PD-1 TARGETED IL-15 / IL-15R ALPHA FC FUSION PROTEINS AND USES THEREOF IN COMBINATION THERAPY

Abstract

PD-1 targeted IL-15 / IL-15R alpha FC fusion proteins and their use in combination therapy. [Solution] The present invention relates to novel PD-1-targeting heterodimeric Fc-fusion proteins, including an IL-15 / IL-15Rα Fc-fusion protein and a PD-1 antibody fragment-Fc fusion protein. In some embodiments, the PD-1-targeting IL-15 / Rα-Fc fusion protein is administered to a patient to treat cancer. In some embodiments, the PD-1-targeting IL-15 / Rα-Fc fusion protein is administered in combination with a PD-1-blocking antibody, such as nivolumab and / or pembrolizumab. In some embodiments, the PD-1-targeting IL-15 / Rα-Fc fusion protein does not compete for antigen binding with a PD-1-blocking antibody, such as nivolumab and / or pembrolizumab.

Description

[Technical field]

[0001] Priority claim This application claims priority to U.S. Provisional Patent Application No. 62 / 744,946, filed October 12, 2018, and U.S. Provisional Patent Application No. 62 / 784,334, filed December 21, 2018. [Background technology]

[0002] Two highly promising approaches in cancer immunotherapy involve cytokine-based therapy and blockade of immune checkpoint proteins such as PD-1.

[0003] Cytokines such as IL-2 and IL-15 function to support the proliferation and differentiation of B cells, T cells, and NK cells. Both cytokines exert their cell signaling functions by binding to a trimeric complex consisting of two common receptors, the common gamma chain (γc, CD132) and the IL-2 receptor beta chain (IL-2Rβ, CD122), as well as an alpha chain receptor specific to each cytokine, the IL-2 receptor alpha (IL-2Rα, CD25) or the IL-15 receptor alpha (IL-15Rα, CD215). Both cytokines are considered potentially valuable therapeutic agents in oncology, with IL-2 approved for use in patients with metastatic renal cell carcinoma and malignant melanoma. Currently, there are no approved uses for recombinant IL-15, although several clinical trials are ongoing. However, as promising drugs, both cytokines suffer from extremely fast clearance, with half-lives measured in minutes. IL-2 immunotherapy is associated with systemic toxicity when administered at high doses to overcome rapid clearance, which has also been reported for IL-15 immunotherapy in recent clinical trials (Guo et al., J Immunol, 2015, 195(5):2353-64).

[0004] Immune checkpoint proteins such as PD-1 are upregulated following T cell activation, and upon binding to immune checkpoint ligands such as PD-L1, eliminate autoimmunity by exhausting activated T cells. However, immune checkpoint proteins are also upregulated in tumor infiltrating lymphocytes (TILs), and immune checkpoint ligands are overexpressed in tumor cells, causing immune escape by tumor cells. De-inhibition of TILs by blocking immune checkpoint interactions with drugs such as Opdivo® (nivolumab) and Keytruda® (pembrolizumab) has proven highly effective in treating cancer. Despite the promise of checkpoint blockade therapies such as nivolumab and pembrolizumab, many patients still fail to achieve a sufficient response with checkpoint blockade alone.

[0005] Therefore, there remains an unmet need in tumor treatment for tumor-targeted cytokine treatment strategies that do not require high doses and avoid systemic toxicity.In addition, there is a need to identify additional therapeutic modalities that are compatible with checkpoint blockade and can increase patient response rates.This can be particularly complicated, as additional therapeutic modalities must not compete with checkpoint blockade.The present invention addresses these needs and caveats by providing PD-1-targeted IL-15 fusion proteins with enhanced half-life and more selective targeting of TILs to improve safety profile, and therefore does not compete with checkpoint blockade antibodies that may be combined. Summary of the Invention

[0006] The present invention generally relates to fusion proteins comprising combinations of IL-15 complexes that include an IL-15 domain, an IL-15Rα domain, and an anti-PD-1 antigen binding domain (ABD) that does not compete for binding with nivolumab or pembrolizumab.

[0007] Thus, in some embodiments, the invention provides a targeted IL-15 / Rα heterodimer Fc fusion protein comprising: a) a first monomer comprising, from N-terminus to C-terminus: i) an IL-15Rα sushi domain protein; ii) a first domain linker; iii) an IL-15 protein; and iv) a first variant Fc domain; b) a second monomer comprising a heavy chain comprising VH-CH1-hinge-CH2-CH3, wherein said CH2-CH3 is a second variant Fc domain; and c) a third monomer comprising a light chain comprising VL-CL, wherein said VH and VL domains form an antigen-binding domain that binds to human PD-1 and does not compete with nivolumab and / or pembrolizumab for human PD-1.

[0008] In an additional embodiment, the targeted IL-15 / Rα heterodimer Fc fusion protein has a VH domain that is a variant of SEQ ID NO:5 comprising the amino acid substitution F32L, and the VL domain is a variant of SEQ ID NO:6 comprising N27dH, and optionally further comprising the amino acid substitutions K30Y / S93T.

[0009] In a further embodiment, the targeted IL-15 / Rα heterodimer Fc fusion protein has an H1 VH and an L1.1 VL.

[0010] In additional embodiments, the targeting IL-15 / Rα heterodimer Fc fusion protein is selected from the group consisting of D30N / E64Q / N65D, D30N / N65D, N1D, N4D, D8N, D30N, D61N, E64Q, N65D, Q108E, N1D / N4D / D8N, N1D / N4D / N65D, N1D / D30N, N1D / D61N, N1D / D61N / E64Q / Q108E, N1D / E64Q, N1D / N65D, N1D / Q108E, N4D, N4D / D30N, N4 and N65D / Q108E (compared to SEQ ID NO:2).

[0011] In a further embodiment, the targeted IL-15 / Rα heterodimeric Fc fusion protein has a first variant Fc domain and a second variant Fc domain comprising the amino acid substitutions L368D / K370S:LS364K / E357Q according to EU numbering.

[0012] In an additional embodiment, the targeted IL-15 / Rα heterodimeric Fc fusion protein of the invention has a first variant Fc domain and a second variant Fc domain, each comprising the amino acid substitutions M428L / N343S according to EU numbering.

[0013] In a further embodiment, the targeted IL-15 / Rα heterodimer Fc fusion protein comprises: a) a first monomer comprising, from N-terminus to C-terminus, i) an IL-15Rα sushi domain protein, ii) a first domain linker, iii) a variant IL-15 protein, iv) a second domain linker, and v) a first variant Fc domain comprising CH2-CH3; and b) a second monomer comprising, from N-terminus to C-terminus, i) an scFv domain, ii) a third domain linker, iii) a second variant Fc domain comprising CH2-CH3, wherein said scFv domain comprises a first variable heavy domain, an scFv linker, and a first variable light domain, wherein said scFv domain binds to human PD-1 and does not compete with nivolumab and / or pembrolizumab for said human PD-1.

[0014] In an additional embodiment, the targeted IL-15 / Rα heterodimer Fc fusion protein comprises: a) a first monomer comprising, from N-terminus to C-terminus, i) an IL-15Rα sushi domain protein, ii) a first domain linker, iii) a first variant Fc domain comprising CH2-CH3; b) a second monomer comprising, from N-terminus to C-terminus, i) an scFv domain, ii) a third domain linker, iii) a second variant Fc domain comprising CH2-CH3, wherein said scFv domain comprises a first variable heavy domain, an scFv linker, and a first variable light domain, wherein said scFv domain binds to human PD-1 and does not compete with nivolumab and / or pembrolizumab for said human PD-1; and c) a third monomer comprising a variant IL-15 domain protein.

[0015] In a further embodiment, the targeted IL-15 / Rα heterodimer Fc fusion protein comprises: a) a first monomer comprising a heavy chain comprising VH-CH1-hinge-CH2-CH3, wherein said CH2-CH3 is a first variant Fc domain; b) a second monomer comprising, from N-terminus to C-terminus, i) an IL-15Rα sushi domain protein; ii) a second domain linker; iii) a second variant Fc domain comprising CH2-CH3; b) a third monomer comprising a variant IL-15 domain protein; and c) a fourth monomer comprising a light chain comprising VL-CL, wherein said VH and VL domains form an antigen-binding domain that binds to human PD-1 and does not compete with nivolumab and / or pembrolizumab for said human PD-1.

[0016] In an additional embodiment, the targeted IL-15 / Rα heterodimer Fc fusion protein comprises: a) a first monomer comprising a heavy chain comprising VH-CH1-hinge-CH2-CH3, wherein said CH2-CH3 is a first variant Fc domain; b) a second monomer comprising VH-CH1-hinge-CH2-CH3-domain linker-IL-15Rα sushi domain protein-domain linker-variant IL-15 domain protein, wherein said CH2-CH3 is a second variant Fc domain; and c) a third monomer comprising a light chain comprising VL-CL, wherein said VH and VL domains form an antigen-binding domain that binds to human PD-1 and does not compete with nivolumab and / or pembrolizumab for said human PD-1.

[0017] In a further embodiment, the targeted IL-15 / Rα heterodimer Fc fusion protein comprises: a) a first monomer comprising a heavy chain comprising VH-CH1-hinge-CH2-CH3, wherein said CH2-CH3 is a first variant Fc domain; b) a second monomer comprising a VH-CH1-hinge-CH2-CH3-domain linker-IL-15Rα sushi domain protein, wherein said CH2-CH3 is a second variant Fc domain; c) a third monomer comprising a variant IL-15 domain protein; and d) a fourth monomer comprising a light chain comprising VL-CL, wherein said VH and VL domains bind to human PD-1 and do not compete with nivolumab and / or pembrolizumab for said human PD-1.

[0018] In an additional embodiment, the targeted IL-15 / Rα heterodimer Fc fusion protein comprises: a) a first monomer comprising, from N-terminus to C-terminus, VH-CH1-domain linker-variant IL-15 domain protein-domain linker-CH2-CH3, wherein said CH2-CH3 is a first variant Fc domain; b) a second monomer comprising, from N-terminus to C-terminus, VH-CH1-domain linker-IL-15Rα sushi domain protein-domain linker-CH2-CH3, wherein said CH2-CH3 is a second variant Fc domain; and c) a third monomer comprising a light chain comprising VL-CL, wherein said VH and VL domains form an antigen binding domain that binds to human PD-1 and does not compete with nivolumab and / or pembrolizumab for said human PD-1.

[0019] In an additional embodiment, the targeted IL-15 / Rα heterodimer Fc fusion protein comprises: a) a first monomer comprising, from N-terminus to C-terminus, VH-CH1-domain linker-variant IL-15Rα sushi domain protein-domain linker-variant IL-15 domain protein-domain linker-CH2-CH3, wherein said CH2-CH3 is a first variant Fc domain; b) a second monomer comprising a heavy chain comprising VH-CH1-hinge CH2-CH3, wherein said CH2-CH3 is a second variant Fc domain; and c) a third monomer comprising a light chain comprising VL-CL, wherein said VH and VL domains form an antigen binding domain that binds to human PD-1 and does not compete with nivolumab and / or pembrolizumab for said human PD-1.

[0020] In an additional aspect, the invention provides a nucleic acid composition comprising: a) a first nucleic acid encoding a first monomer of the embodiment; b) a second nucleic acid encoding a second monomer of the embodiment; and c) a third nucleic acid encoding a third monomer of the embodiment.

[0021] In a further aspect, the invention provides an expression vector composition comprising: a) an expression vector comprising a first nucleic acid; b) an expression vector comprising a second nucleic acid; and c) an expression vector comprising a third nucleic acid.

[0022] In an additional aspect, the invention provides a host cell comprising the expression vector composition.

[0023] In a further aspect, the present invention provides a method for producing a targeted IL-15 / Rα heterodimer Fc fusion protein of the present invention, comprising culturing a host cell under conditions in which said targeted IL-15 / Rα heterodimer Fc fusion protein is expressed, and recovering said protein.

[0024] In an additional aspect, the present invention provides a method of treating cancer comprising administering to a patient in need thereof a pharmaceutical composition comprising a targeted IL-15 / Rα heterodimer Fc fusion protein of the present invention.

[0025] In a further aspect, the present invention provides a method of treating cancer comprising administering to a patient in need thereof a pharmaceutical composition comprising a targeted IL-15 / Rα heterodimeric Fc fusion protein of the present invention in combination with an anti-PD-1 antibody, including nivolumab and / or pembrolizumab. [Brief description of the drawings]

[0026] [Figure 1] FIG. 1A and FIG. 1B show the sequences of IL-15 and its receptor. [Diagram 2] To facilitate clinical development, the sequences of both human and cynomolgus PD-1 are presented to facilitate the development of antigen-binding domains that bind both human and cynomolgus. [Diagram 3] 3A-3E show useful pairs of Fc heterodimerization variant sets (including scubariants and pI variants). There are variants for which there is no corresponding "monomer 2" variant; these are pI variants that can be used alone in either monomer. [Figure 4] A list of isosteric variant antibody constant regions and their respective substitutions is provided. pI_(-) indicates lower pI variants and pI_(+) indicates higher pI variants. These can be optionally and independently combined with other heterodimerization variants of the invention (and other variant types as outlined herein). [Diagram 5] Useful deletion variants that eliminate FcγR binding are shown (sometimes referred to as "knockout" or "KO" variants). Generally, deletion variants are found in both monomers, although in some cases they may only be in one monomer. [Figure 6]6A-6E show particularly useful embodiments of the "non-cytokine" component of the IL-15 / Rα-Fc fusion protein of the invention. [Figure 7] 7A-7F show particularly useful embodiments of the "non-cytokine" / "non-Fv" components of the PD-1-targeted IL-15 / Rα-Fc fusion proteins of the invention. [Figure 8] Several exemplary variable length linkers for use in IL-15 / Rα-Fc fusion proteins are shown. In some embodiments, these linkers are used to link the C-terminus of IL-15 and / or IL-15Rα(sushi) to the N-terminus of the Fc region. In some embodiments, these linkers are used to fuse IL-15 to IL-15Rα(sushi). [Figure 9] 9A-9C show several charged scFv linkers used to increase or decrease the pI of heterodimeric antibodies utilizing one or more scFvs as building blocks. (+H) positive linkers are specifically used herein. A single prior art scFv linker with a single charge is referred to as "Whitlow" from Whitlow et al., Protein Engineering 6(8):989-995 (1993). It should be noted that this linker was used to reduce aggregation and increase proteolytic stability in scFvs. In some embodiments, these linkers are used to link the C-terminus of IL-15 and / or IL-15Rα(sushi) to the N-terminus of the Fc region and / or fuse IL-15 to IL-15 / Rα(sushi). [Figure 10]10A-10D show the sequences of several useful IL-15 / Rα-Fc format scaffolds based on human IgG1 that do not contain cytokine sequences (e.g., Il-15 and / or IL-15Rα (sushi)). It is important to note that these scaffolds may also be used in certain embodiments of PD-1-targeted IL-15 / Rα-Fc fusion proteins. Scaffold 1 is based on human IgG1 (356E / 358M allotype) and contains C220S in both chains, S364K / E357Q:L368D / K370S scaffold variants in one chain, Q295E / N384D / Q418E / N421D pI variants, with L368D / K370S scaffold variants and E233P / L234V / L235A / G236del / S267K deletion variants in both chains. Scaffold 2 is based on human IgG1 (356E / 358M allotype) and contains C220S in both chains, S364K in one chain:L368D / K370S scavariant, Q295E / N384D / Q418E / N421D pI variant, with L368D / K370S scavariant and E233P / L234V / L235A / G236del / S267K deletion variants in both chains. Scaffold 3 is based on human IgG1 (356E / 358M allotype) and contains C220S in both chains, S364K in one chain:L368E / K370S scavariant, Q295E / N384D / Q418E / N421D pI variant, with L368E / K370S scavariant and E233P / L234V / L235A / G236del / S267K deletion variants in both chains. Scaffold 4 is based on human IgG1 (356E / 358M allotype) containing C220S in both chains, D401K in one chain: K360E / Q362E / T411E scavians, Q295E / N384D / Q418E / N421D pI variants, with K360E / Q362E / T411E scavians and E233P / L234V / L235A / G236del / S267K deletion variants in both chains.Scaffold 5 is based on human IgG1 (356D / 358L allotype) and contains C220S in both chains, a scuba variant of S364K / E357Q:L368D / K370S in one chain, a pI variant of Q295E / N384D / Q418E / N421D, and has a scuba variant of L368D / K370S in both chains and deletion variants of E233P / L234V / L235A / G236del / S267K. Scaffold 6 is based on human IgG1 (356E / 358M allotype) and contains C220S in both chains, S364K / E357Q:L368D / K370S scavariant in one chain, Q295E / N384D / Q418E / N421D pI variant, L368D / K370S scavariant in both chains, E233P / L234V / L235A / G236del / S267K deletion variant, and N297A variant in both chains. Scaffold 7 is identical to 6 except that the mutation is N297S. Alternative formats of scaffolds 6 and 7 can exclude the deletion variant E233P / L234V / L235A / G236del / S267K in both chains. Scaffold 8 is based on human IgG4 and contains a scuba variant of S364K / E357Q:L368D / K370S, a pI variant of Q295E / N384D / Q418E / N421D in one chain, a scuba variant of L368D / K370S in both chains and a S228P (according to EU numbering, which is S241P in Kabat) variant, which eliminates Fab arm exchange as known in the art. Scaffold 9 is based on human IgG2 and contains a scuba variant of S364K / E357Q:L368D / K370S, a pI variant of Q295E / N384D / Q418E / N421D in one chain, and a scuba variant of L368D / K370S. Scaffold 10 is based on human IgG2 and contains the S364K / E357Q:L368D / K370S scavariant, the Q295E / N384D / Q418E / N421D pI variant in one chain, the L368D / K370S scavariant, and the S267K variant in both chains. Scaffold 11 is identical to scaffold 1, except that it contains the M428L / N434S Xtend mutations.Scaffold 12 is based on human IgG1 (356E / 358M allotype) with C220S in both identical chains and E233P / L234V / L235A / G236del / S267K deletion variants in both identical chains. Scaffold 13 is based on human IgG1 (356E / 358M allotype) with C220S in both chains, S364K / E357Q:L368D / K370S scavariant in one chain, P217R / P228R / N276K pI variant, S364K / E357Q scavariant in both chains, and E233P / L234V / L235A / G236del / S267K deletion variants. As will be appreciated by those skilled in the art and as outlined below, these sequences may be used with any of the IL-15 and IL-15Rα (sushi) pairs outlined herein, including but not limited to IL-15 / Rα-heteroFc, ncIL-15 / Rα, and scIL-15 / Rα, as shown diagrammatically in Figures 14A-14G. Additionally, any IL-15 and / or IL-15Rα (sushi) variants may be incorporated into these scaffolds of Figures 11A-11C in any combination. Included in each of these scaffolds are sequences that are 90, 95, 98, and 99% identical (as defined herein) to the listed sequences, and / or contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions (compared to the "parent" in the figures, which already contains some amino acid modifications compared to the parent human IgG1 (or IgG2 or IgG4 depending on the scaffold), as will be appreciated by those skilled in the art). That is, the recited backbone may contain additional amino acid modifications (generally amino acid substitutions) in addition to the sqvariants, pI variants and deletion variants contained within the backbone of this figure. [Figure 11]Shown are sequences of several useful PD-1-targeting IL-15 / Rα-Fc fusion format scaffolds based on human IgG1 that do not contain cytokine sequences (e.g., Il-15 and / or IL-15Rα (sushi)) or VH, and further exclude the cognate light chain scaffolds shown in Figure 12. Scaffold 1 is based on human IgG1 (356E / 358M allotype) and contains the S364K / E357Q:L368D / K370S scaffold variants, C220S, and Q295E / N384D / Q418E / N421D pI variants in the chains, with the L368D / K370S scaffold variants and E233P / L234V / L235A / G236del / S267K deletion variants in both chains. Scaffold 2 is based on human IgG1 (356E / 358M allotype) and contains the S364K / E357Q:L368D / K370S scavariant in one chain, the N208D / Q295E / N384D / Q418E / N421D pI variants, the L368D / K370S scavariant in one chain, the C220S, the S364K / E357Q variants in both chains, and the E233P / L234V / L235A / G236del / S267K deletion variants. Scaffold 3 is based on human IgG1 (356E / 358M allotype) with the scavariant S364K / E357Q:L368D / K370S in one chain, the pI variant N208D / Q295E / N384D / Q418E / N421D, with the scavariant L368D / K370S in one chain, the pI variant Q196K / I199T / P217R / P228R / N276K, with the variants S364K / E357Q in both chains, and the deletion variants E233P / L234V / L235A / G236del / S267K. In certain embodiments, these sequences may be of the 356D / 358L allotype. In other embodiments, these sequences may contain either the N297A or N297S substitutions. In some other embodiments, these sequences may contain the M428L / N434S Xtend mutations. In still other embodiments, these sequences may instead be based on human IgG4, with both chains containing the S228P (EU numbering, S241P in Kabat) variant that eliminates Fab arm exchange as known in the art.In still further embodiments, these sequences may instead be based on human IgG2. Additionally, these sequences may instead utilize other scavariant, pI variants, and deletion variants as shown in Figures 4A-4E, 5, and 6. As will be appreciated by those skilled in the art and outlined below, these sequences may be used with any of the IL-15 and IL-15Rα (sushi) pairs outlined herein, including but not limited to scIL-15 / Rα, ncIL-15 / Rα, and dsIL-15Rα, as shown diagrammatically in Figures 34A-34H. Additionally, as will be appreciated by those skilled in the art and outlined below, any IL-15 and / or IL-15Rα (sushi) variants may be incorporated into these scaffolds. Additionally, as will be appreciated by those skilled in the art and outlined below, these sequences may be used with any of the VH and VL pairs outlined herein, including either scFv or Fab. Included in each of these scaffolds are sequences that are 90, 95, 98, and 99% identical (as defined herein) to the listed sequence, and / or contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions (compared to the "parent" in the figure, which already contains some amino acid modifications compared to the parent human IgG1 (or IgG2 or IgG4, depending on the scaffold), as will be appreciated by one of skill in the art). That is, the listed scaffolds can contain additional amino acid modifications (generally amino acid substitutions) in addition to the scubariants, pI variants, and deletion variants contained within the scaffolds of this figure. [Figure 12] 1 shows the "non-Fv" framework of the cognate light chain (i.e., constant light chain) used in the PD-1-targeting IL-15 / Rα-Fc fusion protein of the invention. [Figure 13]Figures 13A-13G show several formats of the "non-targeted" IL-15 / Rα-Fc fusion proteins of the invention. The IL-15Rα heterodimer Fc fusion or "IL-15 / Rα-hetero Fc" (Figure 13A) comprises IL-15 recombinantly fused to one side of the heterodimeric Fc, and IL-15Rα(sushi) recombinantly fused to the other side of the heterodimeric Fc. IL-15 and IL-15Rα(sushi) can have variable length Gly-Ser linkers between the C-terminus and N-terminus of the Fc region. Single chain IL-15 / Rα-Fc fusion or "scIL-15 / Rα-Fc" (Figure 13B) contains IL-15Rα(sushi) fused to IL-15 by a variable length linker (referred to as "single chain" IL-15 / IL-15Rα(sushi) complex or "scIL-15 / Rα"), which is then fused to the N-terminus of the heterodimeric Fc region, with the other side of the molecule being "Fc only" or "empty Fc". Non-covalent IL-15 / Rα-Fc or "ncIL-15 / Rα-Fc" (Figure 13C) contains IL-15Rα(sushi) fused to the heterodimeric Fc region, while IL-15 is transfected separately such that a non-covalent IL-15 / Rα complex is formed, with the other side of the molecule being "Fc only" or "empty Fc". Bivalent non-covalent IL-15 / Rα-Fc fusions or "bivalent ncIL-15 / Rα-Fc" (Figure 13D) contain IL-15Rα(sushi) fused to the N-terminus of the homodimeric Fc region, while IL-15 is transfected separately to form a non-covalent IL-15 / Rα complex. Bivalent single-chain IL-15 / Rα-Fc fusions or "bivalent scIL-15 / Rα-Fc" (Figure 13E) contain IL-15 fused to IL-15Rα(sushi) by a variable length linker (referred to as "single-chain" IL-15 / IL-15Rα(sushi) complex or "scIL-15 / Rα"), which is then fused to the N-terminus of the homodimeric Fc region. The Fc noncovalent IL-15 / Rα fusion or "Fc-ncIL-15 / Rα" (Figure 13F) contains IL-15Rα (sushi) fused to the C-terminus of the heterodimeric Fc region, while IL-15 is transfected separately such that a noncovalent IL-15 / Rα complex is formed, and the other side of the molecule is "Fc only" or "empty Fc."The Fc-single chain IL-15 / Rα fusion or "Fc-scIL-15 / Rα" (Figure 13G) comprises IL-15 fused to IL-15Rα(sushi) by a variable length linker (referred to as "single chain" IL-15 / IL-15Rα(sushi) complex or "scIL-15 / Rα"), which is then fused to the C-terminus of a heterodimeric Fc region, with the other side of the molecule being "Fc only" or "empty Fc". [Figure 14] 8 shows the sequence of an exemplary IL-15 / Rα-Fc fusion protein in the non-targeting "IL-15 / Rα-hetero-Fc" format. IL-15 and IL-15Rα (sushi) are underlined, the linker is double underlined (however, as will be appreciated by those skilled in the art, the linker may be replaced with other linkers, some of which are shown in FIGS. 8 and 9), and a slash ( / ) indicates the boundary(s) between the IL-15, IL-15Rα, linker, and Fc regions. [Figure 15] 8 shows the sequence of an exemplary IL-15 / Rα-Fc fusion protein in the non-targeted "scIL-15 / Rα-Fc" format. IL-15 and IL-15Rα (sushi) are underlined, the linker is double underlined (however, as will be appreciated by those of skill in the art, the linker may be replaced with other linkers, some of which are shown in FIGS. 8 and 9), and a slash ( / ) indicates the boundary(s) between the IL-15, IL-15Rα, linker, and Fc regions. [Figure 16] 8 shows the sequence of an exemplary IL-15 / Rα-Fc fusion protein in the non-targeting "ncIL-15 / Rα-Fc" format. IL-15 and IL-15Rα (sushi) are underlined, the linker is double underlined (however, as will be appreciated by those of skill in the art, the linker may be replaced with other linkers, some of which are shown in FIGS. 8 and 9), and a slash ( / ) indicates the boundary(s) between the IL-15, IL-15Rα, linker, and Fc regions. [Figure 17]17A-17C show exemplary IL-15 / Rα-Fc fusion proteins in scIL-15 / Rα-Fc format (XENP21478) and ncIL-15 / Rα-Fc format (XENP21479) induce proliferation of A) NK (CD56+ / CD16+) cells, B) CD4+ T cells, and C) CD8+ T cells based on Ki67 expression measured by FACS. [Figure 18] 1 shows the structures of IL-15 in complex with IL-15Rα, IL-2Rβ, and the common gamma chain, with the locations of substitutions designed to reduce potency indicated. [Figure 19] 19A-19C show sequences of exemplary IL-15 variants engineered to reduce potency. Included in each of these variant IL-15 sequences are sequences that are 90, 95, 98, and 99% identical (as defined herein) to the listed sequences and / or contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions. As will be apparent to one of skill in the art, IL-15 variants can be used in any of the IL-15 / Rα-Fc fusions and PD-1-targeted IL-15 / Rα-Fc fusion proteins described herein. [Figure 20] 8 shows the sequence of an exemplary IL-15 / Rα-Fc fusion protein (e.g., a non-targeted construct) in the "non-targeted IL-15 / Rα-hetero-Fc" format that includes an IL-15 variant engineered to reduce potency. IL-15 and IL-15Rα (sushi) are underlined, the linker is double underlined (however, as will be appreciated by those of skill in the art, the linker may be replaced with other linkers, some of which are shown in FIGS. 8 and 9), and a slash ( / ) indicates the boundary(s) between the IL-15, IL-15Rα, linker, and Fc regions. [Figure 21]Figures 21A and 21B show the sequence of an exemplary IL-15 / Rα-Fc fusion protein in the non-targeting "scIL-15 / Rα-Fc" format that includes an IL-15 variant engineered to reduce potency. IL-15 and IL-15Rα (sushi) are underlined, the linker is double underlined (although as one of skill in the art will appreciate, the linker may be replaced with other linkers, some of which are shown in Figures 8 and 9), and a slash ( / ) indicates the boundary(s) between the IL-15, IL-15Rα, linker, and Fc regions. [Figure 22] Figures 22A and 22B show the percentage of A) CD4+CD45RA- and b) CD8+CD45RA- cells expressing Ki67 following incubation with the indicated test substances. [Diagram 23] 1 shows the amino acid sequence of XENP15074 (a bivalent anti-RSV mAb based on motavizumab and human IgG1 Fc with E233P / L234V / L235A / G236del / S267K substitutions). CDRs are underlined and slashes indicate the boundary(s) of the variable regions. [Figure 24] 1 shows the amino acid sequences of A) XENP16432 (a bivalent anti-PD-1 mAb based on nivolumab and human IgG1 Fc with E233P / L234V / L235A / G236del / S267K substitutions), B) XENP21641 (pembrolizumab), and C) XENP28437 (a bivalent anti-PD-1 mAb based on pembrolizumab and human IgG1 Fc with E233P / L234V / L235A / G236del / S267K substitutions). CDRs are underlined and slashes indicate the boundary(s) of the variable regions. [Diagram 25]1 shows the sequences of hybridoma clone 1C11 with E233P / L234V / L235A / G236del / S267K substitutions in the heavy chain and chimeric and humanized anti-PD-1 antibodies based on the variable regions of human IgG1, XENP21575. The CDRs are underlined and the slashes indicate the boundaries of the variable domains. As is true for any sequence described herein and containing CDRs herein, the exact identification of the CDR positions may vary slightly depending on the numbering used as shown in Table 1, and thus, not only the underlined CDRs, but also CDRs contained within the VH and VL domains using other numbering systems are included herein. As will be appreciated by those of skill in the art, the VH and VL domains can be formatted as Fab or scFv for use in the PD-1-targeting IL-15 / Rα-Fc fusion proteins of the present invention. [Figure 26] 1 shows the sequences of exemplary humanized variants of anti-PD-1 mAb A and mAb B in a bivalent human IgG1 format with E233P / L234V / L235A / G236del / S267K substitutions in the heavy chain. The CDRs are underlined and the slashes indicate the boundaries of the variable domains. As is true for any sequence described herein and containing CDRs herein, the exact identification of the CDR positions may vary slightly depending on the numbering used as shown in Table 1, and thus, not only the underlined CDRs, but also CDRs contained within the VH and VL domains using other numbering systems are included herein. As will be appreciated by one of skill in the art, the VH and VL domains can be formatted as Fab or scFv for use in the PD-1-targeting IL-15 / Rα-Fc fusion proteins of the invention. [Figure 27] Figure 1 shows epitope binning of XENP16432 (a bivalent anti-PD-1 mAb based on nivolumab), XENP21461 (pembrolizumab), chimeric mAb A (chmAb A), chimeric mAb B (chmAb B), and 1C11-based mAbs as shown by normalized BLI responses Octet. A normalized BLI response above 0.5 indicates that the pair of antibodies does not bind to the same epitope. [Figure 28]Figures 28A-28H show several formats of PD-1-targeted IL-15 / Rα-Fc fusion proteins of the invention. The "scIL-15 / Rα x scFv" format (Figure 28A) comprises IL-15Rα (sushi) fused to IL-15 by a variable length linker (referred to as "scIL-15 / Rα"), which is then fused to the N-terminus of the heterodimeric Fc region, with the scFv fused to the other side of the heterodimeric Fc. The "scFv x ncIL-15 / Rα" format (Figure 28B) comprises an scFv fused to the N-terminus of the heterodimeric Fc region, with the IL-15Rα (sushi) fused to the other side of the heterodimeric Fc, while IL-15 is transfected separately such that a non-covalent IL-15 / Rα complex is formed. The "scIL-15 / Rα x Fab" format (Figure 28C) comprises IL-15Rα (sushi) fused to IL-15 by a variable length linker (referred to as "scIL-15 / Rα"), which is then fused to the N-terminus of the heterodimeric Fc region, and the variable heavy chain (VH) is fused to the other side of the heterodimeric Fc, while the corresponding light chain is transfected separately to form a Fab with the VH. The "ncIL-15 / Rα x Fab" format (Figure 28D) comprises VH fused to the N-terminus of the heterodimeric Fc region, and IL-15Rα (sushi) is fused to the other side of the heterodimeric Fc, while the corresponding light chain is transfected separately to form a Fab with the VH, while IL-15 is transfected separately to form a non-covalent IL-15 / Rα complex. The "mAb-scIL-15 / Rα" format (Figure 28E) comprises a VH fused to the N-terminus of the first and second heterodimeric Fc, where IL-15 is fused to IL-15Rα (sushi), which is then further fused to the C-terminus of one of the heterodimeric Fc regions, while the corresponding light chain is transfected separately to form a Fab with the VH.The "mAb-ncIL-15 / Rα" format (Figure 28F) comprises a VH fused to the N-terminus of the first and second heterodimeric Fc, and IL-15Rα (sushi) is fused to the C-terminus of one of the heterodimeric Fc regions, while the corresponding light chain is transfected separately to form a Fab containing the VH, and IL-15 is transfected separately to form a non-covalent IL-15 / Rα complex. The "Central-IL-15 / Rα" format (Figure 28G) comprises a VH recombinantly fused to the N-terminus of IL-15 (which is then further fused to one side of the heterodimeric Fc) and a VH recombinantly fused to the N-terminus of IL-15Rα (sushi) (which is then further fused to the other side of the heterodimeric Fc), while the corresponding light chain is transfected separately to form a Fab with the VH. The "central-scIL-15 / Rα" format (Figure 28H) contains a VH fused to the N-terminus of IL-15Rα (sushi), which is then fused to one side of a heterodimeric Fc, and a VH fused to the other side of the heterodimeric Fc, while the corresponding light chain is transfected separately to form a Fab containing the VH. [Figure 29]6 shows the sequence of an exemplary [C]PD-1-targeting IL-15 / Rα-Fc fusion protein in the "scIL-15 / Rα x Fab" format. The CDRs are shown in bold. As is true for any sequence described herein and containing CDRs herein, the exact identification of the CDR positions may vary slightly depending on the numbering used as shown in Table 1, and thus, not only the underlined CDRs but also CDRs contained within the VH and VL domains using other numbering systems are included herein. IL-15 and IL-15Rα (sushi) are italicized, the linker is double underlined (however, as will be appreciated by those of skill in the art, the linker may be replaced with other linkers, portions of which are shown in Figures 8 and 9), and a slash ( / ) indicates the boundary(s) between IL-15, IL-15Rα, linker, variable region, and constant / Fc region. As will be apparent to one of skill in the art, each of the described targeted IL-15 / Rα-Fc fusion proteins may include or exclude Xtend Fc (M428L / N434S). [Diagram 30] 1 shows the sequence of an exemplary [NC]PD-1-targeting IL-15 / Rα-Fc fusion protein in the "scIL-15 / Rα x Fab" format. The CDRs are underlined. As is true for any sequence described herein and containing CDRs herein, the exact identification of the CDR positions may vary slightly depending on the numbering used as shown in Table 1, and thus, not only the underlined CDRs but also CDRs contained within the VH and VL domains using other numbering systems are included herein. IL-15 and IL-15Rα (sushi) are italicized, the linker is double underlined (however, as will be appreciated by those of skill in the art, the linker may be replaced with other linkers, some of which are shown in Figures 8 and 9), and a slash ( / ) indicates the boundary(s) between IL-15, IL-15Rα, the linker, the variable region, and the constant / Fc region. As will be apparent to one of skill in the art, each of the described targeted IL-15 / Rα-Fc fusion proteins may include or exclude Xtend Fc (M428L / N434S). [Diagram 31]Figures 31A-31D show the sequence of a control RSV-targeting IL-15 / Rα-Fc fusion. The CDRs are underlined. As is true for any sequence described herein and containing CDRs herein, the exact identification of the CDR positions may vary slightly depending on the numbering used as shown in Table 1, and therefore, not only the underlined CDRs, but also CDRs contained within the VH and VL domains using other numbering systems are included herein. IL-15 and IL-15Rα (sushi) are written in italics, the linker is double underlined (however, as will be understood by those skilled in the art, the linker may be replaced with other linkers, some of which are shown in Figures 8 and 9), and a slash ( / ) indicates the boundary(s) between IL-15, IL-15Rα, the linker, the variable region, and the constant / Fc region. As will be apparent to one of skill in the art, each of the described targeted IL-15 / Rα-Fc fusion proteins may also exclude or include Xtend Fc (M428L / N434S). [Diagram 32] Figures 32A and 32B show proliferation of A) CD8+ T cells and B) CD4+ T cells following incubation with PD-1-targeted IL-15 / Rα-Fc fusions (XENP28532, XENP28692, and XENP25850), as well as a control RSV-targeted IL-15 / Rα-Fc fusion (XENP26007) and an anti-PD-1 mAb (XENP28519). [Diagram 33] Figures 33A and 33B show induction of STAT5 phosphorylation in A) CD4+CD45RA-CD25+ and B) CD8+CD45RA-CD25+ by XENP25850 (an exemplary PD-1-targeted IL-15 / Rα-Fc fusion). Fresh cells are shown with dotted lines and activated cells are shown with solid lines. All fresh cells are CD25-. [Diagram 34]Figures 34A and 34B show induction of STAT5 phosphorylation in CD8+CD45RA-CD25+PD-1+ T cells by A) [C]PD-1-targeting IL-15 / Rα-Fc fusion XENP25937 and B) [NC]PD-1-targeting IL-15 / Rα-Fc fusion XENP28532 after preincubation with either nivolumab-based XENP16432, pembrolizumab, or anti-RSV mAb XENP15074. [Diagram 35] Shown is the change in body weight (as a percentage of initial body weight) of huPBMC-engrafted NSG mice over time following administration of the indicated test substances. [Diagram 36] Figures 36A and 36B show A) CD8+ T cell counts and B) CD4+ T cell counts 14 days after the first dose of the indicated test article in human PBMC engrafted NSG mice. [Figure 37] Figures 37A and 37B show CD25 expression in A) CD8+ T cells and B) CD4+ T cells 10 days after the first dose of the indicated test article in human PBMC-engrafted NSG mice. [Figure 38] Shown are the percentages of CD8+ and CD4+ T cells 10 days after the first dose of the indicated test article in human PBMC engrafted NSG mice. [Figure 39] Figures 39A and 39B show A) CD8+ T cell counts and B) CD4+ T cell counts 10 days after the first dose of the indicated test article in human PBMC engrafted NSG mice. [Diagram 40] Figures 40A and 40B show CD25 expression in A) CD8+ T cells and B) CD4+ T cells 10 days after the first dose of the indicated test article in human PBMC-engrafted NSG mice. [Diagram 41] FIG. 42 shows serum concentrations of the indicated test articles over time in cynomolgus monkeys following an initial dose at the indicated relative concentrations. [Diagram 42]1 shows the sequences of XENP29484 and XENP29485, an exemplary [NC]PD-1 targeting IL-15 / Rα-Fc fusion protein with IL-15(D30N / N65D) variant. The CDRs are underlined. As is true for any sequence described herein and containing CDRs herein, the exact identification of the CDR positions may vary slightly depending on the numbering used as shown in Table 1, and thus, not only the underlined CDRs, but also CDRs contained within the VH and VL domains using other numbering systems are included herein. IL-15 and IL-15Rα (sushi) are italicized, the linker is double underlined (however, as will be appreciated by those of skill in the art, the linker may be replaced with other linkers, some of which are shown in the figures), and a slash ( / ) indicates the boundary(s) between IL-15, IL-15Rα, linker, variable region, and constant / Fc region. [Diagram 43] Figures 43A-43X show the variable heavy and variable light chains of additional exemplary anti-PD-1 ABDs that do not compete with nivolumab or pembrolizumab. The CDRs are underlined. As is true for any sequence described herein and that contains the CDRs herein, the exact identification of the CDR positions may vary slightly depending on the numbering used as shown in Table 1, and thus, not only the underlined CDRs, but also CDRs contained within the VH and VL domains using other numbering systems are included herein. [Diagram 44] Figures 44A and 44B show the percentage of PD-1+ in A) CD8+CD45RA-CD45RO+ T cells and B) CD4+CD45RA-CD45RO+ T cells following treatment with the PD-1-targeting IL-15 / Rα-Fc fusion XENP28532 and controls XENP24306 (non-targeting IL-15 / Rα-Fc fusion) and XENP26007 (RSV-targeting IL-15 / Rα-Fc fusion). [Diagram 45]1 shows the sequence of an exemplary humanized variant of anti-PD-1 mAb C in a bivalent human IgG1 format with E233P / L234V / L235A / G236del / S267K substitutions in the heavy chain. The CDRs are underlined and the slashes indicate the boundaries of the variable domains. As is true for any sequence described herein and containing CDRs herein, the exact identification of the CDR positions may vary slightly depending on the numbering used as shown in Table 1, and thus, not only the underlined CDRs, but also CDRs contained within the VH and VL domains using other numbering systems are included herein. As will be appreciated by one of skill in the art, the VH and VL domains can be formatted as Fab or scFv for use in the PD-1-targeting IL-15 / Rα-Fc fusion proteins of the invention. [Figure 46] 1 shows the affinity of XENP28536, XENP28537, XENP28538, XENP28539, and XENP28519 for human and cynomolgus PD-1 as determined by Octet. [Figure 47] Figure 1 shows epitope binning of XENP16432 (a bivalent anti-PD-1 mAb based on nivolumab), XENP21461 (pembrolizumab), and chimeric mAb C (chmAb C). A normalized BLI response of greater than 0.5 indicates that the antibody pair does not bind to the same epitope. The data indicates that anti-PD-1 mAb C does not bind to the same epitope as nivolumab and pembrolizumab. [Figure 48]Figures 48A-48C show sequences of exemplary [NC]PD-1-targeting IL-15 / Rα-Fc fusion proteins in the "scIL-15 / Rα x Fab" format, including a PD-1-targeting arm based on mAb C and various IL-15 potency variants. The CDRs are underlined. As is true for any sequence described herein and including CDRs herein, the exact identification of the CDR positions may vary slightly depending on the numbering used as shown in Table 1, and thus, not only the underlined CDRs, but also CDRs contained within the VH and VL domains using other numbering systems are included herein. The linker is double underlined (however, as will be appreciated by those of skill in the art, the linker may be replaced with other linkers, portions of which are shown in Figures 65 and 66), and a slash ( / ) indicates the boundary(s) between IL-15, IL-15Rα, linker, variable region, and constant / Fc region. As will be apparent to one of skill in the art, each of the PD-1-targeting IL-15 / Rα-Fc fusion proteins described may also exclude or include Xtend Fc (M428L / N434S). [Figure 49] Figures 49A-49C show sequences of exemplary [NC]PD-1-targeting IL-15 / Rα-Fc fusion proteins in the "scIL-15 / Rα x Fab" format, comprising a PD-1 targeting arm based on mAb C and various IL-15 potency variants, and additionally comprising Xtend Fc (M428L / N434S). CDRs are underlined. As is true for any sequence described herein and containing CDRs herein, the exact identification of the CDR positions may vary slightly depending on the numbering used as shown in Table 1, and thus, not only the underlined CDRs, but also CDRs contained within the VH and VL domains using other numbering systems are included herein. The linker is double underlined (however, as will be appreciated by those of skill in the art, the linker may be replaced with other linkers, portions of which are shown in Figures 65 and 66), and a slash ( / ) indicates the boundary(s) between IL-15, IL-15Rα, the linker, the variable region, and the constant / Fc region. [Figure 50]Figure 1 shows induction of A) CD8+ and B) CD4+ T cell proliferation by [NC]PD-1-targeted IL-15 / Rα-Fc fusions (and controls) as indicated by percentage of proliferating cells (determined based on CFSE dilution). The data show that [NC]PD-1-targeted IL-15 / Rα-Fc fusions are more potent at inducing CD4+ T cell proliferation compared to non-targeted IL-15(D30N / E64Q / N65D) / Rα-Fc fusions (as well as control RSV-targeted IL-15 / Rα-Fc fusions). [Figure 51] Figure 1 shows the induction of LAG-3 positive CD8+ T cells by [NC]PD-1 targeted IL-15 / Rα-Fc fusions (and controls) as indicated by the percentage of proliferating cells (determined based on CFSE dilution). The data show that XENP28532 is more potent in inducing proliferation of CD8+LAG-3+ T cells compared to non-targeted IL-15(D30N / E64Q / N65D) / Rα-Fc fusions (as well as control RSV targeted IL-15 / Rα-Fc fusions). Furthermore, XENP28543 was more potent in inducing CD8+LAG-3+ T cell proliferation than bulk CD8+ T cell proliferation (EC50 276.8 vs. 71.94). Taken together, this supports the idea that [NC]PD-1 targeted IL-15 / Rα-Fc fusions may be selective for T cells expressing checkpoints such as those found in the tumor environment. [Figure 52] Activation of A) CD4+CD45RA- memory T cells and B) CD4+CD45RA+ naive T cells following incubation with [NC]PD-1-targeted IL-15 / Rα-Fc fusions (and controls) as indicated by the percentage of cells expressing CD25. [Diagram 53] [NC] Shows the percentage of A) CD4+CD45RA- and B) CD4+CD45RA+ T cells expressing PD-1 after incubation with PD-1-targeting IL-15 / Rα-Fc fusions (and controls). [Figure 54]Figure 1 shows IFNγ secretion by PBMCs pre-stimulated with A) 50ng / ml, B) 100ng / ml, C) 500ng / ml plate-bound anti-CD3 (OKT3) and incubated with the indicated test substances. The data show that both XENP28532 and XENP28543 were able to potently stimulate IFNγ secretion. Notably, XENP28532 (PD-1 targeting arm based on mAb A) appeared to be more active than XENP28543 (PD-1 targeting arm based on mAb C) in inducing IFNγ secretion. [Figure 55] Figure 1 shows induction of STAT5 phosphorylation in A) CD8+CD45RA-CD25+PD-1+ and B) CD4+CD45RA-CD25+PD-1+ T cells by XENP28543, a [NC]PD-1-targeting IL-15 / Rα-Fc fusion with a PD-1-targeting arm based on mAb C, following preincubation with either nivolumab-based XENP16432, pembrolizumab, or anti-RSV mAb XENP15074. The data show that PD-1 blockade does not interfere with the activity of XENP28543. [Figure 56] Shown is the change in body weight (as a percentage of initial body weight) in huPBMC-engrafted NSG mice over time following administration of the indicated test substances. [Figure 57] Body weights (as a percentage of initial body weight) of huPBMC-engrafted NSG mice are shown A) 11, B) 14, and C) 18 days after the first dose of the indicated test agent. p values ​​were determined using an unpaired t-test. The data show that by day 11, the combination of [NC]PD-1-targeted IL-15 / Rα-Fc fusion with PD-1 blockade significantly enhanced GVHD compared to treatment with [NC]PD-1-targeted IL-15 / Rα-Fc fusion alone. [Figure 58] Numbers of human A) CD45+ cells, B) CD3+ T cells, C) CD4+ T cells, D) CD8+ T cells, E) γδ T cells, and F) NK cells in the blood of huPBMC-engrafted NSG mice 14 days after the first dose of the indicated test article. [Figure 59]Activation of human A) CD8+ and B) CD4+ T cells (as indicated by CD25 MFI) in the blood of huPBMC-engrafted NSG mice 14 days after the first dose of the indicated test substance. [Figure 60] [NC] Tumor volume (as determined by caliper measurements) over time in pp65-MCF7 and huPBMC-engrafted NSG mice administered PD-1-targeting IL-15 / Rα-Fc fusion alone or in combination with PD-1 blockade. [Figure 61] [NC] Tumor volumes (as determined by caliper measurements) at days 26, 28, 30, 33, 35, and 37 (after PBMC transfer and first dose of test article) in pp65-MCF7 and huPBMC-engrafted NSG mice administered PD-1-targeting IL-15 / Rα-Fc fusion alone or in combination with PD-1 blockade. p values ​​were determined by unpaired t-test. Data show that by day 28, the combination of XENP28543 and PD-1 blockade significantly reduced tumor size more than treatment with PD-1 blockade alone. [Figure 62] Figure 1 shows activation of human A) CD8+ and B) CD4+ T cells (as indicated by CD25 MFI) in blood of pp65-MCF7 and huPBMC-engrafted NSG mice 7 days after the first dose of the indicated test substance. The data show that [NC]PD-1-targeted IL-15 / Rα-Fc fusion alone or in combination with PD-1 blockade was able to significantly enhance early activation of CD8+ T cells. Statistics were performed on log-transformed data using unpaired t-tests. [Figure 63]Shown are the numbers of human A) CD45+ cells, B) CD3+ T cells, C) CD4+ T cells, D) CD8+ T cells, and E) NK cells in the blood of pp65-MCF7 and huPBMC-engrafted NSG mice 14 days after the first dose of the indicated test agent. The data show that [NC]PD-1-targeted IL-15 / Rα-Fc fusion alone or in combination with PD-1 blockade was able to significantly enhance proliferation of multiple lymphocyte populations by day 14 more than PD-1 blockade alone. Statistics were performed on log-transformed data using unpaired t-tests. [Figure 64] 1 shows the sequence of an exemplary affinity engineered variant of anti-PD-1 mAb C in a bivalent human IgG1 format with E233P / L234V / L235A / G236del / S267K substitutions in the heavy chain. The CDRs are underlined and the slashes indicate the boundaries of the variable domains. As is true for any sequence described herein and containing CDRs herein, the exact identification of the CDR positions may vary slightly depending on the numbering used as shown in Table 1, and thus, not only the underlined CDRs, but also CDRs contained within the VH and VL domains using other numbering systems are included herein. As will be appreciated by one of skill in the art, the VH and VL domains can be formatted as Fab or scFv for use in the PD-1-targeting IL-15 / Rα-Fc fusion proteins of the invention. [Figure 65]Figures 65A-65I show the apparent dissociation constants (KDapp), association rates (ka), and dissociation rates (kd) of affinity engineered mAb C[PD-1]_H1L1 variants (bivalent IgG1 format with E233P / L234V / L235A / G236_ / S267K deletion variants) as determined by Octet, as well as fold improvement over mAb C[PD-1]_H1L1. Substitutions in the variable heavy or variable light regions, if listed, are based on Xencor numbering (corresponding Kabat positions are listed in the next column). Of the 304 variants with single point mutations in either the variable heavy or variable light regions, we identified only 11 variants (including mAb C[PD-1]_H1_L1.1 and mab_C[PD-1]_H1_L1.3) with mutations greater than 2-fold improved affinity over WT. It should be noted that in the Kabat numbering sequence, some positions are also numbered using letters. For example, in the Kabat numbering, there is one amino acid at position 27d (N27d), but another at position 27e (S27e). Thus, the inclusion of a lower case letter indicates the position, not the specific amino acid at that position. [Figure 66] Figure 1 shows the apparent dissociation constants (KDapp), association rates (ka), and dissociation rates (kd) of affinity engineered mAb C[PD-1]_H1L1 variants combining preferred single substitution VH variants and single substitution VL variants (in the context of PD-1 targeting IL15 / Rα-Fc) as determined by Octet. Substitutions in the variable heavy or variable light regions, when listed, are based on Xencor numbering (corresponding Kabat positions are listed in the next column). H1.132_L1 provides higher affinity than H1.19_L1, yet H1.19_L1.1 allows for higher affinity than H1.132_L1.1. [Figure 67]Figure 1 shows the apparent dissociation constants (KDapp), association rates (ka), and dissociation rates (kd) of affinity engineered (in the context of PD-1 targeting IL15 / Rα-Fc) mAb C[PD-1]_H1L1 variants combining multiple substitutions in VH and / or VL as determined by Octet. Substitutions in the variable heavy or variable light regions, when listed, are based on Xencor numbering (corresponding Kabat positions are listed in the next column). The triple substitution VL variant N31H / K36Y / S99T (L1.140, N27dH / K30Y / S93T in Kabat numbering) shows a 36-fold improvement in KD over wild type and combines well with the VH variant to exert a ~100-fold improvement in KD over wild type. [Figure 68] Figures 68A-68H show sequences of exemplary [NC]PD-1-targeting IL-15 / Rα-Fc fusion proteins in the "scIL-15 / Rα x Fab" format, including a PD-1-targeting arm based on the affinity-optimized mAb C ABD and various IL-15 potency variants. The CDRs are underlined. As is true for any sequence described herein and including CDRs herein, the exact identification of the CDR positions may vary slightly depending on the numbering used as shown in Table 1, and thus, not only the underlined CDRs, but also CDRs contained within the VH and VL domains using other numbering systems are included herein. The linker is double underlined (however, as will be appreciated by those of skill in the art, the linker may be replaced with other linkers, some of which are shown in Figures 8 and 9), and a slash ( / ) indicates the boundary(s) between IL-15, IL-15Rα, the linker, the variable region, and the constant / Fc region. As will be apparent to one of skill in the art, each of the PD-1-targeting IL-15 / Rα-Fc fusion proteins described may also exclude or include Xtend Fc (M428L / N434S). [Figure 69]Figures 69A and 69B show the sequence of an exemplary [NC]PD-1-targeting IL-15 / Rα-Fc fusion protein in the "scIL-15 / Rα x Fab" format, comprising a PD-1 targeting arm based on the affinity-optimized mAb C ABD and various IL-15 potency variants, and additionally comprising Xtend Fc (M428L / N434S). The CDRs are underlined. As is true for any sequence described herein and containing CDRs herein, the exact identification of the CDR positions may vary slightly depending on the numbering used as shown in Table 1, and thus, not only the underlined CDRs, but also CDRs contained within the VH and VL domains using other numbering systems are included herein. The linker is double underlined (although as one of skill in the art would understand, the linker may be replaced with other linkers, some of which are shown in Figures 8 and 9), and a slash ( / ) indicates the boundary(s) between IL-15, IL-15Rα, the linker, the variable region, and the constant / Fc region. [Figure 70] Figure 1 shows induction of A) CD8+ and B) CD4+ T cell proliferation by [NC]PD-1-targeted IL-15 / Rα-Fc fusions (and controls) with various PD-1 affinity and IL-15 potency variants as indicated by percentage of proliferating cells (determined based on CFSE dilution). Data show that XENP30046 (with an affinity-enhanced PD-1 targeting arm) induces proliferation of both CD8+ and CD4+ T cells more potently than XENP28543 (2-fold increase). Furthermore, data show that IL-15(D30N / N65D) variants do not dramatically affect the activity of PD-1-targeted IL-15 / Rα-Fc fusions. [Figure 71] Activation of A) CD8+CD45RA- and B) CD8+CD45RA+ T cells following incubation with [NC]PD-1-targeted IL-15 / Rα-Fc fusions (and controls) with various PD-1 affinity and IL-15 potency variants, as indicated by the percentage of cells expressing CD25. [Figure 72]Activation of A) CD4+CD45RA- and B) CD4+CD45RA+ T cells following incubation with [NC]PD-1-targeted IL-15 / Rα-Fc fusions (and controls) with various PD-1 affinity and IL-15 potency variants, as indicated by the percentage of cells expressing CD25. [Figure 73] The percentage of A) CD4+CD45RA- and B) CD4+CD45RA+ T cells expressing PD-1 after incubation with [NC]PD-1-targeted IL-15 / Rα-Fc fusions with various PD-1 affinity and IL-15 potency variants (and controls) is shown. The data show that [NC]PD-1-targeted IL-15 / Rα-Fc fusions induce downregulation of PD-1 in CD4+ cells, and that downregulation correlates with PD-1 affinity. [Figure 74] Change in body weight (as a percentage of initial body weight) over time in huPBMC-engrafted NSG mice treated with [NC]PD-1-targeted IL-15 / Rα-Fc fusions (and controls) with various PD-1 affinity and IL-15 potency variants. 70% were used for dead mice. [Figure 75] Shown are body weights on days A) 11, B) 14, C) 18, and D) 21 of huPBMC-engrafted NSG mice treated with [NC]PD-1-targeted IL-15 / Rα-Fc fusions (and controls) with various PD-1 affinity and IL-15 potency variants. 70% were used for dead mice. p values ​​were determined using unpaired t-tests. Data show that treatment with XENP30046 alone (with affinity-enhanced PD-1-targeted arm) resulted in significant weight loss compared to PBS treatment, as measured on days 11 and 18, whereas treatment with XENP28543 alone did not result in significant weight loss (compared to PBS treatment). [Figure 76]Figures 76A-76C show serum concentrations of A) IFNγ, B) IL-10, and C) IL-2Rα in huPBMC-engrafted NSG mice 7, 11, and 14 days after administration of [NC]PD-1-targeted IL-15 / Rα-Fc fusions (and controls) with various PD-1 affinity and IL-15 potency variants. [Figure 77] Figure 1 shows A) CD4+ T cell and B) CD8+ T cell activation (as indicated by CD25 MFI) on day 7 in the blood of huPBMC-engrafted NSG mice administered [NC]PD-1-targeted IL-15 / Rα-Fc fusions with various PD-1 affinity and IL-15 potency variants (and controls) (statistics were performed on log-transformed data using unpaired t-tests). [Figure 78] Figure 11 shows A) CD45+ cell, B) CD3+ T cell, C) CD8+ T cell, D) CD4+ T cell, and E) NK cell counts in the blood of huPBMC-engrafted NSG mice treated with [NC]PD-1-targeted IL-15 / Rα-Fc fusions with various PD-1 affinity and IL-15 potency variants (and controls) (statistics were performed on log-transformed data using unpaired t-tests). [Figure 79] Shown are A) CD45+ cell, B) CD3+ T cell, C) CD8+ T cell, D) CD4+ T cell, and E) NK cell counts in the blood of huPBMC-engrafted NSG mice treated with [NC]PD-1-targeted IL-15 / Rα-Fc fusions with various PD-1 affinity and IL-15 potency variants (and controls) on day 14. [Figure 80]Figure 1 shows induction of proliferation of A) CD8+ and B) CD4+ T cells by [NC]PD-1-targeted IL-15 / Rα-Fc fusions (and controls) with various PD-1 affinity and IL-15 potency variants as indicated by percentage of proliferating cells (determined based on CFSE dilution). The data show that XENP30272 (with a KD of 3.1 nM for PD-1) is more potent in inducing proliferation and activation of various T cell populations than XENP30046 (with a KD of 5.4 nM for PD-1). Notably, XENP30429 (PD-1-targeted IL-15 / Rα-Fc fusion with IL-15(D30N / E64Q / N65D) variant) was 1.8-2.5 times less active in CD8+ and CD4+ T cells compared to XENP30046 (PD-1-targeted IL-15 / Rα-Fc fusion with IL-15(N4D / N65D) variant), whereas XENP30432 (surrogate RSV-targeted IL-15 / Rα-Fc with IL-15(D30N / E64Q / N65D) variant) was 12-fold less active in CD8+ T cells and 530-fold less active in CD4+ T cells compared to XENP30046 (based on proliferation activity). This suggests that PD-1-targeted IL-15 / Rα-Fc fusions carrying the IL-15(D30N / E64Q / N65D) variant retain activity in the tumor environment and remain essentially inactive outside the tumor environment. [Figure 81] Activation of A) CD4+CD45RA- and B) CD4+CD45RA+ T cells following incubation with [NC]PD-1-targeted IL-15 / Rα-Fc fusions (and controls) with various PD-1 affinity and IL-15 potency variants as indicated by CD25 MFI. [Figure 82] Activation of A) CD8+CD45RA- and B) CD8+CD45RA+ T cells following incubation with [NC]PD-1-targeted IL-15 / Rα-Fc fusions (and controls) with various PD-1 affinity and IL-15 potency variants as indicated by CD25 MFI. [Figure 83]Shown is PD-1 expression on A) CD4+ T cells, B) CD4+CD45RA- T cells, and C) CD4+CD45RA+ T cells following incubation with [NC]PD-1-targeted IL-15 / Rα-Fc fusions with various PD-1 affinity and IL-15 potency variants (and controls) as indicated by PD-1 MFI. [Figure 84] Shown is PD-1 expression on A) CD8+ T cells, B) CD8+CD45RA- T cells, and C) CD8+CD45RA+ T cells following incubation with [NC]PD-1-targeted IL-15 / Rα-Fc fusions with various PD-1 affinity and IL-15 potency variants (and controls) as indicated by PD-1 MFI. [Figure 85] Figure 1 shows proliferation (as indicated by cell counts) of human A) CD45+ cells, B) CD3+ T cells, C) CD4+ T cells, and D) CD8+ T cells in NSG mice 10 days after the first dose of the indicated test article. The data show that more potent IL-15 results in greater proliferation of various lymphocyte populations. [Figure 86] Activation of A) CD4+ and B) CD8+ T cells (as indicated by CD25 staining) in NSG mice 10 days after the first dose of the indicated test substance is shown. The data indicate that more potent IL-15 results in greater proliferation of various lymphocyte populations. [Figure 87] Figures 87A-87C show induction of A) CD4+ T cell proliferation, B) CD8+ T cell, and C) NK cell proliferation by [NC]PD-1-targeted IL-15 / Rα-Fc fusions with Xtend Fc and control RSV-targeted IL-15 / Rα-Fc fusions as indicated by the percentage of proliferating cells (determined based on CFSE dilution). Notably, the data show that the EC50 of XENP30046 is comparable to that of XENP30290 (the Xtend analog of XENP30046). [Figure 88]Activation of A) CD4+ T cells and B) CD8+ T cells by [NC]PD-1-targeted IL-15 / Rα-Fc fusion with Xtend Fc and control RSV-targeted IL-15 / Rα-Fc fusion as indicated by CD25 MFI. [Figure 89] Figure 1 shows PD-1 regulation in A) CD4+ and B) CD8+ T cells by [NC]PD-1-targeted IL-15 / Rα-Fc fusions with Xtend Fc and control RSV-targeted IL-15 / Rα-Fc fusions as indicated by PD-1 MFI. Consistent with Example 9B, the test agents downregulated PD-1 on T cells. [Figure 90] [NC] Tumor volume (as determined by caliper measurements) over time in pp65-MCF7 and huPBMC-engrafted NSG mice administered PD-1-targeting IL-15 / Rα-Fc fusion alone or in combination with PD-1 blockade. [Figure 91] Figures 91A-91H show tumor volumes (as determined by caliper measurements) at days 11, 14, 17, 19, 21, 24, 26 and 28 (after PBMC transfer and first dose of test substance) in pp65-MCF7 and huPBMC-engrafted NSG mice administered [NC]PD-1-targeted IL-15 / Rα-Fc fusion alone or in combination with PD-1 blockade. Statistics were performed on baseline-corrected data using the Mann-Whitney test. * indicates that treatment significantly (p<0.05) enhanced proliferation compared to PBS control. # indicates that treatment significantly (p<0.05) enhanced proliferation compared to treatment with XENP31123. † indicates that treatment significantly (p<0.05) enhanced proliferation compared to PD-1 blockade (XENP16432) alone. The data show that by day 28, all combinations of XENP30290 (0.1, 0.3 or 1 mg / kg) or XENP30516 (0.3, 1 or 3 mg / kg) with PD-1 blockade significantly reduced tumor size more than treatment with PD-1 blockade alone. [Figure 92]Figures 92A-92F show the numbers of human A) CD45+ cells, B) CD3+ T cells, C) NK cells, D) CD4+ T cells, and E) CD8+ T cells in the blood of pp65-MCF7 and huPBMC-engrafted NSG mice 14 days after the first dose of the indicated test substance, and F) the ratio of CD8 to CD4 T cells. Statistics of CD45+ cell proliferation were performed on log-transformed data using unpaired t-tests. † indicates that treatment significantly enhanced proliferation compared to PD-1 blockade (XENP16432) alone. [Figure 93] Figures 93A-93F show the numbers of human A) CD45+ cells, B) CD3+ T cells, C) NK cells, D) CD4+ T cells, and E) CD8+ T cells in the blood of pp65-MCF7 and huPBMC-engrafted NSG mice 21 days after the first dose of the indicated test agent, and F) the ratio of CD8 to CD4 T cells. † indicates that treatment significantly enhanced proliferation compared to PD-1 blockade (XENP16432) alone. [Figure 94] Figure 1 shows PD-1 expression levels (as indicated by MFI) in various lymphocyte populations in CD34+ Hu-NSG mice (before treatment with any test substance). The data show that the mice have a similar PD-1 expression profile to humans, i.e., higher expression in effector memory populations. [Figure 95] Figure 1 shows the fold increase in various lymphocyte populations in CD34+Hu-NSG mice on day 7 after treatment with 0.3 mg / kg XENP30046 ([NC]PD-1-targeted IL-15 / Rα-Fc with mAb C_H1_L1.1 and IL-15(N4D / N65D) variant). XENP30046 shows over 100-fold expansion of effector memory populations. [Figure 96] Figure 1 shows the fold increase in various lymphocyte populations in CD34+Hu-NSG mice on day 7 after treatment with 0.3 mg / kg XENP30429 ([NC]PD-1-targeted IL-15 / Rα-Fc with mAb C_H1_L1.1 and IL-15(D30N / E64Q / N65D) variant). [Figure 97]Figure 1 shows the fold increase in various lymphocyte populations in CD34+ Hu-NSG mice on day 7 after treatment with 0.3 mg / kg XENP26007 (control RSV-targeted IL-15 / Rα-Fc with IL-15 (N4D / N65D) variant). XENP26007 shows very little proliferation of various lymphocyte populations, indicating minimal proliferation of peripheral lymphocytes for the PD-1-targeted IL-15 / Rα-Fc fusion. [Figure 98] Figure 1 shows the fold increase in various lymphocyte populations in CD34+ Hu-NSG mice on day 7 after treatment with 0.3 mg / kg XENP30432 (control RSV-targeted IL-15 / Rα-Fc with IL-15 (D30N / E64Q / N65D) variant). XENP30432 shows very little proliferation of various lymphocyte populations, indicating minimal proliferation of peripheral lymphocytes for the PD-1-targeted IL-15 / Rα-Fc fusion. [Figure 99] Fold change over time of A) CD45 cells, B) CD3+ cells, C) CD4+ cells, D) CD8+ cells, E) γδ cells, and F) NK cells in CD34+ Hu-NSG mice following treatment with 0.3 mg / kg XENP30046, XENP30429, XENP26007, or XENP30432. [Figure 100] Figure 1 shows the fold change over time of A) CD4 naive cells, B) CD4 central memory cells, C) CD4 terminal effector cells, and D) CD4 effector memory cells in CD34+ Hu-NSG mice following treatment with 0.3 mg / kg XENP30046, XENP30429, XENP26007, or XENP30432. The data show that XENP30046 and XENP30429 are selective for PD-1+ populations, including CD4 effector memory cells. Notably, XENP30429 expansion of CD4 naive cells was minimal, indicating that reducing the potency of the IL-15 arm improves selectivity. [Figure 101]Figure 1 shows the fold change over time of A) CD8 naive cells, B) CD8 central memory cells, C) CD8 terminal effector cells, and D) CD8 effector memory cells in CD34+ Hu-NSG mice following treatment with 0.3 mg / kg XENP30046, XENP30429, XENP26007, or XENP30432. The data show that XENP30046 and XENP30429 are selective for PD-1+ populations, including CD8 effector memory cells. Notably, XENP30429 expansion of CD8 naive cells was minimal, indicating that reducing the potency of the IL-15 arm improves selectivity. [Figure 102] Figures 102A and 102B show the correlation between lymphocyte proliferation and baseline PD-1 expression in CD34+ Hu-NSG mice on day 7 after treatment with A) 0.3 mg / kg XENP30046 or B) 0.3 mg / kg XENP30429. [Figure 103] Figure 1 shows A) CD8+ T cell and B) NK cell proliferation in cynomolgus monkeys administered 0.3x XENP22853, 1x XENP25937, or 0.3x XENP24306. The data show that PD-1 targeted IL-15 / Rα-Fc fusion reduces NK cell proliferation while maintaining CD8+ T cell proliferation. [Figure 104] Figure 1 shows proliferation of A) CD8+ naive T cells and B) CD8+ effector memory T cells in cynomolgus monkeys treated with 0.3x XENP22853, 1x XENP25937, or 0.3x XENP24306. The data show that PD-1 targeted IL-15 / Rα-Fc fusions selectively expand CD8+ effector memory T cells. [Figure 105]Figures 105A-105E show the proliferation of A) CD8+PD1+ T cells, B) CD8 stem cell memory, C) CD8 naive, D) γδ T cells, and E) CD56+ NK cells in cynomolgus monkeys after administration of either XENP30290 (mAb C_H1_L1.1 x IL-15 [N4D / N65D]) or XENP30362 (αRSV x IL-15 [N4D / N65D]). Collectively, the data show that XENP30290 (with high PD-1 affinity and high IL-15 potency) allows for sustained peripheral pharmacodynamics for 2-3 weeks with modest PD1- cell proliferation. Notably, γδ T cells are the most proliferative cell population, CD4+ and CD8+ naive T cells are the least proliferative cell populations, and CD8+ stem cell memory cells are the most expanded relevant population. [Fig. 106] Figures 106A-106E show the proliferation of A) CD8+PD1+ T cells, B) CD8 stem cell memory, C) CD8 naive, D) γδ T cells, and E) CD56+ NK cells in cynomolgus monkeys after administration of either XENP30516 (mAb C_H1_L1.1 x IL-15 [D30N / E64Q / N65D]) or XENP30518 (αRSV x IL-15 [D30N / E64Q / N65D]). Collectively, the data show that XENP30516 (with high PD-1 affinity and low IL-15 potency) allows for sustained peripheral pharmacodynamics with insignificant PD1- cell proliferation. Notably, γδ T cells are the most proliferating cell population, CD4+ and CD8+ naive T cells are the least proliferating cell populations, and CD8+ stem cell memory cells are the most expanded relevant population. [Figure 107]Figure 1 shows the change in serum concentration levels over time in cynomolgus monkeys administered XENP30290, XENP30291, XENP29439, or XENP30516. The data show that XENP30290, which has the highest PD-1 affinity and higher IL-15 potency, resulted in faster clearance than both XENP30291 and XENP29439, which have lower PD-1 affinity. However, XENP30516, which has the highest PD-1 affinity but lower IL-15 potency, resulted in slower clearance than XENP30290. [Figure 108]

[0023] Figure 1 shows the time course of serum concentration levels in cynomolgus monkeys administered XENP30290, XENP30292, or XENP30516. The data show that XENP30516, which has lower IL-15 potency, had slower clearance than XENP30290. [Figure 109] 1 shows the time course of serum concentration levels in cynomolgus monkeys administered XENP30291 or XENP30293. [Figure 110] 1 shows the time course of serum concentration levels in cynomolgus monkeys administered XENP29439 or XENP30302. [Figure 111]

[0023] Figure 1 shows the time course of serum concentration levels in cynomolgus monkeys administered XENP30362 or XENP30518. The data indicate that the more potent IL-15 is associated with more rapid clearance. [Figure 112] Figures 112A-112C show the time course of PD-1 expression in various lymphocyte populations in cynomolgus monkeys administered A) XENP30290, B) XENP30516, or C) XENP30362. The data show that PD-1-targeted IL-15 / Rα-Fc fusions increase PD-1 expression, whereas RSV-targeted IL-15 / Rα-Fc fusions do not. [Figure 113] Correlation of peak fold expansion of all T cell memory subsets with peak PD-1 expression induced by XENP30290 or XENP30516 is shown. [Fig. 114]Figures 114A-114E show proliferation of A) CD8+PD1+ T cells, B) CD8+ stem cell memory, C) CD8 naive (PD1 low), D) gamma delta cells, and E) NK cells in cynomolgus monkeys treated with 1x low dose XENP30290 (PD1xIL15[N4D / N65D]. The data show that XENP30290 induced PD1+ cell proliferation at 1x low dose. [Fig. 115] Figures 115A-115E show proliferation of A) CD8+PD1+ T cells, B) CD8+ stem cell memory, C) CD8 naive (PD1 low), D) γδ cells, and E) NK cells in cynomolgus monkeys administered 10-fold higher dose XENP30290 (PD1×IL15[N4D / N65D]) and the corresponding RSV-targeted surrogate XENP30362. The data show that XENP30290 induced good PD1+ cell proliferation at 10-fold higher doses, but had moderate activity in PD1− cells, as shown by the activity of the RSV-targeted control XENP30362. [Fig. 116] Figures 116A-116E show proliferation of A) CD8+PD1+ T cells, B) CD8+ stem cell memory, C) CD8 naive (PD1 low), D) gamma delta cells, and E) NK cells in cynomolgus monkeys treated with 10-fold higher dose XENP30516 (PD1 x IL15 [D30N / E64Q / N65D]. The data show that XENP30516 induced good PD1+ cell proliferation at the 10-fold higher dose. [Fig. 117] Figures 117A-117E show the proliferation of A) CD8+PD1+ T cells, B) CD8+ stem cell memory, C) CD8 naive (PD1 low), D) γδ cells, and E) NK cells in cynomolgus monkeys administered a 30-fold very high dose of XENP30516 (PD1 x IL15 [D30N / E64Q / N65D]) and the corresponding RSV-targeted surrogate XENP30518. The data show that XENP30516 induced greater PD1+ proliferation at a 30-fold very high dose while maintaining excellent selectivity. [Fig. 118]We summarize the expansion of CD8+PD1+ T cells in cynomolgus macaques administered various concentrations of various PD-1-targeted or RSV-targeted IL-15 / Rα-Fc fusions containing either IL-15[N4D / N65D] or IL-15[D30N / E64Q / N65D]. [Figure 119] Figures 119A-119E show the correlation between selectivity ratio and peak fold expansion of A) CD8+PD1+ T cells, B) CD8+ stem cell memory, C) CD8 naive (PD1 low), D) γδ cells, and E) NK cells in cynomolgus monkeys administered various concentrations of various PD-1-targeted or RSV-targeted IL-15 / Rα-Fc fusions containing either IL-15[N4D / N65D] or IL-15[D30N / E64Q / N65D]. Collectively, the data show that increasing doses result in higher selectivity. [Figure 120] 7 shows the sequences of exemplary IL-15 / Rα-Fc fusion proteins, XENP20818 (WT IL-15), XENP22821 (IL-15[N65D]), and XENP24045 (IL-15[D30N / E64Q / N65D]), in the "IL-15 / Rα-hetero-Fc" format. IL-15 and IL-15Rα (sushi) are underlined, the linker is double underlined (as will be appreciated by those of skill in the art, the linker may be replaced with other linkers, some of which are shown in FIG. 7), and a slash ( / ) indicates the boundary(s) between the IL-15, IL-15Rα, linker, and Fc regions. [Figure 121] Figure 1 shows the proliferation of rapamycin-expanded Tregs after treatment with non-targeted IL-15 / Rα-Fc fusions XENP20818 and XENP24045, and various PD-1-targeted or control RSV-targeted IL-15 / Rα-Fc fusions. The data show that IL-15 / Rα-Fc fusions (targeted and non-targeted) induce proliferation of rapamycin-expanded Tregs (measured by Tag-it Violet dilution). Notably, PD-1-targeted IL-15 / Rα-Fc fusions were much less potent at inducing Treg proliferation compared to non-targeted IL-15 / Rα-Fc fusions. [Figure 122]Figures 122A and 122B show the proliferation (as determined by CFSE dilution) of A) CD8 effector memory T cells and B) CD4 effector memory T cells after incubation of 1x105 CFSE-labeled PBMCs with 5μg / ml of the indicated test substances and increasing numbers of rapamycin-expanded Tregs. The data show that PD-1-targeted IL-15 / Rα-Fc fusions shifted (reduced) the potency of Treg-induced suppression of CD8 and CD4 effector memory T cell proliferation. Notably, the shift by the control RSV-targeted IL-15 / Rα-Fc fusion was less than the reduction in potency induced by the PD-1-targeted IL-15 / Rα-Fc fusion. [Figure 123] Figures 123A and 123B show the ratios of A) Treg to CD8 effector memory T cells and B) Treg to CD4 effector memory T cells after incubation of 1x105 CFSE-labeled PBMCs with 5μg / ml of the indicated test agent and increasing numbers of rapamycin-expanded Tregs. The data show that proliferation of TEM cells is enhanced by PD-1-targeted IL-15 / Rα-Fc fusions, even though PD-1-targeted IL-15 / Rα-Fc fusions increased the Treg / TEM ratio compared to no test agent. This indicates that although Tregs are expanded, expanded Tregs show reduced suppressive capacity. [Figure 124] Figures 124A and 124B show the percentage of proliferation (as determined by CFSE dilution) of A) CD8+ T cells and B) CD4+ T cells following incubation of CD3 stimulated PBMCs with rapamycin-expanded Tregs pre-cultured for 6 days in complete Treg medium (RPMI with 10% FBS, 0.5 μg / ml anti-CD28, 100 U / ml IL-2, 100 ng / ml rapamycin), complete Treg medium without rapamycin, or 100 ng / ml IL-15 (RPMI with 10% FBS, 0.5 μg / ml anti-CD28, no IL-2, no rapamycin). The data show that Tregs pre-treated with IL-15 have impaired suppressive capacity. [Fig. 125]Figures 125A and 125B show CD25 and FOXP3 expression in CD4+ T cells from PBMCs treated without or with 5 μg / ml of IL-15 / Rα-Fc fusion XENP22821 for 14 days. The data show that treatment with XENP22821 reduced FOXP3 expression in the CD4+ T cell population. [Fig. 126] Figures 126A and 126B show CD45RA and FOXP3 expression in CD4+ T cells from PBMCs treated without or with B) 5 μg.ml of IL-15 / Rα-Fc fusion XENP22821 for 14 days. The data show that treatment with XENP22821 shifted the CD4+CD45RA- population from FoxP3 high to FoxP3 low, and that treatment with IL-15 / Rα-Fc fusion actually shifted the population from eTreg (a decreased population of 5.24% to 2.72%) to activated effector CD4 T cells (an increased population of 18.2% to 28.6%). [Figure 127] Figures 127A and 127B show CD25 and CCR4 expression in CD4+ T cells from PBMCs treated without or with 5 μg / ml of IL-15 / Rα-Fc fusion XENP22821 for 14 days. The data show that treatment with XENP22821 reduced CCR4 expression in the CD4+ T cell population. [Figure 128] Figures 128A and 128B show proliferation of A) CD8 T cells and B) CD4 T cells (as determined by CFSE dilution) after incubation of CFSE-labeled PBMCs on 100 ng / ml plate-bound anti-CD3 (OKT3) with 10 μg / ml IL-15 / Rα-Fc fusion XENP24045 and the indicated concentrations of TGFβ1. The data show that TGFβ dose-dependently inhibits T cell proliferation, but notably, the IL-15 / Rα-Fc fusion prevents TGFβ inhibition of T cell proliferation at all doses tested. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0027] I. NomenclatureThe heterodimeric fusion proteins of the present invention are listed in several different formats. Each polypeptide is given a unique "XENP" number, but longer sequences may contain shorter sequences, as understood in the art. For example, a monomeric heavy chain containing a Fab for anti-PD-1 (see, e.g., FIG. 28C) has a first XENP number, but the VH and VL domains may have different XENP numbers. Since some molecules have three polypeptides, the XENP number is used as the name along with the components. Thus, the molecule XENP29484, which is a "scIL-15 / Rα x Fab", contains three sequences, commonly referred to as "XENP29484-chain 1", "XENP29484 chain 2", and "XENP29484 chain 3", or equivalents, which one of skill in the art would be able to easily identify by sequence alignment. These XENP numbers are in the sequence listing as well as identifiers and are used in the figures. Furthermore, a molecule containing three components will generate multiple sequence identifiers. For example, the list of Fab monomers includes three CDRs: full length sequence, variable heavy sequence, and variable heavy sequence; the light chain includes three CDRs: full length sequence, variable light sequence, and variable light sequence; the scFv-Fc domain includes a full length sequence, a scFv sequence, a variable light sequence, three light chain CDRs, a scFv linker, a variable heavy sequence, and three heavy chain CDRs; it should be noted that all molecules herein that include scFv domains use a single charged scFv linker (+H), but other molecules can be used. Furthermore, the nomenclature of certain variable domains uses a format of "Hx.xx_Ly.yy" type, where the number is a unique identifier for the particular variable chain sequence. Thus, the variable domain on the Fab side of XENP30486 (which binds to PD-1) is "H1.132", indicating that the variable heavy chain domain H1.132 was used, which was combined with the light chain domain L1.135 in XENP30486. Thus, the name "mAbC[PD-1]_H1.132_L1.135" indicates that the variable heavy chain domain H1.132 is combined with the light chain domain L1.135.When these sequences are combined into an scFv, the name indicates that the scFv is in the VH-linker-VL orientation from N-terminus to C-terminus. This molecule with identical sequences of heavy and light chain variable domains but in reverse order is named "mAbC[PD-1]_L1.135_H1.132". Similarly, different constructs can "mix and match" heavy and light chains, as is evident from the sequence listing and figures.

[0028] II. Definition In order to facilitate a more complete understanding of this application, certain definitions are provided below. Such definitions are intended to encompass grammatical equivalents.

[0029] As used herein, "eliminate" refers to reducing or eliminating activity. Thus, for example, "eliminate FcγR binding" means that the Fc region amino acid variant has less than 50% of the starting binding compared to an Fc region that does not contain that particular variant, with less than 70-80-90-95-98% loss of activity being preferred, and generally the activity being below the level of binding detectable in a Biacore assay. Particularly useful in eliminating FcγR binding are shown in FIG. 6. However, unless otherwise indicated, the Fc monomers of the present invention retain binding to the FcRn receptor.

[0030] As used herein, "ADCC" or "antibody-dependent cell-mediated cytotoxicity" refers to a cell-mediated reaction in which non-specific cytotoxic cells expressing FcγR recognize bound antibodies on target cells, and subsequently cause lysis of the target cells. ADCC correlates with binding to FcγRIIIa, and increased binding to FcγRIIIa results in increased ADCC activity. As discussed herein, many embodiments of the present invention completely eliminate ADCC activity.

[0031] As used herein, "ADCP" or antibody-dependent cell-mediated phagocytosis refers to a cell-mediated reaction in which non-specific cytotoxic cells expressing FcγR recognize bound antibody on a target cell and subsequently cause phagocytosis of the target cell.

[0032] As used herein, an "antigen binding domain" or "ABD" refers to a set of six complementarity determining regions (CDRs) that, when present as part of a polypeptide sequence, specifically bind to a target antigen as discussed herein. Thus, a "PD-1 antigen binding domain" binds to the human PD-1 antigen as outlined herein (and often also binds to cynomolgus PD-1). As is known in the art, these CDRs are referred to as variable heavy chain CDRs (vhCDRs or VHCDRs). H A first set of variable light chain CDRs (vCDRs or V L The CDRs are present in the variable heavy and variable light chain domains, respectively, and together form the Fv region. Thus, in some cases, the six CDRs of the antigen-binding domain are contributed by the variable heavy and variable light chains. In the "Fab" format, the set of six CDRs is provided by two different polypeptide sequences, the variable heavy chain domain (vh or VCDR1), the variable light chain domain (vlCDR2), and the variable light chain domain (vlCDR3). H , vhCDR1, vhCDR2 and vhCDR3) and a variable light chain domain (vl or V L , vlCDR1, vlCDR2 and vlCDR3), of which the C-terminus of the vh domain is attached to the N-terminus of the CH1 domain of the heavy chain and the C-terminus of the vl domain is attached to the N-terminus of the constant light chain domain (thus forming the light chain). In the scFv format, the VH and VL domains are generally covalently linked into a single polypeptide sequence by the use of a linker, as outlined herein, which can be (starting from the N-terminus) either vh-linker-vl or vl-linker-vh.

[0033] The hypervariable regions generally consist of amino acid residues from about amino acid residues 24-34 (LCDR1, "L" represents light chain), 50-56 (LCDR2), and 89-97 (LCDR3) in the light chain variable region, and amino acid residues from about amino acid residues 31-35B (HCDR1, "H" represents heavy chain), 50-65 (HCDR2), and 95-102 (HCDR3) in the heavy chain variable region; Kabat et al., SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991) and / or those residues which form the hypervariable loops (e.g., residues 26-32 (LCDR1), 50-52 (LCDR2) and 91-96 (LCDR3) in the light chain variable region and 26-32 (HCDR1), 53-55 (HCDR2) and 96-101 (HCDR3) in the heavy chain variable region; Chothia and Lesk (1987) J. Mol. Biol. 196:901-917. Particular CDRs of the invention are described below.

[0034] As will be understood by those skilled in the art, the exact numbering and arrangement of CDRs may vary in different numbering systems. However, it should be understood that the disclosure of a variable heavy and / or variable light sequence includes the disclosure of the associated (unique) CDRs. Thus, the disclosure of each variable heavy region is the disclosure of vhCDRs (e.g., vhCDR1, vhCDR2, and vhCDR3), and the disclosure of each variable light region is the disclosure of vlCDRs (e.g., vlCDR1, vlCDR2, and vlCDR3).

[0035] For a useful comparison of CDR numbering, see Lafranc et al., Dev. Comp. Immunol. 27(1):55-77 (2003). [Table 1]

[0036] Throughout this specification, the Kabat numbering system is generally used when referring to residues within the variable domain (approximately residues 1-107 for light chain variable regions and residues 1-113 for heavy chain variable regions), while the EU numbering system is for the Fc region (e.g., Kabat et al., supra (1991)).

[0037] The present invention provides a number of different CDR sets. In this case, a "complete CDR set" includes three variable light chain and three variable heavy chain CDRs, such as vlCDR1, vlCDR2, vlCDR3, vhCDR1, vhCDR2, and vhCDR3. These may be part of a larger variable light or variable heavy chain domain, respectively. Furthermore, as more fully outlined herein, the variable heavy and variable light chain domains may be on separate polypeptide chains when heavy and light chains are used (e.g., when Fabs are used), or on a single polypeptide chain in the case of scFv sequences.

[0038] CDRs contribute to antigen binding, or more specifically, to the formation of the epitope binding site of an antibody. "Epitope" refers to a determinant that interacts with a specific antigen binding site in the variable region of an antibody molecule known as a paratope. An epitope is a grouping of molecules, such as amino acids or sugar side chains, that usually have specific structural characteristics, as well as specific charge characteristics. A single antigen may have more than one epitope.

[0039] An epitope may include amino acid residues that are directly involved in binding (also referred to as the immunodominant components of the epitope) and other amino acid residues that are not directly involved in binding, such as amino acid residues that are effectively blocked by a specific antigen-binding peptide, in other words, amino acid residues that are within the footprint of a specific antigen-binding peptide.

[0040] Epitopes may be either conformational or linear. Conformational epitopes are generated by the spatial juxtaposition of amino acids from different segments of a linear polypeptide chain. Linear epitopes are those generated by adjacent amino acid residues in a polypeptide chain. Conformational and non-conformational epitopes can be distinguished in that the binding to the former but not the latter is lost in the presence of denaturing solvents.

[0041] An epitope typically comprises at least 3, more usually at least 5, or 8-10 amino acids in a unique spatial conformation. Antibodies that recognize the same epitope can be validated in a simple immunoassay showing the ability of one antibody to block the binding of another antibody to a target antigen, e.g., "binning". As outlined below, the invention includes not only the recited antigen binding domains and antibodies herein, but also those that compete for binding to the epitope bound by the recited antigen binding domains (or, in the case of NC[PD-1]Fv, the anti-PD-1 CDRs of the invention do not compete for binding to the same epitope as the recited antibodies).

[0042] For the antibody whose components are used in the present invention, the carboxy-terminal portion of each chain defines the constant region that is mainly involved in effector function. Kabat et al. collected a large number of primary sequences of the variable regions of heavy and light chains. Based on the degree of sequence conservation, Kabat et al. classified each primary sequence into CDR and framework, and created a list (see SEQUENCES OF IMMUNOLOGICAL INTEREST, 5th edition, NIH publication, No. 91-3242, EA Kabat et al., which is incorporated by reference in its entirety).

[0043] In the IgG subclass of immunoglobulins, there are several immunoglobulin domains in the heavy chain. By "immunoglobulin (Ig) domain" herein is meant a region of an immunoglobulin that has a distinct tertiary structure. The heavy chain domains that include the constant heavy (CH) domain and the hinge domain are of interest in the present invention. In the context of IgG antibodies, IgG isotypes each have three CH regions. Thus, the "CH" domains associated with IgG are as follows: "CH1" refers to positions 118-220 according to the EU index as in Kabat; "CH2" refers to positions 231-340 according to the EU index as in Kabat, and "CH3" refers to positions 341-447 according to the EU index as in Kabat. As shown herein and described below, pI variants may be present in one or more of the CH region and the hinge region, discussed below.

[0044] Another type of Ig domain of the heavy chain is the hinge region. As used herein, "hinge" or "hinge region" or "antibody hinge region" or "immunoglobulin hinge region" refers to a flexible polypeptide that includes the amino acids between the first and second constant domains of an antibody. Structurally, the IgG CH1 domain ends at EU position 215, and the IgG CH2 domain begins at residue EU position 231. Thus, for IgG, the antibody hinge is defined herein to include positions 216 (E216 in IgG1) to 230 (P230 in IgG1), with numbering according to the EU index as in Kabat. In some embodiments, for example in relation to the Fc region, the lower hinge is included, and the start of the "lower hinge" generally refers to position 226. As described herein, pI variants can be made in the hinge region as well.

[0045] As will be appreciated by those skilled in the art, the exact numbering and arrangement of heavy constant region domains may vary in different numbering systems. For a useful comparison of the EU and Kabat heavy constant region numberings, see Edelman et al., 1969, Proc Natl Acad Sci USA 63:78-85, and Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th Ed., United States Public Health Service, National Institutes of Health, Bethesda (incorporated by reference in their entirety). [Table 2]

[0046] A light chain generally comprises two domains: a variable light domain (which contains the light chain CDRs and, together with the variable heavy domain, forms the Fv region), and a constant light region (often referred to as CL or Cκ).

[0047] Another region of interest for additional substitutions, as outlined below, is the Fc region.

[0048] Thus, the present invention provides different protein domains.As described herein and known in the art, the heterodimeric fusion protein of the present invention comprises different domains in heavy and light chains, which may also overlap.These domains include, but are not limited to, Fc domain, CH1 domain, CH2 domain, CH3 domain, hinge domain, heavy chain constant domain (CH1-hinge-Fc domain or CH1-hinge-CH2-CH3), variable heavy chain domain, variable light chain domain, light chain constant domain, Fab domain, and scFv domain.

[0049] As used herein, "Fc" or "Fc region" or "Fc domain" refers to a polypeptide comprising the constant region of an antibody excluding the first constant region immunoglobulin domain (e.g., CH1), and optionally a portion of the hinge. In the case of IgG, the Fc domain comprises immunoglobulin domains CH2 and CH3 (Cγ2 and Cγ3) and the hinge region between CH1 (Cγ1) and CH2 (Cγ2). Although the boundaries of the Fc region can vary, the human IgG heavy chain Fc region is usually defined to include residues C226 or P230 at its carboxyl terminus, numbering according to the EU index as in Kabat. Thus, the "CH" domains relevant to IgG are as follows: "CH1" refers to positions 118-215 according to the EU index as in Kabat. "Hinge" refers to positions 216-230 according to the EU index as in Kabat. "CH2" refers to positions 231-340 according to the EU index as in Kabat, and "CH3" refers to positions 341-447 according to the EU index as in Kabat. Thus, an "Fc domain" comprises a -CH2-CH3 domain, and optionally a hinge domain (hinge-CH2-CH3. In some embodiments, amino acid modifications are made to the Fc region to alter binding to one or more FcγR or FcRn receptors, and to enable heterodimer formation and purification, as described in more detail below, e.g., as outlined herein.

[0050] Thus, the "Fc domain" includes the -CH2-CH3 domain and, optionally, the hinge domain, which often functions as a domain linker. In the embodiments herein, when an scFv is bound to an Fc domain, it is the C-terminus of the scFv construct that is bound to all or part of the hinge of the Fc domain, e.g., it is generally bound to the sequence EPKS (SEQ ID NO: XX), which is the beginning of the hinge. Similarly, when an IL-15 component (IL-15 complex, IL-15 domain, or IL-15Rα domain) is bound to an Fc domain, it is generally similarly bound to all or part of the hinge of the Fc domain (as a domain linker), e.g., it is generally bound to the sequence EPKS (SEQ ID NO: XX), which is the beginning of the hinge.

[0051] The present invention generally relates to Fc domains based on the IgG class, which has several subclasses, including but not limited to IgG1, IgG2, IgG3, and IgG4. In general, IgG1, IgG2, and IgG4 are used more frequently than IgG3. It should be noted that IgG1 has different allotypes with polymorphisms at 356 (D or E) and 358 (L or M). The sequences shown herein use the 356E / 358M allotype, but other allotypes are also included herein. That is, any sequence containing an IgG1 Fc domain included herein can have 356D / 358L instead of the 356E / 358M allotype.

[0052] Additionally, many of the sequences herein have at least one cysteine ​​at position 220 replaced with a serine, generally on the "scFv monomer" or "IL-15 component" side for most of the sequences described herein, but can also be on the "Fab monomer" side to reduce disulfide formation, or both. Specifically included within the sequences herein are those in which one or both of these cysteines have been replaced (C220S).

[0053] By "heavy chain" or "heavy chain domain" herein is meant, from N-terminus to C-terminus, VH-CH1-hinge-CH2-CH3 domains, where CH2-CH3 comprises the Fc domain. The heavy chain comprises a variable heavy domain and a constant domain, which comprises CH1-optionally a hinge-Fc domain that comprises CH2-CH3. The light chain comprises a variable light domain and a light chain constant domain (VL-CL).

[0054] "Modification" herein means substitution, insertion, and / or deletion of amino acids in a polypeptide sequence, or modification to a moiety chemically linked to a protein. For example, the modification may be a modified carbohydrate or PEG structure attached to a protein. "Amino acid modification" herein means substitution, insertion, and / or deletion of amino acids in a polypeptide sequence. For clarity, unless otherwise stated, the amino acid modification is always to the amino acids coded by DNA, e.g., the 20 amino acids that have codons in DNA and RNA.

[0055] "Amino acid substitution" or "substitution" herein refers to replacing an amino acid at a particular position in a parent polypeptide sequence with a different amino acid. In particular, in some embodiments, the substitution is for an amino acid that does not naturally occur (either does not naturally occur in an organism or does not occur in any organism) at the particular position. For example, the substitution E272Y or 272Y refers to a variant polypeptide, in this case an Fc variant, in which glutamic acid at position 272 is replaced with tyrosine. For clarity, a protein that has been engineered to change the nucleic acid coding sequence but not the starting amino acid (e.g., replacing CGG (which codes for arginine) with CGA (which still codes for arginine) to increase host organism expression levels) is not an "amino acid substitution", i.e., despite the creation of a new gene that codes for the same protein, if the protein has the same amino acid at the particular position where it starts, it is not an amino acid substitution.

[0056] As used herein, "amino acid insertion" or "insertion" refers to the addition of an amino acid sequence at a particular position in a parent polypeptide sequence. For example, -233E indicates the insertion of glutamic acid after position 233 and before position 234. Furthermore, -233ADE or A233ADE indicates the insertion of AlaAspGlu after position 233 and before position 234.

[0057] As used herein, "amino acid deletion" or "deletion" refers to removing an amino acid residue or sequence at a particular position in a parent polypeptide sequence. For example, E233-, E233#, E233(), E233_, or E233del indicate a deletion of glutamic acid at position 233. Additionally, EDA233- or EDA233# indicate a deletion of the sequence GluAspAla beginning at position 233.

[0058] As used herein, "variant protein" or "protein variant" or "variant" refers to a protein that differs from that of a parent protein based on at least one amino acid modification. Protein variant may refer to the protein itself, a composition comprising the protein, or the amino sequence encoding it. Preferably, the protein variant has at least one amino acid modification compared to the parent protein, e.g., about 1 to about 70 amino acid modifications compared to the parent, preferably about 1 to about 5 amino acid modifications. As described below, in some embodiments, the parent polypeptide, the Fc parent polypeptide, is a human wild-type sequence, such as an Fc region from IgG1, IgG2, IgG3 or IgG4. The sequence of the protein variant herein preferably has at least about 80% identity, most preferably at least about 90% identity, more preferably at least about 95-98-99% identity to the parent protein sequence. Variant protein may refer to the variant protein itself, a composition comprising the protein variant, or the DNA sequence encoding it.

[0059] Thus, as used herein, "Fc variant" or "variant Fc" refers to a protein that comprises an amino acid modification in the Fc domain. The Fc variants of the present invention are defined according to the amino acid modifications that compose them. Thus, for example, N434S or 434S is an Fc variant with a substitution serine at position 434 relative to the parent Fc polypeptide, where the numbering is according to the EU index. Similarly, M428L / N434S defines an Fc variant with substitutions M428L and N434S relative to the parent Fc polypeptide. The identity of the WT amino acids may not be specified, in which case the aforementioned variant is referred to as 428L / 434S. The order in which the substitutions are provided is arbitrary, i.e., for example, M428L / N434S is the same Fc variant as N434S / M428L, etc. For all positions discussed in the present invention related to antibodies, unless otherwise stated, the numbering of the amino acid positions is according to the EU index. The EU index, or EU index similar to Kabat or EU numbering schemes, refers to the numbering of EU antibodies (Edelman et al, 1969, Proc Natl Acad Sci USA 63:78-85, incorporated herein by reference in its entirety). Modifications may be additions, deletions, or substitutions. Substitutions may include naturally occurring and possibly synthetic amino acids. Examples include U.S. Pat. No. 6,586,207, WO98 / 48032, WO03 / 073238, US2004 / 0214988A1, WO05 / 35727A2, WO05 / 74524A2, JW Hin et al., (2002), Journal of the American Chemical Society 124:9026-9027, JW Hin, & P.G. Schultz, (2002), ChemBioChem 11:1135-1137, JW Hin, et al., (2002), PICAS United States of America 99:11020-11024, and L. Wang, & P.G. Schultz, (2002), Chem. 1-10, all of which are incorporated by reference in their entirety.

[0060] As used herein, "protein" herein refers to at least two covalently linked amino acids, including proteins, polypeptides, oligopeptides, and peptides. "Residue" as used herein refers to the position in a protein and its associated amino acid identity. For example, asparagine 297 (also referred to as Asn297 or N297) is the residue at position 297 in human antibody IgG1.

[0061] As used herein, "Fab" or "Fab region" refers to a polypeptide that comprises the VH, CH1, VL, and CL immunoglobulin domains. Fab can refer to this region in isolation or in the context of a full-length antibody, an antibody fragment, or a Fab fusion protein.

[0062] "Fv" or "Fv fragment" or "Fv region" as used herein refers to a polypeptide comprising the VL and VH domains of a single antibody, forming an ABD. As will be appreciated by those skilled in the art, these are generally composed of two chains, or can be combined (generally by a linker as discussed herein) to form an scFv.

[0063] By "single chain Fv" or "scFv" herein is generally meant a variable heavy domain covalently linked to a variable light domain to form an scFv or scFv domain using an scFv linker as discussed herein. The scFv domain can be in either orientation from N-terminus to C-terminus (vh-linker-vl or vl-linker-vh).

[0064] As used herein, "IgG subclass modification" or "isotype modification" refers to an amino acid modification that converts one amino acid of one IgG isotype to the corresponding amino acid of a different, matching IgG isotype. For example, since IgG1 contains a tyrosine and IgG2 contains a phenylalanine at EU position 296, an F296Y substitution in IgG2 is considered to be an IgG subclass modification.

[0065] Thus, as used herein, "isotype" means any subclass of immunoglobulins defined by the chemical and antigenic characteristics of their constant regions. It is understood that therapeutic antibodies can also include hybrids of isotypes and / or subclasses.

[0066] As used herein, "variable region" refers to a region of an immunoglobulin that includes one or more Ig domains substantially encoded by any of the Vκ, Vλ, and / or VH genes that constitute the κ, λ, and heavy chain immunoglobulin loci, respectively. With respect to the variable heavy and light chain domains of the present invention, the amino-terminal portion of each heavy and light antibody chain comprises a variable region of about 100-110 or more amino acids primarily responsible for antigen recognition, commonly referred to in the art and herein as an "Fv domain" or "Fv region." In the variable region, three loops for each of the heavy and light chain V domains are aggregated to form the antigen binding site. Each of the loops is referred to as a complementarity determining region (hereinafter referred to as a "CDR"), where variation in amino acid sequence is most prominent. "Variable" refers to the fact that certain segments of the variable region vary widely in sequence between antibodies. The variability within the variable region is not evenly distributed. Instead, V regions consist of relatively invariant stretches of 15-30 amino acids called framework regions (FRs) separated by shorter regions of extreme variability called "hypervariable regions", each 9-15 amino acids long or longer.

[0067] Each VH and VL is composed of three CDRs and four framework regions (FRs) arranged from amino-terminus to carboxy-terminus in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Thus, the variable heavy chain domain comprises VHFR1-VHCDR1-VHFR2-VHCDR2-VHFR3-VHCDR3-VHFR4 and the variable light chain domain comprises VLFR1-VLCDR1-VLFR2-VLCDR2-VLFR3-VLCDR3-VLFR4.

[0068] As used herein, a "non-naturally occurring modification" refers to an amino acid modification that is not isotypic. For example, the substitution 434S in IgG1, IgG2, IgG3, or IgG4 (or hybrids thereof) is considered to be a non-naturally occurring modification, since none of the IgGs contain serine at position 434.

[0069] As used herein, "amino acid" and "amino acid identity" refer to one of the 20 naturally occurring amino acids encoded by DNA and RNA.

[0070] As used herein, "effector function" refers to a biochemical event that results from the interaction of an antibody Fc region with an Fc receptor or ligand. Effector functions include, but are not limited to, ADCC, ADCP, and CDC.

[0071] As used herein, "Fc gamma receptor," "FcγR," or "Fc gamma R" refers to any member of a family of proteins that binds to the IgG antibody Fc region and is encoded by the FcγR gene. In humans, this family includes, but is not limited to, FcγRI (CD64), which includes isoforms FcγRIa, FcγRIb, and FcγRIc; FcγRII (CD32), which includes isoforms FcγRIIa (including allotypes H131 and R131), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2), and FcγRIIc; and FcγRIII (CD16), which includes isoforms FcγRIIIa (including allotypes V158 and F158), and FcγRIIIb (including allotypes FcγRIIb-NA1 and FcγRIIb-NA2) (Jefferis et al., 2002, Immunol Lett 82:57-65, entirely incorporated by reference), as well as any undiscovered human FcγR or FcγR isoform or allotype.

[0072] As used herein, "FcRn" or "neonatal Fc receptor" refers to a protein that binds to the Fc region of an IgG antibody and is at least partially encoded by the FcRn gene. As known in the art, a functional FcRn protein contains two polypeptides, often referred to as heavy and light chains. The light chain is β-2-microglobulin, and the heavy chain is encoded by the FcRn gene. Unless otherwise stated herein, FcRn or FcRn protein refers to the complex of FcRn heavy chain and β-2-microglobulin. To increase binding to FcRn receptors, various FcRn variants, which in some cases are used to increase serum half-life, can be used. In general, unless otherwise stated, the Fc monomers of the present invention retain binding to FcRn receptors (and may contain amino acid variants to increase binding to FcRn receptors, as described below).

[0073] "Parent polypeptide" as used herein refers to a starting polypeptide that is subsequently modified to generate a variant. A parent polypeptide may be a naturally occurring polypeptide, or a variant or modified version of a naturally occurring polypeptide. A parent polypeptide may refer to the polypeptide itself, a composition that includes the parent polypeptide, or the amino acid sequence that encodes it.

[0074] By "heavy chain constant region" herein is meant the CH1-hinge-CH2-CH3 portion of an antibody.

[0075] By "Fc fusion protein" or "immunoadhesin" herein is meant a protein comprising an Fc region generally linked (optionally through a linker moiety as described herein) to a different protein, such as IL-15 and / or IL-15Rα as described herein. In some cases, two Fc fusion proteins can form a homodimeric Fc fusion protein or a heterodimeric Fc fusion protein, the latter being preferred. In some cases, one monomer of the heterodimeric Fc fusion protein comprises only an Fc domain (e.g., an empty Fc domain), and the other monomer is an Fc fusion comprising a protein domain, such as a variant Fc domain and an IL-15 complex. As outlined herein, in some embodiments, one monomer of the heterodimeric protein is an Fc fusion protein comprising an IL-15 complex, and the other monomer is a conventional heavy chain (with associated light chain).

[0076] "Position" as used herein means a location in a sequence of a protein. Positions may be numbered sequentially or according to established formats, such as the EU index for antibody numbering.

[0077] "Strandedness" in the present specification in relation to the monomers of the heterodimeric antibodies of the present invention means that the heterodimerization variants are incorporated into each monomer such that they retain the ability to "match" and form heterodimers, similar to "matching" double-stranded DNA. For example, if some pI variants are engineered into monomer A (e.g., to make the pI higher), the steric variants that are "charge-paired" that can be similarly utilized do not interfere with the pI variants, e.g., the charge variants with higher pIs can be placed in the same "strand" or "monomer" and retain both functionality. Similarly, for "skewed" variants that are paired sets, as outlined in more detail below, the skilled artisan will consider the pI when deciding which strand or monomer to incorporate one set of pairs into, similarly using the pI of the skew to maximize pI separation.

[0078] "Target cell," as used herein, means a cell that expresses the target antigen (in this case, the PD-1 and / or IL-15 receptor).

[0079] "Wild-type or WT" herein refers to an amino acid or nucleotide sequence found in nature, including allelic variations. A WT protein has an amino acid or nucleotide sequence that has not been intentionally modified.

[0080] The bispecific heterodimeric proteins of the present invention are generally isolated or recombinant. When used to describe the various polypeptides disclosed herein, "isolated" refers to a polypeptide that has been identified and separated and / or recovered from the cell or cell culture in which it is expressed. Usually, an isolated polypeptide is prepared by at least one purification step. "Isolated protein" refers to a protein that is substantially free of other proteins with different binding specificities. "Recombinant" refers to a protein that is produced using recombinant nucleic acid technology in an exogenous host cell.

[0081] "Percent (%) amino acid sequence identity" with respect to protein sequences is defined as the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in a particular (parent) sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and does not consider any conservative substitutions as part of the sequence identity. Alignment to determine percent amino acid sequence identity can be accomplished in a variety of ways that are within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms required to achieve maximum alignment over the entire length of the sequences being compared. One particular program is the ALIGN-2 program, as outlined in U.S. Patent Application Publication No. 2016 / 0244525, paragraphs

[0279] to

[0280] , which is incorporated herein by reference.

[0082] The degree of identity between an amino acid sequence of the present invention ("sequence of the present invention") and a parent amino acid sequence is calculated as the number of exact matches in matching the two sequences, divided by the shorter of the length of the "sequence of the present invention" or the length of the parent sequence. The result is expressed as a percentage identity.

[0083] In some embodiments, the two or more amino acid sequences are at least 50%, 60%, 70%, 80%, or 90% identical, hi some embodiments, the two or more amino acid sequences are at least 95%, 97%, 98%, 99%, or even 100% identical.

[0084] "Specific binding" or "specifically binds to" or "specific for" a particular antigen or epitope (in this case, human PD-1) refers to binding that is measurably different from non-specific interactions. Specific binding can be measured, for example, by determining the binding of a molecule compared to the binding of a control molecule, which is generally a molecule of similar structure that has no binding activity. For example, specific binding can be determined by competition between the target and a similar control molecule.

[0085] Specific binding to a particular antigen or epitope is, for example, at least about 10 -4 M, at least about 10 -5 M, at least about 10 -6 M, at least about 10 -7 M, at least about 10 -8 M, at least about 10 -9 M, alternatively at least about 10 -10 M, at least about 10 -11 M, at least about 10 -12 M or higher K D where KD refers to the dissociation rate of a particular ABD-antigen interaction. Typically, an ABD that specifically binds to an antigen will have a KD that is 20-fold, 50-fold, 100-fold, 500-fold, 1000-fold, 5,000-fold, 10,000-fold, or more, greater than that of a control molecule compared to the antigen or epitope.

[0086] Specific binding to a particular antigen or epitope may also be exhibited, for example, by an antibody having a K or K for the antigen or epitope that is at least 20-fold, 50-fold, 100-fold, 500-fold, 1000-fold, 5,000-fold, 10,000-fold, or more greater than that of the epitope, where K or K refers to the association rate of a particular antibody-antigen interaction. Binding affinity is generally measured using a surface plasmon resonance (SPR)-based assay (e.g., Biacore) or a biolayer interferometry (BLI)-based assay (e.g., Octet).

[0087] III. Introduction The present invention provides targeted heterodimeric fusion proteins capable of binding to the checkpoint inhibitor PD-1 antigen and complexing with the common gamma chain (γc, CD132) and / or the IL-2 receptor β chain (IL-2Rβ, CD122) due to the presence of the IL-15 complex. In general, the heterodimeric fusion proteins of the present invention have three functional components: an IL-15 / IL-15Rα (sushi) component, generally referred to herein as the "IL-15 complex" or "IL-15 / Rα complex," an anti-PD-1 component that functions as a "targeting" moiety by bringing the fusion protein to cells expressing PD-1, and an Fc component, each of which can take different forms and each of which can be combined with the other components in any configuration.

[0088] However, the anti-PD-1 component of the targeted heterodimeric fusion proteins of the invention does not compete for binding with known anti-PD-1 antibodies, such as nivolumab and pembrolizumab. That is, by including an anti-PD-1 (αPD-1) antigen-binding domain (ABD) that does not compete for binding with an approved αPD-1 antibody ("NC[PD-1]"), the fusion proteins of the invention allow for efficient combination therapy with anti-PD-1 antibody therapy. That is, by including an anti-PD-1 ABD ("NC-αPD-1ABD") that does not compete for binding with an approved therapy, the fusion protein can be targeted to tumors using the non-competing ABD, but allows for therapeutic treatment with an additional anti-PD-1 antibody, since both can bind PD-1 non-competitively. Furthermore, in some embodiments, the NC-αPD-1 ABD also either completely blocks the PD-1:PD-L1 interaction (embodiments based on mAb A variant), partially blocks the interaction (mAbC variant), or does not block the interaction at all (mAbB variant).

[0089] As will be appreciated by those of skill in the art and as outlined herein, several different formats of the different targeting heterodimeric fusion proteins, non-competing constructs, "NC-αPD-1×IL-15 / Rα", are shown in FIG. 28.

[0090] Furthermore, the present invention relies on a comparison of this embodiment with a "non-targeted IL-15 / Rα-Fc fusion protein" that does not contain an antigen-binding domain to human PD-1, as shown in FIG. 13.

[0091] Additionally, either the non-targeting or targeted heterodimeric fusion proteins of the present invention can be combined with other antibodies against checkpoint receptors, including anti-PD-1, anti-TIM-3, anti-LAG-3, anti-TIGIT, etc.

[0092] Thus, the present invention provides several different functional components that can be assembled in several different ways to generate the heterodimeric fusion proteins of the present invention. As described above, the fusion proteins comprise an IL-15 complex that includes an IL-15 domain and an IL-15 receptor component.

[0093] A.IL-15 complex As shown in the figure, the IL-15 complex can take several forms. As mentioned above, the IL-15 protein itself is less stable than when complexed with the IL-15Rα protein. As known in the art, the IL-15Rα protein contains the "sushi domain", which is the shortest region of the receptor that retains IL-15 binding activity and is SEQ ID NO:4. Thus, although heterodimeric fusion proteins can be made that contain the entire extracellular domain of the IL-15Rα protein (SEQ ID NO:3) or a functional fragment (e.g., larger than SEQ ID NO:4, but not all of SEQ ID NO:3), preferred embodiments herein include complexes that only use the sushi domain.

[0094] Thus, the IL-15 complex generally comprises the IL-15 protein and the sushi domain of IL-15Rα (unless otherwise stated that the full-length sequence is used, "IL-15Rα", "IL-15Rα(sushi)", and "sushi" are used interchangeably throughout). To complicate matters, the nomenclature is represented as "IL-15 / Rα" with a "slash", " / ", which means that the IL-15 domain and the IL-15Rα domain are present.

[0095] 1.IL-15 domain As will be appreciated by those of skill in the art, the IL-15 domains can be wild-type human sequences or can be engineered to contain variants, particularly potency variants, as discussed below.

[0096] In some embodiments, the human IL-15 protein has NCBI reference sequence NP_000576.1, or the precursor sequence SEQ ID NO:1. In some cases, the coding sequence for human IL-15 is set forth in NCBI reference sequence NM_000585. An exemplary IL-15 protein of the Fc-fusion heterodimeric protein outlined herein may have the amino acid sequence of SEQ ID NO:2 (mature IL-15), which corresponds to amino acids 49-162 of SEQ ID NO:1. In some embodiments, the IL-15 protein has at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO:2.

[0097] In some embodiments, the IL-15 domain has been engineered to contain amino acid substitutions.

[0098] Additionally, in some embodiments, IL-15 human proteins are engineered to confer reduced potency, as generally described in PCT / US2019 / 028107, which is incorporated herein by reference in its entirety. That is, as described herein, reduced potency of IL-15 in the heterodimeric fusion proteins of the invention (optionally with or without Xtend-Fc substitutions described herein, such as M428L / N434S) may enhance both pharmacodynamics and pharmacokinetics in subjects administered such proteins. Similarly, as shown in Example 7 of PCT / US2019 / 028107, IL-15 / Rα-Fc variants with reduced potency, such as XENP22821, can expand lymphocyte counts for a longer period of time than wild-type IL-15 / Rα-Fc fusion proteins described herein, such as XENP20818. Notably, XENP23343, an Xtend analog of XENP22821, further enhanced the duration of lymphocyte expansion over XENP22821. Furthermore, reducing the potency of IL-15 can improve the therapeutic index (i.e., allowing higher dosing with less toxicity).

[0099] As shown in Example 14, NC-αPD-1×IL-15 / Rα-Fc fusion protein can overcome Treg suppression of effector T cell proliferation.

[0100] Similarly, as shown in Example 4 below, NC-αPD-1×IL-15 / Rα-Fc fusion protein can promote leukocyte proliferation and exacerbate xenogeneic GVHD over a range of dose levels. Notably, combination therapy of NC-αPD-1×IL-15 / Rα-Fc fusion protein with an anti-PD-1 antibody demonstrated synergistic effects (e.g., synergistic effects), especially at low doses.

[0101] As shown in Examples 3 and 8, the PD-1×IL-15 / R-Fc fusion protein is selective for activated lymphocytes (e.g., lymphocytes that may be found in a tumor environment).

[0102] Thus, the present invention provides: N1D, N4D, D8N, D30N, D61N, E64Q, N65D, Q108E, N1D / N4D / D8N, N1D / N4D / N65D, N1D / D30N, N1D / D61N, N1D / D61N / E64Q / Q108E, N1D / E64Q, N1D / N65D, N1D / Q108E, N4D, N4D / D30N, N4D / D61N, N4D / D61N / N65D, N4D / D61N / E64Q / Q108E, N4D / E64Q, N4D / N65D, D8 A number of suitable IL-15 amino acid variants that confer reduced potency and increased pharmacokinetics are provided, including variant IL-15 proteins that include, but are not limited to, amino acid substitution(s) selected from the group of N / D61N, D8N / E64Q, D30N / E64Q, D30N / N65D, D30N / E64Q / N65D, D30N / Q180E, D61N / E64Q / N65D, E64Q, E64Q / N65D, E64Q / Q108E, and N65D / Q108E.

[0103] In some embodiments, the amino acid substitution(s) may be isosteric substitutions at the IL-15:IL-2β and IL-15:common gamma chain interfaces.

[0104] In some embodiments, the human IL-15 protein, such as the human mature IL-15 protein, of the Fc fusion protein is identical to the amino acid sequence of SEQ ID NO: 2. In some cases, the human IL-15 protein, such as the human mature IL-15 protein, has no amino acid substitutions.

[0105] In some embodiments, the human mature IL-15 variant protein has one or more amino acid mutations (e.g., substitutions, insertions, and / or deletions). In some cases, the mutations introduce a cysteine ​​residue that can form a disulfide bond with the human IL-15 receptor alpha (IL-15Rα) protein.

[0106] In some embodiments, the present invention provides a protein comprising a human IL-15 variant having the amino acid variant D30N. In some embodiments, the protein comprises the amino acid sequence of SEQ ID NO:2 and the D30N substitution. In some embodiments, the protein comprises the amino acid sequence of SEQ ID NO:2 and at least the D30N substitution.

[0107] In some embodiments, the present invention provides a protein comprising a human IL-15 variant having an amino acid variant N1D. In some embodiments, the protein comprises the amino acid sequence of SEQ ID NO:2 and an N1D substitution. In some embodiments, the protein comprises the amino acid sequence of SEQ ID NO:2 and at least an N1D substitution.

[0108] In some embodiments, the present invention provides a protein comprising a human IL-15 variant having the amino acid variant N4D. In some embodiments, the protein comprises the amino acid sequence of SEQ ID NO:2 and an N4D substitution. In some embodiments, the protein comprises the amino acid sequence of SEQ ID NO:2 and at least an N4D substitution.

[0109] In some embodiments, the present invention provides a protein comprising a human IL-15 variant having the amino acid variant E64Q. In some embodiments, the protein comprises the amino acid sequence of SEQ ID NO:2 and an E64Q substitution. In some embodiments, the protein comprises the amino acid sequence of SEQ ID NO:2 and at least an E64Q substitution.

[0110] In some embodiments, the present invention provides a protein comprising a human IL-15 variant having the amino acid variant N65D. In some embodiments, the protein comprises the amino acid sequence of SEQ ID NO:2 and the N65D substitution. In some embodiments, the protein comprises the amino acid sequence of SEQ ID NO:2 and at least the N65D substitution.

[0111] In some embodiments, the invention provides a protein comprising a human IL-15 variant having the amino acid substitution N1D / D30N. In some embodiments, the protein comprises the amino acid sequence of SEQ ID NO:2 and the N1D / D30N substitution. In some embodiments, the protein comprises the amino acid sequence of SEQ ID NO:2 and at least the N1D / D30N substitution.

[0112] In some embodiments, the invention provides a protein comprising a human IL-15 variant having the amino acid substitution N4D / D30N. In some embodiments, the protein comprises the amino acid sequence of SEQ ID NO:2 and the N4D / D30N substitution. In some embodiments, the protein comprises the amino acid sequence of SEQ ID NO:2 and at least the N4D / D30N substitution.

[0113] In some embodiments, the invention provides a protein comprising a human IL-15 variant having the amino acid substitution D30N / E64Q. In some embodiments, the protein comprises the amino acid sequence of SEQ ID NO:2 and the D30N / E64Q substitution. In some embodiments, the protein comprises the amino acid sequence of SEQ ID NO:2 and at least the D30N / E64Q substitution.

[0114] In some embodiments, the invention provides a protein comprising a human IL-15 variant having the amino acid substitutions D30N / N65D. In some embodiments, the protein comprises the amino acid sequence of SEQ ID NO:2 and the D30N / N65D substitutions. In some embodiments, the protein comprises the amino acid sequence of SEQ ID NO:2 and at least the D30N / N65D substitutions.

[0115] In some embodiments, the invention provides proteins comprising human IL-15 variants having amino acid substitutions N4D / N65D. In some embodiments, the proteins comprise the amino acid sequence of SEQ ID NO:2 with the N4D / N65D substitutions. In some embodiments, the proteins comprise the amino acid sequence of SEQ ID NO:2 and at least the N4D / N65D substitutions.

[0116] In some embodiments, the invention provides proteins comprising a human IL-15 variant having the amino acid substitutions D30N / E64Q / N65D. In some embodiments, the protein comprises the amino acid sequence of SEQ ID NO:2 having the D30N / E64Q / N65D substitutions. In some embodiments, the protein comprises the amino acid sequence of SEQ ID NO:2 and at least the D30N / E64Q / N65D substitutions.

[0117] 2.IL-15 / Rα domain In addition to the IL-15 domain, optionally containing the amino acid variants outlined above, the heterodimeric fusion proteins of the present invention contain a "sushi" domain.

[0118] In some embodiments, the human IL-15 receptor alpha (IL-15Rα) protein has the amino acid sequence set forth in NCBI reference sequence number NP_002180.1 or SEQ ID NO: 3. In some cases, the coding sequence of human IL-15Rα is set forth in NCBI reference sequence number NM_002189.3. An exemplary IL-15Rα protein of the Fc-fusion heterodimeric protein outlined herein can comprise or consist of the sushi domain of SEQ ID NO: 3 (e.g., amino acids 31-95 of SEQ ID NO: 3), or in other words the amino acid sequence of SEQ ID NO: 4. That is, certain embodiments utilize a truncated version of the extracellular domain of the receptor.

[0119] In some embodiments, the IL-15Rα protein has the amino acid sequence of SEQ ID NO:4 and an amino acid insertion selected from the group consisting of D96, P97, A98, D96 / P97, D96 / C97, D96 / P97 / A98, D96 / P97 / C98, and D96 / C97 / A98, where the amino acid positions are relative to full-length human IL-15Rα protein or SEQ ID NO:3. For example, an amino acid(s) such as D (e.g., Asp), P (e.g., Pro), A (e.g., Ala), DP (e.g., Asp-Pro), DC (e.g., Asp-Cys), DPA (e.g., Asp-Pro-Ala), DPC (e.g., Asp-Pro-Cys), or DCA (e.g., Asp-Cys-Ala) may be added to the C-terminus of the IL-15Rα protein of SEQ ID NO:4. In some embodiments, the IL-15Rα protein has an amino acid sequence of SEQ ID NO: 4 and one or more amino acid substitutions selected from the group consisting of K34C, A37C, G38C, S40C, and L42C, where the amino acid positions are relative to SEQ ID NO: 4. The IL-15Rα (sushi) protein of SEQ ID NO: 4 can have 1, 2, 3, 4, 5, 6, 7, 8, or more amino acid mutations (e.g., substitutions, insertions, and / or deletions).

[0120] 3. IL-15 complexes in different formats Thus, the IL-15 complex generally comprises the IL-15 protein and the sushi domain of IL-15Rα. To complicate matters, the nomenclature is denoted as "IL-15 / Rα" with a "slash", " / ", which means that there is an IL-15 domain and an IL-15Rα domain.

[0121] As shown in Figure 28, the IL-15 / Rα complex can be in two different formats, either non-covalent or covalent. As shown in Figures 28B, 28D, 28F, and 28G, the IL-15 protein and IL-15Rα (sushi) are not covalently bound, but rather self-assemble through normal ligand-ligand interactions. As described more fully herein, it can be either the IL-15 domain or the sushi domain that is covalently linked to the Fc domain (typically using an optional domain linker).

[0122] Alternatively, the IL-15 / Rα complex is covalently linked using a domain linker, as generally shown in Figures 28A, 28C, 28E and 28H. In each of these cases, the orientation of IL-15 and IL-15Rα can be switched from N-terminus to C-terminus. That is, in Figure 28A, the invention also includes the case where the IL-15 domain is N-terminus and the IL-15Rα domain is linked to the Fc domain using a linker. Similarly, in Figure 28C, the invention also includes the case where the IL-15 domain is N-terminus and the IL-15Rα domain is linked to the Fc domain using a linker. Figure 28E of the invention also includes the case where the IL-15 domain is C-terminus of the Fc domain (linked using a domain linker) and the IL-15Rα domain is linked to the IL-15 domain using a linker. Finally, Figure 28H can also include the case where the IL-15 domain is N-terminus of the IL-15Rα domain.

[0123] B. Non-competing anti-PD-1 antigen-binding domain The anti-PD-1 component of the invention (e.g., anti-PD-1 antigen binding domain (ABD)) is generally a set of six CDRs contained within the variable heavy and variable light domains that form an Fv domain that can bind to human PD-1 (the sequence of which is shown in Figure 2) but does not compete with commercially available anti-PD-1 antibodies such as pembrolizumab and nivolumab. This allows for superior targeting of the heterodimeric fusion proteins of the invention to tumors, allowing for localized action of the IL-15 / Rα complex, as well as combination therapy with effective anti-PD-1 antibodies without competing for the same PD-1 epitope.

[0124] The NC-αPD-1 antigen-binding domain of the present invention can take two general formats, either as an scFv domain, as shown in Figures 28A and 28B, or as a Fab domain present on two different polypeptides, as shown in Figures 28C and 28D (monovalent binding of PD-1) and Figures 28E, 28F, 28G and 28H (bivalent binding of PD-1), which are described more fully below.

[0125] There are numerous VH and VL domains that may be used in the present constructs, many of which are shown in the figures, including in particular Figure 43, which contains VH and VL sequences of antigen binding domains that do not compete with pembrolizumab and nivolumab for binding. It should be noted that any VH domain may be combined with any VL domain in the following useful specific combinations:

[0126] 1. VH domain Specifically for the mAbC variants, there is also a set of useful affinity variants of the VH domain, as shown in Figure 65. In these embodiments, compared to mAbC_H1, particularly useful amino acid substitutions in the VH domain include, but are not limited to, F32L, S52aG, R97E, R97Y and R97W, which may be used alone or in combination. In particular, five single amino acid variants plus VH combinations that find particular use in some embodiments include, but are not limited to, F32L / S52aG and F32L / S52aG / R97W.

[0127] In some embodiments, the VH domain of the NC-PD-1 ABD is selected from those shown in Figure 43.

[0128] In some embodiments, the NC-PD-1 ABD is based on the mAbC parent (H1_L1, sequence shown in Figure 43) and the VH domain of the mAbC variant is selected from the group consisting of H1, H1.1, H1.2, H1.3, etc. through H1.168 of Figure 43.

[0129] In some embodiments, the NC-PD-1 ABD is based on the mAbC parent (H1_L1, sequence shown in Figure 43) and the VH domain of the mAbC variant is selected from the group consisting of H1.19, H1.48, H1.125, H1.130, H1.132, H1.169, and H1.175 in Figure 43.

[0130] 2. VL domain Specifically for the mAbC variants, there is also a set of useful affinity variants of the VL, as shown in Figure 65. In these embodiments, particularly useful amino acid substitutions in the VL domain compared to mAbC_L1 include, but are not limited to, N27dH, N27dS, K30Y, S93T and Y94W, which may be used alone or in combination.

[0131] In some embodiments, the VL domain of the mAbC variant is selected from the group consisting of L1.1, L1.3, L1.45, L1.117, L1.135, L1.136 and L1.140.

[0132] 3. VH and VL combinations As generally shown in FIG. 63, within a parent backbone, for example within a mAbC sequence, the VL and VH domains may be interchangeable.

[0133] Of particular use in some embodiments are VH combinations selected from the group consisting of H1, H1.1, H1.2, H1.3, H1.4, H1.5, H1.6, H1.7, H1.8, H1.9, H1.10, etc., to H1.168 (inclusive) and H1.175 and L1.140.

[0134] Of particular use in some embodiments are VH combinations selected from the group consisting of H1, H1.1, H1.2, H1.3, H1.4, H1.5, H1.6, H1.7, H1.8, H1.9, H1.10, etc., to H1.168 (inclusive) and H1.175 and L1.1.

[0135] A particular combination for use in the present invention is mAb C H1.19_L1.140.

[0136] A particular combination for use in the present invention is mAb C H1_L1.1.

[0137] Particularly useful in some embodiments are H1.19_L1.140, H1_L1.1, H1.19_L1, H1.48_L1, H1.125_L1, H1.30_L1, H1.132_L1, H1_L1.1 The VH and VL domains are combinations of H1_L1.3, H1_L1.45, H1_L1.117, H1_L1.129, H1.19_L1.1, H1.32_L1.1, H1.169_L1.1, H1.169_L1.1, H1.175_L1.1, H1.175_L1.1, H1_L1.140, H1_L1.135, H1_L1.136, H1.132_L1.135, H1.132_L1.140, H1.175_L1.135 and H1.175_L1.140.

[0138] Additional combinations of VH and VL domains from mAbC that find use in the present invention include mAb A[PD-1]_H1 and mAb A[PD-1]_L1, mAb B[PD-1]_H1 and mAb B[PD-1]_L1, mAb C[PD-1]_H1 and mAb C[PD-1]_L1, mAb C[PD-1]_H1 and mAb C[PD-1]_L1.1, mAb C[PD-1]_H1 and mAb C[PD-1]_L1.3, mAb C[PD-1]_H1 and mAb C[PD-1]_L1.45, mAb C[PD-1]_H1 and mAb C[PD-1]_L1.117, mAb C[PD-1]_H1 and mAb C[PD-1]_L1.135, mAb C[PD-1]_H1 and mAb C[PD-1]_L1.140, mAb C[PD-1]_H1 and mAb C[PD-1]_L2, mAb C[PD-1]_H1.132 and mAb C[PD-1]_L1, mAb C[PD-1]_H1.132 and mAb C[PD-1]_L1.1, mAb C[PD-1]_H1.132 and mAb C[PD-1]_L1.3, mAb C[PD-1]_H1.132 and mAb C[PD-1]_L1.45, mAb C[PD-1]_H1.132 and mAb C[PD-1]_L1.117, mAb C[PD-1]_H1.132 and mAb C[PD-1]_L1.135, mAb C[PD-1]_H1.132 and mAb C[PD-1]_L1.140, mAb C[PD-1]_H1.132 and mAb C[PD-1]_L2, mAb C[PD-1]_H2 and mAb C[PD-1]_L1, mAb C[PD-1]_H2 and mAb C[PD-1]_L1.1, mAb C[PD-1]_H2 and mAb C[PD-1]_L1.3, mAb C[PD-1]_H2 and mAb C[PD-1]_L1.45, mAb C[PD-1]_H2 and mAb C[PD-1]_L1.117, mAb C[PD-1]_H2 and mAb C[PD-1]_L1.135, mAb C[PD-1]_H2 and mAb C[PD-1]_L1.140, and mAb C[PD-1]_H2 and mAb C[PD-1]_L2, but is not limited to these.

[0139] Some embodiments of the invention comprise at least one scFv domain, which is not naturally occurring, but generally comprises a variable heavy domain and a variable light domain linked together by an scFv linker. As outlined herein, the scFv domain is generally oriented N-terminal to C-terminal as vh-scFv linker-vl, while this can be reversed for any scFv domain (or domain constructed using vh and vl sequences from a Fab) to vl-scFv linker-vh, with optional linkers at one or both ends depending on the format (see generally Figures 4A-4B of US62 / 353,511).

[0140] As shown herein, the anti-PD-1 ABD may alternatively be in the form of an scFv, where the vh and vl domains are linked using an scFv linker, which may optionally be a charged scFv linker. As will be appreciated by those skilled in the art, scFvs can be assembled from the N-terminus to the C-terminus as N-vh-scFv linker-vl-C, or as N-vl-scFv linker-vh-C, with the C-terminus of the scFv domain generally linked to a hinge-CH2-CH3 Fc domain. A suitable Fv (including a CDR set and variable heavy / variable light domains) can be used in scFv format, or Fab format as shown in FIG. 43. As will be further appreciated by those skilled in the art, all or part of the hinge (which can be a wild type hinge from IgG1, IgG2, or IgG4, or a variant thereof such as the IgG4 S241P or S228P hinge variants having a substituted proline at position 228 relative to the IgG4 hinge polypeptide (the numbering S228P is according to the EU index, and S241P is the Kabat numbering)) can be used as a domain linker between the scFv domain and the CH2-CH3 domain, or a domain linker as shown in the figure can be used.

[0141] C. Fc domain In addition to the IL-15 complex and the targeting NC-αPD-1 Fv domain, the present invention further provides heterodimeric Fc domains as building blocks. As shown in Figure 28, these heterodimeric Fc domains serve to bring together the IL-15 / Rα and the targeting anti-PD-1 domains into a single construct, and generally comprise either two polypeptide chains (e.g., in Figure 28A, one monomer comprises the IL-15 / Rα complex, and the other monomer comprises the anti-PD-1 scFv), three polypeptide chains (e.g., in Figure 28C, one monomer comprises the IL-15 / Rα complex, the second monomer comprises the heavy chain, and the third monomer comprises the light chain), etc.

[0142] The Fc domain components of the present invention are as described herein, which generally include scubariant and / or optional pI variants and / or deletion variants as outlined herein. See, for example, the disclosure of WO2017 / 218707 under the heading "IV Heterodimeric Antibodies", including chapters IV.A, IV.B, IV.C, IV.D, IV.E, IV.F, IV.G, IV.H, and IV.I (expressly incorporated by reference in its entirety). Particularly used in the heterodimeric proteins of the present invention are Fc domains including "scubariant", "pI variant", "deletion variant" and FcRn variants as outlined therein. Particularly useful Fc domains are those shown in FIG. 8.

[0143] The Fc domain may be derived from an IgG Fc domain, e.g., an IgG1, IgG2, IgG3 or IgG4 Fc domain, with an IgG1 Fc domain being of particular use in the present invention. The following describes Fc domains useful in the IL-15 / IL-15Rα Fc fusion monomer and checkpoint antibody fragments of the heterodimeric Fc proteins of the present invention.

[0144] Thus, the present invention provides different antibody domains, such as different Fc domains.As described herein and known in the art, the heterodimeric proteins of the present invention comprise different domains, which may also overlap.These domains include, but are not limited to, Fc domain, CH1 domain, CH2 domain, CH3 domain, hinge domain, heavy constant domain (CH1-hinge-Fc domain or CH1-hinge-CH2-CH3).

[0145] Thus, the Fc domain includes the -CH2-CH3 domain and optionally the hinge domain, and can be from human IgG1, IgG2, IgG3, or IgG4, with the Fc domain from IgG1. In some embodiments herein, when a protein fragment, such as IL-15 or IL-15Rα, is bound to the Fc domain, it is the C-terminus of the IL-15 or IL-15Rα construct that is bound to all or part of the hinge of the Fc domain, e.g., it is bound to the sequence EPKS, which is generally the beginning of the hinge. In other embodiments, when a protein fragment, such as IL-15 or IL-15Rα, is bound to the Fc domain, it is the C-terminus of the IL-15 or IL-15Rα construct that is bound to the CH1 domain of the Fc domain.

[0146] In some of the constructs and sequences of Fc domain proteins outlined herein, the C-terminus of the IL-15 or IL-15Rα protein fragment is linked to the N-terminus of a domain linker, which is linked to the N-terminus of the constant Fc domain (N-IL-15 or IL-15Rα protein fragment-linker-Fc domain-C), but this can be switched (N-Fc domain-linker-IL-15 or IL-15Rα protein fragment-C). In other constructs and sequences outlined herein, the C-terminus of the first protein fragment is linked to the N-terminus of the second protein fragment, optionally via a domain linker, which is linked to the N-terminus of the constant Fc domain, optionally via a domain linker. In still other constructs and sequences outlined herein, a constant Fc domain is provided that is not linked to the first or second protein fragment. A heterodimeric Fc fusion protein may comprise two or more of the exemplary monomeric Fc domain proteins described herein.

[0147] Thus, in some embodiments, the present invention provides heterodimeric Fc fusion proteins that rely on the use of two different heavy chain variant Fc sequences that self-assemble to form a heterodimeric Fc domain fusion polypeptide that allows binding to one or more binding partners, ligands or receptors. Heterodimeric Fc constructs are based on the self-assembly nature of two Fc domains of antibody heavy chains, e.g., two "monomers" that assemble into a "dimer". Heterodimeric Fc fusions are created by varying the amino acid sequence of each monomer, as described in more detail below. Thus, the present invention generally relates to creating heterodimeric Fc fusion proteins that can co-bind binding partner(s) or ligand(s) or receptor(s) in several ways, relying on amino acid variants in the constant regions that are different in each chain to promote heterodimer formation and / or to facilitate purification of the heterodimer over homodimers.

[0148] 1. Heterodimerization variants The heterodimeric IL-15 / Rα×NCPD-1-Fc fusion protein comprises two distinct Fc domains (one each for the first and second monomers) that contain modifications that promote heterodimerization of the first and second monomers and / or facilitate purification of the heterodimer over the homodimer, collectively referred to herein as "heterodimerization variants." As discussed below, heterodimerization variants can include scubariants (e.g., variants that "bend" the formation of the heterodimer over the homodimer), as well as "pI variants" that facilitate separation of the homodimer from the heterodimer. As generally described in U.S. Pat. No. 9,605,084, which is incorporated by reference in its entirety, and specifically as follows for the discussion of heterodimerization variants, mechanisms useful for heterodimerization include "knob and hole" ("KIH") as described in U.S. Pat. No. 9,605,084, "electrostatic steering" or "charge pair" as described in U.S. Pat. No. 9,605,084, pI variants as described in U.S. Pat. No. 9,605,084, and further general Fc variants as outlined in U.S. Pat. No. 9,605,084 and below.

[0149] a. Scuba liant In some embodiments, the heterodimeric fusion protein comprises a scubariant, which is one or more amino acid modifications in the first Fc domain (A) and / or the second Fc domain (B) that promote the formation of an Fc heterodimer (an Fc dimer comprising a first and a second Fc domain; AB) over an Fc homodimer (an Fc dimer comprising two of the first Fc domains or two of the second Fc domains; AA or BB). Suitable scubariants are included in Figure 29 of US Patent Application Publication No. 2016 / 0355608, which is incorporated herein by reference in its entirety and, in particular, for its disclosure of scubariants and in Figure 4.

[0150] One mechanism of scubariant is commonly referred to in the art as "knobs and holes" and refers to amino acid manipulations that create steric effects that favor heterodimer formation and not homodimer formation, as described in USSN61 / 596,846, Ridgway et al., Protein Engineering 9(7):617 (1996), Atwell et al., J. Mol. Biol. 1997 270:26, U.S. Patent No. 8,216,805 (all of which are incorporated herein by reference in their entirety, and in particular for their disclosure of "knobs and holes" mutations). This is sometimes referred to herein as a "stereovariant". These figures identify several "monomer A-monomer B" pairs that rely on "knobs and holes". Furthermore, these "knobs and holes" mutations can be combined with disulfide bonds to further promote the generation of Fc heterodimers, as described in Merchant et al., Nature Biotech. 16:677 (1998).

[0151] An additional mechanism of scubariants that find use in the generation of heterodimers is sometimes referred to as "electrostatic steering", as described in Gunasekaran et al., J. Biol. Chem. 285(25):19637 (2010), the entirety of which is incorporated herein by reference. This is sometimes referred to herein as "charge pairing". In this embodiment, electrostatics are used to bend the generation towards heterodimerization. As will be appreciated by those skilled in the art, these may also affect pI, i.e. purification, and therefore may in some cases be considered as pI variants. However, because they were generated to force heterodimerization and were not used as a purification means, they are classified as "scubariants". These include, but are not limited to, D221E / P228E / L368E paired with D221R / P228R / K409R (e.g., these are the "monomer" match set) and C220E / P228E / 368E paired with C220R / E224R / P228R / K409R.

[0152] In some embodiments, the scubariants advantageously and simultaneously promote heterodimerization based on both the "knob-and-hole" mechanism and the "electrostatic steering" mechanism. In some embodiments, the heterodimeric fusion protein comprises one or more sets of such heterodimerizing scubariants. Exemplary scubariants that fall into this category include S364K / E357Q:L368D / K370S; L368D / K370S:S364K; L368E / K370S:S364K; T411T / E360E / Q362E:D401K; L368D / K370S:S364K / E357L; K370S:S364K / E357Q; or T366S / L368A / Y407V:T366W (optionally including a bridging disulfide, T366S / L368A / Y407V / Y349C:T366W / S354C). These variants form "pairs" of "sets." That is, one set of the pair is incorporated into the first monomer, and the other set of the pair is incorporated into the second monomer. In terms of nomenclature, the pair "S364K / E357Q:L368D / K370S" means that one of the monomers contains an Fc domain with amino acid substitutions S364K and E357Q, and the other monomer contains an Fc domain with amino acid substitutions L368D and K370S. As mentioned above, the "twistiness" of these pairs depends on the starting pI. It should be noted that these sets do not necessarily behave as "knob-and-hole" variants, but have a one-to-one correspondence between the residues of one monomer and the residues of the other monomer. That is, these pairs of sets may instead form an interface between the two monomers that promotes heterodimer formation and not homodimer formation, such that the percentage of heterodimers that form spontaneously under biological conditions is greater than 90% instead of the expected 50% (25% homodimer A / A: 50% heterodimer A / B: 25% homodimer B / B). Exemplary heterodimerization "skew" variants are shown in FIG. 4.

[0153] In exemplary embodiments, the heterodimeric fusion protein comprises a "skewed" variant amino acid substitution set: S364K / E357Q:L368D / K370S; L368D / K370S:S364K; L368E / K370S:S364K; T411T / E360E / Q362E:D401K; L368D / K370S:S364K / E357L; K370S:S364K / E357Q; or T366S / L368A / Y407V:T366W (optionally including a bridging disulfide, T366S / L368A / Y407V / Y349C:T366W / S354C). In an exemplary embodiment, the heterodimeric IL-15-Fc fusion protein comprises the "S364K / E357Q:L368D / K370S" amino acid substitution set.

[0154] In some embodiments, the scubariants provided herein are optionally and independently incorporated into either or both of the first and second Fc domains of an IL-15-Fc fusion protein, along with other modifications, including, but not limited to, other scubariants (see, e.g., FIG. 37 of US Patent Application Publication No. 2012 / 0149876, particularly incorporated herein by reference for its disclosure of scubariants), pI variants, isotype variants, FcRn variants, deletion variants, etc. Additionally, individual modifications can also be independently and optionally included in or excluded from the subject IL-15-Fc fusion proteins.

[0155] b. pI (isoelectric point) variants of heterodimers In some embodiments, the heterodimeric fusion protein comprises a purification variant that advantageously allows for separation of the heterodimeric fusion protein from the homodimeric protein.

[0156] There are several basic mechanisms that can facilitate the purification of heterodimeric proteins. One such mechanism relies on the use of pI variants that contain one or more modifications that affect the isoelectric point of one or both of the monomers of the fusion protein, so that each monomer, and subsequently each dimer species, has a different pI, thus allowing isoelectric purification of AA, AB and BB dimeric proteins. Alternatively, some formats also allow for separation based on size. As further outlined below, scuba variants can also be used to "skew" the formation of heterodimers rather than homodimers. Thus, the combination of heterodimerizing scuba variants and pI variants finds particular use in the subject IL-15 fusion proteins provided herein.

[0157] Furthermore, depending on the format of the heterodimeric Fc fusion protein, the pI variants can either be contained within the constant region and / or Fc domain of the monomer, or a domain linker can be used, as outlined more fully below. In some embodiments, the heterodimeric IL-15-Fc fusion proteins can also contain additional modifications for alternative functions to create pI alterations, such as Fc, FcRn and KO variants.

[0158] In embodiments utilizing pI as a separation mechanism to allow purification of the heterodimeric fusion protein, amino acid modifications may be introduced into one or both of the monomers of the heterodimeric fusion protein. That is, the pI of one of the monomers (referred to herein as "monomer A" for simplicity) may be engineered away from monomer B, or both monomers A and B may be altered, increasing the pI of monomer A and decreasing the pI of monomer B. As discussed, the pI change of either or both monomers can be accomplished by removing or adding a charged residue (e.g., replacing a neutral amino acid with a positively or negatively charged amino acid residue, e.g., glutamine to glutamic acid), by changing a charged residue from positive or negative to the opposite charge (e.g., aspartic acid to lysine), or by changing a charged residue to a neutral residue (e.g., loss of charge, lysine to serine). Some of these variants are illustrated in the figures, including Figures 4 and 5.

[0159] Creating a sufficient change in pI in at least one of the monomers to allow separation of heterodimers from homodimers can be done by using a "wild type" heavy chain constant region and a variant region engineered to increase or decrease its pI (wt A:B+ or wt A:B-), or by increasing one region and decreasing the other (A+:B- or A-:B+).

[0160] Thus, in general, a component of some embodiments of the fusion proteins of interest of the present invention are amino acid variants in the Fc domain or constant domain region that are intended to alter the isoelectric point (pI) of at least one, if not both, of the monomers of the dimeric protein by incorporating an amino acid substitution ("pI variant" or "pI substitution") in one or both of the monomers. Separation of the heterodimer from the two homodimers herein can be achieved when the pI of the two monomers differs by as little as 0.1 pH units, with 0.2, 0.3, 0.4, and 0.5 or more differing being all of use in the present invention.

[0161] As will be appreciated by those of skill in the art, the number of pI variants included in each or both monomer(s) of a heterodimeric fusion protein to achieve good separation will depend, in part, on the starting pI of the components. That is, to determine which monomer to engineer or in which "direction" (e.g., more positive or more negative), the sequence of the Fc domain and any IL-15 complex, or linker, included in each monomer is calculated and a decision is made therefrom based on the pI of that monomer. As is known in the art, different Fc domains, linkers, and IL-15 complexes will have different starting pIs. Generally, as outlined herein, the pI is engineered to result in a total pI difference for each monomer of at least about 0.1 log, with 0.2-0.5 being preferred as outlined herein.

[0162] In general, as will be understood by those skilled in the art, there are two general categories of amino acid modifications that affect the pI: those that increase the pI of a protein (basic changes) and those that decrease the pI of a protein (acidic changes). As described herein, all combinations of these variants can be used. One monomer can be wild type or a variant that does not exhibit a significantly different pI from the wild type, and the other monomer contains an Fc domain that is either more basic or more acidic. Alternatively, each monomer can be changed, one to be more basic and one to be more acidic.

[0163] When heterodimerization is achieved using pI variants, a more modular approach to designing and purifying heterodimeric fusion proteins is provided. Thus, in some embodiments, heterodimerization variants (including scubariants and pI variants) must be engineered. Furthermore, in some embodiments, the potential for immunogenicity resulting from pI variants is significantly reduced by importing pI variants from different IgG isotypes such that the pI is changed without introducing significant immunogenicity (see isotype variants below). Thus, an additional problem to be solved is the elucidation of low pI constant domains with high human sequence content, e.g., minimizing or avoiding non-human residues at any particular position. Alternatively or in addition to isotype substitutions, the potential for immunogenicity resulting from pI variants is significantly reduced by utilizing isosteric substitutions (e.g., Asn to Asp and Gln to Glu).

[0164] Potential collateral benefits of this pI engineering are also the extension of serum half-life and the increase of FcRn binding. That is, as described in US Patent Application Publication No. US2012 / 0028304 (incorporated by reference in its entirety, specifically for the disclosure of pI variants that confer additional functions), the reduction of the pI of antibody constant domains (including those found in Fc fusions) can lead to longer serum retention in vivo. These pI variants for extending serum half-life also facilitate pI changes for purification.

[0165] Furthermore, the pI variants of the heterodimerization variants provide additional benefits to the analytical and quality control process of Fc fusion proteins, as the ability to eliminate, minimize, and distinguish homodimers when present is significant. Similarly, the ability to reliably test the reproducibility of the production of heterodimeric Fc fusion proteins is important.

[0166] Exemplary combinations of pI variants are shown in Figures 4 and 5, and Figure 30 of US Patent Application Publication No. 2016 / 0355608, all of which are incorporated herein by reference in their entirety, and in particular for their disclosure of pI variants. As outlined herein and shown in the figures, these changes are shown relative to IgG1, but all isotypes can be modified in this manner, as can isotype hybrids. R133E and R133Q can also be used when the heavy chain constant domain is derived from IgG2-4.

[0167] In one embodiment, the heterodimeric fusion protein comprises a monomer having a variant Fc domain with the pI variant modifications 295E / 384D / 418E / 421D (Q295E / N384D / Q418E / N421D for human IgG1). In one embodiment, the heterodimeric fusion protein comprises a monomer having a variant Fc domain with the pI variant modifications 217R / 228R / 276K (P217R / P228R / N276K for human IgG1). Additional exemplary pI variant modifications that can be incorporated into the Fc domain of interest are shown in FIG. 5.

[0168] In some embodiments, the modifications occur at the hinge of the Fc domain, including positions 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, and 230, based on EU numbering. Thus, pI variants, particularly substitutions, can be made at one or more of positions 216-230, with 1, 2, 3, 4, or 5 mutations finding use. Similarly, all possible combinations are contemplated, alone or with other pI variants in other domains.

[0169] Particular substitutions used to reduce the pI of the hinge domain include, but are not limited to, a deletion at position 221, a non-native valine or threonine at position 222, a deletion at position 223, a non-native glutamic acid at position 224, a deletion at position 225, a deletion at position 235, and a deletion or non-native alanine at position 236. In some cases, only the pI substitutions are made in the hinge domain, while in other instances, these substitution(s) are added in any combination to other pI variants in other domains.

[0170] In some embodiments, mutations can be made in the CH2 region including positions 233, 234, 235, 236, 274, 296, 300, 309, 320, 322, 326, 327, 334 and 339 based on EU numbering. Note that changes at 233-236 can be made to increase effector function (along with 327A) in an IgG2 backbone. Similarly, all possible combinations of these 14 positions can be made, e.g., an IL-15-Fc fusion protein may contain a variant Fc domain with 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 CH2 pI substitutions.

[0171] Specific substitutions used to reduce the pI of the CH2 domain include, but are not limited to, a non-native glutamine or glutamic acid at position 274, a non-native phenylalanine at position 296, a non-native phenylalanine at position 300, a non-native valine at position 309, a non-native glutamic acid at position 320, a non-native glutamic acid at position 322, a non-native glutamic acid at position 326, a non-native glycine at position 327, a native glutamic acid at position 334, a non-native threonine at position 339, and all possible combinations within CH2 and with other domains.

[0172] In this embodiment, modifications may be independently and optionally selected from positions 355, 359, 362, 384, 389, 392, 397, 418, 419, 444 and 447 (EU numbering) of the CH3 region. Specific substitutions used to reduce the pI of the CH3 domain include, but are not limited to, a non-native glutamine or glutamic acid at position 355, a non-native serine at position 384, a non-native asparagine or glutamic acid at position 392, a non-native methionine at position 397, a non-native glutamic acid at position 419, a non-native glutamic acid at position 359, a non-native glutamic acid at position 362, a non-native glutamic acid at position 389, a non-native glutamic acid at position 418, a non-native glutamic acid at position 444, and a deletion or non-native aspartic acid at position 447.

[0173] C isotype variants Furthermore, some embodiments of the fusion proteins provided herein rely on the "import" of pI amino acids at specific positions from one IgG isotype to another, thus reducing or eliminating the possibility of introducing undesirable immunogenicity into the variant. Some of these are shown in Figure 21 of US Patent Application Publication No. 2014 / 0370013, which is incorporated herein by reference, particularly for the disclosure of isotype variants. That is, IgG1 is a common isotype for therapeutic antibodies for a variety of reasons, including high effector function. However, the heavy constant region of IgG1 has a higher pI than that of IgG2 (8.10 vs. 7.31). By introducing IgG2 residues into the IgG1 backbone at specific positions, the pI of the resulting monomer is lowered (or increased) and also exhibits a longer serum half-life. For example, IgG1 has glycine (pI 5.97) at position 137, while IgG2 has glutamic acid (pI 3.22), and the import of glutamic acid affects the pI of the resulting protein. As described below, several amino acid substitutions are generally required to significantly affect the pI of the variant Fc fusion protein, however, it should be noted that even changes in the IgG2 molecule can allow for increased serum half-life, as discussed below.

[0174] In other embodiments, non-isotypic amino acid modifications are made to reduce the overall charge state of the resulting protein (e.g., by changing from a high pI amino acid to a low pI amino acid) or to allow for tuning of the structure, such as for stability, as described in more detail below.

[0175] Furthermore, by pI engineering both the heavy and light chain constant domains, significant modifications can be seen in each monomer of the heterodimer. As discussed herein, the pI of the two monomers differing by at least 0.5 can allow for separation by ion exchange chromatography or isoelectric focusing, or other methods sensitive to isoelectric point.

[0176] d. Calculation of pI The pI of each monomer depends on the pI of the variant heavy chain constant domain and the pI of the entire monomer, which may include the variant heavy chain constant domain and the fusion partner. Thus, in some embodiments, the change in pI is calculated based on the variant heavy chain constant domain using the chart in Figure 19 of US Patent Application Publication No. 2014 / 0370013. As discussed herein, which monomers to engineer is generally determined by the inherent pI of each monomer.

[0177] e. pI variants that also confer better FcRn in vivo binding If the pI variants decrease the pI of the monomer, they may have the added advantage of improving serum retention in vivo.

[0178] Although still under investigation, it is believed that Fc regions have a longer half-life in vivo because Fc is sequestered when bound to FcRn at pH 6 in endosomes (Ghetie and Ward, 1997 Immunol Today 18(12):592-598, incorporated by reference in its entirety). The endosomal compartment then recycles Fc to the cell surface. Once the compartment opens to the extracellular space, a higher pH of about 7.4 induces Fc release into the blood. In mice, Dall'Acqua et al. showed that Fc mutants with increased FcRn binding at pH 6 and pH 7.4 actually have reduced serum concentrations and half-lives identical to wild-type Fc (Dall'Acqua et al. 2002, J. Immunol. 169:5171-5180, incorporated by reference in its entirety). It is believed that the increased affinity of Fc for FcRn at pH 7.4 prevents Fc release into the blood. Therefore, Fc mutations that increase the half-life of Fc in vivo would ideally increase FcRn binding at lower pH while still allowing release of Fc at higher pH. The amino acid histidine changes its charge state in the pH range of 6.0 to 7.4. It is therefore not surprising to find His residues at key positions in the Fc / FcRn complex.

[0179] 2. Additional Fc variants for additional functionality In addition to pI amino acid variants, there are a number of useful Fc amino acid modifications that can be made for a variety of reasons, including but not limited to, altering binding to one or more FcγR receptors, altering binding to the FcRn receptor, etc.

[0180] Thus, the proteins of the invention can include amino acid modifications including the heterodimerization variants outlined herein, including pI variants and conformational variants, each set of variants can be independently and optionally included or excluded from a particular heterodimeric protein.

[0181] A.FcγR variants There are several useful Fc substitutions that can be made to modify binding to one or more of the FcγR receptors. Substitutions that result in increased binding as well as decreased binding can be useful. For example, increased binding to FcγRIIIa is generally known to result in increased ADCC (antibody-dependent cell-mediated cytotoxicity, i.e., a cell-mediated reaction in which non-specific cytotoxic cells expressing FcγR recognize bound antibodies on target cells and subsequently cause lysis of the target cells). Similarly, decreased binding to FcγRIIb (an inhibitory receptor) can also be beneficial in some circumstances. Amino acid substitutions that can be used in the present invention include those listed in USSN11 / 124,620 (especially FIG. 41), USSN11 / 174,287, USSN11 / 396,495, USSN11 / 538,406, all of which are expressly incorporated herein by reference in their entirety, specifically the variants disclosed therein. Specific variants used include, but are not limited to, 236A, 239D, 239E, 332E, 332D, 239D / 332E, 267D, 267E, 328F, 267E / 328F, 236A / 332E, 239D / 332E / 330Y, 239D, 332E / 330L, 243A, 243L, 264A, 264V, and 299T.

[0182] Furthermore, amino acid substitutions that increase affinity for FcγRIIc may also be included in the Fc domain variants outlined herein. For example, the substitutions described in USSN 11 / 124,620 and USSN 14 / 578,305 are useful.

[0183] Furthermore, as specifically disclosed in USSN 12 / 341,769, which is incorporated herein by reference in its entirety, there are additional Fc substitutions that are used to increase binding to the FcRn receptor and increase serum half-life, including, but not limited to, 434S, 434A, 428L, 308F, 259I, 428L / 434S, 259I / 308F, 436I / 428L, 436I or V / 434S, 436V / 428L, and 259I / 308F / 428L.

[0184] B. Deletion variant Similarly, another category of functional variants is "FcγR ablation variants" or "Fc knockout (FcKO or KO)" variants. In these embodiments, for some therapeutic applications, it is desirable to reduce or eliminate the normal binding of the Fc domain to one or more or all Fcγ receptors (e.g., FcγR1, FcγRIIa, FcγRIIb, FcγRIIIa, etc.) to avoid additional mechanisms of action. This is, for example, in many embodiments, particularly in the use of bispecific immunomodulatory antibodies, where it is desirable to remove FcγRIIIa linkage so that one of the Fc domains comprises one or more Fcγ receptor ablation variants to eliminate or significantly reduce ADCC activity. These deletion variants are shown in Figure 31 of USSN 15 / 141,350, all of which are incorporated herein by reference in their entirety, and each may be independently and optionally included or excluded, with preferred embodiments employing deletion variants selected from the group consisting of G236R / L328R, E233P / L234V / L235A / G236del / S239K, E233P / L234V / L235A / G236del / S267K, E233P / L234V / L235A / G236del / S239K / A327G, E233P / L234V / L235A / G236del / S267K / A327G, and E233P / L234V / L235A / G236del, according to the EU index. Note that the ablation variants referred to herein ablate FcγR binding, but do not ablate FcRn binding.

[0185] Exemplary embodiments of pI variants are provided in the figures, including FIG.

[0186] 3. Combination of heterodimers and Fc variants As will be appreciated by those of skill in the art, all of the listed heterodimerization variants (including scubariants and / or pI variants) can be optionally and independently combined as long as they maintain their "strandedness" or "monomer split." Furthermore, all of these variants can be combined in any of the heterodimerization formats.

[0187] In the case of pI variants, while a specifically used embodiment is shown in the figure, other combinations can be generated following the basic rule of varying the pI difference between the two monomers to facilitate purification.

[0188] Additionally, any of the heterodimerization variants, skews, and pIs can be independently and optionally combined with Fc depletion variants, Fc variants, FcRn variants, as generally outlined herein.

[0189] Additionally, the monomeric Fc domain may comprise a set of amino acid substitutions comprising C220S / S267K / L368D / K370S or C220S / S267K / S364K / E357Q.

[0190] Additionally, heterodimeric Fc fusion proteins can include skew mutations (e.g., the set of amino acid substitutions shown in Figures 1A-1C of USSN 15 / 141,350, all of which are incorporated by reference herein in their entireties); particularly useful skew mutations include S364K / E357Q:L368D / K370S, L368D / K370S:S364K, L368E / K370S:S364K, T41 and selected from the group consisting of 1T / E360E / Q362E:D401K, L368D / K370S:S364K / E357L, K370S:S364K / E357Q, T366S / L368A / Y407V:T366W and T366S / L368A / Y407V / Y349C:T366W / S354C, optionally including deletion mutations, optionally including a charged domain linker, and wherein the heavy chain comprises a pI mutation.

[0191] In some embodiments, the Fc domain is 236R, 239D, 239E, 243L, M252Y, V259I, 267D, 267E, 298A, V308F, 328F, 328R, 330L, 332D, 332E, M428L, N434A, N434S, 236R / 328R, 239D / 332E, M428L, 236R / 328F, V259I / V308F, 267E / 328F, M428L / N The Fc domain comprises an amino acid substitution selected from the group consisting of 434S, Y436I / M428L, Y436V / M428L, Y436I / N434S, Y436V / N434S, 239D / 332E / 330L, M252Y / S254T / T256E, V259I / V308F / M428L, E233P / L234V / L235A / G236del / S267K, G236R / L328R, and PVA / S267K. In some cases, the Fc domain comprises the amino acid substitution 239D / 332E. In other cases, the Fc domain comprises the amino acid substitution G236R / L328R or PVA / S267K.

[0192] In one embodiment, a particular combination of skew and pI variants used in the present invention is T366S / L368A / Y407V:T366W (optionally including the bridging disulfide T366S / L368A / Y407V / Y349C:T366W / S354C), with one monomer containing Q295E / N384D / Q418E / N481D and the other a positively charged domain linker. As understood in the art, the "knobs-in-holes" variant does not change the pI and therefore can be used in either monomer.

[0193] In one embodiment, a particular combination of skew and pI variants used in the present invention is L368D / K370S:S364K / E357Q, with one monomer comprising Q295E / N384D / Q418E / N421D.

[0194] Useful pairs of Fc dimerization variant sets (including skew and pI variants) are provided in Figures 4A-4E. Addition pI variants are provided in Figure 5. Useful deletion variants are provided in Figure 6. Useful embodiments of non-cytokine components of the IL-15 / Rα x anti-PD1 ABD heterodimer fusion proteins of the invention are provided in Figures 7A-7E and 8A-8F.

[0195] D. Domain Linker The three components of the invention, the anti-PD-1 Fv, the Il-15 / Rα complex, and the heterodimerized Fc domain of the invention, are optionally linked together using a domain linker. Although direct covalent linkage can be performed (e.g., linking the C-terminus of the IL-15 complex to the N-terminus of the CH2 domain of the Fc domain), generally any linker that provides flexibility and is optionally functional is used.

[0196] In some embodiments, the IL-15 protein is attached to the N-terminus of the Fc domain and the IL-15Rα protein is attached to the N-terminus of the IL-15 protein. In other embodiments, the IL-15Rα protein is attached to the N-terminus of the Fc domain and the IL-15Rα protein is non-covalently attached to the IL-15 protein. In yet other embodiments, the IL-15Rα protein is attached to the C-terminus of the Fc domain and the IL-15Rα protein is non-covalently attached to the IL-15 protein.

[0197] In some embodiments, the IL-15 protein and the IL-15Rα protein are linked together via a linker (e.g., "scIL-15 / Rα" format). Optionally, the proteins are not linked via a linker, but rather utilize either natural self-assembly or disulfide bonds as outlined herein. In other embodiments, the IL-15 protein and the IL-15Rα protein are non-covalently linked. In some embodiments, the IL-15 protein is linked to the Fc domain via a linker. In certain embodiments, the IL-15 protein is directly linked to the Fc domain without a linker. In certain embodiments, the IL-15 protein is linked to the Fc domain via a hinge region or a fragment thereof. In other embodiments, the IL-15Rα protein is linked to the Fc domain via a linker. In other embodiments, the IL-15Rα protein is directly linked to the Fc domain without a linker. In certain embodiments, the IL-15Rα protein is linked to the Fc domain via a hinge region or a fragment thereof. Optionally, no linker is used to attach the IL-15 protein or the IL-15Rα protein to the Fc domain.

[0198] In some cases, the PD-1 ABD is covalently linked to the N-terminus of the Fc domain via a linker, such as a domain linker. In some embodiments, the PD-1 ABD is directly linked to the Fc domain without a linker. In certain embodiments, the PD-1 ABD is linked to the Fc domain via a hinge region or a fragment thereof.

[0199] In some embodiments, the linker is a "domain linker" used to link together any two domains outlined herein. The linker peptide may primarily comprise the following amino acid residues: Gly, Ser, Ala, or Thr. The linker peptide should be of sufficient length to link the two molecules in such a way that they assume the correct conformation relative to each other so that they retain the desired activity. In one embodiment, the linker is about 1-50 amino acids long, preferably about 1-30 amino acids long. In one embodiment, a linker of 1-20 amino acids long may be used, and in some embodiments, about 5 to about 10 amino acids are used. Useful linkers include, for example, glycine-serine polymers, glycine-alanine polymers, alanine-serine polymers, including (GS)n (SEQ ID NO:XX), (GSGGS)n (SEQ ID NO:XX), (GGGGS)n (SEQ ID NO:XX), and (GGGS)n (SEQ ID NO:XX), where n is an integer of at least 1 (and generally 3-4), as well as other flexible linkers. Alternatively, a variety of non-proteinaceous polymers may be useful as linkers, including, but not limited to, polyethylene glycol (PEG), polypropylene glycol, polyoxyalkylenes, or copolymers of polyethylene glycol and polypropylene glycol.

[0200] Other linker sequences include any sequence of the CL / CH1 domain of any length, but may not include all residues of the CL / CH1 domain, for example, the first 5-12 amino acid residues of the CL / CH1 domain. Linkers can be derived from immunoglobulin light chains, for example, Cκ or Cλ. Linkers can be derived from immunoglobulin heavy chains of any isotype, including, for example, Cγ1, Cγ2, Cγ3, Cγ4, Cα1, Cα2, Cδ, Cε, and Cμ. Linker sequences can also be derived from other proteins, such as Ig-like proteins (e.g., TCR, FcR, KIR), hinge region-derived sequences, and other naturally occurring sequences from other proteins.

[0201] In some embodiments, the scFv linker is a charged scFv linker, some of which are shown in FIG. 4A of US62 / 353,511. Thus, the present invention further provides charged scFv linkers to facilitate the separation of pI between the first and second monomers (e.g., IL-15 / IL-15Rα monomer and PD-1 ABD monomer). That is, by incorporating either a positively or negatively charged scFv linker (or both in the case of scaffolds using scFvs on different monomers), this allows the pI of the monomers containing the charged linker to be changed without further altering the Fc domain. These charged linkers can be substituted into any scFv containing standard linkers. Also, as will be appreciated by those skilled in the art, a charged scFv linker is used on the correct "chain" or monomer according to the desired change in pI. For example, as discussed herein, to generate a triple-F format heterodimeric antibody, the original pI of the Fv region for each of the desired antigen binding domains is calculated, one is selected to generate the scFv, and depending on the pI, either a positive or negative linker is selected.

[0202] Charged domain linkers can also be used to increase the pI separation of the monomers of the present invention, and thus those included in FIG. 10 can be used in any embodiment herein where a linker is utilized.

[0203] IV. Useful Formats of the Invention There are several useful formats of the PD-1-targeted IL-15 / IL-15Rα(sushi) Fc fusion proteins of the invention, as shown in Figures 28A-28H. In general, the heterodimeric fusion proteins of the invention have three functional components: an IL-15 / IL-15Rα(sushi) component, an anti-PD-1 component, and an Fc component, each of which can take different forms as outlined herein, and each of which can be combined with the other components in any configuration.

[0204] In any of the following formats, the first and second Fc domains may have a set of amino acid substitutions selected from the group consisting of: a) S267K / L368D / K370S:S267K / LS364K / E357Q, b) S364K / E357Q:L368D / K370S, c) L368D / K370S:S364K, d) L368E / K370S:S364K, e) T411T / E360E / Q362E:D401K, f) L368D / K370S:S364K / E357L, and g) K370S:S364K / E357Q, according to EU numbering.

[0205] In some embodiments, the first and / or second Fc domain has an additional set of amino acid substitutions comprising Q295E / N384D / Q418E / N421D according to EU numbering.

[0206] Optionally, the first and / or second Fc domain has an additional set of amino acid substitutions consisting of G236R / L328R, E233P / L234V / L235A / G236del / S239K, E233P / L234V / L235A / G236del / S267K, E233P / L234V / L235A / G236del / S239K / A327G, E233P / L234V / L235A / G236del / S267K / A327G, and E233P / L234V / L235A / G236del, according to EU numbering.

[0207] Optionally, the first and / or second Fc domain has the M428L / N434S variant for half-life extension. In some embodiments, the first and / or second Fc domain has the 428L / 434S variant for half-life extension.

[0208] Thus, particularly useful variants for any of the following formats have positive monomers that contain the scubariant S364K / E357Q and the deletion variants E233P / L234V / L235A / G236del / S267K, and negative monomers that contain the scubariant L368D / K370S, the deletion variants E233P / L234V / L235A / G236del / S267K and the pI variants N208D / Q295E / N384D / Q418E / N421D (if the monomer contains a CH1 domain) or the pI variants Q295E / N384D / Q418E / N421D (if the monomer does not contain a CH1 domain).

[0209] A.scIL-15 / Rα×scFv One embodiment is shown in Figure 28A and contains two monomers. This is commonly referred to as "scIL-15 / Rα x scFv", where "sc" stands for "single chain" and refers to the attachment of the IL-15 and sushi domains using a covalent linker. The "scIL-15 / Rα x scFv" format (see Figure 28A) contains IL-15Rα (sushi) fused to IL-15 by a variable length linker (referred to as "scIL-15 / Rα"), which is then fused to the N-terminus of the heterodimeric Fc region, and the scFv is fused to the other side of the heterodimeric Fc.

[0210] In the Figure 28A format, some embodiments include an Fc domain having a positive monomer comprising the scubariant S364K / E357Q and the deletion variants E233P / L234V / L235A / G236del / S267K, and a negative monomer comprising the scubariant L368D / K370S, the deletion variants E233P / L234V / L235A / G236del / S267K and the pI variants Q295E / N384D / Q418E / N421D, and optionally the 428L / 434S FcRn variant. In some embodiments, the sushi domain is wild-type (SEQ ID NO: 4) and the anti-PD-1 Fv domain is H1.19_L1.140, H1_L1.1, H1.19_L1, H1.48_L1, H1.125_L1, H1.30_L1, H1.132_L1, H1_L1.1 The mAbC VH and VL domain combinations are selected from the group consisting of H1_L1.3, H1_L1.45, H1_L1.117, H1_L1.129, H1.19_L1.1, H1.32_L1.1, H1.169_L1.1, H1.169_L1.1, H1.175_L1.1, H1.175_L1.1, H1_L1.140, H1_L1.135, H1_L1.136, H1.132_L1.135, H1.132_L1.140, H1.175_L1.135 and H1.175_L1.140.

[0211] In the Figure 28A format, some embodiments include an Fc domain having a positive monomer comprising the scubariant S364K / E357Q and the deletion variants E233P / L234V / L235A / G236del / S267K, and a negative monomer comprising the scubariant L368D / K370S, the deletion variants E233P / L234V / L235A / G236del / S267K and the pI variants Q295E / N384D / Q418E / N421D, and optionally the 428L / 434S FcRn variant. In these embodiments, the sushi domain is wild-type (SEQ ID NO:XX), the anti-PD-1 Fv domain is a combination of the VH and VL domains of H1.19_L1.140, and the IL-15 domain comprises a variant selected from N4D / N65D, D30N / N65D, and D30N / E64Q / N65D.

[0212] In the Figure 28A format, some embodiments include an Fc domain having a positive monomer comprising the scubariant S364K / E357Q and the deletion variants E233P / L234V / L235A / G236del / S267K, and a negative monomer comprising the scubariant L368D / K370S, the deletion variants E233P / L234V / L235A / G236del / S267K and the pI variants Q295E / N384D / Q418E / N421D, and optionally the 428L / 434S FcRn variant. In these embodiments, the sushi domain is wild-type (SEQ ID NO:XX), the anti-PD-1 Fv domain is selected from the group consisting of a combination of the VH and VL domains of H1_L1.1, and the IL-15 domain comprises a variant selected from N4D / N65D, D30N / N65D, and D30N / E64Q / N65D.

[0213] B.scFv×ncIL-15 / Rα This embodiment is shown in Figure 28B and contains three monomers. It is commonly referred to as "ncIL-15 / Rα x scFv" or "scFv x ncIL-15 / Rα", where "nc" stands for "non-covalent" referring to the self-assembled non-covalent association of the IL-15 and sushi domains. The "scFv x ncIL-15 / Rα" format (see Figure 34B) contains an scFv fused to the N-terminus of the heterodimeric Fc region, with IL-15Rα (sushi) fused to the other side of the heterodimeric Fc, while IL-15 is transfected separately such that a non-covalent IL-15 / Rα complex is formed.

[0214] In the Figure 28B format, some embodiments include an Fc domain having a positive monomer comprising the scubariant S364K / E357Q and the deletion variants E233P / L234V / L235A / G236del / S267K, and a negative monomer comprising the scubariant L368D / K370S, the deletion variants E233P / L234V / L235A / G236del / S267K and the pI variants Q295E / N384D / Q418E / N421D, and optionally the 428L / 434S FcRn variant. In these embodiments, the sushi domain is wild-type (SEQ ID NO: 4), the anti-PD-1 Fv domain is a combination of the VH and VL domains of H1.19_L1.140, and the IL-15 domain comprises a variant selected from N4D / N65D, D30N / N65D, and D30N / E64Q / N65D.

[0215] In the Figure 28B format, some embodiments include an Fc domain having a positive monomer comprising the scubariant S364K / E357Q and the deletion variants E233P / L234V / L235A / G236del / S267K, and a negative monomer comprising the scubariant L368D / K370S, the deletion variants E233P / L234V / L235A / G236del / S267K and the pI variants Q295E / N384D / Q418E / N421D, and optionally the 428L / 434S FcRn variant. In these embodiments, the sushi domain is wild-type (SEQ ID NO: 4), the anti-PD-1 Fv domain is selected from the group consisting of a combination of the VH and VL domains of H1_L1.1, and the IL-15 domain comprises a variant selected from N4D / N65D, D30N / N65D, and D30N / E64Q / N65D.

[0216] C.scIL-15 / Rα×Fab This preferred format is shown in Figure 28C and contains three monomers. It is commonly referred to as "scIL-15 / Rα x Fab" or "Fab x scIL-15 / Rα", used interchangeably, with "sc" standing for "single chain". scIL-15 / Rα x Fab (see Figure 28C) contains IL-15Rα (sushi) fused to IL-15 by a variable length linker (referred to as "scIL-15 / Rα"), which is then fused to the N-terminus of a heterodimeric Fc region (including both ends of the hinge). The second monomer is the heavy chain, VH-CH1-hinge-CH2-CH3, while the corresponding light chain (third monomer) is transfected separately to form a Fab with VH.

[0217] In the FIG. 28C format, some embodiments include an Fc domain having a positive monomer that includes the scubariant S364K / E357Q and deletion variants E233P / L234V / L235A / G236del / S267K, the 428L / 434S FcRn variant, and a negative monomer that includes the scubariant L368D / K370S, deletion variants E233P / L234V / L235A / G236del / S267K, pI variants N208D / Q295E / N384D / Q418E / N421D, and the 428L / 434S FcRn variant. In these embodiments, the sushi domain is wild-type (SEQ ID NO:4) and the anti-PD-1 Fv domain comprises a VH that includes the F32L amino acid substitution compared to SEQ ID NO:5, and a VL that includes the amino acid substitutions N27dH / K30Y / S93T compared to SEQ ID NO:6.

[0218] In the FIG. 28C format, some embodiments include an Fc domain having a positive monomer that includes the scubariant S364K / E357Q and the deletion variants E233P / L234V / L235A / G236del / S267K, and a negative monomer that includes the scubariant L368D / K370S, the deletion variants E233P / L234V / L235A / G236del / S267K, and the pI variants N208D / Q295E / N384D / Q418E / N421D. In these embodiments, the sushi domain is wild-type (SEQ ID NO:4), the anti-PD-1 Fv domain comprises a VH that comprises an F32L amino acid substitution compared to SEQ ID NO:5, and a VL that comprises amino acid substitutions N27dH / K30Y / S93T compared to SEQ ID NO:6, and the IL-15 domain comprises a variant selected from N4D / N65D, D30N / N65D, and D30N / E64Q / N65D.

[0219] In the FIG. 28C format, some embodiments include an Fc domain having a positive monomer that includes the scubariant S364K / E357Q and the deletion variants E233P / L234V / L235A / G236del / S267K, the 428L / 434S FcRn variant, and a negative monomer that includes the scubariant L368D / K370S, the deletion variants E233P / L234V / L235A / G236del / S267K, and the pI variants N208D / Q295E / N384D / Q418E / N421D, and the 428L / 434S FcRn variant. In these embodiments, the sushi domain is wild-type (SEQ ID NO: 4), the anti-PD-1 Fv domain is a combination of the VH and VL domains of H1.19_L1.140, and the IL-15 domain comprises a variant selected from N4D / N65D, D30N / N65D, and D30N / E64Q / N65D.

[0220] In the FIG. 28C format, some embodiments include an Fc domain having a positive monomer that includes the scubariant S364K / E357Q and the deletion variants E233P / L234V / L235A / G236del / S267K, the 428L / 434S FcRn variant, and a negative monomer that includes the scubariant L368D / K370S, the deletion variants E233P / L234V / L235A / G236del / S267K, and the pI variants N208D / Q295E / N384D / Q418E / N421D, and the 428L / 434S FcRn variant. In these embodiments, the sushi domain is wild-type (SEQ ID NO: 4), the anti-PD-1 Fv domain is a combination of the VH and VL domains of H1_L1.1, and the IL-15 domain comprises a variant selected from N4D / N65D, D30N / N65D, and D30N / E64Q / N65D.

[0221] In the FIG. 28C format, some embodiments include an Fc domain having a positive monomer that includes the scubariant S364K / E357Q and the deletion variants E233P / L234V / L235A / G236del / S267K, and a negative monomer that includes the scubariant L368D / K370S, the deletion variants E233P / L234V / L235A / G236del / S267K, and the pI variants N208D / Q295E / N384D / Q418E / N421D. In these embodiments, the sushi domain is wild-type (SEQ ID NO: 4), the anti-PD-1 Fv domain is a combination of the VH and VL domains of H1.19_L1.140, and the IL-15 domain comprises a variant selected from N4D / N65D, D30N / N65D, and D30N / E64Q / N65D.

[0222] In the Figure 28C format, some embodiments include an Fc domain having a positive monomer comprising the scubariant S364K / E357Q and the deletion variants E233P / L234V / L235A / G236del / S267K, and a negative monomer comprising the scubariant L368D / K370S, the deletion variants E233P / L234V / L235A / G236del / S267K and the pI variants N208D / Q295E / N384D / Q418E / N421D, and optionally the 428L / 434S FcRn variant. In these embodiments, the sushi domain is wild-type (SEQ ID NO: 4), the anti-PD-1 Fv domain is selected from the group consisting of a combination of the VH and VL domains of H1_L1.1, and the IL-15 domain comprises a variant selected from N4D / N65D, D30N / N65D, and D30N / E64Q / N65D.

[0223] The amino acid sequence of an exemplary non-competitive PD-1-targeting IL-15 / Rα-Fc fusion protein in the scIL-15 / Rα×Fab format ( FIG. 28C ) is provided in FIGS. 30 , 48 , 49 , and 68 .

[0224] In some embodiments, the first monomer comprises, from N-terminus to C-terminus, sushi domain-domain linker-variant IL-15-domain linker-CH2-CH3, the second monomer comprises the heavy chain, VH-CH1-hinge-CH2-CH3, and the third monomer is the light chain, VL-CL. A preferred combination of variants for this embodiment is seen in Figure 7C of PCT / US2017 / 056826.

[0225] In the scIL-15 / Rα×Fab format, one preferred embodiment utilizes the scUB variant pair S364K / E357Q:L368D / K370S.

[0226] In the scIL-15 / Rα×Fab format, one preferred embodiment utilizes scFv variants S364K / E357Q (on the scFv-Fc monomer) and L368D / K370S (on the IL-15 complex monomer), pI variants Q295E / N384D / Q418E / N421D (on the IL-15 complex side), deletion variants E233P / L234V / L235A / G236_ / S267K on both monomers, and optionally 428L / 434S variants on both sides.

[0227] D. ncIL-15 / Rα×Fab This embodiment is shown in Figure 28D and contains three monomers. It is commonly referred to as "ncIL-15 / Rα x Fab" or "Fab x ncIL-15 / Rα" as used interchangeably, with "nc" standing for "non-covalent" referring to the self-assembled non-covalent association of the IL-15 and sushi domains. The ncIL-15 / Rα x Fab format (see Figure 34D) contains a VH fused to the N-terminus of the heterodimeric Fc region, with IL-15Rα (sushi) fused to the other side of the heterodimeric Fc, while the corresponding light chain is transfected separately to form a Fab with the VH, while IL-15 is transfected separately to form a non-covalent IL-15 / Rα complex.

[0228] In some embodiments, the first monomer comprises, from N-terminus to C-terminus, sushi domain-domain linker-CH2-CH3, the second monomer comprises heavy chain, VH-CH1-hinge-CH2-CH3. The third monomer is an IL-15 domain. In the ncIL-15 / Rα×Fab format, one preferred embodiment utilizes the scubariant pair S364K / E357Q:L368D / K370S.

[0229] In the Figure 28D format, some embodiments include an Fc domain having a positive monomer comprising the scubariant S364K / E357Q and the deletion variants E233P / L234V / L235A / G236del / S267K, and a negative monomer comprising the scubariant L368D / K370S, the deletion variants E233P / L234V / L235A / G236del / S267K and the pI variants N208D / Q295E / N384D / Q418E / N421D, and optionally the 428L / 434S FcRn variant. In these embodiments, the sushi domain is wild-type (SEQ ID NO: 4), the anti-PD-1 Fv domain is a combination of the VH and VL domains of H1.19_L1.140, and the IL-15 domain comprises a variant selected from N4D / N65D, D30N / N65D, and D30N / E64Q / N65D.

[0230] In the Figure 28D format, some embodiments include an Fc domain having a positive monomer comprising the scubariant S364K / E357Q and the deletion variants E233P / L234V / L235A / G236del / S267K, and a negative monomer comprising the scubariant L368D / K370S, the deletion variants E233P / L234V / L235A / G236del / S267K and the pI variants N208D / Q295E / N384D / Q418E / N421D, and optionally the 428L / 434S FcRn variant. In these embodiments, the sushi domain is wild-type (SEQ ID NO: 4), the anti-PD-1 Fv domain is selected from the group consisting of a combination of the VH and VL domains of H1_L1.1, and the IL-15 domain comprises a variant selected from N4D / N65D, D30N / N65D, and D30N / E64Q / N65D.

[0231] E. mAb-scIL-15 / Rα This embodiment is shown in Figure 28E and contains three monomers (but the fusion protein is a tetramer). It is commonly referred to as "mAb-scIL-15 / Rα", where "sc" stands for "single chain". The mAb-scIL-15 / Rα format (see Figure 34E) contains a VH fused to the N-terminus of the first and second heterodimeric Fc, where IL-15 is fused to IL-15Rα (sushi), which is then further fused to the C-terminus of one of the heterodimeric Fc regions, while the corresponding light chain is transfected separately to form a Fab with a VH.

[0232] In some embodiments, the first monomer comprises a heavy chain, VH-CH1-hinge-CH2-CH3. The second monomer comprises a heavy chain, VH-CH1-hinge-CH2-CH3-domain linker-sushi domain-domain linker-IL-15, with the scIL-15 complex. The third (and fourth) monomer is a light chain, VL-CL. This is commonly referred to as "mAb-scIL-15 / Rα", with "sc" standing for "single chain". In the mAb-scIL-15 / Rα format, one preferred embodiment utilizes the scavariant pair S364K / E357Q:L368D / K370S.

[0233] In the Figure 28E format, some embodiments include an Fc domain having a positive monomer comprising the scubariant S364K / E357Q and the deletion variants E233P / L234V / L235A / G236del / S267K, and a negative monomer comprising the scubariant L368D / K370S, the deletion variants E233P / L234V / L235A / G236del / S267K and the pI variants N208D / Q295E / N384D / Q418E / N421D, and optionally the 428L / 434S FcRn variant. In these embodiments, the sushi domain is wild-type (SEQ ID NO: 4), the anti-PD-1 Fv domain is a combination of the VH and VL domains of H1.19_L1.140, and the IL-15 domain comprises a variant selected from N4D / N65D, D30N / N65D, and D30N / E64Q / N65D.

[0234] In the Figure 28E format, some embodiments include an Fc domain having a positive monomer comprising the scubariant S364K / E357Q and the deletion variants E233P / L234V / L235A / G236del / S267K, and a negative monomer comprising the scubariant L368D / K370S, the deletion variants E233P / L234V / L235A / G236del / S267K and the pI variants N208D / Q295E / N384D / Q418E / N421D, and optionally the 428L / 434S FcRn variant. In these embodiments, the sushi domain is wild-type (SEQ ID NO: 4), the anti-PD-1 Fv domain is selected from the group consisting of a combination of the VH and VL domains of H1_L1.1, and the IL-15 domain comprises a variant selected from N4D / N65D, D30N / N65D, and D30N / E64Q / N65D.

[0235] F.mAb-ncIL-15 / Rα This embodiment is shown in Figure 28F and contains four monomers (although the heterodimeric fusion protein is a pentamer). It is commonly referred to as "mAb-ncIL-15 / Rα", where "nc" stands for "non-covalent". The mAb-ncIL-15 / Rα format (Figure 34F) contains a VH fused to the N-terminus of the first and second heterodimeric Fc, and IL-15Rα (sushi) is fused to the C-terminus of one of the heterodimeric Fc regions, while the corresponding light chains are transfected separately to form a Fab with the VH, while IL-15 is transfected separately to form a non-covalent IL-15 / Rα complex.

[0236] In some embodiments, the first monomer comprises a heavy chain, VH-CH1-hinge-CH2-CH3. The second monomer comprises a heavy chain with an IL-15Rα (sushi) domain, VH-CH1-hinge-CH2-CH3-domain linker-sushi domain. The third monomer is an IL-15 domain. The fourth (and fifth) monomer is a light chain, VL-CL. In the mAb-ncIL-15 / Rα format, one preferred embodiment utilizes the scubariant pair S364K / E357Q:L368D / K370S.

[0237] In the Figure 28F format, some embodiments include an Fc domain having a positive monomer comprising the scubariant S364K / E357Q and the deletion variants E233P / L234V / L235A / G236del / S267K, and a negative monomer comprising the scubariant L368D / K370S, the deletion variants E233P / L234V / L235A / G236del / S267K and the pI variants N208D / Q295E / N384D / Q418E / N421D, and optionally the 428L / 434S FcRn variant. In these embodiments, the sushi domain is wild-type (SEQ ID NO: 4), the anti-PD-1 Fv domain is a combination of the VH and VL domains of H1.19_L1.140, and the IL-15 domain comprises a variant selected from N4D / N65D, D30N / N65D, and D30N / E64Q / N65D.

[0238] In the Figure 28F format, some embodiments include an Fc domain having a positive monomer comprising the scubariant S364K / E357Q and the deletion variants E233P / L234V / L235A / G236del / S267K, and a negative monomer comprising the scubariant L368D / K370S, the deletion variants E233P / L234V / L235A / G236del / S267K and the pI variants N208D / Q295E / N384D / Q418E / N421D, and optionally the 428L / 434S FcRn variant. In these embodiments, the sushi domain is wild-type (SEQ ID NO: 4), the anti-PD-1 Fv domain is selected from the group consisting of a combination of the VH and VL domains of H1_L1.1, and the IL-15 domain comprises a variant selected from N4D / N65D, D30N / N65D, and D30N / E64Q / N65D.

[0239] G.Central-IL-15 / Rα This embodiment is shown in Figure 28G and contains four monomers forming a tetramer. It is commonly referred to as "Central-IL-15 / Rα". The Central-IL-15 / Rα format (see Figure 28G) contains a VH recombinantly fused to the N-terminus of IL-15 (which is then further fused to one side of the heterodimeric Fc) and a VH recombinantly fused to the N-terminus of IL-15Rα (sushi) (which is then further fused to the other side of the heterodimeric Fc), while the corresponding light chain is transfected separately to form a Fab with the VH.

[0240] In the Figure 28G format, some embodiments include an Fc domain having a positive monomer comprising the scubariant S364K / E357Q and the deletion variants E233P / L234V / L235A / G236del / S267K, and a negative monomer comprising the scubariant L368D / K370S, the deletion variants E233P / L234V / L235A / G236del / S267K and the pI variants N208D / Q295E / N384D / Q418E / N421D, and optionally the 428L / 434S FcRn variant. In these embodiments, the sushi domain is wild-type (SEQ ID NO: 4), the anti-PD-1 Fv domain is a combination of the VH and VL domains of H1.19_L1.140, and the IL-15 domain comprises a variant selected from N4D / N65D, D30N / N65D, and D30N / E64Q / N65D.

[0241] In the Figure 28G format, some embodiments include an Fc domain having a positive monomer comprising the scubariant S364K / E357Q and the deletion variants E233P / L234V / L235A / G236del / S267K, and a negative monomer comprising the scubariant L368D / K370S, the deletion variants E233P / L234V / L235A / G236del / S267K and the pI variants N208D / Q295E / N384D / Q418E / N421D, and optionally the 428L / 434S FcRn variant. In these embodiments, the sushi domain is wild-type (SEQ ID NO: 4), the anti-PD-1 Fv domain is selected from the group consisting of a combination of the VH and VL domains of H1_L1.1, and the IL-15 domain comprises a variant selected from N4D / N65D, D30N / N65D, and D30N / E64Q / N65D.

[0242] H. central scIL-15 / Rα This embodiment is shown in Figure 28H and contains four monomers forming a tetramer. It is commonly referred to as "Central-scIL-15 / Rα", where "sc" stands for "single chain". The "Central-scIL-15 / Rα" format (see Figure 34H) contains a VH fused to the N-terminus of IL-15Rα (sushi), which is then fused to IL-15 and then further fused to one side of the heterodimeric Fc, and a VH fused to the other side of the heterodimeric Fc, while the corresponding light chain is transfected separately to form a Fab containing the VH.

[0243] In the Figure 28H format, some embodiments include an Fc domain having a positive monomer comprising the scubariant S364K / E357Q and the deletion variants E233P / L234V / L235A / G236del / S267K, and a negative monomer comprising the scubariant L368D / K370S, the deletion variants E233P / L234V / L235A / G236del / S267K and the pI variants N208D / Q295E / N384D / Q418E / N421D, and optionally the 428L / 434S FcRn variant. In these embodiments, the sushi domain is wild-type (SEQ ID NO: 4), the anti-PD-1 Fv domain is a combination of the VH and VL domains of H1.19_L1.140, and the IL-15 domain comprises a variant selected from N4D / N65D, D30N / N65D, and D30N / E64Q / N65D.

[0244] In the Figure 28H format, some embodiments include an Fc domain having a positive monomer comprising the scubariant S364K / E357Q and the deletion variants E233P / L234V / L235A / G236del / S267K, and a negative monomer comprising the scubariant L368D / K370S, the deletion variants E233P / L234V / L235A / G236del / S267K and the pI variants N208D / Q295E / N384D / Q418E / N421D, and optionally the 428L / 434S FcRn variant. In these embodiments, the sushi domain is wild-type (SEQ ID NO: 4), the anti-PD-1 Fv domain is selected from the group consisting of a combination of the VH and VL domains of H1.19_L1.140, and the IL-15 domain comprises a variant selected from N4D / N65D, D30N / N65D, and D30N / E64Q / N65D.

[0245] I. Additional Formats As will be appreciated by those of skill in the art, there are additional formats that can be used with an IL-15 conjugate and a non-competing anti-PD-1 Fv sequence. Thus, for example, the invention provides a fusion protein comprising an IL-15 conjugate that includes an IL-15 domain (which can be wild-type or variant), a domain linker, an IL-15R sequence (which can be all or a portion of the extracellular domain, including variants), and a non-competing anti-PD-1 ABD as a Fab or scFv.

[0246] V. Useful Embodiments of the Invention Provided herein are PD-1 targeting IL-15 / Rα-Fc fusion proteins with one or more engineered amino acid substitutions in the IL-15 protein and anti-PD-1 ABDs that do not compete for binding with selected approved anti-PD-1 antibodies. In some embodiments, the IL-15 variant of the Fc fusion protein has an N4D / N65D substitution. In some embodiments, the IL-15 variant of the Fc fusion protein has a D30N substitution. In some embodiments, the IL-15 variant of the Fc fusion protein has a D30N / E64Q / N65D substitution. In some embodiments, the IL-15 variant of the Fc fusion protein has a D30N / N65D substitution. Such IL-15 / Rα-Fc containing fusion proteins are capable of targeting NK cells, CD8 + T cells, and CD4 + It can induce or promote the proliferation of immune cells including T cells. In particular, IL-15 / Rα-Fc, which contains a fusion protein with no linker on the IL-15Fc side (e.g., only the hinge region), showed weaker proliferation activity.

[0247] Provided herein are PD-1-targeted IL-15 / Rα-Fc fusion proteins with lower potency, increased pharmacokinetics, and / or increased serum half-life. The PD-1-targeted IL-15 / Rα-Fc fusion proteins described herein have been engineered to reduce their potency compared to the parent construct. In some embodiments, one or more amino acid substitutions have been introduced into the Fc domain(s) of the IL-15 / Rα complex and / or heterodimeric Fc fusion protein. In some embodiments, the PD-1-targeted IL-15 / Rα-Fc fusion proteins with reduced potency compared to the control construct (e.g., the parent construct) have a substantially longer serum half-life. In certain embodiments, the serum half-life is increased by 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 25-fold, or more.

[0248] Provided herein are PD-1-targeted IL-15 / Rα-Fc fusion proteins that enhanced GVHD in animal models (e.g., human PBMC-engrafted NSG mice) compared to combination therapy of a control scIL-15 / Rα-Fc fusion protein and an anti-PD-1 antibody engineered to reduce efficacy. Administration of an exemplary non-competitive PD-1-targeted IL-15 / Rα-Fc fusion protein resulted in greater efficacy compared to the combination of IL-15 and PD-1 blockade.

[0249] The PD-1-targeted IL-15 / Rα-Fc fusion proteins described herein, including non-competitive PD-1-targeted IL-15 / Rα-Fc fusion proteins, can induce STAT5 phosphorylation in immune cells, including, but not limited to, activated lymphocytes, activated T cells (e.g., activated CD4+ T cells and activated CD8+ cells), and activated tumor-infiltrating lymphocytes (e.g., activated TILs).

[0250] Preferred embodiments utilize the Figure 28C format. In these embodiments, a) positive monomers including (i) scubariant S364K / E357Q, deletion variants E233P / L234V / L235A / G236del / S267K, and 428L / 434S FcRn variants, b) and (ii) scubariant L368D / K370S, deletion variants E233P / L234V / L235A / G236del / S267K, pI variants N208D / Q295E / N384D / Q418E / N421D, and 428L / 434S a) the sushi domain is wild type (SEQ ID NO:4); c) the anti-PD-1 Fv domain comprises a VH that comprises a F32L amino acid substitution compared to SEQ ID NO:5, and a VL that comprises amino acid substitutions N27dH / K30Y / S93T compared to SEQ ID NO:6; and d) the IL-15 domain is D30N / E64Q / N65D.

[0251] Additional preferred embodiments utilize the Figure 28C format. In these embodiments, a) positive monomers including (i) scubariant S364K / E357Q, deletion variants E233P / L234V / L235A / G236del / S267K, and 428L / 434S FcRn variants, (ii) scubariant L368D / K370S, deletion variants E233P / L234V / L235A / G236del / S267K, pI variants N208D / Q295E / N384D / Q418E / N421D, and 428L / 434S FcRn variants, (iii) positive monomers including scubariant L368D / K370S, deletion variants E233P / L234V / L235A / G236del / S267K, pI variants N208D / Q295E / N384D / Q418E / N421D, and 428L / 434S FcRn variants, (iv) positive monomers including scubariant S364K / E357Q, deletion variants E233P / L234V / L235A / G236del / S267K, pI variants N208D / Q295E / N384D / Q418E / N421D, and 428L / 434S FcRn variants, (v) positive monomers including scubariant S364K / E357Q, deletion variants E233P / L234V / L235A / G236del / S267K, pI variants N208D / Q295E / N384D / Q418E / N421D, and 42 a) the sushi domain is wild type (SEQ ID NO: 4); c) the anti-PD-1 Fv domain is a combination of the VH and VL domains of H1_L1.1; and d) the IL-15 domain comprises D30N / E64Q / N65D.

[0252] In some embodiments, particularly useful sequences for the fusion proteins of the invention are based on the framework sequences of Figure 11 for the Figure 34C format. These are sequences of two monomers, "IL-15 complex side" and "Fab side", and do not include the IL-15 complex sequence or the VH of the Fab (light chain constant region sequence is in Figure 12).

[0253] Thus, the invention provides Scaffold 1 sequences in combination with the VH and VL sequences from FIG.

[0254] VI. Nucleic Acids of the Invention The present invention further provides nucleic acid compositions encoding (or, in the case of monomeric Fc domain proteins, nucleic acids encoding) the heterodimeric Fc fusion proteins of the present invention.

[0255] As will be appreciated by those skilled in the art, the nucleic acid composition will depend on the format of the heterodimeric IL-15 / Rα Fc fusion protein. Thus, for example, when a format requires three amino acid sequences, the three nucleic acid sequences can be incorporated into one or more expression vectors for expression. Similarly, some formats require only two nucleic acids, which can be incorporated into one or two expression vectors as well.

[0256] As known in the art, the nucleic acid encoding the components of the present invention can be incorporated into an expression vector, depending on the host cell used to produce the heterodimeric Fc fusion protein of the present invention, as known in the art. In general, the nucleic acid is operably linked to any number of regulatory elements (promoter, origin of replication, selectable marker, ribosome binding site, inducer, etc.). The expression vector can be an extrachromosomal vector or an integrating vector.

[0257] The nucleic acids and / or expression vectors of the invention are then transformed into any number of different types of host cells known in the art, including mammalian cells, bacterial cells, yeast cells, insect cells and / or fungal cells, although mammalian cells (e.g., CHO cells) are used in many embodiments.

[0258] In certain embodiments, the nucleic acids encoding each monomer are contained within a single expression vector, each generally under the control of a different or the same promoter, as applicable depending on the format. In embodiments of particular use in the present invention, each of these two or three nucleic acids is contained within a different expression vector.

[0259] The heterodimeric Fc fusion proteins of the present invention are produced by culturing host cells containing the expression vector(s), as is well known in the art. Once produced, conventional fusion protein or antibody purification steps, including ion exchange chromatography steps, are performed. As discussed herein, the pI of the two monomers differs by at least 0.5, which may allow for separation by ion exchange chromatography or isoelectric focusing, or other methods that are sensitive to isoelectric point. That is, by including pI substitutions that change the isoelectric point (pI) of each monomer so that each monomer has a different pI, and the heterodimer also has a different pI, it facilitates isoelectric purification of the heterodimer (e.g., anion exchange column, cation exchange column). These substitutions also aid in identifying and monitoring any contaminating homodimers after purification (e.g., IEF gels, cIEF, and analytical IEX columns).

[0260] VII. Biological and biochemical functionality of bispecific immune checkpoint antibody x IL-15 / IL-15Rα heterodimer immunomodulatory fusion proteins In general, the Fc-fusion protein of the present invention is administered to a patient with cancer, and efficacy is evaluated in several ways as described herein. Thus, while standard assays of efficacy can be performed, such as assessment of cancer burden, tumor size, presence or extent of metastasis, immuno-oncological treatment can also be evaluated based on immune status assessment. This can be done in several ways, including both in vitro and in vivo assays. For example, assessment of changes in immune status (e.g., presence of ICOS+CD4+T cells after ipi treatment) can be performed in conjunction with "classical" measurements such as tumor burden, size, invasiveness, LN involvement, metastasis, etc. Thus, any or all of the following can be evaluated: CD4 + T cell activation or proliferation, CD8 + T (CTL) cell activation or proliferation, CD8 +the inhibitory effect of the fusion protein on T cell-mediated cytotoxic activity and / or CTL-mediated cell depletion, NK cell activity and NK-mediated cell depletion, the enhancing effect of the fusion protein on Treg cell differentiation and proliferation, and Treg or myeloid-derived suppressor cell (MDSC)-mediated immunosuppression or immune tolerance, and / or the effect of the fusion protein on inflammatory cytokine production by immune cells (e.g., IL-2, IFN-γ or TNF-α production by T cells or other immune cells).

[0261] In some embodiments, evaluation of treatment is performed using, for example, CFSE dilution assays, Ki67 intracellular staining of immune effector cells, and 3 This is performed by assessing immune cell proliferation using the H-thymidine incorporation method.

[0262] In some embodiments, treatment is evaluated by assessing increased gene expression or increased protein levels of activation-associated markers, including one or more of CD25, CD69, CD137, ICOS, PD1, GITR, OX40, and cellular degranulation as measured by surface expression of CD107A.

[0263] In general, gene expression assays are performed as known in the art.

[0264] Generally, protein expression measurements are also performed as known in the art.

[0265] In some embodiments, the evaluation of the treatment is carried out by assessing cytotoxic activity, as measured by target cell viability detection through inferring a number of cellular parameters, such as enzyme activity (including protease activity), cell membrane permeability, cell adhesion, ATP production, coenzyme production, and nucleotide uptake activity. Specific examples of these assays include trypan blue or PI staining, 51 Cr or 35These include, but are not limited to, S release assays, LDH activity, MTT and / or WST assays, calcein-AM assays, luminescence-based assays, and others.

[0266] In some embodiments, evaluation of treatment is performed by assessing T cell activity as measured by cytokine production, either intracellularly, in culture supernatants, using cytokines including, but not limited to, IFNγ, TNFα, GM-CSF, IL2, IL6, IL4, IL5, IL10, IL13, using well known techniques.

[0267] Thus, evaluation of a treatment can be performed using assays that assess one or more of the following: (i) increasing the immune response, (ii) increasing αβ and / or γδ T cell activation, (iii) increasing cytotoxic T cell activity, (iv) increasing NK and / or NKT cell activity, (v) reducing αβ and / or γδ T cell suppression, (vi) increasing pro-inflammatory cytokine secretion, (vii) increasing IL-2 secretion, (viii) increasing interferon-γ production, (ix) increasing Th1 responses, (x) decreasing Th2 responses, (xi) decreasing or eliminating at least one of the cell numbers and / or activity of regulatory T cells (Tregs).

[0268] A. Assays for Measuring Competitive Binding In general, epitope binning assays as described herein, as well as other competitive inhibition assays as known in the art, can be performed to determine whether NCPD-1Fv competes with approved antibodies for binding to PD-1. Epitope binning is a process of testing antibodies in a pairwise combinatorial manner using competitive immunoassays, where antibodies that compete for the same binding region, i.e., the same or closely related epitopes of the antigen, are grouped into bins. Thus, antibodies that bind to epitopes different from nivolumab and / or pembrolizumab are considered to be non-competitive with nivolumab and / or pembrolizumab.

[0269] A non-competitive antibody can be determined by an assay in which the antibody or immunologically functional fragment being tested does not prevent or inhibit the specific binding of the reference antibody to a common antigen. Typically, such an assay involves the use of purified antigen (e.g., PD-1 or a domain or fragment thereof) bound to a solid surface or cell. Competitive inhibition is measured by determining the amount of the primary antibody bound to the solid surface or cell in the presence of a secondary antibody. Usually, when a competing antibody is present in excess, it inhibits the specific binding of the reference antibody to the common antigen by at least 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70% or 75%. In some cases, binding is inhibited by at least 80%, 85%, 90%, 95% or 97% or more. Conversely, when the reference antibody binds, it is preferred that it inhibits the binding of the subsequently added test antibody (i.e., PD-1 antibody) by at least 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70% or 75%. In some cases, binding of the test antibody is inhibited by at least 80%, 85%, 90%, 95% or 97% or more.

[0270] Generally, binning or competitive binding can be determined using various art-recognized techniques, such as immunoassays such as Western blot, radioimmunoassays, enzyme-linked immunosorbent assays (ELISA), "sandwich" immunoassays, immunoprecipitation assays, precipitation reactions, gel diffusion precipitation reactions, immunodiffusion assays, agglutination assays, complement fixation assays, immunoradiometric assays, fluorescent immunoassays and protein A immunoassays. Such immunoassays are conventional and well known in the art (see Ausubel et al, eds, (1994) Current Protocols in Molecular Biology, Vol.1, John Wiley&Sons, Inc., New York). In addition, cross-blocking assays can be used (see, for example, WO2003 / 48731, and Harlow et al. (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and David Lane).

[0271] Other techniques used to determine competitive inhibition (hence "bins") include surface plasmon resonance, e.g., using a BIAcore™ 2000 system (GE Healthcare), biolayer interferometry, e.g., using a ForteBio® Octet RED (ForteBio), or flow cytometry bead arrays, e.g., using a FACSCanto II (BD Biosciences) or multiplex LUMINEX™ detection assays (Luminex). One particular method for determining competitive binding using biolayer interferometry is provided in Examples 2 and 7 herein.

[0272] Luminex is a bead-based immunoassay platform that allows for large-scale multiplex antibody pairing. The assay compares the simultaneous binding patterns of antibody pairs to a target antigen. One antibody of the pair (the capture mAb) binds to a Luminex bead, and each capture mAb binds to a different colored bead. The other antibody (the detector mAb) binds to a fluorescent signal (e.g., phycoerythrin (PE)). The assay analyzes the simultaneous binding (pairing) of antibodies to the antigen and groups antibodies with similar pairing profiles. Similar profiles of the detector mAb and the capture mAb indicate that the two antibodies bind to the same or closely related epitopes. In one embodiment, the Pearson correlation coefficient can be used to determine the pairing profile to identify the antibody that most closely correlates with a particular antibody on the panel of antibodies being tested. In an embodiment, if the Pearson correlation coefficient of the antibody pair is at least 0.9, the test / detector mAb will be determined to be in the same bin as the reference / capture mAb. In other embodiments, the Pearson correlation coefficient is at least 0.8, 0.85, 0.87 or 0.89. In further embodiments, the Pearson correlation coefficient is at least 0.91, 0.92, 0.93, 0.94, 0.95, 0.96, 0.97, 0.98, 0.99 or 1. Other methods of analyzing data obtained from Luminex assays are described in U.S. Patent No. 8,568,992. The ability of Luminex to simultaneously analyze 100 (or more) types of beads provides an almost unlimited antigen and / or antibody surface, improving the throughput and resolution of antibody epitope profiling over biosensor assays (Miller, et al., 2011, PMID:21223970).

[0273] Similarly, binning techniques, including surface plasmon resonance, are compatible with the present invention. As used herein, "surface plasmon resonance" refers to an optical phenomenon that allows the analysis of specific interactions in real time by detecting changes in protein concentration within a biosensor matrix. Whether selected antibodies compete with each other for binding to a defined antigen can be easily determined using commercially available devices such as the BIAcore™ 2000 system.

[0274] In other embodiments, a technique that can be used to determine whether a test antibody "competes" for binding with a reference antibody is "biolayer interferometry," an optical analysis technique that analyzes the interference patterns of white light reflected from two surfaces, a layer of immobilized proteins on a biosensor chip, and an internal reference layer. Changes in the number of molecules that bind to the tip of the biosensor change the interference pattern, which can be measured in real time. Such a biolayer interferometry assay can be performed using a ForteBio® Octet RED machine as follows: A reference antibody (Ab1) is captured on an anti-mouse capture chip, a high concentration of unbound antibody is used to block the chip, and a baseline is collected. Monomeric recombinant target protein is then captured by the specific antibody (Ab1), and the chip is immersed in a well containing the same antibody (Ab1) as the reference, or a well containing a different test antibody (Ab2). If no further binding occurs, as determined by comparing the binding levels with the control Ab1, Ab1 and Ab2 are determined to be "competing" antibodies. If additional binding with Ab2 is observed, Ab1 and Ab2 are determined to not compete with each other. This process can be expanded to screen large libraries of unique antibodies, with entire rows of antibodies in a 96-well plate representing unique bins. In embodiments, a test antibody competes with a reference antibody if the reference antibody inhibits specific binding of the test antibody to a common antigen by at least 40%, 45%, 50%, 55%, 60%, 65%, 70% or 75%. In other embodiments, binding is inhibited by at least 80%, 85%, 90%, 95%, or 97% or more.

[0275] B. Assays for Measuring Efficacy In some embodiments, T cell activation is assessed using a mixed lymphocyte reaction (MLR) assay, as known in the art. Increased activity indicates immune stimulatory activity. Suitable increased activity is outlined below.

[0276] In one embodiment, the signal transduction pathway assay measures the increase or decrease of immune response, for example, by phosphorylation or dephosphorylation of different factors, or by measuring other post-translational modifications. Increased activity indicates immune stimulatory activity. Suitable increases in activity are outlined below.

[0277] In one embodiment, the signaling pathway assay measures an increase or decrease in activation of αβ and / or γδ T cells, e.g., as measured by cytokine secretion, or by proliferation, or by changes in expression of activation markers, such as, for example, CD25, CD69, CD137, CD107a, PD1, etc. Increased activity is indicative of immune stimulatory activity. Suitable increases in activity are outlined below.

[0278] In one embodiment, the signal transduction pathway assay measures the increase or decrease of cytotoxic T cell activity, for example, by direct killing of target cells, for example, cancer cells, or by cytokine secretion, or by proliferation, or by changes in the expression of activation markers, such as, for example, CD25, CD69, CD137, CD107a, PD1, etc. Increased activity indicates immune stimulatory activity. Suitable increases in activity are outlined below.

[0279] In one embodiment, the signal transduction pathway assay measures the increase or decrease of NK and / or NKT cell activity, for example, measured by direct killing of target cells, such as cancer cells, or by cytokine secretion, or by changes in the expression of activation markers, such as CD107a. Increased activity indicates immune stimulatory activity. Suitable increases in activity are outlined below.

[0280] In one embodiment, the signaling pathway assay measures an increase or decrease in αβ and / or γδ T cell suppression, e.g., as measured by cytokine secretion, or by proliferation, or by changes in expression of activation markers, such as, for example, CD137, CD107a, PD1, etc. Increased activity indicates immune stimulatory activity. Suitable increases in activity are outlined below.

[0281] In one embodiment, the signal transduction pathway assay measures the increase or decrease of pro-inflammatory cytokine secretion, for example, by ELISA, or by Luminex, or by multiplex bead-based method, or by intracellular staining and FACS analysis, or by Alispot, etc. Increased activity indicates immune stimulatory activity. Suitable increased activity is outlined below.

[0282] In one embodiment, the signal transduction pathway assay measures the increase or decrease of IL-2 secretion, for example, measured by ELISA, or by Luminex, or by Multiplex bead-based methods, or by intracellular staining and FACS analysis, or by Alispot, etc. Increased activity indicates immune stimulatory activity. Suitable increased activity is outlined below.

[0283] In one embodiment, the signal transduction pathway assay measures the increase or decrease of interferon-gamma production, for example, measured by ELISA, or by Luminex, or by Multiplex bead-based method, or by intracellular staining and FACS analysis, or by Alispot, etc. Increased activity indicates immune stimulatory activity. Suitable increased activity is outlined below.

[0284] In one embodiment, the signal transduction pathway assay measures the increase or decrease of Th1 response, for example, as measured by cytokine secretion or by changes in the expression of activation markers. Increased activity indicates immune stimulatory activity. Suitable increased activity is outlined below.

[0285] In one embodiment, the signal transduction pathway assay measures the increase or decrease of Th2 response, for example, as measured by cytokine secretion or by changes in the expression of activation markers. Increased activity indicates immune stimulatory activity. Suitable increased activity is outlined below.

[0286] In one embodiment, the signal transduction pathway assay measures the increase or decrease in the number and / or activity of at least one of regulatory T cells (Treg), for example, measured by flow cytometry or by IHC. A decrease in response indicates immune stimulatory activity. The appropriate decrease is the same as the increase outlined below.

[0287] In one embodiment, the signal transduction pathway assay measures the increase or decrease in M2 macrophage cell number, for example, by flow cytometry or by IHC. A decrease in response indicates immune stimulatory activity. The appropriate decrease is the same as the increase outlined below.

[0288] In one embodiment, the signal transduction pathway assay measures the increase or decrease of M2 macrophage tumorigenicity-promoting activity, for example, as measured by cytokine secretion or by changes in expression of activation markers. A decrease in response indicates immune stimulatory activity. Suitable decreases are the same as increases as outlined below.

[0289] In one embodiment, the signal transduction pathway assay measures an increase or decrease in N2 neutrophilia, for example, by flow cytometry or by IHC. A decrease in response indicates immune stimulatory activity. The appropriate decrease is the same as the increase outlined below.

[0290] In one embodiment, the signal transduction pathway assay measures the increase or decrease of N2 neutrophil tumorigenicity-promoting activity, for example, as measured by cytokine secretion or by changes in expression of activation markers. A decrease in response indicates immune stimulatory activity. Suitable decreases are the same as increases as outlined below.

[0291] In one embodiment, the signal transduction pathway assay measures the increase or decrease of inhibition of T cell activation, for example, by cytokine secretion, or by proliferation, or by changes in the expression of activation markers, such as, for example, CD137, CD107a, PD1, etc. Increased activity indicates immune stimulatory activity. Suitable increased activity is outlined below.

[0292] In one embodiment, the signal transduction pathway assay measures the increase or decrease of inhibition of CTL activation, for example, by direct killing of target cells, for example, cancer cells, or by cytokine secretion, or by proliferation, or by changes in the expression of activation markers, for example, CD137, CD107a, PD1, etc. Increased activity indicates immune stimulatory activity. Suitable increased activity is outlined below.

[0293] In one embodiment, the signal transduction pathway assay measures an increase or decrease in αβ and / or γδ T cell depletion, for example as measured by changes in expression of activation markers. A decrease in response indicates immune stimulatory activity. Suitable decreases are the same as increases as outlined below.

[0294] In one embodiment, the signaling pathway assay measures an increase or decrease in αβ and / or γδ T cell responses, e.g., as measured by cytokine secretion, or by proliferation, or by changes in expression of activation markers, such as, for example, CD137, CD107a, PD1, etc. Increased activity indicates immune stimulatory activity. Suitable increases in activity are outlined below.

[0295] In one embodiment, the signaling pathway assay measures increased or decreased stimulation of an antigen-specific memory response, e.g., as measured by cytokine secretion, or by proliferation, or by changes in expression of activation markers such as, e.g., CD45RA, CCR7, etc. Increased activity is indicative of immune stimulatory activity. Suitable increases in activity are outlined below.

[0296] In one embodiment, the signal transduction pathway assay measures the increase or decrease of apoptosis or lysis of cancer cells, for example, by cytotoxicity assay such as MTT, Cr release, calcine AM, or by flow cytometry-based assay such as CFSE dilution or propidium iodide staining. Increased activity indicates immune stimulating activity. Suitable increased activity is outlined below.

[0297] In one embodiment, the signal transduction pathway assay measures the increase or decrease of the stimulation of the cytotoxic or cytostatic effect of cancer cells, for example, by cytotoxicity assay such as MTT, Cr release, calcine AM, or by flow cytometry-based assay such as CFSE dilution or propidium iodide staining.The increase in activity indicates immune stimulating activity.Suitable increase in activity is outlined below.

[0298] In one embodiment, the signal transduction pathway assay measures the increase or decrease of direct killing of cancer cells, for example, by cytotoxicity assay such as MTT, Cr release, calcine AM, or by flow cytometry-based assay such as CFSE dilution or propidium iodide staining. Increased activity indicates immune stimulatory activity. Suitable increased activity is outlined below.

[0299] In one embodiment, the signal transduction pathway assay measures the increase or decrease of Th17 activity, for example, by cytokine secretion, or by proliferation, or by the change in expression of activation markers.The increase in activity indicates immune stimulatory activity.Suitable increase in activity is outlined below.

[0300] In one embodiment, the signal transduction pathway assay measures the increase or decrease of the induction of complement-dependent cytotoxicity and / or antibody-dependent cell-mediated cytotoxicity, for example, by cytotoxicity assays such as MTT, Cr release, calcine AM, or by flow cytometry-based assays such as CFSE dilution or propidium iodide staining. Increased activity indicates immune stimulatory activity. Suitable increased activity is outlined below.

[0301] In one embodiment, T cell activation is measured by direct killing of target cells, e.g., cancer cells, or by cytokine secretion, or by proliferation, or by changes in expression of activation markers, examples of which include CD137, CD107a, PD1, etc. For T cells, increases in proliferation, cell surface markers of activation (e.g., CD25, CD69, CD137, PD1), cytotoxicity (ability to kill target cells), and cytokine production (e.g., IL-2, IL-4, IL-6, IFNγ, TNF-a, IL-10, IL-17A) may indicate immune modulation consistent with enhanced cancer cell killing.

[0302] In one embodiment, NK cell activation is measured, for example, by direct killing of target cells, e.g., cancer cells, or by cytokine secretion, or by changes in expression of activation markers, such as, for example, CD107a. For NK cells, proliferation, cytotoxicity (ability to kill target cells and increase CD107a, granzyme, and perforin expression), cytokine production (e.g., IFNγ and TNF), and increased cell surface receptor expression (e.g., CD25) can be indicators of immune modulation consistent with enhanced killing of cancer cells.

[0303] In one embodiment, γδ T cell activation is measured, for example, by cytokine secretion, or by proliferation, or by changes in expression of activation markers.

[0304] In one embodiment, Th1 cell activation is measured, for example, by cytokine secretion or by changes in expression of activation markers.

[0305] A suitable increase (or decrease, as appropriate as outlined above) in activity or response is at least a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 98-99% percent increase compared to the signal in either a reference or control sample, e.g., a test sample that does not contain an anti-PVRIG antibody of the invention. Similarly, an increase of at least 1-, 2-, 3-, 4- or 5-fold compared to a reference subject or control sample indicates efficacy.

[0306] VIII. Checkpoint Blockade Antibodies The PD-1-targeting IL-15 / Rα-Fc fusion proteins of the invention described herein are combined with other therapeutic agents including checkpoint blockade antibodies such as, but not limited to, PD-1 inhibitors, TIM3 inhibitors, CTLA4 inhibitors, PD-L1 inhibitors, TIGIT inhibitors, LAG3 inhibitors, or combinations thereof.

[0307] In addition to the antibodies discussed below, additional disclosure is found in USSN 62 / 784,334, which is incorporated by reference in its entirety, particularly for its discussion of checkpoint antibodies for use in combination.

[0308] A. Anti-PD1 antibody In some embodiments, the PD-1-targeting IL-15 / Rα-Fc fusion proteins described herein can be administered to a subject with cancer in combination with a checkpoint blockade antibody, e.g., an anti-PD-1 antibody or an anti-PD-L1 antibody.

[0309] In many embodiments, the PD-1 inhibitor is an anti-PD-1 antibody selected from those that do not compete for binding with the anti-PD-1 non-competing Fv sequences outlined herein. Particularly useful are anti-PD-1 antibodies approved for human use in the United States or abroad, including but not limited to nivolumab and pembrolizumab.

[0310] In some embodiments, the anti-PD-1 antibody is nivolumab. Other names for nivolumab include MDX-1106, MDX-1106-04, ONO-4538, or BMS-936558. In some embodiments, the anti-PD-1 antibody is nivolumab (CAS Registry Number: 946414-94-4). Nivolumab is a fully human IgG4 monoclonal antibody that specifically blocks PD1. Nivolumab (clone 5C4) and other human monoclonal antibodies that specifically bind to PD1 are disclosed in US8,008,449 and WO2006 / 121168. In one embodiment, the inhibitor of PD-1 is nivolumab and has a sequence disclosed herein (or a sequence substantially identical or similar thereto, e.g., a sequence at least 85%, 90%, 95% or more identical to the specified sequence).

[0311] In some embodiments, the anti-PD-1 antibody is pembrolizumab. Pembrolizumab (also called lambrolizumab, MK-3475, MK03475, SCH-900475, or KEYTRUDA®; Merck) is a humanized IgG4 monoclonal antibody that binds to PD-1. Pembrolizumab and other humanized anti-PD-1 antibodies are disclosed in Hamid, O. et al. (2013) New England Journal of Medicine 369(2):134-44, US8,354,509, and WO2009 / 114335.

[0312] Other anti-PD1 antibodies include, inter alia, AMP514 (Amplimmune), e.g., the anti-PD1 antibodies disclosed in US8,609,089, US2010 / 028330, and / or US2012 / 0114649.

[0313] In some embodiments, the PD-1 inhibitor is an immunoadhesin (e.g., an immunoadhesin that includes an extracellular or PD-1 binding portion of PD-L1 or PD-L2 fused to a constant region (e.g., an Fc region of an immunoglobulin sequence). In some embodiments, the PD-1 inhibitor is AMP-224 (B7-DCIg; Amplimmune; disclosed, for example, in WO2010 / 027827 and WO2011 / 066342), a PD-L2 Fc fused soluble receptor that blocks the interaction between PD-1 and B7-H1.

[0314] In some embodiments, anti-PD-1 antibodies may be used in combination with the IL-15 / Rα×anti-PD1 heterodimer Fc fusion proteins of the present invention. There are several anti-PD-1 antibodies, including but not limited to, two currently FDA approved antibodies, pembrolizumab and nivolizumab, as well as currently, but not limited to, tislelizumab, Sym021, REGN2810 (developed by Rengeneron), JNJ-63723283 (developed by J and J), SHR-1210, pidilizumab, AMP-224, MEDio680, PDR001, and CT-001, and others reviewed in Liu et al., J. Hemat. & Oncol. (2017) 10:136, which antibodies are expressly incorporated by reference.

[0315] B. Anti-TIM3 antibody The IL-15 / Rα×[NC]PD-1 heterodimer fusion proteins of the invention can also be co-administered with an anti-TIM-3 antibody. Exemplary, non-limiting anti-TIM-3 antibody molecules are disclosed in US2015 / 0218274, published August 6, 2015, entitled "Antibody Molecules to TIM-3 and Uses Thereof," which is incorporated by reference in its entirety.

[0316] There are several TIM-3 antibodies in clinical development, including but not limited to MBG453, Sym023, BGB-A425 and TSR-022.

[0317] In one embodiment, the anti-TIM-3 antibody molecule comprises at least one or two heavy chain variable domains (optionally including a constant region), at least one or two light chain variable domains (optionally including a constant region), or both, and is selected from the group consisting of ABTIM3, ABTIM3-hum01, ABTIM3-hum02, ABTIM3-hum03, ABTIM3-hum04, ABTIM3-hum05, ABTIM3-hum06, ABTIM3-hum07, ABTIM3-hum08, ABTIM3-hum09, ABTIM3-hum10, ABTIM3-hum11, ABTIM3-hum12, ABTIM3-hum13, ABTIM3-hum14, ABTIM3-hum15, ABTIM3-hum16, ABTIM3-hum17, ABTIM3-hum18, ABTIM3-hum19, ABTIM3-hum20, ABTIM3-hum21, ABTIM3-hum22, ABTIM3-hum23, ABTIM3-hum24, ABTIM3-hum25, ABTIM3-hum26, ABTIM3-hum27, ABTIM3-hum28, ABTIM3-hum29, ABTIM3-hum30, ABTIM3-hum31, ABTIM3-hum32, ABTIM3-hum33, ABTIM3-hum34, ABTIM3-hum35, ABTIM3-hum36, ABTIM3-hum37, ABTIM3-hum38, ABTIM3-hum39, ABTIM3-hum40, ABTIM3-hum41, ABTIM3-hum42, ABTIM3-hum43, ABTIM3-hum44, ABTIM3-hum45, ABTIM3-hum46, ABTIM3-hum47, ABTIM3-hum48, ABTIM3-hum49, ABTIM3 The anti-TIM-3 antibody molecule optionally includes a leader sequence from the heavy chain, light chain, or both, or a sequence substantially identical thereto, as set forth in US2015 / 0218274.

[0318] In yet another embodiment, the anti-TIM-3 antibody molecule is an antibody described herein, ABTIM3, ABTIM3-hum01, ABTIM3-hum02, ABTIM3-hum03, ABTIM3-hum04, ABTIM3-hum05, ABTIM3-hum06, ABTIM3-hum07, ABTIM3-hum08, ABTIM3-hum09, ABTIM3-hum10, ABTIM3-hum11, ABTIM3-hum12, ABTIM3-hum13, ABTIM3-hum14, ABTIM3-hum15, ABTIM3-hum16, ABTIM3-hum17, ABTIM3-hum1 or as described in Tables 1-4 of US2015 / 0218274, or encoded by the nucleotide sequence of Tables 1-4, or comprising at least one, two, or three complementarity determining regions (CDRs) from the heavy chain variable region and / or light chain variable region of a sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical) to any of the aforesaid sequences.

[0319] In yet another embodiment, the anti-TIM-3 antibody molecule comprises at least one, two, or three CDRs (or collectively all of the CDRs) from a heavy chain variable region comprising an amino acid sequence shown in Tables 1-4 of US2015 / 0218274 or encoded by the nucleotide sequence shown in Tables 1-4. In one embodiment, one or more of the CDRs (or collectively all of the CDRs) have one, two, three, four, five, six or more alterations, e.g., amino acid substitutions or deletions, relative to the amino acid sequence shown in Tables 1-4 or encoded by the nucleotide sequence shown in Tables 1-4.

[0320] In yet another embodiment, the anti-TIM-3 antibody molecule comprises at least one, two, or three CDRs (or collectively all of the CDRs) from a light chain variable region comprising an amino acid sequence as set forth in Tables 1-4 of US2015 / 0218274 or encoded by the nucleotide sequence set forth in Tables 1-4. In one embodiment, one or more of the CDRs (or collectively all of the CDRs) have one, two, three, four, five, six or more alterations, e.g., amino acid substitutions or deletions, relative to the amino acid sequence as set forth in Tables 1-4 or encoded by the nucleotide sequence set forth in Tables 1-4. In certain embodiments, the anti-TIM-3 antibody molecule comprises a substitution in a light chain CDR, e.g., one or more substitutions in CDR1, CDR2 and / or CDR3 of the light chain.

[0321] In another embodiment, the anti-TIM-3 antibody molecule comprises at least one, two, three, four, five or six CDRs (or collectively all of the CDRs) from the heavy and light chain variable regions comprising the amino acid sequences shown in Tables 1-4 of US2015 / 0218274 or encoded by the nucleotide sequences shown in Tables 1-4. In one embodiment, one or more of the CDRs (or collectively all of the CDRs) have one, two, three, four, five, six or more alterations, e.g., amino acid substitutions or deletions, relative to the amino acid sequences shown in Tables 1-4 or encoded by the nucleotide sequences shown in Tables 1-4.

[0322] C. Anti-CTLA4 antibody The IL-15 / Rα×[NC]PD-1 heterodimer fusion protein of the present invention can also be co-administered with an anti-CTLA-4 antibody. Suitable anti-CTLA-4 antibodies for use in combination therapy as outlined herein include, but are not limited to, the currently FDA approved antibody ipilimumab, as well as several more including CP-675,206 and AGEN-1884. Exemplary anti-CTLA4 antibodies include tremelimumab (an IgG2 monoclonal antibody available from Pfizer, formerly known as ticilimumab, CP-675,206), and dim (a CTLA-4 antibody, also known as MDX-010, CAS number 477202-00-9). Other exemplary anti-CTLA-4 antibodies are disclosed, for example, in U.S. Pat. No. 5,811,097.

[0323] In one embodiment, the anti-CTLA4 antibody is ipilimumab, e.g., as disclosed in US5,811,097, US7,605,238, WO00 / 32231, and WO97 / 20574, and has a sequence disclosed herein (or a sequence substantially identical or similar thereto, e.g., a sequence that is at least 85%, 90%, 95% or more identical to the specified sequence).

[0324] In one embodiment, the anti-CTLA4 antibody is tremelimumab, e.g., as disclosed in US 6,682,736 and WO 00 / 37504, and has a sequence disclosed herein (or a sequence substantially identical or similar thereto, e.g., a sequence at least 85%, 90%, 95% identical or more to the specified sequence).

[0325] D. Anti-PD-L1 antibody The IL-15 / Rα×[NC]PD-1 heterodimer fusion proteins of the invention can also be co-administered with an anti-PD-L1 antibody. Exemplary, non-limiting anti-PD-L1 antibody molecules are disclosed in US2016 / 0108123, published April 21, 2016, entitled "Antibody Molecules to PD-L1 and Uses Thereof," which is incorporated by reference in its entirety.

[0326] In one embodiment, the anti-PD-L1 antibody molecule comprises at least one or two heavy chain variable domains (optionally including a constant region), at least one or two light chain variable domains (optionally including a constant region), or both, and is selected from the group consisting of BAP058-hum01, BAP058-hum02, BAP058-hum03, BAP058-hum04, BAP058-hum05, BAP058-hum06, BAP058-hum07, BAP058-hum08, BAP058-hum09, BAP058-hum10, BAP058-hum11, BAP058-hum12, BAP058-hum13, BAP The amino acid sequence of BAP058-hum14, BAP058-hum15, BAP058-hum16, BAP058-hum17, BAP058-Clone-K, BAP058-Clone-L, BAP058-Clone-M, BAP058-Clone-N, or BAP058-Clone-O, as set forth in Table 1 of US2016 / 0108123 or encoded by the nucleotide sequence of Table 1, or a sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical) to any of the foregoing sequences.

[0327] In yet another embodiment, the anti-PD-L1 antibody molecule is an antibody described herein, BAP058-hum01, BAP058-hum02, BAP058-hum03, BAP058-hum04, BAP058-hum05, BAP058-hum06, BAP058-hum07, BAP058-hum08, BAP058-hum09, BAP058-hum10, BAP058-hum11, BAP058-hum12, BAP058-hum13, BAP058-hum14, BAP058-hum15, BAP058-hum16, BAP058-hum17, BAP058-Clone- or an antibody selected from any of BAP058-Clone-K, BAP058-Clone-L, BAP058-Clone-M, BAP058-Clone-N, or BAP058-Clone-O; or as described in Table 1 of US2016 / 0108123, or encoded by the nucleotide sequence of Table 1, or comprising at least one, two, or three complementarity determining regions (CDRs) from a heavy chain variable region and / or a light chain variable region of a sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical) to any of the aforesaid sequences.

[0328] In some embodiments, the PD-L1 inhibitor is an antibody molecule. In some embodiments, the anti-PD-L1 inhibitor is selected from YW243.55.S70, MPDL3280A, MEDI-4736, MSB-0010718C, MDX-1105, atezolizumab, darvalumab, avelumab, or BMS936559.

[0329] In some embodiments, the anti-PD-L1 antibody is atezolizumab. Atezolizumab (also referred to as MPDL3280A and Atezo®; Roche) is a monoclonal antibody that binds to PD-L1. Atezolizumab and other humanized anti-PD-L1 antibodies are disclosed in US 8,217,149 and have sequences disclosed herein (or sequences substantially identical or similar thereto, e.g., sequences that are at least 85%, 90%, 95% or more identical to the specified sequences).

[0330] In some embodiments, the anti-PD-L1 antibody is avelumab. Avelumab (A09-246-2; also known as Merck Serono) is a monoclonal antibody that binds to PD-L1. Avelumab and other humanized anti-PD-L1 antibodies are disclosed in US9,324,298 and WO2013 / 079174 and have sequences disclosed herein (or sequences substantially identical or similar thereto, e.g., sequences that are at least 85%, 90%, 95% or more identical to the specified sequences).

[0331] In some embodiments, the anti-PD-L1 antibody is durvalumab. Durvalumab (also referred to as MEDI4736; AstraZeneca) is a monoclonal antibody that binds to PD-L1. Durvalumab and other humanized anti-PD-L1 antibodies are disclosed in US 8,779,108 and have sequences disclosed herein (or sequences substantially identical or similar thereto, e.g., sequences that are at least 85%, 90%, 95% or more identical to the specified sequences).

[0332] In some embodiments, the anti-PD-L1 antibody is BMS-936559. BMS-936559 (also referred to as MDX-1105; BMS) is a monoclonal antibody that binds to PD-L1. BMS-936559 and other humanized anti-PD-L1 antibodies are disclosed in US 7,943,743 and WO 2007 / 005874 and have sequences disclosed herein (or sequences substantially identical or similar thereto, e.g., sequences that are at least 85%, 90%, 95% or more identical to the specified sequences).

[0333] In some embodiments, anti-PD-L1 antibodies may be used in combination with the IL-15 / Rα×anti-PD1 heterodimeric Fc fusion proteins of the present invention.There are several anti-PD-L1 antibodies currently available, including three FDA approved antibodies, atezolizumab, avelumab, durvalumab, and others reviewed in Liu et al., J. Hemat. & Oncol. (2017) 10:136, which antibodies are expressly incorporated by reference.

[0334] In some embodiments, the PD-1-targeting IL-15 / Rα-Fc fusion proteins described herein may be used in combination with a PD-L1 or PD-L2 inhibitor (e.g., an anti-PD-L1 antibody).

[0335] E. Anti-TIGIT antibody The IL-15 / Rα×[NC]PD-1 heterodimer fusion protein of the present invention can also be co-administered with an anti-TIGIT antibody. In some embodiments, the anti-TIGIT antibody is OMP-313M32. OMP-313M32 (OncoMed Pharmaceuticals) is a monoclonal antibody that binds to TIGIT. OMP-313M32 and other humanized anti-TIGIT antibodies have sequences disclosed in US2016 / 0376365 and WO2016 / 191643 and disclosed herein (or sequences substantially identical or similar thereto, e.g., sequences that are at least 85%, 90%, 95% or more identical to the specified sequences).

[0336] In some embodiments, the anti-TIGIT antibody is BMS-986207. BMS-986207 (also called ONO-4686; Bristol-Myers Squibb) is a monoclonal antibody that binds to TIGIT. BMS-986207 and other humanized anti-TIGIT antibodies are disclosed in US2016 / 0176963 and WO2016 / 106302 and have sequences disclosed herein (or sequences substantially identical or similar thereto, e.g., sequences that are at least 85%, 90%, 95% or more identical to the specified sequences).

[0337] In some embodiments, the anti-TIGIT antibody is MTIG7192. MTIG7192 (Genentech) is a monoclonal antibody that binds to TIGIT. MTIG7192 and other humanized anti-TIGIT antibodies have sequences disclosed in US2017 / 088613, WO2017 / 053748 and WO2016 / 011264 and disclosed herein (or sequences substantially identical or similar thereto, e.g., sequences that are at least 85%, 90%, 95% or more identical to the specified sequences).

[0338] In some embodiments, anti-TIGIT antibodies may be used in combination with the IL-15 / Rα×anti-PD1 heterodimeric Fc proteins of the present invention. Several TIGIT antibodies are in clinical development: BMS-986207, OMP-313M32, and MTIG7192A.

[0339] F. Anti-LAG-3 antibody The IL-15 / Rα×[NC]PD-1 heterodimer fusion proteins of the invention can also be co-administered with an anti-LAG-3 antibody. Exemplary, non-limiting anti-LAG-3 antibody molecules are disclosed in US2015 / 0259420, published September 17, 2015, entitled "Antibody Molecules to LAG-3 and Uses Thereof," which is incorporated by reference in its entirety.

[0340] In one embodiment, the anti-LAG-3 antibody molecule is selected from the group consisting of BAP050-hum01, BAP050-hum02, BAP050-hum03, BAP050-hum04, BAP050-hum05, BAP050-hum06, BAP050-hum07, BAP050-hum08, BAP050-hum09, BAP050-hum10, BAP050-hum11, BAP050-hum12, BAP050-hum13, BAP050-hum14, BAP050-hum15, BAP050-hum16, BAP050-hum17, BAP050-hum18, BAP050-hum19, BAP050-hum20, BAP050-hum21, BAP050-hum22, BAP050-hum23, BAP050-hum24, BAP050-hum25, BAP050-hum26, BAP050-hum27, BAP050-hum28, BAP050-hum29, BAP050-hum30, BAP050-hum31, BAP050-hum32, BAP050-hum33, BAP050-hum34, BAP050-hum35, BAP050-hum36, BAP050-hum37, BAP050-hum38, BAP050-hum39, BAP050-hum40, BAP050-hum41, BAP050-hum42, BAP050-hum43, BAP050-hum44, BAP050-hum45, BAP050-hum46, BAP050-hum47, BAP050-hum48, BAP050-hum49, B AP050-hum17, BAP050-hum18, BAP050-hum19, BAP050-hum20, huBAP050(Ser) (e.g. BAP050-hum01-Ser, BAP050-hum02-Ser, BAP050-hum03 -Ser, BAP050-hum04-Ser, BAP050-hum05-Ser, BAP050-hum06-Ser, BAP050-hum07-Ser, BAP050-hum08-Ser, BAP050-hum09-Ser, BAP050-h um10-Ser, BAP050-hum11-Ser, BAP050-hum12-Ser, BAP050-hum13-Ser, BAP050-hum14-Ser, BAP050-hum15-Ser, BAP050-hum18-Ser, BAP0 50-hum19-Ser, or BAP050-hum20-Ser), BAP050-Clone-F, BAP050-Clone-G, BAP050-Clone-H, BAP050-Clone-I, or BAP050-Clone-J, or At least one or two heavy chain variable domains (optionally including constant regions), at least one or two light chain variable domains (optionally including constant regions), or both, comprising any of the amino acid sequences set forth in Table 1 of US2015 / 0259420, or encoded by the nucleotide sequence of Table 1, or a sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical) to any of the foregoing sequences.

[0341] In yet another embodiment, the anti-LAG-3 antibody molecule is, for example, BAP050-hum01, BAP050-hum02, BAP050-hum03, BAP050-hum04, BAP050-hum05, BAP050-hum06, BAP050-hum07, BAP050-hum08, BAP050-hum09, BAP050-hum10, BAP050-hum11, BAP050-hum12, BAP050-hum13, BAP050-hum14, BAP050-hum15, BAP050-hum16, BAP050-hum17, BAP050-hum18, BAP050-hum19, BAP050-hum20, BAP050-hum21, BAP050-hum22, BAP050-hum23, BAP050-hum24, BAP050-hum25, BAP050-hum26, BAP050-hum27, BAP050-hum28, BAP050-hum29, BAP050-hum30, BAP050-hum31, BAP050-hum32, BAP050-hum33, BAP050-hum34, BAP050-hum35, BAP050-hum36, BAP050-hum37, BAP050-hum38, BAP050-hum39, BAP050-hum40, BAP050-hum41, BAP050-hum42, BAP050-hum43, BAP050-hum44, BAP050-hum45, BAP050-hum46, BAP050-hum47, BAP050-hum48, BAP050-hum49, BAP 50-hum16, BAP050-hum17, BAP050-hum18, BAP050-hum19, BAP050-hum20, huBAP050(Ser) (e.g., BAP050-hum01-Ser, BAP050-hum02-Ser, BAP050-hum03-Ser, BAP050-hum04-Ser, BAP050-hum05-Ser, BAP050-hum06-Ser, BAP050-hum07-Ser, BAP050-hum08-Ser, BAP050-hum09 -Ser, BAP050-hum10-Ser, BAP050-hum11-Ser, BAP050-hum12-Ser, BAP050-hum13-Ser, BAP050-hum14-Ser, BAP050-hum15-Ser, BAP050 -hum18-Ser, BAP050-hum19-Ser, or BAP050-hum20-Ser), BAP050-Clone-F, BAP050-Clone-G, BAP050-Clone-H, BAP050-Clone-I, or BA P050-Clone-J, or any of the amino acid sequences set forth in Table 1 of US2015 / 0259420, or encoded by the nucleotide sequence of Table 1, or substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical) to any of the foregoing sequences.

[0342] In some embodiments, the anti-LAG-3 antibody is BMS-986016. BMS-986016 (also referred to as BMS986016; Bristol-Myers Squibb) is a monoclonal antibody that binds to LAG-3. BMS-986016 and other humanized anti-LAG-3 antibodies are disclosed in US2011 / 0150892, WO2010 / 019570, and WO2014 / 008218.

[0343] In some embodiments, the anti-LAG3 antibody is LAG525. LAG525 (also called IMP701; Novartis) is a monoclonal antibody that binds to LAG3. LAG525 and other humanized anti-LAG3 antibodies are disclosed in US9,244,059 and WO2008 / 132601 and have sequences disclosed herein (or sequences substantially identical or similar thereto, e.g., sequences that are at least 85%, 90%, 95% or more identical to the specified sequences).

[0344] Other exemplary anti-LAG-3 antibodies are disclosed, for example, in US2011 / 150892 and US2018 / 066054.

[0345] In some embodiments, anti-LAG-3 antibodies may be used in combination with the IL-15 / Rα×anti-PD1 bifunctional heterodimer fusion proteins of the present invention. There are several anti-LAG-3 antibodies in clinical development, including REGN3767 and TSR-033 (Tesaro) by Regeneron.

[0346] IX. Treatment Once produced, the compositions of the invention are generally used in a number of oncology applications, including cancer treatment by promoting T cell activation (e.g., T cells are no longer suppressed) using binding of the heterodimeric Fc fusion proteins of the invention.

[0347] Thus, the heterodimeric Fc protein compositions of the present invention are used to treat these cancers.

[0348] A. Management In this context, administered "in combination" as used herein means that two treatments are provided to a subject during the course of the subject's suffering from a disorder, for example, two or more treatments are provided after the subject is diagnosed with a disorder, but before the disorder is cured or resolved, or before the treatment is discontinued for other reasons. In some embodiments, the delivery of one treatment is still occurring when the delivery of the second treatment begins, so there is an overlap in terms of administration. This is sometimes referred to herein as "simultaneous" or "co-delivery". In other embodiments, the delivery of one treatment ends before the delivery of the other treatment begins. In some embodiments in either case, the treatments are more effective due to the combined administration. For example, the second treatment is more effective, for example, a comparable effect is seen with less of the second treatment, or the second treatment reduces symptoms to a greater extent than would be seen if the second treatment were administered in the absence of the first treatment, or a similar situation is seen with the first treatment. In some embodiments, the delivery is such that the reduction in symptoms, or other parameters associated with the disorder, is greater than that observed with one treatment delivered in the absence of the other. The effects of the two treatments can be partially additive, fully additive, or greater than additive. Delivery can be such that the effect of the first treatment delivered is still detectable when the second treatment is delivered.

[0349] B. Formulations for in vivo administration Formulations of antibodies used in accordance with the present invention are prepared for storage in the form of lyophilized formulations or aqueous solutions by mixing the antibody of the desired purity with optional pharma- ceutically acceptable carriers, excipients, or stabilizers (as reviewed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed.

[1980] ).

[0350] The PD-1-targeted IL-15 / Rα-Fc fusion proteins disclosed herein and chemotherapeutic agents of the invention are administered to a subject according to known methods, such as intravenous administration as a bolus or by continuous infusion over a period of time.

[0351] All cited references are expressly incorporated by reference herein in their entirety.

[0352] While particular embodiments of the present invention have been described above for purposes of illustration, those skilled in the art will appreciate that many changes in detail may be made without departing from the invention as set forth in the appended claims.

[0353] X. Example Examples are provided below to illustrate the present invention. These examples are not meant to limit the present invention to any particular application or theory of operation. For all constant region positions discussed in this invention, the numbering follows the EU index as in Kabat (Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th Ed., United States Public Health Service, National Institutes of Health, Bethesda, incorporated by reference in its entirety). Those skilled in the art of antibodies will understand that this rule consists of non-contiguous numbering in certain regions of the immunoglobulin sequence, allowing a standard reference to conserved positions in the immunoglobulin family. Thus, the position of any given immunoglobulin as defined by the EU index does not necessarily correspond to its contiguous sequence.

[0354] The general and specific science and technology are outlined in U.S. Patent Application Publication Nos. 2015 / 0307629, 2014 / 0288275, and WO2014 / 145806, all of which are expressly incorporated by reference in their entirety, particularly with respect to the technology outlined therein. Examples 1 and 2 of U.S. Patent Application No. 62,416,087, filed November 1, 2016, are expressly incorporated by reference in their entirety, including the corresponding figures.

[0355] A. Example 1: IL-15 / Rα-Fc 1. Engineering of IL-15 / Rα-Fc Fusion Protein To address the short half-life of the IL-15 / IL-15Rα heterodimer, the inventors generated IL-15 / IL-15Rα (sushi) complexes as Fc fusions (collectively referred to herein as "IL-15 / Rα-Fc fusion proteins") to facilitate production and promote FcRn-mediated recycling and prolong the half-life of the complex.

[0356] Plasmids encoding IL-15 or IL-15Rα sushi domains were constructed by standard gene synthesis followed by subcloning into pTT5 expression vectors containing Fc fusion partners (e.g., the constant regions shown in Figure 10). Cartoonized schematics of exemplary IL-15 / Rα-Fc fusion protein formats are shown in Figures 13A-G.

[0357] An exemplary protein in the IL-15 / Rα-hetero-Fc format (FIG. 13A) is XENP20818, the sequence of which is shown in FIG. 14, along with the sequences of additional proteins in this format. An exemplary protein in the scIL-15 / Rα-Fc format (FIG. 13B) is XENP21478, the sequence of which is shown in FIG. 15. An exemplary protein in the ncIL-15 / Rα-Fc format (FIG. 13C) is XENP21479, the sequence of which is shown in FIG. 16.

[0358] The protein was produced by transient transfection in HEK293E cells and purified by a two-step purification process involving protein A and ion exchange chromatography.

[0359] Exemplary IL-15 / Rα-Fc fusion proteins in scIL-15 / Rα-Fc format (XENP21478) and ncIL-15 / Rα-Fc format (XENP21479) were tested in a cell proliferation assay. Human PBMCs were treated with test substances at the indicated concentrations. Four days after treatment, PBMCs were stained with anti-CD8-FITC (RPA-T8), anti-CD4-PerCP / Cy5.5 (OKT4), anti-CD27-PE (M-T271), anti-CD56-BV421 (5.1H11), anti-CD16-BV421 (3G8), and anti-CD45RA-BV605 (Hi100) and gated for the following cell types: CD4 + T cells, CD8 + T cells, and NK cells (CD56+ / CD16+). Ki67 is a protein strictly associated with cell proliferation, and staining for intracellular Ki67 was performed using anti-Ki67-APC (Ki-67) and Foxp3 / transcription factor staining buffer set (Thermo Fisher Scientific, Waltham, Mass.). The percentage of Ki67 for the above mentioned cell types was measured using FACS (shown in Figures 17A-C). The data show that the exemplary IL-15 / Rα-Fc fusion protein upregulates CD8 + These results show that it induced strong proliferation of T cells and NK cells.

[0360] 2. IL-15 / Rα-Fc Fusion Proteins Engineered for Reduced Potency To further improve PK and extend half-life, it was thought that reducing the potency of the IL-15 / Rα-Fc fusion would reduce the antigen sink and therefore increase the circulating half-life. By examining the crystal structures of the IL-15:IL-2Rβ and IL-15:common gamma chain interfaces, and by modeling using MOE software, residues in these interfaces that could be substituted to reduce potency were predicted. Figure 18 shows a structural model of the IL-15:receptor complex showing the predicted residue positions that were engineered with isosteric substitutions (to reduce the risk of immunogenicity). The sequences of exemplary IL-15 variants engineered to reduce potency are shown in Figure 19.

[0361] Plasmids encoding IL-15 or IL-15Rα (sushi) were constructed by standard gene synthesis followed by subcloning into pTT5 expression vectors containing Fc fusion partners (e.g., the constant regions shown in FIG. 10). Substitutions identified as described above were incorporated by standard mutagenesis techniques. Exemplary IL-15 / Rα-Fc fusion protein sequences in the "scIL-15 / Rα-Fc" format engineered for reduced potency include XENP29281, XENP24050, XENP29284, XENP29285, XENP29286, the sequences of which are shown in FIG. 21. Proteins were produced and purified as generally described in Example 1A.

[0362] a. In vitro activity of scIL-15 / Rα-Fc fusion proteins, including IL-15 variants engineered to reduce potency Exemplary scIL-15 / Rα-Fc fusion proteins, including IL-15 variants, were tested in a cell proliferation assay. Human PBMCs were incubated with the indicated test substances at the indicated concentrations for 3 days. After incubation, PBMCs were stained with anti-CD3-PE (OKT3), anti-CD4-FITC (RPA-T4), anti-CD8-eF660 (SIDI8BEE), anti-CD16-BV421 (3G8), anti-CD45RA-APC / Fire750 (HI100), anti-CD56-BV605 (5.1H11), and anti-Ki67-PE / Cy7 (Ki-67) and analyzed by flow cytometry. Figure 22 shows the percentage of CD4 and CD8 T cell populations expressing Ki67, which indicates proliferation.

[0363] The data show that several exemplary scIL-15 / Rα-Fc fusion proteins containing IL-15 variants engineered to reduce potency exhibited reduced potency compared to scIL-15 / Rα-Fc fusions containing WT IL-15. In particular, the data show that scIL-15 / Rα-Fc fusions containing the IL-15(D30N / E64Q / N65D) variant had no or dramatically reduced activity in the proliferation of various lymphocyte populations in the context of scIL-15 / Rα-Fc fusions compared to scIL-15 / Rα-Fc fusions containing the IL-15(N4D / N65D) or IL-15(D30N / N65D) variants. On the other hand, scIL-15 / Rα-Fc fusions containing the IL-15(D30N) variant showed little or no reduction in potency compared to scIL-15 / Rα-Fc fusions containing WT IL-15.

[0364] B. Example 2: PD-1 Targeting Arm As mentioned above, PD-1 expression is upregulated on activated tumor-infiltrating lymphocytes. Therefore, targeting the IL-15 / Rα-Fc fusion protein of the present invention to PD-1-expressing lymphocytes may be a useful approach to direct the IL-15 / Rα-Fc fusion to the tumor environment and avoid systemic toxicity. Furthermore, since it is useful to combine the targeting IL-15 / Rα-Fc fusion protein of the present invention with a PD-1 blocking antibody or to administer the targeting IL-15 / Rα-Fc fusion protein of the present invention after treatment with a PD-1 blocking antibody, it is important that the PD-1 targeting arm of the targeting IL-15 / Rα-Fc fusion protein does not bind to the same or similar epitope as the PD-1 blocking antibody. PD-1 blocking antibodies contemplated herein include, but are not limited to, nivolumab and pembrolizumab.

[0365] The sequences of several anti-PD-1 mAbs whose variable regions are contemplated for use herein are shown in Figures 25-26. To investigate whether the anti-PD-1 binding domains described above compete with nivolumab and pembrolizumab, tandem epitope binning was performed on the chimeric mAbs. Tandem epitope binning was performed using an Octet HTX instrument. A HIS1K biosensor was first used to capture PD-1-His, followed by soaking in 100 nM primary antibody and then 100 nM secondary antibody. The antibodies tested were XENP16432 (a bivalent anti-PD-1 mAb based on nivolumab, sequence shown in Figure 24A), XENP21461 (pembrolizumab, sequence shown in Figure 24B), chimeric mAb A, chimeric mAb B, and a 1C11-based mAb. PD-L1-Fc was also included to explore blocking of PD-1:PD-L1 interactions by antibodies. BLI responses were normalized to the BLI response of the biosensor soaked in HBS-EP buffer followed by anti-PD-1 antibodies. If an antibody pair provided a normalized BLI response of less than 0.5, the pair was considered to compete or partially compete and to be in the same epitope bin, i.e., recognizing very similar or largely overlapping epitopes. If an antibody pair provided a normalized BLI response of more than 0.5, the pair was considered to not compete and to bind different epitopes. The normalized BLI responses for each antibody pair are summarized in Figure 27.

[0366] Binning shows that anti-PD-1 mAb and mAb B do not compete with nivolumab or pembrolizumab, whereas the 1C11-based mAb competes with both nivolumab and pembrolizumab. Furthermore, mAb A does not appear to block the PD-1:PD-L1 interaction, whereas mAb B does block the PD-1:PD-L1 interaction.

[0367] For simplicity, the PD-1 binding domain that competes with nivolumab and / or pembrolizumab is hereafter referred to as anti-PD-1[C], and the PD-1 binding domain that does not compete with nivolumab and / or pembrolizumab is hereafter referred to as anti-PD-1[NC].

[0368] C. Example 3: PD-1-targeted IL-15 / Rα-Fc fusions Here we describe the generation and characterization of IL-15 / Rα-Fc fusions that target PD-1 (collectively referred to herein as PD-1-targeted IL-15 / Rα-Fc fusions).

[0369] 1. Generation and Physical Characterization of PD-1-Targeted IL-15 / Rα-Fc Fusions Plasmids encoding IL-15, IL-15Rα sushi domain, or anti-PD-1 variable regions were constructed by standard gene synthesis followed by subcloning into pTT5 expression vectors containing an Fc fusion partner (e.g., the constant region shown in FIG. 11). A cartoonized schematic of an exemplary PD-1-targeted IL-15 / Rα-Fc fusion is shown in FIG.

[0370] A particular exemplary format, the "scIL-15 / Rα x Fab" format (Figure 28C), comprises IL-15Rα (sushi) fused to IL-15 by a variable length linker (referred to as "scIL-15 / Rα"), which is then fused to the N-terminus of a heterodimeric Fc region, and a variable heavy chain (VH) is fused to the other side of the heterodimeric Fc, while the corresponding light chain is transfected separately to form a Fab with the VH.

[0371] PD-1-targeted IL-15 / Rα-Fc fusions were generated in this format using both an anti-PD-1[C] targeting arm and an anti-PD-1[NC] targeting arm. Fusions containing an anti-PD-1[C] targeting arm are referred to herein as [C]PD-1-targeted IL-15 / Rα-Fc fusions, and fusions containing an anti-PD-1[NC] targeting arm are referred to herein as [NC]PD-1-targeted IL-15 / Rα-Fc fusions.

[0372] As described in Example 1B(a), scIL-15 / Rα-Fc fusions containing the IL-I5(D30N / D64N / N65D) potency variant were almost completely inactive in the proliferation of various lymphocyte populations. Therefore, the IL-15(N4D / N65D) variant was used to generate prototype PD-1-targeted IL-15 / Rα-Fc fusions (both [C] and [NC]). The prototype [C]PD-1-targeted IL-15 / Rα-Fc fusion was generated using the variable regions of humanized 1C11, and the prototype [NC]PD-1-targeted IL-15 / Rα-Fc fusion was generated using the variable regions of humanized mAb A and mAb B. The sequence of an exemplary [C]PD-1-targeted IL-15 / Rα-Fc fusion protein itself is shown in FIG. 29, and the sequence of an exemplary [NC]PD-1-targeted IL-15 / Rα-Fc fusion protein itself is shown in FIG. 30. Exemplary [C]PD-1-targeted IL-15 / Rα-Fc fusion proteins XENP25850 and [NC]PD-1-targeted IL-15 / Rα-Fc fusions were also generated with Xtend Fc (M428L / N434S). A control RSV-targeted IL-15 / Rα-Fc fusion protein XENP26007 was also generated and its sequence is shown in FIG.

[0373] The protein was produced by transient transfection in HEK293E cells and purified by a two-step purification process involving protein A and ion exchange chromatography.

[0374] 2.3B: PD-1-targeted IL-15 / Rα-Fc fusions are active in vitro In initial experiments investigating the activity of PD-1-targeted IL-15 / Rα-Fc fusions, human PBMCs were stimulated with 100 ng / ml plate-bound anti-CD3 (OKT3) for 48 h, then CFSE-labeled and incubated with the indicated test substances at 37°C for 4 days. + and CD4 + T cell proliferation was measured by CFSE dilution and dead cells were excluded using Zombie dye. + T cells and CD4 +Data showing the proportion of T cells are shown in FIG.

[0375] The data show that each of the PD-1-targeted IL-15 / Rα-Fc fusions (both [C] and [NC] versions) inhibited CD8 + and CD4 + Furthermore, a control RSV-targeted IL-15 / Rα-Fc fusion was active in proliferating T cells, whereas the anti-PD-1 mAb XENP28519 alone was not.

[0376] 3. PD-1-targeted IL-15 / Rα-Fc fusions are selective for activated lymphocytes After a cytokine binds to its receptor, receptor-associated Janus kinases (JAKs) phosphorylate STAT proteins, which then translocate to the nucleus to regulate further downstream processes. Thus, phosphorylation of STAT proteins (particularly STAT5, including STAT5a and STAT5b) is one of the earliest signaling events triggered by IL-15 binding to its receptor. Therefore, we investigated the ability of PD-1-targeted IL-15 / Rα-Fc fusions to induce STAT5 phosphorylation in various cell types.

[0377] Both fresh and activated PBMCs were used in this experiment. Activated PBMCs, used as a surrogate for activated lymphocytes in the tumor environment with upregulated PD-1 expression, were prepared by stimulating fresh PBMCs with 100 ng / ml plate-bound anti-CD3 (OKT3) for 2 days. Fresh activated PBMCs were incubated with the indicated concentrations of XENP25850 for 15 min at 37 °C. To gate the various cell populations after incubation, PBMCs were stained with anti-CD3-BUV395 (UCHT1), anti-CD4-BV605 (RPA-T4), and anti-CD8-Alexa700 (SK1) for 30–45 min at room temperature. Cells were washed and incubated with pre-chilled (-20 °C) 90% methanol for 20–60 min. After methanol incubation, cells were washed again and stained with anti-CD25-BV421 (M-A251), anti-CD45RA-BV510 (HI100), and anti-pSTAT5-Alexa647 (pY687) to label different cell populations and STAT5 phosphorylation. + and CD4 + Data showing induction of STAT5 phosphorylation in T cell populations is shown in Figure 33. In particular, the data show that the PD-1-targeted IL-15 / Rα-Fc fusion protein XENP25850 showed increased effects on T cells from activated PBMCs while minimizing effects on T cells from fresh PBMCs. This suggests that in the clinical setting, the PD-1-targeted IL-15 / Rα-Fc fusion is selective for tumor-infiltrating lymphocytes activated in the tumor environment.

[0378] D. Example 4: [NC]PD-1-targeted IL-15 / Rα-Fc fusion combined with PD-1 blockade 1. PD-1 blockade does not interfere with the activity of [NC]PD-1-targeted IL-15 / Rα-Fc fusions in vitro Fresh PBMCs were stimulated with 100 ng / ml plate-bound anti-CD3 (OKT3) for 2 days. Activated PBMCs were preincubated with 100 μg / mL XENP16432, pembrolizumab, or XENP15074 (anti-RSV mAb as control) for 30 min. After preincubation, PBMCs were incubated with the indicated test substances at the indicated concentrations for 15 min at 37 °C. To gate the various cell populations after incubation, PBMCs were first stained with anti-CD3-BUV395 (UCHT1), anti-CD4-BV605 (RPA-T4), anti-CD25-BV421 (M-A251), and anti-CD45RA-BV510 (HI100) antibodies. After the first staining, cells were permeabilized using PerFix EXPOSE (Beckman Coulter, Indianapolis, IN). After permeabilization, cells were stained with anti-CD8-PerCP / Cy5.5 (RPA-T8), anti-pSTAT5-AF647, and anti-PD-1-APC-Fire750 (EH12.2H7). After a second staining, cells were analyzed by flow cytometry to investigate STAT5 phosphorylation in CD8+CD45RA-CD25+PD-1+ (as shown in Figure 34) and CD4+CD45RA-CD25+PD-1+ T cells (data not shown).

[0379] The data show that preincubation of PBMCs with XENP16432 or pembrolizumab reduced T cell activation by the [C]PD-1-targeting IL-15 / Rα-Fc fusion XENP25937 compared to preincubation of PBMCs with anti-RSV mAb XENP15074, indicating that, as expected, anti-PD-1 mAbs prevented the binding of the [C]PD-1-targeting IL-15 / Rα-Fc fusion XENP25937 to T cells. On the other hand, preincubation of PBMCs with XENP16432 or pembrolizumab did not affect T cell activation by the [NC]PD-1-targeting IL-15 / Rα-Fc fusion XENP28523 compared to preincubation of PBMCs with anti-RSV mAb XENP15074. This suggests that the [NC]PD-1-targeting IL-15 / Rα-Fc fusion may be stacked with anti-PD-1 mAb without negative effects.

[0380] 2. Synergistic combination of PD-1-targeted IL-15 / Rα-Fc fusion and PD-1 blockade in NC PD-1 targeted IL-15 / Rα-Fc fusions were evaluated in a graft-versus-host disease (GVHD) model performed in NSG (NOD-SCID-gamma) immunodeficient mice. When NSG mice are transplanted with human PBMCs, the human PBMCs induce an autoimmune response against mouse cells and subsequently GVHD. Therefore, GVHD is a model for anti-tumor responses. Treatment of huPBMC-transplanted NSG mice with PD-1 targeted IL-15 / Rα-Fc fusions should promote proliferation of transplanted T cells and enhance GVHD.

[0381] In the first GVHD study, NSG mice were administered 10 × 10 6Human PBMCs were transplanted and the indicated test substances were administered intraperitoneally at the indicated concentrations on days 0, 7, 14, and 21. Body weight was assessed twice weekly as an indicator of GVHD (change in body weight as a percentage of initial body weight shown in FIG. 35) and blood was drawn on days 7, 10, and 14 to assess proliferation of various lymphocytes (data for day 14 are shown in FIG. 36, respectively). Expression of CD25 on CD8+ and CD4+ T cells was also assessed as a T cell activation marker (as shown in FIG. 37).

[0382] The data show that each of the PD-1-targeted IL-15 / Rα-Fc fusions (both [C] and [NC]) enhanced GVHD compared to PD-1 blockade alone (either XENP16432 or XENP28437) and scIL-15 / Rα-Fc XENP24050 alone, as indicated by CD45+, CD3+T, CD8+, T, and CD4+T cell counts. However, consistent with the in vitro data above, [C] combining the PD-1-targeted IL-15 / Rα-Fc fusions XENP25850 and XENP28437 resulted in reductions in various cell counts, and [NC] combining the PD-1-targeted IL-15 / Rα-Fc fusions XENP28532 (based on mAb A) and XENP28692 (based on mAb B) resulted in further enhanced GVHD, as indicated by cell counts. Similar trends are observed using changes in body weight as an indicator of GVHD, as shown in Figure 35, and for CD8+ and CD4+ T cell activation, as shown in Figure 37. Furthermore, there appears to be a high preference for CD4+ T cells by the PD-1-targeted IL-15 / Rα-Fc fusion, as shown by the low ratio of CD8+ to CD4+ T cells, as shown in Figure 38.

[0383] In the second GVHD study, NSG mice were transplanted with 10x106 human PBMCs via IV-OSP on day 1 and administered the indicated test articles intraperitoneally at the indicated concentrations on days 0, 7, 14, and 21. Body weight was assessed twice weekly as an indicator of GVHD, and blood was drawn on days 7, 10, and 14 to assess proliferation of various lymphocytes (day 10 data shown in Figure 39). CD25 expression on CD8+ T and CD4+ cells was also assessed, as shown in Figure 40.

[0384] As noted above, the data show that [NC]PD-1-targeted IL-15 / Rα-Fc fusions expanded T cell numbers compared to anti-PD-1 blockade alone. Additionally, the data show a clear dose response on T cell proliferation as shown by the enhancement of GVHD with higher concentrations of [NC]PD-1-targeted IL-15 / Rα-Fc fusions XENP28532 (0.3 mg / kg vs. 0.1 mg / kg). Notably, the combination of XENP28532 (at 0.3 mg / kg) and the PD-1 blocking mAb XENP28437 enhanced CD25 expression on CD8+ and CD4+ T cells.

[0385] E. Example 5: Generation of [NC]PD-1-targeted IL-15 / Rα-Fc fusions with alternative IL-15 potency variants 1. PD-1-targeted IL-15 / Rα-Fc fusions containing the IL-15 (N4D / N65D) variant show poor pharmacokinetics In a study investigating the pharmacokinetics of IL-15 / Rα-Fc potency variants with Xtend, cynomolgus monkeys were first intravenously (iv) administered a single dose of XENP22853 (WT IL-15 / Rα-hetero Fc with Xtend), XENP24306 (IL-15(D30N / E64Q / N65D) / Rα-hetero Fc with Xtend), XENP24113 (IL-15(N4D / N65D) / Rα-hetero Fc with Xtend), XENP24294 (scIL-15(N4D / N65D) / Rα-Fc with Xtend), and XENP25937 ([C]PD-1-targeted IL-15(N4D / N65D) / Rα-Fc with Xtend) at various concentrations.

[0386] Figure 41 shows the serum concentration of the test material over time after the first dose. As expected, the incorporation of potency variants in addition to Xtend substitutions (as in XENP24306 and XENP24113) significantly improves the pharmacokinetics of IL-15 / Rα-Fc fusions (compared to XENP22583). Unexpectedly, the PD-1-targeted IL-15 / Rα-Fc fusion XENP25937, which contains the IL-15(N4D / N65D) variant, showed substantially reduced pharmacokinetics compared to XENP24306. Similarly, the IL-15 / Rα-hetero Fc fusion XENP24113 and the scIL-15 / Rα-Fc fusion XENP24294 (with the same IL-15(N4D / N65D) potency variant) also showed substantially reduced pharmacokinetics compared to XENP24306. This suggests that the reduced pharmacokinetics may be due to the specific IL-15 potency variants rather than the format of the PD-1-targeted IL-15 / Rα-Fc fusions. The reduced pharmacokinetics of XENP25937 (and XENP24113) was predicted based on IL-15 / Rα-Fc fusions with IL-15(N4D / N65D) variants exhibiting higher in vitro potency than IL-15 / Rα-Fc fusions with IL-15(D30N / E64Q / N65D) variants (as shown in Example 1B(a)), while the reduced pharmacokinetics was unexpectedly disproportionate to the increased potency. Therefore, we sought to identify alternative IL-15 potency variants for use in the PD-1-targeted IL-15 / Rα-Fc fusions of the present invention.

[0387] 2. Engineering PD-1-targeted IL-15 / Rα-Fc fusions containing IL-15 variants modified with the IL-15:CD132 interface It should be noted that the IL-15(N4D / N65D) variant has both substitutions at the IL-15 interface responsible for binding to CD122, while IL-15(D30N / E64Q / N65D) has two substitutions at the IL-15:CD122 interface (E64Q and N65D) and one substitution at the IL-15 interface responsible for binding to CD132 (D30N). Therefore, it was thought that modifications at the IL-15:CD132 interface may contribute to the superior pharmacokinetics observed with XENP24306. Therefore, additional exemplary PD-1-targeting IL-15 / Rα-Fc fusions containing the IL-15(D30N / N65D) variant were generated, the sequences of which are shown in FIG. 42.

[0388] F. Example 6: PD-1-targeted IL-15 / Rα-Fc fusion induces internalization of PD-1 on T cells In another experiment investigating the activity of PD-1-targeted IL-15 / Rα-Fc fusions, CFSE-labeled human PBMCs were incubated with 20 ng / ml plate-bound anti-CD3 (OKT3) and the following test substances, XENP28532 (PD-1-targeted IL-15 / Rα-Fc fusions with an αPD-1 arm based on mAb A), XENP24306 (control non-targeted IL-15 / Rα-Fc fusions), and XENP26007 (control RSV-targeted IL-15 / Rα-Fc fusions) at 37° C. for 4 days. Cells were then labeled with αPD-1XENP16432, which does not compete for binding to the PD-1-targeted arm of XENP28532, and analyzed by flow cytometry, as shown in Example 2C and FIG. 34. Data showing the percentage of various T cell populations that are PD-1+ are shown in FIG. 44. Surprisingly, a dose-dependent reduction in PD-1 in T cells following treatment with PD-1-targeted IL-15 / Rα-Fc fusions was observed compared to treatment with controls XENP24306 and XENP26007, indicating that PD-1 receptors were internalized, suggesting a potential long-term effect of PD-1-targeted IL-15 / Rα-Fc, where immune inhibition by PD-1 ligands (e.g., PD-L1 and PD-L2) may remain reduced following eventual clearance of the PD-1-targeted IL-15 / Rα-Fc molecules.

[0389] G. Example 7: Identification of Cross-Reactive [NC]PD-1 mAb C in Cynomolgus Monkeys To facilitate clinical development, it is useful to investigate various parameters of [NC]PD-1 targeting IL-15 / Rα-Fc fusions, such as pharmacokinetics, pharmacodynamics, and toxicity in cynomolgus monkeys. Therefore, we sought to identify [NC]PD-1 targeting arm cross-reactivity to human and cynomolgus monkey PD-1. We identified additional anti-PD-1 binding domains (herein referred to as mAb C) that were humanized using string content optimization (see, for example, U.S. Patent No. 7,657,380, issued February 2, 2010), sequences shown in Figure 43 (variable domain sequences) and Figure 45 (bivalent mAbs such as XENP28536, XENP28537, XENP28538, and XENP28539).

[0390] 1. Anti-PD-1 mAb C is cross-reactive with human and cynomolgus monkey PD-1 The binding of XENP28536, XENP28537, XENP28538, and XENP28539 to human and cynomolgus PD-1 was investigated using Octet generally as described above. In particular, the antibodies were captured using an anti-human Fc (AHC) biosensor and soaked in multiple concentrations of human and cynomolgus PD-1-His to determine the KD, and the data are shown in Figure 46. The data show that both humanized variants XENP25836 and XENP25837 bound to human and cynomolgus with similar KD. Notably, humanized variants XENP25838 and XENP28539 lost the ability to bind to both human and cynomolgus PD-1. In comparison, XENP28519 (humanized mAb A) binds more strongly to human PD-1 but has no cross-reactivity to cynomolgus PD-1.

[0391] 2. Anti-PD-1 mAb C does not compete for binding with nivolumab and pembrolizumab To investigate whether mAb C competes with nivolumab and pembrolizumab, tandem epitope binning was performed on chimeric mAb C as described in Example 2C. Data for XENP16432, XENP21461 and chimeric mAb C are shown in Figure 47. Binning indicates that anti-PD-1 mAb C does not compete with nivolumab or pembrolizumab and is a partial blocker of the PD-1:PD-L1 interaction.

[0392] H. Example 8: [NC]PD-1-targeted IL-15 / Rα-Fc fusion based on mAb C [NC]PD-1-targeting IL-15 / Rα-Fc fusions in the "scIL-15 / Rα x Fab" format based on mAb C and IL-15 variants were engineered and generated as generally described in Example 3, and exemplary sequences are shown in Figure 48. It should be noted that fusions were generated with the IL-15(N4D / N65D) variant, and the IL-15(D30N / N65D) variant, in line with the notion that IL-15 variants containing modifications at the IL-15:CD132 interface described in Example 5 may have enhanced pharmacokinetics. Additionally, the sequence of the Xtend Fc(M428L / N434S) analog is shown in Figure 49.

[0393] 1.8A: mAb C-based [NC]PD-1-targeted IL-15 / Rα-Fc fusion is active in vitro a.8A(a): Induction of T cell proliferation Human PBMCs were stimulated with 500 ng / ml plate-bound anti-CD3 (OKT3) for 48 hours, labeled with CFSE, and then cultured with the following test substances: XENP28543 (a [NC]PD-1-targeting IL-15 / Rα-Fc based on mAb C_H1L1 and IL-15(N4D / N65D)), XENP28532 (a [NC]PD-1-targeting IL-15 / Rα-Fc based on mAb C_H1L1 and IL-15(N4D / N65D)), The cells were incubated with [NC]PD-1-targeting IL-15 / Rα-Fc based on A_H1L1 and IL-15(N4D / N65D), XENP24306 (non-targeting IL-15 / Rα-hetero-Fc based on IL-15(D30N / E64Q / N65D), and XENP26007 (control RSV-targeting IL-15 / Rα-Fc based on motavizumab and IL-15(N4D / N65D)) at 37°C for 4 days. After incubation, cells were stained with the following antibodies: anti-LAG-3-PE (3DS223H), anti-CD8-PerCP-Cy5.5 (SK1), anti-CD3-PE-Cy7 (OKT3), anti-PD-1-Alexa647 (XENP16432 stained with Alexa Fluor™ 647 antibody labeling kit), anti-CD45RO-APC-Fire750 (UCHL1), anti-HLA-DR-Alexa700 (L243), anti-TIGIT-BV421 (A15153G), anti-CD16-BV605 (3G6), anti-CD56-BV605 (HCD56), anti-CD25-BV711 (M-A251), anti-CD45RA-BV785 (HI100), anti-CD4-BUV395 (SK3), and Zombie. The various cell populations were stained with Aqua-BV510 and analyzed by flow cytometry.

[0394] Proliferation of various T cell populations based on CFSE dilution (Zombie Aqua to exclude dead cells) was examined and the data are shown in Figure 50. Both [NC]PD-1 targeted IL-15 / Rα-Fc fusions induced proliferation of CD8+ and CD4+ T cells. Notably, [NC]PD-1 targeted IL-15 / Rα-Fc fusions induced significantly more proliferation of CD4+ T cells compared to non-targeted IL-15(D30N / E64Q / N65D) / Rα-Fc fusions (as well as control RSV targeted IL-15 / Rα-Fc fusions). +It was more potent in inducing T cell proliferation, but less potent in inducing CD8+ T cell proliferation. Furthermore, XENP28532 (PD-1 targeting arm based on mAb A) appeared to be more potent than XENP28543 (PD-1 targeting arm based on mAb C) in inducing proliferation of both CD8+ and CD4+ T cells.

[0395] Interestingly, as shown in FIG. 51, XENP28532 significantly inhibited CD8+ / α-Fc expression compared to the non-targeted IL-15(D30N / E64Q / N65D) / Rα-Fc fusion (as well as the control RSV-targeted IL-15 / Rα-Fc fusion). + LAG-3 + Moreover, XENP28543 is more potent at inducing proliferation of bulk CD8 + CD8 rather than T cell proliferation + LAG-3 + It was more potent in inducing T cell proliferation (EC50 276.8 vs. 71.94). Taken together, this supports the idea that [NC]PD-1-targeted IL-15 / Rα-Fc fusions may be selective for T cells expressing checkpoints such as those found in the tumor environment.

[0396] We also investigated the expansion of memory (CD45RA-) and naive (CD45RA+) populations, as shown in Figure 52. Notably, the [NC]PD-1-targeted IL-15 / Rα-Fc fusion was more potent than the non-targeted IL-15(D30N / E64Q / N65D) / Rα-Fc fusion in inducing the expansion of CD4+CD45RA- T cells, but was less potent in inducing the expansion of CD4+CD45RA+ T cells, indicating selectivity for memory T cells.

[0397] Finally, expression of PD-1 in various T cell populations was investigated (stained using XENP16432, which binds to a different epitope than mAb A and mAb C, as shown herein). The data are shown in Figure 53, where a dose-dependent reduction of PD-1 in CD4+CD45RA- T cells following treatment with both [NC]PD-1-targeting IL-15 / Rα-Fc fusions was observed compared to treatment with controls XENP24306 and XENP26007, which internalized the PD-1 receptor. Notably, downregulation of PD-1 was more potently induced by XENP28532 using the PD-1-targeting arm based on mAb A.

[0398] b.8A(b): Induction of cytokine secretion Human PBMCs were pre-stimulated with various concentrations of plate-bound anti-CD3 (OKT3) for 48 hours at 37°C, labeled with CFSE, and incubated with the indicated test substances for 4 days at 37°C. Supernatants were collected and evaluated by V-PLEX Proinflammatory Panel 1 Human Kit (Meso Scale Discovery, Rockville, MD, following the manufacturer's protocol). The data shown in Figure 54 indicate that both XENP28532 and XENP28543 were able to potently stimulate IFNγ secretion. Notably, XENP28532 (PD-1 targeting arm based on mAb A) appeared to be more active than XENP28543 (PD-1 targeting arm based on mAb C) in inducing IFNγ secretion.

[0399] c.8A(c): PD-1 blockade does not interfere with the activity of mAb C-based [NC]PD-1-targeted IL-15 / Rα-Fc fusion in vitro As described in Example 4A, the potential interference of PD-1 blockade on the activity of [NC]PD-1-targeting IL-15 / Rα-Fc fusions based on mAb C was investigated. The data shown in Figure 55 show that preincubation of PBMCs with XENP16432 or pembrolizumab did not affect T cell activation by [NC]PD-1-targeting IL-15 / Rα-Fc fusions XENP28543 compared to preincubation of PBMCs with anti-RSV mAb XENP15074. This suggests that [NC]PD-1-targeting IL-15 / Rα-Fc fusions with a PD-1-targeting arm based on mAb C may be stacked with anti-PD-1 mAbs without negative effects.

[0400] 2.8B: mAb C-based [NC]PD-1-targeted IL-15 / Rα-Fc fusion enhances GVHD in vivo and is combined with PD-1 blockade The second GVHD study described in Example 4B also investigated the in vivo activity of XENP28543 at various concentrations, alone and in combination with PD-1 blockade. Data showing the change in body weight over time (as a percentage of initial body weight) is shown in FIG. 56, and data showing body weight (as a percentage of initial body weight) on days 11, 14, and 18 are shown in FIG. 57. The proliferation and activation of various lymphocyte populations was also investigated, and the data are shown in FIG. 58-59 on day 14. Taken together, the data show that the combination of [NC]PD-1-targeted IL-15 / Rα-Fc fusion and PD-1 blockade significantly enhanced GVHD over treatment with [NC]PD-1-targeted IL-15 / Rα-Fc fusion alone. The enhanced effect is particularly notable in the context of the combination of low concentrations (i.e., 0.1 mg / kg) of [NC]PD-1-targeted IL-15 / Rα-Fc fusion and PD-1 blockade.

[0401] 3.8C: mAb C-based [NC]PD-1-targeted IL-15 / Rα-Fc fusion combined with PD-1 blockade enhances antitumor activity in NSG mice NSG mice (10 per group) were inoculated intradermally with 3x106 pp65-transduced MCF-7 cells on day -15. Mice were then injected intraperitoneally with 5x106 human PBMCs (or PBS for controls) and treated with test articles on day 0 and further treated with the indicated test articles on days 7, 14, 21, 29, and 36. Tumor volumes were measured with calipers three times a week, body weights were measured weekly, and blood was drawn weekly.

[0402] Tumor volume over time is shown in Figure 60, and tumor volumes at days 26, 28, 30, 33, 35, and 37 are shown in Figure 61. The data show that by day 28, the combination of XENP28543 and PD-1 blockade significantly reduced tumor size compared to treatment with PD-1 blockade alone. In addition, proliferation and activation of various lymphocyte populations were investigated, and the data are shown in Figures 62-63. [NC]PD-1-targeted IL-15 / Rα-Fc fusion alone or in combination with PD-1 blockade was able to significantly enhance early (day 7 post-administration) induction of CD8+ T cells compared to PD-1 blockade alone, and [NC]PD-1-targeted IL-15 / Rα-Fc fusion combined with PD-1 blockade was able to significantly enhance early (day 7 post-administration) induction of CD4+ T cells compared to PD-1 blockade alone. Furthermore, we show that by day 14, [NC]PD-1-targeted IL-15 / Rα-Fc fusions, alone or in combination with PD-1 blockade, were able to significantly enhance proliferation of multiple lymphocyte populations compared with PD-1 blockade alone.

[0403] I. Example 9: [NC]PD-1-targeted IL-15 / Rα-Fc fusions with affinity-optimized PD-1 targeting arms 1.9A: Affinity optimization of mAb C-based ABD As described in Example 8A, in certain situations, such as in vitro downregulation of PD-1, T cell proliferation, and induction of cytokine secretion, XENP28532 (PD-1 targeting arm based on mAb A) appeared to be more potent and / or active than XENP28543 (PD-1 targeting arm based on mAb C). As described in Example 7A, a bivalent mAb based on humanized mAb A (XENP28519) bound more strongly to human PD-1 than a bivalent mAb based on humanized mAb C (XENP28536). In light of this, we reasoned that the affinity of the PD-1 targeting arm for PD-1 may affect the activity of PD-1-targeted IL-15 / Rα-Fc fusions. Therefore, we engineered affinity-optimized variants of mAb C. A library of variants was constructed by standard mutagenesis to introduce point mutations into the variable heavy or light chain regions of XENP28536. Exemplary sequences are shown in Figures 43 (variable domain sequences) and 64 (bivalent mAbs). Affinity screening of affinity engineered mAb C[PD-1]_H1L1 variants (bivalent IgG1 format with E233P / L234V / L235A / G236_ / S267K deletion variants) was performed in the general Octet described above, and the data is shown in Figure 65. Of 304 variants with single point mutations in either the variable heavy or variable light regions, only 11 variants (including mAbs C[PD-1]_H1_L1.1 and mab_C[PD-1]_H1_L1.3) with mutations greater than 2-fold improved affinity over WT were identified. The preferred VH substitutions were at positions 32, 52A, and 97 (numbering according to Kabat), and the preferred VL substitutions were at positions 27D, 30, 93, and 94 (numbering according to Kabat).

[0404] To further increase affinity, the preferred single-substitution VH variants and single-substitution VL variants were combined. These new VH / VL combo variants were constructed in the context of PD-1-targeting IL15 / Rα-Fc fusions. Data showing the affinity of the fusions to PD-1 are shown in Figure 66. Notably, H1.19_L1.1 showed higher affinity than H1.132_L1.1, although H1.132_L1 provided higher affinity than H1.19_L1, suggesting a synergistic affinity enhancement provided by the F32L substitution in VH (numbering according to Kabat) as in H1.19.

[0405] The preferred single substitutions in VH and / or VL were then combined with the new variants constructed in the context of PD-1-targeted IL15 / Rα-Fc fusions. Data showing the affinity of the fusions to PD-1 are shown in Figure 67. The triple-substituted VL variant L1.140 (containing a histidine at position 27D, a tyrosine at position 30, and a threonine at position 93, numbered according to Kabat) shows a 36-fold improvement in KD compared to wild-type, and in combination with VH variants (e.g., H1.132 and H1.175), exerts an approximately 100-fold improvement in KD compared to wild-type.

[0406] In particular, a ladder of affinity variants was generated by combining single substitutions in VH or VL, and by combining VH and VL variants used to tune the potency and selectivity of PD-1-targeting IL-15 / Rα-Fc fusions.

[0407] [NC]PD-1-targeting IL-15 / Rα-Fc fusions in "scIL-15 / Rα x Fab" format based on affinity-enhanced mAb C and IL-15 variants were engineered and generated as generally described in Example 3, and exemplary sequences are shown in Figure 68 as XENP30046, XENP30047, XENP30049 and XENP30050. Additionally, the sequence of the Xtend Fc(M428L / N434S) analog is shown in Figure 69.

[0408] 2.9B: [NC]PD-1-targeted IL-15 / Rα-Fc fusions with improved PD-1 binding have enhanced activity in vitro Next, the effect of the affinity-enhanced PD-1 targeting arm (and the IL-15(D30N / N65D) variant) on the PD-1-targeted IL-15 / Rα-Fc fusion of the present invention was investigated. Human PBMCs were stimulated with 500 ng / ml plate-bound anti-CD3 (OKT3) for 48 hours, labeled with CFSE, and incubated with test substances for 4 days at 37°C. Cells were stained with the following antibodies: anti-CD8-PerCP-By5.5 (SK1), anti-CD3-PE-Cy7 (OKT3), anti-PD-1-Alexa647 (XENP16432 stained with Alexa Fluor™ 647 antibody labeling kit), anti-CD45RO-APC-Fire750 (UCHL1), anti-HLA-DR-Alexa700 (L243), anti-CD107a-BV421 (H4A3), anti-CD16-BV605 (3G6), anti-CD56-BV605 (HCD56), anti-CD25-BV711 (M-A251), anti-CD45RA-BV785 (M-A251), anti-CD4-BUV395 (SK3), and Zombie Aqua (BV510), and analyzed by flow cytometry of the various cell populations.

[0409] We investigated the proliferation of different T cell populations based on CFSE dilution (Zombie Aqua to exclude dead cells) (data shown in Figure 70), the activation of different T cell populations based on expression of CD25 (a late T cell activation marker) (data shown in Figures 71-72), and the expression of PD-1 in different populations (data shown in Figure 73 for CD4+CD45RA-PD-1+ T cells).

[0410] Taken together, the data indicate that the activity of PD-1-targeted IL-15 / Rα-Fc fusions correlates with PD-1 affinity. For example, as shown in Figure 70, XENP30046 (with an affinity-enhanced PD-1 targeting arm) more potently inhibited CD8 T cells than XENP28543. + and CD4 +Induce both proliferation and proliferation of T cells (2-fold increase). Notably, as shown in Figure 73, even downregulation of PD-1 on T cells by PD-1-targeted IL-15 / Rα-Fc fusions correlates with PD-1 affinity. Furthermore, the data show that the IL-15(D30N / N65D) variant does not dramatically affect the activity of PD-1-targeted IL-15 / Rα-Fc fusions.

[0411] 3.9C: [NC]PD-1-targeted IL-15 / Rα-Fc fusions with improved PD-1 binding have enhanced activity in vivo NSG mice were injected with 10 × 10 6 Human PBMCs were transplanted and the indicated test substances were administered intraperitoneally at the indicated concentrations on days 0, 7, and 14: XENP28437 (an anti-PD-1 mAb based on pembrolizumab and the E233P / L234V / L235A / G236del / S67K deletion variant), and XENP29481 (a control RSV-targeting IL-15 / Rα-Fc fusion with the D30N / N65D IL-15 variant, the sequence of which is shown in FIG. 31). Body weight was assessed twice weekly as an indicator of GVHD, and the data are shown in FIGS. 74-75 as change in initial body weight. Notably, treatment with XENP30046 alone (with an affinity-enhanced PD-1 targeting arm) resulted in significant weight loss compared to PBS treatment, as measured on days 11 and 18, whereas treatment with XENP28543 alone did not result in significant weight loss (compared to PBS treatment).

[0412] Blood was collected on days 7, 10, and 14 to examine human lymphocyte proliferation and to examine cytokine secretion, and the data are shown in Figure 76. Taken together, the data generally show enhanced activity by [NC]PD-1-targeted IL-15 / Rα-Fc fusions with affinity-enhanced PD-1 targeting arms. Furthermore, the data show that [NC]PD-1-targeted IL-15 / Rα-Fc binds productively with PD-1 blockade (XENP28437).

[0413] Additionally, as shown in Figures 78 and 79, XENP30046 significantly enhanced proliferation of CD45+ cells, CD3+ T cells, CD4+ T cells, CD8+ T cells, and NK cells by day 11 compared to administration of XENP28543 which persisted in all populations (except NK cells) through day 14. Additionally, as shown in Figure 77, XENP30046 significantly enhanced early (day 7 post-administration) activation of CD8+ and CD4+ T cells (as indicated by CD25 expression) compared to XENP28543.

[0414] J. Example 10: Fine-tuning the potency and selectivity of PD-1-targeted IL-15 / Rα-Fc fusions by adjusting PD-1 affinity and IL-15 potency Although the PD-1-targeted IL-15 / Rα-Fc fusion was designed to target the tumor environment via the PD-1-targeting arm, the cytokine portion can signal before reaching the tumor site and may contribute to systemic toxicity. Therefore, we sought to further reduce IL-15 potency by constructing PD-1-targeted IL-15 / Rα-Fc fusions with IL-15(D30N / E64Q / N65D) variants with dramatically reduced activity, as shown in Example 1B(a). The sequences of exemplary PD-1-targeted IL-15 / Rα-Fc fusions with IL-15(D30N / E64Q / N65D) variants are shown in Figures 30 and 48-49 as XENP30428, XENP30429, XENP30430, XENP30519, XENP30516, XENP30517, and XENP30455. Additionally, we constructed XENP30432, an RSV-targeted IL-15 / Rα-Fc fusion containing the IL-15(D30N / E64Q / N65D) variant (the sequence of which is shown in FIG. 32) to serve as a surrogate for investigating the behavior of PD-1-targeted IL-15 / Rα-Fc fusions containing the IL-15(D30N / E64Q / N65D) variant outside the tumor environment.

[0415] 1.10A: In vitro activity of [NC]PD-1-targeted IL-15 / Rα-Fc fusions tailored for PD-1 affinity and IL-15 potency In vitro activity of additional [NC]PD-1-targeted IL-15 / Rα-Fc fusions with varying PD-1 binding affinity and IL-15 potency. Human PBMCs were stimulated with 500 ng / ml plate-bound anti-CD3 (OKT3) for 48 h, labeled with CFSE, and incubated with test substances for 4 days at 37°C. Cells were stained with the following antibodies: anti-CD8-PerCP-Cy5.5 (SK1), anti-CD3-PE-Cy7 (OKT3), anti-PD-1-Alexa647 (XENP16432 stained with Alexa Fluor™ 647 antibody labeling kit), anti-CD45RO-APC-Fire750 (UCHL1), anti-HLA-DR-Alexa700 (L243), anti-CD107a-BV421 (H4A3), anti-CD16-BV605 (3G6), anti-CD56-BV605 (HCD56), anti-CD25-BV711 (M-A251), anti-CD45RA-BV785 (M-A251), anti-CD4-BUV395 (SK3), and Zombie Aqua (BV510), and analyzed by flow cytometry of the various cell populations.

[0416] The proliferation of different T cell populations was investigated based on CFSE dilution (Zombie Aqua to exclude dead cells) (data is shown in Figure 80), the activation of different T cell populations based on expression of CD25 (a late T cell activation marker) (data is shown in Figures 81-82), and the expression of PD-1 in different populations (data is shown in Figures 83-84).

[0417] The data show that XENP30272 (with higher affinity PD-1 binding) is more potent than XENP30046 (with lower affinity PD-1 binding) in inducing proliferation and activation of various T cell populations, demonstrating the importance of tuning PD-1 affinity. In particular, XENP30429 (PD-1-targeted IL-15 / Rα-Fc fusion with IL-15(D30N / E64Q / N65D) variant) upregulates CD8 T cell proliferation and activation compared to XENP30046 (PD-1-targeted IL-15 / Rα-Fc fusion with IL-15(N4D / N65D) variant). + and CD4 +Although the activity was lower in CD8 T cells by 1.8-2.5, XENP30432 (a surrogate RSV-targeted IL-15 / Rα-Fc with the IL-15 (D30N / E64Q / N65D) variant) showed a significantly increased CD8 T cell proliferation compared to XENP30046 (based on proliferation activity as shown in Figure 80). + 12 times higher in T cells, and CD4 + It was 530-fold less active in T cells, suggesting that the PD-1-targeted IL-15 / Rα-Fc fusion carrying the IL-15(D30N / E64Q / N65D) variant retains activity in the tumor environment but remains essentially inactive outside the tumor environment.

[0418] 2.10B: In vivo activity of [NC]PD-1-targeted IL-15 / Rα-Fc fusions adjusted for IL-15 potency In vivo lymphocyte proliferation by [NC]PD-1-targeted IL-15 / Rα-Fc fusions adjusted for IL-15 potency was investigated in GvHD studies. NSG mice were transferred with 10x106 human PBMCs via IV-OSP on day 1 and administered the indicated test substances intraperitoneally at the indicated concentrations on days 0, 7, and 14. Blood was collected on days 7, 10, and 14 to assess proliferation and activation of various lymphocytes (data shown in Figures 85-86). Taken together, the data show that when PD-1 affinity is comparable, a more potent IL-15 (e.g., XENP30046) allows for greater proliferation (and earlier activation) of T cells compared to a less potent IL-15 (e.g., XENP30429).

[0419] K. Example 11: [NC]PD-1 Targeted IL-15 / Rα-Fc with Xtend [NC] Xtend analogs of PD-1-targeting IL-15 / Rα-Fc fusions were engineered to further expand pharmacokinetics and ...

Claims

1. 1. A composition comprising an anti-PD-1 binding domain, wherein the anti-PD-1 binding domain comprises a variable heavy domain (VH domain) and a variable light domain (VL domain), wherein the VH domain is SEQ ID NO:5 and the VL domain is SEQ ID NO:6 or SEQ ID NO:368, and the anti-PD-1 binding domain is a Fab or an scFv, wherein the scFv comprises an scFv linker covalently linking the VH domain and the VL domain to form the scFv.

2. The composition described in claim 1, wherein the anti-PD-1 binding domain is a Fab.

3. 2. The composition of claim 1, wherein the anti-PD-1 binding domain is an scFv.

4. 4. The composition of claim 3, wherein the scFv linker is (GGGGS)n (SEQ ID NO: 10), where n is an integer from 1 to 7.

5. The composition of claim 3, wherein the scFv linker is (GKPGS)n (SEQ ID NO:28), where n is an integer from 1 to 6.

6. A nucleic acid encoding the composition of claim 1.

7. a) a first nucleic acid encoding a VH domain according to claim 1; and b) a second nucleic acid encoding the VL domain of claim 1.

8. An expression vector comprising the nucleic acid of claim 6.

9. a) a first expression vector comprising the first nucleic acid of claim 7; and b) a second expression vector comprising the second nucleic acid of claim 7.

10. A host cell comprising the expression vector of claim 8 or the expression vector composition of claim 9.

11. 13. A method for producing the composition of claim 1, comprising culturing a host cell of claim 10 under conditions in which the composition is expressed, and recovering the composition.

12. the composition is a PD-1-targeted IL-15 / Rα heterodimeric Fc fusion protein; The PD-1-targeted IL-15 / Rα heterodimer Fc fusion protein comprises: a) from the N-terminus to the C-terminus: i) IL-15Rα sushi domain protein; ii) a first domain linker; iii) IL-15 protein, and iv) a first monomer comprising a first variant Fc domain; and b) a second monomer comprising a heavy chain comprising VH-CH1-hinge-CH2-CH3, where CH2-CH3 is a second variant Fc domain; and and c) a third monomer comprising a light chain comprising a VL-CL, wherein the VH and VL domains form the anti-PD-1 binding domain, and the anti-PD-1 binding domain does not compete with nivolumab and / or pembrolizumab for human PD-1.

13. The IL-15 protein is D30N / E64Q / N65D, D30N / N65D, N1D, N4D, D8N, D30N, D61N, E64Q, N65D, Q108E, N1D / N4D / D8N, N1D / N4D / N65D, N1D / D3 0N, N1D / D61N, N1D / D61N / E64Q / Q108E, N1D / E64Q, N1D / N65D, N1D / Q108E, N4D, N4D / D30N, N4D / D61N, N4D / D61N / N65 13. The composition of claim 12, wherein the IL-15 protein is a variant IL-15 protein comprising an amino acid substitution(s) selected from the group consisting of D, N4D / D61N / E64Q / Q108E, N4D / E64Q, N4D / N65D, D8N / D61N, D8N / E64Q, D30N / E64Q, D30N / Q180E, D61N / E64Q / N65D, E64Q, E64Q / N65D, E64Q / Q108E, and N65D / Q108E.

14. 14. The composition of claim 12 or 13, wherein the variant IL-15 protein comprises an amino acid substitution(s) selected from the group: N4D / N65D, D30N, D30N / E64Q, D30N / N65D, and D30N / E64Q / N65D.

15. 15. The composition of any one of claims 12 to 14, wherein the first variant Fc domain and the second variant Fc domain comprise the amino acid substitutions L368D / K370S:S364K / E357Q according to EU numbering.

16. 16. The composition of any one of claims 12 to 15, wherein the first variant Fc domain and the second variant Fc domain each comprise the amino acid substitutions M428L / N434S according to EU numbering.

17. a) a first nucleic acid encoding a first monomer according to any one of claims 12 to 16; b) a second nucleic acid encoding a second monomer according to any one of claims 12 to 16; and c) a third nucleic acid encoding a third monomer according to any one of claims 12 to 16.

18. a) a first expression vector comprising a first nucleic acid according to claim 17; b) a second expression vector comprising a second nucleic acid according to claim 17; and c) a third expression vector comprising the third nucleic acid of claim 17.

19. A host cell comprising the expression vector composition of claim 18.

20. 20. A method for producing a composition according to any one of claims 12 to 16, comprising culturing a host cell according to claim 19 under conditions in which the composition is expressed, and recovering the composition.

21. a) a first monomer comprising the amino acid sequence set forth in SEQ ID NO:553; b) a second monomer comprising the amino acid sequence set forth in SEQ ID NO:554; and c) a third monomer comprising the amino acid sequence set forth in SEQ ID NO:

555.

22. a) a first nucleic acid encoding a first monomer according to claim 21; b) a second nucleic acid encoding a second monomer according to claim 21; and c) a third nucleic acid encoding the third monomer of claim 21.

23. a) a first expression vector comprising a first nucleic acid according to claim 22; b) a second expression vector comprising a second nucleic acid according to claim 22; and c) a third expression vector comprising the third nucleic acid of claim 22.

24. A host cell comprising the expression vector composition of claim 23.

25. 22. A method of producing the composition of claim 21, comprising culturing a host cell of claim 24 under conditions in which the composition is expressed, and recovering the composition.

Images