Discovery of broadly-neutralizing antibodies for recombinant antivenoms

Snakebite is an ever-present threat for millions of people around the world, especially those living in impoverished areas of the tropics. Each year, around 125,000 people are killed by venomous snakes, and an additional 400,000 are left permanently disabled or disfigured. The only specific and effective treatment available for snakebite envenoming is antivenom, which is based on animal-derived antibodies obtained after immunization of larger animals with snake venoms. Although these medicines have saved countless lives since their invention, they are associated with a list of drawbacks due to their heterologous nature. On top of adverse effects, such as serum sickness and anaphylactic shock, these medicines are costly to manufacture, have a low therapeutic content, and have varying efficacies.In the last decades, the interest in improving the treatment for snakebite envenoming has spiked amongst researchers worldwide. As a result, several different molecules have been proposed for a new generation of antivenoms. These molecules have different pros and cons in terms of efficacy, safety, manufacturing, and regulatory paths that should be taken into account by researchers aiming to bring antivenoms into the twenty-first century (Paper I).This PhD project aims to aid in developing what has been termed recombinant antivenoms. Recombinant antivenoms can be either polyclonal or oligoclonal. Polyclonal recombinant antivenoms are based on undefined mixtures of antibodies, much like current antivenoms. Oligoclonal recombinant antivenoms, which are the focus of this thesis, are envisioned to be based on mixtures of (human) monoclonal antibodies. Compared to current antivenoms, oligoclonal recombinant antivenoms are likely to be associated with fewer adverse reactions as human or closely related antibodies are compatible with the patient’s immune system, be of higher efficacy as their therapeutic content can be significantly increased, and be cost-competitive to manufacture.In this work, the antibodies constituting these oligoclonal recombinant antivenoms were discovered using antibody phage display technology. Phage display enables many aspects of engineering and tailoring to individual antibody discovery campaigns. These include the discovery of antibodies against antigens with high toxicity or low immunogenicity or cross-panning using similar toxins from different snake species facilitating the discovery of antibodies with broadly neutralizing capabilities (Paper II, Manuscript I, and Manuscript III).To realize the potential of recombinant antivenoms in the future, the cost of treatment is essential, as victims of snakebite envenoming are often poor and living in countries with low healthcare expenditure. This cost will depend on many different variables, including the cost of manufacture and the required dosage. For the dosage to be as low as possible, the antibodies should have very high affinities to their target toxins. The cost of manufacture can likely be minimized if oligoclonal expression and purification are employed; however, for this to be possible, the number of unique antibodies included in the antivenom should be limited.To discover antibodies with high affinities, many strategies can be employed. Naïve antibodies can be affinity matured in vitro using random mutagenesis, rational design, or by employing chain-shuffling. In the work of this thesis, a study was completed illustrating the discovery of the first-ever human monoclonal immunoglobin (Ig) G molecule able to neutralize the whole venom of a snake (the monocled cobra, Naja kaouthia) following intravenous (i.v.) injection in mice (Manuscript II). The work demonstrates that antibodies with sufficiently high affinity to neutralize potent α-neurotoxins can be discovered from naïve human phage display libraries if followed by affinity maturation using chain-shuffling.Building directly upon this work, another study was set up aiming to increase the affinity and cross-reactivity of naïve antibodies simultaneously. This was conducted by combining cross-panning strategies with chain-shuffling, eventually leading to the discovery of an antibody that neutralized N. kaouthia whole venom even at the lowest tested dose of one antibody per toxin in mice (Manuscript III). Beyond this potent neutralization of N. kaouthia venom, the antibody exhibited binding to long chain α-neurotoxins from five other elapid snakes of three different genera from Asia and Africa. Furthermore, the antibody neutralized the effect of three of these toxins in an electrophysiology-based in vitro assay and prolonged survival of mice injected with either black mamba (Dendroaspis polylepis) or king cobra (Ophiophagus hannah) venom.In this thesis, both cross-panning and chain-shuffling have been explored to discover antibodies for recombinant antivenoms from naïve phage display libraries. The work yielded antibodies with sufficiently high affinities to neutralize venom at a very low dose while simultaneously exhibiting broadly-neutralizing capacity to similar toxins from different snake species from distant genera. The research and protocols described in this thesis should guide future research and researchers in their search for new toxin-targeting antibodies with high affinities and broadly-neutralizing potential. Thereby eventually assisting in making recombinant antivenoms a reality, benefitting the millions of people that suffer from snakebites every year. Beyond applications within antivenom research, the protocols presented in this thesis may find utility in other areas, such as research in the treatment of infectious diseases, where broad toxin-neutralization also has an important role to play