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Supplementary Data from Quantifying Antivascular Effects of Monoclonal Antibodies to Vascular Endothelial Growth Factor: Insights from Imaging

CCR Translation for the Article from Quantifying Antivascular Effects of Monoclonal Antibodies to Vascular Endothelial Growth Factor: Insights from Imaging

Purpose: Little is known concerning the onset, duration, and magnitude of direct therapeutic effects of anti–vascular endothelial growth factor (VEGF) therapies. Such knowledge would help guide the rational development of targeted therapeutics from bench to bedside and optimize use of imaging technologies that quantify tumor function in early-phase clinical trials.Experimental Design: Preclinical studies were done using ex vivo microcomputed tomography and in vivo ultrasound imaging to characterize tumor vasculature in a human HM-7 colorectal xenograft model treated with the anti-VEGF antibody G6-31. Clinical evaluation was by quantitative magnetic resonance imaging in 10 patients with metastatic colorectal cancer treated with bevacizumab.Results: Microcomputed tomography experiments showed reduction in perfused vessels within 24 to 48 h of G6-31 drug administration (P ≤ 0.005). Ultrasound imaging confirmed reduced tumor blood volume within the same time frame (P = 0.048). Consistent with the preclinical results, reductions in enhancing fraction and fractional plasma volume were detected in patient colorectal cancer metastases within 48 h after a single dose of bevacizumab that persisted throughout one cycle of therapy. These effects were followed by resolution of edema (P = 0.0023) and tumor shrinkage in 9 of 26 tumors at day 12.Conclusion: These data suggest that VEGF-specific inhibition induces rapid structural and functional effects with downstream significant antitumor activity within one cycle of therapy. This finding has important implications for the design of early-phase clinical trials that incorporate physiologic imaging. The study shows how animal data help interpret clinical imaging data, an important step toward the validation of image biomarkers of tumor structure and function. (Clin Cancer Res 2009;15(21):6674–82)

Further evidence is needed to support the use of plasma amyloid β (Aβ) biomarkers as Alzheimer's disease prescreening tools. This study evaluated the clinical performance and robustness of plasma AβData were collected from 333 BioFINDER and 121 Alzheimer's Disease Neuroimaging Initiative study participants. Risk and predictive values versus percentile of plasma AβElecsys plasma AβImplementing plasma AβPlasma Aβ

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Background We investigated the effect of crenezumab, a humanized anti-amyloid-beta (Aβ) immunoglobulin (Ig)G4 monoclonal antibody, on biomarkers of amyloid pathology, neurodegeneration, and disease progression in patients with mild-to-moderate Alzheimer’s disease (AD). Methods This double-blind, placebo-controlled, randomized phase II study enrolled patients with mild-to-moderate AD and a Mini-Mental State Examination (MMSE) score of 18–26. In part 1 of the study, patients were 2:1 randomized to receive low-dose subcutaneous (SC) 300 mg crenezumab every 2 weeks (q2w) or placebo for 68 weeks; in part 2, patients were 2:1 randomized to receive high-dose intravenous (IV) 15 mg/kg crenezumab every 4 weeks (q4w) or placebo for 68 weeks. The primary endpoint was change in amyloid burden from baseline to week 69 assessed by florbetapir positron emission tomography (PET) in the modified intent-to-treat population. Secondary endpoints were change from baseline to week 69 in cerebrospinal fluid (CSF) biomarkers and fluorodeoxyglucose PET, and change from baseline to week 73 in 12-point Alzheimer’s Disease Assessment Scale cognitive subscale (ADAS-Cog12) and Clinical Dementia Rating Sum of Boxes (CDR-SB). Safety was assessed in patients who received at least one dose of study treatment. Results From August 2011 to September 2012, 91 patients were enrolled and randomized (low-dose SC cohort: crenezumab (n = 26) or placebo (n = 13); high-dose IV cohort: crenezumab (n = 36) or placebo (n = 16)). The primary endpoint was not met using a prespecified cerebellar reference region to calculate standard uptake value ratios (SUVRs) from florbetapir PET. Exploratory analyses using subcortical white matter reference regions showed nonsignificant trends toward slower accumulation of plaque amyloid in the high-dose IV cohort. In both cohorts, a significant mean increase from baseline in CSF Aβ(1–42) levels versus placebo was observed. Nonsignificant trends toward ADAS-Cog12 and CDR-SB benefits were identified in a mild (MMSE 20–26) subset of the high-dose IV cohort. No amyloid-related imaging abnormalities due to edema/effusion were observed. Conclusion The primary endpoint was not met. Exploratory findings suggest potential Aβ target engagement with crenezumab and possible slower accumulation of plaque amyloid. Studies investigating the effects of higher doses of crenezumab on amyloid load and disease progression are ongoing. Trial registration ClinicalTrials.gov, NCT01397578. Registered on 18 July 2011.

ObjectiveTo evaluate the safety and efficacy of crenezumab in patients with mild to moderate Alzheimer disease (AD).MethodsIn this phase 2 trial, 431 patients with mild to moderate AD 50 to 80 years of age were randomized 2:1 (crenezumab:placebo). Patients received low-dose subcutaneous crenezumab 300 mg or placebo every 2 weeks (n = 184) or high-dose intravenous crenezumab 15 mg/kg or placebo every 4 weeks (n = 247) for 68 weeks. Primary outcome measures were change in Alzheimer's Disease Assessment Scale–Cognitive Subscale (ADAS-Cog12) and Clinical Dementia Rating–Sum of Boxes scores from baseline to week 73.ResultsThe primary and secondary endpoints were not met. In an exploratory post hoc analysis, a reduction in decline on the ADAS-Cog12 was observed in the high-dose group. Separation from the placebo group on the ADAS-Cog12 was greatest in the milder subsets of AD patients and reached statistical significance in the group with Mini-Mental State Examination scores of 22 to 26. In both groups, there was a significant increase in CSF β-amyloid1-42 levels that correlated with crenezumab CSF levels. The overall rate of adverse events was balanced between groups. One case of amyloid-related imaging abnormalities indicative of vasogenic edema or effusions was reported.ConclusionsAlthough prespecified criteria for testing treatment effects were not met, these data suggest a potential treatment effect in patients with mild AD treated with high-dose crenezumab. Together with the safety profile for crenezumab, these data support the exploration of crenezumab treatment at even higher doses in patients with early AD.Clinicaltrials.gov identifierNCT 01343966.Classification of evidenceThis study provides Class II evidence that, for people with AD, crenezumab does not significantly improve cognition or function at 18 months. The study is rated Class II because

To identify a cognitive composite that is sensitive to tracking preclinical Alzheimer's disease decline to be used as a primary end point in treatment trials.We capitalized on longitudinal data collected from 1995 to 2010 from cognitively unimpaired presenilin 1 (PSEN1) E280A mutation carriers from the world's largest known early-onset autosomal dominant Alzheimer's disease kindred to identify a composite cognitive test with the greatest statistical power to track preclinical Alzheimer's disease decline and estimate the number of carriers age 30 years and older needed to detect a treatment effect in the Alzheimer's Prevention Initiative's (API) preclinical Alzheimer's disease treatment trial. The mean-to-standard-deviation ratios (MSDRs) of change over time were calculated in a search for the optimal combination of 1 to 7 cognitive tests/subtests drawn from the neuropsychological test battery in cognitively unimpaired mutation carriers during a 2- and 5-year follow-up period (n = 78 and 57), using data from noncarriers (n = 31 and 56) during the same time period to correct for aging and practice effects. Combinations that performed well were then evaluated for robustness across follow-up years, occurrence of selected items within top-performing combinations, and representation of relevant cognitive domains.The optimal test combination included Consortium to Establish a Registry for Alzheimer's Disease (CERAD) Word List Recall, CERAD Boston Naming Test (high frequency items), Mini-Mental State Examination (MMSE) Orientation to Time, CERAD Constructional Praxis, and Raven's Progressive Matrices (Set A), with an MSDR of 1.62. This composite is more sensitive than using either the CERAD Word List Recall (MSDR = 0.38) or the entire CERAD-Col battery (MSDR = 0.76). A sample size of 75 cognitively normal PSEN1 E280A mutation carriers aged 30 years and older per treatment arm allows for a detectable treatment effect of 29% in a 60-month trial (80% power, P = .05).We have identified a composite cognitive test score representing multiple cognitive domains that, compared to the most sensitive single test item, has improved power to track preclinical Alzheimer's disease decline in autosomal dominant Alzheimer's disease mutation carriers and to evaluate preclinical Alzheimer's disease treatments. This API composite cognitive test score will be used as the primary end point in the first API trial in cognitively unimpaired autosomal dominant Alzheimer's disease carriers within 15 years of their estimated age at clinical onset. We have independently confirmed our findings in a separate cohort of cognitively healthy older adults who progressed to the clinical stages of late-onset Alzheimer's disease, described in a separate report, and continue to refine the composite in independent cohorts and compared with other analytic approaches.

Passive immunization against β-amyloid (Aβ) has become an increasingly desirable strategy as a therapeutic treatment for Alzheimer's disease (AD). However, traditional passive immunization approaches carry the risk of Fcγ receptor-mediated overactivation of microglial cells, which may contribute to an inappropriate proinflammatory response leading to vasogenic edema and cerebral microhemorrhage. Here, we describe the generation of a humanized anti-Aβ monoclonal antibody of an IgG4 isotype, known as MABT5102A (MABT). An IgG4 subclass was selected to reduce the risk of Fcγ receptor-mediated overactivation of microglia. MABT bound with high affinity to multiple forms of Aβ, protected against Aβ1–42 oligomer-induced cytotoxicity, and increased uptake of neurotoxic Aβ oligomers by microglia. Furthermore, MABT-mediated amyloid plaque removal was demonstrated using in vivo live imaging in hAPP((V717I))/PS1 transgenic mice. When compared with a human IgG1 wild-type subclass, containing the same antigen-binding variable domains and with equal binding to Aβ, MABT showed reduced activation of stress-activated p38MAPK (p38 mitogen-activated protein kinase) in microglia and induced less release of the proinflammatory cytokine TNFα. We propose that a humanized IgG4 anti-Aβ antibody that takes advantage of a unique Aβ binding profile, while also possessing reduced effector function, may provide a safer therapeutic alternative for passive immunotherapy for AD. Data from a phase I clinical trial testing MABT is consistent with this hypothesis, showing no signs of vasogenic edema, even in ApoE4 carriers.

The British Columbia Center for Disease Control laboratory performs approximately 95% of all hepatitis C virus (HCV) antibody tests for the province's 4 million inhabitants. In 2002, the laboratory tested 96,000 specimens for anti-HCV antibodies, of which 4,800 (5%) were seroreactive and required confirmation of active infection. Although HCV RNA assays with a sensitivity of 50 IU/ml or less are recommended for the confirmation of active HCV infection, given the large number of seroreactive specimens tested annually, we evaluated the Ortho trak-C assay (OTCA) as a second-line confirmatory test and determined its limit of detection (LoD). Of 502 specimens from treatment-naïve anti-HCV-positive individuals, 478 had sufficient volumes for evaluation by the OTCA and HCV RNA tests. Core antigen was not detected in 147 of 478 (30.8%) of these specimens, of which 37 of 147 (25.2%) were shown to be viremic by the VERSANT HCV (version 3.0) (branched-DNA) assay and/or the VERSANT HCV qualitative assay. Testing of 144 replicates of a World Health Organization standard dilution series indicated that the LoD of OTCA was ∼27,000 IU/ml. This LoD is consistent with the inability of OTCA to detect core antigen in clinical specimens with low viral loads. We conclude that OTCA has limited value as a confirmatory test for the diagnosis of active HCV infection because 37 of 367 (10%) of viremic specimens had undetectable core antigen. Qualitative HCV RNA testing remains the present standard for the confirmation of active HCV infection in the diagnostic setting.

Multicenter, open-label, single-dose, dose-escalation Phase Ia study to determine the safety, tolerability, maximum tolerated dose, and immunogenicity of FCFD4514S, an antigen-binding fragment from a humanized monoclonal antibody directed against complement factor D, in patients with geographic atrophy.Eighteen patients with geographic atrophy (lesion size: ≥ 0.75 disk areas; best-corrected visual acuity: 20/125-20/400 Snellen equivalent) were sequentially enrolled and received 1 of 6 escalating doses of intravitreal FCFD4514S subject to dose-limiting toxicity criteria. Follow-up assessments (clinical examination, best-corrected visual acuity, intraocular pressure) were conducted at postadministration Days 1, 3, 7, 14, 30, 60, and 90. Serum pharmacokinetics, immunogenicity, and complement activity were also evaluated.All patients completed the study with no reported FCFD4514S-related dose-limiting toxicities or ocular or systemic adverse events. The maximum tolerated dose for this study was 10 mg, the highest dose tested. No antitherapeutic antibody response or adverse effects on systemic complement activity were observed. Time to maximum serum concentration was 1 day to 3 days postdosing; serum terminal half-life was 5.9 days.Single-dose intravitreal FCFD4514S administrations were safe and well tolerated and not associated with any study drug-related ocular or systemic adverse events. These data support a multidose safety and tolerability assessment of FCFD4514S in geographic atrophy.

Here, we describe an automated nonparametric method for evaluating gadolinium-diethylene triamine pentaacetic acid (GdDTPA) kinetics, based on dynamic contrast-enhanced–MRI scans of glioblastoma patients taken before and after treatment with bevacizumab; no specific model or equation structure is assumed or used. Tumor and venous blood concentration-time profiles are smoothed, using a robust algorithm that removes artifacts due to patient motion, and then deconvolved, yielding an impulse response function. In addition to smoothing, robustness of the deconvolution operation is assured by excluding data that occur prior to the plasma peak; an exhaustive analysis was performed to demonstrate that exclusion of the prepeak plasma data does not significantly affect results. All analysis steps are executed by a single R script that requires blood and tumor curves as the sole input. Statistical moment analysis of the Impulse response function yields the area under the curve (AUC) and mean residence time (MRT). Comparison of deconvolution results to fitted Tofts model parameters suggests that AUC and AUC of the Impulse response function closely approximate fractional clearance from plasma to tissue (K trans ) and fractional interstitial volume (ve). Intervisit variability is shown to be comparable when using the deconvolution method (11%[ AUC ] and 13%[AUC]) compared to the Tofts model (14%[K trans ] and 24%[ve]). AUC and AUC both exhibit a statistically significant decrease (P < 0.005) 1 day after administration of bevacizumab. Magn Reson Med 63:1366–1375, 2010. © 2010 Wiley-Liss, Inc.

Purpose: Little is known concerning the onset, duration, and magnitude of direct therapeutic effects of anti–vascular endothelial growth factor (VEGF) therapies. Such knowledge would help guide the rational development of targeted therapeutics from bench to bedside and optimize use of imaging technologies that quantify tumor function in early-phase clinical trials. Experimental Design: Preclinical studies were done using ex vivo microcomputed tomography and in vivo ultrasound imaging to characterize tumor vasculature in a human HM-7 colorectal xenograft model treated with the anti-VEGF antibody G6-31. Clinical evaluation was by quantitative magnetic resonance imaging in 10 patients with metastatic colorectal cancer treated with bevacizumab. Results: Microcomputed tomography experiments showed reduction in perfused vessels within 24 to 48 h of G6-31 drug administration (P ≤ 0.005). Ultrasound imaging confirmed reduced tumor blood volume within the same time frame (P = 0.048). Consistent with the preclinical results, reductions in enhancing fraction and fractional plasma volume were detected in patient colorectal cancer metastases within 48 h after a single dose of bevacizumab that persisted throughout one cycle of therapy. These effects were followed by resolution of edema (P = 0.0023) and tumor shrinkage in 9 of 26 tumors at day 12. Conclusion: These data suggest that VEGF-specific inhibition induces rapid structural and functional effects with downstream significant antitumor activity within one cycle of therapy. This finding has important implications for the design of early-phase clinical trials that incorporate physiologic imaging. The study shows how animal data help interpret clinical imaging data, an important step toward the validation of image biomarkers of tumor structure and function. (Clin Cancer Res 2009;15(21):6674–82)

A conformational pairing analysis was used to devise nucleobase analogs capable of forming nonselective and energetically favorable base pairs opposite either the purine or the pyrimidine constituents of nucleic acids. 5-methylisocytosine and isoguanine were conceived as a degenerate pyrimidine and a degenerate purine, respectively. Data from previous DNA duplex melting experiments verified that the analogs can act as degenerate nucleobases as hypothesized. Isoguanine also formed unusually stable base pairs with guanine. A quantitative PCR assay yielding equivalent results across hepatitis C virus (HCV) subtypes was created with this system, despite the use of a single probe targeted to a polymorphic region. Amplification curves using probes with 5-methylisocytosine or isoguanine opposite appropriate ambiguous target positions exhibited more signal than curves from similar probes containing common degenerate nucleobase hypoxanthine.

Plasma human immunodeficiency virus type 1 (HIV-1) RNA level is an important parameter for patient management, yet viral load assays from different manufacturers are not standardized.In this study, we evaluated the concordance between test results obtained for 1,000 plasma specimens collected from HIV-1-infected individuals measured with the VERSANT HIV-1 RNA 3.0 assay (bDNA) and the COBAS AMPLICOR HIV-1 MONITOR 1.5 test (PCR). We compared viral load values obtained by each of these assays throughout their dynamic ranges, with particular focus on samples with low viral load (i.e. 50-250 copies/mL), and calculated the estimated distribution of distinct plasma viral load levels for the entire study population modeled from the data observed in the study.We found that these two assays show excellent agreement, with a correlation (R(2)) of 0.957 and a slope of 1.004. The mean difference in viral load values between the two assays was less than 0.10-log(10) throughout the dynamic range and 98.2% of all samples had bDNA and PCR results within 0.5-log(10) of each other, a difference that is within the range considered to be a minimal change in plasma viremia. Moreover, the two assays show very similar results across all assay ranges tested. The estimated prevalence of samples with results50 copies/mL, 50-250 copies/mL, and 250-500,000 copies/mL were 41.6%, 7.7%, and 49.7%, respectively, by the bDNA assay, and 42.4%, 6.9%, and 50.7%, respectively, by the PCR assay.Based on our findings from 1,000 clinical specimens, we do not see the need to re-establish a baseline value or apply a conversion factor when switching from one assay to the other. Since the majority of our patient population likely is infected with subtype B virus, it is unclear if our findings will apply to other patient populations with a greater incidence of infection with non-B subtypes.

We report the results of a multicenter evaluation of a new assay for the detection of hepatitis C virus (HCV) RNA in human serum or plasma based on transcription-mediated amplification (HCV TMA). Analysis of combined data obtained from 15 independent sites, including 4 sites in the United States and 11 in Europe, by using preproduction kits showed a limit of detection of 9.8 IU/ml and an overall mean specificity of 97.9%. In addition, assay runs and samples were valid consistently (97.8% of assay runs and 98.0% of specimen results). Of the 15 sites that participated in the multicenter evaluation, 2 subsequently carried out additional performance studies with production kits in support of the assay's registration in France. Comparison of the findings from these two sites during the multicenter evaluation and during the registration studies showed an overall improvement in assay performance. A statistically significant ( P < 0.001) improvement was achieved for both specificity and specimen validity in the registration studies, which were 99.4 and 98.1%, respectively. Combined data from the two sites showed a lower limit of detection of approximately 2.4 IU/ml and an improved assay validity of 100%, although the sample size was too small to show statistical significance at the 0.05 level. In summary, the performance characteristics of HCV TMA indicate that this assay is a reliable tool for the detection of HCV RNA in serum or plasma. Improvement in assay performance has been demonstrated with refinement of assay reagents, instrumentation, and operator experience.

A preclinical evaluation of a qualitative assay for the detection of hepatitis C virus (HCV) RNA by transcription-mediated amplification (TMA) was conducted according to the guidelines of the National Committee for Clinical Laboratory Standards and the U.S. Food and Drug Administration. Our results showed that this assay, HCV TMA, detected 95% of samples with HCV RNA concentrations of 5.3 IU/ml and 29 copies/ml. HCV TMA showed an overall specificity of 99.6% and was highly reproducible, detecting 99.3% of samples with HCV RNA concentrations of 50 copies/ml across seven different lots of reagents. Experiments with clinical samples showed that HCV TMA detected all HCV genotypes with similar efficiencies, detecting ≥95% of samples at 50 HCV RNA copies/ml from patients infected with HCV genotypes 1a, 2b, 3a, 4a, 5a, and 6a. In experiments with RNA transcripts, HCV TMA detected ≥96.6% of transcripts derived from HCV genotypes 1a, 1b, 2a, 2c, 3a, 4a, 5a, and 6a at 50 HCV RNA copies/ml. Detection of transcripts derived from HCV genotype 2b was slightly lower (88.4%) at 50 copies/ml but was 97.0% at 75 copies/ml. In addition, HCV TMA exhibited robust performance in detecting HCV RNA in samples subjected to various conditions commonly encountered in a clinical laboratory, including long-term storage, multiple freeze-thaw cycles, different collection tubes, and the presence of endogenous substances, commonly prescribed drugs, or other microorganisms and viruses. With its high sensitivity, specificity, reproducibility, and equivalent genotype reactivity, HCV TMA may provide an attractive alternative for routine qualitative HCV RNA testing in clinical laboratories.

2539 Background: DCE-MRI allows for the functional analysis of tumor microcirculation and composition, and has been used to evaluate changes in vascular parameters in response to anti-angiogenic ag...

SummaryNucleic acid duplexes associating through purine-purine base pairing have been constructed and characterized in a remarkable demonstration of nucleic acids with mixed sequence and a natural backbone in an alternative duplex structure. The antiparallel deoxyribose all-purine duplexes associate specifically through Watson-Crick pairing, violating the nucleobase size-complementarity pairing convention found in Nature. Sequence-specific recognition displayed by these structures makes the duplexes suitable, in principle, for information storage and replication fundamental to molecular evolution in all living organisms. All-purine duplexes can be formed through association of purines found in natural ribonucleosides. Key to the formation of these duplexes is the N3-H tautomer of isoguanine, preferred in the duplex, but not in aqueous solution. The duplexes have relevance to evolution of the modern genetic code and can be used for molecular recognition of natural nucleic acids.