Abstract 650: KIR/HLA interactions negatively affect rituximab, but not GA101 (obinutuzumab)-induced ADCC
Authors: Terszowski Grzegorz , Klein Christian , Stern Martin 
Published: May 2015

Abstract
Antibody dependent cellular cytotoxicity (ADCC) - an important therapeutic mechanism of monoclonal antibodies - is triggered by effector cell binding to IgG-Fc. GA101 (obinutuzumab), a novel anti-CD20 antibody, contains a glycoengineered Fc-portion with increased affinity to FcγR3A, the natural killer (NK) cell IgG-Fc receptor. NK cell activation is regulated by inhibitory killer-cell immunoglobulin-like receptors (KIR), which interact with public epitopes on target cell HLA class I antigens (KIR2DL1-HLA-C2; KIR2DL2/3-HLA-C1, KIR3DL1-HLA-Bw4). The KIR/HLA interaction provides a main mechanism of NK cell tolerance. We analyzed how KIR/HLA interactions influence in vitro ADCC induced by both conventional (rituximab, Rituxan/MabThera) and glycoengineered (GA101, obinutuzumab, Gazyva) CD20 antibodies.
We found that KIR/HLA interactions strongly and selectively inhibit rituximab induced ADCC. NK cell activation (degranulation, i.e. CD107a expression) in KIR3DL1+ NK cells decreased from 45 ± 9% to 19 ± 4% using 721.221 B-cells transfected to express a single HLA antigen with Bw4 specificity compared to parental HLA-negative 721.221 cells in rituximab induced ADCC. In contrast, GA101-induced activation was significantly less affected (63 ± 3% vs 58 ± 3%).
FH7 lymphoma cells (carrying ligands to KIR2DL1 and KIR2DL3, but not KIR3DL1) induced rituximab-triggered activation of KIR3DL1+ NK cells (35 ± 6%), but not of KIR2DL1 (6 ± 2%) or of KIR2DL3+ NK cells (5 ± 1%, p&lt;0.001). After incubation with GA101, all NK cell subsets were activated more strongly and to a similar degree (KIR2DL1 49 ± 6%, KIR2DL3 48 ± 8%, KIR3DL1 52 ± 9%, p=0.91).
In agreement with this, target cell lysis negatively correlated with the number of KIR ligands expressed by target B-cells in rituximab induced ADCC (SPL, one ligand, lysis 27 ± 2%; FH7, two ligands, lysis 10 ± 5%; BatJ, three ligands, lysis 1 ± 1%). In contrast, target cell lysis in GA101-induced ADCC was not correlated with number of KIR ligands (SPL lysis 52 ± 1%; FH7 lysis 55 ± 1%, BatJ lysis 50 ± 1%). Similar results were obtained with primary CLL cells.
Finally, total NK cell activation in healthy donors against target B-cells lacking KIR ligands correlated with the frequency of NK cells expressing the cognate KIR in rituximab-induced ADCC (R2 0.34, p=0.04) but not in GA101-induced ADCC (R2 0.01, p=0.79).
Collectively, these data show that KIR/HLA interactions affect ADCC with rituximab. In contrast, the novel glycoengineered Type II CD20 antibody GA101 apparently overrides inhibitory KIR/HLA interactions, activates all NK cell subsets, and induces increased target cell lysis independent from KIR/HLA interactions. Fc-modification to enhance ADCC may therefore be an effective strategy to augment the efficacy of therapeutic antibodies by recruiting effector NK cells irrespective of their KIR expression. This mechanism may in part explain the high efficacy of GA101 recently documented in clinical trials.
Citation Format: Grzegorz Terszowski, Christian Klein, Martin Stern. KIR/HLA interactions negatively affect rituximab, but not GA101 (obinutuzumab)-induced ADCC. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 650. doi:10.1158/1538-7445.AM2014-650