An engineered ultrahigh affinity bi-paratopic uPAR targeting agent confers enhanced tumor targeting.

Urokinase-type plasminogen activator receptor (uPAR) is overexpressed on tumor cells in multiple types of cancer and contributes to disease progression and metastasis. In this work, we engineered a novel bi-paratopic uPAR targeting agent by fusing the binding domains of two native uPAR ligands: uPA and vitronectin, with a flexible peptide linker. The linker length was optimized to facilitate simultaneous engagement of both domains to their adjacent epitopes on uPAR, resulting in a high affinity and avid binding interaction.

Structural Basis of the Differential Binding of Engineered Knottins to Integrins αVβ3 and α5β1.

Targeting both integrins αVβ3 and α5β1 simultaneously appears to be more effective in cancer therapy than targeting each one alone. The structural requirements for bispecific binding of ligand to integrins have not been fully elucidated. RGD-containing knottin 2.

Emerging Strategies for Developing Next-Generation Protein Therapeutics for Cancer Treatment.

Protein-based therapeutics have been revolutionizing the oncology space since they first appeared in the clinic two decades ago. Unlike traditional small-molecule chemotherapeutics, protein biologics promote active targeting of cancer cells by binding to cell-surface receptors and other markers specifically associated with or overexpressed on tumors versus healthy tissue. While the first approved cancer biologics were monoclonal antibodies, the burgeoning field of protein engineering is spawning research on an expanded range of protein formats and modifications that allow tuning of properties such as target-binding affinity, serum half-life, stability, and immunogenicity.

Engineered knottin peptides as diagnostics, therapeutics, and drug delivery vehicles.

Inhibitor cystine-knots, also known as knottins, are a structural family of ultra-stable peptides with diverse functions. Knottins and related backbone-cyclized peptides called cyclotides contain three disulfide bonds connected in a particular arrangement that endows these peptides with high thermal, proteolytic, and chemical stability. Knottins have gained interest as candidates for non-invasive molecular imaging and for drug development as they can possess the pharmacological properties of small molecules and the target affinity and selectively of protein biologics.

Integrin-Targeting Knottin Peptide-Drug Conjugates Are Potent Inhibitors of Tumor Cell Proliferation.

Antibody-drug conjugates (ADCs) offer increased efficacy and reduced toxicity compared to systemic chemotherapy. Less attention has been paid to peptide-drug delivery, which has the potential for increased tumor penetration and facile synthesis. We report a knottin peptide-drug conjugate (KDC) and demonstrate that it can selectively deliver gemcitabine to malignant cells expressing tumor-associated integrins.

Targeted Drug Delivery with an Integrin-Binding Knottin-Fc-MMAF Conjugate Produced by Cell-Free Protein Synthesis.

Antibody-drug conjugates (ADC) have generated significant interest as targeted therapeutics for cancer treatment, demonstrating improved clinical efficacy and safety compared with systemic chemotherapy. To extend this concept to other tumor-targeting proteins, we conjugated the tubulin inhibitor monomethyl-auristatin-F (MMAF) to 2.5F-Fc, a fusion protein composed of a human Fc domain and a cystine knot (knottin) miniprotein engineered to bind with high affinity to tumor-associated integrin receptors.

A Bioengineered Peptide that Localizes to and Illuminates Medulloblastoma: A New Tool with Potential for Fluorescence-Guided Surgical Resection.

Tumors of the central nervous system are challenging to treat due to the limited effectiveness and associated toxicities of chemotherapy and radiation therapy. For tumors that can be removed surgically, extent of malignant tissue resection has been shown to correlate with disease progression, recurrence, and survival. Thus, improved technologies for real-time brain tumor imaging are critically needed as tools for guided surgical resection.

Balancing cationic and hydrophobic content of PEGylated siRNA polyplexes enhances endosome escape, stability, blood circulation time, and bioactivity in vivo.

A family of pH-responsive diblock polymers composed of poly[(ethylene glycol)-b-[(2-(dimethylamino)ethyl methacrylate)-co-(butyl methacrylate)], PEG-(DMAEMA-co-BMA), was reversible addition-fragmentation chain transfer (RAFT) synthesized with 0-75 mol % BMA in the second polymer block. The relative mole % of DMAEMA and BMA was varied in order to identify a polymer that can be used to formulate PEGylated, siRNA-loaded polyplex nanoparticles (NPs) with an optimized balance of cationic and hydrophobic content in the NP core based on siRNA packaging, cytocompatibility, blood circulation half-life, endosomal escape, and in vivo bioactivity. The polymer with 50:50 mol % of DMAEMA:BMA (polymer "50 B") in the RAFT-polymerized block efficiently condensed siRNA into 100 nm NPs that displayed pH-dependent membrane disruptive behavior finely tuned for endosomal escape.