Obinutuzumab (GA101) is a novel, type II, glycoengineered, humanized anti-CD20 monoclonal antibody that has been developed to address the need for new therapeutics with improved efficacy in patients with lymphocytic leukemia and lymphoma of B-cell origin. of obinutuzumab with novel targeted agents. expression of the inhibitory Fc receptor FcRIIb on CGS-15943 manufacture target B-cells and has been shown to predict less durable responses to rituximab therapy in patients with MCL [37]. Management of relapse and resistance in rituximab-treated patients presents a significant challenge [6], and there is a need for treatments with improved activity across B-cell NHL subtypes and CLL. Better understanding of antibody biology and modes of action, together with increased ability to design CGS-15943 manufacture highly efficient therapeutics, has led to the development of novel mAbs with improved activity. As a review article, the following paper does not contain any new studies with human or animal subjects performed by any of the authors. Obinutuzumab, a Novel Humanized Type II mAb Obinutuzumab (GA101) is a novel, type II, MDK glycoengineered, humanized anti-CD20 mAb that has been developed to address the need for novel therapeutics with higher activity than rituximab. The post-translational glycoengineering process used in the development of this agent [resulting in the absence of a fucose sugar residue from immunoglobulin G (IgG) oligosaccharides in the Fc region of the mAb molecule] was developed to increase activity by enhancing binding affinity to the FcRIII receptor on immune effector cells (Fig.?1) [38, 39]. Additionally, obinutuzumab has a modified elbow-hinge amino acid sequence compared to type I agents, which together with the unique epitope recognized by obinutuzumab results in spatial alterations of the CD20-mAb complex on B-cells [39, 40]; this is believed to be the molecular basis for the type II biology of obinutuzumab [40] as both type II character and cell death induction (as described below) can be switched on and off by mutating this elbow-hinge region [39]. Fig.?1 Structure and binding behavior of obinutuzumab. Glycoengineered structure and type II binding properties of obinutuzumab. a Glycoengineering by defucosylation of immunoglobulin G oligosaccharides in the Fc region of obinutuzumab. In Chinese hamster ovary … The type II mechanism of action of obinutuzumab together with glycoengineering acts to enhance direct cell death and ADCC/ADCP, while decreasing CDC (Fig.?2) [41], and differentiates the drug from classical type I anti-CD20 mAbs, such as rituximab and ofatumumab [39, 40, 42C44]. Rituximab, by comparison, works primarily via CDC (by clustering CD20 within lipid rafts) and by ADCC/ADCP, with direct cell death contributing much less to the overall antitumor activity [45]. Ofatumumab also acts primarily via CDC after binding both loop domains of CD20 at a different epitope compared to rituximab [46]. Fig.?2 Putative mechanisms of action of obinutuzumab. Please refer to the text for further information and supporting references. antibody-dependent cell-mediated cytotoxicity, antibody-dependent cellular phagocytosis, complement-dependent cytotoxicity … Increased Direct Cell Death Induction Obinutuzumab has been shown to be faster than and superior to both rituximab and ofatumumab in inducing direct cell death in malignant B-cells. This was demonstrated by phosphatidylserine exposure and propidium iodide staining [with analysis by fluorescence-activated cell sorting (FACS) and time-lapse microscopy] in a panel of CD20-expressing tumor cell lines [39, 43]. While some researchers have questioned the CGS-15943 manufacture validity of using FACS to assess mAb-induced direct cell death (because of potential mechanical interference with mAb-mediated homotypic adhesion) [47], multiple studies, using a variety of methods (including FACS), have confirmed that, overall, obinutuzumab induces greater direct cell death than type I mAbs [48C53] and occurs without disruption of homotypic aggregates [43, 54]. The mechanisms that may underlie the ability of type II anti-CD20 mAbs to directly evoke programmed cell death (PCD) are still poorly understood, but have been investigated in several studies [39, 48, 55]. Honeychurch et al. demonstrated actin-dependent, lysosome-mediated induction of PCD by type II mAbs, such as obinutuzumab or tositumomab, which was directly correlated CGS-15943 manufacture with the production of reactive oxygen species (ROS) [48]. In contrast, type I mAbs, such as rituximab, induced only minimal levels of ROS and PCD. Generation of ROS mediated by nicotinamide adenine dinucleotide phosphate oxidase, independently of mitochondria, was unaffected by B-cell lymphoma 2 (BCL-2) overexpression and took.