Sangeeta Karle

Antibodies as defensive enzymes

Antibodies (Abs) and enzymes are structural and functional relatives. Abs with promiscuous peptidase activity are ubiquitous in healthy humans, evidently derived from germline variable domain immunoglobulin genes encoding the serine protease-like nucleophilic function. Exogenous and endogenous electrophilic antigens can bind the nucleophilic sites covalently, and recent evidence suggests that immunization with such antigens can induce proteolytic antibodies. Previously, Ab catalytic activities have been linked to pathogenic autoimmune reactions, but recent studies indicate that proteolytic Abs may also serve beneficial functions. An example is the rapid and selective cleavage of the HIV-1 coat protein gp120 by IgMs found in uninfected humans. The selectivity of this reaction appears to derive from recognition of gp120 as a superantigen. A second example is the cleavage of amyloid β-peptide by IgM and IgG from aged humans, a phenomenon that may represent a specific proteolytic response to a neurotoxic endogenous peptide implicated in the pathogenesis of Alzheimer’s disease.

VIPase autoantibodies in Fas-defective mice and patients with autoimmune disease

The immunoregulatory neuropeptide vasoactive intestinal peptide (VIP) was cleaved by purified IgG from Fas‐defective C3H/gld mice, lupus patients, and autoimmune thyroiditis patients. No VIPase activity was detected in IgG from control mice and humans. Kinetic analyses of VIPase IgG preparations suggested low‐affinity recognition of VIP. Yet the VIPase activity was VIP selective, judged by lack of correlation with other protease activities expressed by the IgG and by noninterference of unrelated peptides in the activity. Recombinant Fv constructs selected from a human lupus phage show library displayed VIPase activity, confirming that the active site is located in the V domains. Inhibition of the VIPase activity by di‐isopropylfluorophosphate suggested a serine protease‐like mechanism of catalysis. Irreversible binding of a biotinyated phosphonate diester by the IgG and Fv preparations was observed, consistent with the presence of activated nucleophiles similar to those in enzymes capable of covalent catalysis. These observations show that VIP is a target for specific catalytic autoantibodies in autoimmune disease.

Prospects for immunotherapeutic proteolytic antibodies

Monoclonal antibodies are suitable for therapeutic applications by virtue of their excellent target binding characteristics (specificity, affinity) and long half-life in vivo. Catalytic antibodies (CAbs) potentially represent a new generation of therapeutics with enhanced antigen inactivation capability. Here, we describe prospects for development of therapeutic CAbs to the envelope protein gp120 of HIV. The strategy consists of exploiting the natural tendency of the immune system to synthesize germline-encoded, serine protease-like CAbs. Lupus patients were found to develop antibodies to a conserved component of the CD4 binding site of gp120, potentially offering a means to obtain human antibodies expressing broad reactivity with various HIV strains. Covalently reactive antigen analogs (CRAs) capable of selective recognition of nucleophilic Abs were synthesized and applied to isolate Fv and L chain catalysts from lupus phage repertoires. CRA binding by the recombinant Ab fragments was statistically correlated with catalytic cleavage of model peptide substrates. A peptidyl CRA composed of residues 421-431 with a phosphonate diester moiety at its C terminus was validated as a reagent that combines noncovalent and covalent binding interactions in recognition of a gp120ase L chain. A general challenge in the field is the apparent instability of the catalytic conformation of the Abs. In reference to therapy of HIV infection, assurance is required that the Abs recognize the native conformation of gp120 expressed as a trimer on the virus surface.

Covalent reactivity of phosphonate monophenyl esters with serine proteinases: an overlooked feature of presumed transition state analogs.

Phosphonate monoesters have been assumed to serve as noncovalent transition state analogs for enzymes capable of catalyzing transacylation reactions. Here, we present evidence for the covalent reaction of certain serine proteinases and peptidase antibody fragments with monophenyl amino(4-amidinophenyl)methanephosphonate derivatives. Stable adducts of the N-biotinylated monophenyl ester with trypsin and antibody fragments were evident under conditions that disrupt noncovalent interactions. The reaction was inhibited by the active-site-directed reagent diisopropyl fluorophosphate. Mass spectrometry of the fragments from monoester-labeled trypsin indicated phosphonylation of the active site. Irreversible inhibition of trypsin- and thrombin-catalyzed hydrolysis of model substrates was observed. Kinetic analysis of inactivation of trypsin by the N-benzyloxycarbonylated monoester suggested that the first-order rate constant for formation of covalent monoester adducts is comparable to that of the diester adducts (0.47 vs 2.0 min(-1)). These observations suggest that the covalent reactivity of phosphonate monoesters contributes to their interactions with serine proteinases, including certain proteolytic antibodies.

Cross-clade HIV-1 neutralization by an antibody fragment from a lupus phage display library.

A single-chain fragment containing antibody V domains (scFv) isolated from a lupus antibody library displayed the ability to bind gp120 and the conserved gp120 determinant composed of residues 421-436. The scFv neutralized R5 and X4-dependent HIV-1 strains from clades B, C, and D. The lupus repertoire may be useful as a source of neutralizing antibodies to HIV.

Carrier-dependent specificity of antibodies to a conserved peptide determinant of gp120

Amino acid residues 421-436 constitute a comparatively conserved determinant of gp120 that participates in the binding of host cell CD4 receptors by HIV-1. We compared the immunogenicity of synthetic Cys-gp120 (421-436) conjugated to KLH via the N terminal Cys residue (KLH-I) and gp120 (421-436) extended at its N terminus with a 15 residue tetanus toxoid T cell epitope (T-I) in non-autoimmune mice (BALB/cstrain) and Fas-defective autoimmune mice (MRL/lpr strain). Both immunogens elicited high titer Abs detected as the binding to gp120 (421-436) conjugated to bovine serum albumin (BSA-I) immobilized in ELISA plates. Abs from KLH-I immunized mice displayed binding to full-length gp120 but the Abs from T-I immunized mice did not. Proteins unrelated in sequence to gp120 did not bind the Abs. Soluble I and T-I failed to compete with immobilized BSA-I for binding to anti-KLH-I Abs, whereas these peptides inhibited anti-T-I Ab binding by BSA-I (rank potency order: BSA-I > T-I >> I). These results indicate the influence of the carrier protein on the specificity of Abs to synthetic I. Low level BSA-I and gp120 binding Abs were detected in sera from non-immunized MRL/lpr mice. Similar Ab binding titers and specificity profiles were evident in MRL/lpr and BALB/c mice following immunization with KLH-I and T-I, indicating that pre-existing immunity to gp120 in the former strain does not influence the magnitude or specificity of the Ab response.

A mechanism-based probe for gp120-Hydrolyzing antibodies.

An antigenic peptide analogue consisting of HIV gp120 residues 421-431 (an antigen recognition site probe) with diphenyl amino(4-amidinophenyl)methanephosphonate located at the C-terminus (a catalytic site probe) was synthesized and its trypsin and antibody reactivity characteristics were studied. Antibodies to the peptide determinant recognized the peptidyl phosphonate probe. Trypsin was inhibited equipotently by the peptidyl phosphonate and its simple phosphonate counterpart devoid of the peptide determinant. The peptidyl phosphonate inhibited the gp120-hydrolyzing activity of a catalytic antibody light chain. It was bound covalently by the light chain and the binding was inhibited by the classical active-site directed inhibitor of serine proteinase, diisopropyl fluorophosphate. These results reveal that the peptidyl phosphonate ester can serve as a probe for the antigen recognition and catalytic subsites of proteolytic antibodies.

Toward Selective Covalent Inactivation of Pathogenic Antibodies A PHOSPHONATE DIESTER ANALOG OF VASOACTIVE INTESTINAL PEPTIDE THAT INACTIVATES CATALYTIC AUTOANTIBODIES

We report the selective inactivation of proteolytic antibodies (Abs) to an autoantigen, the neuropeptide vasoactive intestinal peptide (VIP), by a covalently reactive analog (CRA) of VIP containing an electrophilic phosphonate diester at the Lys20 residue. The VIP-CRA was bound irreversibly by a monoclonal Ab that catalyzes the hydrolysis of VIP. The reaction with the VIP-CRA proceeded more rapidly than with a hapten CRA devoid of the VIP sequence. The covalent binding occurred preferentially at the light chain subunit of the Ab. Covalent VIP-CRA binding was inhibited by VIP devoid of the phosphonate diester group. These results indicate the importance of noncovalent VIP recognition in guiding Ab nucleophilic attack on the phosphonate group. Consistent with the covalent binding data, the VIP-CRA inhibited catalysis by the recombinant light chain of this Ab with potency greater than the hapten-CRA. Catalytic hydrolysis of VIP by a polyclonal VIPase autoantibody preparation that cleaves multiple peptide bonds located between residues 7 and 22 essentially was inhibited completely by the VIP-CRA, suggesting that the electrophilic phosphonate at Lys20 enjoys sufficient conformational freedom to react covalently with Abs that cleave different peptide bonds in VIP. These results suggest a novel route to antigen-specific covalent targeting of pathogenic Abs.

Antibodies to a Superantigenic Glycoprotein 120 Epitope as the Basis for Developing an HIV Vaccine

Failure to induce synthesis of neutralizing Abs to the CD4 binding determinant (CD4BD) of gp120, a central objective in HIV vaccine research, has been alternately ascribed to insufficient immunogen binding to Abs in their germline V region configuration expressed as BCRs, insufficient adaptive mutations in Ab V regions, and conformational instability of gp120. We employed peptide analogs of gp120 residues 421–433 within the CD4BD (CD4BDcore) to identify Abs produced without prior exposure to HIV (constitutive Abs). The CD4BDcore peptide was recognized by single-chain Fv fragments from noninfected humans with lupus that neutralized genetically diverse strains belonging to various HIV subtypes. Replacing the framework region (FR) of a VH4-family single-chain Fv with the corresponding VH3-family FRs from single-chain Fv JL427 improved the CD4BDcore peptide-binding activity, suggesting a CD4BDcore binding site outside the pocket formed by the CDRs. Replacement mutations in the FR site vicinity suggested the potential for adaptive improvement. A very small subset of serum CD4BDcore-specific serum IgAs from noninfected humans without autoimmune disease isolated by epitope-specific chromatography neutralized the virus potently. A CD4BDcore-specific, HIV neutralizing murine IgM with H and L chain V regions (VH and VL regions) free of immunogen-driven somatic mutations was induced by immunization with a CD4BDcore peptide analog containing an electrophilic group that binds B cells covalently. The studies indicate broad and potent HIV neutralization by constitutive Abs as an innate, germline-encoded activity directed to the superantigenic CD4BDcore epitope that is available for amplification for vaccination against HIV.

Synthesis of a Covalently Reactive Antigen Analog Derived from a Conserved Sequence of HIV-1 gp120

Antibodies (Abs) and their light (L) chain subunits are reported to catalyze the cleavage of VIP, the HIV coat proteins gp41 and gpl20, Arg-vasopressin, thyroglobulin, factor VIII, prothrombin and various model peptidase substrates. The serine protease inhibitor diisopropylfluorophosphate (DFP) consistently inhibits the catalytic activity of the Abs, and a serine protease-like catalytic triad in a model proteolytic Ab L chain has previously been deduced from site-directed mutagenesis studies. The catalytic site appears to be germline encoded [1]. In principle, probes that can specifically recognize the active site of serine protease Abs could be applied for the selection of efficient catalytic Abs (CAbs) from display libraries, and perhaps also as the immunogens capable of recruiting the germline encoded Ab variable region genes.