Optimizing 25-OH-Vitamin D3 Serum Levels for Rituximab- and Obinutuzumab-Mediated Antibody-Dependent Cellular Cytotoxicity

Background:  Vitamin D3 deficiency impairs the rituximab-mediated antibody-dependent cellular cytotoxicity (ADCC) and the outcome of elderly patients with diffuse large B-cell lymphoma (Bittenbring et al., J Clin Oncol. 2014 Oct 10; 32(29):3242-8) treated with R-CHOP and of patients with follicular lymphoma (Kelly et al., J Clin Oncol. 2015 May 1; 33(1):1482-90. The aim of this study was to determine the optimal 25-OH-vitamin D3 (VD3) level for rituximab- and obinutuzumab-mediated ADCC.

Methods:  Ten individuals (5 males, 5 females; mean age: 67.7 years, range 41-79) without malignant disease or immunosuppression were included in this study after written informed consent. PBMC were isolated by density gradient centrifugation. CD16⁺ NK cells were separated by depleting all non-NK-cells magnetically. ADCC activity of the NK cells was tested against CD20-expressing Daudi cells without or after anti-CD20-antibody treatment with rituximab and obinutuzumab, respectively. Cytotoxic activity was assessed by LDH release by the target cells. NK cells were studied at four different VD3 serum levels: 1 (insufficient supply: < 20 ng/ml); 2 (lower normal range; 30 ng/ml), 3 (mid normal range; 65 ng/ml), and 4 (high normal range; 90 ng/ml). To achieve these levels the probands were substituted with cholecalciferol.

Results:  The median VD3 serum level before substitution was 10 ng/ml. The only formula for achieving predefined VD3 serum levels published by van Groningen et al. (Eur J Endocrinol. 2010; 162:805-11) failed to achieve the predetermined VD3 levels by far. Modifying this formula by using a multiplication factor of 1.8-2.2 we achieved the preset dose levels with high precision with median levels of 32.6 ng/ml, 66.4 ng/ml and 92.3 ng/ml aiming at dose levels of 30, 65 and 90 ng/ml, respectively. 2/10 test persons had starting VD3 levels above 30 ng/ml, but 6 of the remaining 8 individuals showed a significantly increased ADCC after VD3 substitution to level 2 (ranging from + 10 % to + 147 % increased Daudi lysis, p < 0.05). Further substitution to 65 ng/ml significantly increased ADCC activity in all 10 persons compared to the ADCC at 30 ng/ml (ranging from + 18.4 % to + 89.2 % increased Daudi lysis, p < 0.05). 8/10 individuals were further substituted to achieve 90 ng/ml. However, in 7/8 of these probands the NK-cell-mediated ADCC was significantly reduced compared to their values at 65 ng/ml (ranging from - 23.1 % to - 58.1 % decreased Daudi lysis, p < 0.05). The extent of the substitution-induced ADCC improvement varied individually and depended on the antibody concentration used to treat the target cells. Rituximab-mediated ADCC was significantly more affected by VD3 levels than obinutuzumab.

Conclusion:  Our study demonstrates for the first time that the ADCC of NK cells is optimal at median VD3 serum levels around 65 ng/ml. Our data strongly argue for a rapid vitamin D3 substitution before/at the beginning of R-CHOP treatment using the modified van Groningen formula. 65 ng/ml was chosen as the target VD3 level in the ongoing OPTIMAL>60 study of the German High-Grade Non-Hodgkin Lymphoma Study Group (DSHNHL) in elderly patients with DLBCL. This study is supported by the Eva Mayr-Stihl-Stiftung (Waiblingen, Germany).