Progress Toward Long-Term Survivors of Glioblastoma.

OBJECTIVE: To identify the frequency and characteristics of long-term survivors of glioblastoma. PATIENTS AND METHODS: Using all cases of glioblastoma with histopathological confirmation in the National Cancer Database from January 1, 2004, through December 31, 2009, clinical, institutional, and treatment-related factors were evaluated with multivariable logistic regression models so as to elucidate factors independently associated with higher than 5-year overall survival after diagnosis. RESULTS: A total of 48,652 patients met the inclusion criteria, with 2249 (4.6%) achieving 5-year survival. Factors associated with odds of improved 5-year overall survival in multivariable analysis were younger age, female sex, less medical comorbidities, nonwhite race, highest median income quartile, left-sided tumors and tumors outside the brainstem, and treatment with radiotherapy (P<.05 for all). The percentage of patients surviving 5 years remained relatively unchanged over the 6-year study period (P=.97). CONCLUSION: Despite improvements in median and short-term overall survival shown in recent large clinical trials for glioblastoma, the percentage of patients with glioblastoma achieving 5-year overall survival remains low. This observation calls for the development of practice-redefining therapies and justifies the increased application of radical novel and experimental treatment paradigms for all patients with glioblastoma.

Camelid heavy chain only antibody fragment domain against ß-site of amyloid precursor protein cleaving enzyme 1 inhibits ß-secretase activity in vitro and in vivo.

UNLABELLED: Accumulation and aggregation of the amyloid-ß (Aß) peptide is associated with Alzheimer's disease (AD). Aß is generated from the amyloid precursor protein by the successive action of two membrane-associated processing enzymes: ß-secretase or ß-site of amyloid precursor protein cleaving enzyme 1 (BACE1) and γ-secretase. Inhibition of one or both of these enzymes prevents Aß generation and the accompanying Aß accumulation. Antigen binding fragments from camelid heavy chain only antibodies (VHHs) were found to exert excellent enzyme inhibition activity. In the present study, we generated VHHs against BACE1 by active immunization of Lama glama with the recombinant BACE1 protein. Two classes of VHHs were selected from a VHH-phage display library by competitive elution with a peptide encoding the Swedish mutation variant of the BACE1 processing site. One VHH was found to inhibit the enzyme activity of BACE1 in vitro and in cell culture, whereas two other VHHs were found to stimulate BACE1 activity under the same conditions in vitro. Furthermore, an in vivo study with a transgenic AD mouse model, using intracisternal injection of the inhibitory VHH, led to acute reduction of the Aß load in the blood and brain. This inhibitory VHH may be considered as a candidate molecule for a therapy directed towards reduction of Aß load and prevention of AD progression. Both the inhibitory and stimulatory VHH may be useful for improving our understanding of the structure-function relationship of BACE1, as well as its role in AD progression. DATABASE: The GenBank sequence accession numbers are KR363186 for VHH B1a; KR363187 for VHH B3a; and KR363188 for VHH B5a.

Fusion of hIgG1-Fc to 111In-anti-amyloid single domain antibody fragment VHH-pa2H prolongs blood residential time in APP/PS1 mice but does not increase brain uptake.

INTRODUCTION: Llama single domain antibody fragments (VHH), which can pass endothelial barriers, are being investigated for targeting amyloid plaque load in Alzheimer's disease (AD). Contrary to conventional human or murine antibodies consisting of IgG or F(ab')2 antibody fragments, VHH are able to effectively pass the blood brain barrier (BBB) in vitro. However, in earlier in vivo studies, anti-amyloid VHH showed poor BBB passage due to their short serum half-lives. It would be of interest to develop a VHH based protein with elongated serum half-life to enhance BBB passage, allowing the VHH to more easily reach the cerebral amyloid deposits. METHODS: To increase serum persistence, the Fc portion of the human IgG1 antibody (hinge plus CH2 and CH3 domains) was fused to the C-terminus of the VHH (VHH-pa2H-Fc). To determine the pharmacokinetics and biodistribution profile of the fusion protein, the chelator p-SCN-Bz-DTPA was linked to the protein and thereafter labeled with radioactive indium-111 ((111)In). Double transgenic APPswe/PS1dE9 and wild type littermates were injected with 20 µg VHH-pa2H-Fc-DTPA-(111)In (10-20 MBq). Pharmacokinetics of the tracer was determined in blood samples at 10 intervals after injection and imaging using microSPECT was performed. The biodistribution of the radioactivity in various excised tissues was measured at 48 h after injection. RESULTS: We succeeded in the expression of the fusion protein VHH-pa2H-Fc in HEK293T cells with a yield of 50mg/L growth medium. The fusion protein showed homodimerization - necessary for successful Fc neonatal receptor recycling. Compared to VHH-pa2H, the Fc tailed protein retained high affinity for amyloid beta on human AD patient brain tissue sections, and significantly improved serum retention of the VHH. However, at 48 h after systemic injection of the non-fused VHH-DTPA-(111)In and the VHH-Fc-DTPA-(111)In fusion protein in transgenic mice, the specific brain uptake of VHH-Fc-DTPA-(111)In was not improved compared to non-fused VHH-DTPA-(111)In. CONCLUSION: Using VHH-Fc conjugates increases the blood half-life of the protein. However, purely extending the time window for brain uptake does not increase BBB passage. Nevertheless, VHH-Fc holds promise for therapeutic applications where a sustained systemic circulation of VHH is advantageous.

Enhanced glutathione PEGylated liposomal brain delivery of an anti-amyloid single domain antibody fragment in a mouse model for Alzheimer's disease.

Treatment of neurodegenerative disorders such as Alzheimer's disease is hampered by the blood-brain barrier (BBB). This tight cerebral vascular endothelium regulates selective diffusion and active transport of endogenous molecules and xenobiotics into and out of the brain parenchyma. In this study, glutathione targeted PEGylated (GSH-PEG) liposomes were designed to deliver amyloid-targeting antibody fragments across the BBB into the brain. Two different formulations of GSH-PEG liposomes based on 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and egg-yolk phosphatidylcholine (EYPC) were produced. Both formulations encapsulate 15kDa amyloid beta binding llama single domain antibody fragments (VHH-pa2H). To follow the biodistribution of VHH-pa2H rather than the liposome, the antibody fragment was labeled with the radioisotope indium-111. To prolong the shelf life of the construct beyond the limit of radioactive decay, an active-loading method was developed to efficiently radiolabel the antibody fragments after encapsulation into the liposomes, with radiolabeling efficiencies of up to 68% after purification. The radiolabeled liposomes were administered via a single intravenous bolus injection to APPswe/PS1dE9 double transgenic mice, a mouse model of Alzheimer's disease, and their wildtype littermates. Both GSH-PEG DMPC and GSH-PEG EYPC liposomes significantly increased the standard uptake values (SUV) of VHH-pa2H in the blood of the animals compared to free VHH-pa2H. Encapsulation in GSH-PEG EYPC liposomes resulted in the highest increase in SUV in the brains of transgenic animals. Overall, these data provide evidence that GSH-PEG liposomes may be suitable for specific delivery of single domain antibody fragments over the BBB into the brain.

In vivo detection of amyloid-ß deposits using heavy chain antibody fragments in a transgenic mouse model for Alzheimer's disease.

This study investigated the in vivo properties of two heavy chain antibody fragments (V(H)H), ni3A and pa2H, to differentially detect vascular or parenchymal amyloid-ß deposits characteristic for Alzheimer's disease and cerebral amyloid angiopathy. Blood clearance and biodistribution including brain uptake were assessed by bolus injection of radiolabeled V(H)H in APP/PS1 mice or wildtype littermates. In addition, in vivo specificity for Aß was examined in more detail with fluorescently labeled V(H)H by circumventing the blood-brain barrier via direct application or intracarotid co-injection with mannitol. All V(H)H showed rapid renal clearance (10-20 min). Twenty-four hours post-injection (99m)Tc-pa2H resulted in a small yet significant higher cerebral uptake in the APP/PS1 animals. No difference in brain uptake were observed for (99m)Tc-ni3A or DTPA((111)In)-pa2H, which lacked additional peptide tags to investigate further clinical applicability. In vivo specificity for Aß was confirmed for both fluorescently labeled V(H)H, where pa2H remained readily detectable for 24 hours or more after injection. Furthermore, both V(H)H showed affinity for parenchymal and vascular deposits, this in contrast to human tissue, where ni3A specifically targeted only vascular Aß. Despite a brain uptake that is as yet too low for in vivo imaging, this study provides evidence that V(H)H detect Aß deposits in vivo, with high selectivity and favorable in vivo characteristics, making them promising tools for further development as diagnostic agents for the distinctive detection of different Aß deposits.

Pre-clinical optical imaging and MRI for drug development in Alzheimer's disease.

Optical and magnetic resonance imaging have the potential to be complementary non-invasive imaging modalities. Yet without advances in imaging technologies and contrast agents both have short-comings that cannot be ignored. In this review we demonstrate the pre-clinical use of the two imaging techniques in Alzheimer's disease, including examples from recent applications and discuss what is needed to improve their applicability for drug discovery.: