SUMMARY: Neuroblastoma (NB) is the most common cancer diagnosed in children younger than one year old and is the most frequent solid tumor in children < 5 years of age. Those with high-risk disease have only a 40- 50% survival rate. For locally advanced and metastatic disease, high-dose chemotherapy is the primary modality of treatment. Irradiation of the primary tumor bed is commonly undertaken after surgery for those with the MYC proto-oncogene family that encodes basic helix-loop-helix transcription factor N (MYCN) amplified tumors, correlated with high-risk cancers and poor prognosis. Despite the introduction of immunotherapies, the five-year survival remains below 50%. NanoPharmaceuticals LLC is developing a novel dual targeting ligand for the treatment of high-risk NB through a combination of a plasma membrane integrin αvβ3 antagonist and uptake of a metabolically stable norepinephrine structural analogue via high norepinephrine transporter (NET) expression typical of NB and other neuroendocrine cancers. BG-P-TAT is our novel chemical entity containing triazole tetrac (TAT), a derivative of L-thyroxine, and benzylguanidine (BG), covalently bound to a 1600 MW polyethylene glycol (PEG), P1600-TAT. P1600-TAT has a substantial panel of anticancer actions (antiproliferation, pro-apoptosis, anti- angiogenesis) mediated by its binding to integrin αvβ3, a plasma membrane protein preferentially expressed and activated by cancer cells. BG binds to the norepinephrine transporter (NET) expressed on the surface of neuroendocrine cells, conferring additional binding affinity and tumor specificity. Our in vivo studies of tumor cell targeting showed that BG-P1600-TAT (NanoPharma 855: NP855) had 70-95% tumor shrinkage in two cell lines. This Direct to Phase II SBIR will establish IND-enabling therapeutic efficacy and safety of NP855 following scaled up manufacturing. Aim 1. Evaluation of efficacy and PK of NP855 using scaled up product. The objective is to confirm in vitro and in vivo efficacy in NB cells and tumors following scaled up manufacturing of non-GMP drug material. Tasks include (1.1) Production of NP855 following process transfer, analytical evaluation, lab demo batch production, and reference standard qualification; (1.2) In vitro confirmation of integrin binding affinity and NET engagement by measuring uptake kinetics in NB cells versus normal cell line; (1.3) In vivo evaluation of anti-cancer efficacy (tumor growth, NP855 tumor uptake, tumor histopathology for cancer viability and anti- angiogenic efficacy using anti-CD31 tumor immunostaining) and PK in mice. Goals: (a) Production of 500g of drug substance for formulation of the drug product (DP) with reference standard qualification; (b) Confirmation of efficacy findings using scaled up DP. Aim 2. Perform IND-enabling toxicology evaluations. We will conduct non-GLP (14 days) and GLP (28 days) dose range finding and repeated dose studies in rodent (rat) and non- rodent (minipig) as well as in vitro assessments of solubility, stability, protein binding, transporter and cytochrome inhibition. In vivo central nervous system, respiratory, and cardiovascular assessments will be completed. Goal: Establish safety and toxicokinetics for NP855 in two species for IND preparation.

NARRATIVE: Neuroblastoma is the most common cancer diagnosed in children younger than one year of age and, for those with high-risk disease, results in only a 40-50% survival rate. Despite progress in treatment for the disease, survival rates have remained relatively unchanged. NanoPharmaceuticals LLC proposes a novel treatment for high-risk neuroblastoma using a drug-drug conjugate that binds to two highly expressed targets in neuroblastoma tumors, thereby providing the potential for improving survival in children suffering from this disease.