*3.5.4 Monoclonal antibodies*

Advances in basic research culminated in the development of the breakthrough hybridoma technology in 1975 by Kohler and Milstein in which hybrid cells producing rodent-derived monoclonal antibodies (mAbs) were generated in unlimited quantities [51]. These hybrid cells were achieved by fusing B cells obtained from an immunized animal with myeloma cells and the resulting selected cell secreting one specific antibody. Thus, the interest in the use of antibodies in therapeutics was again awoken by the hybridoma technology following its discovery. Subsequently, techniques were developed to overcome the safety, efficacy, and immunogenicity problems associated with the use of rodent-derived antibodies by transforming same into structures akin to human antibodies, ensuring that the binding properties to the target are retained. Consequently, the first humanization method resulted in the development of chimeric antibodies through combining sequences of human constant region domain and murine variable domain, with the resulting antibody preserving the specificity and reducing its immunogenicity [52].

## *Perspective Chapter: Diagnostic and Antivenom Immunotherapeutic Approaches… DOI: http://dx.doi.org/10.5772/intechopen.112147*

In a previous study [53], the possibility of antivenom based on monoclonal antibodies was investigated. In this study, neutralizing mAbs were developed and tested against three key toxic constituents of *Bothrops atrox*, the main snake group causing accidents in the Northern region of Brazil. Like the venom of other *Bothrops* species, *B. atrox* venom contains a complex mixture of venom proteins including proteases whose substrates include blood clotting system components such as factors X, XII, and fibrinogen. The venom of *B. atrox* is also made up of proteins that are zinc-dependent metalloproteinases, majority of which are hemorrhagic and phospholipase A2, which has been demonstrated to play a role in myonecrosis and inflammation. Hybridoma cells secreting neutralizing mAbs PLA2 clone A85/9-4, Zn-metalloproteinase clone 59/ 2-E4, and serine proteinase clone 6 AD2-G5 of *B. atrox* were cultured, expanded and cells injected i.p. into BALB/c mice. Ascitic fluid was subsequently collected *via* abdominal puncture and mAbs purified *via* caprylic acid followed by ammonium sulfate precipitation. The results obtained showed that purified mAbs specific to these three venom proteins were successfully generated. When submitted to immunochemical analysis *via* SDS-PAGE, all three mAbs showed two main protein bands, one around 55 kDa and the other approximately 29 kDa, indicative of immunoglobulin heavy and light chains, together with several minor contaminant bands. On performing western blot analysis with anti-mouse IgG as the primary antibody, the two major bands were confirmed to correspond to mouse IgG heavy and light chains. Furthermore, estimation of the purity of the mAb preparations was performed by densitometry analysis of SDS-PAGE, which revealed purity grades of 46, 45, and 37% for A85/9-4, 59/2-E4, and 6 AD2-G5, respectively.

In another study by Laustsen et al. [54], an experimental recombinant antivenom based on a mixture of fully humanized IgG monoclonal antibodies, able to neutralize neurotoxicity facilitated by dendrotoxins of the notorious black mamba (*Dendroaspis polylepis*) is described. The approach to discovering the suite of human IgGs combined among other things toxicovenomics, antibody phage display technology and engineering, and mammalian cell expression. A human antibody phage display library of clones and single-chain variable fragments (scFvs) antibodies were constructed from B-lymphocytes obtained from nonimmunized human donors. ScFv antibodies that produced the highest binding signals from an expression-normalized capture (ENC) assay were selected and converted to IgG format. The results of the study demonstrate the possibility of exploiting oligoclonal cocktails of monoclonal human IgGs to treat black mamba envenoming. The findings further showed that individual monoclonal IgGs targeting neutralization of dendrotoxins cannot do that alone and for which reason it could be beneficial to employ antibody cocktails that neutralize multiple black mamba venom toxins in order to achieve full protection.

In a recent study by Manson et al. [55], anti-3FTxs monoclonal antibodies were developed against *N. ashei* venom in mice. Preliminary activity of the purified mAbs against 3FTx antigen was assessed by testing the ability of the mAbs to identify and bind to the target antigen in an ELISA titration. All three mAbs demonstrated capacity to recognize 3FTxs, and thus were able to bind to the antigen at the lowest concentration tested (0.0002 mg/mL). When compared with the control sample, all three clones were found to bind to the target antigen with comparatively higher efficacy as assessed by optical density. Further confirmation of the recognition, binding, and activity of the mAbs was evaluated using an inhibition ELISA assay previously described [43]. A cocktail of the purified mAbs was tested against two commercial antivenoms available in the Kenyan market (VINS™ and Inoserp™) and 3FTxschallenged mice polyclonal antibodies. Tukey's multiple comparison test showed that

the 3FTxs-induced inhibition by the test mAbs was significantly different when compared with inhibition by the two antivenoms. Both the mAbs and polyclonal antibodies induced comparable inhibition. Similarly, there was a significant difference in the inhibition induced by the polyclonal antibodies relative to both antivenoms. Thus, the results demonstrate that the immunoaffinity-purified test mAbs have higher binding efficacy, and hence higher specificity for the target antigen relative to both the negative control and the two leading commercial brands of antivenoms on the Kenyan market. The results further demonstrate the prospects of developing toxinspecific monoclonal-based antivenoms for snakebite immunotherapy.
