Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2016 Jan 31.
Published in final edited form as: Nanomedicine (Lond). 2015 Apr;10(7):1139–1147. doi: 10.2217/nnm.14.194

Photoimmunotherapy of hepatocellular carcinoma-targeting Glypican-3 combined with nanosized albumin-bound paclitaxel

Hirofumi Hanaoka 1, Takahito Nakajima 1, Kazuhide Sato 1, Rira Watanabe 1, Yen Phung 2, Wei Gao 2, Toshiko Harada 1, Insook Kim 3, Chang H Paik 4, Peter L Choyke 1, Mitchell Ho 2, Hisataka Kobayashi 1,*
PMCID: PMC4420156  NIHMSID: NIHMS685057  PMID: 25929570

Abstract

Aim

Effectiveness of Glypican-3 (GPC3)-targeted photoimmunotherapy (PIT) combined with the nanoparticle albumin-bound paclitaxel (nab-paclitaxel) for hepatocellular carcinoma was evaluated.

Materials & methods

GPC3 expressing A431/G1 cells were incubated with a phthalocyanine-derivative, IRDye700DX (IR700), conjugated to an anti-GPC3 antibody, IR700-YP7 and exposed to near-infrared light. Therapeutic experiments combining GPC3-targeted PIT with nab-paclitaxel were performed in A431/G1 tumor-bearing mice.

Results

IR700-YP7 bound to A431/G1 cells and induced rapid target-specific necrotic cell death by near-infrared light exposure in vitro. IR700-YP7 accumulated in A431/G1 tumors. Tumor growth was inhibited by PIT compared with nontreated control. Additionally, PIT dramatically increased nabpaclitaxel delivery and enhanced the therapeutic effect.

Conclusion

PIT targeting GPC3 combined with nab-paclitaxel is a promising method for treating hepatocellular carcinoma.

Keywords: Glypican-3, hepatoma, monoclonal antibody, nab-paxlitaxel, nanodelivery, photocyanine dye, photoimmunotherapy

Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide, especially in Asia, and is the third leading cause of cancer death worldwide. While surgical resection or liver transplantation are the only curative treatments for HCC, just 15% of patients are eligible for these treatments because HCC frequently grows as multiple foci [1,2]. Percutaneous ethanol injection, radiofrequency ablation, and transarterial chemoembolization are minimally invasive techniques that have shown efficacy in reducing tumor volume; however, they have had only modest impact on patient survival [2,3]. Although HCC is highly resistant to conventional systemic therapies, new targeted drugs offer some hope for the future [4,5]. However, for most patients with HCC, it remains an incurable disease, and new types of therapy are needed.

We recently reported a new form of monoclonal antibody (mAb)-based immunotherapy termed ‘photoimmunotherapy’ (PIT), which utilizes a mAb-bound to a photosen-sitizing phthalocyanine dye IRDye700DX® (IR700) conjugate to target cancer cells via exposure to near-infrared (NIR) light [6]. PIT is a highly selective phototherapy, which employs an antibody conjugated to a highly hydrophilic photosensitizer unlike conventional photodynamic therapy (PDT) that uses highly hydrophobic and nonconjugated photosensitizers. Moreover, PIT induces a rapid cell necrosis based on membrane disruption caused by a combination of photoinduced ligand exchange and reactive oxygen species (ROS), whereas conventional PDT requires cellular internalization, is exclusively dependent on ROS and produces an apoptotic cell death [6,7]. The antibody–photo-sensitizer conjugate (APC) is only active as a therapeutic agent, when it is bound to the target cell membrane; otherwise it has no effect on adjacent nonexpressing cells. Additionally, because PIT does not destroy tumor vessels early after therapy, there is a striking increase in the blood flow and permeability of tumor vessels after PIT. This permits the delivery of relatively high concentrations of nanosized-drugs specifically into treated tumors while allowing only minimal uptake in untreated regions [8]. Thus, the direct effects of PIT can be supplemented with post-PIT delivery of nanodrugs resulting in additional effect on tumor killing.

Glypican-3 (GPC3) represents an attractive target for HCC therapy because it is highly expressed in HCC but not in normal tissue [9-12]. The GPC3 core protein is a 70-kD protein, with a furin cleavage site in the middle. GPC3 has been suggested as a target for antibody and cell-based immunotherapies, and a number of targeted agents based on this target have been proposed [13,14]. In this study, PIT was performed with an APC consisting of IR700 conjugated to the anti-GPC3 mAb (YP7) in an animal model of HCC.

Materials & methods

General

Anti-GPC3 mouse mAb YP7 was developed in house [15]. Nanoparticle albumin-bound paclitaxel (nab-paclitaxel; Abraxane®) was purchased from Celgene (Summit, NJ, USA). A431/G1, a cell line stably expressing human GPC3, was established [15]. IR700 NHS ester was obtained from LI-COR Bioscience (Lincoln, NE, USA). A red light-emitting diode (LED) light source, which emits light at 690 ± 20 nm (antigen wavelength (L690–66–60, Marubeni America Co., CA, USA) was used for NIR light irradiation of PIT in vitro and in vivo. A power density was measured with an optical power meter (PM100, Thorlabs, NJ, USA) and exposure with this LED light for 1 min was calculated as 2.5 J/cm2. Other reagents were of reagent grade and were used as received.

Synthesis of IR700-conjugated YP7 & IR800-conjugated nab-paclitaxel

Conjugation of dyes with YP7 was performed according to a previous report [6]. Typically, YP7 (1 mg, 6.8 nmol) was incubated with IR700 NHS ester (60.2 μg, 30.8 nmol) in 0.1 mol/l Na2HPO4 (pH 8.5) at room temperature for 1 h. The mixture was purified with a Sephadex G50 column (PD-10; GE Healthcare, NJ, USA). The protein concentration was determined with the Coomassie Plus protein assay kit (Thermo Fisher Scientific Inc, IL, USA) by measuring the absorption at 595 nm with spectroscopy (8453 Value System; Agilent Technologies, CA, USA). The concentration of IR700 was determined by absorption at 689 nm with spectroscopy to confirm the number of IR700 molecules conjugated to each YP7. The synthesis was controlled so that an average of three IR700 molecules were bound to a single antibody. For conjugation of dyes with nab-paclitaxel, 5 mg of nab-paclitaxel (containing 4500 μg, 67.2 nmol of albumin and 500 μg of paclitaxel) was incubated with IRDye800CW® (IR800) NHS ester (129.2 μg, 134.3 nmol) in 0.1M Na2HPO4 (pH 8.5) at room temperature for 1 h, followed by purification with PD-10.

Binding assay

125I-YP7 was prepared using the Iodo-Gen procedure. Briefly, 100 μg of YP7 was added to each Iodogen coated vial with 0.5 M phosphate buffer, pH 7.2 and labeled with 37 MBq of 125I-Na at room temperature. After 5 min, the 125I radiolabeled product was purified with a PD-10 size exclusion column. The specific activity of the radiolabeled YP7 was 218 MBq/mg. Quality control was performed with size exclusion-HPLC with TSK SWxl G3000 (TosoHaas, PA, USA) with mobile phase 0.067 M phosphate-buffered saline (PBS) with 100 mM KCl.

A solution of 125I-YP7 (containing 0.003 μg of anti-body) was added to 100 μl of a cell suspension (containing 1 × 106 cells) along with increased amounts of unlabeled (‘cold’) antibody. After incubation for 1 h at room temperature, the cell suspension was centrifuged at 3000 rpm for 5 min. After the supernatant was removed, the radioactivity of the cell fraction was measured with a well-type gamma counter (Wizard 2480®, PerkinElmer, CT, USA). Bmax (antigen expression level) was determined by Scatchard plot analysis.

Flow cytometry

For flow cytometry (FACS), A431/G1 cells (1 × 105) were placed into 24-well plates. Cells were incubated with IR700-YP7 (10 μg/ml) for 1 h or 8 h at 37°C, followed by washing with PBS. Fluorescence from cells was measured using a flow cytometer (FACS Calibur®, BD BioSciences, CA, USA) and CellQuest® software (BD BioSciences). Signals from cells were collected using the FL4 emission filter (661/16, BP).

Fluorescence microscopy studies

To detect the antigen-specific localization of IR700-YP7, fluorescence microscopy was performed (BX61; Olympus America, NY, USA). A431/G1 cells (1 × 104) were plated on cover-glass-bottomed dishes. Cells were incubated with IR700-YP7 (10 μg/ml) over night at 37°C, followed by washing with PBS. The filter was set to detect IR700 fluorescence with a 590–650 nm excitation filter since IR700 has a sub-absorption peak at 600–650 nm, and a 665–740 nm band pass emission filter. The cells were continuously exposed by microscopy light without using a neutral density (ND) filter to induce phototoxicity, and serial images were obtained. Transmitted light differential interference contrast (DIC) images were also acquired before and after the fluorescence imaging.

In vitro photoimmunotherapy

A431/G1 cells (1 × 105) or parental A431 cells were placed into 24-well plates and incubated for 24 h at 37°C. Medium was replaced with fresh culture medium containing 10 μg/ml of IR700-YP7 and incubated for 6 h at 37°C. After washing with PBS, PBS was added again. Then, cells were irradiated with NIR light at 16 J/cm2. Cells incubated with APC but no NIR light exposure were also prepared as a control. Plates were washed with PBS and the cytotoxicity of PIT was determined by quantitative flow cytometry using propidium iodide (PI) as a stain for dead cells.

In vivo fluorescence imaging

All in vivo procedures were conducted in compliance with the Guide for the Care and Use of Laboratory Animal Resources (1996), US National Research Council, and approved by the local Animal Care and Use Committee. Six- to 8-week-old female homozygote athymic nude mice were purchased from Charles River (NCI-Frederick).

Two million A431/G1 cells were injected subcutaneously in the right dorsum of the mice. In order to determine tumor volume, the greatest longitudinal diameter (length) and the greatest transverse diameter (width) were determined with an external caliper. Tumor volume based on caliper measurements was calculated by the following formula: tumor volume = length × width2 × 0.5. Tumors reaching approximately 40 mm3 in volume were selected for the study. Mice were anesthetized with 2% isoflurane, and fluorescence imaging was obtained with a Pearl® Imager (LI-COR Biosciences) using the 700- and 800-nm fluorescence channels for IR700 and IR800, respectively. Fluorescence images of tumor-bearing mice after IR700-YP7 injection, were obtained before and after NIR light irradiation. Resions of interest (ROIs) were placed on the spectral images with a white light reference to measure fluorescence intensities of tumor and left dorsum (i.e., background tissue on the opposite side of the tumor). A Pearl Cam Software (LI-COR Biosciences) was used for calculating average fluorescence intensity within each ROI. Additionally, in some mice undergoing PIT, IR800-nab-paclitaxel (7.5 mg) was intravenously injected 1 h after PIT, and IR800 fluorescence images were obtained 10 min, 30 min, 60 min, 4 h and 24 h after injection. Fluorescence imaging of mice that received NIR light exposure only (50 J/cm2) but no prior APC injection was also obtained as a control. Then, the average IR800 fluorescence intensity of tumor and left dorsum was also calculated.

In vivo therapeutic studies

Based on the pharmacokinetics derived from the fluorescence imaging, we conducted two therapy experiments combining PIT with nab-paclitaxel. First, in order to demonstrate the effect of increased nab-paclitaxel delivery after PIT, a simple study was conducted in which a single exposure to light was either followed by nab-paclitaxel or no nab-paclitaxel (study 1). To increase the therapeutic efficacy of the combination therapy (PIT + nab-paclitaxel), NIR light exposure was repeated on 3 consecutive days after the animal received the APC along with nab-paclitaxel (study 2) with appropriate control groups as shown below (Figure 1). Dose of NIR light exposure was determined according to previous studies [16].

Figure 1. Outline of therapeutic study design.

Figure 1

Open in a new tab

Study 1 groups include (n ≥ 10; 1 time treatment): (1) no treatment (control); (2) 100 μg of IR700-YP7 iv., NIR light exposure at 50 J/cm2 on day 1 after injection (PIT × 1); (3) no PIT, but nab-paclitaxel (7.5 mg) iv. on day 1 (Abrax only × 1); (4) PIT × 1, followed by nab-paclitaxel (7.5 mg) iv. 1 h after light exposure (PIT + Abrax × 1). Study 2 groups include (n ≥ 10; repeated treatment) (1) no treatment (control); (2) 100 μg of IR700-YP7 iv., no NIR light exposure, no nab-paclitaxel (Ab only); (3) no antibody-photosensitizer conjugate, NIR light exposure at 50 J/cm2 on day 1 and 100 J/cm2 on days 2 and 3, no nab-paclitaxel (light only); (4) 100 μg of IR700-YP7 iv., NIR light exposure at 50 J/cm2 day 1 after injection and 100 J/cm2 on day 2 and day 3 after injection, no nab-paclitaxel (PIT); (5) no PIT, nab-paclitaxel (7.5 mg) iv. on days 1, 2, and 3 (Abrax only); (6) PIT, followed by nab-paclitaxel (7.5 mg) iv. 1 h after each light exposure (PIT + Abrax)

Ab: Antibody; iv.: Intravenous(ly); J: J/cm2 ; NIR: Near infrared; PIT: Photoimmunotherapy.

Study 1 (one time treatment) consisted of the following groups: (1) no treatment (control); (2) 100 μg of IR700-YP7 iv., NIR light exposure at 50 J/cm2 on day 1 after injection (PIT × 1); (3) no PIT, but nab-paclitaxel (7.5 mg) iv. on day 1 (Abrax only × 1) and (4) PIT × 1, followed by nab-paclitaxel (7.5 mg) iv. 1 h after light exposure (PIT + Abrax × 1).

Study 2 (repeated treatment) consisted of the following groups: (1) no treatment (control); (2) 100 μg of IR700-YP7 iv., no NIR light exposure, no nab-paclitaxel (Ab only); (3) no APC, NIR light exposure at 50 J/cm2 on day 1 and 100 J/cm2 on day 2 and day 3, no nab-paclitaxel (light only); (4) 100 μg of IR700-YP7 iv., NIR light exposure at 50 J/cm2 on day 1 after injection and 100 J/cm2 on day 2 and day 3 after injection, no nab-paclitaxel (PIT); (5) no PIT, nab-paclitaxel (7.5 mg) iv. on day 1, 2, and 3 (Abrax only); (6) PIT, followed by nab-paclitaxel (7.5 mg) iv. 1 h after each light exposure (PIT + Abrax).

Mice were randomly assigned to each treatment group (at least ten mice per group). Mice were monitored daily, and tumor volumes were measured three times a week until the tumor volume reached 2000 mm3, whereupon the mice were euthanized with carbon dioxide.

Statistical analysis

Data are expressed as means ± standard error of the mean from a minimum of four experiments, unless otherwise indicated. Statistical analysis was performed using the unpaired t-test for comparing differences between two groups and using one-way ANOVA followed by Tukey’s honestly significant difference (HSD) test for comparing differences between multiple groups. The cumulative probability of survival was estimated in each group with the use of Kaplan–Meier survival curve analysis, and the results were compared with the log-rank test. Differences were considered statistically significant when p values were less than 0.05.

Results

In vitro evaluation of IR700-YP7

High and specific binding of 125I-YP7 to A431/G1 cells was observed. The GPC3 expression level was calculated with Scatchard plot analysis to be about 500,000 receptors per cell (Supplementary Figure 1, see online at http://www.futuremedicine.com/doi/full/10.2217/NNM.14.194). Flow cytometry showed that IR700-YP7 bound to A431/G1 cells by 1 h of incubation, which increased at 8 h (Figure 2A). Fluorescence microscopy showed that IR700-YP7 localized both on the surface and within the cell. When these cells were observed during continuous light exposure, almost immediate swelling, budding and rupture of the lysosome was observed (Figure 2B), as observed previously [6]. Small populations of cell death were observed on A431/G1 cells without NIR light exposure due to technical difficulty to detaching cells from the culture dish, however, the ratio of dead/live cells was greatly enhanced by NIR light exposure (Figure 2D). A small proportion of cell death was also observed in A431/G1 cells with NIR light exposure reflecting the low binding of IR700-YP7 (Figure 2C & D). These results indicated that PIT with IR700-YP7 induced highly target-specific cytotoxicity.

Figure 2. In vitro evaluation of IR700-YP7 with GPC-3 positive A431/G1 cells.

Figure 2

Open in a new tab

(A) Histograms for flow cytometry of A431/G1 cells at 1 h or 8 h after incubation with IR700-YP7. IR700-YP7 bound to A431/G1 cells by 1 h of incubation, which increased at 8 h. (B) Serial DIC (upper row) and fluorescence microscopy (lower row) images before or after NIR light irradiation. IR700-YP7 localized both on the surface and within the cell. Necrotic cell death was observed upon excitation with NIR light. Flow cytometry analysis of (C) IR700-YP7 binding and (D) propidium iodide staining after irradiation with NIR light at 16 J/cm2 of A431 cells and A431/G1 cells. Binding of IR700-YP7 to A431 cells was much lower than that of A431/G1 cells. Propidium iodide-positive dead cells were observed in A431/G1 cells without NIR light exposure due to detachment of cells from the culture dish, however, the ratio of dead cells was greatly increased by NIR light exposure. A small proportion of cell death was also observed on A431 cells with NIR light exposure that reflects low binding of IR700-YP7.

DIC: Differential interference contrast; NIR: Near infrared; PIT: Photoimmunotherapy.

In vivo imaging & therapeutic studies

A431/G1 tumors were visualized 1 day after intravenous injection of IR700-YP7 (Figure 3A) and the target-to-background ratio (TBR) of IR700 fluorescence intensity was 1.74 ± 0.11 (Figure 3B). The fluorescence intensity in the tumor and TBR decreased to background level immediately after NIR light irradiation which is likely caused by a combination of wash out of IR700-YP7 from dead cells and photo-bleaching of IR700. However, circulating IR700-YP7 reaccumulated in the tumor bed within a day, leading to a return in fluorescence within the tumor by the time of the second NIR light irradiation. These results indicated that IR700-YP7 accumulated in the surviving cancer cells even after the initial and second treatment with PIT, and therefore, enabling the second and third exposure of NIR light irradiation to be effective.

Figure 3. In vivo fluorescence imaging of tumor-bearing mice.

Figure 3

Open in a new tab

(A) A typical IR700 fluorescence image and (B) the TBR of fluorescence intensity in A431/G1 tumors over time during IR700-YP7 PIT. Images were obtained before and immediately after each NIR irradiation (1 day, 2 days and 3 days after IR700-YP7 injection). Yellow arrows show tumor. TBR data represents means ± standard error of the mean of nine mice. A431/G1 tumors were visualized 1 day after injection of IR700-YP7. The fluorescence intensity in the tumor and TBR decreased to background level immediately after NIR light irradiation, however circulating IR700-YP7 reaccumulated in the tumor bed within a day. (C) Dynamic images and (D) serial TBR of A431/G1 tumors over time of IR800-nab-paclitaxel after IR700-YP7-mediated PIT or NIR light exposure only. Rapid and high accumulation and high TBR of IR800-nab-paclitaxel was observed in acutely treated (PIT) tumor vs untreated tumor.

iv.: Intravenous(ly); J: J/cm2; NIR: Near infrared; PIT: Photoimmunotherapy; TBR: Tumor to background ratio.

Rapid accumulation of IR800-nab-paclitaxel was observed in acutely treated (PIT) animals. The accumulation of IR800-nab-paclitaxel enabled the tumor to be clearly visualized within 60 min after injection in PIT treated animals (Figure 3C) and the TBR of IR800 fluorescence intensity was more than three at 60 min after injection (Figure 3D). In control tumors, not undergoing PIT, uptake of IR800-nab-paclitaxel was minimal at 60 min after injection. Thus, the fluorescence intensity in PIT-treated tumors and TBR at 4 h and 24 h after injection of IR800-nab-paclitaxel was much higher than that in the control tumor. This indicated that PIT with IR700-YP7 can dramatically increase the leakage of nanosized drugs such as nabpaclitaxel into the tumor bed, which should result in increased drug efficacy.

The results of therapeutic study 1 are shown in Figure 4. No significant therapeutic effect was observed in mice after only a single exposure of PIT using 100 μg of IR700-YP7 and 50 J/cm2 of NIR compared with untreated control mice (p > 0.05) (Figure 4A). Tumor inhibition was increased by the addition of 7.5 mg of nab-paclitaxel treatment compared with untreated control mice (p < 0.01). Combinations of single-shot PIT with 7.5 mg of nab-paclitaxel showed significantly prolonged survival compared with mice treated with nab-paclitaxel only (p < 0.05) (Figure 4B).

Figure 4. Therapeutic effect of 1-time treatment (study 1).

Figure 4

Open in a new tab

(A) Tumor growth inhibition by PIT and nab-paclitaxel in A431/G1 tumors. Data are means ± standard error of the mean. (more than ten mice in each group). Tumor inhibition was increased by the addition of 7.5 mg of nab-paclitaxel treatment compared to untreated control mice (*p < 0.01). (B) Analysis using a Kaplan–Meier survival curve of the PIT and nab-paclitaxel in A431/G1 tumors (more than ten mice in each group). Combinations of single-shot PIT with 7.5 mg of nab-paclitaxel showed significantly prolonged survival compared to mice treated with nab-paclitaxel only (#p < 0.05).

PIT: Photoimmunotherapy.

The results of therapeutic study 2 with an optimized regimen of PIT and nab-paclitaxel are shown in Figure 5. Tumor growth was significantly inhibited in PIT treated mice using the three NIR light exposure regimens (50, 100, 100 J/cm2 on 3 consecutive days) compared with untreated control mice (Figure 5A). No significant therapeutic effect was observed in mice receiving IR700-YP7 alone or receiving light only. Tumor growth was inhibited by nab-paclitaxel only or in combination with PIT compared with untreated control mice (p < 0.01, after day 4). However, both tumor inhibition and survival were significantly improved in mice treated with the combination of PIT and nab-paclitaxel compared with mice treated with nab-paclitaxel alone (p < 0.05) (Figure 5B). Therefore, the therapeutic effects of nab-paclitaxel were significantly enhanced by prior PIT.

Figure 5. Therapeutic effect of repeated photoimmunotherapy (study 2).

Figure 5

Open in a new tab

(A) Tumor growth inhibition by PIT and nab-paclitaxel in A431/G1 tumors. Data are means ± standard error of the mean (more than ten mice in each group). Tumor growth was significantly inhibited in PIT treated mice compared to untreated control mice (*p < 0.05). Tumor inhibition was significantly improved in mice treated with the combination of PIT and nab-paclitaxel compared to mice treated with nab-paclitaxel only (#p < 0.05). (B) Analysis using a Kaplan–Meier survival curve of the PIT and nab-paclitaxel in A431/G1 tumors (ten mice in each group). Survival was significantly improved in mice treated with the combination of PIT and nab-paclitaxel compared to mice treated with nab-paclitaxel only (#p < 0.05).

PIT: Photoimmunotherapy.

Discussion

PIT is a highly selective treatment for killing cancer cells which depends on target-specific binding of an APC and exposure to NIR light. In the case of HCC, GPC3 is considered an attractive target for HCC based on its high expression levels and high tumor to background ratios. High affinity anti-GPC3 antibodies were developed by Ho et al. and one of these, YP7 was reported to exhibit high affinity and specific binding for GPC3-expressed tumor cells [15,17]. As shown in Figure 2, IR700-labeled YP7 bound to the GPC3-positive tumor cell line A431/G1, resulted in rapid target-specific necrotic cell death after exposure to NIR light. Furthermore, IR700-YP7 with PIT showed tumor growth inhibition in vivo. Thus, IR700-YP7 is a potential PIT agent for HCC treatment.

An obvious challenge for PIT in HCC is the limited light penetration of NIR within the liver. We estimate that NIR light can penetrate approximately 2 cm from the light source in vivo and still be effective for PIT. Thus, in order to treat HCC with PIT, it will be necessary to introduce a light fiber into the tumor via a percutaneous hollow needle through which the light fiber can enter the tumor. We envision that NIR light would be delivered with a needle-based light diffuser under imaging guidance [18]. Larger tumors may require several needles to create overlapping regions of treatment. This is not an uncommon practice in the treatment of HCC. Many other minimally invasive treatments for HCC exist including percutaneous ethanol injection, radiofrequency ablation and transarterial chemoembolization. All of these have the disadvantage that they indiscriminately kill both tumor and normal cells in a predictable pattern around the needle or catheter tip. The advantage of PIT over the other methods mentioned is that it would selectively kill cancer cells but preserve normal hepatic cells which do not express GPC3.

Our previous data demonstrates that PIT is more effective when there are repeated NIR light exposures, even with a single injection of the APC [16]. In this study, since YP7 is a mouse antibody injected into mice, blood clearance was slow. Thus, we determined that it should be possible to irradiate the tumor at least three times (24 h, 48 h and 72 h) after a single IR700-YP7 injection since there would be considerable amounts of circulating APC even 3 days after injection. Although PIT resulted in loss of fluorescence, reaccumulation of fluorescence was observed within 24 h, and therefore, it was assumed that repeated exposure to NIR light would lead to additional tumor killing. In fact, mice exposed to NIR light three times on consecutive days showed a distinctly better outcome than mice exposed to only one dose of NIR light.

An important aspect of PIT is that there are rapid and dramatic increases in vascular permeability to nanosized molecules after NIR light exposure, an effect that has been termed, ‘super enhanced permeability and retention’ (SUPR) [8]. Nanosized drugs rely on conventional enhanced permeability and retention to ‘selectively’ accumulate in tumors, however, this effect is modest and relatively large doses must be given to the patient to achieve responses. In contrast, the PIT-induced SUPR effect enhances the delivery of nanosized agents by more than tenfold early after PIT. In this study, rapid and high accumulation of IR800-nab-paclitaxel was observed in treated tumors versus untreated tumors, when it was injected immediately after PIT. Furthermore, combination therapy of PIT and nab-paclitaxel showed at least additive therapeutic effects. Since the PIT-induced SUPR effect allows anticancer reagents to penetrate more rapidly and deeply into a tumor, relatively high concentrations of anticancer drugs with homogeneous distribution can be achieved. Therefore, the PIT-induced SUPR effect makes PIT an ideal treatment for combinatorial cancer therapy with nanosized anticancer agents.

Conclusion

IR700-YP7 bound to the GPC3-positive tumor cell line, A431/G1, followed by NIR light exposure led to target-specific necrotic cell death in vitro and in vivo. PIT with IR700-YP7 showed a therapeutic effect and led to increases in the concentration of the nanodrug, nab-paclitaxel, within the tumor. These findings indicate that PIT using IR700-YP7 may be useful for the focal treatment of HCC.

Future perspective

PIT is a new, promising method of cancer therapy because of its high specificity to target-expressing cancer cells. As shown in this and previous studies, PIT is widely applicable to variety of cancers. A first in human clinical trial of PIT is ready and will start in late 2014. Thus, PIT would become a commonly used cancer therapy in clinical practice for a wide variety of cancer in near future. Furthermore, since PIT induced highly enhanced nanodrug delivery due to the PIT-induced SUPR effect, nanosized drugs combined with PIT would be an attractive cancer nanotherapy in future.

Supplementary Material

Executive summary.

In vitro evaluation of IR700-YP7

  • IR700-labeled YP7 bound to the GPC3-positive tumor cell line A431/G1, resulted in rapid target-specific necrotic cell death after exposure to near-infrared light.

Therapeutic effect of PIT with IR700-YP7

  • IR700-YP7 accumulated in the tumor, and tumor growth was significantly inhibited in photoimmunotherapy (PIT) treated A431/G1 tumor-bearing mice compared with untreated control mice.

Improvement nanosized drug delivery & efficacy by PIT with IR700-YP7

  • Rapid accumulation of the nanosized drug, nanoparticle albumin-bound paclitaxel (nab-paclitaxel) was observed in the PIT-treated tumors.

  • The combination of PIT and nab-paclitaxel significantly improved both tumor inhibition and survival in tumor-bearing mice compared with mice treated with nab-paclitaxel only.

Conclusion

  • These findings indicate that PIT with IR700-YP7 is a promising agent for the treatment of hepatocellular carcinoma.

Acknowledgments

This research was supported by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research. KS is supported with JSPS Research Fellowship for Japanese Biomedical and Behavioral Researchers at NIH. In addition, this project has been funded in whole or in part with Federal funds from the National Cancer Institute, NIH, under contract no. HHSN261200800001E.

Footnotes

Disclaimer

The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the US Government.

Financial & competing interests disclosure

The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

No writing assistance was utilized in the production of this manuscript.

Ethical conduct of research

The authors state that they have obtained appropriate institutional review board approval or have followed the principles outlined in the Declaration of Helsinki for all human or animal experimental investigations. In addition, for investigations involving human subjects, informed consent has been obtained from the participants involved.

References

Papers of special note have been highlighted as:

• of interest; •• of considerable interest

  • 1.Roxburgh P, Evans TRJ. Systemic therapy of hepatocellular carcinoma: are we making progress? Adv. Ther. 2008;25(11):1089–1104. doi: 10.1007/s12325-008-0113-z. [DOI] [PubMed] [Google Scholar]
  • 2.Llovet JM, Burroughs A, Bruix J. Hepatocellular carcinoma. Lancet. 2003;362(9399):1907–1917. doi: 10.1016/S0140-6736(03)14964-1. [DOI] [PubMed] [Google Scholar]
  • 3.Forner A, Llovet JM, Bruix J. Hepatocellular carcinoma. Lancet. 2012;379(9822):1245–1255. doi: 10.1016/S0140-6736(11)61347-0. [DOI] [PubMed] [Google Scholar]
  • 4.Huynh H. Molecularly targeted therapy in hepatocellular carcinoma. Biochem. Pharmacol. 2010;80(5):550–560. doi: 10.1016/j.bcp.2010.03.034. [DOI] [PubMed] [Google Scholar]
  • 5.Finn RS. Development of molecularly targeted therapies in hepatocellular carcinoma: where do we go now? Clin. Cancer Res. 2010;16(2):390–397. doi: 10.1158/1078-0432.CCR-09-2084. [DOI] [PubMed] [Google Scholar]
  • 6.Mitsunaga M, Ogawa M, Kosaka N, Rosenblum LT, Choyke PL, Kobayashi H. Cancer cell-selective in vivo near infrared photoimmunotherapy targeting specific membrane molecules. Nat. Med. 2011;17(12):1685–1691. doi: 10.1038/nm.2554. • First report revealed the usefulness of photoimmunotherapy with IR700-labeled antibody. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Shirasu N, Yamada H, Shibaguchi H, Kuroki M. Potent and specific antitumor effect of CEA-targeted photoimmunotherapy. Int. J. Cancer. 2014;135(11):2697–2710. doi: 10.1002/ijc.28907. [DOI] [PubMed] [Google Scholar]
  • 8.Sano K, Nakajima T, Choyke PL, Kobayashi H. Markedly enhanced permeability and retention effects induced by photo-immunotherapy of tumors. ACS Nano. 2013;7(1):717–724. doi: 10.1021/nn305011p. •• First report revealed that photoimmunotherapy induces the super enhanced permeability and retention effect. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Zhu ZW, Friess H, Wang L, et al. Enhanced glypican-3 expression differentiates the majority of hepatocellular carcinomas from benign hepatic disorders. Gut. 2001;48(4):558–564. doi: 10.1136/gut.48.4.558. • Reported that Glypican-3 represents is highly expressed in hepatocellular carcinoma but not in normal tissue. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Baumhoer D, Tornillo L, Stadlmann S, Roncalli M, Diamantis EK, Terracciano LM. Glypican 3 expression in human nonneoplastic, preneoplastic, and neoplastic tissues: a tissue microarray analysis of 4,387 tissue samples. Am. J. Clin. Pathol. 2008;129(6):899–906. doi: 10.1309/HCQWPWD50XHD2DW6. [DOI] [PubMed] [Google Scholar]
  • 11.Capurro M, Wanless IR, Sherman M, et al. Glypican-3: a novel serum and histochemical marker for hepatocellular carcinoma. Gastroenterology. 2003;125(1):89–97. doi: 10.1016/s0016-5085(03)00689-9. [DOI] [PubMed] [Google Scholar]
  • 12.Filmus J, Capurro M. Glypican-3 and alphafetoprotein as diagnostic tests for hepatocellular carcinoma. Mol. Diagn. 2004;8(4):207–212. doi: 10.1007/BF03260065. [DOI] [PubMed] [Google Scholar]
  • 13.Ho M, Kim H. Glypican-3: a new target for cancer immunotherapy. Eur. J. Cancer. 2011;47(3):333–338. doi: 10.1016/j.ejca.2010.10.024. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Filmus J, Capurro M. Glypican-3: a marker and a therapeutic target in hepatocellular carcinoma. FEBS J. 2013;280(10):2471–2476. doi: 10.1111/febs.12126. [DOI] [PubMed] [Google Scholar]
  • 15.Phung Y, Gao W, Man YG, Nagata S, Ho M. High-affinity monoclonal antibodies to cell surface tumor antigen glypican-3 generated through a combination of peptide immunization and flow cytometry screening. mAbs. 2012;4(5):592–599. doi: 10.4161/mabs.20933. • Established anti-Glypican-3 mouse monoclonal antibody YP7 and A431/G1, a cell line stably expressing human GPC3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Mitsunaga M, Nakajima T, Sano K, Choyke PL, Kobayashi H. Near-infrared theranostic photoimmunotherapy (PIT): repeated exposure of light enhances the effect of immunoconjugate. Bioconjug. Chem. 2012;23(3):604–609. doi: 10.1021/bc200648m. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Feng M, Ho M. Glypican-3 antibodies: a new therapeutic target for liver cancer. FEBS Lett. 2014;588(2):377–382. doi: 10.1016/j.febslet.2013.10.002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Kosaka N, Mitsunaga M, Longmire MR, Choyke PL, Kobayashi H. Near infrared fluorescence-guided real-time endoscopic detection of peritoneal ovarian cancer nodules using intravenously injected indocyanine green. Int. J. Cancer. 2011;129(7):1671–1677. doi: 10.1002/ijc.26113. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

NIHMS685057-supplement-01.pdf (77.2KB, pdf)

RESOURCES