Systematic evaluation of antibody-mediated siRNA delivery using an industrial platform of THIOMAB-siRNA conjugates

Abstract

Delivery of siRNA is a key hurdle to realizing the therapeutic promise of RNAi. By targeting internalizing cell surface antigens, antibody-siRNA complexes provide a possible solution. However, initial reports of antibody-siRNA complexes relied on non-specific charged interactions and have not been broadly applicable. To assess and improve this delivery method, we built on an industrial platform of therapeutic antibodies called THIOMABs, engineered to enable precise covalent coupling of siRNAs. We report that such coupling generates monomeric antibody-siRNA conjugates (ARCs) that retain antibody and siRNA activities. To broadly assess this technology, we generated a battery of THIOMABs against seven targets that use multiple internalization routes, enabling systematic manipulation of multiple parameters that impact delivery. We identify ARCs that induce targeted silencing in vitro and extend tests to target prostate carcinoma cells following systemic administration in mouse models. However, optimal silencing was restricted to specific conditions and only observed using a subset of ARCs. Trafficking studies point to ARC entrapment in endocytic compartments as a limiting factor, independent of the route of antigen internalization. Our broad characterization of multiple parameters using therapeutic-grade conjugate technology provides a thorough assessment of this delivery technology, highlighting both examples of success as well as remaining challenges.

Figure 1.

Synthesis and purification of antibody–siRNA conjugates (ARCs). (a) Chemically stabilized siRNAs containing a 3′ amine for coupling and a 5′ Dy547 for tracking (both modifications on the sense strand) were reacted with SMCC (non-reducible) or SPDB (reducible) NHS-linkers to form a thio-reactive adduct (maleimide-oligo). The siRNAs were then reacted with THIOMABs, containing engineered cysteines on the heavy chains (shown) or light chains to form the ARCs. (b) Example of electrospray TOF Mass spectrometry analysis of ARCs (after purification by anion exchange to remove free siRNA and size-exclusion chromatography to remove free antibodies). Purification yields preparations that are free of uncoupled antibodies (mass at 147875) and contain conjugates with either one siRNA (ARC mass at 154451.2) or two siRNAs (ARC mass at 161042.5). In this example, the ratio of ARCs with one siRNA to ARCs with two siRNAs is ∼1:1.

Figure 2.

Antibody and siRNA components function normally when coupled in ARCs. (a) Flow cytometry comparing binding of naked anti-TENB2 antibody (control) compared to anti-TENB2 ARC (with both SMCC and SPDB linkers, as indicated) to PC3-TENB2-High cells; 488xHu, secondary antibody alone; no Ab, control without primary antibody or ARC added. (b) Flow cytometry as in (a) but using PC3-Her2 cells and naked Trastuzumab (anti-Her2) antibody or Trastuzumab ARC. (c) PPIB silencing, expressed as an average of PPIB mRNA levels relative to GAPDH mRNA levels, following lipid transfection of free siRNA or siRNA conjugated in Trastuzumab ARCs, as indicated. siPPIB, siRNA targeting PPIB; siNTC, non-targeting control siRNA against firefly luciferase, ffluc; error bars represent standard deviation, n = 2 independent experiments.

Figure 3.

TENB2 ARCs induce silencing in vitro independent of antibody and linker format, assessed at the mRNA and protein levels. (a) Quantigene expression analysis of PPIB mRNA levels following a 72-h administration to PC3-TENB2-high cells of the indicated ARCs (using non-reducible SMCC or reducible SPDB linkers, as indicated) or, as controls, free antibodies (Naked TENB2) or free siRNAs. Data were normalized to TENB2-siNTC control to allow for comparisons between experiments (dotted line at 1.0); N = 10 independent experiments for all samples, except Her2 ARC where N = 8. Error bars represent SD. (b) Immunoblotting of PPIB protein in PC3-TENB2-high cells following 72 h of treatment with the indicated ARCs. Numbers represent the PPIB protein levels normalized to GAPDH protein levels, expressed relative to the value determined for the TENB2-SPDB-siNTC control. (c) Gene expression analysis as in (a) comparing silencing using TENB2 ARC with siRNAs conjugated to the heavy chain (HC), a second TENB2 ARC using a different anti-TENB2 antibody (ch20d1), and a TENB2 ARC with siRNAs conjugated to the light chain (LC). The dotted line represents the value determined for the TENB2-siNTC control (set to 1.0) to which the values for the other groups were normalized (SD shown, N = 4; ARCs used the SPDB linker).

Figure 4.

5′ RACE assay demonstrates anti-NaPi2b-ARCs induce silencing via an RNAi mediated mechanism. (a) Schematic of the 5′ RACE assay used to determine the cleavage position on PPIB mRNA. (b) Agarose (3%) gel electrophoresis analysis of RT-PCR products using RNA isolated from PC3-NaPi2b cells treated with 100 nM of the indicated ARCs (SPDB linkers) for 72 h. The sample designated with (T) is a positive control that used lipid transfection of the ARC, rather than antigen internalization, to deliver the ARC and induce RNAi. Only use of the anti-NaPi-SPDB-siPPIB ARCs generated the 300 bp product indicative of accurate RNAi-mediated cleavage. (c) DNA sequencing results of the RT-PCR product using RNA isolated from cells treated with the anti-NaPi-SPDB-siPPIB ARC. Sequencing reads mapped the cleavage site to the indicated position (nucleotide 447 in the PPIB mRNA sequence), the site predicted from a true RNAi cleavage mechanism.

Figure 5.

The antibody and siRNA components of both silencing-active and silencing-inactive ARCs traffic to lysosomes. (a) Anti-TENB2 ARCs deliver anti-TENB2 and the siRNA payload to lysosomes. PC3-TENB2-high cells were treated for 40 h with anti-TENB2-SMCC-siPPIB ARCs in the presence of lysosomal protease inhibitors. Antibody (green) and late endosome-lysosome (LAMP1 marker, blue) localizations were visualized using IF, and the siRNA (red) was tracked using a Dy4547 label on the 5′ end of the siRNA sense strand. Top row, localization patterns for each individual component; bottom row, merged patterns, as indicated. All three markers show overlapping localization. (b) Time course of ARC lysosomal delivery. The experiment was performed as in (a) with images captured at the indicated times. LAMP1, green; siRNAs, red. (c) Comparison of antibody internalization. The experiment was performed as in (a), except using antibodies targeting the indicated antigens and the cognate antigen-expressing cell lines, with continuous uptake for ≥20 h. Anti-Mesothelin (MSLN), which is internalized very slowly, served as a control for surface membrane localization after 19 h. Top row, antibody localization alone; bottom row, antibody (red) and LAMP1 (green) localizations merged.

Figure 6.

siRNA screen of endocytic pathway components reveals that HSP4 inhibition improves ARC-mediated silencing efficiency. (a) List of genes targeted in the candidate-based siRNA screen and the observed phenotypes following knock down. (b) Schematic of the experimental approach. We transfected TENB2-PC3-High cells with siRNAs against endocytic pathway genes and incubated for 3 days. We then added anti-TENB2-SPDB-siPPIB ARCs to the cells and assayed ARC-mediated silencing of PPIB mRNA after 3 additional days. (c) ARC-mediated silencing of PPIB mRNA after initial transfection with siRNA targeting HPS4, siRNA targeting TENB2 (control, to reduce levels of the antibody target) or non-targeting control (NTC) siRNA (SD shown, N = 4).

Figure 7.

ARCs are delivered to tumors in vivo and mediate silencing. (a) TENB2 antibody and siRNAs (Dy-547 labeled) are co-delivered to tumor cells within 5 h after intravenous injection of ARCs. (b) Visualization of siRNAs (Dy-547 labeled) and the endothelial cell maker CD31 in tumor sections reveals siRNA delivery to tumor cells near the vasculature in a manner that depends on the targeted antibody–antigen interaction. (c) Schematic of in vivo silencing study. We dosed mice bearing tumors with a volume of ∼400 mm³ three times with 24 mg/kg (total ARC mass in mg per body weight in kg, equivalent to ∼20.6 mg/kg antibody plus 3.4 mg/kg siRNA) of ARCs and harvested tumors on day 5. (d) Assessment of TENB2 ARC induced silencing in vivo (in EpCam+ cells isolated from near the CD31+ tumor vasculature); PPIB mRNA levels, normalized to GAPDH mRNA levels, are shown for each animal (N = 6 for each group, SD shown, Student's t-test: P = 0.0217).