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US-12617829-B2 - Activatable cytokine polypeptides and methods of use thereof

US12617829B2US 12617829 B2US12617829 B2US 12617829B2US-12617829-B2

Abstract

The disclosure features fusion proteins that are conditionally active variants of a cytokine of interest. In one aspect, the full-length polypeptides of the invention have reduced or minimal cytokine-receptor activating activity even though they contain a functional cytokine polypeptide. Upon activation, e.g., by cleavage of a linker that joins a blocking moiety, e.g. a steric blocking polypeptide, in sequence to the active cytokine, the cytokine can bind its receptor and effect signaling. Typically, the fusion proteins further comprise an in vivo half-life extension element, which may be cleaved from the cytokine in the tumor microenvironment.

Inventors

  • William Winston
  • Heather Brodkin
  • Cynthia Seidel-Dugan
  • Daniel Hicklin
  • Jose Andres SALMERON-GARCIA

Assignees

  • WEREWOLF THERAPEUTICS, INC.

Dates

Publication Date
20260505
Application Date
20240311

Claims (13)

  1. 1 . A conditionally active IL-18 comprising a fusion polypeptide comprising at least one of each of: a) A human IL-18 polypeptide [A]; b) an IL-18 blocking moiety [D], wherein the IL-18 blocking moiety [D] comprises an antibody or antigen-binding fragment that binds the IL-18 polypeptide, a ligand-binding domain or fragment of a cognate receptor for the IL-18 polypeptide; c) a half-life extension element [H], wherein the half-life extension element [H] is a human serum albumin, an antigen-binding polypeptide that binds serum albumin, or an immunoglobulin Fc; and d) a protease cleavable polypeptide linker [L]; wherein the conditionally active cytokine has attenuated IL-18 receptor activating activity that is at least about 10-fold less than the IL-18 receptor activating activity of the polypeptide that contains the IL-18 polypeptide that is produced by cleavage of the protease cleavable linker.
  2. 2 . The conditionally active cytokine of claim 1 , wherein the IL-18 blocking moiety [D] is an antibody fragment that binds to the IL-18 polypeptide and the antibody fragment is a single domain antibody, a Fab or scFv that binds the IL-18 polypeptide.
  3. 3 . The conditionally active cytokine of claim 1 , wherein the IL-18 blocking moiety inhibits the cytokine polypeptide from activating its cognate receptor.
  4. 4 . The conditionally active cytokine comprising of claim 1 , wherein the protease cleavable linker comprises a sequence that is capable of being cleaved by a protease selected from the group consisting of a kallikrein, thrombin, chymase, carboxypeptidase A, an elastase, PR-3, granzyme M, a calpain, a matrix metalloproteinase (MMP), an ADAM, a FAP, a plasminogen activator, a cathepsin, a caspase, a tryptase, and a tumor cell surface protease.
  5. 5 . The conditionally active cytokine of claim 1 , wherein the protease cleavable polypeptide linker independently comprises two or more cleavage sites for the same protease, or two or more cleavage sites that are cleaved by different proteases or at least one of the protease-cleavable polypeptides comprises a cleavage site for two or more different proteases.
  6. 6 . The conditionally active cytokine of claim 1 , wherein the protease cleavable polypeptide linker comprises a sequence that is cleaved by cathepsin selected from the group consisting of cathepsin B, cathepsin C, cathepsin D, cathepsin E, cathepsin K, cathepsin L, and cathepsin G.
  7. 7 . The conditionally active cytokine of claim 1 , wherein the protease cleavable polypeptide linker comprises a sequence that is cleaved by a matrix metalloprotease (MMP) selected from the group consisting of MMP1, MMP2, MMP3, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, and MMP14.
  8. 8 . The conditionally active cytokine of claim 1 , comprising a fusion polypeptide having the Formula: [A]-[L1]-[H]-[L2]-[D] or [A]-[L1]-[D]-[L2]-[H] or [D]-[L2]-[H]-[L1]-[A] or [H]-[L2]-[D]-[L1]-[A] or [D]-[L1]-[A]-[L1]-[H] or [H]-[L1]-[A]-[L1]-[D], wherein, L1 is a protease cleavable polypeptide linker, and L2 is a polypeptide linker that is optionally protease cleavable.
  9. 9 . The conditionally active cytokine of claim 1 , wherein the serum half-life of the IL-18 polypeptide that is produced by cleavage of the protease-cleavable linker is comparable to the half-life of naturally occurring IL-18.
  10. 10 . A nucleic acid encoding the conditionally active cytokine of claim 1 .
  11. 11 . A pharmaceutical composition comprising a non-viral delivery system comprising the nucleic acid of claim 10 .
  12. 12 . A pharmaceutical composition comprising a mammalian cell comprising the conditionally active cytokine of claim 1 .
  13. 13 . A pharmaceutical composition comprising a mammalian cell comprising the nucleic acid of claim 10 .

Description

RELATED APPLICATIONS This application is a continuation of Ser. No. 18/312,245, filed May 4, 2023, which is a continuation of Ser. No. 17/208,643, filed Sep. 22, 2020, which is a continuation-in-part of PCT/US2019/032320, filed on May 14, 2019, which claims the benefit of U.S. Provisional Application 62/671,225, filed on May 14, 2018, U.S. Provisional Application No. 62/756,504, filed on Nov. 6, 2018, U.S. Provisional Application No. 62/756,507, filed on Nov. 6, 2018, and U.S. Provisional Application No. 62/756,515, filed on Nov. 6, 2018; and claims the benefit of U.S. Provisional Application No. 62/935,605, filed on Nov. 14, 2019, each of which are incorporated herein by reference in their entireties. SEQUENCE LISTING The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML file, created on May 4, 2023, is named 761146.200011_SL.xml and is 618,247 bytes in size. BACKGROUND The development of mature immunocompetent lymphoid cells from less-committed precursors, their subsequent antigen-driven immune responses, and the suppression of these and unwanted autoreactive responses are highly dependent and regulated by cytokines (including interleukin-2 [IL-2], IL-4, IL-7, IL-9, IL-15, and IL-21) that utilize receptors in the common γ-chain (γc) family (Rochman et al., 2009) and family members including IL-12, 18 and 23. IL-2 is essential for thymic development of Treg cells and critically regulates several key aspects of mature peripheral Treg and antigen-activated conventional T cells. Because of its potent T cell growth factor activity in vitro, IL-2 has been extensively studied in part because this activity offered a potential means to directly boost immunity, e.g., in cancer and AIDS-HIV patients, or a target to antagonize unwanted responses, e.g., transplantation rejection and autoimmune diseases. Although in vitro studies with IL-2 provided a strong rationale for these studies, the function of IL-2 in vivo is clearly much more complex as first illustrated in IL-2-deficient mice, where a rapid lethal autoimmune syndrome, not lack of immunity, was observed (Sadlack et al., 1993, 1995). Similar observations were later made when the gene encoding IL-2Rα (Il2ra) and IL-2Rβ (Il2rb) were individually ablated (Suzuki et al., 1995; Willerford et al., 1995). The present invention refers to conditionally active and/or targeted cytokines for use in the treatment of cancer and other diseases dependent on immune up or down regulation. For example, the antitumoral activity of some cytokines is well known and described and some cytokines have already been used therapeutically in humans. Cytokines such as interleukin-2 (IL-2) and interferon α (IFNα) have shown positive antitumoral activity in patients with different types of tumors, such as kidney metastatic carcinoma, hairy cell leukemia, Kaposi sarcoma, melanoma, multiple myeloma, and the like. Other cytokines like IFNβ, the Tumor Necrosis Factor (TNF) α, TNFβ, IL-1, 4, 6, 12, 15 and the CSFs have shown a certain antitumoral activity on some types of tumors and therefore are the object of further studies. SUMMARY Provided herein are therapeutic proteins, nucleic acids that encode the proteins, and compositions and methods of using the proteins and nucleic acids for the treatment of a disease or disorder, such as proliferative disease, a tumorous disease, an inflammatory disease, an immunological disorder, an autoimmune disease, an infectious disease, a viral disease, an allergic reaction, a parasitic reaction, graft-versus-host disease and the like. In certain embodiments, the protein is one or more of, including any combinations, SEQ ID NOs.: 193-271 and the protein referred to herein as: ACP200ACP201ACP202ACP203ACP204ACP205ACP206ACP207ACP208ACP211ACP213ACP214ACP215ACP240ACP241ACP242ACP243ACP244ACP245ACP247ACP284ACP285ACP286ACP287ACP288ACP289ACP290ACP291ACP292ACP296ACP297ACP298ACP299ACP300ACP302ACP303ACP304ACP305ACP306ACP309ACP310ACP311ACP312ACP313ACP314ACP336ACP337ACP338ACP339ACP340ACP341ACP342ACP343ACP344ACP345ACP346ACP347ACP348ACP349ACP350ACP351ACP352ACP353ACP354ACP355ACP356ACP357ACP358ACP359ACP371ACP372ACP373ACP374ACP375ACP376ACP377ACP378ACP379ACP383ACP384ACP385ACP386ACP387ACP388ACP389ACP390ACP391ACP392ACP393ACP394ACP395ACP396ACP397ACP398ACP399ACP400ACP401ACP402ACP403ACP404ACP405ACP406ACP407ACP408ACP409ACP410ACP411ACP412ACP413ACP414ACP415ACP416ACP417ACP418ACP419ACP420ACP421ACP422ACP423ACP424ACP425ACP426ACP427ACP428ACP429ACP430ACP431ACP432ACP433ACP434ACP439ACP440ACP441ACP442ACP443ACP444ACP445ACP446ACP447ACP451ACP452ACP453ACP454ACP455ACP456ACP457ACP458ACP459ACP460ACP461ACP462ACP463ACP464ACP465ACP466ACP467ACP468ACP469ACP470ACP471 The invention features fusion proteins that are conditionally active variants of a cytokine of interest. In one aspect, the full-length polypeptides of the invention have reduced or minimal cytokine-receptor activating activity even