Envenoming due to Loxosceles spider bites still remains a neglected disease of particular medical concern in the Americas. To date, there is no consensus for the treatment of envenomed patients, yet horse polyclonal antivenoms are usually infused to patients with identified severe medical conditions. It is widely known that venom proteins in the 30–35 kDa range with sphingomyelinase D (SMasesD) activity, reproduce most of the toxic effects observed in loxoscelism. Hence, we believe that monoclonal antibody fragments targeting such toxins might pose an alternative safe and effective treatment. In the present study, starting from the monoclonal antibody LimAb7, previously shown to target SMasesD from the venom of L. intermedia and neutralize its dermonecrotic activity, we designed humanized antibody V-domains, then produced and purified as recombinant single-chain antibody fragments (scFvs). These molecules were characterized in terms of humanness, structural stability, antigen-binding activity, and venom-neutralizing potential. Throughout this process, we identified some blocking points that can impact the Abs antigen-binding activity and neutralizing capacity. In silico analysis of the antigen/antibody amino acid interactions also contributed to a better understanding of the antibody’s neutralization mechanism and led to reformatting the humanized antibody fragment which, ultimately, recovered the functional characteristics for efficient in vitro venom neutralization.

In order to increase the successful development of recombinant antibodies and fragments, it seems fundamental to enhance their expression and/or biophysical properties, such as the thermal, chemical, and pH stabilities. In this study, we employed a method bases on replacing the antibody framework region sequences, in order to promote more particularly single-chain Fragment variable (scFv) product quality. We provide evidence that mutations of the VH- C-C′ loop might significantly improve the prokaryote production of well-folded and functional fragments with a production yield multiplied by 27 times. Additional mutations are accountable for an increase in the thermal (+19.6 °C) and chemical (+1.9 M) stabilities have also been identified. Furthermore, the hereby-produced fragments have shown to remain stable at a pH of 2.0, which avoids molecule functional and structural impairments during the purification process. Lastly, this study provides relevant information to the understanding of the relationship between the antibodies amino acid sequences and their respective biophysical properties.

Organ-on-a-chip devices combine microfabrication, tissue engineering, and microfluidics to recreate physiologically relevant microenvironments for in vitro studies. In this work, we validated a dynamic 2D breast-on-a-chip microfluidic bioassay operated at a controlled infusion rate of 20 µL/h to assess anticancer drug responses under defined flow conditions. Using Palbociclib as a reference compound, we evaluated proliferation, viability/apoptosis, cytoskeleton organization, and differential processing of the resistance-associated marker PARP1 in MCF-7 and T47D breast cancer cells. Under dynamic microfluidic conditions, Palbociclib induced dose-dependent effects, with the higher concentration (20 µM) consistently reducing cell proliferation and viability and increasing late apoptosis compared to 10 µM Palbociclib. Cytoskeletal disorganization was observed at both concentrations, while differential PARP1 processing patterns between MCF-7 and T47D cells were detected across doses. These responses are consistent with known effects of CDK4/6 inhibition and were reproducibly captured under controlled flow conditions. Overall, our results demonstrate that this breast-on-a-chip microfluidic model provides a reproducible and physiologically relevant in vitro platform for integrated assessment of drug efficacy and resistance-associated markers under dynamic perfusion.