Investigation of the role of neutralizing antibodies against bevacizumab as mediators of tachyphylaxis

Editor, T achyphylaxis, defined as a diminished therapeutic response to a drug following repeated administration, has recently been documented for intravitreal bevacizumab (IVB) and ranibizumab in the treatment of exudative age-related macular degeneration (AMD) (Keane et al. 2008; Schaal et al. 2008; Forooghian et al. 2009). Its mechanism in this context is unclear, and we have speculated that the formation of circulating neutralizing antibodies (NAbs) against bevacizumab may be contributory (Forooghian et al. 2009). Systemic NAbs against humanized biologics, including ranibizumab, have previously been documented (Rosenfeld et al. 2006). In the present study, we evaluated the possibility that NAbs against bevacizumab may underlie the tachyphylactic response. We collected serum from patients who were treated with IVB for exudative AMD and had developed tachyphylaxis (n = 11), including five patients who had been described in a previous report (Forooghian et al. 2009). The comparison group included patients with exudative AMD who were naı̈ve to IVB (n = 7) and AMD patients who were being treated with IVB but had not developed tachyphylaxis (n = 5). This prospective study was approved by the National Institutes of Health Institutional Review Board and registered (http://www.clinicaltrials.gov, NCT00605943). An enzyme-linked immunosorbent assay (ELISA) was designed to detect anti-bevacizumab antibodies in patient sera. Anti-bevacizumab antibodies were generated in immunized rabbits, and affinity-purified using standard techniques (Sigma-Genosys, The Woodlands, TX, USA). The Fab fragment of bevacizumab was prepared (Fab Preparation Kit, Pierce, Rockford, IL, USA) and used to coat 96-well high-binding polystyrene microtitre plates for an ELISA assay (1 lg ⁄well). A standard curve was derived using purified rabbit anti-bevacizumab at varying dilutions as the primary antibody, and HRP-conjugated goat anti-rabbit IgG-Fc as the secondary antibody (1:5 000, Jackson ImmunoResearch, West Grove, PA, USA). For patient samples, undiluted sera were placed in the wells, and HRP-conjugated mouse anti-human IgG-Fc used as the secondary antibody (1:1 000 000). Color reaction was developed, and absorbance read at 450 nm. The standard curve of our ELISA assay was linear over the range of 5–40 pg ⁄ml and was used to determine the concentration of antibevacizumab reactivity in patient sera. Basic demographic data, as well as mean anti-bevacizumab titres, are shown in Table 1. The anti-bevacizumab titre in patients with tachyphylaxis was higher (13.6 ± 4.7 pg ⁄ml) than that in the control group (10.9 ± 4.9 pg ⁄ml), a result that approached statistical significance. A one-tailed non-parametric Mann–Whitney test of the hypothesis of greater anti-bevacizumab titres in the tachyphylaxis group compared to the control group yielded a p-value of 0.052. Our results suggest that a NAb response against bevacizumab may develop following repeated IVB. However, these responses were relatively small overall (5–20pg ⁄ml for all patients). Reported serum concentrations of NAbs against other biologic agents, such as natalizumab and infliximab, are 1000–10 000 times greater than observed here (Baert et al. 2003; Calabresi et al. 2007). These other agents are administered by intravenous infusion, achieving much higher serum concentrations than bevacizumab following intravitreal injection. The higher serum concentration of antigen in these cases would be expected to result in a higher concentration of NAbs. Interestingly, a subset of patients receiving intravitreal ranibizumab in the MARINA study also developed circulating serum antibodies to ranibizumab, with their prevalence increasing with longer treatment duration (Rosenfeld et al. 2006). The concentrations of these antibodies were however not described in the report. We determined that a minimal antibody response develops following IVB and that it may be increased in cases of tachyphylaxis. It remains uncertain that this response contributes significantly to the mechanism underlying tachyphylaxis. The small size of the response found and the borderline differences between our treatment and control groups leaves open the possibility that other mechanisms may be at play. The clinical significance of systemic anti-bevacizumab antibodies, as well as those against ranibizumab, deserves further exploration in larger studies.