Titolo della tesi: Development of novel antibody-drug conjugates as immunomodulators for the treatment of inflammatory-related disorders.
The growing incidence of inflammatory and immune-based diseases requires the identification of novel targets and therapeutic strategies to modulate the inflammatory and immune phenomena. Chemokines are endogenous mediators of the flogistic process since they intervene in leukocyte chemotaxis, in the neutralization of bacterial toxins and viral infections, in the induction of cell-mediated cytotoxicity, in allergic responses and in the appearance of delayed hypersensitivity, therefore determining the type, intensity and duration of the immune and inflammatory response. Consequently, the massive expression and release of proinflammatory chemokines by immune cells play a prominent role in the etiology of inflammatory and immune-mediated disorders, including chronic inflammatory diseases and autoimmune diseases such as rheumatoid arthritis, Sjogren's syndrome, autoimmune thyroiditis, multiple sclerosis, psoriasis and uveoretinitis and cutaneous and systemic lupus erythematosus.
This thesis aims at developing a new strategy to directly intervene on immune cells, particularly macrophages which are key players in inflammatory chronicization, and to modulate the aberrant expression and release of pro-inflammatory chemokines.
Among the most innovative strategies adopted in recent years, Antibody-Drug Conjugates (ADCs) represent the most effective method to obtain targeted therapy. The general structure of an ADC includes a monoclonal antibody conjugated to the biologically active molecule through a central linker bearing chemical bonds that allow the selective release and internalization of the drug in target cells. Due to the antibody affinity for the antigen expressed on the target cells, the ADC technology improves the therapeutic window and minimizes the systemic toxicity mostly dependent on the poor selectivity of the active compounds, while allowing the administration of a higher dosage, compared to that normally used for the single unconjugated compound.
In the scenario of inflammatory and immune-based diseases, this thesis is constructed as a proof-of-concept of the ADC strategy potential to intervene on M1 macrophages while minimizing the side effects related to the whole inflammatory and immune systems suppression.
These novel ADCs are constructed with the anti-CD14 mAb conjugated via a Val-Cit cleavable linker to the immunomodulatory agents tacrolimus and MM-401.
The CD14 antigen is selectively expressed on M1 macrophages and allows the endocytosis-mediated internalization of the ADC in macrophages. Upon internalization, the Val-Cit cleavable linker is subject to cleavage by the lysosomal enzyme cathepsin B, followed by the 1,6-elimination of the PABC group and the consequent release of the free active compound in the cell cytoplasm, where it can therefore exercise its therapeutic activity.
Once reached the cytoplasm, tacrolimus and MM-401 could exert their immunomodulatory activities by inhibiting the expression of pro-inflammatory citokines and chemokines, interfering with the phagocytic activity of macrophages or preventing the polarization towards the M1 isotype, therefore representing a potential treatment for immune and inflammatory diseases.
Lastly, compound ML324 has been identified as a potential epigenetic modulator that could inhibit UMLILO-WDR5 interaction and modulate abberant immune and inflammatory processes. In vitro studies demonstrated that ML324 effectively reduces the proinflammatory chemokines transcription and expression, therefore representing a promising candidate for the development of novel immunomodulatory ADCs.