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Published in final edited form as: Bioconjug Chem. 2017 Apr 26;28(5):1458–1469. doi: 10.1021/acs.bioconjchem.7b00144

Near-Infrared Photochemoimmunotherapy by Photoactivatable Bifunctional Antibody–Drug Conjugates Targeting Human Epidermal Growth Factor Receptor 2 Positive Cancer

Kimihiro Ito †, Makoto Mitsunaga *, Takashi Nishimura, Masayuki Saruta, Takeo Iwamoto ‡, Hisataka Kobayashi §, Hisao Tajiri †
PMCID: PMC7920516  NIHMSID: NIHMS1664788  PMID: 28402624

Abstract

Near-infrared photoimmunotherapy (NIR-PIT) is a new class of molecular targeted cancer therapy based on antibody–photoabsorber conjugates and NIR light irradiation. Recent studies have shown effective tumor control, including that of human epidermal growth factor receptor 2 (HER2)-positive cancer, by selective molecular targeting with NIR-PIT. However, the depth of NIR light penetration limits its use. Trastuzumab emtansine (T–DM1) is an antibody–drug conjugate consisting of the monoclonal antibody trastuzumab linked to the cytotoxic agent maytansinoid DM1. Here, we developed bifunctional antibody–drug–photoabsorber conjugates, T–DM1–IR700, that can work as both NIR-PIT and chemoimmunotherapy agents. We evaluated the feasibility of T–DM1–IR700-mediated NIR light irradiation by comparing the in vitro and in vivo cytotoxic efficacy of trastuzumab–IR700 (T–IR700)-mediated NIR light irradiation in HER2-expressing cells. T–IR700 and T–DM1–IR700 showed almost identical binding to HER2 in vitro and in vivo. Owing to the presence of internalized DM1 in the target cells, NIR-PIT using T–DM1–IR700 tended to induce greater cytotoxicity than that of NIR-PIT using T–IR700 in vitro. In vivo NIR-PIT using T–DM1–IR700 did not show a superior antitumor effect to NIR-PIT using T–IR700 in subcutaneous small-tumor models, which could receive sufficient NIR light. In contrast, NIR-PIT using T–DM1–IR700 tended to reduce the tumor volume and showed significant prolonged survival compared to NIR-PIT using T–IR700 in large-tumor models that could not receive sufficient NIR light. We successfully developed a T–DM1–IR700 conjugate that has a similar immunoreactivity to the parental antibody with increased cytotoxicity due to DM1 and potential as a new NIR-PIT agent for targeting tumors that are large and inaccessible to sufficient NIR light irradiation to activate the photoabsorber IR700.

Graphical Abstract

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INTRODUCTION

Human epidermal growth factor receptor 2 (HER2) is a member of the epidermal growth factor receptor family, which regulates cell proliferation, differentiation, and apoptosis through signal transduction by forming homodimers or heterodimers.1 HER2 is commonly expressed on the cell membrane of various types of cancers, and its overexpression is associated with tumor malignancy.2 Trastuzumab, a humanized monoclonal antibody that targets HER2, manifests its antitumor activity by inducing antibody-dependent cellular cytotoxicity, inhibiting ligand-independent HER2 signaling, blocking the active formation of HER2 and preventing the cleavage of the extracellular domain of HER2.3,4 Although trastuzumab is widely used for treating HER2-expressing cancers, its therapeutic effect is rarely curative when it is used as a single agent; therefore, it is mainly used in combination with chemotherapy.5,6 Trastuzumab emtansine (trastuzumab–DM1; T–DM1) is a recently developed antibody-drug conjugate (ADC) composed of a highly potent cytotoxic drug, DM1 derived from maytansine, connected to trastuzumab via a nonreducible thioether linker. In addition to retaining all the mechanisms of action of native unconjugated trastuzumab, T–DM1 also has HER2-targeted cytotoxicity, which depends on DM1.7–9 On binding to HER2, T–DM1 undergoes internalization and lysosomal degradation. This process induces the intracellular release of DM1-containing catabolites, which bind to tubulin and prevent microtubule assembly, resulting in mitotic arrest, cell growth inhibition, and cell death.10,11

Near-infrared photoimmunotherapy (NIR-PIT) is a new class of molecular targeted cancer therapy based on an antibody–photoabsorber conjugate (APC) and NIR light irradiation. A photoabsorbing phthalocyanine dye, IR700, which is conjugated with antibody, induces selective cytotoxicity only to APC-bound cells only when excited by NIR light at a specific wavelength of 690 nm. The APC shows similar immunoreactivity to that of the native unconjugated antibody, resulting in highly selective binding to the target molecules on the cell membrane, rapidly inducing membrane rupture and cellular necrosis by the photo-activated IR700 after NIR light exposure without cytotoxic effects toward nonexpressing cells.12–14 NIR-PIT using trastuzumab–IR700 (T–IR700) conjugates has been shown to cause HER2-targeted phototoxicity in various HER2-expressing cancer mouse models, leading to strong antitumor effects.15–20 However, some cancer cells were found to survive and tumor recurrences were eventually seen in mouse models after a single NIR-PIT treatment. Thus, it is necessary to develop a new method for enhancing the effectiveness of NIR-PIT treatment.

Here, we developed an antibody–drug–photoabsorber conjugate (ADPC), trastuzumab–DM1–IR700 (T–DM1–IR700), which has potential applications in both in NIR-PIT and chemoimmunotherapy. We assumed that NIR-PIT using T–DM1–IR700 is more useful than NIR-PIT using T–IR700 by increased cytotoxicity due to DM1. Therefore, we compared the in vitro and in vivo cytotoxic efficacy of NIR-PIT for HER2-expressing cells using T–IR700 or T–DM1–IR700 and evaluated the utility of T–DM1–IR700 as a new agent for NIR-photochemoimmunotherapy.

RESULTS

In Vitro Characterization of T–IR700 and T–DM1–IR700 Conjugates.

On the basis of the concentrations of trastuzumab and IR700 determined by spectrometry, the conjugates T–IR700 and T–DM1–IR700 were synthesized by covalently conjugating approximately three IR700 molecules each to trastuzumab and T–DM1, respectively. We also examined IR700 conjugation to trastuzumab and T–DM1 by mass spectrometry. Liquid chromatography–electrospray ionization mass spectrometry analyses showed that the peak of the molecular weights of trastuzumab, T–IR700, T–DM1, and T–DM1–IR700 were approximately 148 000, 150 000–156 000, 148 000–152 000, and 152 000–159 000, respectively (Figure S1). Schematic structures of T–IR700 and T–DM1–IR700 are shown in Figure 1.

Figure 1.

Figure 1.

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Schematic structures of T–IR700 and T–DM1–IR700. T–DM1 contains an average of 3–3.5 DM1 molecules linked to trastuzumab via a nonreducible thioether linker (MCC linker). An average of three IR700 molecules are covalently conjugated to trastuzumab and T–DM1 each. MW: molecular weight.

HER2 Expression in Vitro.

After 3 h of incubation with 10 μg/mL T–IR700 or 10 μg/mL T–DM1–IR700, 3T3/HER2 cells showed strong IR700 fluorescence, and these signals were almost completely blocked by the addition of excess unconjugated trastuzumab, suggesting HER2-specific binding of T–IR700 and T–DM1–IR700 (Figure 2A). The ratios of the mean fluorescence intensities (MFIs) compared to that of the isotype control were 248.9 ± 3.4 for T–IR700, 258.0 ± 3.8 for T–DM1–IR700, 5.1 ± 0.1 for T–IR700 with trastuzumab blocking, and 4.9 ± 0.2 for T–DM1–IR700 with trastuzumab blocking, respectively (means ± standard error of the mean [SEM], n = 3). Similar to 3T3/HER2 cells, HCC-1419 cells also showed strong IR700 fluorescence with T–IR700 or T–DM1–IR700, and these signals were blocked by the addition of excess unconjugated trastuzumab (Figure 2B). MFI ratios (treatment/isotype control) were 91.2 ± 3.5 for T–IR700, 90.9 ± 3.0 for T–DM1–IR700, 1.9 ± 0.4 for T–IR700 with trastuzumab blocking, and 4.2 ± 0.8 for T–DM1–IR700 with trastuzumab blocking (means ± SEM, n = 3). In contrast, there were no significant differences in signal intensity between T–IR700 or T–DM1–IR700 treatment and the isotype control in HER2-negative BALB/3T3 cells (Figure 2C).

Figure 2.

Figure 2.

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Expression of human epidermal growth factor receptor 2 and the dose–response binding of T–IR700 and T–DM1–IR700 in vitro. (A–C) Flow cytometry analysis revealed strong human epidermal growth factor receptor 2 (HER2)-specific binding of T–IR700 and T–DM1–IR700 in 3T3/HER2 and HCC-1419 cells but not in NIH/3T3 cells after 3 h of incubation with 10 μg/mL T–IR700 or 10 μg/mL T–DM1–IR700. Specific binding was demonstrated by excess unconjugated trastuzumab blocking. (D) Flow cytometry analysis was performed after 24 h of incubation with several concentrations of T–IR700 or T–DM1–IR700. T–DM1–IR700 and T–IR700 bound to HER2-positive cells in a dose-dependent manner at equal levels. Data are presented as means ± standard error of the mean (SEM; n = 3).

Similar HER2-Specific Binding Capabilities of T–IR700 and T–DM1–IR700 in Vitro.

When cells were exposed to T–IR700 or T–DM1–IR700 for 24 h, IR700 signals increased in a dose-dependent manner but were almost saturated by treatment with 3 μg/mL of IR700 conjugates in both 3T3/HER2 and HCC-1419 cells (Figure 2D). Therefore, we considered 3 μg/mL of IR700 conjugates as the treatment dose for the in vitro study. Despite being HER2-negative, BALB/3T3 cells showed weak IR700 signals after 24 h of exposure to 10 and 30 μg/mL of the IR700 conjugates. These signals were not blocked by the addition of excess unconjugated trastuzumab; therefore, we considered them as nonspecific signals (Figure S2).

Fluorescence Microscopy.

To detect the time-lapse co-localization of T–IR700 or T–DM1–IR700, fluorescence microscopy was performed. IR700 fluorescence signals were detected predominantly on the cell surface of 3T3/HER2 and HCC-1419 cells after 3 h of incubation with T–IR700 or T–DM1–IR700, whereas only partial subcellular localization was observed. Trastuzumab–photoabsorber conjugate showed gradual internalization into the cytoplasm of HER2-positive cells in earlier studies.12,21 Consistent with those studies, when cells were incubated with T–IR700 or T–DM1–IR700 for 24 h, subcellular localization pattern of IR700 was not different between 3T3/HER2 and HCC-1419 cells, indicating similar levels of internalization of T–IR700 and T–DM1–IR700 (Figure 3A,B). In contrast, HER2-negative BALB/3T3 cells did not show any detectable fluorescence for IR700 under the same camera conditions after treatment with T–IR700 or T–DM1–IR700 (Figure 3C).

Figure 3.

Figure 3.

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Fluorescence microscopy showing HER2-specific binding of T–IR700 and T–DM1–IR700 in 3T3/HER2 and HCC-1419 cells but not in BALB/3T3 cells. IR700 fluorescence signals were detected mainly on the cell surface after 3 h of incubation and were gradually internalized. (A) 3T3/HER2, (B) HCC-1419, and (C) BALB/3T3 cells. DIC: differential interference contrast. Scale bar: 30 μm.

Higher Cytotoxicity of T–DM1–IR700-Mediated NIR Light Irradiation Than That of T–IR700-Mediated NIR Light Irradiation in Vitro.

A LIVE/DEAD assay showed that the percentage of cell death by T–DM1–IR700 single treatment was significantly higher than that in the untreated control and by T–IR700 single treatment in HER2-positive 3T3/HER2 and HCC-1419 cells, whereas cytotoxicity of T–IR700 single treatment and the untreated control did not differ significantly (Figure 4A,B). There was no difference in cytotoxicity between untreated control and 1 J/cm2 of NIR light irradiation (without mAbs) in each cell line (Figure S3A). When cells were treated with either T–IR700 plus NIR light or T–DM1–IR700 plus NIR light, the percentage of cell death increased in a NIR-light dose-dependent manner. In addition, cytotoxicity differed significantly between T–IR700 single treatment and T–IR700 plus NIR light treatment, as well as between T–DM1–IR700 single treatment and T–DM1–IR700 plus NIR light treatment. Furthermore, the percentage of cell death by T–DM1–IR700 plus NIR light treatment was significantly higher than that by T–IR700 plus NIR light treatment especially in low NIR-light doses (Figure 4A,B). In contrast, no cytotoxicity associated with T–IR700 single treatment, T–DM1–IR700 single treatment, T–IR700 plus NIR light treatment, or T–DM1–IR700 plus NIR light treatment was detected in HER2-negative BALB/3T3 cells (Figure 4C).

Figure 4.

Figure 4.

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T–DM1–IR700-mediated NIR light irradiation induced greater cytotoxicity in vitro. (A–C) A LIVE/DEAD assay in HER2-positive 3T3/HER2 and HCC-1419 cells showed that the percentage of cell death by T–DM1–IR700 plus NIR light treatment was significantly higher compared to that by T–IR700 plus NIR light treatment, especially in low-NIR light doses. No cytotoxicity associated with T–IR700 or T–DM1–IR700 single treatment or plus NIR light irradiation was found in HER2-negative BALB/3T3 cells. Data are presented as means ± SEM (n = 3; *, P < 0.05 and **, P < 0.01, Student t-test). (D–F) A LDH cytotoxicity assay showed that T–DM1–IR700 plus NIR light treatment induced greater cytotoxicity than that by T–IR700 plus NIR light treatment in both 3T3/HER2 and HCC-1419 cells. No cytotoxicity associated with T–IR700 or T–DM1–IR700 single treatment or plus NIR light irradiation was found in BALB/3T3 cells. Data are presented as means ± SEM (n = 3; *, P < 0.05 and **, P < 0.01, Student t-test).

A LDH cytotoxicity assay showed that T–DM1–IR700 single treatment resulted in greater cytotoxicity than that by T–IR700 or T–DM1–IR700 single treatment in 3T3/HER2 cells (Figure 4D). When cells were treated with either T–IR700 plus NIR light or T–DM1–IR700 plus NIR light, cytotoxicity increased in a NIR-light dose-dependent manner. Furthermore, T–DM1–IR700 plus NIR light treatment resulted in greater cytotoxicity than that by T–IR700 plus NIR light treatment (Figure 4D). Similar cytotoxicity was detected in HCC-1419 cells treated with 1 J/cm2 of NIR light irradiation (Figure 4E). In contrast, no cytotoxicity associated with T–IR700 or T–DM1–IR700 single treatment or plus NIR light irradiation was detected in BALB/3T3 cells (Figure 4F)

To compare the long-term cytotoxic effects of T–IR700 plus NIR light or T–DM1–IR700 plus NIR light treatment, a trypan blue dye exclusion assay and microscopic observation were performed. As shown in Figure 5A, T–DM1–IR700 single treatment resulted in significant growth inhibition compared to that in the untreated control or that by T–IR700 single treatment in 3T3/HER2 cells, whereas there was no significant difference in growth inhibition between T–IR700 single treatment and the untreated control. There was no difference in long-term growth inhibition between untreated control and 1 J/cm2 of NIR light irradiation in both cell lines (Figure S3B,C). T–DM1 and T–IR700 plus NIR light treatment could not enhance cytotoxicity compared with T–DM1–IR700 plus NIR light treatment or T–IR700 plus NIR light treatment, which is caused by competition between T–DM1 and T–IR700 (Figure S4). Long-term growth inhibition was apparent after T–IR700 plus NIR light or T–DM1–IR700 plus NIR light treatment. Importantly, the growth inhibition was more prominent by T–DM1–IR700 plus NIR light treatment than that by T–DM1–IR700 single treatment or T–IR700 plus NIR light treatment. In contrast, no cytotoxicity associated with T–IR700 or T–DM1–IR700 single treatment or plus NIR light irradiation was observed in BALB/3T3 cells (Figure 5B).

Figure 5.

Figure 5.

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Long-term growth inhibition assay and morphological changes in response to T–DM1–IR700-mediated NIR light irradiation in vitro. (A) A trypan blue dye exclusion assay showed significant growth inhibition by T–DM1–IR700 plus NIR light treatment compared to that with T–DM1–IR700 single treatment or T–IR700 plus NIR light treatment in 3T3/HER2 cells. Data are presented as means ± SEM (n = 3; **, P < 0.01 and ***, P < 0.001, Student t-test). (B) No cytotoxicity associated with T–IR700 or T–DM1–IR700 single treatment or plus NIR light irradiation was observed in BALB/3T3 cells. (C) Microscopic changes in response to T–DM1–IR700 plus NIR light treatment in 3T3/HER2 cells. T–DM1–IR700 single treatment induced giant cell formation and a reduction in the number of cells, while T–IR700 single treatment did not cause any visible morphological changes compared to the untreated control. T–IR700 plus NIR light treatment induced cell collapse and a reduction in the number of cells, and T–DM1–IR700 plus NIR light treatment induced giant cell formation, cell collapse, and a reduction in the number of cells. Scale bar: 100 μm.

Microscopic observation revealed that T–DM1–IR700 single treatment induced a tendency of giant cell formation and a reduction in the number of 3T3/HER2 cells, whereas T–IR700 single treatment did not cause any morphological changes or a reduction in cell number compared to the untreated control (Figure 5C). T–IR700 plus NIR light treatment induced cell collapse caused by membrane rupture and a reduction in the number of cells, and T–DM1–IR700 plus NIR light treatment induced giant cell formation, cell collapse, and a reduction in the number of cells.

In Vivo Biodistribution of T–IR700 and T–DM1–IR700.

To examine the biodistribution of T–IR700 and T–DM1–IR700 in a xenograft tumor model, serial fluorescence images were obtained before and after the injection of T–IR700 or T–DM1–IR700. IR700 fluorescence intensities of 3T3/HER2 tumors were strong and similar for T–DM1–IR700 and T–IR700 1 day after the treatment, gradually decreasing thereafter (Figure 6A,B).

Figure 6.

Figure 6.

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In vivo biodistribution of T–IR700 and T–DM1–IR700. (A) 3T3/HER2 tumor xenografts (right dorsum) visualized by IR700 fluorescence were similar after intravenous injection of T–DM1–IR700 and T–IR700. (B) Quantitative analysis showed comparable levels of IR700 fluorescence in 3T3/HER2 tumors between T–DM1–IR700 treatment and T–IR700 treatment. Data are presented as means ± SEM (n = 3).

Comparison of in Vivo Antitumor Effect between T–IR700-Mediated NIR Light Irradiation and T–DM1–IR700-Mediated NIR Light Irradiation.

In large-tumor experiments, tumor xenografts reached 200 mm3 in volume approximately 12 days after subcutaneous injection of 2 000 000 3T3/HER2 cells. Tumors were irradiated with 100 J/cm2 of NIR light twice 1 day after intravenous injection of T–IR700 or T–DM1–IR700 (day 1 and 8 after initial intravenous injection) under the guidance of IR700 fluorescence because the highest IR700 accumulation was observed at that time point. As compared with the non-NIR-PIT groups, significant antitumor effects were observed in the groups of both T–IR700 plus NIR light irradiation and T–DM1–IR700 plus NIR light irradiation (Figure 7A). Comparing the 2 NIR-PIT groups, T–DM1–IR700 plus NIR light irradiation treatment tended to reduce the tumor volume compared to T–IR700 plus NIR light treatment. In addition, T–DM1–IR700 plus NIR light treatment showed significant prolonged survival compared to T–IR700 plus NIR light treatment (Figure 7B). Pathological analysis showed that massive granulation with inflammatory changes and giant cells formation were observed in the tumor nodules treated with T–DM1–IR700 plus NIR light irradiation (Figure S5).

Figure 7.

Figure 7.

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In vivo antitumor effects of T–IR700-mediated NIR light irradiation and T–DM1–IR700-mediated NIR light irradiation. (A) Large-tumor experiments: strong antitumor effects were observed in the groups of T–IR700 plus NIR light irradiation and T–DM1–IR700 plus NIR light irradiation compared to the untreated control group. T–DM1–IR700 plus NIR light irradiation treatment tended to reduce the tumor volume compared to T–IR700 plus NIR light treatment. Data are presented as means ± SEM (n = 10 in each group, 11 days after initial treatment; ***, P = 0.001: T–IR700 plus NIR light irradiation vs untreated control; ****, P < 0.0001: T–DM1–IR700 plus NIR light irradiation vs untreated control; Mann–Whitney U test). (B) Large-tumor experiments: survival was prolonged significantly when mice were treated with T–DM1–IR700 plus NIR light irradiation compared to T–IR700 plus NIR light irradiation. (n = 10 in each group; **, P = 0.0077: T–DM1–IR700 plus NIR light irradiation vs T–IR700 plus NIR light irradiation; log-rank test). (C) Small-tumor experiments: strong antitumor effects were observed in the groups of T–IR700 plus NIR light irradiation and T–DM1–IR700 plus NIR light irradiation compared to the untreated control group. There was no difference in the tumor volume and no significant difference in survival between the 2 NIR-PIT groups. Data are presented as means ± SEM (n = 10 in each group, 11 days after initial treatment; ****, P < 0.0001: T–IR700 plus NIR light irradiation vs untreated control; ****, P < 0.0001: T–DM1–IR700 plus NIR light irradiation vs untreated control; Mann–Whitney U test). (D) Small-tumor experiments: survival was prolonged significantly when mice were treated with T–IR700 plus NIR light irradiation and T–DM1–IR700 plus NIR light irradiation. However, there was no significant difference in survival between the 2 NIR-PIT groups (n = 10 in each group; log-rank test).

In small-tumor experiments, tumor xenografts reached 30 mm3 in volume approximately 6 days after the subcutaneous injection of 1 000 000 3T3/HER2 cells. Consistent with the results of large-tumor experiments, significant antitumor effects were observed in the groups of both T–IR700 plus NIR light irradiation and T–DM1–IR700 plus NIR light irradiation compared with the non-NIR-PIT groups; however, there was no difference in the tumor volume, and there was no significant difference in survival between these 2 NIR-PIT treatment groups (Figure 7C,D).

DISCUSSION

NIR-PIT is a highly selective cancer therapy, which is based on a molecularly targeted monoclonal antibody conjugated to the photoabsorber IR700 and NIR light irradiation. In previous studies, we have reported that necrotic cell death in HER2-positive cells is induced by PIT using T–IR700 in combination with NIR light and that this cell death depends on the dose of T–IR700 and NIR light.12,20 However, some cancer cells were found to survive, and tumor recurrences were eventually seen in mouse models after a single NIR-PIT treatment. Thus, it is necessary to develop a new method for enhancing the effectiveness of NIR-PIT treatment. To this end, we found that the effects of NIR-PIT were enhanced by repeated NIR light exposure, the use of two different antibody–IR700 conjugates, and combination with chemotherapy.20,22,23 For the further development of NIR-PIT, we created a trastuzumab–maytansinoid drug–photoabsorber conjugate (namely, T–DM1–IR700) and evaluated its applicability as a new agent for NIR-PIT. T–DM1 shows greater cytotoxicity owing to internalized DM1 than that of native unconjugated trastuzumab while maintaining selectivity for HER2-expressing cells.8,9,24 Meanwhile, NIR-PIT in combination with 5-fluorouracil chemotherapy results in an enhanced antitumor effect compared to that of NIR-PIT monotherapy in HER2-expressing cancer cells.23 Therefore, we hypothesized that treatment with T–DM1–IR700 might achieve HER2-specific binding equivalent to that of T–IR700 and induce additional cytotoxicity owing to DM1, especially for lesions irradiated with insufficient NIR light to disrupt cells by NIR-PIT. Our findings confirmed our hypothesis, and we obtained a novel candidate agent for NIR-PIT that can target large and deep-seated tumors.

In this study, we used 3T3/HER2 fibroblast cells as HER2-positive cells because of the stable and strong expression of HER2 and the successful experience in previous NIR-PIT experiments12 and HCC-1419 human breast cancer cells as stably HER2-expressing human cancer cells. For comparison with 3T3/HER2, we used BALB/3T3 fibroblast cells as HER2-negative control.

We went on to demonstrate that the HER2-specific binding capabilities of T–IR700 and T–DM1–IR700 were identical in vitro (Figures 2D and 3A,B). In line with our previous study that found that T–IR700 had similar immunoreactivity and target specificity to that of native unconjugated trastuzumab,12 T–DM1–IR700 also had a HER2-specific binding ability similar to that of unconjugated trastuzumab or T–IR700.

A LIVE/DEAD assay can detect damaged cell membrane and evaluate the percentage of cell death; stronger signals obtained when dye can permeate inside the cells. However, LDH cytotoxicity assay can detect LDH released from damaged cells and evaluate the total amount of LDH. The difference of results in between these two cytotoxicity assays shown in Figure 4 might have risen as a result. Elevated LDH release was seen in T–IR700 single treatment and T–DM1–IR700 single treatment in 3T3/HER2 cells but not in both HCC-1419 cells, probably because of the difference in expression levels of HER2 as shown in Figure 2A,B. In addition, cellular permeability could be increased by incubating with T–IR700 or T–DM1–IR700 in 3T3/HER2 cells, leading to LDH release into the medium. As is a tendency throughout in vitro studies, T–DM1–IR700 single treatment resulted in greater cytotoxicity than that of T–IR700 single treatment. Microscopic observation showed that T–DM1–IR700 induced giant cell formation and reduced the number of cells; this was a result of microtubule assembly inhibition and arrest at the G2-M phase of cell cycle by the internalized DM1.25,26 However, T–IR700 did not show any morphological changes and cytotoxicity compared to the untreated control (Figures 4 and 5). Furthermore, T–DM1–IR700 plus NIR light treatment tended to induce greater cytotoxicity than T–IR700 plus NIR light treatment, presumably because of cell membrane damage caused by NIR-PIT itself and additional cytotoxicity owing to the internalized DM1. With high-dose NIR light exposure, however, the cytotoxicity did not differ between these 2 NIR-PIT treatments. NIR-PIT with T–IR700 exerted sufficient cytotoxicity, suggesting that T–DM1–IR700 might be a useful agent for NIR-PIT when using low-dose NIR light exposure, which induces insufficient cell membrane damage.

Consistent with in vitro studies, in vivo quantitative biodistribution analysis showed that HER2-specific IR700 fluorescence accumulation after T–DM1–IR700 injection was almost the same as that after T–IR700 injection, indicating that the conjugation of IR700 to T–DM1 did not hamper the HER2-specificity of trastuzumab (Figure 6A,B). Compared to non-NIR light irradiation controls including T–DM1–IR700 single treatment, both T–IR700 plus NIR light and T–DM1–IR700 plus NIR light treatments led to substantial tumor volume control, suggesting that NIR-PIT using APC or ADPC have the potential to treat patients compared to monoclonal antibody or ADC treatment. Furthermore, T–DM1–IR700 plus NIR light treatment tended to reduce the tumor volume and led to significant prolonged survival compared to T–IR700 plus NIR light treatment in large-tumor models (Figure 7A,B). However, there was no significant difference in antitumor effects between T–IR700 plus NIR light and T–DM1–IR700 plus NIR light treatment in small-tumor models (Figure 7C,D). There are several possible reasons of the difference in the results between large-tumor models and small-tumor models: (1) There was no significant difference in cytotoxicity between T–IR700 plus NIR light irradiation and T–DM1–IR700 plus NIR light irradiation in the case of strong NIR light irradiation conditions (1 J/cm2) in vitro (Figure 4A,B). Similar to this results, in vivo NIR-PIT was conducted with strong NIR light penetration to activate IR700, leading to show no significant differences in antitumor effects by NIR-PIT itself. (2) Recent reports showed that NIR-PIT treatment induces super-enhanced permeability and retention (SUPR) effects that causes an increase in nanodrug delivery into treated tumor compared with untreated tumors.27,28 We expected NIR-PIT using ADPC to take advantage of SUPR delivery and enhanced drug effects, and SUPR effects contributed the results of the prolonged survival in large-tumor models treated with T–DM1–IR700 plus NIR light repeatedly.

In many clinical cases, cancerous tumors are located deep from the surface, limiting the effect of NIR-PIT because of light scattering and absorption by the tissues. Therefore, in addition to endoscopic or laparoscopic NIR light irradiation, which can achieve sufficient tissue penetration,23,29 the clinical applicability of NIR-PIT using ADPC can be extended to cell killing by internalized DM1 in deep-seated tumors where the NIR light is not strong enough to activate the photoabsorber or multiple metastases where NIR light cannot reach sufficiently to all disease sites. Furthermore, recent studies have shown the efficacy of fluorescence-guided surgery (FGS), which is performed under fluorescence navigation; however, it is difficult to remove all microscopic disease with FGS.30–37 FGS using ADPC with NIR light irradiation for tumors located deeper within body cavity might be useful than FGS alone by IR700 fluorescence navigation for tumor resection and sterilizing the residual cancer cells with photochemoimmunotherapy, resulting in complete tumor resection.

CONCLUSIONS

We have developed a new ADPC, T–DM1–IR700, which showed nearly identical binding to targeted HER2 compared to APC, T–IR700 in vitro and in vivo. T–DM1–IR700 plus NIR light treatment tended to induce a greater cytotoxic effect than that of T–IR700 plus NIR light treatment in vitro, and T–DM1–IR700 plus NIR light treatment tended to reduce the tumor volume and the prolonged survival compared to T–IR700 plus NIR light treatment in large-tumor models. T–DM1–IR700 is therefore a potential agent for NIR-PIT in the clinical setting, especially for treating tumors located deep in the tissue where NIR light cannot penetrate efficiently enough. In future, to further investigate the applicability of photochemoimmunotherapy using ADPC, we will examine the NIR-PIT using ADPC for larger animal models with inhomogeneous light doses and multiple metastases models to pave the way for clinical translation.

EXPERIMENTAL PROCEDURES

Reagents.

Trastuzumab (Herceptin) and T–DM1 (Kadcyla) were purchased from Chugai Pharmaceutical Co. Ltd. (Tokyo, Japan). IRDye700DX NHS ester (IR700) was purchased from LI-COR Biosciences (Lincoln, NE).

Synthesis and Purification of IR700-Conjugated Trastuzumab or T–DM1.

On the basis of previous experiments, to synthesize the IR700-conjugates that an average of 3 IR700 molecules were bound to trastuzumab or T–DM1 molecule, trastuzumab (1.0 mg, 6.8 nmol) or T–DM1 (1.0 mg, 6.6 nmol) was incubated with IR700 (66.8 μg, 34.2 nmol) in 0.1 M Na2HPO4 (pH 8.5) at room temperature for 1 h.12,20,23 The mixture was purified with a Sephadex G50 column (PD-10; GE Healthcare, Piscataway, NJ). The concentrations of protein and IR700 were measured by absorption at 280 and 689 nm, respectively, using spectroscopy (UV-1800; Shimadzu Corp., Kyoto, Japan) to confirm the number of IR700 molecules bound to trastuzumab or T–DM1 molecule.

Cell Lines and Culture Conditions.

A total of three cell lines were used in this study. HER2-expressing 3T3/HER2 cells were established by transfection of HER2 into NIH/3T3.38 HER2-negative BALB/3T3 cells and HER2-expressing human breast cancer HCC-1419 cells were obtained from the American Type Culture Collection (Manassas, VA). Cells were cultured with RPMI 1640 (Life Technologies, Gaithersburg, MD) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin (Life Technologies) in tissue culture flasks in a humidified incubator at 37 °C in an atmosphere of 95% air and 5% carbon dioxide.

Determination of HER2 Expression and Dose–response Binding of T–IR700 and T–DM1–IR700 in Vitro.

To determine the HER2 expression of the cells, IR700 fluorescence was measured by flow cytometry analysis (MACS-Qant analyzer; Miltenyi Biotec, Bergisch Gladbach, Germany) after treatment with T–IR700 or T–DM1–IR700. Cells were seeded on 35 mm dishes and incubated for 48 h at 37 °C. The medium was replaced with fresh culture medium containing 10 μg/mL of T–IR700 or 10 μg/mL of T–DM1–IR700 and incubated for 3 h at 37 °C. Cells were washed with PBS, and flow cytometry analysis was performed. To confirm the target specificity of T–IR700 or T–DM1–IR700, excess unconjugated trastuzumab (100 μg/mL) was added to block HER2 before T–IR700 or T–DM1–IR700 treatment. The MFIs were evaluated and compared to the MFI of the isotype control. In addition, to determine IR700 fluorescence intensities after treatment with various doses of T–IR700 or T–DM1–IR700, cells were treated with 1, 3, 10, and 30 μg/mL T–IR700 or 1, 3, 10, and 30 μg/mL T–DM1–IR700 and incubated for 24 h at 37 °C. Next, flow cytometry analysis was performed, and the MFIs were evaluated.

Fluorescence Microscopy.

To confirm HER2-specific binding and time-lapse co-localization of IR700 conjugates, fluorescence microscopy was performed (IX73; Olympus, Tokyo, Japan). Cells were seeded on cover glass-bottomed dishes and incubated for 48 h at 37 °C. The medium was replaced with fresh culture medium containing 10 μg/mL T–IR700 or 10 μg/mL T–DM1–IR700 and incubated for 3 or 24 h at 37 °C. Cells were washed with PBS, and fluorescence microscopy was performed with a 608–648 nm excitation filter and a 672–712 nm emission filter to detect IR700 fluorescence. All fluorescence images were analyzed with ImageJ software (http://rsb.info.nih.gov/ij/).

In Vitro NIR-PIT.

Cells were seeded and incubated at 37 °C. The medium was replaced with fresh culture medium containing 3 μg/mL T–IR700 or 3 μg/mL T–DM1–IR700 and incubated for another 24 h at 37 °C. After cells were washed with PBS, phenol red–free culture medium was added. Then, cells were irradiated with NIR light using a 690 nm continuous wave laser (ML6540–690; Modulight, Inc., Tampere, Finland). A power density of 20 mW/cm2 measured with an optical power meter (PM 100; Thorlabs, Newton, NJ) was used for this study.

Cytotoxicity Assay in Vitro.

The cytotoxic effects in response to PIT with T–IR700 or T–DM1–IR700 were determined by the LIVE/DEAD Fixable Green Dead Cell Stain Kit (Life Technologies) and the LDH cytotoxicity detection kit (TaKaRa Bio Inc., Shiga, Japan). For the LIVE/DEAD assay, which can detect cell membrane damage by flow cytometry, cells were seeded on 35 mm dishes and incubated for 48 h, followed by incubation with T–IR700 or T–DM1–IR700 for another 24 h and then irradiated with NIR light (0.3, 0.5, or 1 J/cm2). Cells were trypsinized just after NIR light irradiation and were washed with PBS. LIVE/DEAD green fluorescent reactive dye was added to the cell suspension and incubated for 30 min at room temperature, followed by flow cytometry analyses. For the LDH cytotoxicity assay, which can detect damaged cells by measuring LDH released into the cell culture medium, cells were seeded in a 96 well microplate and incubated with the culture medium supplemented with 1% FBS for 24 h. This was followed by incubation with T–IR700 or T–DM1–IR700 for another 24 h and irradiation with NIR light. The assay was performed 3 h after NIR light irradiation using a microplate reader (iMark, Bio-Rad, Hercules, CA), and cytotoxicity was calculated according to the manufacturer’s instructions.

Long-Term Growth Inhibition Assay in Vitro.

To further determine the long-term cytotoxic effects of NIR-PIT with T–IR700 or T–DM1–IR700, trypan blue dye exclusion assays and microscopic observation were performed. For the trypan blue dye exclusion assay, 3T3/HER2 cells (0.5 × 105 per well) and BALB/3T3 cells (0.5 × 105 per well) were seeded on 100 mm dishes and incubated for 48 h. The medium was replaced with fresh culture medium containing 3 μg/mL T–IR700 or 3 μg/mL T–DM1–IR700 and incubated for another 24 h at 37 °C. Then, cells were irradiated with NIR light (0.5 J/cm2). At the indicated time point after the NIR light irradiation, cells were collected, and viable cells were counted with an automated cell counter (Countess; Life Technologies) based on trypan blue dye uptake. Microscopic observation on a cover glass-bottomed dish was also carried out.

Xenograft Tumor Model.

Female BALB/c-nu/nu mice (6 weeks old; CAnN.Cg-Foxn1nu/CrlCrlj nu/nu) were obtained from Charles River Laboratories Japan, Inc. (Yokohama, Japan). All mice were allowed to acclimatize and recover from shipping-related stress for 1 week before the studies and were kept under a controlled light–dark cycle (12:12 h). All animal studies were conducted in accordance with the guidelines established by the Animal Care Committee at the Jikei University School of Medicine. A total of 1 000 000 or 2 000 000 cells were injected subcutaneously into the dorsa of the mice. The tumor xenografts were measured with an external caliper, and the tumor volume was calculated using the following formula: length × width × height ×0.5.39

In Vivo Fluorescence Imaging.

To determine the biodistribution of T–IR700 or T–DM1–IR700 and evaluate IR700 fluorescence intensities in the tumors, 3T3/HER2 (right dorsum) tumor-bearing mice were created, and fluorescence images were obtained with the IVIS Imaging System (Caliper Life Sciences, Hopkinton, MA) using a 675 nm excitation filter and a 695–770 nm emission filter. Tumors reaching approximately 200 mm3 in volume were selected and randomized into three groups with three mice each as follows: (i) control (intravenous (iv) injection of PBS), (ii) iv injection of 100 μg T–IR700, and (iii) iv injection of 100 μg T–DM1–IR700. IR700 fluorescence images were obtained 1, 2, 3, and 5 days after the injection using the same settings of exposure time, camera binning, and stage height under isoflurane anesthesia. All fluorescence images were analyzed with ImageJ software. The regions of interest were manually determined on each tumor area depending on the localization of the IR700 fluorescence.

NIR-PIT in Vivo.

To determine the antitumor effects in response to NIR-PIT with T–IR700 or T–DM1–IR700, the following experiments were conducted on large and small-tumor models. 3T3/HER2 tumors reaching approximately 200 mm3 in volume for large-tumor experiments and 30 mm3 in volume for small-tumor experiments were selected and randomized into 6 groups with 10 mice each as follows, respectively: (i) control (iv injection of PBS without NIR light irradiation); (ii) iv injection of PBS followed by NIR light irradiation (100 J/cm2); (iii) iv injection of 100 μg T–IR700 without NIR light irradiation; (iv) iv injection of 100 μg T–DM1–IR700 without NIR light irradiation; (v) iv injection of 100 μg T–IR700 followed by NIR light irradiation (100 J/cm2) 1 day after injection; and (vi) iv injection of 100 μg T–DM1–IR700 followed by NIR light irradiation (100 J/cm2) 1 day after injection. NIR light irradiation was performed under isoflurane anesthesia with a 690 nm continuous wave laser at a power density of 330 mW/cm2. For large-tumor experiments, the above treatments were repeated 7 days after the initial injection. After the treatments, tumor volumes were measured three times a week until the volume reached 3000 mm3 for large-tumor experiments or 500 mm3 for small-tumor experiments.

Statistical Analyses.

Mean ± SEM values from a minimum of three experiments were determined. Statistical analyses were carried out using GraphPad Prism software (GraphPad Software Inc., La Jolla, CA). A Student t-test was used to compare the two treatment groups. For in vivo experiments, the Mann–Whitney U test was used to evaluate the differences in tumor volume between two treatment groups. The cumulative probability of survival was estimated in each treatment group by Kaplan–Meier survival curve analysis, and the results were compared by the log-rank test. P < 0.05 was considered to indicate a statistically significant difference.

Supplementary Material

Supporting figures

ACKNOWLEDGMENTS

This study was supported by a Grant-in-Aid for Young Scientists (A) (JSPS KAKENHI 26710010), funding from the Japan Agency for Medical Research and Development (AMED) and the Cell Science Research Foundation (M.M.), and funding from the Intramural Research Program of the National Institutes of Health, National Cancer Institute, Center for Cancer Research (H.K.).

ABBREVIATIONS

ADC

antibody–drug conjugate

APC

antibody–photoabsorber conjugate

HER2

human epidermal growth factor receptor 2

NIR-PIT

near-infrared photoimmunotherapy

T–DM1

trastuzumab emtansine

T–IR700

trastuzumab–IR700

T–DM1–IR700

trastuzumab emtansine–IR700

Footnotes

The authors declare no competing financial interest.

ASSOCIATED CONTENT

Supporting Information

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.bioconjchem.7b00144.

Figures showing mass spectrometry of the conjugates, flow cytometry analyses, in vitro cytotoxicity analyses, trypan blue dye exclusion assays, and histological results of photochemoimmunotherapy treatment. (PDF)

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Supplementary Materials

Supporting figures
NIHMS1664788-supplement-Supporting_figures.pdf (865.8KB, pdf)

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