Optimization of attenuated Listeria monocytogenes cell wall chemical engineering to increase its anticancer vaccine activity and to use it as metastasis tracer

Attenuated Listeria monocytogenes (Lmat) is widely investigated as anticancer vaccine thanks to its capability to activate host immune-response against the tumour tissues. Lots of genetic engineering strategies have been used to improve its power, such as increasing its immune-stimulation against the cancer tissues by the expression of tumour associated antigens. Since Lmat is capable to selectively accumulate in primary tumours and metastasis, compared with the healthy tissues, we started exploring the possibility of using Lmat as drug carrier and metastasis tracer, by using chemical cell wall modifications that allow us to attach little chemicals onto the bacterial cell wall. In this work we optimized the chemical engineering of Lmat cell wall to let it expose reporter chemical groups, such as alkyne or azido group, that can selectively react with the respective chemical partner (azido and alkyne) by bioorthogonal and bio-compatible “click-reaction”. After comparison of different probes for the functionalization of Lmat cell wall, we optimized the protocol for efficient and totally bio-compatible labelling by using azido-D-alanine probe and both the strain-promoted alkyne-azide cycloaddition (SPAAC) and the copper(I)-catalysed azide-alkyne cycloaddition (CuAAC) reactions. We tested the in vitro features of labelled Lmat and demonstrated that it maintains unaltered proliferative activity, infectivity and intrinsic toxicity effect on 501-mel cell line. We started exploring the chemical conjugation between Lmat and doxorubicin for the drug-carrier strategy, and with Cy7.5 Photoacoustic Dye (PAI) for the metastasis imaging strategy.