Novel chimeric monoclonal antibodies that block fentanyl effects and alter fentanyl biodistribution in mice

Abstract

The prevalence and societal impact of opioid use disorder (OUD) is an acknowledged public health crisis that is further aggravated by the current pandemic. One of the devastating consequences of OUD is opioid overdose deaths. While multiple medications are now available to treat OUD, given the prevalence and societal burden, additional well-tolerated and effective therapies are still needed. To this point, we have developed chimeric monoclonal antibodies (mAb) that will specifically complex with fentanyl and its analogs in the periphery, thereby preventing them from reaching the central nervous system. Additionally, mAb-based passive immunotherapy offers a high degree of specificity to drugs of abuse and does not interfere with an individual's ability to use any of the medications used to treat OUD. We hypothesized that sequestering fentanyl and its analogs in the periphery will mitigate their negative effects on the brain and peripheral organs. This study is the first report of chimeric mAb against fentanyl and its analogs. We have discovered, engineered the chimeric versions, and identified the selectivity of these antibodies, through in vitro characterization and in vivo animal challenge studies. Two mAb candidates with very high (0.1-1.3 nM) binding affinities to fentanyl and its analogs were found to be effective in engaging fentanyl in the periphery and blocking its effects in challenged animals. Results presented in this work constitute a major contribution in the field of novel therapeutics targeting OUD.

Keywords: Fentanyl mAb; fentanyl analogs; opioid use disorder; opioids; passive immunization.

Figure 1.

Description of immunogen and experimental strategy. a) Design of the fentanyl vaccine that was used to immunize mice. The immunogen is composed of tetanus toxoid (TT) carrier protein conjugated to the para-AmFenHap hapten through the NHS-PEG₂-maleimide linker. b) Experimental strategy to generate the chimeric mAb described in this study. The constant IgG domains in the chimeric mAbs originated from human IgG while the variable domains were from mouse

Figure 2.

Characterization of purified mAb. a) SDS-PAGE analysis of purified anti-fentanyl chimeric antibodies. Purification of anti-fentanyl antibodies using protein A affinity chromatography. Lane M represents molecular marker, lane 1 represents P1B6H7 Ab under reduced condition, and lane 2 represents P1C3H9 Ab under reduced condition, respectively. Lanes 3 and 4 represent P1B6H7 and P1C3H9 Abs under non-reducing condition, respectively. HC indicates heavy chain and LC indicates light chain. b) Confirmatory ELISA of chimeric version of anti-fentanyl antibodies derived from hybridomas

Figure 3.

Specificity and cross-reactivity of mAb. Stock mAb solutions in PBS (1.0 mg/mL) were diluted with a buffer that contained 5 nM of indicated drugs and dialyzed against buffer in an equilibrium dialysis plate. Drug concentrations in the sample and buffer chambers were determined after 24 h, and fraction bound was calculated. a) fentanyl, b) acryl fentanyl, c) cyclopropyl fentanyl, d) furanyl fentanyl, e) buprenorphine, f) methadone, g) naloxone, h) naltrexone. Data shown are mean ± std dev of triplicate measurements. Red: mAb P1C3H9, Blue: mAb P1B6H7

Figure 4.

Molecular modeling of mAbs and their interaction with the fentanyl ligand. a) Predicted 3-dimensional (3D) structures alignment of the variable domain of antibodies B6H7 and C3H9 single-chain variable fragments (scFvs) with a comparison of the binding pockets. P1B6H7 is yellow (VL) and dark red (VH). P1C3H9 is purple (VL) and green (VH). The CDRs are indicated using arrow mark, LCDR represents for light chain and HCDR represents for heavy chain, respectively. b) The residues forming binding sites with fentanyl ligand (gray black) of antibodies: P1B6H7 and P1C3H9. The elements of specific protein-ligand interactions are shown. The key residues of mAbs interact with fentanyl ligand are shown in red

Figure 5.

Effect of mAb on the fentanyl-induced antinociception. Mice (n = 5–7/group) were immunized with 1.0 mg of indicated mAb (i.v.) and challenged with 0.1 mg/kg fentanyl (s.c.). Controls did not receive mAb. Antinociception was measured 15 mins post-fentanyl using a tail immersion test. The percentage maximum possible effect (%MPE) was calculated as the posttest latency minus the pretest latency divided by the maximum time (10 seconds) minus the pretest latency times 100. Data shown are mean ± s.e.m. Statistical analysis used ordinary one-way ANOVA with Bonferroni correction for multiple comparisons. * p < .001, ** p < .01. Red: mAb P1C3H9, Blue: mAb P1B6H7, Black: Control

Figure 6.

Blood-brain distribution of fentanyl in mice. Mice (n = 5/group) were immunized with 1.0 mg of indicated mAb (i.v.) and challenged with 0.1 mg/kg fentanyl (s.c.). Controls (black bars) did not receive mAb. Mice were sacrificed and blood and brain were collected for fentanyl measurements using LC-MS/MS. Data shown are mean ± std dev. Statistical analysis used ordinary one-way ANOVA with Bonferroni correction for multiple comparisons. ** p < .001, *** p < .0001. Red: mAb P1C3H9, Blue: mAb P1B6H7