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Gardberg AS, Dice LT, Ou S, Rich RL, Helmbrecht E, Ko J, Wetzel R, Myszka DG, Patterson PH, Dealwis C. Molecular basis for passive immunotherapy of Alzheimer's disease. Proc Natl Acad Sci U S A. 2007 Oct 2;104(40):15659-64. PubMed.
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University of California, Irvine
Anti-amyloid immunotherapy remains one of the front-line strategies for the development of Alzheimer therapeutics. Both passive and active immunization are currently under active development for human clinical application. Antibodies that target the amino terminus of Aβ seem particularly interesting. Not only does this region appear to be an immuno-dominant site, but antibodies that recognize epitopes in this region also seem particularly effective in reversing AD pathogenesis in transgenic animals and in depolymerizing amyloid fibrils in vitro. In this article, Chris Dealwis and colleagues report the crystal structures of two monoclonal antibodies that target the amino terminus of Aβ.
These antibodies, PFA1 and PFA2, are remarkably specific for the EFRHD sequence at residues 3-7 of the Aβ peptide, as substitution of an alanine residue at any position nearly eliminates antibody binding. The crystal structures of the Fab complex with the peptide DAEFRHDS reveals that a WWDDD motif in the heavy chain complementarity determining region (CDR) of the antibodies forms salt bridges, hydrogen bonds, and hydrophobic contacts with the EFRHD sequence of Aβ.
Although both PFA1 and PFA2 are remarkably specific for the EFRHD sequence, the authors show that a similar sequence (AKFRHD) derived from the human protein GRIP1 also reacts with the monoclonal antibodies. This raises the possibility of undesirable cross-reactivity with other human proteins; however, the structure of the antigen combining site suggests that one could redesign the CDRs to eliminate undesired cross-reactivity.
The amino terminus of Aβ is also interesting because it seems to contain a conformational switch associated with aggregation and is the site that some conformation-dependent antibodies recognize. The fact that antibodies directed against this region depolymerize amyloid fibrils suggests that antibody binding induces a structure that is incompatible with the amyloid fibril lattice (1). The amino terminus is also the site of a conformation-dependent epitope recognized by the M16 polyclonal antisera that is specific for Aβ aggregates and fibrils, but does not recognize Aβ monomer or APP (2).
References:
Frenkel D, Balass M, Katchalski-Katzir E, Solomon B. High affinity binding of monoclonal antibodies to the sequential epitope EFRH of beta-amyloid peptide is essential for modulation of fibrillar aggregation. J Neuroimmunol. 1999 Mar 1;95(1-2):136-42. PubMed.
Soreghan B, Pike C, Kayed R, Tian W, Milton S, Cotman C, Glabe CG. The influence of the carboxyl terminus of the Alzheimer Abeta peptide on its conformation, aggregation, and neurotoxic properties. Neuromolecular Med. 2002;1(1):81-94. PubMed.
Tel Aviv University
The paper of Gardberg et al. (1) describes, in an elegant and convincing way, the molecular basis of immunotherapy with Aβ peptide anti-N-terminal antibodies. They report the isolation of two mAbs (PFA1 and PFA2) raised against stabilized protofibrils of Aβ, which recognize Aβ monomers, protofibrils, and fibrils. Importantly, they report the structures of their antigen binding fragments (Fabs) in complex with the Aβ(1-8) peptide DAEFRHDS.
As previously shown, immunization against the EFRH sequence rescues cognitive function in mouse models of Alzheimer disease. The EFRH epitope is available for antibody binding when Aβ peptide is either in solution or is an aggregate, and locking of this epitope by antibodies affects the dynamics of all the molecules, preventing self-aggregation as well as enabling resolubilization of already formed aggregates (2-4). All these prior findings illustrate the importance of understanding the structural basis of antibody recognition of this sequence.
Among the proposed mechanisms of immunotherapy, the catalytic dissolution via antibodies, which act as chaperones catalyzing the structural change of the Aβ peptide from the β-strand to an alternative conformation less prone to aggregation, has an important role. Consistent with this mechanism, the efficacy of a given mAb depends on the Aβ sequence element it binds; thus, the mAb 6C6, which recognizes the Aβ N- terminus, is three times more effective in disaggregating Aβ fibrils than the mAb 1C2 directed to other regions. Antibody binding to Aβ is required in either monomer or aggregated forms. The authors suggest that the most appropriate antibodies are those equally capable of recognizing all assembly forms of Aβ peptides, and this is particularly pointed out in this study. Indeed, antibodies against the EFRHD sequence recognize Aβ in all these conformations. As previously shown (5), only antibodies against this sequence are able to dissolve already formed aggregation.
The high specificity of such antibodies to epitope EFRHD results from studies on crystallization of Fab fragments with Aβ. The Fab fragments exhibit binding to Aβ monomers in the 20–40 nM range, and this binding is significantly impaired or eliminated in Aβ(1–40) mutants where a single residue in the 3–7 segment is replaced with alanine.
The accumulated experience of many efforts to obtain antibodies to Aβ suggests that the N-terminus is the immuno-dominant epitope of this peptide. Furthermore, if aggregated forms of Aβ are to be targeted in therapy, antibodies to the N-terminus will probably be required, given the poor accessibility of other portions of the sequence in aggregates.
As cross-reactivity occurred between these antibodies and other unrelated proteins, a smaller amount of high-affinity antibodies to the N-terminal epitope of Aβ peptide is required for a successful immunotherapy in Alzheimer disease.
References:
Gardberg AS, Dice LT, Ou S, Rich RL, Helmbrecht E, Ko J, Wetzel R, Myszka DG, Patterson PH, Dealwis C. Molecular basis for passive immunotherapy of Alzheimer's disease. Proc Natl Acad Sci U S A. 2007 Oct 2;104(40):15659-64. PubMed.
Frenkel D, Balass M, Solomon B. N-terminal EFRH sequence of Alzheimer's beta-amyloid peptide represents the epitope of its anti-aggregating antibodies. J Neuroimmunol. 1998 Aug 1;88(1-2):85-90. PubMed.
Frenkel D, Balass M, Katchalski-Katzir E, Solomon B. High affinity binding of monoclonal antibodies to the sequential epitope EFRH of beta-amyloid peptide is essential for modulation of fibrillar aggregation. J Neuroimmunol. 1999 Mar 1;95(1-2):136-42. PubMed.
Solomon B. Clinical immunologic approaches for the treatment of Alzheimer's disease. Expert Opin Investig Drugs. 2007 Jun;16(6):819-28. PubMed.
Solomon B, Koppel R, Frankel D, Hanan-Aharon E. Disaggregation of Alzheimer beta-amyloid by site-directed mAb. Proc Natl Acad Sci U S A. 1997 Apr 15;94(8):4109-12. PubMed.
View all comments by Beka SolomonKULeuven
I agree with Charles and Beka that this is excellent work. It's actually long overdue in the AD field, which is at the same time crowded and lacking some essential experts.
Nevertheless, I am more critical than my two learned friends and colleagues about the real meaning of this study for immunotherapy in AD. After careful reading—and discussion with an expert or two—we came to the conclusion that this paper sails under the wrong flag. A more apt title might have read: "Molecular Basis for Recognition of Epitope EFRHD on the Amyloid-β Peptide by a Monoclonal Antibody."
The data highlight in exquisite detail the structure of the peptide-antibody immune complex. But they do not address—and therefore do not answer—the primary question in AD immunotherapy: why do N-terminal-specific antibodies dissociate amyloid peptide aggregates, and thereby improve the cognitive functions of AD mice (and hopefully patients as well)?
I am convinced that this excellent paper will help considerably in paving the way to answer the first part of that question. At the same time, I cannot resist adding this extra level of complexity of why Mabs against "conformational" epitopes are most effective in doing what they do (Muhs et al., 2007)?
References:
Muhs A, Hickman DT, Pihlgren M, Chuard N, Giriens V, Meerschman C, van der Auwera I, Van Leuven F, Sugawara M, Weingertner MC, Bechinger B, Greferath R, Kolonko N, Nagel-Steger L, Riesner D, Brady RO, Pfeifer A, Nicolau C. Liposomal vaccines with conformation-specific amyloid peptide antigens define immune response and efficacy in APP transgenic mice. Proc Natl Acad Sci U S A. 2007 Jun 5;104(23):9810-5. PubMed.
View all comments by Fred Van LeuvenMake a Comment
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