Structure-Based Engineering of Monoclonal Antibodies for Improved Binding to Counteract the Effects of Fentanyl and Carfentanil

Abstract

The opioid overdose epidemic is a growing and evolving public health crisis fueled by the widespread presence of fentanyl and fentanyl analogues (F/FAs) in both street mixtures and counterfeit pills. To expand current treatment options, drug-targeting monoclonal antibodies (mAbs) offer a viable therapeutic for both pre- and postexposure clinical scenarios. This study reports the isolation, in vitro characterization, and in vivo efficacy of two murine mAb families targeting fentanyl, carfentanil, or both. Because humanization of the mAbs by CDR grafting negatively impacted affinity for both fentanyl and carfentanil, crystal structures of mAbs in complex with fentanyl or carfentanil were analyzed to identify key residues involved in ligand binding in murine versus humanized structures, and site-directed mutagenesis was used to verify their functional importance. The structural analysis identified a framework residue, Tyr36, present in the murine germline sequence of two mAbs, which was critical for binding to fentanyl and carfentanil. These studies emphasize the importance of structural considerations in mAb engineering to optimize mAbs targeting small molecules including opioids and other drugs of public health interest.

Figure 1

Efficacy of fentanyl-specific mAbs against F/FA in mice. Mice were passively immunized with 40 mg/kg murine HY6-F9 or HY11-7E1 and challenged with 0.1 mg/kg fentanyl, 1.0 mg/kg acetylfentanyl, or 0.02 mg/kg carfentanil 24 h after passive immunization. Concentrations of (A) fentanyl, (B) acetylfentanyl, or (C) carfentanil in serum (left y-axis) and the brain (right y-axis) were measured 30 min after drug administration. **p < 0.01, ***p < 0.001, ****p < 0.0001 compared to saline control.

Figure 2

Isolation of novel mAbs using fentanyl- and carfentanil-based haptens. (A) Structures of F/FA, fentanyl-based hapten F₁, and carfentanil-based hapten F₁₃. (B) Immunization scheme for the isolation of novel mAbs. Mice were immunized with F₁-CRM or F₁₃-CRM as shown. (C) F₁-specific (fentanyl) ELISA titer in mouse sera after one (day 14) or two (day 32) immunizations with F₁-CRM or F₁₃-CRM. Labels above bars indicate the order of immunization. (D) F₁₃-specific (carfentanil) ELISA titer in mouse serum after one or two immunizations with F₁-CRM or F₁₃-CRM. (E) Relative affinity (IC₅₀) for murine mAbs against fentanyl or carfentanil was determined by competitive ELISA. Values for HY6-F9, HY11-6B2, and HY11-7E1 were previously published and are included here for comparison (www.tandfonline.com). (F) Diagram of the mAb humanization scheme showing regions of murine residues in tan or human residues in blue.

Figure 3

Efficacy of novel mAb against fentanyl and carfentanil. Mice (n = 5/group) were passively immunized with chimeric mAb, 40 mg/kg, s.c. and then challenged with a mixture of 0.05 mg/kg fentanyl and 0.005 mg/kg carfentanil on day 3. Concentrations of (A) fentanyl and (B) carfentanil in serum (left y-axis) and brain (right y-axis) were measured 30 min after drug administration. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 compared to saline control.

Figure 4

Crystal structures and sequence comparisons of 6B2_Mu:fentanyl, 7E1_Mu:fentanyl, and 7E1_Hu (DE):F₁₁. (A) Left: Surface representation of the binding pocket of 6B2_Mu Fab with fentanyl in gray. Surface within 4 Å of fentanyl is colored in tan. Right: Stick representations of the same view. Potential polar bonds are shown in yellow. (B) Left: Surface representation of 7E1_Mu Fab with fentanyl in gray shown in the same manner as panel (A). Right: Stick representation of the same view. Bonds and orientations are the same as described in panel (A). (C) Left: Surface representation of 7E1_Hu (DE) Fab with F₁₁ hapten is shown in the same manner as panel (A). Red dots indicate the positions of water molecules in the structure. Right: Stick representation of the same view. Bonds and orientations are the same as described in panel (A). (D) Structural overlay of 6B2_Mu Fab and 7E1_Mu Fab, with 7E1_Mu Fab shown in transparency. The orientation is the same as panels (A) and (B), and the view is zoomed in. (E) Structural overlay of 7E1_Mu Fab and 7E1_Hu (DE) Fab, with 7E1_Hu (DE) shown in transparency. The orientation is the same as in panels (B) and (C), and the view is zoomed in. (F) Contact surface area (CSA) plot of the ligand when bound to 6B2_Mu, 7E1_Mu, or 7E1_Hu (DE). Sequence alignments of the VH and VL regions of each mAb are shown. Dots show conserved residues with 7E1_Mu as the template. Dashes indicate gaps. Numbering and CDRs are true to 7E1_Mu.

Figure 5

Crystal structures of 5B1_Mu Apo and 5B1_Mu:fentanyl. (A) Left: Surface representation of the 5B1_Mu Fab apo showing the binding pocket of 5B1_Mu. Right: Stick representation of the same view. (B) Left: Surface representation of 5B1_Mu Fab complexed with fentanyl is shown in the same manner as panel (A). Surfaces within 4 Å of fentanyl are colored in tan. Right: Stick representation of the same view. The orientation is equivalent to that in panel (A). Fentanyl is shown in light gray. Bonds are shown in yellow with distances listed. (C) Structural overlay of 5B1_Mu Apo versus 5B1_Mu:fentanyl. The orientation is the same as in panels (A) and (B). Distances and bonds are omitted for clarity. (D) Structural overlay of 5B1_Mu Fab bound to fentanyl (green) and 6B2_Mu Fab bound to fentanyl (blue transparency). The orientation is the same as in panel (B). Distances are omitted for clarity. (E) Structural overlay of 5B1_Mu Fab bound to fentanyl (green) and 7E1_Mu Fab bound to fentanyl (red transparency). The orientation is the same as in panel (B). Distances are omitted for clarity. (F) CSA with a plot of the ligand when bound to 5B1_Mu or 7E1_Mu, with sequence alignments of the VH and VL regions of 5B1_Mu, compared to 7E1_Mu and the murine germline IGKV6-15. Dots show conserved residues with 5B1_Mu as a template. Dashes indicate gaps. Numbering and CDRs are true to 5B1_Mu.