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Germline humanization of a murine Abeta antibody and crystal structure of the humanized recombinant Fab fragment.

ABSTRACT: Alzheimer's disease is the most common form of dementia, affecting 26 million people worldwide. The Abeta peptide (39-43 amino acids) derived from the proteolytic cleavage of the amyloid precursor protein is one of the main constituents of amyloid plaques associated with disease pathogenesis and therefore a validated target for therapy. Recently, we characterized antibody fragments (Fab and scFvs) derived from the murine monoclonal antibody WO-2, which bind the immunodominant epitope ((3)EFRH(6)) in the Abeta peptide at the N-terminus. In vitro, these fragments are able to inhibit fibril formation, disaggregate preformed amyloid fibrils, and protect neuroblastoma cells against oligomer-mediated toxicity. In this study, we describe the humanization of WO-2 using complementary determining region loop grafting onto the human germline gene and the determination of the three-dimensional structure by X-ray crystallography. This humanized version retains a high affinity for the Abeta peptide and therefore is a potential candidate for passive immunotherapy of Alzheimer's disease.

SUBMITTER: Robert R

PROVIDER: S-EPMC2865728 | biostudies-literature | 2010 Feb

REPOSITORIES: biostudies-literature

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Germline humanization of a murine Abeta antibody and crystal structure of the humanized recombinant Fab fragment.

Robert Remy R Streltsov Victor A VA Newman Janet J Pearce Lesley A LA Wark Kim L KL Dolezal Olan O

Protein science : a publication of the Protein Society 20100201 2

Alzheimer's disease is the most common form of dementia, affecting 26 million people worldwide. The Abeta peptide (39-43 amino acids) derived from the proteolytic cleavage of the amyloid precursor protein is one of the main constituents of amyloid plaques associated with disease pathogenesis and therefore a validated target for therapy. Recently, we characterized antibody fragments (Fab and scFvs) derived from the murine monoclonal antibody WO-2, which bind the immunodominant epitope ((3)EFRH(6) ...[more]

PMID: 20014445

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Project description:BACKGROUND:Signal Regulatory Protein ? (SIRP?) is a member of a closely related family of three cell surface receptors implicated in modulating immune/inflammatory responses. SIRP? is expressed on T lymphocytes where it appears to be involved in the integrin-independent adhesion of lymphocytes to antigen-presenting cells. Here we describe the first full length structure of the extracellular region of human SIRP?. RESULTS:We obtained crystals of SIRP? by making a complex of the protein with the Fab fragment of the anti-SIRP antibody, OX117, which also binds to SIRP? and SIRP?. We show that the epitope for FabOX117 is formed at the interface of the first and second domains of SIRP? and comprises residues which are conserved between all three SIRPs. The FabOX117 binding site is distinct from the region in domain 1 which interacts with CD47, the physiological ligand for both SIRP? and SIRP? but not SIRP?. Comparison of the three domain structures of SIRP? and SIRP? showed that these receptors can adopt different overall conformations due to the flexibility of the linker between the first two domains. SIRP? in complex with FabOX117 forms a dimer in the crystal. Binding to the Fab fixes the position of domain 1 relative to domains 2/3 exposing a surface which favours formation of a homotypic dimer. However, the interaction appears to be relatively weak since only monomers of SIRP? were observed in sedimentation velocity analytical ultracentrifugation of the protein alone. Studies of complex formation by equilibrium ultracentrifugation showed that only a 1:1 complex of SIRP?: FabOX117 was formed with a dissociation constant in the low micromolar range (Kd?=?1.2 +/- 0.3 ?M). CONCLUSION:The three-domain extracellular regions of SIRPs are structurally conserved but show conformational flexibility in the disposition of the amino terminal ligand-binding Ig domain relative to the two membrane proximal Ig domains. Binding of a cross-reactive anti-SIRP Fab fragment to SIRP? stabilises a conformation that favours SIRP dimer formation in the crystal structure, though this interaction does not appear sufficiently stable to be observed in solution.

Project description:BackgroundMany antibody crystal structures have been solved. Structural modeling programs have been developed that utilize this information to predict 3-D structures of an antibody based upon its sequence. Because of the problem of self-reference, the accuracy and utility of these predictions can only be tested when a new structure has not yet been deposited in the Protein Data Bank.MethodsWe have solved the crystal structure of the Fab fragment of RAC18, a protective anti-ricin mAb, to 1.9 Å resolution. We have also modeled the Fv structure of RAC18 using publicly available Ab modeling tools Prediction of Immunoglobulin Structures (PIGS), RosettaAntibody, and Web Antibody Modeling (WAM). The model structures underwent energy minimization. We compared results to the crystal structure on the basis of root-mean-square deviation (RMSD), template modeling score (TM-score), Z-score, and MolProbity analysis.FindingsThe crystal structure showed a pocket formed mainly by AA residues in each of the heavy chain complementarity determining regions (CDRs). There were differences between the crystal structure and structures predicted by the modeling tools, particularly in the CDRs. There were also differences among the predicted models, although the differences were small and within experimental error. No one modeling program was clearly superior to the others. In some cases, choosing structures based only on sequence homology to the crystallized Ab yielded RMSDs comparable to the models.ConclusionsMolecular modeling programs accurately predict the structure of most regions of antibody variable domains of RAC18. The hypervariable CDRs proved most difficult to model, particularly H chain CDR3. Because CDR3 is most often involved in contact with antigen, this defect must be considered when using models to identify potential contacts between antibody and antigen. Because this study represents only a single case, the results cannot be generalized. Rather they highlight the utility and limitations of modeling programs.

Dataset Information

Germline humanization of a murine Abeta antibody and crystal structure of the humanized recombinant Fab fragment.

Publications

Germline humanization of a murine Abeta antibody and crystal structure of the humanized recombinant Fab fragment.

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