WP4: Functional proof of principle of MAPS
- Evaluation of the biological activity of MAPS-based nanoparticles on cells.
- In vivo stability and trafficking of polypeptide-coated vesicles.
- Evaluation of the therapeutic efficacy of selected designed nanoparticles in melanoma mouse model.
In Task 4.1, the biological activity of designed nanoparticles will be tested for the selectivity in cell targeting, cell entry, presentation of antigens, cell toxicity and activation of innate immune response. Tests will be performed on different cultured or primary human or mouse cells (e.g. THP-1, RAW, PBMCs, BMDMs, BMDCs,1 HEK203 transfected with different TLRs and cells deficient in different signalling pathways).
In Task 4.2, designed nanoparticles and vesicles reinforced by MAPS will be loaded with doxorubicin or cisplatin prodrugs (for targeted chemotherapy) without and with immunomodulatory drugs/tumour cell-specific antigen (for combining synergistically chemotherapy with immunotherapy effects) and injected into mice by different routes of injection. For monitoring the circulation, biodistribution patterns and lifetimes of such minimal protocells in vivo, the constructs will be radiolabelled immediately prior to their administration. Different labelling strategies for the incorporation of the radioisotope will be assayed. The most appropriate strategy will be selected on a case- by-case basis, taking into consideration the physic-chemical properties of the nanosystems and loaded drug(s). Direct surface recognition processes, use of bifunctional chelators (BFC) for subsequent formation of a chelator-radiometal complex, incorporation of a radiolabelled tag, and formation of covalent bonding will be considered to incorporate a wide variety of radioisotopes, such as 67Ga, 68Ga, 64Cu, 89Zr, 125I, 131I or 124I. Dual labelling for determination of the biological fate and stability in vivo using a combination of imaging techniques or energy discriminant SPECT will be applied (P4). We plan to compare the effect of the designed cytoskeleton on the in vivo stability. The extracellular cytoskeleton will be decorated with peptide domains targeting the melanoma cancer cells (NIC, CICB).
After identification of the best construct based on in vitro activity studies and in vivo behaviour, the anticancer efficacy will be evaluated in tumour-bearing mice (task 4.3). The selected experimental model is the syngeneic B16 melanoma model transfected with OVA (B16-OVA), which is widely used as a tumour model to investigate tumour immunity and in which effective treatment is notoriously difficult. It is also susceptible to different chemotherapy treatments. To study the effect of the immunization with MAPS-derived systems, tumour size will be measured twice a week and tumour metabolism will be measured by administration of fluorine-18 fluorodeoxyglucose (FDG) – a standard probe in the diagnostic and therapeutic management procedure in clinical oncology, which has also been applied successfully for monitoring immune responses in mice. The chemotherapeutic effect will also be analysed by the study of apoptotic cells by TUNEL assay and proliferating cells will be detected by the expression of Ki-67 in the nuclei. The immunotherapeutic properties of the designed systems will also be analysed by the study of the innate and adaptive immune responses induced in tumour-draining lymph nodes and tumour infiltrating lymphocytes (TILs) (CICB). In addition, the frequency of different cell populations will be determined, including that of myeloid derived suppressor cells (MDSC), cells that are involved in tumour progression.