Humanized Lewis-Y Specific Antibody Based Delivery of STAT3 siRNA

Abstract

The clinical application of siRNA is limited largely by the lack of efficient, cell-specific delivery systems. Antibodies are attractive delivery vehicles for targeted therapy due to their high specificity. In this study we describe the use of a humanized monoclonal antibody (mAb), hu3S193, against Lewis-Y (Ley), as a delivery vehicle for STAT3 siRNA. This mAb is rapidly internalized into Ley expressing cancer cells via antigen recognition, and when coupled to STAT3 siRNA, a potentially powerful molecularly targeted delivery agent is created. Selective silencing of STAT3 is associated with tumor suppression. Two hu3S193 based siRNA delivery systems using STAT3 siRNA as a prototype were developed and tested in Ley-positive cancer cells: (a) a covalent construct based on a reductive disulfide linker that is expected to undergo cleavage within cells and (b) a non-covalent construct based on (D-Arginine)9 (9r) modified hu3S193. Ley-specific binding and internalization of both the covalent and non-covalent constructs were confirmed by flow cytometry and confocal microscopy. Both the covalent and the non-covalent system led to efficient STAT3 silencing in Ley-positive cancer cells (A431), but not in Ley-negative cancer cells (MDA-MB-435). The covalent construct, however, required co-treatment with reagents such as chloroquine or 9r that facilitate the escape of the siRNA from endosomes to achieve significant gene silencing. The 9r modified non-covalent construct, induced ~70% STAT3 knockdown at sub-micromolar siRNA concentrations when used at an optimal vehicle-to-siRNA ratio of 5:1. The STAT3 knockdown also led to ~50% inhibition of cell proliferation of Ley-positive cells. Non-covalent linked STAT3 siRNA-hu3S193 has great promise for targeted knockdown of STAT3 in tumor cells.

TLR9-mediated siRNA delivery for targeting of normal and malignant human hematopoietic cells in vivo

“…Previously developed siRNA-delivery strategies used antibodies, oligonucleotides, or RNA aptamers specific to surface receptors on tumor cells. 16,36,42 In contrast, our approach used a ligand to intracellular TLR9 receptor that is ubiquitously expressed in B-cell malignancies and AML. [25][26][27] Dicer RNase, which is critical for uncoupling of CpG-siRNA conjugates, is also commonly expressed in normal and transformed B cells and in the majority of clinical AML samples.…”

“…For the design of the human cell-specific CpG-siRNA (Figure 2A), we used a 25/27mer Dicersubstrate STAT3 siRNA selected from more than 20 sequences based on the high silencing efficacy (Ͼ 95%). 36 The 25/27mer siRNA sequence was selected to enable intracellular uncoupling of both parts of the molecule by Dicer endonuclease. 20 We conjugated STAT3 siRNA to both type-B (CpG7909) and type-A (D19) CpG-ODNs.…”

Subcellular Fate and Off-Target Effects of siRNA, shRNA, and miRNA

“…Monoclonal antibodies targeted against specific cell types can also be used to deliver siRNAs enclosed in liposomes. A recent study has utilized humanized Lewis-Y monoclonal antibody to selectively deliver STAT3 siRNA to Lewis-Y protein expressing cancer cells via antigen recognition (171). Pirollo and colleagues employed cationic immunoliposomes decorated with antitransferrin receptor antibody.…”

Bioconjugates for targeted delivery of therapeutic oligonucleotides

“…A variety of linkages have been used to conjugate ligands and ONs including amide, thioether, thiol-maleimide, ester, and disulfide [23, 28, 29]. Some linkages are readily cleavable inside the cells, including disulfide, some peptides, and hydrazone, and they are needed to attain biological activity of ONs when large ligands such as antibody [30] or carrier proteins such as albumin [31] are linked the ONs.…”

“…This approach of direct conjugation has been extended to ON delivery; however, direct antibody-ONs conjugates haven't accomplished similar success as antibody-drug conjugates. In one study, a humanized monoclonal antibody was linked to siRNA via a reductive disulfide bond that is cleavable within cells [30]. Although antigen-specific binding and internalization of the antibody-ON conjugates were observed, single treatment of the conjugates failed to produce significant gene silencing.…”