Periodic Reporting for period 4 - MyNano (Towards the design of Personalised Polymer-based Combination Nanomedicines for Advanced Stage Breast Cancer Patients)
Reporting period: 2020-01-01 to 2020-12-31
Research into anticancer therapies has provided little progress towards improved survival rates for patients with metastatic disease. There exists growing concern about patient variability regarding the tumor pathophysiology that underlies therapeutic outcomes. Nanomedicine can be optimized for use in rationally-designed targeted combination therapies, a concept that enhances therapeutic efficiency. Biomarkers can select patients likely to respond to these therapies. MyNano's objective is to engineer polypeptide-based combination therapies for personalized metastatic breast cancer (BCa) treatment with interesting targeting properties for distal metastasis treatment and immunoncological approaches. In parallel, to stablish critical physicochemical design features and functional biomarkers towards safe and efficient conjugates, understanding the underlying mechanisms of action in relevant preclinical models representing various BCa subtypes. Novel technological approaches have also been developed, including HTS approaches to understand metastasis progression, while advanced physicochemical techniques have been employed to characterize conjugates in physiological fluids to aid clinical translation. This platform has applications in metastatic BCa therapy, immunotherapy (thanks to lymphotropism), and central nervous system (CNS) disorders including brain metastasis (ability to cross the blood-brain barrier).
We have synthesized polyglutamate (PGA)-based architectures as novel hydrophilic and biodegradable carriers using BTA-derivatized cores as macroinitiators to yield star-shaped PGAs. We developed methodologies and synthesized several families of star-PGAs with different branching and degree of polymerization in a controlled manner with low polydispersity. We performed physicochemical characterization of polymers, derivatives, and conjugates using advanced spectroscopic techniques and developed a self-assembly theory of star-polyelectrolytes in dilute aqueous solutions. We discovered carrier candidates with a not previously described self-assembly behavior. Indeed, this success has been translated into a patent (WO/2017/025298-licensed and in exploitation), a high impact factor publication highlighted as cover page (AdvMat2017), papers describing the formation of star-shaped PGAs with complex architectures (PolymChem2020 & ChemSci2021 Submit.), and two PhD theses with an Extraordinary Award (A Duro-Castaño) and an excellent grade (O Zagorodko).We also note the publication of reviews (BiomatSci2015, MatHorizons2019, WIREsNanoNanobi2019), with the two latter demonstrating the applicability of our polypeptide carriers in immunotherapy and neurological disorders (cover page).The lymphotropism of our polypeptide carriers, apart from apparent applications in the treatment of metastasis, has led to their exploration in immunotherapy. Indeed, this data provided the basis for the ERCPoC Polymmune on melanoma and La Caixa NanoPanther project on pancreatic cancer. After derivatization, we have also explored our carriers for brain metastasis (PhD Thesis F Rodríguez-Otormín, June2021) and CNS pathologies (SciAdv2021). Adequate drug selection and rationale linking chemistries also represent crucial parts of the conjugate design. We have developed and fully characterized 2D and 3D cultures of representative BCa cell lines and performed high throughput screening (HTS) of drug combinations by measuring cell death and exosome release. Exosome characterization via NTA and proteomic analysis has contributed to the identification of biomarkers. We also established an HTS process that combined InCell and AlphaScreen technologies as an easier means of discovering new exosome biogenesis/release inhibitors (manuscript in prep). We have identified synergistic drug combinations for each BCa subtype. We note that our screening platform forms part of the ERIC-EUOpenscreen. We developed effective biodegradable linking chemistries for selected drug combinations to control targeting and release kinetics depending on microenvironmental factors. This work has derived a patent (PCT/EP2020/058940),high-impact articles (Biomaterials 2018, AdvFuncMat 2018), excellent graded PhD thesis (JJ Arroyo-Crespo 2018), several reviews (ADDR2020; IntRevCellMolBiol2019, MacromolBiosci2017) and the current development of at least three more articles regarding different drug combinations and the impact of experimental design in their development. With our first family of combination conjugates, we sought to explore accurate SAR to identify physicochemical descriptors and functional biomarkers through evaluations in the above-described models. We have found that trafficking proteins, intracellular pH, cathepsin B levels, and redox potential correlate with conjugate activity. We have also established a biobank of patient-derived BCa organoids and have started to evaluate selected conjugates (PhD thesis P Boix). Exosomal protein analysis provided interesting biomarkers to aid conjugate development for BCa treatment. We also performed in-depth physico-chemical characterization steps to understand our systems' behavior in complex physiological fluids (PhD thesis S Đorđević). We note relevant reviews (CurrPharmDes2016 cover page, JPersMed2018, ADDR2021). We have evaluated safety/biodistribution/PK/pharmacological activity of selected conjugates in two metastatic orthotopic TNBC models (IntJCancer 2019) for anticancer and antimetastatic activity including brain metastasis through a collaboration with Valiente group at CNIO (PhD thesis F Rodriguez). Our attempts to generate PDX models have not succeeded to this point, although our conjugates' success in other in vivo and 3D models provides a solid platform for their successful evaluation in the future. Our results have been disseminated via over 100 oral and poster presentations (vicentresearchlab.com) YouTube, social media, and other significant interactions, such as Pint of Science and European Researchers Night events.
The design of our new BTA-initiated star-shaped polymers and the patents/manuscripts associated with the project can be viewed as progress beyond state of the art, as the advanced characteristics that these polymer constructs display allow to effectively target BCa primary tumor, lymph nodes, and inhibit metastasis, important role here of the exosome modulation approach. Moreover, our HTS strategies even in spheroid based phenotypic assays capable to evaluate exosome inhibitors can be integrated into standardized large-scale drug test routines and help us identify effective drugs against BCa subtypes. The characterization of BCa organoids from human tissue patients has allowed a better understanding of the tumor microenvironment. Our systematic outlook, with in-depth analysis and understanding of conjugate SARs within the biological environment in preclinically relevant models, will foster these anti-cancer agents' market translation and open new applications for our technological platform. The findings have opened new avenues for the use of polypeptides as delivery systems. Indeed, industrial concerns have begun to explore our platform in gene therapy and immunotherapy, which are expected to solve specific unmet needs. We consider MyNano as a breakthrough as it introduces a paradigm shift in the strategies employed to design nanomedicines in areas of unmet clinical need.