Quantitative cumulative biodistribution of antibodies in mice: effect of modulating binding affinity to the neonatal Fc receptor

Abstract

The neonatal Fc receptor (FcRn) plays an important and well-known role in antibody recycling in endothelial and hematopoietic cells and thus it influences the systemic pharmacokinetics (PK) of immunoglobulin G (IgG). However, considerably less is known about FcRn's role in the metabolism of IgG within individual tissues after intravenous administration. To elucidate the organ distribution and gain insight into the metabolism of humanized IgG1 antibodies with different binding affinities FcRn, comparative biodistribution studies in normal CD-1 mice were conducted. Here, we generated variants of herpes simplex virus glycoprotein D-specific antibody (humanized anti-gD) with increased and decreased FcRn binding affinity by genetic engineering without affecting antigen specificity. These antibodies were expressed in Chinese hamster ovary cell lines, purified and paired radiolabeled with iodine-125 and indium-111. Equal amounts of I-125-labeled and In-111-labeled antibodies were mixed and intravenously administered into mice at 5 mg/kg. This approach allowed us to measure both the real-time IgG uptake (I-125) and cumulative uptake of IgG and catabolites (In-111) in individual tissues up to 1 week post-injection. The PK and distribution of the wild-type IgG and the variant with enhanced binding for FcRn were largely similar to each other, but vastly different for the rapidly cleared low-FcRn-binding variant. Uptake in individual tissues varied across time, FcRn binding affinity, and radiolabeling method. The liver and spleen emerged as the most concentrated sites of IgG catabolism in the absence of FcRn protection. These data provide an increased understanding of FcRn's role in antibody PK and catabolism at the tissue level.

Keywords: FcRn; biodistribution; indium; metabolism; pharmacokinetics; radiolabeled.

Figure 1. Plasma pharmacokinetic curves of the IgG1 WT, FcRn⁺, and FcRn^- variants radiolabeled with (A) iodine-125 and (B) indium-111.

Figure 2. Tissue distributions at 6 (A and B), 24 (C and D), and 168 (E and F) hours of the IgG1 WT, FcRn⁺, and FcRn^- variants radiolabeled with iodine-125 (A, C and E) and indium-111 (B, D and F). Asterisks represent significant (P < 0.05) differences relative to WT by unpaired t test.

Figure 3. Ratios of tissue AUC_0–7 to plasma AUC_0–7 of the IgG1 WT, FcRn⁺, and FcRn^- variants radiolabeled with (A) iodine-125 and (B) indium-111. The abbreviation AUC_0–7 denotes the area under the concentration-time curve from 0 to 7 d. Note that AUC values for indium-111-labeled antibodies do not reflect true exposure of intact antibody since indium-111 is a residualizing radiometal label (Fig. 4).

Figure 4. Schematic diagram of the cellular processing of radiolabeled IgG in the presence and absence of FcRn binding affinity. Both ¹²⁵I- and ¹¹¹In-DOTA-labeled antibodies enter the cell by pinocytosis. In the sorting endosome, FcRn-protected IgG is recycled back to the extracellular space, while non-protected IgG proceeds to lysosomal proteolytic degradation. The radiolabeled catabolite, ¹¹¹In-DOTA-lysine, is residualizing due to its polarity or charge and tends to accumulate within cells. In contrast, radioiodinated protein catabolites are rapidly effluxed from cells.