Abeta targets of the biosimilar antibodies of Bapineuzumab, Crenezumab, Solanezumab in comparison to an antibody against N­truncated Abeta in sporadic Alzheimer disease cases and mouse models.

Solanezumab and Crenezumab are two humanized antibodies targeting Amyloid-ß (Aß) which are currently tested in multiple clinical trials for the prevention of Alzheimer's disease. However, there is a scientific discussion ongoing about the target engagement of these antibodies. Here, we report the immunohistochemical staining profiles of biosimilar antibodies of Solanezumab, Crenezumab and Bapineuzumab in human formalin-fixed, paraffin-embedded tissue and human fresh frozen tissue. Furthermore, we performed a direct comparative immunohistochemistry analysis of the biosimilar versions of the humanized antibodies in different mouse models including 5XFAD, Tg4-42, TBA42, APP/PS1KI, 3xTg. The staining pattern with these humanized antibodies revealed a surprisingly similar profile. All three antibodies detected plaques, cerebral amyloid angiopathy and intraneuronal Aß in a similar fashion. Remarkably, Solanezumab showed a strong binding affinity to plaques. We also reaffirmed that Bapineuzumab does not recognize N-truncated or modified Aß, while Solanezumab and Crenezumab do detect N-terminally modified Aß peptides Aß4-42 and pyroglutamate Aß3-42. In addition, we compared the results with the staining pattern of the mouse NT4X antibody that recognizes specifically Aß4-42 and pyroglutamate Aß3-42, but not full-length Aß1-42. In contrast to the biosimilar antibodies of Solanezumab, Crenezumab and Bapineuzumab, the murine NT4X antibody shows a unique target engagement. NT4X does barely cross-react with amyloid plaques in human tissue. It does, however, detect cerebral amyloid angiopathy in human tissue. In Alzheimer mouse models, NT4X detects intraneuronal Aß and plaques comparable to the humanized antibodies. In conclusion, the biosimilar antibodies Solanezumab, Crenezumab and Bapineuzumab strongly react with amyloid plaques, which are in contrast to the NT4X antibody that hardly recognizes plaques in human tissue. Therefore, NT4X is the first of a new class of therapeutic antibodies.

Do current therapeutic anti-Aß antibodies for Alzheimer's disease engage the target?

Reducing amyloid-ß peptide (Aß) burden at the pre-symptomatic stages of Alzheimer's disease (AD) is currently the advocated clinical strategy for treating this disease. The most developed method for targeting Aß is the use of monoclonal antibodies including bapineuzumab, solanezumab and crenezumab. We have synthesized these antibodies and used surface plasmon resonance (SPR) and mass spectrometry to characterize and compare the ability of these antibodies to target Aß in transgenic mouse tissue as well as human AD tissue. SPR analysis showed that the antibodies were able to bind Aß with high affinity. All of the antibodies were able to bind Aß in mouse tissue. However, significant differences were observed in human brain tissue. While bapineuzumab was able to capture a variety of N-terminally truncated Aß species, the Aß detected using solanezumab was barely above detection limits while crenezumab did not detect any Aß. None of the antibodies were able to detect any Aß species in human blood. Immunoprecipitation experiments using plasma from AD subjects showed that both solanezumab and crenezumab have extensive cross-reactivity with non-Aß related proteins. Bapineuzumab demonstrated target engagement with brain Aß, consistent with published clinical data. Solanezumab and crenezumab did not, most likely as a result of a lack of specificity due to cross-reactivity with other proteins containing epitope overlap. This lack of target engagement raises questions as to whether solanezumab and crenezumab are suitable drug candidates for the preventative clinical trials for AD.

Crystal structure of Alzheimer's disease phospholipase D3 provides a molecular basis for understanding its normal and pathological functions.

Human 5'-3' exonuclease PLD3, a member of the phospholipase D family of enzymes, has been validated as a therapeutic target for treating Alzheimer's disease. Here, we have determined the crystal structure of the luminal domain of the enzyme at 2.3 Å resolution, revealing a bilobal structure with a catalytic site located between the lobes. We then compared the structure with published crystal structures of other human PLD family members which revealed that a number of catalytic and lipid recognition residues, previously shown to be key for phospholipase activity, are not conserved or, are absent. This led us to test whether the enzyme is actually a phospholipase. We could not measure any phospholipase activity but the enzyme shows robust nuclease activity. Finally, we have mapped key single nucleotide polymorphisms onto the structure which reveals plausible reasons as to why they have an impact on Alzheimer's disease.

Structural biology of cell surface receptors implicated in Alzheimer's disease.

Alzheimer's disease is a common and devastating age-related disease with no effective disease-modifying treatments. Human genetics has implicated a wide range of cell surface receptors as playing a role in the disease, many of which are involved in the production or clearance of neurotoxins in the brain. Amyloid precursor protein, a membrane-bound signaling molecule, is at the very heart of the disease: hereditary mutations in its gene are associated with a greatly increased risk of getting the disease. A proteolytic breakdown product of amyloid precursor protein, the neurotoxic Aß peptide, has been the target for many drug discovery efforts. Antibodies have been designed to target Aß production with some success, although they have not proved efficacious in clinical trials with regards to cognitive benefits to date. Many of the recently identified genes associated with late-onset Alzheimer's disease risk are integral to the innate immune system. Some of these genes code for microglial proteins, such as the strongest genetic risk factor for the disease, namely APOE, and the cell surface receptors CD33 and TREM2 which are involved in clearance of the Aß peptide from the brain. In this review, we show how structural biology has provided key insights into the normal functioning of these cell surface receptors and provided a framework for developing novel treatments to combat Alzheimer's disease.

Small Molecule Binding to Alzheimer Risk Factor CD33 Promotes Aß Phagocytosis.

Polymorphism in the microglial receptor CD33 gene has been linked to late-onset Alzheimer disease (AD), and reduced expression of the CD33 sialic acid-binding domain confers protection. Thus, CD33 inhibition might be an effective therapy against disease progression. Progress toward discovery of selective CD33 inhibitors has been hampered by the absence of an atomic resolution structure. We report here the crystal structures of CD33 alone and bound to a subtype-selective sialic acid mimetic called P22 and use them to identify key binding residues by site-directed mutagenesis and binding assays to reveal the molecular basis for its selectivity toward sialylated glycoproteins and glycolipids. We show that P22, when presented on microparticles, increases uptake of the toxic AD peptide, amyloid-ß (Aß), into microglial cells. Thus, the sialic acid-binding site on CD33 is a promising pharmacophore for developing therapeutics that promote clearance of the Aß peptide that is thought to cause AD.

Crystallization and preliminary X-ray diffraction analysis of the Fab fragment of WO2, an antibody specific for the Abeta peptides associated with Alzheimer's disease.

The murine monoclonal antibody WO2 specifically binds the N-terminal region of the amyloid beta peptide (Abeta) associated with Alzheimer's disease. This region of Abeta has been shown to be the immunodominant B-cell epitope of the peptide and hence is considered to be a basis for the development of immunotherapeutic strategies against this prevalent cause of dementia. Structural studies have been undertaken in order to characterize the molecular basis for antibody recognition of this important epitope. Here, details of the crystallization and X-ray analysis of the Fab fragment of the unliganded WO2 antibody in two crystal forms and of the complexes that it forms with the truncated Abeta peptides Abeta(1-16) and Abeta(1-28) are presented. These crystals were all obtained using the hanging-drop vapour-diffusion method at 295 K. Crystals of WO2 Fab were grown in polyethylene glycol solutions containing ZnSO(4); they belonged to the orthorhombic space group P2(1)2(1)2(1) and diffracted to 1.6 A resolution. The complexes of WO2 Fab with either Abeta(1-16) or Abeta(1-28) were cocrystallized from polyethylene glycol solutions. These two complex crystals grew in the same space group, P2(1)2(1)2(1), and diffracted to 1.6 A resolution. A second crystal form of WO2 Fab was grown in the presence of the sparingly soluble Abeta(1-42) in PEG 550 MME. This second form belonged to space group P2(1) and diffracted to 1.9 A resolution.

Ex vivo 18O-labeling mass spectrometry identifies a peripheral amyloid ß clearance pathway.

BACKGROUND: Proteolytic degradation of amyloid ß (Aß) peptides has been intensely studied due to the central role of Aß in Alzheimer's disease (AD) pathogenesis. While several enzymes have been shown to degrade Aß peptides, the main pathway of Aß degradation in vivo is unknown. Cerebrospinal fluid (CSF) Aß42 is reduced in AD, reflecting aggregation and deposition in the brain, but low CSF Aß42 is, for unknown reasons, also found in some inflammatory brain disorders such as bacterial meningitis. METHOD: Using 18O-labeling mass spectrometry and immune-affinity purification, we examined endogenous proteolytic processing of Aß in human CSF. RESULTS: The Aß peptide profile was stable in CSF samples from healthy controls but in CSF samples from patients with bacterial meningitis, showing increased leukocyte cell count, 18O-labeling mass spectrometry identified proteolytic activities degrading Aß into several short fragments, including abundant Aß1-19 and 1-20. After antibiotic treatment, no degradation of Aß was detected. In vitro experiments located the source of the proteolytic activity to blood components, including leukocytes and erythrocytes, with insulin-degrading enzyme as the likely protease. A recombinant version of the mid-domain anti-Aß antibody solanezumab was found to inhibit insulin-degrading enzyme-mediated Aß degradation. CONCLUSION: 18O labeling-mass spectrometry can be used to detect endogenous proteolytic activity in human CSF. Using this technique, we found an enzymatic activity that was identified as insulin-degrading enzyme that cleaves Aß in the mid-domain of the peptide, and could be inhibited by a recombinant version of the mid-domain anti-Aß antibody solanezumab.

Molecular basis for mid-region amyloid-ß capture by leading Alzheimer's disease immunotherapies.

Solanezumab (Eli Lilly) and crenezumab (Genentech) are the leading clinical antibodies targeting Amyloid-ß (Aß) to be tested in multiple Phase III clinical trials for the prevention of Alzheimer's disease in at-risk individuals. Aß capture by these clinical antibodies is explained here with the first reported mid-region Aß-anti-Aß complex crystal structure. Solanezumab accommodates a large Aß epitope (960 Å(2) buried interface over residues 16 to 26) that forms extensive contacts and hydrogen bonds to the antibody, largely via main-chain Aß atoms and a deeply buried Phe19-Phe20 dipeptide core. The conformation of Aß captured is an intermediate between observed sheet and helical forms with intramolecular hydrogen bonds stabilising residues 20-26 in a helical conformation. Remarkably, Aß-binding residues are almost perfectly conserved in crenezumab. The structure explains the observed shared cross reactivity of solanezumab and crenezumab with proteins abundant in plasma that exhibit this Phe-Phe dipeptide.

Crystallization and preliminary X-ray diffraction analysis of the Fab portion of the Alzheimer's disease immunotherapy candidate bapineuzumab complexed with amyloid-ß.

Bapineuzumab (AAB-001) and its derivative (AAB-003) are humanized versions of the anti-Aß murine antibody 3D6 and are immunotherapy candidates in Alzheimer's disease. The common Fab fragment of these immunotherapies has been expressed, purified and crystallized in complex with ß-amyloid peptides (residues 1-8 and 1-28). Diffraction data at high resolution were acquired from crystals of Fab-Aß8 (2.0 Å) and Fab-Aß28 (2.2 Å) complexes at the Australian Synchrotron. Both crystal forms belonged to the primitive orthorhombic space group P21221.

Bapineuzumab captures the N-terminus of the Alzheimer's disease amyloid-beta peptide in a helical conformation.

Bapineuzumab is a humanized antibody developed by Pfizer and Johnson & Johnson targeting the amyloid (Aß) plaques that underlie Alzheimer's disease neuropathology. Here we report the crystal structure of a Fab-Aß peptide complex that reveals Bapineuzumab surprisingly captures Aß in a monomeric helical conformation at the N-terminus. Microscale thermophoresis suggests that the Fab binds soluble Aß(1-40) with a K(D) of 89 (±9) nM. The structure explains the antibody's exquisite selectivity for particular Aß species and why it cannot recognize N-terminally modified or truncated Aß peptides.

An Escherichia coli cell-free system for recombinant protein synthesis on a milligram scale.

In vitro translation systems derived from a wide range of organisms have been described in the literature and are widely used in biomedical research laboratories. Perhaps the most robust and efficient of these cell-free systems is that derived from Escherichia coli. Over the past decade or so, experimental strategies have been developed which have enhanced the efficiency and stability of E. coli cell-free systems such that we can now prepare recombinant proteins on a scale suitable for purification and analysis by biophysical and structural biology techniques, which commonly require relatively large quantities of protein. This chapter describes in detail the protocols employed in our laboratory to prepare translationally active E. coli extracts and to synthesise proteins on a milligram scale from these extracts.

Copper binding to the Alzheimer's disease amyloid precursor protein.

Alzheimer's disease is the fourth biggest killer in developed countries. Amyloid precursor protein (APP) plays a central role in the development of the disease, through the generation of a peptide called A beta by proteolysis of the precursor protein. APP can function as a metalloprotein and modulate copper transport via its extracellular copper binding domain (CuBD). Copper binding to this domain has been shown to reduce A beta levels and hence a molecular understanding of the interaction between metal and protein could lead to the development of novel therapeutics to treat the disease. We have recently determined the three-dimensional structures of apo and copper bound forms of CuBD. The structures provide a mechanism by which CuBD could readily transfer copper ions to other proteins. Importantly, the lack of significant conformational changes to CuBD on copper binding suggests a model in which copper binding affects the dimerisation state of APP leading to reduction in A beta production. We thus predict that disruption of APP dimers may be a novel therapeutic approach to treat Alzheimer's disease.

Amyloid-beta-anti-amyloid-beta complex structure reveals an extended conformation in the immunodominant B-cell epitope.

Alzheimer's disease (AD) is the most common form of dementia. Amyloid-beta (A beta) peptide, generated by proteolytic cleavage of the amyloid precursor protein, is central to AD pathogenesis. Most pharmaceutical activity in AD research has focused on A beta, its generation and clearance from the brain. In particular, there is much interest in immunotherapy approaches with a number of anti-A beta antibodies in clinical trials. We have developed a monoclonal antibody, called WO2, which recognises the A beta peptide. To this end, we have determined the three-dimensional structure, to near atomic resolution, of both the antibody and the complex with its antigen, the A beta peptide. The structures reveal the molecular basis for WO2 recognition and binding of A beta. The A beta peptide adopts an extended, coil-like conformation across its major immunodominant B-cell epitope between residues 2 and 8. We have also studied the antibody-bound A beta peptide in the presence of metals known to affect its aggregation state and show that WO2 inhibits these interactions. Thus, antibodies that target the N-terminal region of A beta, such as WO2, hold promise for therapeutic development.