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 vivo and ex vivo analysis 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 (K_D 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.

Evaluation of the in vivo fate of ultrapure alginate in mice model Introduction: Alginate is an anionic co-polymer composed of (1, 4)-linked β-D-mannuronate (M) and α-L-guluronate (G) residues and is sourced from nature (eg. seaweed)[1]. This polymer has been comprehensively studied for use in various biomedical applications because of its reported biocompatibility, non-toxicity and mild gelation property[2],[3]. Mammals lack the enzyme alginase which is required for the biodegradation of the polymer chains of alginate[4]; however, thus far only one study has investigated the bio-distribution profile of radiolabeled chemically modified alginate (propylene glycol alginate-tyrosinamide)[2]. This study showed that the in vivo fate of modified alginate following systemic administration is dependent on the molecular weight and the higher molecular weight (> 48000 g/mol) alginate fragments remained in circulation with no significant accumulation in organs until 48 hours[3]. The lack of a bio-distribution study on pure alginate and of studies involving prolonged time points on the biodistribution profile of alginate-based polymers in general is a clear knowledge gap in the literature, as this information is crucial for alginate to be used in biomedical applications. Thus the present study which investigates the bio-distribution of cyanine 5-amine conjugated ultrapure alginate is highly topical. Materials and Methods: High purity alginate from FMC BioPolymer was conjugated with cyanine 5-amine using carbodiimide chemistry at a loading of 14 mg cyanine 5-amine/g alginate-dye conjugate. This polymer was administered to Balb/c mice through the tail vein, and imaged using a dual fluorescent X-ray imaging system (Bruker MsFx pro) at 1 hour, 2 hour, 1 day, and 2 days post sample administration under isoflurane anesthesia. At each time point, subsets of animals were euthanized and the organs (spleen, liver, heart, lungs, brain and kidney) were harvested for ex vivo imaging and flow cytometry analysis. Cyanine 5-amine was used as a control. Results and Discussion: From the imaging results at 1st hour, a rapid clearance of some of the alginate through the kidneys (e.g. smaller molecular weight alginate fragments) and liver (e.g. molecular weight alginate fragments) could be observed. Conclusion: The results of this in vivo biodistribution study will form the foundation for future research in the translation of alginate-based materials into different applications such as drug delivery, tissue engineering and wound healing. One of the authors A. Anitha is grateful to The University of Queensland, Australia for providing a postdoctoral research fellowship for carrying out this research work.

References:

[1] Lee KY; Mooney DJ (2012) ProgPolym Sci 37, 106.

[2] Al-Shamkhani A; Duncan R (1995) J Bioactive Compatible Polym 10, 4.

[3] Kolambkar YM et al (2011) Biomaterials 32, 65. [4] Kuo CK; Ma PX (2011) Biomaterials 22, 511.

Development of sensitive molecular imaging agents is one of the major challenges for advancing targeted imaging using MRI. The development of imaging agents that can be directly imaged using MRI holds particular interest since these have the potential to increase sensitivity while being able to directly probe a reaction or process that occurs in vivo. In general, directly observable MR probes are very sensitive to their local environment and as such, can be manipulated as switchable agents in response to a number of endogenous or exogenous stimuli.

In this presentation we report on the development of sensitive hyperbranched polymeric ¹⁹F MRI contrast agents that combine controllable functionality, ability for cell-targeting in vivo and low cytotoxicity. More importantly, owing to the strong dependence of the ¹⁹F MR properties on the local environment, such systems are amenable to the development of responsive probes that give real-time assessment of biological function. We demonstrate this effect with some examples of systems that show a change in signal intensity based on its location in the body. Finally, the application of ¹⁹F MRI as a means to potentially quantify drug delivery in vivo is discussed, with an example that highlights the potential of this technique, but also the challenges that remain.

Polymer and nanoparticle-based devices have evolved to significantly enhance therapeutic efficacy. However, in order to be truly effective, these polymeric devices must maintain their physical and chemical integrity under physiological conditions this can only be achieved by developing a strong understanding of the fundamental properties of the nanomaterial-delivery system, in addition to identifying and successfully delivering new therapies. Central to the development of these future therapeutic platforms, is the field of theranostics. This is the premise that future medical devices need to be capable of delivering a therapeutic dose to the correct site within the body, but must also possess mechanisms for online diagnosis, monitoring of disease progression and visualisation of drug delivery, release and efficacy of treatment. Such materials require significant advancements in chemistry, materials science and engineering such that the nanomedicine is complementary with the biological milieu.

While there are countless examples of polymer or nanoparticle systems that show efficacy in animal models, the ability to rationally-optimise the materials is hindered by the inability to directly assess the behaviour of the materials in vivo. For example, improvements in administration for most biologically-targeted polymeric nanomaterial systems are achieved by monitoring efficacy in animals rather than monitoring the fundamental behaviour of the nanomaterial itself (e.g. measuring efficacy, rather than quantifying how a change in material properties results in a biological response). In this presentation, we describe recent efforts to develop self-reporting nanomedicines for a truly closed-loop approach to medicine, where nanomaterial behaviour is monitored in real-time using molecular imaging as a function of therapy. These materials are based on architectural polymers that form a scaffold allowing combination of imaging and therapeutic modalities. Molecular imaging provides a route to validate how structure and property affects function in animals.

Nanomedicines are a promising addition to the arsenal of new cancer therapies. During development, scientists must precisely track their distribution in the body, a task that can be severely limited by traditional 2D displays. With its stereoscopic capacity and real-time interactivity, virtual reality (VR) provides an encouraging platform to accurately visualize dynamic 3D volumetric data. In this research, we develop a prototype application to track nanomedicines in VR. This platform has the potential to enhance data assessment, comprehension and communication in preclinical research which may ultimately influence the paradigm of future clinical protocols.