Desenho de biobetters baseados no anticorpo anti-Notch1 Brontictuzumabe usando abordagens de otimização in silico

Abstract

Cancer, one of the leading causes of global mortality, represents a set of diseases with multifactorial etiologies that encompass genetic and epigenetic alterations, including mutations in cell signaling pathways such as Notch, crucial for processes such as differentiation and apoptosis. Activating mutations or overexpression of the Notch1 receptor, for example, are found in patients with chronic lymphocytic leukemia, diffuse large B-cell lymphoma, and adenoid cystic carcinoma. In this context, the monoclonal antibody Brontictuzumab (BRON), which blocks Notch1 activation by binding to its Negative Regulatory Region (NRR), emerges as a promising basis for the development of new biobetters with therapeutic potential. This study aimed to computationally design new antibodies based on Brontictuzumab, focusing on affinity maturation and the proposal of single-chain fragment variables (scFvs), with the goal of enhancing its therapeutic potential. Initially, the three-dimensional structure of BRON was modeled and used to predict interactions with NRR through molecular docking. The complex was simulated via molecular dynamics, with interaction and dissociation energy predictions. The predominant conformations identified were used for optimizations through saturation mutagenesis, CDR grafting, inverse folding and generalized language models. Solubility and other development parameters for therapeutic antibodies were evaluated. BRON-based scFvs were also modeled and their interactions analyzed. Thus, the binding mode of the BRON-NRR complex was identified, suggesting a new inhibition mechanism mediated by BRON's CDR-H2, which reduces accessibility to the S2 cleavage site, essential for Notch1 activation. A total of 10,072 mutants were tested, identifying hot spots in residues such as Thr28, Thr74, Gly101, and Gly104 in the heavy chain and Gly52 in the light chain. The AbSeldon R3 design demonstrated stability, solubility and ΔGbind comparable to native BRON, increasing the participation of the light chain at the interface and being compatible with biophysical parameters of other therapeutic antibodies. Among the scFvs constructed, the one with a 12-amino acid linker showed stability and interactions with S2. Thus, this work resulted in the proposal of promising biobetters, paving the way for new anti-Notch1 antineoplastic therapies based on Brontictuzumab. Keywords: Notch; Brontictuzumab; biobetter.