Mathematical Modeling of Combined Oncolytic Immunotherapy

Data of immunotherapeutic treatments (top) generated using in vitro and in vivo tumor models (bottom right) are integrated into a mathematical model (bottom left) to allow for in silico prediction of optimal treatment schedules for further development and ultimately clinical translation.
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PI(s)
Christine E. Engeland, Johannes P. W. Heidbüchel

Contributing CCU members
Jessica Albert, Birgit Hoyler, Stefanie Prien

Cancer immunotherapies, such as immune checkpoint blockade and chimeric antigen receptor (CAR) T cell therapy, have recently proven successful in numerous pre-clinical and clinical studies. However, critical challenges including toxicities and lack of efficacy, especially against solid tumors, have yet precluded benefit for the majority of patients. Combination with oncolytic viruses which directly lyse tumor cells and stimulate an anti-tumor immune response has been shown to be a viable option to increase the therapeutic window of immunotherapy. However, the complexity of the immune system and the plethora of possible combinations rule out comprehensive testing to identify optimal treatment schedules. To address these challenges, we are developing mathematical models of combination immunovirotherapies together with our collaborators from Moffitt Cancer Center in Tampa, FL, Dr. Daniel Abate-Daga and Dr. Heiko Enderling.

Lines of research include parametrization of the model with quantitative experimental data. In interdisciplinary collaborations, we are working together with Jessica Hunger and Dr. Dr. Michael Breckwoldt (DKFZ) on imaging approaches and with Aileen Berger, Dr. Patrick Schmidt, Prof. Dr. Dirk Jäger (University Hospital Heidelberg), Alice de Roia, Dr. Matthias Bozza and Prof. Dr. Richard Harbottle (DKFZ) on CAR design and development.

Following validation and refinement, the models will be used to simulate outcomes of theoretical combination therapies in order to prioritize the most promising treatment approaches for further development. Crucial parameters in the system will be identified as targets for therapeutic intervention. This innovative workflow will accelerate clinical translation of combination immunotherapies for the benefit of cancer patients.

Publications

  • Mathematical modeling of oncolytic virotherapy. Heidbuechel JPW, Abata-Daga D, Engeland CE, Enderling H. In: Engeland CE (ed.) Oncolytic Viruses. Methods in Molecular Biology vol 2058, Humana, New York, NY, ISBN 978-1-4939-9793-0.
  • Targeted BiTE Expression by an Oncolytic Vector Augments Therapeutic Efficacy Against Solid Tumors. Speck T*, Heidbuechel JPW*, Veinalde R, Jaeger D, von Kalle C, Ball CR, Ungerechts G, Engeland CE. Clinical Cancer Research 2018; 24: 2128-37.
  • Fighting cancer with mathematics and viruses. Santiago DN*, Heidbuechel JPW*, Kandell WM, Walker R, Djeu J, Engeland CE, Abate-Daga D, Enderling H. Viruses 2017; 9(9), 239.

Funding
Wilhelm Sander Foundation, Grant No. 2018.058.1 (to CEE), NCT Heidelberg School of Oncology (HSO²), Postdoctoral Fellowship in Clinical Cancer Research (to JPWH)

Mechanisms of Oncolytic Immunotherapy

Measles virus oncolysis and the cancer immunity cycle. Factors supporting the different steps in induction of tumor-specific T cell responses which have been implicated in MeV oncolytic virotherapy are displayed. (Reprinted from Mechanisms of measles virus oncolytic immunotherapy. Pidelaserra Martí G, Engeland CE. Cytokine & Growth Factor Reviews 2020 Jul 3:S1359-6101(20)30175-1, Copyright 2020, with permission from Elsevier.)
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PI(s)
Christine E. Engeland

Contributing CCU members
Gemma Pidelaserra Martí, Maximiliane Finkbeiner, Johannes P.W. Heidbüchel, Jessica Albert, Birgit Hoyler, Stefanie Prien

Previous research from our group and others has demonstrated that oncolytic virotherapy can induce tumor-specific immune responses in terms of an in situ tumor vaccination effect. This projects aims to dissect the underlying mechanisms to define determinants of successful cancer immunotherapy. Using preclinical ex vivo and in vivo models, our goal is to pinpoint the cytokines, chemokines, signaling pathways, and immune cell subsets that govern treatment outcome. To increase translational impact, we collaborate with the groups of Prof. Dr. Richard Harbottle (DKFZ Heidelberg) and PD Dr. Niels Halama (DKFZ and NCT Heidelberg) on preclinical model development. Our results provide a basis for rational further development of immunomodulatory oncolytic therapeutics with enhanced safety and efficacy.

Publications

  • Mechanisms of measles virus oncolytic immunotherapy. Pidelaserra Martí G, Engeland CE. Cytokine & Growth Factor Reviews 2020 Jul 3:S1359-6101(20)30175-1.
  • Immunological Effects and Viral Gene Expression Determine the Efficacy of Oncolytic Measles Vaccines Encoding IL-12 or IL-15 Agonists. Backhaus PS, Veinalde R, Hartmann L, Dunder JE, Jeworowski LM, Albert J, Hoyler B, Poth T, Jäger D, Ungerechts G, Engeland CE. Viruses 2019 Oct 3;11(10):914.
  • Targeted BiTE Expression by an Oncolytic Vector Augments Therapeutic Efficacy Against Solid Tumors. Speck T, Heidbuechel JPW, Veinalde R, Jaeger D, von Kalle C, Ball CR, Ungerechts G, Engeland CE. Clinical Cancer Research 2018; 24: 2128-37.Oncolytic measles virus encoding interleukin-12 mediates potent antitumor effects through T cell activation. Veinalde R, Grossardt C, Hartmann L, Bourgeois-Daigneault MC, Bell JC, Jäger D, von Kalle C, Ungerechts G, Engeland CE. Oncoimmunology 2017 Jan 31;6(4):e1285992.

Funding
Deutsche Forschungsgemeinschaft, EN-1119-2/2 (to CEE)
Else Kröner-Fresenius-Stiftung, Else Kröner Memorial Stipendium 2019_EKMS.02 (to CEE)

RadioViroTherapy for Treatment of Refractory Solid Cancer

Project overview: This project focusses on a systematic development of optimal dosing and scheduling of radiovirotherapy (RVTx) followed by an in-depth molecular and functional characterization of induced mechanisms of action. This will allow the rational modification of RVTx.
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PI(s)
Christine E. Engeland, Mathias F. Leber

Contributing CCU members
Scientific staff: Judith Förster, Katia Günther, Karim Zaoui
Technical assistance: Jessica Albert, Birgit Hoyler, Stefanie Prien

Refractory tumors such as pancreatic ductal adenocarcinoma (PDAC) or head and neck squamous cell carcinoma (HNSCC) remain a major challenge in medical oncology. To address the limited clinical efficacy of current standard therapies, we aim to investigate virotherapy using oncolytic measles virus (MeV) combined with radiotherapy. We hypothesize that combined radiovirotherapy (RVTx) may elicit synergistic anti-cancer effects and induce a sustained anti-tumor immune response.

We have analyzed different dosing schedules of radiation and oncolytic MeV and have confirmed synergy of RVTx in HNSCC and PDAC cell lines in vitro. An in-depth molecular and functional characterization focuses on induction of immunogenic cell death, markers for viral RNA and DNA damage sensing, as well as downstream effects of innate immune activation. To investigate therapeutic effects of RVTx in models of high translational relevance, we employ patient-derived 3D ex vivo tissue samples in collaboration with Dr. Karim Zaoui (Department of Otorhinolaryngology, Head and Neck Surgery, Heidelberg University Hospital). Further, we use syngeneic in vivo tumor models to analyze anti-tumor immune responses. The molecular and functional characterization as well as immunophenotyping allows for rational modification of RVTx in order to develop advanced therapeutic regimens including e.g. immune checkpoint blockade or heavy ion irradiation.

Our research aims to uncover critical mechanisms of anti-tumor immunity in relevant preclinical models. By employing these, we intend to develop a state-of-the-art cancer RVTx to achieve sustained improvements in the treatment of refractory tumors.

Funding
Alois Hirdt-Erben und Wieland-Stiftung Heidelberg

Targeted Immunomodulation

Oncolytic viruses encoding immunomodulatory transgenes such as interleukin-12 (IL-12) activate immune effector cells including CD8+ T cells within the tumor microenvironment (left panel), leading to enhanced anti-tumor efficacy in murine tumor models (right panel).
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PI(s)
Christine E. Engeland

Contributing CCU members
Lukas Kuchernig, Alessia Floerchinger, Jessica E. Klein, Elena Busch, Jessica Albert, Birgit Hoyler, Stefanie Prien

Advances in basic immunology have revealed multiple targets for cancer immunotherapy. However, many potent immunomodulators are highly toxic upon systemic administration. To overcome these limitations, we employ oncotropic viruses as delivery vectors. Moreover, synergistic effects can arise between viral oncolysis and the respective immunomodulator. Immunomodulators under investigation include amongst others cytokines, chemokines, immune checkpoint antibodies, and bispecific molecules. Furthermore, encoding tumor-associated antigens within the viral vector allows to direct the ensuing immune response towards distinct targets. After virological and immunological characterization of novel vectors, we use preclinical ex vivo and in vivo model systems to assess therapeutic effects and mechanisms of action. Active collaborations include Prof. Dr. Stefan Eichmüller and Dr. Wolfram Osen (GMP and T Cell Therapy, DKFZ), Tanja Poth (Institute of Pathology, University Hospital Heidelberg) as well as Prof. Dr. Christian Buchholz and Dr. Michael Mühlebach (Paul-Ehrlich-Institut, Langen).
The ultimate goal of our efforts is to identify the most effective immunomodulatory oncolytic vectors for clinical development.

Publications

  • Measles Vaccines Designed for Enhanced CD8+ T Cell Activation. Busch E, Kubon KD, Mayer JKM, Pidelaserra-Martí G, Albert J, Hoyler B, Heidbuechel JPW, Stephenson KB, Lichty BD, Osen W, Eichmüller SB, Jäger D, Ungerechts G, Engeland CE. Viruses 2020 Feb 21;12(2):242.
  • Immunological Effects and Viral Gene Expression Determine the Efficacy of Oncolytic Measles Vaccines Encoding IL-12 or IL-15 Agonists. Backhaus PS, Veinalde R, Hartmann L, Dunder JE, Jeworowski LM, Albert J, Hoyler B, Poth T, Jäger D, Ungerechts G, Engeland CE. Viruses 2019 Oct 3;11(10):914.
  • Paramyxoviruses for Tumor-targeted Immunomodulation: Design and Evaluation Ex Vivo. Heidbuechel JPW, Engeland CE Journal of Visualized Experiments 2019 Jan 7;(143).
  • Tumor-Specific Delivery of Immune Checkpoint Inhibitors by Engineered AAV Vectors. Reul J, Frisch J, Engeland CE, Thalheimer FB, Hartmann J, Ungerechts G, Buchholz CJ. Frontiers in Oncology 2019 Feb 14;9:52.
  • Targeted BiTE Expression by an Oncolytic Vector Augments Therapeutic Efficacy Against Solid Tumors. Speck T, Heidbuechel JPW, Veinalde R, Jaeger D, von Kalle C, Ball CR, Ungerechts G, Engeland CE. Clinical Cancer Research 2018; 24: 2128-37.
  • Oncolytic measles virus encoding interleukin-12 mediates potent antitumor effects through T cell activation. Veinalde R, Grossardt C, Hartmann L, Bourgeois-Daigneault MC, Bell JC, Jäger D, von Kalle C, Ungerechts G, Engeland CE. Oncoimmunology 2017 Jan 31;6(4):e1285992.
  • CTLA-4 and PD-L1 checkpoint blockade enhances oncolytic measles virus therapy. Engeland CE, Grossardt C, Veinalde R, Bossow S, Lutz D, Kaufmann JK, Shevchenko I, Umansky V, Nettelbeck DM, Weichert W, Jäger D, von Kalle C, Ungerechts G. Molecular Therapy 2014 Nov;22(11):1949-59.
  • Granulocyte-macrophage colony-stimulating factor-armed oncolytic measles virus is an effective therapeutic cancer vaccine. Grossardt C, Engeland CE, Bossow S, Halama N, Zaoui K, Leber MF, Springfeld C, Jaeger D, von Kalle C, Ungerechts G. Human Gene Therapy 2013 Jul;24(7):644-54.

Funding
Deutsche Krebshilfe, Mildred Scheel MD Fellowships (to LK and JEK)

Heterotypic Spheroid Model Development for Preclinical Studies of Virotherapy-induced Changes of Tumor Microenvironment

Establishment of monotypic (A) and heterotypic: double (B), triple (C) and quadruple (D) coculture pancreatic cancer spheroids
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PI(s)
Assia Angelova

Contributing CCU members
Jean Rommelaere (Prof. Emeritus), Alexandra Just, Milena Barf

Rodent protoparvoviruses possess natural tumor-killing (oncolytic) properties. Furthermore, these viruses, H-1PV in particular, are capable of reversing tumor-driven immune suppression through the induction of immunogenic tumor cell death and the establishment of a proinflammatory tumor microenvironment. H-1PV immunostimulating effects have been demonstrated both preclinically and in early phase clinical trials: ParvOryx01 (glioblastoma) (https://clinicaltrials.gov/ct2/show/NCT01301430) and ParvOryx02 (pancreatic cancer) (https://clinicaltrials.gov/ct2/show/NCT02653313).
Parvovirotherapy has potential as anticancer strategy and the prospects of development of parvovirus-based combinatorial treatments urge the need for relevant preclinical models applicable to both basic and translational viro-immunotherapy research. The main goal of our team is to establish in vitro systems, which mimic the structural and functional complexity of the tumor microenvironment, including the interplay between stroma, vasculature and immune cells. We apply a methylcellulose-based hanging drop approach to generate double, triple and quadruple heterotypic spheroids consisting of tumor, fibroblast, endothelial and immune cells. These complex 3D systems may be subjected to various viro-immunotherapeutic treatments and further implemented into transwell co-culture, organotypic or animal models.
We are currently focused on pancreatic cancer spheroid model establishment. We are also pursuing the reproducible generation of non-Hodgkin lymphoma (NHL) spheroids, taking up the challenge of the intrinsically low 3D growth capacity of blood cancer cells. Diffuse large B cell lymphoma and cutaneous T cell lymphoma, two NHL subtypes presently lacking pertinent tumor microenvironment recapitulation in vitro, are in the spotlight of our research activities. The ultimate goal of our team is the establishment of a translationally relevant, patient tumor material-based heterotypic spheroid model as a novel platform for preclinical viro-immunotherapeutic testing.
In collaboration with K. Geletneky (Darmstadt, Germany), B. Leuchs (DKFZ), J. Nüesch (DKFZ), A. Marchini (DKFZ and LIH, Luxembourg).

Publications

  • Fluorescence In Situ Hybridization (FISH) Detection of Viral Nucleic Acids in Oncolytic H-1 Parvovirus-Treated Human Brain Tumors. Kiprianova I, Just A, Leuchs B, Rommelaere J, Angelova AL. Methods Mol Biol 2020; 2058: 295-306.
  • Immune Conversion of Tumor Microenvironment by Oncolytic Viruses: The Protoparvovirus H-1PV Case Study. Marchini A, Daeffler L, Pozdeev VI, Angelova A, Rommelaere J. Front Immunol 2019; 10: 1848.
  • Immune System Stimulation by Oncolytic Rodent Protoparvoviruses. Angelova A, Rommelaere J. Viruses 2019; 11: 415.
  • Oncolytic H-1 Parvovirus Shows Safety and Signs of Immunogenic Activity in a First Phase I/IIa Glioblastoma Trial. Geletneky K, Hajda J, Angelova AL, Leuchs B, ...Just A, ....Rommelaere J. Mol Ther 2017, 25: 2620-34.
  • The Oncolytic Virotherapy Era in Cancer Management: Prospects of Applying H-1 Parvovirus to Treat Blood and Solid Cancers. Angelova AL, Witzens-Harig M, Galabov AS, Rommelaere J. Front Oncol 2017; 7: 93.

Oncolytic Adenoviruses: Selecting and Engineering Adenovirus Serotypes with Improved Oncolytic and Immunostimulatory potency

An efficiency-enhanced adenovirus shows an up to 10-fold increase in killing of low passage cancer cells. Cells were infected with indicated adenoviruses at indicated virus titers. Following virus replication and spread, surviving cells were stained with crystal violet.
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PI(s)
Dirk M. Nettelbeck

Contributing CCU members
N.N.

Oncolytic adenoviruses possess many advantageous features for applications in virotherapy, including unique mechanisms of tumor-selectivity, immunostimulatory activity supporting oncolytic tumor vaccination and various engineering opportunities for adapting or improving oncolytic functions. However, previous work has focused on one of now >100 known adenovirus serotypes.

The aim of this program is to characterize the oncolytic and immunological properties of adenovirus serotypes and engineer lead candidates towards a new generation of efficiency-enhanced oncolytic adenoviruses. To this end, we systematically screen recently established adenovirus libraries (cooperation with Anja Ehrhardt, Univ. of Witten-Herdecke) in tumor and immune cell cultures and in sophisticated patient-derived pre-clinical tumor models (cooperation with our colleagues at NCT and DKFZ, Claudia Ball and Niels Halama). Comprehensive analyses include various virological and immunological parameters at the molecular and cellular level and in the context of tumor tissue. Emerging adenovirus serotypes with superior oncolytic and immunological features will be engineered to incorporate stringent tumor-selectivity. To this end, we utilize new technologies for modification of large viral genomes that are applicable irrespective of adenovirus serotype.

These next generation oncolytic adenoviruses will allow for further exploration of combination immunotherapy and/or "arming" with therapeutic genes, and ultimately for clinical translation within the CCU framework.

Funding
Wilhelm Sander-Stiftung, project grant (to DMN)

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