Design and preclinical evaluation of highly loaded antibody-drug conjugates for anticancer therapy based on tailored PEG linkers

Antibody-drug conjugates (ADCs) have been described as the “magic bullets” of cancer treatment, conceived to fight the lack of tumor selectivity blemishing conventional chemotherapeutics. In the ADC design, the cytotoxic agent, or payload, is preferentially delivered to cancerous cells by means of a monoclonal antibody which serves as targeting molecule towards a tumor-associated antigen. Although simple in the idea, the conjugation of a small drug to an antibody, through a chemical linker, could brutally impact the chemical and physical stability of the antibody and alter the in vivo pharmacokinetic and biodistribution profile of the final ADC, mainly due to the high hydrophobicity characterizing most of the payloads. For this reason, it is believed that only a few (2-4) payload molecules can be attached to the antibody backbone. In this thesis, we have explored the validity of three innovative hydrophilic polyethylene glycol (PEG)-based linkers useful to offset the hydrophobicity coming from eight auristatin E (MMAE) molecules linked to the eight native interchain cysteine residues of the anti-HER2 trastuzumab. Three differently-sized PEG chains, containing two pendant chains of four, eight or twelve ethylene oxide units per PEG chain, branching out of a single point in the structure, were compared in their hydrophobicity shielding and ability to give ADCs physically stable and with improved in vivo properties. The ADCs were synthetized by following a double-step synthesis featuring the fine-tuned reduction of the interchain disulfide bonds of trastuzumab, followed by the alkylation of the released thiols with the linker-drug moiety. By exploiting this synthesis, the highly loaded ADCs resulted to be homogeneous and monomeric. The conjugates were tested for their physical stability through a 28-days in solution stability study under thermal stress conditions. The study revealed that the incorporation of two pendant PEG chains could be a valid tool to obtain highly loaded ADCs with a substantially decreased aggregation tendency. Indeed, all the PEG linkers allowed to double the MMAE cargo while maintaining the same, or even lower, aggregation level of a non-PEGylated ADC reference carrying half of the drug. The key in vitro founding that the inclusion of longer PEG chains in a pendant orientation within the linker promoted hydrophobicity masking properties to the final conjugates, and not only to the single linker-MMAE moiety as such, was translated to the pharmacokinetic, safety and potency of the immunoconjugates, thus establishing a deeply-rooted correlation between the PEG size and in vivo properties exhibited. Indeed, PEG chains sufficient long to mask the MMAE-correlated hydrophobicity gave ADCs with a restored pharmacokinetic profile, more similar to that of the naked trastuzumab, providing for an almost complete tumor regression in an ovarian cancer xenograft model. Moreover, a proper hydrophobicity masking allowed for an improvement of the therapeutic index of the ADCs, very likely due to a reduction of the hydrophobicity-mediated ADC accumulation in off-target tissues. The above-mentioned findings have proved the potential to use a pendant PEG-linker with fine-tuned PEG length as hydrophilicity promoting entity for the formulation of high MMAE-loaded cysteinyl-ADCs with improved in vivo performance. The promising results obtained in vivo provided a strong motivation for proving the validity of our hydrophilic highly-loaded ADCs towards critical tumor subtypes characterized by a lower and more heterogeneous expression of the antigen.