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Behring's serum therapy ... marked the beginning of immunotherapy

Emil von Behring (1854–1917) discovered passive immunization as an effective treatment for diphtheria and tetanus. Moreover, he brought this discovery from bench to bedside, and his serum therapy markedly reduced deaths from diphtheria — the 'strangling angel of children' — which at the time was feared as the deadliest disease of infants. Thus, together with Paul Ehrlich (1854–1915) and Elie Metchnikoff (1845–1916), he is considered one of the founders of immunology. With the subsequent introduction of vaccines and antibiotics, serum therapy left the limelight. However, the emergence of new pathogens with pandemic potential and antibiotic resistance has rekindled interest in serum therapy using tailor-made monoclonal antibodies (http://www.nature.com/milestones/antibodies). At the same time, targeting the immune system with monoclonal antibodies has been a major breakthrough for the treatment of chronic inflammation and cancer. Behring's serum therapy, therefore, marked the beginning of immunotherapy.

Research highlights and career path

During his scientific career, Behring published hundreds of papers. Arguably, the three most important were: the paper he published in 1890 with Shibasaburo Kitasato on immunity to diphtheria and tetanus in experimental animals1; his paper on immunity against diphtheria in experimental animals, published 1 week later2; his paper describing the successful clinical data on active immunization against diphtheria with antigen−antibody complexes published more than 20 years later in 1913 (Ref. 3). Together, these publications provided experimental proof that antisera generated against diphtheria and tetanus toxins could be used to prevent and cure these diseases through serum transfer.

Early career. Behring studied medicine as part of his military service, which he entered because his family could not pay the fees for university studies. During his military service, he was introduced to research and soon became fascinated by the new discipline of infectious diseases. His first research projects as a military doctor focused on disinfectants based on the antiseptic concept brought forward by Sir Joseph Lister. The possibility of harnessing antiseptics for therapy fascinated Behring. Specifically, he showed that iodine-containing chemicals not only killed bacteria but also detoxified bacterial products. Technically, his serum therapy of tetanus and diphtheria benefitted from his insights into the antiseptic and antitoxic activity of iodine compounds. The most successful immunization protocol comprised local treatment with iodine trichloride soon after toxin administration or bacterial infection4. In this way, immunized animals did not suffer excessively from tetanus or diphtheria and still developed strong immunity against the toxin. During his studies on disinfectants, Behring discovered that sera from certain experimental animals possessed natural resistance against distinct bacterial pathogens4. These studies led to his appointment to the centres of excellence on infectious diseases in Berlin, headed by Robert Koch5. Behring's work from this period confirmed that serum from certain animal species showed natural resistance against distinct bacterial pathogens. More exciting was the identification of a new kind of bactericidal activity that was absent in naive animals, and only appeared after immunization. Both entities were distinct — a first hint of the coexistence of innate and acquired immunity.

Translation through public−private partnership. At the time, work by Koch on tuberculosis had helped fuel interest in translational medicine and the chemical industry was actively seeking collaborators in academia. Behring found a partner in the Hoechst Company (Meister, Lucius & Bruening), a major producer of dyes, which had shifted its portfolio towards medical products6. The Chief Scientific Officer at Hoechst, August Laubenheimer, negotiated a contract with Behring to use serum therapy against diphtheria. Behring was responsible for the provision of toxin for immunization and Hoechst for serum production. Later, Behring decided to become an entrepreneur himself and founded a spin-off company for serum and vaccine production, the Behringwerke (Behring Company) in Marburg, which became a major vaccine producer in Germany.

Figure 1: Mentors, colleagues, friends and competitors of Behring.
figure 1

Centre panel: Emil Behring as a young military doctor (left) and as an established professor and entrepreneur (right). Clockwise from upper left: Friedrich Althoff, the German government official responsible for academic research, who supported Behring's academic career; Robert Koch, discoverer of the aetiology of tuberculosis and mentor of Behring; August Laubenheimer, Chief Scientific Officer at Hoechst Company, who promoted serum therapy as a pharmaceutical product; Emile Roux, discoverer of diphtheria toxin and the first to use horses for serum production; Shibasaburo Kitasato, a Japanese researcher based in Berlin who established Clostridium tetani culture and co-authored the first paper on serum therapy with Behring; Paul Ehrlich, the researcher who developed standardization of antitoxin activity for serum therapy; Erich Wernicke, a close collaborator of Behring on serum therapy; Sir Joseph Lister, who developed antiseptics for wound healing and inspired Behring's search for disinfectants, leading to serum therapy. Images reproduced courtesy of S. Kaufmann.

PowerPoint slide

The first step in industrial serum production was to identify an appropriate animal species to generate large quantities of serum. Successful clinical studies by Emile Roux and Alexandre Yersin had used horses for serum production, and Behring and the Hoechst Company also switched to horses to produce antiserum. A standardized method needed to guarantee reproducible titres of high toxin-neutralizing activity in the antiserum, and this was achieved through methods developed by Ehrlich5. Behring and Hoechst contracted Ehrlich for this purpose. There is some dispute about the first experimental study in humans. According to Behring, clinical trials started only in 1893 (Ref. 4). However, Behring's colleague, Wernicke, described the experimental treatment of a child with diphtheria antiserum around Christmas 1891 (Ref. 7). In any case, a statistically sound clinical trial using serum therapy for diphtheria achieved an overall cure rate of near to 77% and was published in 1894 with Ehrlich as first author8.

Regulatory aspects. Early on, it was realized that serum quality was quite variable and often of low activity. Ehrlich had established validation of high-titre serum and it was agreed that continuous quality control of serum batches by a state institution was urgently needed. Most medicines in use were chemicals, yet one of the few biologicals was the smallpox vaccine, which had become compulsory in Germany9. This had raised profound anti-vaccination movements arguing against its safety and efficacy. In response to such criticism, smallpox production became state controlled. Moreover, Koch's tuberculin, which had been praised as the remedy for tuberculosis, had failed in clinical trials and this led to a further decline in confidence in biologicals. Thus, a meeting was convened that led to the founding of a state-controlled institute headed by Ehrlich. To avoid any conflict of interest, Ehrlich terminated his contracts related to serum therapy with Hoechst when he became director of the regulatory agency. He determined the necessary quality standards, and the industry willingly followed his recommendations. Rapidly, serum therapy became an established treatment option, reducing diphtheria mortality by more than 50% in German cities in the single year from 1894 to 1895 (Ref. 7).

Later career. In 1895, Behring became Professor for Hygiene at the University of Marburg, despite the opposition of its faculty5. At the beginning of the 20th century, two major events occurred. First, in 1901, Behring was awarded the Nobel Prize for Physiology and Medicine for his discovery of serum therapy for diphtheria; the first Nobel Prize ever awarded in the field of immunology (http://www.nobelprize.org/nobel_prizes/medicine/laureates/1901/). Second, Behring was endowed with hereditary nobility, which granted him the title 'von Behring' (Ref. 5).

His last scientific dream came true when he succeeded in formulating a vaccine for active immunization to prevent diphtheria3. For this, he took advantage of the principles of immunology and tried antigen−antibody complexes. His antitoxin–toxin diphtheria vaccine was soon shown to be safe and efficient. He published his first experimental vaccination studies in humans in 1913 (Ref. 3). Immediately after, larger clinical trials were performed in regions with epidemic diphtheria outbreaks, which proved the high efficacy and safety of the vaccine: fewer than 1% of vaccinated infants fell ill and adverse events were negligible7. Having provided proof of principle, Behring tried to convince the government to introduce compulsory diphtheria vaccination in neonates. However, his vaccine was hampered by high production costs (both toxin and antitoxin were needed), and the public situation further deteriorated with the outbreak of the First World War. It took another decade before active vaccines against diphtheria and tetanus — composed of toxoids produced by formaldehyde treatment — were created by Gaston Ramon. These vaccines led to the global decline of both diphtheria and tetanus.

Behind the scenes

Behring was highly ambitious and assertive. From early on he claimed that he alone deserved credit for the discovery of serum therapy, announcing this not only in scientific circles but also to the public. Behring's communication style is illustrated well in his statement: “One has to act on emotions rather than reason if one wants to engage the public”. When newspapers confronted him with the claim that the patent he had filed in the United States was for mercenary reasons only and incompatible with the medical profession, he responded harshly, stating that he was no longer a medical doctor but an inventor, and for this he needed financial resources10. He argued that his request for government financial support had fallen on deaf ears and, therefore, he was forced to depend on private funding. Although his style may have facilitated his successful career and given him control of all steps of the pipeline (from discovery via industrial production to medical application), it also led to numerous disputes, not only with his competitors but also with his colleagues, friends and mentors.

Behring understood the art of a deal

As an employee of a state-owned research institute, Behring was obliged to publish all his results9. On several occasions, Behring complained that although he was honoured for scientific achievement, the financial reward was given to others. As a consequence, he seriously considered becoming a full-time employee of the Hoechst Company, although his contract with Hoechst was highly favourable6. Not only did it guarantee substantial financial support for his research, it also granted him a 50% share of income from product sales. Nevertheless, he constantly complained about his contract to Laubenheimer, stating that he needed further financial support. Obviously, Behring understood the art of a deal. Before he started his own company in 1904 (Behring Company), he had negotiated with Hoechst's major competitor (Elberfelder Farbenfabrik, now Bayer Company) — a negotiation that disappointed Laubenheimer profoundly6. Even his patron at the government, Friedrich Althoff, experienced difficulties with Behring5. Although Althoff had pushed through the appointment of Behring as full professor in Marburg, Behring sent him numerous letters of complaint. In a conciliatory letter dated 30 August 1903, Behring wrote to Althoff: “I request to accept my promise that in the future I will be less ruthless not only in pursuing my scientific therapeutic goals but also in my special interests and do my best to become more peaceful”5. Unfortunately, his promise did not last very long. Althoff, therefore, decided to teach Behring a lesson and cut all relations with him and his family (although they later reunited).

Even his mentor Koch and his close colleague Ehrlich were not spared. Adversity with Koch culminated in 1898, when Behring filed a patent about the isolation of compounds from tubercle bacilli in direct competition with Koch's tuberculin. Obviously, Koch was not amused and the two were engaged in lawsuits and numerous quarrels. The conflict between Ehrlich and Behring was exacerbated when Ehrlich produced serum with higher activity by a new technique, which Behring apparently received with humiliation. Reciprocally, when Ehrlich learned about the favourable conditions Behring had negotiated with Hoechst, he felt unfairly treated as his contract was far less alluring. Therefore, it is unsurprising that collaborations between Behring and Ehrlich remained conflict ridden.

Concluding remarks

I return to the question of Behring's impact on immunology. When he entered research as a military doctor, he was fascinated by the new discipline of infectious diseases at the interface between basic research and medical application. Behring stumbled over immunology in his search for clinical application, namely disinfectants to cure infectious diseases. He defined disinfectants as molecules that specifically interfere with disease by neutralizing causative factors (that is, toxins or virulence factors). He was wise enough to immediately realize the enormous potential of his observation. As a result, Behring discovered a novel host mechanism of general importance, which we now call the immune response; a discovery that won him the first ever Nobel Prize in Physiology or Medicine.