Evaluating the Use of Antibody Variable Region (Fv) Charge as a Risk Assessment Tool for Predicting Typical Cynomolgus Monkey Pharmacokinetics

Abstract

The pharmacokinetic (PK) behavior of monoclonal antibodies in cynomolgus monkeys (cynos) is generally translatable to that in humans. Unfortunately, about 39% of the antibodies evaluated for PKs in cynos have fast nonspecific (or non-target-mediated) clearance (in-house data). An empirical model relating variable region (Fv) charge and hydrophobicity to cyno nonspecific clearance was developed to gauge the risk an antibody would have for fast nonspecific clearance in the monkey. The purpose of this study was to evaluate the predictability of this empirical model on cyno nonspecific clearance with antibodies specifically engineered to have either high or low Fv charge. These amino acid changes were made in the Fv region of two test antibodies, humAb4D5-8 and anti-lymphotoxin α. The humAb4D5-8 has a typical nonspecific clearance in cynos, and by making it more positively charged, the antibody acquires fast nonspecific clearance, and making it less positively charged did not impact its clearance. Anti-lymphotoxin α has fast nonspecific clearance in cynos, and making it more positively charged caused it to clear even faster, whereas making it less positively charged caused it to clear slower and within the typical range. These trends in clearance were also observed in two other preclinical species, mice and rats. The effect of modifying Fv charge on subcutaneous bioavailability was also examined, and in general bioavailability was inversely related to the direction of the Fv charge change. Thus, modifying Fv charge appears to impact antibody PKs, and the changes tended to correlate with those predicted by the empirical model.

Keywords: antibody; antibody engineering; charge; clearance; homology modeling; mutant; pharmacokinetics.

FIGURE 1.

Modeling of charge variants onto crystal structures determined for complexes of anti-LTα·LTα and humAb4D5-8:HER2. A, antibody-antigen structures for anti-LTα (Protein Data Bank code 4MXV) (29) and humAb4D5-8 (Protein Data Bank code 1N8Z) (30) are shown with antigens colored purple and the antibody Fabs in green. The colored spheres show the location of the amino acids that were mutated to alter Fv charge. B, electrostatic surfaces calculated (PyMOL, Shrödinger, Inc.) for Fv portions of modeled charge variants. Positive charge is indicated by blue and negative charge by red.

FIGURE 2.

In silico model predictions of where the nonspecific CL of the Fv charge variants would fall relative to their parental counterparts. Fv charge was calculated at pH 5.5, and hydrophobicity product was calculated by taking the product of the hydrophobic to hydrophilic amino acid ratios for CDRs L1, L3, and H3, weighted by their respective lengths (17). The anti-LTα variants are shown in A, with the parental antibody at +8.1 Fv charge. The humAb4D5-8 variants are shown in B, with the parental antibody at +6.1 Fv charge.

FIGURE 3.

Antigen binding kinetics of the parental and Fv charge variants as determined by BIAcore. A, binding kinetics of the anti-LTα antibodies was determined for LTα1b2. The kinetics were determined at concentrations between 0.076 and 500 nm. B, binding kinetics of the humAb4D5-8 antibodies was determined for HER2. The kinetics were determined at concentrations between 1.23 and 100 nm. The data were fit to a simple 1:1 binding model in the BIAevaluation software.

FIGURE 4.

Inhibition of TNFR2 and LTβR signaling by parental and Fv charge variants of anti-LTα antibodies. HeLa/NF-βB-luc reporter cells were stimulated with LTα3 (A) or LTα1β2 (B) to induce activation of the NF-βB signaling pathway mediated through TNFR2 or LTβR, respectively. NF-βB activity was measured in relative luciferase units and normalized to luminescence measured in the absence of antibody blockade. Baseline activity in unstimulated cells (diamond) and activity in stimulated cells in absence of blockade (+ symbol) are indicated. Data are shown as mean ± S.D. of duplicate wells from duplicate plates. Data are representative of at least two experiments.

FIGURE 5.

Concentration-time profiles of the parental antibodies and the Fv charge variants following a single i.v. or s.c. dose at 10 mg/kg to cynos. The parental antibodies are depicted in red, and the more positively charge variants are depicted in blue, and the less positively charge variants are depicted in green. The i.v. dose groups are depicted in A and C for the anti-LTα and humAb4D5-8 antibodies, respectively. The s.c. dose groups are depicted in B and D for the anti-LTα and humAb4D5-8 antibodies, respectively. Each point represents an individual animal (n = 4), and the line represents the average concentration among them.

FIGURE 6.

Concentration-time profiles of the parental antibodies and the Fv charge variants following a single i.v. or s.c. dose at 10 mg/kg to Sprague-Dawley rats. The parental antibodies are depicted in red; the more positively charge variants are depicted in blue, and the less positively charge variants are depicted in green. The i.v. dose groups are depicted in A and C for the anti-LTα and humAb4D5-8 antibodies, respectively. The s.c. dose groups are depicted in B and D for the anti-LTα and humAb4D5-8 antibodies, respectively. Each point represents an individual animal (n = 3), and the line represents the average concentration among them.

FIGURE 7.

Concentration-time profiles of the parental antibodies and the Fv charge variants following a single i.v. or s.c. dose at 10 mg/kg to C57BL/6 mice. The parental antibodies are depicted in red; the more positively charge variants are depicted in blue, and the less positively charge variants are depicted in green. The i.v. dose groups are depicted in A and C for the anti-LTα and humAb4D5-8 antibodies, respectively. The s.c. dose groups are depicted in B and D for the anti-LTα and humAb4D5-8 antibodies, respectively. Each point represents an individual animal (n = 3), and the line represents the average concentration among them.