US2022002370A1PendingUtilityA1
Masked cytokine polypeptides

Assignee: XILIO DEV INCPriority: Sep 27, 2018Filed: Sep 27, 2019Published: Jan 6, 2022
Est. expirySep 27, 2038(~12.2 yrs left)· nominal 20-yr term from priority
Inventors:Margaret Karow Deborah Moore Lai Dheeraj Tomar Parker Johnson Raphael Rozenfeld Ronan O'Hagan Huawei Qiu
A61K 38/00C07K 14/7155C07K 14/5434C07K 2319/50C07K 2319/30C07K 2319/01C07K 14/5443C07K 14/55C07K 2317/52C07K 14/54A61P 35/00C07K 14/52C07K 2317/94
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Claims
Abstract

Provided herein are cytokines or functional fragments thereof that, in some embodiments, are engineered to be masked by a masking moiety at one or more receptor binding site(s) of the cytokine or functional fragment thereof. In some embodiments, the cytokines are engineered to be activatable by a protease at a target site, such as in a tumor microenvironment, by including a proteolytically cleavable linker. In some embodiments, the proteolytically cleavable linker links the cytokine to the masking moiety, links the cytokine to a half-life extension domain, and/or links the masking moiety to a half-life extension domain. The masking moiety blocks, occludes, inhibits (e.g., decreases) or otherwise prevents (e.g., masks) the activity or binding of the cytokine to its cognate receptor or protein. Upon proteolytic cleavage of the cleavable linker at the target site, the cytokine becomes activated, which renders it capable of binding to its cognate receptor or protein with increased affinity.
Claims

exact text as granted — not AI-modified
What is claimed is: 
     
         1 . A masked cytokine comprising:
 a) a masking moiety; and   b) a cytokine or functional fragment thereof,   wherein the masking moiety is linked to the cytokine or functional fragment thereof via a first linker.   
     
     
         2 . A masked cytokine comprising:
 a) a first masking moiety;   b) a cytokine or functional fragment thereof,   wherein the first masking moiety is linked to the cytokine or functional fragment thereof via a first linker; and   c) a second masking moiety,   wherein the second masking moiety is linked to the cytokine or functional fragment thereof via a second linker.   
     
     
         3 . A masked cytokine comprising:
 a) a first half-life extension domain and a second half-life extension domain;   b) a masking moiety; and   c) a cytokine or functional fragment thereof,   wherein the masking moiety is linked to the first half-life extension domain,   wherein the cytokine or functional fragment thereof is linked to the second half-life extension domain, and   wherein the first half-life extension domain and the second half-life extension domain contain modifications promoting the association of the first and the second half-life extension domain.   
     
     
         4 . A masked cytokine comprising:
 a) a first half-life extension domain and a second half-life extension domain;   b) a first masking moiety and a second masking moiety; and   c) a cytokine or functional fragment thereof,   wherein the first masking moiety is linked to the first half-life extension domain,   wherein the second masking moiety is linked to the cytokine or functional fragment thereof,   wherein either the second masking moiety or the cytokine or functional fragment thereof is linked to the second half-life extension domain, and   wherein the first half-life extension domain and the second half-life extension domain contain modifications promoting the association of the first and the second half-life extension domain.   
     
     
         5 . The masked cytokine of  claim 1 , further comprising a half-life extension domain that is linked to either the masking moiety or the cytokine or functional fragment thereof. 
     
     
         6 . The masked cytokine of  claim 2 , further comprising a half-life extension domain that is linked to either the first masking moiety or the second masking moiety. 
     
     
         7 . The masked cytokine of  claim 3 , wherein the masking moiety is linked to the first half-life extension domain via a first linker; and/or wherein the cytokine or functional fragment thereof is linked to the second half-life extension domain via a second linker. 
     
     
         8 . The masked cytokine of  claim 4 , wherein:
 a) the first masking moiety is linked to the first half-life extension domain via a first linker; and/or   b) either the second masking moiety or the cytokine or functional fragment thereof is linked to the second half-life extension domain via a second linker.   
     
     
         9 . The masked cytokine of any one of  claims 1 - 8 , wherein the cytokine or functional fragment thereof is an IL-2 polypeptide or functional fragment thereof. 
     
     
         10 . The masked cytokine of  claim 9 , wherein the IL-2 polypeptide or functional fragment thereof comprises:
 a) an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-8, 160, 230, 243-251, 260, 775-792, and 813-822; or   b) an amino acid sequence produced by introducing one or more of the following amino acid substitutions into any one of SEQ ID NOs: 1-8, 160, 243-251, 260, 775-792, and 813-822: H16I, L18C, D20A, D20L, D20F, N29L, R38A, F42A, F42K, F42E, F43A, Y45A, Y45N, Y45R, E62A, E62R, E62S, L72G, L80F, R81D, L85V, I86V, I92F, and C125S.   
     
     
         11 . The masked cytokine of any one of  claims 1 ,  3 ,  5 ,  7 ,  9 , and  10 , wherein the masking moiety comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 9, 10, 161-165, 187-218, 221-229, 231, and 261. 
     
     
         12 . The masked cytokine of any one of  claims 2 ,  4 ,  6 , and  8 - 10 , wherein the first masking moiety comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 9, 10, 161-165, 187-218, 221-229, 231, and 261, and the second masking moiety comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 9, 10, 161-165, 187-218, 221-229, 231, and 261, and wherein the amino acid sequence of the first masking moiety and the second masking moiety are different. 
     
     
         13 . The masked cytokine of any one of  claims 1 - 8 , wherein the cytokine or functional fragment thereof is an IL-15 polypeptide or functional fragment thereof. 
     
     
         14 . The masked cytokine of  claim 13 , wherein the IL-15 polypeptide or functional fragment thereof comprises the amino acid sequence of SEQ ID NO: 167. 
     
     
         15 . The masked cytokine of any one of  claims 1 ,  3 ,  5 ,  7 ,  13 , and  14 , wherein the masking moiety comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 161-165, 219-229, 232-234, 261, and 823-825. 
     
     
         16 . The masked cytokine of any one of  claims 2 ,  4 ,  6 ,  8 ,  13 , and  14 , wherein:
 a) the first masking moiety comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 232-234, and 823-825, and the second masking moiety comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 161-165, 219-229, and 261;   b) the first masking moiety comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 161-165, 219-229, and 261, and the second masking moiety comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 232-234, and 823-825;   c) the first masking moiety comprises an amino acid sequence produced by introducing one or more of the following amino acid substitutions into the amino acid sequence of any one of SEQ ID NOs: 232-234, and 823-825: R24A, R26A, K34A, 540A, L42A, and P67A, and the second masking moiety comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 161-165, 219-229, and 261; or   d) the first masking moiety comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 161-165, 219-229, and 261, and the second masking moiety comprises an amino acid sequence produced by introducing one or more of the following amino acid substitutions into the amino acid sequence of any one of SEQ ID NOs: 232-234, and 823-825: R24A, R26A, K34A, S40A, L42A, and P67A.   
     
     
         17 . The masked cytokine of any one of  claims 5 ,  6 , and  9 - 16 , wherein the half-life extension domain is an antibody or fragment thereof. 
     
     
         18 . The masked cytokine of  claim 17 , wherein the antibody or fragment thereof is a Fragment crystallizable domain (Fc domain) or fragment thereof. 
     
     
         19 . The masked cytokine of  claim 18 , wherein the Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 154. 
     
     
         20 . The masked cytokine of any one of  claims 3 ,  4 , and  7 - 19 , wherein the first half-life extension domain is a first antibody or fragment thereof, and the second half-life extension domain is a second antibody or fragment thereof. 
     
     
         21 . The masked cytokine of  claim 20 , wherein the first antibody or fragment thereof is a first Fc domain or fragment thereof, and second antibody or fragment thereof is a second Fc domain or fragment thereof. 
     
     
         22 . The masked cytokine of  claim 21 , wherein:
 a) the first Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 155, and the second Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 156;   b) the first Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 156, and the second Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 155;   c) the first Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 154, and the second Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 154;   d) the first Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 265, and the second Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 156;   e) the first Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 156, and the second Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 265;   f) the first Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 155, and the second Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 616;   g) the first Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 616, and the second Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 155;   h) the first Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 157, and the second Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 158;   i) the first Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 158, and the second Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 157;   j) the first Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 796, and the second Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 774;   k) the first Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 774, and the second Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 796;   l) the first Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 721, and the second Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 619;   m) the first Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 619, and the second Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 721;   n) the first Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 721, and the second Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 772;   o) the first Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 772, and the second Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 721;   p) the first Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 793, and the second Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 622;   q) the first Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 622, and the second Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 793;   r) the first Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 793, and the second Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 773;   s) the first Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 773, and the second Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 793;   t) the first Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 796, and the second Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 625;   u) the first Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 625, and the second Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 796;   v) the first Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 156, and the second Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 156;   w) the first Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 796, and the second Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 625; or   x) the first Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 625, and the second Fc domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 796.   
     
     
         23 . The masked cytokine of any one of  claims 1 ,  5 , and  9 - 22 , wherein the first linker comprises a first cleavable peptide. 
     
     
         24 . The masked cytokine of  claim 23 , wherein the first cleavable peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 96-153, 236-242, 264, 270-302, 306-317, 342-347, 356-415, 420-491, 494-501, 504-535, and 538-555. 
     
     
         25 . The masked cytokine of any one of  claims 1 ,  5 , and  9 - 24 , wherein the first linker comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 11-153, 235-242, 262-264, 268-320, 323-338, 340-354, 356-555, 668, 691, 724, 725, 727, 762-771, 794, and 797-812. 
     
     
         26 . The masked cytokine of any one of  claims 2  and  7 - 22 , wherein the first linker comprises a first cleavable peptide, and/or the second linker comprises a second cleavable peptide. 
     
     
         27 . The masked cytokine of  claim 26 , wherein the first cleavable peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 96-153, 236-242, 264, 270-302, 306-317, 342-347, 356-415, 420-491, 494-501, 504-535, and 538-555, and/or the second cleavable peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 96-153, 236-242, 264, 270-302, 306-317, 342-347, 356-415, 420-491, 494-501, 504-535, and 538-555. 
     
     
         28 . The masked cytokine of any one of  claims 2 ,  7 - 22 ,  26 , and  27 , wherein the first linker comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 11-153, 235-242, 262-264, 268-320, 323-338, 340-354, 356-555, 668, 691, 724, 725, 727, 762-771, 794, and 797-812, and/or the second linker comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 11-153, 235-242, 262-264, 268-320, 323-338, 340-354, 356-555, 668, 691, 724, 725, 727, 762-771, 794, and 797-812. 
     
     
         29 . A nucleic acid encoding the masked cytokine of any one of  claims 1 - 28 . 
     
     
         30 . A vector comprising the nucleic acid of  claim 29 . 
     
     
         31 . A host cell comprising the nucleic acid of  claim 29 . 
     
     
         32 . A method of producing a masked cytokine comprising culturing the host cell of  claim 31  under a condition that produces the masked cytokine. 
     
     
         33 . A composition comprising the masked cytokine of any one of  claims 1 - 28 . 
     
     
         34 . A pharmaceutical composition comprising the masked cytokine of any one of  claims 1 - 28 , and a pharmaceutically acceptable carrier. 
     
     
         35 . A kit comprising the masked cytokine of any one of  claims 1 - 28 , or the composition of  claim 33 , or the pharmaceutical composition of  claim 34 . 
     
     
         36 . A method of treating or preventing a neoplastic disease in a subject, the method comprising administering to the subject an effective amount of the masked cytokine of any one of  claims 1 - 28 , or the composition of  claim 33 , or the pharmaceutical composition of  claim 34 . 
     
     
         37 . A method of treating or preventing an inflammatory or autoimmune disease in a subject, the method comprising administering to the subject an effective amount of the masked cytokine of any one of  claims 1 - 28 , or the composition of  claim 33 , or the pharmaceutical composition of  claim 34 .
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