Cancer is a major challenge for modern medicine. Despite extensive research efforts, we still lack tools which specifically target malignant over healthy tissues. Current anti-cancer treatments are often inefficient and imply undesired side-effects. In order to improve this, new strategies are being developed. Harnessing the ability of our own immune system to eliminate malignant cells is promising to revolutionize the treatment of cancer, although the challenges still remain. New targets, a better understanding of their synergies and new modalities for the immunotherapy of cancer are still required. An emerging therapeutic modalities are bispecific antibodies (bsAbs) or antibody drug conjugates (ADC). This type of constructs exploit a combination of the binding specificity of antibodies with cytotoxic drugs or other desired payloads. The challenge stands to improve the efficiency of their production. In this project the aim was to establish a new, chemistry driven, strategy for the construction of bispecific antibodies using site-specific bioorthogonal chemistry.
Despite the potential of bsAbs or ADC's for clinical use, the drawback of this technology currently resides in the linearity of its synthesis which slows down their development. Over the last decade protein site-selective conjugation has become a thriving research field. A plethora of methods have been reported for the construction of protein conjugates with a wide array of applications in chemical biology and medicine. Cysteine (Cys) and lysine(Lys) remain the main target residues in protein modification although recently, methodologies which target alternative residues such as aspartic/glutamic acid, tryptophan, methionine or tyrosine have been described. Cys targeting methods are particularly ubiquitous, due to cysteine’s lower abundance and the intrinsic high nucleophilicity of the thiol at physiological pH. These features contribute to the formation of structurally homogeneous conjugates in proteins containing native or genetically engineered free Cys. Multiple strategies have been reported for the selective conjugation of Cys, usually based on alkylation/arylation reagents or Michael acceptors Michael acceptors and in particular, maleimides remain the most commonly used reagent for the construction of conjugates for biological applications, due to their associated fast reaction kinetics. In fact, a number of Food and Drug Administration approved conjugates such as the antibody-drug conjugates (ADCs) Brentuximab vedotin Trastuzumab emtansine or the PEGylated conjugate Cimzia contain a thio-succinimide adduct derived from maleimide conjugation. However, it is well known that thio-succinimide adducts can undergo fast and uncontrolled disruptive cleavage by thiol-exchange in plasma, ultimately compromising the safety and efficacy of the conjugate. Considerable efforts have been devoted to increase the stability of maleimide based constructs but it remains a challenge. With the increase in applications of modified proteins as therapuetics or visualisation agents the necessity for stable linkages and fast conjugation reagents has become priority in chemical biology. The advancement these methods would allow for the next generation of therapeutics which would treat cancer or autoimmune diseases, reducing the multiple side effects which are currently associated with these types of pharmaceuticals.
This proposal aims to establish a new chemical approach for the synthesis of bsAbs. Our overall objective is to develop new site selective chemistry with comparable kinetics to current methods but superior stability. Key challenges in the field, such as specificity, temporal control of the reactions or the toxicity of catalysts, are addressed in this proposal. The growing importance of these type of constructs in oncology highlights the importance of developing safe methods and tools to synthesize them.
