Exceptional Amyloid β Peptide Hydrolyzing Activity of Nonphysiological Immunoglobulin Variable Domain Scaffolds*

Nucleophilic sites in the paired variable domains of the light and heavy chains (VL and VH domains) of Ig can catalyze peptide bond hydrolysis. Amyloid β (Aβ)-binding Igs are under consideration for immunotherapy of Alzheimer disease. We searched for Aβ-hydrolyzing human IgV domains (IgVs) in a library containing a majority of single chain Fv clones mimicking physiological VL-VH-combining sites and minority IgV populations with nonphysiological structures generated by cloning errors. Random screening and covalent selection of phage-displayed IgVs with an electrophilic Aβ analog identified rare IgVs that hydrolyzed Aβ mainly at His14-Gln15. Inhibition of IgV catalysis and irreversible binding by an electrophilic hapten suggested a nucleophilic catalytic mechanism. Structural analysis indicated that the catalytic IgVs are nonphysiological structures, a two domain heterodimeric VL (IgVL2-t) and single domain VL clones with aberrant polypeptide tags (IgVL-t′). The IgVs hydrolyzed Aβ at rates superior to naturally occurring Igs by 3-4 orders of magnitude. Forced pairing of the single domain VL with VH or VL domains resulted in reduced Aβ hydrolysis, suggesting catalysis by the unpaired VL domain.Ångstrom level amino acid displacements evident in molecular models of the two domain and unpaired VL domain clones explain alterations of catalytic activity. In view of their superior catalytic activity, the VL domain IgVs may help attain clearance of medically important antigens more efficiently than natural Igs.

The antigen-combining sites of immunoglobulins found in higher organisms are composed of the variable domains of light and heavy chain subunits (V L and V H domains). The individual V L and V H domains can bind antigens independently of each other, but the paired V L -V H structure consistently expresses superior antigen binding affinity because of cooperative antigen-binding forces contributed by the two domains (1). High affinity Igs are generated by adaptive V domain sequence diversification over the course of B lymphocyte differentiation, a process in which antigen binding to mutated B cell receptors (surface Igs associated with signal-transducing proteins) drives the selective expansion of the cells. Adaptive Ig maturation entails the use of one each of ϳ50 inherited V L and V H genes, diversification at the junctions of the V L -D L gene segments and V H -D H -J H gene segments, and somatic mutation over the entire length of the V domains.
Following initial noncovalent binding of antigen, some Igs proceed to catalyze its chemical transformation. Examples of Ig-catalyzed reactions include hydrolysis of polypeptide antigens (2,3), hydrolysis of nucleic acids (4,5), and various acyl transfer reactions of other antigen classes (6). Proteolytic Igs that utilize serine protease-like covalent hydrolytic pathways have been described (7,8). Serine protease-like catalytic triads have been identified in the V domains of Igs by site-directed mutagenesis and crystallography (9,10). The catalytic mechanism involves nucleophilic attack on the electrophilic carbonyl of peptide bonds. Electrophilic phosphonate diesters originally developed as covalent probes for the nucleophilic site of serine proteases bind catalytic Igs irreversibly and inhibit their catalytic activity (7,11,12). The strength of Igantigen noncovalent binding often exceeds that of enzyme-substrate binding. An important limitation holding back the application of catalytic Igs for clearance of undesirable antigens is that their catalytic rate constants (turnover number; k cat ) are small compared with enzymes. Evidently, Ig adaptive selection is geared toward noncovalent immune complexation (the ground state stabilization step), and the ability of Igs to recognize the high energy transition state complex that must be stabilized to accelerate chemical reactions is limited. This is supported by observations that IgMs, the first and least diversified Ig class produced during B cell differentiation, express superior catalytic rate constants than IgGs produced by the cells at later stages of their adaptive differentiation (12).
Accumulation of amyloid ␤ peptide (A␤) 2 aggregates in the brain is thought to be a central contributor to neurodegenera-tive changes underlying Alzheimer disease (AD). Administration of monoclonal IgGs that bind A␤ reversibly to transgenic mice overexpressing human A␤ clears brain A␤ deposits and improves cognitive function (13,14). Suggested mechanisms explaining the favorable effect of peripherally administered IgG are as follows: (a) A␤ containing immune complexes formed by small amounts of IgGs that cross the blood-brain barrier are removed by Fc receptor-mediated uptake by resident macrophages in the brains, the microglia (14); (b) A␤ binding to the IgG constrains the peptide into a nonaggregable conformation (15); (c) IgG bound to FcRn receptors on the blood-brain barrier accelerates A␤ exit from the brain to periphery blood (16); and (d) binding of peripherally circulating A␤ by IgG disrupts equilibrium between the central and peripheral compartments, causing compensatory A␤ release from the brain (17). In principle, Igs that catalyze the hydrolysis of A␤ can be applied to clear A␤. We reported naturally occurring IgMs and isolated Ig light chain subunits (IgLs) that hydrolyze A␤ impede A␤ aggregation and inhibit A␤-induced neurotoxicity (18,19). However, these Igs hydrolyze A␤ slowly, and development of more efficient catalysts will help advance the use of catalytic Igs for A␤ clearance.
We report here the search for efficient A␤-hydrolyzing Ig fragments in a human IgV domain (IgV) library in which the majority of clones are single chain Fv constructs (scFv-t; a V L domain attached via a linker peptide to a V H domain). The scFv scaffold mimics the physiological structure of antigen-combining sites. A minority of clones in the library are nonphysiological V domain structures generated by repertoire cloning errors. Unexpectedly, the nonphysiological two domain and single domain IgV L fragments expressed exceptional A␤ hydrolyzing efficiency. scFv-t derivatives obtained by repairing a high activity single domain IgV L displayed reduced catalytic activity. The observations suggest that novel Ig structures freed of constraints imposed by the physiological organization of V domains can be the source of efficient catalysts to medically important antigens.

MATERIALS AND METHODS
Electrophilic Compounds-Syntheses and Ig binding characteristics of these compounds are reported: E-hapten 1 and 2 (20) and E-hapten 3 (12). Bt-E-A␤40 was prepared by reacting biotinylated A␤-(1-40) (A␤40; 10 mg, 2.1 mol) with diphenyl-N-[O-(3-sulfosuccinimidyl)suberoyl]-amino(4-amidinophenyl)methane phosphonate (10.6 mg, 12.5 mol) in DMSO. The reaction mixture was purified by RP-HPLC (Waters) and lyophilized. Its identity and purity were confirmed by RP-HPLC (retention time 36.36 min, purity Ͼ99.9%, Vydac C4 column; 0.05% trifluoroacetic acid in water, 0.05% trifluoroacetic acid in MeCN 90:10 -40:60 in 50 min, 1.0 ml/min; 220 nm absorbance) and electrospray ionization-mass spectrometry (ESI-MS; observed m/z, 1427.6, 1142. 6  Igs-Methods for IgV preparation and characterization have been described (7,21). Briefly, the library consists of 1.4 ϫ 10 7 IgVs cloned in the phagemid vector pHEN2 prepared from the peripheral blood lymphocyte of patients with lupus. A His 6 sequence and a c-myc epitope are located at the IgV C terminus. Expression levels were 1-3 mg of IgV/liter of bacterial culture, determined by anti-c-myc immunoblotting. Soluble IgVs were purified from periplasmic extracts of HB2151 cells by metal affinity chromatography. Further purification was by anion exchange FPLC (MonoQ HR 5/5 column; 0 -1 M NaCl in 50 mM Tris buffer, pH 7.4, containing 0.1 mM CHAPS). Purity was determined by SDS-gel electrophoresis and immunoblotting. IgV phages (10 12 colony-forming units) were packaged using the hyperphage method (22) and incubated (2 h, 37°C) with Bt-E-A␤40 in 0.07 ml of 10 mM sodium phosphate, 137 mM NaCl, 2.7 mM KCl, pH 7.4 (PBS). Phages with bound Bt-E-A␤40 were captured using anti-biotin antibody coupled to agarose gel (0.22 ml settled gel; Sigma) and washed with 100 ml of PBS containing 0.1% bovine serum albumin. Reversibly bound phages were eluted by incubation of the gel in 0.2 ml of 100 M A␤40 for 1 h with slow mixing, and the residual phages covalently complexed to Bt-E-A␤ were eluted with 0.4 ml of 0.1 M glycine, pH 2.7. scFv-t derivatives of single domain IgV L -tЈ 5D3 were prepared by inserting the deleted V H residues 8 -115 (Kabat numbering). For this, full-length V H cDNA was amplified by PCR using as template the IgV-pHEN2 DNA library and back/forward primers containing ApaLI/NotI restriction sites (respectively, GGTAGTGCACTTCAGGTGCAGCTGTTGC-AGTCT/ATGTGCGGCCGCGGGGAAAAGGGTTGGGGG-CATGC), and the cDNA digested with ApaLI/NotI was ligated into similarly digested plasmid IgV L -tЈ 5D3 DNA with T4 DNA ligase (Invitrogen). Full-length light chain L-tЈ 5D3 was prepared by Mutagenex by a chimeragenesis method (23) using as starting materials the V L domain of IgV L -tЈ 5D3 and human chain constant domain (obtained from pLC-huC (24)) and cloned into pHEN2 vector as the NcoI/NotI-digested fragment. cDNAs for the homodimeric IgV L2 -t form of clone 5D3 were prepared by PCR by Mutagenex. Briefly, V L cDNA was amplified by PCR from the IgV L -tЈ5D3 template using back/forward primers containing ApaLI/NotI restriction sites (respectively, AAAGTGCACTTGAAATTGTGTTGACGCAGTCTC/AAA-GCGGCCGCGCGTTTGATCTCCAGCTTGGT), and the cDNA digested with ApaLI/NotI was ligated into pHEN2 vector. The nucleotide sequence of all constructs determined by dideoxy nucleotide sequencing in the 5Ј to 3Ј and 3Ј to 5Ј directions was identical (Applied Biosystems, ABI PRISM 3100 Genetic Analyzer). Following electroporation of IgV phagemid DNA into HB2151 cells, soluble IgVs were purified from periplasmic extracts as before. Total protein was determined by the microBCA kit (Pierce). For mass spectroscopy (25), the IgV band was excised from the SDS-electrophoresis gel stained with GelCode Blue (Pierce), subjected to dehydration in 50% acetonitrile and SpeedVac drying, reduced (dithiothreitol) and alkylated (iodoacetamide), and digested with sequencing grade trypsin (Promega) and Lys-C (Wako) for 20 h at 37°C. Following extraction of gel fragments with acetonitrile/formic acid, digested peptides obtained by ZipTip C18 (Millipore) fractionation using 5 l of aqueous 50% acetonitrile containing 2% formic acid were analyzed by mass spectrometry using a matrix of ␣-cyano-4-hydroxycinnamic acid (ABI 4700 MALDI-TOF/ TOF mass spectrometer). Predicted monoisotopic peptide mass values were obtained using MS-Fit for protein data base searches (Protein Prospector, University of California, San Francisco). IgM was purified from human sera as described (18).
Hydrolysis and Binding Assays-Hydrolysis of 125 I-A␤40 was determined as described (18). Briefly, 125 I-A␤40 prepared by the chloramine-T method was purified by RP-HPLC (2.2 Ci/mol). The 125 I-A␤40 (ϳ0.1 nM, ϳ30,000 cpm/tube) was treated with IgVs in PBS containing 0.1 mM CHAPS and 0.1% (w/v) bovine serum albumin; intact peptide was separated from fragments by precipitation with trichloroacetic acid, and acidsoluble radioactivity was counted and corrected for background values in control assay tubes incubated in diluent without Ig (mean Ϯ S.D., 18 Ϯ 6%; n ϭ 7 assays). This procedure affords estimates of hydrolysis concordant with RP-HPLC separation of the reaction mixtures. Apparent kinetic parameters were estimated by fitting hydrolysis rates observed at varying A␤40 concentrations mixed with a constant amount of 125 I-A␤40 to the following equation , where V max is the maximum velocity at saturating A␤40 concentrations, and K m is the concentration at which half-maximal velocity was observed. To identify the reaction products, reaction mixtures of nonradiolabeled synthetic A␤40 or A␤42 (100 M; American Peptide Co.) incubated with IgVs in PBS/CHAPS were desalted by gel filtration (Bio-Rad micro Bio-spin 6 columns), lyophilized, and subjected to MALDI-TOF MS with ␣-cyano-4-hydroxycinnamic acid as matrix (positive ion mode, 20,000 V). RP-HPLC of A␤40-Ig reaction mixtures and ESI-MS identification of the product have been described previously (18). Hydrolysis of the amide bond linking 7-amino-4-methylcoumarin (AMC) to the C-terminal amino acid of peptide-AMC substrates (Peptides International) was measured in PBS/CHAPS buffer by fluorimetry with authentic AMC as reference (em 470 nm; ex 360 nm (12)). Hydrolysis of biotinylated proteins was determined by SDS-electrophoresis using peroxidase-conjugated streptavidin to stain blots of the gels (18). IgV covalent binding to biotinylated E-hapten 2 or E-hapten 3 was assayed in PBS/CHAPS (12). The reaction mixtures were boiled in SDS in reducing buffer (5 min) and subjected to SDS-electrophoresis, and blots of the gels were stained with the streptavidin-peroxidase conjugate. IgV binding to immobilized Bt-A␤40 was determined by enzyme-linked immunosorbent as described (18) except that anti-c-myc antibody (1:100) was employed to detect IgVs bound to immobilized antigens (26).
IgV Modeling-The two V L domains of the heterodimeric IgV L2 -t 2E6 located, respectively, on the N-and C-terminal side of the linker (designated VL1 and VL2) were initially modeled as monomers by sequence alignment to the most-homologous V L domains in the Protein Data Bank (PDB codes, respectively, 1MCB and 2BX5; 85-95% sequence identity) and homology modeling with DS 1.7 (Accelrys; modeler module followed by minimization in CHARMm force field; 1000 cycles). The VL1 and VL2 structures were then refined in dimeric form using as template the light chain dimer PDB 1MCW. The flexible interdomain linker peptide and C-terminal tag region were incorporated into the model, and minor steric clashes were removed by energy minimization using CNS 1.1 (200 cycles; see Ref. 27).
The V L domain of the single domain IgV L -tЈ 5D3 was initially modeled in its paired V L -V H scFv-t 5D3-E6 form by the WAM server. V L models in the IgV L -tЈ, IgV L2 -t, and full-length light chain L-t forms of the molecule were prepared by superimposing the V L domain from the WAM model to the coordinates of the homodimeric light chain crystal structure (PDB 1B6D; 85% sequence identity). The structures were submitted to steepestdescent energy minimization using the Adopted Basis-set Newton-Raphson method under the CHARMm force field (2000 cycles) until an r.m.s. deviation of 0.1 kcal/mol/Å was obtained. The quality of all structures was checked using PROCHECK. Percent V L residues in the final models located in the most favored or generous regions of the Ramachandran plot were as follows: IgV L2 -t 2E6, 90.8%; IgV L -tЈ 5D3, 96.5%; scFv-t 5D3-E6, 97.7%; IgV L2 -t 5D3 homodimer, 95.5%; L-t 5D3, 96.9%. No unacceptable atomic collisions were detected. The van der Waals energy was negative, suggesting the absence of bad nonbonded contacts. Superimposition and determination of global r.m.s. deviation and translational C␣-C␣ movements were performed using PyMOL (DeLano Scientific LLC). The feasibility of A␤40 interactions with IgV L2 -t 2E6 was assessed by molecular docking as described (18)

RESULTS
A␤40 Hydrolyzing IgVs-We reported previously the hydrolysis of A␤40 by polyclonal IgM purified from humans without dementia (18). Here we searched for A␤40-hydrolyzing human IgVs in a library composed of ϳ10 7 clones. A majority of the clones in the library are scFv-t constructs with the domain organization V L -Li-V H -t, where Li denotes the 16-residue peptide SS(GGGGS) 2 GGSA joining the V L domain C terminus to the V H domain N terminus, and t denotes the 26-residue C-terminal peptide containing the c-myc peptide and His 6 tags (7). A minority of clones possess unusual IgV structures generated by cloning errors (see below). Sixty three IgVs purified from the periplasmic extracts of randomly picked clones by His 6 binding to nickel affinity columns were tested for 125 I-A␤40 hydrolyzing activity. Two IgVs with activity markedly superior to the remaining clones were identified (Fig. 1A). The activity of the empty vector control extract (pHEN2 devoid of an IgV insert) was within the assay error range (50 cpm/h, corresponding to mean background acid soluble radioactivity ϩ 3 S.D.). The phagemid DNA of the high activity IgV clone 2E6 was re-expressed in 15 individual bacterial colonies. All recloned colonies secreted IgV with robust 125 I-A␤40 hydrolyzing activity (Fig.  1B), ruling out trivial sample preparation variations as the cause of proteolytic activity. As before, the purified extract of the control empty vector clone did not hydrolyze 125 I-A␤40.
In previous studies, electrophilic phosphonate groups incorporated within polypeptides were bound covalently by catalytic Ig nucleophilic sites, with noncovalent binding at the peptide epitopes conferring specificity to the reaction (21). We employed the biotinylated A␤40 analog containing phospho-nates at Lys 16 and Lys 28 side chains to isolate A␤40 catalysts displayed on phage surface (Bt-E-A␤40; Fig. 2A). Phage IgVs treated with Bt-E-A␤40 were captured using immobilized antibiotin antibody, and noncovalently bound phage IgVs were eluted by treatment with excess A␤40 (designated noncovalently selected IgVs), and the irreversible phage IgV immune complexes were eluted by acid disruption of the biotin-antibiotin antibody complexes (designated covalently selected IgVs). The frequency of IgVs with robust A␤ hydrolyzing activity was increased by covalent selection (Fig. 2B). Four of 7 IgVs obtained by covalent selection at 2 M A␤40 hydrolyzed 125 I-A␤40 at rates Ͼ400 cpm/h, compared with 2 of 63 IgVs with this level of activity identified by random screening. Phage selections conducted at increased A␤40 concentration (10 M) yielded less active IgVs (Fig. 2B), consistent with the prediction of more efficient selection of catalysts at the lower ligand concentration. Eighteen IgVs recovered by noncovalent selection displayed no or little hydrolytic activity.
IgV Primary Structure and Activity Validation-The cDNAs for IgV clone 2E6 obtained without phage selection and three covalently selected IgVs with the greatest A␤40 hydrolyzing activity (clones 5D3, 1E4, and 5H3) were sequenced. Identical nucleotide sequences were obtained for each clone sequenced from the 5Ј to 3Ј direction and the 3Ј to 5Ј direction. The cDNA sequences indicated that IgV 2E6 is a dimer of two different V L domains with the intervening linker peptide and the expected C-terminal tag (designated heterodimeric IgV L2 -t, Two clones were studied further, IgV L2 -t 2E6 and IgV L -tЈ 5D3. Their deduced protein masses predicted from the cDNA sequences are, respectively, 27 and 17 kDa. Denaturing electrophoresis of the IgVs purified by 2 cycles of nickel-affinity chromatography and anion exchange FPLC (IgV L2 -t 2E6 and IgV L -tЈ 5D3 fractions corresponding, respectively, to retention times 10 -11 and 23-23.5 min; supplemental Fig. S2) revealed silver and anti-c-myc stainable protein bands close to the predicted mass of the monomer proteins (IgV L2 -t 2E6, 29 kDa; IgV L -tЈ 5D3, 18 kDa; Fig. 3B). The presence of the c-myc peptide epitope confirms that these are IgV bands. In view of its unusual structure, the identity of IgV L -tЈ 5D3 monomer band was confirmed further by tryptic digestion and mass spectroscopy (supplemental Table S1). All observed spectroscopic signals originated from peptides within the predicted IgV L -tЈ structure deduced from the cDNA sequence, and the peptide signals were consistent with deletion of V H residues 8 -115 predicted from the cDNA sequence. Additional IgV bands were detected prior to anion exchange chromatography (low mass IgV L2 -t 2E6 band at 18 kDa; high mass IgV L -tЈ 5D3 bands at 36, 50, 58, 67, and 74 kDa). All of these bands were stained by anti-c-myc antibody (Fig. 3B). As irrelevant proteins are not stained by the antic-myc antibody, there is no evidence of non-IgV contaminants. We concluded that the anomalous low mass band is an IgV L2 -t self-degradation product, and the high mass bands are IgV L -tЈ aggregates. 125 I-A␤40 hydrolyzing activities of both IgVs remained constant after one and two cycles of nickel-affinity chro-   DECEMBER 26, 2008 • VOLUME 283 • NUMBER 52 matography but were increased following further FPLC purification that removed the degradation product and aggregates (Table 1; by 3.1-and 31.2-fold, respectively, for the IgV L2 -t and IgV L -tЈ). These observations are consistent with A␤ hydrolysis by the unaggregated IgVs. Sequencing of 24 randomly picked clones indicated that most IgVs in the library are scFv-t constructs (83.3% clones), with only rare representation of IgV L2 -t and IgV L -tЈ constructs (respectively, 12.5 and 4.2% clones; supplemental Table S2). The cumulative probability that all four A␤40-hydrolyzing IgVs identified in the present study are IgV L2 -t or IgV L -tЈ clones by random chance is very small (p ϭ 0.9 ϫ 10 Ϫ5 ; computed as 0.125 ϫ 0.042 3 ). It may be concluded that the rare IgV structures favor expression of A␤ hydrolysis compared with the physiological V L -V H paired structure of scFv-t clones. This is supported by comparisons of IgV catalytic activities with the previously reported polyclonal human IgM preparations and a monoclonal IgM from a patient with Waldenstrom's macroglobulinemia (18). IgV L2 -t 2E6 and IgV L -tЈ 5D3 hydrolyzed 125 I-A␤40 with potencies superior to the IgMs by 3-4 orders of magnitude (Fig. 3C).

Catalytic Antibody Variable Domains
Repaired IgV 5D3 Versions-The aberrant tЈ region of IgV L -tЈ 5D3 contains a deletion of V H domain residues 8 -115 (Kabat numbering). Four scFv-t constructs were generated from the IgV L -tЈ by inserting the deleted residues derived from fulllength V H domains represented in the library (supplemental Fig. S3). The repaired scFv-t 5D3 derivatives migrated at the expected mass in electrophoresis gels (30 kDa, example in Fig.  4A, inset). Their 125 I-A␤40 hydrolyzing activity was consistently lower than the parent IgV L -tЈ (by ϳ82-167-fold, computed by rate comparisons in the linear region of the hydrolysis curves; Fig. 4A). This suggests that A␤40 hydrolysis occurs at an autonomous catalytic site in the IgV L -tЈ V L domain that is suppressed by pairing with V H domains.
Intermolecular noncovalent bonding between isolated light chains can generate dimeric light chain structures (29). To assess whether noncovalently associated IgV L -tЈ dimers containing paired V L -V L structures might account for the hydrolytic activity, we prepared the homodimeric IgV L2 -t molecule containing two 5D3 V L domains connected by the peptide linker. Homodimeric IgV L2 -t 5D3 hydrolyzed 125 I-A␤40 poorly  1 and 2, respectively, SDS-electrophoresis gels of the IgV L -tЈ stained with silver and anti-c-myc antibody. Lanes 3 and 4, respectively, an example scFv-t derivative of 5D3 (clone 5D3-E6) stained with silver and anti c-myc antibody. The bands at 18 and 30 kDa are, respectively, the IgV L -tЈ and scFv-t. B, hydrolytic activity of full-length L-t 5D3 and homodimeric IgV L2 -t 5D3. 125 I-A␤40 hydrolysis assayed as in A. Inset, lanes 1 and 2, respectively, SDS-electrophoresis gels of the L-t stained with silver and anti-c-myc antibody. Lanes 3 and 4, respectively, the IgV L2 -t stained with silver and anti-c-myc antibody. The bands at 30 and 27 kDa are, respectively, the L-t and IgV L2 -t.

TABLE 1 125 I-A␤-(1-40) hydrolyzing activity of IgVs following metal affinity and anion exchange FPLC purification
Recombinant IgV preparations purified by one round of metal affinity chromatography on nickel-agarose (preparation MA1) were subjected either to a second round of metal affinity chromatography (MA2) or anion exchange chromatography (AEQ) on a Mono Q FPLC column. A␤ hydrolysis was assayed as in Fig. 2
The 125 I-A␤40 hydrolysis measurements were conducted using a small amount of the A␤40 substrate (0.1 nM) mixed with excess albumin (1 mg/ml; 15 M), which can serve as an alternate substrate for promiscuous catalysts. As hydrolysis of 125 I-A␤40 was detected readily, the IgVs do not appear to be nonspecific catalysts. In addition, there was no evidence for hydrolysis of several irrelevant biotinylated polypeptides (ovalbumin, soluble extracellular domain of the epidermal growth factor receptor, human immunodeficiency virus gp120, protein A; supplemental Fig S4B). Previous reports have identified promiscuous Igs present in human blood using model fluorigenic peptide substrates (12). IgV L2 -t 2E6 and IgV L -tЈ 5D3 failed to hydrolyze the model peptide substrate appreciably (Table 3), whereas a representative human polyclonal IgM preparation 9010 hydrolyzed large amounts of Arg/Lys-containing peptide substrates. The data indicate specific A␤ hydrolysis by the IgVs. Previous reports have indicated that proteolytic Igs utilize a serine protease-like catalytic mechanism entailing nucleophilic attack on the electrophilic carbonyl of peptide bonds (7,9). This was the basis for the covalent phage IgV selection in this study. To confirm the mechanism, we studied the reactivity of IgV L2 -t 2E6 and IgV L -tЈ 5D3 with the electrophilic phosphonate diester E-hapten-1 (Fig. 6A), a compound originally developed as a covalent inhibitor of serine proteases (30). E-hapten-1 inhibited 125 I-A␤40 hydrolysis by both clones (Fig. 6B). The biotin-containing version of the phosphonate diester,
Molecular Models-Intramolecular H-bonding between triads and dyads of amino acids enhances the nucleophilicity of certain side chains responsible for enzymatic catalysis. Examples are the hydroxyl side chains of Ser, Thr, and Tyr residues activated by spatially neighboring general bases contributed by His, Lys, Arg, Tyr, Glu, and Asp residues (31)(32)(33)(34). We screened molecular models of IgV L2 -t 2E6 and IgV L -tЈ 5D3 for side chain hydroxyls located within 4 Å of atoms that can serve as general bases as described in Ref. 31. One triad and several dyads fulfilling this requirement were found in each of the catalysts (supplemental Fig. S5 and supplemental Table S3). The presence of the potential nucleophiles is consistent with the mechanism of IgV catalysis suggested by electrophilic inhibitor studies. Several complexes containing the candidate nucleophilic residues of IgV L2 -t 2E6 apposed to Gln 15 of the major A␤40 scissile bond were evaluated by molecular modeling. Among these, the complex containing VL1 domain Thr 105 apposed to the scissile bond was the energetically most favored structure. This complex also contained various noncovalent stabilizing interactions between IgV L2 -t 2E6 and A␤40, including VL1 domain Gly 41 and Ala 43 backbone atoms hydrogen bonded with, respectively, A␤40 Asp 23 backbone and side chain atoms.
The V L domain of clone 5D3 is highly catalytic in the unpaired IgV L -tЈ state and poorly catalytic when paired with a second V domain in scFv-t or homodimeric IgV L2 -t states. Subangstrom movements of electronegative atoms can weaken or strengthen H-bonds and thereby modulate the nucleophilic and proteolytic activities (35,36). Frequent backbone displacements on the order of 0.5-1.7 Å were evident by energy minimization of the V L domain modeled in the unpaired state versus the paired scFv-t or IgV L2 -t states (Fig. 7, A and B). Spatial displacement of amino acid side chains that influence H-bonding strength and increase or decrease the nucleophilic reactivity could also occur by virtue of rotation around single bonds (see supplemental Table S3 for changes of inter-residue distances within the potential nucleophilic sites in the unpaired and paired V L domain states). The modeling results therefore suggest the feasibility of altered nucleophilic reactivity and provide a rational explanation for unequal catalysis by various V L domain-containing molecules.

DISCUSSION
Our observations indicate the superior A␤ hydrolyzing activity of V L domains expressed in the IgV L2 -t and IgV L -tЈ scaffolds compared with scFv-t constructs mimicking physiological antigen-combining sites. The catalytic activity is also strikingly superior to previously reported catalytic IgMs that contain fully natural A␤-combining sites (18). Several IgV L2 -t and IgV L -tЈ clones with exceptional A␤ hydrolyzing activity were identified from the library.   Ig light chain homodimers overproduced by cancerous B cells in multiple myeloma patients can bind certain antigens (39,40). There is no naturally occurring Ig homolog of IgV L2 -t 2E6, a heterodimer of two V L domains. Homodimeric IgV L2 -t constructs containing the individual V L domains of IgV L2 -t 2E6 were without appreciable A␤ hydrolyzing activity, suggesting that both V L domains in the heterodimer are important in maintaining the integrity of the catalytic site. Three A␤-hydrolyzing clones with the IgV L -tЈ scaffold were also identified. In energy-minimized molecular models of one such clone, the small V H domain peptide in the C-terminal segment was revealed as a disordered region without the ␤ sheet structure typical of the Ig fold. Moreover, the proximity of the V H peptide region to the V L domain was insufficient to anticipate that it contributes to A␤ recognition by the V L domain catalytic site. Noncovalent intermolecular association of the single domain IgV L -tЈ can be hypothesized to generate homodimeric V L -V Lcombining sites. However, the stable homodimeric IgV L2 -t derivative containing its two V L domains was devoid of catalytic activity, supporting attribution of catalysis to the unpaired V L domain. No natural Igs with an unpaired, functionally active V L domain are known. In extant organisms, the closest functional homolog of the unpaired catalytic V L domain are certain jawed fish and camelid Igs containing a single V H domain, which is thought to bind antigen in its unpaired state (41,42). The V L and V H domains express appreciable sequence identity with each other, and modern Igs have likely evolved by duplication and sequence diversification of a common primordial gene encoding the Ig fold (43). The phylogenic origin of Ig catalysis and deterioration or improvement of the catalytic function over the course of evolution of the immune system remains to be examined.
Electrophilic compounds that react irreversibly with the active site of serine proteases inhibited the A␤ hydrolyzing activity and formed irreversible complexes with the catalytic IgVs. This suggests a nucleophilic catalytic mechanism as deduced for other proteolytic Igs from inhibitor, mutagenesis, and crystallography studies (7,9,10). Protein nucleophilic reac-tivity is generated by intramolecular activation reactions within dyads and triads formed by precisely positioned amino acids, e.g. by hydrogen bonding between the Ser hydroxyl side chain and an imidazole nitrogen. Even small, sub-Å side chain movements can weaken the bonding and induce loss of active site nucleophilic reactivity. Noncovalent antigen binding, on the other hand, is mediated by weak and more numerous interactions at several contact residues in Ig-combining sites (1). Loss of any single contact because of a minor conformational change may weaken noncovalent antigen binding, but an abrupt transition from the binding state to a nonbinding state is less likely. Molecular modeling of the single domain IgV L -tЈ 5D3 suggested the likelihood of minor structural perturbations upon pairing the catalytic V L domain with another V domain, helping explain the poor catalytic activity of the scFv-t and homodimeric IgV L2 -t versions of the molecule. Compared with the suppressive effect of V L -V H and V L -V L pairing, the catalytic activity of the single domain V L was tolerant to inclusion of the C-terminal constant domain. This is significant, because it opens the route to inclusion of C-terminal moieties that reduce IgV clearance, e.g. polyethylene glycol or the Ig Fc fragment (44 -46). Engineering stable catalyst versions with sufficient longevity in vivo is an important goal for clinical applications. scFv-t constructs have short half-lives in peripheral blood (47), and in view of their small size, the IgVs may also be subject to rapid clearance in vivo. Another route to prolonging the lifetime of IgVs is their recloning within the physiological IgG scaffold (48). The two domain IgV L2 -t 2E6 is a heterodimeric structure that should allow development of a IgG-like structure with a combining site formed by the two V L domains.
Drugs currently employed to treat AD do not arrest the underlying pathology and progressive cognitive decline. A␤ oligomer accumulation is thought to be a major cause of neuronal death and dysfunction in the AD brain (49,50). Small amounts of peripherally infused A␤ binding monoclonal IgGs traverse the blood-brain barrier, and IgG-facilitated A␤ clearance has emerged as a novel therapeutic strategy with the potential to halt cognitive decline in AD patients (51). Reversibly binding IgGs can at best bind two antigen molecules. Large quantities of stoichiometrically binding monoclonal IgGs are usually required for immunotherapy. Catalytic Igs hold the potential of clearing A␤ efficiently by virtue of the specific A␤ degrading activity. For example, from its k cat value in Table 1, a single IgV L -tЈ 5D3 molecule is predicted to digest 4320 A␤ molecules in 3 days at excess A␤ concentration. The enzyme neprilysin has received attention as a potential A␤-clearing AD drug (52). The k cat of neprilysin for A␤ is comparable with the IgVs reported here (53). Neprilysin, however, also hydrolyzes irrelevant polypeptides (54), whereas the IgVs did not degrade FIGURE 7. Models of the V L domain of IgV L -t 5D3 (black) superimposed on the VL domains of its heterodimeric scFv-t derivative clone 5D3-E6 (red, A) and its homodimeric IgV L2 -t 5D3 derivative (red, B). C-␣ atoms belonging to amino acids with r.m.s. deviation Ͼ1.5 Å are identified, and overall r.m.s. deviations the superimposed models are indicated. DECEMBER 26, 2008 • VOLUME 283 • NUMBER 52 non-A␤ polypeptides detectably. A␤ degradation by an IgM at a substantially lower rate than the IgVs was previously reported to inhibit A␤ aggregation and A␤-induced neurotoxicity (18). The IgVs hydrolyze the His 14 -Gln 15 bond and, at lower levels, other peptide bonds located in the central A␤ region. The aggregability of various synthetic A␤ fragments is generally weaker than full-length A␤ (55)(56)(57), but the precise functional effects of IgV-catalyzed A␤ hydrolysis remain to be examined. Concerns have been raised that A␤ binding IgGs can induce inflammation (58) and vascular microhemorrhages (59,60) caused, respectively, by immune complex-stimulated release of microglial inflammatory mediators and IgG-stimulated A␤ deposition in cerebral blood vessels. The IgVs reported here do not form immune complexes detectably. They degrade A␤ permanently, minimizing the risks of inflammatory mediator release and A␤ re-deposition in the vascular wall. The recently reported phase II clinical trial of a reversibly binding anti-A␤ monoclonal IgG in patients with mild-to-moderate AD patients highlights the importance of searching for safer and more effective immunotherapeutic reagents (61). A dose-limiting incidence of vasogenic edema in magnetic resonance images was evident in this trial. At lower doses of the IgG, cognitive performance tended to improve, but the effect did not reach statistical significance in the intent-to-treat population. However, upon exclusion of patients homozygous for the apolipoprotein E4 allele, post-hoc analysis suggested significantly improved cognitive functions in the remaining patient subgroup. The apolipoprotein E4 allele is known to predispose AD patients to increased amyloid accumulation (62).

Catalytic Antibody Variable Domains
In summary, the specific and efficient A␤ degrading activity of the IgVs supports evaluation of their efficacy and safety in attaining A␤ clearance. The potential medical utility of catalytic Igs to microbial antigens and cancer-associated antigens has been discussed previously (28), but the low catalytic rate constants of physiological Igs have been a barrier to their clinical application. Our observations suggest that enhanced catalysis can be achieved by placing the V L domains within nonphysiological two domain and single domain scaffolds. If this finding proves generally applicable, development of efficient catalysts specific for other clinically important antigens should be feasible.