Near-infrared photoimmunotherapy (NIR-PIT) is a newly developed and selective cancer treatment that induces necrotic and immunogenic cell death and utilizes a mAb conjugated to a photo-absorber dye, IR700DX, activated by NIR light. Although CD44 is a 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, CD25-targeted NIR-PIT has been reported to achieve selective and local depletion of FOXP3+CD25+CD4+ regulatory T cells (Treg), 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- and CD25-targeted NIR-PIT also resulted in some complete remissions. 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.

Programmed cell death 1 (PD-1) blockade therapy can result in dramatic responses in some patients with cancer. However, about 15% of patients receiving PD-1 blockade therapy experience rapid tumor progression, a phenomenon termed “hyperprogressive disease” (HPD). The mechanism(s) underlying HPD has been difficult to uncover because HPD is challenging to reproduce in animal models. Near-infrared photoimmunotherapy (NIR-PIT) is a method by which specific cells in the tumor microenvironment (TME) can be selectively depleted without disturbing other cells in the TME. In this study, we partially depleted CD8+ T cells with NIR-PIT by targeting the CD8β antigen thereby temporarily changing the balance of T-cell subsets in two different syngeneic tumor models. PD-1 blockade in these models led to rapid tumor progression compared with controls. CD3ε+CD8α+/CD3ε+CD4+FoxP3+ (Teff/Treg) ratios in the PD-1 and NIR-PIT groups were lower than in controls. Moreover, in a bilateral tumor model, low-dose CD8β-targeted NIR-PIT with anti–PD-1 blockade showed rapid tumor progression only in the tumor exposed to NIR light. In this experiment CD8β-targeted NIR-PIT in the exposed tumor reduced local CD8+ T cells resulting in a regulatory T-cell (Treg)–dominant TME. In conclusion, this reports an animal model to simulate the Treg-dominant TME, and the data generated using the model suggest that HPD after PD-1 blockade therapy can be attributed, at least in part, to imbalances between effector T cells and Tregs in the TME.

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). 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). 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. 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 and highly selective cancer treatment that induces necrotic/immunogenic cell death. It employs a monoclonal antibody (mAb) conjugated to a photo-absorber dye, IRDye700DX, which is activated by NIR light. Tumor-targeting NIR-PIT is also at least partly mediated by a profound immune response against the tumor. Cytotoxic T-lymphocyte antigen-4 (CTLA4) is widely recognized as a major immune checkpoint protein, which inhibits the immune response against tumors and is therefore, a target for systemic blockade. We investigated the effect of combining tumor-targeted NIR-PIT against the cell-surface antigen, CD44, which is known as a cancer stem cell marker, with a systemic CTLA4 immune checkpoint inhibitor in three syngeneic tumor models (MC38-luc, LL/2, and MOC1). CD44-targeted NIR-PIT combined with CTLA4 blockade showed greater tumor growth inhibition with longer survival compared with CTLA4 blockade alone in all tumor models. NIR-PIT and CTLA4 blockade produced more complete remission in MOC1 tumors (44%) than NIR-PIT and programmed cell death protein 1 (PD-1) blockade (8%), which was reported in our previous paper. However, the combination of NIR-PIT and CTLA4 blockade was less effective in MC38-luc tumors (11%) than the combination of NIR-PIT and PD-1 blockade (70%). Nonetheless, in many cases ineffective results with NIR-PIT and PD-1 blockade were reversed with NIR-PIT and CTLA4 blockade.

Near-infrared photoimmunotherapy (NIR-PIT) is a highly selective cancer treatment that employs an antibody photoabsorber conjugate (APC) composed of a targeting monoclonal antibody (mAb) conjugated with a photoactivatable phthalocyanine-derivative dye. Once injected and allowed to bind to a tumor, the APC is activated by local near-infrared light which kills cancer cells and induces a strong immune response in the tumor microenvironment by unmasking of new tumor antigens emerging from damaged tumor cells. Due to its ability to incite an immune reaction, even in poorly immunogenic tumors, NIR-PIT has the potential to enhance immunogenicity in tumors especially after immune checkpoint inhibition. In this study, we employ a poorly immunogenic MOC2-luc syngeneic tumor model and evaluate the efficacy of cancer-targeting CD44-targeted NIR-PIT. Increased infiltration of CD8+ T cells observed after NIR-PIT suggested an enhanced immune environment. Next, we evaluated tumor progression and survival after the combination of CD44-targeted NIR-PIT and short-term administration of an anti-PD1 immune checkpoint inhibitor (ICI) to further activate CD8+ T cells. Additionally, in mice in which the tumors were eradicated by this combination therapy, a re-challenge with fresh MOC2-luc cells demonstrated failure of tumor implantation implying acquired long-term immunity against the cancer cells. Combination therapy decreased tumor progression and prolonged survival significantly. Therefore, we concluded that NIR-PIT was able to convert a minimally immunogenic tumor unresponsive to anti-PD-1 ICI into a highly immunogenic tumor responsive to anti-PD-1 ICI, and this therapy was capable of inducing long-term immunity against the treated cancer.

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.