibodies labelling intracellular targets (Helios and Granzyme B) were then added, incubated at room temperature for 30 minutes, and then washed once in 1x Perm buffer. Cells were then resuspended in FACS buffer and analysed. Table 31. Flow cytometry antibodies used in Example 18 Marker Label Clone Cat. Vendor Zombie NIR APC-Cy7 - 77184 Biolegend 170886321.1 Helios PE daz 22F6 137232 Biolegend GranzymeB PE-Cy7 QA16A02 372214 Biolegend uman reg n s expermen were e ne as live hCD45+CD3+CD56-CD8+CD4-KIR2D+. At day 38, a significant increase in KIR+CD8+ Treg cells was seen with KIR-IL-15v9, compared to mice that had received saline (FIG.21). An insufficient number of mice that had received Fc-IL-15v9 remained on study for statistical comparison to KIR-IL-15v9, but levels of CD8+KIR+ Treg were similar to mice receiving saline. s. EXAMPLE 19: Killing of autoimmune TCR expressing target cells by CD8+ Treg To test if CD8+ Tregs expanded in IL-15 mutein conditions were functional, cells were assessed for their ability to kill CD4+ T cells. Total PBMCs (peripheral blood mononuclear cells) from a celiac disease (CeD) donor (Sanguine Biosciences, Woburn, MA) were thawed into human T cell media (huTCM) (X-Vivo 15 (Lonza) containing 5% human AB serum (Gemini Bio), 1x penicillin / streptomycin (Gibco), and 1x GlutaMax (Gibco)). Cells were plated in 6-well plates at 6.6e6 c / well and 4 mL / well. CD8-IL-15 wild-type (WT) or CD8-IL-15v9 (as described in Example 2) were added to cells at a concentration of 0.38 nM. After 5 days in culture 2 mL of media were removed and replaced with 2 mL of media containing either 0.76 nM of CD8-IL-15WT or CD8-IL-15v9. Cells were cultured at 37°C in a humidified incubator for an additional 3 days (8 days total), and then flow sorted for CD5+CD4-NKp46-CCR7- CD28- cells to enrich for the KIR+ CD8+ Treg population. An aliquot of sorted cells were stained for CD8 and KIR to determine the percentage CD8+KIR+ and used to calculate the number of CD8+ Treg present in CD8-IL15 WT or CD8-IL15v9 treated samples. GFP+ SKW cells engineered to express the LS2.8 TCR responsive to gliadin peptide alpha-1a were stimulated overnight with equal numbers of CD14+ cells isolated from PBMC derived from a healthy donor (BloodworksNW) with an HLA-DQ2.5

[0114] 135 170886321.1 haplotype and 10 ug / mL gliadin peptide QLQPFPQPELPYPQPQ (SEQ ID NO.:439) (Elim BioPharmaceuticals) and 5 ng / mL IFNγ (Biolegend) and used as target cells. Activated targets were plated in poly-L-lysine coated 384-well, optically clear plates at 20,000 cells / well in 50 uL of huTCM and allowed to settle for 30 to 60 minutes before placing into the incubator. Equal numbers of either CD8-IL-15WT or CD8-IL-15v9 treated CD8+ Tregs were added on top of 20,000 activated targets in 50 uL, and then transferred to IncuCyte® cell analysis system (Sartorius, Ann Arbor, MI). Every four hours, the GFP fluorescence of the SKW cells was then measured. Level of GFP-fluorescence was then normalized to the 8-hour time point. At time points after 8 hours, co-cultures containing CD8+ Treg that had been cultured in CD8-IL- 15WT or CD8-IL-15v9 showed lower levels of fluorescence, compared to cultures of SKW alone, suggesting that CD8+ Treg are able to kill or suppress the GFP labelled autoimmune TCR expressing SKW cells (FIG.22). Both CD8+ Treg cells cultured in IL-15WT and IL-15v9 were able to reduce the SKW cells to the same level, suggesting that IL-15 mutein is able to function similarly to IL-15WT in expanding functional CD8+ Treg. t. EXAMPLE 20: Expansion of KIR+CD8+ Treg in a Mixed T cell- Engrafted Mouse Model To test the ability of KIR-targeted IL-15 muteins in vivo, a model using mixed CD4+ T cell and enriched CD8 Treg populations was developed. Separately, the CD4+ and CD8+ T cells were negatively selected via bead enrichment (Dynabeads Untouched, Invitrogen, Waltham, MA) from a healthy donor by following the manufacturer's instructions. CCR7- CD28- CD8+ cells, a cell population enriched for KIR+ CD8s, were further selected by removing CCR7 (Biolegend) and CD28 (Biolegend) positive cells. To achieve this, CD8+ cells were incubated with CCR7-PE and CD28-PE antibodies (Biolegend) for 30 minutes on ice in the dark. The cells were then washed once in isolation buffer (PBS (Ca2+ and Mg2+ free) supplemented with 0.1% BSA and 2 mM EDTA), and then mixed anti-PE beads (Miltenyi) according to

[0115] 136 170886321.1 the manufacturer’s instructions. These cells were then washed twice in the isolation buffer and passed through an LS column (Miltenyi Biotech) to remove bead-labelled cells. NSG mice (Jackson Laboratory, Bar Harbor, ME), were injected with a mixture of 1x106CD4+ T cells and 2x105CCR7-CD28-CD8+ on SD0. Cells were enriched and injected by i.v. into irradiated mice on study day 0. Mice then received intraperitoneal injection of either 0.075 mg / kg targeted anti-KIR-IL15 v67 (K10Q Y26A V49G; as described in Example 2) or saline on days 1, 8, 15, 22, 32, 39, 46, 53, 60, 67, 74, and 81. On day 35, mice were bled for flow cytometry. Blood from mice was taken, placed into wells of a 96 deep-well plate, spun down, and subjected to RBC lysis by adding ACK lysis buffer (Gibco) for 5 minutes at room temperature. The lysis was stopped by adding 1ml PBS to each well and the plate washed twice in PBS. The cells were then stained with Zombie NIR live / dead stain and Fc block (Biolegend) for 20 mins at room temperature in the dark. Cells were then washed once in PBS, and once in FACs buffer, followed by addition of antibodies staining extracellular targets (Table 32; all except Helios, Ki67, and Granzyme B), and left at room temperature for 30 minutes in the dark. The cells were then washed again in FACs buffer and placed into Fix / Perm buffer (eBioscience Foxp3 / Transcription factor Fix / Perm kit) for 30 minutes at room temperature, followed by washing in 1x Perm buffer (eBioscience). The antibodies labelling intracellular targets (Helios, Ki67, and Granzyme B) were then added, incubated at room temperature for 30 minutes, and then washed once in 1x Perm buffer. Cells were then resuspended in FACS buffer and analyzed. Table 32. Flow cytometry antibodies used in Example 20 MarkerLabelClone Catalog Company 170886321.1 CD8 BV605 SK1 344742 Biolegend CD4 BV650 OKT4 317436 Biolegend Survival of mice was tracked over the study, with mice which had lost more than 20% of initial body weight being euthanized. Animals that received KIR-IL15 v67 trended towards increased survival relative to those that received saline control (FIG. 23). KIR+ CD8+ Tregs were defined as live, hCD45+CD3+CD4-CD8+KIR+ cells. At study day 35, KIR+ CD8+ Treg were significantly increased in animals that received anti-KIR-IL15 v67 relative to saline control animals (FIG.24A). CD8+KIR+ cells from mice dosed with anti-KIR-IL15 v67 also showed significantly increased Granzyme B relative to saline-treated animals (FIG.24B). u. EXAMPLE 21: Targeting IL-15 to CD8+ Treg results in their selective expansion and activation and is a potential therapeutic approach to ameliorate autoimmune disease in patients with deficient CD8+ Treg populations CD8+ regulatory T cells (CD8+ Treg) are characterized by their capacity to kill self-reactive and pathogenic CD4+ T cells. Control of pathogenic CD4+ T cells appears impaired in autoimmune disease, in part due to reduced CD8+ Treg prevalence and function. A IL-15 mutein with reduced potency (v67 fused to an anti-KIR antibody, as described in Example 6 and including the anti-KIR knob HC sequence according to SEQ ID NO:423) was developed and tethered to a targeting antibody to promote selective expansion, activation, and cytolytic capacity of CD8+ Treg, with the potential of ameliorating disease.

[0116] 138 170886321.1 Flow cytometry was used to characterize CD8+ Treg incubated with wild-type IL-15 or a targeted IL-15 mutein. Wild-type IL-15 expanded and activated CD8+ Tregs in vitro. Wild-type IL-15 showed a higher fold change in CD8+ Treg counts compared to other γ-chain cytokines after 5 days of culture in vitro (FIG.25A), and wild-type IL-2 and wild-type IL-15 showed highest increase in % Granzyme B+ CD8+ Tregs at day 10 of culture (FIG. 25B). Flow cytometry characterization of CD8+ Treg from different patient populations showed increased expression of T-bet (FIG.26A), CD69 (FIG.26B), and ICOS (FIG. 26C) with wild-type IL-15 incubation for 24 hours. CD8+ Treg from Type 1 diabetes (T1D) and rheumatoid arthritis (RA) patients showed statistically significant increases of all three markers. CD8+ Treg from Crohn’s disease (CrD) patients showed a trend of increased expression of all three markers. Pancreatic organoids were incubated with peptide-stimulated PBMCs from a type-1 diabetes patient (T1D) to evaluate the effects of the targeted IL-15 mutein on the CD8+ Treg and Beta cell populations. PBMCs from a T1D patient were thawed and cultured + / - T1D antigenic peptides and + / - targeted IL-15 mutein for 48 hours. PBMCs were added to the culture media of pancreatic organoids from a healthy donor and incubated for 72 hours (FIG.27A).Targeted IL-15 mutein expanded CD8+ Treg in the T1D organoid model and resulted in protection of Beta cells. Anti-KIR-v67IL-15 resulted in greater expansion of CD8+ Tregs in the T1D patient PBMCs, with or without T1D antigenic peptides (FIG.27B). Addition of anti-KIR-v67IL-15 also resulted in fewer apoptotic Beta cells in the pancreatic organoid (FIG.27C). The results of this experimental example, in combination with those described above (e.g., Examples 12, 13, 14, 15, 17, 18, 20), show: (1) Wild-type IL-15 demonstrated higher potential for expansion and activation of the CD8+ Treg population compared to other γ chain cytokines (e.g., FIGS.25A, 25B); (2) Reduction of IL-15 activity via point mutations can be restored by fusion of the IL-15 mutein to a targeting antibody (e.g., Example 12, FIGS.13A, 13B; Example 13, FIGS.14A, 14G); (3) Selective signaling of the IL-15 mutein in the CD8+ Treg population can be

[0117] 139 170886321.1 achieved by fusion to an antibody for an appropriate CD8+ Treg surface marker (e.g., Example 12, FIGS.13A-13D; Example 13, FIGS.14A-14I, 16); (4) Selective signaling of a CD8+ Treg-targeted IL-15 mutein can be enhanced with select combinations of point mutations (e.g., Example 12, FIGS.13A-13D; Example 13, FIGS.14A-14I, 16); (5) the CD8+ Treg-targeted IL-15 mutein demonstrates proliferation of the CD8+ Treg population over other PBMC subsets in vitro (Example 14, FIG.17H); (6) PBMCs incubated with the CD8+ Treg-targeted IL-15 mutein show an increased CD8+ Treg population and prevent Beta cell death in a T1D pancreatic organoid model (FIGS. 27A-27C); and (7) Humanized NSG mice with acute GvHD receiving the CD8+ Treg- targeted IL-15 mutein showed trends towards increased survival and CD8+ Treg prevalence in peripheral blood (Example 20, FIGS.23, 24A, 24B). Anti-KIR-v67IL-15 provided selective pSTAT5 signaling and proliferation of the CD8 Treg population in vitro. GvHD mice treated with this CD8+ Treg-targeted IL-15 mutein showed a trend toward increased survival and higher levels of CD8+ Tregs in peripheral blood. These data suggest that IL-15 muteins targeted to CD8+ Tregs have therapeutic potential for autoimmune diseases that are characterized by a deficiency of the CD8 Treg population.

[0118] 140 170886321.1 EXAMPLE SEQUENCES (ADDITIONAL SEQUENCES IN SEQUENCE LISTING) Table 33. Sequences. SEQ Description Sequence ID N V L I V L I V L I V L I V L I V L I V L I V L I V L I M N S 141 170886321.1 11 v11, N-terminal ISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKC truncation II FLLELQVISLESGDASIHDTVENLIILANNSLSSNGN VTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS V L I V L I V L I V L I V L I V L I V L I V L I V L I V L I V L 170886321.1 SSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFI NTS V L I V L I V L I V L I V L I V L I V L I V L I V L I V L I V L 170886321.1 SSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFI NTS V L I V L I V L I V L I V L I V L I V L I V L I V L I V L I V L 170886321.1 SSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFI NTS V L I V L I V L I V L I V L I V L I V L I V L I V L I V L I V L 145 170886321.1 SSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFI NTS V L I V L I V L I V L I V L I V L I V L I V L I V L I V L I V L I 170886321.1 67 v67, K10Q Y26A NWVNVISDLQKIEDLIQSMHIDATLATESDVHPSCKV V49G TAMKCFLLELQGISLESGDASIHDTVENLIILANNSL SSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFI V L I V L I V L I V L I V L I V L I V L I V L I V L I V L I

[0119] 147 170886321.1 78 v78, V49Y E53R NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKV E64G TAMKCFLLELQYISLRSGDASIHDTVGNLIILANNSL SSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFI V L I V L I V L I V L I V L I V L I V L I V L I V L I V L I

[0120] 148 170886321.1 89 v89, K10Q V49Y NWVNVISDLQKIEDLIQSMHIDATLYTESDVHPSCKV E53A M109A TAMKCFLLELQYISLASGDASIHDTVENLIILANNSL SSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQAFI V L I V L I V L I V L I V L I V L I Q T V T G

[0121] 149 170886321.1 101 anti-CD8α Mb1b GFNIKDT Heavy CDR1 Q T A W D V T G K S D E Q I V L L V T G K S D E Q I V L L

[0122] 150 170886321.1108 (EAAAK)3 linkerGEAAAKEAAAKEAAAK

[0123] 151 170886321.1 112 Anti-CD8a Mb1b EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHFVRQA IgG1r mAb PGKGLEWIGRIDPANDNTLYASKFQGKATISADTSKNTAY (G4S)3 C-t rm LQMNSLRAEDTAVYYCGRGYGYYVFDHWGQGTLVTVSSAS 170886321.1 N65D (v7), knob VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV heavy chain DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY KCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPCRDELT 170886321.1 HIDATLYTESNVHPSCKVTAMKCFLLGLQRISLESGDASI HDTVQNLIILANNSLSSNGNVTESGCKECEELEEKNIKEF LQSFVHIVQMFINTS*

[0124] 154 170886321.1 122 Anti-CD8a Mb1b EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHFVRQA IgG1r mAb PGKGLEWIGRIDPANDNTLYASKFQGKATISADTSKNTAY (G4S)3 C-t rm LQMNSLRAEDTAVYYCGRGYGYYVFDHWGQGTLVTVSSAS 170886321.1 knob heavy VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV chain DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY KCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPCRDELT 170886321.1 HIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASI HDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEF LQSFVHIVQMFINTS*

[0125] 157 170886321.1 132 Anti-CD8a Mb1b EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHFVRQA IgG1r mAb PGKGLEWIGRIDPANDNTLYASKFQGKATISADTSKNTAY (G4S)3 C-t rm LQMNSLRAEDTAVYYCGRGYGYYVFDHWGQGTLVTVSSAS 170886321.1 knob heavy VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV chain DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY KCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPCRDELT 170886321.1 HIDATLYTESDVHPSCKVTAMKCFLLELQGISLESGDASI HDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEF LQSFVHIVQMFINTS*

[0126] 160 170886321.1 142 Anti-CD8a Mb1b EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHFVRQA IgG1r mAb PGKGLEWIGRIDPANDNTLYASKFQGKATISADTSKNTAY (G4S)3 C-t rm LQMNSLRAEDTAVYYCGRGYGYYVFDHWGQGTLVTVSSAS 170886321.1 knob heavy VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV chain DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY KCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPCRDELT 170886321.1 HIDATLATESDVHPSCKVTAMKCFLLKLQVISLESGDASI HDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEF LQSFVHIVQMFINTS*

[0127] 163 170886321.1 152 Anti-CD8 Mb1b EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHFVRQA IgG1r mAb PGKGLEWIGRIDPANDNTLYASKFQGKATISADTSKNTAY (G4S)3 C-t rm LQMNSLRAEDTAVYYCGRGYGYYVFDHWGQGTLVTVSSAS 170886321.1 (v46), knob VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV heavy chain DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY KCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPCRDELT 170886321.1 HIDATLYTESDVHPSCKVTAMKCFLLKLQVISLKSGDASI HDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEF LQSFVHIVQMFINTS*

[0128] 166 170886321.1 162 Anti-CD8 Mb1b EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHFVRQA IgG1r mAb PGKGLEWIGRIDPANDNTLYASKFQGKATISADTSKNTAY (G4S)3 C-t rm LQMNSLRAEDTAVYYCGRGYGYYVFDHWGQGTLVTVSSAS 170886321.1 (v56), knob VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV heavy chain DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY KCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPCRDELT 170886321.1 HIDATLYTESDVHPSCKVTAMKCFLLELQVISLKSGDASI HDTVENLIILANNSLSSNGNVTESGCKECKELEEKNIKEF LQSFVHIVQMFINTS*

[0129] 169 170886321.1 172 Anti-CD8 Mb1b EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHFVRQA IgG1r mAb PGKGLEWIGRIDPANDNTLYASKFQGKATISADTSKNTAY (G4S)3 C-t rm LQMNSLRAEDTAVYYCGRGYGYYVFDHWGQGTLVTVSSAS 170886321.1 knob heavy VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV chain DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY KCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPCRDELT 170886321.1 HIDATLATESDVHPSCKVTAMKCFLLELQGISLESGDASI HDTVGNLIILANNSLSSNGNVTESGCKECEELEEKNIKEF LQSFVHIVQMFINTS*

[0130] 172 170886321.1 182 Anti-CD8 Mb1b EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHFVRQA IgG1r mAb PGKGLEWIGRIDPANDNTLYASKFQGKATISADTSKNTAY (G4S)3 C-t rm LQMNSLRAEDTAVYYCGRGYGYYVFDHWGQGTLVTVSSAS 170886321.1 knob heavy VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV chain DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY KCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPCRDELT 170886321.1 HIDATLYTESDVHPSCKVTAMKCFLLELQYISLESGDASI HDTVENLIILANNSLSSNGNVTESGCKECKELEEKNIKEF LQSFVHIVQMFINTS*

[0131] 175 170886321.1 192 Anti-CD8 Mb1b EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHFVRQA IgG1r mAb PGKGLEWIGRIDPANDNTLYASKFQGKATISADTSKNTAY (G4S)3 C-t rm LQMNSLRAEDTAVYYCGRGYGYYVFDHWGQGTLVTVSSAS 170886321.1 (v86), knob VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV heavy chain DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY KCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPCRDELT 170886321.1 HIDATLYTESDVHPSCKVTAMKCFLLELQYISLASGDASI HDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEF LQSFVHIVQAFINTS*

[0132] 178 170886321.1 202 Anti-CD8 Mb1b EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHFVRQA IgG1r mAb PGKGLEWIGRIDPANDNTLYASKFQGKATISADTSKNTAY (G4S)3 C-t rm LQMNSLRAEDTAVYYCGRGYGYYVFDHWGQGTLVTVSSAS

[0133] 179 170886321.1 206 plasmid signal ATGGGCTGGTCCTGCATCATCCTGTTCCTGGTGGCCACAG sequence CCACCGGCGTGCACAGC (n l i id)

[0134] 180 170886321.1 315 v5 DNA AACTGGGTGAACGTGATCAGCGACCTGAAGAAGATCGAGG sequence ACCTGATCCAGAGCATGCACATCGATGCCACCCTGTACAC CGAGAGCGATGTGCACCCCAGCTGCAAGGTGACAGCTATG 170886321.1 CAACAACAGCCTGTCGAGCAACGGCAATGTGACCGAGAGC GGATGTAAAGAATGCGAGGAGCTGGAAGAGAAAAACATCA AGGAATTCCTGCAAAGCTTTGTGCATATCGTGCAAATGTT

[0135] 182 170886321.1 327 v16 DNA AACTGGGTGAACGTGATCAGCGACCTGAAGAAGATCGAGG sequence ACCTGATCCAGAGCATGCACATCGATGCCACCCTGgccAC CGAGAGCGATGTGCACCCCAGCTGCAAGGTGACAGCTATG 170886321.1 ACCGAGAGCGGATGTAAAGAATGCGAGGAGCTGGAAGAGA AAAACATCAAGGAATTCCTGCAAAGCTTTGTGCATATCGT GCAAATGTTCATCAACACATCT

[0136] 184 170886321.1 338 v27 DNA AACTGGGTGAACGTGATCAGCGACCTGAAGAAGATCGAGG sequence ACCTGATCCAGAGCATGCACATCGATGCCACCCTGTACAC CGAGAGCGATGTGCACCCCAGCTGCAAGGTGACAGCTATG 170886321.1 ACCGAGAGCGGATGTAAAGAATGCGAGGAGCTGGAAGAGA AAAACATCAAGGAATTCCTGCAAAGCTTTGTGCATATCGT GCAAATGTTCATCAACACATCT

[0137] 186 170886321.1 349 v38 DNA AACTGGGTGAACGTGATCAGCGACCTGAAGAAGATCGAGG sequence ACCTGATCCAGAGCATGCACATCGATGCCACCCTGTACAC CGAGAGCGATGTGCACCCCAGCTGCAAGGTGACAGCTATG 170886321.1 ACAGAGAGCGGATGCAAGGAGTGCGAGGAACTGGAGGAGA AGAACATCAAGGAGTTCCTGCAATCTTTTGTGCACATCGT GCAGATGTTCATCAACACCAGC

[0138] 188 170886321.1 360 v49 DNA AATTGGGTCAACGTGATTTCTGATCTGAAAAAGATCGAGG sequence ACCTCATCCAGAGCATGCACATCGACGCTACACTGTACAC CGAGAGCGACGTGCATCCTAGCTGCAAGGTGACCGCCATG 170886321.1 ACAGAGAGCGGCTGCAAGGAATGTGAAGAGCTGGAGGAGA AGAACATCAAGGAGTTCCTGCAATCTTTTGTGCACATTGT GCAGATGTTCATCAACACCAGC

[0139] 190 170886321.1 371 v60 DNA AATTGGGTCAACGTGATCAGCGACCTCAAGAAGATCGAGG sequence ACCTGATCCAGTCTATGCACATCGACGCTACACTGTACAC CGAGTCTGATGTGCATCCTAGCTGCAAGGTGACCGCCATG 170886321.1 ACAGAGAGCGGATGTAAAGAGTGCGAGGAACTGGAAGAGA AGAACATCAAGGAGTTCCTGCAGTCTTTTGTGCACATTGT GCAGATGTTCATCAACACCAGC

[0140] 192 170886321.1 382 v71 DNA AATTGGGTCAACGTGATCTCTGATCTGCAGAAGATCGAGG sequence ACCTGATCCAGTCCATGCACATCGACGCCACCCTGTACAC CGAGAGCGACGTGCATCCTAGCTGCAAGGTGACAGCCATG 170886321.1 ACAGAGTCTGGATGTAAAGAGTGCGAGGAACTGGAAGAGA AGAACATCAAGGAGTTCCTGCAGAGCTTCGTGCACATTGT GCAGATGTTCATCAACACCAGC

[0141] 194 170886321.1 393 v82 DNA AATTGGGTCAACGTTATCTCCGATCTGCAGAAGATCGAGG sequence ACCTGATCCAGAGCATGCACATCGACGCTACACTGTACAC CGAGTCTGATGTGCATCCTAGCTGCAAGGTGACCGCCATG 170886321.1 ACCGAGTCTGGCTGTAAAGAGTGCAAGGAACTGGAGGAGA AGAACATCAAGGAGTTCCTGCAGTCTTTTGTGCACATCGT GCAGATGTTCATCAACACCAGC

[0142] 196 170886321.1 404 v93 DNA AATTGGGTCAACGTGATCAGCGACCTGAAGAAGATCGAGG sequence ACCTGATCCAGAGCATGCACATCGACGCCACCCTGTACAC AGAGTCTGATGTGCATCCTAGCTGCAAGGTGACAGCCATG

[0143] 197 170886321.1 414 anti-KIR EIVLTQSPVTLSLSPGERATLSCRASQSVSSYLAWYQQKP IPH2102 light GQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEP h in EDFAVYYCQQRSNWMYTFGQGTKLEIKRTVAAPSVFIFPP 170886321.1 mutein N1G TQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEA D30N E46G EGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVK V49R E64Q FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWL 170886321.1 HYTQKSLSLSPGGGGGSGGGGSGGGGNWVNVISDLKKIED LIQSMHIDATLATESDVHPSCKVTAMKCFLLELQVISLES GDASIHDTVENLIILANNSLSSNGNVTESGCKECKELEEK

[0144] 200 170886321.1 425 Anti-KIR QVQLVQSGAEVKKPGSSVKVSCKASGGTFSFYAISWVRQA IPH2102 IgG1r PGQGLEWMGGFIPIFGAANYAQKFQGRVTITADESTSTAY mAb (G4S)3 C- MELSSLRSDDTAVYYCARIPSGSYYYDYDMDVWGQGTTVT 201 170886321.1 447 anti-CD8α EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHFVRQA variable heavy PGKCLEWIGRIDPANDNTLYASKVQGKFTISADTSKNTAY d m in (VH) LQMNSLRAEDTAVYYCARGYGYYVFDHWGQGTLVTVSS Q T A W D V E D L L E I K A S D 170886321.1 GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPGK Q T S A Y A A H V Q V R 170886321.1 TTATTACTACGACTACGACATGGACGTGTGGGGCCAAGGCACCA CCGTGACCGTCTCCTCT 170886321.1 CAGCAACACAACGAGAATCCTCTGACCTTCGGCTGCGGAACCAA GGTGGAAATCAAGGGCAGCACCAGCGGCGGCGGCAGCGGCGGAG GCAGCGGCGGAGGTGGTTCATCAGAAGTGCAGCTGGTGGAAAGC

[0145] 205 170886321.1 While specific embodiments have been illustrated and described, it will be readily appreciated that the various embodiments described above can be combined to provide further embodiments, and that various changes can be made therein without departing from the spirit and scope of the invention. All of the U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications referred to in this specification or listed in the Application Data Sheet, including U.S. Provisional Application No.63 / 636,588, filed April 19, 2024, U.S. Provisional Application No.63 / 687,395, filed August 27, 2024, and U.S. Provisional Application No.63 / 720,558, filed November 14, 2024, are incorporated herein by reference, in their entirety, unless explicitly stated otherwise. Aspects of the embodiments can be modified, if necessary to employ concepts of the various patents, applications, and publications to provide yet further embodiments. These and other changes can be made to the embodiments in light of the above- detailed description. In general, in the following claims, the terms used should not be construed to limit the claims to the specific embodiments disclosed in the specification and the claims, but should be construed to include all possible embodiments along with the full scope of equivalents to which such claims are entitled. Accordingly, the claims are not limited by the disclosure.

[0146] 206 170886321.1

Claims

CLAIMS What is claimed is:

1. An isolated IL-15 peptide comprising the amino acid sequence according to any one of SEQ ID NOs:67, 51, 44, 84, 92, 43, 45, 10-43, 46-50, 52-83, 85-91, and 93-94.

2. An isolated IL-15 peptide consisting of the amino acid sequence according to any one of SEQ ID NOs:67, 51, 44, 84, 92, 43, 45, 10-43, 46-50, 52-83, 85-91, and 93-94.

3. An isolated IL-15 peptide having one or more amino acid substitutions or deletions relative to SEQ ID NO:95, wherein the one or more amino acid substitutions or deletions comprises or consists of: a. K10Q, Y26A, and V49G; b. E46K and E89K; c. Y26A and E53R; d. E53A, E64G, and E89K; e. K10Q, V49Y, E89K, and M109A; f. Y26A and E53K; g. Y26A and E89K; h. an N-terminal truncation in which the first three amino acids (NWV) are deleted; i. an N-terminal truncation in which the first five amino acids (NWVNV) are deleted; j. V49Y; k. E46K; l. E53A; m. E53K; n. Y26A; o. Y26K; p. E89K;207 170886321.1q. D8K; r. K10V; s. K11A; t. D61K; u. E64G; v. N65G; w. L69W; x. D30K; y. H105A; z. M109A; aa. V49G; bb. E53S; cc. E53R; dd. T24K; ee. D8A; ff. K10Q; gg. D61A; hh. N65W; ii. Q108A; jj. V49A; kk. Y26A and E46K; ll. Y26A and V49G; mm. Y26A and V49Y; nn. Y26A and E53A; oo. Y26K and E53A; pp. E46K and V49Y; qq. E46K and E53A; rr. E46K and E53K; ss. E46K and E53R;208 170886321.1tt. V49G and E53K; uu. V49G and E89K; vv. V49Y and E53A; ww. V49Y and E53K; xx. V49Y and E53R; yy. V49Y and E89K; zz. E53A and E89K; aaa. E53K and E89K; bbb. E53R and E89K; ccc. E53S and E89K; ddd. K10Q and D61A; eee. K10Q and E64G; fff. K10V and D61A; ggg. K10V and E64G; hhh. D61A and E64G; iii. K10Q, Y26A, V49G, and E64G; jjj. Y26A, V49G, and E64G; kkk. K10Q, V49G, and E53K; lll. K10Q, V49G, E53K, and E64G; mmm. V49G, E53K, and E64G; nnn. K10Q, V49Y, and E53A; ooo. K10Q, V49Y, E53A, and E64G; ppp. V49Y, E53A, and E64G; qqq. K10Q, V49Y, and E53R; rrr. K10Q, V49Y, E53R, and E64G; sss. V49Y, E53R, and E64G; ttt. K10Q, V49Y, and E89K; uuu. K10Q, V49Y, E64G, and E89K; vvv. V49Y, E64G, and E89K;209 170886321.1www. K10Q, E53A, and E89K; xxx. K10Q, E53A, E64G, and E89K; yyy. K10Q, E53S, and E89K; zzz. K10Q, E53S, E64G, and E89K; aaaa. E53S, E64G, and E89K; bbbb. V49Y, E53A, and M109A; cccc. K10Q, V49Y, E53A, and M109A; dddd. V49Y, E53R, and M109A; eeee. V49Y, E89K, and M109A; ffff. E53A, E89K, and M109A; or gggg. K10Q, E53A, E89K, and M109A.

4. The isolated IL-15 peptide of any one of claims 1-3, wherein the peptide comprises SEQ ID NO:

67.

5. The isolated IL-15 peptide of any one of claims 1-3, wherein the one or more amino acid substitutions or deletions relative to SEQ ID NO:95 are K10Q, Y26A, and V49G.

6. The isolated IL-15 peptide of any one of claims 1-3, wherein the peptide comprises SEQ ID NO:

51.

7. The isolated IL-15 peptide of any one of claims 1-3, wherein the one or more amino acid substitutions or deletions relative to SEQ ID NO:95 are E46K and E89K.

8. The isolated IL-15 peptide of any one of claims 1-3, wherein the peptide comprises SEQ ID NO:

44.

9. The isolated IL-15 peptide of any one of claims 1-3, wherein the one or more amino acid substitutions or deletions relative to SEQ ID NO:95 are Y26A and E53R.

10. The isolated IL-15 peptide of any one of claims 1-3, wherein the peptide comprises SEQ ID NO:84.210 170886321.

111. The isolated IL-15 peptide of any one of claims 1-3, wherein the one or more amino acid substitutions or deletions relative to SEQ ID NO:95 are E53A, E64G, and E89K.

12. The isolated IL-15 peptide of any one of claims 1-3, wherein the peptide comprises SEQ ID NO:

92.

13. The isolated IL-15 peptide of any one of claims 1-3, wherein the one or more amino acid substitutions or deletions relative to SEQ ID NO:95 are K10Q, V49Y, E89K, and M109A.

14. The isolated IL-15 peptide of any one of claims 1-3, wherein the peptide comprises SEQ ID NO:

43.

15. The isolated IL-15 peptide of any one of claims 1-3, wherein the one or more amino acid substitutions or deletions relative to SEQ ID NO:95 are Y26A and E53K.

16. The isolated IL-15 peptide of any one of claims 1-3, wherein the peptide comprises SEQ ID NO:

45.

17. The isolated IL-15 peptide of any one of claims 1-3, wherein the one or more amino acid substitutions or deletions relative to SEQ ID NO:95 are Y26A and E89K.

18. An isolated fusion protein comprising (i) a IL-15 peptide comprising or consisting of the amino acid sequence as set forth in any one of claims 1-17 and (ii) an antibody or fragment thereof.

19. The isolated fusion protein of claim 18, wherein the IL-15 peptide is covalently connected to the antibody or fragment thereof by a linker.

20. The isolated fusion protein of claim 19, wherein the linker comprises or consists of SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:437, or SEQ ID NO:

438.

21. The isolated fusion protein of claim 18, wherein the antibody or fragment thereof comprises: a light chain variable region (VL) having CDRL1, CDRL2, and CDRL3 amino acid sequences according to SEQ ID NO:97, SEQ ID NO:98, and SEQ ID NO:99, respectively; and a heavy chain variable region (VH) having CDRH1,211 170886321.1CDRH2, and CDRH3 amino acid sequences according to SEQ ID NO:101, SEQ ID NO:102, and SEQ ID NO:103, respectively.

22. The isolated fusion protein of claim 18 or claim 21, wherein the antibody or fragment thereof comprises: a light chain variable region (VL) comprising or consisting of the amino acid sequence according to SEQ ID NO:96; and a heavy chain variable region (VH) comprising or consisting of the amino acid sequence according to SEQ ID NO:

100.

23. The isolated fusion protein of any one of claims 18, 21, and 22, wherein the antibody or fragment thereof comprises: a first chain, a second chain, and a third chain, wherein: (i) the first chain is a light chain comprising or consisting of the amino acid sequence according to SEQ ID NO:104; (ii) the second chain is a heavy chain comprising or consisting of the amino acid sequence according to SEQ ID NO:106; and (iii) the third chain is a heavy chain comprising or consisting of the amino acid sequence according to SEQ ID NO:105 covalently connected to the IL-15 peptide by a linker.

24. The isolated fusion protein of claim 23, wherein the linker comprises or consists of the amino acid sequence according to SEQ ID NO:107 or 108.

25. The isolated fusion protein of claim 23, wherein the linker comprises or consists of the amino acid sequence according to SEQ ID NO:437 or SEQ ID NO:

438.

26. An isolated antibody-IL-15 fusion protein comprising: a first peptide chain, a second peptide chain, and a third peptide chain, wherein: (i) the first peptide chain comprises or consists of the amino acid sequence according to SEQ ID NO:104; (ii) the second peptide chain comprises or consists of the amino acid sequence according to SEQ ID NO:106; and (iii) the third peptide chain comprises or consists of the amino acid sequence according to any one of SEQ ID NOs:109-204.212 170886321.

127. The fusion protein of claim 26, wherein the third peptide chain comprises or consists of the amino acid sequence according to SEQ ID NO:

153.

28. The fusion protein of claim 26, wherein the third peptide chain comprises or consists of the amino acid sequence according to SEQ ID NO:

176.

29. The fusion protein of claim 26, wherein the third peptide chain comprises or consists of the amino acid sequence according to SEQ ID NO:

193.

30. The fusion protein of claim 26, wherein the third peptide chain comprises or consists of the amino acid sequence according to SEQ ID NO:

201.

31. The fusion protein of claim 26, wherein the third peptide chain comprises or consists of the amino acid sequence according to SEQ ID NO:

160.

32. The fusion protein of claim 26, wherein the third peptide chain comprises or consists of the amino acid sequence according to SEQ ID NO:

152.

33. The fusion protein of claim 26, wherein the third peptide chain comprises or consists of the amino acid sequence according to SEQ ID NO:

154.

34. The isolated fusion protein of claim 18, wherein the antibody or fragment thereof comprises: a light chain variable region (VL) having CDRL1, CDRL2, and CDRL3 amino acid sequences according to SEQ ID NO:407, SEQ ID NO:408, and SEQ ID NO:409, respectively; and a heavy chain variable region (VH) having CDRH1, CDRH2, and CDRH3 amino acid sequences according to SEQ ID NO:411, SEQ ID NO:142, and SEQ ID NO:413, respectively.

35. The isolated fusion protein of claim 18 or claim 34, wherein the antibody or fragment thereof comprises: a light chain variable region (VL) comprising or consisting of the amino acid sequence according to SEQ ID NO:406; and a heavy chain variable region (VH) comprising or consisting of the amino acid sequence according to SEQ ID NO:

410.

36. The isolated fusion protein of any one of claims 18, 34, and 36, wherein the antibody or fragment thereof comprises: a first chain, a second chain, and a third chain, wherein:213 170886321.1(i) the first chain is a light chain comprising or consisting of the amino acid sequence according to SEQ ID NO:414; (ii) the second chain is a heavy chain comprising or consisting of the amino acid sequence according to SEQ ID NO:416; and (iii) the third chain is a heavy chain comprising or consisting of the amino acid sequence according to SEQ ID NO:415 covalently connected to the IL-15 peptide by a linker.

37. The isolated fusion protein of claim 36, wherein the linker comprises or consists of the amino acid sequence according to SEQ ID NO:107, 108, 437, or 438.

38. An isolated antibody-IL-15 fusion protein comprising: a first peptide chain, a second peptide chain, and a third peptide chain, wherein: (i) the first peptide chain comprises or consists of the amino acid sequence according to SEQ ID NO:414; (ii) the second peptide chain comprises or consists of the amino acid sequence according to SEQ ID NO:416; and (iii) the third peptide chain comprises or consists of the amino acid sequence according to any one of SEQ ID NOs:417-425.

39. The fusion protein of claim 38, wherein the third peptide chain comprises or consists of the amino acid sequence according to SEQ ID NO:

419.

40. The fusion protein of claim 38, wherein the third peptide chain comprises or consists of the amino acid sequence according to SEQ ID NO:

420.

41. The fusion protein of claim 38, wherein the third peptide chain comprises or consists of the amino acid sequence according to SEQ ID NO:

421.

42. The fusion protein of claim 38, wherein the third peptide chain comprises or consists of the amino acid sequence according to SEQ ID NO:

422.

43. The fusion protein of claim 38, wherein the third peptide chain comprises or consists of the amino acid sequence according to SEQ ID NO:

423.

44. The fusion protein of claim 38, wherein the third peptide chain comprises or consists of the amino acid sequence according to SEQ ID NO:424.214 170886321.

145. The fusion protein of claim 38, wherein the third peptide chain comprises or consists of the amino acid sequence according to SEQ ID NO:

425.

46. The isolated fusion protein of claim 18, wherein the antibody or fragment thereof is bispecific and comprises: (1) a first light chain variable region (VL) having CDRL1, CDRL2, and CDRL3 amino acid sequences according to SEQ ID NO:407, SEQ ID NO:408, and SEQ ID NO:409, respectively; and a first heavy chain variable region (VH) having CDRH1, CDRH2, and CDRH3 amino acid sequences according to SEQ ID NO:411, SEQ ID NO:142, and SEQ ID NO:413, respectively; and (2) a second light chain variable region (VL) having CDRL1, CDRL2, and CDRL3 amino acid sequences according to SEQ ID NO:97, SEQ ID NO:98, and SEQ ID NO:99, respectively; and a second heavy chain variable region (VH) having CDRH1, CDRH2, and CDRH3 amino acid sequences according to SEQ ID NO:101, SEQ ID NO:102, and SEQ ID NO:103, respectively.

47. The isolated fusion protein of claim 18 or claim 46, wherein the antibody or fragment thereof is bispecific and comprises: (1) a first light chain variable region (VL) comprising or consisting of the amino acid sequence according to SEQ ID NO:406; and a first heavy chain variable region (VH) comprising or consisting of the amino acid sequence according to SEQ ID NO:410; and (2) a second light chain variable region (VL) comprising or consisting of the amino acid sequence according to SEQ ID NO:96; and a second heavy chain variable region (VH) comprising or consisting of the amino acid sequence according to SEQ ID NO:

100.

48. The isolated fusion protein of any one of claims 46 or 47, further comprising a linker connecting the IL-15 to the antibody or fragment thereof.

49. The isolated fusion protein of claim 48, wherein the linker comprises or consists of the amino acid sequence according to SEQ ID NO:107, 108, 437, or 438.

50. The isolated fusion protein of claim 18, wherein the antibody or fragment thereof is bispecific and comprises: (1) a first light chain variable region (VL) having CDRL1, CDRL2, and CDRL3 amino acid sequences according to SEQ ID NO:448, SEQ ID NO:449, and SEQ ID NO:450, respectively; and a first heavy chain215 170886321.1variable region (VH) having CDRH1, CDRH2, and CDRH3 amino acid sequences according to SEQ ID NO:451, SEQ ID NO:452, and SEQ ID NO:453, respectively; and (2) a second light chain variable region (VL) having CDRL1, CDRL2, and CDRL3 amino acid sequences according to SEQ ID NO:440, SEQ ID NO:441, and SEQ ID NO:442, respectively; and a second heavy chain variable region (VH) having CDRH1, CDRH2, and CDRH3 amino acid sequences according to SEQ ID NO:443, SEQ ID NO:444, and SEQ ID NO:445, respectively.

51. The isolated fusion protein of claim 18, wherein the antibody or fragment thereof is bispecific and comprises: (1) a first light chain variable region (VL) comprising or consisting of the amino acid sequence according to SEQ ID NO:454; and a first heavy chain variable region (VH) comprising or consisting of the amino acid sequence according to SEQ ID NO:455; and (2) a second light chain variable region (VL) comprising or consisting of the amino acid sequence according to SEQ ID NO:446; and a second heavy chain variable region (VH) comprising or consisting of the amino acid sequence according to SEQ ID NO:

447.

52. The isolated fusion protein of claim 18, wherein the antibody or fragment thereof is bispecific and comprises: (i) a light chain comprising or consisting of the amino acid sequence according to SEQ ID NO:456; (ii) a heavy chain comprising or consisting of the amino acid sequence according to SEQ ID NO:457; and (iii) a heavy chain comprising or consisting of the amino acid sequence according to SEQ ID NO:

458.

53. The isolated fusion protein of any one of claims 50-52, wherein the heavy chain comprising or consisting of the amino acid sequence according to SEQ ID NO:458 is covalently connected to the IL-15 peptide by a linker.

54. The isolated fusion protein of claim 53, wherein the linker comprises or consists of the amino acid sequence according to SEQ ID NO:107, 108, 437, or 438.216 170886321.

155. An isolated nucleic acid molecule comprising a polynucleotide sequence that encodes the peptide or fusion protein of any one of claims 1-54.

56. The isolated nucleic acid molecule of claim 55, wherein the polynucleotide sequence comprises or consists of any one of SEQ ID NOs:207-405, 426-436, and 460-463.

57. The isolated nucleic acid molecule of claim 55 or claim 56, further comprising a polynucleotide sequence that encodes a signal peptide.

58. The isolated nucleic acid molecule of claim 57, wherein the signal peptide comprises or consists of the amino acid sequence of SEQ ID NO:205 or 206.

59. A recombinant expression vector comprising the nucleic acid molecule according to any one of claims 55-58.

60. A pharmaceutical composition comprising the peptide according to any one of claims 1-17 and a pharmaceutically acceptable carrier.

61. A pharmaceutical composition comprising the fusion protein according to any one of claims 18-54 and a pharmaceutically acceptable carrier.

62. A pharmaceutical composition comprising the nucleic acid molecule or vector according to any one of claims 55-59 and a pharmaceutically acceptable carrier.

63. A method for treating a disease in a subject in need thereof, comprising administering the peptide according to any one of claims 1-17, or the pharmaceutical composition according to claim 60, to the subject.

64. A method for treating a disease in a subject in need thereof, comprising administering the fusion protein according to any one of claims 18-54, or the pharmaceutical composition according to claim 61, to the subject.

65. A method for treating a disease in a subject in need thereof, comprising administering the nucleic acid molecule or vector according to any one of claims 55-59, or the pharmaceutical composition according to claim 62, to the subject.

66. Use of the peptide, fusion protein, nucleic acid molecule, vector, or pharmaceutical composition of any one of claims 1-62 in a method for treating a disease in a subject in need thereof.217 170886321.

167. The peptide, fusion protein, nucleic acid molecule, vector, or pharmaceutical composition of any one of claims 1-62 for use in a method for treating a disease in a subject in need thereof.

68. Use of the peptide, fusion protein, nucleic acid molecule, vector, or pharmaceutical composition of any one of claims 1-62 in the manufacture of a medicament for treating a disease in a subject in need thereof.

69. The method, use, or composition for use of any one of claims 63-68, wherein the disease is an inflammatory disease, an autoimmune disease, or cancer.

70. The method, use, or composition for use of any one of claims 63-68, wherein the disease is celiac disease, Crohn's disease, rheumatoid arthritis, diabetes, Sjögren's syndrome (SS), lupus, systemic lupus erythematosus (SLE), or graft versus host disease (GVHD).

71. A host cell comprising the nucleic acid molecule according to any one of claims 55-58.

72. A fusion protein comprising: (a) a binding agent comprising: (i) a first binding domain that specifically binds to a KIR protein, and (ii) a second binding domain that specifically binds to a protein expressed on CD8+KIR+ T regulatory cells (Tregs) other than a KIR protein; and (b) a cytokine.

73. The fusion protein of claim 72, wherein the cytokine is a IL-15.

74. The fusion protein of claim 73, wherein the IL-15 comprises or consists of an amino acid sequence according to SEQ ID NO:

67.

75. The fusion protein of claim 73, wherein the IL-15 comprises or consists of an amino acid sequence according to SEQ ID NO:

51.

76. The fusion protein of claim 73, wherein the IL-15 comprises or consists of an amino acid sequence according to SEQ ID NO:44.218 170886321.

177. The fusion protein of claim 73, wherein the IL-15 comprises or consists of an amino acid sequence according to SEQ ID NO:

84.

78. The fusion protein of claim 73, wherein the IL-15 comprises or consists of an amino acid sequence according to SEQ ID NO:

92.

79. The fusion protein of claim 73, wherein the IL-15 comprises or consists of an amino acid sequence according to SEQ ID NO:

43.

80. The fusion protein of claim 73, wherein the IL-15 comprises or consists of an amino acid sequence according to SEQ ID NO:

45.

81. The fusion protein of claim 73, wherein the IL-15 comprises or consists of an amino acid sequence according to any one of SEQ ID NO:1-94.

82. A method of increasing CD8+ Treg activity comprising administering to a subject in need thereof a molecule that comprises (1) a binding domain that binds to one or more proteins expressed on the surface of a CD8+ Treg cell and (2) a cytokine.

83. A method of increasing CD8+ Treg proliferation comprising administering to a subject in need thereof a molecule that comprises (1) a binding domain that binds to one or more proteins expressed on the surface of a CD8+ Treg cell and (2) a cytokine.

84. A method of treating an autoimmune disease, comprising administering to a subject in need thereof a molecule that comprises (1) a binding domain that binds to one or more proteins expressed on the surface of a CD8+ Treg cell, and (2) a cytokine.

85. A method of suppressing an immune response mediated by pathogenic immune cells, comprising administering to a subject in need thereof a molecule that comprises (1) a binding domain that binds to one or more proteins expressed on the surface of a CD8+ Treg cell, and (2) a cytokine, whereby the number or activity of pathogenic immune cells is decreased.

86. The method of any one of claims 82-85, wherein the molecule comprises a fusion protein comprising (1) an antibody or antibody fragment that binds to one or more proteins expressed on the surface of a CD8+ Treg cell, and (2) a cytokine.219 170886321.

187. The method of claim 86, wherein the fusion protein comprises the fusion protein according to any one of claims 18-54.220 170886321.1

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