Rhamnose Modification to Enhance the Fc Effector Functions of Trastuzumab
Objective:To investigate whether rhamnose(Rha)modification could improve the Fc effector functions of trastuzumab(Tra).Methods:Tra-Rha conjugate was prepared by conjugating reduced Tra with Rha derivative.SDS-PAGE,Western blotting and HPLC-SEC assays were performed to identify the purified Tra-Rha conjugate.The specific binding affinity of the Tra-Rha conjugate to HER2 was evaluated by flow cytometry.The anti-Rha antibody recruiting capacity of the Tra-Rha conjugate was evaluated by immunofluorescence and flow cytometry.CDC and ADCP assays were performed to assess the level of enhanced Fc effector functions.Results:The Tra-Rha conjugate was successfully synthesized.Binding affinity and anti-Rha antibody recruiting capacity assays showed that Tra-Rha conjugate retained almost full binding affinity to HER2,and was able to selectively redirect high-level anti-Rha antibodies to target cells.In the subsequent CDC assay,Tra-Rha was shown to be capable of inducing potent cell killing in the presence of anti-Rha antibodies,with the maximum cell lysis in Tra-Rha-treated SKBR3 and SKOV3 cells being 75％and 70％,respectively.Notably,Tra failed to eliminate cells via CDC under the same conditions.Finally,the dramatically enhanced ADCP activity of the Tra-Rha conjugate was observed,with the detected phagocytosis rate in Tra-Rha treated SKBR3 and SKOV3 cells being 2.3-fold and 1.2-fold higher,respectively,compared to that of cells treated with Tra.Conclusion:Rha modification is a cost-effective strategy to generate novel Tra with enhanced effector functions,thereby improving the therapeutic index of Tra.
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