• diffuse large B-cell lymphoma
• metronomic chemotherapy
• vinorelbine
• etoposide
• anti-CD19 monoclonal antibody

Introduction. Diffuse large B-cell lymphomas (DLBCL) is the most common lymphoid malignancy in adults, representing almost 30% of all cases of non-Hodgkin’s lymphoma. Metronomic chemotherapy (mCHEMO) can be defined as the frequent, regular administration of drug doses that maintain a low, prolonged, and active, range of plasma concentrations of drugs with a more favorable toxicity profile. mCHEMO in treatment-naïve diffuse large B-cell lymphoma (DLBCL) was recently reported to allow overall and disease-free survivals at 24 months of 54% and 74%, respectively. In non-Hodgkin lymphoma, the combination of targeted therapies with mCHEMO schedules have shown very promising pre-clinical and clinical results. Conversely, the combination of mCHEMO with monoclonal antibodies is almost unexplored in hematological cancers. A novel humanized, anti-CD19 monoclonal antibody (mAb) with an engineered constant fragment (Fc)-domain, has recently been designed to enhance FcγRIIIa binding affinity, for the treatment of B-cell malignancies.
Aim of the study. The aim of this study is to evaluate and enhance the activity of an anti-CD19 mAb in concomitant combination with metronomic vinorelbine (mVNR) and etoposide (mETO) on human DLBCL cells and in mouse xenograft models.
Methods. In vitro proliferation assay was performed on three human CD19+ DLBCL cell lines, named Toledo, OCI-LY3 and SU-DHL10, exposed to a single dose of anti-CD19 mAb, thrice a week mVNR and daily mETO, alone and their concomitant combination for 144h. Synergism was evaluated by the combination index (CI) and the Loewe additivity model. Apoptosis assay was evaluated by ELISA at 48h. The VNR and ETO intracellular concentration were assessed by UPLC-MS/MS technology. Protein phosphorylation in DLBCL cell lysates was investigated by Luminex analysis. In vivo DLBCL subcutaneous xenograft and systemic tumor models in athymic nude mice were treated with anti-CD19 mAb, mVNR, and their concomitant combination.
Results. The single 144h exposure of both mVNR and mETO and anti-CD19 mAb inhibited the DLBCL cell proliferation in a concentration-dependent manner. mETO inhibited the Toledo, OCI-LY3 and SU-DHL10 cell growth with an IC50 of 9.81±1.14 nM, 7.92±3.6 nM and 8.19±0.29 nM, respectively. A higher antiproliferative effect was found after mVNR treatment on Toledo, OCI-LY3 and SU-DHL10 cell lines, as demonstrated by the calculated IC50s of 692.1±134.55 pM, 35.58±12.64 pM and 511.4±133.07 pM, respectively. Interestingly, the anti-CD19 mAb therapy significantly inhibited the Toledo, OCI-LY3 and SU-DHL10 cell proliferation with an IC50 of 1472±351 nM, 906.9±50.87 nM and 57.84±22.14 nM, respectively. In all cell lines, the concomitant treatment anti-CD19 mAb plus mVNR and mETO showed a marked synergism for all the fractions of affected cells (Fa), with a CI<1 and DRI>1, except for anti-CD19 mAb plus mETO on SU-DHL10 cells. These findings were confirmed by Loewe analysis. A significant pro-apoptotic activity was found in all DLBCL cell-lines treated with anti-CD19 mAb alone for 48h, whereas the combination of anti-CD19 mAb with mVNR and mETO further enhanced apoptosis. Furthermore, the anti-CD19 mAb plus mVNR combination significantly inhibited after 24h the phosphorylation of GSK 3α/β, mTOR, p70S6K, RPS6 and TSC2 proteins in DLBCL cells. Interestingly, anti-CD19 therapy significantly increased the VNR and ETO intracellular concentrations in DLBCL cells after 24h, except ETO levels in SU-DHL10. Finally, in all three subcutaneous DLBCL models, the mVNR and anti-CD19 mAb combination significantly reduced the subcutaneous tumor volumes without causing significant toxicity and significantly increase the overall survival of mice affected by a systemic disease.
Conclusions. We report, for the first time, the synergistic activity of anti-CD19 mAb, with mVNR or mETO in DLBCL cells in vitro and in vivo. These results prompt a rapid translation of this combination schedule into future clinical trials.