Lipoplex mediated silencing of membrane regulators (CD46, CD55 and CD59) enhances complement‐dependent anti‐tumor activity of trastuzumab and pertuzumab

Abstract

The therapeutic potential of anticancer antibodies is limited by the resistance of tumor cells to complement-mediated attack, primarily through the over-expression of membrane complement regulatory proteins (mCRPs: CD46, CD55 and CD59). Trastuzumab, an anti- HER2 monoclonal antibody, approved for the treatment of HER2-positive breast and gastric cancers, exerts only minor complement-mediated cytotoxicity (CDC). Pertuzumab is a novel anti-HER2 monoclonal antibody, which blocks HER2 dimerization with other ligand-activated HER family members. Here, we explored the complement-mediated anti-tumor effects of trastuzumab and pertuzumab on HER2-positive tumor cells of various histological origins. Delivery of chemically stabilized anti-mCRP siRNAs using cationic lipoplexes, AtuPLEXes, to HER2-over-expressing BT474, SK-BR-3 (breast), SKOV3 (ovarian) and Calu-3 (lung) cancer cells reduced mCRPs expression by 85-95%. Knockdown of individual complement regulators variably led to increased CDC only upon combined treatment with trastuzumab and pertuzumab. The combined down-regulation of all the three regulators augmented CDC by 48% in BT474, 46% in SK-BR-3 cells, 78% in SKOV3 cells and by 30% in Calu-3 cells and also increased complement-induced apoptosis and caspase activity on mCRP neutralized tumor cells. In addition, antibody-induced C3 opsonization of tumor cells was significantly enhanced after mCRP silencing and further augmented tumor cell killing by macrophages. Our findings suggest that siRNA-induced inhibition of complement regulator expression clearly enhances complement- and macrophage-mediated anti-tumor activity of trastuzumab and pertuzumab on HER2-positive tumor cells. Thus - if selectively targeted to the tumor - siRNA-induced inhibition of complement regulation may serve as an innovative strategy to potentiate the efficacy of antibody-based immunotherapy.

The membrane complement regulatory protein CD59 promotes tumor growth and predicts poor prognosis in breast cancer

“…In most solid tumors, CD59 is overexpressed compared to adjacent normal cells, and inhibiting CD59 function can improve breast cancer treatment (14,30,31). Distant metastases are lethal to breast cancer patients (7).…”

Regulation of complement and modulation of its activity in monoclonal antibody therapy of cancer

“…80,81 A combination of chemicallystabilized anti-mCRP siRNAs using cationic lipoplexes and treatment with a mixture of anti-Her2 mAbs (trastuzumab and pertuzumab) targeting different epitopes resulted in augmented CDC, complement-mediated cell death and caspase activity and CDCC by macrophages of breast, ovarian, and lung cancer cell lines. 82 A potent CD59 inhibitor (rILYd4) sensitized normally rituximab-resistant lymphoma cells to CDC by rituximab 83 and ofatumumab. 84 Alternatively, mCRP function could be blocked by specific "neutralizing" antibodies.…”

CD20 mAb-Mediated Complement Dependent Cytotoxicity of Tumor Cells is Enhanced by Blocking the Action of Factor I

“…However, as noted above, prototype in vitro experiments have in fact demonstrated that inhibition of cell-associated complement control proteins CD55 and CD59, as well as inhibition of the action of soluble Factor H can all enhance CDC mediated by both of these mAbs [20,33,53,54,61], thus indicating there is indeed room for improvement. Moreover, levels of CDC higher than 90% are difficult to achieve with RTX or with OFA, even with cell lines, and this is particular evident when a clinically important target, i.e., primary CLL cells are examined [16,63].…”

“…Alternatively, both soluble factors as well as the complement control proteins expressed on the tumor cells, in common with normal cells, may limit mAb-mediated killing [42,43,53,54]. Therefore, focused strategies that can mitigate or limit exhaustion, or that can block the action of these factors have the potential to increase substantially killing of tumors by mAbs already approved by the FDA [20,22,30,33,53,54,61].…”

“…Moreover, pre-clinical mouse model studies have revealed that the relative contribution of these effector mechanisms depends in part upon the detailed nature of the model under investigation [14,[17][18][19]. However, these mechanisms are critical for clinical efficacy, and therefore many ongoing research efforts have been initiated with a goal of improving and enhancing mAb-based therapies, based on increasing the efficacy of specific effector mechanisms [20][21][22][23][24][25][26][27][28][29][30][31][32][33][34]. Killing of targeted tumor cells can be increased by either developing next generation mAbs with rationally-based differences in primary structure or degree of glycosylation, or strategies can be employed to increase the capacity/efficiency of the effector functions.…”