Abstract P1-19-02: Innovation in diagnosis and treatment of brain metastases using multifunctional nanomedicines
Authors: JM Saunus, M Lim, S Puttick, P Kalita-de Croft, ZH Houston, ML Jones, MJ Latter, LC Campbell, P Thomas, RL Jeffree, SE Rose, SM Mahler, KJ Thurecht, AM Scott, SR Lakhani
Journal: Cancer Research (2019)

Abstract Systemic therapies have limited efficacy against brain metastases, largely because passive delivery of naked compounds via the bloodstream does not achieve sufficiently high or evenly dispersed intratumoural concentrations. Heterogeneous tissue architecture, abnormal perfusion, hypoxic zones and high interstitial fluid pressure are key factors limiting drug delivery, compounded by patchy blood-tumour-barrier permeability. Also, brain metastases are usually detected late, once patients become symptomatic. We are investigating whether engineered biopharmaceuticals might improve diagnostic sensitivity for earlier detection, as well as therapeutic efficacy and side-effect profiles of existing agents through active tumour targeting, delayed clearance and microenvironment-mediated activation. This study is proceeding with parallel preclinical and clinical tracks. Preclinical aims: (1) Develop and characterise monoclonal antibody (mAb) fragments (scFvs) that target the brain metastasis markers HER2 and HER3; (2) Functionalise polyethylene glycol (PEG)-based nanocarriers with the scFvs, along with imaging agents to facilitate in vivoand ex vivoanalysis of tissue distribution; (3) Functionalise HER2/3-targeted carriers with doxorubicin via an acid-labile hydrazone bond for release in hypoxic environments, or the endosome compartment after internalization. Results to date. His-tagged HER2- and HER3-targeted scFvs based on ligand-binding sequences of clinically-approved mAbs were expressed and purified from Expi293 suspension cultures. Binding affinities are an order of magnitude stronger than parent mAbs (KD 2-8x10E-11M), determined using surface plasmon resonance analysis. The scFvs are cytostatic and moderately cytotoxic in vitro, with IC50s in order of 0.4-1.0μM. HER2 and HER3 scFvs exhibited dose-dependent, additive growth inhibition when used in combination, and induced internalisation of their receptor ligands within 4 hours in SKBr3 cells. Conclusions.The scFvs are strong carrier-tethering candidates in terms of both extracellular and intracellular payload release. Carrier synthesis is currently underway and preliminary in vivo data will be presented. Clinical aims: (1) Develop and characterise 89Zirconium-labelled HER2-targeted PET tracers based on parent mAb and scFv; (2) Compare uptake and retention of the tracers in breast cancer patients with brain metastases; (3) Computationally relate tumour uptake to the administered dose, perfusion, tumour size and HER2 expression; (4) Determine the uptake range within and between patients, and the minimum size for reliable detection. Results to date. The mAb tracer has been synthesised, characterised and labelling processes scaled for clinical production. It is stable in physiologic conditions, retains HER2-binding activity and has a favourable biodistribution profile in NOD-SCID mice bearing BT474 xenografts. Conclusions. Australian regulatory approvals are in place and recruitment for the mAb imaging trial ("BoNSAI") has begun. Preliminary data will be presented. Citation Format: Saunus JM, Lim M, Puttick S, Kalita-de Croft P, Houston ZH, Jones ML, Latter MJ, Campbell LC, Thomas P, Jeffree RL, Rose SE, Mahler SM, Thurecht KJ, Scott AM, Lakhani SR. Innovation in diagnosis and treatment of brain metastases using multifunctional nanomedicines [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P1-19-02.