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
at the absorption maximum of 689 nm)17
, than conventional photosensitizers such as the hematoporphyrin derivative; Photofrin®
(1.2 × 103
at 630 nm), meta-tetrahydroxyphenylchlorin; Foscan®
at 652 nm), and mono-L-aspartylchlorin e6; NPe6/Laserphyrin®
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 (). Short term viability assays, as well as long term proliferation assays, demonstrated that the conjugate was capable of inducing specific cell death (). 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 (; 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 (). 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 (, 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 (). 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
. 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 (, and Supplementary video 1a, b
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
. Because both bound and unbound agent fluoresces, there is relatively high background signal at therapeutic doses (). Nevertheless, after PIT, the fluorescence of the treated tumors decreased and eventually disappeared, suggesting a potential means of monitoring the treatment ().
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 ( and Supplementary Fig. 4b
. 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 (). 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 (). 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.