Antibody Fc engineering improves frequency and promotes kinetic boosting of serial killing mediated by NK cells

Abstract

• Fc-engineered mAb promotes NK cell ADCC via better activation, serial killing, and kinetic boosting at higher target cell densities.• Enhanced target killing also increased frequency of NK cell apoptosis, but this effect is donor-dependent.The efficacy of most therapeutic monoclonal antibodies (mAbs) targeting tumor antigens results primarily from their ability to elicit potent cytotoxicity through effectormediated functions. We have engineered the fragment crystallizable (Fc) region of the immunoglobulin G (IgG) mAb, HuM195, targeting the leukemic antigen CD33, by introducing the triple mutation Ser293Asp/Ala330Leu/Ile332Glu (DLE), and developed Time-lapse Imaging Microscopy in Nanowell Grids to analyze antibody-dependent cellmediated cytotoxicity kinetics of thousands of individual natural killer (NK) cells and mAb-coated target cells. We demonstrate that the DLE-HuM195 antibody increases both the quality and the quantity of NK cell-mediated antibody-dependent cytotoxicity by endowing more NK cells to participate in cytotoxicity via accrued CD16-mediated signaling and by increasing serial killing of target cells. NK cells encountering targets coated with DLE-HuM195 induce rapid target cell apoptosis by promoting simultaneous conjugates to multiple target cells and induce apoptosis in twice the number of target cells within the same period as the wild-type mAb. Enhanced target killing was also associated with increased frequency of NK cells undergoing apoptosis, but this effect was donor-dependent. Antibody-based therapies targeting tumor antigens will benefit from a better understanding of cell-mediated tumor elimination, and our work opens further opportunities for the therapeutic targeting of CD33 in the treatment of acute myeloid leukemia. (Blood. 2014;124(22):3241-3249) Introduction Therapeutic monoclonal antibodies (mAbs) elicit functional responses through many different mechanisms, including antibody-dependent cell-mediated cytotoxicity (ADCC), complement dependent cytotoxicity, antibody-dependent cell-mediated phagocytosis (ADCP), and direct induction of apoptosis in tumor cells.1 By using the principles of glycoengineering and mutagenesis, Fc variants have been isolated that show either increased affinity for the activating receptors or altered selectivity for the activating/inhibitory receptors. [2][3][4] Preliminary clinical data with such antibodies Fc-engineered to improve the ADCC/ADCP potential and targeting CD19, CD20, Her2, or CD40 have shown reasonable promise in improving the therapeutic potential of mAb.5-8 Natural killer (NK) cells occupy a pivotal role in immunity: not only can they exert direct cytotoxicity toward infected or tumor cells but they also participate in shaping the adaptive response. 9,10 In the context of mAb treatment, NK cells are unique in that they express only the low-affinity activating FcgR CD16 (FcgRIIIa), and no inhibitory antibody receptors, underscoring a significant role in ADCC.11-13 Several studies using mouse tumor models have established a link between ...

Decitabine enhances anti-CD33 monoclonal antibody BI 836858–mediated natural killer ADCC against AML blasts

“…Targeted Fc engineering either by glycosylation or by mutagenesis increases molecular affinity toward CD16 (Fcg receptor IIIa [FcgRIIIa]) on natural killer (NK) cells and has been shown to potentiate NK-mediated ADCC. 13 Also, coengagement of AML target cells via CD33, and NK cells via CD16, has been shown to result in increased cytotoxicity of the target cells. 14 In addition to CD16 engagement, we evaluated whether receptor-ligand interactions between blasts and effectors can potentiate NK-mediated cytotoxicity against AML blasts.…”

Antigen-specific immunotherapies for acute myeloid leukemia

“…71 Fc-engineered antibodies targeting a variety of AML antigens (eg, CD33, CD38, CD96, CD123, CD133, CD135, CD157, CD300F, and T-cell immunoglobulin mucin receptor 3 [TIM-3]) have been preclinically tested recently, and some constructs targeting CD33 (MAb 33.1, BI 836858), CD123 (CSL362), and CD157 (MEN1112) have entered clinical testing. 70,72 Instead of targeting AML antigens to elicit direct cytotoxic effects, some antibodies aim to amplify native immune responses, for example via modulation of programmed cell death-1 (PD-1)/programmed death ligand-1 (PD-L1) signaling, which may serve as a negative regulatory mechanism in AML.…”

Fc-optimized antibodies quickly pull the trigger

“…1 The presented work gives important novel insights into the mechanism of effector cell-mediated target cell killing triggered by Fc-engineered antibodies and explains how they achieve a higher antibody-dependent cell-mediated cytotoxicity (ADCC) potency than native immunoglobulin G1 (IgG1) antibodies. Using time-lapse imaging microscopy in nanowell grids (TIMING), the authors were able to demonstrate at the single-cell level that antibody Fc engineering improves frequency and promotes kinetic boosting of serial killing mediated by NK cells.…”

“…Romain and colleagues generated an Fc-engineered CD33 antibody variant DLE-HuM195 by introducing 3 amino acid exchanges (S239D-A330L-I332E 5 DLE) in the CH2 domain (see figure) and used TIMING to analyze NK cell-mediated killing of antibody-coated target cells at the single-cell level. 1 This From a clinical perspective, antibodies engineered for higher-affinity FcgRIIIa binding, like obinutuzumab, showed promising activity in clinic trials, but corresponding clinical data with a nonengineered counterpart are not available, making conclusions on the contribution of Fc engineering to the therapeutic effects difficult. 9 In a preclinical nonhuman primate model, the cytolytic potential of an Fc-engineering strategy similar to that applied by Romain and colleagues in the background of an antibody directed against CD19 was convincingly demonstrated.…”