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Supplementary Figure S5 shows the immunohistochemical evaluation of hypoxia in NCI-H1703 subcutaneous tumor model

supplementary materials & methods

Supplementary Table S2 shows EVT801 metabolite potency and selectivity profile

Current autologous cell therapies, with blockbuster products on the market, have been leading for a decade to unprecedented clinical successes in patients with hematological malignancies. However, these patient-derived T-cell therapies are facing many challenges. The use of GMP iPSC lines to produce immune effector cells will reduce the complexity of the manufacturing process and will provide an unlimited source of starting material. The goal of the EVOcells Oncology platform is to offer a truly allogeneic cell therapy platform to treat a broad number of cancer patients with consistent quality and scalability of the final product. Besides, the versatility of our platform to produce different immune cell types combined to customized genetic engineering strategies will bring cell therapy to the level of personalized medicine. Our “off-the-shelf” cell therapy platform has already validated two pillars: iPSC-derived NK cells (iNK) and iPSC-derived Macrophages (iMACs). Through multiple genetic engineering strategies specific to each immune cell type, we are developing a comprehensive portfolio of cell therapy products to address specific tumor escape mechanism in liquid and solid tumors. Our initial effort aimed to develop these two innate immune cell types to propose efficacious cell therapies with an increased safety profile as they have low risk of graft-versus-host disease (GvHD) or CRS (Cytokine Release Syndrome). Thanks to the expression of a broad pattern of activatory receptors, iNK cells form Immunological Synapses with tumor cells leading in turn to efficient killing with and without addition of a CAR construct. Besides, we demonstrated the possibility to combine “naked” iNK cells with marketed therapeutic monoclonal antibodies (mAb) to further improve their efficacy. At the end of the differentiation process, iMACs are showing a M0 like phenotype with high plasticity allowing the in vitro differentiation of the cells towards either a M1 or a M2 polarization in response to the appropriate stimulations. iMACs produce key macrophages cytokines and are able to kill tumor cells via ADCP (Antibody-Dependent-Cell-Phagocytosis) mechanism when combined to a therapeutic mAb. Thanks to our collaboration with clinicians at the IUCT-Oncopole (Toulouse Cancer Hospital), we were able to identify appropriate cancer indications and further demonstrate in a translational fashion that both iNK and iMACs are able to kill primary resistant tumor cells which were isolated from patient’ samples. Taken together, these results are showing the versatility and the breadth of our EVOcells Oncology platform to produce a true arsenal of cell therapies and its potential for future clinical development. Citation Format: Michael Esquerré, Audrey Holtzinger, Nadja Wagner, Monika Braun, Mélanie Pichery, Stefanie Pfaender, Stephanie Sontag, Kathrin Haake, Michela Mirenda, Michael Paillasse, Davide Grandolfo, Chloé Beuraud, Mandy Richter, Philip Hublitz, Julien Bousquet, Marion Fabre, Mylène Gador, Daniel Sommermeyer, Tanja Schneider, Oriane Bombarde, Camille Esquerré, Loic Ysebaert, Fabien Despas, Matthias Austen, Andreas Scheel, Markus Dangl. EVOcells Oncology: Tailored genetic engineering of iPSC-derived immune effector cells and combination with the right biologic therapeutics result in optimal killing of primary tumor cells from patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3203.

Cancer immunotherapy has transformed the oncology landscape and shown promising therapeutic results in the clinic but it is also clearly facing multiple limitations. So far, the focus of immunotherapies has mainly been on the modulation of T-cells. Natural killer (NK) cells offer promising perspectives for immunotherapies as they directly kill tumor cells and produce pro-inflammatory cytokines to activate and recruit T-cells and other immune cells. Thus, NK cells are considered key players in the anti-tumor immune response that can make a “cold” tumor “hot”. However, in the tumor microenvironment (TME) of cancer patients, the low numbers of NK cells present and their exhaustion due to local immunosuppression often limit tumor cell killing. The development of NK based cell therapy products can overcome these limitations, either as single therapy or in combination with biologics such as e.g. NK cell engagers or monoclonal antibodies for boosting ADCC (antibody dependent cell cytotoxicity). To overcome the main hurdle of current autologous cell therapies, the EVOcells Oncology platform aims at developing a streamlined manufacturing process for iPSC-derived immune effector cells. The goal is to develop “off-the-shelf” cell therapy products for clinical use in large patient populations. We are currently establishing a multi-asset portfolio combining multiple immune effector cell types with different genetic editing strategies that serve to optimize this approach further. iPSC-derived NK cells (iNK cells) offer an attractive option for improving both therapeutic efficacy and safety of CAR cell therapies. Our robust, feeder-free production protocol produces iNK cells that can be reliably frozen with good recovery rates. We have generated multiple genetically modified iNK cells and have validated that the genetic modifications boost iNK function. At the end of the production process, iNK cells show phenotypic properties and single cell RNA sequencing profiles comparable to blood-derived NK cells. Importantly, iNK cells are fully functional with the ability to produce key pro-inflammatory cytokines and to form lytic immunological synapses leading to efficient killing of cancer cell lines. In addition to the killing of established tumor cell lines, we also evaluated the ability of iNK cells to kill primary leukemia cells ex vivo by using blood samples from B-cell chronic lymphocytic leukemia patients. In combination with a standard of care anti-CD20 antibody, iNK cells demonstrated the ability to kill primary leukemia cells via ADCC. Taken together, these promising results obtained with iNK cells show the potential of the EVOcells Oncology platform to deliver the next generation of cell therapies in oncology paving the way to develop a portfolio of iPSC-derived immune effector cells. Citation Format: Michael Esquerré, Audrey Holtzinger, Mélanie Pichery, Stefanie Pfaender, Saniye Yumlu, Mandy Richter, Delphine Betous, Oriane Bombarde, Mylène Gador, Nadja Sailer, Michael Paillasse, Loïc Ysebaert, Fabien Despas, Matthias Austen, Andreas Scheel, Markus Dangl. iPSC-derived natural killer cells as the front-runner program of the EVOcells Oncology platform: From inception to translational validation using patient samples [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 2749.

IL-33 (previously known as NF from high endothelial venules) is an IL-1 family cytokine that signals through the ST2 receptor and drives cytokine production in mast cells, basophils, eosinophils, invariant NKT and NK cells, Th2 lymphocytes, and type 2 innate immune cells (natural helper cells, nuocytes, and innate helper 2 cells). Little is known about endogenous IL-33; for instance, the cellular sources of IL-33 in mouse tissues have not yet been defined. In this study, we generated an Il-33–LacZ gene trap reporter strain (Il-33Gt/Gt) and used this novel tool to analyze expression of endogenous IL-33 in vivo. We found that the Il-33 promoter exhibits constitutive activity in mouse lymphoid organs, epithelial barrier tissues, brain, and embryos. Immunostaining with anti–IL-33 Abs, using Il-33Gt/Gt (Il-33–deficient) mice as control, revealed that endogenous IL-33 protein is highly expressed in mouse epithelial barrier tissues, including stratified squamous epithelia from vagina and skin, as well as cuboidal epithelium from lung, stomach, and salivary gland. Constitutive expression of IL-33 was not detected in blood vessels, revealing the existence of species-specific differences between humans and mice. Importantly, IL-33 protein was always localized in the nucleus of producing cells with no evidence for cytoplasmic localization. Finally, strong expression of the Il-33–LacZ reporter was also observed in inflamed tissues, in the liver during LPS-induced endotoxin shock, and in the lung alveoli during papain-induced allergic airway inflammation. Together, our findings support the possibility that IL-33 may function as a nuclear alarmin to alert the innate immune system after injury or infection in epithelial barrier tissues.