Monoclonal antibody testing

To the Editor: The episode in the UK in which six previously healthy men suffered serious side effects while undergoing a trial for a new drug [1], i.e. a humanized monoclonal antibody TGN1412 which activates killer T cells in the immune system, has provoked considerable debate about the future of human drug trials. We believe human drug trials are essential and they must continue. At the same time, the UK trial questions assumptions about the occurrence of exact homologies in immune system functions across human and animal species. In particular, the pre-clinical results from assessing TGN1412 in monkeys, where reported side effects were relatively mild and limited to increased volume of lymph nodes, were not telltale of the massive immune system dysfunction and damage to body organs sustained by the trial participants. We believe that monkeys might not be the appropriate nonhuman primate species for monoclonal-related trials. Instead, chimpanzees may be, in many circumstances, the only adequate model for testing the safety of humanized monoclonal antibodies, and they have been used extensively for this purpose [6]. There are at least three reasons why chimpanzees may serve as the most relevant animal model for monoclonal antibody testing. 1. Generally, the amino acid sites to which the antibodies bind on human proteins are identical in chimpanzees but not in other species. Sometimes, the antibodies do not bind at all to the orthologous protein of other species. In these instances, the chimpanzee is the most relevant model for safety testing. Even when the monoclonal antibodies bind to the orthologous protein of other species, different binding capacities can lead to major differences in the immediate physiological response to the antibody and to the cascade of events following treatment. This may account for the differences in the responses of monkeys and humans after administration of the anti-CD28 antibody in the UK. 2. Many of the monoclonal antibodies currently being developed for clinical use target cells of the immune system. Immunomodulation is risky because the events that may occur consequent to perturbations of the immune system are complex and sometimes unpredictable. In some instances, the immune system of monkeys (and other non-ape species) may not be similar enough to the immune system of humans to provide an accurate model, even if the antibody has a similar binding affinity to monkey cells. 3. Many, if not most, monoclonal antibody therapies require long-term (often lifelong) administration (e.g. to reduce inflammation in autoimmune diseases). Because the antibodies are humanized, chimpanzees and humans are the only species that do not recognize them as foreign. Monkeys quickly clear them after the second administration, so the long-term effects of these antibodies in the circulation can be tested only in chimpanzees. Either or both of the first two points above may have been responsible, at least in part, for the disastrous consequences of the TGN1412 trial in human subjects. In order to assess the likelihood of differential binding of TG412 to human, chimpanzee and macaque CD28 cells, we identified the putative chimpanzee CD28 sequence in GenBank (XM_525998) by blastn and aligned it with human isoform 1 (SwissProt P10747-1) and the deduced macaque CD28 sequence (AAAK37538.1; NM_001042641). The alignment showed identical amino acid sequences in the human and chimpanzee extracellular domain. In contrast, the macaque sequence had four amino acid substitutions in the extracellular domain, including a G86E substitution in the C¢¢D superagonist binding loop [4]. The notion that CD28 is highly conserved in mammals [2] apparently led to that pre-clinical testing in macaques would accurately anticipate human results, specifically because the macaque and human CD28 receptors were assumed to be identical in amino acid sequence [3]. Although we have no data that compare the binding affinities of TG412 with CD28 cells of the three species, the shape and the binding efficacy of the antigenic sites may differ between human/chimpanzee CD28 and macaque CD28 [5]. Such a difference, and/or downstream species differences in the cascade of immunological events elicited by the antibody, may well explain why the results from macaque trials failed to indicate the side effects observed in humans [2, 3]. The risks associated with monoclonal antibody trials in human subjects cannot be entirely eliminated. However, the development of knowledge and understanding J Med Primatol doi:10.1111/j.1600-0684.2006. 00193.x