Rapid, deep and precise profiling of the plasma proteome with multi-nanoparticle protein corona.

Large-scale, unbiased proteomics studies are constrained by the complexity of the plasma proteome. Here we report a highly parallel protein quantitation platform integrating nanoparticle (NP) protein coronas with liquid chromatography-mass spectrometry for efficient proteomic profiling. A protein corona is a protein layer adsorbed onto NPs upon contact with biofluids. Varying the physicochemical properties of engineered NPs translates to distinct protein corona patterns enabling differential and reproducible interrogation of biological samples, including deep sampling of the plasma proteome. Spike experiments confirm a linear signal response. The median coefficient of variation was 22%. We screened 43 NPs and selected a panel of 5, which detect more than 2,000 proteins from 141 plasma samples using a 96-well automated workflow in a pilot non-small cell lung cancer classification study. Our streamlined workflow combines depth of coverage and throughput with precise quantification based on unique interactions between proteins and NPs engineered for deep and scalable quantitative proteomic studies.

Characterization of a multiplex, 12-biomarker test for rheumatoid arthritis.

Rheumatoid arthritis (RA) is a chronic inflammatory disorder that primarily involves the joints. Accurate and frequent assessment of RA disease activity is critical to optimal treatment planning. A novel algorithm has been developed to determine a multi-biomarker disease activity (MBDA) score based upon measurement of the concentrations of 12 serum biomarkers in multiplex format. Biomarker assays from several different platforms were used in feasibility studies to identify biomarkers of potential significance. These assays were adapted to a multiplex platform for training and validation of the algorithm. In this study, the analytical performance of the underlying biomarker assays and the MBDA score was evaluated. Quantification of 12 biomarkers was performed with multiplexed sandwich immunoassays in three panels. Biomarker-specific capture antibodies were bound to specific locations in each well; detection antibodies were labeled with electrochemiluminescent tags. Data were acquired with a Sector Imager 6000, and analyte concentrations were determined. Parallelism, dynamic range, cross-reactivity, and precision were established for each biomarker as well as for the MBDA score. Interference by serum proteins, heterophilic antibodies, and common RA therapies was also assessed. The individual biomarker assays had 3-4 orders of magnitude dynamic ranges, with good reproducibility across time, operators, and reagent lots; the MBDA score had a median coefficient of variation of <2% across the score range. Cross-reactivity as well as interference by serum rheumatoid factor (RF), human anti-mouse antibodies (HAMA), or common RA therapies, including disease-modifying antirheumatic drugs and biologics, was minimal. The same MBDA score was observed in different subjects despite having different biomarker profiles, supporting prior literature reports that multiple pathways contribute to RA.

Pre-analytical effects of blood sampling and handling in quantitative immunoassays for rheumatoid arthritis.

Variability in pre-analytical blood sampling and handling can significantly impact results obtained in quantitative immunoassays. Understanding the impact of these variables is critical for accurate quantification and validation of biomarker measurements. Particularly, in the design and execution of large clinical trials, even small differences in sample processing and handling can have dramatic effects in analytical reliability, results interpretation, trial management and outcome. The effects of two common blood sampling methods (serum vs. plasma) and two widely-used serum handling methods (on the clot with ambient temperature shipping, "traditional", vs. centrifuged with cold chain shipping, "protocol") on protein and autoantibody concentrations were examined. Matched serum and plasma samples were collected from 32 rheumatoid arthritis (RA) patients representing a wide range of disease activity status. Additionally, a set of matched serum samples with two sample handling methods was collected. One tube was processed per manufacturer's instructions and shipped overnight on cold packs (protocol). The matched tube, without prior centrifugation, was simultaneously shipped overnight at ambient temperatures (traditional). Upon delivery, the traditional tube was centrifuged. All samples were subsequently aliquoted and frozen prior to analysis of protein and autoantibody biomarkers. Median correlation between paired serum and plasma across all autoantibody assays was 0.99 (0.98-1.00) with a median % difference of -3.3 (-7.5 to 6.0). In contrast, observed protein biomarker concentrations were significantly affected by sample types, with median correlation of 0.99 (0.33-1.00) and a median % difference of -10 (-55 to 23). When the two serum collection/handling methods were compared, the median correlation between paired samples for autoantibodies was 0.99 (0.91-1.00) with a median difference of 4%. In contrast, significant increases were observed in protein biomarker concentrations among certain biomarkers in samples processed with the 'traditional' method. Autoantibody quantification appears robust to both sample type (plasma vs. serum) and pre-analytical sample collection/handling methods (protocol vs. traditional). In contrast, for non-antibody protein biomarker concentrations, sample type had a significant impact; plasma samples generally exhibit decreased protein biomarker concentrations relative to serum. Similarly, sample handling significantly impacted the variability of protein biomarker concentrations. When biomarker concentrations are combined algorithmically into a single test score such as a multi-biomarker disease activity test for rheumatoid arthritis (MBDA), changes in protein biomarker concentrations may result in a bias of the score. These results illustrate the importance of characterizing pre-analytical methodology, sample type, sample processing and handling procedures for clinical testing in order to ensure test accuracy.

A phase 1 study of SU11248 in the treatment of patients with refractory or resistant acute myeloid leukemia (AML) or not amenable to conventional therapy for the disease.

Fifteen patients with refractory AML were treated in a phase 1 study with SU11248, an oral kinase inhibitor of fms-like tyrosine kinase 3 (Flt3), Kit, vascular endothelial growth factor (VEGF), and platelet-derived growth factor (PDGF) receptors. Separate cohorts of patients received SU11248 for 4-week cycles followed by either a 2- or a 1-week rest period. At the starting dose level of 50 mg (n = 13), no dose-limiting toxicities were observed. The most frequent grade 2 toxicities were edema, fatigue, and oral ulcerations. Two fatal bleedings possibly related to the disease, one from a concomitant lung cancer and one cerebral bleeding, were observed. At the 75 mg dose level (n = 2), one case each of grade 4 fatigue, hypertension, and cardiac failure was observed, and this dose level was abandoned. All patients with FLT3 mutations (n = 4) had morphologic or partial responses compared with 2 of 10 evaluable patients with wild-type FLT3. Responses, although longer in patients with mutated FLT3, were of short duration. Reductions of cellularity and numbers of Ki-67(+), phospho-Kit(+), phospho-kinase domain-containing receptor-positive (phospho-KDR(+)), phospho-signal transducer and activator of transcription 5-positive (phospho-STAT5(+)), and phospho-Akt(+) cells were detected in bone marrow histology analysis. In summary, monotherapy with SU11248 induced partial remissions of short duration in acute myeloid leukemia (AML) patients. Further evaluation of this compound, for example in combination with chemotherapy, is warranted.

Gene expression profiling of human colon xenograft tumors following treatment with SU11248, a multitargeted tyrosine kinase inhibitor.

Biomarkers that indicate biological activity and/or efficacy are a potentially useful tool in the development of molecularly targeted therapeutics. It is useful, though challenging, to identify biomarkers during preclinical development in order to impact decision-making during early clinical development. SU11248 is an oral, selective multitargeted tyrosine kinase inhibitor currently in Phase II oncology clinical trials. It exhibits direct antitumor and antiangiogenic activity via inhibition of the receptor tyrosine kinases PDGFR, VEGFR, KIT and FLT3. To identify clinically translatable biomarkers of SU11248 activity, expression profiling was performed on Colo205 human xenograft tumors following treatment with SU11248. Over 100 transcripts changed in abundance in SU11248 as compared to vehicle-treated tumors. Nine candidate transcripts, chosen based on putative function, were also analysed and validated by TaqMan. One such potential biomarker, cadherin-11, was further evaluated at the protein level and was found to have increased expression in xenograft tumors after SU11248 treatment. Interestingly, cadherin-11 expression was also detected via immunohistochemical analysis of archived solid tumors, indicating the technical feasibility of translating this putative biomarker to clinical studies. Importantly, SU11248 treatment also resulted in increased expression of cadherin-11 protein in human tumor biopsies in three out of seven patients examined and confirms the feasibility of using transcriptional profiling of preclinical models to identify clinically translatable biomarkers.

An innovative phase I clinical study demonstrates inhibition of FLT3 phosphorylation by SU11248 in acute myeloid leukemia patients.

PURPOSE: Obtaining direct and rapid proof of molecular activity in early clinical trials is critical for optimal clinical development of novel targeted therapies. SU11248 is an oral multitargeted kinase inhibitor with selectivity for fms-related tyrosine kinase 3/Flk2 (FLT3), platelet-derived growth factor receptor alpha/beta, vascular endothelial growth factor receptor 1/2, and KIT receptor tyrosine kinases. FLT3 is a promising candidate for targeted therapy in acute myeloid leukemia (AML), because activating mutations occur in up to 30% of patients. We conducted an innovative single-dose clinical study with a primary objective to demonstrate inhibition of FLT3 phosphorylation by SU11248 in AML. EXPERIMENTAL DESIGN: Twenty-nine AML patients each received a single dose of SU11248, escalated from 50 to 350 mg, in increments of 50 mg and cohorts of three to six patients. FLT3 phosphorylation and plasma pharmacokinetics were evaluated at seven time points over 48 h after SU11248 administration, and FLT3 genotype was determined. Study drug-related adverse events occurred in 31% of patients, mainly grade 1 or 2 diarrhea and nausea, at higher dose levels. RESULTS: Inhibition of FLT3 phosphorylation was apparent in 50% of FLT3-wild-type (WT) patients and in 100% of FLT3-mutant patients. FLT3 internal tandem duplication (ITD) mutants showed increased sensitivity relative to FLT3-WT, consistent with preclinical predictions. The primary end point, strong inhibition of FLT3 phosphorylation in >50% patients, was reached in 200 mg and higher dose cohorts. Downstream signaling pathways were also inhibited; signal transducer and activator of transcription 5 (STAT5) was reduced primarily in internal tandem duplication patients and at late time points in FLT3-WT patients, whereas extracellular signal-regulated kinase (ERK) activity was reduced in the majority of patients, independent of FLT3 inhibition. CONCLUSIONS: This novel translational study bridges preclinical models to the patient setting and provides the first evidence of anti-FLT3 activity in patients. Proof of target inhibition accomplishes a crucial milestone in the development of novel oncology therapeutics.

Fibroblast growth factor-2 gene delivery stimulates axon growth by adult retinal ganglion cells after acute optic nerve injury.

Basic fibroblast growth factor (or FGF-2) has been shown to be a potent stimulator of retinal ganglion cell (RGC) axonal growth during development. Here we investigated if FGF-2 upregulation in adult RGCs promoted axon regrowth in vivo after acute optic nerve injury. Recombinant adeno-associated virus (AAV) was used to deliver the FGF-2 gene to adult RGCs providing a sustained source of this neurotrophic factor. FGF-2 gene transfer led to a 10-fold increase in the number of axons that extended past 0.5 mm from the lesion site compared to control nerves. Detection of AAV-mediated FGF-2 protein in injured RGC axons correlated with growth into the distal optic nerve. The response to FGF-2 upregulation was supported by our finding that FGF receptor-1 (FGFR-1) and heparan sulfate (HS), known to be essential for FGF-2 signaling, were expressed by adult rat RGCs. FGF-2 transgene expression led to only transient protection of injured RGCs. Thus the effect of this neurotrophic factor on axon extension could not be solely attributed to an increase in neuronal survival. Our data indicate that selective upregulation of FGF-2 in adult RGCs stimulates axon regrowth within the optic nerve, an environment that is highly inhibitory for regeneration. These results support the hypothesis that key factors involved in axon outgrowth during neural development may promote regeneration of adult injured neurons.

Expression profiling of blood samples from an SU5416 Phase III metastatic colorectal cancer clinical trial: a novel strategy for biomarker identification.

BACKGROUND: Microarray-based gene expression profiling is a powerful approach for the identification of molecular biomarkers of disease, particularly in human cancers. Utility of this approach to measure responses to therapy is less well established, in part due to challenges in obtaining serial biopsies. Identification of suitable surrogate tissues will help minimize limitations imposed by those challenges. This study describes an approach used to identify gene expression changes that might serve as surrogate biomarkers of drug activity. METHODS: Expression profiling using microarrays was applied to peripheral blood mononuclear cell (PBMC) samples obtained from patients with advanced colorectal cancer participating in a Phase III clinical trial. The PBMC samples were harvested pre-treatment and at the end of the first 6-week cycle from patients receiving standard of care chemotherapy or standard of care plus SU5416, a vascular endothelial growth factor (VEGF) receptor tyrosine kinase (RTK) inhibitor. Results from matched pairs of PBMC samples from 23 patients were queried for expression changes that consistently correlated with SU5416 administration. RESULTS: Thirteen transcripts met this selection criterion; six were further tested by quantitative RT-PCR analysis of 62 additional samples from this trial and a second SU5416 Phase III trial of similar design. This method confirmed four of these transcripts (CD24, lactoferrin, lipocalin 2, and MMP-9) as potential biomarkers of drug treatment. Discriminant analysis showed that expression profiles of these 4 transcripts could be used to classify patients by treatment arm in a predictive fashion. CONCLUSIONS: These results establish a foundation for the further exploration of peripheral blood cells as a surrogate system for biomarker analyses in clinical oncology studies.

SU11248 is a novel FLT3 tyrosine kinase inhibitor with potent activity in vitro and in vivo.

FLT3 (fms-related tyrosine kinase/Flk2/Stk-2) is a receptor tyrosine kinase (RTK) primarily expressed on hematopoietic cells. In blasts from acute myelogenous leukemia (AML) patients, 2 classes of FLT3 activating mutations have been identified: internal tandem duplication (ITD) mutations in the juxtamembrane domain (25%-30% of patients) and point mutations in the kinase domain activation loop (7%-8% of patients). FLT3-ITD mutations are the most common molecular defect identified in AML and have been shown to be an independent prognostic factor for decreased survival. FLT3-ITD is therefore an attractive molecular target for therapy. SU11248 is a recently described selective inhibitor with selectivity for split kinase domain RTKs, including platelet-derived growth factor receptors, vascular endothelial growth factor receptors, and KIT. We show that SU11248 also has potent activity against wild-type FLT3 (FLT3-WT), FLT3-ITD, and FLT3 activation loop (FLT3-Asp835) mutants in phosphorylation assays. SU11248 inhibits FLT3-driven phosphorylation and induces apoptosis in vitro. In addition, SU11248 inhibits FLT3-induced VEGF production. The in vivo efficacy of SU11248 was investigated in 2 FLT3-ITD models: a subcutaneous tumor xenograft model and a bone marrow engraftment model. We show that SU11248 (20 mg/kg/d) dramatically regresses FLT3-ITD tumors in the subcutaneous tumor xenograft model and prolongs survival in the bone marrow engraftment model. Pharmacokinetic and pharmacodynamic analysis in subcutaneous tumors showed that a single administration of an efficacious drug dose potently inhibits FLT3-ITD phosphorylation for up to 16 hours following a single dose. These results suggest that further exploration of SU11248 activity in AML patients is warranted.

AAV-mediated expression of vascular endothelial growth factor induces choroidal neovascularization in rat.

PURPOSE: To develop a small-animal model of choroidal neovascularization (CNV) by injecting adeno-associated virus (AAV)-VEGF into the subretinal space (SRS) of rats. METHODS: An adeno-associated viral vector encoding human VEGF(165) was injected into the subretinal space (SRS) of Sprague-Dawley or Long Evans rats. Expression of VEGF was identified by RT-PCR and immunohistochemistry. Physiological and pathologic changes in the retina and choroid were evaluated by electroretinography, fluorescein angiography, light microscopy, and three-dimensional reconstruction of serial sections. RESULTS: Green fluorescent protein (GFP) and VEGF were expressed for at least 20 months in the retina and retinal pigment epithelium (RPE). Histologic sections showed extensive subretinal neovascularization, degenerating photoreceptors, and proliferating RPE at 5 weeks to 20 months after injection of AAV-VEGF. At 2 to 12 months after injection, leaking blood vessels were detected by fluorescein angiography. Electroretinogram a- and b-wave amplitudes were significantly decreased during this time. Three-dimensional reconstruction of serial sections demonstrated that choroidal blood vessels penetrated Bruch's membrane, one of them splitting into three branches in the SRS. In the current model, CNV was produced in 95% of the animals tested (19/20). It persisted for more than 20 months, a necessary requirement for modeling the development of CNV in age-related macular degeneration (AMD). CONCLUSIONS: In this study, a highly reproducible animal model of long-lasting CNV was developed. This model is being used to test antiangiogenic molecules to reduce or inhibit CNV and could be extended to primates.

Tightly regulated long-term erythropoietin expression in vivo using tet-inducible recombinant adeno-associated viral vectors.

Recombinant adeno-associated viral (rAAV) vectors containing an improved tetracycline (tet) system of transcriptional regulation are an efficient strategy for the control of long-term therapeutic gene expression. In vivo studies with the original tet-off and tet-on vectors, while promising, have failed to demonstrate complete repression in the uninduced state. To address this issue, we incorporated the tTS(kid) fusion of the tet repressor and a KRAB-derived transcriptional silencer into the tet-on system in the context of rAAV vectors. The tTS(kid) repressor and rtTA activator were expressed constituitively from a regulator vector, and the repressor and an erythropoietin (Epo) transgene were expressed inducibly via a second vector. Following intramuscular co-injection of these vectors, we observed repeated induction of serum Epo protein following drug administration and undetectable levels after its withdrawal. Four cycles of regulation were achieved over a 32-week period. Thus, the tet-on system plus the tTS(kid) repressor delivered via nonpathogenic rAAV vectors is a powerful tool for controlling the in vivo expression of therapeutic transgenes. In a clinical setting, the repressor could provide a mechanism for abolishing transgene expression if it were no longer needed or if the safety of a patient became compromised.