Improving effector functions of antibodies for cancer treatment: Enhancing ADCC and CDC

Akito Natsume, Rinpei Niwa, Mitsuo SatohAntibody Research Laboratories, Research Division, Kyowa Hakko Kirin Co., Ltd.,/Machida-shi, Tokyo, JapanAbstract: As platforms for therapeutic agents, monoclonal antibodies (MAbs) have already been approved, and several MAbs have demonstrated clinical effectiveness in a variety of malignancies. However, several issues have also been emerging in antibody therapy, such as high cost and insufficient drug action. Recently, to improve MAb activity in humans, effector functions have been subjects of focus, especially antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). Extensive efforts have been made to enhance these effector functions of MAbs, and successful approaches have been reported by us and others, wherein the binding activity of MAbs to FcγRIIIa or C1q is increased by introducing amino acid mutations into heavy chain constant regions or through glyco-modification of Fc-linked oligosaccharides. In addition, one of the next approaches to optimizing therapeutic antibodies would be to combine multiple enhancing modifications into a single antibody platform to overcome the diverse mechanisms of clinical resistance of tumor cells. For this aim, we have recently developed a successful combination composed of ADCC-enhancing modification by the fucose depletion from Fc-linked oligosaccharides and CDC-enhancing modification by IgG1 and IgG3 isotype shuffling in heavy chains, which could be of great value for the development of third-generation antibody therapeutics.Keywords: ADCC, CDC, effector functions, Fc oligosaccharides, IgG isotypes, nonfucosylated Ig

(A) , , , and MEFs were cultured in complete or starvation medium for 60 min

In the recovery experiments, starved MEFs were cultured in fresh complete medium for an additional 60 min (replenishment). The cell lysates were subjected to immunoblot analysis with the indicated antibodies. (B) Wild-type and MEFs were cultured in the complete or starvation medium for indicated time with or without 100 nM bafilomycin A. The cell lysates were subjected to immunoblot analysis with anti-LC3 antibody. (C) Wild-type and MEFs were transfected with retroviral vectors encoding GFP-Atg5 or GFP-LC3. Resulting cells were cultured in the starvation medium for 120 min. The cells were fixed and examined by fluorescence microscopy. Bar, 20 μm. (D–G) Wild-type (D and E) and (F and G) MEFs were cultured in complete (D and F) or starvation (E and G) medium for 120 min and then fixed and subjected to EM analysis. Autophagosome-like structures (open arrowheads), and autolysosomes (closed arrowheads) are indicated. Bar, 1 μm. (H) The ratio of total area of autophagosomes (AP) and autolysosomes (AL) to total cytoplasmic area in D–G was determined by morphometric analysis.<p><b>Copyright information:</b></p><p>Taken from "FIP200, a ULK-interacting protein, is required for autophagosome formation in mammalian cells"</p><p></p><p>The Journal of Cell Biology 2008;181(3):497-510.</p><p>Published online 5 May 2008</p><p>PMCID:PMC2364687.</p><p></p

(A) NIH3T3 cells stably expressing GFP-FIP200 were cultured in complete or starvation medium for 120 min and the GFP signal was observed

(B) NIH3T3 cells stably expressing GFP-FIP200 were cultured in starvation medium for 120 min and then subjected to immunofluorescence microscopy using anti-Atg16L1 antibody and Alexa Fluor 568–conjugated secondary antibody. Bars, 20 μm. More than 90% of GFP-FIP200 dots were positive for Atg16L1. (C) NIH3T3 cells stably expressing GFP-ULK1 were starved for 120 min and then subjected to immunofluorescence microscopy using anti-FIP200 antibody and Alexa Fluor 568–conjugated secondary antibody. Black squares indicate the enlarged areas shown in insets. Bar, 20 μm.<p><b>Copyright information:</b></p><p>Taken from "FIP200, a ULK-interacting protein, is required for autophagosome formation in mammalian cells"</p><p></p><p>The Journal of Cell Biology 2008;181(3):497-510.</p><p>Published online 5 May 2008</p><p>PMCID:PMC2364687.</p><p></p

(A) and MEFs stably expressing GFP-ULK1 or -ULK2 were cultured in starvation medium for 120 min

The cells were fixed and examined by fluorescence microscopy. Bar, 20 μm. (B) and MEFs were cultured in complete medium. The cell lysates were subjected to immunoblot analysis with antibodies against ULK1 or HSP90 (loading control). (C) Phosphatase sensitivity of ULK1. and MEFs were cultured in complete medium. ULK1 was immunoprecipitated from cell lysates and treated with λ phosphatase for 30 min at 30°C in the absence or presence of phosphatase inhibitors (1 mM NaVO, 50 mM KF, 15 mM NaPO, and 1 mM EGTA).<p><b>Copyright information:</b></p><p>Taken from "FIP200, a ULK-interacting protein, is required for autophagosome formation in mammalian cells"</p><p></p><p>The Journal of Cell Biology 2008;181(3):497-510.</p><p>Published online 5 May 2008</p><p>PMCID:PMC2364687.</p><p></p

(A and B) NIH3T3 cells stably expressing GFP-ULK1 (A) and -ULK2 (B) were cultured in complete or starvation medium for 30 min

They were then cultured in fresh complete medium for an additional 30 min (starvation→complete). (C and D) NIH3T3 cells stably expressing GFP-ULK1 (C) and -ULK2 (D) were cultured in starvation medium for 120 min. The cells were fixed, permeabilized, and subjected to immunofluorescence microscopy using anti-Atg16L1 antibody and Alexa Fluor 660–conjugated secondary antibody. More than 90% of GFP-ULKs dots were positive for Atg16L1. (E–H) Wild-type (E and G) and (F and H) MEFs were transfected with retroviral vectors encoding GFP-ULK1 and -ULK2. MEFs stably expressing GFP-ULK1 (E and F) and -ULK2 (G and H) were cultured in complete or starvation medium for 120 min. The cells were fixed and examined by fluorescence microscopy. Bars, 20 μm.<p><b>Copyright information:</b></p><p>Taken from "FIP200, a ULK-interacting protein, is required for autophagosome formation in mammalian cells"</p><p></p><p>The Journal of Cell Biology 2008;181(3):497-510.</p><p>Published online 5 May 2008</p><p>PMCID:PMC2364687.</p><p></p

(A) and MEFs were cultured in the complete or starvation medium for the indicated times with or without 100 nM bafilomycin A

The cell lysates were subjected to immunoblot analysis with the indicated antibodies. (B) and MEFs stably expressing GFP-LC3 were cultured in complete or starvation medium for 120 min. The cells were fixed and examined by fluorescence microscopy. (C) Wild-type MEFs stably expressing GFP-FAK were cultured in complete or starvation medium for 120 min. The cells were fixed and subjected to immunofluorescence microscopy using anti-Atg16 antibody. Black squares indicate the enlarged areas shown in insets. Bars, 20 μm.<p><b>Copyright information:</b></p><p>Taken from "FIP200, a ULK-interacting protein, is required for autophagosome formation in mammalian cells"</p><p></p><p>The Journal of Cell Biology 2008;181(3):497-510.</p><p>Published online 5 May 2008</p><p>PMCID:PMC2364687.</p><p></p

(A) Wild-type and MEFs were treated with 100 ng/ml rapamycin (rapa) or vehicle (DMSO) for 120 min in the presence or absence of 100 nM bafilomycin A

The cell lysates were subjected to immunoblot analysis with anti-LC3 antibody. (B) Wild-type and MEFs stably expressing GFP-Atg5 or GFP-LC3 were cultured in the presence of 100 ng/ml rapamycin for 120 min. The formation of GFP-Atg5 (top) and GFP-LC3 (bottom) puncta was examined by fluorescence microscopy. Bar, 20 μm. (C) Wild-type and MEFs were treated with 10 mM lithium chloride for 24 h or 100 μM C-ceramide for 2 h.<p><b>Copyright information:</b></p><p>Taken from "FIP200, a ULK-interacting protein, is required for autophagosome formation in mammalian cells"</p><p></p><p>The Journal of Cell Biology 2008;181(3):497-510.</p><p>Published online 5 May 2008</p><p>PMCID:PMC2364687.</p><p></p