Therapeutic nanoconjugates comprising Stefin A and uses thereof
Inventors
VILLAVERDE CORRALES, ANTONIO • Vázquez Gómez, Esther • Unzueta Elorza, Ugutz • Mangues Bafalluy, Ramón • CÉSPEDES NAVARRO, María Virtudes • CASANOVA RIGAT, ISOLDA • LÓPEZ LAGUNA, Hector
Assignees
Universitat Autonòma de Barcelona • Fundacio Institut de Recerca de lHospital de La Santa Creu i Sant Pau • Centro de Investigación Biomédica en Red
Interested in licensing this patent?
MTEC can help explore whether this patent might be available for licensing for your application.
Publication Number
US-12540172-B2
Publication Date
2026-02-03
Expiration Date
Abstract
The present invention relates to nanostructured conjugates, more specifically to nanostructured fusion proteins suitable for the selective delivery of their conjugated therapeutic agents to specific cell and tissue types. It also relates to nanoparticles comprising such nanostructured proteins and the therapeutic uses thereof.
Core Innovation
The disclosed invention relates to therapeutic nanoconjugates based on nanostructured Stefin A-based fusion proteins conjugated to auristatin for selective delivery. The fusion architecture includes a polycationic peptide, an intervening polypeptide region that is Stefin A, and a positively charged amino acid-rich region. The Stefin A is human Stefin A and can include a V48D mutation and/or a G4W mutation, with amino acids 71 to 73 replaced by a heterologous peptide insertion consisting of NGP.
The polycationic peptide comprises a sequence selected from SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, or a functionally equivalent variant having at least 70% sequence identity with SEQ ID NO: 5. The intervening Stefin A region is conjugated to at least one auristatin molecule, including monomethylauristatin E. The positively charged amino acid-rich region is defined to support the nanostructured fusion protein design, including embodiments with a polyhistidine region.
Nanoparticles are formed that include multiple copies of the fusion protein and can have a diameter between about 10 and 100 nm. The disclosure includes representative embodiments such as T22-STM-H6-Aur nanoconjugates and T22-GFP-H6-Aur, with characterization of drug-to-nanoparticle loading and in vivo comparisons. The reported results describe reduced leukemia dissemination and increased mitotic catastrophe for auristatin-conjugated constructs versus unconjugated controls in disseminated CXCR4+ AML and subcutaneous colorectal cancer models.
Claims Coverage
The independent claims include one independent claim reciting a fusion protein with a specific architecture of polycationic peptide, intervening human Stefin A region with defined insertion and mutations, and a positively charged amino acid-rich region, with auristatin conjugation. Dependent claims further narrow specific features including the positively charged region composition, auristatin identity, terminal orientation, peptide linkers, and nanoparticle size with multiple copies.
Auristatin-conjugated Stefin A fusion protein with polycationic peptide and positively charged amino acid-rich region
A fusion protein comprising a polycationic peptide comprising a sequence selected from SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and a functionally equivalent variant thereof having at least 70% sequence identity with SEQ ID NO: 5, an intervening polypeptide region which is Stefin A, wherein the Stefin A is human Stefin A and wherein amino acids 71 to 73 are replaced with a heterologous peptide insertion consisting of NGP and wherein the Stefin A comprises a V48D mutation and/or a G4W mutation, and a positively charged amino acid-rich region, wherein the intervening polypeptide region is conjugated to at least one auristatin molecule.
Positively charged polyhistidine region defined length
The fusion protein wherein the positively charged amino acid-rich region is a polyhistidine region made up of between 2 and 10 contiguous histidine residues.
Monomethylauristatin E as the auristatin component
The fusion protein wherein the auristatin is monomethylauristatin E.
Terminal orientation of polycationic peptide and positively charged amino acid-rich region
The fusion protein wherein the polycationic peptide and the positively charged amino acid-rich region are positioned at opposite termini.
First peptide linker sequence options
The fusion protein wherein the first peptide linker includes either the GGSSRSS sequence (SEQ ID NO:35) or the GGGNS sequence (SEQ ID NO:36).
Nanoparticle comprising multiple copies with diameter 10 to 100 nm
A nanoparticle including multiple copies of the fusion protein and having a diameter between 10 and 100 nm.
Across the independent claim and its dependent refinements, the core inventive coverage centers on an auristatin-conjugated fusion protein that combines a defined polycationic peptide, a human Stefin A intervening region with NGP insertion and V48D and/or G4W mutations, and a positively charged amino acid-rich region, with further claim narrowing to polyhistidine length, monomethylauristatin E, terminal orientation, defined linker sequences, and nanoparticle size and multiplicity.
Stated Advantages
Reduced leukemia dissemination for auristatin-conjugated constructs versus unconjugated controls.
Increased mitotic catastrophe for auristatin-conjugated constructs versus unconjugated controls.
Documented Applications
Use in disseminated CXCR4+ AML models, including evaluation of leukemia dissemination and mitotic catastrophe for auristatin-conjugated constructs versus unconjugated controls.
Use in a subcutaneous colorectal cancer model, including evaluation of mitotic catastrophe for auristatin-conjugated constructs versus unconjugated controls.
Interested in licensing this patent?