A strategy for risk mitigation of antibodies with fast clearance

Abstract

A majority of human therapeutic antibody candidates show pharmacokinetic properties suitable for clinical use, but an unexpectedly fast antibody clearance is sometimes observed that may limit the clinical utility. Pharmacokinetic data in cynomolgus monkeys collected for a panel of 52 antibodies showed broad distribution of target-independent clearance values (2.4-61.3 mL/day/kg), with 15 (29%) having clearance > 10 mL/day/kg. Alteration in the interaction with the recycling FcRn receptor did not account for the faster than expected clearance observed for the antibodies; off-target binding was presumed to account for the fast clearance. We developed an assay based on ELISA detection of non-specific binding to baculovirus particles that can identify antibodies having increased risk for fast clearance. This assay can be used during lead generation or optimization to identify antibodies with increased risk of having fast clearance in both humans and cynomolgus monkeys, and thus increase the likelihood of obtaining a suitable drug candidate.

Keywords: FcRn; antibody; baculovirus; non-specific binding; pharmacokinetics.

Figure 1. Correlation of antibody clearance values measured in humans and cynomolgus monkeys (ρ = 0.74, n = 16). For our analyses, antibody doses were chosen that were believed to saturate any target-dependent clearance. The solid line is a linear regression fit of the logarithm of human clearance to the logarithm of cynomolgus monkey clearance [log(Human clearance) = 0.0123 + 0.744 * log(Cyno clearance)]. For most antibodies shown, human clearance is about two-fold slower than the corresponding cynomolgus monkey clearance. By contrast, for anti-NRP1 the clearance in human (9.2 mL/day/kg) is ~2-fold faster than the clearance in cynomolgus monkey (4.3 mL/day/kg).

Figure 2. Clearance values of antibodies (n = 52) in cynomolgus monkeys. Individual animal data and geometric mean values (red bars) are shown from dose groups where contribution of any specific clearance to the reported clearance was assumed to be negligible. Humanized (circle), human (square), synthetic human phage derived (triangle), and chimeric (diamond) antibodies are shown. Filled symbols indicate antibodies with a λ light chain (mAbs 2, 20, 29, 43), all others have a κ light chain. IgG4 isotype antibodies having the hinge-stabilizing mutant S228P are colored blue (mAbs 9, 24), all others are IgG1 isotype. Red symbol indicates an afucosylated antibody (mAb 3). Aglycosylated antibodies obtained via replacement of Asn297 with Ala are colored orange (mAbs 13, 14, 20, 33, 37, 46). Antibodies with Fc amino acid substitutions to modulate FcγR binding are colored green (mAbs 1, 19, 40).

Figure 3. Comparison of clearance values in cynomolgus monkeys of humanized and synthetic human antibodies derived from phage display libraries. Chimeric antibodies (n = 1) and human antibodies from non-phage sources (n = 3) are not shown because of the very small size of the data sets. Plots show individual data values overlaid by boxplots. Boxplot rectangle shows the interquartile range (IQR) between the first quartile (25th percentile) and the third quartile (75th percentile); the thicker horizontal line within the boxplot rectangle is at the median (50th percentile). The lower boxplot whisker extends to the lowest data value that is still within 1.5 IQR of the lower quartile, and the upper whisker extends to the highest data value within 1.5 IQR of the upper quartile.

Figure 4. Relationship between antibody clearance in cynomolgus monkeys and binding affinity (K_D, pH 5.8) for cynomolgus monkey FcRn. Symbols as defined in Figure 2 are used. Dissociation constants (K_D) in nM were determined from steady-state analysis of SPR data as described by Yeung et al.

Figure 5. Association of antibody clearance value in (A) cynomolgus monkey and (B) human with normalized BV ELISA score. A BV ELISA score > 5 is associated with increased risk of fast clearance in cynomolgus monkey (ρ = 0.53, n = 45). The BV score was calculated from the mean of 6 determinations; each determination was normalized by dividing by the signal observed for non-coated wells on the same assay plate. Of antibodies with a BV score < 5, 12% have clearance > 10 mL/day/kg in cynomolgus monkey, while clearance exceeds 10 mL/day/kg for 75% of antibodies with BV score > 5. A maximum likelihood estimate for the odds ratio is 19.5 [Fisher’s Exact Test, 95% Confidence Interval (3.3, 165.7)]. The confidence interval suggests a 3.3 to 166-fold increase in the odds of faster clearance for BV > 5; that the interval does not contain 1 implies statistical significance. (B) BV ELISA score > 5 is associated with increased risk of fast clearance in human (ρ = 0.83, n = 16).

Figure 6. Relationship between antibody clearance in cynomolgus monkeys and antibody (A) pI, (B) hydrophobicity as measured by HIC, and (C) charge calculated for Fv domain.