Opening up new VISTAs: V-domain immunoglobulin suppressor of T cell activation (VISTA) targeted near-infrared photoimmunotherapy (NIR-PIT) for enhancing host immunity against cancers.

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.

Comparison of the Effectiveness of IgG Antibody versus F(ab')2 Antibody Fragment in CTLA4-Targeted Near-Infrared Photoimmunotherapy.

Near-infrared photoimmunotherapy (NIR-PIT) is a novel cancer treatment modality that utilizes antibody-photoabsorber conjugates (APCs) and selectively kills target cells after irradiation with NIR light. Originally, NIR-PIT was targeted against cancer cell surface antigens, but as it became clear that NIR-PIT induced a strong immune response, an effort was made to target selected immune cell populations in the tumor microenvironment to encourage an even stronger immune response. Thus, CD25-targeted NIR-PIT and cytotoxic T-lymphocyte associated protein 4 (CTLA4)-targeted NIR-PIT were developed to kill regulatory T cells (Tregs) in conjunction with cancer-cell-targeted NIR-PIT, in order to amplify the host immune response. It was found that CD25-targeted NIR-PIT, using an antibody with the Fc portion removed, led to better results than the unmodified anti-CD25 antibody-directed NIR-PIT presumably because of a negative effect on activated T cells. The aim of this study was to compare the efficacy of an antibody fragment [anti-CTLA4-F(ab')2] and a whole antibody (anti-CTLA4-IgG) for NIR-PIT. There was no significant difference in NIR-PIT-induced Treg killing between the anti-CTLA4-F(ab')2 and anti-CTLA4-IgG antibodies. Although both the antibody and the antibody fragment resulted in significant tumor growth inhibition, the antibody induced more robust CD8+ T cell activation in ipsilateral lymph nodes and was more effective compared to the antibody fragment. The slower clearance of the anti-CTLA4-IgG APC enhanced antitumor immunity by promoting T cell priming in lymph nodes. In conclusion, unlike the results with CD25 where modified antibodies produced superior results to unmodified antibodies, anti-CTLA4-IgG antibody-based NIR-PIT proved more effective in reducing tumor growth than anti-CTLA4-F(ab')2 antibody-based NIR-PIT.

In vivo imaging of acute physiological responses after treatment of cancer with near-infrared photoimmunotherapy.

PURPOSE: Near-infrared photoimmunotherapy (NIR-PIT) is a new cancer phototherapy using an antibody-photosensitizer conjugate (Ab-IR700). By NIR light irradiation, Ab-IR700 forms a water-insoluble aggregation on the plasma membrane of cancer cells, leading to lethal membrane damage of cancer cells with high selectivity. However, IR700 produces singlet oxygen, which induces non-selective inflammatory responses such as edema in normal tissues around the tumor. Understanding such treatment-emergent responses is important to minimize side effects and improve clinical outcomes. Thus, in this study, we evaluated physiological responses during NIR-PIT by magnetic resonance imaging (MRI) and positron emission tomography (PET). PROCEDURES: Ab-IR700 was intravenously injected into tumor-bearing mice with two tumors on the right and left sides of the dorsum. At 24 h after injection, a tumor was irradiated with NIR light. Edema formation was examined by T1/T2/diffusion-weighted MRI and inflammation was investigated by PET with 2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG). Because inflammation can increase vascular permeability via inflammatory mediators, we evaluated changes in oxygen levels in tumors using a hypoxia imaging probe, [18F]fluoromisonidazole ([18F]FMISO). RESULTS: The uptake of [18F]FDG in the irradiated tumor was significantly decreased compared to the control tumor, indicating the impairment of glucose metabolism induced by NIR-PIT. MRI and [18F]FDG-PET images showed that inflammatory edema with [18F]FDG accumulation was present in the surrounding normal tissues of the irradiated tumor. Furthermore, [18F]FMISO accumulation in the center of the irradiated tumor was relatively low, indicating the enhancement of oxygen supply due to increased vascular permeability. In contrast, high [18F]FMISO accumulation was observed in the peripheral region, indicating enhancement of hypoxia in the region. This could be because inflammatory edema was formed in the surrounding normal tissues, which blocked blood flow to the tumor. CONCLUSIONS: We successfully monitored inflammatory edema and changes in oxygen levels during NIR-PIT. Our findings on the acute physiological responses after light irradiation will help to develop effective measures to minimize the side effects in NIR-PIT.

In Vitro Comparative Study of Near-Infrared Photoimmunotherapy and Photodynamic Therapy.

Near-infrared photoimmunotherapy (NIR-PIT) is a new phototherapy that utilizes a monoclonal antibody (mAb) against cancer antigens and a phthalocyanine dye, IRDye700DX (IR700) conjugate (mAb-IR700). Photodynamic therapy (PDT) is a combination therapy that utilizes photoreactive agents and light irradiation as well as NIR-PIT. In the present study, we compared these therapies in vitro. The characterization of cellular binding/uptake specificity and cytotoxicity were examined using two mAb-IR700 forms and a conventional PDT agent, talaporfin sodium, in three cell lines. As designed, mAb-IR700 had high molecular selectivity and visualized target molecule-positive cells at the lowest concentration examined. NIR-PIT induced necrosis and damage-associated molecular patterns (DAMPs), a surrogate maker of immunogenic cell death. In contrast, talaporfin sodium was taken up by cells regardless of cell type, and its uptake was enhanced in a concentration-dependent manner. PDT induced cell death, with the pattern of cell death shifting from apoptosis to necrosis depending on the concentration of the photosensitizer. Induction of DAMPs was observed at the highest concentration, but their sensitivity differed among cell lines. Overall, our data suggest that molecule-specific NIR-PIT may have potential advantages compared with PDT in terms of the efficiency of tumor visualization and induction of DAMPs.

Antibody Drug Conjugates of Near-Infrared Photoimmunotherapy (NIR-PIT) in Breast Cancers.

Worldwide, the incidence rate of breast cancer is the highest in women. Approximately 2.3 million people were newly diagnosed and 0.685 million were dead of breast cancer in 2020, which continues to grow. Triple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype with a higher risk of recurrence and metastasis, but disappointly, there are no effective and specific therapies clinically, especially for patients presenting with metastatic diseases. Therefore, it is urgent to develop a new type of cancer therapy for survival improvisation and adverse effects alleviation of breast cancers. Near-infrared photoimmunotherapy (NIR-PIT) is a newly developed, photochemistry-based cancer therapy. It was drive by an antibody-photoabsorber conjugate (APC) which is triggered by near-infrared light. The key part of APC is a cancer-targeting monoclonal antibody (mAb) that can bind to receptors or antigens on the surface of tumor cells. Because of this targeted conjugate accumulation, subsequent deployment of focal NIR-light results in functional damage on the targeted cell membranes without harming the immediately adjacent receptor-negative cells and evokes a kind of photochemical, speedy, and highly specific immunogenic cell death (ICD) of cancer cells with corresponding antigens. Subsequently, immature dendritic cells adjacent to dying cancer cells will become mature, further inducing a host-oriented anti-cancer immune response, complicatedly and comprehensively. Currently, NIR-PIT has progressed into phase 3 clinical trial for recurrent head and neck cancer. And preclinical studies have illustrated strong therapeutic efficacy of NIR-PIT targeting various molecular receptors overexpressed in breast cancer cells, including EGFR, HER2, CD44c, CD206, ICAM-1 and FAP-α. Thereby, NIR-PIT is in early trials, but appears to be a promising breast cancer therapy and moving into the future. Here, we present the specific advantages and discuss the most recent preclinical studies against several transmembrane proteins of NIR-PIT in breast cancers.

Distinct sulfated glycans expressed in intrahepatic cholangiocarcinoma: a potential target for new therapy.

Cholangiocytes exhibit morphological and functional heterogeneity, depending on their anatomical localization; however, like other ductal organs, their mucosal surface is covered with mucin, which functions to prevent the entry of foreign substances, lubricate and prevent clogging by bile. Recently, the authors discovered that distinct sulfated glycans recognized by a series of antisulfated glycan antibodies are expressed not only in normal intrahepatic bile ducts but also in intrahepatic cholangiocarcinoma (iCCA). In this review, the authors first describe the anatomy of bile ducts and the biochemical characteristics of bile-duct-associated mucins, and then describe differences in structure and expression patterns of these sulfated glycans in physiological and pathological conditions. Finally, potential therapeutic strategies for iCCA using antisulfated glycan antibodies are discussed.

Endoscopic Applications of Near-Infrared Photoimmunotherapy (NIR-PIT) in Cancers of the Digestive and Respiratory Tracts.

Near-infrared photoimmunotherapy (NIR-PIT) is a newly developed and promising therapy that specifically destroys target cells by irradiating antibody-photo-absorber conjugates (APCs) with NIR light. APCs bind to target molecules on the cell surface, and when exposed to NIR light, cause disruption of the cell membrane due to the ligand release reaction and dye aggregation. This leads to rapid cell swelling, blebbing, and rupture, which leads to immunogenic cell death (ICD). ICD activates host antitumor immunity, which assists in killing still viable cancer cells in the treated lesion but is also capable of producing responses in untreated lesions. In September 2020, an APC and laser system were conditionally approved for clinical use in unresectable advanced head and neck cancer in Japan, and are now routine in appropriate patients. However, most tumors have been relatively accessible in the oral cavity or neck. Endoscopes offer the opportunity to deliver light deeper within hollow organs of the body. In recent years, the application of endoscopic therapy as an alternative to surgery for the treatment of cancer has expanded, providing significant benefits to inoperable patients. In this review, we will discuss the potential applications of endoscopic NIR-PIT, especially in thoracic and gastrointestinal cancers.

CD29 targeted near-infrared photoimmunotherapy (NIR-PIT) in the treatment of a pigmented melanoma model.

Near-infrared photoimmunotherapy (NIR-PIT) is a newly developed cancer treatment that utilizes an antibody-photoabsorber-conjugate (AbPC) combined with NIR light. The AbPC is injected and binds to the tumor whereupon NIR light irradiation causes a photochemical reaction that selectively kills cancer cells. NIR-PIT is ideal for surface-located skin cancers such as melanoma. However, there is concern that the pigment in melanoma lesions could interfere with light delivery, rendering treatment ineffective. We investigated the efficacy of CD29- and CD44-targeted NIR-PIT (CD29-PIT and CD44-PIT, respectively) in the B16 melanoma model, which is highly pigmented. While CD29-PIT and CD44-PIT killed B16 cells invitro and invivo, CD29-PIT suppressed tumor growth more efficiently. Ki67 expression showed that cells surviving CD29-PIT were less proliferative, suggesting that CD29-PIT was selective for more proliferative cancer cells. CD29-PIT did not kill immune cells, whereas CD44-PIT killed both T and NK cells and most myeloid cells, including DCs, which could interfere with the immune response to NIR-PIT. The addition of anti-CTLA4 antibody immune checkpoint inhibitor (ICI) to CD29-PIT increased the infiltration of CD8 T cells and enhanced tumor suppression with prolonged survival. Such effects were less prominent when the anti-CTLA4 ICI was combined with CD44-PIT. The preservation of immune cells in the tumor microenvironment (TME) after CD29-PIT likely led to a better response when combined with anti-CTLA4 treatment. We conclude that NIR-PIT can be performed in pigmented melanomas and that CD29 is a promising target for NIR-PIT, which is amenable to combination therapy with other immunotherapies.

Selective depletion of polymorphonuclear myeloid derived suppressor cells in tumor beds with near infrared photoimmunotherapy enhances host immune response.

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+ T cell activation were enhanced in both intratumoral tissues and tumor-draining lymph nodes, and NK cells demonstrated increased expression of cytotoxic molecules. Host immunity remained activated in the TME for at least one week after NIR-PIT. Abscopal effects in bilateral tumor models were observed. Furthermore, the combination of NIR-PIT targeting cancer cells and PMN-MDSCs yielded synergistic effects and demonstrated highly activated host tumor immunity. In conclusion, we demonstrated that selective local PMN-MDSCs depletion by NIR-PIT could be a promising new cancer immunotherapy.

Combinatory therapy of MRP1-targeted photoimmunotherapy and liposomal doxorubicin promotes the antitumor effect for chemoresistant small cell lung cancer.

Small cell lung cancer (SCLC), considered a mortal recalcitrant cancer, is a severe healthcare issue because of its poor prognosis, early metastasis, drug resistance and limited clinical treatment options. In our previous study, we established a MRP1-targeted antibody-IR700 system (Mab-IR700) for near infrared photoimmunotherapy (NIR-PIT) which exhibited a promising therapeutic effect on drug resistant H69AR cells both in vitro and in vivo, though the tumor growth suppression effect did not last long with a single round of PIT treatment. To achieve a better anticancer effect, we have combined Mab-IR700-mediated NIR-PIT with liposomal doxorubicin (Doxil®) and investigated the in vitro and in vivo cytotoxicity by using a H69AR/3T3 cell co-culture model in which 3T3 cells were used to mimic stromal cells. Cytotoxicity experiments demonstrated the specificity of Mab-IR700 to H69AR cells, while cytotoxicity and flow cytometry experiments confirmed that H69AR cells were doxorubicin-resistant. Compared with Mab-IR700-mediated PIT or Doxil-mediated chemotherapy, the combination therapy exhibited the best cell killing effect in vitro and superior tumor growth inhibition and survival prolongation effect in vivo. Super enhanced permeability and retention (SUPR) effect was observed in both co-culture spheroids and tumor-bearing mice. Owing to an approximately 9-fold greater accumulation of Doxil within the tumors, NIR-PIT combined with Doxil resulted in enhanced antitumor effects compared to NIR-PIT alone. This photoimmunochemotherapy is a practical strategy for the treatment of chemoresistant SCLC and should be further investigated for clinical translation.

Near Infrared Photoimmunotherapy; A Review of Targets for Cancer Therapy.

Near-infrared photoimmunotherapy (NIR-PIT) is a newly developed cancer treatment that uses an antibody-photoabsorber (IRDye700DX) conjugate (APC) that is activated by NIR light irradiation. In September 2020, the first APC and laser system were conditionally approved for clinical use in Japan. A major benefit of NIR-PIT is that only APC-bound cancer cells that are exposed to NIR light are killed by NIR-PIT; thus, minimal damage occurs in adjacent normal cells. These early trials have demonstrated that in addition to direct cell killing, there is a significant therapeutic host immune response that greatly contributes to the success of the therapy. Although the first clinical use of NIR-PIT targeted epidermal growth factor receptor (EGFR), many other targets are suitable for NIR-PIT. NIR-PIT has now been applied to many cancers expressing various cell-surface target proteins using monoclonal antibodies designed to bind to them. Moreover, NIR-PIT is not limited to tumor antigens but can also be used to kill specific host cells that create immune-permissive environments in which tumors grow. Moreover, multiple targets can be treated simultaneously with NIR-PIT using a cocktail of APCs. NIR-PIT can be used in combination with other therapies, such as immune checkpoint inhibitors, to enhance the therapeutic effect. Thus, NIR-PIT has great potential to treat a wide variety of cancers by targeting appropriate tumor cells, immune cells, or both, and can be augmented by other immunotherapies.

Near infrared photoimmunotherapy of cancer; possible clinical applications.

Near-infrared photoimmunotherapy (NIR-PIT) is a new cancer treatment that uses an antibody-photo-absorber conjugate (APC) composed of a targeting monoclonal antibody conjugated with a photoactivatable phthalocyanine-derivative dye, IRDye700DX (IR700). APCs injected into the body can bind to cancer cells where they are activated by local exposure to NIR light typically delivered by a NIR laser. NIR light alters the APC chemical conformation inducing damage to cancer cell membranes, resulting in necrotic cell death within minutes of light exposure. NIR-PIT selectivity kills cancer cells by immunogenic cell death (ICD) with minimal damage to adjacent normal cells thus, leading to rapid recovery by the patient. Moreover, since NIR-PIT induces ICD only on cancer cells, NIR-PIT initiates and activates antitumor host immunity that could be further enhanced when combined with immune checkpoint inhibition. NIR-PIT induces dramatic changes in the tumor vascularity causing the super-enhanced permeability and retention (SUPR) effect that dramatically enhances nanodrug delivery to the tumor bed. Currently, a worldwide Phase 3 study of NIR-PIT for recurrent or inoperable head and neck cancer patients is underway. In September 2020, the first APC and accompanying laser system were conditionally approved for clinical use in Japan. In this review, we introduce NIR-PIT and the SUPR effect and summarize possible applications of NIR-PIT in a variety of cancers.

Comparison of low-molecular-weight ligand and whole antibody in prostate-specific membrane antigen targeted near-infrared photoimmunotherapy.

Near-infrared photoimmunotherapy (NIR-PIT) is a cancer phototherapy that uses antibody-IR700 conjugate (Ab-IR700) and NIR light. Ab-IR700 forms aggregates on the plasma membranes of targeted cancer cells after light exposure, inducing lethal physical damage within the membrane. Low-molecular-weight (LMW) ligands are candidate targeting moieties instead of antibodies, but whether LMW-IR700 conjugates induce cell death by aggregation, the same mechanism as Ab-IR700, is unknown. Thus, we investigated differences in cytotoxicity and mechanisms between LMW-IR700 and Ab-IR700 targeting prostate-specific membrane antigen (PSMA). Both conjugates decreased cell viability to the same degree after light irradiation, but different morphological changes were observed in PSMA-positive LNCaP cells by microscopy. Cell swelling and bleb formation were induced by Ab-IR700, but only swelling was observed in cells treated with LMW-IR700, suggesting the cells were damaged via different cytotoxic mechanisms. However, LMW-IR700 induced bleb formation, a hallmark of NIR-PIT with Ab-IR700, when singlet oxygen was quenched or LMW-IR700 was localized only on the plasma membrane. Moreover, the water-soluble axial ligands of LMW-IR700 were cleaved, consistent with previous reports on Ab-IR700. Thus, the main cytotoxic mechanisms of Ab-IR700 and LMW-IR700 differ, although LMW-IR700 on the plasma membrane can cause aggregation-mediated cytotoxicity as well as Ab-IR700.

MRP1-targeted near infrared photoimmunotherapy for drug resistant small cell lung cancer.

Small cell lung cancer (SCLC), one of the most aggressive cancers, has a high mortality rate and poor prognosis, and the clinical therapeutic outcomes of multidrug resistant SCLC are even worse. Multidrug resistance protein 1 (MRP1), one of the ATP-binding cassette (ABC) transporter proteins that cause decreased drug accumulation in cancer cells, is overexpressed in drug resistant SCLC cells and could be a promising target for treating the patients suffering from this illness. Near infrared photoimmunotherapy (NIR-PIT) is a newly developed approach for targeted cancer treatment which uses a conjugate of a monoclonal antibody and photoabosorber IR700 followed by NIR light irradiation to induce rapid cancer cell death. In the present study, an anti-MRP1 antibody (Mab) -IR700 conjugate (Mab-IR700) was synthesized, purified and used to treat chemoresistant SCLC H69AR cells that overexpressed MRP1, while non-MRP1-expressing H69 cells were used as a control. Then, the photokilling and tumor suppression effect were separately evaluated in H69AR cells both in vitro and in vivo. Higher cellular delivery of Mab-IR700 was detected in H69AR cells, whereas there was little uptake of IgG-IR700 in both H69 and H69AR cells. Due to the targeting activity of Mab, stronger photokilling effect was found both in H69AR cells and spheroids treated with Mab-IR700, while superior tumor suppression effect was also observed in the mice treated with Mab-IR700 and light illumination. Photoacoustic imaging results proved that oxygen was involved in NIR-PIT treatment, and TUNEL staining images showed the occurrence of cell apoptosis, which was also testified by HE staining. This research provides MRP1 as a novel target for PIT and presents a prospective way for treating drug resistant SCLC and, thus, should be further studied.

Implantable wireless powered light emitting diode (LED) for near-infrared photoimmunotherapy: device development and experimental assessment in vitro and in vivo.

PURPOSE: The aim of this study was to develop and assess a novel implantable, wireless-powered, light-emitting diode (LED) for near-infrared photoimmunotherapy (NIR-PIT). NIR-PIT is a recently developed cancer therapy that uses NIR light and antibody-photosensitizer conjugates and is able to induce cancer-specific cell death. Due to limited light penetration depth it is currently unable to treat tumors in deep tissues. Use of implanted LED might potentially overcome this limitation. RESULTS: The wireless LED system was able to emit NIR light up to a distance of 20 cm from the transmitter coil by using low magnetic fields as compliant with limits for use in humans. Results indicated that the LED system was able to kill tumor cells in vitro and to suppress tumor growth in implanted tumor-bearing mice. CONCLUSIONS: Results indicated that the proposed implantable wireless LED system was able to suppress tumor growth in vivo. These results are encouraging as wireless LED systems such as the one here developed might be a possible solution to treat tumors in deep regions in humans. Further research in this area would be important. MATERIALS AND METHODS: An implantable LED system was developed. It consisted of a LED capsule including two LED sources and a receiver coil coupled with an external coil and power source. Wireless power transmission was guaranteed by using electromagnetic induction. The system was tested in vitro by using EGFR-expressing cells and HER2-expressing cells. The system was also tested in vivo in tumor-bearing mice.