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.