Advancing humanized monoclonal antibody for counteracting fentanyl toxicity towards clinical development

Figures & data

Table 1. K_D of murine, chimeric and humanized mAb determined by biolayer interferometry (BLI).

mAb ID	mAb Species	KD, nM for Ligand*
		Fentanyl	Acetylfentanyl	Carfentanil
HY11-2F5	Murine	<0.1	<0.1	0.72
HY11-4E6	Murine	<0.1	<0.1	NDB
HY11-5C1	Murine	<0.1	<0.1	NDB
HY11-6B2	Murine	<0.1	<0.1	NDB
	Chimeric	<0.1	<0.1	NDB
HY11-7E1	Murine	<0.1	<0.1	1.18
	Chimeric	<0.1	0.28	0.51
	Humanized	0.24	0.80	2.48
	Humanized_VH D62E	<0.1	0.88	1.31
HY6-F9	Murine	<0.1	0.63	NDB
	Chimeric	<0.1	0.56	NDB
	Humanized	0.17	1.58	NDB
	Humanized_VL N34Q	<0.1	1.94	NDB

NDB = No Detectable Binding; *hapten.

Table 2. Relative affinity of mAb for fentanyl and other target and off-target compounds as determined by competitive ELISA.

*indicates no detectable binding; detection limit denoted as the highest concentration of competitor used. Color scale to indicate relative affinity; green = IC50 <1E–08; red = IC50 >1E–03.

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.

Table 3. Homology of mAbs to human germline and K_D to fentanyl and carfentanil.

mAb Series	mAb Species	VH Description	VL Description	Homology to Human Sequence			KD by BLI (nM)
				VH*	VL**	Overall***	Fentanyl†	Carfentanil†
HY6-F9	Murine	Murine	Murine	76.2%	80.4%	67.8%	<0.1	N/A
	Chimeric	Murine	Murine	76.2%	80.4%	92.4%	<0.1	N/A
	Intermediate	Low Homology	Murine	91.8%	80.4%	95.2%	<0.1	N/A
	Intermediate	High Homology	Murine	94.3%	80.4%	95.7%	1.31	N/A
	Humanized	Low Homology	Low Homology	91.8%	89.3%	96.7%	0.17	N/A
	Humanized	Low Homology	High Homology	91.8%	91.1%	97.0%	9.24	N/A
HY11-7E1	Murine	Murine	Murine	70.8%	69.2%	64.9%	<0.1	1.18
	Chimeric	Murine	Murine	70.8%	69.2%	89.5%	<0.1	0.51
	Intermediate	Low Homology	Murine	84.2%	69.2%	92.2%	<0.1	1.83
	Intermediate	High Homology	Murine	91.7%	69.2%	93.5%	<0.1	1.33
	Humanized	Low Homology	Low Homology	84.2%	86.9%	95.0%	0.24	2.48
	Humanized	High Homology	Low Homology	91.7%	86.9%	96.4%	<0.1	4.46
	Humanized	High Homology	High Homology	91.7%	91.6%	97.1%	3.26	NDB

*Amino acid homology to human IGHV V-Gene germline sequence chosen for CDR grafting. **Amino acid homology to human IGKV V-Gene germline sequence chosen for CDR grafting. ***Amino acid homology to human IGHV and IGKV V-Gene germline sequence chosen for CDR grafting, with human IgG1 and IgK constant regions. †hapten. Further information on germline sequences used can be found in the Supplemental Information. mAbs chosen as the leads for further development are in bold.

Table 4. Biophysical characterization of chimeric and humanized mAb.

mAb ID	SEC-HPLC		HIC-HPLC	DSF
	% mAb Monomer	% Aggregate	Retention Time (min)*	Fab Tm (°C)
HY6-F9_Ch	98.0	2.0	14.88	77.0
HY6-F9_Hu	>99.5	<0.5	16.17	81.5
HY6-F9_Hu (NQ)	>99.5	<0.5	14.51	78.5
HY6-F9_Hu (GR)	>99.5	<0.5	15.73	78.7
HY11-7E1_Ch	98.5	1.5	13.01	72.5–76.5
HY11-7E1_Hu	>99.5	<0.5	13.11	78.0
HY11-7E1_Hu (DE)	>99.5	<0.5	13.06	80.5
HY11-7E1_Hu (GV)	>99.5	<0.5	13.07	77.6
In-house rituximab	99.2	0.8	13.97	Not Tested

*HIC-HPLC salt gradient ends at 16.67 min.

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.