Natural killer (NK) cells are promising effector cells for adjuvant immunotherapy of cancer. So far, several preclinical studies have shown the feasibility of gene-engineered NK cells, which upon expression of chimeric antigen receptors (CARs) are redirected to otherwise NK cell–resistant tumors. Yet, we reasoned that the efficiency of an immunotherapy using CAR-modified NK cells critically relies on efficient migration to the tumor site and might be improved by the engraftment of a receptor specific for a chemokine released by the tumor. On the basis of the DNAX-activation protein 12 (DAP12), a signaling adapter molecule involved in signal transduction of activating NK cell receptors, we constructed an epidermal growth factor variant III (EGFRvIII)-CAR, designated MR1.1-DAP12 which confers specific cytotoxicity of NK cell towards EGFRvIII⁺ glioblastoma cells in vitro and to established subcutaneous U87-MG^EGFRvIII tumor xenografts. So far, infusion of NK cells with expression of MR1.1-DAP12 caused a moderate but significantly delayed tumor growth and increased median survival time when compared with NK cells transduced with an ITAM-defective CAR. Notably, the further genetic engineering of these EGFRvIII-specific NK cells with the chemokine receptor CXCR4 conferred a specific chemotaxis to CXCL12/SDF-1α secreting U87-MG glioblastoma cells. Moreover, the administration of such NK cells resulted in complete tumor remission in a number of mice and a significantly increased survival when compared with the treatment of xenografts with NK cells expressing only the EGFRvIII-specific CAR or mock control. We conclude that chemokine receptor–engineered NK cells with concomitant expression of a tumor-specific CAR are a promising tool to improve adoptive tumor immunotherapy.

NK cells are emerging as new effectors for immunotherapy of cancer. In particular, the genetic engraftment of chimeric Ag receptors (CARs) in NK cells is a promising strategy to redirect NK cells to otherwise NK cell–resistant tumor cells. On the basis of DNAX-activation protein 12 (DAP12), a signaling adaptor molecule involved in signal transduction of activating NK cell receptors, we generated a new type of CAR targeting the prostate stem cell Ag (PSCA). We demonstrate in this article that this CAR, designated anti–PSCA-DAP12, consisting of DAP12 fused to the anti-PSCA single-chain Ab fragment scFv(AM1) confers improved cytotoxicity to the NK cell line YTS against PSCA-positive tumor cells when compared with a CAR containing the CD3ζ signaling chain. Further analyses revealed phosphorylation of the DAP12-associated ZAP-70 kinase and IFN-γ release of CAR-engineered cells after contact with PSCA-positive target cells. YTS cells modified with DAP12 alone or with a CAR bearing a phosphorylation-defective ITAM were not activated. Notably, infused YTS cells armed with anti–PSCA-DAP12 caused delayed tumor xenograft growth and resulted in complete tumor eradication in a significant fraction of treated mice. The feasibility of the DAP12-based CAR was further tested in human primary NK cells and confers specific cytotoxicity against KIR/HLA-matched PSCA-positive tumor cells, which was further enhanced by KIR-HLA mismatches. We conclude that NK cells engineered with DAP12-based CARs are a promising tool for adoptive tumor immunotherapy.

An intact immune system is essential to prevent the development and progression of neoplastic cells in a process termed immune surveillance. During this process the innate and the adaptive immune systems closely cooperate and especially T cells play an important role to detect and eliminate tumor cells. Due to the mechanism of central tolerance the frequency of T cells displaying appropriate arranged tumor‐peptide‐specific‐T‐cell receptors is very low and their activation by professional antigen‐presenting cells, such as dendritic cells, is frequently hampered by insufficient costimulation resulting in peripheral tolerance. In addition, inhibitory immune circuits can impair an efficient antitumoral response of reactive T cells. It also has been demonstrated that large tumor burden can promote a state of immunosuppression that in turn can facilitate neoplastic progression. Moreover, tumor cells, which mostly are genetically instable, can gain rescue mechanisms which further impair immune surveillance by T cells. Herein, we summarize the data on how tumor cells evade T‐cell immune surveillance with the focus on solid tumors and describe approaches to improve anticancer capacity of T cells.

Malignant glioma represents the most common primary adult brain tumor in Western industrialized countries. Despite aggressive treatment modalities, the median survival duration for patients with glioblastoma multiforme (GBM), the highest grade malignant glioma, has not improved significantly over past decades. One promising approach to deal with GBM is the inactivation of proteins essential for survival or progression of glioma cells by means of RNA interference (RNAi) techniques. A likely candidate for an RNAi therapy of gliomas is the inhibitor of apoptosis protein survivin. Survivin is involved in 2 main cellular processes–cell division and inhibition of apoptosis. We show here that stable RNAi of survivin induced polyploidy, apoptosis, and impaired proliferation of human U343-MG, U373-MG, H4, and U87-MG cells and of primary glioblastoma cells. Proteome profiler arrays using U373-MG cells identified a novel set of differentially expressed genes upon RNAi-mediated survivin knockdown. In particular, the death receptor TRAIL R2/DR5 was strongly upregulated in survivin-depleted glioma cells, inducing an enhanced cytotoxic response of allogeneic human NK cells. Moreover, an experimental in vivo therapy using polyethylenimine (PEI)/siRNA complexes for survivin knockdown efficiently blocked tumor growth of established subcutaneous U373-MG tumors and enhanced survival of NMRI^nu/nu mice orthopically transplanted with U87-MG cells. We conclude that survivin is functionally relevant in gliomas and that PEI-mediated exogenous delivery of siRNA targeting survivin is a promising strategy for glioblastoma therapy.

Background: SOX2, a high mobility group (HMG)-box containing transcription factor, is a key regulator during development of the nervous system and a persistent marker of neural stem cells. Recent studies suggested a role of SOX2 in tumor progression. In our previous work we detected SOX2 in glioma cells and glioblastoma specimens. Herein, we aim to explore the role of SOX2 for glioma malignancy in particular its role in cell proliferation and migration. Methods: Retroviral shRNA-vectors were utilized to stably knockdown SOX2 in U343-MG and U373-MG cells. The resulting phenotype was investigated by Western blot, migration/invasion assays, RhoA G-LISA, time lapse video imaging, and orthotopic xenograft experiments. Results: SOX2 depletion results in pleiotropic effects including attenuated cell proliferation caused by decreased levels of cyclinD1. Also an increased TCF/LEF-signaling and concomitant decrease in Oct4 and Nestin expression was noted. Furthermore, down-regulation of focal adhesion kinase (FAK) signaling and of downstream proteins such as HEF1/NEDD9, matrix metalloproteinases pro-MMP-1 and -2 impaired invasive proteolysis-dependent migration. Yet, cells with knockdown of SOX2 switched to a RhoA-dependent amoeboid-like migration mode which could be blocked by the ROCK inhibitor Y27632 downstream of RhoA-signaling. Orthotopic xenograft experiments revealed a higher tumorigenicity of U343-MG glioma cells transduced with shRNA targeting SOX2 which was characterized by increased dissemination of glioma cells. Conclusion: Our findings suggest that SOX2 plays a role in the maintenance of a less differentiated glioma cell phenotype. In addition, the results indicate a critical role of SOX2 in adhesion and migration of malignant gliomas.

Survivin, belonging to the inhibitor of apoptosis (IAP) gene family, is abundantly expressed in tumors. It has been hypothesized that Survivin facilitates carcinogenesis by inhibition of apoptosis resulting in improved survival of tumorigenic progeny. Additionally, Survivin plays an essential role during mitosis. Together with its molecular partners Aurora B, Borealin and inner centromere protein it secures bipolar chromosome segregation. However, whether increased Survivin levels contribute to progression of tumors by inducing chromosomal instability remains unclear. We overexpressed Survivin in U251-MG, SVGp12, U87-MG, HCT116 and p53-deficient U87-MGshp53 and HCT116p53−/− cells. The resulting phenotype was investigated by FACS-assisted cell cycle analysis, Western Blot analysis, confocal laser scan microscopy, proliferation assays, spectral karyotyping and in a U251-MG xenograft model using immune-deficient mice. Overexpression of Survivin affected cells with knockdown of p53, cells harboring mutant p53 and SV40 large T antigen, respectively, resulting in the increase of cell fractions harboring 4n and >4n DNA contents. Increased γH2AX levels, indicative of DNA damage were monitored in all Survivin-transduced cell lines, but only in p53 wild type cells this was accompanied by an attenuated S-phase entry and activation of p21waf/cip. Overexpression of Survivin caused a DNA damage response characterized by increased appearance pDNA-PKcs foci in cell nuclei and elevated levels of pATM S1981 and pCHK2 T68. Additionally, evolving structural chromosomal aberrations in U251-MG cells transduced with Survivin indicated a DNA-repair by non-homologous end joining recombination. Subcutaneous transplantation of U251-MG cells overexpressing Survivin and mycN instead of mycN oncogene alone generated tumors with shortened latency and decreased apoptosis. Subsequent SKY-analysis of Survivin/mycN-tumors revealed an increase in structural chromosomal aberrations in cells when compared to mycN-tumors. Our data suggest that increased Survivin levels promote adaptive evolution of tumors through combining induction of genetic heterogeneity with inhibition of apoptosis.