The number of publications in the field of chemical cross-linking combined with mass spectrometry (XL-MS) to derive constraints for protein three-dimensional structure modeling and to probe protein–protein interactions has increased during the last years. As the technique is now becoming routine for in vitro and in vivo applications in proteomics and structural biology there is a pressing need to define protocols as well as data analysis and reporting formats. Such consensus formats should become accepted in the field and be shown to lead to reproducible results. This first, community-based harmonization study on XL-MS is based on the results of 32 groups participating worldwide. The aim of this paper is to summarize the status quo of XL-MS and to compare and evaluate existing cross-linking strategies. Our study therefore builds the framework for establishing best practice guidelines to conduct cross-linking experiments, perform data analysis, and define reporting formats with the ultimate goal of assisting scientists to generate accurate and reproducible XL-MS results.
The delivery of nucleic acids into cells is an attractive approach for cancer therapy. Polyethylenimine (PEI) is among the most efficient nonviral carriers. Recent studies have demonstrated that PEI can be conjugated to targeting ligands, such as epidermal growth factor (EGF) and transferrin (Schaffert et al., 2011; Abourbeh et al., 2012; Ogris et al., 1999). Herein we present a simplified protocol for producing homogeneous preparations of PEGylated linear PEI: LPEI-PEG2k. We generated two well-characterized copolymers, with ratios of LPEI to PEG of 1:1 and 1:3. These copolymers were further conjugated through disulfide bonds to a Her-2 targeting moiety, Her-2 affibody. This reaction yielded two triconjugates that target Her-2 overexpressing tumors. Polyplexes were formed by complexing plasmid DNA with the triconjugates. We characterized the biophysical properties of the conjugates, and found that the triconjugate 1:3 polyplex had lower ζ potential, larger particle size, and more heterogeneous shape than the triconjugate 1:1 polyplex. Triconjugate 1:1 and triconjugate 1:3 polyplexes were highly selective toward cells that overexpress Her-2 receptors, but triconjugate 1:1 polyplex was more efficient at gene delivery. Our studies show that the biophysical and biological properties of the conjugates can be profoundly affected by the ratio of LPEI:PEG2k:ligand. The procedure described here can be adapted to generate a variety of triconjugates, simply by changing the targeting moiety.
The development of targeted therapies that affect multiple signaling pathways and stimulate antitumor immunity is greatly needed. About 20% of patients with breast cancer overexpress HER2. Small molecules and antibodies targeting HER2 convey some survival benefits; however, patients with advanced disease succumb to the disease under these treatment regimens, possibly because HER2 is not completely necessary for the survival of the targeted cancer cells. In the present study, we show that a polyinosine/polycytosine (pIC) HER2-homing chemical vector induced the demise of HER2-overexpressing breast cancer cells, including trastuzumab-resistant cells. Targeting pIC to the tumor evoked a number of cell-killing mechanisms, as well as strong bystander effects. These bystander mechanisms included type I IFN induction, immune cell recruitment, and activation. The HER2-targeted pIC strongly inhibited the growth of HER2-overexpressing tumors in immunocompetent mice. The data presented here could open additional avenues in the treatment of HER2-positive breast cancer.
Selective delivery of drugs to tumor cells can increase potency and reduce toxicity. In this study, we describe a novel recombinant chimeric protein, dsRBEC, which can bind polyIC and deliver it selectively into EGFR over-expressing tumor cells. dsRBEC, comprises the dsRNA binding domain (dsRBD) of human PKR (hPKR), which serves as the polyIC binding moiety, fused to human EGF (hEGF), the targeting moiety. dsRBEC shows high affinity towards EGFR and triggers ligand-induced endocytosis of the receptor, thus leading to the selective internalization of polyIC into EGFR over-expressing tumor cells. The targeted delivery of polyIC by dsRBEC induced cellular apoptosis and the secretion of IFN-β and other pro-inflammatory cytokines. dsRBEC-delivered polyIC is much more potent than naked polyIC and is expected to reduce the toxicity caused by systemic delivery of polyIC.
There is an urgent need for an effective treatment for metastatic prostate cancer (PC). Prostate tumors invariably overexpress prostate surface membrane antigen (PSMA). We designed a nonviral vector, PEI-PEG-DUPA (PPD), comprising polyethylenimine-polyethyleneglycol (PEI-PEG) tethered to the PSMA ligand, 2-[3-(1, 3-dicarboxy propyl)ureido] pentanedioic acid (DUPA), to treat PC. The purpose of PEI is to bind polyinosinic/polycytosinic acid (polyIC) and allow endosomal release, while DUPA targets PC cells. PolyIC activates multiple pathways that lead to tumor cell death and to the activation of bystander effects that harness the immune system against the tumor, attacking nontargeted neighboring tumor cells and reducing the probability of acquired resistance and disease recurrence. Targeting polyIC directly to tumor cells avoids the toxicity associated with systemic delivery. PPD selectively delivered polyIC into PSMA-overexpressing PC cells, inducing apoptosis, cytokine secretion, and the recruitment of human peripheral blood mononuclear cells (PBMCs). PSMA-overexpressing tumors in nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice with partially reconstituted immune systems were significantly shrunken following PPD/polyIC treatment, in all cases. Half of the tumors showed complete regression. PPD/polyIC invokes antitumor immunity, but unlike many immunotherapies does not need to be personalized for each patient. The potent antitumor effects of PPD/polyIC should spur its development for clinical use.
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