Design of smart linkers and their applications in controlled-release drug delivery systems

Antibody drug conjugates (ADC) represents an interesting strategy in tumour targeted therapy . The approach is based on the combination of the high affinity of an antibody towards its antigen and the high cytotoxicity of a drug, leading to a selective therapuetic agent with improved efficacy and safety. When considering the development of an ADC, several factors are of great importance. On the one hand, conjugation chemistry defines the properties of the ADC, such as antibody to drug ratio (DAR) and is fundamental to yield stable and homogeneus conjugates. On the other hand, the linker used to connect the drug with the antibody plays an important role in the stability of the final conjugate and its efficiency in terms of cell killing. Considering the importance of these two factors in ADC development, this Thesis has been focused on conjugation reactions and linker chemistry. First, new conjugation strategies have been proposed for the design of homogeneous, stable and efficaceus ADCs. The use of carbonyl acrylamide derivatives has allowed the irreversible cysteine-selective protein modification. Thus, when combined with Thiomab technology, an ADC with DAR 2 was obtained in high yield, using stoichiometric amount of the reagent and under mild conditions. Similarly, a new, ultrafast reagent, based on quaternised vinyl pyridinium scaffold, has been described. After proving that the compound is also cysteine selective in proteins and antibodies, the synthesis of a vinyl pyridinium bearing an alkyne tag for further functionalization was optimized. This approach allowed the introduction of a drug, and the resulting derivative was efficiently used for ADC synthesis. \nConcerning the linker, we have demonstrated that acetals represent an interesting cleavable moiety for the preparation of ADCs as well as small molecules drug conjugates (SMDC). We have prepared acetal linkers featuring coumarin as fluorophore and a duocarmycin analogue as an example of cytotoxic drug. Markedly, it represents the first example a duocarmycin analogue is protected with an acid cleavable moiety. Kinetic studies were performed on these linkers and showed that they are stable in plasma, while being rapidly cleaved under acidic conditons. This methodology was applied then to the design of a small molecule drug conjugate (SMDC), as well as an antibody drug conjugate (ADC). Of note, interesting outcomes emerged from the stability studies performed on the ADC. In fact, our results showed that the stability of the ADC depends not only on the conjugation site, but also different payloads can affect the stability of the acetal linker, depending on the 3D disposition they adopt in the antibody pocket. \nFinally, we have exploited the use of the Grob fragmentation to design ‘self-immolative’ linkers for controlled drug release. Altough the reaction mechanism is well know and presents several synthethic application, no biological application has been described to date. The screening of different substrates allowed the identification of 3-aminocyclohexanol scaffold as a suitable moiety for fragmentation reaction under biological mimicking conditions. The novel methodology has been applied to the controlled release of Crizotinib, a drug used for the treatment of metastatic lung cancer. Thus, blocking the Grob fragmentation pathway, the pro-drug is stable and intact; at the same time, activation of the pathway by removing the amine protecting group of 3-aminocyclohexanol derivative, results in drug release.