Resolving the micro-heterogeneity and structural integrity of monoclonal antibodies by hybrid mass spectrometric approaches

For therapeutic monoclonal antibodies (mAbs), detailed analysis of the structural integrity and heterogeneity, which results from multiple types of post-translational modifications (PTMs), is relevant to various processes, including product characterization, storage stability and quality control. Despite the recent rapid development of new bioanalytical techniques, it is still challenging to completely characterize the proteoform profile of a mAb. As a nearly indispensable tool in mAb analysis, mass spectrometry (MS) provides unique structural information at multiple levels. Here, we tested a hybrid strategy for the comprehensive characterization of micro-heterogeneity by integrating 2 state-of-the-art MS-based approaches, high-resolution native MS and targeted glycan profiling, to perform complementary analysis at the intact protein level and released glycan level, respectively. We compared the performance of these methods using samples of engineered half-body IgG4s and a panel of mAbs approved for human use. The glycosylation characterization data derived from these approaches were found to be mutually consistent in composition profiling, and complementary in identification and relative-quantitation of low-abundant uncommon glycoforms. In addition, multiple other sources of micro-heterogeneity, such as glycation, lack of glycosylation, and loss of light chains, could be detected by this approach, and the contribution of multiple types of modifications to the overall micro-heterogeneity could be assessed using our superposition algorithm. Our data demonstrate that the hybrid strategy allows reliable and thorough characterization of mAbs, revealing product characteristics that would easily be missed if only a single approach were used.     

TNF inhibitor suppresses bone metastasis in a breast cancer cell line

In the evolution of cancer, tumor necrosis factor-alpha (TNF-α) plays a paradoxical role. High doses induce significant anticancer effects, but conversely, physiologic and pathologic levels of TNF-α may be involved in cancer promotion, tumor growth, and metastasis.Infliximab is a chimeric murine monoclonal antibody that binds with high affinity to soluble and membrane TNF-α and inhibits binding of TNF-α to its receptors. In the present study, we investigated the effect of infliximab, a TNF-α antagonist, on breast cancer aggressiveness and bone metastases.Infliximab greatly reduced cell motility and bone metastases in a metastatic breast cancer cell line, MDA-MB-231. The mechanism of bone metastasis inhibition involved decreased expression of CXC chemokine receptor 4 (CXCR4) and increased expression of decorin, which is the prototype of an expanding family of small leucine-rich proteoglycans. These results suggest a novel role for TNF-α inhibition in the reduction or prevention of bone metastases in this breast cancer model. Our study suggests that inhibition of TNF-α using infliximab may become a preventive therapeutic option for breast cancer.     

Increases in body mass index during infliximab therapy in patients with Crohn's disease: an open label prospective study

In the past, the impact of infliximab therapy on nutritional status in patients with Crohn’s disease (CD) has not been assessed. This prospective study was to investigate the effect of infliximab on nutritional status in CD patients. Fifty consecutive patients with active CD received infliximab (5 mg/kg) at weeks 0, 2 and 6 as remission induction therapy, and then at 8 weeks intervals as maintenance therapy. Patients were followed for 60 weeks. CD activity index (CDAI) and body mass index (BMI) were monitored. A fall in CDAI by ?70 was defined as response to therapy, while CDAI <150 meant clinical remission. At week 10, 39 patients (78%) responded to infliximab induction therapy. BMI significantly increased during these 10 weeks (P < 0.0001). The mean increase in BMI was significantly higher in patients who responded to infliximab vs patients who did not (P = 0.03). Further, at weeks 30 and 60, 35 patients (70%) and 33 (66%) were in remission, respectively. The mean increase in BMI was significantly higher in patients who maintained remission vs patients not in remission (week 30, P = 0.02; week 60, P = 0.01). Patients with a low baseline BMI (<18.5) and those with small bowel involvement achieved a higher increase in BMI as compared to patients with BMI ?18.5 or patients without small bowel involvement. In this study, infliximab therapy was associated with improvement of patients’ nutritional status, notably patients who responded to this biologic. Additionally, in patients with malnutrition and small bowel involvement, the nutritional impact of infliximab was higher.     

英夫利昔单抗辅助治疗克罗恩病伴不全性肠梗阻的临床分析

目的:评价英夫利西单抗辅助治疗克罗恩病伴不全性肠梗阻患者中的疗效和安全性。方法:选择22罗恩病伴有不全性肠梗阻的患者,将其随机分为英夫利西单抗治疗组及常规治疗组。常规治疗组患者予禁食或流质饮食、抗感染、补液维持水电平衡等内科常规治疗,并予美沙拉嗪、强的松、硫唑嘌呤长期口服。英夫利昔单抗治疗组在常规治疗的基础上,在治疗第0、2、6、14、22、30周给予英夫利西单抗5mg/kg。治疗30周时,检查和比较2组患者治疗前后的血象、肝功能、血沉、C反应蛋白、记录CD疾病活动指数,行肠镜检查评价疗效。并记录治疗期间患者不良反应的发生情况。结果:治疗第30周末,英夫利昔单抗治疗组的临床总有效率和内镜下总有效率均显著高于常规治疗组(P0.05);且英夫利昔单抗治疗组血沉和CRP水平、CDAI较第0周均显著降低(P0.05)。治疗期间英夫利昔单抗治疗组无严重不良反应发生。结论:在内科常规常规保守治疗的基础上,给予英夫利昔单抗辅助治疗可显著提高CD伴有不全性肠梗阻患者的疗效。     

Monomeric IgA can be produced in planta as efficient as IgG,yet receives different N‐glycans

The unique features of IgA, such as the ability to recruit neutrophils and suppress the inflammatory responses mediated by IgG and IgE, make it a promising antibody isotype for several therapeutic applications. However, in contrast to IgG, reports on plant production of IgA are scarce. We produced IgA1κ and IgG1κ versions of three therapeutic antibodies directed against pro‐inflammatory cytokines in Nicotiana benthamiana: Infliximab and Adalimumab, directed against TNF‐α, and Ustekinumab, directed against the interleukin‐12p40 subunit. We evaluated antibody yield, quality and N‐glycosylation. All six antibodies had comparable levels of expression between 3.5 and 9% of total soluble protein content and were shown to have neutralizing activity in a cell‐based assay. However, IgA1κ‐based Adalimumab and Ustekinumab were poorly secreted compared to their IgG counterparts. Infliximab was poorly secreted regardless of isotype backbone. This corresponded with the observation that both IgA1κ‐ and IgG1κ‐based Infliximab were enriched in oligomannose‐type N‐glycan structures. For IgG1κ‐based Ustekinumab and Adalimumab, the major N‐glycan type was the typical plant complex N‐glycan, biantennary with terminal N‐acetylglucosamine, β1,2‐xylose and core α1,3‐fucose. In contrast, the major N‐glycan on the IgA‐based antibodies was xylosylated, but lacked core α1,3‐fucose and one terminal N‐acetylglucosamine. This type of N‐glycan occurs usually in marginal percentages in plants and was never shown to be the main fraction of a plant‐produced recombinant protein. Our data demonstrate that the antibody isotype may have a profound influence on the type of N‐glycan an antibody receives.     

英夫利西单抗联合复合益生菌对炎症性肠病患者肠黏膜屏障、炎症因子、细胞免疫功能及骨代谢指标的影响

摘要 目的：探讨英夫利西单抗联合复合益生菌对炎症性肠病（IBD）患者肠黏膜屏障、细胞免疫功能及骨代谢指标的影响。方法：研究对象选取2017年3月~2019年12月期间来我院诊治的290例IBD患者,信封抽签法分为对照组和研究组,各145例。研究组采用英夫利西单抗联合复合益生菌治疗,对照组采用英夫利西单抗治疗,比较两组患者疗效、肠黏膜屏障功能、T细胞亚群、骨代谢指标及炎症因子水平,并记录两组治疗期间不良反应情况。结果：对照组治疗2疗程后的临床总有效率为71.72%（104/145）,低于研究组的86.21%（125/145）（P<0.05）。治疗1疗程后、治疗2疗程后,两组患者的C反应蛋白（CRP）、肿瘤坏死因子-α（TNF-α）、白介素-6（IL-6）、骨钙素（BGP）、Ｉ-型胶原Ｃ末端肽（CTX）、CD8⁺及尿乳果糖/甘露醇比值均较治疗前降低,治疗2疗程后上述指标水平低于治疗1疗程后,且研究组低于对照组（P<0.05）。治疗1疗程后、治疗2疗程后,两组的CD4⁺、CD4⁺/ CD8⁺较治疗前升高,治疗2疗程后上述指标水平高于治疗1疗程后,且研究组高于对照组（P<0.05）。两组不良反应发生率对比无统计学差异（P>0.05）。结论：英夫利西单抗联合复合益生菌治疗IBD患者,可减轻患者的炎症反应,提高患者的免疫功能,改善其肠黏膜屏障和骨代谢指标,且安全性较好。     

The effect of a single dose of tumor necrosis factor alpha inhibitor on gut permeability in rats during exposure to a heat stress

Heat stroke is a life threatening illness characterized by a core body temperature of >40 °C, delirium and convulsions, and often results in multi-organ dysfunction, due to the release of endotoxin through the intestinal wall into the circulation. While playing a major role in the gastrointestinal tract permeability changes seen in Crohn's disease, it is not clear whether tumor necrosis factor alpha (TNF-α) mediates the increase in intestinal permeability and the release of endotoxin into the circulation in heat stroke. The aim of the present study was to determine the acute effects of a single dose of TNF-α antibody on gut permeability in rats during heat stress. Fifty-five Sprague-Dawley rats (28 male and 27 female) were treated with either saline or infliximab (a monoclonal antibody to TNF-α), anesthetized with pentobarbitone (50 mg kg⁻¹) and then exposed to either normothermic conditions or an ambient temperature of between 41 and 42 °C for 70 min. Fluorescent isothiocyanate labeled dextrans (FITC-dextrans) were administered intragastrically as a marker of intestinal permeability. Liver enzymes, endotoxin and TNF-α were analyzed in the blood. Exposure to a heat stress significantly increased intestinal permeability to FITC-dextrans compared to the controls (P<0.05). Infliximab did not have an effect on the intestinal permeability to the FITC-dextrans. Heat stress had no significant effect on liver enzymes or endotoxin concentration versus controls (P>0.05). TNF-α was not detectable in any of the samples. TNF-α did not mediate the release of endotoxin into the circulation after an acute bout of heat stroke.     

T cell lymphoproliferative disorders associated with anti-tumor necrosis factor alpha antibody therapy for ulcerative colitis: literature summary

The enhanced risk of development of lymphoproliferative disorders in patients with inflammatory bowel disease has been attributed to immunosuppressive/immunomodulatory therapies. Infliximab is a chimeric monoclonal immunoglobulin G1 antibody directed against tumor necrosis factor alpha (TNF-α) that was approved by the Food and Drug Administration (FDA) in 1998 as an effective therapeutic agent against inflammatory bowel disease. Malignant lymphomas of both B and T cell lineage have been described in patients undergoing therapy involving TNF-α blockade. To date, eight cases of Epstein–Barr virus (EBV)-negative hepatosplenic T cell lymphoma associated with infliximab have been reported to the FDA’s Adverse Event Reporting System, as well as several other T cell lymphoproliferative disorders with aggressive clinical outcomes. We present the histologic, immunophenotypic, and molecular features of a T cell lymphoproliferative disorder involving the axillary lymph node of a 33-year-old male following infliximab treatment for ulcerative colitis. These EBV-negative lymphomas suggest that lymphoproliferative disorders following infliximab treatment for inflammatory bowel disease may involve EBV-independent immune dysregulation. The spectrum of lymphoproliferative disorders associated with infliximab and the potential mechanisms by which they occur are discussed.