Anti-ceramide single-chain variable fragment mitigates radiation GI syndrome mortality independent of DNA repair
Jimmy A. Rotolo, … , Vijay K. Singh, Richard Kolesnick
Jimmy A. Rotolo, … , Vijay K. Singh, Richard Kolesnick
Published March 16, 2021
Citation Information: JCI Insight. 2021;6(8):e145380. https://doi.org/10.1172/jci.insight.145380.
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Research Article Stem cells Vascular biology

Anti-ceramide single-chain variable fragment mitigates radiation GI syndrome mortality independent of DNA repair

Abstract

After 9/11, threat of nuclear attack on American urban centers prompted government agencies to develop medical radiation countermeasures to mitigate hematopoietic acute radiation syndrome (H-ARS) and higher-dose gastrointestinal acute radiation syndrome (GI-ARS) lethality. While repurposing leukemia drugs that enhance bone marrow repopulation successfully treats H-ARS in preclinical models, no mitigator potentially deliverable under mass casualty conditions preserves GI tract. Here, we report generation of an anti-ceramide 6B5 single-chain variable fragment (scFv) and show that s.c. 6B5 scFv delivery at 24 hours after a 90% lethal GI-ARS dose of 15 Gy mitigated mouse lethality, despite administration after DNA repair was complete. We defined an alternate target to DNA repair, an evolving pattern of ceramide-mediated endothelial apoptosis after radiation, which when disrupted by 6B5 scFv, initiates a durable program of tissue repair, permitting crypt, organ, and mouse survival. We posit that successful preclinical development will render anti-ceramide 6B5 scFv a candidate for inclusion in the Strategic National Stockpile for distribution after a radiation catastrophe.

Authors

Jimmy A. Rotolo, Chii Shyang Fong, Sahra Bodo, Prashanth K.B. Nagesh, John Fuller, Thivashnee Sharma, Alessandra Piersigilli, Zhigang Zhang, Zvi Fuks, Vijay K. Singh, Richard Kolesnick

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Figure 1

Generation of anti-ceramide 6B5 scFv.

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Generation of anti-ceramide 6B5 scFv. (A) Monoclonal Ab 6B5 was generate...
(A) Monoclonal Ab 6B5 was generated from mice immunized with BSA-conjugated C16-ceramide. Antigen binding was determined using plates coated with 300 ng/ml C16-ceramide, C16-dihydroceramide, or BSA. Purified monoclonal Ab (0.1, 1, and 10 μg/ml) preferentially binds to C16-ceramide. Negative control preimmune serum did not display antigen binding by ELISA. Data show 1 representative experiment of 3 independent experiments. (B) Jurkat cells (5 × 105/point) were pretreated with or without the indicated concentrations of 6B5 IgG at 15 minutes before 10 Gy. After fixation and staining with bisBenzimide Hoechst 33258, apoptotic death was quantified by fluorescence microscopy at 16 hours, the optimal time for detecting radiation-induced apoptosis in this cell line (3). Data (mean ± 95% CI) were collated from 3 experiments in which 400 cells were analyzed per point. **P < 0.01, ***P < 0.001 vs. control, unpaired t test and multiple-comparison correction using Bonferroni-Dunn correction (α = 0.01). (C) Mouse monoclonal Ab 6B5 dose-dependently protects small intestinal crypts. Purified 6B5 IgG (0–1000 μg) was administered to C57BL/6 mice 15 minutes before 15 Gy WBR. Crypt survival was quantified according to the Withers and Elkind microcolony assay (8). Data are from 1 representative experiment of 3 independent experiments with 2 mice each, analyzing 10–20 intestinal circumferences/ mouse. Only continuous, fully intact circumferences were counted. ***P < 0.001 vs. control, Wilcoxon’s rank sum test with continuity correction. (D) Schematic shows the generation of 6B5 scFv. Briefly, the VH and VL regions of mouse monoclonal 6B5 Ab were codon optimized and cloned into expression vector pX containing a 6x-His tag and a 12x-glycine linker.