CD40L-specific TN3-derived scaffolds and methods of use thereof
Inventors
Coyle, Anthony • BACA, MANUEL • Thisted, Thomas • Drabic, Stacey • Grinberg, Luba • Novarra, Shabazz • Oganesyan, Vaheh • Herbst, Ronald • Spencer, David Kenneth
Assignees
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Publication Number
US-10000553-B2
Publication Date
2018-06-19
Expiration Date
Abstract
The present invention provides Tenascin-3 FnIII domain-based scaffolds that specifically bind to CD40L. The invention further provides engineered variants with increased affinity for the target. The present invention is also related to engineered scaffolds as prophylactic, diagnostic, or therapeutic agents, in particular for therapeutic uses against SLE and other autoimmune diseases and conditions.
Core Innovation
The invention provides a CD40L-specific Tn3 scaffold defined by a monomer subunit having seven beta strands designated A, B, C, D, E, F, and G and six loop regions designated AB, BC, CD, DE, EF, and FG. The scaffold specifically binds CD40L, and specific loop sequence selections are used for the AB, BC, CD, DE, EF, and FG loops, including defined SEQ ID NO assignments and groups of SEQ ID NO options.
The document describes tandem-bivalent architectures, engineered variants, and affinity maturation of CD40L-specific Tn3 scaffolds. It also describes functional characterization of improved binding and potency in CD40L functional assays, with inhibition of CD40L binding to CD40 and disruption of CD40-mediated signaling.
Further description addresses scaffold stabilization and persistence enhancement through engineered disulfide bonds, terminal modifications, and half-life extension strategies using HSA, PEG, and Fc-based components, including FcRn-binding portions. Structural work with co-crystallization and residue-level contact mapping supports epitope/groove binding and higher-affinity or tandem design.
Claims Coverage
The independent claim covers 1 inventive feature: a CD40L-specific Tn3 scaffold defined by a monomer subunit with seven beta strands and six loop regions where the loops are constrained to specific SEQ ID NO selections, and where the scaffold specifically binds CD40L. Dependent claims add tandem architecture, functional disruption of CD40L–CD40 interaction/signaling, and fusion or conjugation features with named moieties such as PEG, HSA, variant HSA, and Fc-related components.
Cd40l-specific Tn3 monomer subunit with defined beta strands and loop sequence selections
A Tn3 scaffold comprising a CD40L-specific monomer subunit having seven beta strands designated A, B, C, D, E, F, and G and six loop regions designated AB, BC, CD, DE, EF, and FG, with the AB loop comprising SEQ ID NO: 4, the BC loop comprising selected SEQ ID NO options, the CD loop comprising SEQ ID NO: 6, the DE loop comprising selected SEQ ID NO options, the EF loop comprising SEQ ID NO: 8, and the FG loop comprising selected SEQ ID NO options, wherein the Tn3 scaffold specifically binds to CD40L.
Tandem cd40l-specific monomer subunits
The Tn3 scaffold includes two CD40L-specific monomer subunits connected in tandem.
Disruption of CD40L-CD40 interaction or CD40-mediated signaling
The Tn3 scaffold binds CD40L and blocks or disrupts CD40L binding to CD40 and/or CD40-mediated signaling.
Fusion or conjugation with a heterologous moiety
At least one CD40L-specific monomer subunit is fused or conjugated to a heterologous moiety including PEG, biotin, albumin/HSA and HSA FcRn binding portions, antibody-related components, targeting ligands, epitope tags, enzymes, cytokines, and drug, toxin, cytotoxic, imaging, or radioactive elements.
Variant HSA with specific sequence identity
The scaffold includes HSA as a variant HSA with the amino acid sequence of SEQ ID NO: 133 within the Tn3 scaffold.
Alternative loop sequence assignments for BC, DE, and FG
The Tn3 scaffold is defined by specific amino-acid sequence assignments for the BC loop, DE loop, and FG loop using multiple alternative combinations of SEQ ID NO options for those loop sequences.
Across the claims, the core coverage is a CD40L-specific Tn3 scaffold defined by constrained loop and strand SEQ ID selections that specifically binds CD40L, with further narrowing through tandem architecture, functional blocking or disruption of CD40L–CD40 interaction and/or CD40-mediated signaling, and incorporation of heterologous fusion or conjugation moieties such as PEG, HSA, variant HSA, and Fc-related components.
Stated Advantages
Extend serum half-life.
Modulate immune interactions.
Alter effector function/ADCC via Fc region mutations.
Increase plasma half-life using albumin/HSA variants.
Improved binding and/or stability via tandem valency and affinity maturation as described.
Inhibition of CD40L–CD40 binding and CD40L–CD40 signaling as described.
Enhanced scaffold persistence and half-life using HSA, PEG, and Fc-based half-life extension strategies as described.
Potency boost from a fortuitous N-terminal constant-region K4E mutation.
Reduced O-glycosylation effects through linker reengineering.
Potency retention for human constructs with serum half-life extension via albumin fusions.
Reduced platelet aggregation for human Tn3 scaffolds.
Documented Applications
Pharmaceutical compositions and uses are described in connection with CD40L-binding Tn3 scaffold constructs.
Diagnostic and imaging uses are described for the disclosed constructs.
Therapeutic uses are described for the disclosed CD40L-binding Tn3 scaffold constructs.
Assays and characterization contexts are described for CD40L-binding and CD40L activity modulation.
Therapeutic, prophylactic, and diagnostic uses for autoimmune diseases, notably systemic lupus erythematosus (SLE), as described.
Targeting approaches addressing platelet aggregation and thromboembolic safety concerns as described.
Inhibition of NFκB, blockade of cellular co-stimulation, inhibition of B-cell proliferation and plasma differentiation, and disruption of the CD40:CD40L axis in vivo.
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