Immune checkpoint inhibition combined with targeted therapy using a novel virus-like drug conjugate induces complete responses in a murine model of local and distant tumors.

Metastases remain the leading cause of cancer-related death worldwide. Therefore, improving the treatment efficacy against such tumors is essential to enhance patient survival. AU-011 (belzupacap sarotalocan) is a new virus-like drug conjugate which is currently in clinical development for the treatment of small choroidal melanoma and high-risk indeterminate lesions in the eye. Upon light activation, AU-011 induces rapid necrotic cell death which is pro-inflammatory and pro-immunogenic, resulting in an anti-tumor immune response. As AU-011 is known to induce systemic anti-tumor immune responses, we investigated whether this combination therapy would also be effective against distant, untreated tumors, as a model for treating local and distant tumors by abscopal immune effects. We compared the efficacy of combining AU-011 with several different checkpoint blockade antibodies to identify optimal treatment regimens in an in vivo tumor model. We show that AU-011 induces immunogenic cell death through the release and exposure of damage-associated molecular patterns (DAMPs), resulting in the maturation of dendritic cells in vitro. Furthermore, we show that AU-011 accumulates in MC38 tumors over time and that ICI enhances the efficacy of AU-011 against established tumors in mice, resulting in complete responses for specific combinations in all treated animals bearing a single MC38 tumor. Finally, we show that AU-011 and anti-PD-L1/anti-LAG-3 antibody treatment was an optimal combination in an abscopal model, inducing complete responses in approximately 75% of animals. Our data show the feasibility of combining AU-011 with PD-L1 and LAG-3 antibodies for the treatment of primary and distant tumors.

M1-derived extracellular vesicles enhance photodynamic therapy and promote immunological memory in preclinical models of colon cancer.

Extracellular vesicles (EVs) are promising drug carriers of photosensitizers for photodynamic therapy (PDT) in cancer treatment, due to their ability to circulate in blood and enter cells efficiently. The therapeutic potential of EVs has been suggested to depend on the type and physiological state of their cell of origin. However, the effects of deriving EVs from various cells in different physiological states on their antitumor capacity are rarely evaluated. In the present study, we compared the antitumor efficacy of EV-mediated PDT by incorporating the photosensitizer Zinc Phthalocyanine (ZnPc) into EVs from multiple cells sources. ZnPc was incorporated by a direct incubation strategy into EVs derived from immune cells (M1-like macrophages and M2-like macrophages), cancer cells (B16F10 melanoma cancer cells) and external sources (milk). Our data show that all EVs are suitable carriers for ZnPc and enable efficient PDT in vitro in co-culture models and in vivo. We observed that EV-mediated PDT initiates immunogenic cell death through the release and exposure of damage associated molecular patterns (DAMPs) on cancer cells, which subsequently induced dendritic cell (DC) maturation. Importantly, of all ZnPc-EVs tested, in absence of light only M1-ZnPc displayed toxicity to MC38, but not to DC, in monoculture and in co-culture, indicating specificity for cancer over immune cells. In MC38 tumor-bearing mice, only M1-ZnPc induced a tumor growth delay compared to control in absence of light. Interestingly, M1- but not M2-mediated PDT, induced complete responses against MC38 tumors in murine models (100% versus 38% of cases, respectively), with survival of all animals up to at least 60 days post inoculation. Finally, we show that all cured animals are protected from a rechallenge with MC38 cells, suggesting the induction of immunological memory after EV-mediated PDT. Together, our data show the importance of the cell type from which the EVs are obtained and highlight the impact of the immunological state of these cells on the antitumor efficacy of EV-mediated PDT.

Tumor Pigmentation Does Not Affect Light-Activated Belzupacap Sarotalocan Treatment but Influences Macrophage Polarization in a Murine Melanoma Model.

Purpose: Pigmentation in uveal melanoma is associated with increased malignancy and is known as a barrier for photodynamic therapy. We investigated the role of pigmentation in tumor behavior and the response to light-activated Belzupacap sarotalocan (Bel-sar) treatment in a pigmented (wild type) and nonpigmented (tyrosinase knock-out [TYR knock-out]) cell line in vitro and in a murine model. Methods: The B16F10 (TYR knock-out) was developed using CRISPR/Cas9. After the treatment with light-activated Bel-sar, cytotoxicity and exposure of damage-associated molecular patterns (DAMPs) were measured by flow cytometry. Treated tumor cells were co-cultured with bone marrow-derived macrophages (BMDMs) and dendritic cells (DCs) to assess phagocytosis and activation. Both cell lines were injected subcutaneously in syngeneic C57BL/6 mice. Results: Knock-out of the tyrosinase gene in B16F10 led to loss of pigmentation and immature melanosomes. Pigmented tumors contained more M1 and fewer M2 macrophages compared with amelanotic tumors. Bel-sar treatment induced near complete cell death, accompanied with enhanced exposure of DAMPs in both cell lines, resulting in enhanced phagocytosis of BMDMs and maturation of DCs. Bel-sar treatment induced a shift to M1 macrophages and delayed tumor growth in both in vivo tumor models. Following treatment, especially the pigmented tumors and their draining lymph nodes contained IFN-gamma positive CD8+T cells. Conclusions: Pigmentation influenced the type of infiltrating macrophages in the tumor, with more M1 macrophages in pigmented tumors. Belzupacap sarotalocan treatment induced immunogenic cell death and tumor growth delay in pigmented as well as in nonpigmented models and stimulated M1 macrophage influx in both models.

Current Challenges and Opportunities of Photodynamic Therapy against Cancer.

BACKGROUND: Photodynamic therapy (PDT) is an established, minimally invasive treatment for specific types of cancer. During PDT, reactive oxygen species (ROS) are generated that ultimately induce cell death and disruption of the tumor area. Moreover, PDT can result in damage to the tumor vasculature and induce the release and/or exposure of damage-associated molecular patterns (DAMPs) that may initiate an antitumor immune response. However, there are currently several challenges of PDT that limit its widespread application for certain indications in the clinic. METHODS: A literature study was conducted to comprehensively discuss these challenges and to identify opportunities for improvement. RESULTS: The most notable challenges of PDT and opportunities to improve them have been identified and discussed. CONCLUSIONS: The recent efforts to improve the current challenges of PDT are promising, most notably those that focus on enhancing immune responses initiated by the treatment. The application of these improvements has the potential to enhance the antitumor efficacy of PDT, thereby broadening its potential application in the clinic.

In Vitro Testing of the Virus-Like Drug Conjugate Belzupacap Sarotalocan (AU-011) on Uveal Melanoma Suggests BAP1-Related Immunostimulatory Capacity.

Purpose: The virus-like drug conjugate belzupacap sarotalocan (AU-011), currently under clinical investigation for first-line treatment of primary uveal melanoma (UM), shows enhanced tumor specificity by targeting heparan sulfate proteoglycans (HSPG). Such a treatment may potentially lead to systemic immune responses. We studied the potential of AU-011 treatment to induce immunogenic cell death as the first step to induce systemic immunity. Methods: We determined binding and uptake of AU-011 in ten primary and metastatic UM cell lines. The subcellular location of AU-011 was assessed by fluorescence microscopy. Following light activation (wavelength 690 nm) of AU-011, the half-maximal effective concentration (EC50) of AU-011 treatment and exposure of damage-associated molecular patterns (DAMPs) were assessed using flow cytometry. DAMPs were measured by RNAseq. Results: Fluorescence microscopy revealed most of the AU-011 was present in the cytoplasm. AU-011 binding and uptake by UM cells increased over time, with a lower uptake in BAP1-negative than in BAP1-positive cell lines. AU-011 activation induced cell death across all UM cell lines with EC50 values at picomolar concentrations. The AU-011 concentration and total light dose (J/cm2) were the most important parameters for the observed cytotoxicity. Finally, light-activated AU-011 induced exposure of DAMPs calreticulin (CRT) and HSP90. CRT exposure by light-activated AU-011 as well as CRT RNA exposure were lower in BAP1-negative compared to BAP1-positive UM cell lines. Conclusions: AU-011 treatment at low picomolar range induces immunogenic cell death in all 10 UM cell lines. The in vitro cytotoxicity was accompanied by exposure of DAMPs (HSP90 and CRT), suggesting AU-011 may contribute to the development of systemic immunity and be a suitable candidate for combination with immunotherapy in vivo. AU-011 treatment was more effective against BAP1-positive cell lines, with a lower EC50 and higher CRT exposure.

Combinatorial Therapeutic Approaches with Nanomaterial-Based Photodynamic Cancer Therapy.

Photodynamic therapy (PDT), in which a light source is used in combination with a photosensitizer to induce local cell death, has shown great promise in therapeutically targeting primary tumors with negligible toxicity and minimal invasiveness. However, numerous studies have shown that noninvasive PDT alone is not sufficient to completely ablate tumors in deep tissues, due to its inherent shortcomings. Therefore, depending on the characteristics and type of tumor, PDT can be combined with surgery, radiotherapy, immunomodulators, chemotherapy, and/or targeted therapy, preferably in a patient-tailored manner. Nanoparticles are attractive delivery vehicles that can overcome the shortcomings of traditional photosensitizers, as well as enable the codelivery of multiple therapeutic drugs in a spatiotemporally controlled manner. Nanotechnology-based combination strategies have provided inspiration to improve the anticancer effects of PDT. Here, we briefly introduce the mechanism of PDT and summarize the photosensitizers that have been tested preclinically for various cancer types and clinically approved for cancer treatment. Moreover, we discuss the current challenges facing the combination of PDT and multiple cancer treatment options, and we highlight the opportunities of nanoparticle-based PDT in cancer therapies.

Zinc-Phthalocyanine-Loaded Extracellular Vesicles Increase Efficacy and Selectivity of Photodynamic Therapy in Co-Culture and Preclinical Models of Colon Cancer.

Photodynamic therapy (PDT) is a promising and clinically approved method for the treatment of cancer. However, the efficacy of PDT is often limited by the poor selectivity and distribution of the photosensitizers (PS) toward the malignant tumors, resulting in prolonged periods of skin photosensitivity. In this work, we present a simple and straightforward strategy to increase the tumor distribution, selectivity, and efficacy of lipophilic PS zinc phthalocyanine (ZnPc) in colon cancer by their stabilization in purified, naturally secreted extracellular vesicles (EVs). The PS ZnPc was incorporated in EVs (EV-ZnPc) by a direct incubation strategy that did not affect size distribution or surface charge. By using co-culture models simulating a tumor microenvironment, we determined the preferential uptake of EV-ZnPc toward colon cancer cells when compared with macrophages and dendritic cells. We observed that PDT promoted total tumor cell death in normal and immune cells, but showed selectivity against cancer cells in co-culture models. In vivo assays showed that after a single intravenous or intratumoral injection, EV-ZnPc were able to target the tumor cells and strongly reduce tumor growth over 15 days. These data expose opportunities to enhance the potential and efficacy of PDT using simple non-synthetic strategies that might facilitate translation into clinical practice.

Photodynamic cancer therapy enhances accumulation of nanoparticles in tumor-associated myeloid cells.

In cancer treatment, nanomedicines may be employed in an attempt to improve the tumor localization of antineoplastic drugs e.g. immunotherapeutic agents either through passive or active targeting, thereby potentially enhancing therapeutic effect and reducing undesired off-target effects. However, a large number of administrated nanocarriers often fail to reach the tumor area. In the present study, we show that photodynamic therapy (PDT) enhances the tumor accumulation of systemically administered lipid-PEG layer coated poly (lactic-co-glycolic acid) (PLGA) nanoparticles (NP). Intravital microscopy and histological analysis of the tumor area reveal that the tumor vasculature was disrupted after PDT, disturbing blood flow and coinciding with entrapment of nanocarriers in the tumor area. We observed that the nanoparticles accumulating after treatment do not confine to specific locations within the tumor, but rather localize to various cells present throughout the tumor area. Finally, we show by flow cytometry that NP accumulation occurred mostly in immune cells of the myeloid lineage present in the tumor microenvironment (TME) as well as in tumor cells, albeit to a lower extent. These data expose opportunities for combination treatments of clinical PDT with NP-based immunotherapy to modulate the TME and improve antitumor immune responses.