Abstract
AbstractThe efficacy of most therapeutic monoclonal antibodies (mAbs) targeting tumor antigens results primarily from their ability to elicit potent cytotoxicity through effector-mediated 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 cell-mediated 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.
Highlights
IntroductionTherapeutic 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, Her[2], or CD40 have shown reasonable promise in improving the therapeutic potential of mAb.[5,6,7,8] Natural killer (NK) cells occupy a pivotal role in immunity: can they exert direct cytotoxicity toward infected or tumor cells but they 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.[11,12,13] Several studies using mouse tumor models have established a link between activating Fc receptors and the efficacy of mAb therapy.[14,15] as CD16 is polymorphic in humans, it has been demonstrated previously that immune cells that harbor the CD16-158V allotype exhibit better binding to human immunoglobulin G1 (IgG1), which in turn leads to more efficient ADCC/ADCP in vitro and to better clinical outcomes.[16,17,18,19]
Therapeutic monoclonal antibodies 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]
To compare the effector functions of DLE-HuM195 with that of wt-HuM195, we first tested their potency to induce ADCC in 3 Acute myeloid leukemia (AML) human cell lines expressing varying amounts of CD33: MV4:11, MOLM13, and HL60.32 For all 3 lines tested, Natural killer (NK) cell-mediated lysis was more pronounced on target cells that were precoated with DLE-HuM195 than on those precoated with wt-HuM195 (Figure 1C-E P1, P2, and P3 values, .0001; details of tests listed in supplemental Table 1)
Summary
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, Her[2], or CD40 have shown reasonable promise in improving the therapeutic potential of mAb.[5,6,7,8] Natural killer (NK) cells occupy a pivotal role in immunity: can they exert direct cytotoxicity toward infected or tumor cells but they 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.[11,12,13] Several studies using mouse tumor models have established a link between activating Fc receptors and the efficacy of mAb therapy.[14,15] as CD16 is polymorphic in humans, it has been demonstrated previously that immune cells that harbor the CD16-158V allotype exhibit better binding to human immunoglobulin G1 (IgG1), which in turn leads to more efficient ADCC/ADCP in vitro and to better clinical outcomes.[16,17,18,19]
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