Construction And Characterization Of A Novel Anti-CD20 MABs’ Nano-comb With Exceptionally Excellent Lymphoma Suppressing Activity

Objective:To fabricate a novel anti-CD20 mAb’s nano-comb with exceptionally excellent tumor suppressing ability, providing new methods and ideas for improving the therapeutic efficacy of targeted therapy against CD20 positive Non-Hodgkin Lymphoma (NHL) in the clinic.Methods:The anti-CD20 mAb’s nano-comb (PPRT) was fabricated through mass arming two different anti-CD20 mAbs of distinct types, Rituximab (type Ⅰ) and Tositumomab (type Ⅱ), to one polyethyleneimine (PEI) polymer. The anchoring of monoclonal antibody to PEI polymer was confirmed by SDS-PAGE after Coomassie brilliant blue (CBB) staining. The characterization of PPRT, including the particle size distribution and morphology, were tested by dvnamic light scattering (DLS) and atomic force microscopy (AFM). respectively. The in vitro lymphoma suppressing ability of PPRT nano-comb was determined by evaluating its CDC (complement dependent cytotoxicity), ADCC (antibody dependent cell-Mediated cytotoxicity) and PCD (programmed cell death) evoking capability against three different NHL cell lines (Raji, Ramos and JeKo-1). The activation of both caspase dependent and independent signal transduction pathways was analyzed by FCM (Flow cytometry), IF (immunofluorescence) and WB (Western Blottong) experiments. The HA (homotypic adhesion) inducing abilities of PPRT and liberal mAbs were compared in NHL cells by observing the cell morphology after incubating with appropriate concentration of therapeutic antibodies for 8 hours. The pharmacokinetics and in vivo distribution of PPRT nano-comb and liberal antibodies were compared in in vivo studies by ELISA (enzyme-linked immunosorbent assay) and IF staining. The in vivo tumor suppressing ability of PPRT was determined in both disseminated and localized human NHL Xeno transplant models. Besides, the Rituximab resistant NHL cell lines, Raji-R and JeKo-1-R, were generated by exposing the wild type (WT) cells to stepwise increased concentration of Rituximab from 0.125 to 128μg/ml with the presence of 10% fresh human serum in RPMI 1640 culture medium. The in vitro and in vivo anti-tumor efficacy against Rituximab resistant NHL cells was also evaluated by the above mentioned experimental methods.Results:PPRT nano-comb owns a relatively high molecule weight (MW) and particle radius (approximately 170nm) when compared with liberal monoclonal antibodies. The morphology of PPRT was clearly observed by AFM. Also, PPRT nano-comb owns a comparable binding avidity and reduced "off-rate" to CD20 antigen on surface of NHL cells comparing with free anti-CD20 mAbs. As treated by PPRT, the caspase dependent apoptosis was remarkably enhanced except for concurrently eliciting of CDC, ADCC and lysosomal mediated programmed cell death (PCD). Also, PPRT treated cells can induce remarkable homotypic adhesion in lymphoma cells by "inter-cell link", which we consider as an important potential factor for its outstanding PCD mediating ability. Pharmacokinetic assays revealed that PPRT experienced a relatively reduced clearance from peripheral blood compared with free mAbs. With the cooperation of all the above mentioned superiorities, PPRT exhibits exceptionally excellent in vivo anti-tumor activities against both wild type and Rituximab resistant lymphoma cells in both disseminated and localized human NHL Xeno-transplant models, which merits further evaluation in the clinic. Conclusions:Our results clearly demonstrated that PPRT nano-comb is more than the sum of its parts, which owns an exceptionally excellent tumor suppression ability against both WT and Rituximab resistant NHL cells in in and ex vivo studies and merits further evaluation in the clinic.