Activatable interleukin 12 polypeptides

• US 10,696,723 B2
• Filed: 06/11/2019
• Issued: 06/30/2020
• Est. Priority Date: 05/14/2018
• Status: Active Grant

• Current Assignee: Werewolf Therapeutics, Inc.
• Original Assignee: Werewolf Therapeutics, Inc.
• Inventors: Winston, William, Hicklin, Daniel, Bhaskar, Vinay, Evnin, Luke, Baeuerle, Patrick, Salmeron Garcia, Jose Andres, Brodkin, Heather, Seidel-Dugan, Cynthia
• Primary Examiner(s): Mertz, Prema M
• Application Number:
  US16/438,166
• Publication Number:
  US 20190367576A1
• Time in Patent Office: 385 Days
• CPC Class Codes: A61K 2039/585 wherein the target is cancer
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: C07K 14/5434 IL-12
• CPC Class Codes: C07K 14/55 IL-2
• CPC Class Codes: C07K 16/18 against material from animals or humans
• CPC Class Codes: C07K 16/244 Interleukins [IL]
• CPC Class Codes: C07K 16/28 against receptors, cell surface antigens or cell surface determinants
• CPC Class Codes: C07K 16/2809 against the T-cell receptor (TcR)-CD3 complex
• CPC Class Codes: C07K 16/2863 against receptors for growth factors, growth regulators
• CPC Class Codes: C07K 16/2866 against receptors for cytokines, lymphokines, interferons
• CPC Class Codes: C07K 16/2887 against CD20
• CPC Class Codes: C07K 16/30 from tumour cells
• CPC Class Codes: C07K 16/3069 Reproductive system, e.g. ovaria, uterus, testes, prostate
• CPC Class Codes: C07K 2317/31 multispecific
• CPC Class Codes: C07K 2317/55 Fab or Fab'
• CPC Class Codes: C07K 2317/569 Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
• CPC Class Codes: C07K 2317/622 Single chain antibody (scFv)
• CPC Class Codes: C07K 2317/76 Antagonist effect on antigen, e.g. neutralization or inhibition of binding
• CPC Class Codes: C07K 2319/30 Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
• CPC Class Codes: C07K 2319/31 fusions, other than Fc, for prolonged plasma life, e.g. albumin
• CPC Class Codes: C07K 2319/33 : fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
• CPC Class Codes: C07K 2319/50 : containing protease site

Abstract

The disclosure features fusion proteins that are conditionally active variants of IL-12. 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.

Claims

1. A fusion polypeptide of the formula:

[A]-[L1]-[D]-[L2]-[B] or [B]-[L1]-[A]-[L1]-[D], wherein,

A is an interleukin 12 (IL-12) polypeptide;

B is a half-life extension element, wherein the half-life extension element is human serum albumin or an antigen-binding polypeptide that binds human serum albumin;

L1 is a protease-cleavable polypeptide linker, L2 is a polypeptide linker that is optionally protease-cleavable, and when protease-cleavable L2 comprises at least one sequence that is cleavable by a protease, wherein for each of L1 and L2, independently, the protease is selected from the group consisting of a kallikrein, thrombin, chymase, carboxypeptidase A, an elastase, PR-3, granzyme M, a calpain, a matrix metalloproteinase (MMP), a fibroblast activation protein (FAP), an ADAM metalloproteinase, a plasminogen activator, a cathepsin, a caspase, a tryptase, and a tumor cell surface protease; and

D is an IL-12 blocking moiety, wherein the blocking moiety is an antibody or antigen-binding fragment of an antibody that binds the IL-12 polypeptide; and

wherein

the fusion polypeptide has attenuated IL-12-receptor activating activity, wherein the IL-12-receptor activating activity of the fusion polypeptide is at least about 10 fold less than the IL-12-receptor activating activity of the polypeptide that comprises the IL-12 polypeptide that is produced by cleavage of the protease-cleavable polypeptide linker L1 or, when L2 is protease-cleavable, by cleavage of both L1 and L2, and wherein the IL-12-receptor activating activity is assessed using a HEK Blue reporter cell assay, with equal amounts on a mole basis of the IL-12 polypeptide and the fusion polypeptide.

2. The fusion polypeptide of claim 1, wherein A comprises the formula:

[A1]-[L3]-[A2] or [A2]-[L3]-[A1], wherein

A1 is an IL-12 p40 subunit polypeptide;

A2 is an IL-12 p35 subunit polypeptide; and

L3 is a polypeptide linker that is optionally protease cleavable.

3. The fusion polypeptide of claim 1, wherein the antibody fragment that binds the IL-12 polypeptide is a single domain antibody, Fab or scFv.

4. The fusion polypeptide of claim 1, wherein L2 is a protease-cleavable polypeptide linker.

5. The fusion polypeptide of claim 1, wherein L1 or L2 or both L1 and L2 are cleaved by two or more different proteases.

6. The fusion polypeptide of claim 1, wherein the IL-12 blocking moiety inhibits activation of the IL-12 receptor by the fusion polypeptide.

7. The fusion polypeptide of claim 1, wherein the cathepsin is cathepsin B, cathepsin C, cathepsin D, cathepsin E, cathepsin K, cathepsin L, or cathepsin G.

8. The fusion polypeptide of claim 1, wherein the matrix metalloprotease (MMP) is MMP1, MMP2, MMP3, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, or MMP14.

9. A nucleic acid encoding the polypeptide of claim 1.

10. An isolated vector comprising the nucleic acid of claim 9.

11. The isolated host cell comprising the vector of claim 10.

12. A method of making a pharmaceutical composition, comprising culturing the isolated host cell of claim 11 under suitable conditions for expression and collection of desired polypeptides.