Nucleic acids encoding a Tn3 scaffold comprising a CD40L-specific monomer subunit
Inventors
Coyle, Anthony • Baca, Manual • Thisted, Thomas • Drabic, Stacey • Grinberg, Luba • Novarra, Shabazz • Oganesyan, Vaheh • Herbst, Ronald • Spencer, David Kenneth
Assignees
Interested in licensing this patent?
MTEC can help explore whether this patent might be available for licensing for your application.
Publication Number
US-11104720-B2
Publication Date
2021-08-31
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 relates to Tenascin-3 (Tn3) FnIII-domain protein scaffolds and engineered variants that bind CD40L. A Tn3 scaffold architecture is defined by a CD40L-specific monomer subunit comprising 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 loop regions specified and/or engineered using defined SEQ ID NOs.
Engineered Tn3 scaffold variants include tandem bivalent formats in which two CD40L-specific monomer subunits are linked, including topologies where the monomers are connected directly or by peptide linkers. Additional scaffold designs include fusions and conjugates where the Tn3 scaffold protein is combined with heterologous moieties such as biotin, albumin, human serum albumin (HSA), an HSA FcRn binding portion, antibody or antibody-related moieties, epitope tags, enzymes, ligands, receptors, binding peptides, non-FnIII scaffolds, and cytokines.
The engineered Tn3 scaffolds are described as producing functional effects on CD40L/CD40 signaling, including blocking and/or disrupting CD40L binding to CD40 and CD40-mediated signaling. The disclosed constructs also include scaffold variants generated using loop randomization and restricted randomization approaches, including phage display panning against human CD40L and CD40L-CHO.
Claims Coverage
The claim coverage includes two independent claims, centered on isolated nucleic acid molecules encoding a defined CD40L-specific Tn3 scaffold monomer architecture and a defined CD40L-specific Tn3 scaffold fusion protein with heterologous moieties. Across these claims, the inventive features are defined by SEQ ID NO-based loop regions within a seven beta-strand Tn3 FnIII scaffold, with fusion protein coverage extending to a listed heterologous moiety.
Isolated nucleic acid encoding a CD40L-specific Tn3 scaffold monomer subunit defined by seven beta strands and six loop regions
An isolated nucleic acid molecule encoding a Tn3 scaffold comprising a CD40L-specific monomer subunit with seven beta strands designated A, B, C, D, E, F, and G, and six loop regions designated AB, BC, CD, DE, EF, and FG, where AB is SEQ ID NO:4; CD is SEQ ID NO:6; EF is SEQ ID NO:8; and BC, DE, and FG loops are selected from enumerated SEQ ID NO alternatives.
Isolated nucleic acid encoding a CD40L-binding Tn3 scaffold fusion protein with a heterologous moiety
An isolated nucleic acid molecule encoding a Tn3 scaffold fusion protein comprising a Tn3 scaffold protein and a heterologous moiety, where the Tn3 scaffold protein includes a CD40L-specific monomer subunit with seven beta strands (A–G) and six loop regions (AB–FG) defined by specified SEQ ID NO sequences, and where the encoded Tn3 scaffold specifically binds to CD40L; the heterologous moiety is one selected from biotin, albumin, HSA, an HSA FcRn binding portion, antibody or antibody-related moieties, non-FnIII scaffold, epitope tag, enzyme, ligand, receptor, binding peptide, and cytokine.
Overall, the claim coverage is directed to nucleic acids encoding CD40L-binding Tn3 scaffold architectures with SEQ ID NO-defined loop region compositions, and to fusion-protein formats that attach heterologous moieties to the CD40L-specific Tn3 scaffold.
Stated Advantages
Improved CD40L binding by engineered variants from affinity maturation libraries.
Maintained thermodynamic stability of the engineered Tn3 variants.
Mostly monodisperse SEC profiles for engineered variants.
Blocks or disrupts CD40L binding to CD40 and/or CD40-mediated signaling.
Functional potency through PBMC assays and neutralization/disruption of the CD40L/CD40 axis in described assays.
Improved serum half-life for engineered HSA fusions via HSA FcRn-related strategies, including mouse and monkey PK half-life changes.
Enables engineered scaffold formats including tandem bivalent scaffolds and scaffold fusion proteins with heterologous moieties.
Provides structurally characterized CD40L:Tn3 complex structures using protein crystals and X-ray crystallography.
Documented Applications
Functional potency testing in PBMC assays, including CD40L pathway functional readouts (described as NFκB reporter, CD86 upregulation, and T/B co-culture inhibition).
Neutralization and disruption assays for CD40L/CD40 axis, including in vivo disruption described as germinal center suppression in mice.
In vivo disruption of CD40L/CD40 axis in cynomolgus monkeys using KLH TDAR, with suppression described.
Use of engineered Tn3 scaffold fusions, including HSA fusions and linker modifications, to affect serum half-life (mouse and monkey PK).
Structural epitope mapping and co-crystal crystallography of CD40L with Tn3 monomers to identify interaction residues and binding sites.
Therapeutic use relating to SLE (systemic lupus erythematosus) and autoimmune and inflammatory disorders.
Use in generating protein scaffolds and engineered variants that bind CD40L for inhibition or blocking of CD40L binding to CD40 and/or CD40-mediated signaling.
Interested in licensing this patent?