Etapas iniciais do desenvolvimento de um anticorpo conjugado a droga: seleção de domínios variáveis anti-CD19 e manipulação dos domínios constantes para conjugação sítio-específica

Abstract

The low specificity of conventional oncologic chemotherapy and the typical tumor cell recalcitrance often require the use of potent cytotoxic agents at dosages close to the maximum tolerated, reaching healthy tissues and causing intense side effects. In the search for more effective and safe therapeutic strategies, biotechnological products have become more and more important, especially monoclonal antibodies (mAbs) and their derivatives. Plasticity and the ability of these proteins to bind with high affinity to specific targets determine their usefulness in proposing new therapies, not only in the cancer context, but also against inflammatory and infectious diseases. A valuable strategy is to take advantage of the specificity of mAb for the targeted delivery of unspecific and potent drugs, so that its release and activity are restricted to the desired cells or microenvironments. Antibody-drug conjugates (ADCs) consist of small cytotoxic molecules covalently linked to a mAb by means of a chemical connector (linker). The 3 components (mAb, linker and drug) play crucial roles in the efficacy and safety of ADCs, with the conjugation method being a determining parameter for their reproducibility. Different protein engineering techniques allow for chemically homogeneous and highly specific ADCs. In this study, sequences of the variable regions of mAbs were selected via phage display as well as mutant sequences of the crystallizable fragment (Fc) were generated by site-directed mutagenesis, aiming to propose an anti-CD19 (target of B cell malignancies) FvFc containing cysteine residues engineered for site-specific conjugation of cytotoxic agent via thiol-reactive linker. A phage library was challenged against cells expressing CD19 protein on their surface, over multiple rounds of selection. The most enriched sequences were obtained by New Generation Sequencing (NGS), being selected for the proposition of 2 anti-CD19 fragments to be produced and characterized. In parallel, points for cysteine mutation in the human IgG1 Fc portion were obtained from the literature and used for generation, by mutagenic PCR, of two independent mutant Fc gene sequences (N421C and S239C). The mutations were confirmed by Sanger sequencing and will be used for the production and characterization of FvFcs regarding the suitability for conjugation and biological activity.