Identification of a common epitope in knottins and phospholipases D present in Loxosceles sp venom by a monoclonal antibody.

In the Americas and specially in Brazil, the Loxosceles intermedia, Loxosceles gaucho and Loxosceles laeta are the three most medically relevant brown spider species, and whose bites can lead to the condition known as loxoscelism. Here, we report the development of a tool capable of identifying a common epitope amongst Loxosceles sp. venom's toxins. A murine monoclonal antibody (LmAb12) and its recombinant fragments (scFv12P and diabody12P) have been produced and characterized. This antibody and its recombinant constructs were able to recognize proteins of Loxosceles spider venoms with specificity. The scFv12P variant was also able to detect low concentrations of Loxosceles venom in a competitive ELISA assay, displaying potential as a venom identification tool. The primary antigenic target of LmAb12 is a knottin, a venom neurotoxin, that has a shared identity of 100 % between the L. intermedia and L. gaucho species and high similarity to L. laeta. Furthermore, we observed LmAb12 was able to partially inhibit in vitro hemolysis, a cellular event typically induced by the Loxosceles sp. venoms. Such behavior might be due to LmAb12 cross-reactivity between the antigenic target of LmAb12 and the venom's dermonecrotic toxins, the PLDs, or even the existence of synergism between these two toxins.

An effective strategy for the humanization of antibody fragments under an accelerated timeline.

The use of monoclonal antibodies (mAbs) in therapy is gradually advancing and discussions entail its safety, rentability and effectiveness. To this date, around a hundred mAbs have been approved by the FDA for the treatment of various diseases. Aiming for their large-scale production, recombinant DNA technology is mainly employed, and antibodies can be expressed in various eukaryotic and prokaryotic systems. Moreover, considering their heterologous origin and potential immunogenicity, various strategies have been developed for mAb humanization, considering that around 50 % of commercial mAbs are humanized. Hence, we introduce LimAb7, a mouse mAb capable of binding and neutralizing brown spider's Loxosceles intermedia dermonecrotic toxins in vivo/in vitro. This antibody has been produced in mouse and humanized scFv and diabody formats, however results indicated losses in antigen-binding affinity, stability, and neutralizing ability. Intending to develop evolved, stable, and neutralizing antibody fragments, we report for the first time the design of humanized antibody V-domains produced as Fab fragments, against spider venom toxins. Improvements in constructs were observed regarding their physicochemical stability, target binding and binding pattern maintenance. As their neutralizing features remain to be characterized, we believe this data sheds new light on antibody humanization by producing a parental molecule in different recombinant formats.