Cetuximab–siRNA Conjugate Linked Through Cationized Gelatin Knocks Down KRAS G12C Mutation in NSCLC Sensitizing the Cells Toward Gefitinib

Abstract

Delivery of small-interfering RNA (siRNA) has been of great interest in the past decade for effective gene silencing. To overcome synthetic and regulatory challenges posed by nanoparticle-mediated siRNA delivery, antibody–siRNA conjugate (ARC) platform is emerging as a potential siRNA delivery system suitable for clinical translation. Herein, we have developed a delivery technology based on the ARC platform for stable delivery of siRNA called as Gelatin-Antibody Delivery System (GADS). In GADS, positively charged gelatin acts as a linker between antibody–siRNA and enables the endosomal escape of siRNA for gene silencing postcellular internalization. For proof of concept, we synthesized a scalable GADS conjugate comprising of Cetuximab (CTB), cationized gelatin (cGel) and NSCLC KRASG12C-specific siRNA. CTB was chemically conjugated to cGel through an amide link to form the CTB–cGel complex. Thereafter, siRNA was chemically conjugated to the cGel moiety of the complex through the thioether link to form CTB–cGel–siRNA conjugate. RP-HPLC analysis was used to monitor the reaction while gel retardation assay was used to determine siRNA loading capacity. SPR analysis showed the preservation of ligand binding affinity of antibody conjugates with KD of ∼0.3 nM. Furthermore, cellular internalization study using florescent microscopy revealed receptor-mediated endocytosis. The conjugate targeted EGFR receptor of KRAS mutant NSCLC to specifically knockdown G12C mutation. The oncogene knockdown sensitized the cells toward small molecule inhibitor—Gefitinib causing ∼70% loss in cell viability. Western blot analysis revealed significant downregulation for various RAS downstream proteins postoncogene knockdown. Comparison of the efficiency of GADS vis-à-vis positive siRNA control and CRISPR–Cas9-based knockout of KRAS Exon 2 in the NCI-H23 NSCLC cell line suggests GADS as a potential technology for clinical translation of gene therapy.

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“…(C) Cetuximab–cationic gelatin–specific siRNA delivery system (CTB-cGel-siRNA conjugate). 1: Cetuximab, 2: cationic gelatin, and 3: KRASG12C-specific siRNA [ 118 ].…”

“…In the cetuximab–cationic gelatin–KRASG12C-specific siRNA gelatin antibody delivery system (GADS; CTB-cGel-siRNA conjugate), positively charged gelatin had a dual role. It acted as a linker between the antibody and the siRNA and as an endosomal escape agent for efficient delivery of the siRNA in the cell cytoplasm due to its charge reversal properties ( Figure 2 , Figure 3 ) [ 118 ]. The second system composed of gefitinib-loaded gelatin-A NPs functionalized with a cetuximab-siRNA conjugate (Ab-siRNA-GelGEF NPs) also efficiently delivered stable siRNA to the cell cytoplasm in vitro and in vivo in a mouse model ( Figure 2 , Figure 3 ).…”

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“…The only major limitation was inefficient endosomal escape of these ARCs. Conjugation via cationic gelatin linker of KRAS G12C -specific siRNA and Cetuximab, allowed efficient KRAS-mutant downregulation in lung cancer cells and their sensitization to the small molecule chemotherapeutic, Gefitinib[ 119 ].…”