Development of Non-Animal Antitoxins

Background: Antitoxins Are Essential Medicines in Need of Modernisation

Antitoxins are life-saving drugs, but the way they are manufactured has remained largely unchanged for more than 100 years. Almost all commercial antitoxins are manufactured using serum from equines who have been hyperimmunised by repeated toxin injections. Large volumes of blood are then drawn from them – up to 15% of their total blood volume at a time – as often as every four weeks.1 The animals are subjected to this process of repeated injections and blood draws for years – or even for their entire lives.

Antitoxins made from the blood of horses and other animals may cause hypersensitivity and serum sickness in humans, when their immune system reacts negatively to foreign material. Since animal blood products are inherently inconsistent, these equine antitoxin products have high batch-to-batch variability and also present the risk of transmitting viruses and other sources of disease between species – important considerations in light of the cross-species origin of COVID-19.2-4 Even in ideal circumstances, equine-derived antitoxins – like all blood products – expire quickly after manufacture and must be continuously harvested from live animals. In many instances, this short shelf life has led to massive global shortages of antitoxins that render them unavailable when they are needed most.5

Antibody Phage Display: A Superior Platform for Antitoxin Development

As with all antibodies and affinity reagents, antitoxins can be manufactured using a number of non-animal approaches,6-9 including antibody phage display. Using cell cultures and human antibody gene libraries in place of horses’ immune systems, antibody phage display can be used to produce antibodies capable of neutralising a target toxin. Selected human antibodies can be optimised for stability and binding affinity (i.e. the strength with which it binds to the toxin molecule). Phage display and other non-animal methods of producing antibodies eliminate many of the practical drawbacks of equine-derived antitoxins, including the possibility of adverse human immune response to foreign material or zoonotic disease transmission. Unlike antitoxins isolated from horse blood, non-animal antitoxins have well-defined structures, are highly pure, and can be designed for a long shelf life amenable to the maintenance of long-lasting stockpiles needed to protect human health.

Starting Points: Diphtheria Antitoxin and Black Widow Spider Antivenom

With this in mind, PETA Science Consortium International e.V. funded the phage display–based creation of fully human recombinant monoclonal antibodies capable of neutralising diphtheria toxin (see the publication here, and additional information here and here). The work was conducted at the Institute for Biochemistry, Biotechnology, and Bioinformatics at the Technische Universität (TU) Braunschweig in Germany, a laboratory with expertise in recombinant antibodies and phage display.

Working with our project partners, the Science Consortium is in discussions with regulatory authorities, pharmaceutical companies, and global health organisations to develop the antibodies into a therapeutic diphtheria antitoxin product that can replace the currently distributed equine-derived therapy. As the new product moves towards human trials, a modern non-animal testing strategy that protects human health better than conventionally required animal tests is being discussed with regulatory agencies. Thus, in addition to replacing equine-derived antitoxin as a therapeutic product, this work will provide a case study for replacing the use of animals during the safety testing that occurs prior to a human pharmaceutical trial.

Based on the success of the aforementioned diphtheria antitoxin project, the Science Consortium has been joined by the Center for Contemporary Equine Studies in co-funding the development of fully human recombinant monoclonal antibodies capable of neutralising black widow spider venom. This work is also being carried out at the Institute for Biochemistry, Biotechnology, and Bioinformatics at TU Braunschweig, and the Ensenada Center for Scientific Research and Higher Education in Mexico is contributing its expertise in the isolation and characterisation of toxins.

Equine-Serum Production Facilities

In 2015, inspections of equine-serum production facilities in India found substandard living conditions, a lack of sufficient veterinary care, and routine violations of basic animal welfare laws. In addition to painful complications caused by being repeatedly injected with toxins and then bled, the animals showed signs of lameness, diseased hooves, eye abnormalities, and malnutrition. Sick or elderly equines were not separated from healthy ones, and the staff often lacked the training and medication necessary to provide basic medical care. These facilities did not provide animals who were too debilitated to recover from advanced illness or injury with humane euthanasia. Video footage of the findings can be viewed below.

Note: The following video contains footage that may be disturbing to viewers.


1Committee for the Purpose of Control and Supervision of Experiments on Animals, Government of India. Care and management of equines used in the production of biologicals. 2001.

2Both L, Banyard AC, van Dolleweerd C, Wright E, Ma JK, Fooks AR. Monoclonal antibodies for prophylactic and therapeutic use against viral infections. Vaccine. 2013;31(12):1553-1559.

3Both L, White J, Mandal S, Efstratiou A. Access to diphtheria antitoxin for therapy and diagnostics. Euro Surveill. 2014;19(24):20830.

4Wenzel EV, Bosnak M, Tierney R, Schubert M, Brown J, Dübel S, Efstratiou A, Sesardic D, Stickings P, Hust M. Human antibodies neutralizing diphtheria toxin in vitro and in vivo. Sci Rep. 2020;10(1):571.

5Both L, White J, Mandal S, Efstratiou A. Access to diphtheria antitoxin for therapy and diagnostics. Euro Surveill. 2014;19(24):20830.

6Bradbury ARM, Sidhu S, Dübel S, McCafferty J. Beyond natural antibodies: the power of in vitro display technologies. Nat Biotechnol. 2011;29(3):245-254.

7Frenzel A, Schirrmann T, Hust M. Phage display–derived human antibodies in clinical development and therapy. MAbs. 2016;8(7):1177-1194.

8Gray AC, Sidhu SS, Chandrasekera PC, Hendriksen CFM, Borrebaeck CAK. Animal-friendly affinity reagents: replacing the needless in the haystack. Trends Biotechnol. 2016;34(12):960-969.

9Groff K, Brown J, Clippinger AJ. Modern affinity reagents: recombinant antibodies and aptamers. Biotechnol Adv. 2015;33(8):1787-1798.