1.

Introduction

Monoclonal antibodies (mAbs) have been used for targeted therapy and imaging because of their high affinity for a diverse number of antigens overexpressed on cancer cells.^1,2 Molecular imaging based on mAbs labeled with radioisotopes can not only localize tumor within the whole body but also characterize it for drug targeting.^3,4. However, the prolonged clearance time of whole mAbs reduces the target-to-background ratio, lowering sensitivity and specificity of labeled mAbs.⁵ To overcome this problem, mAbs have been subjected to enzymatic cleavage or have been genetically modified to create fragments of various sizes, including variable region fragments (Fvs), diabody, fragments, antigen binding (Fabs), minibody, F(ab)’2 fragment, and ${\mathrm{CH}}_2$-deleted mAb.^6,7 If these fragments are too small, their renal clearance will be too rapid, compromising tumor uptake. Additionally, monovalent Fabs have lower binding affinities. Therefore, intermediate-size Fabs with mild prolongation of clearance but with bivalent binding to antigen, such as diabodies (50 to 60 kDa) or minibodies (75 to 80 kDa), would seem more likely to provide the highest tumor-to-background ratios.⁷

Activatable imaging probes upon binding to the target are produced based on a relatively new concept that minimizes background signal, resulting in an increase in the tumor-to-background ratio. These activatable optical imaging probes using mAbs can be activated only when bound to a cell surface receptor followed by internalization.^8–10. Whole immunoglobulin G (IgG)-based activatable probes with a long biological half-life have shown excellent target-specific tumor detection with minimal background signals.^11–13 In this study, we hypothesize that optimal pharmacokinetics of a cys-diabody combined with activatable labeling could improve the specific detection of cancer. Therefore, we have synthesized anti-prostate-specific membrane antigen (PSMA) cys-diabody activatably labeled with indocyanine green (ICG) derivatives¹⁴ and tested them in PSMA-expressing tumor-bearing mice.

2.

Materials and Methods

3.

Results

3.1.

Characterization of PSMA-Cys-Db Modified with ICG Derivatives

By adding 1% SDS to dye-conjugated antibodies, the following dequenching capacities were observed: 37.4-, 10.5-, 16.7-, and 2.5-fold for PSMA-Cys-Db-ICG, PSMA-Cys-Db-PEG4-ICG, PSMA-Cys-Db-PEG8-ICG, and PSMA-Cys-Db-IR700, respectively [Fig. 1(a)]. As defined by SDS-PAGE, the fractions of covalently bound ICG to PSMA-Cys-Db were 4.1, 72.1, and 79.3% for PSMA-Cys-Db-ICG, PSMA-Cys-Db-PEG4-ICG, and PSMA-Cys-Db-PEG8-ICG, respectively [Fig. 1(b)]. Furthermore, PSMA-Cys-Db-ICG showed a 64.1% increase in fluorescence intensity 1 h after incubation in mouse serum [Fig. 1(c)], suggesting significantly lower in vivo stability, compared with PSMA-Cys-Db-PEG4-ICG and PSMA-Cys-Db-PEG8-ICG (17.1% and 10.2%, respectively). The fluorescence of free ICG was dequenched in mouse serum.

Fig. 1

(a) Quenched (left) and chemically dequenched (right) PSMA-Cys-Db-ICG, PSMA-Cys-Db-PEG4-ICG, PSMA-Cys-Db-PEG8-ICG, and PSMA-Cys-Db-IR700. (b) Validation of the covalent conjugation of ICG to antibody by SDS-PAGE (left: colloidal blue staining, right: fluorescence). (c) Stability of probes in mouse serum. Fluorescence recovery was calculated by the following equation: (Fluorescence signal in mouse serum − Fluorescence signal in PBS)/(Fluorescence signal in SDS/PBS–Fluorescence signal in PBS) $\times 100$. Data are presented as $\text{mean}\pm \mathrm{s}.\mathrm{e}.\mathrm{m}.$ The fluorescence of free ICG can be dequenched in mouse serum.

3.2.

Fluorescence Microscopy Studies

Microscopy studies (Fig. 2) using PSMA-Cys-Db-ICG showed intense fluorescence signal within the PC3-PSMA+ and PC3-PSMA− cells 1 and 8 h after incubation regardless of the high dequenching capacity of PSMA-Cys-Db-ICG. On the other hand, PSMA-Cys-Db-PEG4-ICG and PSMA-Cys-Db-PEG8-ICG showed minimal signal 1 h after incubation in both cells and significantly higher signal in PC3-PSMA+ cells than in PC3-PSMA− cells 8 h after incubation.

Fig. 2

PC3-PSMA+ and PC3-PSMA− cells were incubated with PSMA-Cys-Db-ICG, PSMA-Cys-Db-PEG4-ICG, and PSMA-Cys-Db-PEG8-ICG for 1 or 8 h. Although high nonspecific signal was observed in PC3-PSMA− cells when PSMA-Cys-Db-ICG was used, PSMA-Cys-Db-PEG4-ICG and PSMA-Cys-Db-PEG8-ICG demonstrated the specific uptake by PSMA positive cells. Scale $\text{bar}=25\mu \text{m}$.

3.3.

In Vivo Imaging Studies Targeting for PSMA

Figures 3 and 4 show the imaging and quantitative assessment of tumor-bearing mice (PC3-PSMA+ and PC3-PSMA−) administered with PSMA-Cys-Db-ICG, PSMA-Cys-Db-PEG4-ICG, PSMA-Cys-Db-PEG8-ICG, PSMA-Cys-Db-IR700, and J591-ICG using the 700 and 800 nm fluorescence channel of the fluorescence camera for IR700 and ICG, respectively. Covalent conjugation of ICG with short PEG linkers successfully reduced the nonspecific uptake in the liver at early time intervals after injection unlike PSMA-Cys-Db labeled with the original ICG-Sulfo-OSu, which had high liver uptake. However, unexpectedly, there was prolonged high background activity especially in the kidneys, liver, and circulation at 1, 3, and 6 h postinjection with PEG linkers. This resulted in low fluorescence ratios (1.0 to 1.7) of PSMA+ to PSMA− cells for PSMA-Cys-Db-ICG, PSMA-Cys-Db-PEG4-ICG, and PSMA-Cys-Db-PEG8-ICG, although there was a slight improvement in target-to-background ratios after introduction of short PEG linkers to ICG. On the other hand, PSMA-Cys-Db-IR700 (always-on probe) mainly accumulated in the kidneys, but there was low uptake in the liver, which was similar to the biodistribution of intact Cys-Db reported previously.¹⁵ The fluorescence ratios of PSMA+ to PSMA− tumors were increased on PSMA-Cys-Db-IR700 and improved over time. As a comparison, J591-ICG selectively accumulated in PC3-PSMA+ tumors within 24 h with low background signal, except in the liver and bowel. The fluorescence intensity in PC3-PSMA+ tumors was maintained on day 3, as reported previously¹² (data not shown).

Fig. 3

(a) In vivo serial fluorescence images of PC3-PSMA+ (right dorsum) and PC3-PSMA− tumor (left dorsum) bearing mice injected with PSMA-Cys-Db-ICG, PSMA-Cys-Db-PEG4-ICG, PSMA-Cys-Db-PEG8-ICG, PSMA-Cys-Db-IR700, and J591-ICG. (b) Abdominal side of in vivo fluorescence images. (c) Ex vivo fluorescence images of PC3-PSMA+, PC3-PSMA−, liver, and kidneys obtained 24 h (72 h for J591-ICG) after injection of probes. Smallest scale bar indicates 1 mm.

Fig. 4

(a) PSMA+ tumor-to-PSMA and (b) tumor-to-liver signal intensity ratios. PEGylation of ICG could improve the PSMA+ tumor to PSMA− tumor ratios, which was comparable to nonactivatable always-on PSMA-Cys-Db-IR700. J591-ICG achieved the highest target tumor-to-background ratios among five probes. Data are represented as $\text{mean}\pm \mathrm{s}.\mathrm{e}.\mathrm{m}.$ $n=3$.

3.4.

Biodistribution of PSMA-Cys-Db and PSMA-Cys-Db-ICG

Results of in vivo biodistribution studies of ${}^{125}\mathrm{I}$-PSMA-Cys-Db and ${}^{125}\mathrm{I}$-PSMA-Cys-Db-ICG are summarized in Fig. 5. ${}^{125}\mathrm{I}$-PSMA-Cys-Db-ICG showed a biodistribution similar to ${}^{125}\mathrm{I}$-PSMA-Cys-Db. ${}^{125}\mathrm{I}$-PSMA-Cys-Db-ICG was rapidly taken up by the kidneys within 1 h postinjection and showed a fast blood clearance and significantly higher accumulation in PSMA positive tumors than in PSMA negative tumors at 6 and 24 h after probe administration. Notably, the radioactivity observed in the kidneys was sharply reduced within 6 h, while that in the stomach was dramatically increased. These results suggest that the cys-diabody is dehalogenated and possibly catabolized in the kidneys, and the fragment of cys-diabody including the iodine ion can be slowly released into the circulation followed by gradual uptake in the stomach and thyroid.

Fig. 5

In vivo biodistribution of radioactivity at 1, 6, and 24 h after injection of ${}^{125}\mathrm{I}$-PSMA-Cys-Db (a) and ${}^{125}\mathrm{I}$-PSMA-Cys-Db-ICG (b) into mice bearing PC3-PSMA+ and PC3-PSMA− tumors, expressed as % injected dose/g of tissue. Data are represented as $\text{mean}\pm \mathrm{s}.\mathrm{e}.\mathrm{m}.$ $n=3$ to 4. * % injected dose for thyroid.

4.

Discussion

The optimization of pharmacokinetics and stability of activatable optical probes is important for successful implementation. We have shown that ICG with short PEG linkers can be covalently conjugated to IgG with superior efficacy compared with ICG-Sulfo-OSu without a PEG linker.¹⁴ When ICG-Sulfo-OSu was conjugated directly without a PEG linker, there was less covalent binding, resulting in higher liver uptake. By inserting PEG linkers, the activation capacity was slightly reduced due to decreasing quenching efficacy probably because of increased distances between ICG and aromatic residues on mAb molecules.^11,14 However, an activation capacity of at least 10-fold was retained for both PSMA-Cys-Db-PEG4-ICG and PSMA-Cys-Db-PEG8-ICG.

In in vivo fluorescence imaging studies, PSMA-Cys-Db covalently conjugated with ICG with short PEG linkers showed little nonspecific uptake in the liver immediately (5 min) after injection compared with PSMA-Cys-Db conjugated with ICG-Sulfo-OSu. However, an unanticipated increase in background signal was observed especially in the kidneys, liver, and circulation from 1 h to 1 day after administration. The biodistribution study using ${}^{125}\mathrm{I}$-labeled PSMA-Cys-Db and PSMA-Cys-Db-ICG were similar with relatively rapid clearance through the kidneys that is consistent with previous reports. The biodistribution study suggested rapid renal catabolism and deiodination of probes followed by intense accumulation of free radioiodine in the stomach by 6 h after injection. Cys-diabody fragments particularly were subjected to filtration through glomerulus, reabsorption by the proximal convoluted tubule (PCT), and dehalogenated upon internalization in the PCT cells according to the previous reports.^16,17 ICG could be expected to undergo similar catabolism, albeit with a slower release than free iodine. ICG would be released after catabolism of the conjugate within the lysosome. Released ICG would bind to plasma proteins, resulting in increased fluorescence as shown in Fig. 1(c), would then circulate in the blood bound to protein, and would then gradually accumulate into the liver and finally be excreted via the hepatobiliary system as nonconjugate ICG does. Therefore, these fluorescently activated catabolites of ICG, which are formed within a day after injection, persistently increase the background signal in fluorescent images.

PSMA-Cys-Db-IR700 (always-on probe) immediately accumulated in the kidneys by glomerular filtration after injection and then was reabsorbed by PCT similar to radiolabeled diabodies. The difference in the pathway after metabolism in the kidneys between PSMA-Cys-Db-ICG and PSMA-Cys-Db-IR700 might be attributed to the difference in the chemical characteristics of fluorophores. After degradation and release into the circulation, ICG can bind to plasma protein and be excreted into the bile through the liver, whereas IR700 is rapidly excreted into the urine.¹⁸ Therefore, although PSMA-Cys-Db-IR700 is an always-on probe, background activity of IR700 decreases more rapidly than that of ICG. ICG has been approved for human use for 50 years with a good safety profile and is a practical fluorophore for clinical application. However, when conjugated with small proteins such as diabodies, which are catabolized in the kidneys, fluorescently activated ICG and derivatives can be recirculated and excreted through the liver, leading to a prolonged increased background in abdomen on fluorescence images. Therefore, J591(whole IgG)-ICG is apparently a better activatable probe for the PSMA-specific cancer detection than diabody-ICG probes.

In conclusion, optimal design of activatable optical probes should consider the excretion pathway of fluorescent catabolites. A large proportion of such agents can be catabolized in the kidney and, therefore, not reach the target in large amounts. From this point of view, activatable labeling of imaging probes with ICG is better for somewhat larger proteins such as IgG, F(ab)’2, and minibody, which are not filtered through the glomerulus, however, it is relatively inferior for small proteins including cys-diabody, Fab, and Fv fragments, which are filtered rapidly through the glomerulus. When small proteins are used as targeting moieties, we might have to consider more hydrophilic and, therefore, more readily urinary excretable fluorophores such as IR700 for activatable labeling.