Anti-HER2 mAb plus calicheamicin immunoconjugate linked on liposomal vinorelbine induces ADCC and apoptosis in HER2-positive high grade DCIS
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KeywordsVinorelbine Mitotic Rate Immune Effector HER2 Overexpression Membrane Blebbing
Overexpression of the HER2 proto-oncogene which encodes a 185 kDa protein frequently coincides with aggressive and chemoresistant DCIS due to inhibition of PCD after chemotherapy. Also, it is associated with aneuploidy, p53 abnormalities, enhanced DNA repair and synthesis, cell growth, mitotic rate and tumorigenicity. Tissue from high-grade DCIS was excised from a patient, and tumour cells were isolated by the collagenase method. Analysis with IHC showed HER2 overexpression. We prepared immunoconjugates of anti-HER2 mAbs and calicheamicin, which is an apoptotic antibiotic with up to 1000-fold greater potency than the clinically most used anticancer drugs. This immunoconjugate was linked onto pegylated DRV liposomes which contained vinorelbine. After treatment, we observed HER2 downregulation by IHC. TEM exhibited disruption of the microtubular cytoskeleton due to vinorelbine, and mAb(Fc)-directed killing of tumour cells by immune effector cells such as macrophages, neutrophils and lymphoid cells such as K and NK cells, indicating antibody-dependent cellular toxicity (ADCC). Biochemical assays such as MTT exhibited reduced metabolic activity, while BrdU showed great reduction of DNA synthesis. Anti-ssDNA mAbs and binding of Annexine-V with phosphatidyl-serine IHC confirmed that apoptosis was the mechanism of growth inhibition. SEM and TEM showed violent membrane blebbing of apoptotic cells (zeiosis), and fragmentation of DNA creating a vacuolar nucleus due to calicheamicin-damaging action after binding to the third end of oligopurine tracts causing strand breaks. Subsequently, tumour cells break up into apoptotic bodies that adjacent cells recognise and phagocytose due to PS externalisation, indicating a bystander killing effect (BKE). We conclude that by delivering intracellularly cytotoxic agents such as calicheamicin through conjugating to antiHER2 mAbs linked on liposomal vinorelbine, we can circumvent chemoresistance, induce ADCC, inhibit mitosis at metaphase and destroy tumoral DNA, leading to irreversible D2 apoptotic signs with subsequent bystander killing of high grade DCIS.