Biomedical subjects

Ulrik B Nielsen

Publications and source records attributed to Ulrik B Nielsen.

9 recordsLinked to original sources

PubMed2006

A novel assay for monitoring internalization of nanocarrier coupled antibodies.

BACKGROUND: Discovery of tumor-selective antibodies or antibody fragments is a promising approach for delivering therapeutic agents to antigen over-expressing cancers. Therefore it is important to develop methods for the identification of target- and function specific antibodies for effective drug delivery. Here we describe a highly selective and sensitive method for characterizing the internalizing potential of multivalently displayed antibodies or ligands conjugated to liposomes into tumor cells. The assay requires minute amounts of histidine-tagged ligand and relies on the non-covalent coupling of these antibodies to fluorescent liposomes containing a metal ion-chelating lipid. Following incubation of cells with antibody-conjugated liposomes, surface bound liposomes are gently removed and the remaining internalized liposomes are quantitated based on fluorescence in a high throughput manner. We have termed this methodology "Chelated Ligand Internalization Assay", or CLIA. RESULTS: The specificity of the assay was demonstrated with different antibodies to the ErbB-2 and EGF receptors. Antibody-uptake correlated with receptor expression levels in tumor cell lines with a range of receptor expression. Furthermore, Ni-NTA liposomes containing doxorubicin were used to screen for the ability of antibodies to confer target-specific cytotoxicity. Using an anti-ErbB2 single chain Fv (scFv) (F5) antibody, cytotoxicity could be conferred to ErbB2-overexpressing cells; however, a poly(ethylene glycol)-linked lipid (DSPE-PEG-NTA-Ni) was necessary to allow for efficient loading of the drug and to reduce nonspecific drug leakage during the course of the assay. CONCLUSION: The CLIA method we describe here represents a rapid, sensitive and robust assay for the identification and characterization of tumor-specific antibodies capable of high drug-delivery efficiency when conjugated to liposomal nanocarriers.

Antibodies, Neoplasm↗

PubMed2006

Antibody targeting of long-circulating lipidic nanoparticles does not increase tumor localization but does increase internalization in animal models.

We describe evidence for a novel mechanism of monoclonal antibody (MAb)-directed nanoparticle (immunoliposome) targeting to solid tumors in vivo. Long-circulating immunoliposomes targeted to HER2 (ErbB2, Neu) were prepared by the conjugation of anti-HER2 MAb fragments (Fab' or single chain Fv) to liposome-grafted polyethylene glycol chains. MAb fragment conjugation did not affect the biodistribution or long-circulating properties of i.v.-administered liposomes. However, antibody-directed targeting also did not increase the tumor localization of immunoliposomes, as both targeted and nontargeted liposomes achieved similarly high levels (7-8% injected dose/g tumor tissue) of tumor tissue accumulation in HER2-overexpressing breast cancer xenografts (BT-474). Studies using colloidal gold-labeled liposomes showed the accumulation of anti-HER2 immunoliposomes within cancer cells, whereas matched nontargeted liposomes were located predominantly in extracellular stroma or within macrophages. A similar pattern of stromal accumulation without cancer cell internalization was observed for anti-HER2 immunoliposomes in non-HER2-overexpressing breast cancer xenografts (MCF-7). Flow cytometry of disaggregated tumors posttreatment with either liposomes or immunoliposomes showed up to 6-fold greater intracellular uptake in cancer cells due to targeting. Thus, in contrast to nontargeted liposomes, anti-HER2 immunoliposomes achieved intracellular drug delivery via MAb-mediated endocytosis, and this, rather than increased uptake in tumor tissue, was correlated with superior antitumor activity. Immunoliposomes capable of selective internalization in cancer cells in vivo may provide new opportunities for drug delivery.

Animals↗

PubMed2006

The response of human epithelial cells to TNF involves an inducible autocrine cascade.

Tumor necrosis factor (TNF) is a proinflammatory cytokine that induces conflicting pro- and antiapoptotic signals whose relative strengths determine the extent of cell death. TNF receptor (TNFR) has been studied in considerable detail, but it is not known how crosstalk among antagonistic pro- and antiapoptotic signals is achieved. Here we report an experimental and computational analysis of crosstalk between prodeath TNF and prosurvival growth factors in human epithelial cells. By applying classifier-based regression to a cytokine-signaling compendium of approximately 8000 intracellular protein measurements, we demonstrate that cells respond to TNF both directly, via activated TNF receptor, and indirectly, via the sequential release of transforming growth factor-alpha (TGF-alpha), interleukin-1alpha (IL-1alpha), and IL-1 receptor antagonist (IL-1ra). We refer to the contingent and time-varying series of extracellular signals induced by TNF as an "autocrine cascade." Time-dependent crosstalk of synergistic and antagonistic autocrine circuits may serve to link cellular responses to the local environment.

Autocrine Communication↗

PubMed2006

Systems biology and combination therapy in the quest for clinical efficacy.

Combinatorial control of biological processes, in which redundancy and multifunctionality are the norm, fundamentally limits the therapeutic index that can be achieved by even the most potent and highly selective drugs. Thus, it will almost certainly be necessary to use new 'targeted' pharmaceuticals in combinations. Multicomponent drugs are standard in cytotoxic chemotherapy, but their development has required arduous empirical testing. However, experimentally validated numerical models should greatly aid in the formulation of new combination therapies, particularly those tailored to the needs of specific patients. This perspective focuses on opportunities and challenges inherent in the application of mathematical modeling and systems approaches to pharmacology, specifically with respect to the idea of achieving combinatorial selectivity through use of multicomponent drugs.

Drug Therapy, Combination↗

PubMed2005

Using computational modeling to drive the development of targeted therapeutics.

Computational biology is impacting drug design and has the potential to revolutionize the drug development process. Here, we describe how data-driven computational models can optimize the design of targeted therapeutics to yield improved safety and efficacy. We propose that, in the future, this approach will improve drug discovery, clinical development, diagnosis and treatment.

Computational Biology↗

PubMed2004

Multiplexed sandwich assays in microarray format.

Antibody arrays have evolved into powerful tools for quantifying proteins and qualifying their state of activation in complex biological samples. This level of analysis holds tremendous promise as part of a diagnostic or prognostic platform. In particular, multiplex sandwich ELISA assays performed with microarrays in the wells of multi-well plates enable high-throughput analysis of multiple samples with multivariate readouts. Here, we compare and review recent advances in antibody microarray technology and describe its promises towards the systematic analysis of complex biological samples, ranging from profiling patient sera to studying intracellular signaling.

Animals↗

PubMed2003

Profiling receptor tyrosine kinase activation by using Ab microarrays.

Signal transduction in mammalian cells is mediated by complex networks of interacting proteins. Understanding these networks at a circuit level requires devices to measure the amounts and activities of multiple proteins in a rapid and accurate manner. Ab microarrays have previously been applied to the quantification of labeled recombinant proteins and proteins in serum. The development of methods to analyze intracellular signaling molecules on microarrays would make Ab arrays widely useful in systems biology. Here we describe the fabrication of multiplex Ab arrays sensitive to the amounts and modification states of signal transduction proteins in crude cell lysates and the integration of these arrays with 96-well microtiter plate technology to create microarrays in microplates. We apply the Ab arrays to monitoring the activation, uptake, and signaling of ErbB receptor tyrosine kinases in human tumor cell lines. Data obtained from multicolor ratiometric microarrays correlate well with data obtained by using traditional approaches, but the arrays are faster and simpler to use. The integration of microplate and microarray methods for crude cell lysates should make it possible to identify and analyze small molecule inhibitors of signal transduction processes with unprecedented speed and precision. We demonstrate the future potential of this approach by characterizing the action of the epidermal growth factor receptor inhibitor PD153035 on cells by using Ab arrays; direct scale-up to array-based screening in 96- and 384-well plates should allow small molecules to be identified with specific inhibitory profiles against a signaling network.

Animals↗

PubMed2002

Therapeutic efficacy of anti-ErbB2 immunoliposomes targeted by a phage antibody selected for cellular endocytosis.

Many targeted cancer therapies require endocytosis of the targeting molecule and delivery of the therapeutic agent to the interior of the tumor cell. To generate single chain Fv (scFv) antibodies capable of triggering receptor-mediated endocytosis, we previously developed a method to directly select phage antibodies for internalization by recovering infectious phage from the cytoplasm of the target cell. Using this methodology, we reported the selection of a panel of scFv that were internalized into breast cancer cells from a nonimmune phage library. For this work, an immunotherapeutic was generated from one of these scFv (F5), which bound to ErbB2 (HER2/neu). The F5 scFv was reengineered with a C-terminal cysteine, expressed at high levels in Escherichia coli, and coupled to sterically stabilized liposomes. F5 anti-ErbB2 immunoliposomes were immunoreactive as determined by surface plasmon resonance (SPR) and were avidly internalized by ErbB2-expressing tumor cell lines in proportion to the levels of ErbB2 expression. F5-scFv targeted liposomes containing doxorubicin had antitumor activity and produced significant reduction in tumor size in xenografted mice compared to nontargeted liposomes containing doxorubicin. This strategy should be applicable to generate immunotherapeutics for other malignancies by selecting phage antibodies for internalization into other tumor types and using the scFv to target liposomes or other nanoparticles.

Animals↗

PubMed2002

Anti-HER2 immunoliposomes: enhanced efficacy attributable to targeted delivery.

PURPOSE: Anti-HER2 immunoliposomes combine the tumor-targeting of certain anti-HER2 monoclonal antibodies (MAbs) with the pharmacokinetic and drug delivery capabilities of sterically stabilized liposomes. We previously showed that anti-HER2 immunoliposomes bind efficiently to and internalize in HER2-overexpressing cells in vitro, resulting in intracellular drug delivery. EXPERIMENTAL DESIGN: Here we describe the pharmacokinetics and therapeutic efficacy of anti-HER2 immunoliposomes containing doxorubicin (dox) in a series of animal models. RESULTS: Immunoliposomes displayed long circulation that was identical to that of sterically stabilized liposomes in single- and multiple-dose studies in normal rats. Anti-HER2 immunoliposome-dox produced marked therapeutic results in four different HER2-overexpressing tumor xenograft models, including growth inhibition, regression, and cures. These results demonstrated that encapsulation of dox in anti-HER2 immunoliposomes greatly increased its therapeutic index, both by increasing antitumor efficacy and by reducing systemic toxicity. Immunoliposome-dox was significantly superior to all other treatment conditions tested, including free dox, liposomal dox, and anti-HER2 MAb (trastuzumab). When compared with liposomal dox in eight separate therapy studies in HER2-overexpressing models, immunoliposome delivery produced significantly superior antitumor efficacy in each study (P < 0.0001 to 0.04). Anti-HER2 immunoliposome-dox containing either recombinant human MAb HER2-Fab' or scFv C6.5 yielded comparable therapeutic efficacy. Cure rates for immunoliposome-dox reached 50% (11 of 21) with optimized immunoliposomes and Matrigel-free tumors and overall was 16% (18 of 115) versus no cures (0 of 124) with free dox or liposomal dox. Finally, anti-HER2 immunoliposome-dox was also superior to combinations consisting of free MAb plus free dox or free MAb plus liposomal dox. CONCLUSIONS: Anti-HER2 immunoliposomes produced enhanced antitumor efficacy via targeted delivery.

Animals↗