Infliximab–Tumor Necrosis Factor Complexes Elicit Formation of Anti-drug Antibodies

Abstract Background & Aims Some patients develop anti-drug antibodies (ADAs), which reduce the efficacy of infliximab, a monoclonal antibody against tumor necrosis factor (TNF), in treatment of immune-mediated diseases, including inflammatory bowel diseases. ADAs arise inconsistently, and it is not clear what factors determine their formation. We investigated features of the immune system, the infliximab antibody, and its complex with TNF that might contribute to ADA generation. Methods C57BL/6 mice were given injections of infliximab and recombinant human TNF or infliximab F(ab’)2 fragments. Blood samples were collected every 2–3 days for 2 weeks, and weekly thereafter for up to 6 weeks; infliximab–TNF complexes and ADAs were measured by ELISA. Intestinal biopsies and blood samples were obtained from patients undergoing endoscopy who had received infliximab therapy for inflammatory bowel diseases; infliximab–TNF complexes were measured using an ELISA. Infliximab-specific plasma cells were detected in patient tissue samples using mass cytometry. We studied activation of innate immune cells in peripheral blood-mononuclear cells (PBMC, from healthy donors) incubated with infliximab or infliximab–TNF complexes; toll like receptors (TLR) were blocked with antibodies, endocytosis was blocked with the inhibitor PitStop2, and cytokine expression was measured by real-time PCR and ELISAs. Uptake of infliximab and infliximab–TNF complexes by THP-1 cells was measured with confocal microscopy. Results Mice given increasing doses of infliximab dose produced increasing levels of antibodies against the drug (ADAs). Blood samples from mice given injections of human TNF and infliximab contained infliximab–TNF complexes; complex formation associated with ADA formation with an area under the curve of 0.944 (95% CI, 0.851–1.000; P=.003). Intestinal tissues from patients, but not blood samples, contained infliximab–TNF complexes and infliximab-specific plasma cells. Incubation of PBMC with infliximab–TNF complexes resulted in a 4.74-fold increase in level of interleukin 1beta (IL1B) mRNA (P for comparison=.005), increased IL1B protein secretion, and a 2.69-fold increase in expression of TNF mRNA (P for comparison 0.013) compared with control PBMC. Infliximab only reduced IL1B and TNF expression. Antibodies against TLR2 or TLR4 did not block the increases in IL1B or TNF expression, but endocytosis was required. THP-1 cells endocytosed higher levels of infliximab-TNF complexes than infliximab alone. Conclusions In mice, we found ADA formation to increase with dose of infliximab given and concentration of infliximab–TNF complexes detected in blood. Based on studies of human intestinal tissues and blood samples, we propose that infliximab–TNF complexes formed in the intestine are endocytosed by and activate innate immune cells, which increase expression of IL1B and TNF and production of antibodies against the drug complex. It is therefore important to optimize the infliximab dose to a level that is effective but does not activate an innate immune response against the drug–TNF complex.