Activatable interleukin-2 polypeptides and methods of use thereof
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-11981716-B2
Publication Date
2024-05-14
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 a conditionally active cytokine comprising a half-life extension domain comprising an immunoglobulin Fc, an IL-2 binding moiety, and an IL-2 polypeptide. The IL-2 binding moiety comprises a ligand-binding domain or fragment of a cognate receptor for the IL-2 polypeptide, or an antibody or antigen binding fragment of an antibody that binds the IL-2 polypeptide, and the IL-2 binding moiety and the IL-2 polypeptide are operably linked to the immunoglobulin Fc.
The IL-2 binding moiety is linked operably to the immunoglobulin Fc through a linker that comprises a cleavable peptide. In the intact, uncleaved form, the IL-2 polypeptide has attenuated IL-2-receptor activating activity relative to the IL-2 polypeptide produced by cleavage of the cleavable linker, and after protease cleavage of the linker, IL-2 receptor binding and signaling are restored or enabled.
The described IL-2 muteins and fusion constructs include engineered IL-2 variants with attenuated or altered IL-2 receptor activating activity prior to activation, and restored activity after activation events associated with cleavable linkers. The document further describes mutant IL-2 muteins engineered for increased binding to an IL-2 trimeric receptor versus a dimeric receptor, together with conditionally activatable fusion protein examples that include a blocker or masking moiety using a serum albumin-binding dAb blocker/masking domain and protease-cleavable linkage.
Claims Coverage
The claim set includes a conditionally active IL-2 cytokine construct built around an immunoglobulin Fc half-life extension domain, an IL-2 binding moiety operably linked via a cleavable-peptide linker, and an IL-2 polypeptide whose receptor activating activity is attenuated until cleavage restores IL-2-receptor activating activity. Dependent claims refine the IL-2 binding/blocking moiety format, the IL-2 receptor subunit targeting, the IL-2 receptor binding profile, and assay/benchmark comparability for measuring receptor activation and half-life-related constraints.
Conditionally active cytokine with Fc half-life extension and cleavable-peptide linked IL-2 binding moiety
A conditionally active cytokine comprising a half-life extension domain which is an immunoglobulin Fc; an IL-2 binding moiety comprising a ligand-binding domain or fragment of a cognate receptor for the IL-2 polypeptide or an antibody or antigen binding fragment of an antibody that binds the IL-2 polypeptide; and an IL-2 polypeptide, wherein the IL-2 binding moiety and the IL-2 polypeptide are operably linked to the immunoglobulin Fc, and the IL-2 binding moiety is linked operably to the immunoglobulin Fc through a linker that comprises a cleavable peptide.
Attenuated IL-2 receptor activating activity until cleavage activation
The conditionally active cytokine wherein the IL-2 polypeptide has attenuated IL-2-receptor activating activity at least about 10 fold less than the IL-2-receptor activating activity of the polypeptide that is produced by cleavage of the cleavable linker.
IL-2 activity evaluated with receptor-activation cell or signaling assays under a comparability condition
The conditionally active cytokine evaluated for IL-2 receptor activating activity using CTLL-2 proliferation assay, phospho-STAT ELISA, or HEK Blue receptor cell assay, with the IL-2 polypeptide and the cytokine tested at equal molar amounts.
IL-2 binding moiety as an antibody format for IL-2 binding/blocking
The conditionally active cytokine where the IL-2 blocking moiety is an antibody or antibody fragment that binds the IL-2 polypeptide, selected from single domain antibody, Fab, or scFv.
Cognate IL-2 receptor subunit specified as IL-2Rbeta (CD122)
The conditionally active cytokine where the cognate receptor for the IL-2 polypeptide uses IL-2Rbeta (CD122).
IL-2 polypeptide binds one, two, or three IL-2 receptor subunits with altered affinity and/or selectivity
The conditionally active IL-2 cytokine where the IL-2 polypeptide binds one, two, or three IL-2 receptor subunits with an affinity and/or selectivity different from wild-type IL-2.
Cleavage-produced IL-2 polypeptide serum half-life comparable to wild-type
The conditionally active cytokine where a cleavage-produced IL-2 polypeptide has a serum half-life comparable to that of wild-type IL-2.
Overall, the claims cover a conditionally active IL-2 cytokine construct built around an immunoglobulin Fc half-life extension domain, an IL-2 binding moiety operably linked via a cleavable-peptide linker, and an IL-2 polypeptide whose receptor activating activity is attenuated until cleavage restores IL-2-receptor activating activity, with dependent refinements specifying assay evaluation, antibody/fragment moiety formats, IL-2 receptor subunit targeting, receptor binding profile changes, and serum half-life comparison constraints.
Stated Advantages
Reduces systemic toxicity by attenuating IL-2-receptor activating activity in the intact, uncleaved form.
Restores or enables IL-2 receptor binding and signaling after protease cleavage of the linker.
Provides a serum half-life for the cleavage-produced IL-2 polypeptide comparable to wild-type IL-2.
Attenuated IL-2-receptor activating activity until after cleavage of the cleavable linker.
Conditionally restored IL-2-receptor activating activity of the polypeptide produced by cleavage.
Documented Applications
Therapeutic applications are described across cancers and inflammatory/autoimmune or other immune-mediated diseases, using the conditionally active IL-2 cytokine constructs.
Protease cleavage by tumor- or inflammation-associated proteases, including MMPs such as MMP9, is described as enabling localized activity in tumor or inflamed microenvironments.
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