Substructure-Specific Antibodies Against Fentanyl Derivatives

Abstract

Structural variants of the synthetic opioid fentanyl are a major threat to public health. Following an investigation showing that many derivatives are poorly detected by commercial lateral flow and related assays, we created hapten conjugate vaccines using an immunogenic virus-like particle carrier and eight synthetic fentanyl derivatives designed to mimic the structural features of several of the more dangerous analogues. Immunization of mice elicited strong antihapten humoral responses, allowing the screening of hundreds of hapten-specific hybridomas for binding strength and specificity. A panel of 13 monoclonal IgG antibodies were selected, each showing a different pattern of recognition of fentanyl structural variations, and all proving to be highly efficient at capturing parent fentanyl compounds in competition ELISA experiments. These results provide antibody reagents for assay development as well as a demonstration of the power of the immune system to create binding agents capable of both broad and specific recognition of small-molecule targets.

Keywords: antibodies; diagnostics; fentanyl derivatives; immune response; immunization; virus-like particles.

Figure 1

(top) Alkynes of fentanyl-based structures used for conjugation to VLP-azides to construct immunogens for these studies. Compound 1 shows the components of the fentanyl structure. (bottom) Biotinylated reagents for characterization of antibody binding using different linkers than in the immunogens.

Figure 2

VLP-fentanyl conjugate vaccine design and characterization. (a) Bioconjugation scheme for VLP-conjugates. (b–d) Representative characterization data for VLP-fentanyl conjugates (furanyl benzyl fentanyl [2] shown), (b) dynamic light scattering; pd = dispersity of the indicated cluster of peaks. (c) Size-exclusion chromatography (Superose 6). (d) Mass spectrometry of denatured particle showing distribution of coat protein substitution. (e) Summary of functionalization parameters for the indicated vaccines (±10%). Hapten dose = the amount of fentanyl analog delivered in each 50 μg dose of VLP conjugate; mg/kg calculated for mouse weight of 25 g.

Figure 3

Immunogenicity of PP7-fentanyl conjugate vaccines. (a) Representative vaccine schedule of BALB/c mice immunized with 50 μg of PP7-fentanyl conjugates (500 ng of NKT adjuvant included in prime and final boost) (carfentanil and norcarfentanil-2 terminated day 50; norcarfentanil-1 and 4-MeO-Bu terminated on 35). (b) Peak antihapten titer (week 5 postprime, geometric mean ± 95% CI) for each PP7-fentanyl vaccine. (c) Antifentanyl IgG titer of mice immunized with PP7-fentanyl vaccines as described in Figure 2. The last time point (T) represents the terminal bleed. All ELISA assays shown here were performed against the corresponding biotinylated antigen. For example, immune response against the VLP bearing fentanyl alkyne 1 was assayed by ELISA against biotinylated fentanyl 1b. Titers calculated as IC₅₀ by nonlinear regression of 6-fold 5x serum dilutions against specific immunogen by ELISA. Titers reported as box and whiskers ± min/max, line at mean. n = 3–7 mice per group.

Figure 4

Characterization of antifentanyl hybridomas. (a) Hybridoma clones selected from each PP7-fentanyl immunization (dark blue) and number of antigen-specific clones from this population (light blue). Clones chosen by ClonePix based on morphology and degree of IgG-secretion measured by anti-IgG FITC. Antigen specificity determined by binding to the corresponding biotinylated immunogen via ELISA. (b) red = percent of hapten-specific clones from panel a (light blue/dark blue); black = percent of hapten-binding IgG clones that also bound VLPs. (c) Antifentanyl IgG subclass distribution of hybridoma-produced mAbs from six PP7-fentanyl immunizations. Clones derived from immunization with norcarfentanyl derivatives 3 and 4 are not included here and were not carried forward.

Figure 5

Fentanyl analogue cross-reactivity of antigen-specific clones derived from five PP7-fentanyl immunizations. Each panel is labeled at the top with the alkyne used to prepare the VLP-hapten immunogen. Supernatant (1:5 dilution in blocking buffer) from antigen-specific hybridoma clones selected from the indicated splenocytes (rows) were tested for binding to each biotinylated-fentanyl analogue (columns) adhered to a streptavidin-coated ELISA plate. Signals for each hybridoma were normalized to the value obtained for the biotinylated cognate antigen, set to 100%.

Figure 6

Binding profiles of purified monoclonal antibodies to immobilized and solution-phase analytes. (a) Binding of the indicated mAbs to immobilized biotinylated fentanyl derivatives listed at the left, measured by ELISA. In each case, binding of the antibody to its cognate hapten was assigned a relative value of 100%. (b) Competition ELISA of each unfunctionalized fentanyl analyte (vertical axis; “f.” = fentanyl) against the biotinylated analyte indicated across the top, which is the biotinylated cognate structure for each antibody. “good binding” means that the unmodified fentanyl analyte competed well against the biotinylated compound.

Figure 7

Binding of monoclonal antibodies to fentanyl derivatives. Estimated IC₅₀ values from competition ELISA performed as shown in Figure 5 with five concentrations of the indicated fentanyl derivative (0.008–25 ng/mL) in the presence of 0.5 ng/mL of the biotinylated hapten structure used to elicit the antibody, except for (5)-1B1‡, which was performed with biotinylated acetyl-α-methyl fentanyl (8b). Entries boxed in green denote analysis involving the fentanyl derivative corresponding to the hapten used to generate the indicated antibody. Values of >50 indicate the observation of very weak or no competition. “Threat score” denotes how often the indicated fentanyl appeared on DEA and CFSRE publications from 2019 to 2021. Each asterisk denotes 1–2 appearances on these lists. “F kit avg. LOD” denotes the average limit of detection of commercial antifentanyl kit(s) to detect the indicated analyte as reported in ref (18) “CF kit avg LOD” has the same meaning for commercial kits against carfentanil. ND indicates no detection of the indicated compound; the appearance of numerical value appears with (ND) denotes that some of the kits used did not detect the indicated compound at all in repeated tests.