Evaluating TBK1 as a therapeutic target in cancers with activated IRF3.

UNLABELLED: TBK1 (TANK-binding kinase 1) is a noncanonical IκB protein kinase that phosphorylates and activates downstream targets such as IRF3 and c-Rel and, mediates NF-κB activation in cancer. Previous reports demonstrated synthetic lethality of TBK1 with mutant KRAS in non-small cell lung cancer (NSCLC); thus, TBK1 could be a novel target for treatment of KRAS-mutant NSCLC. Here, the effect of TBK1 on proliferation in a panel of cancer cells by both genetic and pharmacologic approaches was evaluated. In KRAS-mutant cancer cells, reduction of TBK1 activity by knockdown or treatment with TBK1 inhibitors did not correlate with reduced proliferation in a two-dimensional viability assay. Verification of target engagement via reduced phosphorylation of S386 of IRF3 (pIRF3(S386)) was difficult to assess in NSCLC cells due to low protein expression. However, several cell lines were identified with high pIRF3(S386) levels after screening a large panel of cell lines, many of which also harbor KRAS mutations. Specifically, a large subset of KRAS-mutant pancreatic cancer cell lines was uncovered with high constitutive pIRF3(S386) levels, which correlated with high levels of phosphorylated S172 of TBK1 (pTBK1(S172)). Finally, TBK1 inhibitors dose-dependently inhibited pIRF3(S386) in these cell lines, but this did not correlate with inhibition of cell growth. Taken together, these data demonstrate that the regulation of pathways important for cell proliferation in some NSCLC, pancreatic, and colorectal cell lines is not solely dependent on TBK1 activity. IMPLICATIONS: TBK1 has therapeutic potential under certain contexts and phosphorylation of its downstream target IRF3 is a biomarker of TBK1 activity.

Human anti-CCR4 minibody gene transfer for the treatment of cutaneous T-cell lymphoma.

BACKGROUND: Although several therapeutic options have become available for patients with Cutaneous T-cell Lymphoma (CTCL), no therapy has been curative. Recent studies have demonstrated that CTCL cells overexpress the CC chemokine receptor 4 (CCR4). METHODOLOGY/PRINCIPAL FINDINGS: In this study, a xenograft model of CTCL was established and a recombinant adeno-associated viral serotype 8 (AAV8) vector expressing a humanized single-chain variable fragment (scFv)-Fc fusion (scFvFc or "minibody") of anti-CCR4 monoclonal antibody (mAb) h1567 was evaluated for curative treatment. Human CCR4+ tumor-bearing mice treated once with intravenous infusion of AAV8 virions encoding the h1567 (AAV8-h1567) minibody showed anti-tumor activity in vivo and increased survival. The AAV8-h1567 minibody notably increased the number of tumor-infiltrating Ly-6G+ FcγRIIIa(CD16A)+ murine neutrophils in the tumor xenografts over that of AAV8-control minibody treated mice. Furthermore, in CCR4+ tumor-bearing mice co-treated with AAV8-h1567 minibody and infused with human peripheral blood mononuclear cells (PBMCs), marked tumor infiltration of human CD16A+ CD56+ NK cells was observed. The h1567 minibody also induced in vitro ADCC activity through both mouse neutrophils and human NK cells. CONCLUSIONS/SIGNIFICANCE: Overall, our data demonstrate that the in vivo anti-tumor activity of h1567 minibody is mediated, at least in part, through CD16A+ immune effector cell ADCC mechanisms. These data further demonstrate the utility of the AAV-minibody gene transfer system in the rapid evaluation of candidate anti-tumor mAbs and the potency of h1567 as a potential novel therapy for CTCL.

Humanization of an anti-CCR4 antibody that kills cutaneous T-cell lymphoma cells and abrogates suppression by T-regulatory cells.

Cutaneous T-cell lymphoma (CTCL) is a heterogeneous group of neoplastic disorders characterized by clonally derived and skin-homing malignant T cells that express high level of chemokine receptor CCR4, which is associated with their skin-homing capacity. CCR4 is also highly expressed on T-regulatory cells (Tregs) that can migrate to several different types of chemotactic ligand CCL17- and CCL22-secreting tumors to facilitate tumor cell evasion from immune surveillance. Thus, its high-level expression on CTCL cells and Tregs makes CCR4 a potential ideal target for antibody-based immunotherapy for CTCL and other types of solid tumors. Here, we conducted humanization and affinity optimization of a murine anti-CCR4 monoclonal antibody (mAb), mAb1567, that recognizes both the N-terminal and extracellular domains of CCR4 with high affinity and inhibits chemotaxis of CCR4(+) CTCL cells. In a mouse CTCL tumor model, mAb1567 exhibited a potent antitumor effect and in vitro mechanistic studies showed that both complement-dependent cytotoxicity (CDC) and neutrophil-mediated antibody-dependent cellular cytotoxicity (ADCC) likely mediated this effect. mAb1567 also exerts human NK cell-mediated ADCC activity in vitro. Moreover, mAb1567 also effectively inhibits chemotaxis of CD4(+)CD25(high) Tregs via CCL22 and abrogates Treg suppression activity in vitro. An affinity-optimized variant of humanized mAb1567, mAb2-3, was selected for further preclinical development based on its higher binding affinity and more potent ADCC and CDC activities. Taken together, this high-affinity humanized mAb2-3 with potent antitumor effect and a broad range of mechanisms of action may provide a novel immunotherapy for CTCL and other solid tumors.

Hepatocyte growth factor regulates the development of highly pure cultured chief cells from rat stomach by stimulating chief cell proliferation in vitro.

The physiology of gastric epithelial cells is often studied by using cancer cell lines, which may or may not provide information relevant to normal cells. Because few models exist to study chief cell physiology in vitro, our purpose was to develop primary cultured chief cells from rodent species that are structurally and functionally similar to native chief cells. For this, isolated chief cells from the rat stomach, purified by counterflow elutriation and density gradient centrifugation, were grown in media with growth factors. Purity and the continuity of tight junctions were determined, and permeability, viability, transepithelial resistance (TER), cell number and proliferation, and pepsinogen secretion in response to carbachol were measured. When plated in media alone or with basic fibroblast growth factor, the isolated chief cells attached by 2 days and were confluent by 4 days after seeding. However, tight junctions were discontinuous, TER was less than 300 Omega cm(2), and permeability was high. In contrast, chief cells incubated with hepatocyte growth factor (HGF) were confluent in 3 days and had a TER greater than 2,000 Omega cm(2), continuous tight junctions, and low permeability. EGF was intermediate. HGF facilitated monolayer development by increasing cell number, which occurred by the proliferation of chief cells. Chief cell cultures, grown with HGF, consisted of more than 99% gastric intrinsic factor-expressing cells and showed robust pepsinogen secretion. Coexpression studies for neck and chief cell markers suggest that the cultures are a mixture of mature, immature, and transitional zone cells. This model will be useful for investigating mechanisms that regulate chief cell physiology in health and disease.

Preparation and characterization of a biodegradable drug targeting system for anticancer drug delivery: microsphere-antibody conjugate.

Targeted delivery of anticancer drugs is one of the most actively pursued goals in anticancer chemotherapy. A major disadvantage of anticancer drugs is their lack of selectivity for tumour tissue, which causes severe side effects and results in low cure rates. Any strategy by which a cytotoxic drug is targeted to the tumour, thus increasing the therapeutic index of the drug, is a way of improving cancer chemotherapy and minimizing systematic toxicity. This study covers the preparation of the gelatin microsphere (GM)-anti-bovine serum albumin (anti-BSA) conjugate for the development of a drug targeting approach for anticancer drug delivery. Microspheres of 5% (w/v) gelatin content were prepared by crosslinking with glutaraldehyde (GTA) at 0.05 and 0.50% (v/v) concentration. Microspheres were in the size range of 71-141?microm. The suitability of these microspheres as drug carriers for anticancer drug delivery was investigated in vitro by studying the release profiles of loaded methotrexate (MTX) and 5-fluorouracil (5-FU) and the cytotoxicities on cancer cell lines. The in vitro MTX release profiles (approximately 22-46% released in 24 h depending on the amount of GTA used) were much slower compared to 5-FU (approximately 42-91% released in 24 h). Both drugs demonstrated an initial fast release, which was followed by gradual, sustained drug release. The MTT cytotoxicity test results of GMs loaded with 5-FU and MTX showed approximately 54-70% and approximately 52-67% cytotoxicities in 4 days. In general, incorporation of MTX and 5-FU in microspheres enhanced the cytotoxic effect in a more prolonged manner compared to the free drugs. Gelatin micospheres were chemically conjugated to anti-BSA and the antigen-antibody activities were studied by immunofluorescence. Results indicated approximately 80% binding with conjugated anti-BSA and BSA-FITC. Based on their low cytotoxicity and the high antigen binding efficiencies, anti-BSA conjugated gelatin microspheres could be suitable targeted drug carrier systems for selective and long-term delivery of anticancer drugs to a specific body compartment (i.e. bladder cancer).

Prolonged cytotoxic effect of colchicine released from biodegradable microspheres.

One the main problems of cancer chemotherapy is the unwanted damage to normal cells caused by the high toxicities of anticancer drugs. Any system of controlled drug delivery that would reduce the total amount of drug required, and thus reduce the side effects, would potentially help to improve chemotherapy. In this respect, biodegradable gelatin microspheres were prepared by water/oil emulsion polymerization and by crosslinking with glutaraldehyde (GTA) as the drug-carrier system. Microspheres were loaded with colchicine, a model antimitotic drug, which was frequently used as an antimitotic agent in cancer research involving cell cultures. Microsphere sizes, swelling and degradation properties, drug-release kinetics, and cytotoxities were studied. Swelling characteristics of microspheres changed upon changing GTA concentration. A decrease in swelling values was recorded as GTA crosslink density was increased. In vitro drug release in PBS (0.01M, pH 7.4) showed rapid colchicine release up to approximately 83% (at t = 92 h) for microspheres with low GTA (0.05% v/v), whereas a slower release profile (only approximately 39%) was obtained for microspheres with high GTA (0.50% v/v) content, for the same period. Cytotoxicity tests with MCF-7, HeLa and H-82 cancer cell lines showed that free colchicine was very toxic, showing an approximately 100% lethal effect in both HeLa and H-82 cell lines and more than 50% decrease in viability in MCF-7 cells in 4 days. Indeed, entrapped colchicine indicated similar initial high toxic effect on cell viability in MCF-7 cell line and this effect became more dominant as colchicine continued to be released from microspheres in the same period. In conclusion, the control of the release rate of colchicine from gelatin microspheres was achieved under in vitro conditions by gelatin through the alteration of crosslinking conditions. Indeed, the results suggested the potential application of gelatin microspheres crosslinked with GTA as a sustained drug-delivery system for anticancer drugs for local chemotherapy administrations.