In the September 25 PNAS online, researchers led by Chris Dealwis at the University of Tennessee in Knoxville published the first crystal structure of a monoclonal antibody to the amyloid-β (Aβ) peptide. Several antibodies have moved into clinical trials for passive immunotherapy of Alzheimer disease. Yet their proponents readily admit that if any of these succeed, a certain dose of good luck will have had a hand in the success, as well. That’s because scientists remain far from fully understanding the particular characteristics of antibody-Aβ binding that make a therapeutic antibody safe and effective in the AD brain. X-ray crystallography is a step in this direction. It provides not only the basis for examining molecular forces and side chains engaged in antibody-antigen recognition, but also a starting point for structure-based design to refine currently available antibodies.


Electrostatic potential surface of antibody with bound Aβ peptide. Blue: positive charge, red: negative charge, white: apolar surface. Aβ(1-8) is drawn with carbon (yellow), nitrogen (blue), and oxygen (red). The Arg 5 residue sits in a pocket of strong negative charge. Glu 3 has no correspondingly positive region around it, making this position susceptible to substitution and cross-reaction. Image credit: Chris Dealwis

Anna Gardberg and colleagues generated several new monoclonal IgGs against stabilized protofibrils of Aβ40. They crystallized the antigen-binding fragments (Fabs) of two of them and found one to be complexed with the immunogenic Aβ (1-8) sequence and the other, to their surprise, with a similar sequence found in the human glutamate receptor interacting protein GRIP1 (Guo and Wang, 2007; Kulangara et al., 2007). The scientists characterize the atomic forces and a WWDDD motif with which the antibodies recognize these two similar peptide sequences. The Alzforum caught Chris Dealwis on the eve of a move. Our thanks to him for shooting off some brief last-minute replies from amid the boxes. See Q&A below.—Gabrielle Strobel.

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. Abstract

Q&A with Chris Dealwis. Questions by Gabrielle Strobel.

Q: Is it possible to do the same research with other Aβ antibodies?
A: Yes, if they have similar binding affinities.

Q: How about the A11?
A: If Charles Glabe's antibody is a monoclonal, then it will be a very interesting mAb to crystallize.

Q: How about the m266?

Q: The 3D6?
A: Possibly.

Q: Are you already working on any of the better-known antibodies in this field, or planning to?
A: Yes.

Q: Would you expect to see the same or similar WWDDD motif in the binding region of other antibodies to Aβ? In other words, do you think this is a common feature of N-terminal Aβ antibodies?
A: Not always.

Q: This is the first public crystal structure of an Aβ-monoclonal antibody complex I am aware of. Crystallography projects can be famously Herculean efforts. Was this one especially difficult to do?
A: Yes!

Q: Can your structure guide “rational drug designers” on how to improve the current crop of therapeutic antibodies? What would the structure suggest they change, for example?
A: Yes, it will improve affinity and specificity.

Q: Can the reactivity to a sequence of the human GRIP1 protein, or to the Ror2 sequence, be engineered out of the antibody without losing binding to Aβ?
A: I think so.

Q: GRIP1 is a post-synaptic scaffolding protein thought to play some role in synaptic activity, possibly through AMPA receptors. Current research also focuses on Aβ's possible role in AMPA receptor regulation. Pure coincidence?
A: Interesting, isn't it?


  1. 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).


    . 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.

    . 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.

    View all comments by Charles Glabe
  2. 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.


    . Molecular basis for passive immunotherapy of Alzheimer's disease. Proc Natl Acad Sci U S A. 2007 Oct 2;104(40):15659-64. PubMed.

    . 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.

    . 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.

    . Clinical immunologic approaches for the treatment of Alzheimer's disease. Expert Opin Investig Drugs. 2007 Jun;16(6):819-28. PubMed.

    . Disaggregation of Alzheimer beta-amyloid by site-directed mAb. Proc Natl Acad Sci U S A. 1997 Apr 15;94(8):4109-12. PubMed.

  3. 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)?


    . 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.

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Paper Citations

  1. . Glutamate stimulates glutamate receptor interacting protein 1 degradation by ubiquitin-proteasome system to regulate surface expression of GluR2. Neuroscience. 2007 Mar 2;145(1):100-9. PubMed.
  2. . Phosphorylation of glutamate receptor interacting protein 1 regulates surface expression of glutamate receptors. J Biol Chem. 2007 Jan 26;282(4):2395-404. PubMed.
  3. . 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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Primary Papers

  1. . Molecular basis for passive immunotherapy of Alzheimer's disease. Proc Natl Acad Sci U S A. 2007 Oct 2;104(40):15659-64. PubMed.