The general distribution patterns of 89Zr-IgG, 89Zr-Fab, [89Zr]Zr-DFO, and [89Zr]Zr-oxalate in cynomolgus macaques were assessed. We aimed to provide basic knowledge regarding the distribution of intact antibodies and the artificial signals produced by their metabolites.
Immuno-PET techniques relying on 89Zr-IgG are advantageous for visualising the whole-body distribution [6]. In this study, we detected 89Zr-IgG accumulation in the liver and kidneys of macaques at 6–10 days post-injection, based on calculated SUV and PET data. In contrast, the blood and other organs showed decreased counts at that time. When 89Zr-Fab, a shortened IgG variant, was administered, considerable retention of the radiolabelled compound was observed in the renal tissues (proximal tubules), and part was delivered to the urinary bladder as intact Fab. In addition, [89Zr]Zr-DFO was mainly delivered to the urinary bladder and, to a lesser extent, to the gallbladder within a few hours, while [89Zr]Zr-oxalate accumulated mainly in the bones.
Accumulation of radiolabelled IgG in the liver has been well documented in humans and other animals [6]; once in the liver, IgG undergoes proteolysis in the reticuloendothelial system. IgG binding to cells of the phagocytic system (macrophages and monocytes) occurs through the Fc portion of the antibody, which interacts with membrane-bound Fcγ receptors to allow subsequent endocytosis and lysosomal degradation [15].
In contrast, the fate of IgG in renal tissue is less known, and previous clinical studies have revealed variable uptake levels of 89Zr-labelled therapeutic antibodies by the kidneys. Some studies conducted in humans revealed that the renal uptake of 89Zr-cetuximab was only one-third of the liver uptake at 6 days p.i. [16, 17]. However, variable ratios between these organs at 4–6 days p.i. were reported for other 89Zr-labelled immunotherapy agents, such as 89Zr-ibritumomab [18], 89Zr-fresolimumab [19], and 89Zr-RG7116 [20]. Only low-molecular-weight proteins, peptides, or amino acids generated during proteolysis in other organs are excreted renally [21]. We hypothesise that renal uptake may differ among IgGs because the amount and size of peptide fragments released from these molecules depend on each degradation process.
We found similar 89Zr-IgG blood clearance in macaques compared to humans [16, 18]. The transportation and retention of intact IgG in the blood closely depend on the uptake and recycling via FcRn, except for the metabolism related to the antigen-antibody response. Suzuki et al. showed that the affinity of therapeutic antibodies for FcRn correlated closely with their serum half-lives [22], and the chimeric IgG serum half-life in mice was shorter than that in humans because of the low affinity of human IgG for mouse FcRn. In contrast, human and macaque IgG have been shown to bind with similar affinities to human and cynomolgus macaque FcRn [23]. Therefore, cynomolgus macaques are more appropriate animal models than rodents for evaluating IgG blood clearance.
We found that 89Zr-Fab immediately accumulated in the kidneys of macaques. Generally, small antibody-derived molecules, such as Fab [24], diabody [25], and scFv [26], are filtered in the glomeruli and reabsorbed in the renal tubules. The mechanisms underlying the renal uptake of radiolabelled proteins have been previously discussed. In this sense, megalin-cubilin receptor involvement in the renal uptake of [111In]DTPA-D-Phe1-octreotide was shown using the opossum kidney cell line [27] and kidney-specific megalin knock-out animals [28]. Because the megalin-cubilin system operates in the proximal convoluted tubules of the renal cortex, radioactivity may localise mainly in that tissue.
The accumulation of 89Zr-Fab and 89Zr-IgG in the renal cortex of macaques showed a similar pattern. This finding indicates that protein/peptide fragments from IgG, including 89Zr, were subjected to reuptake in the renal tubules. In line with this result, the in vitro stability test carried out using macaque serum revealed increases in radioactivity associated with the small molecular-sized fraction after 72 and 144 h of incubation.
In the urine samples from macaques that received 89Zr-Fab, radioactivity was excreted as 89Zr-Fab, revealing that appreciable amounts of intact 89Zr-Fab could be filtered into the urine without previous degradation. Tsai et al. [29] reported that approximately half of the radioactivity in the urine of mice after administration of 111In-Rituxan-Fab corresponded to intact Fab and the remaining part to small metabolites. Conversely, peptide fragments derived from intact 89Zr-IgG did not seem to have been excluded from circulation into the urine; instead, the renal cells retained considerable radioactivity.
Previous reports have documented that radiolabelled proteins undergo proteolysis immediately after their internalisation into renal cells, and radioactivity may be detected in lysosomes [30]. Interestingly, we found that almost all radioactivity existed in the cytosolic fraction of the renal lysates of macaques injected with 89Zr-IgG; the reason for this finding remains unknown. However, because TLC analysis of the renal lysate showed that almost all radioactivity was retained at the origin part of the chromatogram (data not shown), radioactivity as 89Zr4+ might not exist in the renal cytosol.
The liver and kidney lysates of macaques exhibited different lysosome–cytosol radioactivity ratios. Radioactivity in the lysosome fraction obtained from the liver might represent undigested 89Zr-IgG protein, while antibodies internalised into renal cells are expected to be immediately degraded in lysosomes, from which small radiolabelled fragments might go out and reach the cytosol. Rogers et al. indicated that 111In-F(ab)’2 was degraded into [111In]lys at 1 d post-injection in rat kidney lysates, although larger radiolabelled molecules remained in the liver lysates [31].
PET imaging studies performed in macaques injected with [89Zr]Zr-oxalate showed important activity in the bones, especially in the epiphysis of the long bones and vertebral body. [89Zr]Zr-oxalate is thought to be equivalent to 89Zr4+. In mice, [89Zr]Zr-oxalate, [89Zr]Zr-citrate, and [89Zr]Zr-chloride had a strong affinity for bones and joints [11]. It is very likely that 89Zr is chelated by hydroxyapatite, a phosphate constituent of bones and epiphysis; the extracted marrow cells were insignificantly radioactive compared to the calcified tissues (0.1% of the total activity of the bones).
Since 89Zr4+ may be released from the [89Zr]Zr-DFO-antibody, the radioactivity in the bone might increase with time. In this study, 89Zr4+ was scarcely released from DFO-IgG and did not return to circulation, which could be attributed to the decreased bone uptake over time observed in macaques administered with 89Zr-IgG and TLC data in the stability test. Additionally, [89Zr]Zr-DFO was rapidly excreted into the urine and gallbladder and then cleared from the system after the first day. Therefore, after [89Zr]Zr-DFO-antibody administration, it is reasonable to expect that specific cleavage of intact [89Zr]Zr-DFO from the antibody would not lead to 89Zr bone uptake, as it would immediately be excreted.