Molecularly Targeted Cancer Combination Therapy with Near-Infrared Photoimmunotherapy and Near-Infrared Photorelease with Duocarmycin–Antibody Conjugate

Near-infrared photoimmunotherapy (NIR-PIT) is a newly developed cancer treatment that uses a highly targeted monoclonal antibody (mAb)-photoabsorber conjugate (APC). The photoabsorber IRDye700DX (IR700, silicon-phthalocyanine dye) is a highly hydrophilic dye, differentiating it from prior hydrophobic dyes used in photodynamic therapy (PDT; ref. 1). Therefore, mAb–IR700 conjugates behave in the body similar to nonconjugated antibodies. Once the APC is injected and time elapses to allow sufficient binding to target cells, exposure to NIR light results in rapid cell swelling, leading to cell membrane rupture and extrusion of cell contents into the extracellular space. Cell death after NIR-PIT is characterized as necrotic/immunogenic cell death (2). A first-in-human phase I trial of epidermal growth factor receptor (EGFR) targeted NIR-PIT in patients with inoperable head and neck cancer was initiated in June 2015 (https://clinicaltrials.gov/ct2/show/NCT02422979) and has recently advanced to phase II.

A unique advantage of NIR-PIT is that it leads to immediate increases in vascular permeability of treated tumors, which can result in 10- to 24-fold enhancement of macromolecule or nanodrug delivery. This phenomenon has been termed super-enhanced permeability and retention (SUPR) effects because it is substantially greater than conventional “enhanced permeability and retention (EPR),” which is commonly seen in untreated tumors (3–5). SUPR effects result in homogeneous redistribution of already circulating APC or reinjected APCs or other nanosized agents in treated tumors, at least partly overcoming baseline heterogeneity in drug delivery commonly observed in untreated tumors. Therefore, additional exposures of NIR light can further improve therapeutic effects by depositing additional APCs in the tumor bed after initial NIR-PIT (5, 6). To achieve superior NIR-PIT therapeutic effects, repeated NIR light exposures with one APC or a combination of NIR-PIT and nanosized anticancer drugs have been successfully demonstrated (5, 7).

Light in the NIR range (650–900 nm) has several advantages over visible light. NIR light can penetrate deeper into tissue while carrying minimal toxicity. As a consequence, NIR dyes have been used in both diagnostic and therapeutic applications in preclinical and clinical settings (8, 9).

NIR photocaging groups, based on the heptamethine cyanine scaffold, bound to a targeting moiety, and have the ability to accumulate in targeted tissue, enabling both diagnosis by fluorescence imaging and therapy by releasing potent bioactive molecules after NIR light exposure (10–13). Uncaging reactions that are induced with NIR light could site-specifically deliver bioactive compounds to any part of the body. The development of efficient uncaging reactions triggered by the modest photonic energy of NIR light represents a significant chemical challenge and is the subject of ongoing study (14, 15). The most advanced molecule in this area, CyEt–panitumumab–duocarmycin degree of labeling 4 (CyEt-Pan-Duo), releases a derivative of the DNA-alkylating natural product, duocarmycin. This duocarmycin–antibody conjugate shows light-dependent cytotoxic activity in the picomolar range and can be activated with clinically achievable doses of NIR light (16). Studies in mouse models showed that the conjugate was well tolerated, was readily visible with fluorescence imaging, and showed significant antitumor efficacy following external therapeutic doses of NIR light exposure (16).

Superior delivery of target molecules into target tumors prior to NIR-release could enhance therapeutic effects. When combination therapy with pan-IR700 and CyEt-Pan-Duo is used, there is both a large increase in delivered dose and a more homogeneous distribution of CyEt-Pan-Duo based on the SUPR effect after initial NIR-PIT. Thus, a potential strategy is to treat a tumor with conventional NIR-PIT followed by exposure to another dose of NIR to release duocarmycin as an adjuvant therapy. In this study, we investigate the in vivo distribution of CyEt-Pan-Duo after NIR-PIT. Following this, NIR-PIT and NIR-release were performed separately and in combination in a tumor-bearing mouse model in vivo and therapeutic efficacy was established.

Water soluble, silicon-phthalocyanine derivative, IRDye 700DX NHS ester was obtained from LI-COR Biosciences. Panitumumab, a fully humanized clinical IgG₂ mAb directed against EGFR, was purchased from Amgen. All other chemicals were of reagent grade.

Conjugation of dyes with mAb was performed according to a previous report (1). In brief, panitumumab (1.0 mg, 6.8 nmol) was incubated with IR700 NHS ester (60.2 μg, 30.8 nmol) in 0.1 M Na₂HPO₄ (pH 8.6) at room temperature for 1 hour. The mixture was purified with a Sephadex G25 column (PD-10; GE Healthcare). The protein concentration was determined with Coomassie Plus protein assay kit (Thermo Fisher Scientific Inc.) by measuring the absorption at 595 nm with UV-Vis (8453 Value System; Agilent Technologies). The concentration of IR700 was measured by absorption at 689 nm to confirm the number of fluorophore molecules per mAb. The synthesis was controlled so that an average of two IR700 molecules was bound to a single antibody. We abbreviate IR700 conjugated to panitumumab as pan-IR700.

Synthesis is described in a previous report (16). Following synthesis, cyanine-caged duocarmycin was conjugated to panitumumab using conventional conditions (pH 8.5 phosphate buffered saline, PBS, buffer) with 4.5 equivalent of the small molecule and purified using preparative size-exclusion chromatography (SEC) to provide CyEt–panitumumab–duocarmycin degree of labeling degree 4 (CyEt-Pan-Duo). Absorbance of the conjugate was also measured using UV-Vis.

As a quality control for conjugates, we performed SDS-PAGE. The conjugate was separated by SDS-PAGE with a 4% to 20% gradient polyacrylamide gel (Life Technologies). A standard marker (Crystalgen Inc.) was used as a protein molecular weight marker. After electrophoresis at 80 V for 2.5 hours, the gel was imaged with a Pearl Imager (LI-COR Biosciences) using the 700 nm and 800 nm fluorescence channels. We used diluted panitumumab as a control. The gel was stained with Colloidal Blue staining to determine the molecular weight of conjugate.

EGFR-expressing MDAMB468-luc (human breast cancer) cells, which are stably transduced luciferase-transfected cells were used in this study (17, 18). High luciferase expression was confirmed with 10 passages. Cells were grown in RPMI 1640 (Life Technologies) supplemented with 10% fetal bovine serum 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.

To verify in vitro pan-IR700 and CyEt-Pan-Duo binding, fluorescence from cells after incubation with APC was measured using a flow cytometer (FACS Calibur, BD BioSciences) and CellQuest software (BD BioSciences). MDAMB468-luc cells (2 × 10⁵) were seeded into 12-well plates and incubated for 24 hours. Medium was replaced with fresh culture medium containing 3 μg/mL of pan-IR700 or CyEt-Pan-Duo and incubated for 6 hours at 37°C. After washing with PBS, PBS was added. A 488-nm argon ion laser was used for excitation. Signals from cells were collected with a 653- to 669-nm band-pass filter.

Ten thousand MDAMB468-luc cells were seeded on cover-glass-bottomed dishes and incubated for 24 hours. Pan-IR700 or CyEt-Pan-Duo was then added to the culture medium at 3 μg/mL and incubated for 6 hours at 37°C. After incubation, the cells were washed with PBS. To detect the antigen specific localization, fluorescence microscopy was performed (BX61; Olympus America, Inc.) equipped with the following filters: excitation wavelength 590 to 650 nm and 672.5 to 747.5 nm, emission wavelength 665 to 740 nm and 765 to 855 nm for pan-IR700 and CyEt-Pan-Duo, respectively. Transmitted light differential interference contrast (DIC) images were also acquired.

MDAMB468-luc cells (2 × 10⁵) were placed in 12-well plates and incubated for 24 hours. Medium was replaced with fresh culture medium. Cells were divided into 8 groups of at least 3 wells per group for the following treatments: (i) no treatment (control); (ii) NIR light exposure only without conjugate (NIR light); (iii) 6 μg/mL of pan-IR700 (pan-IR700); (iv) 6 μg/mL of CyEt-Pan-Duo (CyEt-Pan-Duo); (v) 3 μg/mL of pan-IR700 and 3 μg/mL of CyEt-Pan-Duo (pan-IR700 + CyEt-Pan-Duo); (vi) 6 μg/mL of CyEt-Pan-Duo, NIR light exposure was administered at 6 J/cm² (CyEt-Pan-Duo + NIR light); (vii) 6 μg/mL of pan-IR700, NIR light exposure was administered at 6 J/cm² (pan-IR700 + NIR light); (viii) 3 μg/mL of pan-IR700 and 3 μg/mL of CyEt-Pan-Duo, NIR light exposure was administered at 6 J/cm² (pan-IR700 + CyEt-Pan-Duo + NIR light). Conjugates were incubated for 6 hours at 37°C. After washing with PBS, phenol red free medium was added. Cells were irradiated with a red light-emitting diode (LED), which emits light at 690 ± 20 nm wavelength (L690-66-60; Marubeni America Co.) at a power density of 50 mW/cm² as measured with an optical power meter (PM 100, Thorlabs). To verify in vitro therapeutic effect of combination therapy, cell count was measured using an automated cell counter (Countess, Invitrogen) 24 hours after treatment.

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. During the procedure, mice were anesthetized with inhaled 3% to 5% isoflurane and/or via intraperitoneal injection of 1 mg of sodium pentobarbital (Nembutal Sodium Solution, Ovation Pharmaceuticals Inc.). In order to determine tumor volume, the greatest longitudinal diameter (length) and the greatest transverse diameter (width) were measured with an external caliper. Tumor volumes were based on caliper measurements and were calculated using the following formula: tumor volume = length × width² × 0.5. Body weight was also measured. Mice were monitored daily for their general health including observation of skin color, weight loss, or loss of appetite. Tumor volumes were measured two times a week until the tumor volume reached 2,000 mm³, whereupon the mice were euthanized with inhalation of carbon dioxide gas.

MDAMB468-luc cells (6 × 10⁶) were injected subcutaneously in the right dorsum of the mice. Tumors were studied after they reached volumes of approximately 50 mm³. To evaluate in vivo CyEt-Pan-Duo biodistribution after NIR-PIT, tumor-bearing mice were randomized into 2 groups of at least 10 animals per group for the following treatments: (i) 100 μg of CyEt-Pan-Duo was injected on day 1 after 100 μg of pan-IR700 intravenously (i.v.), no NIR light was administered (CyEt-Pan-Duo); (ii) NIR light was administered at 50 J/cm² on day 1 after 100 μg of pan-IR700 i.v., 100 μg of CyEt-Pan-Duo was injected immediately after NIR light exposure (NIR-PIT + CyEt-Pan-Duo). Tumors were irradiated with an LED. Serial dorsal fluorescence images of CyEt-Pan-Duo were obtained with a Pearl Imager using a 800 nm fluorescence channel before and 0, 1, 3, 6, 9, and 24 hours after i.v. injection of 100 μg of CyEt-Pan-Duo via the tail vein. Pearl Cam Software (LICOR Biosciences) was used for analyzing fluorescence intensities. Region of interests (ROI) were placed on the tumor. ROIs were also placed in the adjacent nontumor region as background (left dorsum). Average fluorescence intensity of each ROI was calculated. Tumor–background ratios (TBR = fluorescence intensities of target/fluorescence intensities of background) were also calculated (n = 10).

Next, to clarify the in vivo treatment effect of NIR-release with conventional NIR-PIT, we performed combination therapy. MDAMB468-luc cells (6 × 10⁶) were injected subcutaneously in the right dorsum of the mice. Tumors were studied after they reached volumes of approximately 50 mm³. To examine the therapeutic effect of in vivo combination therapy with NIR-PIT and NIR-release, tumor-bearing mice were randomized into 4 groups of at least 10 animals per group for the following treatments: (i) no treatment (control); (ii) 100 μg of pan-IR700 i.v., NIR light was administered at 50 J/cm² on day 1 and 100 J/cm² on day 2 after injection (NIR-PIT); (iii) NIR light was administered at 50 J/cm² on day 1 without pan-IR700 and 100 μg of CyEt-Pan-Duo was injected immediately after NIR light exposure, then NIR light was administered at 100 J/cm² on day 2 (NIR-release); (iv) 100 μg of pan-IR700 i.v., NIR light was administered at 50 J/cm² on day 1 and 100 μg of CyEt-Pan-Duo was injected immediately after NIR exposure, then NIR light was administered at 100 J/cm² on day 2 (NIR-PIT + NIR-release). Tumors were irradiated with an LED. Serial fluorescence images, as well as white light images, were obtained using a Pearl Imager with 700 nm and 800 nm fluorescence channel.

For in vivo bioluminescence imaging (BLI), D-luciferin (15 mg/mL, 200 μL; Gold Biotechnology) was injected intraperitoneally and the mice were analyzed on a BLI system (Photon Imager; Biospace Lab) for luciferase activity (photons/minute). ROIs were set on the entire tumors to quantify the luciferase activities. ROIs were also placed in the adjacent nontumor region as background. Average luciferase activity of each ROI was calculated using M3 Vision Software (Biospace Lab). To measure relative therapeutic effect, luciferase activity of the tumor before NIR-PIT was set to 100%.

Data are expressed as means ± standard error of mean (SEM). Statistical analyses were carried out using GraphPad Prism version 7 (GraphPad Software). For multiple comparisons, a one-way analysis of variance (ANOVA) followed by the Tukey correction for multiple comparisons was used. The cumulative probability of survival based on volume (2,000 mm³) was estimated in each group with a Kaplan–Meier survival curve analysis, and the results were compared using the Gehan–Breslow–Wilcoxon test. Mann–Whitney U test was used to compare the fluorescence intensities and TBRs to controls. A P value of < 0.05 was considered statistically significant.

To characterize both conjugates, each absorbance spectrum was analyzed using UV-Vis (Supplementary Fig. S1) to show that conjugation ratios of both pan-IR700 and CyEt-Pan-Duo Ab-conjugates are identical as ones which we previously reported (1, 16). As defined by SDS-PAGE, pan-IR700, CyEt-Pan-Duo, and nonconjugated control panitumumab showed a nearly identical molecular weight. Fluorescence was seen in the band containing both pan-IR700 and CyEt-Pan-Duo but not the others (Fig. 1A). SEM of CyEt-Pan-Duo was consistent with previous report (16). After a 6-hour incubation with either pan-IR700 or CyEt-Pan-Duo, MDAMB468-luc cells demonstrated fluorescence signal, which was confirmed with flow cytometry (Fig. 1B) and fluorescence microscopy (Fig. 1C).

Based on survived cell counts, both Ab-conjugates bound to target MDAMB468 cells and induced strong cytotoxicity only after exposure of NIR light (NIR-release, NIR-PIT, and combination with NIR-PIT and NIR-release, see Fig. 1D). The combination therapy using both conjugates showed at least additive on-target toxicity. Additionally, mild cell death was observed in CyEt-Pan-Duo alone and pan-IR700 + CyEt-Pan-Duo even without NIR light exposure. In contrast, no treatment effect was shown in NIR light-only and pan-IR700-only groups.

The treatment and imaging regimen is shown in Fig. 2A. 800 nm fluorescence was rapidly seen in tumors undergoing NIR-PIT + CyEt-Pan-Duo tumors (Fig. 2B and C). On the other hand, tumors receiving only CyEt-Pan-Duo showed a gradual increase in 800 nm fluorescence (Fig. 2B and C). Because of the SUPR effect, CyEt-Pan-Duo was able to leak into the tumors more rapidly and the 800 nm fluorescence intensities were significantly higher in the NIR-PIT + CyEt-Pan-Duo group compared with CyEt-Pan-Duo-only group at the most time points (P < 0.01 at 1, 3, 6, and 9 hours; P < 0.05 at 24 hours; Fig. 2C). TBR increased gradually within 1 day in both CyEt-Pan-Duo-only tumors and NIR-PIT + CyEt-Pan-Duo tumors (Fig. 2D). TBRs were also significantly higher in the NIR-PIT + CyEt-Pan-Duo group compared with CyEt-Pan-Duo-only group at all time points (P < 0.01).

The treatment and imaging regimen is shown in Fig. 3A. One day after injection of pan-IR700 followed by NIR-PIT, the tumors had persistently reduced luciferase activity (Fig. 3B). All treatment (NIR-PIT, NIR-release, and NIR-PIT + NIR-release) resulted in decreases in bioluminescence compared with control (Fig. 3B). Luciferase activity significantly decreased after the all treatment groups (P < 0.01 vs. control group; Fig. 3C). In contrast, luciferase activity of tumor in the control group showed an increase due to rapid tumor growth.

The treatment and imaging regimen is shown in Fig. 4A. One day after injection of pan-IR700, the tumors showed higher IR700 fluorescence intensity than did the tumor with no pan-IR700 injection. After exposure to 50 J/cm² of NIR light, pan-IR700 tumor fluorescence signal decreased due to dying cells and partial photobleaching (Fig. 4B). One day after injection of CyEt-Pan-Duo, the tumors in the NIR-PIT + NIR-release group showed higher CyEt-Pan-Duo fluorescence intensity than did the tumor in NIR-release-only group (Fig. 4B). Immediately after exposure to 100 J/cm² of NIR light, CyEt-Pan-Duo fluorescence signal strongly decreased due to photorelease. After photorelease, CyEt-Pan-Duo fluorescence signal gradually increased in both the NIR-release group and the NIR-PIT + NIR-release group; however, the accumulation of CyEt-Pan-Duo was higher in NIR-PIT + NIR-release tumors compared with NIR-release-only tumor. Tumor growth was significantly inhibited in all treatment groups compared with the control group (P < 0.001; Fig. 4C). Tumor growth in the NIR-PIT + NIR-release group was significantly reduced compared with the NIR-release-only group (P < 0.01). Significantly prolonged survival was also achieved in all treatment groups compared with the control group (P < 0.05 for the NIR-release group, P < 0.01 for the NIR-PIT group and NIR-PIT + NIR-release group; Fig. 4D). Survival of the NIR-PIT group was significantly prolonged compared with the NIR-release-only group (P < 0.01). Furthermore, significantly prolonged survival was also achieved in the NIR-PIT + NIR-release group compared with the NIR-PIT-alone group (P < 0.05). From these results, maximal effects were shown with the combination of NIR-PIT and NIR-release. There was no skin necrosis or toxicity attributable to the treatment in any group.

In oncology, mAbs have favorable pharmacokinetics for tumor targeting because of their stable binding to target molecules that leads to high TBRs. However, a limitation of mAb-based therapy is inhomogeneous intratumoral distribution of the antibodies due to their relatively large molecular size (19–21). This occurs especially when a mAb has a high binding affinity for the target receptor and/or the tumor cells express high levels of target antigens. In these cases, mAbs are saturated on the most bioavailable antigen-positive cells which are typically located in the immediate perivascular space. This “binding site barrier” effectively hampers the penetration of mAbs deeper into the tumor (22–25). To achieve sufficient therapeutic effects, new methods for improving the microdistribution of mAbs within the tumor are needed.

We demonstrated therapeutic effect of NIR-release, NIR-PIT, and combination of NIR-PIT and NIR-release in vitro as shown in Fig. 1D. Moreover, we showed that the combination of NIR-PIT and NIR-drug release enabled robust therapeutic effects on EGFR-expressing MDAMB468 tumors compared with NIR-PIT-alone or NIR-release-alone therapy as shown in Fig. 4. After the first NIR-PIT, additional CyEt-Pan-Duo can enter the treated tumor bed more deeply due to the greater permeability and penetration afforded by the SUPR effect which follows NIR-PIT (Fig. 2). There, additional APCs bind homogeneously to the surviving fraction of cancer cells (6). Therefore, the second exposure to NIR light enhances the release of duocarmycin causing local cytotoxicity. On the other hand, when CyEt-Pan-Duo is used without prior NIR-PIT, it tends to accumulate preferentially in the perivascular space with lower concentrations of duocarmycin reaching deeper into the tumor (Fig. 5).

CyEt-Pan-Duo achieved sufficient tumor TBRs as shown in Fig. 2 to be potentially practical for clinical application during surgical, endoscopic, or trans-needle procedures. Efficient binding and distribution of the antibody are important for APCs to be effective as agents for NIR treatment. This also holds for antibody–toxin or antibody–drug conjugates as, to be effective, the drugs and toxins must be internalized after cell binding. Our results demonstrated that once CyEt-Pan-Duo bound to target tumor cells it was internalized within 6 hours of incubation (Fig. 1). Moreover, following application of NIR light exposure, the 800 nm fluorescence intensity of the tumor was nearly completely extinguished, indicating duocarmycin release (16). These results suggest that CyEt-Pan-Duo has favorable characteristics as a NIR-releasing antibody–drug conjugate.

Cyanine-based antibody drug conjugate linkers could enable small-molecule delivery with high precision through the combination of antibody targeting and NIR light-mediated release. Light provides an external stimulus to precisely time and target the release of drugs (26, 27). Our data demonstrate that tumor growth was reduced and survival was prolonged significantly in the NIR-release-alone group compared with the control group. As shown in Fig. 1D, the optimal cyanine conjugate, CyEt-Pan-Duo, displayed significant antitumor efficacy due to the release of duocarmycin after NIR light.

After the initial NIR-PIT, the subsequent SUPR effect permitted deeper penetration of still-circulating APCs into the tumor enabling them to bind uniformly to surviving cancer cells. A second light exposure, thus, results in further cell killing (7). Thus, to maximize the effect of NIR-PIT after a single injection of pan-IR700 two sequential light exposures should be performed. Moreover, to obtain the maximal therapeutic effect from the combination of NIR-PIT and NIR-release, CyEt-Pan-Duo should maximally enter tumor cells with little background uptake. Fluorescence imaging of the tumor also showed that the skin uptake was still high up to one day of incubation (Fig. 2). Thus, we used 1 day of incubation with CyEt-Pan-Duo to achieve a reasonable TBR whereupon the 2nd NIR light exposure released duocarmycin from the CyEt-Pan-Duo. Thus, the 2nd shot of NIR light served two purposes, the first to activate pan-IR700 APCs that had reaccumulated in the tumor and the second to release duocarmycin from CyEt-Pan-Duo.

While the combination therapy with NIR-PIT and NIR-release showed highly selective cytotoxicity, and NIR light can be easily applied to superficial tumors, an obvious limitation is the inability to deliver NIR light to the tumor located deep in the tissue. Skin, fat, and other organs will absorb NIR light before it reaches the tumor. There are several potential solutions to this problem. For instance, NIR light could be delivered to a tumor and to adjacent structures while the tissues are still exposed during a surgical resection, thus treating residual tumor in the tumor margin or in regional lymph nodes. Such procedures have been proposed in the past with PDT (28, 29); however, we believe that NIR-PIT would be much more effective with lower toxicity than PDT. Alternatively, fiber optic light probes could be placed within or nearby tumor using endoscopes, laparoscopes, catheters, or image guided percutaneous needles. Recently, a new type of cancer phototherapy was also reported. Cancer cells expressing specific fluorescent proteins can be treated with exposure of ultra violet C (UVC; refs. 30–33). However, the wavelength of UVC is shorter than that of NIR, therefore, UVC light does not penetrate deep into tissue. Furthermore, fluorescent proteins should be genetically transfected into cancer cells in vivo. Therefore, we think NIR-PIT would be technically simple and easy. Another caveat in this study is that subcutaneously growing human tumors in immunodeficient mice do not sufficiently represent clinical cancer. Superior tumor models such as surgically orthotopic tumor model can clarify the preclinical effect of treatment (34, 35), yet surgical orthotopic injection requires highly trained surgical skills and invasive methods. On the other hand, the cell injection model requires less advanced skills, and is less invasive, so we chose the cell injection model. We used anti-EGFR antibody, panitumumab, in both NIR-PIT and NIR-release. To some extent, pan-IR700 may compete with CyEt-Pan-Duo for EGFR on the remaining cells after initial NIR light exposure (6, 7). As the result, additional binding of CyEt-Pan-Duo might be blocked by prior saturation with pan-IR700. Thus, it may be advantageous to study the effect of performing NIR-PIT with a different antibody than panitumumab. This experiment is planned for the future. Finally, repeated dosing of the APCs with repeated light exposures is likely to increase effectiveness (7, 36). Thus, it would be desirable to extend these studies to include multiple doses of the APCs and multiple NIR light exposures.

CyEt-Pan-Duo accumulates in EGFR-expressing cancer cells and releases duocarmycin after NIR light exposure. Prior treatment with NIR-PIT results in improved microdistribution of CyEt-Pan-Duo and additive therapeutic responses in EGFR-expressing cancers. The combination of NIR-PIT and NIR-release is a promising candidate for the treatment of tumors and could be readily translated to humans.

P.L. Choyke has ownership interest (including patents) in PIT (he does not receive any royalties, as the patent is owned by the U.S. government). No potential conflicts of interest were disclosed by the other authors.

Conception and design: T. Nagaya, P.L. Choyke, H. Kobayashi

Development of methodology: T. Nagaya, A.P. Gorka, M.J. Schnermann, H. Kobayashi

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): T. Nagaya, A.P. Gorka, R.R. Nani, S. Okuyama, H. Kobayashi

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): T. Nagaya, Y. Maruoka, P.L. Choyke, H. Kobayashi

Writing, review, and/or revision of the manuscript: T. Nagaya, A.P. Gorka, F. Ogata, P.L. Choyke, M.J. Schnermann, H. Kobayashi

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): R.R. Nani, F. Ogata, P.L. Choyke, H. Kobayashi

Study supervision: M.J. Schnermann, H. Kobayashi

All the authors were supported by the Intramural Research Program of the NIH, NCI, Center for Cancer Research (ZIA BC011513).

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