IL15 / IL15Rα heterodimeric FC-fusion protein

Abstract

To provide IL15 / IL15Rα heterodimeric FC-fusion proteins.SOLUTION: Disclosed is a heterodimeric protein comprising: a) a first fusion protein comprising a first protein domain and a first Fc domain, where the first protein domain is covalently attached to the N terminus of the first Fc domain using a first domain linker; and b) a second fusion protein comprising a second protein domain and a second Fc domain where the second protein domain is covalently attached to the N terminus of the Fc domain using a second domain linker, where the first Fc domain and the second Fc domain comprises a set of amino acid substitutions selected from a specific group, the first protein domain comprises the IL15 protein and the second protein domain comprises the IL15Rα protein.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] Priority claim This application claims priority to U.S. patent application Ser. Nos. 62 / 408,655, filed Oct. 14, 2016, 62 / 416,087, filed Nov. 1, 2016, 62 / 443,465, filed Jan. 6, 2017, and 62 / 477,926, filed Mar. 28, 2017, which are expressly incorporated herein by reference in their entireties, with particular reference to the drawings, descriptions, and claims therein. [Background technology]

[0002] IL-2 and IL-15 function to support the proliferation and differentiation of B cells, T cells, and NK cells. IL-2 is also essential for the function and survival of regulatory T cells (Tregs). Both cytokines exert their cell signaling functions through binding to a trimeric complex consisting of two shared receptors: a common gamma chain (γc, CD132) and the IL-2 receptor B chain (IL-2Rβ, CD122), as well as an alpha chain specific to each cytokine: IL-2 receptor alpha (IL-2Rα, CD25) or IL-15 receptor alpha (IL-15Rα, CD215). Both cytokines are considered potentially valuable therapeutic agents in oncology, and IL-2 has been 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.

[0003] IL-2 presents several challenges as a therapeutic agent. First, IL-2 preferentially activates T cells expressing a high-affinity receptor complex that is dependent on CD25 expression. Because Treg cells constitutively express CD25, they compete with effector T cells for IL-2 supply, and their activation is favorable for oncological treatment. This imbalance has led to the concept of high-dose IL-2. However, this approach poses additional problems due to IL-2-mediated toxicities, such as vascular leak syndrome.

[0004] IL-2 is primarily secreted by activated T cells, and its receptors are present on activated T cells, Tregs, NK cells, and B cells. In contrast, IL-15 is produced by monocytes and dendritic cells and is presented primarily as a membrane-bound heterodimeric complex with IL-15Rα present on the same cells. Its effects are achieved by trans-presenting the IL-15 / IL-15Rα complex to NK cells and CD8+ T cells that express IL-2Rβ and the common gamma chain.

[0005] As promising drugs, both cytokines suffer from extremely rapid clearance, with half-lives measured in minutes. Furthermore, IL-15 itself has low stability due to its preference for IL-15Rα-associated complexes. It has also been shown that recombinantly produced IL-15 / IL-15Rα heterodimers can potently activate T cells. Nevertheless, the short half-life precludes favorable dosing. The present invention solves this problem by providing a novel IL15 / IL15Rα heterodimeric Fc fusion protein. Summary of the Invention

[0006] Thus, in one aspect, the present invention provides a) a first fusion protein comprising a first protein domain and a first Fc domain, wherein the first protein domain is covalently linked to the N-terminus of the first Fc domain using a first domain linker; and b) a second fusion protein comprising a second protein domain and a second Fc domain. and a second fusion protein, wherein the second protein domain is covalently attached to the N-terminus of the Fc domain by means of a second domain linker, wherein the first and second Fc domains are selected from the group consisting of S267K / L368D / K370S:S267K / LS364K / E357Q, S364K / E357Q:L368D / K370S, L368 and K370S:S364K / E357Q, wherein the first protein domain comprises an IL15 protein and the second protein domain comprises an IL15Rα protein. In some embodiments, the first protein domain is covalently linked directly to the N-terminus of the first Fc domain without a first domain linker, and / or the second protein domain is covalently linked directly to the N-terminus of the second Fc domain without a second domain linker.

[0007] In some embodiments, the heterodimeric protein comprises (i) a first fusion protein having the polypeptide sequence of SEQ ID NO: XX (XENP15902) and a second fusion protein having the polypeptide sequence of SEQ ID NO: XX (XENP5908), (ii) a first fusion protein having the polypeptide sequence of SEQ ID NO: XX (XENP15902) and a second fusion protein having the polypeptide sequence of SEQ ID NO: XX (XENP15909), (iii) a first fusion protein having the polypeptide sequence of SEQ ID NO: XX (XENP16479) and a second fusion protein having the polypeptide sequence of SEQ ID NO: XX (XENP16479). (iv) a first fusion protein having a polypeptide sequence of SEQ ID NO: XX (XENP15902) and a second fusion protein having a polypeptide sequence of SEQ ID NO: XX (XENP16481); (v) a first fusion protein having a polypeptide sequence of SEQ ID NO: XX (XENP15902) and a second fusion protein having a polypeptide sequence of SEQ ID NO: XX (XENP16483); (vi) a first fusion protein having a polypeptide sequence of SEQ ID NO: XX (XENP16479). (vii) a first fusion protein having the polypeptide sequence of SEQ ID NO: XX (XENP15909) and a second fusion protein having the polypeptide sequence of SEQ ID NO: XX (XENP16479) and a second fusion protein having the polypeptide sequence of SEQ ID NO: XX (XENP16481); (viii) a first fusion protein having the polypeptide sequence of SEQ ID NO: XX (XENP16480) and a second fusion protein having the polypeptide sequence of SEQ ID NO: XX (XENP16482); (ix) a first fusion protein having the polypeptide sequence of SEQ ID NO: XX (XENP16 (x) a first fusion protein having a polypeptide sequence of SEQ ID NO: XX (XENP17064) and a second fusion protein having a polypeptide sequence of SEQ ID NO: XX (XENP17038); (xi) a first fusion protein having a polypeptide sequence of SEQ ID NO: XX (XENP17064) and a second fusion protein having a polypeptide sequence of SEQ ID NO: XX (XENP17040);(xii) a first fusion protein having the polypeptide sequence of SEQ ID NO: XX (XENP17062) and a second fusion protein having the polypeptide sequence of SEQ ID NO: XX (V17044); (xiii) a first fusion protein having the polypeptide sequence of SEQ ID NO: XX (XENP17686) and a second fusion protein having the polypeptide sequence of SEQ ID NO: XX (XENP15908); (xiv) a first fusion protein having the polypeptide sequence of SEQ ID NO: XX (XENP17687) and a second fusion protein having the polypeptide sequence of SEQ ID NO: XX (XENP15908); (xv) a first fusion protein having the polypeptide sequence of SEQ ID NO: XX (17688) and a second fusion protein having the polypeptide sequence of SEQ ID NO: XX (XENP15908); (xvi) a first fusion protein having the polypeptide sequence of SEQ ID NO: XX (XENP17689) and a second fusion protein having the polypeptide sequence of SEQ ID NO: XX (XENP15908); (xvii), (xviii) a first fusion protein having a polypeptide sequence of SEQ ID NO: XX (XENP17690) and a second fusion protein having a polypeptide sequence of SEQ ID NO: XX (XENP15908); (xix) a first fusion protein having a polypeptide sequence of SEQ ID NO: XX (XENP17692) and a second fusion protein having a polypeptide sequence of SEQ ID NO: XX (XENP15908); (xx) a first fusion protein having the polypeptide sequence of SEQ ID NO: XX (XENP17693) and a second fusion protein having the polypeptide sequence of SEQ ID NO: XX (XENP15908); (xxi) a first fusion protein having the polypeptide sequence of SEQ ID NO: XX (XENP17694) and a second fusion protein having the polypeptide sequence of SEQ ID NO: XX (XENP15908); (xxii) a first fusion protein having the polypeptide sequence of SEQ ID NO: XX (XENP17695) and a second fusion protein having the polypeptide sequence of SEQ ID NO: XX (XENP17696). (xxiii) a first fusion protein having a polypeptide sequence of SEQ ID NO: XX (XENP17696) and a second fusion protein having a polypeptide sequence of SEQ ID NO: XX (XENP15908); (xxiv) a first fusion protein having a polypeptide sequence of SEQ ID NO: XX (17697) and a second fusion protein having a polypeptide sequence of SEQ ID NO: XX (XENP15908); (xxv) a first fusion protein having a polypeptide sequence of SEQ ID NO: XX (XENP17698). (xxvi) a first fusion protein having a polypeptide sequence of SEQ ID NO: XX (XENP17699) and a second fusion protein having a polypeptide sequence of SEQ ID NO: XX (XENP15908); (xxvii) a first fusion protein having a polypeptide sequence of SEQ ID NO: XX (XENP17701) and a second fusion protein having a polypeptide sequence of SEQ ID NO: XX (XENP15908);(xxviii) a first fusion protein having the polypeptide sequence of SEQ ID NO: XX (XENP17691) and a second fusion protein having the polypeptide sequence of SEQ ID NO: XX (XENP15908); (xxix) a first fusion protein having the polypeptide sequence of SEQ ID NO: XX (XENP17702) and a second fusion protein having the polypeptide sequence of SEQ ID NO: XX (XENP15908); (xxx) a first fusion protein having the polypeptide sequence of SEQ ID NO: XX (XENP17703) and a second fusion protein having the polypeptide sequence of SEQ ID NO: XX (XENP17704). (xxxi) a first fusion protein having a polypeptide sequence of SEQ ID NO: XX (XENP17704) and a second fusion protein having a polypeptide sequence of SEQ ID NO: XX (XENP15908); (xxxii) a first fusion protein having a polypeptide sequence of SEQ ID NO: XX (XENP17705) and a second fusion protein having a polypeptide sequence of SEQ ID NO: XX (XENP15908); (xxxiii) a first fusion protein having a polypeptide sequence of SEQ ID NO: XX (XENP17705) and a second fusion protein having a polypeptide sequence of SEQ ID NO: XX (XENP15908); (xxxiv) the first fusion protein having the polypeptide sequence of SEQ ID NO: XX (XENP18783) and the second fusion protein having the polypeptide sequence of SEQ ID NO: XX (XENP15908); (xxxv) the first fusion protein having the polypeptide sequence of SEQ ID NO: XX (XENP18784) and the second fusion protein having the polypeptide sequence of SEQ ID NO: XX (XENP17761); (xxxvi) the second fusion protein having the polypeptide sequence of SEQ ID NO: XX (XENP18786) and the second fusion protein having the polypeptide sequence of SEQ ID NO: XX (XENP15908); (xxxvii) the first fusion protein having the polypeptide sequence of SEQ ID NO: XX (XENP18788) and the second fusion protein having the polypeptide sequence of SEQ ID NO: XX (XENP15908);(xxxviii) the first fusion protein having the polypeptide sequence of SEQ ID NO: XX (XENP19242) and the second fusion protein having the polypeptide sequence of SEQ ID NO: XX (XENP16481); or (xxxix) the first fusion protein having the polypeptide sequence of SEQ ID NO: XX (XENP19243) and the second fusion protein having the polypeptide sequence of SEQ ID NO: XX (XENP16481).

[0008] In some examples, the heterodimeric protein is selected from the group consisting of XENP20818, XENP20819, XENP21471, XENP21472, XENP21473, XENP21474, XENP21475, XENP21476, XENP21477, XENP22013, XENP22815, XENP22816, XENP22817, XENP22818, XENP22819, XENP22820, XENP22821, XENP22822, XENP22823, XENP22824, XENP22825, XENP22826, XENP22827, XENP22828, XENP22829, XENP22830, XENP22831, XENP22832, XENP22833, XENP22834, XENP22835, XENP22836, XENP22837, XENP22838, XENP22839, XENP22840, XENP22841, XENP22842, XENP22843, XENP22844, XENP22845, XENP22846, XENP22847, XENP22848, XENP22849, XENP22850, XENP22851, XENP22852, XENP22853, XENP22854, XENP22855, XENP22856, XENP22857, XENP22858, XENP22859, XENP22823, XENP22824, XENP22825, XENP22826, XENP22827, XENP22828, XENP22829, XENP22830, XENP22831, XENP22832, XENP22833, XENP22834, XENP23343, XENP23554, XENP23555, XENP23557, XENP23559, XENP24019, and XENP24020.

[0009] In a further aspect, the present invention provides a) a fusion protein comprising a first protein domain, a second protein domain, and a first Fc domain, wherein the first protein domain is covalently linked to the N-terminus of the second protein domain using a first domain linker and the second protein domain is covalently linked to the N-terminus of the first Fc domain using a second domain linker; and b) a heterodimeric protein comprising a second Fc domain, wherein the first and second Fc domains are linked to the N-terminus of the first Fc domain using a second domain linker according to EU numbering. / L368D / K370S:S267K / LS364K / E357Q, S364K / E357Q:L368D / K370S, L368D / K370S:S364K, L368E / K370S:S364K, T411T / E360E / Q362E:D401K, L368D / K370S:S364K / E357L, and K370S:S364K / E357Q, wherein the first protein domain comprises an IL15Rα protein and the second protein domain comprises an IL15 protein.

[0010] In some embodiments, the first fusion protein has the polypeptide sequence of SEQ ID NO:XX(16478) and the Fc domain has the polypeptide sequence of SEQ ID NO:XX(8924). The heterodimeric protein can be XENP21478.

[0011] In another aspect, the present invention provides a heterodimeric protein comprising: a) a fusion protein comprising a first protein domain and a first Fc domain, wherein the first protein domain is covalently linked to the N-terminus of the first Fc domain using a domain linker; b) a second Fc domain; and c) a second protein domain non-covalently linked to the first protein domain, wherein the first and second Fc domains have the amino acid sequence S267K / L368D / K370 according to EU numbering. and K370S:S364K / E357Q, wherein the first protein domain comprises IL15Rα and the second protein domain comprises IL15 protein.

[0012] In some embodiments, the heterodimeric protein comprises (i) a fusion protein having the polypeptide sequence of SEQ ID NO:XX(XENP16481), a second Fc domain having the polypeptide sequence of SEQ ID NO:XX(8793), and a second protein domain having the polypeptide sequence of SEQ ID NO:XX(16484); (ii) a fusion protein having the polypeptide sequence of SEQ ID NO:XX(17034 ), a second Fc domain having the polypeptide sequence of SEQ ID NO:XX(8793), and a second protein domain having the polypeptide sequence of SEQ ID NO:XX(16484), (iii) a fusion protein having the polypeptide sequence of SEQ ID NO:XX(17038), a second Fc domain having the polypeptide sequence of SEQ ID NO:XX(8793), and a second protein domain having the polypeptide sequence of SEQ ID NO:XX(16484), (iv) a fusion protein having the polypeptide sequence of SEQ ID NO:XX(17036), a second Fc domain having the polypeptide sequence of SEQ ID NO:XX(8793), and a second protein domain having the polypeptide sequence of SEQ ID NO:XX(16484), (v) a fusion protein having the polypeptide sequence of SEQ ID NO:XX(17038), a second Fc domain having the polypeptide sequence of SEQ ID NO:XX(8793), and a second protein domain having the polypeptide sequence of SEQ ID NO:XX(17074), (vi) a fusion protein having the polypeptide sequence of SEQ ID NO:XX( (vii) a fusion protein having a polypeptide sequence of SEQ ID NO: XX(17040), a second Fc domain having a polypeptide sequence of SEQ ID NO: XX(8793), and a second protein domain having a polypeptide sequence of SEQ ID NO: XX(17074); (viii) a fusion protein having a polypeptide sequence of SEQ ID NO: XX(17044), a polypeptide of SEQ ID NO: XX(8793). (ix) a fusion protein having a polypeptide sequence of SEQ ID NO: XX(17044), a second Fc domain having a polypeptide sequence of SEQ ID NO: XX(8793), and a second protein domain having a polypeptide sequence of SEQ ID NO: XX(17072); (x) a fusion protein having a polypeptide sequence of SEQ ID NO: XX(17075), a second Fc domain having a polypeptide sequence of SEQ ID NO: XX(8793);and a second protein domain having the polypeptide sequence of SEQ ID NO:XX(17041), (xi) a fusion protein having the polypeptide sequence of SEQ ID NO:XX(17043), a second Fc domain having the polypeptide sequence of SEQ ID NO:XX(8793), and a second protein domain having the polypeptide sequence of SEQ ID NO:XX(17070), (xii) a fusion protein having the polypeptide sequence of SEQ ID NO:XX(17045), a second Fc domain having the polypeptide sequence of SEQ ID NO:XX(8793), and a second protein domain having the polypeptide sequence of SEQ ID NO:XX(17046). (xiii) a fusion protein having the polypeptide sequence of SEQ ID NO:XX(17042), a second Fc domain having the polypeptide sequence of SEQ ID NO:XX(8793), and a second protein domain having the polypeptide sequence of SEQ ID NO:XX(17083), or (xiv) a fusion protein having the polypeptide sequence of SEQ ID NO:XX(15908), a second Fc domain having the polypeptide sequence of SEQ ID NO:XX(8793), and a second protein domain having the polypeptide sequence of SEQ ID NO:XX(16484). The heterodimeric protein can be selected from the group consisting of XENP21479, XENP22357, XENP22354, XENP22355, XENP22356, XENP22357, XENP22358, XENP22359, XENP22360, XENP22361, XENP22362, XENP22363, XENP22364, XENP22365, XENP22366, and XENP22637.

[0013] In a further aspect, the present invention provides a fusion protein comprising: a) a first fusion protein comprising a first protein domain and a first Fc domain, wherein the first protein domain is covalently linked to the N-terminus of said first Fc domain using a domain linker; b) a second fusion protein comprising a second heavy chain comprising a second protein domain and a first second heavy chain comprising a second Fc domain, wherein the second protein domain is covalently attached to the C-terminus of the second Fc domain using a domain linker; and c) a third fusion protein comprising a second heavy chain non-covalently linked to the first protein domain of the first fusion protein. and d) a fourth fusion protein non-covalently linked to a second protein domain of the second fusion protein, wherein the first and second Fc domains are S267K / L368D / K370S:S267K / LS364K / E357Q, S364K / E357Q:L368D / K370S, L368D / K370S:S364K, L368E ... and K370S:S364K / E357Q, wherein the first and second protein domains comprise an IL15Rα protein, and the third and fourth protein domains comprise an IL15 protein.

[0014] In some embodiments, the heterodimeric protein comprises (i) a first fusion protein having the polypeptide sequence of SEQ ID NO:XX(17023), a second fusion protein having the polypeptide sequence of SEQ ID NO:XX(17023), a third protein domain having the polypeptide sequence of SEQ ID NO:XX(16484), and a fourth protein domain having the polypeptide sequence of SEQ ID NO:XX(16484), or (ii) a first fusion protein having the polypeptide sequence of SEQ ID NO:XX(17581), a second fusion protein having the polypeptide sequence of SEQ ID NO:XX(17581), a third protein domain having the polypeptide sequence of SEQ ID NO:XX(17074), and a fourth protein domain having the polypeptide sequence of SEQ ID NO:XX(17074). The heterodimeric protein may be XENP21978 or XENP22634.

[0015] In a further aspect, the present invention provides a heterodimeric protein comprising: a) a first fusion protein comprising a first Fc domain and a first protein domain, wherein the first Fc domain is covalently linked to the N-terminus of the first protein domain using a domain linker; b) a second Fc domain; and c) a second protein domain non-covalently linked to the first protein domain of the first fusion protein, wherein the first and second Fc domains have the amino acid sequence S267K / L368D according to EU numbering. / K370S:S267K / LS364K / E357Q, S364K / E357Q:L368D / K370S, L368D / K370S:S364K, L368E / K370S:S364K, T411T / E360E / Q362E:D401K, L368D / K370S:S364K / E357L, and K370S:S364K / E357Q, wherein the first protein domain comprises an IL15Rα protein and the second protein domain comprises an IL15 protein.

[0016] In some embodiments, the heterodimeric protein comprises (i) a first fusion protein having the polypeptide sequence of SEQ ID NO: XX(17603), a second Fc domain having the polypeptide sequence of SEQ ID NO: XX(8927), and a second protein domain having the polypeptide sequence of SEQ ID NO: XX(16484), or ii) a first fusion protein having the polypeptide sequence of SEQ ID NO: XX(17605), a second Fc domain having the polypeptide sequence of SEQ ID NO: XX(8927), and a second protein domain having the polypeptide sequence of SEQ ID NO: XX(17074).

[0017] In any of the embodiments of the invention, the first and / or second Fc domain may have an additional set of amino acid substitutions including, according to EU numbering, Q295E / N384D / Q418E / N421D. In some cases, the first and / or second Fc domain may have an additional set of amino acid substitutions including, according to EU numbering, 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 an additional set of amino acid substitutions consisting of E233P / L234V / L235A / G236del.

[0018] In some embodiments of the present invention, the IL15 protein has a polypeptide sequence selected from the group consisting of SEQ ID NO:1 (full-length human IL15) and SEQ ID NO:2 (truncated human IL15), and the IL15Rα protein has a polypeptide sequence selected from the group consisting of SEQ ID NO:3 (full-length human IL15Rα) and SEQ ID NO:4 (sushi domain of human IL15Rα). In some cases, the IL15 protein and the IL15Rα protein each have a set of amino acid substitutions or additions selected from the group consisting of E87C:D96 / P97 / C98, E87C:D96 / C97 / A98, V49C:S40C, L52C:S40C, E89C:K34C, Q48C:G38C, E53C:L42C, C42S:A37C, and L45C:A37C.

[0019] In a further aspect, the present invention provides XENP20818, XENP20819, XENP21471, XENP21472, XENP21473, XENP21474, XENP21475, XENP21476, XENP21477, XENP21478, XENP21479, XENP21978, XENP22013, XENP22015, XENP22017, XENP223 54, XENP22355, XENP22356, XENP22357, XENP22358, XENP22359, XENP22360, XENP22361, XENP22362, XENP22363, XENP22364, XENP22365, XENP22366, XENP22637, and XENP22639. In some aspects, the present invention provides XENP20818, XENP20819, XENP21471, XENP21472, XENP21473, XENP21474, XENP21475, XENP21476, XENP21477, XENP22013, XENP22815, XENP22816, XENP22817, XENP22818, XENP22819, XENP22820, XENP22821, XENP22822, XENP22823, XEN Heterodimeric proteins selected from the group consisting of XENP22824, XENP22825, XENP22826, XENP22827, XENP22828, XENP22829, XENP22830, XENP22831, XENP22832, XENP22833, XENP22834, XENP23343, XENP23554, XENP23555, XENP23557, XENP23559, XENP24019, and XENP24020 are provided. Methods of making these proteins and treating patients with the proteins are all provided, as well as nucleic acids, expression vectors, and host cells. [Brief explanation of the drawings]

[0020] [Figure 1] The structure of IL-15 in complex with its receptors IL-15Rα (CD215), IL-15Rβ (CD122), and the common gamma chain (CD132) is shown. [Figure 2A-2B] The sequences of IL-15 and its receptors are shown in Figure 2. Figure 2A shows the sequences of human IL-15, human IL-15Rα, and human IL-15Rβ. Figure 2B shows the sequence of the human common gamma receptor. [Figures 3A-3E] Useful pairs of Fc heterodimer mutation sets (including skew and PI mutations) are shown in Figures 3D and 3E, where there are mutants without the corresponding "monomer 2" mutation, and these are pi mutants that can be used alone in either monomer. [Figure 4] A list of equivalent mutant antibody constant regions and their substitutions is provided below. pI_(-) indicates a low pI mutation and pI_(+) indicates a high pI mutation. These can be optionally and independently combined with other heterodimerization mutations of the invention (and other mutation types as outlined herein). [Figure 5] Useful deletion mutations that eliminate FcγR binding are shown (often called "knockout" or "KO" mutations). Generally, deletion mutations are found in both monomers, although in some cases they may be found in only one monomer. [Figures 6A-6E] 1 illustrates a particularly useful embodiment of the "non-cytokine" component of the present invention. [Figure 7] Some exemplary variable length linkers 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). [Figures 8A-8E]

[0039] Figure 1 shows 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)). Scaffold 1 is based on human IgG1 (356E / 358M allotype) and contains C220S in both chains, skew mutations S364K / E357Q:L368D / K370S in one chain, pI mutations Q295E / N384D / Q418E / N421D, skew mutations L368D / K370S in both chains, and deletion mutations E233P / L234V / L235A / G236del / S267K. Scaffold 2 is based on human IgG1 (356E / 358M allotype) and contains C220S in both chains, S364K:L368D / K370S skew mutations in one chain, and Q295E / N384D / Q418E / N421D pI mutations, with L368D / K370S skew mutations and E233P / L234V / L235A / G236del / S267K deletion mutations in both chains. Scaffold 3 is based on human IgG1 (356E / 358M allotype) and contains C220S in both chains, S364K:L368E / K370S skew mutations in one chain, and Q295E / N384D / Q418E / N421D pI mutations, with L368E / K370S skew mutations and E233P / L234V / L235A / G236del / S267K deletion mutations in both chains. Scaffold 4 is based on human IgG1 (356E / 358M allotype) and contains C220S in both chains, D401K in one chain: K360E / Q362E / T411E skew mutations, Q295E / N384D / Q418E / N421D pI mutations, and K360E / Q362E / T411E skew mutations and E233P / L234V / L235A / G236del / S267K deletion mutations in both chains. Scaffold 5 is based on human IgG1 (356D / 358L allotype) and contains C220S in both chains, skew mutations S364K / E357Q:L368D / K370S in one chain, pI mutations Q295E / N384D / Q418E / N421D, skew mutations L368D / K370S in both chains, and deletion mutations E233P / L234V / L235A / G236del / S267K.Scaffold 6 is based on human IgG1 (356E / 358M allotype) and contains C220S in both chains, skew mutations S364K / E357Q:L368D / K370S in one chain, pI mutations Q295E / N384D / Q418E / N421D, skew mutations L368D / K370S in both chains, deletion mutations E233P / L234V / L235A / G236del / S267K, and an N297A mutation in both chains. Scaffold 7 is identical to 6 except for the N297S mutation. Alternative formats for scaffolds 6 and 7 can eliminate the deletion mutations E233P / L234V / L235A / G236del / S267K in both chains. Scaffold 8 is based on human IgG4 and contains S364K / E357Q:L368D / K370S skew mutations, Q295E / N384D / Q418E / N421D pI mutations in one chain, L368D / K370S skew mutations and S228P (according to EU numbering, which is S241P in Kabat) mutation in both chains, which eliminates Fab arm exchange, as known in the art. Scaffold 9 is based on human IgG2 and contains S364K / E357Q:L368D / K370S skew mutations, Q295E / N384D / Q418E / N421D pI mutations in one chain, and L368D / K370S skew mutations. Scaffold 10 is based on human IgG2 and contains S364K / E357Q:L368D / K370S skew mutations in one chain, Q295E / N384D / Q418E / N421D pI mutations, L368D / K370S skew mutations in both chains, and an S267K mutation. Scaffold 11 is identical to scaffold 1 except that it contains M428L / N434S Xtend mutations. Scaffold 12 is based on human IgG1 (356E / 358M allotype) and contains C220S in both identical chains and E233P / L234V / L235A / G236del / S267K deletion mutations in both identical chains.Scaffold 13 is based on human IgG1 (356E / 358M allotype) and contains C220S in both chains, skew mutations S364K / E357Q:L368D / K370S in one chain, pI mutations P217R / P229R / N276K, skew mutations S364K / E357Q in both chains, and deletion mutations E233P / L234V / L235A / G236del / S267K. As will be appreciated by those skilled in the art and as outlined below, these sequences can 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, scIL-15 / Rα, and dsIL-15 / Rα, as shown schematically in Figures 9A-9G and 39A-39D. Additionally, any IL-15 and / or IL-15Rα(sushi) mutations can be incorporated into these scaffolds of Figures 8A-8E in any combination. Included within 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 (as will be appreciated by those skilled in the art) compared to the "parent" in the diagram, which already contains several amino acid modifications compared to the parent human IgG1 (or IgG2 or IgG4 based on the scaffold). That is, the listed scaffolds can contain further amino acid modifications (generally amino acid substitutions) in addition to the skew, pI, and deletion mutations contained within the scaffold of this diagram (Figure 8). [Figures 9A-9G]Several formats of the IL-15 / Rα-Fc fusion proteins of the present invention are shown. The IL-15Rα heterodimeric Fc fusion or "IL-15 / Rα-hetero-Fc" (FIG. 9A) 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- and N-termini of the Fc region. The single-chain IL-15 / Rα-Fc fusion or "scIL-15 / Rα-Fc" (Figure 9B) 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 either "Fc-only" or "empty Fc" at the other end of the molecule. The noncovalent IL-15 / Rα-Fc or "ncIL-15 / Rα-Fc" (Figure 9C) contains IL-15Rα(sushi) fused to the heterodimeric Fc region, while IL-15 is transfected separately to form the noncovalent IL-15 / Rα complex, with either "Fc-only" or "empty Fc" at the other end of the molecule. The bivalent noncovalent IL-15 / Rα-Fc fusion or "bivalent ncIL-15 / Rα-Fc" (Figure 9D) contains IL-15Rα(sushi) fused to the N-terminus of the homodimeric Fc region, while IL-15 is transfected separately to form a noncovalent IL-15 / Rα complex. The bivalent single-chain IL-15 / Rα-Fc fusion or "bivalent scIL-15 / Rα-Fc" (Figure 9E) contains 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 noncovalently linked IL-15 / Rα fusion or “Fc-ncIL-15 / Rα” ( Fig. 9F ) contains IL-15Rα(sushi) fused to the C-terminus of the heterodimeric Fc region, while IL-15 is transfected separately to form a noncovalently linked IL-15 / Rα complex, 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 9G) 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 "Fc only" or "empty Fc" at the other end of the molecule. [Figure 10] The sequences of exemplary IL-15 / Rα-Fc fusion proteins in the "IL-15 / Rα-hetero-Fc" format, XENP20818 and XENP21475, are shown, with additional sequences listed in the Sequence Listing as XENP20819, XENP21471, XENP21472, XENP21473, XENP21474, XENP21476, and XENP21477. IL-15 and IL-15Rα (sushi) are underlined, the linker is double underlined (as will be understood by those of skill in the art, portions of the linker may be replaced with other linkers shown in Figure 7), and a slash ( / ) indicates the boundary between IL-15, IL-15Rα, linker, and Fc region. [Figure 11] The sequence of XENP21478, an exemplary IL-15 / Rα-Fc fusion protein in the "scIL-15 / Rα-Fc" format, is shown; additional sequences are listed in the Sequence Listing as XENP21993, XENP21994, XENP21995, XENP23174, XENP23175, XENP24477, and XENP24480. IL-15 and IL-15Rα (sushi) are underlined, the linker is double underlined (as will be understood by those of skill in the art, portions of the linker may be replaced with other linkers shown in Figure 7), and a slash ( / ) indicates the boundary between IL-15, IL-15Rα, linker, and Fc region. [Figures 12A-12B]7 shows the sequences of exemplary IL-15 / Rα-Fc fusion proteins, XENP21479, XENP22366, and XENP24348, in the "ncIL-15 / Rα-Fc" format. IL-15 and IL-15Rα (sushi) are underlined, the linker is double underlined (as will be understood by those skilled in the art, portions of the linker may be replaced with other linkers shown in FIG. 7), and a slash ( / ) indicates the boundary between IL-15, IL-15Rα, the linker, and the Fc region. [Figure 13] The sequence of an exemplary IL-15 / Rα-Fc fusion protein, XENP21978, in the "bivalent ncIL-15 / Rα-Fc" format is shown, with an additional sequence listed in the Sequence Listing as XENP21979. IL-15 and IL-15Rα (sushi) are underlined, the linker is double underlined (as will be understood by those skilled in the art, portions of the linker may be replaced with other linkers shown in FIG. 7), and a slash ( / ) indicates the boundary between IL-15, IL-15Rα, the linker, and the Fc region. [Figure 14] 7 shows the sequence of an exemplary IL-15 / Rα-Fc fusion protein in the "bivalent scIL-15 / Rα-Fc" format. IL-15 and IL-15Rα (sushi) are underlined, the linker is double underlined (as will be understood by those skilled in the art, portions of the linker may be replaced with other linkers shown in FIG. 7), and a slash ( / ) indicates the boundary between IL-15, IL-15Rα, linker, and Fc region. [Figure 15] The sequence of an exemplary IL-15 / Rα-Fc fusion protein in the "Fc-ncIL-15 / Rα" format, XENP22637, is shown, with an additional sequence listed in the Sequence Listing as XENP22638. IL-15 and IL-15Rα (sushi) are underlined, the linker is double underlined (as will be understood by those skilled in the art, portions of the linker may be replaced with other linkers shown in FIG. 7), and a slash ( / ) indicates the boundary between IL-15, IL-15Rα, linker, and Fc region. [Figure 16]7 shows the sequence of an exemplary IL-15 / Rα-Fc fusion protein in the "Fc-scIL-15 / Rα" format. IL-15 and IL-15Rα (sushi) are underlined, the linker is double underlined (as will be understood by those skilled in the art, portions of the linker may be replaced with other linkers shown in FIG. 7), and a slash ( / ) indicates the boundary between IL-15, IL-15Rα, linker, and Fc region. [Figures 17A-17E] A) IL-15 / Rα-Fc fusion protein format of XENP20818, B) purity and homogeneity of XENP20818 as determined by SEC and C) CEF, D) affinity of XENP20818 for IL-2Rβ as determined by Octet, and E) stability of XENP20818 as determined by DSF. [Figures 18A-18E] A) IL-15 / Rα-Fc fusion protein format of XENP21478, B) purity and homogeneity of XENP21478 as determined by SEC and C) CEF, D) affinity of XENP21478 for IL-2Rβ as determined by Octet, and E) stability of XENP21478 as determined by DSF. [Figures 19A-19E] A) IL-15 / Rα-Fc fusion protein format of XENP21479, B) purity and homogeneity of XENP21479 as determined by SEC and C) CEF, and D) affinity of XENP21479 for IL-2Rβ as determined by Octet, and E) stability of XENP21479 as determined by DSF. [Figures 20A-20C] Figure 1 shows the induction of proliferation of A) NK (CD56+ / CD16+) cells, B) CD4+ T cells, and C) CD8+ cells based on Ki67 expression measured by FACS by exemplary IL-15 / Rα-Fc fusion proteins in IL-15 / Rα-hetero-Fc format with various linker lengths. [Figures 21A-21C]Figure 1 shows the induction of proliferation of A) NK (CD56+ / CD16+) cells, B) CD4+ T cells, and C) CD8+ cells based on Ki67 expression measured by FACS by exemplary IL-15 / Rα-Fc fusion proteins in scIL-15 / Rα-Fc format (XENP21478) and ncIL-15 / Rα-Fc format (XENP21479). [Figure 22] 1 shows the enhancement of IL-2 secretion by an exemplary IL-15 / Rα-Fc fusion protein, an isotype control, and a bivalent anti-PD-1 antibody relative to a PBS control in an SEB-stimulated PBMC assay. [Figure 23] 1 shows survival curves of PBMC-engrafted NSG mice after treatment with XENP20818 and recombinant IL-15. [Figure 24] 1 shows the engraftment of human PBMCs and the concentration of IFNγ in the serum of NSG mice 7 days after treatment with the indicated concentrations of XENP20818. [Figures 25A-25C] Shown are the numbers of A) CD4+ T cells, B) CD8+ T cells, and C) CD45+ cells in whole blood of human PBMC-engrafted NSG mice on day 7 after treatment with the indicated concentrations of XENP20818. [Figure 26] 1 shows a structural model of the IL-15 / Rα heterodimer showing the location of the engineered disulfide bond pairs. [Figure 27] 1 shows the sequence of an exemplary IL-15Rα(sushi) mutant engineered with additional residues at the C-terminus to serve as a scaffold for engineering cysteine ​​residues. [Figure 28] 1 shows the sequence of an exemplary cysteine ​​engineered IL-15 mutant to form a covalent disulfide bond with a cysteine ​​engineered IL-15Rα (sushi) mutant. [Figure 29] 1 shows the sequence of an exemplary cysteine-engineered IL-15Rα(sushi) mutant to form a covalent disulfide bond with a cysteine-engineered IL-15 mutant. [Figures 30A-30C]IL-15 / Rα heterodimers with or without an engineered disulfide bond between IL-15(sushi) and IL-15Rα are shown. The noncovalently linked IL-15 / Rα heterodimer or "ncIL-15 / Rα heterodimer" (Figure 30A) contains separately transfected, noncovalently linked IL-15Rα(sushi) and IL-15. The disulfide-linked IL-15 / Rα heterodimer or "dsIL-15 / Rα heterodimer" (Figure 30B) contains separately transfected, covalently linked IL-15Rα(sushi) and IL-15 as a result of engineered cysteines. The single-chain IL-15 / Rα heterodimer or "scIL-15 / Rα heterodimer" (Figure 30C) contains IL-15Rα(sushi) fused to IL-15 by a variable-length Gly-Ser linker. [Figure 31] The sequence of an exemplary ncIL-15 / Rα heterodimer, XENP21996, is shown. It is important to note that these sequences were generated using a polyhistidine (His×6 or HHHHHH) C-terminal tag at the C-terminus of IL-15Rα (sushi). [Figure 32] The sequences of exemplary dsIL-15 / Rα heterodimers XENP22004, XENP22005, XENP22006, XENP22008, and XENP22494 are shown, with additional sequences shown in the Sequence Listing as XENP22007, XENP22009, XENP22010, XENP22011, XENP22012, and XENP22493. It is important to note that these sequences were generated using a polyhistidine (Hisx6 or HHHHHH) C-terminal tag at the C-terminus of IL-15Rα(sushi). [Figure 33]The sequence of an exemplary scIL-15 / Rα heterodimer, XENP22049, is shown. It is important to note that these sequences were generated using a polyhistidine (Hisx6 or HHHHHH) C-terminal tag at the C-terminus of IL-15. IL-15 and IL-15Rα (sushi) are underlined, the linker is double underlined (as will be understood by those skilled in the art, portions of the linker may be replaced with other linkers shown in FIG. 7), and a slash ( / ) indicates the boundary between IL-15, IL-15Rα, and the linker. [Figure 34] 1 shows the purity and homogeneity of exemplary IL-15 / Rα heterodimers with and without engineered disulfide bonds as determined by CEF. [Figure 35] 1 shows the purity and homogeneity of exemplary IL-15 / Rα-Fc heterodimers with and without engineered disulfide bonds as determined by CEF. [Figure 36] 1 shows the stability and melting temperature of exemplary IL-15 / Rα heterodimers with and without engineered disulfide bonds as shown by melting curves from DSF. [Figure 37] 1 shows the stability and melting temperature of exemplary IL-15 / Rα heterodimers with and without engineered disulfide bonds as shown by melting curves from DSF. [Figure 38] Expression yield, molecular weight, predicted change in affinity between IL-15 and IL-15Rα(sushi) as calculated by MOE software, melting temperature, and affinity for IL-2Rβ for IL-15 / Rα heterodimers with and without engineered disulfide bonds are shown. Mutations are shown in parentheses after the relevant monomer. [Figures 39A-39D]

[0039] Figure 39A shows additional formats of the IL-15 / Rα-Fc fusion proteins of the invention with engineered disulfide bonds. The disulfide-bonded IL-15 heterodimer Fc fusion or "dsIL-15 / Rα-heterodimer Fc" (Figure 39A) is identical to "IL-15 / Rα-heterodimer Fc," but IL-15Rα(sushi) and IL-15 are additionally covalently attached as a result of engineered cysteines. The disulfide-bonded IL-15 / Rαfc fusion or "dsIL-15 / Rα-Fc" (Figure 39B) is identical to "ncIL-15 / Rα-Fc," but IL-15Rα(sushi) and IL-15 are additionally covalently attached as a result of engineered cysteines. The bivalent disulfide-bonded IL-15 / Rα-Fc or "bivalent dsIL-15 / Rα-Fc" (Figure 39C) is identical to "bivalent ncIL-15 / Rα-Fc" but with the additional covalent attachment of IL-15Rα(sushi) and IL-15 as a result of engineered cysteines. The Fc-disulfide-bonded IL-15 / Rα fusion or "Fc-dsIL-15 / Rα" (Figure 39D) is identical to "Fc-ncIL-15 / Rα" but with the additional covalent attachment of IL-15Rα(sushi) and IL-15 as a result of engineered cysteines. [Figure 40A-40B] 7 shows the sequences of exemplary IL-15 / Rα-Fc fusion proteins in the "dsIL-15 / Rα-heteroFc" format: XENP22013, XENP22014, XENP22015, and XENP22017. IL-15 and IL-15Rα (sushi) are underlined, the linker is double underlined (as will be understood by those skilled in the art, portions of the linker may be replaced with other linkers shown in FIG. 7), and a slash ( / ) indicates the boundary between IL-15, IL-15Rα, linker, and Fc region. [Figure 41A-41B]The sequences of exemplary IL-15 / Rα-Fc fusion proteins in the "dsIL-15 / Rα-Fc" format are shown: XENP22357, XENP22358, XENP22359, XENP22684, and XENP22361. Additional sequences are shown in the Sequence Listing as XENP22360, XENP22362, XENP22363, XENP22364, XENP22365, and XENP22366. IL-15 and IL-15Rα (sushi) are underlined, the linker is double underlined (as will be understood by those skilled in the art, portions of the linker may be replaced with other linkers shown in Figure 7), and a slash ( / ) indicates the boundary between IL-15, IL-15Rα, linker, and Fc region. [Figure 42] The sequences of exemplary IL-15 / Rα-Fc fusion proteins in the "bivalent dsIL-15 / Rα-Fc" format, XENP22634, XENP22635, and XENP22636, are shown. An additional sequence is shown in the Sequence Listing as XENP22687. IL-15 and IL-15Rα (sushi) are underlined, the linker is double underlined (as will be understood by those skilled in the art, portions of the linker may be replaced with other linkers shown in Figure 7), and a slash ( / ) indicates the boundary between IL-15, IL-15Rα, linker, and Fc region. [Figure 43] 7 shows the sequences of exemplary IL-15 / Rα-Fc fusion proteins, XENP22639 and XENP22640, in the "Fc-dsIL-15 / Rα" format. IL-15 and IL-15Rα (sushi) are underlined, the linker is double underlined (as will be understood by those skilled in the art, portions of the linker may be replaced with other linkers shown in FIG. 7), and a slash ( / ) indicates the boundary between IL-15, IL-15Rα, linker, and Fc region. [Figure 44] 1 shows the purity and homogeneity of exemplary IL-15 / Rα-Fc fusion proteins with and without engineered disulfide bonds as determined by CEF. [Figures 45A-45C]1 shows the induction of proliferation of A) NK (CD56+ / CD16+) cells, B) CD8+ T cells, and C) CD4+ T cells by exemplary IL-15 / Rα-Fc fusion proteins with or without engineered disulfide bonds, based on Ki67 expression as measured by FACS. [Figure 46] 1 shows the structures of IL-15 complexed with IL-15Rα, IL-2Rβ, and the common γ chain, with the locations of substitutions designed to reduce potency indicated. [Figures 47A-47C]

[0023] Figures 1A-1C show sequences of exemplary IL-15 mutants engineered to reduce potency. Included within each of these mutant 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. In a non-limiting example, the listed sequences may contain additional amino acid modifications, such as those that contribute to the formation of covalent disulfide bonds, as described in Example 2. [Figures 48A-48D]The sequences of exemplary IL-15 / Rα-Fc fusion proteins in the "IL-15 / Rα-R / R" format engineered for low potency, XENP22821, XENP22822, XENP23554, XENP23557, XENP23561, XENP24018, XENP24019, XENP24045, XENP24051, and XENP24052, are shown. Further sequences are shown in the Sequence Listing as XENP22815, XENP22816, XENP22817, XENP22818, XENP22819, XENP22820, XENP22823, XENP22824, XENP22825, XENP22826, XENP22827, XENP22828, XENP22829, XENP22830, XENP22831, XENP22832, XENP22833, XENP22834, XENP23555, XENP23559, XENP23560, XENP24017, XENP24020, XENP24043, and XENP24048. IL-15 and IL-15Rα (sushi) are underlined, the linker is double underlined (as will be understood by those skilled in the art, portions of the linker may be replaced with other linkers shown in Figure 7), and a slash ( / ) indicates the boundary between IL-15, IL-15Rα, the linker, and the Fc region. [Figures 49A-49C] The sequences of exemplary IL-15 / Rα-Fc fusion proteins in the "SCIL-15 / Rα-Fc" format engineered for low potency are shown in Figure 7. Additional sequences are shown in the Sequence Listing as XENP24013, XENP24014, and XENP24016. IL-15 and IL-15Rα (sushi) are underlined, the linker is double underlined (though one of skill in the art will appreciate that portions of the linker may be replaced with other linkers shown in Figure 7), and a slash ( / ) indicates the boundary between the IL-15, IL-15Rα, linker, and Fc region. [Figure 50A-50B]7 shows the sequences of exemplary IL-15 / Rα-Fc fusion proteins, XENP24349, XENP24890, and XENP25138, in the "ncIL-15 / Rα-Fc" format engineered for low potency. IL-15 and IL-15Rα (sushi) are underlined, the linker is double underlined (as will be understood by those skilled in the art, portions of the linker may be replaced with other linkers shown in FIG. 7), and a slash ( / ) indicates the boundary between IL-15, IL-15Rα, linker, and Fc region. [Figure 51] The sequences of exemplary ncIL-15 / Rα heterodimers engineered to reduce low potency, XENP22801 and XENP22802, are shown. Additional sequences are shown in the Sequence Listing as XENP22791, XENP22792, XENP22793, XENP22794, XENP22795, XENP22796, XENP22803, XENP22804, XENP22805, XENP22806, XENP22807, XENP22808, XENP22809, XENP22810, XENP22811, XENP22812, XENP22813, and XENP22814. It is important to note that these sequences were generated using a polyhistidine (Hisx6 or HHHHHH) C-terminal tag at the C-terminus of IL-15Rα(sushi). [Figure 52] 7 shows the sequence of XENP24342, an exemplary IL-15 / Rα-Fc fusion protein in the "bivalent ncIL-15 / Rα-Fc" format engineered for low potency. IL-15 and IL-15Rα (sushi) are underlined, the linker is double underlined (as will be understood by those skilled in the art, portions of the linker may be replaced with other linkers shown in FIG. 7), and a slash ( / ) indicates the boundary between IL-15, IL-15Rα, linker, and Fc region. [Figure 53]7 shows the sequences of exemplary IL-15 / Rα-Fc fusion proteins, XENP23472 and XENP23473, in the "dsIL-15 / Rα-Fc" format engineered for reduced potency. IL-15 and IL-15Rα (sushi) are underlined, the linker is double underlined (as will be understood by those skilled in the art, portions of the linker may be replaced with other linkers shown in FIG. 7), and a slash ( / ) indicates the boundary between IL-15, IL-15Rα, linker, and Fc region. [Figures 54A-54C] Figure 1 shows the induction of proliferation by mutant IL-15 / Rα-Fc fusion proteins of A) NK cells, B) CD8+ (CD45RA-) T cells, and C) CD4+ (CD45RA-) T cells based on Ki67 expression as measured by FACS. [Figure 55] The EC50 for induction of NK cell and CD8+ T cell proliferation by mutant IL-15 / Rα-Fc fusion proteins and the fold reduction in EC50 compared to XENP20818 are shown. [Figures 56A-56C] Figures 59A-59D show the gating of lymphocytes and subpopulations in the experiment shown. Figure 56A shows the gated lymphocyte population. Figure 56B shows the CD3-negative and CD3-positive subpopulations. Figure 56C shows the CD16-negative and CD16-positive subpopulations of CD3-negative cells. [Figures 57A-57C] Figures 59A-59D show gating of CD3+ lymphocyte subpopulations in the experiment shown. Figure 57A shows CD4+, CD8+, and γδ T cell subpopulations of CD3+ T cells. Figure 57B shows CD45RA(-) and CD45RA(+) subpopulations of CD4+ T cells. Figure 57C shows CD45RA(-) and CD45RA(+) subpopulations of CD8+ T cells. [Figure 58A-58B] The expression of CD69 and CD25 before (FIG. 58A) and after (FIG. 58B) incubation of human PBMC with XENP22821 is shown. [Figures 59A-59D]Figures 59A-C show cell proliferation in human PBMCs incubated with the indicated mutant IL-15 / Rα-Fc fusion proteins for 4 days. Figures 59A-C show the percentage of proliferating NK cells (CD3-CD16+) (Figure 59A), CD8+ T cells (CD3+CD8+CD45RA-) (Figure 59B), and CD4+ T cells (CD3+CD4+CD45RA-) (Figure 59C). Figure 59D shows the fold change in EC50 of various IL15 / IL15Rα Fc heterodimers compared to the control (XENP20818). [Figures 60A-60D] Figures 60A-C show cell proliferation in human PBMCs incubated for 3 days with the indicated mutant IL-15 / Rα-Fc fusion proteins. Figures 60A-C show the percentage of proliferating CD8+ (CD45RA-) T cells (Figure A), CD4+ (CD45RA-) T cells (Figure B), γδ T cells (Figure C), and NK cells (Figure D). [Figures 61A-61C] The percentage of Ki67 expression in (A) CD8+ T cells, (B) CD4+ T cells, and (C) NK cells after treatment with additional IL-15 / Rα mutants is shown. [Figures 62A-62E] The percentage of Ki67 expression in (A) CD8+ (CD45RA-) T cells, (B) CD4+ (CD45RA-) T cells, (C) γδ T cells, (D) NK (CD16+CD8α-) cells, and (E) NK (CD56+CD8α-) cells after treatment with IL-15 / Rα mutants is shown. [Figures 63A-63E] The percentage of Ki67 expression in (A) CD8+ (CD45RA-) T cells, (B) CD4+ (CD45RA-) T cells, (C) γδ T cells, (D) NK (CD16+CD8α-) cells, and (E) NK (CD56+CD8α-) cells after treatment with IL-15 / Rα mutants is shown. [Figure 64A-64D] Shown are the percentages of Ki67 expression in (A) CD8+ T cells, (B) CD4+ T cells, (C) γδ T cells, and (D) NK (CD16+) cells after treatment with additional engineered IL-15 / Rα mutants with various linker lengths for reduced potency. [Figure 65A-65D]The percentage of Ki67 expression in (A) CD8+ T cells, (B) CD4+ T cells, (C) γδ T cells, and (D) NK (CD16+) cells after treatment with additional IL-15 / Rα mutants is shown. [Figures 66A-66D] Gating of lymphocytes and subpopulations for the experiment shown in Figure 67 is shown. Figure 66A shows gating of lymphocyte populations. Figure 66B shows CD4+ and CD8+ T cells. Figure 66C shows CD45RA and CD27 expressing subpopulations of CD4+ T cells. Figure 66D shows CD45RA and CD27 expressing subpopulations of CD8+ T cells. [Figures 67A-67C] Figure 67C shows STAT5 phosphorylation in A) CD8+ T cells (CD45RA-CD27-) and B) CD4+ T cells (CD45RA-CD27-) after 4 days of incubation of PBMCs with the indicated concentrations of mutant IL15 / IL15Rα-Fc fusions. Figure 67C shows the fold change in EC50 of various IL15 / IL15Rα Fc heterodimers compared to the control (XENP20818). [Figure 68] PK of various IL-15 / RαFc fusion proteins or control upon IV-TV administration in C57BL / 6 mice at a single dose of 0.1 mg / kg. [Figure 69] Correlation between half-life and NK cell activity is shown. [Figure 70] This shows that CD45+ cell levels are a predictor of disease. [Figure 71A-71B] Enhancement of engraftment by mutant IL-15 / Rα-Fc fusion proteins as indicated by CD45+ cell counts at A) day 4 and B) day 8. [Figures 72A-72C] shows IFNγ levels (A) 4, (B) 7, and (C) 11 days after treatment of human PBMC-engrafted NSG mice with the indicated mutant IL15 / Rα-Fc fusion proteins or control. [Figures 73A-73C] CD45+ lymphocyte cell counts are shown on days (A) 4, (B) 7, and (C) 11 after treatment of human PBMC-engrafted NSG mice with the indicated mutant IL15 / Rα-Fc fusion proteins or control. [Figures 74A-74C] NK cell (CD16+CD56+CD45RA+) numbers are shown on days (A) 4, (B) 7, and (C) 11 after treatment of human PBMC-transferred NSG mice with the indicated IL15 / Rα-Fc fusion proteins or control. [Figure 75A-75B] CD8+ T cell (CD8+CD45RA+) counts are shown (A) 7 and (B) 11 days after treatment of human PBMC-transferred NSG mice with the indicated IL15 / Rα-Fc fusion proteins or control. [Figure 76A-76B] CD4+ T cell (CD4+CD45RA+) counts are shown (A) 7 and (B) 11 days after treatment of human PBMC-transferred NSG mice with the indicated IL15 / Rα-Fc fusion proteins or control. [Figure 77] IFNγ levels in serum on days 4, 7 and 11 after treatment of huPBMC-transferred mice with additional mutant IL-15 / Rα-Fc fusion proteins are shown. [Figures 78A-78C] CD8+ T cell counts in whole blood after treatment of huPBMC-transferred mice with additional mutant IL-15 / Rα-Fc fusion proteins on days A) 4, B) 7, and C) 11 are shown. [Figures 79A-79C] CD4+ T cell counts in whole blood after treatment of huPBMC-transferred mice with additional mutant IL-15 / Rα-Fc fusion proteins on days A) 4, B) 7, and C) 11 are shown. [Figures 80A-80C] CD45+ cell counts in whole blood after treatment of huPBMC-transferred mice with additional mutant IL-15 / Rα-Fc fusion proteins on days A) 4, B) 7, and C) 11 are shown. [Figures 81A-81C] Body weight as a percentage of initial body weight of huPBMC-transplanted mice after treatment with additional IL-15 / Rα variants on days A) 4, B) 7, and C) 11. Each point represents a single NSG mouse. Mice whose body weight fell below 70% of their initial weight were euthanized. Mice that died are represented as 70%. [Figures 82A-82E]Lymphocyte counts in cynomolgus monkeys after administration of XENP20818 are shown in Figures 82A-E, which show the fold change in absolute counts of CD56+ NK cells (Figure 82A), CD16+ NK cells (Figure 82B), γδ T cells (CD45RA+CD3+CD4-CD8-) (Figure 82C), CD8+ T cells (Figure 82D), and CD4+ T cells (Figure 82E), respectively. [Figures 83A-83E] Figure 83A shows the proliferation of CD56+ NK cells (Figure 83A), CD16+ NK cells (Figure 83B), CD8+ T cells (CD45RA+) (Figure 83C), CD8+ T cells (CD45RA-) (Figure 83D), and CD4+ T cells (CD45RA-) (Figure 83E) in cynomolgus monkeys after administration of XENP20818. [Fig. 84A-84E] Lymphocyte counts in cynomolgus monkeys after administration of XENP22819 are shown in Figures 84A-E, which show the fold change in absolute counts of CD56+ NK cells (Figure 84A), CD16+ NK cells (Figure 84B), γδ T cells (CD45RA+CD3+CD4-CD8-) (Figure 84C), CD8+ T cells (Figure 84D), and CD4+ T cells (Figure 84E), respectively. [Figures 85A-85E] Figure 85 shows proliferation of CD56+ NK cells (Figure 85A), CD16+ NK cells (Figure 85B), CD8+ T cells (CD45RA+) (Figure 85C), CD8+ T cells (CD45RA-) (Figure 85D), and CD4+ T cells (CD45RA-) (Figure 85E) in cynomolgus monkeys after administration of XENP22819. [Figures 86A-86E] Figures 86A-E show lymphocyte counts in cynomolgus monkeys after administration of XENP22821. Figures 86A-E show the fold change in absolute counts of CD56+ NK cells (Figure 86A), CD16+ NK cells (Figure 86B), γδ T cells (CD45RA+CD3+CD4-CD8-) (Figure 86C), CD8+ T cells (Figure 86D), and CD4+ T cells (Figure 86E), respectively. [Figures 87A-87E] Figure 87 shows proliferation of CD56+ NK cells (Figure 87A), CD16+ NK cells (Figure 87B), CD8+ T cells (CD45RA+) (Figure 87C), CD8+ T cells (CD45RA-) (Figure 87D), and CD4+ T cells (CD45RA-) (Figure 87E) in cynomolgus monkeys after administration of XENP22821. [Figures 88A-88E] Figures 88A-E show lymphocyte counts in cynomolgus monkeys after administration of XENP22822. Figures 88A-E show the fold change in absolute counts of CD56+ NK cells (Figure 88A), CD16+ NK cells (Figure 88B), γδ T cells (CD45RA+CD3+CD4-CD8-) (Figure 88C), CD8+ T cells (Figure 88D), and CD4+ T cells (Figure 88E), respectively. [Figures 89A-89E] Figure 89 shows proliferation of CD56+ NK cells (Figure 89A), CD16+ NK cells (Figure 89B), CD8+ T cells (CD45RA+) (Figure 89C), CD8+ T cells (CD45RA-) (Figure 89D), and CD4+ T cells (CD45RA-) (Figure 89E) in cynomolgus monkeys after administration of XENP22822. [Figures 90A-90E] Figures 90A-E show lymphocyte counts in cynomolgus monkeys after administration of XENP22834. Figures 90A-E show the fold change in absolute counts of CD56+ NK cells (Figure 90A), CD16+ NK cells (Figure 90B), γδ T cells (CD45RA+CD3+CD4-CD8-) (Figure 90C), CD8+ T cells (Figure 90D), and CD4+ T cells (Figure 90E), respectively. [Figures 91A-91E] Figure 91A shows the proliferation of CD56+ NK cells (Figure 91A), CD16+ NK cells (Figure 91B), CD8+ T cells (CD45RA+) (Figure 91C), CD8+ T cells (CD45RA-) (Figure 91D), and CD4+ T cells (CD45RA-) (Figure 91E) in cynomolgus monkeys after administration of XENP22834. [Figures 92A-92E] Figures 92A-E show lymphocyte counts in cynomolgus monkeys after administration of XENP23343. Figures 92A-E show the fold change in absolute counts of CD56+ NK cells (Figure 92A), CD16+ NK cells (Figure 92B), γδ T cells (CD45RA+CD3+CD4-CD8-) (Figure 92C), CD8+ T cells (Figure 92D), and CD4+ T cells (Figure 92E), respectively. [Figures 93A-93E]Figure 93A shows the proliferation of CD56+ NK cells (Figure 93A), CD16+ NK cells (Figure 93B), CD8+ T cells (CD45RA+) (Figure 93C), CD8+ T cells (CD45RA-) (Figure 93D), and CD4+ T cells (CD45RA-) (Figure 93E) in cynomolgus monkeys after administration of XENP23343. [Figures 94A-94D] 7 shows the sequences of exemplary IL-15 / Rα-Fc fusion proteins in the "IL-15 / Rα-hetero-Fc" format, XENP23343, XENP23504, XENP24113, XENP24301, XENP24306, and XENP24341, with M428L / N434S substitutions. IL-15 and IL-15Rα (sushi) are underlined, the linker is double underlined (as will be understood by those skilled in the art, portions of the linker may be replaced with other linkers shown in FIG. 7), and a slash ( / ) indicates the boundary between the IL-15, IL-15Rα, linker, and Fc region. FIG. 94D shows the sequence of XENP25938, an exemplary IL-15 / Rα-Fc fusion protein in the “scIL-15 / Rα-Fc” format with M428L / N434S substitutions. [Figure 95] 7 shows the sequence of XENP24383, an exemplary IL-15 / Rα-Fc fusion protein in the "ncIL-15 / Rα-Fc" format with M428L / N434S substitutions. IL-15 and IL-15Rα (sushi) are underlined, the linker is double underlined (as will be understood by those skilled in the art, portions of the linker may be replaced with other linkers shown in FIG. 7), and a slash ( / ) indicates the boundary between IL-15, IL-15Rα, linker, and Fc region. [Figure 96]7 shows the sequences of exemplary IL-15 / Rα-Fc fusion proteins, XENP24346 and XENP24351, in the "bivalent ncIL-15 / Rα-Fc" format with M428L / N434S substitutions. IL-15 and IL-15Rα (sushi) are underlined, the linker is double underlined (as will be understood by those skilled in the art, portions of the linker may be replaced with other linkers shown in FIG. 7), and a slash ( / ) indicates the boundary between IL-15, IL-15Rα, linker, and Fc region. [Figures 97A-97C] Shown are the percentages of Ki67 expression in (A) human CD8+ T cells, (B) human CD4+ T cells, and (C) human NK cells after treatment with an IL-15 / Rα mutant with the M428L / N434S Fc mutation. [Figures 98A-98C] The percentage of Ki67 expression in (A) cyno CD8+ T cells, (B) cyno CD4+ T cells, and (C) cyno NK cells after treatment with an IL-15 / Rα mutant with the M428L / N434S Fc mutation is shown. [Figures 99A-99C] Shown are CD4+ T cell counts in whole blood on days (A) 4 and (B) 7 and CD4+ T cell counts in spleen on day (C) 8 after treatment of huPBMC-transferred mice with additional mutant IL-15 / Rα-Fc fusion proteins. [Figures 100A-100C] Figure 1 shows CD8+ T cell counts in whole blood on days A) 4 and (B) 7 and (C) spleen on day 8 after treatment of huPBMC-transferred mice with additional mutant IL-15 / Rα-Fc fusion proteins. [Figures 101A-101C] Figure 1 shows CD8+ T cell counts in whole blood on days A) 4 and (B) 7 and (C) spleen on day 8 after treatment of huPBMC-transferred mice with additional mutant IL-15 / Rα-Fc fusion proteins. [Figures 102A-102E]Body weight as a percentage of initial body weight of huPBMC-transplanted mice is shown on days (A) -2, (B) 1, (C) 5, (D) 8, and (E) 11 after treatment with additional IL-15 / Rα variants. Each point represents a single NSG mouse. Figure 102F shows the time course of body weight in huPBMC-transplanted mice after treatment with IL-15 / Rα variants. [Figures 103A-103C] The numbers of (A) CD8+ T cells, (B) CD4+ T cells, and (C) NK cells in cynomolgus monkeys after treatment with IL-15 / Rα mutants on day 1 are shown. [Figure 104A-Z.104AA-AZ.104BA-BL] The sequences of the present invention are shown in bold, with the CDRs in bold, IL-15 and IL15-Rα(sushi) underlined, the linker double underlined, and a slash ( / ) between IL-15, IL15-Rα(sushi), the linker, and the Fc domain. [Figure 105] Some preferred embodiments of the present invention are shown: The "Xtend" version contains the 428L / 434S mutations in the Fc domain of each monomer. [Figure 106] A list of engineered heterodimer-skewed (e.g., "stereoheterodimerization") Fc mutants with heterodimer yield (determined by HPLC-CIEX) and thermal stability (determined by DSC) is shown. Thermal stability not determined is indicated by "nd." DETAILED DESCRIPTION OF THE INVENTION

[0021] I. Definition In order to provide a more complete understanding of the present invention, certain definitions are provided below. Such definitions are intended to encompass grammatical equivalents.

[0022] As used herein, "truncating" refers to reducing or eliminating activity. Thus, for example, "removing FcγR linkage" means that an Fc region amino acid mutation has less than 50% of the starting linkage compared to an Fc region that does not contain that particular mutation, with less than 70-80-90-95-98% loss of activity being preferred, and generally activity below the level of detectable activity in a Biacore assay. Particularly useful FcγR linkage removals are shown in Figure 86. However, unless otherwise noted, the Fc monomers of the present invention retain binding to the FcRn receptor.

[0023] As used herein, "ADCC" or "antibody-dependent cell-mediated cytotoxicity" refers to a cell-mediated reaction in which nonspecific cytotoxic cells expressing FcγR recognize ligated antibodies on target cells and subsequently cause lysis of the target cells. ADCC correlates with ligation to FcγRIIIa, and increased ligation to FcγRIIIa results in increased ADCC activity. As discussed herein, many embodiments of the present invention completely eliminate ADCC activity.

[0024] As used herein, "ADCP" or antibody-dependent cell-mediated phagocytosis refers to a cell-mediated reaction in which nonspecific cytotoxic cells expressing FcγR recognize ligated antibody on a target cell and subsequently cause phagocytosis of the target cell.

[0025] As used herein, "modification" refers to the substitution, insertion, and / or deletion of an amino acid in a polypeptide sequence, or to a 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. As used herein, "amino acid modification" refers to the substitution, insertion, and / or deletion of an amino acid in a polypeptide sequence. For clarity, unless otherwise specified, the amino acid modification is always to an amino acid encoded by DNA, e.g., the 20 amino acids that have codons in DNA and RNA.

[0026] As used herein, "amino acid substitution" or "substitution" refers to the replacement of 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 at the particular position (either not naturally occurring in an organism or not occurring in any organism). For example, the substitution E272Y refers to a mutant polypeptide, in this case an Fc mutant, in which glutamic acid at position 272 is replaced with tyrosine. For clarity, an engineered protein that alters the nucleic acid coding sequence but does not change the starting amino acid (e.g., replacing CGG (which encodes arginine) with CGA (which still encodes arginine) to increase host organism expression levels) is not an "amino acid substitution." That is, if a protein has the same amino acid at a particular position where it starts, despite the creation of a new gene encoding the same protein, it is not an amino acid substitution.

[0027] "Amino acid insertion" or "insertion," as used herein, refers to the addition of an amino acid sequence at a particular position in a parent polypeptide sequence. For example, -233E or 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.

[0028] "Amino acid deletion" or "deletion," as used herein, refers to the removal of an amino acid sequence at a particular position in a parent polypeptide sequence. For example, E233- or E233#, E233(), or E233del indicates a deletion of glutamic acid at position 233. Additionally, EDA233- or EDA233# indicates a deletion of the sequence GluAspAla beginning at position 233.

[0029] 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 can refer to the protein itself, a composition containing the protein, or the amino acid sequence encoding it. Preferably, a protein variant has at least one amino acid modification compared to the parent protein, e.g., about 1 to about 70 amino acid modifications, preferably about 1 to about 5 amino acid modifications, compared to the parent. As described below, in some embodiments, the parent polypeptide, e.g., the Fc parent polypeptide, is a human wild-type sequence such as an Fc region from IgG1, IgG2, IgG3, or IgG4; however, human sequences with mutations can also serve as "parent polypeptides," including, for example, IgG1 / 2 hybrids. The sequence of a protein variant herein preferably has at least about 80% identity, most preferably at least about 90% identity, and more preferably at least about 95-98-99% identity to the parent protein sequence. Variant protein can refer to the variant protein itself, a composition containing the protein variant, or the DNA sequence encoding it.

[0030] Thus, as used herein, "antibody variant" or "variant antibody" refers to an antibody that differs from a parent antibody based on at least one amino acid modification; as used herein, "IgG variant" or "variant IgG" refers to an antibody that differs from a parent IgG (again, often derived from a human IgG) based on at least one amino acid modification; and as used herein, "immunoglobulin variant" or "variant immunoglobulin" refers to an immunoglobulin sequence that differs from that of a parent immunoglobulin sequence based on at least one amino acid modification. As used herein, "Fc variant" or "variant Fc" refers to a protein containing an amino acid modification in the Fc domain. Fc variants of the present invention are defined according to the amino acid modifications that comprise them. Thus, for example, N434S or 434S is an Fc variant with a substituted serine at position 434 relative to the parent Fc polypeptide, where numbering is according to the EU index. Similarly, M428L / N434S defines an Fc variant with the substitutions M428L and N434S relative to the parent Fc polypeptide. The identity of the WT amino acid may not be specified, in which case the mutation is referred to as 428L / 434S. The substitutions may be provided in any order; for example, 428L / 434S is the same Fc mutation as M428L / N434S. For all positions discussed in this invention related to antibodies, unless otherwise specified, the numbering of amino acid positions follows the EU index. The EU index, or EU index similar to Kabat or EU numbering scheme, 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 can be additions, deletions, or substitutions. Substitutions can include naturally occurring amino acids and, in some cases, synthetic amino acids.Examples include U.S. Pat. No. 6,586,207, WO98 / 48032, WO03 / 073238, US2004-0214988A1, WO05 / 35727A2, WO05 / 74524A2, JW Chin et al., (2002), Journal of the American Chemical Society 124:9026-9027, JW Chin, & PG Schultz, (2002), ChemBioChem 11:1135-1137, JW Chin, et al., (2002), PICAS United Stat. es of America 99:11020-11024, and L. Wang, & P.G. Schultz, (2002), Chem. 1-10, which are incorporated by reference in their entireties.

[0031] As used herein, "protein" herein means at least two covalently attached amino acids, including proteins, polypeptides, oligopeptides, and peptides. A peptidyl group may be a naturally occurring amino acid and peptide bond, or a synthetic peptidomimetic structure, i.e., a peptoid (Simon, incorporated by reference in its entirety).

[0013] The present invention may also include "analogs" such as those derived from naturally occurring amino acids (see, for example, "analogs" of amino acids derived from naturally occurring amino acids, see ... Variants of the invention may include modifications including the use of synthetic amino acids incorporated using techniques developed by Schultz and coworkers, including, but not limited to, those described by Cropp & Shultz, 2004, Trends Genet. 20(12):625-30; Anderson et al., 2004, Proc Natl Acad Sci USA 101(2):7566-71; Zhang et al., 2003, 303(5656):371-3; and Chin et al., 2003, Science 301(5635):964-7 (incorporated by reference in their entirety). Additionally, polypeptides may include synthetic derivatization of one or more side chains or termini, glycosylation, PEGylation, circular permutation, cyclization, linkers to other molecules, fusion to proteins or protein domains, and addition of peptide tags or labels.

[0032] "Residue," as used herein, refers to a position in a protein and its associated amino acid identity. For example, asparagine 297 (also called Asn297 or N297) is the residue at position 297 in the human antibody IgG1.

[0033] 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, because IgG1 contains tyrosine and IgG2 contains phenylalanine at EU position 296, an F296Y substitution in IgG2 is considered to be an IgG subclass modification.

[0034] As used herein, "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 because none of the IgGs contain serine at position 434.

[0035] As used herein, "amino acid" and "amino acid identity" refer to one of the 20 naturally occurring amino acids encoded by DNA and RNA.

[0036] 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.

[0037] As used herein, the term "IgG Fc ligand" refers to any biologically derived molecule, preferably a polypeptide, that binds to the Fc region of an IgG antibody to form an Fc / Fc ligand complex. Fc ligands include FcγRI, FcγRII, FcγRIII, FcRn, and the like. Fc ligands include, but are not limited to, C1q, C3, mannan-binding lectin, mannose receptor, staphylococcal protein A, streptococcal protein G, and viral FcγRs. Fc ligands also include Fc receptor homologs (FcRHs), a family of Fc receptors that are homologous to FcγRs (Davis, (Et al., 2002, Immunological Reviews 190:123-136, incorporated by reference in its entirety). Fc ligands may include as yet undiscovered molecules that bind to Fc. Specific IgG Fc ligands are FcRn and Fc gamma receptors. As used herein, "Fc ligand" refers to any biologically derived molecule, preferably a polypeptide, that binds to the Fc region of an antibody to form an Fc / Fc ligand complex.

[0038] As used herein, "Fc gamma receptor," "FcγR," or "Fc gamma R" refers to any member of a family of proteins that binds to the Fc region of an IgG antibody and is encoded by the FcγR gene. In humans, this family includes FcγRI (CD64), which includes the isoforms FcγRIa, FcγRIb, and FcγRIc; FcγRII (CD32), which includes the isoforms FcγRIIa (including the allotypes H131 and R131), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2), and FcγRIIc; and FcγRIII (CD16), which includes the isoforms FcγRIIIa (including the allotypes V158 and F158), and FcγRIIIb (including the allotypes FcγRIIb-NA1 and FcγRIIb-NA2) (Jefferis et al., 2002, Immunol Lett 82:57-65, entirely incorporated by reference), and any undiscovered human FcγR or FcγR isoform or allotype. FcγRs can be derived from any organism, including, but not limited to, humans, mice, rats, rabbits, and monkeys. Mouse FcγRs include, but are not limited to, FcγRI (CD64), FcγRII (CD32), FcγRIII (CD16), and FcγRIII-2 (CD16-2), as well as any undiscovered mouse FcγR or FcγR isoform or allotype.

[0039] As used herein, "FcRn" or "neonatal Fc receptor" refers to a protein linked to the Fc region of an IgG antibody and at least partially encoded by the FcRn gene. FcRn can be derived from any organism, including, but not limited to, human, mouse, rat, rabbit, and monkey. As known in the art, a functional FcRn protein comprises two polypeptides, often referred to as a heavy chain and a light chain. The light chain is β-2-microglobulin, and the heavy chain is encoded by the FcRn gene. Unless otherwise specified herein, FcRn or FcRn protein refers to the complex of the FcRn heavy chain and β-2-microglobulin. Various FcRn mutations can be used to increase binding to the FcRn receptor and, in some cases, to increase serum half-life. Generally, unless otherwise specified, the Fc monomers of the present invention retain binding to the FcRn receptor (and may contain amino acid mutations to enhance binding to the FcRn receptor, as described below).

[0040] As used herein, "parent polypeptide" 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 engineered version of a naturally occurring polypeptide. A parent polypeptide may refer to the polypeptide itself, a composition comprising the parent polypeptide, or the amino acid sequence that encodes it. Thus, as used herein, a "parent immunoglobulin" refers to an unmodified immunoglobulin polypeptide that is modified to generate a variant, and a "parent antibody" as used herein refers to an unmodified immunoglobulin polypeptide that is modified to generate a variant antibody. It should be noted that "parent antibody" includes known commercially available recombinantly produced antibodies, as outlined below.

[0041] As used herein, "Fc" or "Fc region" or "Fc domain" refers to a polypeptide comprising the constant region of an antibody, optionally excluding the first constant region immunoglobulin domain (e.g., CH1) or a portion thereof, and optionally a portion of the hinge. Thus, Fc can refer to the last two constant region immunoglobulin domains (e.g., CH2 and CH3) of IgA, IgD, and IgG, the last three constant region immunoglobulin domains of IgE and IgM, and the flexible hinge N-terminal to these domains. In the case of IgA and IgM, Fc may include the J chain. In the case of IgG, the Fc domain comprises immunoglobulin domains Cγ2 and Cγ3 (Cγ2 and Cγ3) and the lower hinge region between Cγ1 (Cγ1) and Cγ2 (Cγ2). In some embodiments, Fc refers to the shortened CH1 domain, and CH2 and CH3 of an immunoglobulin. Although the boundaries of the Fc region might vary, the human IgG heavy chain Fc region is usually defined to include residues E216, C226, or P230 at its carboxy-terminus, where numbering is according to the EU index as in Kabat. In some embodiments, amino acid modifications are made to the Fc region to, for example, alter binding to one or more FcγR or FcRn receptors, as described in more detail below.

[0042] As used herein, "Fc fusion protein" or "immunoadhesin" generally refers to a protein comprising an Fc region linked (optionally via a linker moiety as described herein) to a different protein, e.g., IL-15 and / or IL-15R 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 variant Fc domain and a protein domain such as a receptor, ligand, or other binding partner.

[0043] As used herein, "position" means a location in the sequence of a protein. Positions may be numbered sequentially or according to established formats, such as the EU index for antibody numbering.

[0044] "Strandedness" herein, in reference to the monomers of the heterodimeric antibodies of the present invention, refers to the incorporation of heterodimerization mutations into each monomer such that they retain the ability to "match" and form heterodimers, similar to "matching" double-stranded DNA. For example, if several pI mutations are engineered into monomer A (e.g., to increase the pI), similarly exploitable "charge-paired" steric mutations do not interfere with the pI mutations; for example, the pI-increasing charge mutations can be placed in the same "strand" or "monomer" to retain both functions. Similarly, for "skewed" mutations resulting in paired sets, as outlined in more detail below, those skilled in the art will consider the pI when determining which strand or monomer will incorporate one set of the pair, and similarly use the skewed pI to maximize pI separation.

[0045] As used herein, "wild-type or WT" 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.

[0046] The heterodimeric proteins of the present invention are generally isolated or recombinant. "Isolated" when used to describe the various polypeptides disclosed herein. "Isolated" means a polypeptide that has been identified and separated and / or recovered from the cell or cell culture in which it is expressed. Ordinarily, an isolated polypeptide is prepared by at least one purification step. "Isolated protein" refers to a protein that is substantially free of other antibodies having different antigenic specificities. "Recombinant" means that the protein is produced using recombinant nucleic acid techniques in an exogenous host cell.

[0047] "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 those 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 for purposes of determining percent amino acid sequence identity can be achieved in a variety of ways within the skill of the art, 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 needed to achieve maximum alignment over the entire length of the sequences being compared. One particular program is the ALIGN-2 program, outlined in paragraphs

[0279] -

[0280] of U.S. Patent Application Publication No. 2016 / 0244525, incorporated herein by reference.

[0048] 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 percent identity.

[0049] In some embodiments, two or more amino acid sequences are at least 50%, 60%, 70%, 80%, or 90% identical, hi some embodiments, two or more amino acid sequences are at least 95%, 97%, 98%, 99%, or even 100% identical.

[0050] "Specific binding" to a particular antigen or epitope or "specifically binds to" or "specific for" a particular antigen or epitope means 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 does not have binding activity. For example, specific binding can be determined by competition with a control molecule that is similar to the target.

[0051] Before the present invention is further described, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0052] II. Heterodimeric Fc Fusion Proteins The present invention relates to heterodimeric Fc fusion proteins comprising IL-15 and IL-15 receptor alpha (IL-15Rα) protein domains in different orientations. The Fc domains can be derived from IgG Fc domains, such as IgG1, IgG2, IgG3, or IgG4 Fc domains, with IgG1 Fc domains being of particular use in the present invention.

[0053] The carboxy-terminal portion of each chain defines a constant region primarily responsible for effector function. collected numerous primary sequences of heavy and light chain variable regions. Based on the degree of sequence conservation, Kabat et al. classified each primary sequence into CDR and framework regions and compiled a list (see SEQUENCES OF IMMUNOLOGICAL INTEREST, 5th edition, NIH publication, No. 91-3242, E.A. Kabat et al., which is incorporated by reference in its entirety). Throughout this specification, the Kabat numbering system is generally used when referring to residues within the variable domain (approximately residues 1-107 of the light chain variable region and residues 1-113 of the heavy chain variable region), and the EU numbering system is for the Fc region (see, e.g., Kabat et al., supra (1991)).

[0054] The IgG subclass of immunoglobulins has several immunoglobulin domains in the heavy chain. As used herein, "immunoglobulin (Ig) domain" refers to a region of an immunoglobulin with a distinct tertiary structure. The heavy chain domain, including the constant heavy (CH) domain and the hinge domain, is of interest in the present invention. In the context of IgG antibodies, each IgG isotype has three CH regions. Thus, the "CH" domains in the context of IgG are as follows: "CH1" refers to positions 118-220 according to the EU index as per Kabat; "CH2" refers to positions 237-340 according to the EU index as per Kabat; and "CH3" refers to positions 341-447 according to the EU index as per Kabat. As indicated herein and described below, pI mutations can occur in one or more of the CH regions and, as discussed below, the hinge region.

[0055] Another type of Ig domain in 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 comprising the amino acids between the first and second constant domains of an antibody. Structurally, the IgG CH1 domain ends at EU position 220, and the IgG CH2 domain begins at residue EU position 237. Thus, for IgG, the antibody hinge is defined herein to include positions 221 (D221 in IgG1) to 236 (G236 in IgG1), with numbering according to the EU index as in Kabat. In some embodiments, the lower hinge is included in the context of the Fc region, and "lower hinge" generally refers to positions 226 or 230. As described herein, pI mutations can also be made in the hinge region.

[0056] Thus, the present invention provides different antibody 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, an Fc domain, a CH1 domain, a CH2 domain, a CH3 domain, a hinge domain, and a heavy chain constant domain (CH1-hinge-Fc domain or CH1-hinge-CH2-CH3).

[0057] Thus, an "Fc domain" includes the -CH2-CH3 domains and, optionally, the hinge domain. In some embodiments, the Fc domain also includes a truncated CH1 domain. In embodiments herein, when a protein fragment, e.g., IL-15 or IL-15Rα, is bound to an Fc domain, it is the C-terminus of the IL-15 or IL-15Rα construct that is attached to all or part of the hinge of the Fc domain, e.g., it is attached to the sequence EPKSS, which is generally the beginning of the hinge. In other embodiments, when a protein fragment, e.g., IL-15 or IL-15Rα, is bound to an Fc domain, it is the C-terminus of the IL-15 or IL15Rα construct that is attached to the CH1 domain of the Fc domain.

[0058] In some of the Fc domain protein constructs and sequences outlined herein, the C-terminus of IL-15 or IL-15Rα is linked to the N-terminus of a domain linker, which in turn is linked to the N-terminus of a constant Fc domain (N-IL-15 or IL-15Rα protein fragment-linker-Fc domain-C), although this can be reversed (N-Fc domain-linker-IL-15 or IL-15Rα protein domain-C). In other constructs and sequences outlined herein, the C-terminus of a first protein fragment is linked to the N-terminus of a second protein fragment, optionally via a domain linker, and the C-terminus of the second protein fragment 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 a first or second protein fragment. Heterodimeric Fc fusion proteins can comprise two or more of the exemplary monomeric Fc domain proteins described herein.

[0059] In some embodiments, the linker is a "domain linker" used to link together any two domains outlined herein, some of which are shown in Figure 87. While any suitable linker can be used, many embodiments utilize glycine-serine polymers, including, for example, (GS)n, (GSGGS)n, (GGGGS)n, and (GGGS)n, where n is at least 1 (and generally 0-1-2-3-4-5), as well as any peptide sequence that allows for recombinant attachment of two domains with sufficient length and flexibility so that each domain retains its biological function. In some cases, the linker is a charged domain linker, with attention to "twistiness," as outlined below.

[0060] In one embodiment, the heterodimeric Fc fusion protein comprises at least two constant domains that can be engineered to generate heterodimers, such as by pI engineering. Other Fc domains that can be used include pI-engineered fragments comprising one or more of the CH1, CH2, CH3, and hinge domains of the invention. In particular, the formats shown in Figures 9A-9G and 39A-39D are heterodimeric Fc fusion proteins, meaning that the protein has two linked Fc sequences and at least one protein fragment (e.g., one, two, or more protein fragments) that self-assemble into a heterodimeric Fc domain. In some cases, a first protein fragment is linked to a first Fc sequence, and a second protein fragment is linked to a second Fc sequence. In other cases, a first protein fragment is linked to a first Fc sequence, and the first protein fragment is noncovalently attached to a second protein fragment that is not linked to an Fc sequence. In some cases, the heterodimeric Fc fusion protein comprises a first protein fragment linked to a second protein fragment that is linked to a first Fc sequence, and a second Fc sequence that is not linked to either the first or second protein fragment.

[0061] 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.

[0062] The present invention relates to novel constructs for providing heterodimeric Fc fusion proteins capable of binding to one or more binding partners, ligands, or receptors. Heterodimeric Fc fusion constructs are based on the self-assembly properties of two Fc domains of antibody heavy chains, e.g., two "monomers," which assemble into a "dimer." Heterodimeric Fc fusions are created by altering the amino acid sequence of each monomer, as discussed more fully below. Thus, the present invention generally relates to the fusion of several binding partners, ligands, or receptors, relying on amino acid mutations in different constant regions on each chain to promote heterodimer formation and / or to facilitate purification of heterodimers over homodimers. This invention relates to the creation of heterodimeric Fc fusion proteins that can be co-linked in the above manner.

[0063] There are many mechanisms that can be used to generate the heterodimers of the present invention. Furthermore, as will be understood by those skilled in the art, these mechanisms can be combined to ensure high heterodimerization. Therefore, amino acid mutations that result in the production of heterodimers are called "heterodimerization mutations." As described below, heterodimerization mutations can include steric mutations (e.g., "knob-and-hole" or "skew" mutations and "charge pair" mutations) that allow the purification of homodimers from heterodimers, as well as "pI mutations." As incorporated by reference in its entirety, and specifically incorporated herein below for the discussion of "heterodimerization mutations," useful mechanisms of heterodimerization include "knob-and-hole" ("KIH," sometimes referred to herein as "skew" mutations (see discussion in WO2014 / 145806), "electrostatic steering" or "charge pairing" as described in WO2014 / 145806, pI mutations as described in WO2014 / 145806, and general additional Fc mutations as outlined in WO2014 / 145806 and below.

[0064] In the present invention, there are several basic mechanisms that can facilitate the purification of heterodimeric antibodies. One of these relies on the use of pI mutations, where each monomer has a different pI, thereby enabling isoelectric purification of AA, AB, and BB dimeric proteins. Alternatively, some formats also allow for size-based separation. As further outlined below, it is also possible to "skew" the formation of heterodimers over homodimers. Therefore, the combination of steric heterodimerization mutations with pI or charge pair mutations is particularly useful in the present invention.

[0065] In general, embodiments of particular use in the present invention rely on a set of mutations that include a skewing mutation that favors heterodimer formation over homodimer formation, combined with a pI mutation that increases the pI difference between the two monomers.

[0066] Furthermore, as outlined more fully below, depending on the format of the heterodimeric Fc fusion protein, the pI mutations may either be contained within the constant and / or Fc domains of the monomers, or a domain linker may be used. That is, the present invention also provides pI variants on one or both monomers, and / or charged domain linkers. Furthermore, additional amino acid engineering for alternative functionality may also confer pI changes, such as Fc, FcRn, and KO mutations.

[0067] In the present invention, which utilizes pI as a separation mechanism to enable purification of heterodimeric proteins, amino acid mutations can be introduced into the polypeptides of one or both monomers; i.e., the pI of one of the monomers (referred to herein for simplicity as "monomer A") can be engineered to be different from monomer B, or changes in both monomers A and B can be engineered to increase the pI of monomer A and decrease the pI of monomer B. As discussed, pI changes in either or both monomers can be achieved by removing or adding a charged residue (e.g., substituting a neutral amino acid with a positively or negatively charged amino acid residue, e.g., glycine 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., eliminating a charge, lysine to serine). Some of these mutations are shown in the figures.

[0068] Thus, this embodiment of the invention provides for creating a sufficient pI change in at least one monomer so that the heterodimer can be separated from the homodimer. As will be appreciated by those skilled in the art, and as discussed further below, this is referred to as "wild type" This can be done by using the heavy chain constant region and mutated regions engineered to increase or decrease its pI (wtA-+B or wtA--B), or by increasing one region and decreasing the other (A+-B- or A-B+).

[0069] Thus, in general, a component of some embodiments of the present invention is amino acid mutations in the constant region aimed at altering the isoelectric point (pI) of at least one, if not both, of the monomers of a dimeric protein by incorporating an amino acid substitution ("pI mutation" or "pI substitution") into one or both of the monomers. As shown herein, separation of a heterodimer from two homodimers can be achieved when the pI of the two monomers differs by as little as 0.1 pH units, with differences of 0.2, 0.3, 0.4, and 0.5 or more all being of use in the present invention.

[0070] As will be appreciated by those skilled in the art, the number of pI variations to be included in each or both monomers to obtain good separation will depend in part on the starting pI of the components. As is known in the art, different Fc's will have different starting pIs to be utilized in the present invention. Generally, as outlined herein, the pI is engineered to produce a difference in the total pI of each monomer of at least about 0.1 log, with 0.2-0.5 being preferred as outlined herein.

[0071] As will be appreciated by those skilled in the art, the number of pI variations to be included in each or both monomers to obtain good separation will depend in part on the starting pI of the components. That is, to determine which monomers to engineer or in which "direction" (e.g., more positive or more negative), the sequence of the Fc domain, and in some cases, the protein domains linked to the Fc domain, is calculated and a decision made from there. As is known in the art, different Fc domains and / or protein domains will have different starting pIs to be utilized in the present invention. Generally, as outlined herein, pIs are engineered to produce a difference in the total pI of each monomer of at least about 0.1 log, with 0.2-0.5 being preferred as outlined herein.

[0072] Additionally, as will be appreciated by those skilled in the art and outlined herein, in some embodiments, heterodimers can be separated from homodimers based on size. For example, as shown in the figures, some of the formats allow for separation of heterodimers and homodimers based on size.

[0073] Using the constant region of the Fc domain to achieve heterodimerization using pI mutations provides a more modular approach to designing and purifying heterodimeric Fc fusion proteins. Thus, in some embodiments, heterodimerization mutations (including skew and purified heterodimerization mutations) must be engineered. Furthermore, in some embodiments, the potential for immunogenicity resulting from pI mutations is significantly reduced by incorporating pI mutations from different IgG isotypes so that the pI is altered without introducing significant immunogenicity. Therefore, a further problem to be solved is the elucidation of low pI constant domains with a high human sequence content, for example, minimizing or avoiding non-human residues at any particular position.

[0074] Potential side benefits of this pI engineering are also increased serum half-life and increased FcRn ligation. That is, as described in USSN 13 / 194,904 (incorporated herein by reference in its entirety), lowering the pI of antibody constant domains (including those found in antibodies and Fc fusions) can result in longer serum retention in vivo. These pI mutations to extend serum half-life also facilitate pI changes for purification.

[0075] Furthermore, the pI modification of heterodimerization mutants provides significant benefits to the analytical and quality control processes of Fc fusion proteins, as it allows for the significant ability to eliminate, minimize, and distinguish between homodimers when they are present. Similarly, the ability to reliably test the reproducibility of heterodimeric Fc fusion protein production is important.

[0076] A. Heterodimerization mutations The present invention provides heterodimeric proteins, including heterodimeric Fc fusion proteins in various formats, that utilize heterodimerization mutations to enable heterodimer formation and / or purification from homodimers. Heterodimeric fusion constructs are based on the self-assembly properties of two Fc domains, e.g., two "monomers," that assemble into a "dimer."

[0077] There are several suitable pairs of heterodimerization-skew mutation sets. These mutations are "pairs" of "sets." That is, one set of mutations is incorporated into the first monomer, and the other set of mutations is incorporated into the second monomer. It is noteworthy that these sets do not necessarily behave as "knobs-in-holes" mutations, with a one-to-one correspondence between residues on one monomer and residues on the other. That is, these sets of mutations form an interface between the two monomers that promotes heterodimer formation and prevents homodimer formation, resulting in a heterodimer formation rate of 90% or more under biological conditions, rather than the expected 50% (25% homodimer A / A: 50% heterodimer A / B: 25% homodimer B / B).

[0078] B. Stereomutation In some embodiments, heterodimer formation can be promoted by the addition of conformational mutations, i.e., by changing the amino acids in each heavy chain, different heavy chains are more likely to associate to form heterodimeric structures than to form homodimers with identical Fc amino acid sequences. Suitable conformational mutations are included in Figure 29 of USSN 15 / 141,350, all of which are incorporated herein by reference in their entirety, as well as in Figure 84.

[0079] One mechanism, commonly referred to in the art as "knobs and holes," refers to amino acid manipulations that result in steric effects favoring heterodimer formation and disfavoring homodimer formation and can optionally be used; this is often referred to as "knobs and holes," as described in USSN 61 / 596,846; Ridgway et al., Protein Engineering 9(7):617 (1996); Atwell et al., J. Mol. Biol. 1997 270:26; and U.S. Patent No. 8,216,805, all of which are incorporated herein by reference in their entireties. These figures identify several "monomer A-monomer B" pairs that rely on "knobs and holes." Furthermore, as described in Merchant et al., Nature Biotech. 16:677 (1998), these "knobs and holes" mutations can be combined with disulfide linkages to skew formation toward heterodimerization.

[0080] An additional mechanism used to generate heterodimers is often referred to as "electrostatic steering," as described in Gunasekaran et al., J. Biol. Chem. 285(25):19637 (2010), the entire contents of which are incorporated herein by reference. This is sometimes referred to herein as "charge pairing." In this embodiment, electrostatics are used to skew formation toward heterodimerization. As will be appreciated by those skilled in the art, these may also affect pI and therefore purification, Thus, in some cases, they may be considered pI mutations. However, because they were generated to force heterodimerization and were not used as a purification tool, they are classified as "stereotypic mutations." These include, but are not limited to, D221E / P228E / L368E paired with D221R / P228R / K409R (e.g., these are the "monomer matched set"), and C220E / P228E / 368E paired with C220R / E224R / P228R / K409R.

[0081] The additional monomer A and monomer B mutations can optionally be independently combined in any amount with other mutations, such as the pI mutations outlined herein or other steric mutations shown in Figure 37 of US2012 / 0149876, all of which are expressly incorporated herein by reference.

[0082] In some embodiments, the conformational mutations outlined herein can optionally and independently incorporate any pI mutations (or other mutations such as Fc mutations, FcRn mutations, etc.) into one or both monomers and can independently and optionally be included or excluded from the proteins of the invention.

[0083] A list of suitable scubariant pairs is shown in Figure 84. Particularly useful in many embodiments are pairs from the set including, but not limited to, S364K / E357Q:L368D / K370S, L368D / K370S:S364K, L368E / K370S:S364K, T411T / E360E / Q362E:D401K, L368D / K370S:S364K / E357L, K370S:S364K / E357Q, and T366S / L368A / Y407V:T366W (optionally containing the bridging disulfide T366S / L368A / Y407V / Y349C:T366W / S354C). In terms of nomenclature, the pair "S364K / E357Q:L368D / K370S" means that one monomer has the double mutation set S364K / E357Q and the other has the double mutation set L368D / K370S, and as mentioned above, the "twistiness" of these pairs depends on the starting pI.

[0084] C. pI (isoelectric point) mutation of heterodimer In general, as will be understood by those skilled in the art, there are two general categories of pI mutations: 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 mutations can be made: one monomer can be wild-type or a mutation that does not exhibit a pI that is significantly different from wild-type, and the other can be either more basic or more acidic. Alternatively, each monomer can be changed, one to be more basic and one to be more acidic.

[0085] Preferred combinations of pI variants are shown in Figure 30 of USSN 15 / 141,350, all of which are incorporated herein by reference in their entirety. 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.

[0086] In one embodiment, when one of the Fc monomers comprises a CH1 domain, a preferred combination of pI mutations has one monomer comprising 208D / 295E / 384D / 418E / 421D mutations (N208D / Q295E / N384D / Q418E / N421D when compared to human IgG1). In some cases, the second monomer comprises a positively charged domain linker comprising (GKPGS)4. In some cases, the first monomer comprises a CH1 domain comprising position 208. Thus, constructs that do not comprise a CH1 domain (e.g., heterodimeric Fc fusion proteins that do not utilize a CH1 domain in one of the domains) can be used. In the case of ), a preferred negative pI mutated Fc set comprises 295E / 384D / 418E / 421D mutations (Q295E / N384D / Q418E / N421D when compared to human IgG1).

[0087] In some embodiments, mutations are made in the hinge domain of the Fc domain, including positions 221, 222, 223, 224, 225, 233, 234, 235, and 236. Note that changes at 233-236 can be made to increase effector function (together with 327A) in the IgG2 backbone. Thus, pI mutations, particularly substitutions, can be made at one or more of positions 221-225 with 1, 2, 3, 4, or 5 mutations used in the present invention. Similarly, all possible combinations are contemplated, alone or in combination with other pI mutations in other domains.

[0088] Specific 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 pI substitutions are made in the hinge domain, while in other cases, these substitutions are added in any combination to other pI mutations in other domains.

[0089] In some embodiments, mutations can be made in the CH2 region that include positions 274, 296, 300, 309, 320, 322, 326, 327, 334, and 339. Likewise, all possible combinations of these 10 positions can be made, e.g., a pI antibody can have 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 CH2 pI substitutions.

[0090] Specific substitutions used to reduce the pI of the CH2 domain include, but are not limited to, unnatural glutamine or glutamic acid at position 274, unnatural phenylalanine at position 296, unnatural phenylalanine at position 300, unnatural valine at position 309, unnatural glutamic acid at position 320, unnatural glutamic acid at position 322, unnatural glutamic acid at position 326, unnatural glycine at position 327, native glutamic acid at position 334, unnatural threonine at position 339, and all possible combinations within CH2 and with other domains.

[0091] In this embodiment, mutations may be independently and optionally selected from positions 355, 359, 362, 384, 389, 392, 397, 418, 419, 444, and 447. Specific substitutions used to reduce the pI of the CH3 domain include, but are not limited to, an unnatural glutamine or glutamic acid at position 355, an unnatural serine at position 384, an unnatural asparagine or glutamic acid at position 392, an unnatural methionine at position 397, an unnatural glutamic acid at position 419, an unnatural glutamic acid at position 359, an unnatural glutamic acid at position 362, an unnatural glutamic acid at position 389, an unnatural glutamic acid at position 418, an unnatural glutamic acid at position 444, and a deletion or unnatural aspartic acid at position 447.

[0092] D. Isotype Variation Furthermore, many embodiments of the invention 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 U.S. Patent Application Publication No. 2014 / 0370013, which is incorporated herein by reference. Briefly, IgG1 is a common isotype for therapeutic antibodies for a variety of reasons, including high effector function. However, the heavy chain 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 reduced (or increased). The resulting IgG1 / IgG2 variants exhibit increased serum half-life (pI increase) and thus a longer serum half-life. For example, IgG1 has a glycine at position 137 (pI 5.97) and IgG2 has a glutamic acid (pI 3.22), and introducing 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 mutant Fc-fusion protein. However, it should be noted that even changes in the IgG2 molecule can increase serum half-life, as discussed below.

[0093] In other embodiments, non-isotypic amino acid changes are made to reduce the overall charge state of the resulting protein (e.g., by changing from higher pI amino acids to lower pI amino acids) or to allow for structural tuning, such as for stability, as described in more detail below.

[0094] Furthermore, by pI engineering both the heavy and light chain constant domains, significant changes can be seen in each monomer of the heterodimer. As discussed herein, a difference in pI of at least 0.5 between the two monomers can allow for separation by ion exchange chromatography or isoelectric focusing, or other methods sensitive to isoelectric point.

[0095] Calculation of E.pI The pI of each monomer may depend on the pI of the variant heavy chain constant domain and the pI of the entire monomer, including 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 U.S. Patent Application Publication No. 2014 / 0370013. As discussed herein, which monomers to engineer is generally determined by the intrinsic pI of each monomer.

[0096] The F.pI mutation also confers better FcRn binding in vivo. If the pI mutations decrease the pI of the monomer, they may have the additional benefit of improving serum retention in vivo.

[0097] Although still under investigation, it is believed that the Fc region has a longer half-life in vivo because binding to FcRn at pH 6 in endosomes sequesters Fc (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 approximately 7.4 induces Fc release into the bloodstream. In mice, Dall'Acqua et al. showed that Fc mutants with increased FcRn binding at pH 6 and pH 7.4 actually had 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). The increased affinity of Fc for FcRn at pH 7.4 is thought to prevent Fc release into the bloodstream. Therefore, Fc mutations that increase the in vivo half-life of Fc would ideally increase FcRn ligation at lower pH while still allowing Fc release at higher pH. The amino acid histidine changes its charge state in the pH range of 6.0 to 7.4. Therefore, it is not surprising to find His residues at key positions in the Fc / FcRn complex.

[0098] G. Additional Fc mutations for additional functionality In addition to pI amino acid mutations, there are several useful Fc amino acid modifications that can be made for a variety of reasons, including, but not limited to, altering linkage to one or more FcγR receptors, altering linkage to the FcRn receptor, etc.

[0099] Thus, proteins of the invention can include amino acid modifications, including heterodimerization mutations as outlined herein, including pI mutations and conformational mutations, each set of mutations independently and optionally can be included or excluded from a particular heterodimeric protein.

[0100] H.FcγR mutation There are several useful Fc substitutions that can be made to alter binding to one or more FcγR receptors. Substitutions that result in increased binding as well as decreased binding can be useful. For example, increased binding to FcγRIIIa is known to generally result in increased ADCC (antibody-dependent cell-mediated cytotoxicity, i.e., a cell-mediated reaction in which nonspecific cytotoxic cells expressing FcγR recognize ligated antibodies on target cells and subsequently cause lysis of the target cells). Similarly, under some circumstances, decreased binding to FcγRIIb (an inhibitory receptor) can also be beneficial. Amino acid substitutions useful in the present invention include those listed in USSN 11 / 124,620 (especially Figure 41), USSN 11 / 174,287, USSN 11 / 396,495, and USSN 11 / 538,406, all of which are expressly incorporated herein by reference in their entirety, particularly the mutations disclosed therein. Specific mutations 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.

[0101] Furthermore, amino acid substitutions that increase affinity for FcγRIIc can also be included in the Fc domain mutations outlined herein. For example, the substitutions described in USSN 11 / 124,620 and USSN 14 / 578,305 are useful.

[0102] 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.

[0103] I. Elimination Mutations Similarly, another category of functional mutations are "FcγR ablation mutations" or "Fc knockout (FcKO or KO) mutations. In these embodiments, for some therapeutic applications, it is desirable to reduce or eliminate the normal binding of an 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, particularly in the use of bispecific immunomodulatory antibodies, where it is desirable in many embodiments to ablate FcγRIIIa binding so that one of the Fc domains comprises one or more Fcγ receptor ablation mutations to ablate or significantly reduce ADCC activity. These deletion mutations are shown in Figure 31 of USSN 15 / 141,350, all of which are incorporated herein by reference in their entirety, and are listed in accordance with the EU index. In a preferred embodiment, deletion mutations are used that are 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, each independently can be optionally included or excluded. Note that the deleting mutations referred to herein remove FcγR ligation, but generally do not remove FcRn ligation.

[0104] J. Combining Heterodimers and Fc Mutations As will be appreciated by those skilled in the art, all of the listed heterodimerization mutations (including skew and / or pI mutations) can be optionally combined independently in any way as long as their "twistiness" or "monomer distribution" is preserved. Furthermore, all of these mutations can be combined in any of the heterodimerization formats.

[0105] In the case of pI mutations, specifically used embodiments are shown in the figures, but other combinations can be generated following the basic rule of altering the pI difference between the two monomers to facilitate purification.

[0106] Additionally, any of the heterodimerization mutations, skew, and pI can be independently and optionally combined with Fc depletion mutations, Fc mutations, FcRn mutations, as generally outlined herein.

[0107] Additionally, the monomeric Fc domain may comprise a set of amino acid substitutions including C220S / S267K / L368D / K370S or C220S / S267K / S364K / E357Q.

[0108] 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 herein by reference in their entireties); particularly useful skew mutations include S364K / E357Q:L368D / K370S, L368D / K370S:S364K, L368E / K370S:S364K, T41 and T366S / L368A / Y407V / Y349C:T366W / S354C, optionally comprising deletion mutations, optionally a charged domain linker, and wherein the heavy chain comprises a pI mutation.

[0109] In some embodiments, the Fc domain comprises 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 / In some cases, the Fc domain comprises an amino acid substitution selected from the group consisting of N434S, 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 other 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.

[0110] In one embodiment, the particular combination of skew and pI mutations used in the present invention is T366S / L368A / Y407V:T366W (optionally including the bridging disulfide T366S / L368A / Y407V / Y349C:T366W / S354C), with one monomer comprising Q295E / N384D / Q418E / N481D and the other a positively charged domain linker. As understood in the art, "knob-in-hole" domains are also known. The "mol" mutation does not change the pI and therefore can be used for either monomer.

[0111] III. IL-15 and IL15Rα Protein Domains The present invention provides heterodimeric Fc fusion proteins comprising IL-15 and IL-15Rα proteins. As shown in the figures, IL-15 complexes can take several forms. As noted above, IL-15 protein itself is less stable than when complexed with IL-15Rα protein. As is known in the art, IL-15Rα protein contains a "sushi domain," which is the shortest region of the receptor that retains IL-15 binding activity. Therefore, while heterodimeric fusion proteins comprising the entire IL-15Rα protein can be produced, preferred embodiments herein include complexes using only the sushi domain, the sequence of which is shown in the figures.

[0112] Thus, an IL-15 complex generally comprises the IL-15 protein and the IL-15Rα sushi domain (unless otherwise specified that the full-length sequence is used, "IL-15Rα," "IL-15Rα(sushi)," and "sushi" are used interchangeably throughout). This complex can be used in three different formats. As shown in Figure 9A, the IL-15 protein and IL-15Rα(sushi) are not covalently attached, but rather self-assemble through normal ligand-ligand interactions. As explained more fully herein, either the IL-15 domain or the sushi domain can be covalently linked to the Fc domain (typically using an optional domain linker). Alternatively, they can be covalently linked using a domain linker, as generally shown in Figures 9B, 9E, and 9G. Figure 9B shows the sushi domain as the N-terminal domain, but this can be reversed. Finally, each of the IL-15 domain or sushi domain can be engineered to contain a cysteine ​​amino acid, similarly allowing it to form a complex with either the IL-15 domain or the sushi domain covalently attached (with an optional domain linker) to an Fc domain, as generally shown in Figures 39A-39D.

[0113] In some embodiments, the human IL-15 protein has the amino acid sequence set forth in NCBI Reference SEQ ID NO: NP_000576.1 or SEQ ID NO: 1. In some cases, the coding sequence for human IL-15 is set forth in NCBI Reference SEQ ID NO: 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 or 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. In some embodiments, the IL-15 protein has the amino acid sequence set forth in SEQ ID NO: 2 and the amino acid substitution N72D. In other embodiments, the IL-15 protein has the amino acid sequence of SEQ ID NO:2 and one or more amino acid substitutions selected from the group consisting of C42S, L45C, Q48C, V49C, L52C, E53C, E87C, and E89C. Optionally, the IL-15 protein also has an N72D substitution. The IL-15 protein of the Fc fusion protein can have 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acid substitutions.

[0114] The amino acid substitutions can be equivalent substitutions at the IL-15:IL-2β and IL-15:common gamma chain interfaces. In some embodiments, the human IL-15 protein has one or more amino acid substitutions selected from the group consisting of N1D, N4D, D8N, D30N, D61N, E64Q, N65D, and Q108E. In some cases, the IL-15 protein has the amino acid substitution Q108E. In some cases, the IL-15 protein has 1, 2, 3, 4, 5, 6, 7, 8, or more amino acid substitutions. The IL-15 protein can have one or more amino acid substitutions selected from the group consisting of N1D, N4D, D8N, D30N, D61N, E64Q, N65D, and Q108E. In some embodiments, the amino acid substitutions may include N1D / D61N, N1D / E64Q, N4D / D61N, N4D / E64Q, D8N / D61N, D8N / E64Q, D61N / E64Q, E64Q / Q108E, N1D / N4D / D8N, D61N / E64Q / N65D, N1D / D61N / E64Q, N1D / D61N / E64Q / Q108E, or N4D / D61N / E64Q / Q108E. In some cases, the IL-15 protein has the amino acid substitutions D30N / E64Q / N65D.

[0115] In some embodiments, the human IL-15 receptor alpha (IL-15Rα) protein has the amino acid sequence set forth in NCBI Reference SEQ ID NO: NP_002180.1 or SEQ ID NO: 3. In some cases, the coding sequence for human IL-15Rα is set forth in NCBI Reference SEQ ID NO: 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), in other words, the amino acid sequence of SEQ ID NO: 4. 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 the full-length human IL-15Rα protein or SEQ ID NO: 3. For example, an amino acid 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) can be added to the C-terminus of the IL-15Rα protein of SEQ ID NO: 4. In some embodiments, the IL-15Rα protein has the 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 protein can have 1, 2, 3, 4, 5, 6, 7, 8, or more amino acid mutations (e.g., substitutions, insertions, and / or deletions).

[0116] IV. Domain Linkers In some embodiments, the IL-15 protein and the IL-15Rα protein are linked together via a linker. Optionally, the proteins are not linked via a linker. In other embodiments, the IL-15 protein and the IL-15Rα protein are non-covalently attached. In some embodiments, the IL-15 protein is linked to the Fc domain via a linker. In some embodiments, the IL-15 protein is linked to the Fc domain directly, for example, without a linker. In other embodiments, the IL-15Rα protein is linked to the Fc domain via a linker. In other embodiments, the IL-15Rα protein is linked directly to the Fc domain. In some cases, a linker is not used to link the IL-15 protein or the IL-15Rα protein to the Fc domain.

[0117] In some embodiments, the linker is a "domain linker" used to link together any two domains outlined herein. While any suitable linker can be used, many embodiments utilize glycine-serine polymers, including, for example, (GS)n, (GSGGS)n, (GGGGS)n, and (GGGS)n, where n is at least 0 (and generally 0 to 1 to 2 to 3 to 4 to 5), as well as any peptide sequence that allows for recombinant attachment of two domains with sufficient length and flexibility so that each domain retains its biological function. In some cases, useful linkers include (GGGGS)0 or (GGGGS)1 or (GGGGS)2. In some cases, Thus, with attention to "twist" as outlined below, charged domain linkers can be used as discussed herein and shown in FIG.

[0118] V. Useful Formats of the Invention As shown in Figures 9A-9G and 39A-39D, there are several useful formats for the bispecific heterodimeric fusion proteins of the invention. In general, the heterodimeric fusion proteins of the invention have two functional components: an IL-15 / IL-15Rα (sushi) component and an Fc component, both of which can take different forms as outlined herein and both of which can be combined with other components in any configuration.

[0119] 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.

[0120] 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.

[0121] 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.

[0122] Optionally, the first and / or second Fc domain has a 428L / 434S mutation for half-life extension.

[0123] A. IL-15 / Rα-hetero Fc format In this embodiment, the heterodimeric fusion protein comprises two monomers, as shown in Figure 9A. The first monomer comprises (N- to C-terminus) IL-15-optional domain linker-CH2-CH3, where the domain linker often comprises all or part of a hinge. The second monomer comprises IL-15 / Rα(sushi)-optional domain linker-CH2-CH3, where the domain linker often comprises all or part of a hinge.

[0124] In the IL-15 / Rα-hetero-Fc format, a preferred embodiment utilizes the skewed mutation pair S364K / E357Q:L368D / K370S.

[0125] In the IL-15 / Rα-hetero-Fc format, a preferred embodiment utilizes the IL-15 mutation Q108E.

[0126] In the IL-15 / Rα-hetero-Fc format, a preferred embodiment utilizes the IL-15 mutation Q108E and the skew mutation pair S364K / E357Q:L368D / K370S.

[0127] In the IL-15 / Rα-hetero-Fc format, preferred embodiments utilize the IL-15 mutation Q108E and the skewed pair of mutations S364K / E357Q:L368D / K370S, as well as the 428L / 434S mutations in both monomers.

[0128] In the IL-15 / Rα-hetero-Fc format, a preferred embodiment utilizes the D30N / E64Q / N65D mutations of IL-15.

[0129] In the IL-15 / Rα-hetero-Fc format, a preferred embodiment utilizes the D30N / E64Q / N65D mutations of IL-15 and the skewed mutation pair S364K / E357Q:L368D / K370S.

[0130] In the IL-15 / Rα-hetero Fc format, preferred embodiments utilize the D30N / E64Q / N65D mutations of IL-15, the skewed pair of mutations S364K / E357Q:L368D / K370S, and the 428L / 434S mutations in each Fc monomer.

[0131] In the IL-15 / Rα-hetero-Fc format, a preferred embodiment utilizes the N65D mutation of IL-15.

[0132] In the IL-15 / Rα-hetero-Fc format, a preferred embodiment utilizes the N65D mutation and the skew mutation pair S364K / E357Q:L368D / K370S of IL-15.

[0133] In the IL-15 / Rα-hetero Fc format, a preferred embodiment utilizes the N65D mutation of IL-15, the skewed mutation pair S364K / E357Q:L368D / K370S, and the 428L / 434S mutations in each Fc monomer.

[0134] In the IL-15 / Rα-hetero-Fc format, a preferred embodiment utilizes the N4D / N65D mutations of IL-15.

[0135] In the IL-15 / Rα-hetero-Fc format, a preferred embodiment utilizes the N4D / N65D mutations and the skewed mutation pair S364K / E357Q:L368D / K370S of IL-15.

[0136] In the IL-15 / Rα-hetero Fc format, a preferred embodiment utilizes the N4D / N65D mutations of IL-15, the skewed pair S364K / E357Q:L368D / K370S, and the 428L / 434S mutations in each Fc monomer.

[0137] In the IL-15 / Rα-hetero-Fc format, a preferred embodiment utilizes the N1D / N65D mutations of IL-15.

[0138] In the IL-15 / Rα-hetero-Fc format, a preferred embodiment utilizes the N1D / N65D mutations and the skewed mutation pair S364K / E357Q:L368D / K370S of IL-15.

[0139] In the IL-15 / Rα-hetero-Fc format, a preferred embodiment utilizes the N1D / N65D mutations of IL-15, the skewed mutation pair S364K / E357Q:L368D / K370S, and the 428L / 434S mutations in each Fc monomer.

[0140] In the IL-15 / Rα-hetero Fc format, a preferred embodiment is shown in FIG. (XENP22822 containing strand 1 (17693) and strand 2 (15908)), Figure 94A (XENP23504 containing strand 1 and strand 2), Figure 104AO (XENP24045 containing strand 1 and strand 2), Figure 104AQ (XENP24306 containing strand 1 and strand 2), Figure 48A (XENP22821 containing strand 1 and strand 2), Figure 94A (XENP23343 containing strand 1 and strand 2), Figure 10 4AJ (XENP23557 containing strand 1 and strand 2), FIG. 104AP (XENP24113 containing strand 1 and strand 2), FIG. 104AP (XENP24051 containing strand 1 and strand 2), FIG. 104AR (XENP24341 containing strand 1 and strand 2), FIG. 104AP (XENP24052 containing strand 1 and strand 2), and FIG. 104AP (XENP24301 containing strand 1 and strand 2).

[0141] B.scIL-15-Rα-Fc In this embodiment, as shown in Figure 9B, the heterodimeric fusion protein comprises two monomers. The first monomer comprises (N- to C-terminally): IL-15 / Rα(sushi)-domain linker-IL-15-optional domain linker-CH2-CH3, where the domain linker often includes all or part of a hinge. The second monomer comprises an "empty" Fc, including all or part of hinge-CH2-CH3. This is referred to as "scIL-15 / Rα-Fc," where "sc" stands for "single chain" (e.g., IL-15 / sushi complex).

[0142] In the scIL-15 / Rα-Fc format, a preferred embodiment utilizes the skew mutation pair S364K / E357Q:L368D / K370S.

[0143] In the scIL-15 / Rα-Fc format, a preferred embodiment utilizes the IL-15 mutation Q108E.

[0144] In the scIL-15 / Rα-Fc format, a preferred embodiment utilizes the IL-15 mutation Q108E and the skew mutation pair S364K / E357Q:L368D / K370S.

[0145] In the scIL-15 / Rα-Fc format, preferred embodiments utilize the IL-15 mutation Q108E and the skewed mutation pair S364K / E357Q:L368D / K370S, as well as the 428L / 434S mutations in both monomers.

[0146] In the scIL-15 / Rα-Fc format, a preferred embodiment utilizes the D30N / E64Q / N65D mutations of IL-15.

[0147] In the scIL-15 / Rα-Fc format, a preferred embodiment utilizes the D30N / E64Q / N65D mutations of IL-15 and the skewed mutation pair S364K / E357Q:L368D / K370S.

[0148] In the scIL-15 / Rα-Fc format, preferred embodiments utilize the D30N / E64Q / N65D mutations of IL-15, the skewed pair of mutations S364K / E357Q:L368D / K370S, and the 428L / 434S mutations in each Fc monomer.

[0149] In the scIL-15 / Rα-Fc format, a preferred embodiment utilizes the N65D mutation of IL-15.

[0150] In the scIL-15 / Rα-Fc format, a preferred embodiment is the N65D mutation of IL-15 and the skew mutation pair S364K / E357Q:L368D / K370S Use.

[0151] In the scIL-15 / Rα-Fc format, a preferred embodiment utilizes the N65D mutation of IL-15, the skewed mutation pair S364K / E357Q:L368D / K370S, and the 428L / 434S mutations in each Fc monomer.

[0152] In the scIL-15 / Rα-Fc format, a preferred embodiment utilizes the N4D / N65D mutations of IL-15.

[0153] In the scIL-15 / Rα-Fc format, a preferred embodiment utilizes the N4D / N65D mutations and the skew mutation pair S364K / E357Q:L368D / K370S of IL-15.

[0154] In the scIL-15 / Rα-Fc format, preferred embodiments utilize the N4D / N65D mutations of IL-15, the skewed mutation pair S364K / E357Q:L368D / K370S, and the 428L / 434S mutations in each Fc monomer.

[0155] In the scIL-15 / Rα-Fc format, a preferred embodiment utilizes the N1D / N65D mutations of IL-15.

[0156] In the scIL-15 / Rα-Fc format, a preferred embodiment utilizes the N1D / N65D mutations and the skew mutation pair S364K / E357Q:L368D / K370S of IL-15.

[0157] In the IL-15 / Rα-hetero-Fc format, a preferred embodiment utilizes the N1D / N65D mutations of IL-15, the skewed mutation pair S364K / E357Q:L368D / K370S, and the 428L / 434S mutations in each Fc monomer.

[0158] C.ncIL-15 / Rα-Fc In this embodiment, as shown in Figure 9C, the heterodimeric fusion protein contains three monomers. The first monomer contains (N- to C-terminally) IL-15 / Rα(sushi)-domain linker-CH2-CH3, where the domain linker often includes all or part of the hinge. The second monomer contains an "empty" Fc, including all or part of the hinge-CH2-CH3. The third monomer is IL-15. This is referred to as "ncIL-15 / Rα-Fc," where "nc" stands for "non-covalent."

[0159] In the ncIL-15 / Rα-Fc format, a preferred embodiment utilizes the skew mutation pair S364K / E357Q:L368D / K370S.

[0160] In the ncIL-15 / Rα-Fc format, a preferred embodiment utilizes the IL-15 mutation Q108E.

[0161] In the ncIL-15 / Rα-Fc format, a preferred embodiment utilizes the IL-15 mutation Q108E and the skew mutation S364K / E357Q:L368D / K370S.

[0162] In the ncIL-15 / Rα-Fc format, preferred embodiments utilize the IL-15 mutation Q108E and the skewed mutation pair S364K / E357Q:L368D / K370S, as well as the 428L / 434S mutations in both monomers.

[0163] In the ncIL-15 / Rα-Fc format, a preferred embodiment utilizes the D30N / E64Q / N65D mutations of IL-15.

[0164] In the ncIL-15 / Rα-Fc format, a preferred embodiment utilizes the D30N / E64Q / N65D mutations and the skewed mutation pair S364K / E357Q:L368D / K370S of IL-15.

[0165] In the ncIL-15 / Rα-Fc format, preferred embodiments utilize the D30N / E64Q / N65D mutations of IL-15, the skewed mutation pair S364K / E357Q:L368D / K370S, and the 428L / 434S mutations in each Fc monomer.

[0166] In the ncIL-15 / Rα-Fc format, a preferred embodiment utilizes the N65D mutation of IL-15.

[0167] In the ncIL-15 / Rα-Fc format, a preferred embodiment utilizes the N65D mutation and the skew mutation pair S364K / E357Q:L368D / K370S of IL-15.

[0168] In the ncIL-15 / Rα-Fc format, a preferred embodiment utilizes the N65D mutation of IL-15, the skewed mutation pair S364K / E357Q:L368D / K370S, and the 428L / 434S mutations in each Fc monomer.

[0169] In the ncIL-15 / Rα-Fc format, a preferred embodiment utilizes the N4D / N65D mutations of IL-15.

[0170] In the ncIL-15 / Rα-Fc format, a preferred embodiment utilizes the N4D / N65D mutations and the skew mutation pair S364K / E357Q:L368D / K370S of IL-15.

[0171] In the ncIL-15 / Rα-Fc format, preferred embodiments utilize the N4D / N65D mutations of IL-15, the skewed mutation pair S364K / E357Q:L368D / K370S, and the 428L / 434S mutations in each Fc monomer.

[0172] In the ncIL-15 / Rα-Fc format, a preferred embodiment utilizes the N1D / N65D mutations of IL-15.

[0173] In the ncIL-15 / Rα-Fc format, a preferred embodiment utilizes the N1D / N65D mutations and the skew mutation pair S364K / E357Q:L368D / K370S of IL-15.

[0174] In the ncIL-15 / Rα-Fc format, preferred embodiments utilize the N1D / N65D mutations of IL-15, the skewed mutation pair S364K / E357Q:L368D / K370S, and the 428L / 434S mutations in each Fc monomer.

[0175] In the ncIL-15 / Rα-hetero-Fc format, preferred embodiments are shown in Figure 104AS (XENP24349 comprising chain 1 and chain 2) and Figure 104AT (XENP24383 comprising chain 1 and chain 2).

[0176] D. Bivalent ncIL-15 / Rα-Fc In this embodiment, as shown in Figure 9D, the heterodimeric fusion protein contains four monomers. The first and second monomers contain (N- to C-terminally) IL-15 / Rα(sushi)-domain linker-CH2-CH3, where the domain linker often includes all or part of a hinge. The third and fourth monomers contain IL-15. This is referred to as "bivalent ncIL-15 / Rα-Fc," where "nc" stands for "non-covalent."

[0177] In the bivalent ncIL-15 / Rα-Fc format, a preferred embodiment utilizes the skew mutation pair S364K / E357Q:L368D / K370S.

[0178] In the bivalent ncIL-15 / Rα-Fc format, a preferred embodiment utilizes the IL-15 mutation Q108E.

[0179] In the bivalent ncIL-15 / Rα-Fc format, a preferred embodiment utilizes the IL-15 mutation Q108E and the skew mutation S364K / E357Q:L368D / K370S.

[0180] In the bivalent ncIL-15 / Rα-Fc format, preferred embodiments utilize the IL-15 mutation Q108E and the skewed mutation pair S364K / E357Q:L368D / K370S, as well as the 428L / 434S mutations in both monomers.

[0181] In the bivalent ncIL-15 / Rα-Fc format, a preferred embodiment utilizes the D30N / E64Q / N65D mutations of IL-15.

[0182] In the bivalent ncIL-15 / Rα-Fc format, a preferred embodiment utilizes the D30N / E64Q / N65D mutations and the skewed mutation pair S364K / E357Q:L368D / K370S of IL-15.

[0183] In the bivalent ncIL-15 / Rα-Fc format, preferred embodiments utilize the D30N / E64Q / N65D mutations of IL-15, the skewed mutation pair S364K / E357Q:L368D / K370S, and the 428L / 434S mutations in each Fc monomer.

[0184] In the bivalent ncIL-15 / Rα-Fc format, a preferred embodiment utilizes the N65D mutation of IL-15.

[0185] In the bivalent ncIL-15 / Rα-Fc format, a preferred embodiment utilizes the N65D mutation and the skew mutation pair S364K / E357Q:L368D / K370S of IL-15.

[0186] In the bivalent ncIL-15 / Rα-Fc format, a preferred embodiment utilizes the N65D mutation of IL-15, the skewed mutation pair S364K / E357Q:L368D / K370S, and the 428L / 434S mutations in each Fc monomer.

[0187] In the bivalent ncIL-15 / Rα-Fc format, a preferred embodiment utilizes the N4D / N65D mutations of IL-15.

[0188] In the bivalent ncIL-15 / Rα-Fc format, a preferred embodiment utilizes the N4D / N65D mutations and the skew mutation pair S364K / E357Q:L368D / K370S of IL-15.

[0189] In the bivalent ncIL-15 / Rα-Fc format, a preferred embodiment utilizes the N4D / N65D mutations of IL-15, the skewed mutation pair S364K / E357Q:L368D / K370S, and the 428L / 434S mutations in each Fc monomer.

[0190] In the bivalent ncIL-15 / Rα-Fc format, a preferred embodiment utilizes the N1D / N65D mutations of IL-15.

[0191] In the bivalent ncIL-15 / Rα-Fc format, a preferred embodiment utilizes the N1D / N65D mutations and the skew mutation pair S364K / E357Q:L368D / K370S of IL-15.

[0192] In the bivalent ncIL-15 / Rα-Fc format, a preferred embodiment utilizes the N1D / N65D mutations of IL-15, the skewed mutation pair S364K / E357Q:L368D / K370S, and the 428L / 434S mutations in each Fc monomer.

[0193] In the bivalent ncIL-15 / Rα-Fc format, preferred embodiments are shown in Figure 104AR (XENP24342 comprising chain 1 and chain 2) and (XENP24346 comprising chain 1 and chain 2).

[0194] VI. Useful Embodiments of the Invention As will be appreciated by those of skill in the art and described more fully below, the heterodimeric fusion proteins of the present invention can take on a wide variety of configurations, as generally shown in Figures 9A-9G and 39A-39D. The amino acid sequences of exemplary fusion proteins are provided in Figures 8A-8E, 10, 11, 12A, 12B, 13-15, 40A, 40B, 41A, 41B, 42, 43, 48A-48D, 49A-49C, 50A, 50B, 51, 52, 53, and 94A-94D.

[0195] Many of the embodiments outlined herein generally rely on a format comprising a first monomer (first fusion protein) comprising an IL-15 protein domain covalently linked to the N-terminus of a first Fc domain using a first domain linker, and a second monomer (second fusion protein) comprising an IL-15Rα protein domain covalently linked to the N-terminus of a second Fc domain using a second domain linker. Exemplary embodiments of this format ("IL-15 / Rα hetero-Fc" and "dsIL-15 / Rα hetero-Fc") include XENP20818, XENP20819, XENP21471, XENP21472, XENP21473, XENP21474, XENP21475, XENP21476, XENP21477, and XENP22013. , XENP22815, XENP22816, XENP22817, XENP22818, XENP22819, XENP22820, XENP22821, XENP228 22, XENP22823, XENP22824, XENP22825, XENP22826, XENP22827, XENP22828, XENP22829, XENP22 830, XENP22831, XENP22832, XENP22833, XENP22834, XENP22815, XENP22816, XENP22817, XENP 22818, XENP22819, XENP22820, XENP22821, XENP23343, XENP23554, XENP23555, XENP23557, XE These include, but are not limited to, NP23559, XENP23561, XENP24018, XENP24019, XENP24020, XENP24051, XENP24052, XENP23504, XENP24306, XENP24306, XENP23343, XENO24113, XENP24341, and XENP24301.

[0196] A useful format for heterodimeric Fc fusion proteins is a heterodimeric Fc fusion protein in which the N-terminus of the first Fc domain is The fusion protein includes a first protein domain covalently attached via a first domain linker to the N-terminus of a second protein domain covalently attached via a second domain linker to the N-terminus of the second protein domain, and a second Fc domain (e.g., an empty Fc domain). In some cases, the first protein domain is an IL-15Rα protein domain and the second protein domain is an IL-15 protein domain. Exemplary embodiments of this format ("scIL-15 / Rα-Fc") include, but are not limited to, XENP21478.

[0197] Yet another useful heterodimeric Fc-fusion protein outlined herein includes a fusion protein comprising a first protein domain covalently attached to the N-terminus of the first Fc domain via a domain linker, a second Fc domain (e.g., an empty Fc domain), and a second protein domain non-covalently attached to the first protein domain. In some cases, the first protein domain is an IL-15 protein domain and the second protein domain is an IL-15Rα protein domain. Exemplary embodiments of this format ("ncIL-15 / Rα-Fc" or "dsIL-15 / Rα-Fc") include, but are not limited to, XENP21479, XENP22357, XENP22354, XENP22355, XENP22356, XENP22357, XENP22358, XENP22359, XENP22360, XENP22361, XENP22362, XENP22363, XENP22364, XENP22365, XENP22366, XENP22637, XENP24348, XENP24349, and XENP24383.

[0198] Another useful format of the heterodimeric Fc-fusion proteins outlined herein comprises a first fusion protein comprising a first protein domain covalently attached to the N-terminus of the first Fc domain via a first domain linker, a second fusion protein comprising a second protein domain covalently attached to the N-terminus of the second Fc domain via a second domain linker, a third protein domain non-covalently attached to the first protein domain of the first fusion protein, and a fourth protein domain non-covalently attached to the second protein domain of the second fusion protein. In some cases, the first and second protein domains are IL-15Rα protein domains, and the third and fourth protein domains are IL-15 protein domains. Exemplary embodiments of this format (“bivalent ncIL-15 / Rα-Fc” or “bivalent dsIL-15 / Rα-Fc”) include, but are not limited to, XENP21978, XENP22634, XENP24342, and XENP24346.

[0199] Another useful format ("bivalent scIL-15 / Rα-Fc") is outlined in FIG. 14 herein.

[0200] Yet another useful format for the heterodimeric Fc fusion proteins outlined herein includes a fusion protein comprising a first Fc domain covalently attached to the N-terminus of the first protein domain using a domain linker, a second Fc domain (e.g., an empty Fc domain), and a second protein domain noncovalently attached to the first protein domain. Exemplary embodiments of this format ("Fc-ncIL-15 / Rα" or "Fc-dsIL-15 / Rα") include, but are not limited to, XENP22637 and XENP22639, and those shown in Figure 16. In some embodiments, the first and second proteins are linked via a linker (Figure 9G).

[0201] For any of the heterodimeric Fc fusion proteins outlined herein, the first domain The in-linker and second domain linker can be the same or different. Furthermore, the first Fc domain and the second Fc domain of the heterodimeric protein can have different amino acid sequences.

[0202] The Fc domain of the present invention comprises an IgG Fc domain, such as an IgG1 Fc domain. In some embodiments, the first and second Fc domains have a set of amino acid substitutions selected from the group consisting of L368D / K370S and S364K, L368D / K370S and S364K / E357L, L368D / K370S and S364K / E357Q, T411E / K360E / Q362E and D401K, L368E / K370S and S364K, K370S and S364K / E357Q, K370S and S364K / E357Q, or S267K / L368D / K370S and S267K / S364K / E357Q, according to EU numbering. In some examples, the first and / or second Fc domain of any of the heterodimeric Fc fusion formats outlined herein may have an additional set of amino acid substitutions including Q295E / N384D / Q418E / N421D according to EU numbering. In some embodiments, 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.

[0203] Additional heterodimerization mutations are independently and optionally included and can be selected from the mutations outlined in the figure. These compositions can further include deletion mutations, pI mutations, charge mutants, isotype mutations, etc.

[0204] VII. Nucleic Acids of the Invention The present invention further provides nucleic acid compositions encoding the heterodimeric Fc fusion proteins of the present invention (or, in the case of monomeric Fc domain proteins, nucleic acids encoding them).

[0205] As those skilled in the art will understand, the nucleic acid composition will depend on the format of heterodimeric protein.Therefore, for example, when a format requires three amino acid sequences, three nucleic acid sequences can be incorporated into one or more expression vectors for expression.Similarly, some formats only require two nucleic acids, and can be incorporated into one or two expression vectors.

[0206] As is known in the art, nucleic acids encoding the components of the present invention can be incorporated into expression vectors, as is known in the art and depending on the host cell used to produce the heterodimeric Fc-fusion proteins of the present invention. Generally, the nucleic acid is operably linked to any number of regulatory elements (promoter, origin of replication, selectable marker, ribosome binding site, inducer, etc.). Expression vectors can be extrachromosomal or integrating vectors.

[0207] 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.

[0208] In some embodiments, the nucleic acids encoding each monomer are expressed in a single expression vector, each generally under the control of 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 in a different expression vector.

[0209] The heterodimeric Fc-fusion proteins of the present invention are produced by culturing host cells containing an expression vector, as is well known in the art. Once produced, conventional fusion protein or antibody purification processes, including ion exchange chromatography, are performed. As discussed herein, the pIs of the two monomers differ by at least 0.5, which can enable separation by ion exchange chromatography or isoelectric focusing, or other methods sensitive to isoelectric point. That is, by including pI substitutions that alter the isoelectric point (pI) of each monomer so that the pI of each monomer is different and the heterodimer also has a different pI, isoelectric purification of the heterodimer (e.g., anion exchange column, cation exchange column) is facilitated. These substitutions also aid in the identification and monitoring of any contaminating homodimers after purification (e.g., IEF gel, cIEF, and analytical IEX columns).

[0210] VIII. Biological and Biochemical Functionality of IL-15 / IL15Rα Heterodimeric Immunomodulatory Fc Fusion Proteins Generally, the heterodimeric Fc-fusion proteins of the present invention are administered to patients with cancer, and efficacy is assessed by several methods, as described herein. Thus, while standard assays of efficacy, such as assessment of cancer burden, tumor size, and the presence or extent of metastases, can be performed, immuno-oncological treatments can also be evaluated based on immune status assessment. This can be done by several methods, including both in vitro and in vivo assays. For example, assessment of changes in immune status (e.g., the 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. To this end, any or all of the following can be assessed: the inhibitory effect of PVRIG on CD4+ T cell activation or proliferation, CD8+ T (CTL) cell activation or proliferation, CD8+ T cell-mediated cytotoxic activity and / or CTL-mediated cell depletion, NK cell activity and NK-mediated cell depletion, the enhancing effect of PVRIG on Treg cell differentiation and proliferation and Treg cell- or myeloid-derived suppressor cell (MDSC)-mediated immunosuppression or immune tolerance, and / or the effect of PVRIG on inflammatory cytokine production by immune cells, e.g., IL-2, IFN-γ, or TNF-α production by T cells or other immune cells.

[0211] In some embodiments, evaluation of treatment is performed by assessing immune cell proliferation using, for example, the CFSE dilution method, Ki67 intracellular staining of immune effector cells, and 3H-thymidine incorporation.

[0212] In some embodiments, treatment is evaluated by assessing increased gene expression or increased protein levels of activation-associated markers, including one or more of cellular degranulation as measured by surface expression of CD25, CD69, CD137, ICOS, PD1, GITR, OX40, and CD107A.

[0213] Generally, gene expression assays are performed as known in the art.

[0214] Generally, protein expression measurements are also performed as known in the art.

[0215] In some embodiments, evaluation of treatment involves cell damage as measured by detecting target cell viability 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, but are not limited to, trypan blue or PI staining, 51Cr or 35S release assays, LDH activity, MTT and / or WST assays, calcein-AM assays, luminescence-based assays, and others.

[0216] 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.

[0217] Thus, evaluation of a treatment can be performed using an assay that assesses 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 proinflammatory cytokine secretion, (vii) increasing IL-2 secretion, (viii) increasing interferon-γ production, (ix) increasing Th1 responses, (x) decreasing Th2 responses, (xi) reducing or eliminating the number and / or activity of at least one regulatory T cell (Treg).

[0218] A. 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 immunostimulatory activity. Suitable increases in activity are outlined below.

[0219] In one embodiment, the signal transduction pathway assay measures an increase or decrease in immune response, for example, measured by phosphorylation or dephosphorylation of different factors or by measuring other post-translational modifications. Increased activity indicates immunostimulatory activity. Suitable increases in activity are outlined below.

[0220] 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, e.g., CD137, CD107a, PD1, etc. Increased activity is indicative of immunostimulatory activity. Suitable increases in activity are outlined below.

[0221] In one embodiment, the signaling pathway assay measures an increase or decrease in cytotoxic T cell activity, e.g., as 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, e.g., CD137, CD107a, PD1, etc. Increased activity indicates immunostimulatory activity. Suitable increases in activity are outlined below.

[0222] In one embodiment, the signaling pathway assay measures an increase or decrease in NK and / or NKT cell activity, e.g., as measured by direct killing of target cells, e.g., cancer cells, or by cytokine secretion, or by changes in expression of activation markers, e.g., CD107a, etc. Increased activity indicates immunostimulatory activity. Suitable increases in activity are outlined below.

[0223] In one embodiment, the signaling pathway assay is, for example, by cytokine secretion, or by proliferation, or by activity of, for example, CD137, CD107a, PD1, etc. The antibody or antibody fragments measure an increase or decrease in αβ and / or γδ T cell suppression, as measured by changes in expression of immunostimulatory markers. Increased activity indicates immunostimulatory activity. Suitable increases in activity are outlined below.

[0224] In one embodiment, the signaling pathway assay measures an increase or decrease in pro-inflammatory cytokine secretion, as measured, for example, by ELISA, by Luminex, by multiplex bead-based methods, by intracellular staining and FACS analysis, or by Alispot, etc. Increased activity indicates immunostimulatory activity. Suitable increases in activity are outlined below.

[0225] In one embodiment, the signaling pathway assay measures an increase or decrease in IL-2 secretion, as measured, for example, by ELISA, by Luminex, by multiplex bead-based methods, by intracellular staining and FACS analysis, or by Alispot, etc. Increased activity indicates immunostimulatory activity. Suitable increases in activity are outlined below.

[0226] In one embodiment, the signaling pathway assay measures an increase or decrease in interferon-γ production, as measured, for example, by ELISA, by Luminex, by multiplex bead-based methods, by intracellular staining and FACS analysis, or by Alispot, etc. Increased activity indicates immunostimulatory activity. Suitable increases in activity are outlined below.

[0227] In one embodiment, the signal transduction pathway assay measures an increase or decrease in Th1 response, for example, as measured by cytokine secretion or by changes in the expression of activation markers. Increased activity indicates immunostimulatory activity. Suitable increases in activity are outlined below.

[0228] In one embodiment, the signal transduction pathway assay measures an increase or decrease in Th2 response, for example, as measured by cytokine secretion or by changes in the expression of activation markers. Increased activity indicates immunostimulatory activity. Suitable increases in activity are outlined below.

[0229] In one embodiment, the signal transduction pathway assay measures the increase or decrease in the number and / or activity of at least one regulatory T cell (Treg), for example, measured by flow cytometry or IHC. A decrease in response indicates immunostimulatory activity. Suitable decrease is the same as the increase outlined below.

[0230] In one embodiment, the signal transduction pathway assay measures the increase or decrease in the number of M2 macrophage cells, for example, by flow cytometry or IHC. A decrease in response indicates immunostimulatory activity. The appropriate decrease is the same as the increase described below.

[0231] In one embodiment, the signaling pathway assay measures an increase or decrease in M2 macrophage pro-tumorigenic activity, as measured, for example, by cytokine secretion or by changes in the expression of activation markers. A decrease in response indicates immunostimulatory activity. Suitable decreases are the same as those for increases, as outlined below.

[0232] In one embodiment, the signal transduction pathway assay measures an increase or decrease in N2 neutrophilia, for example, measured by flow cytometry or IHC. A decrease in response indicates immunostimulatory activity. Suitable decreases are the same as those for increases, as outlined below.

[0233] In one embodiment, the signaling pathway assay is, for example, by cytokine secretion: Alternatively, an increase or decrease in N2 neutrophil tumorigenicity activity, as measured by changes in expression of activation markers, is measured. A decrease in response indicates immunostimulatory activity. Suitable decreases are the same as increases, as outlined below.

[0234] In one embodiment, the signaling pathway assay measures an increase or decrease in the suppression of T cell activation, e.g., as measured by cytokine secretion, or by proliferation, or by changes in expression of activation markers such as, e.g., CD137, CD107a, PD1, etc. Increased activity indicates immunostimulatory activity. Suitable increases in activity are outlined below.

[0235] In one embodiment, the signaling pathway assay measures an increase or decrease in inhibition of CTL activation, e.g., as 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, e.g., CD137, CD107a, PD1, etc. Increased activity indicates immunostimulatory activity. Suitable increases in activity are outlined below.

[0236] In one embodiment, the signaling pathway assay measures an increase or decrease in αβ and / or γδ T cell depletion, e.g., as measured by changes in expression of activation markers. A decrease in response indicates immunostimulatory activity. Suitable decreases are the same as for increases, as outlined below.

[0237] 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, e.g., CD137, CD107a, PD1, etc. Increased activity is indicative of immunostimulatory activity. Suitable increases in activity are outlined below.

[0238] In one embodiment, the signaling pathway assay measures increased or decreased stimulation of an antigen-specific memory response, as measured, for example, by cytokine secretion, or by proliferation, or by changes in expression of activation markers such as, for example, CD45RA, CCR7, etc. Increased activity indicates immunostimulatory activity. Suitable increases in activity are outlined below.

[0239] In one embodiment, the signal transduction pathway assay measures an increase or decrease in apoptosis or lysis of cancer cells, for example, as measured 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 immunostimulatory activity. Suitable increased activity is outlined below.

[0240] In one embodiment, the signal transduction pathway assay measures an increase or decrease in the stimulation of the cytotoxic or cytostatic effect of cancer cells, for example, as measured by a cytotoxicity assay such as MTT, Cr release, calcine AM, or by a flow cytometry-based assay such as CFSE dilution or propidium iodide staining. Increased activity indicates immunostimulatory activity. Suitable increased activity is outlined below.

[0241] In one embodiment, the signal transduction pathway assay measures an increase or decrease in direct killing of cancer cells, for example, as measured 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 immunostimulatory activity. Suitable increased activity is outlined below.

[0242] In one embodiment, the signaling pathway assay measures, for example, cytokine secretion. The increase or decrease in Th17 activity is measured by measuring the activity of the cells, by proliferation, or by changes in the expression of activation markers. Increased activity indicates immune stimulatory activity. Suitable increases in activity are outlined below.

[0243] In one embodiment, the signal transduction pathway assay measures, for example, an increase or decrease in the induction of complement-dependent cytotoxicity and / or antibody-dependent cell-mediated cytotoxicity, as measured by a cytotoxicity assay such as MTT, Cr release, calcine AM, or by a flow cytometry-based assay such as CFSE dilution or propidium iodide staining. Increased activity indicates immunostimulatory activity. Suitable increases in activity are outlined below.

[0244] In one embodiment, T cell activation is measured, for example, 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 such as, for example, CD137, CD107a, PD1, etc. For T cells, increases in proliferation, cell surface markers of activation (e.g., CD25, CD69, CD137, PD1), cytotoxicity (the ability to kill target cells), and cytokine production (e.g., IL-2, IL-4, IL-6, IFNγ, TNF-α, IL-10, IL-17A) can be indicative of immune modulation consistent with enhanced killing of cancer cells.

[0245] 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 (the 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.

[0246] 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.

[0247] In one embodiment, Th1 cell activation is measured, for example, by cytokine secretion or by changes in expression of activation markers.

[0248] A suitable increase (or decrease, as outlined above, as appropriate) 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 the reference or control sample indicates efficacy.

[0249] IX. Treatment Once produced, the compositions of the invention are used in a number of oncology applications, generally by treating cancer by promoting T cell activation (e.g., T cells are no longer suppressed), generally through binding of the heterodimeric Fc fusion proteins of the invention.

[0250] Therefore, the heterodimeric compositions of the present invention are used to treat these cancers.

[0251] A. Heterodimeric Protein Compositions for In Vivo Administration Formulations of antibodies used in accordance with the present invention may be prepared by dissolving antibodies of the desired purity in a solution containing an appropriate amount of pharmaceutically acceptable carriers, excipients, or stabilizers (e.g., Remington's Pharmaceuticals). The drug may be prepared in the form of a lyophilized formulation or aqueous solution for storage by mixing with an appropriate carrier, buffer, excipient, or stabilizer (as generally reviewed in Remington's Pharmaceutical Sciences, 16th edition, Osol, A. Ed., 1980). Acceptable carriers, buffers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed and include buffers such as phosphate, citric acid, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl, or benzyl alcohol; alkylparabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; and blood proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, and lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as TWEEN™, PLURONICS™, or polyethylene glycol (PEG).

[0252] B. Mode of Administration The heterodimeric protein of the present invention and the chemotherapeutic agent 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.

[0253] C.Treatment In the method of the present invention, treatment is used to provide a positive therapeutic response for disease or pathology. "Positive therapeutic response" refers to the improvement of disease or pathology and / or the improvement of symptoms associated with disease or pathology. For example, a positive therapeutic response can refer to one or more of the following improvements in disease: (1) a decrease in the number of tumor cells; (2) an increase in tumor cell death; (3) inhibition of tumor cell survival; (5) inhibition (i.e., some slowing, preferably cessation) of tumor growth; (6) an increase in patient survival rate; and (7) some relief from one or more symptoms associated with disease or pathology.

[0254] Positive therapeutic response in any given disease or condition can be determined by standardized response criteria specific to that disease or condition.Tumor response can be evaluated for changes in tumor morphology (i.e., tumor burden, tumor size, etc.) using screening techniques such as magnetic resonance imaging (MRI) scan, X-ray, computed tomography (CT) scan, bone scan, endoscopy, and tumor biopsy sampling, including bone marrow aspiration (BMA) and counting circulating tumor cells.

[0255] In addition to these positive therapeutic responses, subjects undergoing treatment may experience the beneficial effect of amelioration of symptoms associated with the disease.

[0256] Treatment according to the present invention includes a "therapeutically effective amount" of the pharmaceutical agent used. A "therapeutically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result.

[0257] A therapeutically effective amount may vary depending on factors such as the disease state, age, sex, and weight of the individual, and the ability of the agent to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the antibody or antibody portion are outweighed by the therapeutically beneficial effects.

[0258] A "therapeutically effective amount" for tumor therapy can also be measured by its ability to stabilize disease progression. The ability of a compound to inhibit cancer may also be evaluated in animal model systems predictive of efficacy in human tumors.

[0259] Alternatively, this property of the composition can be evaluated by examining the ability of the compound to inhibit cell proliferation or induce apoptosis by in vitro assays known to those skilled in the art. A therapeutically effective amount of a therapeutic compound can reduce tumor size or otherwise alleviate symptoms in a subject. Those skilled in the art will be able to determine such an amount based on factors such as the size of the subject, the severity of the subject's symptoms, and the specific composition or route of administration selected.

[0260] Dosage regimen is adjusted to provide the optimum desired response (e.g., therapeutic response).For example, a single bolus can be administered, or several divided doses can be administered over time, or the dose can be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation.Non-oral compositions can be formulated in unit dosage form for ease of administration and uniformity of dosage.As used herein, unit dosage form refers to a physically discrete unit suitable as a single dosage for the subject to be treated, each unit containing a predetermined amount of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.

[0261] The specifications for the unit dosage forms of the present invention are influenced by and directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of formulating such active compounds for the treatment of susceptible individuals.

[0262] The effective dosage and dosage regimen of the bispecific antibodies used in the present invention depends on the disease or condition being treated and can be determined by one skilled in the art.

[0263] An exemplary, non-limiting range for a therapeutically effective amount of a bispecific antibody used in the present invention is about 0.1 to 100 mg / kg.

[0264] All cited references are expressly incorporated herein by reference in their entirety.

[0265] 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. [Example]

[0266] 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 that of Kabat (Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th Ed., United States Public The EU index is similar to that of the National Institutes of Health, Bethesda, MD (National Health Service, National Institutes of Health, Bethesda, MD, incorporated by reference in its entirety). Those skilled in the art of antibodies will understand that this convention consists of non-contiguous numbering in certain regions of the immunoglobulin sequence, allowing for a standard reference to conserved positions within the immunoglobulin family. Thus, the positions of any given immunoglobulin as defined by the EU index do not necessarily correspond to its contiguous sequence.

[0267] General and specific science and technology are described in U.S. Patent Application Publication No. 2015 / 0307629; No. 2014 / 0288275, and WO2014 / 145806, all of which are expressly incorporated by reference in their entirety, particularly with respect to the technology outlined therein.

[0268] Example 1: IL-15 / IL-15Rα(sushi)Fc fusion protein To address the short half-life of the IL-15 / IL-15Rα heterodimer, we generated the IL-15 / IL-15Rα(sushi) complex as an Fc fusion (hereafter referred to as IL-15 / Rα-Fc fusion) to enhance production and promote FcRn-mediated recycling of the complex and prolong its half-life.

[0269] A. Example 1A: Engineering an IL-15 / Rα-Fc Fusion Protein Plasmids encoding the IL-15 or IL-15Rα sushi domain 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 8). Cartoon schematics of exemplary IL-15 / Rα-Fc fusion protein formats are shown in Figures 9A-9G.

[0270] The IL-15Rα heterodimeric Fc fusion or "IL-15 / Rα-hetero-Fc" format comprises IL-15 recombinantly fused to one side of the heterodimeric Fc and an IL-15Rα sushi domain recombinantly fused to the other side of the heterodimeric Fc (Figure 9A). IL-15 and IL-15Rα can have variable-length linkers between the C- and N-termini of their respective Fc regions (see Figure 7). Exemplary proteins in this format include XENP20818 and XENP21475, whose sequences are shown in Figure 10 (see also Table 1). The sequences of additional proteins in this format are listed in the figures and sequence listing as XENP20819, XENP21471, XENP21472, XENP21473, XENP21474, XENP21476, and XENP21477. [Table 1]

[0271] The single-chain IL-15 / Rα-Fc fusion or "scIL-15 / Rα-Fc" format comprises an IL-15Rα sushi domain fused to IL-15 by a variable-length linker (referred to as a "single-chain" IL-15 / IL-15Rα complex or "scIL-15 / Rα"), which is then fused to the N-terminus of a heterodimeric Fc region, with either an "Fc-only" or "empty Fc" heterodimeric Fc at the other end of the molecule (Figure 9B). An exemplary linker sequence is shown in Figure 7. An exemplary protein of this format is XENP21478, the sequence of which is shown in Figure 11 (see also Table 2). Further variations of this format are possible. The protein sequences are listed in the figures and sequence listing as XENP21993, XENP21994, XENP21995, XENP23174, XENP23175, XENP24477, and XENP24480. [Table 2]

[0272] The noncovalent IL-15 / Rα-Fc fusion or "ncIL-15 / Rα-Fc" format contains the IL-15Rα sushi domain fused to the heterodimeric Fc region, while IL-15 is transfected separately to form a noncovalent IL-15 / IL-15Rα complex, with the other end of the molecule being an "Fc-only" or "empty Fc" heterodimeric Fc (Figure 9C). Exemplary proteins of this format include XENP21479, XENP22366, and XENP24348, whose sequences are shown in Figure 12.

[0273] The bivalent noncovalent IL-15 / Rα-Fc fusion or "bivalent ncIL-15 / Rα-Fc" format (Figure 9D) contains IL-15Rα(sushi) fused to the N-terminus of the homodimeric Fc region, while IL-15 is transfected separately to form a noncovalent IL-15 / Rα complex. An exemplary protein of this format is XENP21978, the sequence of which is shown in Figure 13. The sequence of an additional protein of this format is listed as XENP21979 in the figures and sequence listing.

[0274] The bivalent single-chain IL-15 / Rα-Fc fusion or "bivalent scIL-15 / Rα-Fc" format (Figure 9E) comprises IL-15 fused to IL-15Rα(sushi) by a variable-length linker (referred to as a "single-chain" IL-15 / IL-15Rα(sushi) complex or "scIL-15 / Rα"), which is then fused to the N-terminus of a homodimeric Fc region. An exemplary linker sequence is shown in Figure 7. An exemplary protein sequence for this format is shown in Figure 14.

[0275] The Fc non-covalent IL-15 / Rα fusion or "Fc-ncIL-15 / Rα" format (Figure 9E) contains IL-15Rα(sushi) fused to the C-terminus of the heterodimeric Fc region, while IL-15 is transfected separately to form a non-covalent IL-15 / Rα complex, with "Fc only" or "empty Fc" on the other side of the molecule. An exemplary protein in this format is XENP22637, the sequence of which is shown in Figure 15. The sequence of an additional protein in this format is listed as XENP22638 in the figures and sequence listing.

[0276] Fc-single chain IL-15 / Rα fusion or "Fc-scIL-15 / Rα" format The format (Figure 9G) contains IL-15 fused to IL-15Rα(sushi) by a variable length linker ("scIL-15 / Rα"), which is then fused to the C-terminus of a heterodimeric Fc region, with "Fc only" or "empty Fc" at the other end of the molecule. Exemplary linker sequences are shown in Figure 7. Exemplary protein sequences for this format are shown in Figure 16.

[0277] The protein was produced by transient transfection in HEK293E cells and purified by a two-step purification process including protein A chromatography (GE Healthcare) and anion exchange chromatography (HiTrapQ 5 mL column with a 5–40% gradient of 50 mM Tris pH 8.5 and 50 mM Tris pH 8.5 containing 1 M NaCl).

[0278] B. Example 1B: Engineering an IL-15 / Rα-Fc Fusion Protein IL-15 / Rα-Fc fusion proteins produced in several formats as described above were analyzed for purity and homogeneity by size exclusion chromatography (SEC) and capillary isoelectric focusing (CEF), as generally described below.

[0279] Proteins were analyzed using SEC to measure their size (i.e., hydrodynamic volume) and determine the native behavior of purified samples. Analysis was performed on an Agilent 1200 high-performance liquid chromatography (HPLC) system. Samples were injected onto a Superdex™ 200 10 / 300GL column (GE Healthcare Life Sciences) using 1×PBS, pH 7.4, as the mobile phase at 1.0 mL / min for 25 minutes at 4°C with a UV detection wavelength of 280 nM. Analysis was performed using the Agilent OpenLab Chromatography Data System (CDS) ChemStation Edition AIC version C.01.07. Chromatograms of selected IL-15 / Rα-Fc fusion proteins are shown in Figures 17B, 18B, and 19B.

[0280] Protein Express Assay performed using the manufacturer's instructions LabChip and Protein Express Assay Reagent Proteins were analyzed electrophoretically by CEF using a LabChip GXII Touch HT (PerkinElmer, Waltham, Massachusetts) with a kit. Samples were run in duplicate, one under reducing (with dithiothreitol) and the other under non-reducing conditions. Gel images for selected IL-15 / Rα-Fc fusion proteins are shown in Figures 17C, 18C, and 19C.

[0281] For each fusion protein, the symmetry of the peaks and the relatively small populations of other species indicate that the various formats were robust.

[0282] C. Example 1C: Analysis of IL-15 / Rα-Fc Fusion Protein for Affinity and Stability Affinity screening of IL-15 / Rα-Fc fusion proteins was performed using Octet, a method based on BioLayer Interferometry (BLI). The Octet experimental steps generally included immobilization (capture of the ligand or test sample onto the biosensor), association (immersion of the ligand- or test sample-coated biosensor into wells containing serial dilutions of the corresponding test sample or ligand), and dissociation (replacement of the biosensor in wells containing buffer) to determine the affinity of the test sample. Reference wells containing buffer only were also included in the method for background correction during data processing. Specifically, an anti-human Fc (AHC) biosensor was used to capture the test sample, followed by multiple concentrations of IL-2Rβ (R&D Systems, M) for KD determination. The constructs were immersed in a 500-well plate (McClms Page number 17) containing 1000 mAb of IL-1Rβ (McClms Page number 17). The affinity results and corresponding sensorgrams are shown in Figures 17D, 18D, and 19D. Each of the three constructs exhibited high affinity binding (3-8 nM) to IL-1Rβ.

[0283] The stability of the IL-15 / Rα-Fc fusion protein was assessed using differential scanning fluorometry (DSF). DSF experiments were performed using a Bio-Rad CFX Connect Real-Time PCR Detection System. The protein was mixed with SYPRO Orange fluorescent dye and diluted to 0.2 mg / mL in PBS. The final concentration of SYPRO Orange was 10x. After an initial 10-minute incubation period at 25°C, the protein was heated from 25°C to 95°C at a heating rate of 1°C / min. The melting temperature (Tm) was calculated using the instrument's software. The stability results and corresponding melting curves are shown in Figures 17E, 18E, and 19E. Each construct demonstrated good overall stability with a Tm of approximately 68°C.

[0284] D. Example 1D: Activity of IL-15 / Rα-Fc Fusion Protein in a Cell Proliferation Assay Various formats of IL-15 / Rα-Fc fusion proteins, as described above, were tested in a cell proliferation assay. Human PBMCs were treated with the test samples 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, Massachusetts). The percentage of Ki67 in the above cell types was measured using FACS (shown in Figures 20A-20C and 21A-21C).

[0285] Various IL-15 / Rα-Fc fusion proteins induced strong proliferation of CD8+ T cells and NK cells. The difference in proliferation activity depended on the length of the linker on the IL-15-Fc side. Linker-less (hinge-only) constructs, including XENP21471, XENP21474, and XENP21475, showed weaker proliferation activity.

[0286] E. Example 1E: Activity of IL-15 / Rα-Fc Fusion Protein in SEB-Stimulated PBMC Assay As described above, the IL-15 / Rα heterodimer can potently activate T cells. Various formats of IL-15 / Rα-Fc fusion proteins, as described above, were tested in an SEB-stimulated PBMC assay. Staphylococcal enterotoxin B (SEB) is a superantigen that triggers T cell activation and proliferation, similar to that achieved by T cell receptor (TCR)-mediated activation. Stimulation of human PBMCs with SEB is a common method for assaying T cell activation and proliferation.

[0287] Human PBMCs from multiple donors were stimulated with 10 ng / mL SEB in combination with 20 μg / mL of various IL-15 / Rα-Fc fusion proteins or controls (PBS, isotype control, and bivalent anti-PD-1 antibody) for 72 hours. After treatment, supernatants were collected and assayed for IL-2, and the data are shown in Figure 22. The data clearly demonstrate that the IL-15 / Rα-Fc fusion proteins enhanced IL-2 secretion over PBS and the isotype control. Notably, several IL-15 / Rα-Fc fusion proteins enhanced IL-2 secretion compared to PBS and the isotype control. The substance has activity equivalent to or greater than that of anti-PD-1 antibodies.

[0288] F. Example 1F: IL-15 / Rα-Fc fusion protein enhances engraftment and disease activity in NSG mice transplanted with human PBMCs The IL-15 / Rα-Fc fusion protein XENP20818 was evaluated in a graft-versus-host disease (GVHD) model performed in female NSG (NOD-SCID-gamma) immunodeficient mice. When NSG mice were injected with human PBMCs, the human PBMCs elicited an autoimmune response against mouse cells. Subsequent treatment of NSG mice injected with human PBMCs with the IL-15 / Rα-Fc fusion protein enhanced the proliferation of transplanted T cells.

[0289] Ten million human PBMCs were transferred into NSG mice via IV-OSP on day 0, followed by administration of XENP20818 (1 mg / kg on day 1, then weekly) and recombinant IL-15 (Biolegend, 0.17 mg / kg on day 1, then weekly). Survival curves are shown in Figure 23. The data show that mice administered IL-15 / Rα-Fc fusion protein demonstrated rapid morbidity and mortality (all died by day 10) compared to mice administered recombinant IL-15 (all survived until day 14). This is likely due to the longer half-life predicted for the IL-15 / Rα-Fc fusion protein.

[0290] In another experiment, NSG mice were transferred with 10 million human PBMCs via IV-OSP on day 0, followed by XENP20818 (1 mg / kg, 0.3 mg / kg, 0.1 mg / kg, or 0.03 mg / kg, weekly thereafter) or PBS on day 1. A control group in which mice were not transferred with PBMCs was included to examine the effect of XENP20818 on wild-type NSG mice. Blood was collected on day 7 to measure IFNγ, the data of which are shown in Figure 24, and the number of CD4+ T cells, CD8+ T cells, and CD45+ cells, the data of which are shown in Figure 25. The data demonstrate a clear dose response to XENP20818.

[0291] XI. Example 2: IL-15 / Rα-Fc Heterodimeric Fusion Protein with Engineered Disulfide Bonds To further improve the stability and extend the half-life of the IL-15 / Rα-Fc fusion protein, a disulfide bond was engineered at the IL-15 / Rα interface.

[0292] A. Example 2A: Engineering and Analysis of IL-15 / Rα Heterodimers with Engineered Disulfide Bonds By examining the crystal structure of the IL-15 / Rα complex, and Molecular Modeling using the Chemical Computing Group Operating Environment (MOE, Montreal, Quebec, Canada) software predicted residues at the IL-15 / Rα interface that could be substituted with cysteine ​​to form a covalent disulfide bond, as shown in Figure 26.

[0293] Plasmids encoding IL-15 or IL-15Rα(sushi) were constructed by standard gene synthesis followed by subcloning into the pTT5 expression vector. The IL-15Rα(sushi) chain contained a C-terminal polyhistidine tag. Residues identified as described above were substituted with cysteines by standard mutagenesis techniques. Additionally, up to three amino acids following the sushi domain of IL-15Rα were added to the C-terminus of IL-15Rα(sushi) as a scaffold for engineered cysteines (exemplary sequences are shown in Figure 27). Exemplary cysteine-engineered IL-15 and IL-15Rα(sushi) i) The sequences of the mutants are shown in Figures 28 and 29, respectively.

[0294] Cartoon schematics of IL-15 / Rα heterodimers with and without engineered disulfides are shown in Figures 30A-C. The sequence of an exemplary ncIL-15 / Rα heterodimer, XENP21996, is shown in Figure 31. The sequences of exemplary dsIL-15 / Rα heterodimers, XENP22004, XENP22005, XENP22006, XENP22008, and XENP22494, are shown in Figure 32. The sequence of an exemplary scIL-15 / Rα heterodimer is shown in Figure 33. "Wild-type" IL-15 / Rα heterodimers with additional residues at the C-terminus but without the engineered cysteine ​​were generated as controls. The sequences of these control IL-15 / Rα heterodimers are listed in the figures and sequence listing as XENP22001, XENP22002, and XENP22003. The protein was produced by transient transfection in HEK293E cells and purified by Ni-NTA chromatography.

[0295] After the proteins were purified, generally as described in Example 1B, they were analyzed for purity and homogeneity by capillary isoelectric focusing (CEF), and the gel images are shown in Figures 34-35. The proteins were then screened for stability using DSF, generally as described in Example 1C, and the data are shown in Figures 36-38. Finally, the proteins were screened for binding to IL-2Rβ by Octet, generally as described in Example 1C, and the data are shown in Figure 38.

[0296] Many disulfide bonds were correctly formed, as shown by denatured, non-reduced CEF, where a larger molecular weight of the covalent complex could be observed when compared to a control without engineered disulfide bonds (Figures 34-35). The disulfide-linked IL-15 / Rα heterodimer had high thermal stability up to +13°C (Figure 38). Binding to IL-2Rβ was unaffected by the inclusion of engineered disulfide bonds (Figure 38). Preferred disulfide-linked pairs were XENP22005, XENP22006, XENP22008, and XENP22494, which were constructed as Fc fusion proteins as described below.

[0297] B. Example 2B: Analysis of IL-15 / Rα-Fc Fusion Proteins with Engineered Disulfide Bonds Plasmids encoding the IL-15 or IL-15Rα sushi domains with the above-mentioned mutations were subcloned into pTT5 expression vectors containing Fc fusion partners (e.g., the constant regions shown in Figure 8). Cartoon schematics of IL-15 / Rα-Fc fusion proteins with engineered disulfide bonds are shown in Figures 39A-D.

[0298] The disulfide-bonded IL-15 heterodimeric Fc fusion or "dsIL-15 / Rα-heteroFc" (Figure 39A) is identical to "IL-15 / Rα-heteroFc," but IL-15Rα (sushi) and IL-15 are additionally covalently attached as a result of engineered cysteines. Exemplary proteins of this format include XENP22013, XENP22014, XENP22015, and XENP22017, whose sequences are shown in Figure 40.

[0299] The disulfide-linked IL-15 / Rαfc fusion or "dsIL-15 / Rα-Fc" (Figure 39B) is identical to "ncIL-15 / Rα-Fc," but IL-15Rα (sushi) and IL-15 are additionally covalently attached as a result of engineered cysteines. Exemplary proteins in this format include XENP22357, XENP22358, XENP22359, XENP22684, and XENP22361, the sequences of which are shown in Figure 41. Additional proteins in this format are The sequences are listed in the figures and sequence listing as XENP22360, XENP22362, XENP22363, XENP22364, XENP22365 and XENP22366.

[0300] The bivalent disulfide-bonded IL-15 / Rα-Fc or "bivalent dsIL-15 / Rα-Fc" (Figure 39C) is identical to the "bivalent ncIL-15 / Rα-Fc," but with the additional covalent attachment of IL-15Rα (sushi) and IL-15 as a result of engineered cysteines. Exemplary proteins of this format include XENP22634, XENP22635, and XENP22636, the sequences of which are shown in Figure 42. The sequence of an additional protein of this format is listed in the figures and sequence listing as XENP22687.

[0301] The Fc-disulfide-linked IL-15 / Rα fusion or "Fc-dsIL-15 / Rα" (Figure 39D) is identical to "Fc-ncIL-15 / Rα," but with the additional covalent attachment of IL-15Rα (sushi) and IL-15 as a result of engineered cysteines. Exemplary proteins of this format include XENP22639 and XENP22640, whose sequences are shown in Figure 43.

[0302] "Wild-type" IL-15 / Rα-Fc fusion proteins with additional residues at the C-terminus but no engineered cysteine ​​were generated as controls. The sequences of these control IL-15 / Rα-Fc fusion proteins are listed in the figures and sequence listing as XENP21988, XENP21989, XENP21990, XENP21991, XENP21992, XENP22354, XENP22355, and XENP22356.

[0303] The protein was produced by transient transfection in HEK293E cells and purified by a two-step purification process including protein A chromatography (GE Healthcare) and anion exchange chromatography (HiTrapQ 5 mL column with a 5–40% gradient of 50 mM Tris pH 8.5 and 50 mM Tris pH 8.5 containing 1 M NaCl).

[0304] After the proteins were purified, generally as described in Example 1B, they were analyzed for purity and homogeneity by capillary isoelectric focusing (CEF). As noted above, many disulfide bonds were correctly formed, as shown by denaturing, non-reduced CEF, where a higher molecular weight of the covalent complex can be observed when compared to a control that does not contain engineered disulfide bonds (Figure 44).

[0305] The proteins were then tested in a cell proliferation assay. IL-15 / Rα-Fc fusion proteins (with or without engineered disulfide bonds) or a control were incubated with PBMCs for 4 days. After incubation, PBMCs were stained with anti-CD4-PerCP / Cy5.5 (RPA-T4), anti-CD8-FITC (RPA-T8), anti-CD45RA-BV510 (HI100), anti-CD16-BV421 (3G8), anti-CD56-BV421 (HCD56), anti-CD27-PE (O323), and anti-Ki67-APC (Ki-67) to mark various cell populations and analyzed by FACS, generally as described in Example 1D. The proliferation of NK cells, CD4+ T cells, and CD8+ T cells, as indicated by Ki67 expression, is shown in Figures 45A-C. The IL-15 / Rα-Fc fusion protein and the IL-15 control each induced robust proliferation of NK cells, CD8+ T cells, and CD4+ T cells.

[0306] XII. Example 3: IL-15 / Rα-Fc Fusion Protein Engineered for Lower Potency and Increased PK and Half-Life To further improve PK and extend half-life, we reasoned that decreasing the potency of IL-15 would decrease the antigen sink and, therefore, increase half-life.

[0307] A. Example 3A: Engineering and Production of Mutant IL-15 / Rα-Fc Fusion Proteins By examining the crystal structures of the IL-15:IL-2Rβ β and IL-15:common gamma chain interfaces and by modeling using MOE software, residues at these interfaces that could be substituted to reduce potency were predicted. Figure 46 shows a structural model of the IL-15:receptor complex, showing the locations of predicted residues that were engineered with equivalent substitutions (to reduce the risk of immunogenicity). The sequences of exemplary IL-15 mutants engineered to reduce potency are shown in Figure 47.

[0308] 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 Figure 8). Substitutions identified as described above were incorporated by standard mutagenesis techniques. The sequences of exemplary IL-15 / Rα-Fc fusion proteins in the "IL-15 / Rα-hetero-Fc" format engineered to reduce potency are shown in Figure 48, and additional sequences are listed in the figures and sequence listing as XENP22815, XENP22816, XENP22817, XENP22818, XENP22819, XENP22820, XENP22823, XENP22824, XENP22825, XENP22826, XENP22827, XENP22828, XENP22829, XENP22830, XENP22831, XENP22832, XENP22833, XENP22834, XENP22835, XENP22836, XENP22837, XENP22838, XENP22839, XENP22840, XENP22841, XENP22842, XENP22843, XENP22844, XENP22845, XENP22846, XENP22847, XENP22848, XENP22849, XENP22850, XENP22851, XENP22852, XENP22853, XENP22854, XENP22855, XENP22856, XENP22857, X 825, XENP22826, XENP22827, XENP22828, XENP22829, XENP22830, XENP22831, XENP22832, XENP22833, XENP22834, XENP23555, XENP23559, XENP23560, XENP24017, XENP24020, XENP24043, and XENP24048.

[0309] The sequence of an exemplary IL-15 / Rα-Fc fusion protein in the "scIL-15 / Rα-Fc" format engineered for reduced potency is shown in Figure 49, and additional sequences are listed in the figure and in the sequence listing as XENP24013, XENP24014, and XENP24016. The sequence of an exemplary IL-15 / Rα-Fc fusion protein in the "ncIL-15 / Rα-Fc" format engineered for reduced potency is shown in Figure 50. The sequence of an exemplary ncIL-15 / Rα heterodimer engineered for reduced potency is shown in Figure 51, and additional sequences are listed in the figure and in the sequence listing as XENP22791, XENP22792, XENP22793, XENP22794, XENP22795, XENP22796, XENP22803, XENP22804, XENP22805, XENP22806, XENP22807, XENP22808, XENP22809, XENP22810, XENP22811, XENP22812, XENP22813, and XENP22814. The sequence of an exemplary IL-15 / Rα-Fc fusion protein in a "bivalent ncIL-15 / Rα-Fc" format engineered for reduced potency is shown in Figure 52. The sequence of an exemplary IL-15 / Rα-Fc fusion protein in a "dsIL-15 / Rα-Fc" format engineered for reduced potency is shown in Figure 53.

[0310] The protein was produced by transient transfection in HEK293E cells and purified by a two-step purification process including protein A chromatography (GE Healthcare) and anion exchange chromatography (HiTrapQ 5 mL column with a 5–40% gradient of 50 mM Tris pH 8.5 and 50 mM Tris pH 8.5 containing 1 M NaCl).

[0311] B. Example 3B: In Vitro Activity of Mutant IL-15 / Rα-heteroFc and scIL-15 / Rα-Fc Fusion Proteins Engineered to Reduce Potency The mutant IL-15 / Rα-Fc fusion proteins were tested in several cell proliferation assays.

[0312] In the initial cell proliferation assay, IL-15 / Rα-Fc fusion proteins (with or without engineered substitutions) or controls were incubated with PBMCs for 4 days. After incubation, PBMCs were stained with anti-CD4-Evolve605 (SK-3), anti-CD8-PerCP / Cy5.5 (RPA-T8), anti-CD45RA-APC / Cy7 (HI100), anti-CD16-eFluor450 (CB16), anti-CD56-eFluor450 (TULY56), anti-CD3-FITC (OKT3), and anti-Ki67-APC (Ki-67) to mark various cell populations and analyzed by FACS generally as described in Example 1D. The proliferation of NK cells, CD8+ T cells, and CD4+ T cells, as indicated by Ki67 expression, is shown in Figures 54-55. Most IL-15 / Rα-Fc fusion proteins induced proliferation of each cell population, although activity varied depending on the specific engineered substitutions.

[0313] In a second cell proliferation assay, IL-15 / Rα-Fc fusion proteins (with or without engineered substitutions) were incubated with PBMCs for 3 days. After incubation, PBMCs were stained with anti-CD3-FITC (OKT3), anti-CD4-Evolve604 (SK-3), anti-CD8-PerCP / Cy5.5 (RPA-T8), anti-CD16-eFluor450 (CB16), anti-CD56-eFluor450 (TULY56), anti-CD27-PE (O323), anti-CD45RA-APC / Cy7 (H1100), and anti-Ki67-APC (20Raj1) antibodies to mark various cell populations. Figures 56-57 represent the selection of various cell populations after incubation with XENP22821 by FACS. Lymphocytes were first gated based on side scatter (SSC) and forward scatter (FSC) (Figure 56A). Lymphocytes were then gated based on CD3 expression (Figure 56B). Cells negative for CD3 expression were further gated based on CD16 expression to identify NK cells (CD16+) (Figure 56C). CD3+ T cells were further gated based on CD4 and CD8 expression to identify CD4+ T cells, CD8+ T cells, and γδ T cells (CD3+CD4-CD8-) (Figure 57A). CD4+ and CD8+ T cells were gated for CD45RA expression, as shown in Figures 57B-C, respectively. Finally, proliferation of various cell populations was determined based on the percentage of Ki67 expression, and the data are shown in Figures 59A-D. NK and CD8+ T cells were more sensitive to IL-15 / Rα-Fc fusion protein than CD4+ T cells, and as described above, proliferative activity varied depending on the specific engineered substitutions. Figure 59D shows the fold change in EC50 of various IL-15 / Rα-Fc fusion proteins compared to the control XENP20818. Figures 58A and B further demonstrate lymphocyte activation after treatment with IL-15 / Rα-Fc fusion proteins by gating on the expression of CD69 and CD25 (T cell activation markers) before and after incubation of PBMCs with XENP22821.

[0314] In a third experiment, additional mutant IL-15 / Rα-Fc fusion proteins were incubated with human PBMCs for 3 days at 37°C. After incubation, PBMCs were stained with anti-CD3-FITC (OKT3), anti-CD4-SB600 (SK-3), anti-CD8-PerCP / Cy5.5 (RPA-T8), anti-CD45RA-APC / Cy7 (HI100), anti-CD16-eFluor450 (CB16), anti-CD25-PE (M-A251), and anti-Ki67-APC (Ki-67) to mark various cell populations and analyzed by FACS generally as described in Example 1D. The proliferation of CD8+ (CD45RA-) T cells, CD4+ (CD45RA-) T cells, γδ T cells, and NK cells, as indicated by Ki67 expression, is shown in Figures 60A-D.

[0315] In the fourth experiment, human PBMCs were treated with the indicated concentrations of additional IL-15 / Rα-Fc variants. The PBMCs were incubated with the allogeneic antibody for 3 days. After incubation, PBMCs were stained with anti-CD3-FITC (OKT3), anti-CD4 (SB600), anti-CD8-PerCP / Cy5.5 (RPA-T8), anti-CD16-eFluor450 (CB16), anti-CD25-PE (M-A251), anti-CD45RA-APC / Cy7 (H1100), and anti-Ki67-APC (Ki67) and analyzed by FACS as generally described in Example 1D. The percentages of Ki67 on CD8+ T cells, CD4+ T cells, and NK cells after treatment are shown in Figure 61.

[0316] In a fifth experiment, mutant IL-15 / Rα-Fc fusion proteins were incubated with human PBMCs at 37°C for 3 days. After incubation, cells were stained with anti-CD3-PE (OKT3), anti-CD4-FITC (RPA-T4), anti-CD8α-BV510 (SK1), anti-CD8β-APC (2ST8.5H7), anti-CD16-BV421 (3G8), anti-CD25-PerCP / Cy5.5 (M-A251), anti-CD45RA-APC / Cy7 (HI100), anti-CD56-BV605 (NCAM16.2), and anti-Ki67-PE / Cy7 (Ki-67) and analyzed by FACS generally as described in Example 1D. The percentages of Ki67 for CD8+ T cells, CD4+ T cells, γδ T cells, and NK cells are shown in Figures 62A-E.

[0317] In the sixth experiment, mutant IL-15 / Rα-Fc fusion proteins were incubated with human PBMCs at 37°C for 3 days. After incubation, cells were stained with anti-CD3-PE (OKT3), anti-CD4-FITC (RPA-T4), anti-CD8α-BV510 (SK1), anti-CD8β-APC (SIDI8BEE), anti-CD16-BV421 (3G8), anti-CD25-PerCP / Cy5.5 (M-A251), anti-CD45RA-APC / Cy7 (HI100), anti-CD56-BV605 (NCAM16.2), and anti-Ki67-PE / Cy7 (Ki-67) and analyzed by FACS generally as described in Example 1D. The percentages of Ki67 for CD8+ T cells, CD4+ T cells, γδ T cells, and NK cells are shown in Figures 63A-E.

[0318] C. Example 3C: In vitro activity of mutant scIL-15 / Rα-Fc fusion proteins engineered to reduce potency with various linker lengths between IL-15 and IL-15Rα IL-15 / Rα-Fc fusion proteins (shown in Table 3) containing some of the above-mentioned substitutions and various lengths of linker between IL-15 and IL-15Rα were incubated with human PBMCs at the indicated concentrations for 3 days at 37°C. After incubation, PBMCs were stained with anti-CD3-PE (OKT3), anti-CD4-FITC (RPA-T4), anti-CD8-APC (RPA-T8), anti-CD16-BV605 (3G8), anti-CD25-PerCP / Cy5.5 (M-A251), anti-CD45RA-APC / Fire750 (HI100), and anti-Ki67-PE / Cy7 (Ki-67) and analyzed by FACS generally as described in Example 1D. The percentages of Ki67 for CD8+ T cells, CD4+ T cells, γδ T cells, and NK (CD16+) cells are shown in Figures 64A–D. The data show that the ncIL-15 / Rα-Fc fusion protein XENP21479 is the most potent inducer of proliferation of CD8+ T cells, CD4+ T cells, NK (CD16+) cells, and γδ T cells. Each scIL-15 / Rα-Fc fusion protein was less potent than XENP21479 in inducing proliferation, although the differences depended on both linker length and the specific engineered substitutions. [Table 3]

[0319] D. Example 3D: In Vitro Activity of Mutant IL-15 / Rα-Fc Fusion Proteins Engineered to Reduce Potency in Additional Formats Different formats of mutant IL-15 / Rα-Fc fusion proteins (as shown in Table 4) were incubated with human PBMCs at the indicated concentrations at 37°C for 3 days. After incubation, PBMCs were stained with anti-CD3-PE (OKT3), anti-CD4-FITC (RPA-T4), anti-CD8-APC (RPA-T8), anti-CD16-BV605 (3G8), anti-CD25-PerCP / Cy5.5 (M-A251), anti-CD45RA-APC / Fire750 (HI100), and anti-Ki67-PE / Cy7 (Ki-67) and analyzed by FACS generally as described in Example 1D. The percentages of Ki67 for CD8+ T cells, CD4+ T cells, γδ T cells, and NK (CD16+) cells are shown in Figures 65A-D, respectively. As mentioned above, our data demonstrate that the ncIL-15 / Rα-Fc fusion protein XENP21479 is the most potent inducer of proliferation of CD8+ T cells, CD4+ T cells, NK (CD16+) cells, and γδ T cells. Notably, introduction of the Q108E substitution into the ncIL-15 / Rα-Fc format (XENP24349) dramatically reduces its proliferative activity compared to the wild-type (XENP21479). [Table 4]

[0320] E. Example 3E: STAT5 Phosphorylation by Mutant IL-15 / Rα-Fc Fusion Proteins Transpresentation of IL-15 and IL-15Rα promotes STAT5 phosphorylation and subsequent proliferation of NK cells and T cells (CD4+ and CD8+). Therefore, CD8+ and CD4+ T cells were analyzed for STAT5 phosphorylation after 15 minutes of incubation with the indicated IL-15 / Rα-Fc test samples. PBMCs were stained with anti-CD4-BV421 (RPA-T4) and anti-CD8-A700 (SK1) at room temperature for 30–45 minutes. Cells were washed and incubated with pre-chilled (-20°C) 90% methanol for 20–60 minutes. After incubation with methanol, cells were washed again and stained with anti-CD45RA-BV510 (HI100), anti-CD27-BV605 (L128), anti-CD25-PE (M-A251), and anti-pSTAT5-Alexa647 (pY68). 7) and anti-FoxP3-Alexa488 (259D) to mark various cell populations and STAT5 phosphorylation. Figures 66A-D show the selection of various cell populations after incubation with XENP22821. Lymphocytes were first gated based on SSC and FSC (Figure 66A). Lymphocytes were then gated based on CD4 and CD8 expression to identify CD4+ and CD8+ T cells (Figure 66B). CD4+ and CD8+ T cells were then further gated based on CD45RA and CD27 expression to identify additional subpopulations, shown in Figures 66C-D, respectively. Finally, STAT5 phosphorylation in various cell populations was determined, and the data are shown in Figures 67A-C. STAT5 phosphorylation in T cells was induced in a dose-dependent manner and also varied depending on the specific engineered substitutions. Figure 67C shows the fold change in EC50 for STAT5 phosphorylation of the mutant IL-15 / Rα-Fc fusion protein compared to the control.

[0321] F. Example 3F: PK of mutant IL-15 / Rα-Fc fusion proteins engineered for reduced potency To determine whether IL-15 / Rα-Fc fusion proteins engineered to reduce potency had improved half-life and PK, we examined these variants in a PK study in C57BL / 6 mice. On day 0, two cohorts of mice (five mice per test sample per cohort) were administered 0.1 mg / kg of the indicated test sample via IV-TV. Serum was collected 60 minutes after administration, then on days 2, 4, and 7 for cohort 1 and days 1, 3, and 8 for cohort 2. Serum levels of IL-15 / Rα-Fc fusion protein were determined using anti-IL-15 and anti-IL-15Rα antibodies in a sandwich ELISA. The results are shown in Figure 68. Figure 69 shows the correlation between potency and half-life of the test samples.

[0322] As expected, the reduced potency mutants exhibited substantially longer half-lives, notably improving the half-life to approximately 9 days compared to 0.5 days for the wild-type control XENP20818 (see XENP22821 and XENP22822).

[0323] G. Example 3G: IL-15 / Rα-Fc fusion protein enhances engraftment and disease activity in NSG mice transplanted with human PBMCs The mutant IL-15 / Rα-Fc fusion proteins were evaluated in a GVHD model performed in female NSG immunodeficient mice generally as described in Example 1F.

[0324] In the first study, 10 million human PBMCs were transferred into NSG mice via IV-OSP on day 0, followed by administration of IL-15 / Rα-Fc fusion proteins at the indicated concentrations on day 1. CD45+ proliferation correlated with weight loss (as shown in Figure 70), and therefore CD45+ cells were measured on days 4 and 8 as an indicator of disease activity in this study (Figures 71A-B). The data show that each IL-15 / Rα-Fc fusion protein enhanced CD45+ cell proliferation in human PBMC-transplanted NSG mice compared to control (PBS).

[0325] In another study, 10 million human PBMCs were transferred into NSG mice via IV-OSP on day 0, followed by administration of IL-15 / Rα-Fc fusion protein at the indicated concentrations on day 1. IFNγ levels and the numbers of human NK cells, CD45+ lymphocytes, CD8+ T cells, and CD4+ T cells were measured on days 4, 7, and 11 (Figures 72-76). The data show that the mutant IL-15 / Rα-Fc fusion proteins dose-dependently enhance IFNγ secretion and proliferation of human NK cells and T cells. Notably, the observed activity correlates with the in vitro potency of each mutation.

[0326] In yet another study, NSG mice were transferred with 10 million human PBMCs via IV-OSP on day -8, followed by administration of the indicated test samples at the indicated concentrations on day 0. IFNγ levels and the numbers of human NK cells, CD45+ lymphocytes, CD8+ T cells, and CD4+ T cells were measured on days 4, 7, and 11. Figure 77 shows IFNγ levels in mouse serum on days 4, 7, and 11. Figures 78A-C show CD8+ T cell counts on days 4, 7, and 11, respectively. Figures 79A-C show CD4+ T cell counts on days 4, 7, and 11, respectively. Figures 80A-C show CD45+ cell counts on days 4, 7, and 11, respectively. Mouse body weights were also measured on days 4, 7, and 11 and are shown as a percentage of initial body weight in Figure 81.

[0327] H. Example 3H: IL-15 / Rα-Fc fusion protein is active in cynomolgus monkeys Cynomolgus monkeys were administered a single intravenous (iv) dose of XENP20818 (n=3), XENP22819 (n=1), XENP22821 (n=3), XENP22822 (n=3), XENP22834 (n=3), and XENP23343 (n=3). Lymphocyte counts (Figures 82, 84, 86, 88, 90, and 92) and proliferation (Figures 83, 85, 87, 89, 91, and 93) were assessed over time. The data show significant changes in CD56+ NK cells (Figure 86A), CD16+ NK cells (Figure 86B), γδ T cells (Figure 86C), CD8+ T cells (CD45RA+) (Figure 86D), CD8+ T cells (CD45RA-) (Figure 86E), and CD4+ T cells (Figure 86F) after treatment with XENP22821, which peaked at day 6 and then recovered and normalized. Finally, the figures show significant expression of Ki67 in CD56+ NK cells (Figure 87A), CD16+ NK cells (Figure 87B), CD8+ T cells (CD45RA+) (Figure 87C), CD8+ T cells (CD45RA-) (Figure 87D), and CD4+ T cells (Figure 87E), indicating proliferative activity after treatment with XENP22821. Similar proliferative activity was observed after treatment with XENP20818, XENP22819, XENP22822, and XENP23343, demonstrating that most IL-15 / Rα-Fc fusion proteins of the invention are active in cynomolgus monkeys.

[0328] XIII. Example 4: Xtend Fc Engineered IL-15 / Rα-Fc Fusion Protein IL-15 / Rα-Fc variants engineered to reduce potency as described above were further engineered with Xtend Fc to further increase half-life by subcloning plasmids encoding IL-15 and / or IL-15Rα(sushi) into pTT5 expression vectors (see Figure 8, framework 11) containing Fc fusion partners with M428L / N434S substitutions (referred to herein as "IL-15 / Rα-XtendFc" fusion proteins). Exemplary IL-15 / Rα-XtendFc sequences are shown in Figures 94-96 (see also Table 5). [Table 5]

[0329] A. Example 4A: In Vitro Activity of Additional IL-15 / Rα-Fc Variants Human PBMCs were incubated with the indicated concentrations of IL-15 / Rα-XtendFc variants for 3 days. After incubation, PBMCs were stained with anti-CD3-FITC (OKT3), anti-CD4-PE (RPA-T4), anti-CD8-eFluor450 (SK-1), anti-CD45RA-PE / Cy7 (HI100), anti-CD16-PerCP / Cy5.5 (3G8), anti-CD25-APC / Fire750 (M-A251), and anti-Ki67-APC (Ki-67) to mark various cell populations and analyzed by FACS generally as described in Example 1D. The proliferation of CD8+ T cells, CD4+ T cells, and NK cells, as indicated by Ki67 expression, after treatment is shown in Figure 97.

[0330] Xtend variants were selected for activity in cynomolgus monkeys, and their ability to expand cynomolgus monkey T cells was examined. Cyno PBMCs were incubated with the indicated concentrations of selected test samples for 3 days. After incubation, PBMCs were stained with anti-CD3-FITC (SP34), anti-CD4-PE / Cy7 (OKT4), anti-CD8-APC (RPA-T8), anti-CD45RA-APC / Fire750 (HI100), anti-CD16-BV605 (3G8), anti-CD25-BV421 (M-A251), and anti-Ki67-PerCP / Cy5.5 (Ki-67) to mark various cell populations and analyzed by FACS, generally as described in Example 1D. The proliferation of CD8+ T cells, CD4+ T cells, and NK cells after treatment, as indicated by Ki67 expression, is shown in Figure 98.

[0331] B. Example 4B: In Vivo Activity of IL-15 / Rα-XtendFc Variants in a GVHD Model Ten million human PBMCs were transferred into NSG mice via IV-OSP on day -7, followed by administration of the indicated test sample (0.3 mg / kg) on ​​day 0. Whole blood was collected on days 4 and 7, and mice were sacrificed for spleens on days 5-8 or 11 to measure the number of CD4+ T cells, CD8+ T cells, and CD45+ cells using FACS. Figures 99A-C show the CD4+ T cell counts in whole blood on days 4 and 7, respectively, and in the spleen on day 8. Figures 100A-C show the CD8+ T cell counts in whole blood on days 4 and 7, respectively, and in the spleen on day 8. Figures 101A-C show the CD4+ T cell counts in whole blood on days 4 and 7, respectively, and in the spleen on day 8. Mouse body weights were also measured on days -8, -2, 1, 5, 8, and 11, and are shown in Figures 102A-102F. Each dot represents one female NSG mouse.

[0332] C. Example 4C: In vivo activity of mutant IL-15 / Rα-XtendFc fusion proteins in cynomolgus monkeys Monkeys (n=3) were administered a single intravenous (iv) dose of the indicated test sample (day 1), and blood was drawn daily. The numbers of CD8+ T cells, CD4+ T cells, and NK cells in the blood were assessed over time as shown in Figures 103A-C, respectively. Each point is the average of three cynomolgus monkeys. The data show that each of the variants is active in proliferating immune cells, indicating that the IL-15 / Rα-Fc fusion proteins of the present invention may be useful as therapeutic agents for human cancer.

[0333] The above examples are provided to provide those of skill in the art with a complete disclosure and description of how to make and use embodiments of the compositions, systems, and methods of the present invention, and are not intended to limit the scope of what the inventors regard as their invention. Modifications of the above-described modes for carrying out the invention that are obvious to those of skill in the art are intended to be within the scope of the appended claims. All patents and publications mentioned herein are indicative of the level of skill of those skilled in the art to which this invention pertains. All references cited in this disclosure are incorporated by reference to the same extent as if each reference was individually incorporated by reference in its entirety.

[0334] All heading and section designations are used for clarity and reference purposes only and should not be considered limiting in any way. For example, those skilled in the art will recognize the utility of combining various aspects from different headings and sections as appropriate in accordance with the spirit and scope of the invention described herein.

[0335] All references cited in this specification are incorporated by reference herein in their entirety for all purposes to the same extent as if each individual publication or patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety for all purposes.

[0336] It will be apparent to those skilled in the art that many modifications and variations of this application can be made without departing from the spirit and scope thereof. The specific embodiments and examples described herein are offered by way of example only and should be limited only by the terms of the appended claims, along with the full scope of equivalents to which such claims are entitled.

Claims

1. a) a first fusion protein comprising an IL15Rα protein having a polypeptide sequence selected from SEQ ID NO:3 or SEQ ID NO:4 and a first Fc domain, wherein the IL15Rα protein is covalently attached to the N-terminus of the Fc domain using a first domain linker; b) a second fusion protein comprising a human interleukin-15 (IL-15) variant comprising an amino acid substitution at amino acid position N65 of SEQ ID NO:6 and further comprising one or more amino acid substitutions at amino acid positions N1, N4, D30, D61, E64 and / or Q108 of SEQ ID NO:6, and a second Fc domain, wherein the IL-15 variant is covalently attached to the N-terminus of the second Fc domain using a second domain linker, wherein the amino acid substitution is a) N1D and N65D; b) N4D and N65D; c) D30N and N65D; d) E64Q and N65D; e) N65D and Q108E; f) N1D, N4D, and N65D; g) N4D, D61N, and N65D; and h) D30N, E64Q, and N65D is selected from the group consisting of The composition, wherein the IL-15 variant is non-covalently complexed with the IL15Rα protein.

2. a) a first nucleic acid encoding the IL15Rα protein of claim 1; and b) a second nucleic acid encoding the IL-15 variant of claim 1. A nucleic acid composition comprising:

3. a) a first expression vector comprising the first nucleic acid of claim 2; and b) a second expression vector comprising the second nucleic acid of claim 2. An expression vector composition comprising:

4. A host cell comprising the nucleic acid composition of claim 2 or the expression vector composition of claim 3.

5. a) a first fusion protein comprising an IL15Rα protein having a polypeptide sequence selected from SEQ ID NO:3 or SEQ ID NO:4 and a first Fc domain, wherein the IL15Rα protein is covalently attached to the N-terminus of the Fc domain using a first domain linker; b) a second fusion protein comprising a human interleukin-15 (IL-15) variant comprising the amino acid substitutions D30N / E64Q / N65D in SEQ ID NO: 6 and a second Fc domain, wherein the IL-15 variant is covalently attached to the N-terminus of the second Fc domain using a second domain linker; The composition, wherein the IL-15 variant is non-covalently complexed with the IL15Rα protein.

6. The composition of claim 5, wherein the IL-15 variant comprises the amino acid sequence of SEQ ID NO:

383.

7. a) a first nucleic acid encoding the IL15Rα protein of claim 5; and b) a second nucleic acid encoding the IL-15 variant of claim 5. A nucleic acid composition comprising:

8. 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; An expression vector composition comprising:

9. A host cell comprising the nucleic acid composition of claim 7.

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

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