Christer Nordstedt

Gantenerumab: A Novel Human Anti-Aβ Antibody Demonstrates Sustained Cerebral Amyloid-β Binding and Elicits Cell-Mediated Removal of Human Amyloid-β

The amyloid-β lowering capacity of anti-Aβ antibodies has been demonstrated in transgenic models of Alzheimers disease (AD) and in AD patients. While the mechanism of immunotherapeutic amyloid-β removal is controversial, antibody-mediated sequestration of peripheral Aβ versus microglial phagocytic activity and disassembly of cerebral amyloid (or a combination thereof) has been proposed. For successful Aβ immunotherapy, we hypothesized that high affinity antibody binding to amyloid-β plaques and recruitment of brain effector cells is required for most efficient amyloid clearance. Here we report the generation of a novel fully human anti-Aβ antibody, gantenerumab, optimized in vitro for binding with sub-nanomolar affinity to a conformational epitope expressed on amyloid-β fibrils using HuCAL(®) phage display technologies. In peptide maps, both N-terminal and central portions of Aβ were recognized by gantenerumab. Remarkably, a novel orientation of N-terminal Aβ bound to the complementarity determining regions was identified by x-ray analysis of a gantenerumab Fab-Aβ(1-11) complex. In functional assays gantenerumab induced cellular phagocytosis of human amyloid-β deposits in AD brain slices when co-cultured with primary human macrophages and neutralized oligomeric Aβ42-mediated inhibitory effects on long-term potentiation in rat brain. In APP751(swedish)xPS2(N141I) transgenic mice, gantenerumab showed sustained binding to cerebral amyloid-β and, upon chronic treatment, significantly reduced small amyloid-β plaques by recruiting microglia and prevented new plaque formation. Unlike other Aβ antibodies, gantenerumab did not alter plasma Aβ suggesting undisturbed systemic clearance of soluble Aβ. These studies demonstrated that gantenerumab preferentially interacts with aggregated Aβ in the brain and lowers amyloid-β by eliciting effector cell-mediated clearance.

Endogenous proteins controlling amyloid beta-peptide polymerization. Possible implications for beta-amyloid formation in the central nervous system and in peripheral tissues.

We report that certain plasma proteins, at physiological concentrations, are potent inhibitors of amyloid β-peptide (Aβ) polymerization. These proteins are also present in cerebrospinal fluid, but at low concentrations having little or no effect on Aβ. Thirteen proteins representing more than 90% of the protein content in plasma and cerebrospinal fluid were studied. Quantitatively, albumin was the most important protein, representing 60% of the total amyloid inhibitory activity, followed by α1-antitrypsin and immunoglobulins A and G. Albumin suppressed amyloid formation by binding to the oligomeric or polymeric Aβ, blocking a further addition of peptide. This effect was also observed when the incorporation of labeled Aβ into genuine β-amyloid in tissue section was studied. The Aβ and the anti-diabetic drug tolbutamide apparently bind to the same site on albumin. Tolbutamide displaces Aβ from albumin, increasing its free concentration and enhancing amyloid formation. The present results suggest that several endogenous proteins are negative regulators of amyloid formation. Plasma contains at least 300 times more amyloid inhibitory activity than cerebrospinal fluid. These findings may provide one explanation as to why β-amyloid deposits are not found in peripheral tissues but are only found in the central nervous system. Moreover, the data suggest that some drugs that display an affinity for albumin may enhance β-amyloid formation and promote the development of Alzheimer’s disease.

Controlling Polymerization of β-Amyloid and Prion-derived Peptides with Synthetic Small Molecule Ligands

The Alzheimer β-amyloid peptide (Aβ) and a fragment of the prion protein have the capacity of forming amyloid-like fibrils when incubated under physiological conditions in vitro. Here we show that a small amyloid ligand, RO-47-1816/001, enhances this process severalfold by binding to amyloid molecules and apparently promote formation of the peptide-to-peptide bonds that join the monomers of the amyloid fibrils. This effect could be antagonized by other ligands, including analogues of RO-47-1816/001, as well as the structurally unrelated ligand Congo red. Analogues of RO-47-1816/001 with low affinity for amyloid did not display any antagonistic effect. In conclusion, these data suggest that synthetic molecules, and possibly also small natural substances present in the brain, may act in a chaperone-like fashion, promoting Aβ polymerization and growth of amyloid fibrils in vitro and possibly also in vivo. Furthermore, we demonstrate that small organic molecules can be used to inhibit the action of amyloid-enhancing compounds.

A novel recombinant human amyloid plaque-specific antibody for the treatment of Alzheimer's disease: Molecular properties and preclinical efficacy

Background: Preclinical profile of Ab binding properties and efficacy of a fully human monoclonal anti-Ab antibody. Methods: Binding properties were assessed by Biacore and X-ray crystallography. In life efficacy for plaque binding and Ab clearance was characterized by immunoassays and quantitative image analysis. Results: Anti Ab antibodies are currently explored as promising therapeutics to treat Alzheimer’s disease (AD). We have generated a new type of a human recombinant antibody with amyloid plaque specificity by phage display and in vitro maturation technologies. Antibody binding properties were optimized to achieve subnanomolar affinity and strong reactivity with human amyloid plaques and maximal target occupancy in vivo. A conformation specific binding mode was indicated by higher binding affinity to fibrillar versus monomeric Ab40/42. Epitope mapping with linear Ab decapeptides showed reactivity against N-terminal (EFRHDSGYE) and central (FFAEDVGS) amino acids, suggesting recognition of a unique structure present on Ab fibrils. X-ray crystallography revealed an interesting binding mode involving multiple amino acids of heavy and light chain CDRs and all residues of Ab 3-11. In vivo, the human antibody entered the brain and efficiently co-localized to all types of plaques in APPswe x PS2N141I (PS2APP) mice. Plaque binding was dose-dependent with a minimal effective dose of 0.1 mg/kg and detectable over 2 months after single administration. Chronic treatment of PS2APP mice revealed a significant decrease of amyloid plaque load by partial clearance of pre-existing plaques and substantial prevention of de novo plaque formation. Efficacy of amyloid lowering was directly dependent on the ability of the antibody to cross the blood-brain-barrier and to bind to amyloid plaques. Remarkably, peripheral Ab40 and Ab42 levels were not increased after administration of the human antibody to PS2APP mice indicating that clearance of peripheral Ab was not affected. The absence of stable antibody-Ab complexes in blood may also have facilitated the pronounced and long lasting target occupancy observed in vivo in PS2APP mice. Conclusions: The fully human antibody described represents a novel, centrally-active anti-Ab antibody with a broad and sensitive Ab-amyloid specificity. Its binding mode and amyloid lowering capacity are promising features supporting further development as an immunotherapeutic treatment for Alzheimer’s Disease.