Activatable interleukin 12 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-11453710-B2
Publication Date
2022-09-27
Expiration Date
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.
Core Innovation
The invention provides a pharmaceutical composition that includes a human interleukin 12 (IL-12) polypeptide having an IL-12 subunit polypeptide selected from IL-12p35 and IL-12p40. The composition further includes an IL-12 blocking moiety that comprises either a ligand-binding domain or fragment of a cognate receptor for the IL-12 polypeptide, or an antibody or antigen-binding fragment that binds the IL-12 polypeptide, operably linked by a protease-cleavable polypeptide linker.
The IL-12 polypeptide exhibits attenuated IL-12-receptor activating activity prior to cleavage, at least about 10 fold less than the IL-12-receptor activating activity of the polypeptide produced by cleavage of the protease-cleavable polypeptide linker. 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 pharmaceutical composition.
The composition also includes a half-life extension element that is human serum albumin, an antigen-binding polypeptide that binds human serum albumin, or an immunoglobulin Fc. The described architecture is intended to extend circulating half-life while remaining conditionally activatable upon protease cleavage, with receptor agonist activity restored in the cleaved IL-12 form.
Claims Coverage
The consolidated disclosure contains one independent claim. The claim combines a human IL-12 subunit polypeptide, an IL-12 blocking moiety linked by a protease-cleavable polypeptide linker, and a half-life extension element, with attenuated IL-12-receptor activating activity before cleavage and restored activity after cleavage.
Protease-cleavable IL-12 blocking fusion with attenuated receptor activation
A pharmaceutical composition that includes a human IL-12 subunit polypeptide selected from IL-12p35 and IL-12p40; an IL-12 blocking moiety comprising a ligand-binding domain or fragment of a cognate receptor for the IL-12 polypeptide, or an antibody or antigen-binding fragment of an antibody that binds the IL-12 polypeptide; and a protease-cleavable polypeptide linker, wherein the IL-12 subunit polypeptide and the IL-12 blocking moiety are operably linked by the protease-cleavable polypeptide linker and the IL-12 polypeptide has attenuated IL-12-receptor activating activity at least about 10 fold less than the IL-12-receptor activating activity of the polypeptide produced by cleavage of the protease-cleavable polypeptide linker.
Half-life extension element coupled to conditional IL-12 activation
The pharmaceutical composition further includes a half-life extension element wherein the half-life extension element is human serum albumin, an antigen-binding polypeptide that binds human serum albumin, or an immunoglobulin Fc, and the composition is assessed for IL-12 receptor activating activity using a HEK Blue reporter cell assay with equal amounts on a mole basis of the IL-12 polypeptide and the pharmaceutical composition.
Independent claim covers a pharmaceutical composition that conditionally activates IL-12 receptor agonist activity through protease-cleavable linkage of an IL-12 subunit to an IL-12 blocking moiety, with receptor activation attenuated before cleavage and assessed by a HEK Blue reporter assay, together with a half-life extension element selected from human serum albumin, an HSA-binding antigen-binding polypeptide, or an immunoglobulin Fc.
Stated Advantages
Extends circulating half-life while remaining activatable in vivo upon protease cleavage.
Reduces IL-12 receptor activation prior to cleavage and restores higher IL-12 receptor activating activity after cleavage.
Aims to limit systemic toxicity by attenuating IL-12 receptor activating activity before cleavage.
Protease-cleavable masking that activates tumor-local therapeutic activity while maintaining in vivo stability.
Documented Applications
Protease-activated activation in the tumor microenvironment, with cleavage-assessed increases in IL-12 reporter cell activity and associated tumor efficacy in xenograft tumor models.
Evaluation of IL-12 receptor activating activity using a HEK Blue reporter cell assay (HEK Blue IL-12 reporter).
Use of ProTriTAC anti-tumor activity testing in an HCT116 xenograft model with expanded human T cells.
Pharmacokinetic comparison studies using a PSMA-targeting ProTriTAC (cleavable) versus non-cleavable controls and a non-cleavable active drug-mimicking ProTriTAC.
Functional testing of soluble ProTriTACs on human primary T cells by flow cytometry and measurement of T cell-dependent cellular cytotoxicity using luciferase-expressing HCT116 target cells.
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