Il-15 variants and uses thereof

TWI776024BInactive Publication Date: 2022-09-01PFIZER INC
View PDF 0 Cites 0 Cited by

Patent Information

Application Number
TW108106585
Authority / Receiving Office
TW · TW
Patent Type
Patents
Current Assignee / Owner
Priority Date
2018-12-21
Filing Date
2019-02-26
Publication Date
2022-09-01
Estimated Expiration
Not applicable · inactive patent

Smart Images

  • Figure TWG2TB001665887_001
    Figure TWG2TB001665887_001
  • Figure TWG2TB001665887_002
    Figure TWG2TB001665887_002
  • Figure TWG2TB001665887_003
    Figure TWG2TB001665887_003
Patent Text Reader

Abstract

This invention relates to human interleukin-15 (IL-15) variants with therapeutic and diagnostic uses, and methods for manufacturing the same. The invention also provides fusion proteins comprising human IL-15 variants. Methods for stimulating or inhibiting immune responses in mammals and methods for treating conditions (e.g., cancer) using IL-15 variants or fusion proteins of such IL-15 variants are also provided.
Need to check novelty before this filing date? Find Prior Art

Description

[Technical Field] This invention relates to interleukin-15 (IL-15) variants with therapeutic and diagnostic uses, and methods for manufacturing the same. The invention also provides fusion proteins comprising such IL-15 variants. Methods for stimulating or inhibiting immune responses in mammals and methods for treating conditions (e.g., cancer) using IL-15 variants or fusion proteins of such IL-15 variants are also provided. [Previous Technology] Cytokines are potent modulators of the immune response and remain a significant potential influence on the outcomes of immuno-oncology treatments. However, previous efforts to utilize cytokines in human individuals have yielded only modest efficacy and significant toxicity. Recent studies have demonstrated that "targeting cytokines," such as antibody-cytokine fusion proteins, can deliver cytokines to the desired cell types while minimizing peripheral exposure and thus toxicity. See, for example, Guo et al., Cytokine Growth Factor Rev. 38:10-21 (2017); Jakobisiak M et al., Cytokine Growth Factor Rev. 22(2):99-108 (2011); Robinson, T. & Schluns, KS, Immunol. Lett. 190:159-168 (2017); Rhode et al., Cancer Immunol. Res. 4(1): 49-60 (2016); Conlon et al., J Clin. Oncol. 33(1): 74-82 (2015). Therefore, the development of therapeutics based on targeted cytokines will be of great value in the treatment of various diseases such as cancer. [Summary of the Invention] The present invention disclosed herein relates to human interleukin-15 (IL-15) variants and fusion proteins comprising them. It has been demonstrated that, compared to wild-type human IL-15 peptides or wild-type IL-15 receptor α-IL-15 fusion peptides, the IL-15 variants of the present invention exhibit reduced or no binding to IL-15 receptor α (CD215), and / or reduced interaction between IL-15 and its signal transduction receptors (composed of IL-2 receptor β (CD122) and a common γ chain (CD132)). In a second embodiment of the present invention, these reduced-affinity IL-15 variants (when presented as antibody fusion chimeric proteins) selectively target desired cell types (cells expressing antibody targets). Cell types expressing the IL-15 receptor complex (rather than antibody targets) are less activated or unactivated compared to cells expressing both components. Therefore, the IL-15 variants and IL-15 fusion proteins of the present invention selectively modulate the activation of cell subsets to effectively and safely promote biological activities, such as antitumor activity. In the third embodiment of the present invention, these reduced-affinity IL-15 variants and IL-15 fusion proteins (in secretory or membrane-bound form when represented as polynucleotides in CAR (chimeric antigen receptor) T cells) are used to enhance CAR T function, including activity and proliferation. Therefore, in one embodiment, the present invention provides an isolated human interleukin-15 (IL-15) variant comprising amino acid substitutions at positions a) V49 and I51 or b) V49, I50 and S51 of SEQ ID NO: 1, and further comprising one or more amino acid substitutions at positions N1, N4, S7, K10, K11, Y26, S29, D30, V31, H32, E53, G55, E64, I68, L69, E89, L91, M109 and / or I111 of SEQ ID NO: 1, wherein, compared to wild-type human IL-15 polypeptide or wild-type IL-15 receptor α-IL-15 fusion polypeptide, the IL-15 variant has substitutions for human IL-15 receptor α (IL-15Rα) and human IL-2 receptor β / γ. The binding affinity of (IL-2Rβγ) is reduced or absent, and the amino acid substitution at position V49 is glycosylated. In another embodiment, a separated fusion protein is provided, comprising: 1) an antibody containing an Fc domain; and b) a human interleukin-15 (IL-15) variant comprising amino acid substitutions at positions a) V49 and I51 or b) V49, I50 and S51 of SEQ ID NO: 1, wherein the amino acid substitution at position V49 of SEQ ID NO: 1 is glycosylated, and further comprises one or more amino acid substitutions at positions N1, N4, S7, K10, K11, Y26, S29, D30, V31, H32, E53, G55, E64, I68, L69, E89, L91, M109 and / or I111 of SEQ ID NO: 1, wherein the IL-15 variant is covalently linked to the Fc domain of the antibody, and wherein the antibody-dependent cytotoxicity (ADCC) activity of the Fc domain is reduced or absent compared to wild-type Fc. In some embodiments, the IL-15 variant comprises an amino acid substitution at V49N, wherein V49N is glycosylated. In some embodiments, the amino acid substitutions at E53 and / or E89 of SEQ ID NO: 1 are also glycosylated. In some embodiments, the IL-15 variant comprises an amino acid substitution at the position of SEQ ID NO: 1 selected from the group consisting of: a) V49, I50, S51, N4, D30, and E64; b) V49, I50, S51, N4, D30, E64, and I68; c) V49, I50, S51, N4, D30, E64, and M109; d) V49, I50, S51, N4, D30, E64, I68, and M109; e) V49, I50, S51, D30, E64, and I68; f) V49, I50, S51, D30, E64, and M109; g) V49, I50, S51, D30, E64, I68, and M109; h) N1, V49, I50 and S51; i) N4, V49, I50 and S51; j) S7, V49, I50 and S51; k) K10, V49, I50 and S51; l) K11, V49, I50 and S51; m) S29, V49, I50 and S51; n) V31, V49, I50 and S51; o) H32, V49, I50 and S51; p) V49, I50, S51 and E64; q) V49, I50, S51 and I68; r) V49, I50, S51 and L69; s) V49, I50, S51 and I111; t) N4, V49, I50, S51 and E64; u) N1, D30, V49, I50 and S51; v) N4, D30, V49, I50 and S51; w) S7 D30, V49, I50 and S51; x) K10, D30, V49, I50 and S51; y) K11, D30, V49, I50 and S51; z) S29, D30, V49, I50 and S51; aa) D30, V49, I50, S51 and E64; bb) D30, V49, I50, S51 and I68; cc) D30, V49, I50, S51 and L69; and dd) D30, V49, I50, S51 and I111. In some embodiments, the IL-15 variant comprises amino acid substitutions, such amino acid substitutions comprising one or more specific substitutions at the following positions: a) V49N, V49K, V49E, V49H, V49Q, or V49R; b) I50A or I50G; c) S51T; d) N1K, N1G, N1Q, N1R, N1E, N1A, or N1D; e) N4K, N4G, N4A, N4S, N4D, N4E, N4L, N4R, N4T, or N4Q; f) S7E, S7G, S7D, S7K, S7N, S7R, S7H, or S7T; g) K10A, K10S, K10E, K10L, K10M, K10D, or K10G; h) K11D, K11S, or K11W; i) D30N; j) E64Q, E64K, E64A, E64S, E64N, E64H, E64T, or E64R; k) E53N; l) G55S or G55T; m) E89N; n) L91S or L91T; o) Y26K, Y26R, or Y26H; p) S29N; q) V31S, V31D, or V31K; r) H32G; s) I68S, I68A, I68R, I68T, I68K, I68N, I68M, I68F, I68Y, I68E, or I68H; t) L69A, L69S, L69D, L69T, L69M, L69G, L69Q, L69I, L69E, or L69V; u) M109A, M109S, M109D or M109K; and / or v) I111A, I111K, I111S or I111D. In some embodiments, the IL-15 variant comprises amino acid substitutions selected from the group consisting of: a) N4K, D30N, V49N, I50A, S51T, and E64Q; b) N4Q, D30N, V49N, I50A, and S51T; c) D30N, V49N, I50A, S51T, and E64Q; d) N4Q, D30N, V49N, I50A, S51T, and E64Q; e) N4Q, V49N, I50A, and S51T; f) V49N, I50A, S51T, and E64Q; and g) N4Q, V49N, I50A, S51T, and E64Q. In another embodiment, an isolated human interleukin-15 (IL-15) variant is provided, comprising amino acid substitutions at positions E46 and V49 of SEQ ID NO: 1 and at least one or more amino acid substitutions at positions N1, N4, S7, K10, K11, D22, Y26, S29, D30, V31, H32, E53, G55, E64, I68, L69, E89, E93, M109 and / or I111 of SEQ ID NO: 1, wherein the IL-15 variant has no binding affinity to human IL-15 receptor α (IL-15Rα) and reduced binding affinity to human IL-2 receptor β / γ (IL-2Rβγ) compared to wild-type human IL-15 polypeptide or wild-type IL-15 receptor α-IL-15 fusion polypeptide. In another embodiment, a separated fusion protein is provided, comprising: 1) an antibody containing an Fc domain; and b) a human interleukin-15 (IL-15) variant comprising amino acid substitutions at positions E46 and V49 of SEQ ID NO: 1 and at least one or more amino acid substitutions at positions N1, N4, S7, K10, K11, D22, Y26, S29, D30, V31, H32, E53, G55, E64, I68, L69, E89, E93, M109 and / or I111 of SEQ ID NO: 1, wherein the IL-15 variant is covalently linked to the Fc domain of the antibody, and wherein the antibody-dependent cytotoxicity (ADCC) activity of the Fc domain is reduced or absent compared to wild-type Fc. In some embodiments, the IL-15 variant comprises an amino acid substitution at the position selected from the group consisting of: a) N1, E46, and V49; b) N4, E46, and V49; c) S7, E46, and V49; d) K10, E46, and V49; e) K11, E46, and V49; f) S29, E46, and V49; g) V31, E46, and V49; h) H32, E46, and V49; i) E46, V49, and E64; j) E46, V49, and I68; k) E46, V49, and L69; l) E46, V49, and I111; m) N4, E46, V49, and E64; n) E46, V49, N4, D30, and E64; o) E46, V49, N4, D30, E64 and I68; p) E46, V49, N4, D30, E64 and M109; q) E46, V49, N4, D30, E64, I68 and M109; r) N1, D30, E46 and V49; s) N4, D30, E46 and V49; t) S7, D30, E46 and V49; u) K10, D30, E46 and V49; v) K11, D30, E46 and V49; w) S29, D30, E46 and V49; x) D30, E46, V49 and E64; y) D30, E46, V49R and I68; z) D30, E46, V49R and L69; aa) D30, E46, V49R and I111; bb) N1, D30, E46, V49 and M109; cc) N4, D30, E46, V49 and M109; dd) S7, D30, E46, V49 and M109; ee) K10, D30, E46, V49 and M109; ff) K11, D30, E46, V49 and M109; gg) D30, E46, V49, E64 and M109; hh) D30, E46, V49, I68 and M109; ii) D30, E46, V49, L69 and M109; jj) D30, E46, V49, M109 and I111; kk) D30, E46, V49, E64, I68 and M109; ll) E46, V49, D30, E64 and I68; mm) E46, V49, E64 and M109; nn) E46, V49, D30, E64, I68 and M109; oo) D22, Y26, V49, E46, E53, E89 and E93; and pp) N1, D30, E46, V49 and E64. In some embodiments, the IL-15 variant comprises amino acid substitutions, which include one or more specific substitutions at the following positions: a) N1Q, N1K, N1R, N1E, N1A, N1D, or N1G; b) N4K, N4G, N4A, N4S, N4D, N4E, N4R, N4T, N4I, N4L, N4W, or N4Q; c) S7E, S7G, S7D, S7K, S7N, S7R, S7H, or S7T; d) K10D, K10A, K10S, K10E, K10L, K10M, K10D, or K10G; e) K11D, K11S, or K11W; f) D22N; g) Y26K, Y26R, or Y26H; h) S29N; i) D30N; j) V31S, V31D, or V31K; k) H32G; l) E46G or E46Q; m) V49N, V49K, or V49R, V49E, V49H, or V49Q; n) E53Q; o) G55S or G55T; p) E64Q, E64K, E64A, E64S, E64N, E64H, E64T, or E64R; q) I68S, I68A, I68R, I68T, I68K, I68N, I68M, I68F, I68Y, I68E, or I68H; r) L69S, L69A, L69D, L69T, L69M, L69G, L69Q, L69I, L69E, or L69V; s) E89Q; t) E93Q; u) M109A, M109S, M109D or M109K; and / or v) I111A, I111K, I111S or I111D.In some embodiments, the IL-15 variant comprises amino acid substitutions selected from the group consisting of: a) N1K, E46G, and V49R; b) N4K, E46G, and V49R; c) N4Q, E46G, and V49R; d) S7T, E46G, and V49R; e) V31S, E46G, and V49R; f) V31K, E46G, and V49R; g) E46G, V49R, and E64Q; h) E46G, V49R, and E64K; i) N4Q, E46G, V49R, and E64Q; j) N1G, D30N, E46G, and V49R; k) N1K, D30N, E46G, and V49R; l) N1Q, D30N, E46G, and V49R; m) N4G, D30N, E46G and V49R; n) N4K, D30N, E46G and V49R; o) N4Q, D30N, E46G and V49R; p) S7E, D30N, E46G and V49R; q) S7G, D30N, E46G and V49R; r) S7T, D30N, E46G and V49R; s) K10D, D30N, E46G and V49R; t) D30N, E46G, V49R and E64A; u) D30N, E46G, V49R and E64Q; v) D30N, E46G, V49R and E64K; w) D30N, E46G, V49R and I68S; x) D30N, E46G, V49R and I68K; y) N4K, D30N, E46G, V49R and E64K; z) N4Q, D30N, E46G, V49R and E64K; aa) N4K, D30N, E46G, V49R and E64Q; bb) N4Q, D30N, E46G, V49R and E64Q; cc) N4K, D30N, E46G, V49R and I68S; dd) D30N, E46G, V49R, E64Q and I68S; ee) N1A, D30N, E46G and V49R; and ff) N1G, D30N, E46G, V49R and E64Q. In another embodiment, an isolated human IL-15 variant is provided, comprising one or more amino acid substitutions at positions N1, N4, S7, K10, K11, D22, Y26, S29, D30, V31, H32, E46, E53, E64, I68, L69, E89, E93, M109 and / or I111 of SEQ ID NO: 1, wherein the IL-15 variant has reduced or no binding to human IL-15 receptor α (IL-15Rα) and / or human IL-2 receptor β (IL-2Rβ) and / or IL-2 receptor γ (IL-2Rγ) compared to wild-type human IL-15 polypeptide or wild-type IL-15 receptor α-IL-15 fusion polypeptide. In another embodiment, an isolated human IL-15 variant is provided, comprising the amino acid sequence shown in SEQ ID NO:93. In some embodiments, the IL-15 variant further comprises a transmembrane domain. In another embodiment, an isolated fusion protein is provided comprising: 1) an antibody containing an Fc domain; and b) a human interleukin-15 (IL-15) variant comprising one or more amino acid substitutions at positions N1, N4, S7, K10, K11, D22, Y26, S29, D30, V31, H32, E46, E53, E64, I68, L69, E89, E93, M109 and / or I111 of SEQ ID NO: 1, wherein the IL-15 variant is covalently linked to the Fc domain of the antibody, and wherein the antibody-dependent cytotoxicity (ADCC) activity of the Fc domain is reduced or absent compared to wild-type Fc. In another embodiment, a separated fusion protein is provided, comprising: 1) an IL-15 antibody containing an Fc domain; and b) a human interleukin-15 (IL-15) protein of SEQ ID NO: 1, wherein IL-15 is covalently linked to the Fc domain of the antibody. In some embodiments, the antibody in the IL-15 fusion protein of the present invention may be a human antibody, a humanized antibody, a chimeric antibody, or a bispecific antibody. In some embodiments, the antibody in the IL-15 fusion protein of the present invention has subclasses of human IgG1, IgG2, IgG2Δa, IgG3, IgG4, IgG4Δb, IgG4Δc, IgG4S228P, IgG4ΔbS228P and IgG4ΔcS228P. In some embodiments, the antibody of the IL-15 fusion protein of the present invention is a) IgG2, and an antibody variable domain modified with amino acids at positions 223, 225 and 228 in the hinge region of human IgG2 (SEQ ID NO: 3) and at positions 409 or 368 (EU numbering scheme) in the CH3 region; b) IgG1, and an antibody variable domain modified with amino acids at positions 221 and 228 in the hinge region of human IgG1 (SEQ ID NO: 2) and at positions 409 or 368 (EU numbering scheme) in the CH3 region; or c) IgG1, and an antibody variable domain modified with amino acids at positions 349, 354, 366, 368 and / or 407 (EU numbering scheme) in the CH3 region of human IgG1 (SEQ ID NO: 2). In some embodiments, the antibody further comprises amino acid modifications at the following locations: a) one or more locations 265, 330, and / or 331 of human IgG2 (SEQ ID NO: 3); or b) one or more locations 234, 235, 237, and / or 322 of human IgG1 (SEQ ID NO: 2). In some embodiments, the antibody further comprises amino acid modifications at one or more locations 234, 235, 237, 349, 354, 366, 368, and / or 407 of human IgG1 (SEQ ID NO: 74 and 75). In some embodiments, the antibody against the IL-15 fusion protein is selected from the group consisting of: anti-CTLA-4 antibody, anti-CD3 antibody, anti-CD4 antibody, anti-CD8 antibody, anti-4-1BB antibody, anti-PD-1 antibody, anti-PD-L1 antibody, anti-TIM3 antibody, anti-LAG3 antibody, anti-TIGIT antibody, anti-OX40 antibody, anti-IL-7Rα (CD127) antibody, anti-IL-8 antibody, anti-IL-15 antibody, anti-HVEM antibody, anti-BTLA antibody, anti-CD40 antibody, anti-CD40L antibody, anti-CD47 antibody, anti-CSF1R antibody, anti-CSF1 antibody, anti-MARCO antibody, anti-CXCR4 antibody, anti-VEGF antibody, anti-VEGFR1 antibody, anti-VEGFR2 antibody, anti-TNFR1 antibody, anti-TNFR2 antibody, anti-CD3 bispecific antibody, anti-CD19 antibody, anti-CD20, anti-Her2 antibody, anti-EGFR antibody, anti-ICOS antibody, anti-CD22 antibody, anti-CD Antibody 52, anti-CCR4 antibody, anti-CCR8 antibody, anti-CD200R antibody, anti-VISG4 antibody, anti-CCR2 antibody, anti-LILRb2 antibody, anti-CXCR4 antibody, anti-CD206 antibody, anti-CD163 antibody, anti-KLRG1 antibody, anti-FLT3 antibody, anti-B7-H4 antibody, anti-B7-H3 antibody, KLRG1 antibody and anti-GITR antibody. In some embodiments, the IL-15 fusion protein of the present invention is covalently linked to an antibody via a peptide linker and / or a peptide marker. In another embodiment, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of an IL-15 variant or IL-15 fusion protein as described herein and a pharmaceutically acceptable carrier. In another embodiment, the present invention provides a separated polynucleotide comprising a nucleotide sequence encoding an IL-15 variant or IL-15 fusion protein as described herein. In yet another embodiment, the present invention provides a carrier comprising the polynucleotide. In another embodiment, the present invention provides isolated host cells or cell lines that recombinantly produce IL-15 variants or IL-15 fusion proteins as described herein. In some embodiments, the host cells or cell lines are engineered immune cells, wherein the engineered immune cells comprise chimeric antigen receptors (CARs). In some embodiments, the CAR-expressing cells are T cells, and the T cells express IL-15 variants or IL-15 fusion proteins as described herein, either in a secretory or membrane-bound form. In another embodiment, the present invention provides a method for producing an IL-15 variant or an IL-15 fusion protein, the method comprising: culturing a cell line that recombinantly produces an IL-15 variant or an IL-15 fusion protein as described herein under conditions in which the protein variant or fusion protein is produced; and recovering the protein variant or fusion protein. In another embodiment, the present invention provides a method for treating a condition in an individual, comprising administering to the individual in need an effective amount of a pharmaceutical composition as described herein. In some embodiments, the condition is cancer. In some embodiments, the cancer is liquid cancer or solid cancer. In some embodiments, the cancer is recurrent, refractory, or metastatic. In another embodiment, the present invention provides a method for inhibiting tumor growth or progression in an individual suffering from a tumor, comprising administering to the individual an effective amount of a pharmaceutical composition as described herein. In another embodiment, the present invention provides a method for inhibiting or preventing cancer metastasis of cancer cells in an individual, comprising administering to an individual in need an effective amount of a pharmaceutical composition as described herein. In another embodiment, the present invention provides a method for inducing tumor regression in an individual in need, comprising administering to the individual an effective amount of a pharmaceutical composition as described herein. In some embodiments, the IL-15 variants and IL-15 fusion proteins described herein may be administered non-enterally to individuals. In some embodiments, the individuals are humans. In some embodiments, the method may further include administering an effective amount of a second therapeutic agent. In some embodiments, the second therapeutic agent is a biological therapeutic agent. In some embodiments, the second therapeutic agent is a cytokine, an immune cytokine (e.g., an anti-EDA-IL10 fusion protein), TNFα, a PAP inhibitor, an oncolytic virus, a kinase inhibitor, an ALK inhibitor (e.g., sunitinib or crizotinib), a MEK inhibitor, an IDO inhibitor, a GLS1 inhibitor, a tyrosine kinase inhibitor (e.g., axitinib or palbociclib), a CAR (chimeric antigen receptor)-T cell or T cell therapy, a PRR (pattern recognition receptor) agonist (such as a TLR (Toll-like receptor) agonist (e.g., TLR3, TLR4, TLR5, TLR7, TLR9)), or a tumor vaccine. This document also provides the use of any of the IL-15 variants or IL-15 fusion proteins described herein for the manufacture of medicaments for the treatment of cancer or for inhibiting tumor growth or progression in individuals in need. In one embodiment, the present invention provides a method for treating cancer in an individual, comprising administering to the individual a combination therapy comprising a first therapeutic agent and a second therapeutic agent, wherein the first therapeutic agent is an IL-15 variant or an IL-15 fusion protein. In some embodiments, the first therapeutic agent is an IL-15 variant. In some embodiments, the first therapeutic agent is an IL-15 fusion protein. In some embodiments, the first therapeutic agent is any IL-15 variant or IL-15 fusion protein of the present invention. In some embodiments, the first therapeutic agent comprises the amino acid sequence shown in SEQ ID NO: 84, 85, 86, 87, 89 or 90. In some embodiments, the second therapeutic agent is an immunocytokine. In some embodiments, the second therapeutic agent is an immunocytokine comprising an antibody or a fragment thereof conjugated or fused to a cytokine (e.g., a fusion protein). In some embodiments, the antibody or a fragment thereof binds to an additional region-A (EDA) isoform of fibronectin (e.g., an anti-EDA antibody). In some embodiments, the fragment of the anti-EDA antibody comprises CDR1, CDR2, and CDR3 of the heavy chain variable (VH) region shown in SEQ ID NO:94 and / or CDR1, CDR2, and CDR3 of the light chain variable (VI) region shown in SEQ ID NO:96. In some embodiments, the anti-EDA antibody or a fragment thereof comprises a VH region having the amino acid sequence of SEQ ID NO:94 and / or a VL region having the amino acid sequence of SEQ ID NO:96. In some embodiments, the cytokine is IL-10. In some embodiments, IL-10 comprises the amino acid sequence of SEQ ID NO:98. In some embodiments, the immunocytokine comprises at least one linker. In some embodiments, the linker comprises SEQ ID NO:95 and / or 97. In some embodiments, the immune intercellular interleukin is an anti-EDA-IL-10 fusion protein comprising the amino acid sequence shown in SEQ ID NO:99. In some embodiments, the present invention provides a method for treating cancer in an individual, comprising administering to the individual a combination therapy comprising a first therapeutic agent and a second therapeutic agent, wherein the first therapeutic agent is an IL-15 fusion protein, and wherein the second therapeutic agent is an anti-EDA-IL-10 fusion protein. In some embodiments, the IL-15 fusion protein comprises the amino acid sequence shown in SEQ ID NO: 84, 85, 86, 87, 89, or 90, and the anti-EDA-IL-10 fusion protein comprises the amino acid sequence shown in SEQ ID NO: 99. In some embodiments, the combination therapy may further comprise 1, 2, 3, 4, or 5 additional therapeutic agents. In some embodiments, the combination therapy further comprises an anti-PD-1 or anti-PD-L1 antibody. In some embodiments, the anti-PD-1 antibody is BCD-100, camrelizumab (SHR-1210), cemiplimab (REGN2810), genolimzumab (CBT-501), MEDI0680, nivolumab (OPDIVO®), pembrolizumab (KEYTRUDA®), PF-06801591 (RN888), sintilimab (IBI-308), spartalizumab (PDR-001), STI-A1110, tislelizumab (BGB-A317), or TSR-042. In some embodiments, the anti-PD-L1 antibody is atezolizumab (TECENTRIQ®), durvalumab (IMFINZI®), BMS-936559 (MDX-1105), or LY3300054. In some embodiments, the therapeutic agents in a combination therapy may be administered simultaneously (e.g., in the same drug or at the same time), in parallel (i.e., in individual drugs administered one after another in any order), or sequentially in any order.

Implementation Method

Claims

1. An isolated fusion protein comprising: 1) an antibody containing an Fc domain; and 2) a human interleukin-15 (IL-15) variant comprising amino acid substitutions at E46G and V49R of SEQ ID NO: 1 and at least one or more amino acid substitutions at positions N1, N4, S7, K10, K11, D22, Y26, S29, D30, V31, H32, E53, G55, E64, I68, L69, E89, E93, M109 and / or I111 of SEQ ID NO: 1, wherein the IL-15 variant is covalently linked to the Fc domain of the antibody, and wherein the antibody-dependent cytotoxicity (ADCC) activity of the Fc domain is reduced or absent compared to the wild-type Fc.

2. The fusion protein of claim 1, wherein the IL-15 variant comprises amino acid substitutions selected from the following groups at positions: a) N1, E46, and V49; b) N4, E46, and V49; c) S7, E46, and V49; d) K10, E46, and V49; e) K11, E46, and V49; f) S29, E46, and V49; g) V31, E46, and V49; h) H32, E46, and V49; i) E46, V49, and E64; j) E46, V49, and I68; k) E46, V49, and L69; l) E46, V49, and I111; m) N4, E46, and V49. V49 and E64; n) E46, V49, N4, D30 and E64; o) E46, V49, N4, D30, E64 and I68; p) E46, V49, N4, D30, E64 and M109; q) E46, V49, N4, D30, E64, I68 and M109; r) N1, D30, E46 and V49; s) N4, D30, E46 and V49; t) S7, D30, E46 and V49; u) K10, D30, E46 and V49; v) K11, D30, E46 and V49; w) S29, D30, E46 and V49; x) D3 0, E46, V49 and E64; y) D30, E46, V49R and I68; z) D30, E46, V49R and L69; aa) D30, E46, V49R and I111; bb) N1, D30, E46, V49 and M109; cc) N4, D30, E46, V49 and M109; dd) S7, D30, E46, V49 and M109; ee) K10, D30, E46, V49 and M109; ff) K11, D30, E46, V49 and M109; gg) D30, E46, V49, E64 and M109; hh) D30 ii) D30, E46, V49, I68 and M109; jj) D30, E46, V49, L69 and M109; kk) D30, E46, V49, E64, I68 and M109; ll) E46, V49, D30, E64 and I68; mm) E46, V49, E64 and M109; nn) E46, V49, D30, E64, I68 and M109; oo) D22, Y26, V49, E46, E53, E89 and E93; and pp) N1, D30, E46, V49 and E64.

3. The fusion protein of claim 1, wherein the IL-15 variant comprises amino acid substitutions selected from the group consisting of: a) N1K, E46G, and V49R; b) N4K, E46G, and V49R; c) N4Q, E46G, and V49R; d) S7T, E46G, and V49R; e) V31S, E46G, and V49R; f) V31K, E46G, and V49R; g) E46G, V49R, and E64Q; h) E46G, V49R, and E64K; i ... E46G, and V49R; d) S7T, E46G, and V49R; e) V31S, E46G, and V49R; f) V31K, E46G, and V49R; g) E46G, E46G, and V49R; d) S7T, E46G, and V49R; e) S7T, E46G, and V49R; e) S7T, E46G, and V49R; e) S7T, E46G, and V49R; e 6G, V49R and E64Q; j) N1G, D30N, E46G and V49R; k) N1K, D30N, E46G and V49R; l) N1Q, D30N, E46G and V49R; m) N4G, D30N, E46G and V49R; n) N4K, D30N, E46G and V49R; o) N4Q, D30N, E46G and V49R; p) S7E, D30N, E46G and V49R; q) S7G, D30N, E46G and V49R; r S7T, D30N, E46G and V49R; S10D, D30N, E46G and V49R; T30N, E46G, V49R and E64A; U10D, E46G, V49R and E64Q; V10D, E46G, V49R and E64K; W10D, D30N, E46G, V49R and I68S; X10D, D30N, E46G, V49R and I68K; Y10D, N4K, D30N, E46G, V49R and E64K; Z20D, N46G, D30N, E46G, V49R and E64K; Z30D, N46G, D30N, E46G, V49R and E64K; Z4 ... 4Q, D30N, E46G, V49R and E64K; aa) N4K, D30N, E46G, V49R and E64Q; bb) N4Q, D30N, E46G, V49R and E64Q; cc) N4K, D30N, E46G, V49R and I68S; dd) D30N, E46G, V49R, E64Q and I68S; ee) N1A, D30N, E46G and V49R; and ff) N1G, D30N, E46G, V49R and E64Q.

4. The fusion protein of claim 1, comprising the amino acid sequence of SEQ ID NO: 86, 87, 89 or 90.

5. The fusion protein of claim 1, wherein the antibody has isotypes selected from the group consisting of: IgG1, IgG2, IgG2△a, IgG4, IgG4△b, IgG4△c, IgG4 S228P, IgG4△b S228P and IgG4△c S228P.

6. The fusion protein of claim 5, wherein the antibody is a) IgG2, and an antibody variable domain comprising amino acid modifications at positions 223, 225 and / or 228 in the hinge region and at positions 409 and / or 368 (EU numbering scheme) in the CH3 region of human IgG2 (SEQ ID NO: 3); b) IgG1, and an antibody variable domain comprising amino acid modifications at positions 221 and / or 228 in the hinge region and at positions 409 and / or 368 (EU numbering scheme) in the CH3 region of human IgG1 (SEQ ID NO: 2); or c) IgG1, and an antibody variable domain comprising amino acid modifications at positions 349, 354, 366, 368 and / or 407 (EU numbering scheme) in the CH3 region of human IgG1 (SEQ ID NO: 2).

7. The fusion protein of claim 6, wherein the antibody further comprises amino acid modifications at one or more positions 265, 330 and / or 331 of human IgG2 (SEQ ID NO: 3); or at one or more positions 234, 235 and / or 237 of human IgG1 (SEQ ID NO: 2).

8. The fusion protein of claim 1, wherein the antibody is selected from the group consisting of: anti-CTLA-4 antibody, anti-CD3 antibody, anti-CD4 antibody, anti-CD8 antibody, anti-4-1BB antibody, anti-PD-1 antibody, anti-PD-L1 antibody, anti-TIM3 antibody, anti-LAG3 antibody, anti-TIGIT antibody, anti-OX40 antibody, anti-IL-8 antibody, anti-IL-7Rα(CD127) antibody, anti-IL15 antibody, anti-HVEM antibody, anti-BTLA antibody, anti-CD40 antibody, anti-CD40L antibody, anti-CD47 antibody, anti-CSF1R antibody, anti-CSF1 antibody, anti-MARCO antibody, anti-CXCR4 antibody, anti-VEGFR1 antibody, anti-VEGFR2 antibody, anti-TNFR1 antibody, anti-TNFR2 antibody, anti-CD3 bispecific antibody, anti-CD19 antibody, anti-CD20, anti-Her2 antibody, anti-EGFR antibody, anti-ICOS antibody, anti-CD22 antibody, anti-CD Antibody 52, anti-CCR4 antibody, anti-CCR8 antibody, anti-CD200R antibody, anti-VISG4 antibody, anti-CCR2 antibody, anti-LILRb2 antibody, anti-CXCR4 antibody, anti-CD206 antibody, anti-CD163 antibody, anti-KLRG1 antibody, anti-FLT3 antibody, anti-B7-H4 antibody, anti-B7-H3 antibody, KLRG1 antibody and anti-GITR antibody.

9. The fusion protein of claim 8, wherein the antibody is an anti-PD-1 antibody, comprising: a heavy chain variable (VH) region comprising a VH complementarity-determining region 1 (CDR1) comprising an amino acid sequence SEQ ID NO: 14, 15, 80, 81, 91 or 92; a VH CDR2 comprising an amino acid sequence SEQ ID NO: 16, 17, 82 or 83; and a VH CDR3 comprising an amino acid sequence shown in SEQ ID NO: 18 or 62; and / or a VL CDR1 comprising an amino acid sequence shown in SEQ ID NO: 19 or 31; a VL CDR2 comprising an amino acid sequence shown in SEQ ID NO: 20 or 32; and a VL CDR3 comprising an amino acid sequence shown in SEQ ID NO: 21 or 33.

10. The fusion protein of claim 8, wherein the antibody is an anti-PD-1 antibody, the antibody comprising: a VH region comprising: CDR1, CDR2 and CDR3 of the VH having the amino acid sequence of SEQ ID NO: 12, 34, 78 or 36; and / or a VL region comprising: CDR1, CDR2 and CDR3 of the VL having the amino acid sequence of SEQ ID NO: 13, 35, 79 or 37.

11. The fusion protein of claim 1, wherein the IL-15 variant is covalently linked to the antibody via a polypeptide linker and / or a polypeptide marker.

12. An isolated cell line that produces an IL-15 fusion protein as claimed in any one of claims 1 to 11.

13. An isolated nucleic acid encoding a fusion protein as described in any one of claims 1 to 11.

14. A recombinant expression vector comprising the nucleic acid as claimed in claim 13.

15. A host cell comprising isolated nucleic acid as claimed in claim 13 or an expression vector as claimed in claim 14.

16. A pharmaceutical composition comprising a fusion protein as claimed in any one of claims 1 to 11, and a pharmaceutically acceptable carrier.

17. A kit for treating cancer, comprising the pharmaceutical composition as claimed in claim 16.

18. Use of a fusion protein as claimed in any one of claims 1 to 11 or a pharmaceutical composition as claimed in claim 16 for manufacturing a medicament for treating cancer in an individual in need, wherein the medicament improves one or more symptoms in the individual associated with the cancer.

19. As used in claim 18, wherein the cancer is a solid cancer or a liquid cancer.

20. As claimed in claim 19, wherein the solid cancer is selected from the group consisting of: gastric cancer, small intestine cancer, sarcoma, head and neck cancer, thymic cancer, epithelial cancer, salivary gland cancer, liver cancer, gallbladder cancer, neuroendocrine tumors, stomach cancer, thyroid cancer, lung cancer, mesothelioma, ovarian cancer, breast cancer, prostate cancer, esophageal cancer, pancreatic cancer, glioma, kidney cancer, bladder cancer, cervical cancer, uterine cancer, vulvar cancer, penile cancer, testicular cancer, anal cancer, choriocarcinoma, colorectal cancer, oral cancer, skin cancer, Merkel cell carcinoma, glioblastoma, brain tumor, bone cancer, eye cancer, and melanoma.

21. As claimed in claim 19, wherein the liquid cancer is selected from the group consisting of: multiple myeloma, malignant plasmacytoma, Hodgkin's lymphoma, Hodgkin's lymphoma predominantly nodular lymphocyte type, Kahler's disease, myeloid leukemia, plasma cell leukemia, plasmacytoma, B-cell prolymphocytic leukemia, hairy cell leukemia, B-cell non-Hodgkin's lymphoma (NHL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), chronic myeloid leukemia (CML), follicular lymphoma, Burkitt's lymphoma, marginal zone lymphoma, mantle cell lymphoma, large cell lymphoma, precursor B lymphoblastic lymphoma, myeloid leukemia, Waldenstrom's macroglobulinemia. Macroglobulienemia, diffuse large B-cell lymphoma, follicular lymphoma, marginal zone lymphoma, mucosa-associated lymphoid tissue lymphoma, small cell lymphoma, mantle cell lymphoma, Burkitt lymphoma, primary mediastinal (thymic) large B-cell lymphoma, lymphoplasmacytic lymphoma, Waldenström macroglobulinemia. Macroglobulinemia, nodal-marginal B-cell lymphoma, splenic-marginal B-cell lymphoma, intravascular large B-cell lymphoma, primary exudative lymphoma, lymphomatoid granuloma, T-cell-rich / histiocytic large B-cell lymphoma, primary central nervous system lymphoma, primary cutaneous diffuse large B-cell lymphoma (leg type), EBV-positive diffuse large B-cell lymphoma in the elderly, inflammation-associated diffuse large B-cell lymphoma, intravascular large B-cell lymphoma, ALK-positive large B-cell lymphoma, plasmablastic lymphoma, large B-cell lymphoma in HHV8-associated multicentric Castleman disease, B-cell lymphoma not classified using the characteristic intermediate between diffuse large B-cell lymphoma and Burkitt lymphoma, B-cell lymphoma not classified using the characteristic intermediate between diffuse large B-cell lymphoma and typical Hodgkin lymphoma, and other hematopoietic cell-related cancers.

22. The use of any of claims 18 to 21, wherein the cancer is recurrent, refractory, or metastatic.

23. The use as claimed in any of claims 18 to 21, wherein the drug further comprises a second therapeutic agent or is administered in combination with a second therapeutic agent.

24. As claimed in claim 23, wherein the second therapeutic agent is an antibody selected from the group consisting of: anti-CTLA-4 antibody, anti-CD3 antibody, anti-CD4 antibody, anti-CD8 antibody, anti-4-1BB antibody, anti-PD-1 antibody, anti-PD-L1 antibody, anti-TIM3 antibody, anti-LAG3 antibody, anti-TIGIT antibody, anti-OX40 antibody, anti-IL-7Rα(CD127) antibody, anti-IL-8 antibody, anti-IL-15 antibody, anti-HVEM antibody, anti-BTLA antibody, anti-CD40 antibody, anti-CD40L antibody, anti-CD47 antibody, anti-CSF1R antibody, anti-CSF1 antibody, anti-IL-7R antibody, anti-MARCO antibody, anti-CXCR4 antibody, anti-VE antibody. GF antibody, anti-VEGFR1 antibody, anti-VEGFR2 antibody, anti-TNFR1 antibody, anti-TNFR2 antibody, anti-CD3 bispecific antibody, anti-CD19 antibody, anti-CD20, anti-Her2 antibody, anti-EGFR antibody, anti-ICOS antibody, anti-CD22 antibody, anti-CD52 antibody, anti-CCR4 antibody, anti-CCR8 antibody, anti-CD200R antibody, anti-VISG4 antibody, anti-CCR2 antibody, anti-LILRb2 antibody, anti-CXCR4 antibody, anti-CD206 antibody, anti-CD163 antibody, anti-KLRG1 antibody, anti-FLT3 antibody, anti-B7-H4 antibody, anti-B7-H3 antibody, KLRG1 antibody, BTN1A1 antibody, and anti-GITR antibody.

25. As claimed in claim 23, wherein the second therapeutic agent is a cytokine, an immune cytokine, TNFα, a PAP inhibitor, an oncolytic virus, a kinase inhibitor, an ALK inhibitor, a MEK inhibitor, an IDO inhibitor, a GLS1 inhibitor, a tyrosine kinase inhibitor, a CAR-T cell or T cell therapy, a TLR agonist, or a tumor vaccine.

26. Use of a fusion protein as claimed in any one of claims 1 to 11 or a pharmaceutical composition as claimed in claim 16 for the manufacture of a medicament for the treatment of cancer.

27. An use as claimed in claim 26, wherein the cancer is a solid cancer or a liquid cancer and / or the cancer is recurrent, refractory, or metastatic.

28. The use as claimed in claim 26, wherein the drug further comprises a second therapeutic agent or is administered in combination with a second therapeutic agent.

29. The use as claimed in claim 23, wherein the drug and the second therapeutic agent are in separate compositions and are administered sequentially or simultaneously.

30. The use as claimed in claim 28, wherein the drug and the second therapeutic agent are in separate compositions and are administered sequentially or simultaneously.

31. An isolated fusion protein comprising: 1) an anti-PD-1 antibody comprising an Fc domain; and 2) a human interleukin-15 (IL-15) variant comprising amino acid substitutions at positions E46G, V49R, N1G, E64Q and D30N of SEQ ID NO: 1, wherein the IL-15 variant is covalently linked to the Fc domain of the antibody.

32. The fusion protein of claim 31, wherein the antibody comprises: a heavy chain variable (VH) region comprising: a VH complementarity-determining region 1 (CDR1) comprising an amino acid sequence SEQ ID NO: 80, 81 or 91; a VH CDR2 comprising an amino acid sequence SEQ ID NO: 82 or 83; and a VH CDR3 comprising an amino acid sequence shown in SEQ ID NO: 62; and / or a VL CDR1 comprising an amino acid sequence shown in SEQ ID NO: 31; a VL CDR2 comprising an amino acid sequence shown in SEQ ID NO: 32; and a VL CDR3 comprising an amino acid sequence shown in SEQ ID NO:

33.

33. The fusion protein of claim 31, wherein the antibody comprises: a VH region comprising: CDR1, CDR2 and CDR3 of VH having the amino acid sequence of SEQ ID NO: 36; and / or a VL region comprising: CDR1, CDR2 and CDR3 of VL having the amino acid sequence of SEQ ID NO: 37.