Pre-Clinical Intravenous Serum Pharmacokinetics of Albumin Binding and Non-Half-Life Extended Nanobodies®

Abstract

Nanobodies are antigen-binding, single variable domain proteins derived from naturally-occurring, heavy chain only antibodies. They are highly soluble, stable, and can be linked to build multi-specific formats. Several Nanobodies are currently in clinical development in different therapeutic areas, for both chronic and acute applications. For the former, prolonged exposure is achieved by half-life extending moieties that target endogenous albumin, while for the latter, non-half-life extended constructs are preferable. To demonstrate the general pharmacokinetic behavior of both formats, serum levels of seven intravenously administered Nanobodies were analyzed in cynomolgus monkeys, mice or rabbits. In monkeys, the total clearance of a monomeric irrelevant Nanobody was rapid (2.0 mL/(min*kg)) and approximated the species glomerular filtration rate, indirectly suggesting that the Nanobody was mainly eliminated via the kidneys. When linked to an anti-albumin Nanobody, a 376-fold decrease in clearance was observed, resulting in a terminal half-life of 4.9 days, corresponding to the expected species albumin half-life. Similar conclusions were drawn for (non-) half-life extended mono-, bi-and trimeric Nanobodies in mice or rabbits, suggesting that these kinetic principles apply across species. Applying this knowledge to species translation and study design is crucial for successful pre-clinical development of novel therapeutic Nanobody candidates.

Influence of protein properties and protein modification on biodistribution and tumor uptake of anticancer antibodies, antibody derivatives, and non‐Ig scaffolds

“…Similarly, conjugation of an albumin-binding VHH (Hle2) to a nontargeted VHH (Irr3) increased the half-life of Irr3 in cynomolgus monkeys. 198 Introducing albumin-binding capacity increased the half-life from 0.080 days (Irr3) to 4.9 days (Hle2-Irr3), which was estimated to be in the same range of serum albumin in cynomolgus monkeys. The half-life of serum albumin in cynomolgus monkeys was estimated to be ∼5.2 days, as was based on a weight of 2.4 kg and the formula: albumin half-life (days) = 3.75 × body weight (kg) 0.368 .…”

“…Similarly, conjugation of an albumin‐binding VHH (Hle2) to a nontargeted VHH (Irr3) increased the half‐life of Irr3 in cynomolgus monkeys . Introducing albumin‐binding capacity increased the half‐life from 0.080 days (Irr3) to 4.9 days (Hle2‐Irr3), which was estimated to be in the same range of serum albumin in cynomolgus monkeys.…”

“…The above studies have been performed in mice. Given the fact that albumin and HSA half‐life in mice (respectively, ∼1.5 days and ∼5.2 days) differ from that in humans (∼19 days), translating these results to a clinical setting is difficult . Due to the difference in albumin half‐life, the effect of albumin binding on the clearance of protein drugs might be more pronounced in humans than in mice.…”

“…Given the fact that albumin and HSA half-life in mice (respectively, ∼1.5 days and ∼5.2 days) differ from that in humans (∼19 days), translating these results to a clinical setting is difficult. 192,198,200,201 Due to the difference in albumin half-life, the effect of albumin binding on the clearance of protein drugs might be more pronounced in humans than in mice. This may also count for biodistribution and tumor uptake, as these parameters are affected by the clearance of protein drugs.…”

Oral delivery of the anti-tumor necrosis factor α domain antibody, V565, results in high intestinal and fecal concentrations with minimal systemic exposure in cynomolgus monkeys

“…Variable domain antibodies retain the potency and specificity of conventional antibodies, and have unique properties including their small size (12)(13)(14)(15), that make them a useful starting point for developing an oral therapy [8]. This scaffold can be engineered to produce domain antibodies that are resistant to intestinal proteases and are suitable for oral delivery (Vorabodies).…”