PD-1 targeted il-15 / il-alpha 15r fc fusion proteins with improved properties

Abstract

Provided are PD-1 - targeted IL-15 fusion proteins that do not compete with checkpoint blocking antibodies, including cytokine-based treatments and blockade of immune checkpoint proteins such as PD-1, two very promising approaches in cancer immunotherapy.SOLUTION: Fusion proteins comprising a variant IL-15 protein, fusion proteins comprising a variant anti-PD-1 antigen binding domain, and fusion proteins comprising a variant IL-15 protein and a variant anti-PD-1 antigen binding domain are provided. Also provided are nucleic acid molecules, expression vectors, host cells and methods for making such fusion proteins and the use of such fusion proteins in the treatment of cancer.SELECTED DRAWING: Figure 154C

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 62 / 914,265, filed October 11, 2019; U.S. Provisional Application No. 62 / 914,317, filed October 11, 2019; and U.S. Provisional Application No. 63 / 011,208, filed April 16, 2020, the contents of each of which are incorporated herein by reference in their entirety. Sequence Listing

[0002] This application has been submitted electronically in ASCII format and contains a Sequence Listing, which is incorporated herein by reference in its entirety. The ASCII copy, created on October 9, 2020, is titled 000218-0005-WO1_-_Sequence_Listing.txt and is 764,863 bytes in size. [Background technology]

[0003] Two highly promising approaches in cancer immunotherapy include cytokine-based treatments and blockade of immune checkpoint proteins such as PD-1.

[0004] Cytokines such as IL-2 and IL-15 function to support the proliferation and differentiation of B cells, T cells, and NK cells. Both cytokines exert their cell signaling functions by binding to a trimeric complex consisting of two shared receptors: the common gamma chain (γc; CD132) and the IL-2 receptor beta chain (IL-2Rβ; CD122), as well as a unique alpha chain receptor for each cytokine: IL-2 receptor alpha (IL-2Rα; CD25) or IL-15 receptor alpha (IL-15Rα; CD215). Both cytokines are considered potentially beneficial therapeutic agents in oncology, and IL-2 has been approved for use in patients with metastatic renal cell carcinoma and malignant melanoma. Currently, there is no approved use for recombinant IL-15, although several clinical trials are underway. However, as potential drugs, both cytokines undergo very rapid clearance, with half-lives measured in minutes. IL-2 immunotherapy is associated with systemic toxicity when administered at high doses to overcome rapid clearance. Such systemic toxicity has also been reported in recent clinical trials of IL-15 immunotherapy (Guo et al., J Immunol, 2015, 195(5):2353-64).

[0005] Immune checkpoint proteins, such as PD-1, are upregulated after T cell activation to eliminate autoimmunity by depleting activated T cells upon binding to immune checkpoint ligands, such as PD-L1. However, immune checkpoint proteins are also upregulated in tumor-infiltrating lymphocytes (TILs), and immune checkpoint ligands are overexpressed on tumor cells, contributing to immune evasion by tumor cells. De-repression of TILs by blocking immune checkpoint interactions with drugs such as Opdivo® (nivolumab) and Keytruda® (pembrolizumab) has proven highly effective in treating cancer. Despite the promise of checkpoint blockade therapies such as nivolumab and pembrolizumab, many patients fail to achieve an adequate response to checkpoint blockade alone.

[0006] Thus, there is an unmet need for oncological treatments for therapeutic strategies using cytokines that do not require high doses and that target tumors to avoid systemic toxicity. Furthermore, there is a need to identify additional therapeutic modalities to stack with checkpoint blockade that could increase patient response rates. This can be particularly complicated, as the additional therapeutic modality should not compete with checkpoint blockade. The present invention addresses these needs and caveats by providing PD-1-targeted IL-15 fusion proteins that have an improved half-life for an improved safety profile, more selective targeting of TILs, and do not compete with checkpoint blockade antibodies with which they may be combined. Summary of the Invention

[0007] A first aspect of the present invention provides a targeted IL-15 / Rα heterodimeric Fc fusion protein comprising: a) a first monomer comprising, from N-terminus to C-terminus: i) an IL-15 / Rα sushi domain; ii) a first domain linker; iii) an IL-15 domain; and iv) a first variant Fc domain; b) a second monomer comprising a heavy chain comprising VH-CH1-hinge-CH2-CH3, where CH2-CH3 is a second variant Fc domain; and c) a third monomer comprising a light chain comprising VL-CL, wherein the VH and VL domains form an antigen-binding domain (ABD) that binds to human PD-1, and wherein the VH is a variant variable heavy domain comprising F32L / W100F amino acid substitutions, Kabat numbering, compared to SEQ ID NO:5, and the VL is a variant variable light domain comprising N27dH / K30Y / S93T, Kabat numbering, compared to SEQ ID NO:168.

[0008] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 318 and the VL comprises the amino acid sequence of SEQ ID NO: 176.

[0009] In some embodiments, the IL-15 domain is D30N / E64Q / N65D, N1D / N4D / D8N, N1D / N4D / N65D, N1D / D30N, N1D / D61N, N1D / D61N / E64Q / Q108E, N1D / E64Q, N1D / N65D, N1D / Q108E, N4D / D30N, N4D / D61N, N4D / D61N / N65D, N4D / D6 In some embodiments, the IL-15 domain is a variant IL-15 domain comprising an amino acid substitution selected from the group consisting of N71Q, N79Q, N112Q, S114del, and S114A, or a combination thereof. In some embodiments, the IL-15 domain is a variant IL-15 domain comprising the amino acid substitutions D30N / E64Q / N65D and N71Q / N79Q / N112Q.

[0010] In some embodiments, the first variant Fc domain comprises all or a portion of a hinge domain, hi some embodiments, the first monomer further comprises a second domain linker between the IL-15 domain and the first variant Fc domain.

[0011] A second aspect of the present invention provides a targeted IL-15 / Rα heterodimeric Fc fusion protein comprising: a) a first monomer comprising: i) an IL-15 Rα sushi domain protein; ii) a first domain linker; iii) a variant IL-15 protein comprising amino acid substitutions D30N / E64Q / N65D and N71Q / N79Q / N112Q compared to SEQ ID NO: 2; and iv) a first variant Fc domain; b) a second monomer comprising a heavy chain comprising VH-CH1-hinge-CH2-CH3, where CH2-CH3 is a second variant Fc domain; and c) a third monomer comprising a light chain comprising VL-CL, wherein the VH domain and VL domain form an antigen-binding domain (ABD) that binds to human PD-1.

[0012] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 5 and the VL comprises the amino acid sequence of SEQ ID NO: 168. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 318 and the VL comprises the amino acid sequence of SEQ ID NO: 176. In some embodiments, the ABD does not compete with nivolumab and / or pembrolizumab for binding to human PD-1.

[0013] In some embodiments, the first variant Fc domain comprises all or a portion of a hinge domain, hi some embodiments, the first monomer further comprises a second domain linker between the IL-15 domain and the first variant Fc domain.

[0014] A third aspect of the present invention provides a) a first monomer comprising: i) an IL-15Rα sushi domain protein; ii) a first domain linker; iii) a variant IL-15 protein comprising amino acid substitutions D30N / E64Q / N65D and N71Q / N79Q / N112Q compared to SEQ ID NO: 2; and iv) a first variant Fc domain; b) a second monomer comprising a heavy chain comprising VH-CH1-hinge-CH2-CH3, where CH2-CH3 is a second variant Fc domain; and c) a third monomer comprising a light chain comprising VL-CL. The present invention provides a targeted IL-15 / Rα heterodimeric Fc fusion protein comprising three monomers, wherein the VH domain and the VL domain form an antigen-binding domain (ABD) that binds to human PD-1, wherein the VH is a variant variable heavy domain comprising F32L / W100F amino acid substitutions, Kabat numbering, compared to SEQ ID NO:5, and the VL is a variant variable light domain comprising N27dH / K30Y / S93T, Kabat numbering, compared to SEQ ID NO:168.

[0015] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 318 and the VL comprises the amino acid sequence of SEQ ID NO: 176.

[0016] In some embodiments, the first variant Fc domain comprises all or a portion of a hinge domain, hi some embodiments, the first monomer further comprises a second domain linker between the IL-15 domain and the first variant Fc domain.

[0017] A fourth aspect of the present invention provides a variant IL-15 protein comprising: a) a first monomer comprising, from N-terminus to C-terminus: i) an IL-15 Ra sushi domain protein; ii) a first domain linker; iii) a variant IL-15 protein comprising an amino acid substitution selected from the group consisting of N71Q, N79Q, N112Q, S114del, and S114A relative to SEQ ID NO: 2; and iv) a first variant Fc domain; b) a second monomer comprising a heavy chain comprising VH-CH1-hinge-CH2-CH3, where CH2-CH3 is a second variant Fc domain. and c) a third monomer comprising a light chain comprising VL-CL, wherein the VH domain and the VL domain form an antigen-binding domain that binds to human PD-1, and the VH domain contains the following amino acid substitutions, relative to SEQ ID NO: 5, according to the Kabat numbering: W100F, F32L, S52aG, R97E, R97Y, R97W, L98R, S100aT, R97A, V99T, V99L, S100aT, R97A, V99T, V99L, S100bT, S100cT, S100dT, S100dT, S100eT, S100fT, S100fT, S100g ... and at least one further amino acid substitution selected from the group consisting of: aA, L98Q, R97Q, V99F, V99L, S100aN, V99I, P100bS, G96H, L98V, V99A, V99Q, G96V, R97K, L98S, L98F, R97T, L98K, L98S, V99I, R97L, G96A, R97A, V99S, R97S, V99Y, R97H, L98R, wherein the VL domain comprises i) the sequence and ii) a variant light domain comprising an amino acid substitution selected from the group consisting of N27dH, N27dS, K30Y, S93T, and Y94W, Kabat numbering, relative to SEQ ID NO: 168, wherein the variant IL-15 protein comprises an amino acid substitution selected from the group consisting of N71Q / N79Q, N71Q / N79Q / N112Q, N71Q / N79Q / S114del, and N71Q / N79Q / S114A.

[0018] In some embodiments, the variant IL-15 protein is D30N / E64Q / N65D, N1D / N4D / D8N, N1D / N4D / N65D, N1D / D30N, N1D / D61N, N1D / D61N / E64Q / Q108E, N1D / E64Q, N1D / N65D, N1D / Q108E, N4D / D30N, N4D / D61N, N4D / D61N / N and N65D / Q108E.

[0019] In some embodiments, the variant heavy domain comprises the amino acid sequence of SEQ ID NO:318 and the variant light domain comprises the amino acid sequence of SEQ ID NO:176.

[0020] In some embodiments, the variant IL-15 protein comprises the amino acid substitutions N71Q / N79Q / N112Q. In some embodiments, the variant IL-15 protein comprises the amino acid substitutions N71Q / N79Q / N112Q and D30N / N65D. In some embodiments, the variant IL-15 protein comprises the amino acid substitutions N71Q / N79Q / N112Q and D30N / E64Q / N65D. In some embodiments, the variant heavy domain is H1.176 (SEQ ID NO: 318), the variant light domain is L1.140 (SEQ ID NO: 176), and the variant IL-15 protein comprises the amino acid substitutions N71Q / N79Q / N112Q and D30N / N65D. In some embodiments, the variant heavy domain is H1.176 (SEQ ID NO: 318), the variant light domain is L1.140 (SEQ ID NO: 176), and the variant IL-15 protein comprises amino acid substitutions N71Q / N79Q / N112Q and D30N / E64Q / N65D.

[0021] In some embodiments, the first domain linker comprises GGGGA (SEQ ID NO: 8). In some embodiments, the first variant Fc domain comprises all or a portion of a hinge domain. In some embodiments, the first monomer further comprises a second domain linker between the IL-15 domain and the first variant Fc domain.

[0022] A fifth aspect of the present invention provides a polypeptide comprising: a) from N-terminus to C-terminus: i) an IL-15 Ra sushi domain protein; ii) a first domain linker; iii) a variant IL-15 protein; and and c) a third monomer comprising a light chain comprising VL-CL, wherein the VH domain and the VL domain form an antigen-binding domain that binds to human PD-1, and the VH has the following amino acid substitutions, according to the Kabat numbering, relative to SEQ ID NO: 5: W100F, F32L, S52aG, R97E, R97Y, R97W, L98R, S100aT, R97A, V99T, V99L, S100aA, L98Q. , R97Q, V99F, V99L, S100aN, V99I, P100bS, G96H, L98V, V99A, V99Q, G96V, R97K, L98S, L98F, R97T, L98K, L98S, V99I, R97L, G96A, R97A, V99S, R97S, V99Y, R97H, L98R, and wherein the VL domain is selected from the group consisting of: i) SEQ ID NO: 168; and ii) a variant light domain comprising an amino acid substitution selected from the group consisting of N27dH, N27dS, K30Y, S93T and Y94W according to the Kabat numbering compared to SEQ ID NO: 168.

[0023] In some embodiments, the variant heavy domain is selected from the group consisting of H1.176, H1.177, H1.178, H1.179, H1.180, H1.181, H1.182, H1.183, H1.184, H1.185, H1.186, H1.187, H1.188, H1.189, H1.190, H1.191, H1.192, H1.193, H1.194, H1.195, H1.196, H1.197, H1.198, H1.199 In some embodiments, the variant light domain is selected from the group consisting of L1.1, L1.3, L1.45, L1.117, L1.129, L1.135, L1.136, and L1.140. In some embodiments, the variant heavy domain comprises the amino acid sequence of SEQ ID NO:318 and the variant light domain comprises the amino acid sequence of SEQ ID NO:176.

[0024] In some embodiments, the first variant Fc domain comprises all or a portion of a hinge domain, hi some embodiments, the first monomer further comprises a second domain linker between the IL-15 domain and the first variant Fc domain.

[0025] A sixth aspect of the present invention provides a targeted IL-15 / Rα heterodimeric Fc fusion protein comprising: a) a first monomer comprising, from N-terminus to C-terminus: i) an IL-15 Rα sushi domain protein; ii) a first domain linker; iii) a variant IL-15 protein comprising an amino acid substitution selected from the group consisting of N71Q, N79Q, N112Q, S114del, and S114A relative to SEQ ID NO:2; and iv) a first variant Fc domain; b) a second monomer comprising a heavy chain comprising VH-CH1-hinge-CH2-CH3, where CH2-CH3 is a second variant Fc domain; and c) a third monomer comprising a light chain comprising VL-CL, wherein the VH domain and the VL domain form an antigen-binding domain that binds to human PD-1 and does not compete with nivolumab and / or pembrolizumab for binding to human PD-1.

[0026] In some embodiments, the variant IL-15 protein comprises an amino acid substitution selected from the group consisting of N71Q / N79Q, N71Q / N79Q / N112Q, N71Q / N79Q / S114del, and N71Q / N79Q / S114A. In some embodiments, the variant IL-15 protein comprises an amino acid substitution selected from the group consisting of N1D / N4D / D8N, N1D / N4D / N65D, N1D / D30N, N1D / D61N, N1D / D61N / E64Q / Q108E, N1D / E64Q, N1D / N65D, N1D / Q108E, N4D / D30N, N4D / D61N, N4D / D61N / N65D, N4D / D61N / In some embodiments, the variant IL-15 protein comprises the amino acid substitutions N71Q / N79Q / N112Q. In some embodiments, the variant IL-15 protein comprises the amino acid substitutions N71Q / N79Q / N112Q and D30N / N65D. In some embodiments, the variant IL-15 protein comprises the amino acid substitutions N71Q / N79Q / N112Q and D30N / E64Q / N65D.

[0027] In some embodiments, the first domain linker is GGGGA (SEQ ID NO: 8). In some embodiments, the first variant Fc domain comprises all or a portion of a hinge domain. In some embodiments, the first monomer further comprises a second domain linker between the IL-15 domain and the first variant Fc domain.

[0028] In some embodiments of any of the above aspects, the first variant Fc domain and the second variant Fc domain comprise amino acid substitutions selected from the group consisting of S364K / E357Q:L368D / K370S; L368D / K370S:S364K; L368E / K370S:S364K; T411E / K360E / Q362E:D401K; L368D / K370S:S364K / E357L,K370S:S364K / E357Q,T366S / L368A / Y407V:T366W and T366S / L368A / Y407V / Y349C:T366W / S354C (according to EU numbering). In some embodiments of any of the above aspects, the first variant Fc domain comprises L368D / K370S and the second variant Fc domain comprises S364K / E357Q (according to EU numbering).

[0029] In some embodiments of any of the above aspects, the first variant Fc domain and the second variant Fc domain each independently comprise an amino acid substitution selected from the group consisting of: G236R / L328R, E233P / L234V / L235A / G236del / S239K, E233P / L234V / L235A / G236del / S267K, E233P / L234V / L235A / G236del / S239K / A327G, E233P / L234V / L235A / G236del / S267K / A327G, and E233P / L234V / L235A / G236del, according to EU numbering. In some embodiments of any of the above aspects, the first variant Fc domain and the second variant Fc domain each comprise amino acid substitutions E233P / L234V / L235A / G236del / S267K (EU numbering).

[0030] In some embodiments of any of the above aspects, the first Fc domain comprises amino acid substitutions Q295E / N384D / Q418E / N481D (EU numbering).

[0031] In some embodiments of any of the above aspects, the first variant Fc domain and the second variant Fc domain each comprise amino acid substitutions M428L / N434S (EU numbering).

[0032] In some embodiments of any of the above aspects, the first monomer comprises the amino acid sequence of SEQ ID NO: 225. In some embodiments of any of the above aspects, the second monomer comprises the amino acid sequence of SEQ ID NO: 244. In some embodiments of any of the above aspects, the third monomer comprises the amino acid sequence of SEQ ID NO: 196. In some embodiments of any of the above aspects, the first monomer comprises the amino acid sequence of SEQ ID NO: 225, the second monomer comprises the amino acid sequence of SEQ ID NO: 244, and the third monomer comprises the amino acid sequence of SEQ ID NO: 196.

[0033] A seventh aspect of the present invention relates to a method for the preparation of a nucleotide sequence comprising: a) a nucleotide sequence which, compared to SEQ ID NO: 5, has the following amino acid substitutions, according to the Kabat numbering: W100F; F32L, S52aG, R97E, R97Y, R97W, L98R, S100aT, R97A, V99T, V99L, S100aA, L98Q, R97Q, V99F, V99L, S100aN, V99I, P100bS, G96H, L98V, V99A, V99Q, G96V, R97K, L98S, L98F, R97T, L98K, L98S, V99I, R97L, G96A, R97A, V99S, R97S, and b) a variant variable heavy domain comprising at least one additional amino acid substitution selected from the group consisting of V99Y, R97H, L98R, and at least one additional amino acid substitution selected from the group consisting of: i) SEQ ID NO: 168; and ii) a variant light domain comprising an amino acid substitution selected from the group consisting of N27dH, N27dS, K30Y, S93T, and Y94W, Kabat numbering, relative to SEQ ID NO: 168, wherein the ABD binds to human PD-1.

[0034] In some embodiments, the variant heavy domain has the amino acid substitutions F32L / W100F according to the Kabat numbering, and the variant light domain has the amino acid substitutions N27dH / K30Y / S93T according to the Kabat numbering.

[0035] In some embodiments, the variant heavy domain is selected from the group consisting of H1.176, H1.177, H1.178, H1.179, H1.180, H1.181, H1.182, H1.183, H1.184, H1.185, H1.186, H1.187, H1.188, H1.189, H1.190, H1.191, H1.192, H1.193, H1.194, H1.195, H1.196, H1.197, H1.198, H1.199 In some embodiments, the variant light domain is selected from the group consisting of L1.1, L1.3, L1.45, L1.117, L1.129, L1.135, L1.136, and L1.140. In some embodiments, the variant heavy domain comprises the amino acid sequence of SEQ ID NO:318 and the variant light domain comprises the amino acid sequence of SEQ ID NO:176.

[0036] In some embodiments, the composition comprises a full-length anti-PD-1 antibody. In some embodiments, the composition comprises a fusion protein. In some embodiments, the fusion protein is XENP32435.

[0037] An eighth aspect of the invention provides a composition comprising a variant IL-15 protein, relative to SEQ ID NO: 2, wherein the variant IL-15 protein comprises an amino acid substitution selected from the group consisting of N71Q, N79Q, N112Q, S114del and S114A.

[0038] In some embodiments, the variant IL-15 protein further comprises an amino acid substitution selected from the group consisting of N1D, N4D, D8N, D30N, D61N, E64Q, N65D and Q108E.

[0039] In some embodiments, the variant IL-15 protein comprises amino acid substitutions selected from the group consisting of N71Q / N79Q, N71Q / N79Q / N112Q, N71Q / N79Q / S114del, and N71Q / N79Q / S114A. In some embodiments, the variant IL-15 protein comprises amino acid substitutions selected from the group consisting of N1D / N4D / D8N, N1D / N4D / N65D, N1D / D30N, N1D / D61N, N1D / D61N / E64Q / Q108E, N1D / E64Q, N1D / N65D, N1D / Q108E, N4D / D30N, N4D / D61N, N4D / D61N / N65D, N4D / D61N / E64Q / Q108E , N4D / E64Q, N4D / N65D, D8N / D61N, D8N / E64Q, D30N / E64Q, D30N / N65D, D30N / E64Q / N65D, D30N / Q180E, D61N / E64Q / N65D, E64Q / N65D, E64Q / Q108E, and N65D / Q108E. In some embodiments, the variant IL-15 protein comprises the amino acid substitutions N71Q / N79Q / N112Q. In some embodiments, the variant IL-15 protein comprises the amino acid substitutions N71Q / N79Q / N112Q and D30N / N65D. In some embodiments, the variant IL-15 protein comprises the amino acid substitutions N71Q / N79Q / N112Q and D30N / E64Q / N65D.

[0040] A ninth aspect of the present invention provides a heterodimeric protein comprising: a) a first fusion protein comprising i) a variant IL-15 protein comprising amino acid substitutions D30N / E64Q / N65D and N71Q / N79Q / N112Q compared to SEQ ID NO: 2; ii) a domain linker; and iii) a first variant Fc domain; and b) a second fusion protein comprising i) an IL-15Rα sushi domain; ii) a domain linker; and iii) a second variant Fc domain.

[0041] In some embodiments, the variant IL-15 fusion protein comprises the amino acid sequence of SEQ ID NO:319.

[0042] In some embodiments, the first variant Fc domain and the second variant Fc domain comprise amino acid substitutions selected from the group consisting of S364K / E357Q:L368D / K370S; L368D / K370S:S364K; L368E / K370S:S364K; T411E / K360E / Q362E:D401K; L368D / K370S:S364K / E357L,K370S:S364K / E357Q,T366S / L368A / Y407V:T366W and T366S / L368A / Y407V / Y349C:T366W / S354C (according to EU numbering). In some embodiments, the first variant Fc domain comprises L368D / K370S and the second variant Fc domain comprises S364K / E357Q (according to EU numbering).

[0043] In some embodiments, the first variant Fc domain and the second variant Fc domain comprise 428L / 434S.

[0044] In some embodiments, the first fusion protein comprises the amino acid sequence of SEQ ID NO: 208, and the second fusion protein comprises the amino acid sequence of SEQ ID NO: 95. In some embodiments, the first fusion protein comprises the amino acid sequence of SEQ ID NO: 211, and the second fusion protein comprises the amino acid sequence of SEQ ID NO: 206.

[0045] Further aspects of the invention provide nucleic acid molecules, nucleic acid compositions, expression vectors, expression vector compositions, host cells, and methods for expressing (a) any of the above fusion proteins; (b) any of the above anti-PD-1 ABDs; (c) any of the above compositions comprising a variant IL-15 protein; or (d) any of the above heterodimeric proteins.

[0046] A further aspect of the invention provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and (a) any of the fusion proteins described above; (b) any of the anti-PD-1 ABDs described above; (c) any of the compositions described above comprising a variant IL-15 protein; or (d) any of the heterodimeric proteins described above.

[0047] A further aspect of the present invention provides a method of treating cancer in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of (a) any of the fusion proteins described above; (b) any of the anti-PD-1 ABDs described above; (c) any of the compositions comprising a variant IL-15 protein described above; (d) any of the heterodimeric proteins described above; or (e) any of the pharmaceutical compositions described above.

[0048] In some embodiments, the method further comprises administering a therapeutically effective amount of a checkpoint blockade antibody. In some embodiments, the checkpoint blockade antibody is an anti-PD-1 antibody or an anti-PD-L1 antibody. In some embodiments, the checkpoint blockade antibody is nivolumab or pembrolizumab.

[0049] Further aspects of the invention provide for the use of (a) any of the above fusion proteins; (b) any of the above anti-PD-1 ABDs; (c) any of the above compositions comprising a variant IL-15 protein; (d) any of the above heterodimeric proteins; or (e) any of the above pharmaceutical compositions in the manufacture of a medicament for treating cancer in a subject in need thereof.

[0050] In some embodiments, the medicament is formulated to be administered in combination with a therapeutically effective amount of a checkpoint blockade antibody. In some embodiments, the checkpoint blockade antibody is an anti-PD-1 antibody or an anti-PD-L1 antibody. In some embodiments, the checkpoint blockade antibody is nivolumab or pembrolizumab.

[0051] Further aspects of the invention provide (a) any of the above fusion proteins; (b) any of the above anti-PD-1 ABDs; (c) any of the above compositions comprising a variant IL-15 protein; (d) any of the above heterodimeric proteins; or (e) any of the above pharmaceutical compositions, for use in treating cancer in a subject in need thereof.

[0052] In some embodiments, the fusion protein anti-PD-1 ABD, composition comprising a variant IL-15 protein, heterodimeric protein, or pharmaceutical composition is administered in combination with a therapeutically effective amount of a checkpoint blockade antibody. In some embodiments, the checkpoint blockade antibody is an anti-PD-1 antibody or an anti-PD-L1 antibody. In some embodiments, the checkpoint blockade antibody is nivolumab or pembrolizumab. [Brief explanation of the drawings]

[0053] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0054] [Figure 1] 1A and 1B show the sequences of IL-15 and its receptor.

[0055] [Figure 2] Figure 2 shows the sequences of both human and cynomolgus PD-1 to facilitate the development of antigen-binding domains that bind to both human and cynomolgus monkeys to facilitate clinical development.

[0056] [Figure 3] Figures 3A-3E show useful pairs of Fc heterodimerization variant sets (including scuba variants and pI variants). There are variants for which there is no corresponding "monomer 2" variant. These are pI variants that can be used alone on either monomer.

[0057] [Figure 4] Figure 4 shows a list of isosteric variant antibody constant regions and their respective substitutions. pI_(-) indicates a lower pI variant and pI_(+) indicates a higher pI variant. These can be optionally and independently combined with other heterodimerization variants of the invention (and other variant types as well as outlined herein).

[0058] [Figure 5] Figure 5 shows useful deletion variants (sometimes called "knockout" or "KO" variants) that abolish FcγR binding. Generally, deletion variants are found in both monomers, but in some cases may be found in only one monomer.

[0059] [Figure 6] 6A-6E show particularly useful embodiments of the "non-cytokine" component of the IL-15 / Rα-Fc fusion protein of the invention.

[0060] [Figure 7] 7A-7F show particularly useful embodiments of the "non-cytokine" / "non-Fv" component of the IL-15 / Rα x anti-PD-1 bifunctional protein of the present invention.

[0061] [Figure 8]Figure 8 shows some exemplary variable length domain linkers for use in IL-15 / Rα-Fc fusion proteins. In some embodiments, these domain linkers find use linking the C-terminus of IL-15 and / or IL-15Rα(sushi) to the N-terminus of the Fc region. In some embodiments, these domain linkers find use fusing IL-15 to IL-15Rα(sushi). In some embodiments, these domain linkers find use linking a single-chain Fv to an Fc chain. In some embodiments, the domain linker is an scFv linker used to link the VH and VL domains and may optionally be charged. In some embodiments, these linkers may be combined. For example, a GGGGS linker may be combined with a "half-hinge" linker.

[0062] [Figure 9] Figure 9 shows several charged scFv linkers that can be used to increase or decrease the pI of heterodimeric antibodies that utilize one or more scFv components. (+H) positive linkers find particular use herein. A single prior art scFv linker bearing a single charge is referred to as "Whitlow" after Whitlow et al., Protein Engineering 6(8):989-995 (1993). It should be noted that this linker was used in scFvs to reduce aggregation and enhance proteolytic stability. In some embodiments, these linkers find use linking the C-terminus of IL-15 and / or IL-15Rα(sushi) to the N-terminus of the Fc region; and / or linking IL-15 to IL-15 / Rα(sushi).

[0063] [Figure 10]Figures 10A-10D show the sequences of several useful IL-15 / Rα-Fc format scaffolds based on human IgG1 that do not contain cytokine sequences (e.g., IL-15 and / or IL-15Rα(sushi)). It is important to note that these scaffolds may also find use in certain embodiments of PD-1-targeted IL-15 / Rα-Fc fusion proteins. Scaffold 1 is based on human IgG1 (356E / 358M allotype) and contains the C220S, S364K / E357Q:L368D / K370S scuba variants on both chains, the Q295E / N384D / Q418E / N421D pI variant on one chain with the L368D / K370S scuba variant, and the E233P / L234V / L235A / G236del / S267K deletion variant on both chains. Scaffold 2 is based on human IgG1 (356E / 358M allotype) and contains the C220S, S364K:L368D / K370S scubariant on both chains, the Q295E / N384D / Q418E / N421D pI variant on the chain with the L368D / K370S scubariant, and the E233P / L234V / L235A / G236del / S267K deletion variants on both chains. Scaffold 3 is based on human IgG1 (356E / 358M allotype) and contains the C220S, S364K:L368E / K370S scubariant on both chains, the Q295E / N384D / Q418E / N421D pI variant on the chain with the L368E / K370S scubariant, and the E233P / L234V / L235A / G236del / S267K deletion variants on both chains. Scaffold 4 is based on human IgG1 (356E / 358M allotype) and contains C220S on both chains, D401K:K360E / Q362E / T411E scubariant, Q295E / N384D / Q418E / N421D pI variants on the chain with the K360E / Q362E / T411E scubariant, and E233P / L234V / L235A / G236del / S267K deletion variants on both chains.Scaffold 5 is based on human IgG1 (356D / 358L allotype) and contains C220S on both chains, S364K / E357Q:L368D / K370S scubariant, Q295E / N384D / Q418E / N421D pI variants on the chain with the L368D / K370S scubariant, and E233P / L234V / L235A / G236del / S267K deletion variants on both chains. Scaffold 6 is based on human IgG1 (356E / 358M allotype) and contains C220S on both chains, S364K / E357Q:L368D / K370S variants, Q295E / N384D / Q418E / N421D pI variants on the chain with the L368D / K370S variant, and E233P / L234V / L235A / G236del / S267K deletion variants on both chains, and N297A variants on both chains. Scaffold 7 is identical to 6 except for the N297S mutation. Alternative formats of scaffolds 6 and 7 can omit the deletion variants E233P / L234V / L235A / G236del / S267K on both chains. Scaffold 8 is based on human IgG4 and contains the S364K / E357Q:L368D / K370S scuba variant, the Q295E / N384D / Q418E / N421D pI variant on the chain with the L368D / K370S scuba variant, and the S228P (EU numbering, which is S241P in Kabat) variant on both chains, which eliminates Fab arm exchange as known in the art. Scaffold 9 is based on human IgG2 and contains the S364K / E357Q:L368D / K370S scuba variant, the Q295E / N384D / Q418E / N421D pI variant on the chain with the L368D / K370S scuba variant. Scaffold 10 is based on human IgG2 and contains the S364K / E357Q:L368D / K370S scubariant, the Q295E / N384D / Q418E / N421D pI variant on the chain with the L368D / K370S scubariant, and the S267K variant on both chains. Scaffold 11 is identical to Scaffold 1 except that it contains the M428L / N434S Xtend mutations.Scaffold 12 is based on human IgG1 (356E / 358M allotype) and contains C220S on both identical chains, E233P / L234V / L235A / G236del / S267K deletion variants on both identical chains, and Scaffold 13 is based on human IgG1 (356E / 358M allotype) and contains C220S on both chains, S364K / E357Q:L368D / K370S scuba variants, P217R / P228R / N276K pI variants on one chain with the S364K / E357Q scuba variant, and E233P / L234V / L235A / G236del / S267K deletion variants on both chains.

[0064] As will be appreciated by those skilled in the art and outlined below, these sequences can be used with any of the IL-15 and IL-15Rα(sushi) pairs outlined herein, including, but not limited to, IL-15 / Rα-heteroFc, ncIL-15 / Rα, and scIL-15 / Rα, as shown schematically in Figures 14A-14G. Additionally, any IL-15 and / or IL-15Rα(sushi) variants can be incorporated into these scaffolds of Figures 10A-10C in any combination.

[0065] Each of these scaffolds includes sequences that are 90, 95, 98, and 99% identical (as defined herein) to the listed sequence, and / or 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions (compared to the "parent" in the diagram, which already includes a number of amino acid modifications compared to the parent human IgG1 (or IgG2 or IgG4, depending on the scaffold), as will be understood by those skilled in the art). That is, the listed scaffolds may include additional amino acid modifications (generally amino acid substitutions) in addition to the skew, pI, and deletion variants included within the scaffold in this diagram.

[0066] [Figure 11]Figure 11 shows the sequences of several useful PD-1-targeting IL-15 / Rα-Fc fusion format scaffolds based on human IgG1 that do not include cytokine sequences (e.g., IL-15 and / or IL-15Rα(sushi)) or VH, and further exclude the cognate light chain scaffolds shown in Figure 12. Scaffold 1 is based on human IgG1 (356E / 358M allotype) and contains the S364K / E357Q:L368D / K370S scuba variant, the C220S and Q295E / N384D / Q418E / N421D pI variants on the chain with the L368D / K370S scuba variant, and the E233P / L234V / L235A / G236del / S267K deletion variants on both chains. Scaffold 2 is based on human IgG1 (356E / 358M allotype) and contains the S364K / E357Q:L368D / K370S scubariant, N208D / Q295E / N384D / Q418E / N421D pI variants on the chain with the L368D / K370S scubariant, C220S within the chain with the S364K / E357Q variant, and E233P / L234V / L235A / G236del / S267K deletion variants on both chains. Scaffold 3 is based on human IgG1 (356E / 358M allotype) and contains the S364K / E357Q:L368D / K370S scuba variant, the N208D / Q295E / N384D / Q418E / N421D pI variants on the chain carrying the L368D / K370S scuba variant, the Q196K / I199T / P217R / P228R / N276K pI variants on the chain carrying the S364K / E357Q variant, and the E233P / L234V / L235A / G236del / S267K deletion variants on both chains.

[0067] In certain embodiments, these sequences may be of the 356D / 358L allotype. In other embodiments, these sequences may include either the N297A or N297S substitution. In some other embodiments, these sequences may include the M428L / N434S Xtend mutation. In still other embodiments, these sequences may instead be based on human IgG4 and include the S228P (EU numbering, which is S241P in Kabat) variant on both chains, which eliminates Fab arm exchange as known in the art. In still further embodiments, these sequences may instead be based on human IgG2. Furthermore, these sequences may instead utilize other scuba variants, pI variants, and deletion variants shown in Figures 3A-3E, 4, and 5.

[0068] As will be appreciated by those of skill in the art and outlined below, these sequences can be used with any IL-15 and IL-15Rα(sushi) pair outlined herein, including, but not limited to, scIL-15 / Rα, ncIL-15 / Rα, and dsIL-15Rα, as shown schematically in Figure 53. Furthermore, as will be appreciated by those of skill in the art and outlined below, any IL-15 and / or IL-15Rα(sushi) variant can be incorporated into these scaffolds. Furthermore, as will be appreciated by those of skill in the art and outlined below, these sequences can be used with any VH and VL pair outlined herein, including either scFv or Fab.

[0069] Each of these scaffolds includes sequences that are 90, 95, 98, and 99% identical (as defined herein) to the listed sequence, and / or 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions (compared to the "parent" in the diagram, which already includes a number of amino acid modifications compared to the parent human IgG1 (or IgG2 or IgG4, depending on the scaffold), as will be understood by those skilled in the art). That is, the listed scaffolds may include additional amino acid modifications (generally amino acid substitutions) in addition to the skew, pI, and deletion variants included within the scaffold in this diagram.

[0070] [Figure 12] FIG. 12 shows a "non-Fv" scaffold of the cognate light chain (ie, constant light chain) that finds use in the PD-1-targeting IL-15 / Rα-Fc fusion proteins of the invention.

[0071] [Figure 13]Figures 13A-13G show several formats for the IL-15 / Rα-Fc fusion proteins of the present invention. IL-15Rα heterodimeric Fc fusions or "IL-15 / Rα-hetero-Fc" (Figure 13A) contain IL-15 recombinantly fused to one side of the heterodimeric Fc and IL-15Rα(sushi) recombinantly fused to the other side of the heterodimeric Fc. IL-15 and IL-15Rα(sushi) can have Gly-Ser linkers of variable length between the C- and N-termini of the Fc regions. The single-chain IL-15 / Rα-Fc fusion or "scIL-15 / Rα-Fc" (Figure 13B) contains IL-15Rα(sushi) fused to IL-15 by a variable-length linker (referred to as "single-chain" IL-15 / IL-15Rα(sushi) complex or "scIL-15 / Rα"), which is then fused to the N-terminus of a heterodimeric Fc region; the other side of the molecule is "Fc-only" or "empty Fc." The noncovalent IL-15 / Rα-Fc or "ncIL-15 / Rα-Fc" (Figure 13C) contains IL-15Rα(sushi) fused to a heterodimeric Fc region, but IL-15 is transfected separately to form a noncovalent IL-15 / Rα complex; the other side of the molecule is "Fc-only" or "empty Fc." The bivalent noncovalent IL-15 / Rα-Fc fusion or "bivalent ncIL-15 / Rα-Fc" (Figure 13D) contains IL-15Rα(sushi) fused to the N-terminus of the homodimeric Fc region, whereas IL-15 is transfected separately to form a noncovalent IL-15 / Rα complex. The bivalent single-chain IL-15 / Rα-Fc fusion or "bivalent scIL-15 / Rα-Fc" (Figure 13E) contains IL-15 fused to IL-15Rα(sushi) by a variable-length linker (referred to as "single-chain" IL-15 / IL-15Rα(sushi) complex or "scIL-15 / Rα"), which is then fused to the N-terminus of the homodimeric Fc region. The Fc-noncovalent IL-15 / Rα fusion or "Fc-ncIL-15 / Rα" (Figure 13F) contains IL-15Rα(sushi) fused to the C-terminus of the heterodimeric Fc region, whereas IL-15 is transfected separately such that a noncovalent IL-15 / Rα complex is formed, and the other side of the molecule is "Fc only" or "empty Fc."The Fc-single-chain IL-15 / Rα fusion or "Fc-scIL-15 / Rα" (Figure 13G) comprises IL-15 fused to IL-15Rα(sushi) by a variable length linker (referred to as "single-chain" IL-15 / IL-15Rα(sushi) complex or "scIL-15 / Rα"), which is then fused to the C-terminus of a heterodimeric Fc region, with the other side of the molecule being "Fc only" or "empty Fc."

[0072] [Figure 14] Figure 14 shows the sequence of an exemplary IL-15 / Rα-Fc fusion protein in the "IL-15 / Rα-hetero-Fc" format. IL-15 and IL-15Rα (sushi) are underlined, the linker is double underlined (those skilled in the art will appreciate that the linker can be replaced with other linkers, some of which are shown in Figure 8), and a slash ( / ) indicates the boundaries of the IL-15, IL-15Rα, linker, and Fc regions.

[0073] [Figure 15] Figure 15 shows the sequence of an exemplary IL-15 / Rα-Fc fusion protein in the "scIL-15 / Rα-Fc" format. IL-15 and IL-15Rα (sushi) are underlined, the linker is double underlined (those skilled in the art will appreciate that the linker can be replaced with other linkers, some of which are shown in Figure 8), and a slash ( / ) indicates the boundaries of the IL-15, IL-15Rα, linker, and Fc regions.

[0074] [Figure 16] Figure 16 shows the sequence of an exemplary IL-15 / Rα-Fc fusion protein in the "ncIL-15 / Rα-Fc" format. IL-15 and IL-15Rα (sushi) are underlined, the linker is double underlined (those skilled in the art will appreciate that the linker can be replaced with other linkers, some of which are shown in Figure 8), and a slash ( / ) indicates the boundaries of the IL-15, IL-15Rα, linker, and Fc regions.

[0075] [Figure 17]Figures 17A-17C show induction of A) NK (CD56+ / CD16+) cell, B) CD4+ T cell, and C) CD8+ T cell proliferation by exemplary IL-15 / Rα-Fc fusion proteins in scIL-15 / Rα-Fc format (XENP21478) and ncIL-15 / Rα-Fc format (XENP21479) based on Ki67 expression measured by FACS.

[0076] [Figure 18] Figure 18 shows the structure of IL-15 complexed with IL-15Rα, IL-2Rβ, and the common γ chain. The locations of substitutions designed to reduce potency are indicated.

[0077] [Figure 19] 19A-19C show the sequences of exemplary IL-15 variants engineered for reduced potency. These variant IL-15 sequences include sequences that are 90, 95, 98, and 99% identical (as defined herein) to the listed sequences and / or sequences that contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions. As will be apparent to one of skill in the art, IL-15 variants can be used in any of the IL-15 / Rα-Fc fusion proteins and PD-1-targeted IL-15 / Rα-Fc fusion proteins described herein.

[0078] [Figure 20] Figure 20 shows the sequence of an exemplary IL-15 / Rα-Fc fusion protein in the "IL-15 / Rα-hetero-Fc" format, which contains an IL-15 variant engineered to reduce potency. IL-15 and IL-15Rα (sushi) are underlined, the linker is double underlined (those skilled in the art will appreciate that the linker can be replaced with other linkers, some of which are shown in Figure 65), and a slash ( / ) indicates the boundaries of the IL-15, IL-15Rα, linker, and Fc regions.

[0079] [Figure 21]Figures 21A-21B show the sequence of an exemplary IL-15 / Rα-Fc fusion protein in the "scIL-15 / Rα-Fc" format, containing an IL-15 variant engineered to reduce potency. IL-15 and IL-15Rα (sushi) are underlined, the linker is double underlined (those skilled in the art will appreciate that the linker can be replaced with other linkers, some of which are shown in Figure 65), and a diagonal line ( / ) indicates the boundaries of the IL-15, IL-15Rα, linker, and Fc regions.

[0080] [Figure 22] 22A-B show the percentage of A) CD4+CD45RA- cells and B) CD8+CD45RA- cells that express Ki67 after incubation with the indicated test articles.

[0081] [Figure 23] Figure 23 shows the amino acid sequence of XENP15074 (a bivalent anti-RSV mAb based on human IgG1 Fc with moatvisumab and E233P / L234V / L235A / G236del / S267K substitutions). CDRs are underlined, and diagonal lines indicate the boundaries of the variable regions.

[0082] [Figure 24] Figure 24 shows the amino acid sequences of A) XENP16432 (a bivalent anti-PD-1 mAb based on nivolumab and a human IgG1 Fc with E233P / L234V / L235A / G236del / S267K substitutions), B) XENP21641 (pembrolizumab), and C) XENP28437 (a bivalent anti-PD-1 mAb based on pembrolizumab and a human IgG1 Fc with E233P / L234V / L235A / G236del / S267K substitutions). CDRs are underlined, and diagonal lines indicate the boundaries of the variable regions.

[0083] [Figure 25]Figure 25 shows the sequences of hybridoma clone 1C11 and XENP21575, chimeric and humanized anti-PD-1 antibodies based on the variable region of human IgG1 with E233P / L234V / L235A / G236del / S267K substitutions in the heavy chain. CDRs are underlined, and diagonal lines indicate the boundaries of the variable domains. As noted herein, and as is true for all sequences herein that contain CDRs, the exact identification of the CDR positions may vary slightly depending on the numbering used, as shown in Table 1; therefore, this specification includes not only the underlined CDRs, but also CDRs contained within the VH and VL domains using other numbering systems. As will be appreciated by those of skill in the art, the VH and VL domains can be formatted as Fab or scFv for use in the PD-1-targeting IL-15 / Rα-Fc fusion proteins of the invention.

[0084] [Figure 26] Figure 26 shows the sequences of exemplary humanized variants of anti-PD-1 mAb A and mAb B in a bivalent human IgG1 format with E233P / L234V / L235A / G236del / S267K substitutions in the heavy chain. The CDRs are underlined, and the diagonal lines indicate the boundaries of the variable domains. As noted herein, and as is true for all sequences herein that contain CDRs, the exact identification of the CDR positions may vary slightly depending on the numbering used, as shown in Table 1; therefore, this specification includes not only the underlined CDRs, but also CDRs contained within the VH and VL domains using other numbering systems. As will be appreciated by those of skill in the art, the VH and VL domains can be formatted as Fab or scFv for use in the PD-1-targeting IL-15 / Rα-Fc fusion proteins of the invention.

[0085] [Figure 27]Figure 27 shows epitope binning of mAbs based on XENP16432 (a bivalent anti-PD-1 mAb based on nivolumab), XENP21461 (pembrolizumab), chimeric mAb A (chmAb A), chimeric mAb B (chmAb B), and 1C11, as indicated by the normalized BLI response Octet. A normalized BLI response greater than 0.5 indicates that the antibody pair does not bin to the same epitope.

[0086] [Figure 28]Figures 28A-H show several formats for the PD-1-targeted IL-15 / Rα-Fc fusion proteins of the invention. The "scIL-15 / Rα x scFv" format (Figure 28A) comprises IL-15Rα(sushi) fused to IL-15 by a variable-length linker (referred to as "scIL-15 / Rα"), which is then fused to the N-terminus of the heterodimeric Fc region, with an scFv fused to the other side of the heterodimeric Fc. The "scFv x ncIL-15 / Rα" format (Figure 28B) comprises an scFv fused to the N-terminus of the heterodimeric Fc region, with IL-15Rα(sushi) fused to the other side of the heterodimeric Fc, and IL-15 is transfected separately to form a noncovalent IL-15 / Rα complex. The "scIL-15 / Rα x Fab" format (Figure 28C) comprises IL-15Rα(sushi) fused to IL-15 by a variable length linker (termed "scIL-15 / Rα"), which is then fused to the N-terminus of a heterodimeric Fc region; the variable heavy chain (VH) is fused to the other side of the heterodimeric Fc; the corresponding light chain is transfected separately to form Fab with the VH. The "ncIL-15 / Rα x Fab" format (Figure 28D) comprises VH fused to the N-terminus of a heterodimeric Fc region; IL-15Rα(sushi) is fused to the other side of the heterodimeric Fc; the corresponding light chain is transfected separately to form Fab with the VH; and IL-15 is transfected separately to form a non-covalent IL-15 / Rα complex. The "mAb-scIL-15 / Rα" format (Figure 28E) comprises a VH fused to the N-terminus of the first and second heterodimeric Fc, where IL-15 is fused to IL-15Rα (sushi), which is then further fused to the C-terminus of one of the heterodimeric Fc regions, and the corresponding light chain is transfected separately to form the VH and Fab.The "mAb-ncIL-15 / Rα" format (Figure 28F) comprises a VH fused to the N-terminus of the first and second heterodimeric Fc regions, IL-15Rα(sushi) is fused to the C-terminus of the heterodimeric Fc region, the corresponding light chain is transfected separately to form a Fab with the VH, and IL-15 is transfected separately to form a non-covalent IL-15 / Rα complex. The "central-IL-15 / Rα" format (Figure 28G) comprises a VH recombinantly fused to the N-terminus of IL-15, which is then fused to one side of the heterodimeric Fc, and the VH is recombinantly fused to the N-terminus of IL-15Rα(sushi), which is then fused to the other side of the heterodimeric Fc, the corresponding light chain is transfected separately to form a Fab with the VH. The "central-scIL-15 / Rα" format (Figure 28H) comprises a VH fused to the N-terminus of IL-15Rα (sushi) fused to IL-15, which is then further fused to one side of a heterodimeric Fc, the VH fused to the other side of the heterodimeric Fc, and the corresponding light chain transfected separately to form the VH and Fab.

[0087] [Figure 29]Figure 29 shows the sequence of an exemplary [C]PD-1-targeting IL-15 / Rα-Fc fusion protein in the "scIL-15 / Rα x Fab" format. The CDRs are in bold. As noted herein, and as is true for all sequences herein that contain CDRs, the exact identification of the CDR positions may vary slightly depending on the numbering used as shown in Table 1; therefore, this specification includes not only the underlined CDRs, but also CDRs contained within the VH and VL domains using other numbering systems. IL-15 and IL-15Rα (sushi) are in italics, the linker is double underlined (as will be appreciated by those of skill in the art, the linker can be substituted with other linkers, some of which are shown in Figures 65 and 66), and a diagonal line ( / ) indicates the boundaries of IL-15, IL-15Rα, linker, variable region, and constant / Fc region. As will be apparent to one of skill in the art, each of the described targeting IL-15 / Rα-Fc fusion proteins can include or exclude Xtend Fc (M428L / N434S). Figures 30A-B show the affinity of XENP22553 for PD-1 as determined by Octet (as well as associated sensorgrams).

[0088] [Figure 30]Figures 30A-30C show the sequence of an exemplary [NC]PD-1-targeting IL-15 / Rα-Fc fusion protein in the "scIL-15 / Rα x Fab" format. CDRs are underlined. As noted herein, and as is true for all sequences herein that contain CDRs, the exact identification of the CDR positions may vary slightly depending on the numbering used as shown in Table 1; therefore, this specification includes not only the underlined CDRs, but also CDRs contained within the VH and VL domains using other numbering systems. IL-15 and IL-15Rα (sushi) are italicized, the linker is double underlined (as will be appreciated by those skilled in the art, the linker can be replaced with other linkers, some of which are shown in Figures 65 and 66), and a diagonal line ( / ) indicates the boundaries of IL-15, IL-15Rα, linker, variable region, and constant / Fc region. As will be apparent to one of skill in the art, each of the described targeting IL-15 / Rα-Fc fusion proteins can include or exclude Xtend Fc (M428L / N434S). Figures 32A-32D show the amino acid sequences of (A) XENP21641 (pembrolizumab) and (B) XENP28437 (pembrolizumab and a bivalent anti-PD-1 mAb based on a human IgG1 Fc with E233P / L234V / L235A / G236del / S267K substitutions). CDRs are underlined, and diagonal lines indicate the boundaries of the variable regions.

[0089] [Figure 31]Figures 31A-31B show the sequence of a control RSV-targeting IL-15 / Rα-Fc fusion. The CDRs are underlined. As noted herein, and as is true for all sequences herein containing CDRs, the exact identification of the CDR positions may vary slightly depending on the numbering used, as shown in Table 1; therefore, the present specification includes not only the underlined CDRs, but also CDRs contained within the VH and VL domains using other numbering systems. IL-15 and IL-15Rα (sushi) are italicized, the linker is double underlined (as will be understood by those skilled in the art, the linker can be replaced with other linkers, some of which are shown in Figures 65 and 66), and the diagonal line ( / ) indicates the boundaries of IL-15, IL-15Rα, the linker, the variable region, and the constant / Fc region. As will be apparent to one of skill in the art, each of the described targeted IL-15 / Rα-Fc fusion proteins can also include Xtend Fc (M428L / N434S).

[0090] [Figure 32] Figures 32A-B show the proliferation of A) CD8+ T cells and B) CD4+ T cells after incubation with PD-1-targeting IL-15 / Rα-Fc fusions (XENP28532, XENP28692, and XENP25850) and a control RSV-targeting IL-15 / Rα-Fc fusion (XENP26007) and an anti-PD-1 mAb (XENP28519).

[0091] [Figure 33] Figures 33A-B show the induction of STAT5 phosphorylation by XENP25850 (an exemplary PD-1-targeting IL-15 / Rα-Fc fusion) on A) CD4+CD45RA-CD25+ and B) CD8+CD45RA-CD25+ cells. Fresh cells are indicated by dotted lines, and activated cells are indicated by solid lines. All fresh cells are CD25-negative.

[0092] [Figure 34]Figures 34A-B show induction of STAT5 phosphorylation in CD8+CD45RA-CD25+PD-1+ T cells by A) the [C]PD-1-targeting IL-15 / Rα-Fc fusion XENP25937 and B) the [NC]PD-1-targeting IL-15 / Rα-Fc fusion XENP28532 after pre-incubation with either the nivolumab-based XENP16432, pembrolizumab, or the anti-RSV mAb XENP15074.

[0093] [Figure 35] FIG. 35 shows the change in body weight (as a percentage of initial body weight) of huPBMC-engrafted NSG mice over time after administration of the indicated test articles.

[0094] [Figure 36] Figures 36A-B show A) CD8+ T cell counts and B) CD4+ T cell counts 14 days after the first dose of the indicated test article in human PBMC-engrafted NSG mice.

[0095] [Figure 37] Figures 37A-B show CD25 expression on A) CD8+ T cells and B) CD4+ T cells 10 days after the first dose of the indicated test article in human PBMC-engrafted NSG mice.

[0096] [Figure 38] FIG. 38 shows the ratio of CD8+ to CD4+ T cells 10 days after the first dose of the indicated test articles in human PBMC-engrafted NSG mice.

[0097] [Figure 39] Figures 39A-B show A) CD8+ T cell counts and B) CD4+ T cell counts 10 days after the first dose of the indicated test article in human PBMC-engrafted NSG mice.

[0098] [Figure 40]Figures 40A-B show CD25 expression on A) CD8+ T cells and B) CD4+ T cells 10 days after the first dose of the indicated test article in human PBMC-engrafted NSG mice.

[0099] [Figure 41] FIG. 41 shows the serum concentrations of the indicated test articles over time in cynomolgus monkeys after the first dose at the indicated relative concentrations.

[0100] [Figure 42] Figure 42 shows the sequences of exemplary [NC]PD-1-targeting IL-15 / Rα-Fc fusion proteins, XENP29484 and XENP29485, with the IL-15(D30N / N65D) variant. CDRs are underlined. As noted herein, and as is true for all sequences herein that contain CDRs, the exact identification of the CDR positions may vary slightly depending on the numbering used, as shown in Table 1; therefore, this specification includes not only the underlined CDRs, but also CDRs contained within the VH and VL domains using other numbering systems. IL-15 and IL-15Rα (sushi) are italicized, the linker is double underlined (as will be understood by those skilled in the art, the linker can be replaced with other linkers, some of which are shown in the figure), and a diagonal line ( / ) indicates the boundaries of IL-15, IL-15Rα, linker, variable region, and constant / Fc region.

[0101] [Figure 43] Figures 43A-B show the variable heavy and variable light chains of additional exemplary anti-PD-1 ABDs that do not compete with nivolumab or pembrolizumab. The CDRs are underlined. As noted herein, and as is true for all sequences herein that contain CDRs, the exact identification of the CDR positions may vary slightly depending on the numbering used, as shown in Table 1; therefore, the present specification includes not only the underlined CDRs, but also CDRs contained within the VH and VL domains using other numbering systems.

[0102] [Figure 44] Figures 44A-B show the percentage of PD-1+ A) CD8+ CD45RA- CD45RO+ T cells and B) CD4+ CD45RA- CD45RO+ T cells after treatment with the PD-1-targeting IL-15 / Rα-Fc fusion XENP28532 and controls XENP24306 (non-targeting IL-15 / Rα-Fc fusion) and XENP26007 (RSV-targeting IL-15 / Rα-Fc fusion).

[0103] [Figure 45] Figures 45A-B show the sequence of an exemplary humanized variant of anti-PD-1 mAb C in a bivalent human IgG1 format with E233P / L234V / L235A / G236del / S267K substitutions in the heavy chain. The CDRs are underlined, and the diagonal lines indicate the boundaries of the variable domains. As noted herein, and as is true for all sequences herein that contain CDRs, the exact identification of the CDR positions may vary slightly depending on the numbering used, as shown in Table 1; therefore, the present specification includes not only the underlined CDRs, but also CDRs contained within the VH and VL domains using other numbering systems. As will be appreciated by one of skill in the art, the VH and VL domains can be formatted as Fab or scFv for use in the PD-1-targeting IL-15 / Rα-Fc fusion proteins of the invention. [Figure 46] Figure 46 shows (A) CD45+ cell counts, (B) CD3+ T cell counts, (C) CD8+ T cell counts, and (D) CD4+ T cell counts 14 days after the first dose of the indicated test article in human PBMC-engrafted NSG mice.

[0104] Figure 46 shows the affinity of XENP28536, XENP28537, XENP28538, XENP28539 and XENP28519 for human PD-1 and cynomolgus monkey PD-1 as determined by Octet.

[0105] [Figure 47]Figure 47 shows epitope binning of XENP16432 (a bivalent anti-PD-1 mAb based on nivolumab), XENP21461 (pembrolizumab), and chimeric mAb C (chmAb C). A normalized BLI response greater than 0.5 indicates that the antibody pair does not bin to the same epitope. The data show that anti-PD-1 mAb C does not bin to the same epitope as nivolumab and pembrolizumab.

[0106] [Figure 48] Figures 48A-48C show the sequences of exemplary [NC]PD-1-targeted IL-15 / Rα-Fc fusion proteins in the "scIL-15 / Rα x Fab" format and various IL-15 potency variants, including a PD-1-targeting arm based on mAb C. CDRs are underlined. As noted herein and as applies to all sequences herein that contain CDRs, the exact identification of the CDR positions may vary slightly depending on the numbering used, as shown in Table X; therefore, this specification includes not only the underlined CDRs, but also CDRs contained within the VH and VL domains using other numbering systems. The linker is double underlined (though those skilled in the art will appreciate that the linker can be substituted with other linkers, some of which are shown in Figures 65 and 66), and a diagonal line ( / ) indicates the boundaries of IL-15, IL-15Rα, linker, variable region, and constant / Fc region. As will be apparent to one of skill in the art, each of the PD-1-targeting IL-15 / Rα-Fc fusion proteins described can also include Xtend Fc (M428L / N434S).

[0107] [Figure 49]Figures 49A-C show sequences of exemplary [NC]PD-1-targeting IL-15 / Rα-Fc fusion proteins in the "scIL-15 / Rα x Fab" format and various IL-15 potency variants (also including Xtend Fc(M428L / N434S)) containing a PD-1-targeting arm based on mAb C. CDRs are underlined. As noted herein, and as is true for all sequences herein that contain CDRs, the exact identification of CDR positions may vary slightly depending on the numbering used, as shown in Table 1; therefore, the present specification includes not only the underlined CDRs, but also CDRs contained within the VH and VL domains using other numbering systems. The linker is double underlined (those skilled in the art will appreciate that the linker can be replaced with other linkers, some of which are shown in Figures 65 and 66), and the diagonal line ( / ) indicates the boundaries of IL-15, IL-15Rα, linker, variable region, and constant / Fc region. [Figure 50] Figures 50A-B show (A) CD45+ cell counts, (B) CD3+ T cell counts, (C) CD8+ T cell counts, and (D) CD4+ T cell counts in human PBMC-engrafted NSG mice 10 days after the first administration of the indicated test article.

[0108] Figures 50A-B show the effects of [NC]PD-1-targeted IL-15 / Rα-Fc fusions (and controls) on the proliferation of CD8 + T cells and B)CD4 + The data show that the [NC]PD-1-targeted IL-15 / Rα-Fc fusion induces CD4 T cell proliferation compared to the non-targeted IL-15 (D30N / E64Q / N65D) / Rα-Fc fusion (as well as the control RSV-targeted IL-15 / Rα-Fc fusion). + These results show that the IL-14-14 mAb was more potent in inducing T cell proliferation.

[0109] [Figure 51]Figure 51 shows the induction of LAG-3+ CD8+ T cells by [NC]PD-1-targeted IL-15 / Rα-Fc fusions (and controls), as indicated by the percentage of proliferating cells (determined based on CFSE dilution). The data show that XENP28532 was more potent at inducing CD8+ LAG-3+ T cell proliferation compared to non-targeted IL-15(D30N / E64Q / N65D) / Rα-Fc fusions (as well as control RSV-targeted IL-15 / Rα-Fc fusions). Furthermore, XENP28543 was more potent at inducing CD8+ LAG-3+ T cell proliferation than bulk CD8+ T cell proliferation (EC50 of 276.8 vs. 71.94). Taken together, this supports the idea that [NC]PD-1-targeted IL-15 / Rα-Fc fusions may be selective for checkpoint-expressing T cells such as those found in the tumor environment.

[0110] [Figure 52] Figures 52A-B show activation of A) CD4+CD45RA- memory T cells and B) CD4+CD45RA+ naive T cells after incubation with [NC]PD-1-targeted IL-15 / Rα-Fc fusions (and controls), as indicated by the percentage of cells expressing CD25.

[0111] [Figure 53] Figures 53A-B show the percentage of A) CD4+CD45RA- T cells and B) CD4+CD45RA+ T cells expressing PD-1 after incubation with [NC]PD-1-targeting IL-15 / Rα-Fc fusion (and control).

[0112] [Figure 54]Figures 54A-C show IFNγ secretion by PBMCs pre-stimulated with A) 50 ng / ml, B) 100 ng / ml, and C) 500 ng / ml plate-bound anti-CD3 (OKT3) and incubated with the indicated test articles. The data show that both XENP28532 and XENP28543 were able to potently stimulate IFNγ secretion. Notably, XENP28532 (PD-1-targeting arm based on mAb A) appeared to be more active in inducing IFNγ secretion than XENP28543 (PD-1-targeting arm based on mAb C).

[0113] [Figure 55] Figures 55A-B show the induction of STAT5 phosphorylation in A) CD8+CD45RA-CD25+PD-1+ T cells and B) CD4+CD45RA-CD25+PD-1+ T cells by [NC]PD-1-targeting IL-15 / Rα-Fc fusion with mAb C-based PD-1 targeting arm (XENP28543) after pre-incubation with either nivolumab-based XENP16432, pembrolizumab, or anti-RSV mAb XENP15074. The data show that PD-1 blockade does not interfere with the activity of XENP28543.

[0114] [Figure 56] FIG. 56 shows the change in body weight (as a percentage of initial body weight) of huPBMC-engrafted NSG mice over time after administration of the indicated test articles.

[0115] [Figure 57] Figures 57A-C show the body weights (as a percentage of initial body weight) of huPBMC-engrafted NSG mice on A) day 11, B) day 14, and C) day 18 after the first dose with the indicated test article. p values ​​were determined using an unpaired t-test. The data show that by day 11, the combination of [NC]PD-1-targeted IL-15 / Rα-Fc fusion with PD-1 blockade significantly enhanced GVHD compared to treatment with [NC]PD-1-targeted IL-15 / Rα-Fc fusion alone.

[0116] [Figure 58] Figures 58A-F show the number of human A) CD45+ cells, B) CD3+ T cells, C) CD4+ T cells, D) CD8+ T cells, E) γδ T cells, and F) NK cells in the blood of huPBMC-engrafted NSG mice 14 days after the first dose with the indicated test article.

[0117] [Figure 59] Figures 59A-B show activation of human A) CD8+ T cells and B) CD4+ T cells (as indicated by CD25 MFI) in the blood of huPBMC-engrafted NSG mice 14 days after first dose with the indicated test articles.

[0118] [Figure 60] Figure 60 shows tumor volume (determined by caliper measurement) over time in pp65-MCF7 mice and huPBMC-engrafted NSG mice administered [NC]PD-1-targeted IL-15 / Rα-Fc fusion alone or in combination with PD-1 blockade.

[0119] [Figure 61] Figures 61A-61F show tumor volumes (determined by caliper measurements) on days 26 (Figure 61A), 28 (Figure 61B), 30 (Figure 61C), 33 (Figure 61D), 35 (Figure 61E), and 37 (Figure 61F) (after PBMC engraftment and the first dose of test article) in pp65-MCF7 mice and huPBMC-engrafted NSG mice administered [NC]PD-1-targeted IL-15 / Rα-Fc fusion alone or in combination with PD-1 blockade. p values ​​were determined by unpaired t-test. The data show that by day 28, the combination of XENP28543 and PD-1 blockade significantly reduced tumor size more than treatment with PD-1 blockade alone.

[0120] [Figure 62]Figures 62A-62B show activation of human A) CD8+ T cells and B) CD4+ T cells (as indicated by CD25 MFI) in the blood of pp65-MCF7 mice and huPBMC-engrafted NSG mice 7 days after initial dosing with the indicated test articles. The data show that [NC]PD-1-targeted IL-15 / Rα-Fc fusions, alone or in combination with PD-1 blockade, enabled significant enhancement of initial activation of CD8+ T cells. Statistics were performed on log-transformed data using unpaired t-tests.

[0121] [Figure 63] Figures 63A-63E show the numbers of human A) CD45+ cells, B) CD3+ T cells, C) CD4+ T cells, D) CD8+ T cells, and E) NK cells in the blood of pp65-MCF7 mice and huPBMC-engrafted NSG mice 14 days after initial dosing with the indicated test articles. The data show that [NC]PD-1-targeted IL-15 / Rα-Fc fusions, alone or in combination with PD-1 blockade, enabled significantly enhanced proliferation of multiple lymphocyte populations by day 14 compared to PD-1 blockade alone. Statistics were performed on log-transformed data using unpaired t-tests.

[0122] [Figure 64]Figures 64A-64B show the sequence of an exemplary affinity-engineered variant of anti-PD-1 mAb C in a bivalent human IgG1 format with E233P / L234V / L235A / G236del / S267K substitutions in the heavy chain. The CDRs are underlined, and the diagonal lines indicate the boundaries of the variable domains. As noted herein, and as is true for all sequences herein that contain CDRs, the exact identification of the CDR positions may vary slightly depending on the numbering used, as shown in Table X; therefore, this specification includes not only the underlined CDRs, but also CDRs contained within the VH and VL domains using other numbering systems. As will be appreciated by one of skill in the art, the VH and VL domains can be formatted as Fab or scFv for use in the PD-1-targeting IL-15 / Rα-Fc fusion proteins of the invention. Figure 67A shows epitope binning of XENP16432 (a bivalent anti-PD-1 mAb based on nivolumab), XENP21461 (pembrolizumab), and chimeric mAb C (chmAb C). A normalized BLI response greater than 0.5 indicates that the antibody pair does not bin to the same epitope. The data show that anti-PD-1 mAb C does not bin to the same epitope as nivolumab and pembrolizumab.

[0123] [Figure 65]Figures 65A-65I show the apparent dissociation constants (KDapp), association rates (ka), and dissociation rates (kd) of affinity-engineered mAb C[PD-1]_H1L1 variants (bivalent IgG1 format containing the E233P / L234V / L235A / G236_ / S267K deletion variant) as determined by Octet, as well as the fold improvement over mAb C[PD-1]_H1L1. Substitutions in the variable heavy or variable light regions (if listed) are based on Xencor numbering (corresponding Kabat positions are listed in the next column). Of 304 variants with single point mutations in either the variable heavy or light regions, we identified only 11 variants (including mAb C[PD-1]_H1_L1.1 and mab_C[PD-1]_H1_L1.3) with affinity that was more than 2-fold improved over WT.

[0124] [Figure 66] Figure 66 shows the apparent dissociation constants (KDapp), association rates (ka), and dissociation rates (kd) of affinity-engineered mAb C[PD-1]_H1L1 variants combining preferred single-substitution VH and VL variants (in the context of PD-1-targeting IL15 / Rα-Fc) as determined by Octet. Substitutions in the variable heavy or variable light regions (if listed) are based on Xencor numbering (corresponding Kabat positions are listed in the next column). H1.19_L1.1 allows for higher affinity than H1.132_L1.1, although H1.132_L1 provides higher affinity than H1.19_L1.

[0125] [Figure 67]Figure 67 shows the apparent dissociation constants (KDapp), association rates (ka), and dissociation rates (kd) of affinity-engineered mAb C[PD-1]_H1L1 variants combining multiple substitutions in the VH and / or VL (in the context of PD-1-targeting IL15 / Rα-Fc) as determined by Octet. Substitutions in the variable heavy or variable light regions (if listed) are based on Xencor numbering (corresponding Kabat positions are listed in the next column). The triple-substituted VL variant N31H / K36Y / S99T (L1.140; N27dH / K30Y / S93T in Kabat numbering) exhibits a 36-fold improvement in KD over wild-type and binds well to the VH variant, exerting an approximately 100-fold improvement in KD over wild-type.

[0126] [Figure 68] Figures 68A-68J show the sequences of exemplary [NC]PD-1-targeting IL-15 / Rα-Fc fusion proteins in the "scIL-15 / Rα x Fab" format and various IL-15 potency variants, including a PD-1-targeting arm based on the affinity-optimized mAb C ABD. CDRs are underlined. As noted herein and applicable to all sequences herein that contain CDRs, the exact identification of the CDR positions may vary slightly depending on the numbering used, as shown in Table 1; therefore, this specification includes not only the underlined CDRs, but also CDRs contained within the VH and VL domains using other numbering systems. The linker is double-underlined (those skilled in the art will understand that the linker can be replaced with other linkers, some of which are shown in Figures 65 and 66), and a diagonal line ( / ) indicates the boundaries of IL-15, IL-15Rα, linker, variable region, and constant / Fc region. As will be apparent to one of skill in the art, each of the described PD-1-targeting IL-15 / Rα-Fc fusion proteins can also include or exclude Xtend Fc (M428L / N434S).

[0127] [Figure 69]Figures 69A-69C show the sequences of exemplary [NC]PD-1-targeting IL-15 / Rα-Fc fusion proteins in the "scIL-15 / Rα x Fab" format and various IL-15 potency variants (also including Xtend Fc(M428L / N434S)) containing a PD-1-targeting arm based on the affinity-optimized mAb C ABD. CDRs are underlined. As noted herein, and as is true for all sequences herein that contain CDRs, the exact identification of CDR positions may vary slightly depending on the numbering used, as shown in Table X; therefore, the present specification includes not only the underlined CDRs, but also CDRs contained within the VH and VL domains using other numbering systems. The linker is double underlined (those skilled in the art will appreciate that the linker can be replaced with other linkers, some of which are shown in Figures 65 and 66), and the diagonal line ( / ) indicates the boundaries of IL-15, IL-15Rα, linker, variable region, and constant / Fc region.

[0128] [Figure 70] Figures 70A-70B show the induction of A) CD8+ T cell and B) CD4+ T cell proliferation by [NC]PD-1-targeted IL-15 / Rα-Fc fusions with various PD-1 affinities and IL-15 potency variants (and controls), as indicated by the percentage of proliferating cells (determined based on CFSE dilution). The data show that XENP30046 (with an affinity-enhanced PD-1-targeting arm) more potently induces proliferation of both CD8+ and CD4+ T cells than XENP28543 (a 2-fold increase). Furthermore, the data show that the IL-15(D30N / N65D) variant does not dramatically affect the activity of PD-1-targeted IL-15 / Rα-Fc fusions.

[0129] [Figure 71]Figures 71A-71B show activation of A) CD8+CD45RA- T cells and B) CD8+CD45RA+ T cells after incubation with [NC]PD-1-targeted IL-15 / Rα-Fc fusions with various PD-1 affinities and IL-15 potency variants (and controls), as indicated by the percentage of cells expressing CD25.

[0130] [Figure 72] Figures 72A-B show activation of A) CD4+CD45RA- T cells and B) CD4+CD45RA+ T cells after incubation with [NC]PD-1-targeted IL-15 / Rα-Fc fusions with various PD-1 affinities and IL-15 potency variants (and controls), as indicated by the percentage of cells expressing CD25.

[0131] [Figure 73] Figures 73A-B show the percentage of A) CD4+CD45RA- T cells and B) CD4+CD45RA+ T cells expressing PD-1 after incubation with [NC]PD-1-targeted IL-15 / Rα-Fc fusions and IL-15 potency variants (and controls) with various PD-1 affinities. The data show that [NC]PD-1-targeted IL-15 / Rα-Fc fusions induce downregulation of PD-1 in CD4+ cells, and that downregulation correlates with PD-1 affinity.

[0132] [Figure 74] Figure 74 shows the change in body weight (as a percentage of initial body weight) over time in huPBMC-engrafted NSG mice treated with [NC]PD-1-targeted IL-15 / Rα-Fc fusions and IL-15 potency variants with varying PD-1 affinity (and control). 70% of the mice were used for death.

[0133] [Figure 75]Figures 75A-75D show the body weights of huPBMC-engrafted NSG mice treated with [NC]PD-1-targeted IL-15 / Rα-Fc fusions with various PD-1 affinities and IL-15 potency variants (and controls) on A) day 11, B) day 14, C) day 18, and D) day 21. 70% of the mice died. p values ​​were determined using an unpaired t-test. The data show that treatment with XENP30046 alone (with the affinity-enhanced PD-1-targeted arm) resulted in significant weight loss compared to PBS treatment, as measured on days 11 and 18, whereas treatment with XENP28543 alone did not result in significant weight loss (compared to PBS treatment).

[0134] [Figure 76] Figures 76A-C show serum concentrations of A) IFNγ, B) IL-10, and C) IL-2Rα in huPBMC-engrafted NSG mice on days 7, 11, and 14 after administration of [NC]PD-1-targeted IL-15 / Rα-Fc fusions with various PD-1 affinities and IL-15 potency variants (and controls).

[0135] [Figure 77] Figures 77A-B show activation of A) CD4+ T cells and B) CD8+ T cells (as indicated by CD25 MFI) on day 7 in the blood of huPBMC-engrafted NSG mice administered [NC]PD-1-targeted IL-15 / Rα-Fc fusions with various PD-1 affinities and IL-15 potency variants (and controls) (statistics were performed on log-transformed data using unpaired t-tests).

[0136] [Figure 78]Figures 78A-78E show the numbers of A) CD45+ cells, B) CD3+ T cells, C) CD8+ T cells, D) CD4+ T cells, and E) NK cells in the blood of huPBMC-engrafted NSG mice administered [NC]PD-1-targeted IL-15 / Rα-Fc fusions with various PD-1 affinities and IL-15 potency variants (and controls) on day 11 (statistics were performed on log-transformed data using unpaired t-tests).

[0137] [Figure 79] Figures 79A-79E show the numbers of A) CD45+ cells, B) CD3+ T cells, C) CD8+ T cells, D) CD4+ T cells, and E) NK cells in the blood of huPBMC-engrafted NSG mice treated with [NC]PD-1-targeted IL-15 / Rα-Fc fusions with various PD-1 affinities and IL-15 potency variants (and controls) on day 14.

[0138] [Figure 80]Figures 80A-80B show the induction of A) CD8+ T cell and B) CD4+ T cell proliferation by [NC]PD-1-targeted IL-15 / Rα-Fc fusions and IL-15 potency variants (and controls) with varying PD-1 affinities, as indicated by the percentage of proliferating cells (determined based on CFSE dilution). The data show that XENP30272 (with a KD of 3.1 nM for PD-1) is more potent than XENP30046 (with a KD of 5.4 nM for PD-1) in inducing proliferation and activation of various T cell populations. Notably, XENP30429 (a PD-1-targeted IL-15 / Rα-Fc fusion with the IL-15 (D30N / E64Q / N65D) variant) was only 1.8-2.5 times less active against CD8+ and CD4+ T cells than XENP30046 (a PD-1-targeted IL-15 / Rα-Fc fusion with the IL-15 (N4D / N65D) variant), whereas XENP30432 (a surrogate RSV-targeted IL-15 / Rα-Fc with the IL-15 (D30N / E64Q / N65D) variant) was 12-fold less active against CD8+ T cells and 530-fold less active against CD4+ T cells (based on proliferation activity) than XENP30046. This suggests that PD-1-targeted IL-15 / Rα-Fc fusions with the IL-15(D30N / E64Q / N65D) variant should retain activity in the tumor environment while remaining essentially inactive outside the tumor environment.

[0139] [Figure 81] Figures 81A-C show activation of A) CD4+CD45RA- and B) CD4+CD45RA+ T cells after incubation with [NC]PD-1-targeted IL-15 / Rα-Fc fusions and IL-15 potency variants (and controls) with varying PD-1 affinities, as indicated by CD25 MFI.

[0140] [Figure 82]Figures 82A-C show activation of A) CD8+CD45RA- and B) CD8+CD45RA+ T cells after incubation with [NC]PD-1-targeted IL-15 / Rα-Fc fusions and IL-15 potency variants (and controls) with varying PD-1 affinities, as indicated by CD25 MFI.

[0141] [Figure 83] Figures 83A-C show the expression of PD-1 on A) CD4+ T cells, B) CD4+CD45RA- T cells, and C) CD4+CD45RA+ T cells after incubation with [NC]PD-1-targeted IL-15 / Rα-Fc fusions and IL-15 potency variants (and controls) with various PD-1 affinities, as indicated by PD-1 MFI.

[0142] [Figure 84] Figures 84A-C show the expression of PD-1 on A) CD8+ T cells, B) CD8+CD45RA- T cells, and C) CD8+CD45RA+ T cells after incubation with [NC]PD-1-targeted IL-15 / Rα-Fc fusions and IL-15 potency variants (and controls) with various PD-1 affinities, as indicated by PD-1 MFI.

[0143] [Figure 85] Figures 85A-D show proliferation (as indicated by cell counts) of A) CD45 cells, B) CD3+ T cells, C) CD4+ T cells, and D) CD8+ T cells in NSG mice 10 days after initial dosing with the indicated test article. The data show that more potent IL-15 leads to greater expansion of various lymphocyte populations.

[0144] [Figure 86] Figures 86A-B show activation of A) CD4+ T cells and B) CD8+ T cells (as indicated by CD25 staining) in NSG mice 10 days after initial dosing with the indicated test articles. The data show that more potent IL-15 leads to greater expansion of various lymphocyte populations.

[0145] [Figure 87] Figures 87A-C show the induction of A) CD4+ T cell proliferation, B) CD8+ T cell proliferation, and C) NK cell proliferation by [NC]PD-1-targeted IL-15 / Rα-Fc fusions with Xtend Fc and control RSV-targeted IL-15 / Rα-Fc fusions as indicated by the percentage of proliferating cells (determined based on CFSE dilution). Notably, the data show that the EC50 of XENP30046 is comparable to the EC50 of XENP30290 (the Xtend analog for XENP30046).

[0146] [Figure 88] Figures 88A-B show activation of A) CD4+ T cells and B) CD8+ T cells by [NC]PD-1-targeted IL-15 / Rα-Fc fusion with Xtend Fc and control RSV-targeted IL-15 / Rα-Fc fusion as indicated by CD25 MFI.

[0147] [Figure 89] Figures 89A-89B show PD-1 modulation on A) CD4+ T cells and B) CD8+ T cells by [NC]PD-1-targeting IL-15 / Rα-Fc fusions with Xtend Fc and control RSV-targeting IL-15 / Rα-Fc fusions as indicated by PD-1 MFI. Consistent with Example 9B, the test articles downregulated PD-1 on T cells.

[0148] [Figure 90] Figure 90 shows tumor volume (determined by caliper measurement) over time in pp65-MCF7 mice and huPBMC-engrafted NSG mice administered [NC]PD-1-targeted IL-15 / Rα-Fc fusion alone or in combination with PD-1 blockade.

[0149] [Figure 91]Figures 91A-91H show tumor volumes (determined by caliper measurements) on days 11, 14, 17, 19, 21, 24, 26, and 28 (after PBMC engraftment and the first dose of test article) in pp65-MCF7 mice and huPBMC-implanted NSG mice treated with [NC]PD-1-targeting IL-15 / Rα-Fc fusion alone or in combination with PD-1 blockade. Statistics were performed on baseline-corrected data using the Mann-Whitney test. * Indicates that treatment significantly (p<0.05) enhanced expansion compared to the PBS control. # Indicates that treatment significantly (p<0.05) enhanced expansion compared to treatment with XENP31123. † Indicates that treatment significantly (p<0.05) enhanced expansion compared to PD-1 blockade (XENP16432) alone. The data show that by day 28, all combinations of XENP30290 (0.1, 0.3, or 1 mg / kg) or XENP30516 (0.3, 1, or 3 mg / kg) with PD-1 blockade significantly reduced tumor size more than treatment with PD-1 blockade alone.

[0150] [Figure 92] Figures 92A-92F show the numbers of human A) CD45+ cells, B) CD3+ T cells, C) NK cells, D) CD4+ T cells, and E) CD8+ T cells in the blood of pp65-MCF7 mice and huPBMC-engrafted NSG mice 14 days after initial dosing with the indicated test articles, and F) the ratio of CD8 to CD4 T cells. Statistics of CD45+ cell proliferation performed on log-transformed data using an unpaired t-test. † Indicates that treatment significantly enhanced expansion compared to PD-1 blockade (XENP16432) alone.

[0151] [Figure 93]Figures 93A-93F show the numbers of human A) CD45+ cells, B) CD3+ T cells, C) NK cells, D) CD4+ T cells, and E) CD8+ T cells in the blood of pp65-MCF7 mice and huPBMC-engrafted NSG mice 21 days after initial dosing with the indicated test articles, and F) the ratio of CD8 to CD4 T cells. †Treatment significantly enhanced expansion compared to PD-1 blockade (XENP16432) alone.

[0152] [Figure 94] Figure 94 shows PD-1 expression levels (as indicated by MFI) on various lymphocyte populations in CD34+ Hu-NSG mice (prior to treatment with any test article). The data indicate that the mice have a similar PD-1 expression profile to humans, with higher expression on effector memory populations.

[0153] [Figure 95] Figure 95 shows the fold increase in various lymphocyte populations in CD34+ Hu-NSG mice at day 7 after treatment with 0.3 mg / kg of XENP30046 (mAb C_H1_L1.1 and [NC]PD-1-targeted IL-15 / Rα-Fc containing IL-15(N4D / N65D) variant). XENP30046 shows a greater than 100-fold expansion in the effector memory population.

[0154] [Figure 96] Figure 96 shows the fold increase in various lymphocyte populations in CD34+ Hu-NSG mice at day 7 after treatment with 0.3 mg / kg of XENP30429 ([NC]PD-1-targeted IL-15 / Rα-Fc containing mAb C_H1_L1.1 and IL-15(D30N / E64Q / N65D) variant).

[0155] [Figure 97]Figure 97 shows the fold increase in various lymphocyte populations in CD34+ Hu-NSG mice on day 7 after treatment with 0.3 mg / kg of XENP26007 (control RSV-targeted IL-15 / Rα-Fc containing the IL-15 (N4D / N65D) variant). XENP26007 showed very low proliferation of various lymphocyte populations, indicating that the PD-1-targeted IL-15 / Rα-Fc fusion has minimal peripheral lymphocyte proliferation.

[0156] [Figure 98] Figure 98 shows the fold increase in various lymphocyte populations in CD34+ Hu-NSG mice on day 7 after treatment with 0.3 mg / kg of XENP30432 (control RSV-targeted IL-15 / Rα-Fc containing the IL-15 (D30N / E64Q / N65D) variant). XENP30432 showed very low proliferation of various lymphocyte populations, indicating that the PD-1-targeted IL-15 / Rα-Fc fusion has minimal peripheral lymphocyte proliferation.

[0157] [Figure 99] Figures 99A-99F show the fold change over time of A) CD45 cells, B) CD3+ cells, C) CD4+ cells, D) CD8+ cells, E) γδ cells, and F) NK cells in CD34+ Hu-NSG mice after treatment with 0.3 mg / kg of XENP30046, XENP30429, XENP26007, or XENP30432.

[0158] [Figure 100]Figures 100A-100D show the fold change over time of A) CD4 naive cells, B) CD4 central memory cells, C) CD4 terminal effector cells, and D) CD4 effector memory cells in CD34+ Hu-NSG mice after treatment with 0.3 mg / kg of XENP30046, XENP30429, XENP26007, or XENP30432. The data show that XENP30046 and XENP30429 are selective for PD-1+ populations, including CD4 effector memory cells. Notably, XENP30429 expansion of CD4 naive cells was minimal, indicating that reducing the potency of the IL-15 arm improves selectivity.

[0159] [Figure 101] Figures 101A-101D show the fold change over time of A) CD8 naive cells, B) CD8 central memory cells, C) CD8 terminal effector cells, and D) CD8 effector memory cells in CD34+ Hu-NSG mice after treatment with 0.3 mg / kg of XENP30046, XENP30429, XENP26007, or XENP30432. The data show that XENP30046 and XENP30429 are selective for PD-1+ populations, including CD8 effector memory cells. Notably, XENP30429 expansion of CD8 naive cells was minimal, indicating that reducing the potency of the IL-15 arm improves selectivity.

[0160] [Figure 102] Figures 102A-B show the correlation between lymphocyte expansion and baseline PD-1 expression in CD34+ Hu-NSG mice 7 days after treatment with A) 0.3 mg / kg XENP30046 or B) 0.3 mg / kg XENP30429.

[0161] [Figure 103]Figures 103A-B show A) CD8+ T cell and B) NK cell proliferation in cynomolgus monkeys administered 0.3xXENP22853, 1xXENP25937, or 0.3xXENP24306. The data show that PD-1-targeted IL-15 / Rα-Fc fusions reduce NK cell proliferation while maintaining CD8+ T cell proliferation.

[0162] [Figure 104] Figures 104A-B show the proliferation of A) CD8+ naive T cells and B) CD8+ effector memory T cells in cynomolgus monkeys treated with 0.3xXENP22853, 1xXENP25937, or 0.3xXENP24306. The data show that PD-1-targeted IL-15 / Rα-Fc fusions selectively expand CD8+ effector memory T cells.

[0163] [Figure 105] Figures 105A-105E show the expansion of A) CD8+PD1+ T cells, B) CD8 stem cell memory, C) CD8 naive, D) γδ T cells, and E) CD56+ NK cells in cynomolgus monkeys after administration of either XENP30290 (mAb C_H1_L1.1 x IL-15[N4D / N65D]) or XENP30362 (αRSV x IL-15[N4D / N65D]). Together, the data show that XENP30290 (with high PD-1 affinity and higher IL-15 potency) enabled sustained peripheral pharmacodynamics over 2-3 weeks with moderate PD1- cell expansion. In particular, γδ T cells are the cell population with the highest fold expansion; CD4+ and CD8+ naive T cells are the cell population with the lowest fold expansion; and CD8+ stem cell memory cells are the most highly proliferative population.

[0164] [Figure 106]Figures 106A-106E show the expansion of A) CD8+PD1+ T cells, B) CD8 stem cell memory, C) CD8 naive, D) γδ T cells, and E) CD56+ NK cells in cynomolgus monkeys after administration of either XENP30516 (mAb C_H1_L1.1 x IL-15[D30N / E64Q / N65D]) or XENP30518 (αRSV x IL-15[D30N / E64Q / N65D]). Together, the data show that XENP30516 (with high PD-1 affinity and lower IL-15 potency) enabled sustained peripheral pharmacodynamics without significant PD1- cell expansion. In particular, γδ T cells are the cell population with the highest fold expansion; CD4+ and CD8+ naive T cells are the cell population with the lowest fold expansion; and CD8+ stem cell memory cells are the most highly proliferative population.

[0165] [Figure 107] Figure 107 shows the change in serum concentration levels over time in cynomolgus monkeys administered XENP30290, XENP30291, XENP29439, or XENP30516. The data show that XENP30290, which has the highest PD-1 affinity and higher IL-15 potency, resulted in faster clearance than both XENP30291 and XENP29439, which have lower PD-1 affinity. However, XENP30516, which has the highest PD-1 affinity but lower IL-15 potency, resulted in slower clearance than XENP30290.

[0166] [Figure 108] Figure 108 shows the change in serum concentration levels over time in cynomolgus monkeys administered XENP30290, XENP30292, or XENP30516. The data show that XENP30516, which has lower IL-15 potency, resulted in slower clearance than XENP30290.

[0167] [Figure 109]FIG. 109 shows the change in serum concentration levels over time in cynomolgus monkeys administered XENP30291 or XENP30293.

[0168] [Figure 110] FIG. 110 shows the change in serum concentration levels over time in cynomolgus monkeys administered XENP29439 or XENP30302.

[0169] [Figure 111] Figure 111 shows the change in serum concentration levels over time in cynomolgus monkeys administered XENP30362 or XENP30518. The data show that higher IL-15 potency correlates with faster clearance.

[0170] [Figure 112] Figures 112A-C show the time course of PD-1 expression in various lymphocyte populations in cynomolgus monkeys administered A) XENP30290, B) XENP30516, or C) XENP30362. The data show that the PD-1-targeted IL-15 / Rα-Fc fusion increases PD-1 expression, whereas the control RSV-targeted IL-15 / Rα-Fc fusion does not.

[0171] [Figure 113] Figure 113 shows the correlation between peak fold expansion of all T cell memory subsets and peak PD-1 expression induced by XENP30290 or XENP30516.

[0172] [Figure 114]Figure 114 shows the sequences of exemplary IL-15 variants engineered to remove glycosylation. These variant IL-15 sequences include sequences that are 90, 95, 98, and 99% identical (as defined herein) to the listed sequences and / or sequences that contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions. As will be apparent to one of skill in the art, IL-15 variants can be used in any of the IL-15 / Rα-Fc fusion proteins and PD-1-targeted IL-15 / Rα-Fc fusion proteins described herein. Additionally, each of the IL-15 modifications described herein can be used alone or in combination with any other IL-15 modifications as described herein.

[0173] [Figure 115] Figures 115A-C show the sequences of exemplary IL-15 / Rα-Fc fusion proteins in the "IL-15 / Rα-hetero-Fc" format engineered to eliminate glycosylation. 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 with other linkers, some of which are shown in Figure 7), and a slash ( / ) indicates the boundaries of IL-15, IL-15Rα, linker, and Fc region. As will be apparent to those skilled in the art, each of the IL-15 / Rα-Fc fusion proteins described can include or exclude Xtend Fc (M428L / N434S).

[0174] [Figure 116]Figures 116A-G show the sequence of an exemplary PD-1-targeting IL-15 / Rα-Fc fusion protein in the "scIL-15 / Rα x Fab" format engineered to eliminate glycosylation. CDRs are underlined. As noted herein, and as is true for all sequences herein that contain CDRs, the exact identification of the CDR positions may vary slightly depending on the numbering used as shown in Table 1; therefore, this specification includes not only the underlined CDRs, but also CDRs contained within the VH and VL domains using other numbering systems. IL-15 and IL-15Rα (sushi) are italicized, the linker is double underlined (as will be understood by those skilled in the art, the linker can be replaced with other linkers, some of which are shown in Figures 9 and 10A-G), and a diagonal line ( / ) indicates the boundaries of IL-15, IL-15Rα, linker, variable region, and constant / Fc region. As will be apparent to one of skill in the art, each of the targeted IL-15 / Rα-Fc fusion proteins described can include or exclude Xtend Fc (M428L / N434S).

[0175] [Figure 117]Figures 117A-117F show the sequence of a control RSV-targeting IL-15 / Rα-Fc fusion protein in the "scIL-15 / Rα x Fab" format, engineered to eliminate glycosylation. CDRs are underlined. As noted herein and as applies to all sequences herein containing CDRs, the exact identification of the CDR positions may vary slightly depending on the numbering used, as shown in Table 1; therefore, this specification includes not only the underlined CDRs, but also CDRs contained within the VH and VL domains using other numbering systems. IL-15 and IL-15Rα (sushi) are italicized, the linker is double-underlined (as will be understood by those skilled in the art, the linker can be replaced with other linkers, some of which are shown in Figures 9 and 10), and a diagonal line ( / ) indicates the boundaries of IL-15, IL-15Rα, the linker, the variable region, and the constant / Fc region. As will be apparent to one of skill in the art, each of the targeted IL-15 / Rα-Fc fusion proteins described can include or exclude Xtend Fc (M428L / N434S).

[0176] [Figure 118]Figures 118A-118B show i) chromatograms showing purification part 2 of XENP30516, XENP31981, XENP31982, XENP31984, XENP31985, XENP31986, and XENP31987, and ii) analytical anion exchange chromatography (analytical AIEX) characterization of the main peaks from the anion exchange separation as shown in Figure 114A. Figure 118A shows XENP30516, XENP31981, XENP31982, XENP31984, and XENP31985. Figure 118B shows XENP31986 and XENP31987. The constructs were XENP30516 (mAb C H1_L1.1 x IL-15[D30N / E64Q / N65D] w / (G4S) linker (SEQ ID NO: 7)), XENP31981 (mAb C H1_L1.1 x IL-15[D30N / E64Q / N65D / N71Q / N79Q] ​​w / (G4S) linker (SEQ ID NO: 7)), XENP31982 (mAb C H1_L1.1 x IL-15[D30N / E64Q / N65D / N71Q / N79Q / N112Q] w / (G4S) linker (SEQ ID NO: 7)), XENP31984 (mAb C H1_L1.1 x XENP31985 (mAb C H1_L1.1 x IL-15[D30N / E64Q / N65D / N71Q / N79Q / S114A] w / (G4A) linker (SEQ ID NO: 8)), XENP31986 (mAb C H1_L1.1 x IL-15[D30N / E64Q / N65D / N71Q / N79Q / N112Q] w / (G4A) linker (SEQ ID NO: 8)), and XENP31987 (mAb C H1_L1.1 x IL-15[D30N / E64Q / N65D / N71Q / N79Q / N112Q] w / (G4A) linker (SEQ ID NO: 8)). IL-15[D30N / E64Q / N65D / N71Q / N79Q / S114]w / (G4A) linker (SEQ ID NO: 8).

[0177] [Figure 119]Figure 119 shows induction of CD8 effector memory T cell proliferation by glycoengineered PD-1-targeted IL-15 / Rα-Fc fusions. Each of the test articles, including IL-15 variants engineered to remove glycosylation-retaining activity, was unexpectedly more potent than XENP30516 (and the corresponding XENP31984 with a Gly-Ala linker), which has not been engineered to remove IL-15 glycosylation. The "(G4S) linker" is SEQ ID NO: 7, and the "(G4A) linker" is SEQ ID NO: 8.

[0178] [Figure 120] Figure 120 shows the induction of CD4 and CD8 effector memory T cell proliferation by glycoengineered PD-1-targeted IL-15 / Rα-Fc fusions and glycoengineered RSV-targeted IL-15 / Rα-Fc fusions. The fold increase in potency of the PD-1-targeted IL-15 / Rα-Fc fusions compared to the corresponding RSV-targeted IL-15 / Rα-Fc fusions is shown to demonstrate selectivity (for TILs over peripheral lymphocytes).

[0179] [Figure 121] Figures 121A-121D show the expansion of CD8 effector memory T cells by A) XENP30290 vs. XENP30362; B) XENP31979 vs. XENP32163; C) XENP30516 vs. XENP30518; and D) XENP31986 vs. XENP32169. The data show that removal of N-linked glycosylation increases potency but also improves selectivity. The "(G4S) linker" is SEQ ID NO: 7, and the "(G4A) linker" is SEQ ID NO: 8.

[0180] [Figure 122]Figure 122 shows tumor volume (determined by caliper measurement) over time in pp65-MCF7 mice and huPBMC-engrafted NSG mice administered glycoengineered PD-1-targeted IL-15 / Rα-Fc fusion alone or in combination with PD-1 blockade. The glycoengineered variant XENP319816 showed enhanced antitumor activity (even at low doses) and combined productively with PD-1 blockade. The "(G4S) linker" is shown as SEQ ID NO: 7, and the "(G4A) linker" is shown as SEQ ID NO: 8.

[0181] [Figure 123] Figure 123 shows the change in tumor volume (determined by caliper measurement) by day 17 in pp65-MCF7 mice and huPBMC-engrafted NSG mice administered glycoengineered PD-1-targeted IL-15 / Rα-Fc fusion alone or in combination with PD-1 blockade.

[0182] [Figure 124] Figure 124 shows the CD8+ cell counts in the blood of pp65-MCF7 mice and huPBMC-engrafted NSG mice administered glycoengineered PD-1-targeted IL-15 / Rα-Fc fusion alone or in combination with PD-1 blockade on day 14.

[0183] [Figure 125] Figures 125A-125E show proliferation of A) CD8+PD1+ T cells, B) CD8+ stem cell memory, C) CD8 naive (PD1low), D) γδ cells, and E) NK cells in cynomolgus monkeys treated with a 1x low dose of XENP30290 (PD1 x IL15[N4D / N65D]. The data show that XENP30290 induced PD1+ cell proliferation at a 1x low dose.

[0184] [Figure 126]Figures 126A-126E show the proliferation of A) CD8+ PD1+ T cells, B) CD8+ stem cell memory, C) CD8 naive (PD1low), D) γδ cells, and E) NK cells in cynomolgus monkeys administered a 10-fold higher dose of XENP30290 (PD1 x IL15[N4D / N65D]) and the corresponding RSV-targeted surrogate XENP30362. The data show that XENP30290 induced good PD1+ cell proliferation at the 10-fold higher dose, but had moderate activity against PD1− cells, as indicated by the activity of the RSV-targeted control XENP30362. The "(G4S) linker" is SEQ ID NO: 7.

[0185] [Figure 127] Figures 127A-127E show the proliferation of A) CD8+PD1+ T cells, B) CD8+ stem cell memory, C) CD8 naive (PD1low), D) γδ cells, and E) NK cells in cynomolgus monkeys treated with a 10-fold higher dose of XENP30516 (PD1 x IL15[D30N / E64Q / N65D]). The data show that XENP30516 induced good PD1+ cell proliferation at the 10-fold higher dose.

[0186] [Figure 128] Figures 128A-128E show the proliferation of A) CD8+ PD1+ T cells, B) CD8+ stem cell memory, C) CD8 naive (PD1 low), D) γδ cells, and E) NK cells in cynomolgus monkeys administered a 30-fold higher dose of XENP30516 (PD1 x IL15 [D30N / E64Q / N65D]) and the corresponding RSV-targeting surrogate XENP30518. The data show that XENP30516 induced greater PD1+ expansion at a 30-fold higher dose while maintaining excellent selectivity. The "(G4S) linker" is SEQ ID NO: 7.

[0187] [Figure 129]Figures 129A-129E show the proliferation of A) CD8+ PD1+ T cells, B) CD8+ stem cell memory, C) CD8 naive (PD1 low), D) γδ cells, and E) NK cells in cynomolgus monkeys administered a 1x low dose of XENP31986 (PD1 x IL15 [D30N / E64Q / N65D / N71Q / N79Q / N112Q]) and the corresponding RSV-targeted surrogate XENP32169. The data show that XENP31986 induced good PD1+ cell proliferation at a 1x low dose with excellent selectivity. The "(G4A) linker" is SEQ ID NO: 8.

[0188] [Figure 130] Figures 130A-130E show the proliferation of A) CD8+ PD1+ T cells, B) CD8+ stem cell memory, C) CD8 naive (PD1 low), D) γδ cells, and E) NK cells in cynomolgus monkeys administered a 3-fold intermediate dose of XENP31986 (PD1xIL15 [D30N / E64Q / N65D / N71Q / N79Q / N112Q]) and the corresponding RSV-targeted surrogate XENP32169. The data show that XENP31986 induced enhanced PD1+ cell proliferation at a 3-fold intermediate dose while maintaining excellent selectivity. The "(G4A) linker" is SEQ ID NO: 8.

[0189] [Figure 131] Figures 131A-131E show the expansion of A) CD8+ PD1+ T cells, B) CD8+ stem cell memory, C) CD8 naive (PD1 low), D) γδ cells, and E) NK cells in cynomolgus monkeys administered a 10-fold higher dose of XENP31986 (PD1 x IL15 [D30N / E64Q / N65D / N71Q / N79Q / N112Q]) and the corresponding RSV-targeted surrogate XENP32169. The data show that XENP31986 induced significantly greater PD1+ cell expansion at the 10-fold higher dose while maintaining excellent selectivity. The "(G4A) linker" is SEQ ID NO: 8.

[0190] [Figure 132]Figure 132 summarizes the proliferation of CD8+PD1+ T cells in cynomolgus monkeys administered various concentrations of various PD-1-targeted or RSV-targeted IL-15 / Rα-Fc fusions containing either IL-15[N4D / N65D], IL-15[D30N / E64Q / N65D], or IL-15[D30N / E64Q / N65D / N71Q / N79Q / N112Q].

[0191] [Figure 133] Figures 133A-133E show the correlation between the selectivity ratio and peak fold expansion of A) CD8+PD1+ T cells, B) CD8+ stem cell memory, C) CD8 naive (PD1low), D) γδ cells, and E) NK cells in cynomolgus monkeys administered various concentrations of various PD-1- or RSV-targeted IL-15 / Rα-Fc fusions containing either IL-15[N4D / N65D], IL-15[D30N / E64Q / N65D], or IL-15[D30N / E64Q / N65D / N71Q / N79Q / N112Q]. Together, the data show that increasing dose results in greater selectivity. In addition, PD-1-targeted IL-15 / Rα-Fc fusions containing the IL-15[D30N / E64Q / N65D / N71Q / N79Q / N112Q] variant were more selective than PD-1-targeted IL-15 / Rα-Fc fusions containing the IL-15[D30N / E64Q / N65D] variant, which were more selective than PD-1-targeted IL-15 / Rα-Fc fusions containing the IL-15[N4D / N65D] variant.

[0192] [Figure 134]Figure 134 shows the time course of serum concentrations of the indicated test articles in cynomolgus monkeys. Surprisingly, at the same dose, deglycosylated PD-1-targeting IL-15 / Rα-Fc XENP31896 containing the IL-15 [D30N / E64Q / N65D / N71Q / N79Q / N112Q] variant exhibited enhanced pharmacokinetics compared to glycosylated PD-1-targeting IL-15 / Rα-Fc XENP30516 containing the IL-15 [D30N / E64Q / N65D] variant, even though XENP31896 has enhanced potency / pharmacodynamics compared to XENP30516. The "(G4S) linker" is SEQ ID NO: 7, and the "(G4A) linker" is SEQ ID NO: 8.

[0193] [Figure 135] Figure 135 shows the time course of serum concentrations of the indicated test articles in cynomolgus monkeys. At the same dose, deglycosylated RSV-targeted IL-15 / Rα-Fc XENP32169 containing the IL-15 [D30N / E64Q / N65D / N71Q / N79Q / N112Q] variant showed enhanced pharmacokinetics compared to glycosylated RSV-targeted IL-15 / Rα-Fc XENP32169 containing the IL-15 [D30N / E64Q / N65D] variant. The "(G4S) linker" is SEQ ID NO: 7, and the "(G4A) linker" is SEQ ID NO: 8.

[0194] [Figure 136] Figure 136 shows Octet sensorgrams demonstrating binding of Trp-engineered mAb C[PD-1]_H1L1 variants compared to WT mAb C[PD-1]_H1L1. Numbering is according to Kabat. The data show that repair of the labile tryptophan resulted in complete loss of binding to PD-1 or substantially weaker binding.

[0195] [Figure 137]Figures 137A-G show the sequence of an exemplary Trp-engineered and affinity-repaired variant of mAb C[PD-1]_H1L1 in a bivalent human IgG1 format with E233P / L234V / L235A / G236del / S267K substitutions in the heavy chain. CDRs are underlined, and diagonal lines indicate the boundaries of the variable domains. As noted herein, and as applies to all sequences herein that contain CDRs, the exact identification of the CDR positions may vary slightly depending on the numbering used, as shown in Table 1; therefore, this specification includes not only the underlined CDRs, but also CDRs contained within the VH and VL domains using other numbering systems. As will be appreciated by those of skill in the art, the VH and VL domains can be formatted as Fabs or scFvs for use in the PD-1-targeting IL-15 / Rα-Fc fusion proteins of the invention.

[0196] [Figure 138] Figures 138A-G show the sequence of an exemplary PD-1-targeting IL-15 / Rα-Fc fusion protein of "scIL-15 / Rα x Fab" with exemplary Trp-engineered and affinity-repaired variants of mAb C[PD-1]_H1L1. CDRs are underlined. As noted herein, and as is true for all sequences herein that contain CDRs, the exact identification of the CDR positions may vary slightly depending on the numbering used, as shown in Table 1; therefore, this specification includes not only the underlined CDRs, but also CDRs contained within the VH and VL domains using other numbering systems. IL-15 and IL-15Rα (sushi) are in italics, the linker is double underlined (as one of skill in the art will appreciate, the linker can be replaced with other linkers, some of which are shown in Figures 9 and 10), and a slash ( / ) indicates the boundaries of IL-15, IL-15Rα, linker, variable region, and constant / Fc region. As will be apparent to one of skill in the art, each of the targeting IL-15 / Rα-Fc fusion proteins described can include or exclude Xtend Fc (M428L / N434S).

[0197] [Figure 139] Figure 139 shows the apparent dissociation constants (KDapp), association rates (ka), and dissociation rates (kd) of Trp-engineered mAb C[PD-1]_H1L1 variants with restored PD-1 binding as determined by Octet. Substitutions in the variable heavy or variable light chain regions (if listed) are based on Xencor numbering (corresponding Kabat positions are listed in the next column). Several variants were identified that restore PD-1 affinity binding close to that of mAb C_H1_L1.1, including mAb C_H1.176_L1.140, mAb C_H1.177_L1.140, and mAb C_H1.180_L1.140.

[0198] [Figure 140] Figures 140A-B show Octet sensorgrams showing the binding of A) XENP31986 (with mAb C[PD1]_H1_L1.1) and B) XENP32435 (with Trp-engineered / affinity-repaired mAb C[PD1]_H1.176_L1.140) to PD-1. The data show that the repaired molecules have comparable affinity for PD-1.

[0199] [Figure 141] Figure 141 shows induction of CD8+ effector memory T cell proliferation by Trp-engineered PD-1-targeted IL-15 / Rα-Fc fusions. XENP32435, XENP32436, and XENP32439, which have PD-1 binding domains engineered to remove Trp, are as or more potent than XENP31986 in expanding CD8+ effector memory T cells (despite having similar affinity for PD-1).

[0200] [Figure 142]Figure 142 shows the sequences of exemplary IL-15 / Rα-Fc fusion proteins in the "IL-15 / Rα-hetero-Fc" format: XENP20818 (WT IL-15), XENP22821 (IL-15[N65D]), and XENP24045 (IL-15[D30N / E64Q / N65D]). IL-15 and IL-15Rα (sushi) are underlined, the linker is double underlined (those skilled in the art will appreciate that the linker can be replaced with other linkers, some of which are shown in Figure 7), and a diagonal line ( / ) indicates the boundaries of the IL-15, IL-15Rα, linker, and Fc regions.

[0201] [Figure 143] Figure 143 shows the proliferation of rapamycin-expanded Tregs after treatment with non-targeted IL-15 / Rα-Fc fusions XENP20818 and XENP24045 and various PD-1-targeted or control RSV-targeted IL-15 / Rα-Fc fusions. The data show that IL-15 / Rα-Fc fusions (targeted and non-targeted) induce proliferation of rapamycin-expanded Tregs (measured by Tag-it Violet dilution). Notably, the PD-1-targeted IL-15 / Rα-Fc fusion was much less potent at inducing Treg proliferation than the non-targeted IL-15 / Rα-Fc fusion.

[0202] [Figure 144] Figures 144A-B show the proliferation of A) CD8 effector memory T cells and B) CD4 effector memory T cells (determined by CFSE dilution) after incubation of 1 x 10 CFSE-labeled PBMCs with 5 μg / ml of the indicated test article and increasing numbers of rapamycin-expanded Tregs. The data show that PD-1-targeted IL-15 / Rα-Fc fusions shifted (reduced) the potency of Treg-induced suppression of CD8 and CD4 effector memory T cell proliferation. Notably, the shift by the control RSV-targeted IL-15 / Rα-Fc fusion was smaller than the decrease in potency induced by the PD-1-targeted IL-15 / Rα-Fc fusion.

[0203] [Figure 145] Figures 145A-B show the ratios of A) Tregs to CD8 effector memory T cells and B) Tregs to CD4 effector memory T cells after incubation of 1 x 10 CFSE-labeled PBMCs with 5 μg / ml of the indicated test article and increasing numbers of rapamycin-expanded Tregs. The data show that, compared with no test article, PD-1-targeted IL-15 / Rα-Fc fusions increased the Treg / TEM ratio, yet TEM cell proliferation was enhanced by PD-1-targeted IL-15 / Rα-Fc fusions. This indicates that although Tregs expand, expanded Tregs exhibit reduced suppressive capacity.

[0204] [Figure 146] Figures 146A-B show the proliferation percentages of A) CD8+ T cells and B) CD4+ T cells (determined by CFSE dilution) after incubation of CD3-stimulated PBMCs with rapamycin-expanded Tregs precultured for 6 days in complete Treg medium (RPMI containing 10% FBS, 0.5 μg / ml anti-CD28, 100 U / ml IL-2, 100 ng / ml rapamycin); complete Treg medium without rapamycin; or 100 ng / ml IL-15 (RPMI containing 10% FBS, 0.5 μg / ml anti-CD28; no IL-2; no rapamycin). The data show that Tregs pretreated with IL-15 exhibit impaired suppressive capacity.

[0205] [Figure 147] Figures 147A-B show the expression of CD25 and FOXP3 on CD4+ T cells in PBMCs treated for 14 days without (A) or with (B) 5 μg / ml IL-15 / Rα-Fc fusion XENP22821. The data show that treatment with XENP22821 reduced FOXP3 expression on the CD4+ T cell population.

[0206] [Figure 148]Figures 148A-B show CD45RA and FOXP3 expression on CD4+ T cells in PBMCs treated for 14 days without (A) or with (B) 5 μg.ml IL-15 / Rα-Fc fusion XENP22821. The data show that treatment with XENP22821 shifts the CD4+CD45RA- population from FoxP3high to FoxP3low, indicating that treatment with IL-15 / Rα-Fc fusion indeed shifts the population from eTregs (a decreased population from 5.24% to 2.72%) to activated effector CD4 T cells (an increased population from 18.2% to 28.6%).

[0207] [Figure 149] Figures 149A-B show the expression of CD25 and CCR4 on CD4+ T cells in PBMCs treated for 14 days without (A) or with (B) 5 μg / ml IL-15 / Rα-Fc fusion XENP22821. The data show that treatment with XENP22821 reduced CCR4 expression on the CD4+ T cell population.

[0208] [Figure 150] Figures 150A-B show the proliferation of A) CD8 T cells and B) CD4 T cells (determined by CFSE dilution) after incubation of CFSE-labeled PBMCs on 100 ng / ml plate-bound anti-CD3 (OKT3) with 10 μg / ml of the IL-15 / Rα-Fc fusion XENP24045 and the indicated concentrations of TGFβ1. The data show that TGFβ dose-dependently suppresses T cell proliferation, but notably, the IL-15 / Rα-Fc fusion prevents TGFβ suppression of T cell proliferation at all doses tested.

[0209] [Figure 151]Figures 151A-B show the apparent dissociation constants (KDapp), association rates (ka), and dissociation rates (kd) of Trp-engineered mAb C[PD-1]_H1L1 variants with restored PD-1 binding as determined by Octet. Substitutions in the variable heavy chain region (if listed) are based on Xencor numbering (corresponding Kabat positions are listed in the next column).

[0210] [Figure 152] Figures 152A-152N show the variable heavy and variable light chains of additional exemplary anti-PD-1 ABDs that do not compete with nivolumab or pembrolizumab. The CDRs are underlined. As noted herein, and as is true for all sequences herein that contain CDRs, the exact identification of the CDR positions may vary slightly depending on the numbering used as shown in Table 1; therefore, the present specification includes not only the underlined CDRs, but also CDRs contained within the VH and VL domains using other numbering systems.

[0211] [Figure 153]Figures 153A-B show sequences of exemplary PD-1-targeted IL-15 constructs. Figure 153A shows XENP31326. Figure 153B shows XENP31329. It is noted herein, and as is true for all sequences herein that contain CDRs, the exact identification of the CDR positions may vary slightly depending on the numbering used as shown in Table 1; therefore, this specification includes not only the underlined CDRs, but also CDRs contained within the VH and VL domains using other numbering systems. IL-15 and IL-15Rα (sushi) are in italics, the linker is double underlined (as will be appreciated by those skilled in the art, the linker can be replaced with other linkers, some of which are shown in Figures 9 and 10), and a diagonal line ( / ) indicates the boundaries of IL-15, IL-15Rα, linker, variable region, and constant / Fc region. As will be apparent to one of skill in the art, each of the targeted IL-15 / Rα-Fc fusion proteins described can include or exclude Xtend Fc (M428L / N434S).

[0212] [Fig. 154] Figures 154A-C show the induction of CD3 effector memory T cell proliferation by A) XENP31326 vs. XENP31329, B) XENP30516 and XENP32927 vs. XENP30518, and C) XENP31986 and XENP32435 vs. XENP32169. The data show that targeting alone (as shown by a comparison of XENP31326 vs. XENP31329) provides limited selectivity (a 2.4-fold difference in EC50 between RSV targeting and PD1 targeting). PD1 targeting combined with reduced IL-15 potency (as shown by XENP30516 and XENP32927 vs. XENP30518; XENP31986 and XENP32435 vs. XENP32169) provides significantly enhanced selectivity. Remarkably and surprisingly, the deglycosylated variants further enhance selectivity (246-fold selectivity with the deglycosylated variant compared to 105-fold selectivity without it).

[0213] [Figure 155] Figures 155A and 155B show the proliferation of (Figure 155A) CD8+ and (Figure 155B) CD4+ responder cells in the presence of XmAb24306 and various concentrations of rapamycin-expanded Tregs.

[0214] [Figure 156] Figure 156 shows the proliferation of rapamycin-expanded Tregs after treatment with IL-15 / Rα-Fc fusions XENP20818 and XENP24045. The data show that IL-15 / Rα-Fc fusions induce proliferation of rapamycin-expanded Tregs (measured by Tag-it Violet dilution). Notably, XENP24045 demonstrates reduced potency in inducing Treg proliferation compared to XENP20818.

[0215] [Figure 157] Figures 157A and 157B show the proliferation of A) CD8 effector memory T cells and B) CD4 effector memory T cells (determined by CFSE dilution) after incubation of 1 x 10 CFSE-labeled PBMCs with 5 μg / ml of the indicated test article and increasing numbers of rapamycin-expanded Tregs. The data show that IL-15 / Rα-Fc fusions shifted (reduced) the potency of Treg-induced suppression of CD8 and CD4 effector memory T cell proliferation.

[0216] [Figure 158]Figures 158A and 158B show the ratios of A) Tregs to CD8 effector memory T cells and B) Tregs to CD4 effector memory T cells after incubation of 1 x 10 CFSE-labeled PBMCs with 5 μg / ml of the indicated test article and increasing numbers of rapamycin-expanded Tregs. The data show that, compared with no test article, IL-15 / Rα-Fc fusion increased the Treg / TEM ratio, yet TEM cell proliferation was enhanced by IL-15 / Rα-Fc fusion. This indicates that although Tregs expand, expanded Tregs exhibit reduced suppressive capacity.

[0217] [Figure 159] Figures 159A and 159B show the proliferation percentages A) CD8+ T cells and B) CD4+ T cells (determined by CFSE dilution) after incubation of CD3-stimulated PBMCs with rapamycin-expanded Tregs precultured for 6 days in complete Treg medium (RPMI containing 10% FBS, 0.5 μg / ml anti-CD28, 100 U / ml IL-2, 100 ng / ml rapamycin); complete Treg medium without rapamycin; or 100 ng / ml IL-15 (RPMI containing 10% FBS, 0.5 μg / ml anti-CD28; no IL-2; no rapamycin). The data show that Tregs pretreated with IL-15 exhibit impaired suppressive capacity.

[0218] [Figure 160] Figures 160A and 160B show the expression of CD25 and FOXP3 on CD4+ T cells in PBMCs treated for 14 days without (A) or with (B) 5 μg / ml IL-15 / Rα-Fc fusion XENP22821. The data show that treatment with XENP22821 reduced FOXP3 expression on the CD4+ T cell population.

[0219] [Figure 161]Figures 161A and 161B show the expression of CD45RA and FOXP3 on CD4+ T cells in PBMCs treated for 14 days without (A) or with (B) 5 μg / ml IL-15 / Rα-Fc fusion XENP22821. The data show that treatment with XENP22821 shifts the CD4+CD45RA- population from FoxP3high to FoxP3low, indicating that treatment with IL-15 / Rα-Fc fusion indeed shifted the population from eTregs (a decreased population from 5.24% to 2.72%) to activated effector CD4 T cells (an increased population from 18.2% to 28.6%).

[0220] [Figure 162] Figures 162A and 162B show the expression of CD25 and CCR4 on CD4+ T cells in PBMCs treated for 14 days without (A) or with (B) 5 μg / ml IL-15 / Rα-Fc fusion XENP22821. The data show that treatment with XENP22821 reduced CCR4 expression on the CD4+ T cell population.

[0221] [Figure 163] Figures 163A and 163B show the proliferation of A) CD8 T cells and B) CD4 T cells (determined by CFSE dilution) after incubation of CFSE-labeled PBMCs on 100 ng / ml plate-bound anti-CD3 (OKT3) with 10 μg / ml of the IL-15 / Rα-Fc fusion XENP24045 and the indicated concentrations of TGFβ1. The data show that TGFβ dose-dependently suppresses T cell proliferation, but notably, the IL-15 / Rα-Fc fusion prevents TGFβ suppression of T cell proliferation at all doses tested.

[0222] [Fig. 164]Figure 164 shows tumor volume (determined by caliper measurement) over time in pp65-MCF7-implanted huCD34+ NSG mice treated with single-agent PD-1 blockade, non-targeted, reduced-potency IL-15 / Rα-Fc fusion XENP24045 (as a single agent or in combination with PD-1 blockade), or [NC]PD-1-targeted, reduced-potency glycoengineered IL-15 / Rα-Fc. All groups showed significantly enhanced tumor activity by day 15 compared to treatment with PBS control (p≦0.05), with a clear benefit on anti-tumor activity from combining with PD-1 blockade.

[0223] [Figure 165] Figure 165 shows the proliferation of CD4+ and CD8+ T cells by days 7 and 13 in pp65-MCF7-engrafted huCD34+ NSG mice administered single-agent PD-1 blockade, non-targeted, reduced-potency IL-15 / Rα-Fc fusion XENP24045 (as a single agent or in combination with PD-1 blockade), or [NC]PD-1-targeted, reduced-potency glycoengineered IL-15 / Rα-Fc. PD-1-targeted IL-15 in combination with PD-1 blockade significantly enhanced the proliferation of both CD8+ and CD4+ T cells compared to non-targeted IL-15-Fc fusion in combination with PD-1 blockade. The combination of PD-1-targeted IL-15 and PD-1 blockade significantly increased lymphocyte proliferation compared to single-agent PD-1-targeted IL-15.

[0224] [Figure 166] Figure 166 shows the CD8 T cell to Treg ratio on day 13 in pp65-MCF7-engrafted huCD34+ NSG mice administered single-agent PD-1 blockade, non-targeted reduced-potency IL-15 / Rα-Fc fusion XENP24045 (as a single agent or in combination with PD-1 blockade), or [NC]PD-1-targeted reduced-potency glycoengineered IL-15 / Rα-Fc. The CD8:Treg ratio is improved with PD1-targeted IL15 (further in combination with PD-1 blockade). Statistics performed on log-transformed data using unpaired t-tests.

[0225] [Figure 167] Figures 167A-E show: A) percentage of CD3+ T cells in the total CD45 lymphocyte population, B) percentage of effector memory CD8 T cells in the total CD8 population, C) percentage of CD8 naive in the total CD8 population, D) percentage of Tregs in the total CD3 population, and E) ratio of effector memory CD8 T cells to Tregs on day 9 in tumor tissue of pp65-MCF7-implanted huCD34+ NSG mice treated with single-agent PD-1 blockade or [NC]PD-1-targeted reduced efficacy glycoengineered IL-15 / Rα-Fc (as a single agent or in combination with PD-1 blockade). The data show that XENP32986, alone or in combination with PD-1 blockade, increased total CD3+ T cells and shifted T cell phenotype to effector memory CD8+ in tumors; XENP32986, alone or in combination with PD-1 blockade, increased the CD8:Treg ratio in tumors; and a low dose of 0.01 mg / kg of XENP32986 enabled significant pharmacodynamics in combination with PD-1 blockade.

[0226] [Figure 168] Figures 168A-L show baseline-corrected changes in tumor volume (determined by caliper measurement) over time in huPBMC- and pp65-MCF7-engrafted NSG-DKO mice administered single-agent PD-1 blockade, PD-1-targeted IL-15 [D30N / E64Q / N65D] XENP32927 (as a single agent at 0.1, 0.3, or 1 mg / kg, or in combination with PD-1 blockade at 0.3 mg / kg), PD-1-targeted IL-15 [D30N / E64Q / N65D / N71Q / N79Q / N112Q] XENP32435 (as a single agent at 0.03, 0.1, or 0.3 mg / kg, or in combination with PD-1 blockade at 0.1 mg / kg), and RSV-targeted IL-15 control in combination with PD-1 blockade.

[0227] [Figure 169]Figure 169 shows the baseline-corrected median change in tumor volume (determined by caliper measurement) over time in huPBMC- and pp65-MCF7-implanted NSG-DKO mice treated with single-agent PD-1 blockade, PD-1-targeted IL-15 [D30N / E64Q / N65D] XENP32927 (as a single agent at 0.1, 0.3, or 1 mg / kg or in combination with PD-1 blockade at 0.3 mg / kg), and RSV-targeted IL-15 [D30N / E64Q / N65D] control in combination with PD-1 blockade. XENP32927 induced significant tumor regression at 0.3 mg / kg in combination with PD-1 blockade (p<0.05 on days 17, 20, 22, and 27) and at 1 mg / kg in combination with PD-1 blockade (p<0.05 on days 15 and 17) compared to PD-1 blockade alone (statistics performed on baseline-corrected data using the Mann-Whitney test).

[0228] [Figure 170] Figure 170 shows the baseline-corrected median change in tumor volume (determined by caliper measurement) over time in huPBMC- and pp65-MCF7-implanted NSG-DKO mice treated with single-agent PD-1 blockade, PD-1-targeted IL-15 [D30N / E64Q / N65D / N71Q / N79Q / N112Q] XENP32435 (as a single agent at 0.03, 0.1, or 0.3 mg / kg or in combination with PD-1 blockade at 0.1 mg / kg), and RSV-targeted IL-15 [D30N / E64Q / N65D / N71Q / N79Q / N112Q] control in combination with PD-1 blockade. XENP32435 induced significant tumor regression at 0.1 mg / kg in combination with PD-1 blockade compared to PD-1 blockade alone (p<0.05 on days 13 and 15) and at 0.3 mg / kg in combination with PD-1 blockade (p<0.05 on days 13, 15, and 17). Notably, XENP32435 showed significant tumor regression as a single agent in this model (p<0.05 on day 17 compared to PD-1 blockade alone). Statistics performed on baseline-corrected data using the Mann-Whitney test.

[0229] [Figure 171] Figure 171 shows the change in tumor volume by day 17 in huPBMC- and pp65-MCF7-engrafted NSG-DKO mice treated with single-agent PD-1 blockade, PD-1-targeted IL-15 [D30N / E64Q / N65D] XENP32927 (as a single agent at 0.1, 0.3, or 1 mg / kg, or in combination with PD-1 blockade at 0.3 mg / kg), PD-1-targeted IL-15 [D30N / E64Q / N65D / N71Q / N79Q / N112Q] XENP32435 (as a single agent at 0.03, 0.1, or 0.3 mg / kg, or in combination with PD-1 blockade at 0.1 mg / kg), and RSV-targeted IL-15 control combined with PD-1 blockade. Statistics performed on baseline-corrected data using the Mann-Whitney test. The data show that RSV-targeted IL-15 (including either IL-15 variant) did not have enhanced activity in combination with PD-1 blockade compared to PD-1 blockade alone.

[0230] [Fig. 172] Figures 172A-D show: A) CD4+ T cell activation at day 7 and B) CD8+ T cell activation (indicated by CD25 expression) in huPBMC- and pp65-MCF7-engrafted NSG-DKO mice treated with single-agent PD-1 blockade, PD-1-targeted IL-15 [D30N / E64Q / N65D] XENP32927 (as a single agent at 0.1, 0.3, or 1 mg / kg or in combination with PD-1 blockade at 0.3 mg / kg), and RSV-targeted IL-15 [D30N / E64Q / N65D] control combined with PD-1 blockade; and C) CD4+ T cell counts and D) CD8+ T cell counts at day 14. The data show that XENP32927 was active at 0.1-1 mg / kg.

[0231] [Figure 173]Figures 173A-D show: A) CD4+ T cell activation on day 7 and B) CD8+ T cell activation (indicated by CD25 expression); and C) CD4+ T cell counts on day 14 and D) CD8+ T cell counts in huPBMC- and pp65-MCF7-transplanted NSG-DKO mice treated with single-agent PD-1 blockade, PD-1-targeted IL-15 [D30N / E64Q / N65D / N71Q / N79Q / N112Q] XENP32435 (as a single agent at 0.03, 0.1, or 0.3 mg / kg or in combination with PD-1 blockade at 0.1 mg / kg), and RSV-targeted IL-15 [D30N / E64Q / N65D / N71Q / N79Q / N112Q] control combined with PD-1 blockade. The data show that XENP32435 was active at many concentrations, from 0.03 to 0.3 mg / kg.

[0232] [Fig. 174] Figure 174 shows serum IFNγ concentrations over time in huPBMC- and pp65-MCF7-engrafted NSG-DKO mice treated with single-agent PD-1 blockade, PD-1-targeted IL-15[D30N / E64Q / N65D]XENP32927 (as a single agent at 0.1, 0.3, or 1 mg / kg, or in combination with PD-1 blockade at 0.3 mg / kg), and RSV-targeted IL-15[D30N / E64Q / N65D] control in combination with PD-1 blockade.

[0233] [Figure 175] Figure 175 shows serum IFNγ concentrations over time in huPBMC- and pp65-MCF7-engrafted NSG-DKO mice treated with single-agent PD-1 blockade, PD-1-targeted IL-15 [D30N / E64Q / N65D / N71Q / N79Q / N112Q] XENP32435 (as a single agent at 0.03, 0.1, or 0.3 mg / kg or in combination with PD-1 blockade at 0.1 mg / kg), and RSV-targeted IL-15 [D30N / E64Q / N65D / N71Q / N79Q / N112Q] control in combination with PD-1 blockade.

[0234] [Figure 176]Figures 176A-E show the proliferation of A) CD8+ T cells, B) CD8+ stem cell memory T cells, C) CD8+ naive T cells, D) γδ T cells, and E) NK cells in cynomolgus monkeys treated with XENP32435PD1xIL15[D30N / E64Q / N65D / N71Q / N79Q / N112Q]) administered intravenously at a 1x dose.

[0235] [Figure 177] Figures 177A-177E show the proliferation of A) CD8+ T cells, B) CD8+ stem cell memory T cells, C) CD8+ naive T cells, D) γδ T cells, and E) NK cells in cynomolgus monkeys treated with XENP32435PD1xIL15[D30N / E64Q / N65D / N71Q / N79Q / N112Q]) administered intravenously at a 12-fold dose compared to Figures 175A-175E.

[0236] [Figure 178] Figures 178A-E show the proliferation of A) CD8+ T cells, B) CD8+ stem cell memory T cells, C) CD8+ naive T cells, D) γδ T cells, and E) NK cells in cynomolgus monkeys treated with XENP32927 (PD1xIL15[D30N / E64Q / N65D]) administered intravenously at a 1x dose.

[0237] [Figure 179] Figures 179A-179E show proliferation of A) CD8+ T cells, B) CD8+ stem cell memory T cells, C) CD8+ naive T cells, D) γδ T cells, and E) NK cells in cynomolgus monkeys treated with XENP32927 (PD1xIL15[D30N / E64Q / N65D]) administered intravenously at a 20-fold dose compared to Figures 177A-177E.

[0238] [Figure 180] FIG. 180 shows the time course of serum concentrations of test article in cynomolgus monkeys treated with deglycosylated XENP32435 intravenously at 1× and 12× doses.

[0239] [Figure 181] FIG. 181 shows the time course of serum concentrations of test article in cynomolgus monkeys treated with glycosylated XENP32927 intravenously at 1× and 20× doses.

[0240] [Figure 182] Figures 182A-B show: A) CD8+ T cell numbers and B) percentage of Ki-67 expression on CD8+ T cells in mouse splenocytes after incubation with the indicated test articles. The data show that the mouse surrogate PD-1-targeting IL-15 molecule was highly selective for PD1+ mouse T cells.

[0241] [Figure 183] Figure 183A and Figure 183B show: A) CD8+ T cell numbers and B) percentage of Ki-67 expression on CD8+ T cells in mouse splenocytes after incubation with the indicated test articles, including XENP36217, an alternative mouse surrogate PD-1-targeting IL-15 molecule that binds to a different muPD-1 epitope than (i.e., does not compete with) XENP33869. The data show that XENP36217 was also highly selective for PD1+ mouse T cells.

[0242] [Figure 184]Figures 184A-184K show the anti-tumor and pharmacodynamic responses to administration of the PD-L1 blockade muPD1xIL15 surrogate XENP33869. A) Resected tumor weights recorded on days 3 and 6 after group out; B) Enumeration of CD8, CD4, Treg, NK, and NKT cells measured by flow cytometry in the tumor; C) CD8:Treg ratio in the tumor; D) Frequency of CD8, CD4, Treg, NK, and NKT cells measured by flow cytometry in the tumor; E) Frequency of CD8, CD4, Treg, NK, and NKT cells measured by flow cytometry in the spleen; F) Enumeration of CD8, CD4, Treg, NK, and NKT cells measured by flow cytometry in the spleen; G H) Frequency of Granzyme B+CD8, CD4, Treg, NK, and NKT cells measured by flow cytometry in the tumor; I) MFI of Granzyme B+CD8, CD4, Treg, NK, and NKT cells measured by flow cytometry in the tumor; J) Frequency of Granzyme B+CD8, CD4, Treg, NK, and NKT cells measured by flow cytometry in the spleen; K) MFI of Granzyme B+CD8, CD4, Treg, NK, and NKT cells measured by flow cytometry in the spleen.

[0243] [Figure 185]Figures 185A-H show the anti-tumor and pharmacodynamic responses to administration of the PD-L1 blocking muPD1xIL15 surrogate XEN33869 compared to the non-PD-L1 blocking muPD1xIL15 surrogates XENP36213, XENP36216, and XENP36217. A) Resected tumor weight recorded 6 days after group-out; B) Enumeration of CD8, CD4, Treg, NK, and NKT cells measured by flow cytometry in tumors; C) Frequencies of CD8, CD4, Treg, NK, and NKT cells measured by flow cytometry in tumors; D) CD8:Treg ratio in tumors; E) Enumeration of CD8, CD4, Treg, NK, and NKT cells measured by flow cytometry in spleens; F) CD8:Treg ratio in spleens; G) Frequencies of CD8, CD4, Treg, NK, and NKT cells measured by flow cytometry in spleens; H) Frequencies of Granzyme B+ CD8, CD4, Treg, NK, and NKT cells measured by flow cytometry in tumors and spleens.

[0244] [Figure 186] Figure 186A and Figure 186B show the effect of PD-L1 blocking muPD1xIL15 XENP33869 treatment on the growth of the MC38 syngeneic tumor model in female C57B1 / 6 mice after intravenous administration, compared to control and mouse reactive aPD-L1. A) Raw data and fitting. B) Overlay of fitted growth curves for each group.

[0245] [Figure 187] Figure 187A and Figure 187B show the effect of PD-L1 blocking muPD1xIL15 XENP33869 treatment on the growth of the MC38 syngeneic tumor model in female C57B1 / 6 mice after intravenous administration at various dose levels, compared to control and mouse-reactive aPD-L1. The combined activity of XENP33869 and anti-PDL1 was also assessed. A) Raw data and fitting. B) Overlay of fitted growth curves for each group.

[0246] [Figure 188]Figures 188A and 188B show the effect of treatment with the PD-L1 blocking muPD1xIL15 surrogate XENP33869, compared to treatment with the non-PD-L1 blocking muPD1xIL15 XENP36217, on the growth of the MC38 syngeneic tumor model in female C57B1 / 6 mice after intravenous administration, compared to control and mouse reactive aPD-L1. The combined activity of XENP33869 and XENP36217 with anti-PDL1 was also assessed. A) Raw data and fitting. B) Overlay of fitted growth curves for each group.

[0247] [Figure 189] Figure 189A-H show positron emission tomography imaging and quantification of 18F-labeled anti-mouse CD8 tracer following treatment with the muPD1xIL15 surrogate XENP33869 in female C57B1 / 6 mice bearing the MC38 syngeneic tumor model via intravenous administration, compared to treatment with vehicle or mouse-reactive aPD-L1. A) Representative images from each treatment group, with tumor location indicated by arrows. B) CD8 tracer uptake tumor compared to unbound control tracer measured 12 days after treatment. C) Time course of CD8 tracer uptake in blood, D) tumor, E) spleen, F) axillary lymph nodes, G) inguinal lymph nodes, and H) liver. DETAILED DESCRIPTION OF THE INVENTION

[0248] I. Name The heterodimeric fusion proteins of the present invention are listed in several different formats. Each polypeptide is given a unique "XENP" number, although longer sequences may contain shorter sequences, as is understood in the art. For example, a monomeric heavy chain containing an anti-PD-1 Fab (see, e.g., Figure 28C) has a first XENP number, but the VH and VL domains may have different XENP numbers. Some molecules contain three polypeptides, and therefore the XENP numbers of the components are used as the name. Thus, the molecule XENP29484 in "scIL-15 / Rα X Fab" contains three sequences or equivalents commonly referred to as "XENP29484 chain 1," "XENP29484 chain 2," and "XENP29484 chain 3," which one of skill in the art would readily identify by sequence alignment. These XENP numbers are found in the sequence listing as well as identifiers and used in the figures. Furthermore, a molecule containing three components results in multiple sequence identifiers. For example, the list of Fab monomers includes a full-length sequence, a variable heavy sequence, and three CDRs from a variable heavy sequence; a light chain includes three CDRs from a full-length sequence, a variable light sequence, and a variable light sequence; an scFv-Fc domain includes a full-length sequence, an scFv sequence, a variable light sequence, three light CDRs, an scFv linker, a variable heavy sequence, and three heavy CDRs; and all molecules herein that include scFv domains use a single charged scFv linker (+H), although other linkers may be used. Furthermore, the designation of specific variable domains uses a format of "Hx.xx_Ly.yy," where the numbers are unique identifiers for the specific variable chain sequences. Thus, the Fab variable domain (binding to PD-1) of XENP30486 is "H1.132," indicating that the variable heavy domain H1.132 was used and combined with the light domain L1.135 in XENP30486. Thus, the designation "mAbC[PD-1]_H1.132_L1.135" indicates that the variable heavy domain H1.132 has been combined with the light domain L1.134. When these sequences are combined into an scFv, this designation indicates that the scFv is in a VH-linker-VL orientation from N- to C-terminus.This molecule, with the same sequences of the heavy and light variable domains but in reverse order, is designated "mAbC[PD-1]_L1.135_H1.132". Similarly, different constructs can be "mixed and matched" with heavy and light chains, as is evident from the sequence listing and figures. II. Definition

[0249] In order that this application may be more fully understood, certain definitions are set forth below. Such definitions are meant to encompass grammatical equivalents.

[0250] As used herein, "ablation" refers to reducing or eliminating an activity. Thus, for example, "eliminating FcγR binding" means that the Fc region amino acid variant has less than 50% of the binding onset compared to an Fc region not comprising the specific variant, with a loss of activity of greater than 70-80-90-95-98% being preferred, and generally the activity being below the level of binding detectable in a Biacore assay. Of particular use in ablation of FcγR binding is that shown in Figure 6. However, unless otherwise specified, the Fc monomers of the present invention retain binding to the FcRn receptor.

[0251] As used herein, "ADCC" or "antibody-dependent cell-mediated cytotoxicity" refers to a cell-mediated reaction in which nonspecific cytotoxic cells expressing FcγR recognize bound antibody on target cells and subsequently cause lysis of the target cells. ADCC correlates with binding to FcγRIIIa, and increased binding to FcγRIIIa results in increased ADCC activity. As discussed herein, many embodiments of the present invention completely eliminate ADCC activity.

[0252] As used herein, "ADCP" or antibody-dependent cell-mediated phagocytosis refers to a cell-mediated reaction in which nonspecific cytotoxic cells expressing FcγR recognize bound antibody on target cells and subsequently cause phagocytosis of the target cells.

[0253] As used herein, "antigen binding domain" or "ABD" refers to a set of six complementarity determining regions (CDRs) that, when present as part of a polypeptide sequence, specifically binds to a target antigen as discussed herein. Thus, a "PD-1 antigen binding domain" binds to the human PD-1 antigen as outlined herein. As is known in the art, these CDRs generally comprise a first set of variable heavy CDRs (vhCDRs or VhCDRs). H CDRs) and a second set of variable light CDRs (vlCDRs or V L Each CDR is present in a variable heavy domain (vh or V), each of which contains three CDRs (vhCDR1, vhCDR2, vhCDR3 for the heavy chain, and vlCDR1, vlCDR2, and vlCDR3 for the light chain). The CDRs are present in the variable heavy domain and the variable light domain, respectively, and together form the Fv region. Thus, in some cases, the six CDRs of an antigen-binding domain are provided by the variable heavy chain and the variable light chain. In the "Fab" format, the set of six CDRs is provided by two different polypeptide sequences, the variable heavy domain (vh or V), and the variable light domain (vlCDR1, vlCDR2, and vlCDR3). H ; including vhCDR1, vhCDR2 and vhCDR3) and a variable light domain (vl or V L ; vlCDR1, vlCDR2, and vlCDR3), with the C-terminus of the vh domain attached to the N-terminus of the CH1 domain of the heavy chain and the C-terminus of the vl domain attached to the N-terminus of the constant light domain (thus forming the light chain). In the scFv format, the VH and VL domains are generally covalently linked into a single polypeptide sequence by the use of a linker as outlined herein, which can be either vh-linker-vl or vl-linker-vh (starting from the N-terminus).

[0254] The hypervariable regions generally comprise amino acid residues from about 24-34 (LCDR1; "L" indicates light chain), 50-56 (LCDR2), and 89-97 (LCDR3) in the light chain variable region and about 31-35B (HCDR1; "H" indicates heavy chain), 50-65 (HCDR2), and 95-102 (HCDR3) in the heavy chain variable region; Kabat et al., SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991) and / or the amino acid residues thereof that form the hypervariable loops (e.g., residues 26-32 (LCDR1), 50-52 (LCDR2), and 91-96 (LCDR3) in the light chain variable region, and 26-32 (HCDR1), 53-55 (HCDR2), and 96-101 (HCDR3) in the heavy chain variable region; Chothia and Lesk (1987) J. Mol. Biol. 196:901-917). Specific CDRs of the present invention are described below.

[0255] As will be understood by those of skill in the art, the exact numbering and arrangement of CDRs may vary between different numbering systems. However, it should be understood that the disclosure of a variable heavy and / or variable light sequence includes the disclosure of the associated (unique) CDRs. Thus, the disclosure of each variable heavy region is a disclosure of the vhCDRs (e.g., vhCDR1, vhCDR2, and vhCDR3), and the disclosure of each variable light region is a disclosure of the vlCDRs (e.g., vlCDR1, vlCDR2, and vlCDR3).

[0256] A useful comparison of CDR numbering is as follows (see Lafranc et al., Dev. Comp. Immunol. 27(1):55-77 (2003): [Table 1]

[0257] Throughout this specification, when referring to variable domain residues (approximately residues 1-107 for light chain variable regions and residues 1-113 for heavy chain variable regions), the Kabat numbering system is generally used, whereas for the Fc region, the EU numbering system is used (e.g., Kabat et al. (1991) supra).

[0258] The present invention provides a number of different CDR sets. In this case, a "complete CDR set" comprises three variable light CDRs and three variable heavy CDRs, e.g., vlCDR1, vlCDR2, vlCDR3, vhCDR1, vhCDR2, and vhCDR3. These may be part of a larger variable light or variable heavy domain, respectively. Furthermore, as more fully outlined herein, the variable heavy and variable light domains may be on separate polypeptide chains, when heavy and light chains are used (e.g., when a Fab is used), or on a single polypeptide chain, in the case of an scFv sequence.

[0259] CDRs contribute to the formation of the antigen-binding site of an antibody, more specifically, the epitope-binding site. "Epitope" refers to a determinant that interacts with a specific antigen-binding site in the variable region of an antibody molecule known as a paratope. An epitope is a group of molecules, such as amino acids or sugar side chains, that usually have specific structural and charge characteristics. A single antigen can have two or more epitopes.

[0260] An epitope can include amino acid residues that are directly involved in binding (also called the immunodominant component of the epitope) and other amino acid residues that are not directly involved in binding, e.g., amino acid residues that are effectively blocked by the specific antigen-binding peptide, in other words, amino acid residues within the footprint of the specific antigen-binding peptide.

[0261] Epitopes can be either conformational or linear. Conformational epitopes are produced by spatially juxtaposed amino acids from different segments of a linear polypeptide chain. Linear epitopes are produced by adjacent amino acid residues in a polypeptide chain. Conformational and nonconformational epitopes can be distinguished in that binding to conformational epitopes, but not nonconformational epitopes, is lost in the presence of denaturing solvents.

[0262] An epitope typically includes at least 3, and more usually at least 5 or 8-10 amino acids in a unique spatial conformation. Antibodies that recognize the same epitope can be verified in a simple immunoassay (e.g., "binning") showing the ability of one antibody to block the binding of another antibody to a target antigen. As outlined below, the present invention includes not only the antigen-binding domains and antibodies listed herein, but also those that compete for binding with the epitope bound by the listed antigen-binding domain (or, in the case of NC[PD-1]Fv, the anti-PD-1 CDRs of the invention do not compete for binding to the same epitope as the listed antibody).

[0263] For antibodies whose components are used in the present invention, the carboxy-terminal portion of each chain defines a constant region primarily responsible for effector function. Kabat et al. collected a large number of primary sequences of heavy and light chain variable regions. Based on the degree of sequence conservation, they classified each primary sequence into CDRs and frameworks and compiled a list thereof (see SEQUENCES OF IMMUNOLOGICAL INTEREST, 5th edition, NIH publication, No. 91-3242, E.A. Kabat et al., incorporated by reference in its entirety).

[0264] In the IgG subclass of immunoglobulins, several immunoglobulin domains are present in the heavy chain. As used herein, "immunoglobulin (Ig) domain" refers to a region of an immunoglobulin with a defined tertiary structure. Of interest in the present invention are heavy chain domains that contain the constant heavy (CH) domain and the hinge domain. In the context of IgG antibodies, each IgG isotype has three CH regions. Thus, the "CH" domains in the context of IgG are as follows: "CH1" refers to positions 118-220 according to the EU index as in Kabat; "CH2" refers to positions 237-340 according to the EU index as in Kabat; and "CH3" refers to positions 341-447 according to the EU index as in Kabat. As shown herein and described below, pI variants can be present in one or more of the CH and hinge regions described below.

[0265] Another type of Ig domain in the heavy chain is the hinge region. As used herein, "hinge" or "hinge region" or "antibody hinge region" or "immunoglobulin hinge region" refers to a flexible polypeptide comprising the amino acids between the first and second constant domains of an antibody. Structurally, the IgG CH1 domain ends at EU215, and the IgG CH2 domain begins at EU231. Thus, for IgG, the antibody hinge is defined herein as comprising positions 216 (E216 in IgG1) to 230 (P230 in IgG1), numbering according to the EU index as in Kabat. In some embodiments, the lower hinge is included, e.g., in the context of the Fc region, and the start of the "lower hinge" generally refers to position 226. As described herein, pI variants can also be made in the hinge region.

[0266] As will be appreciated by those skilled in the art, the exact numbering and arrangement of heavy constant region domains may vary between different numbering systems. A useful comparison of EU and Kabat heavy constant region numbering follows: See Edelman et al., 1969, Proc Natl Acad Sci USA 63:78-85 and Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th Ed., United States Public Health Service, National Institutes of Health, Bethesda, which are incorporated by reference in their entireties. [Table 2]

[0267] Light chains generally comprise two domains, a variable light domain (which contains the light chain CDRs and which, together with the variable heavy domain, form the Fv region), and a constant light chain region (often called CL or Cκ).

[0268] Another region of interest for further substitutions as outlined herein is the Fc region.

[0269] Thus, the present invention provides different protein domains. As described herein and known in the art, the heterodimeric proteins of the present invention comprise different, optionally overlapping, domains within the heavy and light chains. These domains include, but are not limited to, an Fc domain, a CH1 domain, a CH2 domain, a CH3 domain, a hinge domain, a heavy constant domain (CH1-hinge-Fc domain or CH1-hinge-CH2-CH3), a variable heavy domain, a variable light domain, a light constant domain, a Fab domain, and an scFv domain.

[0270] As used herein, "Fc" or "Fc region" or "Fc domain" refers to a polypeptide comprising the constant region of an antibody excluding the first constant region immunoglobulin domain (e.g., CH1), and optionally a portion of the hinge. In the case of an IgG, the Fc domain includes immunoglobulin domains CH2 and CH3 (Cγ2 and Cγ3) and the hinge region between CH1 (Cγ1) and CH2 (Cγ2). Although the boundaries of the Fc region can vary, the human IgG heavy chain Fc region is usually defined to include residues C226 or P230 at its carboxyl terminus, numbering according to the EU index as in Kabat. Thus, the "CH" domains in the context of an IgG are as follows: "CH1" refers to positions 118-215 according to the EU index as in Kabat; and "hinge" refers to positions 216-230 according to the EU index as in Kabat. "CH2" refers to positions 231 to 340 according to the EU index as in Kabat, and "CH3" refers to positions 341 to 447 according to the EU index as in Kabat. Thus, an "Fc domain" includes a -CH2-CH3 domain and, optionally, a hinge domain (hinge-CH2-CH3).

[0271] Thus, an "Fc domain" includes the -CH2-CH3 domain and, optionally, a hinge domain, which often serves as a domain linker. In embodiments herein, when an scFv is linked to an Fc domain, it is the C-terminus of the scFv construct that is linked to all or part of the hinge of the Fc domain. For example, it is generally linked to the sequence EPKS (SEQ ID NO: 9), which is the beginning of the hinge. Similarly, when an IL-15 component (IL-15 complex, IL-15 domain, or IL-15 Ra domain) is linked to an Fc domain, it is generally similarly linked (as a domain linker) to all or part of the hinge of the Fc domain. For example, it is generally linked to the sequence EPKS (SEQ ID NO: 9), which is the beginning of the hinge.

[0272] The present invention generally relates to Fc domains based on the IgG class, which has several subclasses, including, but not limited to, IgG1, IgG2, IgG3, and IgG4. Generally, IgG1, IgG2, and IgG4 are more frequently used than IgG3. It should be noted that IgG1 has different allotypes with polymorphisms at 356 (D or E) and 358 (L or M). The sequences shown herein use the 356E / 358M allotype, but other allotypes are also encompassed herein. That is, any sequence comprising an IgG1 Fc domain encompassed herein can have the 356D / 358L allotype instead of the 356E / 358M allotype.

[0273] Additionally, many of the sequences herein have at least one cysteine ​​substituted with serine at position 220. Generally, this is on the "scFv monomer" or "IL-15 complex" side for most of the sequences shown herein, but it may also be on the "Fab monomer" side or both to reduce disulfide formation. Specifically included within the sequences herein are one or both of these substituted cysteines (C220S).

[0274] As used herein, "heavy chain" or "heavy chain domain" refers to, from N- to C-terminus, VH-CH1-hinge-CH2-CH3 domains (CH2-CH3 includes the Fc domain). The heavy chain includes a variable heavy domain and a constant domain including CH1 (optional)-hinge-Fc domain including CH2-CH3. The light chain includes a variable light domain and a light constant domain (VL-CL).

[0275] As used herein, "modification" refers to an amino acid substitution, insertion, and / or deletion in a polypeptide sequence, or a change to a moiety chemically linked to a protein. For example, a modification can be an altered carbohydrate or PEG structure attached to a protein. As used herein, an "amino acid modification" refers to an amino acid substitution, insertion, and / or deletion in a polypeptide sequence. For clarity, unless otherwise specified, amino acid modifications are always to amino acids encoded by DNA, e.g., the 20 amino acids for which DNA and RNA have codons.

[0276] As used herein, "amino acid substitution" or "substitution" refers to the replacement of an amino acid at a particular position in a parent polypeptide sequence with a different amino acid. In particular, in some embodiments, the substitution is for an amino acid that does not naturally occur at the particular position and does not naturally occur in the organism or any organism. For example, the substitution E272Y or 272Y refers to a variant polypeptide (in this case, an Fc variant) in which glutamic acid at position 272 has been replaced with tyrosine. For clarity, an engineered protein that alters the nucleic acid coding sequence but does not change the starting amino acid (e.g., replacing CGG (which encodes arginine) with CGA (which still encodes arginine) to increase expression levels in the host organism) is not an "amino acid substitution." That is, if a new gene encoding the same protein is created, but the protein still has the same amino acid at the starting position, it is not an amino acid substitution.

[0277] As used herein, "amino acid insertion" or "insertion" refers to the addition of an amino acid sequence at a particular position in a parent polypeptide sequence. For example, -233E or 233E indicates the insertion of glutamic acid after position 233 and before position 234. Furthermore, -233ADE or A233ADE indicates the insertion of AlaAspGlu after position 233 and before position 234.

[0278] As used herein, "amino acid deletion" or "deletion" refers to the removal of an amino acid residue or sequence at a particular position in a parent polypeptide sequence. For example, E233-, E233#, E233(), E233_, or E233del indicates a deletion of glutamic acid at position 233. Additionally, EDA233- or EDA233# indicates a deletion of the sequence GluAspAla beginning at position 233.

[0279] As used herein, "variant protein" or "protein variant" or "variant" refers to a protein that differs from that of a parent protein by at least one amino acid modification. Protein variant can refer to the protein itself, a composition containing the protein, or the amino acid sequence encoding it. Preferably, a protein variant has at least one amino acid modification compared to the parent protein, for example, about 1 to about 70 amino acid modifications, preferably about 1 to about 5 amino acid modifications, compared to the parent protein. As described below, in some embodiments, the parent polypeptide, e.g., the Fc parent polypeptide, is a human wild-type sequence, such as the Fc region from IgG1, IgG2, IgG3, or IgG4. The protein variant sequence herein preferably has at least about 80% identity, most preferably at least about 90% identity, and more preferably at least about 95-98-99% identity to the parent protein sequence. Variant protein can refer to the variant protein itself, a composition containing the protein variant, or the DNA sequence encoding it.

[0280] Thus, as used herein, "Fc variant" or "variant Fc" refers to a protein comprising an amino acid modification within the Fc domain. Fc variants of the present invention are defined according to the amino acid modification that constitutes them. Thus, for example, N434S or 434S is an Fc variant with a substituted serine at position 434 relative to the parent Fc polypeptide, numbering according to the EU index. Similarly, M428L / N434S defines an Fc variant with the substitutions M428L and N434S relative to the parent Fc polypeptide. The identity of the WT amino acids may be unspecified, in which case the variant is referred to as 428L / 434S. Note that the substitutions may be provided in any order; i.e., for example, M428L / N434S is the same Fc variant as N434S / M428L, etc. For all positions discussed in this invention related to antibodies, unless otherwise specified, amino acid position numbering is according to the EU index. The EU index, or EU index as in the Kabat or EU numbering scheme, refers to the numbering of EU antibodies (Edelman et al., 1969, Proc Natl Acad Sci USA 63:78-85, incorporated herein by reference in its entirety). Modifications can be additions, deletions, or substitutions. Substitutions can include naturally occurring amino acids, and in some cases, synthetic amino acids. Examples include U.S. Pat. No. 6,586,207; WO 98 / 48032; WO 03 / 073238; U.S. Patent Application Publication No. 2004 / 0214988A1; WO 05 / 35727A2; WO 05 / 74524A2; JW Hin et al., (2002), Journal of the American Chemical Society 124:9026-9027; JW Hin, & P.G. Schultz, (2002), ChemBioChem 11:1135-1137; JW Hin, et al., (2002), PICAS United States of America 99:11020-11024; and L. Wang, & P.G. Schultz, (2002), Chem. 1-10, all of which are incorporated by reference in their entireties.

[0281] As used herein, "protein" herein refers to at least two covalently attached amino acids, including proteins, polypeptides, oligopeptides, and peptides. As used herein, "residue" refers to the position in the protein and its associated amino acid identity. For example, asparagine 297 (also called Asn297 or N297) is the residue at position 297 in the human antibody IgG1.

[0282] As used herein, "Fab" or "Fab region" refers to a polypeptide comprising the VH, CH1, VL, and CL immunoglobulin domains. Fab can refer to this region in isolation or in the context of a full-length antibody, antibody fragment, or Fab fusion protein.

[0283] As used herein, "Fv" or "Fv fragment" or "Fv region" refers to a polypeptide comprising the VL and VH domains of a single antibody, forming an ABD. As will be appreciated by those skilled in the art, these generally consist of two chains, or can be combined (generally using a linker as discussed herein) to form an scFv.

[0284] As used herein, "single-chain Fv" or "scFv" refers to a variable heavy domain covalently linked to a variable light domain using an scFv linker, generally as discussed herein, to form an scFv or scFv domain. The scFv domain may be in either N-terminal to C-terminal orientation (vh-linker-vl or vl-linker-vh).

[0285] As used herein, "IgG subclass modification" or "isotype modification" refers to an amino acid modification that converts one amino acid of one IgG isotype to the corresponding amino acid of a different aligned IgG isotype. For example, because IgG1 contains a tyrosine at EU296 and IgG2 contains a phenylalanine, an F296Y substitution in IgG2 is considered an IgG subclass modification.

[0286] Thus, as used herein, "isotype" means any of the subclasses of immunoglobulins defined by the chemical and antigenic properties of their constant regions. It is understood that therapeutic antibodies can also include hybrids of isotypes and / or subclasses.

[0287] As used herein, "variable region" refers to a region of an immunoglobulin containing one or more Ig domains substantially encoded by any of the Vκ, Vλ, and / or VH genes that constitute the κ, λ, and heavy chain immunoglobulin loci, respectively. With respect to the variable heavy and variable light domains of the present invention, the amino-terminal portions of each heavy and light antibody chain contain a variable region of approximately 100 to 110 or more amino acids primarily responsible for antigen recognition, and are generally referred to in the art and herein as "Fv domains" or "Fv regions." Within the variable region, three loops assemble to form the antigen-binding site for each V domain of the heavy and light chains. Each loop is referred to as a complementarity-determining region (hereinafter referred to as "CDR"), where amino acid sequence variation is most prominent. "Variable" refers to the fact that certain segments of the variable region vary extensively in sequence among antibodies. The variability within the variable region is not uniformly distributed. Instead, V regions consist of relatively invariant stretches of 15-30 amino acids called framework regions (FRs) separated by short regions of extreme variability called "hypervariable regions", each 9-15 amino acids long or longer.

[0288] Each VH and VL is composed of three CDRs and four framework regions (FRs), arranged from amino-terminus to carboxy-terminus in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Thus, the variable heavy domain comprises VHFR1-VHCDR1-VHFR2-VHCDR2-VHFR3-VHCDR3-VHFR4, and the variable light domain comprises VLFR1-VLCDR1-VLFR2-VLCDR2-VLFR3-VLCDR3-VLFR4.

[0289] As used herein, a "non-naturally occurring modification" refers to a non-isotypic amino acid modification. For example, the substitution 434S in IgG1, IgG2, IgG3, or IgG4 (or hybrids thereof) is considered a non-naturally occurring modification because none of the IgGs contain serine at position 434.

[0290] As used herein, "amino acid" and "amino acid identity" refer to one of the 20 naturally occurring amino acids encoded by DNA and RNA.

[0291] As used herein, "effector function" refers to a biochemical event that results from the interaction of an antibody Fc region with an Fc receptor or ligand. Effector functions include, but are not limited to, ADCC, ADCP, and CDC.

[0292] As used herein, "Fc gamma receptor," "FcγR," or "Fc gamma R" refers to any member of a family of proteins that binds to the Fc region of an IgG antibody and are encoded by the FcγR gene. In humans, this family includes, but is not limited to, FcγRI (CD64), which includes the isoforms FcγRIa, FcγRIb, and FcγRIc; FcγRII (CD32), which includes the isoforms FcγRIIa (including allotypes H131 and R131), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2), and FcγRIIc; FcγRIII (CD16), which includes the isoforms FcγRIIIa (including allotypes V158 and F158) and FcγRIIIb (including allotypes FcγRIIb-NA1 and FcγRIIb-NA2) (Jefferis et al., 2002, Immunol Lett 82:57-65 (incorporated by reference in its entirety)), and any unidentified human FcγR or FcγR isoform or allotype.

[0293] As used herein, "FcRn" or "fetal Fc receptor" refers to a protein that binds to the Fc region of an IgG antibody and is at least partially encoded by the FcRn gene. As known in the art, a functional FcRn protein comprises two polypeptides, often referred to as a heavy chain and a light chain. The light chain is β2-microglobulin, and the heavy chain is encoded by the FcRn gene. Unless otherwise specified herein, FcRn or FcRn protein refers to the complex of the FcRn heavy chain and β2-microglobulin. Various FcRn variants can be used to increase binding to the FcRn receptor and, in some cases, increase serum half-life. Generally, unless otherwise specified, the Fc monomers of the present invention retain binding to the FcRn receptor (and can include amino acid variants that increase binding to the FcRn receptor, as described below).

[0294] As used herein, "parent polypeptide" refers to a starting polypeptide that is subsequently modified to generate a variant. A parent polypeptide can be a naturally occurring polypeptide, or a variant or engineered version of a naturally occurring polypeptide. A parent polypeptide may refer to the polypeptide itself, a composition comprising the parent polypeptide, or the amino acid sequence that encodes it.

[0295] As used herein, the term "heavy constant region" refers to the CH1-hinge-CH2-CH3 portion of an antibody.

[0296] As used herein, the term "Fc fusion protein" or "immunoadhesin" refers to a protein comprising an Fc region typically linked (optionally via a linker moiety as described herein) to a different protein, such as IL-15 and / or IL-15R, as described herein. In some instances, two Fc fusion proteins can form a homodimeric Fc fusion protein or a heterodimeric Fc fusion protein, the latter being preferred. Optionally, one monomer of the heterodimeric Fc fusion protein comprises only an Fc domain (e.g., an empty Fc domain), and the other monomer is an Fc fusion comprising a variant Fc domain and a protein domain, e.g., an IL-15 complex. As outlined herein, in some embodiments, one monomer of the heterodimeric protein is an Fc fusion protein comprising an IL-15 complex, and the other monomer is a traditional heavy chain (with an associated light chain).

[0297] As used herein, "position" means a location in the sequence of a protein. Positions may be numbered consecutively or according to established formats, such as the EU index for antibody numbering.

[0298] "Strandedness," as used herein in the context of monomers of heterodimeric antibodies of the invention, means that heterodimerization variants are incorporated into each monomer in a manner that maintains their ability to "match" to form heterodimers, similar to two strands of "matched" DNA. For example, if several pI variants are engineered into monomer A (e.g., to increase the pI), a similarly available "charge-paired" steric variant will not interfere with the pI variant; e.g., a charge variant that increases the pI will be placed on the same "strand" or "monomer" to maintain the functionality of both. Similarly, for sets of paired "skewed" variants, as outlined more fully below, one skilled in the art would consider the pI when determining which strands or monomers to incorporate one set of pairs, such that the pI of the skew is also used to maximize pI separation.

[0299] As used herein, "target cell" refers to a cell that expresses a target antigen, in this case the PD-1 and / or IL-15 receptor.

[0300] As used herein, "wild-type or WT" refers to an amino acid sequence or nucleotide sequence found in nature, including allelic variations. A WT protein has an amino acid sequence or nucleotide sequence that has not been intentionally modified.

[0301] The bispecific heterodimeric proteins of the present invention are generally isolated or recombinant. "Isolated," when used to describe the various polypeptides disclosed herein, refers to a polypeptide that has been identified, separated, and / or recovered from the cell or cell culture in which the antibody is expressed. Typically, an isolated polypeptide is prepared by at least one purification step. "Isolated protein" refers to a protein that is substantially free of other proteins with different binding specificities. "Recombinant" means that the protein is produced using recombinant nucleic acid technology in an exogenous host cell.

[0302] "Percent (%) amino acid sequence identity" with respect to protein sequences is defined as the percentage of amino acid residues in a candidate sequence that are identical to those in a particular (parent) sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, without considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in a variety of ways within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms necessary to achieve maximal alignment across the full length of the sequences being compared. One particular program is the ALIGN-2 program, outlined in paragraphs 0279-0280 of U.S. Patent Application Publication No. 20160244525, incorporated herein by reference.

[0303] The degree of identity between an amino acid sequence of the present invention ("invention sequence") and a parent amino acid sequence is calculated as the number of exact matches in an alignment of the two sequences divided by the length of the "invention sequence" or the length of the parent sequence, whichever is shorter. The result is expressed as a percent identity.

[0304] In some embodiments, two or more amino acid sequences are at least 50%, 60%, 70%, 80%, or 90% identical, hi some embodiments, two or more amino acid sequences are at least 95%, 97%, 98%, 99%, or even 100% identical.

[0305] "Specific binding" or "binds specifically" or "specific" for a particular antigen or epitope (in this case, human PD-1) refers to 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 generally a molecule of similar structure that has no binding activity. For example, specific binding can be determined by competition with a control molecule that is similar to the target.

[0306] Specific binding to a particular antigen or epitope is, for example, at least about 10 -4 M, at least about 10 -5 M, at least about 10 -6 M, at least about 10 -7 M, at least about 10 -8 M, at least about 10 -9 M, or at least about 10 -10 M, at least about 10 -11 M, at least about 10 -12 This can be demonstrated by an ABD having a KD for the antigen or epitope that is at or above M, where KD refers to the dissociation rate of a particular ABD-antigen interaction. Typically, an ABD that specifically binds to an antigen has a KD that is 20-fold, 50-fold, 100-fold, 500-fold, 1000-fold, 5,000-fold, 10,000-fold, or more greater than that of a control molecule compared to the antigen or epitope.

[0307] Specific binding to a particular antigen or epitope can also be demonstrated by an antibody having a K or K for the antigen or epitope that is at least 20-fold, 50-fold, 100-fold, 500-fold, 1000-fold, 5,000-fold, 10,000-fold, or more higher than a control, where K or K refers to the association rate of a particular antibody-antigen interaction. Binding affinity is generally measured using a surface plasmon resonance (SPR)-based assay (e.g., Biacore) or a biolayer interferometry (BLI)-based assay (e.g., Octet). III. Introduction

[0308] Some embodiments of the present invention provide targeting heterodimeric fusion proteins that can bind to the checkpoint inhibitor PD-1 antigen and complex with the common gamma chain (γc; CD132) and / or the IL-2 receptor beta chain (IL-2Rβ; CD122) due to the presence of the IL-15 complex. Generally, the heterodimeric fusion proteins of the present invention have three functional components: an IL-15 / IL-15Rα (sushi) component, generally referred to herein as the "IL-15 complex" or "IL-15 / Rα complex," an anti-PD-1 component that serves as a "targeting" moiety by delivering the fusion protein to cells that express PD-1, and an Fc component, each of which can take different forms and each of which can be combined with the other components in any configuration.

[0309] However, the anti-PD-1 component of the targeted heterodimeric fusion proteins of the invention does not compete for binding with known anti-PD-1 antibodies, such as nivolumab or pembrolizumab. That is, by including an anti-PD-1 (αPD-1) antigen-binding domain (ABD) that does not compete for binding with an approved αPD-1 antibody ("NC[PD-1]"), the fusion proteins of the invention enable efficient combination therapy with anti-PD-1 antibody therapy. That is, by including an anti-PD-1 ABD ("NC-αPD-1 ABD") that does not compete for binding with the approved treatment, the non-competing ABD can be used to target the fusion protein to tumors while still allowing therapeutic treatment with an additional anti-PD-1 antibody (because both can bind to PD-1 non-competitively). Furthermore, in some embodiments, the NC-αPD-1 ABD can completely block the PD-1:PD-L1 interaction (embodiments based on mAb A variants), partially block the interaction (mAb C variants), or not block the interaction at all (mAb B variants).

[0310] As will be appreciated by those skilled in the art and outlined herein, several different formats of different targeting heterodimeric fusion proteins, the non-competing construct "NC-αPD-1 X IL-15 / Rα", are shown in FIG. 28.

[0311] Furthermore, some aspects of the present invention rely on a comparison of this embodiment to a "non-targeted IL-15 / Rα-Fc fusion protein" that does not contain an antigen-binding domain for human PD-1, as shown in FIG. 13.

[0312] Additionally, either the non-targeting or targeting heterodimeric fusion proteins of the present invention can be combined with other antibodies against checkpoint receptors, including anti-PD-1, anti-TIM-3, anti-LAG-3, anti-TIGIT, etc.

[0313] Thus, the present invention provides several different functional components that can be assembled in several different ways to generate the heterodimeric fusion proteins of the present invention. As described above, the fusion proteins comprise an IL-15 complex comprising an IL-15 domain and an IL-15 receptor component.

[0314] Some embodiments provide IL-15 / Rα-Fc fusion proteins that can induce the proliferation of regulatory T cells (Tregs) with reduced or minimal immunosuppressive activity. In one embodiment, such heterodimeric fusion proteins induce the proliferation of effector memory T cells (T EM ) proliferation. In one embodiment, the heterodimeric fusion protein EM The ratio of Treg to Treg (Treg / T EM In some cases, treatment with any one of the IL-15 / Rα-Fc fusion proteins outlined herein converts Tregs from a suppressive Treg cell type to a non-suppressive activated effector CD4 T cell. In one embodiment, FOXP3 hi CD45RA - CD4 + Effector Tregs differentiate and express FOXP3 lo CD45RA - CD4 + In some embodiments, the activated effector CD4 T cells have reduced CCR4 expression.

[0315] Also provided herein are IL-15 / Rα-Fc fusion proteins that reverse TGFβ suppression of T cell proliferation. In the tumor environment, TGFβ is expressed by malignant cells and immune cells, including Tregs. TGFβ also functions to suppress T cell proliferation and inhibit anti-tumor immune responses. Treatment with the IL-15 / Rα-Fc fusion proteins of the present invention prevents TGFβ suppression of T cell proliferation. In one embodiment, administration of the IL-15 / Rα-Fc fusion protein counters TGFβ activity on T cells in the tumor environment, thus promoting T cell proliferation and anti-tumor immune responses.

[0316] Some aspects of the present invention relate to heterodimeric Fc fusion proteins comprising IL-15 and IL-15 receptor alpha (IL-15Rα) protein domains in different orientations. The Fc domains can be derived from IgG Fc domains, e.g., IgG1, IgG2, IgG3, or IgG4 Fc domains, with IgG1 Fc domains finding particular use in the present invention.

[0317] Thus, some embodiments of the present invention provide different antibody domains. As described herein and known in the art, the heterodimeric proteins of the present invention comprise different, optionally overlapping, domains. These domains include, but are not limited to, an Fc domain, a CH1 domain, a CH2 domain, a CH3 domain, a hinge domain, and a heavy constant domain (CH1-hinge-Fc domain or CH1-hinge-CH2-CH3).

[0318] In some constructs and sequences of Fc domain proteins outlined herein, the C-terminus of the IL-15 or IL-15Rα protein fragment is linked to the N-terminus of a domain linker, which in turn is linked to the N-terminus of the 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 fragment-C). In other constructs and sequences outlined herein, the C-terminus of a first protein fragment is linked to the N-terminus of a second protein fragment, optionally via a domain linker, and the C-terminus of the second protein fragment is linked to the N-terminus of the constant Fc domain, optionally via a domain linker. In still other constructs and sequences outlined herein, a constant Fc domain is provided that is not linked to the first or second protein fragment. Heterodimeric Fc fusion proteins may contain two or more of the exemplary monomeric Fc domain proteins described herein. In yet another construct, the N-terminus of a first protein fragment is joined, optionally via a domain linker, to the C-terminus of a second protein fragment, and the N-terminus of the second protein fragment is joined, optionally via a domain linker, to the C-terminus of a constant Fc domain. A.IL-15 complex

[0319] As shown in the figure, IL-15 complexes can take several forms. As noted above, IL-15 protein alone is less stable than when complexed with IL-15Rα protein. As known in the art, IL-15Rα protein contains a "sushi domain," the shortest region of the receptor that retains IL-15 binding activity. Therefore, while heterodimeric fusion proteins containing the entire IL-15Rα protein can be made, preferred embodiments herein involve complexes that simply use the sushi domain, the sequence of which is shown in the figure.

[0320] Thus, an IL-15 complex generally comprises the IL-15 protein and the sushi domain of IL-15Rα ("IL-15Rα," "IL-15Rα(sushi)," and "sushi" are used interchangeably throughout unless otherwise noted where the full-length sequence is used). When complexed together, the nomenclature is indicated by a "slash," " / ," as "IL-15 / Rα," signifying the presence of the IL-15 domain and the IL-15Rα domain. 1.IL-15 domain

[0321] As will be appreciated by one of skill in the art, the IL-15 domain may be the wild-type human sequence or may be engineered to contain variants, particularly potency variants as described below.

[0322] In some embodiments, the human IL-15 protein has the amino acid sequence set forth in NCBI Reference SEQ ID NO: NP_000576.1, or SEQ ID NO: 1, which is the precursor sequence. Optionally, the coding sequence for human IL-15 is set forth in NCBI Reference SEQ ID NO: NM_000585. An exemplary IL-15 protein of the Fc-fusion heterodimeric proteins outlined herein can have the amino acid sequence of SEQ ID NO: 2 (mature IL-15), which corresponds to amino acids 49-162 of SEQ ID NO: 1. In some embodiments, the IL-15 protein has at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, sequence identity to SEQ ID NO: 2.

[0323] In some embodiments, the IL-15 domain is engineered to contain amino acid substitutions.

[0324] Thus, in some embodiments, the IL-15 protein is a variant of 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. The IL-15 protein of the heterodimeric fusion protein can have 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acid substitutions. a.IL-15 potency variant

[0325] Furthermore, in some embodiments, the IL-15 human protein is engineered to confer reduced potency, as generally described in PCT / US2019 / 028107, which is incorporated herein by reference in its entirety. That is, as described herein, reducing the potency of IL-15 in the heterodimeric fusion proteins of the invention (optionally with or without Xtend-Fc substitutions, such as M428L / N434S, as described herein) can enhance both the pharmacodynamics and pharmacokinetics in subjects to whom such proteins are administered. Similarly, as shown in Example 7 of PCT / US2019 / 028107, IL-15 / Rα-Fc variants with reduced potency (such as XENP22821) can increase lymphocyte counts for a longer period of time than wild-type IL-15 / Rα-Fc fusion proteins described herein (such as XENP20818). Notably, XENP23343, an Xtend analog of XENP22821, further extended the duration of lymphocyte proliferation beyond XENP22821. Furthermore, reduced IL-15 potency can improve the therapeutic index (i.e., allow higher dosing with less toxicity).

[0326] As shown in Example 8 of PCT / US2019 / 028107 for "non-targeting molecules" such as XmAb24306, IL-15 / Rα-Fc fusion proteins such as those incorporated herein into NC-αPD-1 X IL-15 / Rα-Fc fusion protein can overcome effector T cell proliferation induced by Treg suppression.

[0327] Similarly, as shown in Example 4 below, NC-αPD-1 x IL-15 / Rα-Fc fusion protein can promote leukocyte proliferation and exacerbate xenogeneic GVHD across a range of dose levels. Notably, combination therapy of NC-αPD-1 x IL-15 / Rα-Fc fusion protein with an anti-PD-1 antibody demonstrated synergy (e.g., a potentiating effect), particularly at low doses.

[0328] Therefore, the present invention provides: N1D;N4D;D8N;D30N;D61N;E64Q;N65D;Q108E;N1D / N4D / D8N;N1D / N4D / N65D;N1D / D30N;N1D / D61N;N1D / D61N / E64Q / Q108E;N1D / E64Q;N1D / N65D;N1D / Q108E;N4D;N4D / D30N;N4D / D61N;N4D / D61N / N65D;N4D / D61N / E64Q / Q108E;N4D / E64Q;N4D / N65D Several suitable IL-15 amino acid variants that result in reduced potency and increased pharmacokinetics are provided, including, but not limited to, variant IL-15 proteins comprising amino acid substitutions selected from the group of: D8N / D61N; D8N / E64Q; D30N / E64Q; D30N / N65D; D30N / E64Q / N65D; D30N / Q180E; D61N / E64Q / N65D; E64Q; E64Q / N65D; E64Q / Q108E; and N65D / Q108E. In some embodiments, the variant IL-15 fusion protein comprises the amino acid sequence of SEQ ID NO: 319.

[0329] In some embodiments, the amino acid substitutions can be isosteric substitutions at the IL-15:IL-2β and IL-15:common γ chain interfaces.

[0330] In some embodiments, the human IL-15 protein, such as the human mature IL-15 protein, of the Fc fusion protein is identical to the amino acid sequence of SEQ ID NO: 2. In some cases, the human IL-15 protein, such as the human mature IL-15 protein, does not have an amino acid substitution.

[0331] In some embodiments, the human mature IL-15 variant protein has one or more amino acid mutations (e.g., substitutions, insertions, and / or deletions). In some examples, the mutations introduce a cysteine ​​residue that can form a disulfide bond with the human IL-15 receptor alpha (IL-15Rα) protein.

[0332] In some embodiments, the invention provides proteins comprising a human IL-15 variant having the amino acid variant D30N. In some embodiments, the protein comprises the amino acid sequence of SEQ ID NO: 2 and the D30N substitution. In some embodiments, the protein comprises the amino acid sequence of SEQ ID NO: 2 and at least the D30N substitution.

[0333] In some embodiments, the invention provides proteins comprising a human IL-15 variant having the amino acid substitutions N4D / N65D. In some embodiments, the protein comprises the amino acid sequence of SEQ ID NO: 2 with the N4D / N65D substitutions. In some embodiments, the protein comprises the amino acid sequence of SEQ ID NO: 2 and at least the N4D / N65D substitutions.

[0334] In some embodiments, the present invention provides proteins comprising a human IL-15 variant having the amino acid variant N1D. In some embodiments, the protein comprises the amino acid sequence of SEQ ID NO:2 and an N1D substitution. In some embodiments, the protein comprises the amino acid sequence of SEQ ID NO:2 and at least an N1D substitution.

[0335] In some embodiments, the present invention provides proteins comprising a human IL-15 variant having the amino acid variant N4D. In some embodiments, the protein comprises the amino acid sequence of SEQ ID NO:2 and the N4D substitution. In some embodiments, the protein comprises the amino acid sequence of SEQ ID NO:2 and at least the N4D substitution.

[0336] In some embodiments, the present invention provides proteins comprising a human IL-15 variant having the amino acid variant E64Q. In some embodiments, the protein comprises the amino acid sequence of SEQ ID NO:2 and the E64Q substitution. In some embodiments, the protein comprises the amino acid sequence of SEQ ID NO:2 and at least the E64Q substitution.

[0337] In some embodiments, the invention provides proteins comprising a human IL-15 variant having the amino acid variant N65D. In some embodiments, the protein comprises the amino acid sequence of SEQ ID NO:2 and the N65D substitution. In some embodiments, the protein comprises the amino acid sequence of SEQ ID NO:2 and at least the N65D substitution.

[0338] In some embodiments, the invention provides proteins comprising a human IL-15 variant having the amino acid substitutions N1D / D30N. In some embodiments, the protein comprises the amino acid sequence of SEQ ID NO: 2 and the N1D / D30N substitutions. In some embodiments, the protein comprises the amino acid sequence of SEQ ID NO: 2 and at least the N1D / D30N substitutions.

[0339] In some embodiments, the invention provides proteins comprising a human IL-15 variant having the amino acid substitution N4D / D30N. In some embodiments, the protein comprises the amino acid sequence of SEQ ID NO: 2 and the N4D / D30N substitution. In some embodiments, the protein comprises the amino acid sequence of SEQ ID NO: 2 and at least the N4D / D30N substitution.

[0340] In some embodiments, the present invention provides proteins comprising a human IL-15 variant having the amino acid substitution D30N / E64Q. In some embodiments, the protein comprises the amino acid sequence of SEQ ID NO: 2 and the D30N / E64Q substitution. In some embodiments, the protein comprises the amino acid sequence of SEQ ID NO: 2 and at least the D30N / E64Q substitution.

[0341] In some embodiments, the invention provides proteins comprising a human IL-15 variant having the amino acid substitutions D30N / N65D. In some embodiments, the protein comprises the amino acid sequence of SEQ ID NO: 2 and the D30N / N65D substitutions. In some embodiments, the protein comprises the amino acid sequence of SEQ ID NO: 2 and at least the D30N / N65D substitutions.

[0342] In some embodiments, the invention provides proteins comprising a human IL-15 variant having the amino acid substitutions D30N / E64Q / N65D. In some embodiments, the protein comprises the amino acid sequence of SEQ ID NO: 2 with the D30N / E64Q / N65D substitutions. In some embodiments, the protein comprises the amino acid sequence of SEQ ID NO: 2 and at least the D30N / E64Q / N65D substitutions. b. IL-15 glycosylation variants

[0343] Additionally, the IL-15 domain can have amino acid variants to reduce glycosylation and / or glycosylation heterogeneity, either alone or in combination with potency variants. Suitable glycosylation variants include, but are not limited to, N71Q, N79Q, N112Q, S114del, and S114A relative to SEQ ID NO:2, for use alone or in combination with each other. Sets of glycosylation variants that find particular use in many embodiments of the invention are N71Q / N79Q, N71Q / N79Q / N112Q, N71Q / N79Q / S114del, and N71Q / N79Q / S114A. In some embodiments, the variant IL-15 fusion protein comprises the amino acid sequence of SEQ ID NO:319. c. Potency and glycosylation variant combinations

[0344] In many embodiments, a combination of potency and glycosylation variants is used in the IL-15 domain that forms part of an IL-15 complex with the sushi domain (generally a truncated wild-type sequence of SEQ ID NO:20).

[0345] Thus, in some embodiments, the IL-15 domain comprises the potency variant D30N / N65D and the glycosylation variants N71Q / N79Q / N112Q.

[0346] In some embodiments, the IL-15 domain comprises the potency variant D30N / N65D and the glycosylation variant N71Q / N79Q.

[0347] In some embodiments, the IL-15 domain comprises the potency variant D30N / E64Q / N65D and the glycosylation variants N71Q / N79Q / N112Q.

[0348] In some embodiments, the IL-15 domain comprises the potency variant D30N / E64Q / N65D and the glycosylation variant N71Q / N79Q.

[0349] In some embodiments, the variant IL-15 fusion protein comprises the amino acid sequence of SEQ ID NO:319. 2.IL-15 / Rα domain

[0350] In addition to the IL-15 domain (optionally containing an amino acid variant as outlined above), the heterodimeric fusion proteins of the present invention contain a "sushi" domain.

[0351] In some embodiments, the human IL-15 receptor alpha (IL-15Rα) protein has the amino acid sequence set forth in NCBI Reference SEQ ID NO: NP_002180.1 or SEQ ID NO: 3. Optionally, the coding sequence for human IL-15Rα is set forth in NCBI Reference SEQ ID NO: NM_002189.3. An exemplary IL-15Rα protein of the Fc-fusion heterodimeric proteins outlined herein can comprise or consist of the sushi domain of SEQ ID NO: 3 (e.g., amino acids 31-95 of SEQ ID NO: 3), or in other words, the amino acid sequence of SEQ ID NO: 20. That is, certain embodiments utilize a truncated version of the extracellular domain of the receptor.

[0352] In some embodiments, the IL-15Rα protein has the amino acid sequence of SEQ ID NO: 20 with one or more amino acid insertions selected from the group consisting of D96, P97, A98, D96 / P97, D96 / C97, D96 / P97 / A98, D96 / P97 / C98, and D96 / C97 / A98, where the amino acid positions are relative to the full-length human IL-15Rα protein or SEQ ID NO: 3. For example, an amino acid such as D (e.g., Asp), P (e.g., Pro), A (e.g., Ala), DP (e.g., Asp-Pro), DC (e.g., Asp-Cys), DPA (e.g., Asp-Pro-Ala), DPC (e.g., Asp-Pro-Cys), or DCA (e.g., Asp-Cys-Ala) can be added to the C-terminus of the IL-15Rα protein of SEQ ID NO: 20. In some embodiments, the IL-15Rα protein has the amino acid sequence of SEQ ID NO: 20 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: 20. The IL-15Rα (sushi) protein of SEQ ID NO: 20 can have 1, 2, 3, 4, 5, 6, 7, 8, or more amino acid mutations (e.g., substitutions, insertions, and / or deletions). If amino acid modifications are made to the sushi domain, the variant sushi domain should retain biological activity, e.g., binding to IL-15.

[0353] In some embodiments, the IL-15Rα protein has the amino acid sequence of SEQ ID NO: 4. In some embodiments, the IL-15Rα protein has the amino acid sequence of SEQ ID NO: 4 and an amino acid insertion selected from the group consisting of D96, P97, A98, D96 / P97, D96 / C97, D96 / P97 / A98, D96 / P97 / C98, and D96 / C97 / A98, where the amino acid positions are relative to the full-length human IL-15Rα protein or SEQ ID NO: 3. 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. When amino acid modifications are made to the IL-15Rα protein, the variant IL-15Rα protein should retain biological activity, e.g., binding to IL-15. 3. Different formats of IL-15 complexes

[0354] Thus, the IL-15 complex generally comprises the IL-15 protein and the sushi domain of IL-15Rα. When complexed together, the nomenclature is designated by a "slash", " / ", as "IL-15 / Rα", signifying the presence of the IL-15 domain and the IL-15Rα domain.

[0355] As shown in Figure 28, the IL-15 / Rα complex can be in two different formats: either a non-covalent association or a covalent complex. As shown in Figures 28B, 28D, 28F, and 28G, the IL-15 protein and IL-15Rα (sushi) are not covalently linked, but rather self-assemble through ordered ligand-ligand interactions. As described more fully herein, it can be either the IL-15 domain or the sushi domain covalently linked to an Fc domain (generally using any domain linker).

[0356] Alternatively, the IL-15 / Rα complex is covalently linked using a domain linker, as generally shown in Figures 28A, 28C, 28E, and 28H. In each of these cases, the N- to C-terminal orientation of IL-15 and IL-15Rα can be switched. That is, in Figure 28A, the invention also encompasses the case where the IL-15 domain is N-terminal and the IL-15Rα domain is linked to the Fc domain using a linker. Similarly, in Figure 28C, the invention also encompasses the case where the IL-15 domain is N-terminal and the IL-15Rα domain is linked to the Fc domain using a linker. In Figure 28E, the invention also encompasses the case where the IL-15 domain is C-terminal to the Fc domain (linked using a domain linker) and the IL-15Rα domain is linked to the IL-15 domain using a linker. Finally, Figure 28H can also encompass the case where the IL-15 domain is N-terminal to the IL-15Rα domain. B. Non-competitive anti-PD-1 antigen-binding domain

[0357] The anti-PD-1 moiety (e.g., anti-PD-1 antigen-binding domain (ABD)) of the invention is generally a set of six CDRs contained within a variable heavy domain and a variable light domain that form an Fv domain capable of binding to human PD-1 (the sequence of which is shown in Figure 2) but that does not compete with commercially available anti-PD-1 antibodies such as pembrolizimab and nivolimab. This allows for superior targeting of the heterodimeric fusion proteins of the invention to tumors, thus allowing for localized action of the IL-15 / Rα complex, but also for combination treatment with effective anti-PD-1 antibodies without competing for the same PD-1 epitope.

[0358] The NC-αPD-1 antigen-binding domains of the present invention can take two general formats, as scFv domains such as those shown in Figures 28A and 28B, or as Fab domains present on two different polypeptides such as those shown in Figures 28C and 28D (monovalent binding of PD-1) and Figures 28E, 28F, 28G and 28H (bivalent binding of PD-1), which are described more fully below.

[0359] Compared to the starting mAbC H1_L1 sequence, several useful amino acid variants have been identified. 1. Oxidized variant

[0360] An embodiment of the present invention relates to the fact that a particularly useful non-competitive anti-PD-1 Fv, "[NC]mAb C," contains a tryptophan in VH-CDR3 (W112 in the Xencor numbering system; W100 in the Kabat numbering system), making it prone to oxidation (data not shown) and subsequent loss of PD-1 binding. Thus, as shown in Figure 151, amino acid substitutions that eliminate potential oxidation at this position reduce the binding affinity of the ABD for human PD-1. Therefore, additional variants that confer increased binding affinity, as described above, were combined with the W100F variant to examine whether this binding affinity could be restored, as described below. 2. Affinity Variants

[0361] As shown in Figures 65A-65I, the variable heavy domain contains, but is not limited to, F34L, S52aG, R97E, R97Y, R97W, L98R, S100aT, R97A, V99T, V99L, S100aA, L98Q, R97Q, V99F, V99L, S100aN, V99I, P100bS, G96H, L98V, V99A ... There are several suitable variants that can be used to increase the affinity of the W100F oxidation variant, each alone or in combination, including 9Q, G96V, R97K, L98S, L98F, R97T, L98K, L98S, V99I, R97L, G96A, R97A, V99S, R97S, V99Y, R97H and L98R (Kabat numbering).

[0362] Additionally, there are several suitable variants in the variable light domain compared to SEQ ID NO: 168, including, but not limited to, N27dH, N27dS, K30Y, S93T and Y94W (Kabat numbering) that can be used alone or in combination. 3. Combination of Oxidative and Affinity Variants

[0363] Thus, the present invention provides NC-mAbC ABDs with variants including the oxidation variant W100F in the VH domain in combination with one or more affinity variants to restore appropriate affinity binding.

[0364] In some embodiments, the VH amino acid substitutions (compared to the parent VH domain (SEQ ID NO: 5)) are F32L / W100F; F32L / S52aG / W100F; F32L / S52aG / R97F / W100F; F32L / S52aG / R97Y / W100F; F32L / S52aG / R97E / W100F; F32L / S52aG / R97Q / W100F; F32L / S52aG / R97L / W100F; F32L / S52aG / R97V / W100F; F32L / S 52aG / R97D / W100F;F32L / S52aG / R97H / W100F);F32L / S52aG / R97A / W100F;F32L / S52aG / R97W / W100F;F32L / S52aG / R97T / W100 F;L98R / W100F / S100aT;R97A / W100F;V99T / W100F;V99L / W100F / S100aA;L98Q / V99L / W100F;R97Q / W100F;L98Q / W100F;V99F / W 100F;V99L / W100F;W100F / S100aN;V99I / W100F / P100bS;G96H / L98V / W100F;V99A / W100F;V99Q / W100F / S100aT;G96V / R97A / V 99A / W100F / P100bS;R97Q / L98Q / W100F / S100aA;R97K / W100F / S100aA;W100F;W100F / S100aT;L98S / SW100F;L98F / W100F;R97W / L98H / W100F;W100F / S100aA;R97T / L98K / W100F;L98S / V99I / W100F;R97L / V99I / W100F;G96A / W100F;R97S / L98V / V99L / W100F;V99S / W100F;L98Q / W100F / S100aT;R97S / V99Y / W100F;V99Y / W100F;L98R / W100F;W100F / P100bS;R97H / L98Q / W100F;H1.224 L98R / V99L / W100F.

[0365] In some embodiments, the VL of NCPD-1 mAbC ABD has the amino acid substitution N27dH (compared to the parent VL domain (SEQ ID NO: 168)), and the VH amino acid substitutions (compared to the parent VH domain (SEQ ID NO: 5)) are F32L / W100F; F32L / S52aG / W100F; F32L / S52aG / R97F / W100F; F32L / S52aG / R97Y / W100F; F32L / S52aG / R97E / W100F; F32L / S52aG / R97Q / W100F; F32L / S52aG / R97L / W100F; F32L / S52aG / R97V / W100F;F32L / S52aG / R97D / W100F;F32L / S52aG / R97H / W100F);F32L / S52aG / R97A / W100F;F32L / S52aG / R97W / W100F; F32L / S52aG / R97T / W100F;L98R / W100F / S100aT;R97A / W100F;V99T / W100F;V99L / W100F / S100aA;L98Q / V99L / W100F;R97Q / W100F;L 98Q / W100F;V99F / W100F;V99L / W100F;W100F / S100aN;V99I / W100F / P100bS;G96H / L98V / W100F;V99A / W100F;V99Q / W100F / S100aT ;G96V / R97A / V99A / W100F / P100bS;R97Q / L98Q / W100F / S100aA;R97K / W100F / S100aA;W100F;W100F / S100aT;L98S / SW100F;L98F / W1 00F;R97W / L98H / W100F;W100F / S100aA;R97T / L98K / W100F;L98S / V99I / W100F;R97L / V99I / W100F;G96A / W100F;R97S / L98V / V99L / W100F;V99S / W100F;L98Q / W100F / S100aT;R97S / V99Y / W100F;V99Y / W100F;L98R / W100F;W100F / P100bS;R97H / L98Q / W100F;H1.224 L98R / V99L / W100F.

[0366] In some embodiments, the VL of NCPD-1 mAbC ABD has the amino acid substitutions N27dH / K30Y / S93T (compared to the parent VL domain (SEQ ID NO: 168)), and the VH amino acid substitutions (compared to the parent VH domain (SEQ ID NO: 5)) are F32L / W100F; F32L / S52aG / W100F; F32L / S52aG / R97F / W100F; F32L / S52aG / R97Y / W100F; F32L / S52aG / R97E / W100F; F32L / S52aG / R97Q / W100F; F32L / S52aG / R97L / W100F 00F;F32L / S52aG / R97V / W100F;F32L / S52aG / R97D / W100F;F32L / S52aG / R97H / W100F);F32L / S52aG / R97A / W100F;F32L / S52aG / R97W / W100F;F32L / S52aG / R97T / W100F;L98R / W100F / S100aT;R97A / W100F;V99T / W100F;V99L / W100F / S100aA;L98Q / V99L / W100F;R97Q / W1 00F;L98Q / W100F;V99F / W100F;V99L / W100F;W100F / S100aN;V99I / W100F / P100bS;G96H / L98V / W100F;V99A / W100F;V99Q / W100F / S1 00aT;G96V / R97A / V99A / W100F / P100bS;R97Q / L98Q / W100F / S100aA;R97K / W100F / S100aA;W100F;W100F / S100aT;L98S / SW100F;L98F / W100F;R97W / L98H / W100F;W100F / S100aA;R97T / L98K / W100F;L98S / V99I / W100F;R97L / V99I / W100F;G96A / W100F;R97S / L98V / V99L / W100F;V99S / W100F;L98Q / W100F / S100aT;R97S / V99Y / W100F;V99Y / W100F;L98R / W100F;W100F / P100bS;R97H / L98Q / W100F;H1.224 L98R / V99L / W100F.

[0367] In some embodiments, the NC-PD-1 mAbC ABD is selected from the group consisting of H1.176, H1.177, H1.178, H1.179, H1.180, H1.181, H1.182, H1.183, H1.184, H1.185, H1.186, H1.187, H1.188, H1.189, H1.190, H1.191, H1.192, H1.193, H1.194, H1.195, H1.196, H1.197, H1.198, H1.199, H1.200, H1.201, H1.202, H1.203, H1.204, H1.205, H1.206, H1.207, H1.208, H1.209, H1.210, H1.211, H1.212, H1.213, H1.214, H1.215, H1.216, H1.217, H1.218, H1.219, H1.220, H1.221, H1.222, H1.223, H1.224, H1.225, H1.226, H1.227, H1.228, H1.229, H1.230, H1.231, H1.232, H1.233, H1.234, H1.235, H1.236, H1.237, H1.238, H1.239, H1.240, H1.241, H1.242, H1.243, H1.244, H1.2 2, H1.203, H1.204, H1.205, H1.206, H1.207, H1.208, H1.209, H1.210, H1.211, H1.212, H1.213, H1.214, H1.215, H1.216, H1.217, H1.218, H1.219, H1.220, H1.221, H1.222, H1.223, H1.224.

[0368] In some embodiments, the NC-PD-1 mAbC ABD has a VL selected from those shown in Figure 43, including but not limited to L1.1, L1.3, L1.45, L1.117, L1.129, L1.135, L1.136, and L1.140.

[0369] In some embodiments, the NC-PD-1 mAbC ABD has the sequence H1.176_L1.140.

[0370] In some embodiments, the NC-PD-1 mAbC ABD has the sequence H1.176_L1.1.

[0371] It should be noted that any of the VH domains can be combined with any of the VL domains in specific combinations useful herein.

[0372] In addition to using anti-PD-1 ABDs in a "Fab" format as shown herein, anti-PD-1 ABDs may alternatively be in the form of scFvs, in which the vh and vl domains are linked using an scFv linker, which may optionally be a charged scFv linker. As will be appreciated by those of skill in the art, scFvs can be assembled N-terminus to C-terminus as N-vh-scFv linker-vl-C or N-vl-scFv linker-vh-C, with the C-terminus of the scFv domain generally linked to a hinge-CH2-CH3 Fc domain. Suitable Fvs (comprising a set of CDRs and variable heavy / variable light domains) can be used in either scFv or Fab format, as shown in Figure 43. As will be further understood by those skilled in the art, all or part of the hinge (which may also be a wild-type hinge from IgG1, IgG2 or IgG4 or a variant thereof, such as the IgG4 S241P or S228P hinge variants which have a substituted proline at position 228 relative to the parent IgG4 hinge polypeptide (numbering S228P is according to the EU index, S241P is the Kabat numbering)) can be used as a domain linker between the scFv and the CH2-CH3 domain, or a different domain linker can be used as shown in the figures. C. Fc domain

[0373] In addition to the IL-15 complex and the targeting NC-αPD-1 Fv domain, the present invention further provides heterodimeric Fc domains as components. As shown in Figure 28, these heterodimeric Fc domains serve to bring together the IL-15 / Rα and targeting anti-PD-1 domains in a single construct, generally comprising either two polypeptide chains (e.g., in Figure 28A, one monomer comprises the IL-15 / Rα complex, and the other monomer comprises the anti-PD-1 scFv), three polypeptide chains (e.g., in Figure 28C, one monomer comprises the IL-15 / Rα complex, the second monomer comprises the heavy chain, and the third monomer is the light chain), etc.

[0374] The Fc domain components of the present invention are as described herein and generally include scubariant and / or any pI variant and / or deletion variant. See, for example, the disclosure of WO 2017 / 218707 under the heading "IV Heterodimeric Antibodies" (including sections IV.A, IV.B, IV.C, IV.D, IV.E, IV.F, IV.G, IV.H, and IV.I), all of which sections are expressly incorporated by reference in their entirety. Of particular use in the heterodimeric proteins of the present invention are Fc domains including "scubariant," "pI variant," "deletion variant," and FcRn variants as outlined therein. Particularly useful Fc domains are those shown in Figure 8.

[0375] The Fc domain can be derived from an IgG Fc domain, e.g., an IgG1, IgG2, IgG3, or IgG4 Fc domain, with IgG1 Fc domains finding particular use in the present invention. Below are described Fc domains useful in the IL-15 / IL-15RαFc fusion monomer and checkpoint antibody fragments of the heterodimeric Fc proteins of the invention.

[0376] The carboxy-terminal portion of each chain defines a constant region primarily responsible for effector function. Kabat et al. collected numerous primary sequences of heavy and light chain variable regions. Based on the degree of sequence conservation, they classified each primary sequence into CDR and framework regions and compiled a list of these regions (see SEQUENCES OF IMMUNOLOGICAL INTEREST, 5th edition, NIH publication, No. 91-3242, E.A. Kabat et al., incorporated by reference in its entirety). Throughout this specification, when referring to residues in the variable domain (approximately residues 1-107 in the light chain variable region and residues 1-113 in the heavy chain variable region), the Kabat numbering system is generally used, and for the Fc region, the EU numbering system is used (e.g., Kabat et al., supra, 1991).

[0377] In the IgG subclass of immunoglobulins, several immunoglobulin domains are present in the heavy chain. As used herein, "immunoglobulin (Ig) domain" refers to a region of an immunoglobulin with a defined tertiary structure. Of interest in the present invention are heavy chain domains that contain the constant heavy (CH) domain and the hinge domain. In the context of IgG antibodies, each IgG isotype has three CH regions. Thus, the "CH" domains in the context of IgG are as follows: "CH1" refers to positions 118-220 according to the EU index as in Kabat; "CH2" refers to positions 237-340 according to the EU index as in Kabat; and "CH3" refers to positions 341-447 according to the EU index as in Kabat. As shown herein and described below, pI variants can be present in one or more of the CH and hinge regions described below.

[0378] Another type of Ig domain in the heavy chain is the hinge region. As used herein, "hinge" or "hinge region" or "antibody hinge region" or "immunoglobulin hinge region" refers to a flexible polypeptide comprising the amino acids between the first and second constant domains of an antibody. Structurally, the IgG CH1 domain ends at EU220, and the IgG CH2 domain begins at EU237. Thus, for IgG, the antibody hinge is defined herein as comprising positions 221 (D221 in IgG1) to 236 (G236 in IgG1), numbering according to the EU index as in Kabat. In some embodiments, the lower hinge is included, e.g., in the context of the Fc region, and "lower hinge" generally refers to positions 226 or 230. As described herein, pI variants can also be made in the hinge region.

[0379] Thus, the present invention provides different antibody domains, e.g., different Fc domains. As described herein and known in the art, the heterodimeric proteins of the present invention comprise different, optionally overlapping, domains. These domains include, but are not limited to, an Fc domain, a CH1 domain, a CH2 domain, a CH3 domain, a hinge domain, and a heavy constant domain (CH1-hinge-Fc domain or CH1-hinge-CH2-CH3).

[0380] Thus, an "Fc domain" comprises the -CH2-CH3 domains and, optionally, the hinge domain, and may be derived from human IgG1, IgG2, IgG3, or IgG4, with the Fc domain being derived from IgG1. In some embodiments herein, when a protein fragment (e.g., IL-15 or IL-15Rα) is attached to the Fc domain, it is the C-terminus of the IL-15 or IL-15Rα construct that is attached to all or part of the hinge of the Fc domain. For example, it is generally attached to the sequence EPKS (SEQ ID NO: 9), which is the beginning of the hinge. In other embodiments, when a protein fragment (e.g., IL-15 or IL-15Rα) is attached to the Fc domain, it is the C-terminus of the IL-15 or IL-15Rα construct that is attached to the CH1 domain of the Fc domain.

[0381] In some constructs and sequences of Fc domain proteins outlined herein, the C-terminus of the IL-15 or IL-15Rα protein fragment is linked to the N-terminus of a domain linker, which in turn is linked to the N-terminus of the 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 fragment-C). In other constructs and sequences outlined herein, the C-terminus of a first protein fragment is linked to the N-terminus of a second protein fragment, optionally via a domain linker, and the C-terminus of the second protein fragment is linked to the N-terminus of the constant Fc domain, optionally via a domain linker. In still other constructs and sequences outlined herein, a constant Fc domain is provided that is not linked to the first or second protein fragment. Heterodimeric Fc fusion proteins may contain two or more of the exemplary monomeric Fc domain proteins described herein.

[0382] In some embodiments, the linker is a "domain linker," as discussed more fully below, used to link together any two domains outlined herein, some of which are shown in Figure 8. While any suitable linker can be used, many embodiments utilize glycine-serine polymers, including, for example, (GS)n (SEQ ID NO: 10), (GSGGS)n (SEQ ID NO: 11), (GGGGS)n (SEQ ID NO: 12), and (GGGS)n (SEQ ID NO: 13), where n is an integer of at least 1 (and generally 1 to 2 to 3 to 4 to 5), as well as any peptide sequence that allows for recombinant attachment of two domains with sufficient length and flexibility to allow each domain to retain its biological function. In some cases, charged domain linkers are used, with attention to "chaining" as outlined herein.

[0383] Thus, in some embodiments, the present invention provides heterodimeric Fc-fusion proteins that rely on the use of two different heavy chain variant Fc sequences that self-assemble to form a heterodimeric Fc domain fusion polypeptide.

[0384] The present invention relates to novel constructs for providing heterodimeric Fc fusion proteins capable of binding to one or more binding partners, ligands, or receptors. Heterodimeric Fc fusion constructs are based on the self-assembly properties of two Fc domains of antibody heavy chains, e.g., two "monomers," which assemble into a "dimer." Heterodimeric Fc fusions are created by varying the amino acid sequence of each monomer, as discussed more fully below. Thus, the present invention generally relates to the creation of heterodimeric Fc fusion proteins that can co-engage with binding partners, ligands, or receptors in several ways, depending on amino acid variants in the different constant regions on each chain to promote heterodimer formation and / or to facilitate purification of the heterodimer over homodimers.

[0385] There are many mechanisms that can be used to generate the heterodimers of the present invention. Furthermore, as will be understood by those skilled in the art, these mechanisms can be combined to ensure high heterodimerization. Thus, amino acid variants that result in the production of heterodimers are referred to as "heterodimerization variants." As discussed below, heterodimerization variants can include steric variants (e.g., "knob and hole" or "skew" variants, described below, and "charge pair" variants, described below) as well as "pI variants," which allow for the purification of homodimers from heterodimers. As generally described in WO 2014 / 145806, which is incorporated by reference in its entirety, and specifically described below with respect to the discussion of "heterodimerization variants," mechanisms useful for heterodimerization include "knobs and holes" ("KIH"; sometimes referred to herein as "skew" variants (see discussion in WO 2014 / 145806), "electrostatic steering" or "charge pairing" as described in WO 2014 / 145806, pI variants as described in WO 2014 / 145806, and additional Fc variants in general as outlined in WO 2014 / 145806 and below).

[0386] There are several basic mechanisms that can facilitate the purification of heterodimeric antibodies in the present invention. These rely on the use of pI variants, such that each monomer has a different pI, thus allowing for isoelectric purification of AA, AB, and BB dimeric proteins. Alternatively, some formats also allow for separation based on size. As further outlined below, it is also possible to "skew" the formation of heterodimers over homodimers. Thus, the combination of conformational heterodimerization variants with pI variants or charge pair variants finds particular use in the present invention.

[0387] In general, specific use embodiments of the present invention rely on a set of variants that includes a scuba variant that favors heterodimerization over homodimerization, in combination with a pI variant that increases the pI difference between the two monomers.

[0388] Furthermore, as outlined more fully below, depending on the format of the heterodimeric Fc fusion protein, the pI variants can be contained within the constant domains and / or Fc domains of the monomers, or a domain linker can be used. That is, the present invention also provides pI variants present in one or both of the monomers and / or the charged domain linker. Furthermore, additional amino acid engineering for alternative functionality can also confer pI changes, such as Fc, FcRn, and KO variants.

[0389] In the present invention, which utilizes pI as a separation mechanism to enable purification of heterodimeric proteins, amino acid variants can be introduced into one or both of the monomer polypeptides. That is, the pI of one of the monomers (referred to herein for simplicity as "monomer A") can be designed to be different from monomer B, or both monomers A and B can be altered to increase the pI of monomer A and decrease the pI of monomer B. As discussed, pI changes in either or both monomers can be achieved by removing or adding a charged residue (e.g., a neutral amino acid is substituted for a positively or negatively charged amino acid residue, e.g., glycine to glutamic acid), changing a charged residue from positive or negative to the opposite charge (e.g., aspartic acid to lysine), or changing a charged residue to a neutral residue (e.g., loss of charge; lysine to serine). Some of these variants are illustrated in the figures.

[0390] Thus, this embodiment of the invention provides for making a sufficient change in pI in at least one of the monomers so that the heterodimers can be separated from the homodimers. As will be appreciated by those of skill in the art, and as discussed further below, this can be done by using a "wild-type" heavy chain constant region and a variant region engineered to increase or decrease its pI (wt A-+B or wt A--B), or by increasing one region and decreasing the other (A+B- or A-B+).

[0391] Thus, in general, a component of some embodiments of the present invention is an amino acid variant in the constant region intended to alter the isoelectric point (pI) of at least one, if not both, of the monomers of a dimeric protein by incorporating an amino acid substitution (a "pI variant" or "pI substitution") into one or both of the monomers. As shown herein, separation of a heterodimer from two homodimers can be achieved if the pI of the two monomers differs by 0.1 pH units, with 0.2, 0.3, 0.4, and 0.5 or greater all finding use in the present invention.

[0392] As will be appreciated by those skilled in the art, the number of pI variants that should be included in each or both monomers to obtain good separation will depend in part on the starting pI of the components. As is known in the art, different Fc's will have different starting pIs that are utilized in the present invention. Generally, as outlined herein, the pI is engineered to result in a total pI difference for each monomer of at least about 0.1 log, with 0.2 to 0.5 being preferred as outlined herein.

[0393] As will be appreciated by those skilled in the art, the number of pI variants to be included in each or both monomers to obtain good separation will depend in part on the starting pI of the components. That is, to determine which monomers to engineer, or in which "direction" (e.g., more positive or more negative), the sequence of the Fc domain, and optionally the protein domain linked to the Fc domain, is calculated and a decision made therefrom. As is known in the art, different Fc domains and / or protein domains will have different starting pIs to be utilized in the present invention. Generally, as outlined herein, the pI is engineered to result in a total pI difference for each monomer of at least about 0.1 log, with 0.2 to 0.5 being preferred as outlined herein.

[0394] Furthermore, as will be appreciated by those of skill in the art and outlined herein, in some embodiments, heterodimers can be separated from homodimers based on size. For example, as shown in the figures, some formats allow for the separation of heterodimers and homodimers based on size.

[0395] When heterodimerization is achieved using pI variants, the use of constant regions of the Fc domain provides a more modular approach to designing and purifying heterodimeric Fc fusion proteins. Thus, in some embodiments, heterodimerization variants (including scuba variants and purified heterodimerization variants) must be engineered. Furthermore, in some embodiments, the potential for immunogenicity resulting from pI variants is significantly reduced by incorporating pI variants from different IgG isotypes such that the pI is varied without introducing significant immunogenicity. Thus, a further problem to be solved is the elucidation of low pI constant domains with high human sequence content, e.g., minimizing or avoiding non-human residues at any particular position.

[0396] Potential side benefits of this pI engineering are also increased serum half-life and increased FcRn binding. That is, lowering the pI of antibody constant domains (including those found in antibodies and Fc fusions) can result in longer serum retention in vivo, as described in U.S. Patent Application No. 13 / 194,904 (incorporated by reference in its entirety). These pI variants to increase serum half-life also facilitate pI changes for purification.

[0397] Furthermore, it should be noted that the pI variants of heterodimeric variants provide additional advantages to the analytical and quality control processes of Fc fusion proteins, as the ability to eliminate, minimize, and distinguish from homodimers, if present, is important. Similarly, the ability to reliably test the reproducibility of heterodimeric Fc fusion protein production is important. 1. Heterodimerization variants

[0398] The present invention provides heterodimeric proteins, including heterodimeric Fc fusion proteins in various formats, that utilize heterodimer variants to enable heterodimer formation and / or purification from homodimers. The heterodimeric fusion constructs are based on the self-assembly of two Fc domains, e.g., two "monomers," which assemble into a "dimer."

[0399] Several suitable pairs of heterodimerization scuba variants exist. These variants are provided as "pairs" of "sets." That is, one set of the pair is incorporated into the first monomer, and the other set of the pair is incorporated into the second monomer. Note that these sets do not necessarily behave as "knobs-in-holes" variants; there is a one-to-one correspondence between residues on one monomer and residues on the other. That is, these paired sets form an interface between the two monomers that promotes heterodimer formation and suppresses homodimer formation, allowing the rate of spontaneous heterodimer formation under biological conditions to exceed 90% rather than the expected 50% (25% homodimer A / A: 50% heterodimer A / B: 25% homodimer B / B).

[0400] A list of suitable scuba variants is shown in Figure 3. Of particular use in many embodiments are pairs in the following sets, including, but not limited to: S364K / E357Q:L368D / K370S; L368D / K370S:S364K; L368E / K370S:S364K; T411E / K360E / Q362E:D401K; L368D / K370S:S364K / E357L, K370S:S364K / E357Q, and T366S / L368A / Y407V:T366W (optionally including a bridging disulfide, T366S / L368A / Y407V / Y349C:T366W / S354C). In terms of nomenclature, the pair "S364K / E357Q:L368D / K370S" means that one of the monomers has the double variant set S364K / E357Q and the other has the double variant set L368D / K370S. As noted above, the "chaining" of these pairs depends on the starting pI. 2. Stereovariants

[0401] In some embodiments, heterodimer formation can be promoted by the addition of conformational variants, i.e., by varying the amino acids in each heavy chain, different heavy chains are more likely to associate to form heterodimeric structures than to form homodimers with the same Fc amino acid sequence. Suitable conformational variants are included in Figure 29 of U.S. Patent Application No. 15 / 141,350, all of which, like Figure 8, are incorporated herein by reference in their entirety.

[0402] One mechanism, commonly referred to in the art as "knobs and holes," refers to amino acid engineering that creates steric effects that favor heterodimerization and disfavor homodimerization, and can be used optionally. This is sometimes referred to as "knobs and holes," as described in U.S. Patent Application No. 61 / 596,846; Ridgway et al., Protein Engineering 9(7):617 (1996); Atwell et al., J. Mol. Biol. 1997 270:26; and U.S. Patent No. 8,216,805 (all of which are incorporated herein by reference in their entireties). The diagram identifies several "monomer A-monomer B" pairs that rely on "knobs and holes." Furthermore, as described in Merchant et al., Nature Biotech. 16:677 (1998), these "knobs and holes" mutations can be combined with disulfide bonds to distort heterodimerization.

[0403] Another mechanism used to generate heterodimers is sometimes referred to as "electrostatic steering," as described in Gunasekaran et al., J. Biol. Chem. 285(25):19637 (2010), incorporated herein by reference in its entirety. This is sometimes referred to herein as "charge pairing." In this embodiment, electrostatics are used to skew the formation toward heterodimerization. As will be appreciated by those skilled in the art, these may also affect pI and, therefore, purification, and therefore may also be considered pI variants in some cases. However, because these were generated to force heterodimerization and were not used as a purification tool, they are classified as "steric variants." These include, but are not limited to, D221E / P228E / L368E paired with D221R / P228R / K409R (e.g., these are a "monomer matching set"), and C220E / P228E / 368E paired with C220R / E224R / P228R / K409R.

[0404] Additional Monomer A and Monomer B variants, which may be optionally and independently combined with other variants in any amount, such as the pI variants outlined herein or other steric variants shown in Figure 37 of U.S. Patent Application Publication No. 2012 / 0149876, are all expressly incorporated herein by reference.

[0405] In some embodiments, the steric variants outlined herein can be optionally and independently incorporated into one or both monomers along with any pI variants (or other variants, e.g., Fc variants, FcRn variants, etc.) and can be independently and optionally included or excluded from the proteins of the invention. 3. pI (isoelectric point) variants of heterodimers

[0406] In general, as will be understood by those skilled in the art, there are two general categories of pI variants: those that increase the pI of a protein (basic changes) and those that decrease the pI of a protein (acidic changes). As described herein, all combinations of these variants can be made, with one monomer being wild-type or a variant that does not exhibit a pI that is significantly different from the wild-type, and the other being more basic or more acidic. Alternatively, each monomer can be changed, one to be more basic and one to be more acidic.

[0407] Preferred combinations of pI variants are shown in Figure 30 of U.S. Patent Application No. 15 / 141,350, all of which are incorporated herein by reference in their entirety. As outlined herein and shown in the figures, these changes are shown relative to IgG1, but all isotypes can be altered in this manner, as can isotype hybrids. When the heavy chain constant domain is derived from IgG2-4, R133E and R133Q can also be used.

[0408] In one embodiment, a preferred combination of pI variants, when one of the Fc monomers comprises a CH1 domain, has one monomer comprising the 208D / 295E / 384D / 418E / 421D variants (N208D / Q295E / N384D / Q418E / N421D when compared to human IgG1). In some examples, the second monomer comprises a positively charged domain linker comprising (GKPGS)4 (SEQ ID NO: 14). In some cases, the first monomer comprises a CH1 domain comprising position 208. Thus, in constructs that do not comprise a CH1 domain (e.g., in the case of heterodimeric Fc fusion proteins that do not utilize a CH1 domain in one of the domains), a preferred negative pI variant Fc set comprises the 295E / 384D / 418E / 421D variants (Q295E / N384D / Q418E / N421D when compared to human IgG1).

[0409] In some embodiments, mutations are made in the hinge domain of the Fc domain, including positions 221, 222, 223, 224, 225, 233, 234, 235, and 236. Note that changes at 233-236 can be made to increase effector function (together with 327A) in an IgG2 backbone. Thus, pI mutations and particularly substitutions can be made at one or more of positions 221-225, with 1, 2, 3, 4, or 5 mutations being used in the present invention. Again, all possible combinations are contemplated, alone or in combination with other pI variants in other domains.

[0410] Particular substitutions used to reduce the pI of the hinge domain include, but are not limited to, a deletion at position 221, a non-native valine or threonine at position 222, a deletion at position 223, a non-native glutamic acid at position 224, a deletion at position 225, a deletion at position 235, and a deletion or non-native alanine at position 236. In some cases, the only pI substitutions are made in the hinge domain, and in other cases, these substitutions are in addition to other pI variants in other domains in any combination.

[0411] In some embodiments, mutations can be made in the CH2 region including positions 274, 296, 300, 309, 320, 322, 326, 327, 334, and 339. Again, all possible combinations of these 10 positions can be made. For example, a pI antibody can have 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 CH2 pI substitutions.

[0412] Specific substitutions used to reduce the pI of the CH2 domain include, but are not limited to, unnatural glutamine or glutamic acid at position 274, unnatural phenylalanine at position 296, unnatural phenylalanine at position 300, unnatural valine at position 309, unnatural glutamic acid at position 320, unnatural glutamic acid at position 322, unnatural glutamic acid at position 326, unnatural glycine at position 327, unnatural glutamic acid at position 334, unnatural threonine at position 339, and all possible combinations within CH2 and with other domains.

[0413] In this embodiment, the mutations can be independently and optionally selected from positions 355, 359, 362, 384, 389, 392, 397, 418, 419, 444, and 447. Particular substitutions used to reduce the pI of the CH3 domain include, but are not limited to, an unnatural glutamine or glutamic acid at position 355, an unnatural serine at position 384, an unnatural asparagine or glutamic acid at position 392, an unnatural methionine at position 397, an unnatural glutamic acid at position 419, an unnatural glutamic acid at position 359, an unnatural glutamic acid at position 362, an unnatural glutamic acid at position 389, an unnatural glutamic acid at position 418, an unnatural glutamic acid at position 444, and a deletion or unnatural aspartic acid at position 447. Exemplary embodiments of pI variants are provided in the figures, including FIG.

[0414] Additionally, in some cases the domain linker between the IL-15 and IL-15Rα domains can be charged, and the scFv linker (if present in certain formats) can be charged. 4. Isotype Variants

[0415] Furthermore, many embodiments of the present invention rely on the "incorporation" of pI amino acids at specific positions from one IgG isotype to another, thus reducing or eliminating the possibility of introducing undesirable immunogenicity into the variant. Many of these are illustrated in Figure 21 of U.S. Patent Application Publication No. 2014 / 0370013, incorporated herein by reference. Specifically, IgG1 is a common isotype for therapeutic antibodies for a variety of reasons, including its high effector function. However, the heavy constant region of IgG1 has a higher pI than the heavy constant region of IgG2 (8.10 vs. 7.31). By introducing IgG2 residues at specific positions into the IgG1 backbone, the pI of the resulting monomer is lowered (or increased) and exhibits a longer serum half-life. For example, IgG1 has glycine (pI 5.97) at position 137, while IgG2 has glutamic acid (pI 3.22). Introducing glutamic acid affects the pI of the resulting protein. As described below, several amino acid substitutions are generally required to significantly affect the pI of a variant Fc fusion protein, however, it should be noted that even changes in the IgG2 molecule can allow for increased serum half-life, as discussed below.

[0416] In other embodiments, non-isotypic amino acid changes are made to lower the overall charge state of the resulting protein (e.g., by changing higher pI amino acids to lower pI amino acids), or to allow for accommodation within the structure for stability, etc., as further described below.

[0417] Furthermore, by pI engineering both the heavy and light constant domains, significant changes can be observed in each monomer of the heterodimer. As discussed herein, a pI difference of at least 0.5 between the two monomers can allow for separation by ion exchange chromatography or isoelectric focusing or other methods sensitive to isoelectric point. 5. Calculation of pI

[0418] The pI of each monomer may depend on the pI of the variant heavy chain constant domain and the pI of all monomers, including the variant heavy chain constant domain and fusion partner. Thus, in some embodiments, the change in pI is calculated based on the variant heavy chain constant domain using the chart in Figure 19 of U.S. Patent Application Publication No. 2014 / 0370013. As discussed herein, which monomers to engineer is generally determined by the intrinsic pI of each monomer. 6. pI variants that also confer better FcRn binding in vivo

[0419] If the pI variants decrease the pI of the monomer, they may have the added benefit of improving serum retention in vivo.

[0420] Although still under investigation, it is believed that Fc regions have a longer half-life in vivo because binding to FcRn at pH 6 in endosomes traps Fc (Ghetie and Ward, 1997 Immunol Today. 18(12):592-598, incorporated by reference in its entirety). The endosomal compartment then recycles Fc to the cell surface. Once the compartment opens to the extracellular space, a higher pH (approximately 7.4) induces the release of Fc back into the blood. In mice, Dall'Acqua et al. showed that Fc mutants with increased FcRn binding at pH 6 and pH 7.4 actually had reduced serum concentrations and the same half-life as wild-type Fc (Dall'Acqua et al. 2002, J. Immunol. 169:5171-5180, incorporated by reference in its entirety). The increased affinity of Fc for FcRn at pH 7.4 is thought to prevent Fc from being released into the blood. Therefore, Fc mutations that increase the half-life of Fc in vivo ideally increase FcRn binding at lower pH while still allowing Fc release at higher pH. The amino acid histidine changes its charge state in the pH range of 6.0 to 7.4. Therefore, it is not surprising to find His residues at key positions in the Fc / FcRn complex. 7. Additional Fc variants for added functionality

[0421] In addition to pI amino acid variants, there are several useful Fc amino acid modifications that can be made for a variety of reasons, including, but not limited to, altering binding to one or more FcγR receptors, altering binding to the FcRn receptor, etc.

[0422] Thus, the proteins of the invention can comprise amino acid modifications including the heterodimerization variants outlined herein, including pI variants and conformational variants, each set of variants can independently and optionally be included or excluded from any particular heterodimeric protein. 8. FcγR variants

[0423] Thus, there are several useful Fc substitutions that can be made to alter binding to one or more FcγR receptors. Substitutions that result in increased and decreased binding can be useful. For example, increased binding to FcγRIIIa is known to result in ADCC (antibody-dependent cell-mediated cytotoxicity; a cell-mediated reaction in which nonspecific cytotoxic cells expressing FcγR recognize bound antibody on target cells and subsequently cause lysis of the target cells). Similarly, decreased binding to FcγRIIb (an inhibitory receptor) can also be beneficial in some circumstances. Amino acid substitutions that find use in the present invention include those listed in U.S. Patent Application Nos. 11 / 124,620 (especially Figure 41), 11 / 174,287, 11 / 396,495, and 11 / 538,406, all of which are expressly incorporated by reference in their entirety, particularly with respect to the variants disclosed therein. Specific variants that find use 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.

[0424] Additionally, amino acid substitutions that increase affinity for FcγRIIc can also be included in the Fc domain variants outlined herein, e.g., the substitutions described in U.S. Patent Application Nos. 11 / 124,620 and 14 / 578,305 are useful.

[0425] Furthermore, as specifically disclosed in U.S. Patent Application No. 12 / 341,769, which is incorporated herein by reference in its entirety, there are additional Fc substitutions that are used to increase binding to the FcRn receptor and increase serum half-life, including, but not limited to, 434S, 434A, 428L, 308F, 259I, 428L / 434S, 259I / 308F, 436I / 428L, 436I or V / 434S, 436V / 428L, and 259I / 308F / 428L. 9. Elimination Variant

[0426] Similarly, another category of functional variants are "FcγR-depleted variants" or "Fc knockout (FcKO or KO)" variants. In these embodiments, for some therapeutic applications, it is desirable to reduce or eliminate the normal binding of the Fc domain to one or more or all of the Fcγ receptors (e.g., FcγR1, FcγRIIa, FcγRIIb, FcγRIIIa, etc.) to avoid additional mechanisms of action. Thus, for example, in many embodiments, one of the Fc domains comprises one or more Fcγ receptor-depleted variants, particularly in the use of bispecific immunomodulatory antibodies where it is desirable to eliminate FcγRIIIa binding to eliminate or significantly reduce ADCC activity. These deletion variants are shown in Figure 31 of U.S. Patent Application No. 15 / 141,350, all of which are incorporated herein by reference in their entirety and which may each independently and optionally be included or excluded, with preferred embodiments utilizing a deletion variant selected from the group consisting of G236R / L328R, E233P / L234V / L235A / G236del / S239K, E233P / L234V / L235A / G236del / S267K, E233P / L234V / L235A / G236del / S239K / A327G, E233P / L234V / L235A / G236del / S267K / A327G and E233P / L234V / L235A / G236del, according to the EU index. Note that the ablation variants referred to herein ablate FcγR binding, but generally do not ablate FcRn binding.

[0427] Exemplary embodiments of pI variants are provided in the figures, including FIG. 10. Combinations of Heterodimer Variants and Fc Variants

[0428] As will be appreciated by those skilled in the art, all of the listed heterodimerization variants (including scubariant and / or pI variants) can be combined in any way, and independently, as long as they retain their "chaining" or "monomer distribution." Furthermore, all of these variants can be combined into any of the heterodimerization formats.

[0429] In the case of pI variants, embodiments that find particular use are shown in the figures, but other combinations can be generated following the basic principle of altering the pI difference between the two monomers to facilitate purification.

[0430] Additionally, any of the heterodimerization variants, skews and pIs may also be independently and optionally combined with Fc ablation variants, Fc variants, FcRn variants as generally outlined herein.

[0431] Additionally, the monomeric Fc domain may comprise a set of amino acid substitutions comprising C220S / S267K / L368D / K370S or C220S / S267K / S364K / E357Q.

[0432] Additionally, heterodimeric Fc fusion proteins can include scubariant (e.g., a set of amino acid substitutions as shown in Figures 1A-1C of U.S. Patent Application No. 15 / 141,350, all of which are incorporated herein by reference in their entirety), with a particularly useful scubariant being S364K / E357Q:L368D / K370S;L368D / K370S:S364K;L368E / K370S; S:S364K; T411E / K360E / Q362E:D401K; L368D / K370S:S364K / E357L, K370S:S364K / E357Q, T366S / L368A / Y407V:T366W and T366S / L368A / Y407V / Y349C:T366W / S354C, optionally a deletion variant, optionally a charged domain linker and heavy chain comprising a pI variant.

[0433] In some embodiments, the Fc domain comprises 236R, 239D, 239E, 243L, M252Y, V259I, 267D, 267E, 298A, V308F, 328F, 328R, 330L, 332D, 332E, M428L, N434A, N434S, 236R / 328R, 239D / 332E, M428L, 236R / 328F, V259I / V308F, 267E / 328F, M428L / The Fc domain comprises an amino acid substitution selected from the group consisting of N434S, Y436I / M428L, Y436V / M428L, Y436I / N434S, Y436V / N434S, 239D / 332E / 330L, M252Y / S254T / T256E, V259I / V308F / M428L, E233P / L234V / L235A / G236del / S267K, G236R / L328R, and PVA / S267K. In some cases, the Fc domain comprises the amino acid substitution 239D / 332E. In other cases, the Fc domain comprises the amino acid substitution G236R / L328R or PVA / S267K.

[0434] In one embodiment, a specific combination of scubariant and pI variants for use in the present invention is T366S / L368A / Y407V:T366W (optionally including a bridging disulfide, T366S / L368A / Y407V / Y349C:T366W / S354C), where one monomer contains Q295E / N384D / Q418E / N481D and the other monomer contains a positively charged domain linker. As understood in the art, "knobs-in-hole" variants do not alter the pI and therefore can be used with either monomer.

[0435] In one embodiment, a particular combination of scubariant and pI variant for use in the present invention is L368D / K370S:S364K / E357Q, with one monomer comprising Q295E / N384D / Q418E / N421D.

[0436] Useful pairs of sets of Fc dimerization variants (including scuba variants and pI variants) are provided in Figures 4A-4E. Addition pI variants are shown in Figure 5. Useful deletion variants are shown in Figure 6. Useful embodiments of non-cytokine components of the IL-15 / Rαx anti-PD1 ABD heterodimeric fusion proteins of the invention are provided in Figures 7A-7E and 8A-8F. D. Domain Linker

[0437] The three components of the invention (the anti-PD-1 Fv of the invention, the IL-15 / Rα complex, and the heterodimerized Fc domain) are optionally linked together using a domain linker. While direct covalent linkage can be achieved (e.g., linking the C-terminus of the IL-15 complex to the N-terminus of the CH2 domain of the Fc domain), typically a linker is used that confers flexibility and sometimes function.

[0438] In some embodiments, the IL-15 protein is attached to the N-terminus of the Fc domain and the IL-15Rα protein is attached to the N-terminus of the IL-15 protein. In other embodiments, the IL-15Rα protein is attached to the N-terminus of the Fc domain and the IL-15Rα protein is non-covalently attached to the IL-15 protein. In yet other embodiments, the IL-15Rα protein is attached to the C-terminus of the Fc domain and the IL-15Rα protein is non-covalently attached to the IL-15 protein.

[0439] In some embodiments, the IL-15 protein and the IL-15Rα protein are linked together via a domain linker (e.g., a "scIL-15 / Rα" format). Optionally, the proteins are not linked via a linker, but rather utilize either natural self-assembly or disulfide bonding as outlined herein. In other embodiments, the IL-15 protein and the IL-15Rα protein are non-covalently linked. In some embodiments, the IL-15 protein is linked to the Fc domain via a linker. In certain embodiments, the IL-15 protein is directly linked to the Fc domain, e.g., without a linker. In certain embodiments, the IL-15 protein is linked to the Fc domain via a hinge region or a fragment thereof. In other embodiments, the IL-15Rα protein is linked to the Fc domain via a linker. In other embodiments, the IL-15Rα protein is directly linked to the Fc domain, e.g., without a linker. In certain embodiments, the IL-15Rα protein is linked to the Fc domain via a hinge region or a fragment thereof. Optionally, no linker is used to attach the IL-15 protein or IL-15Rα protein to the Fc domain.

[0440] In some examples, the PD-1 ABD is covalently linked to the N-terminus of the Fc domain via a domain linker. In some embodiments, the PD-1 ABD is directly linked to the Fc domain, e.g., without a linker. In certain embodiments, the PD-1 ABD is linked to the Fc domain via the hinge region or a fragment thereof.

[0441] In some embodiments, the linker is a "domain linker" used to link any two domains outlined herein together. The linker peptide may primarily comprise the following amino acid residues: Gly, Ser, Ala, or Thr. The linker peptide should be long enough to link the two molecules so that they assume the correct conformation relative to each other to retain the desired activity. In one embodiment, the linker is about 1 to 50 amino acids in length, preferably about 1 to 30 amino acids in length. In one embodiment, linkers of 1 to 20 amino acids in length may be used, and in some embodiments, about 5 to about 10 amino acids are used. Useful linkers include glycine-serine polymers (where n is an integer of at least 1 (typically 3 to 4)), including (GS)n (SEQ ID NO: 15), (GSGGS)n (SEQ ID NO: 16), (GGGGS)n (SEQ ID NO: 17), and (GGGS)n (SEQ ID NO: 18), where n is an integer of at least 1 (typically 3 to 4), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers. Alternatively, a variety of non-proteinaceous polymers may find use as linkers, including, but not limited to, polyethylene glycol (PEG), polypropylene glycol, polyoxyalkylenes, or copolymers of polyethylene glycol and polypropylene glycol.

[0442] In some embodiments, the domain linker comprises all or part of the hinge region of IgG1, IgG2, and IgG4, the former being useful in many embodiments. Some hinge domain linkers are shown in Figure 8. As will be appreciated by those skilled in the art, domain linkers according to Figure 8 can also be combined.

[0443] In some embodiments, the domain linker is an scFv linker. Generally, the scFv linker has sufficient flexibility and length to allow the VH and VL domains to associate in the correct format. In some cases, the scFv linker may be charged, depending on the format of the heterodimeric protein and the pI of the components, as generally outlined in Figure 8 and discussed below.

[0444] In some embodiments, the scFv linker is a charged scFv linker, some of which are shown in Figure 7 of WO 2017 / 218707. Accordingly, the present invention further provides charged scFv linkers to facilitate separation in pI between a first and second monomer (e.g., an IL-15 / IL-15Rα monomer and a PD-1 ABD monomer). That is, by incorporating either a positively or negatively charged scFv linker (or both, in the case of scaffolds using scFvs on different monomers and / or using one charged linker in the scFv to link the IL-15 and sushi domains or the IL-15 / Rα component to the Fc domain), the pI of the charged linker-containing monomer can be altered without further changes to the Fc domain. These charged linkers can be substituted into any scFv containing a standard linker. Again, as will be appreciated by those skilled in the art, charged scFv linkers are used on the correct "chains" or monomers according to the desired change in pI. For example, as discussed herein, to generate the heterodimeric fusion proteins of the invention, the original pI of the Fv region for each of the desired domains is calculated, one is selected to generate the scFv, and depending on the pI, a positive or negative linker is selected.

[0445] Charged domain linkers can also be used to increase the pI separation of the monomers of the present invention, and thus those included in Figure 10 can be used in any embodiment herein where a linker is utilized.

[0446] Other linker sequences can include any sequence of the CL / CH1 domain of any length (e.g., the first 5-12 amino acid residues of the CL / CH1 domain), but not all residues of the CL / CH1 domain. Linkers can be derived from immunoglobulin light chains, such as Cκ or Cλ. Linkers can be derived from immunoglobulin heavy chains of any isotype, including, for example, Cγ1, Cγ2, Cγ3, Cγ4, Cα1, Cα2, Cδ, Cε, C, and Cμ. Linker sequences can also be derived from other proteins, such as Ig-like proteins (e.g., TCR, FcR, KIR), sequences derived from hinge regions, and other naturally occurring sequences from other proteins. 1. Glycosylation-variant Linkers

[0447] As noted above for anti-PD-1 Fv, glycosylation can result in heterogeneity that may be undesirable in some cases. Thus, in some cases, a (GGGGA)n linker (SEQ ID NO: 19), where n is 1 to 5, is used as the domain linker connecting the IL-15 domain and the IL-15Rα domain to form the IL-15 complex, as shown in Figure 8. IV. Useful Formats of the Invention

[0448] As shown in Figures 28A-28H, there are several useful formats for the PD-1-targeted IL-15 / IL-15Rα(sushi)Fc fusion proteins of the invention. In general, the heterodimeric fusion proteins of the invention have three functional components: an IL-15 / IL-15Rα(sushi) component, an anti-PD-1 component, and an Fc component, each of which can take different forms as outlined herein, and each of which can be combined with the other components in any configuration.

[0449] In any of the following formats, the first and second Fc domains may have a set of amino acid substitutions selected from the group consisting of: a) S267K / L368D / K370S:S267K / S364K / E357Q; b) S364K / E357Q:L368D / K370S; c) L368D / K370S:S364K; d) L368E / K370S:S364K; e) T411E / K360E / Q362E:D401K; f) L368D / K370S:S364K / E357L and g) K370S:S364K / E357Q (according to EU numbering).

[0450] In some embodiments, the first and / or second Fc domain has an additional set of amino acid substitutions comprising Q295E / N384D / Q418E / N421D according to EU numbering.

[0451] Optionally, the first and / or second Fc domain has a further set of amino acid substitutions consisting of, according to EU numbering: G236R / L328R, E233P / L234V / L235A / G236del / S239K, E233P / L234V / L235A / G236del / S267K, E233P / L234V / L235A / G236del / S239K / A327G, E233P / L234V / L235A / G236del / S267K / A327G and E233P / L234V / L235A / G236del.

[0452] Optionally, the first and / or second Fc domains have the M428L / N434S variant for half-life extension, hi some embodiments, the first and / or second Fc domains have the 428L / 434S variant for half-life extension.

[0453] Thus, particularly useful variants of any of the following formats have positive monomers containing the scubariant S364K / E357Q and the deletion variants E233P / L234V / L235A / G236del / S267K, and negative monomers containing the scubariant L368D / K370S, the deletion variants E233P / L234V / L235A / G236del / S267K, and the pI variants N208D / Q295E / N384D / Q418E / N421D (if the monomer contains a CH1 domain) or the pI variants Q295E / N384D / Q418E / N421D (if the monomer does not contain a CH1 domain). A.scIL-15 / Rα X scFv

[0454] One embodiment is shown in Figure 28A and comprises two monomers. This is commonly referred to as "scIL-15 / Rα x scFv," where "sc" stands for "single chain," referring to the linkage of the IL-15 and sushi domains using a covalent domain linker. The "scIL-15 / Rα x scFv" format (see Figure 28A) comprises IL-15Rα (sushi) fused to IL-15 by a variable length linker (referred to as "scIL-15 / Rα"), which is then fused to the N-terminus of the heterodimeric Fc region, with the scFv fused to the other side of the heterodimeric Fc.

[0455] In the format of Figure 28A, some embodiments include an Fc domain having a positive monomer comprising scubariant S364K / E357Q and deletion variants E233P / L234V / L235A / G236del / S267K, and a negative monomer comprising scubariant L368D / K370S, deletion variants E233P / L234V / L235A / G236del / S267K and pI variants Q295E / N384D / Q418E / N421D and, optionally, the 428L / 434S FcRn variant. In these embodiments, the sushi domain is wild-type (SEQ ID NO: 20) and the anti-PD-1 Fv domain is selected from the group consisting of mAbC H.176_L1.140, H1.19_L1.140, H1_L1.1, H1.19_L1, H1.48_L1, H1.125_L1, H1.30_L1, H1.132_L1, H1_L1.1 The VH and VL domains are selected from the group consisting of combinations of VH and VL domains of H1_L1.3, H1_L1.45, H1_L1.117, H1_L1.129, H1.19_L1.1, H1.32_L1.1, H1.169_L1.1, H1.169_L1.1, H1.175_L1.1, H1.175_L1.1, H1_L1.140, H1_L1.135, H1_L1.136, H1.132_L1.135, H1.132_L1.140, H1.175_L1.135 and H1.175_L1.140.

[0456] In the format of Figure 28A, some embodiments include an Fc domain having a positive monomer comprising scubariant S364K / E357Q and deletion variants E233P / L234V / L235A / G236del / S267K, and a negative monomer comprising scubariant L368D / K370S, deletion variants E233P / L234V / L235A / G236del / S267K and pI variants Q295E / N384D / Q418E / N421D and, optionally, the 428L / 434S FcRn variant. In these embodiments, the sushi domain is wild-type (SEQ ID NO: 20), the anti-PD-1 Fv domain is a combination of the VH and VL domains of H.176_L1.140, and the IL-15 domain comprises a variant selected from N4D / N65D, D30N / N65D, and D30N / E64Q / N65D.

[0457] In the format of Figure 28A, some embodiments include an Fc domain having a positive monomer comprising scubariant S364K / E357Q and deletion variants E233P / L234V / L235A / G236del / S267K, and a negative monomer comprising scubariant L368D / K370S, deletion variants E233P / L234V / L235A / G236del / S267K and pI variants Q295E / N384D / Q418E / N421D and, optionally, the 428L / 434S FcRn variant. In these embodiments, the sushi domain is wild-type (SEQ ID NO: 20), the anti-PD-1 Fv domain is selected from the group consisting of a combination of the VH and VL domains of H1_L1.1, and the IL-15 domain comprises a variant selected from N4D / N65D, D30N / N65D, and D30N / E64Q / N65D. B.scFv X ncIL-15 / Rα

[0458] This embodiment is shown in Figure 28B and contains three monomers. It is commonly referred to as "ncIL-15 / Rα x scFv" or "scFv x ncIL-15 / Rα," where "nc" stands for "non-covalent," referring to the self-assembling non-covalent association of the IL-15 and sushi domains. The "scFv x ncIL-15 / Rα" format (see Figure 34B) contains an scFv fused to the N-terminus of the heterodimeric Fc region, with IL-15Rα (sushi) fused to the other side of the heterodimeric Fc, and IL-15 transfected separately to form a non-covalent IL-15 / Rα complex.

[0459] In the format of Figure 28B, some embodiments include an Fc domain having a positive monomer comprising scubariant S364K / E357Q and deletion variants E233P / L234V / L235A / G236del / S267K, and a negative monomer comprising scubariant L368D / K370S, deletion variants E233P / L234V / L235A / G236del / S267K and pI variants Q295E / N384D / Q418E / N421D and, optionally, the 428L / 434S FcRn variant. In these embodiments, the sushi domain is wild-type (SEQ ID NO: 20), the anti-PD-1 Fv domain is a combination of the VH and VL domains of H.176_L1.140, and the IL-15 domain comprises a variant selected from N4D / N65D, D30N / N65D, and D30N / E64Q / N65D.

[0460] In the format of Figure 28B, some embodiments include an Fc domain having a positive monomer comprising scubariant S364K / E357Q and deletion variants E233P / L234V / L235A / G236del / S267K, and a negative monomer comprising scubariant L368D / K370S, deletion variants E233P / L234V / L235A / G236del / S267K and pI variants Q295E / N384D / Q418E / N421D and, optionally, the 428L / 434S FcRn variant. In these embodiments, the sushi domain is wild-type (SEQ ID NO: 20), the anti-PD-1 Fv domain is selected from the group consisting of a combination of the VH and VL domains of H1_L1.1, and the IL-15 domain comprises a variant selected from N4D / N65D, D30N / N65D, and D30N / E64Q / N65D. C.scIL-15 / Rα X Fab

[0461] This embodiment is shown in Figure 28C and contains three monomers. It is commonly referred to as "scIL-15 / Rα x Fab" or "Fab x scIL-15 / Rα," used interchangeably, with "sc" standing for "single chain." The scIL-15 / Rα x Fab format (see Figure 28C) contains IL-15Rα (sushi) fused to IL-15 by a variable-length linker (referred to as "scIL-15 / Rα"), which is then fused to the N-terminus of a heterodimeric Fc region (including a hinge that serves as the second domain linker). That is, from N- to C-terminus, the first monomer is variant IL-15-first domain linker-IL-15Rα sushi domain-second domain linker-CH2-CH3. In some cases, the second domain linker is a complete hinge domain, as shown in Figure 8. The second monomer is the heavy chain, VH-CH1-hinge-CH2-CH3, and the corresponding light chain (third monomer) is transfected separately to form VH and Fab.

[0462] In some Figure 28C embodiments, the anti-PD-1 Fv domain is selected from the group consisting of mAbC H.176_L1.140, H1_L1, H1.19_L1.140, H1_L1.1, H1.19_L1, H1.48_L1, H1.125_L1, H1.30_L1, H1.132_L1, H1_L1.1 The VH and VL domains are selected from the group consisting of combinations of VH and VL domains of H1_L1.3, H1_L1.45, H1_L1.117, H1_L1.129, H1.19_L1.1, H1.32_L1.1, H1.169_L1.1, H1.169_L1.1, H1.175_L1.1, H1.175_L1.1, H1_L1.140, H1_L1.135, H1_L1.136, H1.132_L1.135, H1.132_L1.140, H1.175_L1.135 and H1.175_L1.140.

[0463] In the format of Figure 28C, some embodiments include an Fc domain having a positive monomer comprising scubariant S364K / E357Q and deletion variants E233P / L234V / L235A / G236del / S267K, and a negative monomer comprising scubariant L368D / K370S, deletion variants E233P / L234V / L235A / G236del / S267K, and pI variants N208D / Q295E / N384D / Q418E / N421D. In these embodiments, the sushi domain is wild-type (SEQ ID NO: 20), the anti-PD-1 Fv domain is a combination of the VH and VL domains of H1.176_L1.140, the IL-15 domain contains amino acid substitutions D30N / N65D for potency and N71Q / N79Q / N112Q for glycosylation, and the domain linker between the IL-15 and sushi domains is GGGGA (SEQ ID NO: 8).

[0464] In the format of Figure 28C, some embodiments include an Fc domain having positive monomers comprising scubariant S364K / E357Q, deletion variants E233P / L234V / L235A / G236del / S267K, and Xtend 428L / 434S variant, and negative monomers comprising scubariant L368D / K370S, deletion variants E233P / L234V / L235A / G236del / S267K, Xtend 428L / 434S variant, and pI variants N208D / Q295E / N384D / Q418E / N421D. In these embodiments, the sushi domain is wild-type (SEQ ID NO: 20), the anti-PD-1 Fv domain is a combination of the VH and VL domains of H1.176_L1.140, the IL-15 domain contains amino acid substitutions D30N / N65D for potency and N71Q / N79Q / N112Q for glycosylation, and the domain linker between the IL-15 and sushi domains is GGGGA (SEQ ID NO: 8).

[0465] In the format of Figure 28C, some embodiments include an Fc domain having a positive monomer comprising scubariant S364K / E357Q and deletion variants E233P / L234V / L235A / G236del / S267K, and a negative monomer comprising scubariant L368D / K370S, deletion variants E233P / L234V / L235A / G236del / S267K, and pI variants N208D / Q295E / N384D / Q418E / N421D. In these embodiments, the sushi domain is wild-type (SEQ ID NO: 20), the anti-PD-1 Fv domain is a combination of the VH and VL domains of H1.176_L1.140, the IL-15 domain contains amino acid substitutions D30N / E64Q / N65D for potency and N71Q / N79Q / N112Q for glycosylation, and the domain linker between the IL-15 and sushi domains is GGGGA (SEQ ID NO: 8).

[0466] In the format of Figure 28C, some embodiments include an Fc domain having positive monomers comprising scubariant S364K / E357Q, deletion variants E233P / L234V / L235A / G236del / S267K, and Xtend 428L / 434S variant, and negative monomers comprising scubariant L368D / K370S, deletion variants E233P / L234V / L235A / G236del / S267K, Xtend 428L / 434S variant, and pI variants N208D / Q295E / N384D / Q418E / N421D. In these embodiments, the sushi domain is wild-type (SEQ ID NO: 20), the anti-PD-1 Fv domain is a combination of the VH and VL domains of H1.176_L1.140, the IL-15 domain contains amino acid substitutions D30N / E64Q / N65D for potency and N71Q / N79Q / N112Q for glycosylation, and the domain linker between the IL-15 and sushi domains is GGGGA (SEQ ID NO: 8).

[0467] In a particularly useful embodiment, the fusion protein is XENP32435, having the sequence shown in Figure 138A.

[0468] In the format of Figure 28C, some embodiments include an Fc domain having a positive monomer comprising scubariant S364K / E357Q and deletion variants E233P / L234V / L235A / G236del / S267K, and a negative monomer comprising scubariant L368D / K370S, deletion variants E233P / L234V / L235A / G236del / S267K and pI variants N208D / Q295E / N384D / Q418E / N421D and, optionally, the 428L / 434S FcRn variant. In these embodiments, the sushi domain is wild-type (SEQ ID NO: 20), the anti-PD-1 Fv domain is selected from the group consisting of a combination of the VH and VL domains of H1_L1.1, and the IL-15 domain comprises a variant selected from D30N / N65D and D30N / E64Q / N65D.

[0469] The amino acid sequence of an exemplary non-competitive PD-1-targeting IL-15 / Rα-Fc fusion protein in the scIL-15 / Rα x Fab format (Figure 28C) is provided in Figures 30, 48, 49, and 68.

[0470] In some embodiments, the first monomer comprises, from N- to C-terminus, sushi domain-domain linker-variant IL-15-domain linker-CH2-CH3, the second monomer comprises heavy chain VH-CH1-hinge-CH2-CH3, and the third monomer is light chain VL-CL. A preferred combination of variants for this embodiment is found in Figure 7C of PCT / US2017 / 056826.

[0471] In the scIL-15 / Rα X Fab format, one preferred embodiment utilizes the scUB variant pair S364K / E357Q:L368D / K370S.

[0472] In the scIL-15 / Rα X Fab format, one preferred embodiment utilizes scFv variants S364K / E357Q (on the scFv-Fc monomer) and L368D / K370S (on the IL-15 complex monomer), pI variants Q295E / N384D / Q418E / N421D (on the IL-15 complex side), deletion variants E233P / L234V / L235A / G236_ / S267K on both monomers, and optionally 428L / 434S variants on both sides. D. ncIL-15 / Rα X Fab

[0473] This embodiment is shown in Figure 28D and contains three monomers. It is commonly referred to interchangeably as "ncIL-15 / Rα x Fab" or "Fab x ncIL-15 / Rα," where "nc" stands for "non-covalent," referring to the self-assembling non-covalent association of the IL-15 and sushi domains. The ncIL-15 / Rα x Fab format (see Figure 34D) comprises a VH fused to the N-terminus of a heterodimeric Fc region, IL-15Rα (sushi) is fused to the other side of the heterodimeric Fc, the corresponding light chain is transfected separately to form the VH and Fab, and IL-15 is transfected separately to form a non-covalent IL-15 / Rα complex.

[0474] In some embodiments, the first monomer comprises, from N- to C-terminus, sushi domain-domain linker-CH2-CH3, the second monomer comprises heavy chain VH-CH1-hinge-CH2-CH3, and the third monomer is an IL-15 domain. In the ncIL-15 / Rα X Fab format, one preferred embodiment utilizes the scuba variant pair S364K / E357Q:L368D / K370S.

[0475] In the format of Figure 28D, some embodiments include an Fc domain having a positive monomer comprising scubariant S364K / E357Q and deletion variants E233P / L234V / L235A / G236del / S267K, and a negative monomer comprising scubariant L368D / K370S, deletion variants E233P / L234V / L235A / G236del / S267K and pI variants N208D / Q295E / N384D / Q418E / N421D and, optionally, the 428L / 434S FcRn variant. In these embodiments, the sushi domain is wild-type (SEQ ID NO: 20), the anti-PD-1 Fv domain is a combination of the VH and VL domains of H.176_L1.140, and the IL-15 domain comprises a variant selected from D30N / N65D and D30N / E64Q / N65D.

[0476] In the format of Figure 28D, some embodiments include an Fc domain having a positive monomer comprising scubariant S364K / E357Q and deletion variants E233P / L234V / L235A / G236del / S267K, and a negative monomer comprising scubariant L368D / K370S, deletion variants E233P / L234V / L235A / G236del / S267K and pI variants N208D / Q295E / N384D / Q418E / N421D and, optionally, the 428L / 434S FcRn variant. In these embodiments, the sushi domain is wild-type (SEQ ID NO: 20), the anti-PD-1 Fv domain is selected from the group consisting of a combination of the VH and VL domains of H1_L1.1, and the IL-15 domain comprises a variant selected from D30N / N65D and D30N / E64Q / N65D. E. mAb-scIL-15 / Rα

[0477] This embodiment is shown in Figure 28E and contains three monomers (although the fusion protein is a tetramer). It is commonly referred to as "mAb-scIL-15 / Rα," with "sc" standing for "single chain." The mAb-scIL-15 / Rα format (see Figure 34E) comprises a VH fused to the N-terminus of a first and second heterodimeric Fc, where IL-15 is fused to IL-15Rα (sushi), which is then further fused to the C-terminus of one of the heterodimeric Fc regions, and the corresponding light chain is transfected separately to form the VH and Fab.

[0478] In some embodiments, the first monomer comprises the heavy chain VH-CH1-hinge-CH2-CH3. The second monomer comprises the heavy chain having the scIL-15 complex: VH-CH1-hinge-CH2-CH3-domain linker-sushi domain-domain linker-IL-15. The third (and fourth) monomer is the light chain VL-CL. This is commonly referred to as "mAb-scIL-15 / Rα," with "sc" standing for "single chain." In the mAb-scIL-15 / Rα format, one preferred embodiment utilizes the sc variant pair S364K / E357Q:L368D / K370S.

[0479] In the format of Figure 28E, some embodiments include an Fc domain having a positive monomer comprising scubariant S364K / E357Q and deletion variants E233P / L234V / L235A / G236del / S267K, and a negative monomer comprising scubariant L368D / K370S, deletion variants E233P / L234V / L235A / G236del / S267K and pI variants N208D / Q295E / N384D / Q418E / N421D and, optionally, the 428L / 434S FcRn variant. In these embodiments, the sushi domain is wild-type (SEQ ID NO: 20), the anti-PD-1 Fv domain is a combination of the VH and VL domains of H.176_L1.140, and the IL-15 domain comprises a variant selected from D30N / N65D and D30N / E64Q / N65D.

[0480] In the format of Figure 28E, some embodiments include an Fc domain having a positive monomer comprising scubariant S364K / E357Q and deletion variants E233P / L234V / L235A / G236del / S267K, and a negative monomer comprising scubariant L368D / K370S, deletion variants E233P / L234V / L235A / G236del / S267K and pI variants N208D / Q295E / N384D / Q418E / N421D and, optionally, the 428L / 434S FcRn variant. In these embodiments, the sushi domain is wild-type (SEQ ID NO: 20), the anti-PD-1 Fv domain is selected from the group consisting of a combination of the VH and VL domains of H1_L1.1, and the IL-15 domain comprises a variant selected from D30N / N65D and D30N / E64Q / N65D. F. mAb-ncIL-15 / Rα

[0481] This embodiment is shown in Figure 28F and contains four monomers (although the heterodimeric fusion protein is a pentamer). It is commonly referred to as "mAb-ncIL-15 / Rα," with "nc" standing for "non-covalent." The mAb-ncIL-15 / Rα format (Figure 34F) contains a VH fused to the N-terminus of the first and second heterodimeric Fc regions, IL-15Rα (sushi) fused to the C-terminus of one of the heterodimeric Fc regions, the corresponding light chain transfected separately to form an Fab with the VH, and IL-15 transfected separately to form a non-covalent IL-15 / Rα complex.

[0482] In some embodiments, the first monomer comprises a heavy chain VH-CH1-hinge-CH2-CH3. The second monomer comprises a heavy chain having an IL-15Rα (sushi) domain: VH-CH1-hinge-CH2-CH3-domain linker-sushi domain. The third monomer is an IL-15 domain. The fourth (and fifth) monomers are light chains VL-CL. In the mAb-ncIL-15 / Rα format, one preferred embodiment utilizes the scuba variant pair S364K / E357Q:L368D / K370S.

[0483] In the format of Figure 28F, some embodiments include an Fc domain having a positive monomer comprising scubariant S364K / E357Q and deletion variants E233P / L234V / L235A / G236del / S267K, and a negative monomer comprising scubariant L368D / K370S, deletion variants E233P / L234V / L235A / G236del / S267K and pI variants N208D / Q295E / N384D / Q418E / N421D and, optionally, the 428L / 434S FcRn variant. In these embodiments, the sushi domain is wild-type (SEQ ID NO: 20), the anti-PD-1 Fv domain is a combination of the VH and VL domains of H.176_L1.140, and the IL-15 domain comprises a variant selected from D30N / N65D and D30N / E64Q / N65D.

[0484] In the format of Figure 28F, some embodiments include an Fc domain having a positive monomer comprising scubariant S364K / E357Q and deletion variants E233P / L234V / L235A / G236del / S267K, and a negative monomer comprising scubariant L368D / K370S, deletion variants E233P / L234V / L235A / G236del / S267K and pI variants N208D / Q295E / N384D / Q418E / N421D and, optionally, the 428L / 434S FcRn variant. In these embodiments, the sushi domain is wild-type (SEQ ID NO: 20), the anti-PD-1 Fv domain is selected from the group consisting of a combination of the VH and VL domains of H1_L1.1, and the IL-15 domain comprises a variant selected from D30N / N65D and D30N / E64Q / N65D. G.Central-IL-15 / Rα

[0485] This embodiment is shown in Figure 28G and includes four monomers forming a tetramer. This is commonly referred to as "Central-IL-15 / Rα." The central-IL-15 / Rα format (see Figure 28G) includes a VH recombinantly fused to the N-terminus of IL-15, which is then fused to one side of a heterodimeric Fc, the VH recombinantly fused to the N-terminus of IL-15Rα(sushi), which is then fused to the other side of a heterodimeric Fc, and the corresponding light chain is transfected separately to form the VH and Fab.

[0486] In the format of Figure 28G, some embodiments include an Fc domain having a positive monomer comprising scubariant S364K / E357Q and deletion variants E233P / L234V / L235A / G236del / S267K, and a negative monomer comprising scubariant L368D / K370S, deletion variants E233P / L234V / L235A / G236del / S267K and pI variants N208D / Q295E / N384D / Q418E / N421D and, optionally, the 428L / 434S FcRn variant. In these embodiments, the sushi domain is wild-type (SEQ ID NO: 20), the anti-PD-1 Fv domain is a combination of the VH and VL domains of H.176_L1.140, and the IL-15 domain comprises a variant selected from D30N / N65D and D30N / E64Q / N65D.

[0487] In the format of Figure 28G, some embodiments include an Fc domain having a positive monomer comprising scubariant S364K / E357Q and deletion variants E233P / L234V / L235A / G236del / S267K, and a negative monomer comprising scubariant L368D / K370S, deletion variants E233P / L234V / L235A / G236del / S267K and pI variants N208D / Q295E / N384D / Q418E / N421D and, optionally, the 428L / 434S FcRn variant. In these embodiments, the sushi domain is wild-type (SEQ ID NO: 20), the anti-PD-1 Fv domain is selected from the group consisting of a combination of the VH and VL domains of H1_L1.1, and the IL-15 domain comprises a variant selected from D30N / N65D and D30N / E64Q / N65D. H.Central scIL-15 / Rα

[0488] This embodiment is shown in Figure 28H and contains four monomers forming a tetramer. It is commonly referred to as "central-scIL-15 / Rα," with "sc" standing for "single chain." The central-scIL-15 / Rα format (see Figure 34H) contains a VH fused to the N-terminus of IL-15Rα (sushi) fused to IL-15, which is then further fused to one side of a heterodimeric Fc, the VH fused to the other side of the heterodimeric Fc, and the corresponding light chain transfected separately to form the VH and Fab.

[0489] In the format of Figure 28H, some embodiments include an Fc domain having a positive monomer comprising scubariant S364K / E357Q and deletion variants E233P / L234V / L235A / G236del / S267K, and a negative monomer comprising scubariant L368D / K370S, deletion variants E233P / L234V / L235A / G236del / S267K and pI variants N208D / Q295E / N384D / Q418E / N421D and, optionally, the 428L / 434S FcRn variant. In these embodiments, the sushi domain is wild-type (SEQ ID NO: 20), the anti-PD-1 Fv domain is a combination of the VH and VL domains of H.176_L1.140, and the IL-15 domain comprises a variant selected from D30N / N65D and D30N / E64Q / N65D.

[0490] In the format of Figure 28H, some embodiments include an Fc domain having a positive monomer comprising scubariant S364K / E357Q and deletion variants E233P / L234V / L235A / G236del / S267K, and a negative monomer comprising scubariant L368D / K370S, deletion variants E233P / L234V / L235A / G236del / S267K and pI variants N208D / Q295E / N384D / Q418E / N421D and, optionally, the 428L / 434S FcRn variant. In these embodiments, the sushi domain is wild-type (SEQ ID NO: 20), the anti-PD-1 Fv domain is selected from the group consisting of a combination of the VH and VL domains of H1_L1.1, and the IL-15 domain comprises a variant selected from D30N / N65D and D30N / E64Q / N65D. V. Useful Embodiments of the Invention

[0491] Provided herein are PD-1-targeting IL-15 / Rα-Fc fusion proteins having one or more engineered amino acid substitutions in the IL-15 protein and anti-PD-1 ABD that do not compete for binding with a selected approved anti-PD-1 antibody. In some embodiments, the IL-15 variant of the Fc fusion protein has N4D / N65D substitutions. In some embodiments, the IL-15 variant of the Fc fusion protein has D30N substitutions. In some embodiments, the IL-15 variant of the Fc fusion protein has D30N / E64Q / N65D substitutions. In some embodiments, the IL-15 variant of the Fc fusion protein has D30N / N65D substitutions. Such IL-15 / Rα-Fc-containing fusion proteins are useful for targeting NK cells, CD8 + T cells and CD4 + It can induce or promote the proliferation of immune cells, including T cells. Notably, IL-15 / Rα-Fc-containing fusion proteins without an exogenous linker (e.g., the hinge domain is the only domain linker) on the IL-15 Fc side showed weaker proliferation activity.

[0492] Provided herein are PD-1-targeted IL-15 / Rα-Fc fusion proteins with lower potency, increased pharmacokinetics, and / or increased serum half-life. The PD-1-targeted IL-15 / Rα-Fc fusion proteins described herein have been engineered to reduce their potency compared to the parent construct. In some embodiments, one or more amino acid substitutions have been introduced into the IL-15 / Rα complex and / or the Fc domain of the heterodimeric Fc fusion protein. In some embodiments, PD-1-targeted IL-15 / Rα-Fc fusion proteins with reduced potency compared to a control construct (e.g., the parent construct) have substantially longer serum half-lives. In certain embodiments, the serum half-life is increased by 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 25-fold, or more.

[0493] Provided herein are PD-1-targeted IL-15 / Rα-Fc fusion proteins that enhanced GVHD in animal models (e.g., human PBMC-engrafted NSG mice) compared to combination therapy with an anti-PD-1 antibody and a control scIL-15 / Rα-Fc fusion protein engineered to be less potent. Administration of an exemplary non-competitive PD-1-targeted IL-15 / Rα-Fc fusion protein resulted in a greater effect compared to the combination of IL-15 and PD-1 blockade.

[0494] The PD-1-targeted IL-15 / Rα-Fc fusion proteins described herein, including non-competing PD-1-targeted IL-15 / Rα-Fc fusion proteins, can induce STAT5 phosphorylation in immune cells, including, but not limited to, activated lymphocytes, activated T cells (e.g., activated CD4+ T cells and activated CD8+ cells), and activated tumor-infiltrating lymphocytes (e.g., activated TILs). VI. Non-targeting heterodimeric Fc fusion proteins

[0495] One aspect of the present invention provides a heterodimeric protein comprising: a) a first fusion protein comprising i) a variant IL-15 protein comprising amino acid substitutions D30N / E64Q / N65D and N71Q / N79Q / N112Q compared to SEQ ID NO: 2; ii) a domain linker; and iii) a first variant Fc domain; and b) a second fusion protein comprising i) an IL-15Rα sushi domain; ii) a domain linker; and iii) a second variant Fc domain.

[0496] In some embodiments, the variant IL-15 fusion protein comprises the amino acid sequence of SEQ ID NO:319.

[0497] In some embodiments, the first variant Fc domain and the second variant Fc domain comprise amino acid substitutions selected from the group consisting of S364K / E357Q:L368D / K370S; L368D / K370S:S364K; L368E / K370S:S364K; T411E / K360E / Q362E:D401K; L368D / K370S:S364K / E357L,K370S:S364K / E357Q,T366S / L368A / Y407V:T366W and T366S / L368A / Y407V / Y349C:T366W / S354C (according to EU numbering). In some embodiments, the first variant Fc domain comprises L368D / K370S and the second variant Fc domain comprises S364K / E357Q (according to EU numbering).

[0498] In some embodiments, the first variant Fc domain and the second variant Fc domain each independently comprise 428L / 434S, hi some embodiments, the first variant Fc domain and the second variant Fc domain both comprise 428L / 434S.

[0499] In some embodiments, the IL-15 / Rα heterodimeric Fc fusion protein is selected from the group of IL-15 / Rα heterodimeric Fc fusion proteins in Figures 115A-115C. In some embodiments, the IL-15 / Rα heterodimeric Fc fusion protein is selected from the group consisting of XENP31967, XENP31968, XENP31969, XENP31970, XENP31971, XENP31972, and XENP31973. In some embodiments, the IL-15 / Rα heterodimeric Fc fusion protein is XENP31969. In some embodiments, the IL-15 / Rα heterodimeric Fc fusion protein is XENP31972. In some embodiments, the first fusion protein comprises the amino acid sequence of SEQ ID NO:208, and the second fusion protein comprises the amino acid sequence of SEQ ID NO:95. In some embodiments, the first fusion protein comprises the amino acid sequence of SEQ ID NO:211 and the second fusion protein comprises the amino acid sequence of SEQ ID NO:206.

[0500]

[0013] In one aspect, provided herein is a method of inducing the expansion of regulatory T cells (Tregs) with reduced or minimized immunosuppressive activity in a patient in need thereof, the method comprising administering to the patient a therapeutically effective amount of an IL-15 / Rα heterodimeric Fc fusion protein, the IL-15 / Rα heterodimeric Fc fusion protein comprising: (a) a first monomer comprising, from N-terminus to C-terminus: i) a variant IL-15 protein; ii) a first domain linker; and iii) a first variant Fc domain comprising CH2-CH3; and (b) a second monomer comprising, from N-terminus to C-terminus: i) an IL-15Rα sushi domain protein; ii) a second domain linker; and iii) a second variant Fc domain comprising CH2-CH3.

[0501] In some embodiments, the variant IL-15 protein of the heterodimeric Fc fusion protein has the amino acid sequence of SEQ ID NO: 2 and N1D;N4D;D8N;D30N;D61N;E64Q;N65D;Q108E;N1D / N4D / D8N;N1D / N4D / N65D;N1D / D30N;N1D / D61N;N1D / D61N / E64Q / Q108E;N1D / E64Q;N1D / N65D;N1D / Q108E;N4D;N4D / D30N ;N4D / D61N;N4D / D61N / N65D;N4D / D61N / E64Q / Q108E;N4D / E64Q;N4D / N65D;D8N / D61N;D8N / E64Q;D30N / E64Q;D30N / N65D;D30N / E64Q / N65D;D30N / Q180E;D61N / E64Q / N65D;E64Q;E64Q / N65D;E64Q / Q108E; and N65D / Q108E. In one embodiment, the variant IL-15 protein comprises the amino acid sequence of SEQ ID NO: 2 and amino acid substitutions selected from the group consisting of N65D; D30N / E64Q / N65D; N4D / N65D; D30N / E64Q; and D30N / N65D. In some embodiments, the variant IL-15 protein comprises amino acid substitutions D30N / E64Q / N65D and N71Q / N79Q / N112Q compared to SEQ ID NO: 2. In some embodiments, the variant IL-15 fusion protein comprises the amino acid sequence of SEQ ID NO: 319.

[0502] In some embodiments, the IL-15Rα sushi domain protein comprises the amino acid sequence of SEQ ID NO: 20. In some embodiments, the IL-15Rα protein comprises the amino acid sequence of SEQ ID NO: 4.

[0503] In some embodiments, the first variant Fc domain and the second variant Fc domain have a set of amino acid substitutions selected from the group consisting of S267K / L368D / K370S:S267K / S364K / E357Q; S364K / E357Q:L368D / K370S; L368D / K370S:S364K; L368E / K370S:S364K; T411E / K360E / Q362E:D401K; L368D / K370S:S364K / E357L and K370S:S364K / E357Q (according to EU numbering). In certain embodiments, the first variant Fc domain and the second variant Fc domain comprise amino acid substitutions S267K / L368D / K370S:S267K / S364K / E357Q (according to EU numbering). In some embodiments, the first variant Fc domain and the second variant Fc domain each comprise amino acid substitutions M428L / N434S (according to EU numbering).

[0504] In some embodiments, the IL-15 / Rα heterodimeric Fc fusion protein is selected from the group of IL-15 / Rα heterodimeric Fc fusion proteins in Figures 115A-C. In some embodiments, the IL-15 / Rα heterodimeric Fc fusion protein is selected from the group consisting of XENP31967, XENP31968, XENP31969, XENP31970, XENP31971, XENP31972, and XENP31973. In some embodiments, the IL-15 / Rα heterodimeric Fc fusion protein is XENP31969. In some embodiments, the IL-15 / Rα heterodimeric Fc fusion protein is XENP31972.

[0505] In some embodiments, the non-targeting heterodimeric Fc-fusion protein comprises a first monomer comprising a first fusion protein comprising a variant IL-15 protein (comprising amino acid substitutions D30N / E64Q / N65D and N71Q / N79Q / N112Q compared to SEQ ID NO: 2) linked to a first variant Fc domain using a domain linker. The second monomer comprises a second fusion protein comprising an IL-15Rα sushi domain linked to a second variant Fc domain using a domain linker. In some embodiments, the heterodimeric protein is selected from XENP31969 and XENP31972.

[0506] In some embodiments, such heterodimeric fusion proteins are capable of inducing effector memory T cells (T EM ) proliferation. In one embodiment, the heterodimeric fusion protein EM The ratio of Treg to Treg (Treg / T EM In some cases, treatment with any one of the IL-15 / Rα-Fc fusion proteins outlined herein converts Tregs from a suppressive Treg cell type to a non-suppressive activated effector CD4 T cell. In one embodiment, FOXP3 hi CD45RA - CD4 + Effector Tregs differentiate and express FOXP3 lo CD45RA - CD4 + In some embodiments, the activated effector CD4 T cells have reduced CCR4 expression.

[0507] In some embodiments, the patient has cancer.

[0508] Useful non-targeted IL-15 / IL-15Rα heterodimeric Fc fusion proteins are described, for example, in U.S. Provisional Patent Application No. 62 / 408,655, filed October 15, 2016; U.S. Provisional Patent Application No. 62 / 416,087, filed November 1, 2016; U.S. Provisional Patent Application No. 62 / 443,465, filed January 6, 2017; U.S. Provisional Patent Application No. 62 / 477,926, filed March 28, 2017; U.S. Patent Application Publication No. 2018 / 0118805, filed October 16, 2017; U.S. Provisional Patent Application No. 2018 / 0118805, filed October 1, 2017; No. 62 / 684,143, filed June 12, 2018; U.S. Provisional Application No. 62 / 724,396, filed August 29, 2018; U.S. Provisional Application No. 62 / 756,800, filed November 7, 2018; U.S. Patent Application No. 16 / 388,174, filed April 18, 2019; and PCT Application No. PCT / US 19 / 28107, filed April 18, 2019, the disclosures of which are incorporated by reference in their entireties, with particular reference to the drawings, legends, sequence listings, and claims therein.

[0509] A particularly useful non-targeted IL-15 / IL-15Rα heterodimeric Fc fusion protein for use in reversing TGFβ-mediated inhibition of T cell proliferation in patients in need thereof is XmAb24306 (also known as "XENP24306"), the sequence of which is shown in Figure 20.

[0510] Additionally, targeted IL-15 / Rα X PD-1 heterodimeric Fc fusion proteins are also used in methods for inducing the expansion of regulatory T cells (Tregs) with reduced or minimal immunosuppressive activity in patients in need thereof. Any of the targeted constructs in the figures may find use in the present application, with XENP32435 being of particular use.

[0511] In one aspect, provided herein is a method of reversing TGFβ-mediated inhibition of T cell proliferation in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of an IL-15 / Rα heterodimeric Fc fusion protein, the IL-15 / Rα heterodimeric Fc fusion protein comprising: (a) a first monomer comprising, from N-terminus to C-terminus: i) a variant IL-15 protein; ii) a first domain linker; and iii) a first variant Fc domain comprising CH2-CH3; and (b) a second monomer comprising, from N-terminus to C-terminus: i) an IL-15Rα sushi domain protein; ii) a second domain linker; and iii) a second variant Fc domain comprising CH2-CH3.

[0512] In some embodiments, the variant IL-15 protein of the heterodimeric Fc fusion protein has the amino acid sequence of SEQ ID NO: 2 and N1D;N4D;D8N;D30N;D61N;E64Q;N65D;Q108E;N1D / N4D / D8N;N1D / N4D / N65D;N1D / D30N;N1D / D61N;N1D / D61N / E64Q / Q108E;N1D / E64Q;N1D / N65D;N1D / Q108E;N4D;N4D / D30N ;N4D / D61N;N4D / D61N / N65D;N4D / D61N / E64Q / Q108E;N4D / E64Q;N4D / N65D;D8N / D61N;D8N / E64Q;D30N / E64Q;D30N / N65D;D30N / E64Q / N65D;D30N / Q180E;D61N / E64Q / N65D;E64Q;E64Q / N65D;E64Q / Q108E; and N65D / Q108E. In one embodiment, the variant IL-15 protein comprises the amino acid sequence of SEQ ID NO: 2 and amino acid substitutions selected from the group consisting of N65D; D30N / E64Q / N65D; N4D / N65D; D30N / E64Q; and D30N / N65D. In some embodiments, the variant IL-15 protein comprises amino acid substitutions D30N / E64Q / N65D and N71Q / N79Q / N112Q compared to SEQ ID NO: 2. In some embodiments, the variant IL-15 fusion protein comprises the amino acid sequence of SEQ ID NO: 319.

[0513] In some embodiments, the IL-15Rα sushi domain protein comprises the amino acid sequence of SEQ ID NO: 20. In some embodiments, the IL-15Rα protein comprises the amino acid sequence of SEQ ID NO: 4.

[0514] In some embodiments, the first variant Fc domain and the second variant Fc domain have a set of amino acid substitutions selected from the group consisting of S267K / L368D / K370S:S267K / S364K / E357Q; S364K / E357Q:L368D / K370S; L368D / K370S:S364K; L368E / K370S:S364K; T411E / K360E / Q362E:D401K; L368D / K370S:S364K / E357L and K370S:S364K / E357Q (according to EU numbering). In certain embodiments, the first variant Fc domain and the second variant Fc domain comprise amino acid substitutions S267K / L368D / K370S:S267K / S364K / E357Q (according to EU numbering). In some embodiments, the first variant Fc domain and the second variant Fc domain each comprise amino acid substitutions M428L / N434S (according to EU numbering).

[0515] In some embodiments, the IL-15 / Rα heterodimeric Fc fusion protein is selected from the group of IL-15 / Rα heterodimeric Fc fusion proteins in Figures 115A-C. In some embodiments, the IL-15 / Rα heterodimeric Fc fusion protein is selected from the group consisting of XENP31967, XENP31968, XENP31969, XENP31970, XENP31971, XENP31972, and XENP31973. In some embodiments, the IL-15 / Rα heterodimeric Fc fusion protein is XENP31969. In some embodiments, the IL-15 / Rα heterodimeric Fc fusion protein is XENP31972.

[0516] In some embodiments, the non-targeting heterodimeric Fc-fusion protein comprises a first monomer comprising a first fusion protein comprising a variant IL-15 protein (comprising amino acid substitutions D30N / E64Q / N65D and N71Q / N79Q / N112Q compared to SEQ ID NO: 2) linked to a first variant Fc domain using a domain linker. The second monomer comprises a second fusion protein comprising an IL-15Rα sushi domain linked to a second variant Fc domain using a domain linker. In some embodiments, the heterodimeric protein is selected from XENP31969 and XENP31972.

[0517] In some embodiments, the patient exhibits increased T cell proliferation after administration. In one embodiment, the T cell proliferation is CD4 T cell proliferation. In one embodiment, the T cell proliferation is CD8 T cell proliferation. In some embodiments, the T cell proliferation is CD4 and CD8 T cell proliferation.

[0518] In some embodiments, the patient has cancer.

[0519] Useful non-targeted IL-15 / IL-15Rα heterodimeric Fc fusion proteins are described, for example, in U.S. Provisional Patent Application No. 62 / 408,655, filed October 15, 2016; U.S. Provisional Patent Application No. 62 / 416,087, filed November 1, 2016; U.S. Provisional Patent Application No. 62 / 443,465, filed January 6, 2017; U.S. Provisional Patent Application No. 62 / 477,926, filed March 28, 2017; U.S. Patent Application Publication No. 2018 / 0118805, filed October 16, 2017; International Publication No. WO 2018071919, filed October 16, 2017; Nos. 62 / 659,563, filed June 12, 2018; 62 / 684,143, filed August 29, 2018; 62 / 724,396, filed August 29, 2018; 62 / 756,800, filed November 7, 2018; 16 / 388,174, filed April 18, 2019; and PCT Application No. PCT / US19 / 28107, filed April 18, 2019, the disclosures of which are incorporated by reference in their entireties, with particular reference to the drawings, legend, sequence listing, and claims therein.

[0520] A particularly useful non-targeted IL-15 / IL-15Rα heterodimeric Fc fusion protein for use in reversing TGFβ-mediated inhibition of T cell proliferation in patients in need thereof is XmAb24306 (also known as "XENP24306"), the sequence of which is shown in Figure 20.

[0521] Additionally, targeted IL-15 / Rα X PD-1 heterodimeric Fc fusion proteins are also used in methods for reversing TGFβ-mediated inhibition of T cell proliferation in patients in need thereof. Any of the targeted constructs in the figures may find use in the present application, with XENP32435 being of particular use.

[0522]

[0013] In another aspect, provided herein is a method of reducing the expression level of FOXP3 in T cells in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of an IL-15 / Rα heterodimeric Fc fusion protein, the IL-15 / Rα heterodimeric Fc fusion protein comprising: (a) a first monomer comprising, from N-terminus to C-terminus: i) a variant IL-15 protein; ii) a first domain linker; and iii) a first variant Fc domain comprising CH2-CH3; and (b) a second monomer comprising, from N-terminus to C-terminus: i) an IL-15Rα sushi domain protein; ii) a second domain linker; and iii) a second variant Fc domain comprising CH2-CH3.

[0523] In some embodiments, the variant IL-15 protein of the heterodimeric Fc fusion protein has the amino acid sequence of SEQ ID NO: 2 and N1D;N4D;D8N;D30N;D61N;E64Q;N65D;Q108E;N1D / N4D / D8N;N1D / N4D / N65D;N1D / D30N;N1D / D61N;N1D / D61N / E64Q / Q108E;N1D / E64Q;N1D / N65D;N1D / Q108E;N4D;N4D / D30N ;N4D / D61N;N4D / D61N / N65D;N4D / D61N / E64Q / Q108E;N4D / E64Q;N4D / N65D;D8N / D61N;D8N / E64Q;D30N / E64Q;D30N / N65D;D30N / E64Q / N65D;D30N / Q180E;D61N / E64Q / N65D;E64Q;E64Q / N65D;E64Q / Q108E; and N65D / Q108E. In one embodiment, the variant IL-15 protein comprises the amino acid sequence of SEQ ID NO: 2 and amino acid substitutions selected from the group consisting of N65D; D30N / E64Q / N65D; N4D / N65D; D30N / E64Q; and D30N / N65D. In some embodiments, the variant IL-15 protein comprises amino acid substitutions D30N / E64Q / N65D and N71Q / N79Q / N112Q compared to SEQ ID NO: 2. In some embodiments, the variant IL-15 fusion protein comprises the amino acid sequence of SEQ ID NO: 319.

[0524] In some embodiments, the IL-15Rα sushi domain protein comprises the amino acid sequence of SEQ ID NO: 20. In some embodiments, the IL-15Rα protein comprises the amino acid sequence of SEQ ID NO: 4.

[0525] In some embodiments, the first variant Fc domain and the second variant Fc domain have a set of amino acid substitutions selected from the group consisting of S267K / L368D / K370S:S267K / S364K / E357Q; S364K / E357Q:L368D / K370S; L368D / K370S:S364K; L368E / K370S:S364K; T411E / K360E / Q362E:D401K; L368D / K370S:S364K / E357L and K370S:S364K / E357Q (according to EU numbering). In certain embodiments, the first variant Fc domain and the second variant Fc domain comprise amino acid substitutions S267K / L368D / K370S:S267K / S364K / E357Q (according to EU numbering). In some embodiments, the first variant Fc domain and the second variant Fc domain each comprise amino acid substitutions M428L / N434S (according to EU numbering).

[0526] In some embodiments, the IL-15 / Rα heterodimeric Fc fusion protein is selected from the group of IL-15 / Rα heterodimeric Fc fusion proteins in Figures 115A-C. In some embodiments, the IL-15 / Rα heterodimeric Fc fusion protein is selected from the group consisting of XENP31967, XENP31968, XENP31969, XENP31970, XENP31971, XENP31972, and XENP31973. In some embodiments, the IL-15 / Rα heterodimeric Fc fusion protein is XENP31969. In some embodiments, the IL-15 / Rα heterodimeric Fc fusion protein is XENP31972.

[0527] In some embodiments, the patient has an expanded population of non-suppressive regulatory T cells (Tregs) after administration.

[0528] In some embodiments, the patient has an expanded population of activated effector CD4 T cells after administration.

[0529] In some embodiments, patients are monitored for the ratio of Tregs to effector memory T cells (Treg / T EM ) is increasing.

[0530] In any embodiment of the present invention, the patient has cancer.

[0531] Useful non-targeted IL-15 / IL-15Rα heterodimeric Fc fusion proteins are described, for example, in U.S. Provisional Patent Application No. 62 / 408,655, filed October 15, 2016; U.S. Provisional Patent Application No. 62 / 416,087, filed November 1, 2016; U.S. Provisional Patent Application No. 62 / 443,465, filed January 6, 2017; U.S. Provisional Patent Application No. 62 / 477,926, filed March 28, 2017; U.S. Patent Application Publication No. 2018 / 0118805, filed October 16, 2017; International Publication No. WO 2018071919, filed October 16, 2017; Nos. 62 / 659,563, filed June 12, 2018; 62 / 684,143, filed August 29, 2018; 62 / 724,396, filed August 29, 2018; 62 / 756,800, filed November 7, 2018; 16 / 388,174, filed April 18, 2019; and PCT Application No. PCT / US19 / 28107, filed April 18, 2019, the disclosures of which are incorporated by reference in their entireties, with particular reference to the drawings, legend, sequence listing, and claims therein.

[0532] A particularly useful non-targeted IL-15 / IL-15Rα heterodimeric Fc fusion protein for use in reducing FOXP3 expression levels in T cells in patients in need thereof is XmAb24306 (also known as "XENP24306"), the sequence of which is shown in Figure 20.

[0533] Additionally, the targeted IL-15 / Rα X PD-1 heterodimeric Fc fusion proteins are also used in methods for reducing FOXP3 expression levels in T cells in patients in need thereof. Any of the targeted constructs in the figures may find use in the present application, with XENP32435 being of particular use.

[0534] In yet another aspect, provided herein is a method for expanding activated effector CD4 T cells in a patient with cancer, the method comprising administering to the patient a therapeutically effective amount of an IL-15 / Rα heterodimeric Fc fusion protein, the IL-15 / Rα heterodimeric Fc fusion protein comprising: (a) a first monomer comprising, from N-terminus to C-terminus: i) a variant IL-15 protein; ii) a first domain linker; and iii) a first variant Fc domain comprising CH2-CH3; and (b) a second monomer comprising, from N-terminus to C-terminus: i) an IL-15Rα sushi domain protein; ii) a second domain linker; and iii) a second variant Fc domain comprising CH2-CH3.

[0535] In some embodiments, the variant IL-15 protein of the heterodimeric Fc fusion protein has the amino acid sequence of SEQ ID NO: 2 and N1D;N4D;D8N;D30N;D61N;E64Q;N65D;Q108E;N1D / N4D / D8N;N1D / N4D / N65D;N1D / D30N;N1D / D61N;N1D / D61N / E64Q / Q108E;N1D / E64Q;N1D / N65D;N1D / Q108E;N4D;N4D / D30N ;N4D / D61N;N4D / D61N / N65D;N4D / D61N / E64Q / Q108E;N4D / E64Q;N4D / N65D;D8N / D61N;D8N / E64Q;D30N / E64Q;D30N / N65D;D30N / E64Q / N65D;D30N / Q180E;D61N / E64Q / N65D;E64Q;E64Q / N65D;E64Q / Q108E; and N65D / Q108E. In one embodiment, the variant IL-15 protein comprises the amino acid sequence of SEQ ID NO: 2 and amino acid substitutions selected from the group consisting of N65D; D30N / E64Q / N65D; N4D / N65D; D30N / E64Q; and D30N / N65D. In some embodiments, the variant IL-15 protein comprises amino acid substitutions D30N / E64Q / N65D and N71Q / N79Q / N112Q compared to SEQ ID NO: 2. In some embodiments, the variant IL-15 fusion protein comprises the amino acid sequence of SEQ ID NO: 319.

[0536] In some embodiments, the IL-15Rα sushi domain protein comprises the amino acid sequence of SEQ ID NO: 20. In some embodiments, the IL-15Rα protein comprises the amino acid sequence of SEQ ID NO: 4.

[0537] In some embodiments, the first variant Fc domain and the second variant Fc domain have a set of amino acid substitutions selected from the group consisting of S267K / L368D / K370S:S267K / S364K / E357Q; S364K / E357Q:L368D / K370S; L368D / K370S:S364K; L368E / K370S:S364K; T411E / K360E / Q362E:D401K; L368D / K370S:S364K / E357L and K370S:S364K / E357Q (according to EU numbering). In certain embodiments, the first variant Fc domain and the second variant Fc domain comprise amino acid substitutions S267K / L368D / K370S:S267K / S364K / E357Q (according to EU numbering). In some embodiments, the first variant Fc domain and the second variant Fc domain each comprise amino acid substitutions M428L / N434S (according to EU numbering).

[0538] In some embodiments, the IL-15 / Rα heterodimeric Fc fusion protein is selected from the group of IL-15 / Rα heterodimeric Fc fusion proteins in Figures 115A-C. In some embodiments, the IL-15 / Rα heterodimeric Fc fusion protein is selected from the group consisting of XENP31967, XENP31968, XENP31969, XENP31970, XENP31971, XENP31972, and XENP31973. In some embodiments, the IL-15 / Rα heterodimeric Fc fusion protein is XENP31969. In some embodiments, the IL-15 / Rα heterodimeric Fc fusion protein is XENP31972.

[0539] In some embodiments, activated effector CD4 T cells express FOXP3 lo CD45RA - CD4 + T cells.

[0540] In some embodiments, activated effector CD4 T cells have reduced CCR4 expression or CCR4 lo / -is.

[0541] In some embodiments, patients are monitored for the ratio of Tregs to effector memory T cells (Treg / T EM ) is increasing.

[0542] Useful non-targeted IL-15 / IL-15Rα heterodimeric Fc fusion proteins are described, for example, in U.S. Provisional Patent Application No. 62 / 408,655, filed October 15, 2016; U.S. Provisional Patent Application No. 62 / 416,087, filed November 1, 2016; U.S. Provisional Patent Application No. 62 / 443,465, filed January 6, 2017; U.S. Provisional Patent Application No. 62 / 477,926, filed March 28, 2017; U.S. Patent Application Publication No. 2018 / 0118805, filed October 16, 2017; International Publication No. WO 2018071919, filed October 16, 2017; Nos. 62 / 659,563, filed June 12, 2018; 62 / 684,143, filed August 29, 2018; 62 / 724,396, filed August 29, 2018; 62 / 756,800, filed November 7, 2018; 16 / 388,174, filed April 18, 2019; and PCT Application No. PCT / US19 / 28107, filed April 18, 2019, the disclosures of which are incorporated by reference in their entireties, with particular reference to the drawings, legend, sequence listing, and claims therein.

[0543] A particularly useful non-targeted IL-15 / IL-15Rα heterodimeric Fc fusion protein for use in expanding activated effector CD4 T cells in patients in need thereof is XmAb24306 (also known as "XENP24306"), the sequence of which is shown in Figure 20.

[0544] Additionally, targeted IL-15 / Rα X PD-1 heterodimeric Fc fusion proteins are also used in methods for expanding activated effector CD4 T cells in patients in need thereof. Any of the targeted constructs in the figures may find use in the present application, with XENP32435 being of particular use. VII. Nucleic Acids of the Invention

[0545] The present invention further provides nucleic acid compositions encoding the heterodimeric Fc fusion proteins of the present invention (or, in the case of monomeric Fc domain proteins, also the nucleic acids encoding them).

[0546] As will be understood by those skilled in the art, the nucleic acid composition will depend on the format of the heterodimeric IL-15 / RαFc fusion protein. Thus, for example, if the format requires three amino acid sequences, the three nucleic acid sequences can be incorporated into one or more expression vectors for expression. Similarly, in some formats, only two nucleic acids are required. Similarly, they can be placed in one or two expression vectors.

[0547] As is known in the art, nucleic acids encoding the components of the present invention can be incorporated into expression vectors, as known in the art and depending on the host cell used to produce the heterodimeric Fc-fusion proteins of the present invention. Generally, the nucleic acid is operably linked to any number of regulatory elements (promoter, origin of replication, selectable marker, ribosome binding site, inducer, etc.). Expression vectors can be extrachromosomal or integrating vectors.

[0548] The nucleic acids and / or expression vectors of the invention are then transformed into any number of different types of host cells known in the art, including mammalian cells, bacterial cells, yeast cells, insect cells, and / or fungal cells, with mammalian cells (e.g., CHO cells) finding use in many embodiments.

[0549] In some embodiments, the nucleic acids encoding each monomer are contained within a single expression vector, generally under the control of different or the same promoter, as applicable depending on the format. In particular embodiments of use in the present invention, each of these two or three nucleic acids is contained in a different expression vector.

[0550] The heterodimeric Fc-fusion proteins of the present invention are produced by culturing host cells containing expression vectors known in the art. Once produced, traditional fusion protein or antibody purification processes, including ion exchange chromatography steps, are performed. As discussed herein, a difference in pI between the two monomers of at least 0.5 can enable separation by ion exchange chromatography or isoelectric focusing or other methods sensitive to isoelectric point. That is, the inclusion of pI substitutions that alter the isoelectric point (pI) of each monomer so that each monomer has a different pI and the heterodimer also has a different pI, facilitates isoelectric purification of the heterodimer (e.g., anion exchange column, cation exchange column). These substitutions also aid in the determination and monitoring of any contaminating homodimers after purification (e.g., IEF gels, cIEF, and analytical IEX columns). VIII. Biological and Biochemical Functionality of Bispecific Immune Checkpoint Antibody x IL-15 / IL-15Rα Heterodimeric Immunomodulatory Fusion Protein

[0551] Generally, the Fc-fusion proteins of the present invention are administered to patients with cancer, and efficacy is assessed by several methods described herein. Thus, while standard efficacy assays such as assessing cancer burden, tumor size, and the presence or extent of metastases can be performed, immuno-oncology treatments can also be evaluated based on immune status assessments. This can be done in several ways, including both in vitro and in vivo assays. For example, assessment of changes in immune status (e.g., the presence of ICOS+CD4+ T cells after ipi treatment) can be performed in conjunction with "old-fashioned" measurements such as tumor burden, size, invasiveness, LN involvement, metastasis, etc. Thus, any or all of the following can be assessed: CD4+ T cell activation or proliferation, CD8 + T (CTL) cell activation or proliferation, CD8 + The inhibitory effect of the fusion protein on T cell-mediated cytotoxic activity and / or CTL-mediated cell depletion, NK cell activity and NK-mediated cell depletion, the enhancing effect of the fusion protein on Treg cell differentiation and proliferation, Treg or myeloid-derived suppressor cell (MDSC)-mediated immunosuppression or immune tolerance, and / or the effect of the fusion protein on pro-inflammatory cytokine production by immune cells, such as IL-2, IFN-γ or TNF-α production by T cells or other immune cells.

[0552] In some embodiments, treatment assessment is performed using, for example, CFSE dilution assays, Ki67 intracellular staining of immune effector cells, and 3 This is done by assessing immune cell proliferation using the H-thymidine incorporation method.

[0553] In some embodiments, treatment is evaluated by assessing increased gene expression or increased protein levels of activation-associated markers, including one or more of the following: cellular degranulation as measured by surface expression of CD25, CD69, CD137, ICOS, PD1, GITR, OX40, and CD107A.

[0554] Generally, gene expression assays are performed as known in the art.

[0555] Generally, protein expression measurements are similarly performed as known in the art.

[0556] In some embodiments, treatment evaluation is performed by assessing cytotoxic activity as measured by target cell viability detection, by estimating a number of cellular parameters, such as enzyme activity (including protease activity), cell membrane permeability, cell adhesion, ATP production, coenzyme production, and nucleotide uptake activity. Specific examples of these assays include, but are not limited to, trypan blue or PI staining, 51 Cr or 35These include the S release method, LDH activity, MTT and / or WST assays, calcein-AM assays, luminescence-based assays, and the like.

[0557] In some embodiments, treatment is evaluated by assessing T cell activity as measured by cytokine production, measured intracellularly in the culture supernatant using well-known techniques using cytokines including, but not limited to, IFNγ, TNFα, GM-CSF, IL2, IL6, IL4, IL5, IL10, IL13.

[0558] Thus, treatment can be evaluated using assays that assess one or more of the following: (i) an increase in immune response, (ii) an increase in αβ and / or γδ T cell activation, (iii) an increase in cytotoxic T cell activity, (iv) an increase in NK and / or NKT cell activity, (v) relief of αβ and / or γδ T cell suppression, (vi) an increase in pro-inflammatory cytokine secretion, (vii) an increase in IL-2 secretion; (viii) an increase in interferon-γ production, (ix) an increase in Th1 response, (x) a decrease in Th2 response, or (xi) a decrease or elimination of at least one of the number and / or activity of regulatory T cells (Tregs). A. Assays for Measuring Competitive Binding

[0559] Generally, epitope binning assays, such as those described herein, as well as other competitive inhibition assays known in the art, can be performed to determine whether NCPD-1 Fvs compete with approved antibodies for binding to PD-1. Epitope binning is a process that uses competitive immunoassays to test antibodies in a pairwise combinatorial manner, where antibodies that compete for the same binding region, i.e., the same or closely related epitopes of an antigen, are grouped together in bins. Thus, antibodies that bin to epitopes different from nivolumab and / or pembrolizumab are not expected to compete with nivolumab and / or pembrolizumab.

[0560] A non-competing antibody can be determined by an assay in which the antibody or immunologically functional fragment being tested does not prevent or inhibit specific binding of a reference antibody to a common antigen. Typically, such an assay involves the use of purified antigen (e.g., PD-1 or a domain or fragment thereof) bound to a solid surface or cells. Competitive inhibition is measured by determining the amount of a first antibody bound to the solid surface or cells in the presence of a second antibody. Typically, when a competing antibody is present in excess, it inhibits specific binding of the reference antibody to a common antigen by at least 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75%. In some instances, binding is inhibited by at least 80%, 85%, 90%, 95%, or 97% or more. Conversely, if the reference antibody binds, it preferably inhibits binding of a subsequently added test antibody (i.e., a PD-1 antibody) by at least 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75%. In some instances, binding of the test antibody is inhibited by at least 80%, 85%, 90%, 95%, or 97% or more.

[0561] Generally, binning or competitive binding can be determined using a variety of art-recognized techniques, such as immunoassays, such as Western blots, radioimmunoassays, enzyme-linked immunosorbent assays (ELISAs), "sandwich" immunoassays, immunoprecipitation assays, precipitation reactions, gel diffusion precipitation reactions, immunodiffusion assays, agglutination assays, complement fixation assays, immunoradiometric assays, fluorescent immunoassays, and protein A immunoassays. Such immunoassays are routine and well known in the art (see, e.g., WO 2003 / 48731 and Harlow et al. (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and David Lane). In addition, cross-blocking assays can be used (see, e.g., WO 2003 / 48731 and Harlow et al. (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and David Lane).

[0562] Other techniques used to determine competitive inhibition (and thus "bins") include surface plasmon resonance, e.g., using a BIAcore™ 2000 system (GE Healthcare); biolayer interferometry, e.g., using ForteBio® Octet RED (ForteBio); or flow cytometry bead arrays, e.g., using a FACSCanto II (BD Biosciences) or multiplexed LUMINEX™ detection assay (Luminex). One specific method for determining competitive binding using biolayer interferometry is provided in Examples 2 and 7 herein.

[0563] Luminex is a bead-based immunoassay platform that enables large-scale multiplexed antibody pairing. This assay compares the simultaneous binding patterns of antibody pairs to a target antigen. One antibody (capture mAb) of the pair is bound to Luminex beads, and each capture mAb is bound to a different colored bead. The other antibody (detection mAb) binds a fluorescent signal (e.g., phycoerythrin (PE)). This assay analyzes the simultaneous binding (pairing) of antibodies to the antigen and groups together antibodies with similar pairing profiles. Similar profiles of the detection mAb and capture mAb indicate that the two antibodies bind to the same or closely related epitopes. In one embodiment, the pairing profile can be determined using a Pearson correlation coefficient to identify the antibody that most closely correlates...

Claims

1. 1. A targeted IL-15 / Rα heterodimeric Fc fusion protein comprising: a) a first monomer comprising, from N-terminus to C-terminus: i) IL-15 / Rα sushi domain; ii) a first domain linker; iii) the IL-15 domain; and iv) a first variant Fc domain; b) a second monomer comprising a heavy chain comprising VH-CH1-hinge-CH2-CH3, wherein said CH2-CH3 is a second variant Fc domain; and c) a third monomer comprising a light chain comprising VL-CL; Including, a targeting IL-15 / Rα heterodimeric Fc fusion protein, wherein the VH domain and the VL domain form an antigen binding domain (ABD) that binds to human PD-1, wherein the VH is a variant variable heavy domain comprising F32L / W100F amino acid substitutions, Kabat numbering, compared to SEQ ID NO:5; and the VL is a variant variable light domain comprising N27dH / K30Y / S93T, Kabat numbering, compared to SEQ ID NO:

168.

2. 2. The fusion protein of claim 1, wherein the VH comprises the amino acid sequence of SEQ ID NO: 318 and the VL comprises the amino acid sequence of SEQ ID NO:

176.

3. the IL-15 domain is D30N / E64Q / N65D, N1D / N4D / D8N, N1D / N4D / N65D, N1D / D30N, N1D / D61N, N1D / D61N / E64Q / Q108E, N1D / E64Q, N1D / N65D, N1D / Q108E, N4D / D30N, N4D / D61N, N4D / D61N / N65D, N4D / D61N / E64Q / Q108E, 3. The fusion protein of claim 1 or 2, which is a variant IL-15 domain comprising amino acid substitutions selected from the group consisting of N4D / E64Q, N4D / N65D, D8N / D61N, D8N / E64Q, D30N / E64Q, D30N / N65D, D30N / Q180E, D61N / E64Q / N65D, E64Q / N65D, E64Q / Q108E and N65D / Q108E.

4. 4. The fusion protein of claim 1, wherein the IL-15 domain is a variant IL-15 domain comprising an amino acid substitution selected from the group consisting of N71Q, N79Q, N112Q, S114del, and S114A, or a combination thereof.

5. The fusion protein of any one of claims 1 to 4, wherein the IL-15 domain is a variant IL-15 domain comprising the amino acid substitutions D30N / E64Q / N65D and N71Q / N79Q / N112Q.

6. The fusion protein of any one of claims 1 to 5, wherein the first variant Fc domain comprises all or part of a hinge domain.

7. The fusion protein of any one of claims 1 to 6, wherein the first monomer further comprises a second domain linker between the IL-15 domain and the first variant Fc domain.

8. 1. A targeted IL-15 / Rα heterodimeric Fc fusion protein comprising: a) a first monomer comprising: i) IL-15 Rα sushi domain protein; ii) a first domain linker; iii) a variant IL-15 protein comprising the amino acid substitutions D30N / E64Q / N65D and N71Q / N79Q / N112Q compared to SEQ ID NO: 2; and iv) a first variant Fc domain; b) a second monomer comprising a heavy chain comprising VH-CH1-hinge-CH2-CH3, wherein said CH2-CH3 is a second variant Fc domain; and c) a third monomer comprising a light chain comprising VL-CL; Including, A targeting IL-15 / Rα heterodimeric Fc fusion protein, wherein the VH domain and the VL domain form an antigen binding domain (ABD) that binds to human PD-1.

9. The fusion protein of claim 8, wherein the VH comprises the amino acid sequence of SEQ ID NO: 5 and the VL comprises the amino acid sequence of SEQ ID NO:

168.

10. The fusion protein of claim 8, wherein the VH comprises the amino acid sequence of SEQ ID NO: 318 and the VL comprises the amino acid sequence of SEQ ID NO:

176.

11. 9. The fusion protein of claim 8, wherein the ABD does not compete with nivolumab and / or pembrolizumab for binding to the human PD-1.

12. The fusion protein of any one of claims 8 to 11, wherein the first variant Fc domain comprises all or part of a hinge domain.

13. The fusion protein of any one of claims 8 to 12, wherein the first monomer further comprises a second domain linker between the IL-15 domain and the first variant Fc domain.

14. 1. A targeted IL-15 / Rα heterodimeric Fc fusion protein comprising: a) a first monomer comprising: i) IL-15 Rα sushi domain protein; ii) a first domain linker; iii) a variant IL-15 protein comprising the amino acid substitutions D30N / E64Q / N65D and N71Q / N79Q / N112Q compared to SEQ ID NO: 2; and iv) a first variant Fc domain; b) a second monomer comprising a heavy chain comprising VH-CH1-hinge-CH2-CH3, wherein said CH2-CH3 is a second variant Fc domain; and c) a third monomer comprising a light chain comprising VL-CL; Including, a targeting IL-15 / Rα heterodimeric Fc fusion protein, wherein the VH domain and the VL domain form an antigen binding domain (ABD) that binds to human PD-1, wherein the VH is a variant variable heavy domain comprising F32L / W100F amino acid substitutions, Kabat numbering, compared to SEQ ID NO:5; and the VL is a variant variable light domain comprising N27dH / K30Y / S93T, Kabat numbering, compared to SEQ ID NO:

168.

15. The fusion protein of claim 14, wherein the VH comprises the amino acid sequence of SEQ ID NO: 318 and the VL comprises the amino acid sequence of SEQ ID NO:

176.

16. 16. The fusion protein of claim 14 or 15, wherein the first variant Fc domain comprises all or part of a hinge domain.

17. The fusion protein of any one of claims 14 to 16, wherein the first monomer further comprises a second domain linker between the IL-15 domain and the first variant Fc domain.

18. 18. The fusion protein of any one of claims 1 to 17, wherein the first variant Fc domain and the second variant Fc domain comprise amino acid substitutions selected from the group consisting of: S364K / E357Q:L368D / K370S; L368D / K370S:S364K; L368E / K370S:S364K; T411E / K360E / Q362E:D401K; L368D / K370S:S364K / E357L,K370S:S364K / E357Q, T366S / L368A / Y407V:T366W and T366S / L368A / Y407V / Y349C:T366W / S354C (according to EU numbering).

19. 19. The fusion protein of claim 18, wherein the first variant Fc domain comprises L368D / K370S and the second variant Fc domain comprises S364K / E357Q, according to EU numbering.

20. 20. The fusion protein of any one of claims 1 to 19, wherein the first variant Fc domain and the second variant Fc domain each independently comprise amino acid substitutions selected from the group consisting of G236R / L328R, E233P / L234V / L235A / G236del / S239K, E233P / L234V / L235A / G236del / S267K, E233P / L234V / L235A / G236del / S239K / A327G, E233P / L234V / L235A / G236del / S267K / A327G, and E233P / L234V / L235A / G236del, according to EU numbering.

21. 21. The fusion protein of claim 20, wherein the first variant Fc domain and the second variant Fc domain each comprise the amino acid substitutions E233P / L234V / L235A / G236del / S267K according to EU numbering.

22. 22. The fusion protein of claim 1, wherein the first Fc domain comprises the amino acid substitutions Q295E / N384D / Q418E / N481D according to EU numbering.

23. 23. The fusion protein of any one of claims 1 to 22, wherein the first variant Fc domain and the second variant Fc domain each comprise amino acid substitutions M428L / N434S according to EU numbering.

24. 15. The fusion protein of any one of claims 1, 8 and 14, wherein the first monomer comprises the amino acid sequence of SEQ ID NO:225, the second monomer comprises the amino acid sequence of SEQ ID NO:244, and the third monomer comprises the amino acid sequence of SEQ ID NO:

196.

25. 1. A nucleic acid composition comprising: a) a first nucleic acid molecule encoding the first monomer according to any one of claims 1 to 24; b) a second nucleic acid molecule encoding the second monomer according to any one of claims 1 to 24; and / or c) a third nucleic acid molecule encoding the third monomer according to any one of claims 1 to 24. and each of the nucleic acid compositions comprises:

26. 1. An expression vector composition comprising: a) a first expression vector comprising the first nucleic acid molecule of claim 25; b) a second expression vector comprising the second nucleic acid molecule of claim 25; and / or c) A third expression vector comprising the third nucleic acid molecule of claim 25. An expression vector composition comprising:

27. 26. An expression vector comprising the first nucleic acid molecule of claim 25, the second nucleic acid molecule of claim 25, and the third nucleic acid molecule of claim 25.

28. 28. A host cell comprising the expression vector composition of claim 26 or the expression vector of claim 27.

29. 30. A method of producing a fusion protein, comprising culturing the host cell of claim 28 under conditions in which the fusion protein is produced, and recovering the fusion protein.

30. 1. A targeted IL-15 / Rα heterodimeric Fc fusion protein comprising: a) a first monomer comprising, from N-terminus to C-terminus: i) IL-15 Rα sushi domain protein; ii) a first domain linker; iii) a variant IL-15 protein comprising an amino acid substitution selected from the group consisting of N71Q, N79Q, N112Q, S114del and S114A compared to SEQ ID NO: 2; and iv) a first variant Fc domain; b) a second monomer comprising a heavy chain comprising VH-CH1-hinge-CH2-CH3, wherein said CH2-CH3 is a second variant Fc domain; and c) a third monomer comprising a light chain comprising VL-CL; Including, the VH domain and the VL domain form an antigen-binding domain that binds to human PD-1, and the VH domain contains, relative to SEQ ID NO: 5, the following amino acid substitutions, according to Kabat numbering: W100F, F32L, S52aG, R97E, R97Y, R97W, L98R, S100aT, R97A, V99T, V99L, S100aA, L98Q, R97Q, V99F, V99L, S100aN, and at least one further amino acid substitution selected from the group consisting of: V99I, P100bS, G96H, L98V, V99A, V99Q, G96V, R97K, L98S, L98F, R97T, L98K, L98S, V99I, R97L, G96A, R97A, V99S, R97S, V99Y, R97H, L98R, wherein said VL domain comprises i) SEQ ID NO: 168; and ii) a variant light domain comprising an amino acid substitution selected from the group consisting of N27dH, N27dS, K30Y, S93T, and Y94W, Kabat numbering, compared to SEQ ID NO: 168; Including, The variant IL-15 protein is a targeted IL-15 / Rα heterodimeric Fc fusion protein comprising amino acid substitutions selected from the group consisting of N71Q / N79Q, N71Q / N79Q / N112Q, N71Q / N79Q / S114del and N71Q / N79Q / S114A.

31. The variant IL-15 protein is selected from the group consisting of D30N / E64Q / N65D, N1D / N4D / D8N, N1D / N4D / N65D, N1D / D30N, N1D / D61N, N1D / D61N / E64Q / Q108E, N1D / E64Q, N1D / N65D, N1D / Q108E, N4D / D30N, N4D / D61N, N4D / D61N / N65D, N4D / D61N / 31. The fusion protein of claim 30, further comprising amino acid substitutions selected from the group consisting of E64Q / Q108E, N4D / E64Q, N4D / N65D, D8N / D61N, D8N / E64Q, D30N / E64Q, D30N / N65D, D30N / Q180E, D61N / E64Q / N65D, E64Q / N65D, E64Q / Q108E and N65D / Q108E.

32. 32. The fusion protein of claim 30 or 31, wherein the variant heavy domain comprises the amino acid sequence of SEQ ID NO: 318 and the variant light domain comprises the amino acid sequence of SEQ ID NO:

176.

33. The fusion protein of any one of claims 30 to 32, wherein the variant IL-15 protein comprises the amino acid substitutions N71Q / N79Q / N112Q.

34. 34. The fusion protein of any one of claims 30 to 33, wherein the variant IL-15 protein comprises the amino acid substitutions N71Q / N79Q / N112Q and D30N / N65D.

35. 35. The fusion protein of any one of claims 30 to 34, wherein the variant IL-15 protein comprises the amino acid substitutions N71Q / N79Q / N112Q and D30N / E64Q / N65D.

36. 36. The fusion protein of any one of claims 30-35, wherein the variant heavy domain is H1.176, the variant light domain is L1.140, and the variant IL-15 protein comprises amino acid substitutions N71Q / N79Q / N112Q and D30N / N65D.

37. 37. The fusion protein of any one of claims 30-36, wherein the variant heavy domain is H1.176, the variant light domain is L1.140, and the variant IL-15 protein comprises amino acid substitutions N71Q / N79Q / N112Q and D30N / E64Q / N65D.

38. 38. The fusion protein of any one of claims 30 to 37, wherein the first domain linker comprises GGGGA (SEQ ID NO: 8).

39. 39. The fusion protein of any one of claims 30 to 38, wherein the first variant Fc domain comprises all or part of a hinge domain.

40. 40. The fusion protein of any one of claims 30 to 39, wherein the first monomer further comprises a second domain linker between the IL-15 domain and the first variant Fc domain.

41. 41. The fusion protein of any one of claims 30 to 40, wherein the first variant Fc domain and the second variant Fc domain comprise amino acid substitutions selected from the group consisting of: S364K / E357Q:L368D / K370S; L368D / K370S:S364K; L368E / K370S:S364K; T411E / K360E / Q362E:D401K; L368D / K370S:S364K / E357L,K370S:S364K / E357Q, T366S / L368A / Y407V:T366W and T366S / L368A / Y407V / Y349C:T366W / S354C (according to EU numbering).

42. 42. The fusion protein of claim 41 , wherein the first variant Fc domain comprises L368D / K370S and the second variant Fc domain comprises S364K / E357Q, according to EU numbering.

43. 43. The fusion protein of any one of claims 30 to 42, wherein the first variant Fc domain and the second variant Fc domain each independently comprise an amino acid substitution 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.

44. 44. The fusion protein of claim 43, wherein the first variant Fc domain and the second variant Fc domain each comprise the amino acid substitutions E233P / L234V / L235A / G236del / S267K according to EU numbering.

45. 45. The fusion protein of any one of claims 30 to 44, wherein the first Fc domain comprises the amino acid substitutions Q295E / N384D / Q418E / N481D according to EU numbering.

46. 46. ​​The fusion protein of any one of claims 30 to 45, wherein the first variant Fc domain and the second variant Fc domain each comprise the amino acid substitutions M428L / N434S according to EU numbering.

47. 47. The fusion protein of any one of claims 30 to 46, wherein the first monomer comprises the amino acid sequence of SEQ ID NO:

225.

48. 48. The fusion protein of any one of claims 30 to 47, wherein the second monomer comprises the amino acid sequence of SEQ ID NO:

244.

49. 49. The fusion protein of any one of claims 30 to 48, wherein the third monomer comprises the amino acid sequence of SEQ ID NO:

196.

50. 50. The fusion protein of any one of claims 30-49, wherein the first monomer comprises the amino acid sequence of SEQ ID NO:225, the second monomer comprises the amino acid sequence of SEQ ID NO:244, and the third monomer comprises the amino acid sequence of SEQ ID NO:

196.

51. 1. A nucleic acid composition comprising: a) a first nucleic acid molecule encoding the first monomer according to any one of claims 30 to 50; b) a second nucleic acid molecule encoding the second monomer according to any one of claims 30 to 50; and / or c) a third nucleic acid molecule encoding the third monomer according to any one of claims 30 to 50; A nucleic acid composition comprising:

52. 1. An expression vector composition comprising: a) a first expression vector comprising the first nucleic acid molecule of claim 51; b) a second expression vector comprising the second nucleic acid molecule of claim 51; and / or c) a third expression vector comprising the third nucleic acid molecule of claim 51; An expression vector composition comprising:

53. 52. An expression vector comprising the first nucleic acid molecule of claim 51, the second nucleic acid molecule of claim 51, and the third nucleic acid molecule of claim 51.

54. 54. A host cell comprising the expression vector composition of claim 52 or the expression vector of claim 53.

55. 55. A method of making a fusion protein, comprising culturing the host cell of claim 54 under conditions in which the fusion protein is produced, and recovering the fusion protein.

56. 1. A targeted IL-15 / Rα heterodimeric Fc fusion protein comprising: a) a first monomer comprising, from N-terminus to C-terminus: i) IL-15 Rα sushi domain protein; ii) a first domain linker; iii) a variant IL-15 protein; and iv) a first variant Fc domain; b) a second monomer comprising a heavy chain comprising VH-CH1-hinge-CH2-CH3, wherein said CH2-CH3 is a second variant Fc domain; and c) a third monomer comprising a light chain comprising VL-CL; Including, the VH domain and the VL domain form an antigen-binding domain that binds to human PD-1, and the VH domain contains, relative to SEQ ID NO: 5, the following amino acid substitutions, according to Kabat numbering: W100F, F32L, S52aG, R97E, R97Y, R97W, L98R, S100aT, R97A, V99T, V99L, S100aA, L98Q, R97Q, V99F, V99L, S100aN, and at least one further amino acid substitution selected from the group consisting of: V99I, P100bS, G96H, L98V, V99A, V99Q, G96V, R97K, L98S, L98F, R97T, L98K, L98S, V99I, R97L, G96A, R97A, V99S, R97S, V99Y, R97H, L98R, wherein said VL domain comprises i) SEQ ID NO: 168; and ii) a variant light domain comprising an amino acid substitution selected from the group consisting of N27dH, N27dS, K30Y, S93T, and Y94W, Kabat numbering, compared to SEQ ID NO: 168; A targeted IL-15 / Rα heterodimeric Fc fusion protein selected from the group consisting of:

57. The variant heavy domain is selected from the group consisting of H1.176, H1.177, H1.178, H1.179, H1.180, H1.181, H1.182, H1.183, H1.184, H1.185, H1.186, H1.187, H1.188, H1.189, H1.190, H1.191, H1.192, H1.193, H1.194, H1.195, H1.196, H1.197, H1.198, H1.199, H1.200, H1.201, H1.202, H1.203, H1.204, H1.205, H1.206, H1.207, H1.208, H1.209, H1.210, H1.211, H1.212, H1.213, H1.214, H1.215, H1.216, H1.217, H1.218, H1.219, H1.220, H1.221, H1.222, H1.223, H1.224, H1.225, H1.226, H1.227, H1.228, H1.229, H1.230, H1.231, H1.232, H1.233, H1.234, H1.235, H1.236, H1.237, H1.238, H1.239, H1.240, H1.241, H1.242, H1.243, H1.244, H1.245, H1.246, H1.

57. The fusion protein of claim 56, wherein the fusion protein is selected from the group consisting of H1.01, H1.202, H1.203, H1.204, H1.205, H1.206, H1.207, H1.208, H1.209, H1.210, H1.211, H1.212, H1.213, H1.214, H1.215, H1.216, H1.217, H1.218, H1.219, H1.220, H1.221, H1.222, H1.223 and H1.

224.

58. 58. The fusion protein of claim 56 or 57, wherein the variant light domain is selected from the group consisting of L1.1, L1.3, L1.45, L1.117, L1.129, L1.135, L1.136 and L1.

140.

59. 59. The fusion protein of any one of claims 56 to 58, wherein the variant heavy domain comprises the amino acid sequence of SEQ ID NO: 318 and the variant light domain comprises the amino acid sequence of SEQ ID NO:

176.

60. 60. The fusion protein of any one of claims 56 to 59, wherein the first variant Fc domain comprises all or part of a hinge domain.

61. 61. The fusion protein of any one of claims 56 to 60, wherein the first monomer further comprises a second domain linker between the IL-15 domain and the first variant Fc domain.

62. 62. The fusion protein of any one of claims 56-61, wherein the first variant Fc domain and the second variant Fc domain comprise amino acid substitutions selected from the group consisting of: S364K / E357Q:L368D / K370S; L368D / K370S:S364K; L368E / K370S:S364K; T411E / K360E / Q362E:D401K; L368D / K370S:S364K / E357L,K370S:S364K / E357Q, T366S / L368A / Y407V:T366W and T366S / L368A / Y407V / Y349C:T366W / S354C (according to EU numbering).

63. 63. The fusion protein of claim 62, wherein the first variant Fc domain comprises L368D / K370S and the second variant Fc domain comprises S364K / E357Q, according to EU numbering.

64. 64. The fusion protein of any one of claims 56-63, wherein the first variant Fc domain and the second variant Fc domain each independently comprise an amino acid substitution selected from the group consisting of: G236R / L328R, E233P / L234V / L235A / G236del / S239K, E233P / L234V / L235A / G236del / S267K, E233P / L234V / L235A / G236del / S239K / A327G, E233P / L234V / L235A / G236del / S267K / A327G, and E233P / L234V / L235A / G236del, according to EU numbering.

65. 65. The fusion protein of claim 64, wherein the first variant Fc domain and the second variant Fc domain each comprise the amino acid substitutions E233P / L234V / L235A / G236del / S267K according to EU numbering.

66. 66. The fusion protein of any one of claims 56 to 65, wherein the first Fc domain comprises the amino acid substitutions Q295E / N384D / Q418E / N481D according to EU numbering.

67. 67. The fusion protein of any one of claims 56 to 66, wherein the first variant Fc domain and the second variant Fc domain each comprise the amino acid substitutions M428L / N434S according to EU numbering.

68. 1. A nucleic acid composition comprising: a) a first nucleic acid molecule encoding the first monomer according to any one of claims 56 to 67; b) a second nucleic acid molecule encoding the second monomer according to any one of claims 56 to 67; and / or c) a third nucleic acid molecule encoding the third monomer according to any one of claims 56 to 67; A nucleic acid composition comprising:

69. 1. An expression vector composition comprising: a) a first expression vector comprising the first nucleic acid molecule of claim 68; b) a second expression vector comprising the second nucleic acid molecule of claim 68; and / or c) a third expression vector comprising the third nucleic acid molecule of claim 68; An expression vector composition comprising:

70. 69. An expression vector comprising the first nucleic acid molecule of claim 68, the second nucleic acid molecule of claim 68, and the third nucleic acid molecule of claim 68.

71. 71. A host cell comprising the expression vector composition of claim 69 or the expression vector of claim 70.

72. 72. A method of making a fusion protein, comprising culturing the host cell of claim 71 under conditions in which the fusion protein is produced, and recovering the fusion protein.

73. 1. A targeted IL-15 / Rα heterodimeric Fc fusion protein comprising: a) a first monomer comprising, from N-terminus to C-terminus: i) IL-15 Rα sushi domain protein; ii) a first domain linker; iii) a variant IL-15 protein comprising an amino acid substitution selected from the group consisting of N71Q, N79Q, N112Q, S114del and S114A compared to SEQ ID NO: 2; and iv) a first variant Fc domain; b) a second monomer comprising a heavy chain comprising VH-CH1-hinge-CH2-CH3, wherein said CH2-CH3 is a second variant Fc domain; and c) a third monomer comprising a light chain comprising VL-CL; Including, A targeted IL-15 / Rα heterodimeric Fc fusion protein, wherein the VH domain and the VL domain form an antigen-binding domain that binds to human PD-1 and does not compete with nivolumab and / or pembrolizumab for binding to said human PD-1.

74. 74. The fusion protein of claim 73, wherein the variant IL-15 protein comprises amino acid substitutions selected from the group consisting of N71Q / N79Q, N71Q / N79Q / N112Q, N71Q / N79Q / S114del, and N71Q / N79Q / S114A.

75. The variant IL-15 protein is selected from the group consisting of N1D / N4D / D8N, N1D / N4D / N65D, N1D / D30N, N1D / D61N, N1D / D61N / E64Q / Q108E, N1D / E64Q, N1D / N65D, N1D / Q108E, N4D / D30N, N4D / D61N, N4D / D61N / N65D, N4D / D61N / E64Q / Q108E, N4D / E 75. The fusion protein of claim 73 or 74, comprising amino acid substitutions selected from the group consisting of 64Q, N4D / N65D, D8N / D61N, D8N / E64Q, D30N / E64Q, D30N / N65D, D30N / E64Q / N65D, D30N / Q180E, D61N / E64Q / N65D, E64Q / N65D, E64Q / Q108E and N65D / Q108E.

76. 76. The fusion protein of any one of claims 73 to 75, wherein the variant IL-15 protein comprises the amino acid substitutions N71Q / N79Q / N112Q.

77. 77. The fusion protein of any one of claims 73 to 76, wherein the variant IL-15 protein comprises the amino acid substitutions N71Q / N79Q / N112Q and D30N / N65D.

78. 78. The fusion protein of any one of claims 73 to 77, wherein the variant IL-15 protein comprises the amino acid substitutions N71Q / N79Q / N112Q and D30N / E64Q / N65D.

79. 79. The fusion protein of any one of claims 73 to 78, wherein the first domain linker is GGGGA (SEQ ID NO: 8).

80. 80. The fusion protein of any one of claims 73 to 79, wherein the first variant Fc domain comprises all or part of a hinge domain.

81. 81. The fusion protein of any one of claims 73 to 80, wherein the first monomer further comprises a second domain linker between the IL-15 domain and the first variant Fc domain.

82. 82. The fusion protein of any one of claims 73-81, wherein the first variant Fc domain and the second variant Fc domain comprise amino acid substitutions selected from the group consisting of: S364K / E357Q:L368D / K370S; L368D / K370S:S364K; L368E / K370S:S364K; T411E / K360E / Q362E:D401K; L368D / K370S:S364K / E357L,K370S:S364K / E357Q, T366S / L368A / Y407V:T366W and T366S / L368A / Y407V / Y349C:T366W / S354C (according to EU numbering).

83. 83. The fusion protein of claim 82, wherein the first variant Fc domain comprises L368D / K370S and the second variant Fc domain comprises S364K / E357Q, according to EU numbering.

84. 84. The fusion protein of any one of claims 73-83, wherein the first variant Fc domain and the second variant Fc domain each independently comprise an amino acid substitution 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.

85. 85. The fusion protein of claim 84, wherein the first variant Fc domain and the second variant Fc domain each comprise the amino acid substitutions E233P / L234V / L235A / G236del / S267K according to EU numbering.

86. 86. The fusion protein of any one of claims 73 to 85, wherein the first Fc domain comprises the amino acid substitutions Q295E / N384D / Q418E / N481D according to EU numbering.

87. 87. The fusion protein of any one of claims 73 to 86, wherein the first variant Fc domain and the second variant Fc domain each comprise the amino acid substitutions M428L / N434S according to EU numbering.

88. 1. A nucleic acid composition comprising: a) a first nucleic acid molecule encoding the first monomer according to any one of claims 73 to 87; b) a second nucleic acid molecule encoding the second monomer according to any one of claims 73 to 87; and / or c) A third nucleic acid molecule encoding the third monomer according to any one of claims 73 to 87. A nucleic acid composition comprising:

89. 1. An expression vector composition comprising: a) a first expression vector comprising the first nucleic acid molecule of claim 88; b) a second expression vector comprising the second nucleic acid molecule of claim 88; and / or c) a third expression vector comprising the third nucleic acid molecule of claim 88; An expression vector composition comprising:

90. 89. An expression vector comprising the first nucleic acid molecule of claim 88, the second nucleic acid molecule of claim 88, and the third nucleic acid molecule of claim 88.

91. 91. A host cell comprising the expression vector composition of claim 89 or the expression vector of claim 90.

92. 92. A method of making a fusion protein, comprising culturing the host cell of claim 91 under conditions in which the fusion protein is produced, and recovering the fusion protein.

93. A composition comprising an anti-PD-1 antigen binding domain (ABD), a) a variant variable heavy domain comprising a W100F amino acid substitution and at least one further amino acid substitution selected from the group consisting of F32L, S52aG, R97E, R97Y, R97W, L98R, S100aT, R97A, V99T, V99L, S100aA, L98Q, R97Q, V99F, V99L, S100aN, V99I, P100bS, G96H, L98V, V99A, V99Q, G96V, R97K, L98S, L98F, R97T, L98K, L98S, V99I, R97L, G96A, R97A, V99S, R97S, V99Y, R97H, L98R, according to Kabat numbering, compared to SEQ ID NO: 5; and b) a variable light domain, i) SEQ ID NO: 168; and ii) a variant light domain comprising an amino acid substitution selected from the group consisting of N27dH, N27dS, K30Y, S93T, and Y94W, Kabat numbering, compared to SEQ ID NO: 168; Including, A composition wherein the ABD binds to human PD-1.

94. 94. The composition of claim 93, wherein the variant heavy domain has the amino acid substitutions F32L / W100F according to Kabat numbering, and the variant light domain has the amino acid substitutions N27dH / K30Y / S93T according to Kabat numbering.

95. The variant heavy domain is selected from the group consisting of H1.176, H1.177, H1.178, H1.179, H1.180, H1.181, H1.182, H1.183, H1.184, H1.185, H1.186, H1.187, H1.188, H1.189, H1.190, H1.191, H1.192, H1.193, H1.194, H1.195, H1.196, H1.197, H1.198, H1.199, H1.200, H1.201, H1.202, H1.203, H1.204, H1.205, H1.206, H1.207, H1.208, H1.209, H1.210, H1.211, H1.212, H1.213, H1.214, H1.215, H1.216, H1.217, H1.218, H1.219, H1.220, H1.221, H1.222, H1.223, H1.224, H1.225, H1.226, H1.227, H1.228, H1.229, H1.230, H1.231, H1.232, H1.233, H1.234, H1.235, H1.236, H1.237, H1.238, H1.239, H1.240, H1.241, H1.242, H1.243, H1.244, H1.245, H1.246, H1.

95. The composition of claim 93 or 94, wherein the compound is selected from the group consisting of H1.01, H1.202, H1.203, H1.204, H1.205, H1.206, H1.207, H1.208, H1.209, H1.210, H1.211, H1.212, H1.213, H1.214, H1.215, H1.216, H1.217, H1.218, H1.219, H1.220, H1.221, H1.222, H1.223 and H1.

224.

96. 96. The composition of any one of claims 93-95, wherein the variant light domain is selected from the group consisting of L1.1, L1.3, L1.45, L1.117, L1.129, L1.135, L1.136 and L1.

140.

97. 97. The composition of any one of claims 93 to 96, wherein the variant heavy domain comprises the amino acid sequence of SEQ ID NO: 318 and the variant light domain comprises the amino acid sequence of SEQ ID NO:

176.

98. The composition of any one of claims 93 to 97, wherein the composition comprises a full-length anti-PD-1 antibody.

99. 99. The composition of any one of claims 93 to 98, wherein the composition comprises a fusion protein.

100. 100. The composition of claim 99, wherein the fusion protein is XENP32435.

101. 1. A nucleic acid composition comprising: a) a first nucleic acid molecule encoding the variable light domain of any one of claims 93 to 100; and / or b) a second nucleic acid molecule encoding the variable heavy domain of any one of claims 93 to 100. A nucleic acid composition comprising:

102. 1. An expression vector composition comprising: a) a first expression vector comprising the first nucleic acid molecule of claim 101; and / or b) a second expression vector comprising the second nucleic acid molecule of claim 101. An expression vector composition comprising:

103. 102. An expression vector comprising the first nucleic acid molecule of claim 101 and the second nucleic acid molecule of claim 101.

104. 104. A host cell comprising the expression vector composition of claim 102 or the expression vector of claim 103.

105. 105. A method of making a composition comprising an anti-PD-1 antigen binding domain (ABD), comprising culturing the host cell of claim 104 under conditions such that the fusion protein is produced, and recovering the fusion protein.

106. 1. A composition comprising a variant IL-15 protein compared to SEQ ID NO:2, wherein the variant IL-15 protein comprises an amino acid substitution selected from the group consisting of N71Q, N79Q, N112Q, S114del, and S114A.

107. 107. The composition of claim 106, wherein the variant IL-15 protein further comprises an amino acid substitution selected from the group consisting of N1D, N4D, D8N, D30N, D61N, E64Q, N65D, and Q108E.

108. 108. The composition of claim 106 or 107, wherein the variant IL-15 protein comprises amino acid substitutions selected from the group consisting of N71Q / N79Q, N71Q / N79Q / N112Q, N71Q / N79Q / S114del and N71Q / N79Q / S114A.

109. The variant IL-15 protein is selected from the group consisting of N1D / N4D / D8N, N1D / N4D / N65D, N1D / D30N, N1D / D61N, N1D / D61N / E64Q / Q108E, N1D / E64Q, N1D / N65D, N1D / Q108E, N4D / D30N, N4D / D61N, N4D / D61N / N65D, N4D / D61N / E64Q / Q108E, N4D / E64 109. The composition of any one of claims 106 to 108, comprising amino acid substitutions selected from the group consisting of Q, N4D / N65D, D8N / D61N, D8N / E64Q, D30N / E64Q, D30N / N65D, D30N / E64Q / N65D, D30N / Q180E, D61N / E64Q / N65D, E64Q / N65D, E64Q / Q108E and N65D / Q108E.

110. 110. The composition of any one of claims 106 to 109, wherein the variant IL-15 protein comprises the amino acid substitutions N71Q / N79Q / N112Q.

111. 111. The composition of any one of claims 106 to 110, wherein the variant IL-15 protein comprises the amino acid substitutions N71Q / N79Q / N112Q and D30N / N65D.

112. 112. The composition of any one of claims 106 to 111, wherein the variant IL-15 protein comprises the amino acid substitutions N71Q / N79Q / N112Q and D30N / E64Q / N65D.

113. 113. A nucleic acid composition comprising a nucleic acid molecule encoding the variant IL-15 protein of any one of claims 106 to 112.

114. 114. An expression vector comprising the nucleic acid molecule of claim 113.

115. A host cell comprising the expression vector of claim 114.

116. 116. A method of making a composition comprising a variant IL-15 protein, the method comprising culturing the host cell of claim 115 under conditions in which said fusion protein is produced, and recovering said fusion protein.

117. A heterodimeric protein, a) a first fusion protein comprising: i) a variant IL-15 protein comprising the amino acid substitutions D30N / E64Q / N65D and N71Q / N79Q / N112Q compared to SEQ ID NO: 2; ii) a domain linker; and iii) a first variant Fc domain; and b) a second fusion protein comprising: i) IL-15Rα sushi domain; ii) a domain linker; and iii) a second variant Fc domain; A heterodimeric protein comprising:

118. 118. The heterodimeric protein of claim 117, wherein said variant IL-15 protein comprises the amino acid sequence of SEQ ID NO:

319.

119. 119. The heterodimeric protein of claim 117 or 118, wherein the first variant Fc domain and the second variant Fc domain comprise amino acid substitutions selected from the group consisting of: S364K / E357Q:L368D / K370S; L368D / K370S:S364K; L368E / K370S:S364K; T411E / K360E / Q362E:D401K; L368D / K370S:S364K / E357L,K370S:S364K / E357Q, T366S / L368A / Y407V:T366W and T366S / L368A / Y407V / Y349C:T366W / S354C (according to EU numbering).

120. 120. The heterodimeric protein of claim 119, wherein the first variant Fc domain comprises L368D / K370S and the second variant Fc domain comprises S364K / E357Q, according to EU numbering.

121. 121. The fusion protein of any one of claims 117 to 120, wherein the first variant Fc domain and the second variant Fc domain comprise 428L / 434S.

122. The fusion protein of claim 117, wherein the first fusion protein comprises the amino acid sequence of SEQ ID NO: 208 and the second fusion protein comprises the amino acid sequence of SEQ ID NO:

95.

123. The fusion protein of claim 117, wherein the first fusion protein comprises the amino acid sequence of SEQ ID NO: 211 and the second fusion protein comprises the amino acid sequence of SEQ ID NO:

206.

124. A pharmaceutical composition comprising the fusion protein of any one of claims 1 to 24, 30 to 50, 56 to 67, and 73 to 87, and a pharmaceutically acceptable carrier.

125. A pharmaceutical composition comprising a composition comprising the anti-PD-1 antigen binding domain (ABD) of any one of claims 93 to 100, and a pharmaceutically acceptable carrier.

126. 113. A pharmaceutical composition comprising a composition comprising the variant IL-15 protein of any one of claims 106 to 112 and a pharmaceutically acceptable carrier.

127. A pharmaceutical composition comprising the heterodimeric protein of any one of claims 117 to 123 and a pharmaceutically acceptable carrier.

128. 125. A method of treating cancer in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of the fusion protein of any one of claims 1 to 24, 30 to 50, 56 to 67, and 73 to 87, or the pharmaceutical composition of claim 124.

129. 129. The method of claim 128, further comprising administering a therapeutically effective amount of a checkpoint blockade antibody.

130. 130. The method of claim 129, wherein the checkpoint blockade antibody is an anti-PD-1 antibody or an anti-PD-L1 antibody.

131. 131. The method of claim 130, wherein the checkpoint blockade antibody is nivolumab or pembrolizumab.

132. 126. A method of treating cancer in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of a composition comprising the anti-PD-1 antigen binding domain (ABD) of any one of claims 93 to 100 or the pharmaceutical composition of claim 125.

133. 133. The method of claim 132, further comprising administering a therapeutically effective amount of a checkpoint blockade antibody.

134. 134. The method of claim 133, wherein the checkpoint blockade antibody is an anti-PD-1 antibody or an anti-PD-L1 antibody.

135. 135. The method of claim 134, wherein the checkpoint blockade antibody is nivolumab or pembrolizumab.

136. 127. A method of treating cancer in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of a composition comprising the variant IL-15 protein of any one of claims 106 to 112 or the pharmaceutical composition of claim 126.

137. 137. The method of claim 136, further comprising administering a therapeutically effective amount of a checkpoint blockade antibody.

138. 138. The method of claim 137, wherein the checkpoint blockade antibody is an anti-PD-1 antibody or an anti-PD-L1 antibody.

139. 139. The method of claim 138, wherein the checkpoint blockade antibody is nivolumab or pembrolizumab.

140. A method for treating cancer in a subject, comprising administering a therapeutically effective amount of the heterodimeric protein of any one of claims 117 to 123 or the pharmaceutical composition of claim 127 to a subject in need of cancer treatment.

141. 141. The method of claim 140, further comprising administering a therapeutically effective amount of a checkpoint blockade antibody.

142. 142. The method of claim 141, wherein the checkpoint blockade antibody is an anti-PD-1 antibody or an anti-PD-L1 antibody.

143. 143. The method of claim 142, wherein the checkpoint blockade antibody is nivolumab or pembrolizumab.

144. 126. Use of the fusion protein of any one of claims 1 to 24, 30 to 50, 56 to 67 and 73 to 87 or the pharmaceutical composition of claim 124 in the manufacture of a medicament for the treatment of cancer in a subject in need thereof.

145. 145. The use of claim 144, wherein the medicament is formulated to be administered in combination with a therapeutically effective amount of a checkpoint blockade antibody.

146. The use of claim 145, wherein the checkpoint blockade antibody is an anti-PD-1 antibody or an anti-PD-L1 antibody.

147. 147. The use of claim 146, wherein the checkpoint blockade antibody is nivolumab or pembrolizumab.

148. 126. Use of a composition comprising the anti-PD-1 antigen binding domain (ABD) of any one of claims 93 to 100 or the pharmaceutical composition of claim 124 in the manufacture of a medicament for the treatment of cancer in a subject in need thereof.

149. 149. The use of claim 148, wherein the medicament is formulated to be administered in combination with a therapeutically effective amount of a checkpoint blockade antibody.

150. The use of claim 149, wherein the checkpoint blockade antibody is an anti-PD-1 antibody or an anti-PD-L1 antibody.

151. 151. The use of claim 150, wherein the checkpoint blockade antibody is nivolumab or pembrolizumab.

152. 127. Use of a composition comprising a variant IL-15 protein according to any one of claims 106 to 112 or a pharmaceutical composition according to claim 126 in the manufacture of a medicament for the treatment of cancer in a subject in need thereof.

153. 153. The use of claim 152, wherein the medicament is formulated to be administered in combination with a therapeutically effective amount of a checkpoint blockade antibody.

154. The use of claim 153, wherein the checkpoint blockade antibody is an anti-PD-1 antibody or an anti-PD-L1 antibody.

155. 155. The use of claim 154, wherein the checkpoint blockade antibody is nivolumab or pembrolizumab.

156. 128. Use of the heterodimeric protein of any one of claims 117 to 123 or the pharmaceutical composition of claim 127 in the manufacture of a medicament for the treatment of cancer in a subject in need thereof.

157. 157. The use of claim 156, wherein the medicament is formulated to be administered in combination with a therapeutically effective amount of a checkpoint blockade antibody.

158. The use of claim 157, wherein the checkpoint blockade antibody is an anti-PD-1 antibody or an anti-PD-L1 antibody.

159. 159. The use of claim 158, wherein the checkpoint blockade antibody is nivolumab or pembrolizumab.

160. 125. The fusion protein of any one of claims 1 to 24, 30 to 50, 56 to 67 and 73 to 87 or the pharmaceutical composition of claim 124 for use in the treatment of cancer in a subject in need thereof.

161. 161. The fusion protein or pharmaceutical composition for use of claim 160, administered in combination with a therapeutically effective amount of a checkpoint blockade antibody.

162. 162. The fusion protein or pharmaceutical composition for use of claim 161, wherein said checkpoint blockade antibody is an anti-PD-1 antibody or an anti-PD-L1 antibody.

163. 163. The fusion protein or pharmaceutical composition for use of claim 162, wherein said checkpoint blockade antibody is nivolumab or pembrolizumab.

164. 126. A composition comprising the anti-PD-1 antigen binding domain (ABD) of any one of claims 93 to 100 or the pharmaceutical composition of claim 125 for use in treating cancer in a subject in need thereof.

165. 165. A composition or pharmaceutical composition comprising an anti-PD-1 ABD for use according to claim 164, administered in combination with a therapeutically effective amount of a checkpoint blockade antibody.

166. 166. The composition or pharmaceutical composition comprising an anti-PD-1 ABD for use according to claim 165, wherein said checkpoint blockade antibody is an anti-PD-1 antibody or an anti-PD-L1 antibody.

167. 167. The composition or pharmaceutical composition comprising an anti-PD-1 ABD for use according to claim 166, wherein said checkpoint blockade antibody is nivolumab or pembrolizumab.

168. 127. A composition comprising a variant IL-15 protein according to any one of claims 106 to 112 or a pharmaceutical composition according to claim 126 for use in the treatment of cancer in a subject in need thereof.

169. 169. A composition or pharmaceutical composition comprising a variant IL-15 protein for use according to claim 168, administered in combination with a therapeutically effective amount of a checkpoint blockade antibody.

170. 170. The composition or pharmaceutical composition comprising a variant IL-15 protein for use according to claim 169, wherein said checkpoint blockade antibody is an anti-PD-1 antibody or an anti-PD-L1 antibody.

171. 171. The composition or pharmaceutical composition comprising a variant IL-15 protein for use according to claim 170, wherein said checkpoint blockade antibody is nivolumab or pembrolizumab.

172. 127. The heterodimeric protein of any one of claims 117 to 123 or the pharmaceutical composition of claim 126 for use in the treatment of cancer in a subject in need thereof.

173. 173. The heterodimeric protein or pharmaceutical composition for use according to claim 172, administered in combination with a therapeutically effective amount of a checkpoint blockade antibody.

174. The heterodimeric protein or pharmaceutical composition for use according to claim 173, wherein said checkpoint blockade antibody is an anti-PD-1 antibody or an anti-PD-L1 antibody.

175. 175. The heterodimeric protein or pharmaceutical composition for use according to claim 174, wherein said checkpoint blockade antibody is nivolumab or pembrolizumab.

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