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Abstract: Antibodies (Abs) with proteolytic and other catalytic activities have been characterized in the blood and mucosal secretions of humans and experimental animals. The catalytic activity can be traced to nucleophilic sites of innate origin located in Ab germline variable regions. Discoveries of the natural chemical reactivity of Abs were initially met with bewilderment, as the notion had taken hold that catalytic activities can be introduced into Abs by artificial means, but somatically operative selection pressures are designed only to adapt non-covalent Ab binding to antigen ground states. Unsurprisingly, initial efforts to engineer Abs with catalytic activity were oriented towards improving the non-covalent binding at the atoms immediately within the transition state reaction center. Slowly, however, dogmatic approaches to Ab catalysis have given way to the realization that efficient and specific catalytic Abs can be prepared by improving the natural nucleophilic reactivity combined with non-covalent recognition of epitope regions remote from the reaction center. The field remains beset, however, with controversy. This article attempts to provide a rational basis for natural Ab catalysis, in the hope that understanding this phenomenon will stimulate medical and basic science advances in the field. [Copyright &y& Elsevier]

We report the selective catalytic cleavage of the HIV coat protein gp120, a B cell superantigen, by IgM antibodies (Abs) from uninfected humans and mice that had not been previously exposed to gp120. The rate of IgM-catalyzed gp120 cleavage was greater than of other polypeptide substrates, including the bacterial superantigen protein A. The kinetic parameters of gp120 cleavage varied over a broad range depending on the source of the IgMs, and turnover numbers as great as 2.1/min were observed, suggesting that different Abs possess distinct gp120 recognition properties. IgG Abs failed to cleave gp120 detectably. The Fab fragment of a monoclonal IgM cleaved gp120, suggesting that the catalytic activity belongs to the antibody combining site. The electrophoretic profile of gp120 incubated with a monoclonal human IgM suggested hydrolysis at several sites. One of the cleavage sites was identified as the Lys432-Ala433 peptide bond, located within the region thought to be the Ab-recognizable superantigenic determinant. A covalently reactive peptide analog (CRA) corresponding to gp120 residues 421–431 with a C-terminal amidino phosphonate diester mimetic of the Lys432-Ala433 bond was employed to probe IgM nucleophilic reactivity. The peptidyl CRA inhibited the IgM-catalyzed cleavage of gp120 and formed covalent IgM adducts at levels exceeding a control hapten CRA devoid of the peptide sequence. These observations suggest that IgMs can selectively cleave gp120 by a nucleophilic mechanism and raise the possibility of their role as defense enzymes. [ABSTRACT FROM AUTHOR]

Examines the natural nucleophilic activity of antibodies for the purpose of specific cleavage of the human immunodeficiency virus-1 coat protein gp120. Enzyme-linked immunoabsorbent assay; Nucleophilic reactivity; Proteolysis.

Catalytic antibodies (CAbs) occur naturally in healthy individuals where they may form part of the innate immune system, but are preferentially found in those with autoimmune disease. CAbs can also be artificially engineered or elicited by immunizations. Their mechanisms of action include nucleophilic catalysis, induction of conformational strain, coordination with metal ions, and stabilization of transition states. Recent applications of CAbs with clinical significance include the conversion of cocaine to a non-psychoactive form, the degradation of nicotine, activation of prodrugs for targeted chemotherapy, protection from ultraviolet radiation, inhibition of HIV infectivity, and the destruction of aggregates of β-amyloid implicated in Alzheimer''s disease. Artificial CAbs are likely to find increasing applications in research, clinical medicine, diagnostics and manufacturing. [Copyright &y& Elsevier]

Classical immunization methods do not generate catalytic antibodies (catabodies), but recent findings suggest that the innate antibody repertoire is a rich catabody source. We describe the specificity and amyloid β (Aβ)-clearing effect of a catabody construct engineered from innate immunity principles. The catabody recognized the AβC terminus noncovalently and hydrolyzed Aβ rapidly, with no reactivity to the Aβ precursor protein, transthyretin amyloid aggregates, or irrelevant proteins containing the catabody-sensitive Aβ dipeptide unit. The catabody dissolved preformed Aβ aggregates and inhibited Aβ aggregation more potently than an Aβ-binding IgG. Intravenous catabody treatment reduced brain Aβ deposits in amouse Alzheimer disease model without inducing microgliosis or microhemorrhages. Specific Aβ hydrolysis appears to be an innate immune function that could be applied for therapeutic Aβ removal. [ABSTRACT FROM AUTHOR]

Peptide bond-hydrolyzing catalytic antibodies (catabodies) could degrade toxic proteins, but acquired immunity principles have not provided evidence for beneficial catabodies. Transthyretin (TTR) forms misfolded β-sheet aggregates responsible for age-associated amyloidosis. We describe nucleophilic catabodies from healthy humans without amyloidosis that degraded misfolded TTR (misTTR) without reactivity to the physiological tetramericTTR(phyTTR). IgM classBcell receptors specifically recognized the electrophilic analog of misTTR but not phyTTR. IgM but not IgG class antibodies hydrolyzed the particulate and soluble misTTR species. No misTTR-IgM binding was detected. The IgMs accounted for essentially all of the misTTR hydrolytic activity of unfractionated human serum. The IgMs did not degrade non-amyloidogenic, non-superantigenic proteins. Individual monoclonal IgMs (mIgMs) expressed variable misTTR hydrolytic rates and differing oligoreactivity directed to amyloid β peptide and microbial superantigen proteins. A subset of the mIgMs was monoreactive for misTTR. Excess misTTR was dissolved by a hydrolytic mIgM. The studies reveal a novel antibody property, the innate ability of IgMs to selectively degrade and dissolve toxic misTTR species as a first line immune function. [ABSTRACT FROM AUTHOR]

Some antibodies contain variable (V) domain catalytic sites. We report the superior amide and peptide bond-hydrolyzing activity of the same heavy and light chain V domains expressed in the IgM constant domain scaffold compared with the IgG scaffold. The superior catalytic activity of recombinant IgM was evident using two substrates, a small model peptide that is hydrolyzed without involvement of high affinity epitope binding, and HIV gp120, which is recognized specifically by noncovalent means prior to the hydrolytic reaction. The catalytic activity was inhibited by an electrophilic phosphonate diester, consistent with a nucleophilic catalytic mechanism. All 13 monoclonal IgMs tested displayed robust hydrolytic activities varying over a 91-fold range, consistent with expression of the catalytic functions at distinct levels by different V domains. The catalytic activity of polyclonal IgM was superior to polyclonal IgG from the same sera, indicating that on average IgMs express the catalytic function at levels greater than IgGs. The findings indicate a favorable effect of the remote IgM constant domain scaffold on the integrity of the V-domain catalytic site and provide a structural basis for conceiving antibody catalysis as a first line immune function expressed at high levels prior to development of mature IgG class antibodies. [ABSTRACT FROM AUTHOR]

Antibodies that recognize microbial B lymphocyte superantigenic epitopes are produced constitutively with no requirement for adaptive immune maturation. We report cleavage of the Staphylococcus aureus virulence factor extracellular fibrinogen-binding protein (Efb) by catalytic antibodies produced with no exposure to the bacterium and reduction of the catalytic antibody activity following infection. IgG catalytic antibodies that specifically hydrolyzed Efb via a nucleophilic catalytic mechanism were found in the blood of healthy humans and aseptic mice free of S. aureus infection. IgG hydrolyzed peptide bonds on the C-terminal side of basic amino acids, including a bond located within the C3b-binding domain of Efb. Efb digested with the IgG lost its ability to bind C3b and inhibit complement-dependent antibody-mediated red blood cell lysis. In addition to catalysis, the IgG expressed saturable Efb binding activity. IgG from S. aureus-infected mice displayed reduced Efb cleaving activity and increased Efb binding activity compared with uninfected controls, suggesting differing effects of the infection on the antibody subsets responsible for the two activities. IgG from children hospitalized for S. aureus infection also displayed reduced Efb cleavage compared with healthy children. These data suggest a potential defense function for constitutively produced catalytic antibodies to a putative superantigenic site of Efb, but an adaptive catalytic response appears to be proscribed. [ABSTRACT FROM AUTHOR]

We describe murine monoclonal antibodies (mAbs) raised by immunization with an electrophilic gp120 analog (E-gp120) expressing the rare ability to neutralize genetically heterologous human immunodeficiency virus (HIV) strains. Unlike gp120, E-gp120 formed covalent oligomers. The reactivity of gp120 and E-gp120 with mAbs to reference neutralizing epitopes was markedly different, indicating their divergent structures. Epitope mapping with synthetic peptides and electrophilic peptide analogs indicated binary recognition of two distinct gp120 regions by anti-E-gp120 mAbs, the 421-433 and 288-306 peptide regions. Univalent Fab and single chain Fv fragments expressed the ability to recognize both peptides. X-ray crystallography of an anti-E-gp120 Fab fragment revealed two neighboring cavities, the typical antigen-binding cavity formed by the complementarity determining regions (CDRs) and another cavity dominated by antibody heavy chain variable (VH) domain framework (FR) residues. Substitution of the FR cavity VH Lys-19 residue by an Ala residue resulted in attenuated binding of the 421-433 region peptide probe. The CDRs and VH FR replacement/silent mutation ratios exceeded the ratio for a random mutation process, suggesting adaptive development of both putative binding sites. All mAbs studied were derived from VH1 family genes, suggesting biased recruitment of the V gene germ line repertoire by E-gp 120. The conserved 421- 433 region of gp120 is essential for HIV binding to host CD4 receptors. This region is recognized weakly by the FR of antibodies produced without exposure to HIV, but it usually fails to induce adaptive synthesis of neutralizing antibodies. We present models accounting for improved CD4-binding site recognition and broad HIV neutralizing activity of the mAbs, long sought goals in HIV vaccine development. [ABSTRACT FROM AUTHOR]

The antigen recognition site of antibodies consists of the heavy and light chain variable domains (VL and VH domains). VL domains catalyze peptide bond hydrolysis independent of VH domains (Mei, S., Mody, B., Ekiund, S. H., and Paul, S. (1991) I. Biol. Chem. 266, 15571-15574). VH domains bind antigens noncovalently independent of VL domains (Ward, E. S., Güssow, D., Griffiths, A. D., Jones, P. T., and Winter, G. (1989) Nature 341, 544-546). We describe specific hydrolysis of fusion proteins of the hepatitis C virus E2 protein with glutathione S-transferase (GST-E2) or FLAG peptide (FLAG-E2) by antibodies containing the VH domain of an anti-E2 IgG paired with promiscuously catalytic VL domains. The hybrid IgG hydrolyzed the E2 fusion proteins more rapidly than the unpaired light chain. An active site-directed inhibitor of serine proteases inhibited the proteolytic activity of the hybrid IgG, indicating a serine protease mechanism. The hybrid IgG displayed noncovalent E2 binding in enzyme-linked immunosorbent assay tests. Immunoblotting studies suggested hydrolysis of FLAG-E2 at a bond within E2 located ∼11 kDa from the N terminus. GST-E2 was hydrolyzed by the hybrid lgG at bonds in the GST tag. The differing cleavage pattern of FLAG-E2 and GST-E2 can be explained by the split-site model of catalysis, in which conformational differences in the E2 fusion protein substrates position alternate peptide bonds in register with the antibody catalytic subsite despite a common noncovalent binding mechanism. These studies provide proof-of-principle that the catalytic activity of a light chain can be rendered antigen-specific by pairing with a noncovalently binding heavy chain subunit. [ABSTRACT FROM AUTHOR]

Nucleophilic sites in the paired variable domains of the light and heavy chains (V[subL] and V[subH] 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 V[subL]-V[subH]-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 His[sup14]-Gln[sup15]. 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 V[subL] (IgV[subL2]-t) and single domain V[subL] clones with aberrant polypeptide tags (IgV[subL]-t'). The IgVs hydrolyzed Aβ at rates superior to naturally occurring Igs by 3-4 orders of magnitude. Forced pairing of the single domain V[subL] with V[subH] or V[subL] domains resulted in reduced Aβ hydrolysis, suggesting catalysis by the unpaired V[subL] domain. Angstrom level amino acid displacements evident in molecular models of the two domain and unpaired V[subL] domain clones explain alterations of catalytic activity. In view of their superior catalytic activity, the V[subL] domain IgVs may help attain clearance of medically important antigens more efficiently than natural Igs. [ABSTRACT FROM AUTHOR]

Abstract: Immunoglobulins (Igs) in uninfected humans recognize residues 421–433 located in the B cell superantigenic site (SAg) of the HIV envelope protein gp120 and catalyze its hydrolysis by a serine protease-like mechanism. The catalytic activity is encoded by germline Ig variable (V) region genes, and is expressed at robust levels by IgMs and IgAs but poorly by IgGs. Mucosal IgAs are highly catalytic and neutralize HIV, suggesting that they constitute a first line of defense against HIV. Lupus patients produce the Igs at enhanced levels. Homology of the 421–433 region with an endogenous retroviral sequence and a bacterial protein may provide clues about the antigen driving anti-SAg synthesis in lupus patients and uninfected subjects. The potency and breadth of HIV neutralization revives hopes of clinical application of catalytic anti-421–433 Igs as immunotherapeutic and topical microbicide reagents. Adaptive improvement of anti-SAg catalytic Igs in HIV infected subjects is not customary. Further study of the properties of the naturally occurring anti-SAg catalytic Igs should provide valuable guidance in designing a prophylactic vaccine that amplifies protective catalytic immunity to HIV. [Copyright &y& Elsevier]

The antigen-binding sites of antibodies (Abs) can express enzyme-like nucleophiles that react covalently with electrophilic compounds. We examined the irreversible and specific inactivation of antibodies (Abs) to Factor VIII (FVIII) responsible for failure of FVIII replacement therapy in hemophilia A (HA) patients. Electrophilic analogs of FVIII (E-FVIII) and its C2 domain (E-C2) were prepared by placing the strongly electrophilic phosphonate groups at surface-exposed Lys side chains of diverse antigenic epitopes. IgG Abs to FVIII from HA patients formed stable immune complexes with E-FVIII and E-C2 that were refractory to dissociation by SDS treatment and boiling, procedures that dissociate noncovalent Ab-antigen complexes. The rate-limiting step in the reaction was formation of the initial noncovalent complexes. Conversion of the initial complexes to the irreversible state occurred rapidly. The antigenic epitopes of E-FVIII were largely intact, and most of the Abs were consumed covalently. E-FVIII expressed poor FVIII cofactor activity in clotting factor assays. Nonspecific interference by E-FVIII in clotting factor function was not evident. Treatment with E-FVIII, and to a lesser extent E-C2, irreversibly relieved the FVIII inhibitory effect of HA IgG in clotting factor assays. Small FVIII peptides did not display useful reactivity, highlighting the diverse epitope specificities of the Abs and the conformational character of FVIII epitopes. E-FVIII is a prototype reagent able to attain irreversible and specific inactivation of pathogenic Abs. [ABSTRACT FROM AUTHOR]

Abstract: Immunoglobulins (Igs) that bind amyloid β peptide (Aβ) are under clinical trials for immunotherapy of Alzheimer disease (AD). We have identified IgMs and recombinant Ig fragments that hydrolyze Aβ. Hydrolysis of peripheral Aβ by the IgMs may induce increased Aβ release from the brain. The catalytic IgMs are increased in AD patients, presumably reflecting a protective autoimmune response. Reduced Aβ aggregation and neurotoxicity attributable to the catalytic function were evident. These findings provide a foundation for development of catalytic Igs for AD immunotherapy. [Copyright &y& Elsevier]

We describe IgM class human autoantibodies that hydrolyze amyloid β peptide 1-40 (Aβ40). A monoclonal IgM from a patient with Waldenström's macroglobulinemia hydrolyzed Aβ340 at the Lys-28-Gly-29 bond and Lys-16-Ala-17 bonds. The catalytic activity was inhibited stoichiometrically by an electrophilic serine protease inhibitor. Treatment with the catalytic IgM blocked the aggregation and toxicity of Aβ40 in neuronal cell cultures. IgMs purified from the sera of patients with Alzheimer disease (AD) hydrolyzed Aβ40 at rates superior to IgMs from age-matched humans without dementia. IgMs from non-elderly humans expressed the least catalytic activity. The reaction rate was sufficient to afford appreciable degradation at physiological Aβ and IgM concentrations found in peripheral circulation. Increased Aβ concentrations in the AD brain are thought to induce neurodegenerative effects. Peripheral administration of Aβ binding antibodies has been suggested as a potential treatment of AD. Our results suggest that catalytic IgM autoantibodies can help clear Aβ, and they open the possibility of using catalytic Abs for AD immunotherapy. [ABSTRACT FROM AUTHOR]

Rare monoclonal antibodies (Abs) can form irreversible complexes with antigens by enzyme-like covalent nucleophile-electrophile pairing. To determine the feasibility of applying irreversible antigen inactivation by Abs as the basis of vaccination against microbes, we studied the polyclonal nucleophilic Ab response induced by the electrophilic analog of a synthetic peptide corresponding to the principal neutralizing determinant (PND) of human immunodeficiency virus type-1 (HIV) gp120 located in the V3 domain. Abs from mice immunized with the PND analog containing electrophilic phosphonates (E-PND) neutralized a homologous HIV strain (MN) ∼50-fold more potently than control Abs from mice immunized with PND. The IgG fractions displayed binding to intact HIV particles. HIV complexes formed by anti-E-PND IgG dissociated noticeably more slowly than the complexes formed by anti-PND IgG. The slower dissociation kinetics are predicted to maintain long-lasting blockade of host cell receptor recognition by gp120. Pre-treatment of the anti-PND IgG with a haptenic electrophilic phosphonate compound resulted in more rapid dissociation of the HIV-IgG complexes, consistent with the hypothesis that enhanced Ab nucleophilic reactivity induced by electrophilic immunization imparts irreversible character to the complexes. These results suggest that electrophilic immunization induces a sufficiently robust nucleophilic Ab response to enhance the anti-microbial efficacy of candidate polypeptide vaccines. [ABSTRACT FROM AUTHOR]

Abstract: Antibodies (Abs) to the superantigenic determinant of HIV gp120 (gp120SAg) are potential protective agents against HIV infection. We report that the light chain subunits of Abs cloned from lupus patients using phage library methods bind and hydrolyze gp120SAg independent of the heavy chain. Unlike frequent gp120SAg recognition by intact Abs attributable to VH domain structural elements, the isolated light chains expressed this activity rarely. Four light chains capable of gp120SAg recognition were identified by fractionating phage displayed light chains using peptide probes containing gp120 residues 421–433, a gp120SAg component. Three light chains expressed non-covalent gp120SAg binding and one expressed gp120SAg hydrolyzing activity. The hydrolytic light chain was isolated by covalent phage fractionation using an electrophilic analog of residues 421–433. This light chain hydrolyzed a reporter gp120SAg substrate and full-length gp120. Other peptide substrates and proteins were hydrolyzed at lower rates or not at all. Consistent with the expected nucleophilic mechanism of hydrolysis, the light chain reacted selectively and covalently with the electrophilic gp120SAg peptide analog. The hydrolytic reaction entailed a fast initial step followed by a slower rate limiting step, suggesting rapid substrate acylation and slow deacylation. All four gp120SAg-recognizing light chains contained sequence diversifications relative to their germline gene counterparts. These observations indicate that in rare instances, the light chain subunit can bind and hydrolyze gp120SAg without the participation of the heavy chain. The pairing of such light chains with heavy chains capable of gp120SAg recognition represents a potential mechanism for generating protective Abs with enhanced HIV binding strength and anti-viral proteolytic activity. [Copyright &y& Elsevier]

A panel of human monoclonal and recombinant antibody light chains was screened for cleavage of the synthetic peptide corresponding to a neutralizing epitope of hepatitis C virus (residues 192–205 of envelope glycoprotein E1). One of the 39 light chains studied hydrolyzed the Val197–Ser198 bond of the peptide with Km and kcat values of 223 ± 7 μM and 0.087 ± 0.001 min-1. [Copyright &y& Elsevier]

We report the chemical activity of immunoglobulin μ and κ/λ subunits expressed on the surface of B cells and in secreted IgM antibodies (Abs) found in the preimmune repertoire. Most of the nucleophilic reactivity of B cells measured by formation of covalent adducts of a hapten amidino phosphonate diester was attributed to and κ/λ subunits of the B cell receptor. Secreted IgM Abs displayed superior nucleophilic reactivity than IgG Abs. IgM Abs catalyzed the cleavage of model peptide substrates at rates up to 344-fold greater than IgG Abs. Catalytic activities were observed in polyclonal IgM Abs from immunologically naive mice and humans without immunological disease, as well as monoclonal IgM Abs to unrelated antigens. Comparison of several IgM Abs indicated divergent activity levels and substrate preferences, with the common requirement of a basic residue flanking the cleavage site. Fab fragments of a monoclonal IgM Ab expressed catalytic activity, confirming the V domain location of the catalytic site. The catalytic reaction was inhibited by the covalently reactive hapten probe and diisopropylfluorophosphate, suggesting a setine protease-like mechanism. These observations indicate the existence of serine protease-like BCRs and secreted IgM Abs as innate immunity components with potential roles in B cell development and Ab effector functions. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Examines the chemical reactivity of natural antibodies expressed in coordination with the specific antigen binding activity. Enzyme-linked immunoabsorbent assay; Covalently reactive antigen analog; Proteolysis assay.

Autoantibodies capable of binding the immunoregulatory neuropeptide vasoactive intestinal peptide (VIP) were detected in the sera of a mouse strain prone to autoimmune disease due to the lpr mutation (MRL/lpr). The autoantibodies were not present in control wildtype MRL/lpr mice, but they were readily detected in humans without autoimmune disease. The binding was due to low affinity VIP recognition. Increased VIP binding activity was evident in patients with systemic lupus erythematosus but not systemic sclerosis, Sjo¨gren’s syndrome (SS), rheumatoid arthritis or autoimmune thyroiditis. Recombinant VIP binding Fv clones (fragment variable; the variable domains of the light and heavy chains antibody subunits joined with a peptide linker) were isolated from a phage display library prepared from lupus patients. One Fv clone displaying VIP-selective binding and several clones displaying cross-reactivity with unrelated peptides were identified. Replacement mutations in the VIP-selective clone were preferentially localized in the regions known to make contacts with the antigen, i.e. the complementarity determining regions, suggesting that the selective binding activity is due to immunological maturation of the antibodies. Frequent occurrences of autoantibody responses to VIP indicate that immunological tolerance to this neuropeptide can be readily broken. The depletion of VIP by specific antibodies in autoimmune disease may interfere with VIP regulation of T cells and inflammatory cells and result in further amplification of autoreactive immunological responses. [Copyright &y& Elsevier]

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. [Copyright &y& Elsevier]

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. [Copyright &y& Elsevier]