Fusa Ogata

Near infrared photoimmunotherapy targeting prostate cancer with prostate-specific membrane antigen (PSMA) antibody

Prostate-specific membrane antigen (PSMA) is a membrane protein that is overexpressed manifold in prostate cancer and provides an attractive target for molecular therapy. Near-infrared photoimmunotherapy (NIR-PIT) is a highly selective tumor treatment that employs an antibody-photoabsorber conjugate (APC). Here, we describe the efficacy of NIR-PIT, using a fully human IgG1 anti-PSMA monoclonal antibody (mAb), conjugated to the photoabsorber, IR700DX, in a PSMA-expressing PC3 prostate cancer cell line. Anti-PSMA-IR700 showed specific binding and cell-specific killing was observed after exposure of the cells to NIR light in vitro. In the in vivo study, anti-PSMA-IR700 showed high tumor accumulation and high tumor–background ratio. Tumor-bearing mice were separated into 4 groups: (i) no treatment; (ii) 100 μg of anti-PSMA-IR700 i.v.; (iii) NIR light exposure; (iv) 100 μg of anti-PSMA-IR700 i.v., NIR light exposure was administered. These were performed every week for up to 3 weeks. Tumor growth was significantly inhibited by NIR-PIT treatment compared with the other control groups (P < 0.001), and significantly prolonged survival was achieved (P < 0.0001 vs. other control groups). More than two thirds of tumors were cured with NIR-PIT. In conclusion, the anti-PSMA antibody is suitable as an APC for NIR-PIT. Furthermore, NIR-PIT with the anti-PSMA-IR700 antibody is a promising candidate of the treatment of PSMA-expressing tumors and could be readily translated to humans. Implications: NIR-infrared photoimmunotherapy (NIR-PIT) using a fully human anti-PSMA-IR700 conjugate showed potential therapeutic effects against a PSMA-expressing prostate cancer that is readily translated to humans. Mol Cancer Res; 15(9); 1153–62. ©2017 AACR.

Near-infrared photoimmunotherapy: a comparison of light dosing schedules

Near infrared photoimmunotherapy (NIR-PIT) is a newly-developed cancer therapy in which a monoclonal antibody is conjugated to a near-infrared photoabsorber, IR700 to form an antibody photoabsorber conjugate (APC). After the APC binds to cancer cells expressing the cognate antigen, exposure to NIR light results in rapid, highly selective necrotic cell death of the cancer cells with minimal off-target effects. Several hours after NIR-PIT, the tumor vessels become supraphysiologically permeable and circulating APC can therefore readily leak into the already-treated tumor space where it can bind with viable cancer cells that is called super-enhanced permeability and retention effect. The presence of the SUPR effect after NIR-PIT has prompted regimens in which there is a repeat exposure of NIR light 24 hours after the initial NIR-PIT to take advantage of the leakage of additional APC deeper into the tumor. However, this post-treatment APC penetration was fully induced within 3 hours, therefore, it is possible that repeated exposures of NIR light could be administered much earlier than 24 hours and still produce the same effects. To test this idea, we compared several modes of delivering additional doses of light after initial NIR-PIT. We found that repeated exposures of NIR light starting 3 hours after initial NIR-PIT produced equal or superior results to more delayed exposures of NIR light. This finding has practical implications of an easy-to-perform regimen as repeated light exposures could be performed during a single day rather than extending the procedure over two days which is the current recommendation.

Near Infrared Photoimmunotherapy in a Transgenic Mouse Model of Spontaneous Epidermal Growth Factor Receptor (EGFR)-expressing Lung Cancer.

Near infrared photoimmunotherapy (NIR-PIT) is a new cancer treatment that combines the specificity of antibodies for targeting tumors with the toxicity induced by a sensitive photoabsorber following exposure to NIR light. Most studies of NIR-PIT have been performed in xenograft models of cancer. The purpose of this study was to evaluate the therapeutic effects of NIR-PIT in a transgenic model of spontaneous lung cancer expressing human EGFR (hEGFR-TL). Mice were separated into 3 groups for the following treatments: (1) no treatment (control); (2) 150 μg of photoabsorber, IR700, conjugated to panitumumab, an antibody targeting EGFR [antibody–photoabsorber conjugate (APC)] intravenously (i.v.) only; (3) 150 μg of APC i.v. with NIR light administration. Each treatment was performed every week up to three weeks. MRI was performed 1 day before and 3, 6, 13, 20, 27, and 34 days after first NIR-PIT. The relative volume of lung tumors was calculated from the tumor volume at each MRI time point divided by the initial volume. Steel test for multiple comparisons was used to compare the tumor volume ratio with that of control. Tumor volume ratio was inhibited significantly in the NIR-PIT group compared with control group (P < 0.01 at all time points). In conclusion, NIR-PIT effectively treated a spontaneous lung cancer in a hEGFR-TL transgenic mouse model. MRI successfully monitored the therapeutic effects of NIR-PIT. Mol Cancer Ther; 16(2); 408–14. ©2016 AACR.

Syngeneic Mouse Models of Oral Cancer Are Effectively Targeted by Anti–CD44-Based NIR-PIT

Oral cavity squamous cell carcinoma (OSCC) is considered one of the most aggressive subtypes of cancer. Anti-CD44 monoclonal antibodies (mAb) are a potential therapy against CD44 expressing OSCC; however, to date the therapeutic effects have been disappointing. Here, a new cancer treatment is described, near-infrared photoimmunotherapy (NIR-PIT), that uses anti-CD44 mAbs conjugated to the photoabsorber IR700DX. This conjugate is injected into mice harboring one of three CD44 expressing syngeneic murine oral cancer cell (MOC) lines, MOC1 (immunogenic), MOC2 mKate2 (moderately immunogenic), and MOC2-luc (poorly immunogenic). Binding of the anti-CD44–IR700 conjugate was shown to be specific and cell-specific cytotoxicity was observed after exposure of the cells to NIR light in vitro. The anti-CD44–IR700 conjugate, when assessed in vivo, demonstrated deposition within the tumor with a high tumor-to-background ratio. Tumor-bearing mice were separated into four cohorts: no treatment; 100 μg of anti-CD44–IR700 i.v. only; NIR light exposure only; and 100 μg of anti-CD44–IR700 i.v. with NIR light exposure. NIR-PIT therapy, compared with the other groups, significantly inhibited tumor growth and prolonged survival in all three cell model systems. In conclusion, these data reveal that anti-CD44 antibodies are suitable as mAb–photoabsorber conjugates for NIR-PIT in MOC cells. Implications: This study using syngeneic mouse models, which better model the disease in humans than conventional xenografts, suggests that NIR-PIT with anti-CD44–IR700 is a potential candidate for the treatment of OSCC. Mol Cancer Res; 15(12); 1667–77. ©2017 AACR.

Near-infrared photoimmunotherapy with galactosyl serum albumin in a model of diffuse peritoneal disseminated ovarian cancer

Near-infrared photoimmunotherapy (NIR-PIT) is a highly cell-selective cancer therapy based on an armed antibody conjugated with a phthalocyanine-based photo-absorber, IRDye700DX (IR700). NIR-PIT can quickly kill target cells that express specific proteins on the cellular membrane but only when the antibody-IR700 conjugate binds to the cell membrane and is then exposed to NIR light. NIR-PIT is highly selective based on the specificity of the antibody. Galactosyl serum albumin (GSA) is composed of albumin decorated with galactose molecules conjugated to the carboxyl groups of albumin. GSA binds to beta-D-galactose receptors, a surface lectin, which are overexpressed on the cell surface of many cancers, including ovarian cancers and is quickly internalized after binding. Here, we demonstrate the feasibility of NIR-PIT in a model of disseminated peritoneal ovarian cancer (SHIN3 cells) using GSA-IR700 that binds to beta-D-galactose receptors. GSA-IR700 bound quickly to SHIN3 cells, then accumulated in the endo-lysosomes. Cell-specific killing was observed in vitro, yet a relatively large dose of NIR light exposure was required for cell killing compared to antibody-IR700 conjugates. To evaluate in vivo therapeutic effects of GSA-IR700 NIR-PIT, peritoneal disseminated SHIN3 tumor-bearing mice were separated into four groups: no treatment; NIR light only; GSA-IR700 only; and GSA-IR700 NIR-PIT. Repeated NIR-PIT showed significant suppression of tumor based on bioluminescence compared to the other groups (p < 0.05). Thus, repeated NIR-PIT using GSA-IR700 can achieve efficient antitumor effects, although GSA-IR700 NIR-PIT was less effective than antibody-IR700 NIR-PIT conjugates likely due to the rapid internalization of GSA-IR700.

Interstitial near-infrared photoimmunotherapy: effective treatment areas and light doses needed for use with fiber optic diffusers

Near-infrared photoimmunotherapy (NIR-PIT), a promising cancer therapy utilizing an antibody-photoabsorber conjugate (APC) and NIR light, which induces rapid necrotic cell death only in APC-bound cells. Effective NIR-PIT in mouse models has been achieved using superficial light illumination (SLI) with light emitting diodes (LEDs) or lasers, but in the clinical setting, fiber optic diffusers have been employed to deliver light to deeper tumors. However, the performance of NIR light in tissue delivered by fiber optic diffusers is poorly understood. Here, we investigated NIR-PIT using a cylindrical fiber optic diffuser in a mouse model of A431 tumors. NIR-PIT with 100 J/cm, the same light dose used in clinical trials of NIR-PIT, was applied after insertion of the diffuser within the tumor bed, and then both bioluminescence and fluorescence imaging were analyzed to assess the therapeutic efficacy. The diffuser can deliver adequate NIR light dose for effective NIR-PIT to the A431 tumor at a distance of approximately 1 cm around the light source at 100 J/cm. At 50 J/cm NIR light effective NIR-PIT was reduced to a distance of 5 – 7 mm diameter around the light source. These results indicate that the energy of interstitial light (measured in Joules/cm) administered via a fiber diffuser determines the depth of effective NIR-PIT around the diffuser and determines the spacing at which such diffusers should be placed to entirely cover the tumor. Thermal measurements demonstrate that interstitial light for NIR-PIT does not cause damage to the skin overlying the diffuser.

Characteristics of ovarian cancer detection by a near-infrared fluorescent probe activated by human NAD(P)H: quinone oxidoreductase isozyme 1 (hNQO1)

Near-infrared (NIR) fluorescent probes are ideal for in vivo imaging, because they offer deeper tissue penetration by the light and lower background autofluorescence than fluorophores that emit in the visible range. Q3STCy is a newly synthesized, NIR light-emitting probe that is activated by an enzyme commonly overexpressed in tumor cells, human nicotinamide adenine dinucleotide (phosphate): quinone oxidoreductase isozyme 1, known as hNQO1 or DT-diaphorase. The purpose of this study is to compare the sensitivity of detecting peritoneal ovarian cancer metastasis (POCM) with Q3STCy and gGlu-HMRG, a green fluorescent probe, upon their surface application. In vitro uptake of Q3STCy was significantly higher than that of gGlu-HMRG. Using a red fluorescence protein (RFP)-labeled in vivo tumor model of POCM, the Q3STCy probe provided high sensitivity (96.9%) but modest specificity (61.0%), most likely the result of albumin-probe interactions and non-specific activation in nearby altered but healthy cells. Three types of kinetic maps based on maximum fluorescence signal (MF), wash-in rate (WIR), and area under the curve (AUC) allowed for differentiation of the activated fluorescence signal associated with POCM from the background signal of the small intestine, thereby significantly improving the specificity of Q3STCy to 80%, 100%, and 100% for MF, WIR, and AUC, as well yielding a moderate improvement in sensitivity (100% for all approaches) that is comparable to that with gGlu-HMRG, but with the added advantages of NIR fluorescence as the transduction modality. Such a new methodology has the potential to afford identification of cancerous lesions deeper within tissue.Near-infrared (NIR) fluorescent probes are ideal for in vivo imaging, because they offer deeper tissue penetration by the light and lower background autofluorescence than fluorophores that emit in the visible range. Q3STCy is a newly synthesized, NIR light-emitting probe that is activated by an enzyme commonly overexpressed in tumor cells, human nicotinamide adenine dinucleotide (phosphate): quinone oxidoreductase isozyme 1, known as hNQO1 or DT-diaphorase. The purpose of this study is to compare the sensitivity of detecting peritoneal ovarian cancer metastasis (POCM) with Q3STCy and gGlu-HMRG, a green fluorescent probe, upon their surface application. In vitro uptake of Q3STCy was significantly higher than that of gGlu-HMRG. Using a red fluorescence protein (RFP)-labeled in vivo tumor model of POCM, the Q3STCy probe provided high sensitivity (96.9%) but modest specificity (61.0%), most likely the result of albumin-probe interactions and non-specific activation in nearby altered but healthy cells. Three types of kinetic maps based on maximum fluorescence signal (MF), wash-in rate (WIR), and area under the curve (AUC) allowed for differentiation of the activated fluorescence signal associated with POCM from the background signal of the small intestine, thereby significantly improving the specificity of Q3STCy to 80%, 100%, and 100% for MF, WIR, and AUC, as well yielding a moderate improvement in sensitivity (100% for all approaches) that is comparable to that with gGlu-HMRG, but with the added advantages of NIR fluorescence as the transduction modality. Such a new methodology has the potential to afford identification of cancerous lesions deeper within tissue.

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

Near-infrared photoimmunotherapy (NIR-PIT) is a highly selective tumor treatment that uses an antibody–photoabsorber conjugate (APC). However, the effect of NIR-PIT can be enhanced when combined with other therapies. NIR photocaging groups, based on the heptamethine cyanine scaffold, have been developed to release bioactive molecules near targets after exposure to light. Here, we investigated the combination of NIR-PIT using panitumumab–IR700 (pan-IR700) and the NIR-releasing compound, CyEt–panitumumab–duocarmycin (CyEt-Pan-Duo). Both pan-IR700 and CyEt-Pan-Duo showed specific binding to the EGFR-expressing MDAMB468 cell line in vitro. In in vivo studies, additional injection of CyEt-Pan-Duo immediately after NIR light exposure resulted in high tumor accumulation and high tumor–background ratio. To evaluate the effects of combination therapy in vivo, tumor-bearing mice were separated into 4 groups: (i) control, (ii NIR-PIT, (iii) NIR-release, (iv) combination of NIR-PIT and NIR-release. Tumor growth was significantly inhibited in all treatment groups compared with the control group (P < 0.05), and significantly prolonged survival was achieved (P < 0.05 vs. control). The greatest therapeutic effect was shown with NIR-PIT and NIR-release combination therapy. In conclusion, combination therapy of NIR-PIT and NIR-release enhanced the therapeutic effects compared with either NIR-PIT or NIR-release therapy alone. Mol Cancer Ther; 17(3); 661–70. ©2017 AACR.

Cerenkov radiation-induced photoimmunotherapy with 18F-FDG

Near-infrared photoimmunotherapy (NIR-PIT) is a new cancer treatment that combines the specificity of antibodies for targeting tumors with toxicity induced by photoabsorbers after irradiation with NIR light. A limitation of NIR-PIT is the inability to deliver NIR light to a tumor located deep inside the body. Cerenkov radiation (CR) is the ultraviolet and blue light that is produced by a charged particle traveling through a dielectric medium faster than the speed of light in that medium and is commonly produced during radioactive decay. Here, we demonstrate the feasibility of using CR generated by 18F-FDG accumulated in tumors to induce photoimmunotherapy. Methods: Using A431-luc cells, we evaluated the therapeutic effects of CR-PIT in vitro and in vivo using bioluminescence imaging. Results: CR-PIT showed significant suppression of tumor size, but the decrease of bioluminescence after CR-PIT was not observed consistently over the entire time course. Conclusion: Although CR-PIT can induce tumor killing deep within body, it is less effective than NIR-PIT, possibly related to the relatively lower efficiency of short wavelength light than NIR.

A topically-sprayable, activatable fluorescent and retaining probe, SPiDER-βGal for detecting cancer: Advantages of anchoring to cellular proteins after activation

SPiDER-βGal is a newly-developed probe that is activated by β-galactosidase and is then retained within cells by anchoring to intracellular proteins. Previous work has focused on gGlu-HMRG, a probe activated by γ-glutamyltranspeptidase, which demonstrated high sensitivity for the detection of peritoneal ovarian cancer metastases in an animal model. However, its fluorescence, after activation by γ-glutamyltranspeptidase, rapidly declines over time, limiting the actual imaging window and the ability to define the border of lesions. The purpose of this study is to compare the fluorescence signal kinetics of SPiDER-βGal with that of gGlu-HMRG using ovarian cancer cell lines in vitro and ex vivo tissue imaging. In vitro removal of gGlu-HMRG resulted in a rapid decrease of fluorescence intensity followed by a more gradual decrease up to 60 min while there was a gradual increase in fluorescence up to 60 min after removal of SPiDER-βGal. This is most likely due to internalization and retention of the dye within cells. This was also confirmed ex vivo tissue imaging using a red fluorescence protein (RFP)-labeled tumor model in which the intensity of fluorescence increased gradually after activation of SPiDER-βGal. Additionally, SPiDER-βGal resulted in intense enhancement within the tumor due to the high target-to-background ratio, which extended up to 60 min after activation. In contrast, gGlu-HMRG fluorescence resulted in decreasing fluorescence over time in extracted tumors. Thus, SPiDER-βGal has the advantages of higher signal with more signal retention, resulting in improved contrast of the tumor margin and suggesting it may be an alternative to existing activatable probes.