Beneficial catalytic immunity to abeta peptide

Abstract

We review attempts to treat Alzheimer disease with antibodies that bind amyloid beta peptide (Abeta) and the feasibility of developing catalytic antibodies for this purpose. Naturally occurring immunoglobulin M (IgM) class antibodies that hydrolyze Abeta and inhibit Abeta aggregation were identified. The production of these antibodies increases as a function of age, ostensibly reflecting an attempt by the immune system to protect against the deleterious effect of Abeta accumulation in old age. A search for catalytic antibodies in a library of human immunoglobulins variable (IgV) domains yielded catalysts that hydrolyzed Abeta specifically at exceptionally rapid rates. The catalytic IgVs contained the light-chain variable domains within scaffolds that are structurally reminiscent of phylogenetically ancient antibodies. Inclusion of the heavy-chain variable domain in the IgV constructs resulted in reduced catalysis. We present our view that catalytic antibodies are likely to emerge as more efficacious and safer immunotherapy reagents compared to traditional Abeta-binding antibodies.

FIG. 1.

Passive immunotherapy of Alzheimer disease (AD) with amyloid β peptide (Aβ)-binding immunoglobulin G (IgG). Reversibly binding IgG injected into peripheral blood can enter the brain in small amounts and help clear Aβ by mechanisms listed in the text. Following binding of IgG to Aβ monomers (a) or aggregates (b), the immune complexes are ingested by an Fcγ receptor-dependent uptake process into microglia (c). The IgG bound by FcRn receptors at the blood–brain barrier may help clear Aβ from the brain and efflux to the periphery via transcytosis (d). Microglial ingestion of the immune complexes is accompanied by release of inflammatory mediators, which could exacerbate the already inflamed state of the AD brain. In mouse AD models, clearance of amyloid plaques from the brain parenchyma induced by Aβ-binding IgGs can be accompanied by Aβ deposition in the blood vessels and microhemorrhages. FcRn, Neonatal Fc receptor; LRP-1, low-density lipoprotein receptor-related protein 1; RAGE, receptor for advanced glycation end products.

FIG. 2.

Identification and structure of amyloid βpeptide (Aβ)-hydrolyzing immunoglobulins variable (IgV) clones. (A) Structure of biotinylated electrophilic Aβ1–40 (Bt-E-Aβ40). The electrophilic phosphonates placed within the peptide permit specific covalent binding to nucleophilic immunoglobulins (Igs) facilitated by noncovalent epitope recognition. (B) Catalytic IgV identification by random screening and covalent selection with Bt-E-Aβ40. For IgV screening, 63 IgVs were purified by metal affinity chromatography of periplasmic extracts of bacterial colonies picked randomly from the IgV library. Aliquots of eluates (75 μL) obtained by metal affinity chromatography of the periplasmic extracts (total eluate volume 0.9 mL) were tested for Aβ hydrolyzing activity using ¹²⁵I-Aβ40 substrate (0.1 nM; 18 h incubation). For covalent selection, the IgV–phage library was treated with Bt-E-Aβ40 (2 μM), covalently bound phages were captured using immobilized anti-biotin antibody, noncovalently bound phage IgVs were removed by treatment with excess Aβ40, and the covalent phage IgV immune complexes were recovered by acid disruption of the biotin–anti-biotin antibody complexes (designated covalently selected IgVs). Hydrolysis of ¹²⁵I-Aβ40 (0.1 nM) by IgVs was measured using ¹²⁵I-Aβ40 substrate (0.1 nM; 18 h incubation). Each point represents an individual IgV clone. (C) Schematic representation of IgV_L2–t 2E6 and IgV_L–t′ 5D3 structures. scFv–t, V_L and V_H domains connected by a peptide linker with t, the His6/c-myc tag located at the carboyl terminus; IgV_L2–t 2E6, a heterodimer of two V_L domains with the linker and t; IgV_L–t′ 5D3, a single domain V_L with t′ at the carboxyl terminus, corresponding to the peptide linker, a short peptide region in place of the V_H domain and the t tag. Data are from ref. .

FIG. 3.

Immunoglobulins variable (IgV) catalytic properties. (A) Cleavage and noncovalent recognition sites. Cleavage sites in Aβ40 (indicated with black arrows) or Aβ42 (white arrows) were determined by matrix-assisted laser desorption/ionization–time-of-flight (MALDI-TOF) mass spectrometric analysis of nonradiolabeled synthetic Aβ peptides (100 μM) incubated with IgV_L2–t 2E6 (1 μM; 89 h). Bigger and smaller arrows denote, respectively, the major and minor cleavage sites in individual peptides. The noncovalent binding determinant for IgV_L2–t 2E6 (indicated with a green horizontal bar) was determined by screening synthetic Aβ peptide fragments for the ability to inhibit IgV-mediated ¹²⁵I-Aβ40 hydrolysis competitively. (B) Inhibition of IgV_L2–t 2E6 and IgV_L–t′ 5D3 catalyzed ¹²⁵I-Aβ 40 hydrolysis by E-hapten-1. IgV_L2–t 2E6 (0.55 μg/mL) or IgV_L–t′ 5D3 (0.075 μg/mL) treated with E-hapten-1 (R1 = benzyloxycarbonyl, R2 = phenyl) for 2 h were allowed to hydrolyze ¹²⁵I-Aβ40 as in Fig. 2B. (Inset) Streptavidin-peroxidase stained blots of sodium dodecyl sulfate (SDS)-gels showing IgV_L2–t 2E6 (20 μg/mL) treated for 18 h with 0.1 mM Bt-E-hapten-1 (R1 = biotinamidohexanoyl, R2 = C6H5; lane 2) or poorly electrophilic control hapten-2 (R1 = biotinamidohexanoyl, R2 = H; lane 3). Lane 1, IgV_L2–t 2E6 stained with silver. (C) Putative nucleophilic triad in the IgV_L2–t deduced by molecular modeling. The amino acid triad contains an H-bonded network that can impart nucleophilic reactivity to the Ser residue. Data are from ref. .

FIG. 4.

Phylogenic origin of antibody catalysis. The structure of highly catalytic immunoglobulins variable (IgVs) is reminiscent of primordial antibodies, suggesting that catalysis is a primitive function that may have developed prior to the evolution of adaptive immunity mechanisms responsible for the high affinity antigen binding capability of modern antibodies. Efficient catalysts could potentially be obtained from primordial antibody libraries or by reverse-engineering of modern antibodies to mimic primordial antibodies. IgNAR, immunoglobulin new antigen receptor.