To address the challenge of drug accumulation and penetration at the tumor site(s), herein we describe a first-in-class nanocarrier containing 24 copies each of two bioactive peptides (BAPs) genetically fused in frame to the 24 N-termini of a human ferritin H-type construct, named THE-10. The two BAPs are specific for PD-L1 and integrin αVβ3/αVβ5 plus Neuropilin (iRGD) respectively, conferring immune checkpoint blockade and drug-internalization properties. In turn, the THE-10 backbone brings 48 BAPs contiguous for synergism, prolonged blood half-life, and release into the tumor microenvironment upon conditional cleavage of a metalloprotease-sensitive site. Predicted THE-10 multitasking activity was experimentally supported as follows. Size-exclusion chromatography and surface plasmon resonance demonstrated BAP cleavage/release and receptor binding (nanomolar KD). Live-cell/time-lapse imaging demonstrated 4-fold-increased internalization of naked therapeutic antibodies, mirrored by enhanced cytotoxicity of the corresponding Antibody-Drug Conjugate. Slight antitumor effects were observed in vivo by treating immune checkpoint-sensitive syngeneic mouse colorectal model with THE-10 alone. Drug boosting was instead considerable on colorectal and pancreatic tumor allografts when THE-10 was co-administered with both small and large chemotherapeutic agents, outperforming the original iRGD cyclic peptide. Thus, THE-10 may enhance target therapy, chemotherapy and immunotherapy altogether, e.g. it candidates as a multitasking, all-round, antineoplastic therapy booster.

Center for Advanced Studies and Technology Italy

Department of Biochemical Sciences Sapienza University Rome Italy

Department of Carbohydrates and Cereals Faculty of Food and Biochemical Technology University of Chemistry and Technology Prague Technická 5 166 28 Prague Czech Republic

Department of Cell Biology Faculty of Science Charles University Prague Czech Republic

Department of Innovative Technologies in Medicine and Dentistry University of Chieti Pescara Chieti Italy

Dipartimento di Scienze Biotecnologiche di Base Cliniche Intensivologiche e Perioperatorie Università Cattolica del Sacro Cuore Largo F Vito 1 Rome Italy

Fondazione Policlinico Universitario A Gemelli IRCCS Largo Agostino Gemelli 8 00168 Rome Italy

Institute of Histology and Embryology Faculty of Medicine University of Ostrava Syllabova 19 Ostrava Vítkovice Czech Republic

Institute of Molecular Biology and Pathology Italian National Research Council IBPM CNR Rome Italy

Laboratory of Immunotherapy Institute of Microbiology Czech Academy of Sciences Prague Czech Republic

Thena Biotech Latina Italy

Translational Oncology Unit IRCCS National Cancer Institute Regina Elena Via Elio Chianesi 53 Rome Italy

UOSD Medicina di Precisione in Senologia Fondazione Policlinico Universitario A Gemelli IRCCS Largo Agostino Gemelli 8 Rome Italy

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