Molecular patterning processes taking place in biological systems are challenging to study in vivo because of their dynamic behavior, subcellular size, and high degree of complexity. In vitro patterning of biomolecules using nanolithography allows simplification of the processes and detailed study of the dynamic interactions. Parallel dip-pen nanolithography (DPN) is uniquely capable of integrating functional biomolecules on subcellular length scales due to its constructive nature, high resolution, and high throughput. Phospholipids are particularly well suited as inks for DPN since a variety of different functional lipids can be readily patterned in parallel. Here DPN is used to spatially pattern multicomponent micro- and nanostructured supported lipid membranes and multilayers that are fluid and contain various amounts of biotin and/or nitrilotriacetic acid functional groups. The patterns are characterized by fluorescence microscopy and photoemission electron microscopy. Selective adsorption of functionalized or recombinant proteins based on streptavidin or histidine-tag coupling enables the semisynthetic fabrication of model peripheral membrane bound proteins. The biomimetic membrane patterns formed in this way are then used as substrates for cell culture, as demonstrated by the selective adhesion and activation of T-cells.
The driving force behind oncoproteomics is to identify protein signatures that are associated with a particular malignancy. Here, we have used a recombinant scFv antibody microarray in an attempt to classify sera derived from pancreatic adenocarcinoma patients versus healthy subjects. Based on analysis of nonfractionated, directly labeled, whole human serum proteomes we have identified a protein signature based on 19 nonredundant analytes, that discriminates between cancer patients and healthy subjects. Furthermore, a potential protein signature, consisting of 21 protein analytes, could be defined that was shown to be associated with cancer patients having a life expectancy of <12 months. Taken together, the data suggest that antibody microarray analysis of complex proteomes will be a useful tool to define disease associated protein signatures.
Immunotherapy with intratumoral injection of adenoviral vectors expressing CD40L has yielded positive results in experimental and clinical bladder cancer. We therefore hypothesized that anti-CD40 antibody would be effective in this setting. Agonistic CD40 antibodies were developed as vaccine adjuvants but have later been used as treatment of advanced solid tumors and hematologic cancers. Systemic anti-CD40 therapy has been associated with immune-related adverse events, such as cytokine release syndrome and liver toxicity, and local delivery is an attractive approach that could reduce toxicity. Herein, we compared local and systemic anti-CD40 antibody delivery to evaluate efficacy, toxicity, and biodistribution in the experimental MB49 bladder cancer model. Antitumor effects were confirmed in the B16 model. In terms of antitumor efficacy, local anti-CD40 antibody stimulation was superior to systemic therapy at an equivalent dose and CD8 T cells were crucial for tumor growth inhibition. Both administration routes were dependent on host CD40 expression for therapeutic efficacy. In vivo biodistribution studies revealed CD40-specific antibody accumulation in the tumor-draining lymph nodes and the spleen, most likely reflecting organs with frequent target antigen-expressing immune cells. Systemic administration led to higher antibody concentrations in the liver and blood compared with local delivery, and was associated with elevated levels of serum haptoglobin. Despite the lack of a slow-release system, local anti-CD40 therapy was dependent on tumor antigen at the injection site for clearance of distant tumors. To summarize, local low-dose administration of anti-CD40 antibody mediates antitumor effects in murine models with reduced toxicity and may represent an attractive treatment alternative in the clinic. Cancer Immunol Res; 2(1); 80-90. Ó2013 AACR.
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