Chemokines as novel and versatile reagents for flow cytometry and cell sorting.

Cell therapy regimens are frequently compromised by low-efficiency cell homing to therapeutic niches. Improvements in this regard would enhance effectiveness of clinically applicable cell therapy. The major regulators of tissue-specific cellular migration are chemokines, and therefore selection of therapeutic cellular populations for appropriate chemokine receptor expression would enhance tissue-homing competence. A number of practical considerations preclude the use of Abs in this context, and alternative approaches are required. In this study, we demonstrate that appropriately labeled chemokines are at least as effective in detecting their cognate receptors as commercially available Abs. We also demonstrate the utility of biotinylated chemokines as cell-sorting reagents. Specifically, we demonstrate, in the context of CCR7 (essential for lymph node homing of leukocytes), the ability of biotinylated CCL19 with magnetic bead sorting to enrich for CCR7-expressing cells. The sorted cells demonstrate improved CCR7 responsiveness and lymph node-homing capability, and the sorting is effective for both T cells and dendritic cells. Importantly, the ability of chemokines to detect CCR7, and sort for CCR7 positivity, crosses species being effective on murine and human cells. This novel approach to cell sorting is therefore inexpensive, versatile, and applicable to numerous cell therapy contexts. We propose that this represents a significant technological advance with important therapeutic implications.

Treatment of inflammatory bowel disease by chemokine receptor-targeted leukapheresis.

Leukapheresis removes circulating leukocytes en route to the target organ. Hitherto unspecific matrixes have been used to remove leukocytes in inflammatory bowel disease (IBD). This report describes a novel selective leukapheresis column based on chemokine-chemokine receptor interaction. We found an increased expression of the gut homing chemokine receptor CCR9 on CD14(+) monocytes and on CD3(+) T lymphocytes from IBD patients. Biologically active CCL25 was coupled to a Sepharose matrix and demonstrated to selectively remove CCR9-expressing cells leaving other cell populations largely unaffected. A patient with active ulcerative colitis, was subjected to CCL25-column leukapheresis. Four days after treatment, he experienced clinical improvement and stable disease improvement ensued. The study illustrates that specific cells can be targeted using high affinity interactions, i.e., CCL25-CCR9 interactions to remove pathogenic gut-homing cells. Leukapheresis using the bCCL25 column should be investigated in a clinical phase I trial of patients with inflammatory bowel disease.

Recombinant protein hydrazides: application to site-specific protein PEGylation.

Here, we describe a novel method for the site-specific C-terminal PEGylation of recombinant proteins. This general approach exploits chemical cleavage of precursor intein-fusion proteins with hydrazine to directly produce recombinant protein hydrazides. This unique functionality within the protein sequence then facilitates site-specific C-terminal modification by hydrazone-forming ligation reactions. This approach was used to generate folded, site-specifically C-terminal PEGylated IFNalpha2b and IFNbeta1b, which retained excellent antiviral activity, demonstrating the utility of this technology in the PEGylation of therapeutic proteins. As this methodology is straightforward to perform, is compatible with disulfide bonds, and is exclusively selective for the protein C-terminus, it shows great potential as general technology for the site-specific engineering and labeling of recombinant proteins.

A fluorescence lifetime-based assay for serine and threonine kinases that is suitable for high-throughput screening.

We describe the development of a novel method for the assay of serine/threonine protein kinases based on fluorescence lifetime. The assay consists of three generic peptides (which have been used by others in the assay of >140 protein kinases in various assay formats) labeled with a long lifetime fluorescent dye (14 or 17 ns) that act as substrates for protein kinases and an iron(III) chelate that modulates the fluorescence lifetime of the peptide only when it is phosphorylated. The decrease in average fluorescence lifetime as measured in a recently developed fluorescence lifetime plate reader (Edinburgh Instruments) is a measure of the degree of phosphorylation of the peptide. We present data showing that the assay performs as well as, and in some cases better than, the "gold standard" radiometric kinase assays with respect to Z' values, demonstrating its utility in high-throughput screening applications. We also show that the assay gives nearly identical results in trial screening to those obtained by radiometric assays and that it is less prone to interference than simple fluorescence intensity measurements.

Single-Domain Antibody-Functionalized pH-Responsive Amphiphilic Block Copolymer Nanoparticles for Epidermal Growth Factor Receptor Targeted Cancer Therapy.

Biocompatible antibody-nanoparticle conjugates have attracted interest as anticancer agents due to their potential to selectively target therapeutic agents at disease sites. However, new formulation and conjugation approaches are urgently needed to improve their uniformity for clinical applications. Here, a pH-responsive benzaldehyde-functionalized poly[oligo(ethylene glycol) methacrylate-st-para-formyl phenyl methacrylate]-b-poly[2-(diisopropyl)aminoethyl methacrylate] [P(OEGMA-st-pFPMA)-b-PDPA] block copolymer, prepared by reversible addition-fragmentation chain transfer polymerization, produced PEGylated nanoparticles (pH ∼ 7.4) by a single emulsion-solvent evaporation formulation approach. Efficient site-specific attachment of an aminooxy-functionalized anti-EGFR single-domain antibody (sdAb) on these benzaldehyde-decorated nanoparticles is achieved by oxime bond formation. These nanoconjugates can specifically bind EGFR (modified ELISA) and have enhanced uptake over nonfunctionalized controls in EGFR-positive HeLa cells. Encapsulation of rhodamine 6G dye and its dispersion upon cellular uptake, consistent with nanoparticle stability loss at pH < 5.7, prove their ability to facilitate triggered release in endosomal compartments and highlight their potential for use as next-generation antibody-drug nanoconjugates for therapeutic drug delivery.

A Comparative Study of Fluorescence Assays in Screening for BRD4.

Fluorescence assay technologies are commonly used in high-throughput screening because of their sensitivity and ease of use. Different technologies have their characteristics and the rationale for choosing one over the other can differ between projects because of factors such as availability of reagents, assay performance, and cost. Another important factor to consider is the assay susceptibility to artifacts, which is almost as important as the ability of the assay to pick up active compounds. Spending time and money on false positives or missing the opportunity to build chemistry around false negatives is something that every drug project tries to avoid. We used a BET family Bromodomain, BRD4(1), to explore the outcome of a screening campaign using three fluorescent assay technologies as primary assays. A diverse 7,038 compound set was screened in fluorescence lifetime, fluorescence polarization, and homogeneous time-resolved fluorescence to look at primary hit rates, compound overlap, and hit confirmation rates. The results show a difference between the fluorescence assay technologies with three separate hit lists and some overlap. The confirmed hits from each assay were further evaluated for translation into cells (NanoBRET™). Most of the actives confirmed in cells originated from compounds that overlapped between the assays. In addition, a well-annotated set of compounds with undesirable mechanism of inhibition was screened against BRD4(1) to compare the ability to discriminate true hits from artifact compounds. The results indicate a difference between the assays in their ability to generate false positives and negatives.

FKBPL and peptide derivatives: novel biological agents that inhibit angiogenesis by a CD44-dependent mechanism.

PURPOSE: Antiangiogenic therapies can be an important adjunct to the management of many malignancies. Here we investigated a novel protein, FKBPL, and peptide derivative for their antiangiogenic activity and mechanism of action. EXPERIMENTAL DESIGN: Recombinant FKBPL (rFKBPL) and its peptide derivative were assessed in a range of human microvascular endothelial cell (HMEC-1) assays in vitro. Their ability to inhibit proliferation, migration, and Matrigel-dependent tubule formation was determined. They were further evaluated in an ex vivo rat model of neovascularization and in two in vivo mouse models of angiogenesis, that is, the sponge implantation and the intravital microscopy models. Antitumor efficacy was determined in two human tumor xenograft models grown in severe compromised immunodeficient (SCID) mice. Finally, the dependence of peptide on CD44 was determined using a CD44-targeted siRNA approach or in cell lines of differing CD44 status. RESULTS: rFKBPL inhibited endothelial cell migration, tubule formation, and microvessel formation in vitro and in vivo. The region responsible for FKBPL's antiangiogenic activity was identified, and a 24-amino acid peptide (AD-01) spanning this sequence was synthesized. It was potently antiangiogenic and inhibited growth in two human tumor xenograft models (DU145 and MDA-231) when administered systemically, either on its own or in combination with docetaxel. The antiangiogenic activity of FKBPL and AD-01 was dependent on the cell-surface receptor CD44, and signaling downstream of this receptor promoted an antimigratory phenotype. CONCLUSION: FKBPL and its peptide derivative AD-01 have potent antiangiogenic activity. Thus, these agents offer the potential of an attractive new approach to antiangiogenic therapy.

9-Aminoacridine peptide derivatives as versatile reporter systems for use in fluorescence lifetime assays.

A novel long lifetime fluorescence reporter based on 9-aminoacridine was designed, the lifetime of which can be modulated in a defined manner when in proximity to a tryptophan residue enabling fluorescence lifetime based biochemical assays to be configured.