Effect Of Near-infrared Photoimmunotherapy Research Articles

Preclinical analysis of the efficacy of near-infrared photoimmunotherapy and anti-PD-1 antibody combination therapy for treating CD73-positive lung cancer.

e14521 Background: The CD73/adenosine pathway, which converts extracellular ATP to adenosine, is emerging as a promising target for immunotherapy in various cancers. In the tumor microenvironment, hypoxia triggers upregulation of adenosine-activating molecules, including CD73, a membrane-anchored enzyme expressed on cancer cells. CD73 induces the expression of PD-1 and CTLA4, and all of this leads to immunosuppression. Therefore, we focused utilizing near-infrared photoimmunotherapy (NIR-PIT) to kill CD73-expressing cells and alleviate immunosuppression. NIR-PIT is an innovative cancer treatment approach which involves irradiating NIR light after the administrated antibody-photosensitizer conjugate binds to the target cells. Methods: In vitro, anti-CD73 antibody-IR700 conjugate was prepared and added to the CMT167 and LLC cell lines, which were irradiated with NIR light under a microscope. In vivo, a mouse model was established using the CMT167 cell line. The mice were divided into 6 groups: 1) NIR-PIT group against CD73; 2) anti-PD-1 antibody group; 3) combined treatment group of 1) and 2); 4) control group; 5) anti-CD73 antibody-IR700 conjugate group; and 6) near-infrared light irradiation group. Tumor volume and survival rate were compared among these groups. Additionally, after 2 weeks of treatment, tumors in the four groups 1) to 4) above were pathologically evaluated using two different AI-equipped software programs. Results: Microscope observation revealed that NIR-PIT induced immunogenic cell death of CD73-positive mouse lung cancer cell lines in vitro. In vivo, the effect of NIR-PIT on this mouse model was also demonstrated. Although previous studies have shown the efficacy of treatment with anti-CD73 antibodies, the present study compared the CD73-IR700 conjugate group to the NIR-PIT to CD73 group, and the latter group showed better therapeutic outcomes. Additionally, the combined treatment group displayed the highest anti-tumor efficacy in terms of survival and tumor volume. Pathological evaluation of the tumor interior showed a significant increase in tumor-infiltrating lymphocytes compared to the other groups. Additionally, the percentage of tumor cells was significantly reduced, while the percentage of stroma, including fibrous tissue, was significantly increased. In line with other studies that have already reported the additive effect of combination therapy with anti-CD73 antibodies and immune checkpoint inhibitors, the present study showed that the combination therapy resulted in further activation of immune cells and enhanced the therapeutic effect. Conclusions: CD73 represents a promising target for NIR-PIT, with potential for enhanced effectiveness when used in combination with immune checkpoint inhibitors.

Near-infrared photoimmunotherapy and anti-cancer immunity.

The activation of the anti-cancer immune system is an important strategy to control cancer. A new form of cancer phototherapy, near-infrared photoimmunotherapy (NIR-PIT), was approved for clinical use in 2020 and uses IRDye® 700DX (IR700)-conjugated antibodies and NIR light. After irradiation with NIR light, the antibody-IR700 conjugate forms water-insoluble aggregations on the plasma membrane of target cells. This aggregation causes lethal damage to the plasma membrane, and effectively leads to immunogenic cell death (ICD). Subsequently, ICD activates anti-cancer immune cells such as dendritic cells and cytotoxic T cells. Combination therapy with immune-checkpoint blockade has synergistically improved the anti-cancer effects of NIR-PIT. Additionally, NIR-PIT can eliminate immunosuppressive immune cells in light-irradiated tumors by using specific antibodies against regulatory T cells and myeloid-derived suppressor cells. In addition to cancer-cell-targeted NIR-PIT, such immune-cell-targeted NIR-PIT has shown promising results by activating the anti-cancer immune system. Furthermore, NIR-PIT can be used to manipulate the tumor microenvironment by eliminating only targeted cells in the tumor, and thus it also can be used to gain insight into immunity in basic research.

Near-Infrared Photoimmunotherapy for Oropharyngeal Cancer.

Human papillomavirus (HPV)-associated oropharyngeal cancer has a better prognosis than other head and neck cancers. However, rates of recurrence and metastasis are similar and the prognosis of recurrent or metastatic HPV-associated oropharyngeal cancer is poor. Near-infrared photoimmunotherapy (NIR-PIT) is a treatment involving administration of a photosensitizer (IRDye®700DX) conjugated to a monoclonal antibody followed by activation with near-infrared light illumination. It is a highly tumor-specific therapy with minimal toxicity in normal tissues. Moreover, NIR-PIT is expected to have not only direct effects on a treated lesion but also immune responses on untreated distant lesions. NIR-PIT with cetuximab-IR700 (AlluminoxTM) has been in routine clinical use since January 2021 for unresectable locally advanced or locally recurrent head and neck cancer in patients that have previously undergone radiotherapy in Japan. NIR-PIT for head and neck cancer (HN-PIT) is expected to provide a curative treatment option for the locoregional recurrent or metastatic disease after radiotherapy and surgery. This article reviews the mechanism underlying the effect of NIR-PIT and recent clinical trials of NIR-PIT for head and neck cancers, treatment-specific adverse events, combination treatment with immune checkpoint inhibitors, illumination approach and posttreatment quality of life, and provides a case of series of two patients who receive NIR-PIT for oropharyngeal cancer at our institution.

Optimal Light Dose for hEGFR-Targeted Near-Infrared Photoimmunotherapy.

Simple SummaryNear-infrared photoimmunotherapy (NIR-PIT) is a cancer therapy that selectively destroys target cells by first injecting monoclonal antibodies conjugated with a photon absorber (IRDye700DX) into the subject and then activating it at the tumor site by applying nonthermal doses of NIR light at 690 nm. NIR-PIT causes immediate immunogenic cell death but also induces a slightly delayed activation of anti-tumor host immunity which can result in complete responses. The immediate therapeutic effect of NIR-PIT can be enhanced by increasing the dose of near-infrared light irradiation; however, this can cause local side effects such as edema. Since the activation of host immunity also adds to the anti-tumor effect it might be possible to reduce the light dose to avoid immediate side effects while maintaining efficacy of the therapy. In this study, we varied the light dose needed to achieve the maximum therapeutic effect in an immunocompetent mouse model. We show that higher-than-needed light doses caused significant local transient edema that could be avoided with lower but still effective light doses. Here, we present our strategy for optimizing the light dose for NIR-PIT.Near-infrared photoimmunotherapy (NIR-PIT) is a newly developed cancer therapy that targets cancer cells using a monoclonal antibody-photon absorber conjugate (APC) that is bound to the target cell surface. Subsequent application of low levels of NIR light results in immediate cancer cell death. The anti-tumor effect of NIR-PIT in immunocompromised mice depends on immediate cancer cell death; therefore, the efficacy increases in a light-dose-dependent manner. However, NIR-PIT also induces a strong anti-tumor immune activation in immunocompetent mice that begins soon after therapy. Thus, it may be possible to reduce the light dose, which might otherwise cause local edema while maintaining therapeutic efficacy. In this study, we determined the optimal dose of NIR light in NIR-PIT based on a comparison of the therapeutic and adverse effects. Either one of two monoclonal antibodies (mAbs) against human epidermal growth factor receptor (hEGFR), Cetuximab or Panitumumab, were conjugated with a photo-absorbing chemical, IRDye700DX (IR700), and then injected in hEGFR-expressing mEERL (mEERL-hEGFR) tumor-bearing C57BL/6 immunocompetent mice or A431-GFP-luc tumor-bearing athymic immunocompromised mice. NIR light was varied between 0 to 100 J/cm2 one day after administration of APC. In an immunocompromised mouse model, tumor growth was inhibited in a light-dose-dependent manner, yet extensive local edema and weight loss were observed at 100 J/cm2. On the other hand, in an immunocompetent mouse model using the mEERL-hEGFR cell line, maximal tumor response was achieved at 50 J/cm2, with a commensurate decrease in local edema. In this study, we show that a relatively low dose of NIR light is sufficient in an immunocompetent mouse model and avoids side effects seen with higher light doses required in immunocompetent mice. Thus, light dosing can be optimized in NIR-PIT based on the expected immune response.

Abstract 5328: Near-infrared photoimmunotherapy for pancreatic cancer: Targeting cell surface glycans using lectin-photoabsorber conjugate (LPC)

Abstract Introduction: Since protein targets in cancer therapy largely have been explored, glycans would be attractive alternative targets. We had previously discovered a fucosylated glycan epitope highly expressed on pancreatic cancer cells, and a lectin probe called rBC2LCN, which can be used as a specific targeting bullet to this glycan (Shimomura O, Oda T, et al: Mol Cancer Ther 2018). We now then hypothesized that, this lectin-based cancer-targeting strategies should be reasonably applicable to near-infrared photoimmunotherapy (NIR-PIT). NIR-PIT have already developed using a conjugate of a photoabsorber, IRdye700DX (IR700), and a monoclonal antibody which recognizes protein antigens on the target cells. In this study, we firstly developed a lectin-photoabsorber conjugate (BC2-IR700) and investigated a new cancer therapy targeting cancer cell surface glycans. Material and Methods: We developed BC2-IR700 by conjugation of rBC2LCN lectin with IR700 NHS ester. A pancreatic cancer cell line Capan-1 was used as a model which is bound by rBC2LCN. The cytotoxic effect of NIR-PIT with BC2-IR700 was first evaluated in vitro. We then evaluated the anti-cancer effect of BC2-IR700 in vivo using pancreatic cancer xenograft mice including subcutaneous tumor model. Results: The product of BC2-IR700 was confirmed by SDS-PAGE, that showed reasonable 16KDa sharp band with clear fluorescence intensity. After incubation with BC2-IR700, Capan-1 cells showed high fluorescence signal, which was confirmed with flow cytometry and fluorescence microscopy. BC2-IR700 was detected on the cell surface of Capan-1 after incubation for 1h. Immediately after exposure to NIR light, the cell death of Capan-1 was induced and the cytotoxicity significantly increased in a light-dose-dependent manner. After intravenous administration of BC2-IR700 into pancreatic cancer xenograft mice, BC2-IR700 was observed in vivo fluorescence imaging to be accumulated in tumor. NIR-PIT was performed by exposing to NIR light (100 J/cm2) at 6 h following BC2-IR700 intravenous administration (20 μg/mice). Tumor growth was significantly inhibited in the NIR-PIT treatment group compared with the other control groups such as no treatment, BC2-IR700 only, and NIR light exposure only (P < 0.05). Conclusions: The lectin-photoabsorber conjugate, BC2-IR700, was able to be sufficiently applied for NIR-PIT in in vivo mouse model. The novel NIR-PIT targeting cell surface glycans could be a promising therapeutic strategy for the treatment of pancreatic cancer. Citation Format: Yukihito Kuroda, Osamu Shimomura, Hiromitsu Nakahashi, Tomoaki Furuta, Yoshihiro Miyazaki, Takahito Nakajima, Hiroaki Tateno, Tatsuya Oda. Near-infrared photoimmunotherapy for pancreatic cancer: Targeting cell surface glycans using lectin-photoabsorber conjugate (LPC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5328.

Near-infrared photoimmunotherapy targeting GPR87: Development of a humanised anti-GPR87 mAb and therapeutic efficacy on a lung cancer mouse model.

BackgroundGPR87 is a G-protein receptor that is specifically expressed in tumour cells, such as lung cancer, and rarely expressed in normal cells. GPR87 is a promising target for cancer therapy, but its ligand is controversial. Near-infrared photoimmunotherapy (NIR-PIT) is a novel cancer therapy in which a photosensitiser, IRDye700DX (IR700), binds to antibodies and specifically destroys target cells by irradiating them with near-infrared-light. Here, we aimed to develop a NIR-PIT targeting GPR87.MethodsWe evaluated the expression of GPR87 in resected specimens of lung cancer and malignant pleural mesothelioma (MPM) resected at Nagoya University Hospital using immunostaining. Humanised anti-GPR87 antibody (huGPR87) was generated by introducing CDRs from mouse anti-GPR87 antibody generated by standard hybridoma method. HuGPR87 was conjugated with IR700 and the therapeutic effect of NIR-PIT was evaluated in vitro and in vivo using lung cancer or MPM cell lines.FindingsAmong the surgical specimens, 54% of lung cancer and 100% of MPM showed high expression of GPR87. It showed therapeutic effects on lung cancer and MPM cell lines in vitro, and showed therapeutic effects in multiple models in vivo.InterpretationThese results suggest that NIR-PIT targeting GPR87 is a promising therapeutic approach for the treatment of thoracic cancer.FundingThis research was supported by the Program for Developing Next-generation Researchers (Japan Science and Technology Agency), KAKEN (18K15923, 21K07217, JSPS), FOREST-Souhatsu, CREST (JST).

Near-Infrared Photoimmunotherapy Targeting GPR87: Development of a Humanized Anti-GPR87 Mab and Therapeutic Efficacy on a Lung Cancer Mouse Model

Background: GPR87 is a G protein receptor that is specifically expressed in tumor cells, such as lung cancer, and rarely expressed in normal cells. GPR87 is a promising target for cancer therapy, but its ligand is controversial. Near-infrared photoimmunotherapy (NIR-PIT) is a novel cancer therapy in which a photosensitizer, IRDye700DX (IR700), binds to antibodies and specifically destroys target cells by irradiating them with near-infrared light. Here, we aimed to develop a NIR-PIT targeting GPR87. Methods: We evaluated the expression of GPR87 in resected specimens of lung cancer and malignant pleural mesothelioma (MPM) resected at Nagoya University Hospital using immunostaining. Humanized anti GPR87 antibody (huGPR87) was generated by introducing VH and VL genes into mouse anti GPR87 antibody which generated by standard hybridoma method. HuGPR87 was conjugated with IR700 and the therapeutic effect of NIR-PIT was evaluated in vitro and in vivo using lung cancer or MPM cell lines. Findings: Among the surgical specimens, 54% of lung cancer and 100% of MPM showed high expression of GPR87. It showed therapeutic effects on lung cancer and MPM cell lines in vitro, and showed therapeutic effects in multiple models in vivo. Interpretation: These results suggest that NIR-PIT targeting GPR87 is a promising therapeutic approach for the treatment of thoracic cancer. Funding: Funding: This research was supported by the Program for Developing Next-generation Researchers (Japan Science and Technology Agency), KAKEN (18K15923, JSPS), Souhatsu (JST). Declaration of Interest: None to declare Ethical Approval: All in vivo procedures were performed in accordance with the Nagoya University Animal Care and Use Committee's Guide for the Management and Use of Laboratory Animal Resources (approval numbers 2017-29438, #2018-30096, # 2019-31234, #2020-20104). The use of specimens from patients was approved by the Ethics Committee of the Nagoya University Clinical Research Committee (Approval No. 2018-0046).

Near infrared photoimmunotherapy targeting the cutaneous lymphocyte antigen for mycosis fungoides

ABSTRACT Background: Mycosis fungoides (MF) is a low-grade T-cell lymphoma with primary cutaneous involvement accounting for more than half of all primary cutaneous lymphomas. The treatment of MF is very challenging due to the limited therapies available. Near‐infrared photoimmunotherapy (NIR-PIT) is a newly developed and highly selective cancer treatment that employs a monoclonal antibody conjugated to a photo-absorber dye, the hydrophilic phthalocyanine IRdye 700DX® (IR700), and near infrared light. In this study, we investigated the effect of NIR-PIT on MF targeting the cell-surface antigen cutaneous lymphocyte antigen (CLA) Matherial and methods: MF derived My-La CD4+ cells were incubated with the anti-CLA antibody conjugated to IR700 and then irradiated with a 690 nm near-infrared light. Cell death was evaluated by propidium iodide staining and flow cytometry 24 hours after irradiation. Results: Treatment with anti-CLA or light irradiation exhibited very modest pro-death effects, whereas treatment with the anti-CLA antibody conjugated to IR700 and then irradiation with a 690 nm near-infrared light induced a substantial increase in death in the MF cell line. Conclusions: NIR-PIT targeting CLA to treat MF showed marked antitumour effects. As such, CLA-targeted NIR-PIT could be a promising treatment for MF and, possibly, other cutaneous diseases characterized by CLA+ skin infiltrating T-cells.

Wound healing after excision of subcutaneous tumors treated with near-infrared photoimmunotherapy.

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 of cancer: a new approach that kills cancer cells and enhances anti-cancer host immunity.

Near-infrared photoimmunotherapy (NIR-PIT) is a recently developed hybrid cancer therapy that directly kills cancer cells as well as producing a therapeutic host immune response. Conventional immunotherapies, such as immune-activating cytokine therapy, checkpoint inhibition, engineered T cells and suppressor cell depletion, do not directly destroy cancer cells, but rely exclusively on activating the immune system. NIR-PIT selectively destroys cancer cells, leading to immunogenic cell death that initiates local immune reactions to released cancer antigens from dying cancer cells. These are characterized by rapid maturation of dendritic cells and priming of multi-clonal cancer-specific cytotoxic T cells that kill cells that escaped the initial direct effects of NIR-PIT. The NIR-PIT can be applied to a wide variety of cancers either as monotherapy or in combination with conventional immune therapies to further activate anti-cancer immunity. A global Phase 3 clinical trial (https://clinicaltrials.gov/ct2/show/NCT03769506) of NIR-PIT targeting the epidermal growth factor receptor (EGFR) in patients with recurrent head and neck cancer is underway, employing RM1929/ASP1929, a conjugate of anti-EGFR antibody (cetuximab) plus the photo-absorber IRDye700DX (IR700). NIR-PIT has been given fast-track recognition by regulators in the USA and Japan. A variety of imaging methods, including direct IR700 fluorescence imaging, can be used to monitor NIR-PIT. As experience with NIR-PIT grows, additional antibodies will be employed to target additional antigens on other cancers or to target immune-suppressor cells to enhance host immunity. NIR-PIT will be particularly important in patients with localized and locally advanced cancers and may help such patients avoid side-effects associated with surgery, radiation and chemotherapy.

CD47-Targeted Near-Infrared Photoimmunotherapy for Human Bladder Cancer.

Near-infrared photoimmunotherapy (NIR-PIT) is a localized molecular cancer therapy combining a photosensitizer-conjugated mAb and light energy. CD47 is an innate immune checkpoint widely expressed on bladder cancer cells, but absent from luminal normal urothelium. Targeting CD47 for NIR-PIT has the potential to selectively induce cancer cell death and minimize damage to normal urothelium. The cytotoxic effect of NIR-PIT with anti-CD47-IR700 was investigated in human bladder cancer cell lines and primary human bladder cancer cells derived from fresh surgical samples. Phagocytosis assays were performed to evaluate macrophage activity after NIR-PIT. Anti-CD47-IR700 was administered to murine xenograft tumor models of human bladder cancer for in vivo molecular imaging and NIR-PIT. Cytotoxicity in cell lines and primary bladder cancer cells significantly increased in a light-dose-dependent manner with CD47-targeted NIR-PIT. Phagocytosis of cancer cells significantly increased with NIR-PIT compared with antibody alone (P = 0.0002). In vivo fluorescence intensity of anti-CD47-IR700 in tumors reached a peak 24-hour postinjection and was detectable for at least 14 days. After a single round of CD47-targeted NIR-PIT, treated animals showed significantly slower tumor growth compared with controls (P < 0.0001). Repeated CD47-targeted NIR-PIT treatment further slowed tumor growth (P = 0.0104) and improved survival compared with controls. CD47-targeted NIR-PIT increased direct cancer cell death and phagocytosis resulting in inhibited tumor growth and improved survival in a murine xenograft model of human bladder cancer.

Near Infrared Photoimmunotherapy with Combined Exposure of External and Interstitial Light Sources

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 cm) 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 2 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.

Evaluation of Early Therapeutic Effects after Near-Infrared Photoimmunotherapy (NIR-PIT) Using Luciferase-Luciferin Photon-Counting and Fluorescence Imaging.

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.

Dynamic changes in the cell membrane on three dimensional low coherent quantitative phase microscopy (3D LC-QPM) after treatment with the near infrared photoimmunotherapy.

Near infrared photoimmunotherapy (NIR-PIT) is a newly developed cancer therapy that relies on the binding of a near-infrared antibody photoabsorber conjugate (APC) to a cancer cell. Subsequent exposure to NIR light selectively induces rapid necrotic cell death on target-expressing cells with minimal off-target effects. When treated with NIR-PIT, targeted cells become swollen, develop blebs and burst within minutes of light exposure. Detailed spatial and temporal morphological changes of the cellular membrane of targeted cells treated with NIR-PIT have not been fully explored with state-of-the-art microscopic methods. In this study, we investigated the morphologic and kinetic effects of PIT on two types of cells, a spindle-shaped 3T3/Her cell and a spheric-shaped MDA-MB468 cell, after NIR-PIT using three-dimensional low-coherent quantitative phase microscopy (3D LC-QPM). Adhesive cells treated with NIR-PIT demonstrated region-specific cell membrane rupture occurring first on the distal free edge of the cell near the site of adhesion, in a process that was independent of cell shape. The results show that the peripheral portions of the cell membrane near the site of adhesion are particularly vulnerable to the effects of NIR-PIT, likely because these sites exhibit higher baseline surface tension.

Avoiding thermal injury during near-infrared photoimmunotherapy (NIR-PIT): the importance of NIR light power density.

Near-infrared photoimmunotherapy (NIR-PIT) is a newly-established cancer treatment which employs the combination of an antibody-photoabsorber conjugate (APC) and NIR light. When NIR light is absorbed by APC-bound tissues, a certain amount of heat is generated locally. For the most part this results in a subclinical rise in skin temperature, however, excessive light exposure may cause non-specific thermal damage. In this study, we investigated the potential for thermal damage caused by NIR-PIT by measuring surface temperature. Two sources of light, laser and light emitting diode (LED), were compared in a mouse tumor model. First, we found that the skin was heated rapidly by NIR light regardless of whether laser or LED light sources were used. Air cooling at the surface reduced the rise in temperature. There were no associations between the rise of skin temperature and tumor volume of the treated tumor, or APC concentration. Second, we investigated the extent of thermal damage to the skin at various light doses. We detected burn injuries 1 day after NIR-PIT, when the NIR light was at a power density higher than 600 mW/cm2. Successful treatments at lower power density could be achieved if the total light energy absorbed by the tumor was the same, i.e. by extending the duration of light exposure. In conclusion, this study demonstrates that thermal injury after NIR-PIT can be avoided by either employing a cooling system or by lowering the power density of the light source and prolonging the exposure time such that the total energy is constant. Thus, thermal damage is preventable side effect of NIR-PIT.

Near infrared photoimmunotherapy of B-cell lymphoma

Near infrared photoimmunotherapy (NIR-PIT) is a new, highly-selective cancer theranostics that employs an antibody-photo absorber conjugate (APC). NIR-PIT has successfully treated preclinical tumor models with APCs and is now in the first-in-human phase 1 clinical trial for head and neck cancer patients against EGFR. CD20 is highly expressed in many B-cell lymphomas and is emerging as a molecular target for this disease. Here, we describe the use of the anti-CD20 monoclonal antibody (mAb), rituximab-IR700 APC for NIR-PIT of B-cell lymphoma in two CD20-expressing lymphoma mouse models. CD20 expressing B-cell lymphoma cell lines (Daudi and Ramos) were used in this study. Rituximab-IR700, rituximab conjugated with IRDye700DX, showed specific binding, and cell-specific killing only after exposure of NIR light to both cells in vitro. To evaluate effects of NIR-PIT in vivo, tumor-bearing mice were separated into 4 groups: (1) control; (2) APC i.v. only; (3) NIR light exposure only; (4) APC and NIR light (NIR-PIT). These were performed every week for up to 3 weeks. Rituximab-IR700 showed high tumor accumulation and high target-to-background ratio in vivo. Tumor growth was significantly inhibited by NIR-PIT in comparison with the other groups (p < 0.001 for both tumors), and survival was significantly prolonged in both tumors (p < 0.001 for Daudi tumors and p < 0.0001 for Ramos tumors vs other groups). More than half of tumors were cured with this single regimen of NIR-PIT. In conclusion, anti-CD20 rituximab-IR700 works as a highly effective APC for NIR-PIT against B-cell lymphoma.

Abstract 4118: Near infrared photoimmunotherapy for lung metastases

Abstract Introduction and Purpose Lung metastases are a leading cause of cancer related deaths. Current therapies include surgery, chemotherapy and radiotherapy but cause considerable side effects in the remaining normal lung, chest wall and heart. Therefore, a new-targeted therapy for lung metastases that results in minimal damage to adjacent structures, yet effectively treats lung metastases, is needed. Since one interesting property of the lung is that it transmits light better than other organs, light therapy is a viable alternative therapy. Near infrared photoimmunotherapy (NIR-PIT) is a new cancer treatment that combines the specificity of intravenously injected antibodies that target tumors with the toxicity induced by photosensitizers activated by NIR light. Herein, we demonstrate the efficacy of NIR-PIT in a mouse model of lung metastases. Experimental Design In vitro and in vivo experiments were conducted with a HER2, luciferase and GFP expressing cell line (3T3/HER2-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 a mouse model of lung metastases as well as a flank model, and assessed by tumor volume and/or bioluminescence (BLI). Results In vitro NIR-PIT-induced cytotoxicity was light dose dependent. In vivo anti-tumor effects of NIR-PIT included significant reductions in both tumor volume (p = 0.0141 vs. APC) and BLI (p = 0.0086 vs. APC) in the flank model, and prolonged survival (p &amp;lt; 0.0001). BLI demonstrated a significant reduction in lung metastases volume (p = 0.0117 vs. APC). Multiple NIR-PIT doses significantly prolonged survival in the lung metastases model (p &amp;lt; 0.0001). Conclusions NIR-PIT prolonged survival suggesting that NIR-PIT is a potential new therapy for the local control of lung metastases. Citation Format: Kazuhide Sato, Tadanobu Nagaya, Peter L. Choyke, Hisataka Kobayashi. Near infrared photoimmunotherapy for lung metastases. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4118. doi:10.1158/1538-7445.AM2015-4118

Near infrared photoimmunotherapy for lung metastases

Lung metastases are a leading cause of cancer related deaths; nonetheless current treatments are limited. Near infrared photoimmunotherapy (NIR-PIT) is a new cancer treatment that combines the specificity of intravenously injected antibodies that target tumors with the toxicity induced by photosensitizers activated by NIR-light. Herein, we demonstrate the efficacy of NIR-PIT in a mouse model of lung metastases.Experiments were conducted with a HER2, luciferase and GFP expressing cell line (3T3/HER2-luc-GFP). An antibody–photosensitizer conjugate (APC) consisting of trastuzumab and a phthalocyanine dye, IRDye700DX, was synthesized. In vitro NIR-PIT-induced cytotoxicity was light dose dependent. With 3D culture, repeated NIR-PIT could eradicate entire spheroids. In vivo anti-tumor effects of NIR-PIT included significant reductions in both tumor volume (p = 0.0141 vs. APC) and bioluminescence image (BLI) (p = 0.0086 vs. APC) in the flank model, and prolonged survival (p < 0.0001). BLI demonstrated a significant reduction in lung metastases volume (p = 0.0117 vs. APC). Multiple NIR-PIT doses significantly prolonged survival in the lung metastasis model (p < 0.0001). These results suggested that NIR-PIT is a potential new therapy for the local control of lung metastases.