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This review serves to highlight approaches that may improve the access of antibody drugs to regions of the brain affected by Alzheimer's Disease. While previous antibody drugs have been unsuccessful in treating Alzheimer's disease, recent work demonstrates that Alzheimer's pathology can be modified if these drugs can penetrate the brain parenchyma with greater efficacy. Research in antibody blood-brain barrier drug delivery predominantly follows one of three distinct directions: (1) enhancing influx with reduced antibody size, addition of Trojan horse modules, or blood-brain barrier disruption; (2) modulating trancytotic equilibrium and/or kinetics of the neonatal Fc Receptor; and (3) manipulation of antibody glycan carbohydrate composition. In addition to these topics, recent studies are discussed that reveal a role of glycan sialic acid in suppressing antibody efflux from the brain.

Understanding protein tertiary and quaternary structures is crucial to a better understanding of their biological functions. Here we illustrate for tryptophan synthase that tandem mass spectrometry (MS/MS) reveals not only protein subunit architectures, but also protein unfolding behavior when coupled with ion mobility (IM). In the present study, we verified the subunit arrangement with surface induced dissociation (SID). We are able to correlate experimental results by IM with those obtained in unfolding simulations for the hetero-tetramer Tryptophan Synthase (TS) protein complex by identifying the presence of at least three stable intermediates (I1, I2, and I3) during the unfolding process in collision induced dissociation (CID). We illustrate that the unfolding of the TS complex is likely due to the initial unfolding of an α-monomer subunit, followed by the unfolding of the second α-monomers. We also illustrate the ability of this combination of techniques to not only identify conformational changes of TS upon addition of D,L-α-glycerol phosphate (GP), but also to determine the location of the ligand, which is buried within the α-monomer of the TS.

Background Drug delivery to the brain is a major roadblock to treatment of Alzheimer's disease. Recent results of the PRIME study indicate that increasing brain penetration of antibody drugs improves Alzheimer's treatment outcomes. New approaches are needed to better accomplish this goal. Based on prior evidence, the hypothesis that glycan modification alters antibody blood-brain barrier permeability was tested here. Methods The blood-brain barrier permeability coefficient Pe of different glycosylated states of anti-amyloid IgG was measured using in vitro models of brain microvascular endothelial cells. Monoclonal antibodies 4G8, with sialic acid, and 6E10, lacking sialic acid, were studied. The amount of sialic acid was determined using quantitative and semi-quantitative surface plasmon resonance methods. Results Influx of IgG was not saturable and was largely insensitive to IgG species and glycosylation state. By contrast, efflux of 4G8 efflux was significantly lower than both albumin controls and 6E10. Removal of α2,6-linked sialic acid group present on 12% of 4G8 completely restored efflux to that of 6E10 but increasing the α2,6-sialylated fraction to 15% resulted in no change. Removal of the Fc glycan from 4G8 partially restored efflux. Alternate sialic acid groups with α2,3 and α2,8 linkages, nor on the Fc glycan, were not detected at significant levels on either 4G8 or 6E10. Conclusions These results support a model in which surface-sialylated 4G8 inhibits its own efflux and that of asialylated 4G8. General significance Glycan modification has the potential to increase antibody drug penetration into the brain through efflux inhibition.

Background The literature contains conflicting results regarding the status of serum anti-Aβ antibody concentrations in Alzheimer's disease (AD). Reduced levels of these antibodies have been suggested to contribute to the development of this disorder. The conflicting results may be due to polyvalent antibodies, antibody "masking" due to Aβ binding, methodological differences, and/or small sample sizes. The objectives of this pilot study were to compare serum anti-Aβ antibody concentrations between AD, mild cognitive impairment (MCI), and elderly noncognitively impaired (NCI) subjects while addressing these issues, and to perform power analyses to determine appropriate group sizes for future studies employing this approach. Methods Serum antibodies to Aβ1-42 monomer and soluble oligomers in AD, MCI, and NCI subjects (10/group) were measured by ELISA, subtracting polyvalent antibody binding and dissociating antibody-antigen complexes. Differences in mean antibody levels were assessed for significance with repeated measures ANOVA using restricted maximum likelihood estimation, using Tukey-Kramer tests and confidence intervals for multiple comparisons. Spearman's rank correlation was used to determine associations between anti-monomer and anti-oligomer antibody concentrations. Estimated sample sizes required to detect effects of various sizes were calculated. Results There were no significant differences between groups for mean anti-Aβ antibody levels, although these tended to be higher in AD than NCI specimens. Estimated group sizes of 328 and 150 for anti-Aβ monomer and oligomer antibodies, respectively, would have been required for 80% power for significance at 0.05 for a 25% increase in the AD mean relative to the NCI mean. Serum antibody concentrations to Aβ monomer and oligomers were strongly associated (correlations: 0.798 for undissociated sera, 0.564 for dissociated sera). Antibody-antigen dissociation significantly increased anti-Aβ monomer but not anti-Aβ oligomer antibody levels. Conclusions The findings in this pilot study are consistent with relatively similar concentrations of specific, non-antigen-bound antibodies to Aβ1-42 monomer and soluble oligomers in AD, MCI, and NCI sera. The differences between groups for these antibodies would have required approximate group sizes of 328 and 150, respectively, for a high probability for statistical significance. These findings do not support the hypothesis that reduced levels of anti-Aβ antibodies might contribute to AD's pathogenesis.

Abeta soluble oligomers are believed to play a key role in the development of Alzheimer's disease (AD). An enzyme-linked immunosorbent assay (ELISA) commonly used to measure these proteins uses the same monoclonal antibody as both capture and reporter antibody. The objective of this study was to examine the specificity and sensitivity of this procedure, using monoclonal anti-Abeta antibody 6E10 as capture antibody and biotinylated 6E10 as reporter antibody. At comparable concentrations of Abeta soluble oligomers and low molecular weight (LMW) Abeta peptides, optical density (OD) values were four- to five-fold higher for the oligomer preparation than for the LMW Abeta. The LMW Abeta preparation, when evaluated by western blots of gels run under native conditions, showed only one band even after storage at 4 °C for more than two months, suggesting that the ELISA was detecting Abeta monomer as well as Abeta oligomers. Possible explanations for these results are that (1) the LMW Abeta preparation may contain Abeta oligomer species below the limit of detection of western blot, but still detectable by ELISA, or (2) some nonspecific binding of the LMW Abeta to the ELISA plate may have occurred, allowing its relevant epitope to remain available for binding by the reporter antibody. Because of the possibility that this ELISA may not be oligomer-specific, it seems prudent to suggest that it should be used in combination with other methods, rather than as the sole technique, for measuring Abeta oligomers in biological specimens.

Improvement in cognitive scores in patients with Alzheimer's disease (AD) has been reported in two trials in which intravenous immunoglobulin (IvIg) preparations were administered. IvIg's benefits in AD patients have been suggested to be due to antibodies to amyloid-beta (Abeta). Our previous study using indirect enzyme-linked immunosorbent assay (ELISA) indicated that much of IvIg's apparent binding to Abeta1-42 is nonspecific; it is detectable even when IvIg is incubated on "specificity controls" (bovine serum albumin [BSA] and Abeta reverse sequence Abeta42-1) rather than Abeta1-42. The objective of this study was to evaluate procedures that might reduce this nonspecific binding. The IvIg preparation used was Gamunex (Talecris Biotherapeutics). Multiple blocking agents were evaluated, but even the most effective blocker only reduced nonspecific binding by 48%. Dissociating Gamunex's antibody-antigen complexes had no effect on specific binding when Abeta42-1 was used as the specificity control, although it increased this binding when BSA was the specificity control. Decreasing Gamunex's dilution from 1:1,500 to 1:500 resulted in a slight (7.4%) but significant (p=0.027) increase in specific binding. Using a sandwich ELISA to measure Gamunex's anti-Abeta antibodies resulted in even less specific binding to Abeta1-42 than with the indirect ELISA. Despite Gamunex's low percentage of specific binding to Abeta1-42, it inhibited Abeta oligomer formation. We conclude that, when anti-Abeta antibodies in IvIg are measured by indirect ELISA, extensive nonspecific binding occurs despite procedures taken to prevent it. This must be subtracted from total binding to accurately measure specific anti-Abeta antibody concentrations.

Cognitive improvement in Alzheimer's disease (AD) patients treated with intravenous immunoglobulin (IvIg) has been attributed to its antibodies to amyloid beta (Abeta). We compared the concentrations of specific antibodies to soluble Abeta1-42 conformations, namely Abeta1-42 monomer and Abeta1-42 soluble oligomers, between three IvIg preparations, Gamunex, Gammagard, and Flebogamma. To determine specific antibody concentrations to these Abeta1-42 conformations, nonspecific binding of the IvIg preparations to the Abeta reverse sequence, Abeta42-1, was subtracted. These antibodies were measured in untreated IvIg preparations and also after they were treated to dissociate antibody-antigen complexes, because this procedure has been reported to increase the detectable levels of serum anti-Abeta antibodies. Antibody levels to Abeta1-42 monomer were significantly higher in untreated Gamunex than in the other two IvIg preparations, and antibody-antigen dissociation increased the measured anti-Abeta monomer concentrations in Gamunex and Gammagard. Dissociated Gamunex and Gammagard had higher anti-Abeta monomer levels than Flebogamma. Generally similar results were found for antibodies to soluble Abeta1-42 oligomers, with the exception that after antibody-antigen dissociation, only Gammagard had significantly higher antibody levels than Flebogamma. These differences in antibody concentrations to Abeta1-42 conformations (particularly to Abeta1-42 soluble oligomers, thought to be the most neurotoxic conformation of soluble Abeta) and the increased availability of these antibodies after antibody-antigen complex dissociation have important implications for IvIg treatment of AD patients.

Kinetic simulations of the folding and unfolding of the mammalian TIM barrel protein aldolase were conducted to determine if a minimalist monomeric Gō model, using the native structure to determine attractive energies in the protein chain, could capture the experimentally determined folding pathway. The folding order, that is, the order in which different secondary structures fold, between the Gō model simulations and that from hydrogen-deuterium exchange experiments, did not agree. To explain this discrepancy, two alternate variant of the basic Gō model were simulated: (1) a monomer Gō model with native contact energies weighted by a statistical potential (SP model) and (2) a monomer Gō model with native contact energies inversely weighted by crystallographic B factors (B model). The B model demonstrated the best agreement between simulation and experiments. The success of the B model is attributed to the ability of B factors to highlight local energetic frustration in the aldolase structure which results in weaker native contacts in these frustrated regions. The predictive success of the B model also reveals the potential use of B factor information for energetic weighting in general protein modeling.

The fraction of IgG antibodies with anti-oligomeric Aβ affinity and surface sialic acid was compared between Octagam and Gammagard intravenous immunoglobulin (IVIG) using two complementary surface plasmon resonance methods. These comparisons were performed to identify if an elevated fraction existed in Gammagard, which reported small putative benefits in a recent Phase III clinical trial for Alzheimer’s Disease. The fraction of anti-oligomeric Aβ IgG was found to be higher in Octagam, for which no cognitive benefits were reported. The fraction and location of surface-accessible sialic acid in the Fab domain was found to be similar between Gammagard and Octagam. These findings indicate that anti-oligomeric Aβ IgG and total surface sialic acid alone cannot account for reported clinical differences in the two IVIG products. A combined analysis of sialic acid in anti-oligomeric Aβ IgG did reveal a notable finding that this subgroup exhibited a high degree of surface sialic acid lacking the conventional α2,6 linkage. These results demonstrate that the IVIG antibodies used to engage oligomeric Aβ in both Gammagard and Octagam clinical trials did not possess α2,6-linked surface sialic acid at the time of administration. Anti-oligomeric Aβ IgG with α2,6 linkages remains untested as an AD treatment.