IL15 / IL15Rα heterodimer FC-fusion protein

Abstract

We provide a fusion protein engineered to address the short half-life of the IL-15 / IL-15Rα heterodimer, which can potently activate T cells. [Solution] A novel IL15 / IL15Rα heterodimer Fc fusion protein having a specific amino acid sequence is provided. Methods for producing these proteins and methods for treating patients with these proteins are also provided, along with nucleic acids, expression vectors, and host cells.

Description

Technical Field

[0001] Priority Claim This application claims the priority of U.S. Patent Application No. 62 / 408,655 filed on October 14, 2016, U.S. Patent Application No. 62 / 416,087 filed on November 1, 2016, U.S. Patent Application No. 62 / 443,465 filed on January 6, 2017, and U.S. Patent Application No. 62 / 477,926 filed on March 28, 2017, which are hereby expressly incorporated by reference in their entirety, particularly referring to the drawings, descriptions, and claims therein.

Background Art

[0002] IL-2 and IL-15 function to assist in 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, the 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, namely the IL-2 receptor alpha (IL-2Rα, CD25) or the IL-15 receptor alpha (IL-15Rα, CD215). Both cytokines are considered potentially valuable therapeutic agents in oncology, and IL-2 has been approved for use in patients with metastatic renal cell carcinoma and malignant melanoma. Currently, several clinical trials are underway, but there is no approved use of recombinant IL-15.

[0003] IL-2 presents several challenges as a therapeutic agent. First, IL-2 preferentially activates T cells that express a high-affinity receptor complex that depends on CD25 expression. Since Treg cells constitutively express CD25, they compete with effector T cells for IL-2 supply, and their activation is not favorable for oncological treatment. This imbalance leads to the concept of high-dose IL-2. However, this approach causes further 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 heterodimer complex with IL-15Rα, which is also present on the same cells. Its effect is achieved by trans-presenting the IL-15 / IL-15Rα complex to NK cells and CD8+ T cells that express IL-2Rβ and a common gamma chain.

[0005] As promising drugs, both cytokines suffer from very rapid clearance, with half-lives measured in minutes. Furthermore, IL-15 itself has low stability due to its preference for IL-15Rα-associated complexes. Recombinantly produced IL-15 / IL-15Rα heterodimers have also been shown to potently activate T cells. Nevertheless, their short half-lives hinder favorable drug delivery. This invention solves this problem by providing a novel IL15 / IL15Rα heterodimer Fc fusion protein. [Overview of the project]

[0006] Accordingly, in one embodiment, the present invention relates to a) a first fusion protein comprising a first protein domain and a first Fc domain, wherein the first protein domain is covalently bonded 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. The second protein domain provides a heterodimer protein containing a second fusion protein covalently attached to the N-terminus of the Fc domain using a second domain linker, where the first and second Fc domains are designated according to EU numbering as S267K / L368D / K370S:S267K / LS364K / E357Q, S364K / E357Q:L368D / K370S, L368 The protein has a set of amino acid substitutions selected from the group consisting of D / K370S:S364K, L368E / K370S:S364K, T411T / E360E / Q362E:D401K, L368D / K370S:S364K / E357L, and K370S:S364K / E357Q, wherein the first protein domain contains the IL15 protein and the second protein domain contains the IL15Rα protein. In some embodiments, the first protein domain is directly covalently bonded to the N-terminus of the first Fc domain without the use of a first domain linker, and / or the second protein domain is directly covalently bonded to the N-terminus of the second Fc domain without the use of a second domain linker.

[0007] In some embodiments, the heterodimer protein is (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 sequence (iv) A second fusion protein having polypeptide sequence number XX (XENP15908), (v) A first fusion protein having polypeptide sequence number XX (XENP15902) and a second fusion protein having polypeptide sequence number XX (XENP16481), (v) A first fusion protein having polypeptide sequence number XX (XENP15902) and a second fusion protein having polypeptide sequence number XX (XENP16483), (vi) A second fusion protein having polypeptide sequence number XX (XENP16479) (vii) A fusion protein having 1 and a second fusion protein having the polypeptide sequence of SEQ ID NO: XX (XENP15909), (viii) A 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), (ix) SEQ ID NO: XX (XENP16480) (x) A first fusion protein having the polypeptide sequence of 480) and a second fusion protein having the polypeptide sequence of SEQ ID NO: XX (XENP15909), (x) A first fusion protein having the polypeptide sequence of SEQ ID NO: XX (XENP17064) and a second fusion protein having the polypeptide sequence of SEQ ID NO: XX (XENP17038), (xi) A first fusion protein having the polypeptide sequence of SEQ ID NO: XX (XENP17064) and a second fusion protein having the polypeptide sequence of SEQ ID NO: XX (XENP17040),(xii) A first fusion protein (XENP17062) having the polypeptide sequence of SEQ ID NO: XX 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 the polypeptide sequence of SEQ ID NO: XX (XENP17690) and a second fusion protein having the polypeptide sequence of SEQ ID NO: XX (XENP15908), (xviii) 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), (xix) A first fusion protein having the polypeptide sequence of SEQ ID NO: XX (XENP17692) and the polypeptide sequence of SEQ ID NO: XX (XENP15908) A second fusion protein having a peptide sequence, (xx) a protein having the polypeptide sequence of the first fusion sequence number XX (XENP17693) and a second fusion protein having the polypeptide sequence of sequence number XX (XENP15908), (xxi) a first fusion protein having the polypeptide sequence of sequence number XX (XENP17694) and a second fusion protein having the polypeptide sequence of sequence number XX (XENP15908), (xxii) a first fusion protein having the polypeptide sequence of sequence number XX (XENP17695) and (xxiii) A second fusion protein having the polypeptide sequence of SEQ ID NO: XX (XENP15908), (xxiii) A first fusion protein having the polypeptide sequence of SEQ ID NO: XX (XENP17696), and a second fusion protein having the polypeptide sequence of SEQ ID NO: XX (XENP15908), (xxiv) A first fusion protein having the polypeptide sequence of SEQ ID NO: XX (17697), and a second fusion protein having the polypeptide sequence of SEQ ID NO: XX (XENP15908), (xxv) Poly (xxvi) A first fusion protein having a peptide sequence and a second fusion protein having the polypeptide sequence of SEQ ID NO: XX (XENP15908), (xxvi) A first fusion protein having the polypeptide sequence of SEQ ID NO: XX (XENP17699) and a second fusion protein having the polypeptide sequence of SEQ ID NO: XX (XENP15908), (xxvii) A first fusion protein having the polypeptide sequence of SEQ ID NO: XX (XENP17701) and a second fusion protein having the 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 sequence (xxxi) A second fusion protein having the polypeptide sequence of sequence number XX (XENP15908), a first fusion protein having the polypeptide sequence of sequence number XX (XENP17704) and a second fusion protein having the polypeptide sequence of sequence number XX (XENP15908), (xxxii) A first fusion protein having the polypeptide sequence of sequence number XX (XENP17705) and a second fusion protein having the polypeptide sequence of sequence number XX (XENP15908), (xxxiii) Sequence number The first fusion protein having the polypeptide sequence of XX(XENP18295) and the second fusion protein having the polypeptide sequence of SEQ ID NO: XX(XENP17761), the first fusion protein having the polypeptide sequence of (xxxiv)SEQ ID NO: XX(XENP18783) and the second fusion protein having the polypeptide sequence of SEQ ID NO: XX(XENP15908), the first fusion protein having the polypeptide sequence of (xxxv)SEQ ID NO: XX(XENP18784) and sequence The second fusion protein having the polypeptide sequence of number XX (XENP15908), the first fusion protein having the polypeptide sequence of sequence number XX (XENP18786) and the second fusion protein having the polypeptide sequence of sequence number XX (XENP15908), the first fusion protein having the polypeptide sequence of sequence number XX (XENP18788) and the second fusion protein having the polypeptide sequence of sequence number XX (XENP15908),(xxxviii) The first fusion protein having the polypeptide sequence of Sequence ID XX (XENP19242) and the second fusion protein having the polypeptide sequence of Sequence ID XX (XENP16481), The first fusion protein having a polypeptide sequence of (xxxix) Sequence ID XX (XENP19243) and the second fusion protein having a polypeptide sequence of Sequence ID XX (XENP16481).

[0008] In some cases, heterodimer proteins are XENP20818, XENP20819, XENP21471, XENP21472, XENP21473, XENP21474, XENP21475, XENP21476, XENP21477, XENP22013, XENP22815, XENP22816, XENP22817, XENP22818, XENP22819, XENP22820, XENP22821, XENP22822, X The selection is made from the group consisting of ENP22823, XENP22824, XENP22825, XENP22826, XENP22827, XENP22828, XENP22829, XENP22830, XENP22831, XENP22832, XENP22833, XENP22834, XENP23343, XENP23554, XENP23555, XENP23557, XENP23559, XENP24019, and XENP24020.

[0009] In a further embodiment, 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 bonded to the N-terminus of the second protein domain using a first domain linker, and the second protein domain is covalently bonded to the N-terminus of the first Fc domain using a second domain linker, and b) a heterodimer protein comprising a second Fc domain, wherein the first and second Fc domains are designated S267K according to EU numbering. The protein has a set of amino acid substitutions selected from the group consisting of / 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 contains the IL15Rα protein and the second protein domain contains the 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 heterodimer protein may be XENP21478.

[0011] In another aspect, the present invention provides a fusion protein comprising a) a first protein domain and a first Fc domain, wherein the first protein domain is covalently bonded to the N-terminus of the first Fc domain using a domain linker, b) a second Fc domain, and c) a heterodimer protein comprising a second protein domain noncovalently bonded to the first protein domain, wherein the first and second Fc domains are designated S267K / L368D / K370 according to EU numbering. The protein has a set of amino acid substitutions selected from the group consisting of S: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 contains IL15Rα and the second protein domain contains the IL15 protein.

[0012] In some embodiments, the heterodimer 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), and (ii) SEQ ID NO: XX(17034) (iii) A fusion protein having the polypeptide sequence of ) 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(17039) (vii) A fusion protein having the polypeptide sequence of 8793) and a second protein domain having the polypeptide sequence of SEQ ID NO: XX(17074), (viii) A fusion protein having the polypeptide sequence of SEQ ID NO: XX(17040), 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), (viii) A fusion protein having the polypeptide sequence of SEQ ID NO: XX(17044), polypeptide of SEQ ID NO: XX(8793) (ix) A fusion protein having a polypeptide sequence of (ix) Sequence ID XX(17044), a second Fc domain having a polypeptide sequence of Sequence ID XX(8793), and a second protein domain having a polypeptide sequence of Sequence ID XX(17072), (x) A fusion protein having a polypeptide sequence of Sequence ID XX(17075), a second Fc domain having a polypeptide sequence of Sequence ID 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 SEQ ID NO: XX(17 The protein comprises a second protein domain having the polypeptide sequence of (073), a fusion protein having the polypeptide sequence of (xiii) 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 a fusion protein having the polypeptide sequence of (xiv) SEQ ID NO: XX(15908), a second Fc domain having the polypeptide sequence of SEQ ID NO: XX(8793), and SEQ ID NO: XX(16484). The heterodimer 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 further embodiments, the present invention relates to a) a first fusion protein comprising a first protein domain and a first Fc domain, wherein the first protein domain is covalently bonded to the N-terminus of the 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 bonded to the C-terminus of the second Fc domain using a domain linker; and c) a third fusion protein noncovalently bonded to the first protein domain of the first fusion protein. The invention provides a heterodimer protein comprising a fusion protein and d) a fourth fusion protein non-covalently bonded to the second protein domain of the second fusion protein, where the first and second Fc domains are, according to EU numbering, S267K / L368D / K370S:S267K / LS364K / E357Q, S364K / E357Q:L368D / K370S, L368D / K370S:S364K, L368E / K3 It has a set of amino acid substitutions selected from the group consisting of 70S:S364K, T411T / E360E / Q362E:D401K, L368D / K370S:S364K / E357L, and K370S:S364K / E357Q, wherein the first and second protein domains contain IL15Rα protein, and the third and fourth protein domains contain IL15 protein.

[0014] In some embodiments, the heterodimer protein includes (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 heterodimer protein may be XENP21978 or XENP22634.

[0015] In a further embodiment, the present invention provides a first fusion protein comprising a) a first Fc domain and a first protein domain, wherein the first Fc domain is covalently bonded to the N-terminus of the first protein domain using a domain linker, b) a second Fc domain, and c) a heterodimer protein comprising a second protein domain noncovalently bonded to the first protein domain of the first fusion protein, wherein the first and second Fc domains are designated S267K / L368D according to EU numbering. The protein has a set of amino acid substitutions selected from the group consisting of / 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 contains the IL15Rα protein and the second protein domain contains the IL15 protein.

[0016] In some embodiments, the heterodimer 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 embodiment of the present invention, the first and / or second Fc domain may have an additional set of amino acid substitutions, including Q295E / N384D / Q418E / N421D, according to EU numbering. In some cases, the first and / or second Fc domain may have G236R / L328R, E233P / L234V / L235A / G236del / S239K, E233P / L234V / L235A / G236del / S267K, E233P / L234V / L235A / G236del / S239K / A327G, E233P / L234V / L235A / G236del / S267K / A327G It also has an additional set of amino acid substitutions consisting of E233P / L234V / L235A / G236del.

[0018] In any embodiment 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 (shortened 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 further embodiments, the present invention relates to XENP20818, XENP20819, XENP21471, XENP21472, XENP21473, XENP21474, XENP21475, XENP21476, XENP21477, XENP21478, XENP21479, XENP21978, XENP22013, XENP22015, XENP22017, and XENP223. The present invention provides heterodimer proteins selected from the group consisting of 54, XENP22355, XENP22356, XENP22357, XENP22358, XENP22359, XENP22360, XENP22361, XENP22362, XENP22363, XENP22364, XENP22365, XENP22366, XENP22637, and XENP22639. In some embodiments, the present invention relates to XENP20818, XENP20819, XENP21471, XENP21472, XENP21473, XENP21474, XENP21475, XENP21476, XENP21477, XENP22013, XENP22815, XENP22816, XENP22817, XENP22818, XENP22819, XENP22820, XENP22821, XENP22822, XENP22823, XEN We provide heterodimer proteins selected from the group consisting of P22824, XENP22825, XENP22826, XENP22827, XENP22828, XENP22829, XENP22830, XENP22831, XENP22832, XENP22833, XENP22834, XENP23343, XENP23554, XENP23555, XENP23557, XENP23559, XENP24019, and XENP24020. Methods for producing these proteins and methods for treating patients with these proteins are also provided, as are nucleic acids, expression vectors, and host cells. [Brief explanation of the drawing]

[0020] [Figure 1] The structure of the complex of IL-15 with its receptors IL-15Rα (CD215), IL-15Rβ (CD122), and a common gamma chain (CD132) is shown. [Figure 2A-2B] The sequences of IL-15 and its receptor are shown. Figure 2A shows the sequences of human IL-15, human IL-15Rα, and human IL-15Rβ. Figure 2A also shows the sequence of the human common gamma receptor. [Figure 3A-3E] Useful pairs of Fc heterodimer mutations (including skew and PI mutations) are shown. In Figures 3D and 3E, there are mutants that lack the corresponding "monomer 2" mutation, and these are pI mutants that can be used individually with either monomer. [Figure 4] A list of equivalent mutant antibody constant regions and their substitutions is shown. pI_(-) indicates a low pI mutation, and pI_(+) indicates a high pI mutation. These can be optionally and independently combined with other heterodimerizing mutations (and other types of mutations as outlined herein) of the present invention. [Figure 5] These mutations exhibit useful removal mutations that eliminate the FcγR bond (often referred to as "knockout" or "KO" mutations). Generally, removal mutations are found in both monomers, but in some cases they may be present in only one monomer. [Figures 6A-6E] Particularly useful embodiments of the "non-cytokine" components of the present invention are shown. [Figure 7] Several 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]The following are sequences of several useful IL-15 / Rα-Fc format skeletons based on human IgG1 that do not contain cytokine sequences (e.g., IL-15 and / or IL-15Rα(sushi)). Skeleton 1 is based on human IgG1 (356E / 358M allotype) and contains C220S in both strands, a skew mutation S364K / E357Q:L368D / K370S in one strand, a pI mutation Q295E / N384D / Q418E / N421D in the other, and a skew mutation L368D / K370S and an excision mutation E233P / L234V / L235A / G236del / S267K in both strands. Skeleton 2 is based on human IgG1 (356E / 358M allotype) and contains C220S in both strands, a skew mutation S364K:L368D / K370S in one strand, a pI mutation Q295E / N384D / Q418E / N421D in the other strand, and a skew mutation L368D / K370S and an excision mutation E233P / L234V / L235A / G236del / S267K in both strands. Skeleton 3 is based on human IgG1 (356E / 358M allotype) and contains C220S in both strands, a skew mutation S364K:L368E / K370S in one strand, a pI mutation Q295E / N384D / Q418E / N421D in the other strand, and a skew mutation L368E / K370S and an excision mutation E233P / L234V / L235A / G236del / S267K in both strands. Skeleton 4 is based on human IgG1 (356E / 358M allotype) and contains C220S in both strands, D401K:K360E / Q362E / T411E skew mutations and Q295E / N384D / Q418E / N421D pI mutations in both strands, and has K360E / Q362E / T411E skew mutations and E233P / L234V / L235A / G236del / S267K removal mutations in both strands. Skeleton 5 is based on human IgG1 (356D / 358L allotype) and contains C220S in both strands, a skew mutation S364K / E357Q:L368D / K370S in one strand, a pI mutation Q295E / N384D / Q418E / N421D in the other strand, and a skew mutation L368D / K370S and an excision mutation E233P / L234V / L235A / G236del / S267K in both strands.Skeleton 6 is based on human IgG1 (356E / 358M allotype) and contains C220S in both strands, the skew mutation S364K / E357Q:L368D / K370S in one strand, the pI mutation Q295E / N384D / Q418E / N421D in the other strand, the skew mutation L368D / K370S in both strands, the removal mutation E233P / L234V / L235A / G236del / S267K, and the N297A mutation in both strands. Skeleton 7 is identical to 6 except that the mutation is N297S. Alternative formats for skeletons 6 and 7 can exclude the removal mutation E233P / L234V / L235A / G236del / S267K in both strands. Skeleton 8 is based on human IgG4 and contains the S364K / E357Q:L368D / K370S skew mutation, the Q295E / N384D / Q418E / N421D pI mutation in the strand, and has the L368D / K370S skew mutation and the S228P (according to EU numbering, which is S241P in Kabat) mutation in both strands, which eliminates Fab arm exchange as is known in the art. Skeleton 9 is based on human IgG2 and contains the S364K / E357Q:L368D / K370S skew mutation and the Q295E / N384D / Q418E / N421D pI mutation in the strand, and has the L368D / K370S skew mutation. Skeleton 10 is based on human IgG2 and contains the skew mutation S364K / E357Q:L368D / K370S, the pI mutation Q295E / N384D / Q418E / N421D, the L368D / K370S skew mutation, and the S267K mutation on both strands. Skeleton 11 is identical to skeleton 1 except that it contains the Xtend mutation M428L / N434S. Skeleton 12 is based on human IgG1 (356E / 358M allotype) and contains the removal mutation C220S on both identical strands and the E233P / L234V / L235A / G236del / S267K on both identical strands.The framework 13 is based on human IgG1 (356E / 358M allotype), contains C220S in both chains, skew mutations of S364K / E357Q:L368D / K370S in one chain, pI mutations of P217R / P229R / N276K, and has skew mutations of S364K / E357Q in both chains and deletion mutations of E233P / L234V / L235A / G236del / S267K. As will be understood by those skilled in the art, as outlined below, these sequences can be used with any IL-15 and IL-15Rα (sushi) pair described herein, including but not limited to IL-15 / Rα-hetero Fc, ncIL-15 / Rα, scIL-15 / Rα, and dsIL-15 / Rα, as schematically shown in FIGS. 9A-9G and 39A-39D. Additionally, any IL-15 and / or IL-15Rα (sushi) mutations can be incorporated into these frameworks of FIGS. 8A-8E in any combination. Each of these frameworks contains sequences that are 90%, 95%, 98%, and 99% identical to the listed sequences (as defined herein), and / or (as will be understood by those skilled in the art, compared to the "parent" figure that already contains some amino acid modifications compared to the parent human IgG1 (or IgG2 or IgG4 based on the framework)), sequences containing 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions. That is, the listed frameworks can contain additional amino acid modifications (generally amino acid substitutions) in addition to the skew, pI, and deletion mutations contained within the framework of this figure (FIG. 8). [Figures 9A-9G]The present invention presents several formats of the IL-15 / Rα-Fc fusion protein. The IL-15Rα heterodimer Fc fusion, or "IL-15 / Rα-heteroFc" (Figure 9A), comprises IL-15 recombinantly fused to one side of the heterodimer Fc and IL-15Rα(sushi) recombinantly fused to the other side of the heterodimer Fc. IL-15 and IL-15Rα(sushi) may have a variable-length Gly-Ser linker between the C-terminus and N-terminus 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 the "single-chain" IL-15 / IL-15Rα(sushi) complex or "scIL-15 / Rα"), which is then fused to the N-terminus of the heterodimeric Fc region, with the other end of the molecule being "Fc only" or "empty Fc". The non-covalent 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 a non-covalent IL-15 / Rα complex, with the other end of the molecule being "Fc only" or "empty Fc". The divalent non-covalent IL-15 / Rα-Fc fusion, or "divalent 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 non-covalent IL-15 / Rα complex. The divalent single-chain IL-15 / Rα-Fc fusion, or "divalent scIL-15 / Rα-Fc" (Figure 9E), contains IL-15 fused to IL-15Rα(sushi) by a variable-length linker (referred to as the "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 non-covalent IL-15 / Rα fusion of Fc, or "Fc-ncIL-15 / Rα" (Figure 9F), contains IL-15Rα(sushi) fused to the C-terminus of the heterodimer Fc region, while IL-15 is transfected separately to form a non-covalent IL-15 / Rα complex, with the other end of the molecule being "Fc only" or "empty Fc".The Fc-single chain IL-15 / Rα fusion or "Fc-scIL-15 / Rα" (Figure 9G) 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 C-terminus of the heterodimeric Fc region, and the opposite side of the molecule is "Fc only" or "empty Fc". [Figure 10] Sequences of exemplary IL-15 / Rα-Fc fusion proteins in the "IL-15 / Rα-hetero Fc" format, XENP20818 and XENP21475, are shown, and additional sequences are 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 skilled in the art, the linker can be replaced in part by other linkers shown in Figure 7), and the slash ( / ) indicates the boundary between IL-15, IL-15Rα, the linker, and the Fc region. [Figure 11] The sequence of an exemplary IL-15 / Rα-Fc fusion protein in the "scIL-15 / Rα-Fc" format, XENP21478, is shown, and 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 skilled in the art, the linker can be replaced in part by other linkers shown in Figure 7), and the slash ( / ) indicates the boundary between IL-15, IL-15Rα, the linker, and the Fc region. [Figures 12A-12B]The sequences of XENP21479, XENP22366, and XENP24348, which are exemplary IL-15 / Rα-Fc fusion proteins in the "ncIL-15 / Rα-Fc" format, are shown. IL-15 and IL-15Rα(sushi) are underlined, the linker is double-underlined (as will be understood by those skilled in the art, the linker may be partially replaced by other linkers shown in Figure 7), and a slash ( / ) indicates the boundary between IL-15, IL-15Rα, the linker, and the Fc region. [Figure 13] The sequence of XENP21978, an exemplary IL-15 / Rα-Fc fusion protein in the "bivalent ncIL-15 / Rα-Fc" format, is shown, with further sequences 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, the linker may be partially replaced by other linkers shown in Figure 7), and a slash ( / ) indicates the boundary between IL-15, IL-15Rα, the linker, and the Fc region. [Figure 14] The sequence of an exemplary IL-15 / Rα-Fc fusion protein in the "bivalent scIL-15 / Rα-Fc" format is shown. IL-15 and IL-15Rα(sushi) are underlined, the linker is double-underlined (as will be understood by those skilled in the art, the linker may be partially replaced by other linkers shown in Figure 7), and a slash ( / ) indicates the boundary between IL-15, IL-15Rα, the linker, and the Fc region. [Figure 15] The sequence of XENP22637, an exemplary IL-15 / Rα-Fc fusion protein in the “Fc-ncIL-15 / Rα” format, is shown, with further sequences 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, the linker may be partially replaced by other linkers shown in Figure 7), and a slash ( / ) indicates the boundary between IL-15, IL-15Rα, the linker, and the Fc region. [Figure 16]The sequence of an exemplary IL-15 / Rα-Fc fusion protein in the "Fc-scIL-15 / Rα" format is shown. IL-15 and IL-15Rα(sushi) are underlined, the linker is double-underlined (as will be understood by those skilled in the art, the linker may be partially replaced by other linkers shown in Figure 7), and a slash ( / ) indicates the boundary between IL-15, IL-15Rα, the linker, and the Fc region. [Figures 17A-17E] A) The format of the IL-15 / Rα-Fc fusion protein of XENP20818, B) the purity and homogeneity of XENP20818 as determined by SEC and C) CEF, D) the affinity of XENP20818 for IL-2Rβ as determined by Octet, and E) the stability of XENP20818 as determined by DSF. [Figures 18A-18E] A) The format of the IL-15 / Rα-Fc fusion protein of XENP21478, B) the purity and homogeneity of XENP21478 as determined by SEC and C) CEF, D) the affinity of XENP21478 for IL-2Rβ as determined by Octet, and E) the stability of XENP21478 as determined by DSF. [Figures 19A-19E] A) The format of the IL-15 / Rα-Fc fusion protein of XENP21479, B) the purity and homogeneity of XENP21479 as determined by SEC and C) CEF, D) the affinity of XENP21479 for IL-2Rβ as determined by Octet, and E) the stability of XENP21479 as determined by DSF. [Figures 20A-20C] This shows the induction of proliferation of A) NK (CD56+ / CD16+) cells, B) CD4+ T cells, and C) CD8+ cells based on Ki67 expression as measured by FACS, by exemplary IL-15 / Rα-Fc fusion proteins of IL-15 / Rα-hetero-Fc formats with various linker lengths. [Figure 21A-21C]This shows the induction of proliferation of exemplary IL-15 / Rα-Fc fusion proteins in the scIL-15 / Rα-Fc format (XENP21478) and ncIL-15 / Rα-Fc format (XENP21479) of A) NK (CD56+ / CD16+) cells, B) CD4+ T cells, and C) CD8+ cells based on Ki67 expression as measured by FACS. [Figure 22] This shows enhancement of IL-2 secretion by a bivalent anti-PD-1 antibody against exemplary IL-15 / Rα-Fc fusion protein, isotype control, and PBS control in an SEB-stimulated PBMC assay. [Figure 23] The survival curves of PBMC-transplanted NSG mice after treatment with XENP20818 and recombinant IL-15 are shown. [Figure 24] This 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] The following shows the number of A) CD4+ T cells, B) CD8+ T cells, and C) CD45+ cells in the whole blood of human PBMC-transplanted NSG mice 7 days after treatment with the indicated concentrations of XENP20818. [Figure 26] A structural model of the IL-15 / Rα heterodimer is shown, illustrating the positions of the manipulated disulfide bond pairs. [Figure 27] The sequence of an exemplary IL-15Rα(sushi) mutant, which has been manipulated by additional residues at the C-terminus to serve as a scaffold for manipulating cysteine ​​residues, is shown. [Figure 28] The sequences of the cysteine-modified IL-15Rα(sushi) mutant and an exemplary IL-15 mutant modified with cysteine ​​to form a covalent disulfide bond are shown. [Figure 29] The sequences of a cysteine-modified IL-15 mutant and an exemplary IL-15Rα(sushi) mutant modified with cysteine ​​to form a covalent disulfide bond are shown. [Figures 30A-30C]This shows IL-15 / Rα heterodimers with or without an engineered disulfide bond between IL-15(sushi) and IL-15Rα. The non-covalent IL-15 / Rα heterodimer or "ncIL-15 / Rα heterodimer" (Figure 30A) contains IL-15Rα(sushi) and IL-15 that are transfected separately and are non-covalently bonded. The disulfide-bonded IL-15 / Rα heterodimer or "dsIL-15 / Rα heterodimer" (Figure 30B) contains IL-15Rα(sushi) and IL-15 that are transfected separately and are covalently bonded as a result of engineered cysteine. 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 XENP21996, an exemplary ncIL-15 / Rα heterodimer, 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 further sequences listed 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 (His×6 or HHHHHH) C-terminal tag at the C-terminus of IL-15Rα(sushi). [Figure 33]The sequence of XENP22049, an exemplary scIL-15 / Rα heterodimer, 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-15. IL-15 and IL-15Rα(sushi) are underlined, the linker is double-underlined (as will be understood by those skilled in the art, the linker may be partially replaced by other linkers shown in Figure 7), and a slash ( / ) indicates the boundary between IL-15, IL-15Rα, and the linker. [Figure 34] This shows the purity and homogeneity of exemplary IL-15 / Rα heterodimers with or without manipulated disulfide bonds, as determined by CEF. [Figure 35] This shows the purity and homogeneity of exemplary IL-15 / Rα-Fc heterodimers with or without manipulated disulfide bonds, as determined by CEF. [Figure 36] This shows the stability and melting temperature of exemplary IL-15 / Rα heterodimers with or without manipulated disulfide bonds, as indicated by the melting curve from DSF. [Figure 37] The stability and melting temperatures of exemplary IL-15 / Rα heterodimers with and without manipulated disulfide bonds are shown, as indicated by the melting curve from the 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 or without the manipulated disulfide bond are shown. Mutations are indicated in parentheses after the associated monomer. [Figures 39A-39D]Further formats of the IL-15 / Rα-Fc fusion protein of the present invention having an engineered disulfide bond are shown. The disulfide-bonded IL-15 heterodimer Fc fusion ("dsIL-15 / Rα-heteroFc") (Figure 39A) is identical to "IL-15 / Rα-heteroFc", but IL-15Rα(sushi) and IL-15 are further covalently attached as a result of the engineered cysteine. The disulfide-bonded IL-15 / Rαfc fusion ("dsIL-15 / Rα-Fc") (Figure 39B) is identical to "ncIL-15 / Rα-Fc", but IL-15Rα(sushi) and IL-15 are further covalently attached as a result of the engineered cysteine. IL-15 / Rα-Fc with a divalent disulfide bond, or "divalent dsIL-15 / Rα-Fc" (Figure 39C), is identical to "divalent ncIL-15 / Rα-Fc," but as a result of the manipulated cysteine, IL-15Rα(sushi) and IL-15 are further covalently attached. IL-15 / Rα fusion with an Fc-disulfide bond, or "Fc-dsIL-15 / Rα" (Figure 39D), is identical to "Fc-ncIL-15 / Rα," but as a result of the manipulated cysteine, IL-15Rα(sushi) and IL-15 are further covalently attached. [Figures 40A-40B] The sequences of XENP22013, XENP22014, XENP22015, and XENP22017, which are exemplary IL-15 / Rα-Fc fusion proteins in the "dsIL-15 / Rα-heteroFc" format, are shown. IL-15 and IL-15Rα(sushi) are underlined, the linker is double-underlined (as will be understood by those skilled in the art, the linker may be partially replaced by other linkers shown in Figure 7), and a slash ( / ) indicates the boundary between IL-15, IL-15Rα, the linker, and the Fc region. [Figure 41A-41B]The sequences of XENP22357, XENP22358, XENP22359, XENP22684, and XENP22361, which are exemplary IL-15 / Rα-Fc fusion proteins in the “dsIL-15 / Rα-Fc” format, are shown. Further 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, the linker may be partially replaced by other linkers shown in Figure 7), and a slash ( / ) indicates the boundary between IL-15, IL-15Rα, the linker, and the Fc region. [Figure 42] The sequences of XENP22634, XENP22635, and XENP22636, exemplary IL-15 / Rα-Fc fusion proteins in the "bivalent dsIL-15 / Rα-Fc" format, are shown. Further sequences are listed 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, the linker may be partially replaced by other linkers shown in Figure 7), and a slash ( / ) indicates the boundary between IL-15, IL-15Rα, the linker, and the Fc region. [Figure 43] The sequences of XENP22639 and XENP22640, exemplary IL-15 / Rα-Fc fusion proteins in the "Fc-dsIL-15 / Rα" format, are shown. IL-15 and IL-15Rα(sushi) are underlined, the linker is double-underlined (as will be understood by those skilled in the art, the linker may be partially replaced by other linkers shown in Figure 7), and a slash ( / ) indicates the boundary between IL-15, IL-15Rα, the linker, and the Fc region. [Figure 44] This shows the purity and homogeneity of exemplary IL-15 / Rα-Fc fusion proteins with or without manipulated disulfide bonds, as determined by CEF. [Figures 45A-45C]This shows the induction of proliferation of A) NK (CD56+ / CD16+) cells, B) CD8+ T cells, and C) CD4+ T cells based on Ki67 expression measured by FACS, by exemplary IL-15 / Rα-Fc fusion proteins with or without manipulated disulfide bonds. [Figure 46] The structures of IL-15Rα, IL-2Rβ, and IL-15 in complex with a common γ chain are shown. The positions of substitutions designed to reduce potency are indicated. [Figures 47A-47C] The sequences of exemplary IL-15 variants manipulated to reduce potency are shown. Each of these variant IL-15 sequences contains sequences that are 90%, 95%, 98%, and 99% identical (as defined herein) to the enumerated sequences and / or contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions. In non-limiting examples, the enumerated sequences may include further amino acid modifications, such as those contributing to the formation of a covalent disulfide bond, as described in Example 2. [Figures 48A-48D]The sequences of XENP22821, XENP22822, XENP23554, XENP23557, XENP23561, XENP24018, XENP24019, XENP24045, XENP24051, and XENP24052 are shown, which are exemplary IL-15 / Rα-Fc fusion proteins in the "IL-15 / Rα-R / R" format that have been manipulated for lower potency. Further sequences are listed 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, the linker may be partially replaced by 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 XENP24015, XENP24050, XENP24475, XENP24476, XENP24478, XENP24479, and XENP24481 are shown, which are exemplary IL-15 / Rα-Fc fusion proteins in the "SCIL-15 / Rα-Fc" format manipulated for lower potency. Further 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 (as will be understood by those skilled in the art, the linker may be partially replaced by other linkers shown in Figure 7), and a slash ( / ) indicates the boundary between IL-15, IL-15Rα, the linker, and the Fc region. [Figures 50A-50B]The sequences of XENP24349, XENP24890, and XENP25138, exemplary IL-15 / Rα-Fc fusion proteins in the "ncIL-15 / Rα-Fc" format manipulated for lower potency, are shown. IL-15 and IL-15Rα(sushi) are underlined, the linker is double-underlined (as will be understood by those skilled in the art, the linker may be partially replaced by other linkers shown in Figure 7), and a slash ( / ) indicates the boundary between IL-15, IL-15Rα, the linker, and the Fc region. [Figure 51] The sequences of XENP22801 and XENP22802, exemplary ncIL-15 / Rα heterodimers manipulated for reduced low potency, are shown. Further 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 (His×6 or HHHHHH) C-terminal tag at the C-terminus of IL-15Rα(sushi). [Figure 52] The sequence of XENP24342, an exemplary IL-15 / Rα-Fc fusion protein in the “bivalent ncIL-15 / Rα-Fc” format manipulated for low potency, is shown. IL-15 and IL-15Rα(sushi) are underlined, the linker is double-underlined (as will be understood by those skilled in the art, the linker may be partially replaced by other linkers shown in Figure 7), and a slash ( / ) indicates the boundary between IL-15, IL-15Rα, the linker, and the Fc region. [Figure 53]The sequences of XENP23472 and XENP23473, exemplary IL-15 / Rα-Fc fusion proteins in the "dsIL-15 / Rα-Fc" format, which has been manipulated for lower potency, are shown. IL-15 and IL-15Rα(sushi) are underlined, the linker is double-underlined (as will be understood by those skilled in the art, the linker may be partially replaced by other linkers shown in Figure 7), and a slash ( / ) indicates the boundary between IL-15, IL-15Rα, the linker, and the Fc region. [Figures 54A-54C] This shows the induction of proliferation of A) NK cells, B) CD8+(CD45RA-)T cells, and C) CD4+(CD45RA-)T cells based on Ki67 expression measured by FACS, by the mutant IL-15 / Rα-Fc fusion protein. [Figure 55] This shows the EC50 of induced proliferation of NK cells and CD8+ T cells by mutant IL-15 / Rα-Fc fusion protein, and the ratio of the decrease in EC50 compared to XENP20818. [Figures 56A-56C] Figures 59A-59D show the gating of lymphocytes and subpopulations in the experiment. Figure 56A shows the gated lymphocyte population. Figure 56B shows CD3-negative and CD3-positive subpopulations. Figure 56C shows CD16-negative and CD16-positive subpopulations of CD3-negative cells. [Figures 57A-57C] Figures 59A-59D show the gating of CD3+ lymphocyte subpopulations in the experiment. 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. [Figures 58A-58B] Figure 58B shows the expression of CD69 and CD25 in human PBMCs before (Figure 58A) and after (Figure 58B) incubation with XENP22821. [Figures 59A-59D]Figures 59A-C show cell proliferation in human PBMCs incubated with the shown mutant IL-15 / Rα-Fc fusion protein for 4 days. Figures 59A-C show the proportions 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 change in EC50 of various IL15 / IL15Rα Fc heterodimers compared to the control (XENP20818). [Figures 60A-60D] The images show cell proliferation in human PBMCs incubated with the mutant IL-15 / Rα-Fc fusion protein for 3 days. Figures 60A-C show the proportions of proliferating CD8+(CD45RA-)T cells (Figure A), CD4+(CD45RA-)T cells (Figure 60B), γδT cells (Figure 60C), and NK cells (Figure 60D). [Figure 61A-61C] The percentages of Ki67 expression in (A) CD8+ T cells, (B) CD4+ T cells, and (C) NK cells after further treatment with IL-15 / Rα mutants are shown. [Figures 62A-62E] The percentage of Ki67 expression in (A) CD8+(CD45RA-)T cells, (B) CD4+(CD45RA-)T cells, (C) γδT cells, (D) NK(CD16+CD8α-) cells, and (E) NK(CD56+CD8α-) cells after treatment with the IL-15 / Rα mutant is shown. [Figures 63A-63E] The percentage of Ki67 expression in (A) CD8+(CD45RA-)T cells, (B) CD4+(CD45RA-)T cells, (C) γδT cells, (D) NK(CD16+CD8α-) cells, and (E) NK(CD56+CD8α-) cells after treatment with the IL-15 / Rα mutant is shown. [Figures 64A-64D] 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 further manipulated IL-15 / Rα mutants with various linker lengths for reduced potency are shown. [Figures 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 further IL-15 / Rα mutants is shown. [Figures 66A-66D] Figure 67 shows the gating of lymphocytes and subpopulations in the experiment shown. Figure 66A shows the gating of the lymphocyte population. Figure 66B shows CD4+ and CD8+ T cells. Figure 66C shows subpopulations of CD4+ T cells expressing CD45RA and CD27. Figure 66D shows subpopulations of CD8+ T cells expressing CD45RA and CD27. [Figures 67A-67C] Figure 67C shows STAT5 phosphorylation in A) CD8+ T cells (CD45RA-CD27-) and B) CD4+ T cells (CD45RA-CD27-) after incubation of PBMCs with mutant IL15 / IL15Rα-Fc fusions at the indicated concentrations for 4 days. Figure 67C shows the ratio of change in EC50 for various IL15 / IL15Rα Fc heterodimers compared to the control (XENP20818). [Figure 68] This shows the pharmacokinetics (PK) of various IL-15 / RαFc fusion proteins or controls in C57BL / 6 mice after a single dose of 0.1 mg / kg, compared to IV-TV administration. [Figure 69] This shows a correlation between half-life and NK cell activity. [Figure 70] This demonstrates that CD45+ cell levels are an indicator that predicts disease. [Figure 71A-71B] A) The number of CD45+ cells on day 4 and B) on day 8 demonstrates enhanced engraftment by the mutant IL-15 / Rα-Fc fusion protein. [Figures 72A-72C] The graphs show IFNγ levels at (A)4, (B)7, and (C)11 days after treatment of NSG mice transplanted with human PBMCs using the shown mutant IL15 / Rα-Fc fusion protein or a control. [Figures 73A-73C] The (A)4, (B)7, and (C)11 day CD45+ lymphocyte counts of NSG mice transplanted with human PBMCs, after treatment with the shown mutant IL15 / Rα-Fc fusion protein or control. [Figures 74A-74C] The numbers of NK cells (CD16+CD56+CD45RA+) on days (A)4, (B)7, and (C)11 in NSG mice transplanted with human PBMCs, indicated by the IL15 / Rα-Fc fusion protein or a control, are shown. [Figures 75A-75B] The numbers of CD8+ T cells (CD8+CD45RA+) in NSG mice transplanted with human PBMCs are shown (A) 7 and (B) 11 days after treatment with the indicated IL15 / Rα-Fc fusion protein or control. [Figures 76A-76B] The numbers of CD4+ T cells (CD4+CD45RA+) in NSG mice transplanted with human PBMCs are shown (A) 7 and (B) 11 days after treatment with the indicated IL15 / Rα-Fc fusion protein or control. [Figure 77] Further mutant IL-15 / Rα-Fc fusion proteins are used to show serum IFNγ levels at days 4, 7, and 11 in mice transplanted with huPBMCs. [Figures 78A-78C] A) shows the number of CD8+ T cells in the whole blood of mice transplanted with huPBMCs using further mutant IL-15 / Rα-Fc fusion proteins on days 4, 7, and 11. [Figures 79A-79C] A) shows the number of CD4+ T cells in whole blood of mice transplanted with huPBMCs after treatment with further mutant IL-15 / Rα-Fc fusion proteins on days 4, 7, and 11. [Figures 80A-80C] A) shows the number of CD45+ cells in whole blood of mice transplanted with huPBMCs after treatment with further mutant IL-15 / Rα-Fc fusion proteins on days 4, 7, and 11. [Figure 81A-81C] A) shows body weight as a percentage of initial body weight of mice transplanted with huPBMCs at days 4, 7, and 11 after further treatment with IL-15 / Rα mutants. Each point represents a single NSG mouse. Mice whose body weight fell below 70% of their initial body weight were euthanized. Mice that died are represented as 70%. [Figures 82A-82E]Figures 82A-E show the lymphocyte counts in cynomolgus monkeys after administration of XENP20818. Figures 82A-82E show the magnification changes in the absolute number 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 83E 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. [Figures 84A-84E] Figures 84A-E show the lymphocyte counts in cynomolgus monkeys after administration of XENP22819. Figures 84A-84E show the magnification changes in the absolute number 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 85E shows the 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 the lymphocyte counts in cynomolgus monkeys after administration of XENP22821. Figures 86A-E show the magnification changes in the absolute number 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 87E shows the 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 the lymphocyte counts in cynomolgus monkeys after administration of XENP22822. Figures 88A-E show the magnification changes in the absolute number 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 89E shows the 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 the lymphocyte counts in cynomolgus monkeys after administration of XENP22834. Figures 90A-90E show the magnification changes in the absolute number 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. [Figure 91A-91E] Figure 91E 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 the lymphocyte counts in cynomolgus monkeys after administration of XENP23343. Figures 92A-92E show the magnification changes in the absolute number 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 93E 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. [Figure 94A-94D] The sequences of XENP23343, XENP23504, XENP24113, XENP24301, XENP24306, and XENP24341 are exemplary IL-15 / Rα-Fc fusion proteins in the “IL-15 / Rα-heteroFc” format with the M428L / N434S substitution. IL-15 and IL-15Rα(sushi) are underlined, the linker is double-underlined (as will be understood by those skilled in the art, the linker may be partially replaced by other linkers shown in Figure 7), and a slash ( / ) indicates the boundary between IL-15, IL-15Rα, the linker, and the Fc region. Figure 94D shows the sequence of XENP25938, an exemplary IL-15 / Rα-Fc fusion protein in the "scIL-15 / Rα-Fc" format with the M428L / N434S substitution. [Figure 95] The sequence of XENP24383, an exemplary IL-15 / Rα-Fc fusion protein in the “ncIL-15 / Rα-Fc” format with the M428L / N434S substitution, is shown. IL-15 and IL-15Rα(sushi) are underlined, the linker is double-underlined (as will be understood by those skilled in the art, the linker may be partially replaced by other linkers shown in Figure 7), and a slash ( / ) indicates the boundary between IL-15, IL-15Rα, the linker, and the Fc region. [Figure 96]The sequences of XENP24346 and XENP24351, exemplary IL-15 / Rα-Fc fusion proteins in the "bivalent ncIL-15 / Rα-Fc" format with the M428L / N434S substitution, are shown. IL-15 and IL-15Rα(sushi) are underlined, the linker is double-underlined (as will be understood by those skilled in the art, the linker may be partially replaced by other linkers shown in Figure 7), and a slash ( / ) indicates the boundary between IL-15, IL-15Rα, the linker, and the Fc region. [Figures 97A-97C] The percentage 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 containing the M428L / N434S Fc mutation is shown. [Figures 98A-98C] The percentages 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 containing the M428L / N434S Fc mutation are shown. [Figures 99A-99C] The images show (A) the number of CD4+ T cells in whole blood on day 4 and (B) day 7, and (C) the number of CD4+ T cells in the spleen on day 8, after treatment of mice transplanted with huPBMCs using a further mutant IL-15 / Rα-Fc fusion protein. [Figures 100A-100C] The following images show the number of CD8+ T cells in the whole blood (A) on day 4 and (B) on day 7, and the number of CD8+ T cells in the spleen (C) on day 8, after treatment of mice transplanted with huPBMCs using a further mutant IL-15 / Rα-Fc fusion protein. [Figure 101A-101C] The following images show the number of CD8+ T cells in the whole blood (A) on day 4 and (B) on day 7, and the number of CD8+ T cells in the spleen (C) on day 8, after treatment of mice transplanted with huPBMCs using a further mutant IL-15 / Rα-Fc fusion protein. [Figure 102A-102E]Figure 102F shows body weight as a percentage of initial body weight in mice transplanted with huPBMCs at (A)-2, (B)1, (C)5, (D)8, and (E)11 days after further treatment with the IL-15 / Rα mutant. Each point represents a single NSG mouse. Figure 102F shows the time course of body weight in mice transplanted with huPBMCs after treatment with the IL-15 / Rα mutant. [Figure 103A-103C] The numbers of (A) CD8+ T cells, (B) CD4+ T cells, and (C) NK cells in cynomolgus monkeys after treatment with the IL-15 / Rα mutant on day 1 are shown. [Figure 104A-Z.104AA-AZ.104BA-BL] The sequence of the present invention is shown. CDR is shown in bold, IL-15 and IL15-Rα(sushi) are underlined, the linker is double underlined, and a slash ( / ) is between IL-15, IL15-Rα(sushi), the linker, and the Fc domain. [Figure 105] The following are some preferred embodiments of the present invention. The "Xtend" version contains the 428L / 434S mutation in the Fc domain of each monomer. [Figure 106] This lists manipulated heterodimer skew (e.g., "stereoheterodimization") Fc mutations that have heterodimer yield (determined by HPLC-CIEX) and thermal stability (determined by DSC). Undetermined thermal stability is indicated by "nd". [Modes for carrying out the invention]

[0021] I. Definition To facilitate a more complete understanding of the present invention, several definitions are provided below. These definitions are intended to encompass grammatical equivalents.

[0022] In this specification, “excision” means reduction or removal of activity. Therefore, for example, “removal of FcγR linkage” means that the Fc region amino acid mutation has less than 50% of the starting linkage compared to an Fc region without that particular mutation, with a preferred loss of activity of less than 70-80-90-95-98%, and generally, the activity is below the level detectable by the Biacore assay. Particularly useful FcγR linkage removals are shown in Figure 86. However, unless otherwise specified, 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 a linked antibody on target cells, subsequently causing lysis of the target cells. ADCC correlates with linkage to FcγRIIIa, and increased linkage 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 response in which nonspecific cytotoxic cells expressing FcγR recognize a linked antibody on a target cell, subsequently triggering phagocytosis of the target cell.

[0025] In this specification, “modification” means the substitution, insertion, and / or deletion of amino acids in a polypeptide sequence, or an alteration of a portion chemically linked to a protein. For example, the modification may be an altered carbohydrate or PEG structure linked to a protein. In this specification, “amino acid modification” means the substitution, insertion, and / or deletion of amino acids in a polypeptide sequence. For clarity, unless otherwise stated, amino acid modification always refers to amino acids encoded by DNA, such as the 20 amino acids that have codons in DNA and RNA.

[0026] In this specification, “amino acid substitution” or “substitution” means replacing an amino acid at a specific position in the parent polypeptide sequence with a different amino acid. In particular, in some embodiments, the substitution is for an amino acid that does not naturally exist at a particular position (either not naturally present in an organism or not present in any organism). For example, substitution E272Y refers to a mutant polypeptide in which glutamic acid at position 272 is replaced with tyrosine, in this case the Fc mutant. To clarify, a protein that has been manipulated to alter the nucleic acid coding sequence but not the starting amino acid (for example, replacing CGG (which codes for arginine) with CGA (which still codes for arginine) to increase the expression level in a host organism) is not an “amino acid substitution.” That is, despite the creation of a new gene that codes for the same protein, if the protein has the same amino acid at the specific position where it starts, it is not an amino acid substitution.

[0027] When used herein, "amino acid insertion" or "insertion" means the addition of an amino acid sequence to a specific position in a parent polypeptide sequence. For example, -233E or 233E indicates an insertion of glutamic acid after position 233 and before position 234. Furthermore, -233ADE or A233ADE indicates an insertion of AlaAspGlu after position 233 and before position 234.

[0028] When used herein, "amino acid deletion" or "deletion" means the removal of an amino acid sequence at a specific position in the parent polypeptide sequence. For example, E233- or E233#, E233() or E233del indicate a deletion of glutamic acid at position 233. Furthermore, EDA233- or EDA233# indicates a deletion of the sequence GluAspAla beginning at position 233.

[0029] As used herein, “variant protein,” “protein variant,” or “variant” means a protein that differs from that of a parent protein based on at least one amino acid modification. A protein variant may refer to the protein itself, a composition containing the protein, or the amino 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 compared to the parent, preferably about 1 to about 5 amino acid modifications. In some embodiments, as described below, the parent polypeptide, e.g., Fc parent polypeptide, is a human wild-type sequence such as an Fc region derived from IgG1, IgG2, IgG3, or IgG4, but a mutated human sequence can also serve as the “parent polypeptide,” e.g., an IgG1 / 2 hybrid. The sequences of protein variants herein preferably have at least about 80% identity with the parent protein sequence, most preferably at least about 90% identity, and more preferably at least about 95-98-99% identity. A variant protein may refer to the variant protein itself, a composition containing the protein variant, or the DNA sequence encoding it.

[0030] Therefore, as used herein, “antibody variant” or “variant antibody” means an antibody that differs from the parent antibody based on at least one amino acid modification; “IgG variant” or “variant IgG” means an antibody that differs from the parent IgG (again, often derived from human IgG) based on at least one amino acid modification; and “immunoglobulin variant” or “variant immunoglobulin” means an immunoglobulin sequence that differs from that of the parent immunoglobulin sequence based on at least one amino acid modification. As used herein, “Fc variant” or “variant Fc” means a protein that contains amino acid modifications in its Fc domain. The Fc variants of the present invention are defined according to the amino acid modifications that constitute them. For example, N434S or 434S is an Fc mutation having a substituted serine at position 434 relative to the parent Fc polypeptide, where the numbering follows the EU index. Similarly, M428L / N434S defines an Fc mutation having substitutions M428L and N434S relative to the parent Fc polypeptide. The identity of the WT amino acid does not need to be specified, in which case the aforementioned mutation is called 428L / 434S. The order in which substitutions are provided is arbitrary, i.e., 428L / 434S is the same Fc mutation as M428L / N434S, for example. For all positions discussed in this invention relating to antibodies, unless otherwise specified, the numbering of amino acid positions follows the EU index. The EU index, or an EU index similar to Kabat or the EU numbering scheme, refers to the numbering of EU antibodies (Edelman et al., 1969, Proc Natl Acad Sci USA 63:78-85, the whole of which is incorporated herein by reference). Modifications may be additions, deletions, or substitutions. Substitutions may include naturally occurring amino acids and, in some cases, synthetic amino acids.Examples include U.S. Patent Nos. 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, This includes & PG Schultz, (2002), Chem. 1-10, which are incorporated in their entirety by reference.

[0031] As used herein, “protein” means at least two covalently bonded amino acids, including proteins, polypeptides, oligopeptides, and peptides. The peptidyl group is a naturally occurring amino acid and peptide bond, or a synthetic peptide-mimicking structure, i.e., a peptoid (the whole is incorporated by reference by Simon This may include “analogs” such as et al., PNAS USA 89(20):9367 (1992). The amino acids may be either naturally occurring or synthetic (e.g., not DNA-encoded amino acids), as will be understood by those skilled in the art. For example, homophenylalanine, citrulline, ornithine, and noreoleucine are considered synthetic amino acids for the purposes of this invention, and both D- and L-(R or S) stereoconfigurations of amino acids can be utilized. The variants of the present invention may include modifications involving the use of synthetic amino acids incorporated using techniques developed by Schultz and collaborators, including but not limited to the methods 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 (the entire text is incorporated by reference). Furthermore, polypeptides may include synthetic derivatization of one or more side chains or terminals, glycosylation, PEGylation, circular permutation, cyclization, linking to other molecules, fusion to proteins or protein domains, and addition of peptide tags or labels.

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

[0033] As used herein, “IgG subclass modification” or “isotype modification” means an amino acid modification that converts one amino acid of one IgG isotype to a corresponding amino acid of a different, matching IgG isotype. For example, since IgG1 contains tyrosine at EU position 296 and IgG2 contains phenylalanine, the F296Y substitution in IgG2 is considered an IgG subclass modification.

[0034] As used herein, “non-naturally occurring modification” means an amino acid modification that is not isotypic. For example, since none of the IgGs contain serine at position 434, the substitution 434S in IgG1, IgG2, IgG3, or IgG4 (or their hybrids) is considered a non-naturally occurring modification.

[0035] As used herein, “amino acid” and “amino acid identity” mean one of the 20 naturally occurring amino acids encoded by DNA and RNA.

[0036] As used herein, “effector function” refers to a biochemical event resulting from the interaction between an antibody Fc region and an Fc receptor or ligand. Effector functions include, but are not limited to, ADCC, ADCP, and CDC.

[0037] As used herein, "IgG Fc ligand" means any biologically derived molecule, preferably polypeptide, that binds to the Fc region of an IgG antibody to form an Fc / Fc ligand complex. Examples of Fc ligands include FcγRI, FcγRII, FcγRIII, and FcRn. Fc ligands include, but are not limited to, C1q, C3, mannan-binding lectins, mannose receptors, staphylococcal protein A, streptococcal protein G, and viral FcγR. Fc ligands also include Fc receptor homologs (FcRH), which are a family of Fc receptors that are homogeneous with FcγR (Davis et al., 2002, Immunological Reviews 190:123-136 (the whole text is incorporated by reference). Fc ligands may include undiscovered molecules that bind to Fc. Specific IgG Fc ligands are FcRn and Fc gamma receptors. As used herein, “Fc ligand” means 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” means any member of the protein family that binds to the Fc region of an IgG antibody and is encoded by the FcγR gene. In humans, this family includes, but is not limited to, FcγRI(CD64), which contains the isoforms FcγRIa, FcγRIb, and FcγRIc; FcγRII(CD32), which contains the isoforms FcγRIIa (including the allotypes H131 and R131), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2), and FcγRIIc; and FcγRIII(CD16), which contains the isoforms FcγRIIIa (including the allotypes V158 and F158), and FcγRIIIb (including the allotypes FcγRIIb-NA1 and FcγRIIb-NA2) (the whole is incorporated by reference Jefferis et al., 2002, Immunol Lett 82:57-65), as well as any undiscovered human FcγR or FcγR isoform or allotype. FcγR can originate from any organism, including but not limited to humans, mice, rats, rabbits, and monkeys. Mouse FcγR includes, but is 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” means a protein that ligates to the Fc region of an IgG antibody and is at least partially encoded by the FcRn gene. FcRn may be derived from any organism, including but not limited to humans, mice, rats, rabbits, and monkeys. As is known in the art, a functional FcRn protein often comprises two polypeptides, called 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 stated 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 the serum half-life. In general, unless otherwise stated, the Fc monomers of the present invention retain binding to the FcRn receptor (and may include amino acid mutations to increase binding to the FcRn receptor, as described below).

[0040] As used herein, “parent polypeptide” means a starting polypeptide that is subsequently modified to produce a variant. The parent polypeptide may be a naturally occurring polypeptide, or a variant or manipulated version of a naturally occurring polypeptide. The parent polypeptide may refer to the polypeptide itself, a composition containing the parent polypeptide, or the amino acid sequence encoding it. Therefore, as used herein, “parent immunoglobulin” means an unmodified immunoglobulin polypeptide that is modified to produce a variant, and as used herein, “parent antibody” means a starting polypeptide that is modified to produce a variant antibody. This refers to unmodified antibodies that are modified by [a specific agent]. It should be noted that "parent antibodies" include known commercially available recombinant antibodies, as outlined below.

[0041] As used herein, “Fc” or “Fc region” or “Fc domain” means a polypeptide comprising, optionally, the constant region of an antibody excluding the first constant region immunoglobulin domain (e.g., CH1) or a portion thereof, and optionally a portion of the hinge. Thus, Fc may 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 mobile hinge N-terminus 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 the immunoglobulin domains Cγ2 and Cγ3 (Cγ2 and Cγ3), as well as the lower hinge region between Cγ1 (Cγ1) and Cγ2 (Cγ2). In some embodiments, Fc refers to the shortened CH1 domain, as well as CH2 and CH3, of the immunoglobulin. Although the boundaries of the Fc region may differ, the human IgG heavy chain Fc region is typically defined as containing residues E216, C226, or P230 at its carboxyl terminus, with numbering following the EU index similar to that of Kabat. In some embodiments, amino acid modifications are made to the Fc region, for example, to alter its binding to one or more FcγR or FcRn receptors, as will be described in more detail below.

[0042] In this specification, “Fc fusion protein” or “immunoadhesin” generally means a protein containing an Fc region linked to a different protein, for example, IL-15 and / or IL-15R, as described herein, via a linker moiety (optionally 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 contains only an Fc domain (e.g., an empty Fc domain), while the other monomer is an Fc fusion containing a mutant Fc domain and a protein domain such as a receptor, ligand, or other binding partner.

[0043] As used herein, “position” means a location within a protein sequence. Positions may be numbered sequentially or according to an established format, such as the EU index for antibody numbering.

[0044] In relation to the monomers of the heterodimer antibodies of the present invention as used herein, “strandedness” means incorporating heterodimerizing mutations into each monomer so as to “match” double-stranded DNA, while retaining the ability to “match” and form heterodimers. For example, if several pI mutations are manipulated into monomer A (e.g., to increase pI), then steric mutations, which are “charge pairs” that can be similarly utilized, do not interfere with the pI mutations, and for example, the charge mutations that increase pI are placed on the same “strand” or “monomer” and retain both functions. Similarly, with “skew” mutations that form sets of pairs, as outlined in more detail below, those skilled in the art will consider pI when determining which strand or monomer will incorporate one set of pairs, and similarly use the pI of the skew to maximize pI separation.

[0045] In this specification, “wild-type” or “WT” means an amino acid or nucleotide sequence found in nature, including allelic mutations. WT proteins have an amino acid or nucleotide sequence that is not intentionally modified.

[0046] The heterodimeric proteins of the present invention are generally isolated or recombinant. When used to describe the various polypeptides disclosed herein, "isolated" "Isolated" means a polypeptide identified, isolated, and / or recovered from the cells or cell cultures on which it is expressed. Typically, isolated polypeptides are prepared by at least one purification step. "Isolated protein" refers to a protein that is substantially free of other antibodies with different antigen specificities. "Recombinant" means a protein produced in exogenous host cells using recombinant nucleic acid technology.

[0047] The “amino acid sequence identity percentage (%)” for a protein sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in a particular (parent) sequence after the sequence has been aligned to achieve the maximum possible sequence identity percentage and gaps have been introduced where necessary, and no conservative substitutions are considered as part of the sequence identity. Alignment for the purpose of determining the amino acid sequence identity percentage can be achieved in various ways within the scope of the art using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. A person skilled in the art can determine appropriate parameters for measuring alignment, including any algorithm required to achieve the maximum alignment over the entire length of the sequences being compared. One particular program is the ALIGN-2 program outlined in paragraphs

[0279] to

[0280] of U.S. Patent Application Publication 2016 / 0244525, which is incorporated herein by reference.

[0048] The degree of identity between the amino acid sequence of the present invention ("the sequence of the present invention") and the parent amino acid sequence is calculated as the number of exact matches in the consistency of 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 an identity percentage.

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

[0050] "Specific binding" to a particular antigen or epitope, or "specific to," or "specific for" a particular antigen or epitope, means a binding that is measurably different from nonspecific 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 a similarly structured molecule that generally does not possess binding activity. For example, specific binding can be determined by competition with a control molecule that is similar to the target.

[0051] Before further describing the present invention, it should be understood that the present invention is not limited to the specific embodiments described and is therefore naturally subject to change. Since the scope of the present invention is limited only by the appended claims, it should also be understood that the terms used herein are for the purpose of describing only specific embodiments and are not intended to limit them.

[0052] II. Heterodimeric Fc Fusion Proteins This invention relates to a heterodimer Fc fusion protein containing IL-15 and IL-15 receptor alpha (IL-15Rα) protein domains in different directions. The Fc domain can be derived from an IgG Fc domain, such as IgG1, IgG2, IgG3, or IgG4Fc domain, with the IgG1Fc domain being particularly used in this invention.

[0053] The carboxyl terminus of each chain defines the constant region that is primarily involved in the effector function. Kabat et al. collected numerous primary sequences of the variable regions of the heavy and light chains. Based on the degree of sequence conservation, Kabat et al. classified the individual primary sequences into CDRs and frameworks and compiled a list (see SEQUENCES OF IMMUNOLOGICAL INTEREST, 5th edition, NIH publication, No.91-3242, EA Kabat et al., which incorporates the entire list by reference). 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), while the EU numbering system is for the Fc region (see, e.g., Kabat et al., above (1991)).

[0054] The IgG subclass of immunoglobulins contains several immunoglobulin domains in the heavy chain. As used herein, “immunoglobulin (Ig) domain” means a region of immunoglobulin having a distinctly different tertiary structure. The heavy chain domains, including the constant heavy (CH) domain and hinge domains, are of interest in this invention. In relation to IgG antibodies, each IgG isotype has three CH domains. Therefore, in relation to IgG, the “CH” domains are as follows: “CH1” points to positions 118–220 according to the same EU index as Kabat; “CH2” points to positions 237–340 according to the same EU index as Kabat; and “CH3” points to positions 341–447 according to the same EU index as Kabat. As shown herein and described below, pI mutations may be present in one or more of the CH domains and, as discussed below, the hinge domains.

[0055] Another type of Ig domain in the heavy chain is the hinge region. In this specification, “hinge,” “hinge region,” “antibody hinge region,” or “immunoglobulin hinge region” means a mobile polypeptide containing amino acids between the first and second constant domains of an antibody. Structurally, the IgG CH1 domain terminates at EU position 220, and the IgG CH2 domain begins at residue EU position 237. Therefore, for IgG, the antibody hinge is defined herein as encompassing positions 221 (D221 in IgG1) through 236 (G236 in IgG1), with numbering following the EU index, as in Kabat. In some embodiments, a lower hinge is included in relation to the Fc region, “lower hinge” generally refers to position 226 or 230. As described herein, pI mutations can also be produced in the hinge region.

[0056] Accordingly, 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, Fc domains, CH1 domains, CH2 domains, CH3 domains, hinge domains, and heavy chain constant domains (CH1-hinge-Fc domain or CH1-hinge-CH2-CH3).

[0057] Therefore, the “Fc domain” includes the -CH2-CH3 domain and, optionally, the hinge domain. In some embodiments, the Fc domain also includes a shortened CH1 domain. In embodiments herein, when a protein fragment, e.g., IL-15 or IL-15Rα, is bound to the Fc domain, it is the C-terminus of the IL-15 or IL-15Rα construct that is bound to all or part of the hinge of the Fc domain, for example, it is generally bound to the sequence EPKSS, which is the beginning of the hinge. In other embodiments, when a protein fragment, e.g., IL-15 or IL-15Rα, is bound to the Fc domain, it is the C-terminus of the IL-15 or IL-15Rα construct that is bound to the CH1 domain of the Fc domain.

[0058] In some of the Fc domain protein constructs and sequences outlined herein, the C-terminus of IL-15 or IL-15Rα is bound to the N-terminus of a domain linker, and its C-terminus is bound 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 switched (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 optionally bound to the N-terminus of a second protein fragment via a domain linker, and the C-terminus of the second protein fragment is optionally bound to the N-terminus of a constant Fc domain via a domain linker. In yet another construct and sequence outlined herein, a constant Fc domain not bound to the first or second protein fragment is provided. A heterodimeric Fc fusion protein may comprise two or more exemplary monomeric Fc domain proteins described herein.

[0059] In some embodiments, the linker is a “domain linker” used to link any two domains outlined herein together, some of which are shown in Figure 87. While any suitable linker can be used, many embodiments utilize glycine-serine polymers, 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 enables recombination of two domains having sufficient length and flexibility so that each domain retains its biological function. In some cases, it is a charged domain linker, with attention paid to the “twist” as outlined below.

[0060] In one embodiment, the heterodimer Fc fusion protein comprises at least two constant domains that can be manipulated to generate a heterodimer, such as through pI manipulation. Other Fc domains that may be used include fragments comprising one or more of the CH1, CH2, CH3, and hinge domains of the present invention that have been pI-manipulated. In particular, the formats shown in Figures 9A-9G and 39A-39D are heterodimer Fc fusion proteins, meaning that this protein has two bound Fc sequences and at least one protein fragment (e.g., 1, 2, or more protein fragments) that self-assemble into a heterodimer Fc domain. In some cases, the first protein fragment is ligated to the first Fc sequence, and the second protein fragment is ligated to the second Fc sequence. In other cases, the first protein fragment is ligated to the first Fc sequence, and the first protein fragment is non-covalently attached to the second protein fragment that is not ligated to the Fc sequence. In some cases, a heterodimeric Fc fusion protein includes a first protein fragment linked to a second protein fragment linked to a first Fc sequence, and a second Fc sequence that is not linked to either the first or second protein fragment.

[0061] Therefore, in some embodiments, the present invention provides a heterodimer Fc fusion protein that relies on the use of two different heavy chain mutant Fc sequences to self-assemble to form a heterodimer Fc domain fusion polypeptide.

[0062] The present invention relates to a novel construct for providing a heterodimer Fc fusion protein that enables binding to one or more binding partners, ligands, or receptors. The heterodimer Fc fusion construct is based on the self-assembly properties of two "monomers" that assemble into, for example, a "dimer," two Fc domains of an antibody heavy chain. Heterodimer Fc fusions are made by altering the amino acid sequence of each monomer, as will be fully discussed below. Thus, the present invention generally relates to several binding partners or ligands or receptors, depending on amino acid mutations in different constant regions on each chain, in order to promote heterodimer formation and / or to facilitate the purification of heterodimers more easily than homodimers. This relates to the creation of heterodimeric Fc fusion proteins that can be co-bonded using the method described above.

[0063] Many mechanisms exist 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 "heterodimerizing mutations." As will be discussed later, heterodimerizing mutations may include stereomutations (e.g., the "knob-and-hole" or "skew" mutations and "charge pair" mutations described below) and "pI mutations" that enable the purification of homodimers from heterodimers. The entirety of which is incorporated herein by reference, specifically as is incorporated herein by reference below in the discussion of “heterodimerizing mutations,” useful mechanisms of heterodimerization include “knob and hole” (“KIH”), often referred herein as “skew” mutations (see discussion in WO2014 / 145806), “electrostatic steering” or “charge pair” as described in WO2014 / 145806, pI mutations as described in WO2014 / 145806, and more general Fc mutations as outlined in WO2014 / 145806 and below.

[0064] In the present invention, several fundamental mechanisms exist that can facilitate the purification of heterodimer antibodies, one of which relies on the use of pI mutations, which enable isoelectric focusing of AA, AB, and BB dimer proteins by having different pIs for each monomer. Alternatively, several formats also allow for separation based on size. It is also possible to "skew" the formation of heterodimers rather than homodimers, as will be further outlined below. Therefore, combinations of steric heterodimerization mutations and pI or charge-pair mutations are particularly used in the present invention.

[0065] Generally, embodiments particularly used in the present invention rely on a set of mutations that include skew mutations that promote heterodimerization over homodimerization, combined with pI mutations that increase the pI difference between two monomers.

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

[0067] In this invention, which utilizes pI as a separation mechanism to enable the purification of heterodimeric proteins, amino acid mutations can be introduced into one or both monomer polypeptides; that is, the pI of one monomer (hereinafter referred to as "monomer A" for simplicity) can be manipulated to be different from that of monomer B, or the changes in both monomers A and B can be modified to increase the pI of monomer A and decrease the pI of monomer B. As will be discussed, pI changes in either or both monomers can be carried out by removing or adding charged residues (e.g., substituting a neutral amino acid with a positively or negatively charged amino acid residue, e.g., glycine to glutamic acid), by changing charged residues from positive or negative to the opposite charge (e.g., aspartic acid to lysine), or by changing charged residues to neutral residues (e.g., loss of charge, lysine to serine). Some of these mutations are shown in the figure.

[0068] Therefore, this embodiment of the present invention provides causing a sufficient change in pI in at least one monomer so that the heterodimer can be separated from the homodimer. As will be understood by those skilled in the art, and as will be discussed further below, this is the "wild type" This can be done by using a heavy chain constant region and a mutant region that has been manipulated 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] Therefore, generally, components of some embodiments of the present invention are amino acid mutations in a constant region aimed at altering the isoelectric point (pI) of at least one, if not both, monomers of a dimeric protein by incorporating an amino acid substitution ("pI mutation" or "pI substitution") into one or both monomers. As shown herein, the separation of heterodimers from two homodimers can be achieved when the pIs of the two monomers differ by a small amount of 0.1 pH units, and differences of 0.2, 0.3, 0.4, and 0.5 or more are all used in the present invention.

[0070] As those skilled in the art will understand, the number of pI mutations to be present in each monomer or both monomers to obtain good separation will depend in part on the starting pIs of the constituents. As is known in the art, different Fc will have different starting pIs to be utilized in this invention. Generally, as outlined herein, the pIs are manipulated to produce a difference of at least about 0.1 log in the total pI of each monomer, and preferably 0.2 to 0.5 as outlined herein.

[0071] As those skilled in the art will understand, the number of pI mutations to be present in each monomer or both monomers to obtain good separation will depend in part on the starting pIs of the constituents. That is, the sequences of the Fc domains, and in some cases the protein domains linked to the Fc domains, are calculated and the decision is made therefrom in order to determine which monomers to manipulate or in which "direction" (e.g., more positive or more negative). As 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, the pIs are manipulated to produce a difference of at least about 0.1 log in the total pI of each monomer, and preferably 0.2 to 0.5 as outlined herein.

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

[0073] When heterodimerization is achieved using pI mutations by utilizing the constant region of the Fc domain, a more modular approach is provided for designing and purifying heterodimerized Fc fusion proteins. Therefore, in some embodiments, heterodimerizing mutations (including skewed and purified heterodimerizing mutations) must be manipulated. Furthermore, in some embodiments, the potential for immunogenicity arising from pI mutations is significantly reduced by introducing pI mutations from different IgG isotypes so that pI is altered without introducing significant immunogenicity. Therefore, a further problem to be solved is the elucidation of the constant domain of low pI with a high human sequence content, e.g., minimizing or avoiding non-human residues at any particular position.

[0074] The secondary benefits that may result from this pI manipulation are also the extension of serum half-life and increased FcRn ligation. Specifically, as described in USSN 13 / 194,904 (which is incorporated herein by reference in its entirety), reducing the pI of the antibody constant domain (including those found in antibodies and Fc fusions) can result in longer serum retention in vivo. These pI mutations for extending serum half-life also facilitate pI changes for purification.

[0075] Furthermore, the pI mutation in heterodimerization mutations offers significant advantages to the analysis and quality control processes of Fc fusion proteins because of its remarkable ability to exclude, minimize, and distinguish homodimers when present. Similarly, the ability to reliably test the reproducibility of heterodimerized Fc fusion protein production is crucial.

[0076] A. Heterodimerative mutations The present invention provides heterodimer proteins, including heterodimer Fc fusion proteins in various formats, that utilize heterodimerization mutations to enable heterodimerization and / or purification from homodimers. Heterodimer fusion constructs are based on the self-assembly properties of two "monomers" that are assembled into, for example, a "dimer" from two Fc domains.

[0077] There are several appropriate pairs of sets of heterodimerizing skew mutations. These mutations form "pairs" of "sets," that is, a pair from one set is incorporated into the first monomer, and a pair from another set is incorporated into the second monomer. It is noteworthy that these sets do not necessarily behave as "knob-in-hole" mutations with a one-to-one correspondence between residues on one monomer and residues on the other monomer; rather, these pairs of sets form an interface between two monomers that promotes heterodimerization and hinders homodimerization, resulting in a rate of heterodimers spontaneously formed under biological conditions of over 90% (25% homodimer A / A: 50% heterodimer A / B: 25% homodimer B / B) rather than the expected 50%.

[0078] B. Stereomorphism In some embodiments, heterodimer formation can be facilitated by the addition of stereovariates. That is, by altering the amino acids in each heavy chain, different heavy chains are more likely to associate to form a heterodimer structure than to form a homodimer with the same Fc amino acid sequence. Appropriate stereovariates are included in Figure 29 of USSN 15 / 141,350, all of which are incorporated herein in their entirety by reference and are also included in Figure 84.

[0079] One mechanism, commonly referred to in the art as "knob and hole," refers to amino acid manipulations that produce steric effects favoring heterodimerization and unfavoring homodimerization, and can be used selectively; this is often referred to as "knob and hole" and is 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 entirety. These figures identify several "monomer A-monomer B" pairs that depend on "knob and hole." Furthermore, as described in Merchant et al., Nature Biotech. 16:677 (1998), these "knob and hole" mutations can be combined with disulfide linkage for skew formation against heterodimerization.

[0080] Further mechanisms used for the formation of heterodimers, as described by reference in Gunasekaran et al., J. Biol. Chem. 285(25):19637(2010), are often referred to as “electrostatic steering.” This is often referred to herein as “charge pairing.” In this embodiment, electrostatics is used to skew the formation toward heterodimerization. As will be understood by those skilled in the art, these may also affect pI, and therefore purification. Therefore, in some cases, they can be considered pI mutations. However, since these were generated to force heterodimerization and were not used as a means of purification, they are classified as "stereovariations." These include, but are not limited to, D221E / P228E / L368E paired with D221R / P228R / K409R (for example, these are "monomer correspondence sets"), and C220E / P228E / 368E paired with C220R / E224R / P228R / K409R.

[0081] Further monomer A and monomer B mutations can be independently and arbitrarily 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 stereovariates outlined herein can be optionally and independently incorporated into one or both monomers any pI mutation (or other mutations such as Fc mutations, FcRn mutations, etc.) and can be optionally included in or excluded from the proteins of the present invention.

[0083] A list of suitable scubarians is shown in Figure 84. Particularly useful in many embodiments are pairs of sets 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 including the cross-linked disulfide T366S / L368A / Y407V / Y349C:T366W / S354C). Regarding 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 "degree of twist" of these pairs depends on the starting pI.

[0084] C. Heterodactyl heterodimer pI (isoelectric point) mutations Generally, as those skilled in the art will understand, there are two common 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 are possible: one monomer may be wild-type or a mutation that does not show a significantly different pI from the wild type, while the other may be either more basic or more acidic. Alternatively, each monomer may change, one becoming more basic and the other more acidic.

[0085] Preferred combinations of pI variants are shown in Figure 30 of USSN15 / 141,350, all of which are incorporated herein by reference in their entirety. While these modifications are shown in comparison to IgG1, as outlined herein and shown in the figure, all isotypes can be modified in this manner, as can isotype hybrids. R133E and R133Q may also be used when the heavy chain constant domain is derived from IgG2-4.

[0086] In one embodiment, when one of the Fc monomers contains a CH1 domain, a preferred combination of pI mutations is having one monomer containing the 208D / 295E / 384D / 418E / 421D mutation (N208D / Q295E / N384D / Q418E / N421D when compared to human IgG1). In some cases, the second monomer contains a positively charged domain linker containing (GKPGS)4. In some cases, the first monomer contains a CH1 domain including position 208. Thus, constructs that do not contain a CH1 domain (e.g., heterodimer Fc fusion proteins that do not utilize a CH1 domain in one of their domains) are also possible. In this case, the preferred negative pI mutation Fc set includes the 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. It should be noted that changes at 233–236 may be made to increase effector function (along with 327A) in the IgG2 skeleton. Thus, pI mutations, particularly substitutions, can be made at one or more of positions 221–225 by the 1, 2, 3, 4, or 5 mutations used in this invention. Similarly, all possible combinations are intended to be used 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, deletions at position 221, non-natural valine or threonine at position 222, deletion at position 223, non-natural glutamate at position 224, deletion at position 225, deletion at position 235, and deletion or non-natural alanine at position 236. In some cases, only pI substitutions occur in the hinge domain, while in others, these substitutions are added in any combination to other pI mutations in other domains.

[0089] In some embodiments, mutations including positions 274, 296, 300, 309, 320, 322, 326, 327, 334, and 339 can be introduced within the CH2 region. Similarly, all possible combinations of these 10 positions can be made, and for example, a pI antibody may 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 glutamate at position 274, unnatural phenylalanine at position 296, unnatural phenylalanine at position 300, unnatural valine at position 309, unnatural glutamate at position 320, unnatural glutamate at position 322, unnatural glutamate at position 326, unnatural glycine at position 327, natural glutamate at position 334, unnatural threonine at position 339, and all possible combinations within CH2 and with other domains.

[0091] In this embodiment, mutations can 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, unnatural glutamine or glutamate at position 355, unnatural serine at position 384, unnatural asparagine or glutamate at position 392, unnatural methionine at position 397, unnatural glutamate at position 419, unnatural glutamate at position 359, unnatural glutamate at position 362, unnatural glutamate at position 389, unnatural glutamate at position 418, unnatural glutamate at position 444, and deletion or unnatural aspartate at position 447.

[0092] D. Isotype variation Furthermore, many embodiments of the present invention rely on the “transfer” 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 mutant. Some of these are shown in Figure 21 of U.S. Patent Application Publication 2014 / 0370013, which is incorporated herein by reference. That is, 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). This results in an increased serum half-life and a longer serum half-life. For example, IgG1 has glycine at position 137 (pI 5.97), and IgG2 has glutamic acid (pI 3.22). Transposing 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 mutant Fc fusion proteins. However, it should be noted that even changes within the IgG2 molecule can lead to an increase in serum half-life, as will be discussed below.

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

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

[0095] Calculation of E.pI The pI of each monomer may depend on the pI of the mutant heavy chain constant domain as well as the pI of the entire monomer, including the mutant heavy chain constant domain and its fusion partner. Therefore, in some embodiments, the change in pI is calculated based on the mutant heavy chain constant domain using the chart in Figure 19 of U.S. Patent Application Publication 2014 / 0370013. As discussed herein, the choice of which monomer to work with is generally determined by the intrinsic pI of each monomer.

[0096] F.pI mutations also confer favorable FcRn upon in vivo binding. If pI mutations reduce monomeric pI, they may have the additional benefit of improving serum retention in vivo.

[0097] Although still under investigation, it is thought that the Fc region has a longer half-life in vivo because Fc is sequestered when it binds to FcRn at pH 6 within the endosome (Ghetie and Ward, 1997 Immunol Today. 18(12):592-598, entire reference incorporated). The endosomal compartment then recycles Fc to the cell surface. When the compartment opens to the extracellular space, a higher pH of approximately 7.4 induces the release of Fc into the bloodstream. In mice, Dall'Acqua et al. showed that Fc mutations with increased FcRn binding at pH 6 and pH 7.4 actually resulted in reduced serum concentrations and the same half-life as wild-type Fc (Dall'Acqua et al. 2002, J.Immunol. 169:5171-5180, entire reference incorporated). The increased affinity of Fc to FcRn at pH 7.4 is thought to hinder the release of Fc into the bloodstream. Therefore, Fc mutations that increase the half-life of Fc in vivo ideally increase FcRn ligation at lower pH levels while still allowing Fc release at higher pH levels. The amino acid histidine changes its charge state in the pH range of 6.0–7.4. Thus, 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 performed for various reasons, including but not limited to alterations of binding to one or more FcγR receptors and alterations of binding to FcRn receptors.

[0099] Accordingly, the proteins of the present invention may include amino acid modifications, including pI mutations and stereomutae, as well as heterodimerizing mutations outlined herein. Each set of mutations may be independently and optionally included in or excluded from a particular heterodimer protein.

[0100] H.FcγR mutation Several useful Fc substitutions exist that can be performed to modify binding to one or more FcγR receptors. Substitutions that result in increased or decreased binding may be useful. For example, increased binding to FcγRIIIa is generally known to result in increased ADCC (antibody-dependent cell-mediated cytotoxicity, i.e., a cell-mediated reaction in which nonspecific cytotoxic cells expressing FcγR recognize a bound antibody on a target cell, subsequently causing lysis of the target cell). Similarly, under some circumstances, decreased binding to FcγRIIb (an inhibitory receptor) may also be beneficial. Useful amino acid substitutions in this invention include those listed in USSN11 / 124,620 (particularly Figure 41), USSN11 / 174,287, USSN11 / 396,495, and USSN11 / 538,406, all of which, in whole, and specifically the mutations disclosed therein, are expressly incorporated herein by reference. 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 may also be included in the Fc domain mutations outlined herein. For example, the substitutions described in USSN11 / 124,620 and USSN14 / 578,305 are useful.

[0102] Furthermore, as specifically disclosed in USSN 12 / 341,769, which is incorporated entirely herein by reference, there are additional Fc substitutions 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. Removal Mutations Similarly, another category of functional mutations is “FcγR removal mutation” or “Fc knockout (FcKO or KO) mutation.” In these embodiments, for some therapeutic applications, it is desirable to reduce or remove the normal binding of the Fc domain to one or more or all Fcγ receptors (e.g., FcγR1, FcγRIIa, FcγRIIb, FcγRIIIa, etc.) in order to avoid further mechanisms of action. This is particularly desirable in many embodiments of using bispecific immunomodulatory antibodies that remove FCγRIIIa binding to remove or significantly reduce ADCC activity, such that one of the Fc domains contains one or more Fcγ receptor removal mutations. In this case, these removal mutations are all shown in Figure 31 of USSN 15 / 141,350, which is incorporated herein by reference in its entirety, and in a preferred embodiment, removal mutations 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 are used, each independently Mutations can be included or excluded at will. Note that the removal mutations mentioned herein remove FcγR linkages but generally do not remove FcRn linkages.

[0104] J. Heterodimer and Fc mutation combination As those skilled in the art will understand, all listed heterodimerizing mutations (including skew and / or pI mutations) can be combined independently and at will in any way that preserves their "twist" or "monomer distribution." Furthermore, all of these mutations can be combined into any of the heterodimerizing formats.

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

[0106] Furthermore, heterodimerizing mutations, skew, and pI can be arbitrarily combined with Fc removal mutations, Fc mutations, and FcRn mutations, as generally outlined herein.

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

[0108] Furthermore, heterodimer Fc fusion proteins can contain skew mutations (e.g., sets of amino acid substitutions shown in Figures 1A-1C of USSN 15 / 141,350, all of which are incorporated herein by reference in their entirety), and particularly useful skew mutations include S364K / E357Q:L368D / K370S, L368D / K370S:S364K, L368E / K370S:S364K, T41 Selected from the group consisting of 1T / E360E / Q362E:D401K, L368D / K370S:S364K / E357L, K370S:S364K / E357Q, T366S / L368A / Y407V:T366W, and T366S / L368A / Y407V / Y349C:T366W / S354C, and may optionally include removal mutations and optionally charged domain linkers, with the heavy chain containing pI mutations.

[0109] In some embodiments, the Fc domains are 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 / The Fc domain contains amino acid substitutions 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 some cases, the Fc domain contains the amino acid substitution 239D / 332E. In other cases, the Fc domain contains the amino acid substitution G236R / L328R or PVA / S267K.

[0110] In one embodiment, a particular combination of skew and pI mutations used in the present invention is T366S / L368A / Y407V:T366W (optionally including the cross-linked disulfide T366S / L368A / Y407V / Y349C:T366W / S354C), where one monomer contains Q295E / N384D / Q418E / N481D and the other is a positively charged domain linker. As understood in the art, "nobuinho The "ru" mutation does not alter pI and therefore can be used with either monomer.

[0111] III. IL-15 and IL15Rα protein domains The present invention provides a heterodimer Fc fusion protein containing IL-15 and IL-15Rα proteins. As shown in the figure, the IL-15 complex can take several forms. As mentioned above, the IL-15 protein itself is less stable when it forms a complex with the IL-15Rα protein. As is known in the art, the IL-15Rα protein contains a "sushi domain," which is the shortest region of the receptor that retains IL-15 binding activity. Thus, heterodimer fusion proteins containing the entire IL-15Rα protein can be constructed, but preferred embodiments herein include a complex using only the sushi domain, the sequence of which is shown in the figure.

[0112] Therefore, the IL-15 complex generally contains the IL-15 protein and the sushi domain of IL-15Rα (unless otherwise stated that the full-length sequence is used, "IL-15Rα", "IL-15Rα(sushi)", and "sushi" are used interchangeably throughout). This complex can be used in three different formats. As shown in Figure 9A, the IL-15 protein and IL-15Rα(sushi) self-assemble by normal ligand-ligand interactions rather than being covalently attached. As will be more fully described herein, either the IL-15 domain or the sushi domain can be covalently linked to the Fc domain (generally using an optional domain linker). Alternatively, they can be covalently linked using a domain linker, generally as shown in Figures 9B, 9E, and 9G. Figure 9B shows the sushi domain as the N-terminal domain, although this can also be reversed. Finally, each of the IL-15 domain or the sushi domain can be manipulated to contain a cysteine ​​amino acid and, similarly, can form a complex with either the IL-15 domain or the sushi domain covalently attached to the Fc domain (using an optional domain linker), generally as shown in Figures 39A–39D.

[0113] In some embodiments, the human IL-15 protein is NCBI reference sequence number NP_000576.1 or sequence number 1. In some cases, the coding sequence of human IL-15 is shown in NCBI reference sequence number No. NM_000585. Exemplary IL-15 proteins of the Fc fusion heterodimer protein outlined herein may have the amino acid sequence of sequence number 2 or amino acids 49-162 of sequence number 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 with respect to sequence number 2. In some embodiments, the IL-15 protein has the amino acid sequence and amino acid substitution N72D described in sequence number 2. 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. In some cases, the IL-15 protein also has the N72D substitution. The IL-15 protein of an Fc fusion protein may have 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acid substitutions.

[0114] The amino acid substitutions may be equivalent substitutions at the interface between IL-15:IL-2β and IL-15:common gamma chain. 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 has N1D, N4D, D8N, D30N, D61N, E64Q, N 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 substitution D30N / E64Q / N65D.

[0115] In some embodiments, the human IL-15 receptor α (IL-15Rα) protein has the amino acid sequence shown in NCBI reference sequence number NP_002180.1 or sequence number 3. In some cases, the coding sequence for human IL-15Rα is shown in NCBI reference sequence number NM_002189.3. Exemplary IL-15Rα proteins of Fc fusion heterodimer proteins outlined herein may include or consist of the sushi domain of sequence number 3 (e.g., amino acids 31-95 of sequence number 3), in other words, the amino acid sequence of sequence number 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 position is relative to the full-length human IL-15Rα protein or SEQ ID NO: 3. For example, amino acids such as D (e.g., Asp), P (e.g., Pro), A (e.g., Ala), DP (e.g., Asp-Pro), DC (e.g., Asp-Cys), DPA (e.g., Asp-Pro-Ala), DPC (e.g., Asp-Pro-Cys), or DCA (e.g., Asp-Cys-Ala) may be added to the C-terminus of the IL-15Rα protein of SEQ ID NO: 4. In some embodiments, the IL-15Rα protein has 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 may have 1, 2, 3, 4, 5, 6, 7, 8, or more amino acid mutations (e.g., substitutions, insertions, and / or deletions).

[0116] IV. Domain Linker In some embodiments, the IL-15 protein and the IL-15Rα protein are bound together via a linker. In some cases, the proteins are not bound via a linker. In other embodiments, the IL-15 protein and the IL-15Rα protein are attached non-covalently. In some embodiments, the IL-15 protein is bound to the Fc domain via a linker. In some embodiments, the IL-15 protein is bound directly to the Fc domain, for example, without a linker. In other embodiments, the IL-15Rα protein is bound to the Fc domain via a linker. In other embodiments, the IL-15Rα protein is bound directly to the Fc domain. In some cases, a linker is not used to bind 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 any two domains outlined herein together. While any suitable linker can be used, many embodiments utilize glycine-serine polymers, for example, (GS)n, (GSGGS)n, (GGGGS)n, and (GGGS)n, where n is at least 0 (and generally 0-1-2-3-4-5), as well as any peptide sequence that enables recombination of two domains having 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 With attention paid to the "degree of twist" as outlined below, charged domain linkers can be used as discussed herein and shown in Figure 7.

[0118] V. Useful Format of the Invention As shown in Figures 9A-9G and 39A-39D, several useful formats of the bispecific heterodimer fusion protein of the present invention exist. Generally, the heterodimer fusion protein of the present invention has two functional components: the IL-15 / IL-15Rα(sushi) component and the Fc component, both of which can take different forms as outlined herein, and both 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, including Q295E / N384D / Q418E / N421D, according to EU numbering.

[0121] Optionally, the first and / or second Fc domains may have an additional set of amino acid substitutions, according to EU numbering, consisting of G236R / L328R, E233P / L234V / L235A / G236del / S239K, E233P / L234V / L235A / G236del / S239K / A327G, E233P / L234V / L235A / G236del / S267K / A327G, and E233P / L234V / L235A / G236del.

[0122] In some cases, the first and / or second Fc domains have a 428L / 434S variant for half-life extension.

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

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

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

[0126] In the IL-15 / Rα-heteroFc format, preferred embodiments utilize the IL-15 mutation Q108E and the skew mutation pair S364K / E357Q:L368D / K370S.

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

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

[0129] In the IL-15 / Rα-heteroFc format, preferred embodiments utilize the D30N / E64Q / N65D mutation and the skew mutation pair S364K / E357Q:L368D / K370S of IL-15.

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

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

[0132] In the IL-15 / Rα-heteroFc format, preferred embodiments utilize the N65D mutation and skew mutation pair S364K / E357Q:L368D / K370S of IL-15.

[0133] In the IL-15 / Rα-heteroFc format, preferred embodiments utilize the N65D mutation of IL-15, the skew mutation versus S364K / E357Q:L368D / K370S, and the 428L / 434S mutation in each Fc monomer.

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

[0135] In the IL-15 / Rα-heteroFc format, preferred embodiments utilize the N4D / N65D mutation and skew mutation pair S364K / E357Q:L368D / K370S of IL-15.

[0136] In the IL-15 / Rα-heteroFc format, preferred embodiments utilize the N4D / N65D mutation of IL-15, the skew mutation versus S364K / E357Q:L368D / K370S, and the 428L / 434S mutation in each Fc monomer.

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

[0138] In the IL-15 / Rα-heteroFc format, preferred embodiments utilize the N1D / N65D mutation and skew mutation pair S364K / E357Q:L368D / K370S of IL-15.

[0139] In the IL-15 / Rα-heteroFc format, preferred embodiments utilize the N1D / N65D mutation of IL-15, the skew mutation versus S364K / E357Q:L368D / K370S, and the 428L / 434S mutation in each Fc monomer.

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

[0141] B.scIL-15-Rα-Fc In this embodiment, as shown in Figure 9B, the heterodimer fusion protein contains two monomers. The first monomer contains (from N-terminus to C-terminus) IL-15 / Rα(sushi)-domain linker-IL-15-optional domain linker-CH2-CH3, where the domain linkers often contain all or part of the hinge. The second monomer contains an "empty" Fc containing all or part of the hinge-CH2-CH3. This is called "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 skew mutation pair S364K / E357Q:L368D / K370S, as well as the 428L / 434S mutation in both monomers.

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

[0147] In the scIL-15 / Rα-Fc format, preferred embodiments utilize the D30N / E64Q / N65D mutation and the skew mutation pair S364K / E357Q:L368D / K370S of IL-15.

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

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

[0150] In the scIL-15 / Rα-Fc format, preferred embodiments include the N65D mutation and skew mutation of IL-15 versus S364K / E357Q:L368D / K370S Use it.

[0151] In the scIL-15 / Rα-Fc format, preferred embodiments utilize the N65D mutation of IL-15, the skew mutation versus S364K / E357Q:L368D / K370S, and the 428L / 434S mutation in each Fc monomer.

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

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

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

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

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

[0157] In the IL-15 / Rα-heteroFc format, preferred embodiments utilize the N1D / N65D mutation of IL-15, the skew mutation versus S364K / E357Q:L368D / K370S, and the 428L / 434S mutation in each Fc monomer.

[0158] C.ncIL-15 / Rα-Fc In this embodiment, as shown in Figure 9C, the heterodimer fusion protein contains three monomers. The first monomer contains IL-15 / Rα(sushi)-domain linker-CH2-CH3 (from N-terminus to C-terminus), where the domain linker often contains all or part of the hinge. The second monomer contains an "empty" Fc, which contains all or part of the hinge-CH2-CH3. The third monomer is IL-15, which is called "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, preferred embodiments utilize the IL-15 mutant Q108E and the skew mutant S364K / E357Q:L368D / K370S.

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

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

[0164] In the ncIL-15 / Rα-Fc format, preferred embodiments utilize the D30N / E64Q / N65D mutation and the skew mutation pair S364K / E357Q:L368D / K370S of IL-15.

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

[0168] In the ncIL-15 / Rα-Fc format, preferred embodiments utilize the N65D mutation of IL-15, the skew mutation versus S364K / E357Q:L368D / K370S, and the 428L / 434S mutation in each Fc monomer.

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

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

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

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

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

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

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

[0176] D. Divalent ncIL-15 / Rα-Fc In this embodiment, as shown in Figure 9D, the heterodimer fusion protein contains four monomers. The first and second monomers contain IL-15 / Rα(sushi)-domain linker-CH2-CH3 (from N-terminus to C-terminus), where the domain linker often contains all or part of the hinge. The third and fourth monomers contain IL-15, which is called "divalent ncIL-15 / Rα-Fc," where "nc" stands for "non-covalent."

[0177] In the divalent 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 divalent ncIL-15 / Rα-Fc format, preferred embodiments utilize the IL-15 mutant Q108E and the skew mutant S364K / E357Q:L368D / K370S.

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

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

[0182] In the divalent ncIL-15 / Rα-Fc format, preferred embodiments utilize the D30N / E64Q / N65D mutation and the skew mutation pair S364K / E357Q:L368D / K370S of IL-15.

[0183] In the divalent ncIL-15 / Rα-Fc format, preferred embodiments utilize the D30N / E64Q / N65D mutation of IL-15, the skew mutation versus S364K / E357Q:L368D / K370S, and the 428L / 434S mutation 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, preferred embodiments utilize the N65D mutation and skew mutation pair S364K / E357Q:L368D / K370S of IL-15.

[0186] In the bivalent ncIL-15 / Rα-Fc format, preferred embodiments utilize the N65D mutation of IL-15, the skew mutation versus S364K / E357Q:L368D / K370S, and the 428L / 434S mutation in each Fc monomer.

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

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

[0189] In the divalent ncIL-15 / Rα-Fc format, preferred embodiments utilize the N4D / N65D mutation of IL-15, the skew mutation versus S364K / E357Q:L368D / K370S, and the 428L / 434S mutation in each Fc monomer.

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

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

[0192] In the divalent ncIL-15 / Rα-Fc format, preferred embodiments utilize the N1D / N65D mutation of IL-15, the skew mutation versus S364K / E357Q:L368D / K370S, and the 428L / 434S mutation in each Fc monomer.

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

[0194] VI. Useful Embodiments of the Invention As will be understood by those skilled in the art and will be described more fully below, the heterodimer fusion proteins of the present invention can take on a wide variety of configurations, generally as shown in Figures 9A-9G and 39A-39D. The amino acid sequences of exemplary fusion proteins are shown in 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 depend on a format comprising a first monomer (first fusion protein) containing an IL-15 protein domain covalently attached to the N-terminus of a first Fc domain using a first domain linker, and a second monomer (second fusion protein) containing an IL-15Rα protein domain covalently attached to the N-terminus of a second Fc domain using a second domain linker. Exemplary embodiments of this format ("IL-15 / Rα heteroFc" and "dsIL-15 / Rα heteroFc") 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 This includes, but is 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 the N-terminus of the first Fc domain. The fusion protein comprises 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 one end, 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] Another useful heterodimeric Fc fusion protein outlined herein includes a fusion protein comprising a first protein domain covalently attached to the N-terminus of a first Fc domain via 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. 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 heterodimer Fc fusion proteins outlined herein includes 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 noncovalently attached to the first protein domain of the first fusion protein, and a fourth protein domain noncovalently 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 ("divalent ncIL-15 / Rα-Fc" or "divalent dsIL-15 / Rα-Fc") include, but are not limited to, XENP21978, XENP22634, XENP24342, and XENP24346.

[0199] Another useful format ("divalent scIL-15 / Rα-Fc") is outlined in Figure 14 of this specification.

[0200] Another useful format of heterodimer Fc fusion proteins outlined herein includes a fusion protein comprising a first Fc domain covalently attached to the N-terminus of a 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, as 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 the second domain linker may be the same or different. Furthermore, the first and second Fc domains of the heterodimeric protein may have different amino acid sequences.

[0202] The Fc domain of the present invention includes an IgG Fc domain, for example, an IgG1Fc 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, S267K / L368D / K370S and S267K / S364K / E357Q according to EU numbering. In some examples, the first and / or second Fc domains of any of the heterodimer Fc fusion formats outlined herein may have a further 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, according to EU numbering, consisting of G236R / L328R, E233P / L234V / L235A / G236del / S239K, E233P / L234V / L235A / G236del / S267K / A327G, E233P / L234V / L235A / G236del / S267K / A327G and E233P / L234V / L235A / G236del.

[0203] Additional heterodimerizing mutations may be included independently and optionally, and may be selected from the mutations outlined in the figure. These compositions may further include removal mutations, pI mutations, charged mutations, isotype mutations, and the like.

[0204] VII. The present invention: nucleic acids The present invention further provides a nucleic acid composition encoding the heterodimeric Fc fusion protein of the present invention (or, in the case of a monomeric Fc domain protein, the encoding nucleic acid).

[0205] As those skilled in the art will understand, nucleic acid compositions will depend on the format of the 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 require only two nucleic acids, and likewise, they can be incorporated into one or two expression vectors.

[0206] As is known in the art, the nucleic acids encoding the components of the present invention can be incorporated into an expression vector, as is known in the art, and in a host cell-dependent manner, to produce the heterodimeric Fc fusion protein of the present invention. Generally, the nucleic acids are operably ligated to any number of regulatory elements (promoters, origins of replication, selectable markers, ribosome binding sites, inducers, etc.). The expression vector can be an extrachromosomal vector or an embedded vector.

[0207] Next, the nucleic acids and / or expression vectors of the present invention are transformed into any number of different 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 under generally different or the same promoter control, so as to be applicable depending on the format. It is contained within. In embodiments particularly used in the present invention, each of these two or three nucleic acids is contained within a different expression vector.

[0209] The heterodimeric Fc fusion protein of the present invention is produced, as is well known in the art, by culturing a host cell containing an expression vector. Once produced, conventional fusion protein or antibody purification steps, including an ion exchange chromatography step, are carried out. As discussed herein, the at least 0.5 difference in the pI of the two monomers can allow separation by ion exchange chromatography or isoelectric focusing, or other methods sensitive to the isoelectric point. That is, by including pI substitutions that vary the isoelectric point (pI) of each monomer such that each monomer has a different pI and the heterodimer also has a different pI, isoelectric point purification of the heterodimer (e.g., anion exchange column, cation exchange column) is facilitated. These substitutions also serve to identify and monitor any contaminating homodimers after purification (e.g., IEF gel, cIEF, and analytical IEX column).

[0210] VIII. Biological and biochemical functionality of the IL-15 / IL15Rα heterodimeric immunomodulatory Fc fusion protein Generally, the heterodimeric Fc fusion protein of the present invention is administered to a patient having cancer, and the efficacy is evaluated by several methods as described herein. Thus, standard assays of efficacy can be performed, such as assessment of cancer load, tumor size, presence or extent of metastasis, etc., but immuno-oncology treatments can also be evaluated based on immune status assessment. This can be done in several ways, including both in vitro assays and in vivo assays. For example, in conjunction with "classical" measurements such as tumor load, size, invasiveness, LN involvement, metastasis, etc., changes in immune status (e.g., presence of ICOS+CD4+T cells after ipi treatment) can be evaluated. Thus, any or all of the following can be evaluated: 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, the evaluation of treatment is performed by assessing immune cell proliferation, for example, using the CFSE dilution method, intracellular staining of Ki67 in immune effector cells, and the 3H-thymidine incorporation method.

[0212] In some embodiments, the evaluation of treatment is done by assessing increased protein levels of markers associated with increased gene expression or activation, including one or more of the cell degranulation 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 in the same manner as is known in the art.

[0215] In some embodiments, the evaluation of treatment is measured by detecting target cell viability through inferring numerous cellular parameters such as enzyme activity (including protease activity), cell membrane permeability, cell adhesion, ATP production, coenzyme production, and nucleotide uptake activity, thereby assessing cell damage. This is done by evaluating the harmful activity. Specific examples of these assays include, but are not limited to, trypan blue or PI staining, 51Cr or 35S release methods, LDH activity, MTT and / or WST assays, calcein-AM assays, luminescence assays, and others.

[0216] In some embodiments, the evaluation of treatment is performed by assessing T cell activity as measured by cytokine production, which is measured intracellularly in the culture supernatant using cytokines including, but not limited to, IFNγ, TNFα, GM-CSF, IL2, IL6, IL4, IL5, IL10, and IL13, using well-known techniques.

[0217] Therefore, the evaluation of treatment can be carried out using assays that assess one or more of the following: (i) increasing the immune response; (ii) increasing the activation of αβ and / or γδ T cells; (iii) increasing cytotoxic T cell activity; (iv) increasing NK and / or NKT cell activity; (v) reducing αβ and / or γδ T cell suppression; (vi) increasing pro-inflammatory cytokine secretion; (vii) increasing IL-2 secretion; (viii) increasing interferon-γ production; (ix) increasing the Th1 response; (x) decreasing the Th2 response; (xi) decreasing or eliminating the number and / or activity of at least one regulatory T cell (Treg).

[0218] A. Assays for measuring effectiveness In some embodiments, T cell activation is assessed using a mixed lymphocyte reaction (MLR) assay, as is known in the art. An increase in activity indicates immunostimulatory activity. Appropriate increases in activity are outlined below.

[0219] In one embodiment, a signaling pathway assay measures an increase or decrease in the immune response, for example, by measuring phosphorylation or dephosphorylation of different factors or by measuring other post-translational modifications. An increase in activity indicates immunostimulatory activity. A suitable increase in activity is outlined below.

[0220] In one embodiment, a signaling pathway assay measures an increase or decrease in αβ and / or γδ T cell activation, for example, by cytokine secretion, proliferation, or changes in the expression of activation markers such as CD137, CD107a, PD1, etc. An increase in activity indicates immunostimulatory activity. Appropriate increases in activity are outlined below.

[0221] In one embodiment, a signaling pathway assay measures an increase or decrease in cytotoxic T cell activity, which is measured, for example, by direct killing of target cells such as cancer cells, or by cytokine secretion, or by proliferation, or by changes in the expression of activating markers such as CD137, CD107a, PD1, etc. An increase in activity indicates immunostimulatory activity. Appropriate increases in activity are outlined below.

[0222] In one embodiment, a signaling pathway assay measures an increase or decrease in NK and / or NKT cell activity, which is measured, for example, by direct killing of target cells such as cancer cells, or by cytokine secretion, or by changes in the expression of an activating marker such as CD107a. An increase in activity indicates immunostimulatory activity. Appropriate increases in activity are outlined below.

[0223] In one embodiment, the signal transduction pathway assay is performed, for example, by cytokine secretion, or by proliferation, or by activity such as CD137, CD107a, PD1, etc. This method measures the increase or decrease in αβ and / or γδ T cell suppression, as measured by changes in the expression of immunoassay markers. Increased activity indicates immunostimulatory activity. A suitable increase in activity is outlined below.

[0224] In one embodiment, the signaling pathway assay measures, for example, an increase or decrease in pro-inflammatory cytokine secretion by ELISA, Luminex, Multiplex bead system, intracellular staining and FACS analysis, or Alispot, etc. An increase in activity indicates immunostimulatory activity. Appropriate increases in activity are outlined below.

[0225] In one embodiment, the signaling pathway assay measures an increase or decrease in IL-2 secretion, for example, by ELISA, Luminex, Multiplex bead system, intracellular staining and FACS analysis, or Alispot, etc. An increase in activity indicates immunostimulatory activity. An appropriate increase in activity is outlined below.

[0226] In one embodiment, the signaling pathway assay measures an increase or decrease in interferon-γ production, for example, by ELISA, Luminex, Multiplex bead system, intracellular staining and FACS analysis, or Alispot, etc. An increase in activity indicates immunostimulatory activity. Appropriate increases in activity are outlined below.

[0227] In one embodiment, a signaling pathway assay measures an increase or decrease in the Th1 response, for example, by cytokine secretion or by changes in the expression of activation markers. An increase in activity indicates immunostimulatory activity. Appropriate increases in activity are outlined below.

[0228] In one embodiment, the signal transduction pathway assay measures an increase or decrease in Th2 response, which is measured, for example, by cytokine secretion or by a change in the expression of activation markers. An increase in activity indicates immunostimulatory activity. An appropriate increase in activity is outlined below.

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

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

[0231] In one embodiment, the signal transduction pathway assay measures an increase or decrease in the M2 macrophage tumorigenesis promoting activity, which is measured, for example, by cytokine secretion or by a change in the expression of activation markers. A decrease in response indicates immunostimulatory activity. An appropriate decrease is the same as in the case of the increase outlined below.

[0232] In one embodiment, the signal transduction pathway assay measures an increase or decrease in the increase of N2 neutrophils, which is measured, for example, by flow cytometry or by IHC. A decrease in response indicates immunostimulatory activity. An appropriate decrease is the same as in the case of the increase outlined below.

[0233] In one embodiment, the signal transduction pathway assay is, for example, by cytokine secretion or measures an increase or decrease in the N2 neutrophil tumorigenesis promoting activity measured by a change in the expression of activation markers. A decrease in response indicates immunostimulatory activity. An appropriate decrease is the same as in the case of the increase outlined below.

[0234] In one embodiment, a signaling pathway assay measures an increase or decrease in the suppression of T cell activation, for example, by cytokine secretion, proliferation, or changes in the expression of activating markers such as CD137, CD107a, PD1, etc. An increase in activity indicates immunostimulatory activity. Appropriate increases in activity are outlined below.

[0235] In one embodiment, a signaling pathway assay measures an increase or decrease in CTL activation inhibition, which is measured, for example, by direct killing of target cells such as cancer cells, or by cytokine secretion, or by proliferation, or by changes in the expression of activating markers such as CD137, CD107a, PD1, etc. An increase in activity indicates immunostimulatory activity. Appropriate increases in activity are outlined below.

[0236] In one embodiment, a signaling pathway assay measures an increase or decrease in αβ and / or γδ T cell depletion, measured, for example, by changes in the expression of activation markers. A decrease in response indicates immunostimulatory activity. A suitable decrease is the same as that for an increase, which are outlined below.

[0237] In one embodiment, a signaling pathway assay measures an increase or decrease in αβ and / or γδ T cell responses, for example, by cytokine secretion, proliferation, or changes in the expression of activating markers such as CD137, CD107a, PD1, etc. An increase in activity indicates immunostimulatory activity. Appropriate increases in activity are outlined below.

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

[0239] In one embodiment, the signaling pathway assay measures the increase or decrease in apoptosis or lysis of cancer cells, for example, by cytotoxic assays such as MTT, Cr release, and calsine AM, or by flow cytometry assays such as CFSE dilution or propidium iodide staining. An increase in activity indicates immunostimulatory activity. Appropriate increases in activity are outlined below.

[0240] In one embodiment, a signaling pathway assay measures the increase or decrease in the cytotoxic or inhibitory effect on cancer cells, measured by cytotoxic assays such as MTT, Cr release, and calsine AM, or by flow cytometry-based assays such as CFSE dilution or propidium iodide staining. An increase in activity indicates immunostimulatory activity. Appropriate increases in activity are outlined below.

[0241] In one embodiment, the signaling pathway assay measures an increase or decrease in direct killing of cancer cells, for example, by cytotoxic assays such as MTT, Cr release, and calsine AM, or by flow cytometry assays such as CFSE dilution or propidium iodide staining. An increase in activity indicates immunostimulatory activity. Appropriate increases in activity are outlined below.

[0242] In one embodiment, the signal transduction pathway assay is performed, for example, by cytokine secretion. The increase or decrease in Th17 activity is measured by proliferation or by changes in the expression of activation markers. An increase in activity indicates immunostimulatory activity. A suitable increase in activity is outlined below.

[0243] In one embodiment, the signaling pathway assay measures the increase or decrease in the induction of complement-dependent cytotoxicity and / or antibody-dependent cell-mediated cytotoxicity, for example, by cytotoxicity assays such as MTT, Cr release, and calsine AM, or by flow cytometry assays such as CFSE dilution or propidium iodide staining. An increase in activity indicates immunostimulatory activity. Appropriate increases in activity are outlined below.

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

[0245] In one embodiment, NK cell activation is measured, for example, by direct killing of target cells, such as cancer cells, or by cytokine secretion, or by changes in the expression of activation markers such as CD107a. For NK cells, increased proliferation, cytotoxicity (ability to kill target cells and increase CD107a, granzyme, and perforin expression), cytokine production (e.g., IFNγ and TNF), and cell surface receptor expression (e.g., CD25) can be indicators of immunomodulation consistent with enhanced killing of cancer cells.

[0246] In one embodiment, γδ T cell activation is measured, for example, by cytokine secretion, proliferation, or changes in the expression of activation markers.

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

[0248] A suitable increase (or decrease, as outlined above) in activity or response is an increase of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 98–99% compared to the signal in either a reference or control sample, e.g., a test sample without the anti-PVRIG antibody of the present invention. Similarly, an increase of at least 1-fold, 2-fold, 3-fold, 4-fold, or 5-fold compared to a reference or control sample indicates efficacy.

[0249] IX. Treatment Once manufactured, the compositions of the present invention are used in several oncological applications by treating cancer, generally by promoting T cell activation (e.g., T cells are no longer suppressed) through the binding of the heterodimeric Fc fusion protein of the present invention.

[0250] Therefore, the heterodimer composition of the present invention can be used to treat these cancers.

[0251] A. Heterodimeric protein compositions for in vivo administration The antibody formulation used in accordance with the present invention comprises an antibody of desired purity, optionally pharmaceutically acceptable carrier, excipient, or stabilizer (Remington's Pharmaceut). For preservation, it is prepared in the form of a lyophilized preparation or aqueous solution by mixing with (as generally outlined in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed.

[1980] ). Acceptable carriers, buffers, excipients, or stabilizers are non-toxic to the recipient at the dosage and concentration used and include buffers such as phosphoric acid, citrate, 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; blood The material contains proteins such as clear albumin, gelatin, or immunoglobulin; 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 dextrin; 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 nonionic surfactants such as TWEEN®, PLURONICS®, or polyethylene glycol (PEG).

[0252] B. Dosage Method The heterodimeric protein and chemotherapeutic agent of the present invention are administered to a subject according to known methods, such as intravenous administration as a bolus or continuous infusion over a period of time.

[0253] C.Treatment In the method of the present invention, the treatment is used to provide a positive therapeutic response with respect to a disease or medical condition. A “positive therapeutic response” is intended to be an improvement in the disease or medical condition and / or an improvement in symptoms associated with the disease or medical condition. For example, a positive therapeutic response may refer to one or more of the following improvements in the disease: (1) a decrease in the number of tumor cells, (2) an increase in tumor cell death, (3) an inhibition of tumor cell survival, (5) an inhibition of tumor growth (i.e., a slowdown, preferably a halt), (6) an increase in patient survival rate, and (7) some relief from one or more symptoms associated with the disease or medical condition.

[0254] A positive treatment response in any given disease or condition may be determined by standardized response criteria specific to that disease or condition. Tumor response may be assessed for changes in tumor morphology (i.e., systemic tumor tissue volume, tumor size, etc.) using screening techniques such as magnetic resonance imaging (MRI) scans, radiography, computed tomography (CT) scans, bone scans, endoscopy, and tumor biopsy sampling, including bone marrow aspiration (BMA) and counting of circulating tumor cells.

[0255] In addition to these positive treatment responses, patients receiving treatment may experience beneficial effects such as improvement in disease-related symptoms.

[0256] The treatment according to this invention includes the “therapeutic effective dose” of the pharmaceutical product used. The “therapeutic effective dose” refers to the amount that is effective in the required dosage and duration to achieve the desired treatment outcome.

[0257] The therapeutically effective dose may vary depending on factors such as the individual's disease state, age, sex, and weight, as well as the drug's ability to induce the desired response in the individual. The therapeutically effective dose is also the amount in which the therapeutically beneficial effects outweigh the toxic or harmful effects of either the antibody or its moiety.

[0258] The "therapeutic effective dose" for tumor therapy can also be measured by its ability to stabilize disease progression. The ability of cancer-inhibiting compounds may be evaluated in animal model systems to predict efficacy in human tumors.

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

[0260] The drug regimen is adjusted to provide the optimal desired response (e.g., therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the urgency of the treatment situation. Parenteral compositions may be formulated in unit dosage forms for ease of administration and uniformity of dosage. As used herein, unit dosage forms refer to physically distinct units suitable as a single dose for the subject being treated, each unit containing a predetermined amount of the active compound calculated to produce the desired therapeutic effect in relation to the required pharmaceutical carrier.

[0261] The specifications of the unit dosage form of the present invention are influenced by, and directly depend upon, (a) the inherent characteristics of the active compound and the specific therapeutic effect to be achieved, and (b) the limitations inherent in the field of formulating such active compounds for the treatment of susceptibility in an individual.

[0262] The efficient dosage and administration regimen of the bispecific antibody used in this invention depend on the disease or condition being treated and can be determined by those skilled in the art.

[0263] The exemplary and non-limiting therapeutic dose range of the bispecific antibody used in this invention is approximately 0.1 to 100 mg / kg.

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

[0265] While specific embodiments of the present invention have been described above for illustrative purposes, those skilled in the art will understand that numerous modifications can be made to the details without departing from the present invention as described in the appended claims. [Examples]

[0266] Examples are provided below to illustrate the present invention. These examples are not intended to limit the present invention to any particular application or operating theory. All steady-state region locations discussed in the present invention are numbered as follows: Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th Ed., United States Public It follows an EU index similar to that of the Health Service, National Institutes of Health, Bethesda (the entire index is incorporated by reference). Those skilled in the art of antibody technology will understand that this rule consists of discontinuous numbering in specific regions of immunoglobulin sequences, enabling a standardized reference to conserved locations within the immunoglobulin family. Thus, the location of any given immunoglobulin as defined by the EU index does not necessarily correspond to its continuity sequence.

[0267] General and specific scientific and technological information is provided in U.S. Patent Application Publication No. 2015 / 0307629. This is outlined in the same document No. 2014 / 0288275 and WO2014 / 145806, all of which are explicitly incorporated by reference, particularly with respect to the technologies 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, the inventors have created the IL-15 / IL-15Rα(sushi) complex (hereinafter referred to as the IL-15 / Rα-Fc fusion) as an Fc fusion, with the aim of promoting production, facilitating FcRn-mediated recycling of the complex, and extending its half-life.

[0269] A. Example 1A: Manipulation of IL-15 / Rα-Fc fusion protein Plasmids encoding IL-15 or the IL-15Rαsushi domain were constructed by standard gene synthesis, followed by subcloning into a pTT5 expression vector containing an Fc fusion partner (e.g., the constant region shown in Figure 8). Schematic cartoon diagrams of exemplary IL-15 / Rα-Fc fusion protein formats are shown in Figures 9A–9G.

[0270] The IL-15Rα heterodimer Fc fusion, or "IL-15 / Rα-heteroFc" format, contains IL-15 recombinantly fused to one side of the heterodimer Fc and an IL-15Rα sushi domain recombinantly fused to the other side of the heterodimer Fc (Figure 9A). IL-15 and IL-15Rα may have a variable-length linker between the C-terminus and N-terminus of their respective Fc regions (see Figure 7). Exemplary proteins of this format include XENP20818 and XENP21475, whose sequences are shown in Figure 10 (see also Table 1). Sequences of further proteins of this format are listed in the figures and sequence listings 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, contains an IL-15Rαsushi domain fused to IL-15 by a variable-length linker (referred to as the "single-chain" IL-15 / IL-15Rα complex or "scIL-15 / Rα"), which is then fused to the N-terminus of a heterodimer Fc region, with the other end of the molecule being a heterodimer Fc of "Fc only" or "empty Fc" (Figure 9B). An exemplary linker sequence is shown in Figure 7. An exemplary protein of this format is XENP21478, whose sequence is shown in Figure 11 (see also Table 2). Further examples of this format... The protein sequences are listed in the figure and sequence listing as XENP21993, XENP21994, XENP21995, XENP23174, XENP23175, XENP24477, and XENP24480. [Table 2]

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

[0273] The divalent non-covalent IL-15 / Rα-Fc fusion, or "divalent 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 non-covalent IL-15 / Rα complex. An exemplary protein of this format is XENP21978, whose sequence is shown in Figure 13. The sequence of a further protein of this format is listed as XENP21979 in the figure and sequence listing.

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

[0275] The 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 heterodimer Fc region, while IL-15 is transfected separately to form a non-covalent IL-15 / Rα complex, with the other end of the molecule being "Fc only" or "empty Fc". An exemplary protein of this format is XENP22637, whose sequence is shown in Figure 15. The sequence of a further protein of this format is listed as XENP22638 in the figure and sequence listing.

[0276] Fc-single-chain IL-15 / Rα fusion or "Fc-scIL-15 / Rα" 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 the heterodimer Fc region, with the other end of the molecule being "Fc only" or "empty Fc". An exemplary linker sequence is shown in Figure 7. An exemplary protein sequence in this format is 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 5mL column with a 5-40% gradient between 50mM Tris pH 8.5 and 1M NaCl).

[0278] B. Example 1B: Manipulation of IL-15 / Rα-Fc fusion protein IL-15 / Rα-Fc fusion proteins produced in the various formats 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 non-denaturing behavior of the purified samples. The analysis was performed using an Agilent 1200 high-performance liquid chromatography (HPLC) system. Samples were injected into a Superdex® 200 10 / 300GL column (GE Healthcare Life Sciences) at a UV detection wavelength of 280 nM using 1.0 mL / min of 1× PBS, pH 7.4 as the mobile phase for 25 minutes at 4°C. The analysis was performed using the Agilent OpenLab Chromatography Data System (CDS) ChemStation Edition AIC version C.01.07. Chromatograms of the 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 for use. LabChip and Protein Express Assay Reagent Proteins were analyzed electrophoretically by CEF using a LabChip GXII Touch HT (PerkinElmer, Waltham, Mass) kit. Samples were tested in a dual manner: one under reducing conditions (using 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 peak symmetry and the relatively small population 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 procedure generally included: immobilization (capture of ligand or test sample onto biosensor), association (immersion of ligand or test sample-coated biosensor into wells containing serial dilutions of the corresponding test sample or ligand), and dissociation to determine the affinity of the test sample (returning the biosensor to a well containing buffer). A reference well containing only buffer was also included in the method for background correction during data processing. In particular, test samples were captured using an anti-human Fc (AHC) biosensor, followed by multiple concentrations of IL-2Rβ (R&D Systems, M) for KD determination. The structures were immersed in inneapolis (Min.). The affinity results and corresponding sensorgrams are shown in Figures 17D, 18D, and 19D. Each of the three constructs showed high affinity binding (3–8 nM) to IL-1Rβ.

[0283] The stability of the IL-15 / Rα-Fc fusion protein was evaluated using differential scanning fluorescence (DSF). DSF experiments were performed using the 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 10-fold. 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 showed 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 using cell proliferation assays. Human PBMCs were treated with the indicated concentrations in the test samples. Four days after treatment, the 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 closely associated with cell proliferation, and intracellular Ki67 staining was performed using anti-Ki67-APC (Ki-67) and Foxp3 / transcription factor staining buffer set (Thermo Fisher Scientific, Waltham, Mass). The proportion of Ki67 in the aforementioned 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. In particular, the differences in proliferation activity depended on the length of the linker on the IL-15-Fc side. Specifically, constructs without a linker (hinge only), 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, IL-15 / Rα heterodimers 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. Staphylococcus enterotoxin B (SEB) is a superantigen that induces T cell activation and proliferation, as is achieved through 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 for 72 hours in combination with various IL-15 / Rα-Fc fusion proteins at 20 μg / mL or controls (PBS, isotype controls, and bivalent anti-PD-1 antibody). After treatment, the supernatant was collected and assayed for IL-2, and the data are shown in Figure 22. The data clearly show that IL-15 / Rα-Fc fusion proteins enhanced IL-2 secretion more than PBS and isotype controls. In particular, several IL-15 / Rα-Fc fusion proteins Its quality is equivalent to or greater than that of anti-PD-1 antibodies.

[0288] F. Example 1: F: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 human PBMCs were injected into NSG mice, the human PBMCs induced an autoimmune response against mouse cells. 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 transplanted 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 with the IL-15 / Rα-Fc fusion protein exhibited a rapid increase in morbidity and mortality (all died by day 10) compared to mice administered with recombinant IL-15 (all survived until day 14). This is likely due to the longer half-life predicted by the IL-15 / Rα-Fc fusion protein.

[0290] In another experiment, 10 million human PBMCs were transplanted into NSG mice via IV-OSP on day 0, followed by administration of XENP20818 (1 mg / kg, 0.3 mg / kg, 0.1 mg / kg, or 0.03 mg / kg, then weekly) or PBS on day 1. The effect of XENP20818 on wild-type NSG mice was examined, including a control group that did not receive PBMC transplantation. Blood was collected on day 7 to measure IFNγ, and the data are shown in Figure 24. The number of CD4+ T cells, CD8+ T cells, and CD45+ cells was also measured, and the data are shown in Figure 25. The data show a clear dose-response to XENP20818.

[0291] XI. Example 2: Manipulated IL-15 / Rα-Fc heterodimer fusion protein with disulfide bond To further improve the stability of the IL-15 / Rα-Fc fusion protein and extend its half-life, we manipulated the disulfide bond at the IL-15 / Rα interface.

[0292] A. Example 2A: Manipulation and analysis of IL-15 / Rα heterodimers having manipulated disulfide bonds By investigating the crystal structure of the IL-15 / Rα complex, and the molecular structure By modeling using Operating Environment (MOE, Chemical Computing Group, Montreal, Quebec, Canada) software, we predicted the IL-15 / Rα interface residues that can be substituted with cysteine ​​to form covalent disulfide bonds, as shown in Figure 26.

[0293] Plasmids encoding IL-15 or IL-15Rα(sushi) were constructed by standard gene synthesis followed by subcloning into a pTT5 expression vector. The IL-15Rα(sushi) chain contained a C-terminal polyhistidine tag. The residues identified above were replaced with cysteine ​​using standard mutagenesis techniques. Furthermore, 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 the cysteine ​​to be manipulated (an exemplary sequence is shown in Figure 27). Exemplary IL-15 and IL-15Rα(sushi) manipulated with cysteine ​​are shown. i) The sequences of the mutants are shown in Figures 28 and 29, respectively.

[0294] Schematic cartoons of IL-15 / Rα heterodimers with and without the manipulated disulfide 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 sequences of exemplary scIL-15 / Rα heterodimers are shown in Figure 33. "Wild-type" IL-15 / Rα heterodimers with an additional residue at the C-terminus but without the manipulated cysteine ​​were generated as controls. The sequences of these control IL-15 / Rα heterodimers are listed as XENP22001, XENP22002, and XENP22003 in the figures and sequence listings. The protein was produced by transient transfection in HEK293E cells and then purified by Ni-NTA chromatography.

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

[0296] As shown by denatured, unreduced CEF, many disulfide bonds were correctly formed, and a larger molecular weight of the covalent complex could be observed compared to a control without the manipulated disulfide bonds (Figures 34-35). The disulfide-bonded IL-15 / Rα heterodimer exhibited high thermal stability up to +13°C (Figure 38). Binding to IL-2Rβ was not affected by the presence of the manipulated disulfide bonds (Figure 38). Preferred disulfide bond pairs were XENP22005, XENP22006, XENP22008, and XENP22494, which were constructed as Fc fusion proteins as described later.

[0297] B. Example 2B: Analysis of IL-15 / Rα-Fc fusion protein with manipulated disulfide bond Plasmids encoding the IL-15 or IL-15Rαsushi domain, possessing the aforementioned mutations, were subcloned into pTT5 expression vectors containing an Fc fusion partner (e.g., the constant region shown in Figure 8). Schematic cartoon diagrams of the manipulated disulfide bond-containing IL-15 / Rα-Fc fusion protein are shown in Figures 39A-D.

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

[0299] 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 further covalently attached as a result of the manipulated cysteine. Exemplary proteins of this format include XENP22357, XENP22358, XENP22359, XENP22684, and XENP22361, whose sequences are shown in Figure 41. Further proteins of this format... The sequences are listed in the figure and sequence listing as XENP22360, XENP22362, XENP22363, XENP22364, XENP22365, and XENP22366.

[0300] Divalent disulfide-bonded IL-15 / Rα-Fc or "divalent dsIL-15 / Rα-Fc" (Figure 39C) is identical to "divalent ncIL-15 / Rα-Fc," but IL-15Rα(sushi) and IL-15 are further covalently attached as a result of the manipulated cysteine. Exemplary proteins of this format include XENP22634, XENP22635, and XENP22636, whose sequences are shown in Figure 42. The sequence of further proteins of this format is listed as XENP22687 in the figure and sequence listing.

[0301] 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 addition of IL-15Rα(sushi) and IL-15 as a result of the manipulated cysteine. Exemplary proteins of this format include XENP22639 and XENP22640, whose sequences are shown in Figure 43.

[0302] A "wild-type" IL-15 / Rα-Fc fusion protein with an additional residue at the C-terminus but without the manipulated cysteine ​​was generated as a control. The sequences of these control IL-15 / Rα-Fc fusion proteins are listed in the figure 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 between 50 mM Tris pH 8.5 and 1 M NaCl).

[0304] Generally as described in Example 1B, after the proteins were purified, they were analyzed for purity and homogeneity by capillary isoelectric focusing (CEF). As mentioned above, many disulfide bonds were correctly formed, as shown by denatured, unreduced CEF, and a larger molecular weight of the covalent complex can be observed here compared to a control that does not contain the manipulated disulfide bonds (Figure 44).

[0305] Next, the proteins were tested in a cell proliferation assay. IL-15 / Rα-Fc fusion protein (with or without an engineered disulfide bond) or a control was 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, which were generally analyzed by FACS as described in Example 1D. The proliferation of NK cells, CD4+ T cells, and CD8+ T cells indicated by Ki67 expression is shown in Figures 45A-C. The IL-15 / Rα-Fc fusion protein and the IL-15 control induced strong proliferation of NK cells, CD8+ T cells, and CD4+ T cells, respectively.

[0306] XII. Example 3: IL-15 / Rα-Fc fusion protein modified for lower potency and increased PK and half-life To further improve PK and extend the half-life, we reasoned that reducing the potency of IL-15 would decrease antigen sink and therefore increase the half-life.

[0307] A. Example 3A: Manipulation and production of mutant IL-15 / Rα-Fc fusion protein By examining the crystal structures of the IL-15:IL-2Rβ β and IL-15:common gamma chain interfaces, and by modeling them using MOE software, we predicted residues at these interfaces that could be substituted to reduce potency. Figure 46 shows a structural model of the IL-15:receptor complex showing the locations of predicted residues manipulated for equivalent substitution (to reduce the risk of immunogenicity). The sequences of exemplary IL-15 mutants manipulated 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 a pTT5 expression vector containing an Fc fusion partner (e.g., the constant region shown in Figure 8). The substitutions identified above were incorporated using standard mutagenesis techniques. Exemplary sequences of IL-15 / Rα-Fc fusion proteins in the "IL-15 / Rα-heteroFc" format, manipulated to reduce potency, are shown in Figure 48. Additional sequences are shown in the figure and sequence listing as XENP22815, XENP22816, XENP22817, XENP22818, XENP22819, XENP22820, XENP22823, XENP22824, XENP22 Listed as 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, manipulated for reduced potency, is shown in Figure 49, with further sequences listed as XENP24013, XENP24014, and XENP24016 in the figure and sequence listing. The sequence of an exemplary IL-15 / Rα-Fc fusion protein in the "ncIL-15 / Rα-Fc" format, manipulated for reduced potency, is shown in Figure 50. An exemplary sequence of ncIL-15 / Rα heterodimers manipulated for reduced potency is shown in Figure 51, and additional sequences are listed in the figure and sequence listing as XENP22791, XENP22792, XENP22793, XENP22794, XENP22795, XENP22796, XENP22803, XENP22804, XENP22805, XENP22806, XENP22807, XENP22808, XENP22809, XENP22810, XENP22811, XENP22812, XENP22813, and XENP22814. Figure 52 shows an exemplary sequence of an IL-15 / Rα-Fc fusion protein in the "divalent ncIL-15 / Rα-Fc" format, which has been manipulated for reduced potency. Figure 53 shows an exemplary sequence of an IL-15 / Rα-Fc fusion protein in the "dsIL-15 / Rα-Fc" format, which has been manipulated for reduced potency.

[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 5mL column with a 5-40% gradient between 50mM Tris pH 8.5 and 1M NaCl).

[0311] B. Example 3B: In vitro activity of mutant IL-15 / Rα-heteroFc and scIL-15 / Rα-Fc fusion proteins manipulated to reduce potency The mutant IL-15 / Rα-Fc fusion protein was tested in several cell proliferation assays.

[0312] In the initial cell proliferation assay, IL-15 / Rα-Fc fusion protein (with or without the manipulated substitution) or a control was 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, which were generally analyzed by FACS as described in Example 1D. The proliferation of NK cells, CD8+ T cells, and CD4+ T cells indicated by Ki67 expression is shown in Figures 54-55. Most IL-15 / Rα-Fc fusion proteins induced proliferation in each cell population, but their activity varied depending on specific manipulated substitutions.

[0313] In the second cell proliferation assay, IL-15 / Rα-Fc fusion protein (with or without the manipulated substitution) was 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 (Hl100), and anti-Ki67-APC (20Raj1) antibodies to mark various cell populations. Figures 56-57 show the selection of various cell populations after incubation with XENP22821 by FACS. Lymphocytes were first gated based on lateral scattering (SSC) and forward scattering (FSC) (Figure 56A). Next, lymphocytes were 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, the 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 the IL-15 / Rα-Fc fusion protein than CD4+ T cells, and as described above, their proliferative activity was altered in response to specific manipulated substitutions. Figure 59D shows the EC50 change ratio 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 the expression of CD69 and CD25 (T cell activation markers) before and after incubation of PBMCs with XENP22821.

[0314] In the third experiment, additional mutant IL-15 / Rα-Fc fusion proteins were incubated with human PBMCs at 37°C for 3 days. 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, which were then analyzed by FACS generally as described in Example 1D. The proliferation of CD8+(CD45RA-)T cells, CD4+(CD45RA-)T cells, γδT cells, and NK cells indicated by Ki67 expression is shown in Figures 60A-D.

[0315] In the fourth experiment, human PBMCs were subjected to additional IL-15 / Rα-Fc modification at the indicated concentrations. The cells were incubated with the allogene for 3 days. After incubation, the 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 (Hl100), and anti-Ki67-APC (Ki67), and analyzed by FACS generally as described in Example 1D. The percentage of Ki67 in CD8+ T cells, CD4+ T cells, and NK cells after treatment is shown in Figure 61.

[0316] In the fifth experiment, mutant IL-15 / Rα-Fc fusion protein was 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 relative to 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 protein was 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 relative to 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 manipulated to reduce potency, having various linker lengths between IL-15 and IL-15Rα. IL-15 / Rα-Fc fusion proteins (shown in Table 3) having some of the above substitutions and further having linkers of varying lengths between IL-15 and IL-15Rα 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 relative to 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 in 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, but the differences depended on both linker length and specific manipulated substitutions. [Table 3]

[0319] D. Example 3D: In vitro activity of a mutant IL-15 / Rα-Fc fusion protein manipulated to further reduce the potency of the format. Mutant IL-15 / Rα-Fc fusion proteins in different formats (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 proportions of Ki67 to CD8+ T cells, CD4+ T cells, γδ T cells, and NK (CD16+) cells are shown in Figures 65A-D, respectively. As described above, the data show that the ncIL-15 / Rα-Fc fusion protein XENP21479 is the most potent inducer of proliferation in CD8+ T cells, CD4+ T cells, NK (CD16+) cells, and γδ T cells. In particular, the introduction of the Q108E substitution into the ncIL-15 / Rα-Fc format (XENP24349) dramatically reduces its proliferation activity compared to the wild type (XENP21479). [Table 4]

[0320] E. Example 3E: STAT5 phosphorylation by mutant IL-15 / Rα-Fc fusion protein Trans-presentation 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 incubation with the indicated IL-15 / Rα-Fc test sample for 15 minutes. 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-cooled (-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) staining were performed 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 SSCs and FSCs (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 further 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 dose-dependently induced and also varied in response to specific manipulated substitutions. Figure 67C shows the EC50 change ratio for STAT5 phosphorylation of mutant IL-15 / Rα-Fc fusion protein compared to controls.

[0321] F. Example 3F: PK of a mutant IL-15 / Rα-Fc fusion protein manipulated for reduced potency To investigate whether IL-15 / Rα-Fc fusion proteins, modified to reduce potency, improved half-life and PK, we examined these variants in a PK study in C57BL / 6 mice. On day 0, mice in two cohorts (5 mice per test sample per cohort) were administered the test sample indicated by IV-TV at 0.1 mg / kg. Serum was collected 60 minutes after administration, then on days 2, 4, and 7 for cohort 1, and on 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 the potency and half-life of the test sample.

[0322] As expected, the less potent mutants exhibited substantially longer half-lives. In particular, the half-life improved 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 protein was evaluated in a GVHD model performed in female NSG immunodeficient mice, generally as described in Example 1F.

[0324] In the initial trial, 10 million human PBMCs were transplanted into NSG mice via IV-OSP on day 0, followed by administration of IL-15 / Rα-Fc fusion protein at the concentrations indicated 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 the control (PBS).

[0325] In another study, 10 million human PBMCs were transplanted into NSG mice via IV-OSP on day 0, followed by administration of IL-15 / Rα-Fc fusion protein at the concentrations shown on day 1. IFNγ levels and the number 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 indicate that the mutant IL-15 / Rα-Fc fusion protein dose-dependently enhances IFNγ secretion and the proliferation of human NK and T cells. Notably, the observed activity correlated with the in vitro potency of each mutation.

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

[0327] H. Example 3: H: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 evaluated 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 on day 6 and then recovered and normalized. Finally, the figures show the 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), demonstrating the proliferative activity after treatment with XENP22821. Similar growth activity was observed after treatment with XENP20818, XENP22819, XENP22822, and XENP23343, demonstrating that most of the IL-15 / Rα-Fc fusion proteins of the present invention are active in cynomolgus monkeys.

[0328] XIII. Example 4: IL-15 / Rα-Fc fusion protein manipulated with Xtend Fc As described above, the IL-15 / Rα-Fc mutant, which was manipulated to reduce potency, was further modified with Xtend Fc to further increase its half-life by subcloning the plasmid encoding IL-15 and / or IL-15Rα(sushi) into a pTT5 expression vector containing an Fc fusion partner with an M428L / N434S substitution (see Figure 8, skeleton 11). (This is referred to herein as the "IL-15 / Rα-XtendFc" fusion protein.) 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 further IL-15 / Rα-Fc mutants Human PBMCs were kept with the indicated concentration of the IL-15 / Rα-XtendFc mutant for 3 days. The cells were incubated. 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, which were then analyzed by FACS, generally as described in Example 1D. Post-treatment proliferation of CD8+ T cells, CD4+ T cells, and NK cells, as indicated by Ki67 expression, is shown in Figure 97.

[0330] Since Xtend mutants were selected to investigate their activity in cynomolgus monkeys, their ability to proliferate cynomolgus monkey T cells was examined. Cyno PBMCs were incubated with selected test samples at the indicated concentrations 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 were generally analyzed by FACS as described in Example 1D. Post-treatment proliferation of CD8+ T cells, CD4+ T cells, and NK cells, as indicated by Ki67 expression, is shown in Figure 98.

[0331] B. Example 4B: In vivo activity of IL-15 / Rα-XtendFc mutant in a GVHD model 10 million human PBMCs were transplanted into NSG mice via IV-OSP on day -7, followed by administration of the test sample (0.3 mg / kg) shown on day 0. Whole blood was collected on days 4 and 7, and mice were sacrificed for spleen on days 5-8 or 11, with the number of CD4+ T cells, CD8+ T cells, and CD45+ cells measured using FACS. Figures 99A-C show the number of CD4+ T cells in whole blood on days 4 and 7, and in the spleen on day 8, respectively. Figures 100A-C show the number of CD8+ T cells in whole blood on days 4 and 7, and in the spleen on day 8, respectively. Figures 101A-C show the number of CD4+ T cells in whole blood on days 4 and 7, and in the spleen on day 8, respectively. Mouse body weight was also measured on days -8, -2, 1, 5, 8, and 11, and is 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 protein in cynomolgus monkeys Three monkeys (n=3) were administered a single intravenous (iv) dose of the test sample shown (day 1), and blood samples were collected daily. The number of CD8+ T cells, CD4+ T cells, and NK cells in the blood was evaluated over time as shown in Figures 103A-C, respectively. Each point represents the average of the three cynomolgus monkeys. The data show that each of the variants is active in proliferative immune cells, indicating that the IL-15 / Rα-Fc fusion protein of the present invention may be useful as a therapeutic agent for human cancer.

[0333] The above embodiments are provided to give those skilled in the art a complete disclosure and explanation of how embodiments of the compositions, systems, and methods of the present invention can be prepared and used, and are not intended to limit the scope of what the inventors consider to be their invention. Modifications of the above forms for carrying out the invention that are obvious to those skilled in the art are intended to be within the scope of the appended claims. All patents and publications referenced herein represent the state of the art to those skilled in the art to which the invention relates. All references cited herein are incorporated by reference to the same extent as each reference is incorporated by reference in whole.

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

[0335] All references cited herein are incorporated by reference in their entirety for the same degree as each individual publication or patent or patent application is specifically and individually indicated so as to be incorporated by reference in its entirety for the entire purpose.

[0336] As will be apparent to those skilled in the art, many modifications and variations of this application can be made without departing from the spirit and scope of this application. The specific embodiments and examples described herein are provided for illustrative purposes only and should be limited only by the terminology of the appended claims, together with the entire scope of the equivalent for which the claims are granted.

Claims

1. It is a heterodimer protein, a) A first fusion protein comprising a first protein domain and a first Fc domain, wherein the first protein domain is covalently attached to the N-terminus of the first Fc domain using a first domain linker, b) A second fusion protein comprising a second protein domain and a second Fc domain, wherein the second protein domain is covalently attached to the N-terminus of the Fc domain using a second domain linker, A heterodimer protein in which the first Fc domain and the second Fc domain have a set of amino acid substitutions selected from the group consisting of S267K / 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 contains IL15 protein and the second protein domain contains IL15Rα protein.

2. The heterodimer protein according to claim 1, wherein the first Fc domain and / or the second Fc domain have an additional set of amino acid substitutions comprising Q295E / N384D / Q418E / N421D according to EU numbering.

3. The heterodimer protein according to claim 1 or 2, wherein the first Fc domain and / or the second Fc domain have an additional set of amino acid substitutions consisting of G236R / L328R, E233P / L234V / L235A / G236del / S239K, E233P / L234V / L235A / G236del / S267K / A327G, E233P / L234V / L235A / G236del / S267K / A327G, and E233P / L234V / L235A / G236del.

4. The heterodimer protein according to any one of claims 1 to 3, wherein 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 (shortened 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).

5. The heterodimer protein according to any one of claims 1 to 4, wherein the IL15 protein has one or more amino acid substitutions selected from the group consisting of N1D, N4D, D8N, D30N, D61N, E64Q, N65D, and Q108E.

6. The heterodimer protein according to any one of claims 1 to 5, wherein 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.

7. The aforementioned heterodimer protein, i) The first fusion protein having the polypeptide sequence of SEQ ID NO: XX (XENP15902), and the first fusion protein having the polypeptide sequence of SEQ ID NO: XX (XENP15908) The second fusion protein, ii) The first fusion protein having the polypeptide sequence of Sequence ID XX (XENP15902), and the second fusion protein having the polypeptide sequence of Sequence ID XX (XENP15909), iii) The first fusion protein having the polypeptide sequence of Sequence ID XX (XENP16479), and the second fusion protein having the polypeptide sequence of Sequence ID XX (XENP15908), iv) The first fusion protein having the polypeptide sequence of Sequence ID XX (XENP15902), and the second fusion protein having the polypeptide sequence of Sequence ID XX (XENP16481), v) The first fusion protein having the polypeptide sequence of Sequence ID XX (XENP15902), and the second fusion protein having the polypeptide sequence of Sequence ID XX (XENP16483), vi) The first fusion protein having the polypeptide sequence of Sequence ID XX (XENP16479), and the second fusion protein having the polypeptide sequence of Sequence ID XX (XENP15909), vii) The first fusion protein having the polypeptide sequence of Sequence ID XX (XENP16479), and the second fusion protein having the polypeptide sequence of Sequence ID XX (XENP16481), viiii) The first fusion protein having the polypeptide sequence of Sequence ID XX (XENP16480), and the second fusion protein having the polypeptide sequence of Sequence ID XX (XENP16482), ix) The first fusion protein having the polypeptide sequence of Sequence ID XX (XENP16480), and the second fusion protein h having the polypeptide sequence of Sequence ID XX (XENP15909), x) The first fusion protein having the polypeptide sequence of Sequence ID XX (XENP17064), and the second fusion protein having the polypeptide sequence of Sequence ID XX (XENP17038), xi) The first fusion protein having the polypeptide sequence of SEQ ID NO: XX (XENP17064), and the second fusion protein having the polypeptide sequence of SEQ ID NO: XX (XENP17040), or xi) The first fusion protein having the polypeptide sequence of SEQ ID NO: XX(17062), and the second fusion protein having the polypeptide sequence of SEQ ID NO: XX(17044), The first fusion protein having the polypeptide sequence of sequence number XX (XENP17686), and the second fusion protein having the polypeptide sequence of sequence number XX (XENP15908), The first fusion protein having the polypeptide sequence of sequence number XX (XENP17687), and the second fusion protein having the polypeptide sequence of sequence number XX (XENP15908), xv) The first fusion protein having the polypeptide sequence of Sequence ID XX (XENP17688), and the second fusion protein having the polypeptide sequence of Sequence ID XX (XENP15908), xvi) The first fusion protein having the polypeptide sequence of Sequence ID XX (XENP17689), and the second fusion protein having the polypeptide sequence of Sequence ID XX (XENP15908), xvii) The first fusion protein having the polypeptide sequence of Sequence ID XX (XENP17690), and the second fusion protein having the polypeptide sequence of Sequence ID XX (XENP15908), The polypeptide sequence having sequence number XX (XENP17691) xviiii) A fusion protein 1, and the second fusion protein having the polypeptide sequence of Sequence ID XX (XENP15908), The first fusion protein having the polypeptide sequence of sequence number XX (XENP17692), and the second fusion protein having the polypeptide sequence of sequence number XX (XENP15908), The first fusion protein having the polypeptide sequence of sequence number XX (XENP17693), and the second fusion protein having the polypeptide sequence of sequence number XX (XENP15908), The first fusion protein having the polypeptide sequence of sequence number XX (XENP17694), and the second fusion protein having the polypeptide sequence of sequence number XX (XENP15908), The first fusion protein having the polypeptide sequence of sequence number XX (XENP17695), and the second fusion protein having the polypeptide sequence of sequence number XX (XENP15908), The first fusion protein having the polypeptide sequence of sequence number XX(XENP17696), and the second fusion protein having the polypeptide sequence of sequence number XX(XENP15908), The first fusion protein having the polypeptide sequence of sequence number XX(XENP17697) and the second fusion protein having the polypeptide sequence of sequence number XX(XENP15908), The first fusion protein having the polypeptide sequence of sequence number XX (XENP17698), and the second fusion protein having the polypeptide sequence of sequence number XX (XENP15908), The first fusion protein having the polypeptide sequence of sequence number XX (XENP17699), and the second fusion protein having the polypeptide sequence of sequence number XX (XENP15908), The first fusion protein having the polypeptide sequence of sequence number XX (XENP17701), and the second fusion protein having the polypeptide sequence of sequence number XX (XENP15908), The first fusion protein having the polypeptide sequence of sequence number XX (XENP17691), and the second fusion protein having the polypeptide sequence of sequence number XX (XENP15908), The first fusion protein having the polypeptide sequence of sequence number XX (XENP17702), and the second fusion protein having the polypeptide sequence of sequence number XX (XENP15908), The first fusion protein having the polypeptide sequence of sequence number XX (XENP17703), and the second fusion protein having the polypeptide sequence of sequence number XX (XENP15908), The first fusion protein having the polypeptide sequence of sequence number XX (XENP17704), and the second fusion protein having the polypeptide sequence of sequence number XX (XENP15908), The first fusion protein having the polypeptide sequence of sequence number XX (XENP17705), and the second fusion protein having the polypeptide sequence of sequence number XX (XENP15908), The first fusion protein having the polypeptide sequence of sequence number xxxiii)XX (XENP18295), and the second fusion protein having the polypeptide sequence of sequence number XX (XENP17761), The first fusion protein having the polypeptide sequence of sequence number xxxiv) XX (XENP18783), and the second fusion protein having the polypeptide sequence of sequence number XX (XENP15908), The first fusion protein having the polypeptide sequence of sequence number XX (XENP18784), and the second fusion protein having the polypeptide sequence of sequence number XX (XENP15908), The first fusion protein having the polypeptide sequence of sequence number XX (XENP18786), and the second fusion protein having the polypeptide sequence of sequence number XX (XENP15908), The first fusion protein having the polypeptide sequence of sequence number XX (XENP18788), and the second fusion protein having the polypeptide sequence of sequence number XX (XENP15908), The first fusion protein having the polypeptide sequence of sequence number XX (XENP19242), and the second fusion protein having the polypeptide sequence of sequence number XX (XENP16481), or A heterodimer protein according to any one of claims 1 to 6, comprising the first fusion protein having the polypeptide sequence of sequence number XX (XENP19243) and the second fusion protein having the polypeptide sequence of sequence number XX (XENP16481).

8. The heterodimer protein according to any one of claims 1 to 7, wherein the first protein domain is directly and covalently attached to the N-terminus of the first Fc domain without using the first domain linker, and / or the second protein domain is directly and covalently attached to the N-terminus of the second Fc domain without using the second domain linker.

9. The aforementioned heterodimer proteins are XENP20818, XENP20819, XENP21471, XENP21472, XENP21473, XENP21474, XENP21475, XENP21476, XENP21477, XENP22013, XENP22815, XE NP22816, XENP22817, XENP22818, XENP22819, XENP22820, XENP22821, XENP2 2822, XENP22823, XENP22824, XENP22825, XENP22826, XENP22827, XENP22828 A heterodimer protein according to any one of claims 1 to 6, selected from the group consisting of XENP22829, XENP22830, XENP22831, XENP22832, XENP22833, XENP22834, XENP23343, XENP23504, XENP23554, XENP23555, XENP23557, XENP23559, XENP24019, XENP24020, XENP24045, XENP24051, XENP24052, XENP24113, XENP24301, XENP24306, and XENP24341.

10. A nucleic acid composition encoding the first fusion protein according to any one of claims 1 to 9.

11. A nucleic acid composition encoding a second fusion protein according to any one of claims 1 to 10.

12. An expression vector comprising the nucleic acid composition according to claim 10.

13. An expression vector comprising the nucleic acid composition according to claim 11.

14. The expression vector according to claim 13, further comprising the nucleic acid composition according to claim 10.

15. A host cell comprising one or more expression vectors according to any one of claims 12 to 14.

16. It is a heterodimer protein, 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 attached to the N-terminus of the second protein domain using a first domain linker, and the second protein domain is covalently attached to the N-terminus of the first Fc domain using a second domain linker, b) Including a second Fc domain, A heterodimer protein in which the first Fc domain and the second Fc domain have a set of amino acid substitutions selected from the group consisting of S267K / 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 contains IL15Rα protein and the second protein domain contains IL15 protein.

17. The heterodimer protein according to claim 16, wherein the first Fc domain and / or the second Fc domain have an additional set of amino acid substitutions, including Q295E / N384D / Q418E / N421D according to EU numbering.

18. The heterodimer protein according to claim 16 or 17, wherein the first Fc domain and / or the second Fc domain have an additional set of amino acid substitutions consisting of G236R / L328R, E233P / L234V / L235A / G236del / S239K, E233P / L234V / L235A / G236del / S267K / A327G, E233P / L234V / L235A / G236del / S267K / A327G, and E233P / L234V / L235A / G236del.

19. The heterodimer protein according to any one of claims 16 to 18, wherein 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 (shortened 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).

20. The heterodimer protein according to any one of claims 16 to 19, wherein the IL15 protein and the IL15Rα protein each have a set of amino acid substitutions 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.

21. The heterodimer protein according to any one of claims 16 to 20, wherein 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).

22. The heterodimer protein according to any one of claims 16 to 21, wherein the heterodimer protein is XENP21478.

23. A nucleic acid composition encoding a fusion protein according to any one of claims 16 to 22.

24. nucleic acid composition encoding a second Fc domain according to any one of claims 16 to 22 。

25. An expression vector comprising the nucleic acid composition according to claim 23.

26. An expression vector comprising the nucleic acid composition according to claim 24.

27. The expression vector according to claim 26, further comprising the nucleic acid composition according to claim 23.

28. A host cell comprising one or two expression vectors according to any one of claims 25 to 27.

29. It is a heterodimer protein, a) A fusion protein comprising a first protein domain and a first Fc domain, wherein the first protein domain is covalently attached to the N-terminus of the first Fc domain using a domain linker, b) The second FC domain, c) comprising a second protein domain non-covalently attached to the first protein domain, A heterodimer protein in which the first Fc domain and the second Fc domain have a set of amino acid substitutions selected from the group consisting of S267K / 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 contains IL15Rα and the second protein domain contains IL15 protein.

30. The heterodimer protein according to claim 29, wherein the first Fc domain and / or the second Fc domain have an additional set of amino acid substitutions comprising Q295E / N384D / Q418E / N421D according to EU numbering.

31. The heterodimer protein according to claim 29 or 30, wherein the first Fc domain and / or the 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.

32. The heterodimer protein according to any one of claims 29 to 31, wherein 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 (shortened 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).

33. The heterodimer protein according to any one of claims 29 to 32, wherein the IL15 protein and the IL15Rα protein each have a set of amino acid substitutions 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.

34. The heterodimer protein according to any one of claims 29 to 33, wherein the fusion protein has a polypeptide sequence selected from the group consisting of SEQ ID NO: XX(16481), SEQ ID NO: XX(17034), SEQ ID NO: XX(17038), SEQ ID NO: XX(17036), SEQ ID NO: XX(17039), SEQ ID NO: XX(17040), SEQ ID NO: XX(17044), SEQ ID NO: XX(17041), SEQ ID NO: XX(17043), SEQ ID NO: XX(17045), SEQ ID NO: XX(17042), SEQ ID NO: XX(15908), and SEQ ID NO: XX(17603).

35. The heterodimer protein according to any one of claims 29 to 34, wherein the second Fc domain has the polypeptide sequence of SEQ ID NO: XX(8793) or SEQ ID NO: XX(8927).

36. The heterodimer protein according to any one of claims 29 to 35, wherein the fusion protein has a polypeptide sequence selected from the group consisting of SEQ ID NO: XX (16484), SEQ ID NO: (17074), SEQ ID NO: (17071), SEQ ID NO: (17072), SEQ ID NO: (17075), SEQ ID NO: (17070), SEQ ID NO: (17073), and SEQ ID NO: (17083).

37. The aforementioned heterodimer protein, i) The fusion protein having the polypeptide sequence of SEQ ID NO: XX(16481), the second Fc domain having the polypeptide sequence of SEQ ID NO: XX(8793), and the second protein domain having the polypeptide sequence of SEQ ID NO: XX(16484), ii) The fusion protein having the polypeptide sequence of SEQ ID NO: XX(17034), the second Fc domain having the polypeptide sequence of SEQ ID NO: XX(8793), and the second protein domain having the polypeptide sequence of SEQ ID NO: XX(16484), iii) The fusion protein having the polypeptide sequence of SEQ ID NO: XX(17038), the second Fc domain having the polypeptide sequence of SEQ ID NO: XX(8793), and the second protein domain having the polypeptide sequence of SEQ ID NO: XX(16484), iv) The fusion protein having the polypeptide sequence of SEQ ID NO: XX(17036), the second Fc domain having the polypeptide sequence of SEQ ID NO: XX(8793), and the second protein domain having the polypeptide sequence of SEQ ID NO: XX(16484), v) The fusion protein having the polypeptide sequence of SEQ ID NO: XX(17038), the second Fc domain having the polypeptide sequence of SEQ ID NO: XX(8793), and the second protein domain having the polypeptide sequence of SEQ ID NO: XX(17074), vi) The fusion protein having the polypeptide sequence of SEQ ID NO: XX(17039), the second Fc domain having the polypeptide sequence of SEQ ID NO: XX(8793), and the second protein domain having the polypeptide sequence of SEQ ID NO: XX(17074), vii) The fusion protein having the polypeptide sequence of SEQ ID NO: XX(17040), the second Fc domain having the polypeptide sequence of SEQ ID NO: XX(8793), and the second protein domain having the polypeptide sequence of SEQ ID NO: XX(17074), viiii) The fusion protein having the polypeptide sequence of SEQ ID NO: XX(17044), the second Fc domain having the polypeptide sequence of SEQ ID NO: XX(8793), and the second protein domain having the polypeptide sequence of SEQ ID NO: XX(17071), ix) The fusion protein having the polypeptide sequence of SEQ ID NO: XX(17044), the second Fc domain having the polypeptide sequence of SEQ ID NO: XX(8793), and the second protein domain having the polypeptide sequence of SEQ ID NO: XX(17072), x) The fusion protein having the polypeptide sequence of SEQ ID NO: XX(17075), the second Fc domain having the polypeptide sequence of SEQ ID NO: XX(8793), and A second protein domain having the polypeptide sequence of sequence number XX (17041), xi) The fusion protein having the polypeptide sequence of SEQ ID NO: XX(17043), the second Fc domain having the polypeptide sequence of SEQ ID NO: XX(8793), and the second protein domain having the polypeptide sequence of SEQ ID NO: XX(17070), xi) The fusion protein having the polypeptide sequence of SEQ ID NO: XX(17045), the second Fc domain having the polypeptide sequence of SEQ ID NO: XX(8793), and the second protein domain having the polypeptide sequence of SEQ ID NO: XX(17073), xiiii) The fusion protein having the polypeptide sequence of SEQ ID NO: XX(17042), the second Fc domain having the polypeptide sequence of SEQ ID NO: XX(8793), and the second protein domain having the polypeptide sequence of SEQ ID NO: XX(17083), or A heterodimer protein according to any one of claims 29 to 36, comprising: the fusion protein having the polypeptide sequence of sequence number XX(15908); the second Fc domain having the polypeptide sequence of sequence number XX(8793); and the second protein domain having the polypeptide sequence of sequence number XX(16484).

38. The heterodimer protein according to any one of claims 29 to 37, wherein the heterodimer protein is selected from the group consisting of XENP21479, XENP22357, XENP22354, XENP22355, XENP22356, XENP22357, XENP22358, XENP22359, XENP22360, XENP22361, XENP22362, XENP22363, XENP22364, XENP22365, XENP22366, XENP22637, XENP24349, and XENP24383.

39. A nucleic acid composition encoding a fusion protein according to any one of claims 29 to 38.

40. A nucleic acid composition encoding a second Fc domain according to any one of claims 29 to 37.

41. An expression vector comprising the nucleic acid composition according to claim 39.

42. An expression vector comprising the nucleic acid composition described in claim 40.

43. The expression vector according to claim 42, further comprising the nucleic acid composition according to claim 39.

44. The expression vector according to any one of claims 41 to 43, further comprising a nucleic acid composition encoding the second protein domain.

45. A host cell comprising one or more expression vectors according to any one of claims 41 to 44.

46. It is a heterodimer protein, a) A first fusion protein comprising a first protein domain and a first Fc domain, wherein the first protein domain is covalently attached to the N-terminus of the first Fc domain using a domain linker, b) A second fusion protein comprising a second heavy chain containing a second protein domain and a first second heavy chain containing 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, c) Non-covalent attachment to the first protein domain of the first fusion protein. The third protein domain, d) comprising a fourth protein domain non-covalently attached to the second protein domain of the second fusion protein, A heterodimer protein in which the first Fc domain and the second Fc domain have a set of amino acid substitutions selected from the group consisting of S267K / 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 according to EU numbering, the first protein domain and the second protein domain contain IL15Rα protein, and the third protein domain and the fourth protein domain contain IL15 protein.

47. The heterodimer protein according to claim 46, wherein the first Fc domain and / or the second Fc domain have an additional set of amino acid substitutions comprising Q295E / N384D / Q418E / N421D according to EU numbering.

48. The heterodimer protein according to claim 46 or 47, wherein the first Fc domain and / or the 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.

49. The heterodimer protein according to any one of claims 46 to 48, wherein 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 (shortened 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).

50. The heterodimer protein according to any one of claims 46 to 49, wherein the IL15 protein and the IL15Rα protein each have a set of amino acid substitutions 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.

51. The aforementioned heterodimer protein, i) The first fusion protein has the polypeptide sequence of SEQ ID NO: XX(17023), the second fusion protein has the polypeptide sequence of SEQ ID NO: XX(17023), the third protein domain has the polypeptide sequence of SEQ ID NO: XX(16484), and the fourth protein domain has the polypeptide sequence of SEQ ID NO: XX(16484), or ii) The heterodimer protein according to any one of claims 46 to 50, wherein the first fusion protein has the polypeptide sequence of SEQ ID NO: XX(17581), the second fusion protein has the polypeptide sequence of SEQ ID NO: XX(17581), the third protein domain has the polypeptide sequence of SEQ ID NO: XX(17074), and the fourth protein domain has the polypeptide sequence of SEQ ID NO: XX(17074).

52. The heterodimer protein according to any one of claims 46 to 51, wherein the heterodimer protein is XENP21978, XENP22634, XENP24342, and XENP24306.

53. A nucleic acid composition encoding the first fusion protein according to any one of claims 46 to 52.

54. A nucleic acid composition encoding a second fusion protein according to any one of claims 46 to 53.

55. An expression vector comprising the nucleic acid composition according to claim 39.

56. An expression vector comprising the nucleic acid composition according to claim 55.

57. The expression vector according to claim 56, further comprising the nucleic acid composition according to claim 53.

58. The expression vector according to any one of claims 55 to 57, further comprising a nucleic acid composition encoding the third protein domain.

59. The expression vector according to any one of claims 55 to 58, further comprising a nucleic acid composition encoding the fourth protein domain.

60. A host cell comprising one or more expression vectors according to any one of claims 56 to 59.

61. It is a heterodimer protein, a) A first fusion protein comprising a first Fc domain and a first protein domain, wherein the first Fc domain is covalently attached to the N-terminus of the first protein domain using a domain linker, b) The second FC domain, c) a second protein domain non-covalently attached to the first protein domain of the first fusion protein, A heterodimer protein in which the first Fc domain and the second Fc domain have a set of amino acid substitutions selected from the group consisting of S267K / 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 contains IL15Rα protein and the second protein domain contains IL15 protein.

62. The heterodimer protein according to claim 61, wherein the first Fc domain and / or the second Fc domain have an additional set of amino acid substitutions comprising Q295E / N384D / Q418E / N421D according to EU numbering.

63. Claim that the first Fc domain and the second Fc domain have 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 A heterodimer protein as described in item 61 or 62.

64. The heterodimer protein according to any one of claims 61 to 63, wherein 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 (shortened 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).

65. The heterodimer protein according to any one of claims 61 to 64, wherein the IL15 protein and the IL15Rα protein each have a set of amino acid substitutions 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.

66. The aforementioned heterodimer protein, i) The fusion protein having the polypeptide sequence of SEQ ID NO: XX(17603), the second Fc domain having the polypeptide sequence of SEQ ID NO: XX(8927), and the second protein domain having the polypeptide sequence of SEQ ID NO: XX(16484), or ii) A heterodimer protein according to any one of claims 61 to 65, comprising the first fusion protein having the polypeptide sequence of SEQ ID NO: XX(17605), the second Fc domain having the polypeptide sequence of SEQ ID NO: XX(8927), and the second protein domain having the polypeptide sequence of SEQ ID NO: XX(17074).

67. The heterodimer protein according to any one of claims 61 to 66, wherein the heterodimer protein is XENP22637 or XENP22639.

68. A nucleic acid composition encoding the first fusion protein according to any one of claims 61 to 67.

69. A nucleic acid composition encoding a second Fc domain according to any one of claims 61 to 67.

70. An expression vector comprising the nucleic acid composition according to claim 68.

71. An expression vector comprising the nucleic acid composition according to claim 69.

72. The expression vector according to claim 71, further comprising the nucleic acid composition according to claim 68.

73. The expression vector according to any one of claims 70 to 72, further comprising a nucleic acid composition encoding the second protein domain.

74. A host cell comprising one or more expression vectors according to any one of claims 70 to 73.

75. 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, XENP2 2831, XENP22832, XENP22833, XENP22834, XENP23343, XENP23504, XENP23554, X ENP23555, XENP23557, XENP23559, XENP24019, XENP24020, XENP24045, XENP240 51, XENP24052, XENP24113, XENP24301, XENP24306, XENP24341, XENP21478, XEN P21479, XENP22357, XENP22354, XENP22355, XENP22356, XENP22357, XENP22358 , XENP22359, XENP22360, XENP22361, XENP22362, XENP22363, XENP22364, XENP2 A heterodimer protein selected from the group consisting of 2365, XENP22366, XENP22637, XENP24349, XENP24383, XENP21978, XENP22634, XENP24342, XENP24306, XENP22637, and XENP22639.

76. XENP20818, XENP20819, XENP21471, XENP21472, XENP21473, XENP21474, XENP21475, XENP21476, XENP21477, XEN P22013, XENP22815, XENP22816, XENP22817, XENP22818, XENP22819, XENP22820, XENP22821, XENP22822, XENP228 23, XENP22824, XENP22825, XENP22826, XENP22827, XENP22828, XENP22829, XENP22830, XENP22831, XENP 22833, XENP 22834, XENP 23343, XENP 23504, XENP 23554, XENP 23555, XENP 23557, 24020, XENP24045, XENP24051, XENP24052, XENP24113, XENP24301, XENP24306, XENP24341, XENP21478, XENP214 79, XENP22357, XENP22354, XENP22355, XENP22356, XENP22357, XENP22358, XENP22359, XENP22360, XENP22361, X A nucleic acid composition comprising one or more nucleic acids encoding a heterodimer protein selected from the group consisting of ENP22362, XENP22363, XENP22364, XENP22365, XENP22366, XENP22637, XENP24349, XENP24383, XENP21978, XENP22634, XENP24342, XENP24306, XENP22637, and XENP22639.

77. An expression vector composition comprising one or more expression vectors, wherein the one or more expression vectors are XENP20818, XENP20819, XENP21471, XENP21472, XENP21473, XENP21474, XENP21475, XENP21476, XENP21477, XENP22013, XENP22 815, XENP22816, XENP22817, XENP22818, XENP22819, XENP22820, XENP22821, XENP22822, X ENP22823, XENP22824, XENP22825, XENP22826, XENP22827, XENP22828, XENP22829, XENP22 830, XENP22831, XENP22832, XENP22833, XENP22834, XENP23343, XENP23504, XENP23554, X ENP23555, XENP23557, XENP23559, XENP24019, XENP24020, XENP24045, XENP24051, XENP24 052, XENP24113, XENP24301, XENP24306, XENP24341, XENP21478, XENP21479, XENP22357, X ENP22354, XENP22355, XENP22356, XENP22357, XENP22358, XENP22359, XENP22360, XENP22 An expression vector composition, each containing nucleic acid, to encode a heterodimer protein selected from the group consisting of 361, XENP22362, XENP22363, XENP22364, XENP22365, XENP22366, XENP22637, XENP24349, XENP24383, XENP21978, XENP22634, XENP24342, XENP24306, XENP22637, and XENP22639.

78. A host cell comprising the nucleic acid composition according to claim 76.

79. A host cell comprising the expression vector composition according to claim 77.

80. A method for producing the heterodimer protein according to claim 75, comprising culturing the host cells according to claim 78 or 79 under appropriate conditions for the expression of the heterodimer protein, and recovering the protein.

81. A method for treating cancer in a patient who requires treatment for cancer, comprising administering to the patient a therapeutically effective amount of the heterodimer protein described in claim 75.

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