Activatable cytokine polypeptides and methods of use thereof

Inventors

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

Assignees

Werewolf Therapeutics Inc

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.

Core Innovation

The invention relates to activatable cytokine fusion polypeptides defined by a modular formula [D]-[L1]-[A]-[L2]-[H]. In the construct, [A] is an interferon alpha (IFNa) polypeptide, a mutein, or an active fragment thereof, [D] is a blocking moiety, and [H] is a half-life extension moiety. [L1] and [L2] are protease-cleavable polypeptide linkers comprising the amino acid sequence of SEQ ID NO: 609, 612, or 615.

The blocking moiety is configured to sterically reduce receptor activation before cleavage, and protease cleavage restores cytokine activity. The blocking moiety and the half-life extension moiety each independently comprise human serum albumin (HSA) or an antibody or antibody fragment that binds HSA. The disclosure also describes fusion orientations, construct permutations, and specific fusion polypeptide sequences defined by SEQ ID numbers.

The document further describes cleavage in connection with protease activity enriched or selective for a target setting, including the tumor microenvironment, and includes examples of protease activity such as MMP9 and PSA. It also describes cytokine or mutein behavior pre- and post-cleavage, including binding and functional cell assays, and identifies additional fusion formats involving HSA or HSA-binding antibody or fragment components and specified IFNa sequence options.

Claims Coverage

The claim coverage centers on one modular fusion polypeptide architecture and additional claims directed to specific fusion polypeptide sequences. The inventive features combine IFNa polypeptides with protease-cleavable linkers and HSA-based blocking and half-life extension moieties, with sequence-based refinements for linkers, IFNa, and full fusion polypeptide sequences.

Overall, the claims cover a protease-activatable IFNa fusion polypeptide defined by a modular [D]-[L1]-[A]-[L2]-[H] architecture, with specified SEQ ID protease-cleavable linkers, HSA or HSA-binding antibody or fragment blocking and half-life extension moieties, selected IFNa sequence options, specific fusion polypeptide sequences, and pharmaceutical compositions containing the fusion polypeptide.

Stated Advantages

Documented Applications