Report

Teaser, summary, work performed and final results

Periodic Reporting for period 3 - MyNano (Towards the design of Personalised Polymer-based Combination Nanomedicines for Advanced Stage Breast Cancer Patients)

Teaser

Research on anti-cancer therapies has provided little progress towards improved survival rates for patients with metastatic disease. The intrinsic advantages of nanomedicines, and in particular polymer conjugates, can be optimized for applications in rationally-designed...

Summary

Research on anti-cancer therapies has provided little progress towards improved survival rates for patients with metastatic disease. The intrinsic advantages of nanomedicines, and in particular polymer conjugates, can be optimized for applications in rationally-designed targeted combination therapies, a concept that allows enhanced therapeutic efficiency. Early clinical trials involving polymer conjugates demonstrated activity in chemotherapy-refractory patients and reduced drug-related toxicity. However, there is growing concern about patient variability regarding tumor patho-physiology that underlies successful therapeutic outcomes. Specific biomarkers are required to select those patients most likely to show heightened clinical response to these therapies.
The objective of MyNano is to engineer polymer-based combination therapies designed to treat metastatic breast cancer in a patient personalized manner. Therefore, we are working towards the development of novel multicomponent polymer conjugates with precise control over size, shape, solution conformation, multifunctionality and bioresponsiveness. In parallel, we are also studying their structure-activity relationships to underlying proposed mechanisms of action in clinically relevant models. We are employing primary breast cancer patient tissue to generate in vitro cell and in vivo models representing different clinical molecular subtypes. Crosslinked star-shaped polyglutamates obtained by controlled polymerization and self-assembly strategies have already been obtained and used as carriers. These architectures have shown high architectural versatility, excellent biocompatibility, and very interesting targeting properties, including an important accumulation in the lymph nodes with a direct effect in distal metastasis and immunoncological approaches currently being explored. MyNano is investigating new drug combination strategies using current treatments together with inhibitors of tumor-derived exosome release pathways, a phenomenon related to metastasis and resistance mechanisms. The aim is to provide a novel methodological approach that would allow, by reiterative design, the design optimization of next generation nanoconjugates for the treatment of specific metastatic cancer clinical subtypes. MyNano will be a breakthrough as it introduces a paradigm shift in the strategy to design nanomedicines in areas of unmet clinical need.

Work performed

During the first 36 months, the chemistry section of MyNano has developed “WP1. Rational design and controlled synthesis of PGA-based combination conjugate - WP1.1. Polypeptide-based carriers”, related to the synthesis of polyglutamate (PGA)-based architectures, novel hydrophilic and fully biodegradable carriers using benzene-1,3,5-tricarboxamide (BTA)-derivatized cores as macroinitiators to yield star-shaped polyglutamates, a task which we have accomplished. In this regard, due to the success obtained in the development of star-shaped polymers with different initiators and the initial failures in di-block copolymer self-assembly strategy, this latter part has been abandoned, and the star-shaped approach has been pushed forward. We developed methodologies and synthesized several families of star-polyglutamates with different branching and degree of polymerization in a perfectly controlled manner with polymer polydispersity approaching the theoretically-possible optimal level. We performed exhaustive physico-chemical characterization of polymers, derivatives, and conjugates using advanced spectroscopic techniques. Based on this data, we developed a theory of self-assembly of star-polyelectrolytes in dilute aqueous solution. We discovered carrier candidates with a new, not previously described self-assembly behavior. Indeed, this success has been translated into a patent (REF. PCT/EP2016/067554- WO/2017/025298) and a high impact factor paper under the title “Capturing “Extraordinary” Soft-Assembled Charge-Like Polypeptides as a Strategy for Nanocarrier Design” highlighted as cover page (Advanced Materials 2017 29(39),170288)(See Figures for Front page and Abstract). We also note here the publication of relevant review articles - “Smart branched polymer drug conjugates as nano-sized drug delivery systems” and “Polypeptide-Based Conjugates as Therapeutics: Opportunities and Challenges” also highlighted as a cover page (See Figures).

During this period, we have also focused on in vitro experiments in different cell models, devoted to “WP1. Rational design and controlled synthesis of PGA-based combination conjugate - WP1.2. Selection of drug combinations”. This included the characterization of four breast cancer cell lines representing the four different patient subtypes for the screening platform: including their Cathepsin B activity, intracellular pH, as well as estrogen, progesterone, Her2 receptors, glutathione, and exosomes levels. We also performed high throughput screening (HTS) of drug combinations by measuring their ability to promote cell death (MTS assays) and their influence on exosome release. This task is still ongoing, past its deadline, due, in part, to the inclusion of previously uncontemplated but interesting drug combinations. Nonetheless, we have discovered many synergistic drug combinations and we already selected specific drug duos for each cell line.

Furthermore, we have purified tumor exosomes from the four different metastatic human breast cancer cell lines treated with drugs and control conditions by serial differential ultracentrifugation. Exosome concentration and size from drug-treated and non-treated breast cancer cells have been determined by Nanoparticle Tracking Analysis and we are currently assessing protein content towards WP2.2. To this end, we are also working towards the optimization of a HTS process to discover new exosome biogenesis/release inhibitors combining InCell and Alpha-Screen technologies, which represent a less time-consuming and cheaper alternative to ultracentrifugation. We have also begun to test drug combinations in breast cancer-spheroids screens, as a more reliable measure of the potential in vivo response to our synergistic drug combinations.

Towards “WP1. Rational design and controlled synthesis of PGA-based combination conjugate - WP1.3. Synthesis of polymer-based combination conjugates/Linking chemistry”, we have developed effective biodegrad

Final results

The design of our new BTA initiated star-shaped polymers and the patents/manuscripts associated can be viewed as progress beyond the state of the art and we hope the advanced characteristics that these polymer constructs exhibit will allow us to effectively target primary tumor, target lymph nodes, and inhibit metastasis. This could provide a revolution in the treatment of various forms of breast cancer and we hope our upcoming in vitro and in vivo work and the associated amalgamation of the biological and chemical arms of the project will synergize to create a truly effective treatment option. To this end, we believe that the inclusion of the study of exosomes also has the potential to have impact towards the ultimate aim of treating metastatic breast cancer.

Furthermore, our systematic outlook, with deep analysis and understanding of conjugate structure-activity relationships with biological environment, will promote the market translation of these anti-cancer agents and will advance our understanding of the molecular basis of life-threatening diseases such as metastatic breast cancer. We believe the MyNano project will contribute enormously towards personalized treatment of metastatic breast cancer.

In the next period, we will finalize the choices of drug combinations, linker use, and work towards the construction of advanced combination conjugates and then move towards in vitro and, importantly, in vivo analysis, including in patient-derived models of human breast cancer (organoids and PDXs models). At the end of the project, we expect to have discovered synergistic combinations for the treatment of different human breast cancer subtypes, working towards personalized therapy-patient and the prevention of metastasis. At the same time we expect to identify relevant biomarkers and descriptors that would allow, on one hand, to accelerate the process of nanoconjugate design and on the other hand to stratify patients, at least from the triple negative breast cancer subtype, that would benefit from treatment with the designed nanoconjugates.

Moreover, we expect to contribute to the scientific community with our HTS platform to test exosome inhibitors, which can be integrated into standardized large-scale drug test routines. The characterization of spheroid models both human cell lines derived and organoids from human tissue patients will allow us a better understanding regarding the tumor microenvironment and the design of spheroid-based HTS will help us to identify effective drugs against BC subtypes.

Website & more info

More info: http://vicentresearchlab.com.