Department of Gastroenterology, Rambam Health Care Campus, Haifa, Israel; Bruce Rappaport School of Medicine, Technion Israel Institute of Technology, Haifa, Israel.
Department of Gastroenterology, Rambam Health Care Campus, Haifa, Israel.
Gastroenterology. 2019 Nov;157(5):1338-1351.e8. doi: 10.1053/j.gastro.2019.08.009. Epub 2019 Aug 8.
BACKGROUND & AIMS: Some patients develop anti-drug antibodies (ADAs), which reduce the efficacy of infliximab, a monoclonal antibody against tumor necrosis factor (TNF), in the 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.
C57BL/6 mice were given injections of infliximab and recombinant human TNF or infliximab F(ab') 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 enzyme-linked immunosorbent assay (ELISA). Intestinal biopsy and blood samples were obtained from patients having endoscopy who had received infliximab therapy for inflammatory bowel diseases; infliximab-TNF complexes were measured with ELISA. Infliximab-specific plasma cells were detected in patient tissue samples by using mass cytometry. We studied activation of innate immune cells in peripheral blood mononuclear cells (PBMCs) from healthy donors incubated with infliximab or infliximab-TNF complexes; toll-like receptors (TLRs) were blocked with antibodies, endocytosis was blocked with the inhibitor PitStop2, and cytokine expression was measured by real-time polymerase chain reaction and ELISAs. Uptake of infliximab and infliximab-TNF complexes by THP-1 cells was measured with confocal microscopy.
Mice given increasing doses of infliximab produced increasing levels of ADAs. Blood samples from mice given injections of human TNF and infliximab contained infliximab-TNF complexes; complex formation was associated with ADA formation with an area under the curve of 0.944 (95% confidence interval, 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 PBMCs with infliximab-TNF complexes resulted in a 4.74-fold increase in level of interleukin (IL) 1β (IL1B) messenger RNA (P for comparison = .005), increased IL1B protein secretion, and a 2.69-fold increase in the expression of TNF messenger RNA (P for comparison = 0.013) compared with control PBMCs. Infliximab reduced only 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.
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.
一些患者会产生针对药物的抗体（ADAs），从而降低肿瘤坏死因子（TNF）单克隆抗体英夫利昔单抗在治疗免疫介导性疾病（包括炎症性肠病）中的疗效。ADAs 的产生不一致，目前尚不清楚是什么因素决定了它们的形成。我们研究了可能导致 ADA 产生的免疫系统、英夫利昔单抗抗体及其与 TNF 的复合物的特征。
C57BL/6 小鼠接受英夫利昔单抗和重组人 TNF 或英夫利昔单抗 F(ab') 片段的注射。每隔 2-3 天采集一次血液样本，持续 2 周，此后每周采集一次，持续 6 周；通过酶联免疫吸附试验（ELISA）测量英夫利昔单抗-TNF 复合物和 ADA。从接受英夫利昔单抗治疗炎症性肠病的接受内镜检查的患者中获得肠活检和血液样本；通过 ELISA 测量英夫利昔单抗-TNF 复合物。使用质谱流式细胞术检测患者组织样本中的英夫利昔单抗特异性浆细胞。我们研究了健康供体外周血单核细胞（PBMCs）与英夫利昔单抗或英夫利昔单抗-TNF 复合物孵育后固有免疫细胞的激活；用抗体阻断 Toll 样受体（TLRs），用抑制剂 PitStop2 阻断内吞作用，并通过实时聚合酶链反应和 ELISA 测量细胞因子表达。使用共聚焦显微镜测量 THP-1 细胞对英夫利昔单抗和英夫利昔单抗-TNF 复合物的摄取。
给予递增剂量英夫利昔单抗的小鼠产生的 ADA 水平逐渐增加。给予 TNF 和英夫利昔单抗注射的小鼠的血液样本中含有英夫利昔单抗-TNF 复合物；复合物的形成与 ADA 的形成相关，曲线下面积为 0.944（95%置信区间，0.851-1.000；P =.003）。患者的肠道组织，但不是血液样本，含有英夫利昔单抗-TNF 复合物和英夫利昔单抗特异性浆细胞。与对照 PBMCs 相比，用英夫利昔单抗-TNF 复合物孵育 PBMCs 会导致白细胞介素（IL）1β（IL1B）信使 RNA 水平增加 4.74 倍（P 比较=.005），IL1B 蛋白分泌增加，TNF 信使 RNA 表达增加 2.69 倍（P 比较=0.013）。英夫利昔单抗仅降低 IL1B 和 TNF 的表达。TLR2 或 TLR4 的抗体并不能阻断 IL1B 或 TNF 表达的增加，但内吞作用是必需的。THP-1 细胞内吞的英夫利昔单抗-TNF 复合物水平高于单独的英夫利昔单抗。
在小鼠中，我们发现 ADA 的形成随着给予的英夫利昔单抗剂量的增加和血液中检测到的英夫利昔单抗-TNF 复合物浓度的增加而增加。基于对人类肠道组织和血液样本的研究，我们提出在肠道中形成的英夫利昔单抗-TNF 复合物被内吞并激活固有免疫细胞，从而增加 IL1B 和 TNF 的表达和对药物复合物的抗体产生。因此，重要的是要优化英夫利昔单抗剂量，使其达到有效但不激活针对药物-TNF 复合物的固有免疫反应的水平。
