Humanized Mice with Subcutaneous Human Solid Tumors for Immune Response Analysis of Vaccinia Virus-Mediated Oncolysis

Abstract

Oncolytic vaccinia virus (VACV) therapy is an alternative cancer treatment modality that mediates targeted tumor destruction through a tumor-selective replication and an induction of anti-tumor immunity. We developed a humanized tumor mouse model with subcutaneous human tumors to analyze the interactions of VACV with the developing tumors and human immune system. A successful systemic reconstitution with human immune cells including functional T cells as well as development of tumors infiltrated with human T and natural killer (NK) cells was observed. We also demonstrated successful in vivo colonization of such tumors with systemically administered VACVs. Further, a new recombinant GLV-1h376 VACV encoding for a secreted human CTLA4-blocking single-chain antibody (CTLA4 scAb) was tested. Surprisingly, although proving CTLA4 scAb’s in vitro binding ability and functionality in cell culture, beside the significant increase of CD56bright NK cell subset, GLV-1h376 was not able to increase cytotoxic T or overall NK cell levels at the tumor site. Importantly, the virus-encoded β-glucuronidase as a measure of viral titer and CTLA4 scAb amount was demonstrated. Therefore, studies in our “patient-like” humanized tumor mouse model allow the exploration of newly designed therapy strategies considering the complex relationships between the developing tumor, the oncolytic virus, and the human immune system.

Humanized Mice as Unique Tools for Human-Specific Studies

“…Currently, there are three ways to study tumor growth and cancer immunology in humanized mice. First, tumor cell lines can be engrafted into humanized mice reconstituted with HSCs or PBMCs (Ito et al 2009; Tsoneva et al 2017; Wege et al 2014). Breast cancer was modelled in mice by concurrently transplanting CD34 + HSCs and tumor cells into newborn mice or engrafting both PBMCs and tumor cells into BRG mice (Wege et al 2014).…”

“…Upon treatment of KM2760, tumor-infiltrating CD56 + NK cells were increased and T-regulatory cells were decreasedIto et al (2009)MelanomaNewborn NSG were intrahepatically injected with CD34 + UBC and injected with human melanoma cell lines (1935-MEL and 888-MEL)Mice were successfully engrafted with a functional human immune system. Oncolytic vaccinia virus therapy, particularly CTLA4 scAb increased CD56 + NK cells and decreased virus titersTsoneva et al (2017)MyelomaNOG mice were intravenously engrafted with human myeloma cell lines (U266)U266 myeloma cells homed to the BM and resulted in paralysis of NOG miceMiyakawa et al (2004)OvarianNSG mice were intraperitoneally engrafted with patient-derived xenografts of primary and metastatic ovarian solid tumor tissue and ovarian ascites fluidSimilar to clinical patients, tumors engrafted in these mice established in the omentum, ovaries, liver, spleen, uterus, and pancreasBankert et al (2011)PancreaticNSG mice were engrafted with patient-derived pancreatic cancer tumors by subcutaneous, intravenous or intra-pancreatic injectionsActivated allogenic and autologous NK cells were able to selectively kill cancer stem cells in NSG mice engrafted with pancreatic cancerAmes et al (2015)ProstateNSG mice were injected with PBMCs with subsets of CD4 +, CD8 + and autologous DCs and subcutaneously injected with human prostate cancer cells (PC3) into the right flankTumor-infiltrating lymphocytes in NSG mice with a functional human immune system and prostate cancer cells were similar to clinical scenariosRoth and Harui (2015) HNSCC head and neck squamous cell carcinoma, RCC renal cell carcinoma, AML acute myeloid leukemia, PDX patient-derived xenografts, mAbs monoclonal antibodies, scAb single-chain antibody …”

“…For example, functional Toll-like receptor 10 (TLR10) is absent in mice (Oosting et al 2014) and cell expression marker CD28 is expressed on 100% of CD4 + and CD8 + T cells in mice but only on 80% of CD4 + and 50% CD8 + T cells in humans (Beyersdorf et al 2015). Due to these differences, it is common that animal models are refractory to many infectious (Bäumler and Fang 2013; Carlton et al 2008; Fauci 1988; Pain et al 2008; Ploss et al 2009), therapeutic (McKenzie et al 1995; Rehman et al 2011), or immunomodulatory agents (Attarwala 2010; Tsoneva et al 2017) that are human-specific.…”

Oncolytic vaccinia virus GLV-1h68 exhibits profound antitumoral activities in cell lines originating from neuroendocrine neoplasms

“…This effect was observed earlier when it was found that not only VACV-injected melanoma metastases decreased in size, but also non-injected distant lesions responded to virotherapy with a granulocyte-macrophage colony-stimulating factor (GM-CSF)-expressing vaccinia virus [40]. Both, a response of the innate immune system mediated by NK-cells, neutrophils and macrophages as well as an adaptive immunity facilitated by antigen-presenting cells and subsequent tumor-in ltrating CD8+ cells have been described after GLV-1h68 treatment [41]. Obviously, this secondary immune-mediated mechanism is complicated to mimic in an in vitro setting.…”

GLV-1h68 vaccinia virus therapy of neuroendocrine tumors

“…This effect was observed earlier when it was found that not only VACV-injected melanoma metastases decreased in size, but also non-injected distant lesions responded to virotherapy with a granulocyte-macrophage colony-stimulating factor (GM-CSF)expressing vaccinia virus [40]. Both, a response of the innate immune system mediated by NK-cells, neutrophils and macrophages as well as an adaptive immunity facilitated by antigen-presenting cells and subsequent tumor-infiltrating CD8 + cells have been described after GLV-1h68 treatment [41].…”