An agonist of toll-like receptor 5 has radioprotective activity in mouse and primate models

Abstract

The toxicity of ionizing radiation is associated with massive apoptosis in radiosensitive organs. Here, we investigate whether a drug that activates a signaling mechanism used by tumor cells to suppress apoptosis can protect healthy cells from the harmful effects of radiation. We studied CBLB502, a polypeptide drug derived from Salmonella flagellin that binds to Toll-like receptor 5 (TLR5) and activates nuclear factor-kappaB signaling. A single injection of CBLB502 before lethal total-body irradiation protected mice from both gastrointestinal and hematopoietic acute radiation syndromes and resulted in improved survival. CBLB502 injected after irradiation also enhanced survival, but at lower radiation doses. It is noteworthy that the drug did not decrease tumor radiosensitivity in mouse models. CBLB502 also showed radioprotective activity in lethally irradiated rhesus monkeys. Thus, TLR5 agonists could potentially improve the therapeutic index of cancer radiotherapy and serve as biological protectants in radiation emergencies.

Fig. 1

Flagellin-mediated radioprotection of mice (18). (A) Groups of 20 NIH-Swiss mice were injected with flagellin or PBS 30 min before TBI. Representative results from one of three independent experiments are shown. The difference in survival between control and experimental groups was statistically significant (P < 0.05 by two-tailed Fisher’s test) on days 15 to 30, 9 to 30, and 8 to 13 after 10, 13 and 17 Gy TBI, respectively. (B) Generation of an optimized flagellin derivative. (Bottom) Schematic of the domain composition of flagellin. Flagellin derivatives were composed from sequences in the conserved N- and C-terminal domains required for TLR5 activation (A, B, and C and A′ and B′, respectively). Amino acid numbering is from flagellin of Salmonella enterica serovar Dublin (GenBank accession no. AAA27081). (Top) Assessment of NF-κB activation by flagellin (FliC) derivatives using electrophoretic mobility shift assay. Flagellin itself and variants AA′, AB′, and BA′ induced NF-κB, whereas other combinations of N and C termini (BB′, CA′, and CB′); isolated N termini (A, B, and C); glutathione-S-transferase (GST)-fusions of C termini (GST-A′, GST-B′); and mixtures of isolated N and C termini (A+A′, A+B′) were not active. Positive control: TNFα; negative controls: untreated cells (Un) and cells incubated with GST alone (GST). Flagellin variant AA′ (red arrow) was renamed CBLB502. (C) NIH-Swiss mice were treated with CBLB502 (0.2 mg/kg), flagellin derivative CB′ (0.2 mg/kg), amifostine (150 mg/kg), 5-AED (30 mg/kg), or PBS before receiving 13 Gy TBI. *P < 0.03 by two-tailed Fisher’s test for comparison of survival in CBLB502- and amifostine-treated groups. (D) ICR mice (15 per group) were injected with CBLB502 at the indicated times relative to 9, 10, or 13 Gy TBI (red arrow). Control mice received PBS 30 min before TBI. The percentage of mice surviving at day 30 post-irradiation is plotted.

Fig. 2

CBLB502-mediated protection of radiosensitive tissues (18). (A) Representative terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick end labeling (TUNEL) staining of apoptotic cells (5 hours postirradiation) in the small intestine of NIH-Swiss mice injected with CBLB502 or PBS 30 min before 15 Gy TBI. Apoptotic endothelial cells displayed yellow fluorescence resulting from overlap of green TUNEL staining and red CD31-specific antibody (endothelial marker) staining. Nuclei were stained with 4′,6′-diamidino-2-phenylindole (DAPI, blue). (B) Morphology of the small intestine in NIH-Swiss and MOLF/Ei mice either untreated or 5 days after 15 Gy TBI with or without prior injection of CBLB502. Representative hematoxylin and eosin (H&E) stained sections are shown. (C) Immunohistochemical detection of in vivo BrdU incorporation in the crypts of the small intestine. Mice (three per group) were left untreated (U/t), given CBLB502 without TBI (CBLB502), or exposed to 13 Gy TBI 30 min after injection of PBS (13Gy)orCBLB502(13Gy+CBLB502). BrdU-positive cells were counted in 12 complete, well-oriented cross sections for each animal. Dashed line: number of BrdU-positive cells considered critical for crypt survival (36). P < 0.05 for the differences between “13 Gy” and other groups. (D) Granulocyte-macrophage colony-forming units were quantified in bone marrow cells obtained from NIH-Swiss mice (n = 3) 3 hours after 0, 10, or 13 Gy TBI (with or without CBLB502 injection 30 min before TBI). P < 0.05 for the differences between irradiated CBLB502- and vehicle-treated groups. (E) Immunohistochemical detection of SOD2 expression (green staining) in sections of small intestine collected 5 hours post-irradiation from NIH-Swiss mice injected with either PBS (13 Gy) or CBLB502 (13Gy + CBLB502) 30 min before 13 Gy TBI. Red staining, smooth muscle actin. (F) Cytokine induction by CBLB502 treatment in the absence of irradiation. Plasma was prepared at the indicated times (0.5 to 24 hours) after intramuscular injection of CBLB502 into ICR mice (n = 3). KC, keratinocyte-derived chemokine; IP-10, interferon-inducible protein 10.

Fig. 3

A single injection of CBLB502 improves survival of lethally irradiated rhesus macaques. (A) In a single pilot experiment, rhesus macaques were injected with 0.04 mg/kg CBLB502 (n = 11) or PBS (n = 8) 45 min before 6.5 Gy TBI (LD_70/40), a dose lethal for 70% of monkeys within 40 days after irradiation. *P < 0.03 by two-tailed Fisher’s test. (B) Platelet counts in the peripheral blood of irradiated monkeys. Days with statistically significant (P < 0.05 by two-tailed t test) differences between control and CBLB502 groups are indicated by filled symbols.

Fig. 4

CBLB502 does not affect the radiosensitivity of mouse tumors (18). (A) The effect of CBLB502 on radiation treatment of NIH 3T3–derived sarcomas grown in NIH-Swiss mice. Tumor bearing mice (n = 5) were left untreated (U/t) or treated three times daily (red arrows) with 4 Gy TBI (IR) alone, CBLB502 alone, or IR + CBLB502. Tumor volumes were measured every second day until the number of live animals dropped significantly. (B) Thirty-day survival curves for the tumor-bearing mice treated with IR alone or with IR + CBLB502. *P < 0.03 by two-way repeated measures analysis of variance (ANOVA). (C) CBLB502 does not enhance the carcinogenicity of irradiation. Tumor development in two groups of p53^+/− C57BL6 mice that received a single dose of 4 Gy TBI, with or without injection of CBLB502 30 min before irradiation, was followed for 49 weeks.