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Photoimmunotherapy (PIT), carried out using an Ab conjugated to the near infrared dye IRDye700DX, is achieving significant success in target-specific elimination of cells. Fibroblast activation protein alpha (FAP-α) is an important target in cancer because of its expression by cancer-associated fibroblasts (CAFs) as well as by some cancer cells. Cancer-associated fibroblasts that express FAP-α have protumorigenic and immune suppressive functions. Using immunohistochemistry of human breast cancer tissue microarrays, we identified an increase of FAP-α

Near-infrared photoimmunotherapy (NIR-PIT) selectively kills tumor cells to which the photo-absorber dye IR700DX-conjugated antibodies are bound and induces a systemic anti-tumor immune response. NIR-PIT induces immunogenic cell death (ICD), releases damage-associated molecular patterns (DAMPs) molecules from dying tumor cells, and activates dendritic cells (DCs). However, it is unclear whether NIR-PIT affects migration of tumor-infiltrating (Ti)-DCs to draining lymph nodes (dLNs), where a systemic anti-tumor response is induced. Here, we utilized in vivo photolabeling of Ti-DCs in tumors in photoconvertible protein Kikume Green-Red (KikGR) mice to show that NIR-PIT enhanced migration of Ti-DCs including cDC1s, cDC2s, and CD326

V-domain immunoglobulin suppressor of T cell activation (VISTA) is an inhibitory immune checkpoint molecule that is broadly expressed on lymphoid and myeloid cells, including regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs). Near-infrared photoimmunotherapy (NIR-PIT) is a cancer treatment that utilizes an antibody-photoabsorber (IRDye 700DX NHS ester) conjugate to selectively kill target cells after the local application of NIR light. Depletion of VISTA-expressing cells in the tumor microenvironment (TME) using NIR-PIT could enhance anti-tumor immune responses by removing immune suppressive cells. The purpose of this study was to evaluate the anti-tumor efficacy of VISTA-targeted NIR-PIT using two murine tumor models, MC38-luc and LL2-luc. VISTA was expressed on T cells including Tregs and MDSCs in the TME of these tumors. In contrast, CD45 - cells, including cancer cells, did not express VISTA. VISTA-targeted NIR-PIT depleted VISTA-expressing cells ex vivo. In vivo VISTA-targeted NIR-PIT inhibited tumor progression and prolonged survival in both models. After VISTA-targeted NIR-PIT, augmented CD8 + T cell and dendritic cell activation were observed in regional lymph nodes. In conclusion, VISTA-targeted NIR-PIT can effectively treat tumors by decreasing VISTA-expressing immune suppressor cells in the TME. Local depletion of VISTA-expressing cells in the tumor bed using NIR-PIT is a promising new cancer immunotherapy for treating various types of tumors.

The immune system is recognized as an important factor in regulating the development, progression, and metastasis of cancer. Myeloid-derived suppressor cells (MDSCs) are a major immune-suppressive cell type by interfering with T cell activation, promoting effector T cell apoptosis, and inducing regulatory T cell expansion. Consequently, reducing or eliminating MDSCs has become a goal of some systemic immunotherapies. However, by systemically reducing MDSCs, unwanted side effects can occur. Near-infrared photoimmunotherapy (NIR-PIT) is a newly developed treatment that selectively kills targeted cells without damaging adjacent normal cells. The aim of this study is to evaluate the antitumor efficacy of MDSC-directed NIR-PIT utilizing anti-Ly6G antibodies to specifically destroy polymorphonuclear (PMN)-MDSCs in the tumor microenvironment (TME) in syngeneic mouse models. PMN-MDSCs were selectively eliminated within tumors by Ly6G-targeted NIR-PIT. There was significant tumor growth suppression and prolonged survival in three treated tumor models. In the early phase after NIR-PIT, dendritic cell maturation/activation and CD8

Near-infrared photoimmunotherapy (NIR-PIT) is a new cancer treatment that utilizes antibody–IRDye700DX (IR700) conjugates. The clinical use of NIR-PIT has recently been approved in Japan for patients with inoperable head and neck cancer targeting human epidermal growth factor receptor (hEGFR). Previously, cytotoxic T-lymphocyte antigen 4 (CTLA4)–targeted NIR-PIT has been shown to strongly inhibit tumor progression and prolonged survival was seen in different tumor models due to enhanced T-cell–mediated antitumor immunity. In this study, combined NIR-PIT targeting CTLA4 expressing cells and cancer cells was investigated in four tumor models including a newly established hEGFR-expressing murine oropharyngeal cancer cell (mEERL-hEGFR). While single molecule–targeted therapy (NIR-PIT targeting hEGFR or CTLA4) did not inhibit tumor progression in poorly immunogenic mEERL-hEGFR tumor, dual (CTLA4/hEGFR)-targeted NIR-PIT significantly suppressed tumor growth and prolonged survival resulting in a 38% complete response rate. After the dual-targeted NIR-PIT, depletion of CTLA4 expressing cells, which were mainly regulatory T cells (Tregs), and an increase in the CD8+/Treg ratio in the tumor bed were observed, suggesting enhanced host antitumor immunity. Furthermore, dual-targeted NIR-PIT showed antitumor immunity in distant untreated tumors of the same type. Thus, simultaneous cancer cell–targeted NIR-PIT and CTLA4-targeted NIR-PIT is a promising new cancer therapy strategy, especially in poorly immunogenic tumors where NIR-PIT monotherapy is suboptimal.

Near‐infrared photoimmunotherapy (NIR‐PIT) is a cell selective cancer therapy that uses an antibody‐photoabsorber (IRDye700DX, IR700) conjugate (APC) and NIR light. NIR‐PIT targeting epidermal growth factor receptor (EGFR) in head and neck cancer (HNC) was conditionally approved in Japan in 2020. APC‐bound tumors can be detected using endoscopic fluorescence imaging, whereas NIR light can be delivered using endoscopic fiber optics. The aims of this study were: (1) to assess the feasibility of endoscopic NIR‐PIT in an orthotopic HNC model using a CD44‐expressing MOC2‐luc cell line; and (2) to evaluate quantitative fluorescence endoscopic imaging prior to and during NIR‐PIT. The results were compared in 3 experimental groups: (1) untreated controls, (2) APC injection without light exposure (APC‐IV), and (3) APC injection followed by NIR light exposure (NIR‐PIT). APC injected groups showed significantly higher fluorescence signals for IR700 compared with the control group prior to therapeutic NIR light exposure, and the fluorescence signal significantly decreased in the NIR‐PIT group after light exposure. After treatment, the NIR‐PIT group showed significantly attenuated bioluminescence compared with the control and the APC‐IV groups. Histology demonstrated diffuse necrotic death of the cancer cells in the NIR‐PIT group alone. In conclusion, endoscopically delivered light combined with quantitative fluorescence imaging can be used to “see and treat” HNC. This method could also be applied to other types of cancer approachable with endoscopy., We have developed a florescence endoscopy system that can detect the IR700 fluorescence signal and deliver therapeutic NIR light through the endoscope to treat IR700 fluorescent lesions with NIR‐PIT. In this study, we established an oral cancer model using a murine oral squamous cell cancer‐derived MOC2 cell and performed NIR‐PIT using the fluorescence endoscopy system.

Fluorogenic probes in the near-infrared (NIR) region have the potential to provide stimuli-dependent information in living organisms. Here, we describe a new class of fluorogenic probes based on the heptamethine cyanine scaffold, the most broadly used NIR chromophore. These compounds result from modification of heptamethine norcyanines with stimuli-responsive carbamate linkers. The resulting cyanine carbamates (CyBams) exhibit exceptional turn-ON ratios (~170×) due to dual requirements for NIR emission: carbamate cleavage through 1,6-elimination and chromophore protonation. Illustrating their utility in complex in vivo settings, a γ-glutamate substituted CyBam was applied to imaging γ-glutamyl transpeptidase (GGT) activity in a metastatic model of ovarian cancer. Overall, CyBams have significant potential to extend the reach of fluorogenic strategies to intact tissue and live animal imaging applications.

Objective Cancer-associated fibroblasts (CAFs) play a significant role in tumor progression within the tumor microenvironment. Previously, we have used near-infrared photoimmunotherapy (NIR-PIT), a next-generation cancer cell-targeted phototherapy, to establish CAF-targeted NIR-PIT. In this study, we investigated whether dual-targeted NIR-PIT targeting cancer cells and CAFs could be a therapeutic strategy. Methods A total of 132 cases of esophageal cancer were analyzed for epidermal growth factor receptor (EGFR), human epidermal growth factor 2 (HER2), and fibroblast activation protein (FAP) expression using immunohistochemistry. Human esophageal cancer cells and CAFs were co-cultured and treated with single- or dual-targeted NIR-PIT in vitro. These cells were co-inoculated into BALB/c-nu/nu mice and the tumors were treated with single-targeted NIR-PIT or dual-targeted NIR-PIT in vivo. Results Survival analysis showed FAP- or EGFR-high patients had worse survival than patients with low expression of FAP or EGFR (log-rank, p p = 0.074, respectively), while no difference was observed in HER2 status. In vitro, dual (EGFR/FAP)-targeted NIR-PIT induced specific therapeutic effects in cancer cells and CAFs along with suppressing tumor growth in vivo, whereas single-targeted NIR-PIT did not show any significance. Moreover, these experiments demonstrated that dual-targeted NIR-PIT could treat cancer cells and CAFs simultaneously with a single NIR light irradiation. Conclusion We demonstrated the relationship between EGFP/FAP expression and prognosis of patients with esophageal cancer and the stronger therapeutic effect of dual-targeted NIR-PIT than single-targeted NIR-PIT in experimental models. Thus, dual-target NIR-PIT might be a promising therapeutic strategy for cancer treatment.

Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer, and conventional chemotherapy and molecular-targeted therapies show limited efficacy. Near-infrared photoimmunotherapy (NIR-PIT) is a new anticancer treatment that selectively damages the cell membrane of cancer cells based on NIR light-induced photochemical reactions of the antibody (Ab)-photoabsorber (IRDye700Dx) conjugate and the cell membrane. TNBC is known to express several adhesion molecules on the cell surface providing a potential new target for therapy. Here, we investigated the therapeutic efficacy of intercellular adhesion molecule-1 (ICAM-1)-targeted NIR-PIT using xenograft mouse models subcutaneously inoculated with two human ICAM-1-expressing TNBC cell lines, MDAMB468-luc and MDAMB231 cells. In vitro ICAM-1-targeted NIR-PIT damaged both cell types in a NIR light dose-dependent manner. In vivo ICAM-1-targeted NIR-PIT in both models showed early histological signs of cancer cell damage, such as cytoplasmic vacuolation. Even among the cancer cells that appeared to be morphologically intact within 2 h post treatment, abnormal distribution of the actin cytoskeleton and a significant decrease in Ki-67 positivity were observed, indicating widespread cellular injury reflected in cytoplasmic degeneration. Such damage to cancer cells by NIR-PIT significantly inhibited subsequent tumor growth and improved survival. This study suggests that ICAM-1-targeted NIR-PIT could have potential clinical application in the treatment of TNBC.

Regulatory T cells (Tregs) are known to inhibit antitumor immunity, yet the specific mechanism by which intratumoral Tregs promote tumor growth remains unclear. To better understand the roles of intratumoral Tregs, we selectively depleted tumor-infiltrating Tregs using anti-CD25-F(ab′)2 near-infrared photoimmunotherapy. Depletion of tumor-infiltrating Tregs induced transient but synchronized IFNγ expression in CD8 T and natural killer (NK) cells. Despite the small fraction of CD8 T and NK cells contained within examined tumors, IFNγ produced by these CD8 T and NK cells led to efficient and rapid tumor vessel regression, intratumoral ischemia, and tumor necrosis/apoptosis and growth suppression. IFNγ receptor expression on vascular endothelial cells was required for these effects. Similar findings were observed in the early phase of systemic Treg depletion in tumor-bearing Foxp3DTR mice; combination with IL15 therapy further inhibited tumor growth and achieved increased complete regression. These results indicate the pivotal roles of intratumoral Tregs in maintaining tumor vessels and tumor growth by suppressing CD8 T and NK cells from producing IFNγ, providing insight into the mechanism of Treg-targeting therapies. Significance: Intratumoral Treg depletion induces synchronized intratumoral CD8 T- and NK-cell activation, IFNγ-dependent tumor vessel regression, and ischemic tumor necrosis/apoptosis, indicating the roles of intratumoral Tregs to support the tumor vasculature.

Near‐infrared photoimmunotherapy (NIR‐PIT) is a new type of cancer treatment, which was recently approved in Japan for patients with inoperable head and neck cancer. NIR‐PIT utilizes antibody‐IRDye700DX (IR700) conjugates and NIR light at a wavelength of 690 nm. NIR light exposure leads to physicochemical changes in the antibody‐IR700 conjugate cell receptor complex, inducing rapid necrotic cell death. Just as fluorescence guided surgery is useful for surgeons to resect tumors completely, real‐time information of tumor locations would help clinicians irradiate NIR light more precisely. IR700 is a fluorescence dye that emits at 702 nm; however, there is no clinically available device optimized for detecting this fluorescence. On the other hand, many indocyanine green (ICG) fluorescence imaging devices have been approved for clinical use. Therefore, we investigated whether LIGHTVISION, one of the clinically available ICG cameras, could be employed for tumor detection. We hypothesized that irradiation with even low‐power 690‐nm laser light, attenuated by 99% with a neutral‐density filter, could be detected with LIGHTVISION without fluorescence decay or therapeutic effect because of the long emission tail of IR700 beyond 800 nm (within the detection range of LIGHTVISION). We demonstrated that the LIGHTVISION camera, originally designed for ICG detection, can detect the tail of IR700 fluorescence in real time, thus enabling the visualization of target tumors., A diagnostic imaging method is developed for accurately localizing tumor and IR700 accumulation based on IR700 fluorescence, using weakened therapeutic laser light at a nontherapeutic dose and a commercially available clinical fluorescence camera for indocyanine green (LIGHTVISION).

Near‐infrared photoimmunotherapy (NIR‐PIT) is a novel cancer therapy that employs a combination of infrared light and tumor‐targeted monoclonal antibody‐photoabsorber conjugates to cause both direct tumor necrosis and immunogenic cell death. NIR‐PIT may have potential in the perioperative setting before surgery, and therefore it is important to know the effect of NIR‐PIT on wound healing. Fifty mice were implanted with subcutaneous xenografts of N87 human gastric cancer cells, and tumors were excised after reaching a predetermined size. After excision, 30 mice were split into three groups: Controls, NIR‐PIT 1 day prior to surgery and NIR‐PIT 3 days prior to surgery. The quantity of reactive oxygen species (ROS) in each wound was measured on Postoperative Days 2 and 4, and mice were monitored weekly for 4 weeks for evidence of local tumor recurrence as well as clinical evidence of wound healing complications (eg, dehiscence, infection). The remaining 20 mice (10 controls, 10 treated with NIR‐PIT 1 day prior to surgery) were sacrificed on either Postoperative Day 7 or 14, the skin around wounds were excised, and tensile strength was measured with a digital force gauge. There were no significant differences between treatment and control groups with respect to wound ROS levels, wound tensile strength, local tumor recurrence, or postoperative complication rates (P > .05). In conclusion, neoadjuvant (pre‐operative) NIR‐PIT shows no evidence of adverse wound healing effects, and it is likely a safe adjunctive treatment to surgery. Postoperative use of NIR‐PIT merits investigation., Near‐infrared photoimmunotherapy (NIR‐PIT), which is now under world‐wide phase 3 trial including in Japan, may be useful in the perioperative setting either before or after tumor excision that should be rational because NIR‐PIT could minimize tumor volume and initiate host antitumor immunity for safe and successful outcome. In order to apply this neoadjuvant NIR‐PIT before the surgery, only the concern of surgeons is if wound healing is hampered by preoperative NIR‐PIT. Therefore, in this study, in order to respond high demands from many surgeons, the effect of NIR‐PIT on wound healing was measured using reactive oxygen species monitoring and wound tensile strength measurements and shown to have no difference between treatment and control mice.

Near-infrared photoimmunotherapy (NIR-PIT) is a newly developed and selective cancer treatment that induces necrotic and immunogenic cell death and utilizes a monoclonal antibody conjugated to a photo-absorber dye, IR700DX, activated by NIR light. Although CD44 is surface cancer marker associated with drug resistance, anti-CD44-IR700 NIR-PIT results in inhibited cell growth and prolonged survival in multiple tumor types. Meanwhile, anti-CD25-IR700–targeted NIR-PIT has been reported to achieve selective and local depletion of FOXP3(+)CD25(+)CD4(+) regulatory T cells (Tregs), which are primary immunosuppressive cells in the tumor microenvironment (TME), resulting in activation of local antitumor immunity. Combined NIR-PIT with CD44- and CD25-targeted agents has the potential to directly eliminate tumor cells and also amplify the immune response by removing FOXP3(+)CD25(+)CD4(+) Tregs from the TME. We investigated the difference in therapeutic effects of CD44-targeted NIR-PIT alone, CD25-targeted NIR-PIT alone, and the combination of CD44- and CD25-targeted NIR-PIT in several syngeneic tumor models, including MC38-luc, LL/2, and MOC1. The combined NIR-PIT showed significant tumor growth inhibition and prolonged survival compared with CD44-targeted NIR-PIT alone in all tumor models and showed prolonged survival compared with CD25-targeted NIR-PIT alone in MC38-luc and LL/2 tumors. Combined CD44/CD25 NIR-PIT also resulted in some complete remissions, whereas this was not achieved with either type of NIR-PIT alone. Therefore, combined NIR-PIT simultaneously targeting cancer antigens and immunosuppressive cells in the TME may be more effective than either type of NIR-PIT alone and may have potential to induce prolonged immune responses in treated tumors.

Recombinant immunotoxins (RIT) are chimeric proteins containing an Fv that binds to tumor cells, fused to a fragment of Pseudomonas exotoxin (PE) that kills the cell. Their efficacy is limited by their short half-life in the circulation. Chemical modification with polyethylene glycol (PEG) is a well-established method to extend the half-lives of biologics. Our goal was to engineer RITs with an increase in half-life and high cytotoxic activity. We introduced single cysteines at different locations in five anti-mesothelin RITs and employed site-specific PEGylation to conjugate them to 20-kDa PEG. Because our previous PEGylation method using β-mercaptoethanol reduction gave poor yields of PEG-modified protein, we employed a new method using tris(2-carboxyethyl)phosphine to reduce the protein and could PEGylate RITs at approximately 90% efficiency. The new proteins retained 19% to 65% of cytotoxic activity. Although all proteins are modified with the same PEG, the radius of hydration varies from 5.2 to 7.1, showing PEG location has a large effect on protein shape. The RIT with the smallest radius of hydration has the highest cytotoxic activity. The PEGylated RITs have a 10- to 30-fold increase in half-life that is related to the increase in hydrodynamic size. Biodistribution experiments indicate that the long half-life is due to delayed uptake by the kidney. Antitumor experiments show that several PEG-RITs are much more active than unmodified RIT, and the PEG location greatly affects antitumor activity. We conclude that PEGylation is a useful approach to improve the half-life and antitumor activity of RITs.

Photoimmunotherapy (PIT) is a new type of tumor‐specific treatment utilizing monoclonal antibody (mAb)‐photosensitizer conjugates and near‐infrared (NIR) light irradiation. One potential PIT target, the type I transmembrane protein TROP2, is expressed at high levels in many cancers, including pancreatic carcinoma (PC) and cholangiocarcinoma (CC), in which its expression is correlated with poor prognosis and tumor aggressiveness. In this study, we assessed the efficacy of PIT utilizing newly developed humanized anti‐TROP2 mAb conjugated to the photosensitizer IR700 (TROP2‐IR700) for PC and CC. Immunohistochemistry on PC and CC tissue microarrays confirmed that TROP2 is overexpressed in about half of PC and CC specimens. Using cultured PC and CC cells, TROP2‐IR700 localized TROP2‐specific and target‐specific cell killing was observed after NIR light irradiation. In addition, TROP2‐IR700 was localized to mouse xenograft tumors expressing TROP2 after intravenous injection. PC and CC xenograft tumor growth was significantly inhibited by TROP2‐targeted PIT relative to controls. The efficacy of TROP2‐targeted PIT in vitro and against xenografted tumors in vivo suggests promise as a therapy for human PC and CC, both of which currently have dismal prognoses and limited therapeutic options., The recently developed photoimmunotherapy (PIT) method targets and disrupts cancer cell membranes by combining monoclonal antibody (mAb)‐based targeting with a photosensitizer IR700. By using newly developed humanized anti‐TROP2 mAb conjugated to the IR700 (TROP2‐IR700), both pancreatic cancer and cholangiocarcinoma cells were successfully treated with near‐infrared light both in vitro and in vivo. Given that there is no molecular‐targeted therapy in the standard of care for pancreatic cancer and cholangiocarcinoma patients, TROP2‐targeted PIT is an attractive candidate for clinical trials and ultimately treatment.

Regulatory T (Treg) cells play a major role in immune suppression permitting tumors to evade immune surveillance. Depletion of intra-tumoral Treg cells can result in tumor regression. However, systemic depletion of Tregs may also induce autoimmune adverse events. Near-infrared photoimmunotherapy (NIR-PIT) is a newly developed cell-specific cancer therapy that locally kills specific cells in the tumor. Antibody-photoabsorber (IRDye700DX) conjugates (APC) are injected and bind to the tumor and subsequent administration of NIR light to the tumor results in rapid cell death only in targeted cells. CD25-targeted NIR-PIT has been shown to induce spatially selective depletion of tumor-associated Treg cells. In this study, we compared the efficacy of an antibody fragment, anti-CD25-F(ab’)(2) and a full antibody, anti-CD25-IgG as agents for NIR-PIT. Tumor-bearing mice were divided into four groups: (1) no treatment; (2) anti-CD25-IgG-IR700 i.v. only; (3) anti-CD25-F(ab’)(2)-IR700 i.v. with NIR light exposure and (4) anti-CD25-IgG-IR700 i.v. with NIR light exposure. Although both anti-CD25-targeted NIR-PITs resulted in significant tumor growth inhibition, the anti-CD25-F(ab’)(2)-IR700 based NIR-PIT was superior to the anti-CD25-IgG-IR700 NIR-PIT. The anti-CD25-F(ab’)(2)-IR700 demonstrated faster clearance from the body than the anti-CD25-IgG-IR700. Sustained circulation of anti-CD25-IgG-IR700 may block IL-2 binding on activated effector T-cells decreasing immune response. In conclusion, anti-CD25-F(ab’)(2) based NIR-PIT was more effective in reducing tumor growth than anti-CD25-IgG based NIR-PIT. Absence of the Fc portion of the APC leads to faster clearance and therefore promotes a superior activated T cell response in tumors.

Conspectus This Account is the first comprehensive review article on the newly developed, photochemistry-based cancer therapy near-infrared (NIR) photoimmunotherapy (PIT). NIR-PIT is a molecularly targeted phototherapy for cancer that is based on injecting a conjugate of a near-infrared, water-soluble, silicon-phthalocyanine derivative, IRdye700DX (IR700), and a monoclonal antibody (mAb) that targets an expressed antigen on the cancer cell surface. Subsequent local exposure to NIR light turns on this photochemical “death” switch, resulting in the rapid and highly selective immunogenic cell death (ICD) of targeted cancer cells. ICD occurs as early as 1 min after exposure to NIR light and results in irreversible morphologic changes only in target-expressing cells based on the newly discovered photoinduced ligand release reaction that induces physical changes on conjugated antibody/antigen complex resulting in functional damage on cell membrane. Meanwhile, immediately adjacent receptor-negative cells are totally unharmed. Because of its highly targeted nature, NIR-PIT carries few side effects and healing is rapid. Evaluation of the tumor microenvironment reveals that ICD induced by NIR-PIT results in rapid maturation of immature dendritic cells adjacent to dying cancer cells initiating a host anticancer immune response, resulting in repriming of polyclonal CD8+T cells against various released cancer antigens, which amplifies the therapeutic effect of NIR-PIT. NIR-PIT can target and treat virtually any cell surface antigens including cancer stem cell markers, that is, CD44 and CD133. A first-in-human phase 1/2 clinical trial of NIR-PIT using cetuximab-IR700 (RM1929) targeting EGFR in inoperable recurrent head and neck cancer patients successfully concluded in 2017 and led to “fast tracking” by the FDA and a phase 3 trial (https://clinicaltrials.gov/ct2/show/NCT03769506) that is currently underway in 3 countries in Asia, US/Canada, and 4 countries in EU. The next step for NIR-PIT is to further exploit the immune response. Preclinical research in animals with intact immune systems has shown that NIT-PIT targeting of immunosuppressor cells within the tumor, such as regulatory T-cells, can further enhance tumor-cell-selective systemic host-immunity leading to significant responses in distant metastatic tumors, which are not treated with light. By combining cancer-targeting NIR-PIT and immune-activating NIR-PIT or other cancer immunotherapies, NIR-PIT of a local tumor, could lead to responses in distant metastases and may also inhibit recurrences due to activation of systemic anticancer immunity and long-term immune memory without the systemic autoimmune adverse effects often associated with immune checkpoint inhibitors. Furthermore, NIR-PIT also enhances nanodrug delivery into tumors up to 24-fold superior to untreated tumors with conventional EPR effects by intensively damaging cancer cells behind tumor vessels. We conclude by describing future advances in this novel photochemical cancer therapy that are likely to further enhance the efficacy of NIR-PIT.

Near-infrared photoimmunotherapy (NIR-PIT) is a cell-specific cancer therapy that uses an antibody-photoabsorber (IRDye700DX, IR700) conjugate (APC) and NIR light. Intravenously injected APC binds the target cells, and subsequent NIR light exposure induces immunogenic cell death only in targeted cells. Panitumumab and cetuximab are antibodies that target human epidermal growth factor receptor (hEGFR) and are suitable for NIR-PIT. In athymic nude mouse models, panitumumab-based NIR-PIT showed superior therapeutic efficacy compared to cetuximab-based NIR-PIT because of the longer half-life of panitumumab-IR700 (pan-IR700) compared with cetuximab-IR700 (cet-IR700). Two light exposures on two consecutive days have also been shown to induce superior effects compared to a single light exposure in the athymic nude mouse model. However, the optimal regimen has not been assessed in immunocompetent mice. In this study, we compared panitumumab and cetuximab in APCs for NIR-PIT, and single and double light exposures using a newly established hEGFR-expressing cancer cell line derived from immunocompetent C57BL/6 mice (mEERL-hEGFR cell line). Fluorescence imaging showed that the decline of pan-IR700 was slower than cet-IR700 confirming a longer clearance time. Among all the combinations tested, mice receiving pan-IR700 and double light exposure showed the greatest tumor growth inhibition. This group was also shown to activate CD8

The continuous emergence of microbial pathogens for which there are no effective antimicrobials threatens global health, necessitating novel antimicrobial approaches. Here, we present a targeted antimicrobial strategy that can be applied to various microbial pathogens. A photoimmuno-conjugate composed of an antibody against the target pathogen and a photoplastic phthalocyanine-derivative probe that generates photo-induced mechanical stress was developed based on photoimmuno-technology. This strategy, named as photoimmuno-antimicrobial strategy (PIAS), eliminates targeted pathogens, regardless of the target species or drug-resistance status. Specifically, PIAS acts on a broad range of microbes, including the bacterial pathogen Staphylococcus aureus, fungal pathogen Candida albicans, including their drug-resistant strains, and viral pathogen SARS-CoV-2, the causative agent of COVID-19. Furthermore, PIAS protects mice from fatal infections without damaging the non-targeted host microbiota and tissues. This study may contribute to the development of next-generation anti-infective therapies.

Background near-infrared photoimmunotherapy (NIR-PIT) is a cancer treatment that uses antibody-photoabsorber (IRDye700DX, IR700) conjugates (APCs) which bind to target cells and are photoactivated by NIR light inducing rapid necrotic cell death. NIR-PIT targeting human epidermal growth factor receptor (hEGFR) has been shown to destroy hEGFR expressing human tumor cells and to be effective in immunodeficient mouse models. NIR-PIT can also be targeted to cells in the tumor microenvironment, for instance, CD25-targeted NIR-PIT can be used to selectively deplete regulatory T cells (Tregs) within a tumor. The aim of this study was to evaluate the combined therapeutic efficacy of hEGFR and CD25-targeted NIR-PIT in a newly established hEGFR expressing murine oropharyngeal cell line (mEERL-hEGFR). Methods panitumumab conjugated with IR700 (pan-IR700) was used as the cancer cell-directed component of NIR-PIT and anti-CD25-F(ab′)2-IR700 was used as the tumor microenvironment-directed component of NIR-PIT. Efficacy was evaluated using tumor-bearing mice in four groups: (1) non-treatment group (control), (2) pan-IR700 based NIR-PIT (pan-PIT), (3) anti-CD25-F(ab′)2-IR700 based NIR-PIT (CD25-PIT), (4) combined NIR-PIT with pan-IR700 and anti-CD25- F(ab′)2-IR700 (combined PIT). Findings the combined PIT group showed the greatest inhibition of tumor growth. Destruction of cancer cells likely leads to an immune response which is amplified by the loss of Tregs in the tumor microenvironment. Interpretation combined hEGFR and CD25-targeted NIR-PIT is a promising treatment for hEGFR expressing cancers in which Treg cells play an immunosuppressive role.

Near-infrared photoimmunotherapy (NIR-PIT) is a newly developed cancer treatment that uses antibody-IRDye700DX (IR700) conjugates and was recently approved in Japan for patients with inoperable head and neck cancer. Exposure of the tumor with NIR light at a wavelength of 690 nm leads to physicochemical changes in the antibody-IR700 conjugate-cell receptor complex, resulting in increased hydrophobicity and damage to the integrity of the cell membrane. However, it is important that the tumor be completely exposed to light during NIR-PIT, and thus, a method to provide real-time information on tumor location would help clinicians direct light more accurately. IR700 is a fluorophore that emits at 702 nm; however, there is no clinically available device optimized for detecting this fluorescence. On the other hand, many indocyanine green (ICG) fluorescence imaging devices have been approved for clinical use in operating rooms. Therefore, we investigated whether LIGHTVISION, one of the clinically available ICG cameras, could be employed for NIR-PIT target tumor detection. Due to the limited benefits of adding IR700 molecules, the additional conjugation of IRDye800CW (IR800) or ICG-EG4-Sulfo-OSu (ICG-EG4), which has an overlapping spectrum with ICG, to trastuzumab-IR700 conjugates was performed. Conjugation of second NIR dyes did not interfere the efficacy of NIR-PIT. The dual conjugation of IR800 and IR700 to trastuzumab clearly visualized target tumors with LIGHTVISION by detecting emission light of IR800. We demonstrated that the conjugation of second NIR dyes enables us to provide a real-time feedback of tumor locations prior to NIR-PIT.

Cancer-associated fibroblasts (CAFs) have an important role in the tumor microenvironment. CAFs have the multifunctionality which strongly support cancer progression and the acquisition of therapeutic resistance by cancer cells. Near-infrared photoimmunotherapy (NIR-PIT) is a novel cancer treatment that uses a highly selective monoclonal antibody (mAb)-photosensitizer conjugate. We developed fibroblast activation protein (FAP)-targeted NIR-PIT, in which IR700 was conjugated to a FAP-specific antibody to target CAFs (CAFs-targeted NIR-PIT: CAFs-PIT). Thus, we hypothesized that the control of CAFs could overcome the resistance to conventional chemotherapy in esophageal cancer (EC). In this study, we evaluated whether EC cell acquisition of stronger malignant characteristics and refractoriness to chemoradiotherapy are mediated by CAFs. Next, we assessed whether the resistance could be rescued by eliminating CAF stimulation by CAFs-PIT in vitro and in vivo. Cancer cells acquired chemoradiotherapy resistance via CAF stimulation in vitro and 5-fluorouracil (FU) resistance in CAF-coinoculated tumor models in vivo. CAF stimulation promoted the migration/invasion of cancer cells and a stem-like phenotype in vitro, which were rescued by elimination of CAF stimulation. CAFs-PIT had a highly selective effect on CAFs in vitro. Finally, CAF elimination by CAFs-PIT in vivo demonstrated that the combination of 5-FU and NIR-PIT succeeded in producing 70.9% tumor reduction, while 5-FU alone achieved only 13.3% reduction, suggesting the recovery of 5-FU sensitivity in CAF-rich tumors. In conclusion, CAFs-PIT could overcome therapeutic resistance via CAF elimination. The combined use of novel targeted CAFs-PIT with conventional anticancer treatments can be expected to provide a more effective and sensible treatment strategy.

Recombinant immunotoxins (RITs) are chimeric proteins composed of an Fv and a protein toxin being developed for cancer treatment. The Fv brings the toxin to the cancer cell, but most of the RITs do not reach the tumor and are removed by other organs. To identify cells responsible for RIT removal, and the pathway by which RITs reach these cells, we studied SS1P, a 63-kDa RIT that targets mesothelin-expressing tumors and has a short serum half-life. The major organs that remove RIT were identified by live mouse imaging of RIT labeled with FNIR-Z-759. Cells responsible for SS1P removal were identified by immunohistochemistry and intravital two-photon microscopy of kidneys of rats. The primary organ of SS1P removal is kidney followed by liver. In the kidney, SS1P passes through the glomerulus, is taken up by proximal tubular cells, and transferred to lysosomes. In the liver, macrophages are involved in removal. The short half-life of SS1P is due to its very rapid filtration by the kidney followed by degradation in proximal tubular cells of the kidney. In mice treated with SS1P, proximal tubular cells are damaged and albumin in the urine is increased. SS1P uptake by kidney is reduced by coadministration of l-lysine. Our data suggests that l-lysine administration to humans might prevent SS1P-mediated kidney damage, reduce albumin loss in urine, and alleviate capillary leak syndrome.

Near infrared photoimmunotherapy (NIR-PIT) is a new target-cell specific cancer treatment that induces highly selective necrotic/immunogenic cell death after systemic administration of a photoabsorber antibody conjugate and subsequent NIR light exposure. However, the depth of NIR light penetration in tissue (approximately 2 centimeters) with external light sources, limits the therapeutic effects of NIR-PIT. Interstitial light exposure using cylindrical diffusing optical fibers can overcome this limitation. The purpose in this study was to compare three NIR light delivery methods for treating tumors with NIR-PIT using a NIR laser system at an identical light energy; external exposure alone, interstitial exposure alone, and the combination. Panitumumab conjugated with the photoabsorber, IRDye-700DX (pan-IR700) was intravenously administered to mice with A431-luc xenografts which are epithelial growth factor receptor (EGFR) positive. One and two days later, NIR light was administered to the tumors using one of three methods. Interstitial exposure alone and in combination with external sources showed the greatest decrease in bioluminescence signal intensity. Additionally, the combination of external and interstitial NIR light exposure showed significantly greater tumor size reduction and prolonged survival after NIR-PIT compared to external exposure alone. This result suggested that the combination of external and interstitial NIR light exposure was more effective than externally applied light alone. Although external exposure is the least invasive means of delivering light, the combination of external and interstitial exposures produces superior therapeutic efficacy in tumors greater than 2 cm in depth from the tissue surface.

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 (NIR-PIT) is a newly developed cancer treatment that induces highly selective immunogenic cell death. It is based on an antibody–photoabsorber conjugate (APC) that is activated by NIR light. The purpose of this study was to investigate the effects of NIR-PIT as measured by luciferase–luciferin photon-counting and fluorescence imaging. Six days after subcutaneous injection of A431-luc-GFP cells tumors formed in a xenograft mouse model. The EGFR-targeting antibody, panitumumab, was conjugated to the photoabsorber, IRDye-700DX (pan–IR700), and was intravenously administered to tumor-bearing mice. Serial luciferase–luciferin photon-counting images and both green fluorescent protein (GFP) and IR700 fluorescence images were obtained from the same mice before and after NIR-PIT treatment (0, 10, 20, 30 min (early phase), and 24, 48 h (late phase) after NIR light exposure). Optical signal intensities were compared for each modality. IR700 fluorescence and luciferase–luciferin photon-counting images showed decreased intensities in both the early and late phases after NIR-PIT (p < 0.01). On the other hand, GFP fluorescence images showed decreased intensities only in the late phase (p < 0.01). In the early phase, GFP fluorescence images showed smaller intensity reductions compared to IR700 fluorescence and luciferase–luciferin photon-counting (p < 0.01), while in the late phase, IR700 fluorescence showed smaller intensity reductions than luciferase–luciferin photon-counting and GFP fluorescence (p < 0.05), due to redistribution of pan–IR700 within the tumor bed. In conclusion, luciferase–luciferin photon-counting imaging is suitable to evaluate early phase NIR-PIT effects, while both luciferase–luciferin photon-counting and GFP reflected later phase effects.

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.

Fast, selective, and noninvasive reporting of intracellular cancer-associated events and species will lead to a better understanding of tumorigenesis at the molecular level and development of precision medicine approaches in oncology. Overexpressed reductase presence in solid tumor cells is key to cancer progression and protection of those diseased cells from the oxidative effects of therapeutics meant to kill them. Human NAD(P)H:quinone oxidoreductase isozyme I (hNQO1), a cytoprotective 2-electron-specific reductase found at unusually high activity levels in cancer cells of multiple origins, has attracted significant attention due to its major role in metastatic pathways and its link to low survival rates in patients, as well as its ability to effectively activate quinone-based, anticancer drugs. Accurate assessment of hNQO1 activities in living tumor models and ready differentiation of metastases from healthy tissue by fluorescent light-based protocols requires creation of hNQO1-responsive, near-infrared probes that offer deep tissue penetration and low background fluorescence. Herein, we disclose a quinone-trigger-based, near-infrared probe whose fluorescence is effectively turned on several hundred-fold through highly selective reduction of the quinone trigger group by hNQO1, with unprecedented, catalytically efficient formation of a fluorescent reporter. hNQO1 activity-specific production of a fluorescence signal in two-dimensional cultures of respiring human cancer cells that harbor the reductase enzyme allows for their quick (30 min) high-integrity recognition. The characteristics of the near-infrared probe make possible the imaging of clinically relevant three-dimensional colorectal tumor models possessing spatially heterogeneous hNQO1 activities and provide for fluorescence-assisted identification of submillimeter dimension metastases in a preclinical mouse model of human ovarian serous adenocarcinoma.

Cancer-associated fibroblasts (CAFs) are strongly implicated in tumor progression, including in the processes of tumorigenesis, invasion, and metastasis. The targeting of CAFs using various therapeutic approaches is a novel treatment strategy; however, the efficacy of such therapies remains limited. Recently, near-infrared photoimmunotherapy (NIR-PIT), which is a novel targeted therapy employing a cell-specific mAb conjugated to a photosensitizer, has been introduced as a new type of phototherapy. In this study, we have developed a novel NIR-PIT technique to target CAFs, by focusing on fibroblast activation protein (FAP), and we evaluate the treatment efficacy in vitro and in vivo. Esophageal carcinoma cells exhibited enhanced activation of fibroblasts, with FAP over-expressed in the cytoplasm and on the cell surface. FAP-IR700-mediated PIT showed induced rapid cell death specifically for those cells in vitro and in vivo, without adverse effects. This novel therapy for CAFs, designed as local control phototherapy, was safe and showed a promising inhibitory effect on FAP(+) CAFs. PIT targeting CAFs via the specific marker FAP may be a therapeutic option for CAFs in the tumor microenvironment in the future.

Near‐infrared photoimmunotherapy (NIR‐PIT) is a molecularly targeted cancer phototherapy that is based on injecting a conjugate of a silicon‐phthalocyanine derivative, IRdye 700DX (IR700), and a monoclonal antibody that targets an expressed antigen on the cancer cell surface. Subsequent local exposure to NIR light results in the rapid and highly selective immunogenic cell death of targeted cancer cells. Because many cancers grow in bones through which light does not penetrate well, the goal of this study was to determine if NIR‐PIT can effectively treat cancers in bone. A bovine rib was used as a bone sample. Because the sample’s NIR light transmittance was shown to be approximately 4.52% in preliminary tests, it was hypothesized that a maximum radiation dosage of 128 and 1500 J/cm2 would be sufficient to induce cell death in in vitro target cells and in vivo mouse tumor models, respectively. Cell viability was measured through bioluminescence studies comparing relative luciferase activity, as well as a cytotoxicity assay. In the in vitro model, tumor cell viability was significantly decreased after 64 and 128 J/cm2 NIR light irradiation through the bone. An in vivo mouse tumor model also showed that 1500 J/cm2 NIR light irradiation through the bone significantly reduced tumor viability at both 24 and 48 hours posttreatment compared to the control group (P = .026 and .040 respectively). Therefore, despite limitations in light transmission, NIR‐PIT nevertheless is capable of effectively treating cancers within bone., Tumors were successfully treated through a bovine bone sample.

// Mikako Ogawa 1, 7, * , Yusuke Tomita 2, * , Yuko Nakamura 3 , Min-Jung Lee 2 , Sunmin Lee 2 , Saori Tomita 2 , Tadanobu Nagaya 3 , Kazuhide Sato 3 , Toyohiko Yamauchi 4 , Hidenao Iwai 4 , Abhishek Kumar 5 , Timothy Haystead 6 , Hari Shroff 5 , Peter L. Choyke 3 , Jane B. Trepel 2 , Hisataka Kobayashi 3 1 Medical Photonics Research Center, Hamamatsu University School of Medicine, Hamamatsu 431-3192, Japan 2 Developmental Therapeutics Branch, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, MD 20892, USA 3 Molecular Imaging Program, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, MD 20892, USA 4 Central Research Laboratory, Hamamatsu Photonics K. K., Hamamatsu 434-8601, Japan 5 Section on High Resolution Optical Imaging, NIBIB/NIH, Bethesda, MD 20892, USA 6 Department of Pharmacology and Cancer Biology, Duke University, Durham, NC 27710, USA 7 Laboratory for Bioanalysis and Molecular Imaging, Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo 060-0812, Japan * Co-first author Correspondence to: Hisataka Kobayashi, email: Kobayash@mail.nih.gov Keywords: near infrared photoimmunotherapy, immunogenic cell death Received: October 12, 2016 Accepted: December 13, 2016 Published: January 02, 2017 ABSTRACT Immunogenic cell death (ICD) is a form of cell death that activates an adaptive immune response against dead-cell-associated antigens. Cancer cells killed via ICD can elicit antitumor immunity. ICD is efficiently induced by near-infrared photo-immunotherapy (NIR-PIT) that selectively kills target-cells on which antibody-photoabsorber conjugates bind and are activated by NIR light exposure. Advanced live cell microscopies showed that NIR-PIT caused rapid and irreversible damage to the cell membrane function leading to swelling and bursting, releasing intracellular components due to the influx of water into the cell. The process also induces relocation of ICD bio markers including calreticulin, Hsp70 and Hsp90 to the cell surface and the rapid release of immunogenic signals including ATP and HMGB1 followed by maturation of immature dendritic cells. Thus, NIR-PIT is a therapy that kills tumor cells by ICD, eliciting a host immune response against tumor.

// Toshiko Harada 1 , Yuko Nakamura 1 , Kazuhide Sato 1 , Tadanobu Nagaya 1 , Shuhei Okuyama 1 , Fusa Ogata 1 , Peter L. Choyke 1 , Hisataka Kobayashi 1 1 Molecular Imaging Program, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, Maryland, 20892, USA Correspondence to: Hisataka Kobayashi, email: kobayash@mail.nih.gov Keywords: near-infrared photoimmunotherapy, ovarian cancer, peritoneal cancer metastases, galactosyl serum albumin, beta-D-galactose receptor Received: August 15, 2016 Accepted: September 30, 2016 Published: October 17, 2016 ABSTRACT 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.

// Yuko Nakamura 1 , Toshiko Harada 1 , Tadanobu Nagaya 1 , Kazuhide Sato 1 , Shuhei Okuyama 1 , Peter L. Choyke 1 , Hisataka Kobayashi 1 1 Molecular Imaging Program, Center for Cancer Research, National Cancer Institute, Bethesda, MD 20892, USA Correspondence to: Hisataka Kobayashi, email: kobayash@mail.nih.gov Keywords: kinetic map, green emitting probe, autofluorescence, γ-glutamyltranspeptidase, peritoneal cancer metastases Received: April 25, 2016 Accepted: May 22, 2016 Published: June 07, 2016 ABSTRACT Optical fluorescence-guided imaging is increasingly used to guide surgery and endoscopic procedures. Activatable probes are particularly useful because of high target-to-background ratios that increase sensitivity for tiny cancer foci. However, green fluorescent activatable probes suffer from interference from autofluorescence found in biological tissue. The purpose of this study was to determine if dynamic imaging can be used to differentiate specific fluorescence arising from an activated probe in a tumor from autofluorescence in background tissues especially when low concentrations of the dye are applied. Serial fluorescence imaging was performed using various concentrations of γ-glutamyl hydroxymethyl rhodamine green (gGlu-HMRG) which was sprayed on the peritoneal surface with tiny implants of SHIN3-DsRed ovarian cancer tumors. Temporal differences in signal between specific green fluorescence in cancer foci and non-specific autofluorescence in background tissue were measured at 5, 10, 20 and 30 min after application of gGlu-HMRG and were processed into three kinetic maps reflecting maximum fluorescence signal (MF), wash-in rate (WIR), and area under the curve (AUC), respectively. Using concentrations up to 10 μM of gGlu-HMRG, the fluorescence intensity of cancer foci was significantly higher than that of small intestine but only at 30 min. However, on kinetic maps derived from dynamic fluorescence imaging, the signal of cancer foci was significantly higher than that of small intestine after only 5 min even at concentrations as low as 2.5 μM of gGlu-HMRG ( p < 0.01). At lower concentrations, kinetic maps derived from dynamic fluorescence imaging were superior to unprocessed images for cancer detection.

Near-infrared photoimmunotherapy (NIR-PIT), which employs monoclonal antibody (mAb)-phototoxic phthalocyanine dye IR700 conjugates, permits the specific, image-guided and spatiotemporally controlled elimination of tumor cells. Here, we report the highly efficient NIR-PIT of human tumor xenografts initiated from patient-derived cancer stem cells (CSCs). Using glioblastoma stem cells (GBM-SCs) expressing the prototypic CSC marker AC133/CD133, we also demonstrate here for the first time that NIR-PIT is highly effective against brain tumors. The intravenously injected theranostic AC133 mAb conjugate enabled the non-invasive detection of orthotopic gliomas by NIR fluorescence imaging, and reached AC133+ GBM-SCs at the invasive tumor front. AC133-targeted NIR-PIT induced the rapid cell death of AC133+ GBM-SCs and thereby strong shrinkage of both subcutaneous and invasively growing brain tumors. A single round of NIR-PIT extended the overall survival of mice with established orthotopic gliomas by more than a factor of two, even though the harmless NIR light was applied through the intact skull. Humanised versions of this theranostic agent may facilitate intraoperative imaging and histopathological evaluation of tumor borders and enable the highly specific and efficient eradication of CSCs.

Near Infrared-Photoimmunotherapy (NIR-PIT) is a new, highly selective tumor treatment that employs an antibody-photon absorber conjugate (APC). When the APC attaches to its target cell and is exposed to NIR light, highly selective cell killing is observed. NIR-PIT has been demonstrated with a limited number of antibodies. Mesothelin is overexpressed in several malignancies and is emerging as a therapeutic target. A recently humanized antibody (hYP218) has been generated against mesothelin that demonstrates high affinity binding. Here, we describe the efficacy of NIR-PIT, using hYP218 as the antibody within the APC to target a mesothelin expressing A431/H9 cell. The hYP218 antibody was conjugated to a photo-absorber, IR700 and incubated with the cells. The hYP218-IR700 showed specific binding to cells and cell-specific killing was observed in vitro. After implanting A431/H9 cells in an athymic nude mouse, tumor-bearing mice were treated with the following regimen of NIR-PIT; 100 μg of hYP218-IR700 i.v., NIR light was administered at 50 J/cm2 on day 1 after injection and 100 J/cm2 of light on day 2 after injection. The hYP218-IR700 showed high tumor accumulation and a high tumor-background ratio (TBR). Tumor growth was significantly inhibited by NIR-PIT treatment compared with the other control groups (p < 0.001), and significantly prolonged survival (p < 0.0001 vs other groups). Thus, the new anti-mesothelin antibody, hYP218, is suitable as an antibody-drug conjugate for NIR-PIT. Furthermore, NIR-PIT with hYP218-IR700 is a promising candidate for the treatment of mesothelin-expressing tumors that could be readily translated to humans.

Near infrared photoimmunotherapy(NIR-PIT)is a new molecularly-targeted cancer photo-therapy based on conjugating a near infrared silica-phthalocyanine dye, IRDye700DX(IR700), to a monoclonal antibody(mAb)targeting cell-surface molecules. NIR-PIT targetingEGFR usingthe cetuximab-IR700 conjugate is now in transition to fast-track global Phase Ⅲ clinical trial in late-stage head and neck squamous cell cancer patients. When exposed to NIR light, the conjugate induces a highlyselective necrotic/immunogenic cell death(ICD)only in target-positive, mAb-IR700-bound cancer cells. This cell death occurs as early as 1 minute after exposure to NIR light. Meanwhile, immediately adjacent target-negative cells are unharmed. ICD induced by NIR-PIT promoted maturation of immature dendritic cells(DCs)to maturated DCs that primed cytotoxic Tcells to react with cancer-related antigens released from destroyed cancer cells. Inhibition of immuno-checkpoints including PD-1/PD-L1 axis or CTLA-4 is one of several major methods for enhancingcytotoxicity of tumor-infiltratingT -cells. In this section, I will also discuss NIR-PIT combined with an immuno-checkpoint inhibitor that could treat not only local tumors but also distant untreated metastatic tumors without recurrence in some syngeneic cancer models. Additionally, NIR-PIT to a local tumor combined with local tumor-infiltratingneg ative-regulatory T-cells by CD25-targeted NIR-PIT that is another major methods for enhancingcytotoxicity of tumor-infiltratingT -cells similarly operated in different tumor models. The regimens of NIR-PIT combined with methods for enhancinghost tumor immunity could cure primary and metastatic tumor and yield vaccination effect against treated cancer cells that would be the ultimate form of NIR-PIT in oncology clinic.

Near infrared (NIR) fluorescent probes are attractive tools for biomedical in vivo imaging due to the relatively deeper tissue penetration and lower background autofluorescence. Activatable probes are turned on only after binding to their target, further improving target to background ratios. However, the number of available activatable NIR probes is limited. In this study, we introduce two types of activatable NIR fluorophores derived from bacteriochlorin; chlorin-bacteriochlorin energy-transfer dyads and boron-dipyrromethene (BODIPY)-bacteriochlorin energy-transfer dyads. These fluorophores are characterized by multiple narrow excitation bands with relatively strong emission in the NIR. Targeted bacteriochlorin-based antibody or peptide probes have been previously limited by aggregation after conjugation. Polyethylene glycol (PEG) chains were added to improve the hydrophilicity without altering pharmacokinetics of the targeting moieties. These PEGylated bacteriochlorin-based activatable fluorophores have potential as targeted activatable, multi-color NIR fluorescent probes for in vivo applications.

Near-infrared photoimmunotherapy (NIR-PIT) induces immunogenic cell death but has mostly failed to induce durable antitumor responses in syngenic tumor mouse models. We hypothesized that adaptive immune resistance could be limiting durable responses after treatmemt with NIR-PIT. We investigated the effects of combining NIR-PIT targeting cell-surface CD44 and PD-1 blockade in multiple syngeneic tumor models. In two of three models, NIR-PIT monotherapy halted tumor growth, enhanced dendritic cell tumor infiltration, and induced de novo tumor antigen–specific T-cell responses absent at baseline. The addition of PD-1 blockade reversed adaptive immune resistance, resulting in both enhanced preexisting tumor antigen–specific T-cell responses and enhanced de novo T-cell responses induced by NIR-PIT. Enhanced immune responses correlated with shared tumor antigen expression, suggesting that antigenicity is a major determinant of response to combination NIR-PIT and PD-1 blockade. Combination treatment induced complete rejection of MC38 tumors treated with NIR-PIT, as well as untreated, distant tumors. Accordingly, tumor antigen–specific T-cell responses were measured in both treated and untreated tumors, validating the development of systemic antitumor immunity. Mice that cleared tumors resisted subsequent tumor challenge, indicating the presence of systemic immune memory. Cumulatively, these results demonstrate reversal of adaptive immune resistance following induction of innate and adaptive immunity by NIR-PIT, resulting in high rates of tumor rejection and/or significant tumor growth control in antigenic syngeneic models of cancer.

As a novel low-side-effect cancer therapy, photo-immunotherapy (PIT) is based on conjugating monoclonal antibody (mAb) with a near-infrared (NIR) phthalocyanine dye IRDye700DX (IR 700). IR700 is not only fluorescent to be used as an imaging agent, but also phototoxic. When illuminating with NIR light, PIT can induce highly-selective cancer cell death while leaving most of tumor blood vessels unharmed, leading to an effect termed super-enhanced permeability and retention (SUPR), which can significantly improve the effectiveness of anti-cancer drug. Currently, the therapeutic effects of PIT are monitored using 2D macroscopic fluorescence reflectance imager, which lacks the resolution and depth information to reveal the 3D distribution of mAb-IR700. In the study, we applied a multi-modal optical imaging approach including high-resolution optical coherence tomography (OCT) and high-sensitivity fluorescence laminar optical tomography (FLOT), to provide 3D tumor micro-structure and micro-distribution of mAb-IR700 in the tumor simultaneously during PIT in situ and in vivo. The multi-wavelength FLOT can also provide the blood vessels morphology of the tumor. Thus, the 3D FLOT reconstructed images allow us to evaluate the IR700 fluorescence distribution change with respect to the blood vessels and at different tumor locations/depths non-invasively, thereby enabling evaluation of the therapeutic effects in vivo and optimization of treatment regimens accordingly. The mAb-IR700 can access more tumor areas after PIT treatment, which can be explained by increased vascular permeability immediately after NIR-PIT. Two-photon microscopy was also used to record the mAb-IR700 on the tumor surface near the blood vessels to verify the results.

Photoimmunotherapy is a new class of molecular targeted cancer therapy based on a monoclonal antibody (mAb) conjugated to a photosensitizer and irradiation with near-infrared (NIR) light for both imaging and therapy. Here, we sought to determine the feasibility of combining photoimmunotherapy using conjugates of human epidermal growth factor receptor 2 (HER2)-specific mAb-photosensitizer IR700, trastuzumab-IR700 and pertuzumab-IR700. HER2-expressing and non-expressing cells were treated with mAb-IR700 conjugates and irradiated with NIR light. Fluorescence imaging and cytotoxic effects were examined in cultured HER2-expressng cancer cell lines and in a mouse tumor xenograft model. Trastuzumab-IR700 and pertuzumab-IR700 could specifically bind to HER2 without competing, and the combination treatment of both agents yielded stronger HER2-specific IR700 fluorescence signals than with the treatment with either agent singly. A cytotoxicity assay showed that the combination treatment of both trastuzumab-IR700 and pertuzumab-IR700 followed by NIR light irradiation induced stronger cytotoxic effect than with treatment of either agent plus NIR light irradiation. Furthermore, the phototoxic and cytotoxic effects of mAb depended on HER2-specific IR700 signal intensities. Consistent with in vitro studies, in xenograft tumor models also, IR700 fluorescence imaging-guided NIR light irradiation after the combination treatment of trastuzumab-IR700 and pertuzumab-IR700 led to stronger antitumor effects than by treatment with either agent followed by NIR light irradiation. In conclusion, fluorescence molecular imaging can facilitate the assessment of treatment outcomes of molecular targeted photoimmunotherapy, which holds great potential in facilitating better outcomes in cancer patients.

Near infrared photoimmunotherapy (NIR-PIT) is a new cancer treatment that combines the specificity of antibodies for targeting tumors with the toxicity induced by photosensitizers after exposure to near infrared (NIR) light. Herein we compare two NIR-light sources; light emitting diodes (LEDs) and Lasers, for their effectiveness in NIR-PIT. A photosensitizer, IRDye-700DX, conjugated to panitumumab (pan-IR700), was incubated with EGFR-expressing A431 and MDA-MB-468-luc cells. NIR-light was provided by LEDs or Lasers at the same light dose. Laser-light produced more cytotoxicity and greater reductions in IR700-fluorescence intensity than LED-light. Laser-light also produced more cytotoxicity in vivo in both cell lines. Assessment of super-enhanced permeability and retention (SUPR) effects were stronger with Laser than LED. These results suggest that Laser-light produced significantly more cytotoxic effects compared to LEDs. Although LED is less expensive, Laser-light produces superior results in NIR-PIT.

Near infrared photoimmunotherapy (NIR-PIT) is a new cancer treatment that combines the specificity of antibodies for targeting tumors with the toxicity induced by photon absorbers after irradiation with NIR light. The purpose of this study was to determine if MR imaging can detect changes in the MR properties of tumor within several hours of NIR-PIT. A431 cells were injected subcutaneously in the right and left dorsi of 12 mice. Six days later, the mice were injected with a photon absorber, IR700, conjugated to panitumumab, an antibody targeting epidermal growth factor receptor. One day later, only right sided tumor was exposed to NIR light (treated tumor). MRI was performed 1 day before and 1-2 hours after NIR-PIT using gadofosveset for six mice and gadopentetate dimeglumine for another six mice. T2 relaxation times, the apparent diffusion coefficient (ADC) for the following combinations of b-values: 0-1000, 200-1000 and 500-1000 s/mm2 and enhancement indices were compared before and after NIR-PIT using a two-sided paired t-test. For treated tumors, T2 relaxation time increased after NIR-PIT (p < 0.01) and all three ADC values decreased after NIR-PIT (p < 0.01). Moreover, the enhancement area under the curve (AUC) using gadofosveset increased after NIR-PIT (p = 0.02). In conclusion, prolongation of T2, reductions in ADC and increased enhancement using gadofosveset are seen within 2 hours of NIR-PIT treatment of tumors. Thus, MRI can be a useful imaging biomarker for detecting early therapeutic changes after NIR-PIT.

Glypican-3 (GPC3) represents an attractive target for hepatocellular carcinoma (HCC) therapy because it is highly expressed in HCC but not in adult normal tissue. Recently, high affinity anti-GPC3 antibodies have been developed; however, full antibodies may not penetrate evenly into tumor parenchyma, reducing their effectiveness. In this study, we compared a whole IgG antibody, anti-GPC3 YP7, with an anti-GPC3 human heavy chain antibody, HN3, with regard to their relative therapeutic effects. Both YP7 and HN3 bound to GPC3-positive A431/G1 cells and were internalized by the cells by in vitro evaluation with (125)I- and (111)In-radiolabeling antibodies. In vivo biodistribution and tumor accumulation was performed with (111)In-labeled antibodies, and intratumoral microdistribution was evaluated using fluorescently labeled antibodies (IR700). HN3 showed similar high tumor accumulation but superior homogeneity within the tumor compared with YP7. Using the same IR700 conjugated antibodies photoimmunotherapy (PIT) was performed in vitro and in a tumor-bearing mouse model in vivo. PIT with IR700-HN3 and IR700-YP7 demonstrated that comparable results could be achieved despite of low reaccumulation 24 h after the first NIR light exposure. These results indicated that a heavy-chain antibody, HN3, showed more favorable characteristics than YP7, a conventional IgG, as a therapeutic antibody platform for designing molecularly targeted agents against HCC.

BACKGROUND: Peritoneal dissemination (PD) of abdominal malignancies is a common form of metastasis and its presence signals a poor prognosis. New treatment is required for patients with PD. Near infrared photoimmunotherapy (NIR-PIT) is a highly selective tumor treatment that employs an antibody-photo-absorber conjugate (APC). In this study, we investigate in vitro and in vivo efficacy of trastuzumab (tra)-IR700DX NIR-PIT on a human epidermal growth factor receptor type 2 (HER2)-positive gastric cancer cell line. METHODS: NIR-PIT effects were investigated in vitro and in vivo. Disseminated peritoneal implants mice were separated into 5 groups: (1) no treatment; (2) tra-IR700 i.v. only; (3) NIR light only; (4) NIR-PIT; (5) repeated NIR-PIT. The peritoneal cavity was irradiated with NIR light using a fiber optic diffuser delivered through the catheter. RESULTS: Specific binding and cell-specific killing was observed after NIR-PIT in vitro. In the in vivo study, fluorescence endoscopy showed high tumor accumulation of tra-IR700 within tumors. Significantly prolonged survival was achieved in the three treatment groups (tra-IR700 i.v. only, NIR-PIT, and repeated NIR-PIT groups) compared with control and NIR light only group (p < 0.05 for tra-IR700 i.v. only, p < 0.01 for NIR-PIT, and p < 0.0001 for repeated NIR-PIT). Moreover, most prolonged survival was shown for the repeated NIR-PIT group (p < 0.0001 vs tra-IR700 i.v. only, p < 0.01 vs NIR-PIT). CONCLUSION: NIR-PIT using a fiber optic diffuser to deliver light is a promising candidate for the treatment of disseminated peritoneal metastases and could be readily translated to humans.

Objectives Tumour-positive resection margins are a major problem during oral cancer surgery. gGlu-HMRG is a tracer that becomes fluorescent upon activation by gamma-glutamyltranspeptidase (GGT). This study aims to investigate the combination of gGlu-HMRG and a clinical fluorescence imaging system for the detection of tumour-positive resection margins. Materials and methods The preclinical Maestro and clinical Artemis imaging systems were compared in vitro and ex vivo with cultured human head and neck cancer cells (OSC19, GGT-positive; and FaDu, GGT negative) and tumour-bearing nude mice. Subsequently, frozen sections of normal and oral cancer tissues were ex vivo sprayed with gGlu-HMRG to determine the sensitivity and specificity. Finally, resection margins of patients with suspected oral cancer were ex vivo sprayed with gGlu-HMRG to detect tumour-positive resection margins. Results Both systems could be used to detect gGlu-HMRG activation in vitro and ex vivo in GGT positive cancer cells. Sensitivity and specificity of gGlu-HMRG and the Artemis on frozen tissue samples was 80% and 87%, respectively. Seven patients undergoing surgery for suspected oral cancer were included. In three patients fluorescence was observed at the resection margin. Those margins were either tumour-positive or within 1 mm of tumour. The margins of the other patients were clear (≥8 mm). Conclusion This study demonstrates the feasibility to detect tumour-positive resection margins with gGlu-HMRG and a clinical fluorescence imaging system. Applying this technique would enable intraoperative screening of the entire resection margin and allow direct re-resection in case of tumour-positivity.

Pleural metastases are common in patients with advanced thoracic cancers and are a cause of considerable morbidity and mortality yet is difficult to treat. Near Infrared Photoimmunotherapy (NIR-PIT) is a cancer treatment that combines the specificity of intravenously injected antibodies for targeting tumors with the toxicity induced by photosensitizers after exposure to NIR-light. Herein, we evaluate the efficacy of NIR-PIT in a mouse model of pleural disseminated non-small cell lung carcinoma (NSCLC). In vitro and in vivo experiments were conducted with a HER2, luciferase and GFP expressing NSCLC cell line (Calu3-luc-GFP). An antibody-photosensitizer conjugate (APC) consisting of trastuzumab and a phthalocyanine dye, IRDye-700DX, was synthesized. In vitro NIR-PIT cytotoxicity was assessed with dead staining, luciferase activity, and GFP fluorescence intensity. In vivo NIR-PIT was performed in mice with tumors implanted intrathoracic cavity or in the flank, and assessed by tumor volume and/or bioluminescence and fluorescence thoracoscopy. In vitro NIR-PIT-induced cytotoxicity was light dose dependent. In vivo NIR-PIT led significant reductions in both tumor volume (p = 0.002 vs. APC) and luciferase activity (p = 0.0004 vs. APC) in a flank model, and prolonged survival (p < 0.0001). Bioluminescence indicated that NIR-PIT lead to significant reduction in pleural dissemination (1 day after PIT; p = 0.0180). Fluorescence thoracoscopy confirmed the NIR-PIT effect on disseminated pleural disease. In conclusion, NIR-PIT has the ability to effectively treat pleural metastases caused by NSCLC in mice. Thus, NIR-PIT is a promising therapy for pleural disseminated tumors.

The use of light as a means of therapy for bladder cancer has a long history but has been hampered by a lack of tumor specificity and therefore, damage to the normal bladder mucosa. Here, we describe a targeted form of phototherapy called photoimmunotherapy (PIT), which targets EGFR-expressing bladder cancer. Anti-EGFR antibody panitumumab was labeled with the photoabsorber (PA), IRDye 700Dx (IR700), to create a panitumumab-IR700 antibody–PA conjugate that is activated by near-infrared radiation (NIR). Bladder cancer tissue microarray (TMA) and bladder cancer cell lines were analyzed for expression of EGFR. Mechanism of PIT-induced cell death was studied using proliferation assays, transmission electron microscopy (TEM), and production of reactive oxygen species. Finally, the in vivo effect was studied in xenografts. EGFR staining of TMAs showed that while most bladder cancers have expression of EGFR to a varying degree, squamous cell carcinomas (SCC) have the highest expression of EGFR. Panitumumab-IR700 activated by NIR light rapidly killed UMUC-5 cells, a bladder SCC line. Panitumumab alone, panitumumab-IR700 without NIR, or NIR alone had no effect on cells. TEM demonstrated that cell death is due to necrosis. Singlet oxygen species contributed toward cell death. NIR-PIT with panitumumab-IR700 reduced growth compared with only panitumumab-IR700–treated UMUC-5 xenograft tumors. PIT is a new targeted treatment for bladder cancer. Panitumumab-IR700–induced PIT selectively kills EGFR-expressing bladder cancer cells in vitro and in vivo and therefore warrants further therapeutic studies in orthotopic xenografts of bladder cancer and ultimately in patients. Mol Cancer Ther; 16(10); 2201–14. ©2017 AACR.

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.

Employing NOD/SCID/Jak3(−/−) mice transplanted with human PBMCs (hNOJ mice) and replication-competent, red-fluorescent-protein (mCherry; mC)-labeled HIV-1(JR-FL) (HIV(mC)), we examined whether early antiretroviral treatment blocked the establishment of HIV-1 infection. The use of hNOJ mice and HIV(mC) enabled us to visually locate infection foci and to examine the early dynamics of HIV(mC) infection without using a large amount of antiretroviral unlike in non-human primate models. Although when raltegravir (RAL) administration was begun 1 day after intraperitoneal (ip) inoculation of HIV(mC), no plasma p24 or plasma HIV-1-RNA (pRNA) were detected in 10 of 12 hNOJ [Formula: see text] mice as assessed on the last day of the 14-day continuous twice-daily RAL administration, all 10 untreated hNOJ(mC) [Formula: see text] mice became positive for p24 and pRNA and had significantly swollen lymph nodes in peritoneal cavity and abundant p24(+)/mC(+)/CD3(+)/CD4(+) T cells and p24(+)/mC(+)/CD68(+) monocytes/macrophages were identified in their omenta and mesenteric lymphoid tissues/lymph nodes upon necropsy of the mice on day 14. In 12 [Formula: see text] mice, no significantly swollen lymph nodes were seen compared to [Formula: see text] mice; however, in the omentum of the 2 [Formula: see text] mice that were positive for pRNA and in site RNA, mC(+)/p24(+)/CD3(+)/CD83(+) cells were identified, suggesting that viral breakthrough occurred later in the observation period. The present data suggest that the use of hNOJ mouse model and HIV(mC) may shed light on the study of early-phase dynamics of HIV-1 infection and cellular events in post-exposure/pre-exposure prophylaxis.

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.