Activatable interleukin-2 polypeptides
Inventors
Winston, William • Hicklin, Daniel • Bhaskar, Vinay • Evnin, Luke • Baeuerle, Patrick • SALMERON GARCIA, Jose Andres • Brodkin, Heather • Seidel-Dugan, Cynthia
Assignees
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Publication Number
US-10696724-B2
Publication Date
2020-06-30
Expiration Date
Abstract
The disclosure features fusion proteins that are conditionally active variants of IL-2. 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.
Core Innovation
The invention relates to engineered interleukin 2 (IL-2) fusion polypeptides in which IL-2 is combined with a half-life extension element, protease-cleavable linker(s), and an IL-2 blocking moiety. The fusion polypeptides are configured so that IL-2-receptor activating activity is attenuated prior to protease cleavage, and the activity is defined by comparison to the activity of an IL-2 polypeptide produced by cleavage of the protease-cleavable linker(s).
In the described constructs, IL-2 (A) is fused to a half-life extension element (B), a protease-cleavable polypeptide linker (L1), and an IL-2 blocking moiety (D), with an additional polypeptide linker (L2) that can optionally be protease-cleavable. L1 and L2 each include at least one sequence cleavable by a protease selected from a defined group of proteases, and the architecture uses blocking of IL-2 receptor activation by an antibody or antigen-binding fragment that binds the IL-2 polypeptide.
A conditional activation concept is used in which protease cleavage modulates IL-2 accessibility and activity. The described approach includes protease-cleavable masking and activation of protease-cleaved IL-2 activity, with IL-2 fusion proteins evaluated for increased detected IL-2 mutein activity upon cleavage and for biological anti-tumor effects in tumor growth inhibition or delay models. The document also introduces a broader conditionally active fusion protein concept using albumin-binding masking domains and protease-cleavable linkers, evaluated with in vitro and in vivo assessment elements and pharmacokinetic considerations such as half-life extension.
Claims Coverage
The partial claim set includes one independent claim. It defines a single fusion-polypeptide architecture combining IL-2, a half-life extension element, protease-cleavable linkers, and an IL-2 blocking moiety, with an explicit attenuation requirement and specified assay readouts.
Protease-activated attenuated IL-2 fusion polypeptide with IL-2 blocking
A fusion polypeptide comprising A (an IL-2 polypeptide), B (a half-life extension element that is human serum albumin or an antigen-binding polypeptide that binds human serum albumin), protease-cleavable polypeptide linker(s) L1 and optional protease-cleavable L2, and D (an IL-2 blocking moiety that is an antibody or antigen-binding fragment binding the IL-2 polypeptide), arranged in one of the specified segment orders, wherein the fusion polypeptide has attenuated IL-2-receptor activating activity that is at least about 10 fold less than the IL-2-receptor activating activity of the IL-2 polypeptide produced by cleavage of the protease-cleavable linker(s), with activity assessed using a CTLL-2 proliferation assay, a phospho STAT ELISA, or HEK Blue reporter cell assay using equal amounts on a mole basis of IL-2 and the fusion polypeptide.
The independent claim centers on a protease-cleavable, half-life extended IL-2 fusion polypeptide containing an IL-2 blocking moiety and an explicit attenuation requirement, with activity evaluated using CTLL-2 proliferation, phospho STAT ELISA, or HEK Blue reporter assays.
Stated Advantages
Attenuated IL-2-receptor activating activity prior to cleavage, at least about 10 fold less than IL-2-receptor activating activity of IL-2 polypeptide produced by cleavage of the protease-cleavable linker(s).
Conditional activation concept in which protease cleavage modulates IL-2 accessibility and activity.
Reduced tumor growth in vivo and induction of anti-tumor memory are described in the document summary of the disclosed examples.
Reduced systemic toxicity.
Enables tumor-localized activity.
Shortened active cytokine half-life after cleavage.
Documented Applications
Tumor growth inhibition/delay in vivo in tumor models, including MC38, Colon 38, xenograft, and NOD/scid mice.
Re-challenge anti-tumor memory.
In vitro evaluation of protease-cleaved IL-2 activity using sandwich ELISA, CTLL-2 proliferation assay, phospho STAT ELISA, and HEK Blue reporter assay.
Pharmacokinetics and half-life extension evaluation, including serum half-life extension and a cynomolgus monkey PK study.
Use of protease-cleavable IL-2 fusion polypeptides in tumor and inflammation contexts, with tumor-localized activity as described in the document.
Discussion of nucleic acids, vectors, host cells, and pharmaceutical formulations for administration across multiple disease indications is described in the document.
Tumor xenograft model outcomes are described, including comparisons of cleavable fusions versus non-cleavable controls.
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