Theravision – Platform Technology for the Development, Production and Testing of oncolytic Viruses

Viruses are capable of efficiently penetrating cells. There they produce their own proteins, multiply, and ultimately kill the infected cells. Based on these characteristics viruses have great potential in cancer therapy. Clinical studies on oncolytic (cancer-killing) viruses are very promising. However, there is a significant need for optimization with regard to virus specificity and efficacy, in production methods, as well as in models for preclinical testing. Therefore, based on a clinical isolate of the Herpes simplex virus type 1 (HSV1), MAVO TheraVision is establishing a broadly applicable platform technology for combinatorial oncolytic virus-immunotherapy based on three pillars a) engineering of therapeutic virus vectors, b) production of viruses with robust and scalable methods, and c) testing using novel and meaningful preclinical in vitro and in vivo models.

Cancer is a disease that affects many of us directly or indirectly. It is the second most frequent cause of death in Germany, preceded only by cardiovascular diseases. Worldwide, about 8 million people were victims of cancer in 2018 with an increasing tendency especially because of the growing population and the increasing life-expectancy.

Conventional therapies (surgery, radio- and chemotherapy) have dominated cancer treatment over many years with moderate success. Only in the last decade have alternatives and increasingly personalized therapy concepts led to significant progress in cancer therapy. Very promising approaches include immunotherapies and oncolytic viruses. With the help of these treatments, it is the goal to reduce side effects that arise with conventional therapies, to increase the number of patients who respond to therapy, as well as to achieve sustainable, long-lasting therapy success. 

Although, it has long been known that viruses can kill cancer cells, their therapeutic potential has only come into focus in the last decade. First results of preclinical and clinical studies with oncolytic viruses are promising. A HSV1-based virus called Imlygic^® was approved in 2015 by the Food and Drug Administration (FDA) as the first oncolytic viral therapeutic for the treatment of malignant melanomas. Subsequently, it was approved by the European Medicines Agency (EMA). This breakthrough has a stimulating effect on the development of new viral therapeutics. So called immunomodulators, particularly in the form of immunocheckpoint-inhibitors, are in the focus of research and development as an alternative to virus therapies.  Currently, combining immunomodulatory therapies with alternative treatment approaches is stirring up hope. Especially, the combination of virus and immune therapies by co-application of these treatment forms is a promising possibility. It is currently being tested in first clinical studies.

In the present project, an oncolytic platform virus was developed. By integrating transgenes in the virus vector it is possible to express immunomodulators in the cancer cells during virus multiplication. Thus, the groundwork has been lain for combinatorial virus-immunotherapy where at the site of infection both forms of treatment can function in a concentrated and synergistic way. It is expected that this highly-innovative combination therapy will effectively, safely, and sustainably kill tumors as well as metastases and at the same time minimize the risk of systemic side effects.

The aim of MAVO TheraVision is to establish a broadly applicable platform technology for combinatorial oncolytic virus-immunotherapy based on three pillars i) engineering of next generation therapeutic Herpes simplex virus type 1 (HSV1)-vectors, ii) development of suitable production procedures for the use  of viruses as pharmaceutical agents, and iii) testing using novel and meaningful preclinical models. An oncolytic virus is being developed as a proof of concept study to treat non-small cell lung cancer (NSCLC), which has a high mortality rate.

Vector / Virus Engineering

A clinical isolate with high potency and acyclovir sensitivity was chosen to establish an HSV1-platform virus. The HSV1-isolate’s genome was cloned as a bacterial artificial chromosome(BAC) in E. coli to make easy and targeted manipulations using bacterial genetics possible. The reconstructed viruses from these BACs had comparable growth characteristics to the clinical isolate. Two insertion sites were identified in the herpes viral genome to integrate the foreign genes into this HSV1-BAC. The result was the so called platform vector used for modular insertion of foreign genes and thus for the diverse functionalization of the virus. Potent functionalized viruses develop as a result of the insertion of transgenes, which express the coded proteins when the virus multiplies.

Production Process Development

As part of MAVO TheraVision a robust and scalable production process for therapeutic viruses will be established. The project will take the necessary GMP-requirements (Good Manufacturing Practice) for the subsequent production of clinical investigational products into account. In particular, critical process parameters in up- and down-stream processing, the use of resources, and the impurity profile, as well as the stability of intermediates and of the product itself will be addressed. The virus production is based on bioreactor cultivation of adherent vero-cells on so called microcarriers. By optimizing the processes, which focus on a finely-tuned balance between cell density, infection time point, and ratio of infecting viruses and living cell number, we have achieved virus titers of up to 6x107 pfu/mL culture supernatant. The procedure is characterized by high robustness and scalability on a scale that is necessary for the clinical material required. Process-associated modeling can also help to optimize the infection and harvesting time points with regard to the target function’s yield, performance, and purity. The corresponding purification sequence will include the combined use of classic and novel chromatography materials. The total process yield reaches purified bulk amounts, which are sufficient for phase I and II clinical studies.

Preclinical Test Systems for a Combinatorial Virus-Immunotherapy

New preclinical models, which are as comparable as possible to the situation in the patient, are necessary to test the treatment efficacy of viruses with combined virus and immunotherapy. Proof of concept for combinatorial oncolytic virus immunotherapy will be done using the exemplary NSCLC model. 

TP3.1 in vitro 3D Tumor Model

As part of the preclinical test platform of the TheraVision-consortium, new tumor models used on intestinal tissue matrices (small intestinal submucosa with mucosa = SISmuc) are being implemented to closely test the in vivo situation. After about 10 days, models develop which have a tissue-like homeostasis. Using bioreactors, the tissue structure can be improved and the culture and therapy duration extended to several weeks. For the project, a disposable inexpensive silicon-bioreactor is being developed that can be used in S2-situations. The efficacy of the HSV1 was dose and time dependently demonstrated in a 3D lung carcinoma model in cell lines over a treatment period of three to seven days. The need for the virus optimization sought by the consortium becomes apparent by the incomplete killing of tumor cells in the deeper tissue structures. Quantification of the virus efficacy in a 3D model is possible using HE-staining, MTT-testing, and with the help of luminescence detection following luciferase-transduction.

TP3.2. Mouse Model with Tumor Cell Lines

The newly established platform virus is being tested with the help of immunodeficient mouse models with bioluminesced lung carcinoma cell lines. The luminescence is measured in the mouse with a highly sensitive light camera. Furthermore, transplantation of human hematopoietic stem cells into these immunodeficient mice can generate a partially functional immune system.  The combination of humanized mice with human tumor growth offers the chance to investigate effects, which are based on immune activation. All three methods have already been established with model viruses. They can now be used with the functionalized HSV1 for in vivo testing of the new therapy. In the future, this platform-testing-technology can be implemented in many different tumor therapy approaches.

TP3.3 PDX-Mouse Model

To test the efficacy of the virus directly on patient material, patient-derived xenograft (PDX) models were generated from primary tumors of patients with NSCLC. These models can be cultivated permanently, and the expression of the target molecules necessary for the virus therapy has been characterized. In addition to the transplanted PDX models, hematopoietic stem cells from umbilical cord blood samples were injected into immunodeficient animals to generate an additional human immune system (humanized mice).  Using these models, the efficacy of the oncolytic viruses including immune modulatory effects on primary tumors and metastases in the presence of human immune cells (especially T-cells) can now be analyzed directly on patient material.