Development of new biological drugs for the treatment of fungal infections

The microbiological world and especially the fungal panorama is currently facing hard times with the rapid development and spread of resistance and the birth of new species intrinsically and sometimes multidrug- resistant. Noted the poor drug arsenal and the lack of effectiveness, an innovative approach had to be found. The application of biological drugs in this field has always been scarce and limited to a few attempts that almost always ended up with failures. In this study, we started from a candidate rich in activity potential but poor in safety and clinical future, the murine monoclonal antibody 2G8. 2G8 demonstrated to be able to bind selectively β-1,3-glucans, vital components of the cell wall of pathogenic fungi, and to be efficient in controlling fungal infections. Nevertheless, the murine nature was its major bottleneck, therefore I started my Ph.D. project with the aim to develop, produce and characterize a humanized antibody starting from 2G8. Two different antibody formats were evaluated: the single-chain fragment variable (scFv) and the full-length. These choices reflected two different ways of administration, hence diverse infection severity and urgency level. The full-length format was designed for systemic applications while the scFv, with its briefer half-life, for topical use. The canonical CDR-grafting permitted the birth of a humanized single-chain fragment variable (hscFv). The orientation as VL-linker-VH and the presence of two ubiquitin monomers at the N-terminus and the His- tag at the C-terminus granted higher quantity, stability, and solubility. With many efforts, we managed to have a clean product (~40 mg from 1L of bacterial culture) with two steps of purification, a negative passage in Q Sepharose and a positive passage in Ni2+ Sepharose. hscFv is still able to bind β-1,3-glucans both as coated antigens and on C. auris and C. albicans cells and to affect the growth of C. auris when combined with caspofungin and amphotericin B. A black mark is represented by its tendency to aggregate forming disulphide bonds and, in a minor way, aspecific interactions. Studies on the best formulation led to the substitution of the reducing agent and to relatively long stability and retention of activity when stored at 4°, -20° and -80° C. In the meanwhile, with a novel humanisation approach combining different humanization methods, the humanised full-length monoclonal antibody H5K1 was generated. It was soon compared to the murine 2G8 from which it took immediate distance in terms of performances showing better IC50, AUC and Kd. Among pathogenic fungi, Candida spp. are the causes of many nosocomial infections and even though C. albicans is still the prevalent specie, over the past few years a mycological shift toward the non-albicans Candida (NAC) species has been recorded. Apropos Candida auris is one of the three leading causes of morbidity and mortality worldwide and is often associated with intrinsic multidrug-resistance and Candida glabrata is associated with higher mortality compared to other NACs. Both these species were considered as a fungal model to evaluate hmAb H5K1 effectiveness. H5K1 can bind selectively β-1,3-glucans on C. auris, C. albicans and C. glabrata cells and on Aspergillus fumigatus and Fusarium solani conidia. In particular, C. auris, C. albicans and C. glabrata had almost 100% of positive cells with a major binding propensity for budded cells and hyphae. Surprisingly H5K1 demonstrated activity alone in C. auris growth and adhesion inhibition and cell wall perturbation and in C. glabrata biofilm matrix alteration. However, the best activity was revealed in combination with amphotericin B and echinocandins on both C. auris and C. glabrata (of the latter two strains had low susceptibility for echinocandins and one was a biofilm hyper producer). H5K1 establishes additivity with echinocandins and synergy with amphotericin B potentiating its fungicidal activity as well. This happens also at low concentrations of both hmAb H5K1 and of the antifungal drugs being an advantage considering the side effects of the available antifungal drugs used for prolonged periods and at high doses. In addition to its intrinsic and combinatorial efficacy, hmAb H5K1 keeps being a full-length antibody then able to be recognized by macrophages. As a matter of fact, after C. auris opsonization, macrophage efficiency increased, and the phagocytosis was enhanced. In conclusion, hscFv and hmAb H5K1, both born from the murine hmAb 2G8, demonstrated comparable and sometimes better performances than their parental. They are effective in vitro especially in combination with amphotericin B and echinocandins showing to be able to modulate not just the fungal growth and adhesion but also the biofilm formation. The two different antibody formats are destined for different applications but in view of this fact, the full-length H5K1, suitable for systemic use, can also enhance the activity of phagocytic cells of the immune system. These data showed suggest that hscFv and hmAb H5K1 could be new drug candidates for the treatment of fungal infections and especially, candidiasis. Although the encouraging results, hscFv must be further explored while, noted the promising preclinical outcomes of hmAb H5K1, we are optimistic and hopeful about its rapid moving to clinical phases with the name of Dia-T51.