Separation moieties and methods of use thereof
Inventors
Winston, William • Evnin, Luke • Bhaskar, Vinay • KNUDSEN, Giselle • Hicklin, Daniel J. • Seidel-Dugan, Cynthia • SALMERON GARCIA, Jose Andres • Brodkin, Heather R.
Assignees
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Publication Number
US-11739132-B2
Publication Date
2023-08-29
Expiration Date
Abstract
Provided herein are separation moieties that are suitable for use in conjunction with a variety of therapeutic payloads. The separation moieties serve to generate conditionally active macromolecules whereby the macromolecules have reduced or minimal biological activity until the separation moieties are modified under specific conditions.
Core Innovation
The invention relates to a recombinant pro-protein comprising a recombinant polypeptide with a cleavable moiety that is a substrate for a protease, and a polypeptide with biological activity. The cleavable moiety comprises the amino acid sequence of SEQ ID NO:198 or a functional variant of SEQ ID NO:198 in which one or two amino acids are substituted in SEQ ID NO:198, and the cleavable moiety is defined to be cleavable in a protease-dependent manner.
The recombinant pro-protein links the protease-cleavable moiety to a polypeptide with biological activity so that biological activity is modulated through protease cleavage. The pro-protein can have attenuated biological activity, and cleavage of the cleavable moiety produces a polypeptide with biological activity that is not attenuated. Dependent refinements specify that the functional variant acts as a substrate for Cathepsin L and that positions 4 and 5 are not substituted.
The document also describes a FRET-based protease substrate screening approach that establishes tumor-enriched secreted protease activity using conditioned media and cell lysates from murine colon tumor and stromal/control cell lines. Reported results include distinct enrichment toward protease motifs associated with ADAM17, MMP14, MMP9, FAPα, and CTSL1, along with motif order/spacing effects tested using an MSP-MS tandem linker library and counter-screening against thrombin, Factor Xa, and hepsin.
Beyond the screening, the document reports serum/plasma and tissue stability testing and human tumor-cell cleavage and IL-2 induction data for Linker-2 (GPAGLYAQ; SEQ ID NO:195) and Linker-3 (ALFKSSFP; SEQ ID NO:198). The described constructs include antibody/Fc- or scFv-like variable regions and cytokine cargoes, including IL-12, IL-2, IFNa, IFG, and chimeric IL-12/IL-2 constructs, consistent with protease-cleavable pro-protein frameworks.
Claims Coverage
The independent claim coverage centers on a recombinant pro-protein with a SEQ ID NO:198-based protease-cleavable moiety linked to a polypeptide with biological activity, with dependent refinements adding substitution limits, Cathepsin L substrate relationship, attenuation/restoration of activity, blocking moieties, and payload definitions. In total, the claim coverage highlights six inventive features.
Protease-cleavable SEQ ID NO: 198 substrate linked to biologically active polypeptide
A recombinant pro-protein comprising a cleavable moiety that is a substrate for a protease, wherein the cleavable moiety comprises the amino acid sequence of SEQ ID NO: 198 or a functional variant of SEQ ID NO: 198 in which one or two amino acids are substituted in SEQ ID NO: 198, and a polypeptide with biological activity.
Cathepsin L substrate relationship for SEQ ID NO: 198 variant
The functional variant of SEQ ID NO: 198 acts as a substrate for Cathepsin L.
Allowed substitutions constrained for SEQ ID NO: 198
The cleavable moiety corresponding to SEQ ID NO: 198 allows one or two substitutions, except that positions 4 and 5 are not substituted.
Attenuated biological activity before cleavage; not attenuated after cleavage
The recombinant pro-protein has attenuated biological activity, and cleavage of the cleavable moiety by the protease produces a polypeptide with biological activity that is not attenuated.
Specific blocking moiety that specifically binds the biologically active polypeptide
The blocking moiety is a specific blocking moiety comprising either an antibody or antigen-binding fragment, or a receptor ligand-binding portion or ligand-binding fragment, that specifically binds the biologically active polypeptide.
Biologically active polypeptide includes cell surface receptor, CAR, or TCR subunit
The biologically active polypeptide includes a cell surface receptor, a chimeric antigen receptor (CAR) subunit, or a T Cell Receptor (TCR) subunit.
The claim framework is anchored on a SEQ ID NO: 198-based protease-cleavable moiety coupled to a biological-activity polypeptide, with limited substitution patterns, Cathepsin L substrate specificity, cleavage-linked restoration of non-attenuated activity, optional blocking moieties that specifically bind the payload, and receptor/CAR/TCR-related payload types.
Stated Advantages
Cleavage converts an attenuated biological payload into a polypeptide with biological activity that is not attenuated.
Provides attenuated biological activity that becomes not attenuated upon cleavage by the protease.
Uncleaved forms can have improved pharmacokinetics, described as an extended half-life.
After cleavage, the active form can have a shorter half-life.
Documented Applications
Conditionally active recombinant pro-proteins and fusion proteins for activation at target sites such as the tumor microenvironment.
Cytokine/cytokine-receptor system configurations using inducible cytokines including IL-2, IL-12, and IFNγ/IFNα.
Engineered antigen-binding formats including CAR/TCR-related receptor subunits and tetravalent/trispecific antibody formats.
Xenograft model use is described, including tumor growth and survival outcomes.
Reporter/proliferation activity is described using reporter assays for IL-12/IL-2 and IFNα.
Documented recombinant fusion/protein conjugate constructs include antibody/Fc- or scFv-like variable regions and cytokine cargoes such as IL-12, IL-2, IFNa, IFG, and chimeric IL-12/IL-2 formats.
Documented protease-cleavable substrate motif sequence tables include protease-substrate motifs for MMP14 and CTSL1, including GPLGLx variants and ALFKSSPP variants, used as cleavable moieties in the pro-protein framework.
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