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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Nat Med. Author manuscript; available in PMC 2012 June 1.
Published in final edited form as:
Published online 2011 November 6. doi:  10.1038/nm.2554
PMCID: PMC3233641

Cancer Cell-Selective In Vivo Near Infrared Photoimmunotherapy Targeting Specific Membrane Molecules


Three major modes of cancer therapies, surgery, radiation and chemotherapy, have been the mainstay of modern oncologic therapy. To minimize side effects, molecular targeted cancer therapies including armed antibody therapy have been developed with limited success. In this study, we developed a new type of molecular targeted cancer therapy, photoimmunotherapy (PIT), employing a target-specific photosensitizer based on a near infrared (NIR) phthalocyanine dye, IR700, conjugated to monoclonal antibodies (MAb) targeting epidermal growth factor receptors (EGFR). Cell death was induced immediately only upon irradiating, MAb-IR700 bound, target cells with NIR light. In vivo tumor shrinkage after irradiation with NIR light was observed only in target EGFR-expressing cells. The MAb-IR700 conjugates were most effective when bound to the cell membrane, producing no phototoxicity when not bound, suggesting a different mechanism for PIT compared with conventional photodynamic therapies. Target selective PIT enables treatment of cancer based on MAb binding on the cell membrane.

In order to minimize the side effects of conventional cancer therapies, including surgery, radiation and chemotherapy, molecular targeted cancer therapies have been developed. Among the existing targeted therapies, monoclonal antibodies (MAb) therapy have the longest history, and to date, over 25 therapeutic MAbs have been approved by the Food and Drug Administration (FDA)1,2. Effective MAb therapy traditionally depends on three mechanisms: antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and receptor blockade and requires multiple high doses of the MAb. MAbs have also been used at lower doses as vectors to deliver therapies such as radionuclides3 or chemical or biological toxins4. Ultimately, however, dose limiting toxicity relates to the biodistribution and catabolism of the antibody conjugates.

Conventional photodynamic therapy (PDT), which combines a photosensitizing agent with the physical energy of non-ionizing light to kill cells, has been less commonly employed for cancer therapy because the current non-targeted photosensitizers are also taken up in normal tissues, thus, causing serious side effects, although the excitation light itself is harmless in the near infrared (NIR) range (Fig. 1). Were it feasible to design a highly targeted photosensitizer, toxicity could be greatly reduced. Although the targeted photosensitizer may distribute throughout the body, it will only be active where intense light is applied, reducing the likelihood of off-target effects. Most existing photosensitizers are poorly selective small molecules which bind not only to cancer cells but also to normal cells including the skin and other epithelial surfaces resulting in unwanted phototoxicity. These agents are generally hydrophobic, therefore, permeate into cells and produce reactive oxygen species intracellularly, leading to cell death. Thus, target specific delivery of conventional photosensitizers is theoretically difficult because, after reaching the cell, the agent must still be internalized into organelles, such as mitochondria, to be most effective. Various combinations of conventional photosensitizers and MAbs have been tested to improve selectivity with limited success especially when measured by in vivo therapeutic effects5-11. There are several reasons for unsuccessful outcomes; 1. Conventional photosensitizers have low extinction coefficients that requires conjugation of large numbers of photosensitizers to a single antibody molecule thus, potentially decreasing binding affinity. 2. Conventional photosensitizers are mostly hydrophobic leading to difficulties in conjugating photosensitizers to antibodies without compromising the immunoreactivity and in vivo target accumulation. 3. Conventional photosensitizers generally absorb light in the visible range reducing tissue penetration.

Figure 1
(a) A schema for explaining selective cancer therapy with photoimmunotherapy (PIT) in the context of other physical cancer therapies employing electro-magnetic wave irradiation. Although other physical cancer therapies induce different types of damages ...

In this study, we develop a MAb-based photosensitizer, which is activated by NIR light for targeted photoimmunotherapy (PIT), only when bound to the target molecule on the cancer cellular membrane. Further, because this agent also emits a diagnostic fluorescence, it can be used to direct the application of light to minimize light exposure to non-relevant tissues and non-invasively monitor therapeutic effects.


In vitro characterization of MAb-IR700 conjugates

Conjugation of trastuzumab, a MAb directed against human epidermal growth factor receptor 2 (HER2), or panitumumab, a MAb directed against human epidermal growth factor receptor (HER1), to IRDye 700DX N-hydroxysuccinimide (NHS) ester (IR700) resulted in ~3 IR700 molecules conjugated to each MAb molecule. The trastuzumab-IR700 (Tra-IR700) and panitumumab-IR700 (Pan-IR700) preparations demonstrated strong association and contained no detectable MAb aggregates as determined by high performance liquid chromatography (HPLC) and sodium dodecyl sulfate polyacrylamidegel elctrophoresis SDS-PAGE (Supplementary Fig. 1a, b). In vitro immunoreactivity of MAb-IR700 conjugates were analyzed with a binding assay using 125I-labeled MAb-IR700 conjugates, and revealed that 73.38 ± 0.39% (125I-Tra-IR700) and 78.61 ± 0.89% (125I-Pan-IR700) of binding was achieved with each MAb conjugate respectively and the specificity of binding was confirmed by blocking with excess native unconjugated MAb (less than 1.4%) (Supplementary Fig. 1c). Since immunoreactivity of 125I-Tra and 125I-Pan measured with the same method were 78 ± 2%, and 82 ± 3%, respectively, minimal loss of MAbs with IR700 conjugation was confirmed.

MAb-IR700 mediated phototoxicity leads rapidly to necrotic cell death in vitro

Fluorescence microscopy was performed to visualize the cellular binding location of the conjugates. Consistent with previous studies, IR700 fluorescence was detected mainly on the cell surface of 3T3/HER2 cells, which express HER2 protein, after 1 h incubation at 4 °C with Tra-IR700, whereas the fluorescence was also detected inside the cells after 6 h incubation at 37 °C with Tra-IR700, indicating gradual internalization12 (Fig. 2a). Co-staining with LysoTracker-Green revealed co-localization of IR700 with the endolysosomal compartment (Fig. 2b). After 1 h and 6 h of incubation with Tra-IR700, excitation light (fluorescence microscope; power density of 2.2 mW cm-2) induced fluorescence as well as cellular swelling, bleb formation, and rupture of vesicles representing necrotic cell death (Fig. 2c and Supplementary Video 1a, b). Necrotic cell death was also observed using HER1 positive A431 cells incubated with Pan-IR700 which were exposed to excitation light as described above (Fig. 3a).

Figure 2
Target specific cell death in response to Tra-IR700 mediated photoimmunotherapy for 3T3/HER2 cells
Figure 3
Target specific cell death in response to Pan-IR700 mediated photoimmunotherapy for EGFR expressing A431 cells

To further explore the nature of the phototoxicity of Tra-IR700 on 3T3/HER2 cells we used the LIVE/DEAD assay to analyze acute phototoxicity and trypan blue dye exclusion assay to analyze the effect on proliferation. As cell death was induced rapidly upon irradiation (Supplementary Video 1a, b), the LIVE/DEAD assay, which can detect the cells with damaged membranes was performed within 1 h after the treatment. The percentage of cell death in target cells vs. untreated control cells was significantly influenced by excitation light dose (Fig. 2d). In addition, there was no significant cytotoxicity associated with exposure to Tra-IR700 without excitation light or with light exposure without Tra-IR700. Similar results were obtained for A431 cells with Pan-IR700 (Fig. 3b), however, panitumumab itself had a noticeable treatment effect against A431 cells due to down regulation and signal inhibition of HER113. In addition, parental 3T3 cells served as HER2 receptor-negative controls, therefore, we chose 3T3/HER2 cells with Tra-IR700 for further in vitro study. Proliferation assay revealed that long term growth inhibition was confirmed only when cells were treated with Tra-IR700 and exposed to light (Fig. 2e, f and Supplementary Fig. 2a).

Target-specific phototoxic cell death induced only when MAb-IR700 is bound to the target membrane antigen

There was no significant difference in phototoxicity between 1 h and 6 h incubation with Tra-IR700 (Fig. 2g), indicating that membrane binding of Tra-IR700 was sufficient to induce cell death. When Tra-IR700 was localized to the endolysosomal compartment (Fig. 2b), it also induced rupture of the vesicle with cellular swelling and bleb formation after irradiation (Supplementary Video 1a). However, this did not appear to be a major cause of cell death, since cell death could be observed without endolysosomal localization of Tra-IR700 within 1 h of incubation at 4 °C (Supplementary Video 1b). Interestingly, failure to wash the cells prior to irradiation did not influence the phototoxic effect indicating that cellular membrane binding was important to the phototoxic effects of the conjugate, not merely the presence of the conjugate. Further, the IR700 dye alone (200 nM; equivalent IR700 concentration of Tra-IR700 conjugates) did not incorporate into the cells or induce phototoxcity in cells (Fig. 2h and Supplementary Fig. 2b). Additionally, phototoxcity was dose-dependently blocked by the excess unconjugated trastuzumab (Supplementary Fig. 2c, d). Furthermore, Tra-IR700 did not induce therapeutic effect to A431 cells (Fig. 2i). These results confirm that cell death is dependent on specific membrane binding of Tra-IR700.

To confirm that the phototoxicity was target specific, we next treated 3T3/HER2 and Balb/3T3/DsRed, which is a parental HER2 negative Balb/3T3 transfected with DsRed fluorescent protein. Tra-IR700 was distributed in a HER2 specific manner while DsRed expressing Balb/3T3 cells did not show phototoxicity upon irradiation (Fig. 4a and Supplementary Video 2). In addition, LIVE/DEAD Green staining demonstrated HER2 specific induction of cell death as determined by multi-color fluorescence microscopy (Fig. 4b) and flowcytometry analysis (Fig. 4c). Target-specific phototoxicity was also confirmed with Pan-IR700 mediated PIT in A431 cells and Balb/3T3/DsRed (HER1 negative) co-cultured cells (Fig. 3c). Overall, Tra-IR700 and Pan-IR700 showed identical therapeutic effects to HER2 positive (3T3/HER2) and HER1 positive (A431) cells, respectively, except that unconjugated panitumumab showed noticeable growth inhibition but unconjugated trastzumab did not reduce growth with the dose used.

Figure 4
Tra-IR700 mediated photoimmunotherapy for HER2 expressing and non expressing co-cultured cells

Role of reactive oxygen species on Tra-IR700 mediated photoimmunotherapy

Reactive oxygen species (ROS) have been implicated in the cell death associated with conventional PDT. To clarify the role of photon-induced redox reactions (e.g. singlet oxygen (1O2)) in producing phototoxicity with Tra-IR700, a redox quencher, sodium azide (NaN3)14, was added to the medium when cells were irradiated. The percentage of cell death was partially decreased in the presence of sodium azide, in a dose dependent manner (Fig. 2j).

Target specific accumulation of MAb-IR700 in vivo

To examine the conjugate distribution in vivo, we prepared a xenograft tumor model bearing A431 (HER1 positive) and 3T3/HER2 (HER1 negative) tumors in each dorsum of a mouse. A431 tumors were visualized with IR700 fluorescence 1 d after intravenous injection of Pan-IR700 (50 μg) (Fig. 5a). The fluorescence intensity of Pan-IR700 in a A431 tumor decreased gradually over days, while tumor to background ratios (TBRs) increased (Fig. 5b, c). The fluorescence intensity of the 3T3/HER2 tumor was the same as that of background (non-tumor lesions). When 300 μg of Pan-IR700 was administered intravenously, fluorescence intensity of the A431 tumor was more than 3 times higher than 50 μg injection at 1 d after injection, however, TBR was lower because of high background signal (Fig. 5b, c). As less antitumor activity was found in mice receiving 50 μg (vs. 300 μg) of Pan-IR700 injection following irradiation (Supplementary Fig. 3), therefore, we chose the higher injection dose for the treatment study. Biodistribution of Tra-IR700 was determined with IR700 fluorescence because tissue levels of radioactivity and fluorescence might be different due to their different excretion routes and catabolism when using dual-labeled radiolabeled-Pan-IR70015. There was no other specific localization of IR700 except for bladder accumulation on day 1 probably due to excretion of catabolized and unbound dye (Fig. 5d, and Supplementary Fig. 4a).

Figure 5
Pan-IR700 mediated photoimmunotherapy for HER1 expressing tumors in vivo

Target-specific tumor shrinkage in response to MAb-IR700 mediated photoimmunotherapy

A431 tumors were treated with a single dose of light at 1 d after Pan-IR700 administration. The efficacy of Pan-IR700 mediated PIT was studied in 8 groups of A431 tumor bearing mice (at least n = 12 mice in each group). All treated tumors were less than 500 mm3 as larger tumors were associated with side effects (i.e. subcutaneous bleeding, tumor bleeding or weakened state) requiring euthanasia in accordance with our institution's animal care and use guidelines. Tumor volume was significantly reduced in A431 tumors treated with Pan-IR700 PIT compared with non treatment control mice (Fig. 5e), and survival was significantly prolonged in Pan-IR700-PIT treated mice (Fig. 5f). No significant therapeutic effect was observed in other control groups of mice. Similar results were obtained in 3T3/HER2 tumors treated with Tra-IR700 PIT (Supplementary Fig. 4b). Pathological analysis revealed that only scant viable A431 tumor cells were present after Pan-IR700 mediated PIT and massive granulation with inflammatory change was observed in the tumor nodule (Fig. 5g). It was also observed that tissue edema developed superficially. To assess the acute phase toxicity of Pan-IR700, we repeatedly administrated 300 μg of Pan-IR700 intravenously twice a week for 4 w, but there were no adverse effects observed up to 8 w (n = 4) compared with the control group.


Conventional PDT for cancer therapy is based on the preferential accumulation of a photosensitizer in tumor to produce phototoxicity with minimal damage to surrounding tissue16. Traditionally, PDT is thought to be mediated by the generation of ROS, especially singlet oxygen, in the presence of oxygen16. However, to the extent that existing photosensitizers lack tumor selectivity, considerable damage can be seen in normal tissues leading to dose limiting toxicity. Thus, current methods of PDT would be improved if more selective targeting of the photosensitizer and more efficient phototoxicity per photon absorbed was possible.

Various combinations of conventional photosensitizers and MAbs have been tested to improve selectivity5-11. Although a large number of photosensitizers have been evaluated, IR700 has several favorable chemical properties. Amino-reactive IR700 is a relatively hydrophilic dye and can be covalently conjugated with MAb using the NHS ester of IR700 which leads to high tumor uptake. IR700 also has more than 5–fold higher extinction coefficient (2.1×105 M-1cm-1 at the absorption maximum of 689 nm)17, than conventional photosensitizers such as the hematoporphyrin derivative; Photofrin® (1.2 × 103 M-1cm-1 at 630 nm), meta-tetrahydroxyphenylchlorin; Foscan® (2.2×104 M-1cm-1 at 652 nm), and mono-L-aspartylchlorin e6; NPe6/Laserphyrin® (4.0×104 M-1cm-1 at 654 nm)18.

The selectivity of the MAb-IR700 is derived from its activation after binding to the cell membrane of target cells; unbound conjugate does not contribute to phototoxicity (Fig. 2a, g, h). Short term viability assays, as well as long term proliferation assays, demonstrated that the conjugate was capable of inducing specific cell death (Fig. 2d–f). When co-cultures of receptor-positive and -negative cells were treated, only the receptor-positive cells were killed despite the presence of unbound MAb-IR700 in the culture medium (Fig. 4a–c; see also Supplementary Video 2). This selective cell killing minimizes damage to normal cells.

Although the mechanism of phototoxicity with MAb-IR700 is not completely clear, the agent must be bound to the cellular membrane to be active. Treatment with sodium azide, a well known redox and singlet oxygen scavenger, only partially reduced the phototoxicity but did not totally eliminate the effectiveness of the conjugate (Fig. 2j). This indicates that ROS generation is a minor part of the phototoxic effect. The observation that phototoxicity was induced after incubation with MAb-IR700 after only 1 h at 4 °C, indicates that internalization of the conjugate is not required for activity (Fig. 2g, and Supplementary Video 1b). This differs from the current generation of PDT agents that require intracellular localization to be effective. Video microscopy demonstrated rapid visible damage to the membrane and lysosomes after exposure to light, following incubation for more than 6 h at 37 °C, when the MAb-conjugate was internalized (Supplementary Video 1a). Previous studies have indicated that intracellular uptake of MAb-photosensitizer conjugates is responsible for inducing phototoxicity6-10. While this new MAb-IR700 conjugate does not require internalization for phototoxic cell death, cell surface antigen binding is required (Fig. 2g, h). For instance, the rupture of endolysosome occurred within a second of light exposure (Supplementary video 1a). Cell death induced by singlet oxygen generally induces a slower apoptotic cell death (Supplementary Fig. 5a, b)19,20. Since cell membrane damage was so quickly induced even at 4 °C by this method, it is hypothesized that cell death is caused by the rapid expansion of locally heated water with relatively minor effects due to singlet oxygen effects (Fig.2g, j, and Supplementary video 1a, b)21.

Another desirable feature of PIT using fluorescent MAb-IR700 conjugate is that it permitted the detection of targeted tissue. Theoretically, this would allow specific lesions to be identified with PIT rather than irradiating the entire field. Doses required for diagnosis (50 μg), however, were significantly lower than those required for therapy (300 μg) Improved intratumoral distribution of antibody occurred with the therapeutic dose (Supplementary Fig. 6)22,23. Because both bound and unbound agent fluoresces, there is relatively high background signal at therapeutic doses (Fig. 5a–c). Nevertheless, after PIT, the fluorescence of the treated tumors decreased and eventually disappeared, suggesting a potential means of monitoring the treatment (Fig. 5d).

One difficulty in interpreting the in vivo results of this study was that trastuzumab, but not panitumumab, demonstrated minor therapeutic effects by itself without the application of light. Since panitumumab is an IgG2 and trastuzumab is an IgG1, this was likely mediated by ADCC and CDC effects even though non-saturating doses were administered (Fig. 5e, f and Supplementary Fig. 4b)24. Based on the similarity of the phototoxicity induced with three different MAbs against several different cells expressing various numbers of respective target molecules and considering the potentially additive benefits from immunotherapy (Supplementary Fig. 7), we believe that this method may be generally applicable to other MAbs25,26.

The NIR excitation light wavelength (peaking at 689 nm) allows penetration of at least several centimeters into tissues16. By using fiber-coupled laser diodes with diffuser tips, NIR light can be delivered within several centimeters of otherwise inaccessible tumors located deep to the body surface. Using such fibers, PDT has been used to treat brain tumors and peritoneal metastasis of ovarian cancer27. In addition to treating solid cancers it may be possible to target circulating tumor cells since they could be excited when they traverse superficial vessels. Although no toxicity was observed in our experiments, clinical translation will require formal toxicity studies. In addition, free IR700 and catabolized IR700, are readily excreted into urine within 1 hour without accumulation in any specific organ (Supplementary Fig. 8). The other component of PIT, light irradiation with NIR at 690 nm wavelength is unlikely to be toxic except at thermal doses. Theoretically, there should be no limitations on the cumulative irradiation dose of NIR light, unlike ionizing radiation such as x-ray or gamma-ray (Fig. 1). Therefore, repeated PIT might be possible for long term management of some cancer patients. In reality, repeated PIT with 3 different regimens (3 or 4 fractionated NIR irradiations at a single dose of MAb-IR700 and 4 cycles of PIT every 2 weeks after multiple doses of antibody) controlled tumor re-growth, resulting in tumor free survival of more than 4 months.

In conclusion, we have developed a target specific PIT based on MAb-IR700 conjugate (Fig. 1). The photosensitizer, IR700, is excited in the NIR range leading to deeper tissue penetration resulting in successful eradication of subcutaneously xenografted tumors after only a single dose of external NIR light irradiation. Targeted phototoxicity seems to be primarily dependent on binding of the MAb-IR700 to the cell membrane and to a lesser extent on internalization and ROS formation. The ability to covalently conjugate any number of different antibodies to IR700 means that this may be a highly flexible theranostic platform. The fluorescence induced by the conjugate can be used to non-invasively guide both PIT and monitor the results of therapy. Thus, the MAb-IR700 conjugate is a promising therapeutic and diagnostic agent for the treatment of cancer.

Supplementary Material


This research was supported by the Intramural Research Program of the US National Institutes of Health, National Cancer Institute, Center for Cancer Research. We would like to thank C. Regino, T. Hirano and C. Paik for their technical support.


Author Contributions: M.M. conducted experiments, performed analysis and wrote the manuscript; M.O., N.K. and L.T.R conducted experiments and performed analysis; P.L.C. wrote the manuscript and supervised the project; and H.K. planned and initiated the project, designed and conducted experiments, wrote the manuscript, and supervised the entire project.


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