AAV-mediated gene transfer of a checkpoint inhibitor in combination with HER2-targeted CAR-NK cells as experimental therapy for glioblastoma.

Glioblastoma (GB) is the most common primary brain tumor, which is characterized by low immunogenicity of tumor cells and prevalent immunosuppression in the tumor microenvironment (TME). Targeted local combination immunotherapy is a promising strategy to overcome these obstacles. Here, we evaluated tumor-cell specific delivery of an anti-PD-1 immunoadhesin (aPD-1) via a targeted adeno-associated viral vector (AAV) as well as HER2-specific NK-92/5.28.z (anti-HER2.CAR/NK-92) cells as components for a combination immunotherapy. In co-culture experiments, target-activated anti-HER2.CAR/NK-92 cells modified surrounding tumor cells and bystander immune cells by triggering the release of inflammatory cytokines and upregulation of PD-L1. Tumor cell-specific delivery of aPD-1 was achieved by displaying a HER2-specific designed ankyrin repeat protein (DARPin) on the AAV surface. HER2-AAV mediated gene transfer into GB cells correlated with HER2 expression levels, without inducing anti-viral responses in transduced cells. Furthermore, AAV-transduction did not interfere with anti-HER2.CAR/NK-92 cell-mediated tumor cell lysis. After selective transduction of HER2+ cells, aPD-1 expression was detected at the mRNA and protein level. The aPD-1 immunoadhesin was secreted in a time-dependent manner, bound its target on PD-1-expressing cells and was able to re-activate T cells by efficiently disrupting the PD-1/PD-L1 axis. Moreover, high intratumoral and low systemic aPD-1 concentrations were achieved following local injection of HER2-AAV into orthotopic tumor grafts in vivo. aPD-1 was selectively produced in tumor tissue and could be detected up to 10 days after a single HER2-AAV injection. In subcutaneous GL261-HER2 and Tu2449-HER2 immunocompetent mouse models, administration of the combination therapy significantly prolonged survival, including complete tumor control in several animals in the GL261-HER2 model. In summary, local therapy with aPD-1 encoding HER2-AAVs in combination with anti-HER2.CAR/NK-92 cells may be a promising novel strategy for GB immunotherapy with the potential to enhance efficacy and reduce systemic side effects of immune-checkpoint inhibitors.

Cell type-dependent pathogenic functions of overexpressed human cathepsin B in murine breast cancer progression.

The cysteine protease cathepsin B (CTSB) is frequently overexpressed in human breast cancer and correlated with a poor prognosis. Genetic deficiency or pharmacological inhibition of CTSB attenuates tumor growth, invasion and metastasis in mouse models of human cancers. CTSB is expressed in both cancer cells and cells of the tumor stroma, in particular in tumor-associated macrophages (TAM). In order to evaluate the impact of tumor- or stromal cell-derived CTSB on Polyoma Middle T (PyMT)-induced breast cancer progression, we used in vivo and in vitro approaches to induce human CTSB overexpression in PyMT cancer cells or stromal cells alone or in combination. Orthotopic transplantation experiments revealed that CTSB overexpression in cancer cells rather than in the stroma affects PyMT tumor progression. In 3D cultures, primary PyMT tumor cells showed higher extracellular matrix proteolysis and enhanced collective cell invasion when CTSB was overexpressed and proteolytically active. Coculture of PyMT cells with bone marrow-derived macrophages induced a TAM-like macrophage phenotype in vitro, and the presence of such M2-polarized macrophages in 3D cultures enhanced sprouting of tumor spheroids. We employed a doxycycline (DOX)-inducible CTSB expression system to selectively overexpress human CTSB either in cancer cells or in macrophages in 3D cocultures. Tumor spheroid invasiveness was only enhanced when CTSB was overexpressed in cancer cells, whereas CTSB expression in macrophages alone did not further promote invasiveness of tumor spheroids. We conclude that CTSB overexpression in the PyMT mouse model promotes tumor progression not by a stromal effect, but by a direct, cancer cell-inherent mode of action: CTSB overexpression renders the PyMT cancers more invasive by increasing proteolytic extracellular matrix protein degradation fostering collective cell invasion into adjacent tissue.

Transgenic expression of human cathepsin B promotes progression and metastasis of polyoma-middle-T-induced breast cancer in mice.

Elevated expression of the cysteine protease cathepsin B (CTSB) has been correlated with a poor prognosis for cancer patients. In order to model high CTSB expression in mammary cancer, transgenic mice expressing human CTSB were crossed with transgenic polyoma virus middle T oncogene breast cancer mice (mouse mammary tumor virus-PymT), resulting in a 20-fold increase in cathepsin B activity in the tumors of double-transgenic animals. CTSB expression did not affect tumor onset, but CTSB transgenic mice showed accelerated tumor growth with significant increase in weight for end-stage tumors, as well as an overall worsening in their histopathological grades. Notably, the lung metastases in the CTSB transgenic animals were found to be both significantly larger and to occur at a significantly higher frequency. Ex vivo analysis of primary PymT tumor cells revealed no significant effects from elevated CTSB levels on tumor cell characteristics, that is, the formation of tumor cell colonies and the sprouting of invasive strands from PymT cell spheroids. However, tumors from CTSB-overexpressing mice showed increased numbers of tumor-associated B cells and mast cells. In addition, more CD31+ endothelial cells were detected in these tumors, correlating with higher levels of vascular endothelial growth factor (VEGF) being present in the tumor and serum. We conclude that elevated proteolytic CTSB activity facilitates progression and metastasis of PymT-induced mammary carcinomas, and is associated with increased immune cell infiltration, enhanced VEGF levels and the promotion of tumor angiogenesis.

Reduced tumour cell proliferation and delayed development of high-grade mammary carcinomas in cathepsin B-deficient mice.

Expression levels of the papain-like cysteine protease cathepsin B (Ctsb) have been positively correlated with mammary tumour progression and metastasis; however, its roles in the hallmark processes of malignant growth remain poorly defined. Using Ctsb-deficient mice we investigated tumour cell differentiation, proliferation and apoptosis in the Tg(MMTV-PyMT) mouse mammary cancer model. Absence of Ctsb significantly impaired development of high-grade invasive ductal carcinomas and reduced the metastatic burden in the lungs. Mice lacking Ctsb exhibited reduced cell proliferation in mammary carcinomas and their lung metastases. Notably, intravenous injection of primarily isolated, Ctsb-expressing tumour cells into congenic Ctsb-deficient mice revealed impaired cell proliferation in the resulting experimental lung metastases, providing evidence for the involvement of Ctsb in paracrine regulation of cancer cell proliferation. No Ctsb genotype-dependent difference in tumour cell death was observed in vivo or by treatment of isolated PyMT cancer cells with tumour necrosis factor-alpha. However, cancer cells lacking Ctsb exhibited significantly higher resistance to apoptosis induction by the lysosomotropic agent Leu-Leu-OMe. Thus, our results indicate an in vivo role for Ctsb in promoting cellular anaplasia in mammary cancers and proliferation in lung metastases.