Background:The epitope and the TNF␣ inhabitation mechanism of Adalimumab remain unclear. Results: The crystal structure of the TNF␣ in complex with Adalimumab is reported at a resolution of 3.1 Å. Conclusion:The epitope of Adalimumab provided information that Adalimumab may have clinical advantage compared with Infliximab. Significance: These data reveal the Adalimumab's mechanism of TNF␣ inhibition and its advantages compared with other TNF inhibitors in clinical practice. TNF␣-targeting therapy with the use of the drugs Etanercept, Infliximab, and Adalimumab is used in the clinical treatment of various inflammatory and immune diseases. Although all of these reagents function to disrupt the interaction between TNF␣ and its receptors, clinical investigations showed the advantages of Adalimumab treatment compared withEtanercept and Infliximab. However, the underlying molecular mechanism of action of Adalimumab remains unclear. In our previous work, we presented structural data on how Infliximab binds with the E-F loop of TNF␣ and functions as a TNF␣ receptorbinding blocker. To further elucidate the variations between TNF␣ inhibitors, we solved the crystal structure of TNF␣ in complex with Adalimumab Fab. The structural observation and the mutagenesis analysis provided direct evidence for identifying the Adalimumab epitope on TNF␣ and revealed the mechanism of Adalimumab inhibition of TNF␣ by occupying the TNF␣ receptor-binding site. The larger antigenantibody interface in TNF␣ Adalimumab also provided information at a molecular level for further understanding the clinical advantages of Adalimumab therapy compared with Infliximab.TNF is an immunity-modulating cytokine required for immune processes. The unregulated activities of TNFs can lead to the development of inflammatory diseases. Excess amounts of TNF␣ expressed in cells are associated with the development of immune diseases, including rheumatoid arthritis, Crohn's disease, psoriatic arthritis, and inflammatory bowel disease (1, 2). The function of TNF␣ requires smooth interaction with its two receptors, TNF receptor 1 (TNFR1) 4 and TNF receptor 2 (TNFR2). Blocking the interaction between TNF␣ and TNFRs has successfully been developed as a therapy in treating inflammatory or autoimmune diseases (3,4). TNF␣ neutralization therapies, including the use of a soluble TNFR2-Fc recombinant (Etanercept), a mouse-human chimera mAb (Infliximab), or a human mAb (Adalimumab), have been introduced in the past decades for the management of rheumatoid arthritis and other immune diseases (5).Although all of these TNF␣ blockers function by interrupting the TNF␣-TNFR interaction, information on whether the different TNF␣ inhibitors have similar clinical efficacy remains controversial because of the lack of randomized clinical trial meta-analyses. In the early stages of clinical usage of Infliximab, its discontinuation was reported to result in loss of response. This largely affected patients who received long term treatment and later discontinued use (6). Approximately 10% of...

There is an urgent need for improved therapy for advanced ovarian carcinoma, which may be met by administering immune-modulatory monoclonal antibodies (mAbs) to generate a tumor-destructive immune response. Using the ID8 mouse ovarian cancer model, we investigated the therapeutic efficacy of various mAb combinations in mice with intraperitoneal (i.p.) tumor established by transplanting 3 × 106 ID8 cells 10 days previously. While most of the tested mAbs were ineffective when given individually or together, the data confirm our previous finding that 2 i.p. injections of a combination of anti-CD137 with anti-PD-1 mAbs doubles overall survival. Mice treated with this mAb combination have a significantly increased frequency and total number of CD8+ T cells both in the peritoneal lavage and spleens, and these cells are functional as demonstrated by antigen-specific cytolytic activity and IFN-γ production. While administration of anti-CD137 mAb as a single agent similarly increases CD8+ T cells, these have no functional activity, which may be attributed to up-regulation of co-inhibitory PD-1 and TIM-3 molecules induced by CD137. Addition of the anti-cancer drug cisplatin to the 2 mAb combination increased overall survival >90 days (and was probably curative) by a mechanism which included a systemic CD8+ T cell response with tumor specificity and immunological memory. Strikingly, combined treatment of cisplatin and CD137/PD-1 mAb also gave rise to the long-term survival of mice with established TC1 lung tumors. A similar combination of the 2 mAbs and cisplatin should be considered for clinical ‘translation’.

Despite widespread use of the anti-CD20 monoclonal antibody (mAb), rituximab, in treating B-cell lymphomas, its efficacy remains variable and often modest. A better understanding of rituximabmediated killing mechanisms is essential to develop more effective therapeutic agents. In this study, we modulated the binding property of rituximab by introducing several point mutations in its complementarity-determining regions. The data showed that changing the binding avidity of rituximab in the range from 10 ؊8 to 10 ؊10 M could regulate its antibodydependent cellular cytotoxicity but not affect its complement-dependent cytotoxicity and apoptosis-inducing activity in B-lymphoma cells. Contradictory to previous findings, we found that the complementdependent cytotoxicity potency of CD20 mAb was independent of the off-rate. Despite still being a type I CD20 mAb, a rituximab triple mutant (H57DE/H102YK/ L93NR), which had a similar binding avidity to a double mutant (H57DE/H102YK), was unexpectedly found to have extremely potent apoptosis-inducing activity. Moreover, this triple mutant, which was demonstrated to efficiently initiate both caspase-dependent and -independent apoptosis, exhibited potent in vivo therapeutic efficacy, even in the rituximabresistant lymphoma model, suggesting that it might be a promising therapeutic agent for B-cell lymphomas. (Blood. 2009; 114:5007-5015) IntroductionThe CD20 molecule is a 30-to 35-kDa integral membrane protein expressed by B lymphocytes in early stages of differentiation and by most B-cell lymphomas. 1,2 CD20 is an ideal target for monoclonal antibodies (mAbs), as it is expressed at high levels on most B-cell malignancies but does not become internalized or shed from the plasma membrane after mAb treatment. 3,4 The mouse/human chimeric anti-CD20 antibody, rituximab, is the first therapeutic mAb approved for the treatment of relapsed/refractory low-grade or follicular B-cell non-Hodgkin lymphomas. 5,6 Previous studies have suggested that several mechanisms might be involved in providing therapeutic efficacy, including complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC), and the induction of apoptosis. 4,7 The relative contributions of these different mechanisms of action are still a matter of debate. 4,7 Anti-CD20 mAbs are usually defined as either type I or II, based on their ability to redistribute CD20 into lipid rafts. 8,9 Type I mAbs (rituximab and most anti-CD20 mAbs) are able to efficiently shift CD20 complexes into rafts, but the type II mAbs (B1 and 11B8) are not. The in vitro assays further indicate that type I mAbs usually exhibit potent CDC activity and relatively low level of apoptosis unless extensively cross-linked by antibody, 8 whereas type II mAbs are relatively inactive in complement activation but tend to promote more apoptosis. 9,10 Both types of mAb are equally potent in ADCC with FcR-bearing myeloid effectors.Although rituximab has been widely used in the treatment of lymphoma, only 48% of patients respond to the treatme...

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