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.

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. Based on the DNAX-activation protein 12 (DAP12), a signaling adapter molecule involved in signal transduction of activating NK cell receptors, we constructed an EGFRvIII-CAR, designated MR1.1-DAP12 which confers specific cytotoxicity of NK cell towards EGFRvIII+ glioblastoma cells in vitro and to established subcutaneous U87-MGEGFRvIII 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 to 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 to 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.

Therapeutics based on small interfering RNAs (siRNAs) offer a great potential to treat so far incurable diseases or metastatic cancer. However, the broad application of siRNAs using various nonviral carrier systems is hampered by unspecific toxic side effects, poor pharmacokinetics due to unwanted delivery of siRNA-loaded nanoparticles into nontarget organs, or rapid renal excretion. In order to overcome these obstacles, several targeting strategies using chemically linked antibodies and ligands have emerged. This study reports a new modular polyplex carrier system for targeted delivery of siRNA, which is based on transfection-disabled maltose-modified poly(propyleneimine)-dendrimers (mal-PPI) bioconjugated to single chain fragment variables (scFvs). To achieve targeted delivery into tumor cells expressing the epidermal growth factor receptor variant III (EGFRvIII), monobiotinylated anti-EGFRvIII scFv fused to a Propionibacterium shermanii transcarboxylase-derived biotinylation acceptor (P-BAP) is bioconjugated to mal-PPI through a novel coupling strategy solely based on biotin-neutravidin bridging. In contrast to polyplexes containing an unspecific control scFv-P-BAP, the generated EGFRvIII-specific polyplexes are able to exclusively deliver siRNA to tumor cells and tumors by receptor-mediated endocytosis. These results suggest that receptor-mediated uptake of otherwise noninternalized mal-PPI-based polyplexes is a promising avenue to improve siRNA therapy of cancer, and introduce a novel strategy for modular bioconjugation of protein ligands to nanoparticles.

Application of Toll-like receptor (TLR) agonists is a promising approach to treat cancer. In particular, nucleic acid-based TLR agonists such as short ssRNA and dsRNA molecules, which activate endosomal TLRs, can be delivered to tumors by use of nanoparticle delivery systems. However, such delivery systems bear unspecific side effects and poor pharmacokinetics. To overcome these limitations we developed a system for targeted delivery of a 50 bp dsRNA TLR3 agonist (Riboxxol) to treat PSCA-positive tumor cells, which consists of neutravidin conjugated to mono-biotinylated dsRNA and to humanized mono-biotinylated anti-PSCA single chain antibody derivative scFv(h-AM1)-BAP. The assembly of the components resulted in the formation of nanoparticle-like immunoconjugates designated Rapid Inducer of Cellular Inflammation and Apoptosis (RICIA). Anti-PSCA-RICIA exclusively delivered Riboxxol to PSCA-positive tumor cells as well as subcutaneous tumors. Uptake of anti-PSCA-RICIA induced a type I-interferon response and apoptosis in HEK-Blue hTLR3/PSCA reporter cells and PSCA-positive HT1376 bladder cancer cells in vitro . No such effects were observed when using RICIA coupled to an unspecific control antibody or when using Riboxxol alone. Treatment of HT1376 xenografts in immune-deficient hosts with targeted delivery of TLR3 agonist did not induce adverse effects and only modestly inhibited tumor growth when compared to controls. These results suggest promising activation of innate immune response and apoptosis upon selective delivery of TLR3 agonists in tumor cells. Yet, further studies using syngeneic and orthotopic tumor models are needed to fully exploit the potential of RICIA immunoconjugates.

BackgroundSurvivin, 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.MethodsWe 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.ResultsOverexpression 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.ConclusionsOur data suggest that increased Survivin levels promote adaptive evolution of tumors through combining induction of genetic heterogeneity with inhibition of apoptosis.Electronic supplementary materialThe online version of this article (10.1186/s12885-017-3932-y) contains supplementary material, which is available to authorized users.

Synthetic platforms for mimicking artificial organelles or for designing multivalent protein therapeutics for targeting cell surface, extracellular matrix, and tissues are in the focus of this study. Furthermore, the availability of a multi‐functionalized and stimuli‐responsive carrier system is required that can be used for sequential in situ and/or post loading of different proteins combined with post‐functionalization steps. Until now, polymersomes exhibit excellent key characteristics to fulfill those requirements, which allow specific transport of proteins and the integration of proteins in different locations of polymeric vesicles. Herein, different approaches to fabricate multivalent protein‐loaded, pH‐responsive, and pH‐stable polymersomes are shown, where a combination of therapeutic action and targeting can be achieved, by first choosing two model proteins such as human serum albumin and avidin. Validation of the molecular parameters of the multivalent biohybrids is performed by dynamic light scattering, cryo‐TEM, fluorescence spectroscopy, and asymmetrical flow‐field flow fractionation combined with light scattering techniques. To demonstrate targeting functions of protein‐loaded polymersomes, avidin post‐functionalized polymersomes are used for the molecular recognition of biotinylated cell surface receptors. These versatile protein‐loaded polymersomes present new opportunities for designing sophisticated biomolecular nanoobjects in the field of (extracellular matrix) protein therapeutics.

Molecular reprogramming of stromal microarchitecture by tumour-derived extracellular vesicles (EVs) is proposed to favour pre-metastatic niche formation. We elucidated the role of extravesicular tissue inhibitor of matrix metalloproteinase-1 (TIMP1EV) in pro-invasive extracellular matrix (ECM) remodelling of the liver microenvironment to aid tumour progression in colorectal cancer (CRC). Immunohistochemistry analysis revealed a high expression of stromal TIMP1 in the invasion front that was associated with poor progression-free survival in patients with colorectal liver metastases. Molecular analysis identified TIMP1EV enrichment in CRC-EVs as a major factor in the induction of TIMP1 upregulation in recipient fibroblasts. Mechanistically, we proved that EV-mediated TIMP1 upregulation in recipient fibroblasts induced ECM remodelling. This effect was recapitulated by human serum-derived EVs providing strong evidence that CRC release active EVs into the blood circulation of patients for the horizontal transfer of malignant traits to recipient cells. Moreover, EV-associated TIMP1 binds to HSP90AA, a heat-shock protein, and the inhibition of HSP90AA on human-derived serum EVs attenuates TIMP1EV-mediated ECM remodelling, rendering EV-associated TIMP1 a potential therapeutic target. Eventually, in accordance with REMARK guidelines, we demonstrated in three independent cohorts that EV-bound TIMP1 is a robust circulating biomarker for a non-invasive, preoperative risk stratification in patients with colorectal liver metastases.

Background aims: Natural killer (NK) cells are promising cells for immunotherapy of cancer, and there are ongoing efforts to improve their ex vivo expansion to clinically relevant numbers. This study focused on the development of a C1-, C2-, Bw4 killer cell immunoglobulin-like receptor (KIR) ligand and NKG2A ligand-containing feeder cell line for autonomous expansion of functional NK cells. Methods: PC3 PSCA -derived feeder cells expressing IL-2, 4-1BBL and membrane-bound IL-15-mutDAP12 (mIL-15d) fusion protein in combinations or alone were generated and used for expansion. Expanded NK cells were analyzed with respect to subpopulations, expression of NK cell receptors and immune checkpoint molecules as well as their cytotoxicity against K562 cells, cetuximab-marked tumor cells and autologous B cells. Results: Only combinatorial expression of IL-2 plus 4-1BBL or IL-2, 4-1BBL plus mIL-15d in feeder cells efficiently expanded NK cells and supported selective outgrowth of NK cells from peripheral blood mononuclear cell samples. Best expansion of NK cells was achieved using PC3 PSCA -IL-2-4-1BBL-mIL-15d feeder cells. Such expanded NK cells exhibited upregulation of natural cytotoxicity receptors, DNAM-1 and NKG2C and induced expression of high affinity IL-2 receptor, which were paralleled by attenuated KIR and increased expression of NKG2A and ILT2. In addition, elevated TIM-3 levels were noted and PD-1 and T cell immunoreceptor with Ig and ITIM domain (TIGIT) levels remained low. Expanded NK cells were highly cytolytic when encountering K562 cells and cetuximab-marked target cells but remained unresponsive to autologous B cells and target cells with protective levels of human leukocyte antigen. Conclusions: Collectively, the results demonstrate the feasibility of PC3 PSCA -IL-2-4-1BBL-mIL-15d feeder cells for robust expansion of NK cells, which remain tolerant to self and could be used in the future for adoptive cell therapy of cancer.