A novel anti-galectin-9 immunotherapy limits the early progression of pancreatic neoplastic lesions in transgenic mice.

Background: Pancreatic adenocarcinoma (PDAC) is a devastating disease with an urgent need for therapeutic innovation. Immune checkpoint inhibition has shown promise in a variety of solid tumors, but most clinical trials have failed to demonstrate clinical efficacy in PDAC. This low efficacy is partly explained by a highly immunosuppressive microenvironment, which dampens anti-tumor immunity through the recruitment or induction of immunosuppressive cells, particularly regulatory T cells (Tregs). In this context, our laboratory has developed a novel immunotherapeutic strategy aimed at inhibiting the suppressive activity of Tregs, based on a patented (EP3152234B1) monoclonal antibody (mAb) targeting galectin-9 (LGALS9). Materials and methods: CD4+ conventional T cells (TCD4 or Tconv), Treg ratio, and LGALS9 expression were analyzed by immunohistochemistry (IHC) and cytometry in blood and pancreas of K-rasLSL.G12D/+;Pdx-1-Cre (KC) and K-rasWildType (WT);Pdx1-Cre (WT) mice aged 4-13 months. Pancreatic intraepithelial neoplasm (PanIN) progression and grade were quantified using FIJI software and validated by pathologists. The anti-galectin-9 mAb was validated for its use in mice on isolated murine C57BL/6 Treg by immunofluorescence staining and cytometry. Its specificity and functionality were validated in proliferation assays on rLGALS9-immunosuppressed murine Tconv and in suppression assays between murine Treg and Tconv. Finally, 2-month-old KC mice were treated with anti-LGALS9 and compared to WT mice for peripheral and infiltrating TCD4, Treg, and PanIN progression. Results: IHC and cytometry revealed a significant increase in LGALS9 expression and Treg levels in the blood and pancreas of KC mice proportional to the stages of precancerous lesions. Although present in WT mice, LGALS9 is expressed at a basal level with low and restricted expression that increases slightly over time, while Treg cells are few in number in their circulation and even absent from the pancreas over time. Using our anti-LGALS9 mAb in mice, it is shown that (i) murine Treg express LGALS9, (ii) the mAb could target and inhibit recombinant murine LGALS9, and (iii) neutralize murine Treg suppressive activity. Finally, the anti-LGALS9 mAb in KC mice reduced (i) LGALS9 expression in pancreatic cancer cells, (ii) the Treg ratio, and (iii) the total surface area and grade of PanIN. Conclusion: We demonstrate for the first time that an anti-LGALS9 antibody, by specifically targeting endogenous LGALS9 tumor and exogenous LGALS9 produced by Treg, was able to limit the progression of pancreatic neoplastic lesions in mice, opening up new prospects for its use as an immunotherapeutic tool in PDAC.

Folate Receptor Targeted Photodynamic Therapy: A Novel Way to Stimulate Anti-Tumor Immune Response in Intraperitoneal Ovarian Cancer.

Photodynamic therapy (PDT) has shown improvements in cancer treatment and in the induction of a proper anti-tumor immune response. However, current photosensitizers (PS) lack tumor specificity, resulting in reduced efficacy and side effects in patients with intraperitoneal ovarian cancer (OC). In order to target peritoneal metastases of OC, which overexpress folate receptor (FRα) in 80% of cases, we proposed a targeted PDT using a PS coupled with folic acid. Herein, we applied this targeted PDT in an in vivo mouse model of peritoneal ovarian carcinomatosis. The efficacy of the treatment was evaluated in mice without and with human peripheral blood mononuclear cell (PBMC) reconstitution. When mice were reconstituted, using a fractionized PDT protocol led to a significantly higher decrease in the tumor growth than that obtained in the non-reconstituted mice (p = 0.0469). Simultaneously, an immune response was reflected by an increase in NK cells, and both CD4+ and CD8+ T cells were activated. A promotion in cytokines IFNγ and TNFα and an inhibition in cytokines TGFß, IL-8, and IL-10 was also noticed. Our work showed that a fractionized FRα-targeted PDT protocol is effective for the treatment of OC and goes beyond local induction of tumor cell death, with the promotion of a subsequent anti-tumor response.

An Efficient 5-Aminolevulinic Acid Photodynamic Therapy Treatment for Human Hepatocellular Carcinoma.

Photodynamic therapy (PDT) is a two-stage treatment relying on cytotoxicity induced by photoexcitation of a nontoxic dye, called photosensitizer (PS). Using 5-aminolevulinic acid (5-ALA), the pro-drug of PS protoporphyrin IX, we investigated the impact of PDT on hepatocellular carcinoma (HCC). Optimal 5-ALA PDT dose was determined on three HCC cell lines by analyzing cell death after treatment with varying doses. HCC-patient-derived tumor hepatocytes and healthy donor liver myofibroblasts were treated with optimal 5-ALA PDT doses. The proliferation of cancer cells and healthy donor immune cells cultured with 5-ALA-PDT-treated conditioned media was analyzed. Finally, therapy efficacy on humanized SCID mice model of HCC was investigated. 5-ALA PDT induced a dose-dependent decrease in viability, with an up-to-four-fold reduction in viability of patient tumor hepatocytes. The 5-ALA PDT treated conditioned media induced immune cell clonal expansion. 5-ALA PDT has no impact on myofibroblasts in terms of viability, while their activation decreased cancer cell proliferation and reduced the tumor growth rate of the in vivo model. For the first time, 5-ALA PDT has been validated on primary patient tumor hepatocytes and donor healthy liver myofibroblasts. 5-ALA PDT may be an effective anti-HCC therapy, which might induce an anti-tumor immune response.

An Efficient Photodynamic Therapy Treatment for Human Pancreatic Adenocarcinoma.

To date, pancreatic adenocarcinoma (ADKP) is a devastating disease for which the incidence rate is close to the mortality rate. The survival rate has evolved only 2-5% in 45 years, highlighting the failure of current therapies. Otherwise, the use of photodynamic therapy (PDT), based on the use of an adapted photosensitizer (PS) has already proved its worth and has prompted a growing interest in the field of oncology. We have developed a new photosensitizer (PS-FOL/PS2), protected by a recently published patent (WO2019 016397-A1, 24 January 2019). This photosensitizer is associated with an addressing molecule (folic acid) targeting the folate receptor 1 (FOLR1) with a high affinity. Folate binds to FOLR1, in a specific way, expressed in 100% of ADKP or over-expressed in 30% of cases. The first objective of this study is to evaluate the effectiveness of this PS2-PDT in four ADKP cell lines: Capan-1, Capan-2, MiapaCa-2, and Panc-1. For this purpose, we first evaluated the gene and protein expression of FOLR1 on four ADKP cell lines. Subsequently, we evaluated PS2's efficacy in our cell lines and we assessed the impact of PDT on the secretome of cancer cells and its impact on the immune system. Finally, we evaluate the PDT efficacy on a humanized SCID mouse model of pancreatic cancer. In a very interesting way, we observed a significant increase in the proliferation of activated-human PBMC when cultured with conditioned media of ADKP cancer cells subjected to PDT. Furthermore, to evaluate in vivo the impact of this new PS, we analyzed the tumor growth in a humanized SCID mice model of pancreatic cancer. Four conditions were tested: Untreated, mice (nontreated), mice with PS (PS2), mice subjected to illumination (Light only), and mice subjected to illumination in the presence of PS (PDT). We noticed that the mice subjected to PDT presented a strong decrease in the growth of the tumor over time after illumination. Our investigations have not only suggested that PS2-PDT is an effective therapy in the treatment of PDAC but also that it activates the immune system and could be considered as a real adjuvant for anti-cancer vaccination. Thus, this new study provides new treatment options for patients in a therapeutic impasse and will provide a new arsenal in the fight against PDAC.