The impact of immunogenicity on therapeutic antibody pharmacokinetics: A preclinical evaluation of the effect of immune complex formation and antibody effector function on clearance

Figures & data

Figure 1. Analysis of dosing solutions yy SEC and ELISA. (a) Size exclusion Chromatograms (absorbance) of all dosing solutions. (b/c) Dosing solution with drug^PGLALA + pADA_<CDR> (b) and drug^PGLALA + mADA_<Fc> (c): Absorbance (solid line) and reconstructed ELISA profile (dashed line). No monomeric drug detectable in the reconstructed ELISA IC profiles. Elution of monomeric IgG at ~ 22.5 min

Table 1. Composition of the IC dosing solutions. The percentages of defined IC sizes in the dosing solutions were determined by analyzing the area under the curve of the UV trace of the SEC chromatograms. The “% monomeric ADA” listed in the table is the value of the area under the curve of the ADA monomer UV peak

	% dimeric IC	% tetrameric IC	% hexameric IC	% HMW IC	% monomeric ADA
drug^PGLALA + pADA<CDR>	7	13	10	53	17
drug^WT + pADA<CDR>	8	16	13	49	14
drug^PGLALA + mADA<CDR>	3	30	22	32	13
drug^WT + mADA<CDR>	2	23	18	46	11
drug^PGLALA + mADA<Fc>	2	8	83		7
drug^WT + mADA<Fc>	2	18	64		16

Figure 2. Comparison of the total drug concentration in serum and plasma. Blood from groups dosed with drug^PGLALA or drug^PGLALA + pADA_<CDR> was divided for serum and plasma preparation and the total drug concentration was determined yy acid dissociation ELISA to study potential influences of serum/plasma preparation on drug concentration when complexed with ADAs

Figure 3. Total drug concentration in serum samples of all groups (PK data was fitted to a 2 compartment model for intra venous bolus injection by statistical method, with relative standard deviation (coefficient of variation), n = 3)

Figure 4. Reconstructed drug + pADA_<CDR> IC profiles from serum samples (mean values of all three animals). (a) ICs were separated by SEC, fractionated, and he drug concentration in every collected fraction was determined by total drug ELISA to reconstruct the IC profiles. Potential elution of monomeric drug is marked with a dashed line. Elution time [min] refers to the elution of drug/ICs from the SEC column. (b) Defined time points were selected for comparison of drug^WT + pADA_<CDR> and drug^PGLALA + pADA_<CDR> indicating clearance differences of the respective dimeric and tetrameric ICs

Figure 5. Reconstructed drug + mADA_<CDR> IC profiles from serum samples (mean values of all three animals). (a) ICs were separated by SEC, fractionated, and the drug concentration in every collected fraction was determined by total drug ELISA to reconstruct the IC profiles. Potential elution of monomeric drug is marked with a dashed line. Elution time [min] refers to the elution of drug/ICs from the SEC column. (b) Defined time points were selected for comparison of drug^WT + mADA_<CDR> and drug^PGLALA + mADA_<CDR> indicating clearance differences of the respective dimeric and tetrameric ICs

Figure 6. Reconstructed drug + mADA_<Fc> IC profiles in serum samples (mean values of all three animals). ICs were separated by SEC, fractionated, and the drug concentration in every collected fraction was determined by total drug ELISA to reconstruct the IC profiles. Potential elution of monomeric drug is marked with a dashed line. Elution time [min] refers to the elution of drug/ICs from the SEC column. IC profiles for later time points (8 hr – 72 hr) are not illustrated because of very low concentration

Table 2. Clearance values of monomeric drug and drug complexed in defined IC species

		clearance			clearance
		[mg/day/kg]			[mg/day/kg]
drug^WT	total	20.1	drug^PGLALA	total	8.78
drug^WT + pADA<CDR>	total	125	drug^PGLALA + pADA<CDR>	total	44.6
	dimeric IC	11.2		dimeric IC	4.44
	tetrameric IC	209		tetrameric IC	13.8
	hexameric IC	784		hexameric IC	112
	HMW IC	3600		HMW IC	2520
drug^WT + mADA<CDR>	total	621	drug^PGLALA + mADA<CDR>	total	236
	dimeric IC	52.3		dimeric IC	13.0
	tetrameric IC	309		tetrameric IC	59.2
	hexameric IC	1460		hexameric IC	943
	HMW IC	6800		HMW IC	12700
drug^WT + mADA<Fc>	total	1200	drug^PGLALA + mADA<Fc>	total	493
	dimeric IC	941		dimeric IC	46.5
	tetrameric IC	1220		tetrameric IC	964
	hexameric IC	1170		hexameric IC	1920
	HMW IC	11900		HMW IC	6650