Advancing humanized monoclonal antibody for counteracting fentanyl toxicity towards clinical development

Abstract

Innovative therapies to complement current treatments are needed to curb the growing incidence of fatal overdoses related to synthetic opioids. Murine and chimeric monoclonal antibodies (mAb) specific for fentanyl and its analogs have demonstrated pre-clinical efficacy in preventing and reversing drug-induced toxicity in rodent models. However, mAb-based therapeutics require extensive engineering as well as in vitro and in vivo characterization to advance to first-in-human clinical trials. Here, novel murine anti-fentanyl mAbs were selected for development based on affinity for fentanyl, and efficacy in counteracting the pharmacological effects of fentanyl in mice. Humanization and evaluation of mutations designed to eliminate predicted post-translational modifications resulted in two humanized mAbs that were effective at preventing fentanyl-induced pharmacological effects in rats. These humanized mAbs showed favorable biophysical properties with respect to aggregation and hydrophobicity by chromatography-based assays, and thermostability by dynamic scanning fluorimetry. These results collectively support that the humanized anti-fentanyl mAbs developed herein warrant further clinical development for treatment of fentanyl toxicity.

Keywords: PTM mitigation; antibody characterization; antibody engineering; clinical development; fentanyl; humanized antibody; mAb; manufacturability; opioid use disorder; synthetic opioids.

Figure 1.

In vivo efficacy against fentanyl of murine mAbs. Mice (n=4 per group, 2 males and 2 females) were passively immunized with anti-fentanyl mAb (40 mg/kg, s.c.), and 24 hours later were challenged with 0.25 mg/kg fentanyl. Fentanyl-induced effects on: (a) antinociception measured by hot plate; (b) heart rate and (c) breath rate measured by pulse oximetry. (d) One week after challenge, serum concentration of mAb measured by ELISA. Data are expressed as mean ± SEM; *p≤.05; **p≤.01; ***p≤.001.

Figure 2.

In vivo comparison of murine and chimeric anti-fentanyl mAbs. Mice (n=3 male mice per group) were passively immunized with anti-fentanyl mAb (40 mg/kg, s.c.), and 24 hours later were challenged with 0.1 mg/kg fentanyl. Concentration of fentanyl in (a) brain and (b) serum measured by LCMS. (c) Serum concentration of mAb and chAb prior to fentanyl challenge measured by ELISA. Data are expressed as mean ± SEM; *p≤.05; ***p≤.001, ****p≤.0001.

Figure 3.

In vivo comparison of murine and chimeric HY6-F9 in rats. Rats (n=3–4 male rats per group) were passively immunized with anti-fentanyl mAb (40 mg/kg, i.p.), and 24 hours later were challenged with 0.1 mg/kg fentanyl. (a) Fentanyl-induced antinociception as latency to respond on a hot plate; (b) oxygen saturation, (c) heart rate, and (d) breath rate measured by pulse oximetry. Concentrations of fentanyl in (e) brain and (f) serum measured by LCMS. Data are expressed as mean ± SEM; *p≤.05; **p≤.01; ***p≤.001.

Figure 4.

Fab T_m comparison of chimeric and humanized mAb. HY6-F9 mAbs (a) and HY11-7E1 mAbs (b) at 1 mg/ml in PBS, pH 7.4 were combined with Protein Thermal Shift^TM assay reagents and subjected to a continuous 0.3% (0.45°C/min) temperature ramp from 25 to 95°C. The T_m of each mAb fragment is determined by the temperature measurement at the derivative peak (dPeak). The initial, broad dPeak in each sample corresponds to the CH2domain, while the second dPeak in each sample corresponds to the Fab domain. All humanized HY6-F9 and HY11-7E1 mAbs show an increased temperature shift in Fab T_m upon humanization, indicating increased thermal stability compared to the murine chimeric counterpart.

Figure 5.

Efficacy of humanized lead mAb in rats. Rats (n=4 male rats per group) were passively immunized with saline, HY6-F9_Ch as positive control, HY6-F9_Hu (NQ), or HY11-7E1_Hu (DE) (40 mg/kg, i.p.), and 24 hours later were challenged with cumulative fentanyl doses up to 0.3 mg/kg. (a) Fentanyl-induced antinociception as latency to respond on a hot plate; (b) oxygen saturation, and (c) heart rate measured by pulse oximetry. (d) Concentration of mAb 1 hour prior to challenge measured by ELISA; and concentrations of fentanyl in (e) serum and (f) brain, and (g) concentration of norfentanyl in serum 15 min after final fentanyl dose measured by LCMS. Data are expressed as mean ± SEM; *, # or † indicate significance of indicated groups vs saline, or bars to indicate significance between groups; [*,#,†]p≤.05; [**,##,††]p≤.01; [***,###,†††]p≤.001; [****,####,††††]p≤.0001.