Catalytic antibody (catabody) platform for age-associated amyloid disease: From Heisenberg's uncertainty principle to the verge of medical interventions.

Quantum mechanics-based design of useful catalytic antibodies (catabodies) failed because of the uncertain structure of the dynamic catalyst-substrate complex. The Catabody Platform emerged from discovery of beneficial germline gene catabodies that hydrolyzed self-proteins by transient covalent pairing of the strong catabody nucleophile with a weak target protein electrophile. Catabodies have evolved by Darwinian natural selection for protection against misfolded self-proteins that threatened survival by causing amyloid disease. Ancient antibody scaffolds upregulate the catalytic activity of the antibody variable (V) domains. Healthy humans universally produce beneficial catabodies specific for at least 3 misfolded self-proteins, transthyretin, amyloid ß peptide and tau protein. Catabody are superior to ordinary antibodies because of catalyst reuse for thousands of target destruction cycles with little or no risk of causing inflammation, a must for non-toxic removal of abundant targets such as amyloids. Library mining with electrophilic target analogs (ETAs) isolates therapy-grade catabodies (fast, specific). Ex vivo- and in vivo-verified catabodies specific for the misfolded protein are available to dissolve brain, cardiac and vertebral amyloids. Immunization with ETAs overcomes important ordinary vaccine limitations (no catabody induction, poor immunogenicity of key target epitopes). We conceive electrophilic longevity vaccines that can induce catabody synthesis for long-lasting protection against amyloid disease.

Hydrolysis and Dissolution of Amyloids by Catabodies.

Catalytic antibodies (catabodies) hold potential for superior immunotherapy because of their turnover capability and no or minimal induction of inflammatory responses. Catabodies neutralize and remove target antigens more potently than conventional antibodies. Depending on the catalytic rate constant, a single catabody molecule degrades thousands to millions of target molecules over its useful lifespan, whereas conventional antibodies only form reversibly associated, stoichiometric complexes with the target. Thus, removal of the antibody-bound target requires accessory phagocytic cells that ingest the immune complexes, which is usually accompanied by release of inflammatory mediators. In comparison, catabodies bind the target only transiently, and the rapid and direct target destruction reduces the concentration of immune complexes that can activate inflammatory processes. These features are especially pertinent when large target amounts at anatomically vulnerable sites must be removed, e.g., amyloids. We reported specific catabodies to misfolded transthyretin (misTTR) amyloid and amyloid ß peptide (Aß). Accumulation of the oligomeric and fibrillized amyloid TTR forms causes diverse systemic pathologies, including cardiomyopathy, polyneuropathy, and skeletal diseases. Brain Aß aggregates are thought to cause central nervous system degenerative disease, chiefly Alzheimer's disease. We describe methods for testing catabody-mediated degradation and dissolution of Aß and TTR.

Specific amyloid ß clearance by a catalytic antibody construct.

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 ß (Aß)-clearing effect of a catabody construct engineered from innate immunity principles. The catabody recognized the Aß C terminus noncovalently and hydrolyzed Aß rapidly, with no reactivity to the Aß precursor protein, transthyretin amyloid aggregates, or irrelevant proteins containing the catabody-sensitive Aß dipeptide unit. The catabody dissolved preformed Aß aggregates and inhibited Aß aggregation more potently than an Aß-binding IgG. Intravenous catabody treatment reduced brain Aß deposits in a mouse Alzheimer disease model without inducing microgliosis or microhemorrhages. Specific Aß hydrolysis appears to be an innate immune function that could be applied for therapeutic Aß removal.

Catalytic immunoglobulin gene delivery in a mouse model of Alzheimer's disease: prophylactic and therapeutic applications.

Accumulation of amyloid beta-peptide (Aß) in the brain is hypothesized to be a causal event leading to dementia in Alzheimer's disease (AD). Aß vaccination removes Aß deposits from the brain. Aß immunotherapy, however, may cause T cell- and/or Fc-receptor-mediated brain inflammation and relocate parenchymal Aß deposits to blood vessels leading to cerebral hemorrhages. Because catalytic antibodies do not form stable immune complexes and Aß fragments produced by catalytic antibodies are less likely to form aggregates, Aß-specific catalytic antibodies may have safer therapeutic profiles than reversibly-binding anti-Aß antibodies. Additionally, catalytic antibodies may remove Aß more efficiently than binding antibodies because a single catalytic antibody can hydrolyze thousands of Aß molecules. We previously isolated Aß-specific catalytic antibody, IgVL5D3, with strong Aß-hydrolyzing activity. Here, we evaluated the prophylactic and therapeutic efficacy of brain-targeted IgVL5D3 gene delivery via recombinant adeno-associated virus serotype 9 (rAAV9) in an AD mouse model. One single injection of rAAV9-IgVL5D3 into the right ventricle of AD model mice yielded widespread, high expression of IgVL5D3 in the unilateral hemisphere. IgVL5D3 expression was readily detectable in the contralateral hemisphere but to a much lesser extent. IgVL5D3 expression was also confirmed in the cerebrospinal fluid. Prophylactic and therapeutic injection of rAAV9-IgVL5D3 reduced Aß load in the ipsilateral hippocampus of AD model mice. No evidence of hemorrhages, increased vascular amyloid deposits, increased proinflammatory cytokines, or infiltrating T-cells in the brains was found in the experimental animals. AAV9-mediated anti-Aß catalytic antibody brain delivery can be prophylactic and therapeutic options for AD.

Deficient synthesis of class-switched, HIV-neutralizing antibodies to the CD4 binding site and correction by electrophilic gp120 immunogen.

OBJECTIVE: HIV is vulnerable to antibodies that recognize a linear CD4 binding site epitope of gp120 (C), but inducing C-directed antibody synthesis by traditional vaccine principles is difficult. We wished to understand the basis for deficient C-directed antibody synthesis and validate correction of the deficiency by an electrophilic gp120 analog (E-gp120) immunogen that binds B-cell receptors covalently. METHODS: Serum antibody responses to a C peptide and full-length gp120 epitopes induced by HIV infection in humans and immunization of mice with gp120 or E-gp120 were monitored. HIV neutralization by monoclonal and variable domain-swapped antibodies was determined from tissue culture and humanized mouse infection assays. RESULTS: We describe deficient C-directed IgG but not IgM antibodies in HIV-infected patients and mice immunized with gp120 accompanied by robust synthesis of IgGs to the immunodominant gp120 epitopes. Immunization with the E-gp120 corrected the deficient C-directed IgG synthesis without overall increased immunogenicity of the C or other gp120 epitopes. E-gp120-induced monoclonal IgGs neutralized diverse HIV strains heterologous to the immunogen. A C-directed IgG neutralized HIV more potently compared to its larger IgM counterpart containing the same variable domains, suggesting obstructed access to HIV surface-expressed C. An E-gp120-induced IgG suppressed HIV infection in humanized mice, validating the tissue culture neutralizing activity. CONCLUSION: A C-selective physiological defect of IgM→IgG class-switch recombination (CSR) or restricted post-CSR B-cell development limits the functional utility of the humoral immune response to gp120. The E-gp120 immunogen is useful to bypass the restriction and induce broadly neutralizing C-directed IgGs (see Supplemental Video Abstract, http://links.lww.com/QAD/A551).

Metal-dependent amyloid ß-degrading catalytic antibody construct.

Catalytic antibodies (catabodies) that degrade target antigens rapidly are rare. We describe the metal-dependence of catabody construct 2E6, an engineered heterodimer of immunoglobulin light chain variable domains that hydrolyzes amyloid ß peptides (Aß) specifically. In addition to the electrophilic phosphonate inhibitor of serine proteases, the metal chelators ethylenediaminetetraacetic acid (EDTA) and 1,10-phenanthroline completely inhibited the hydrolysis of Aß by catabody 2E6. Formation of catabody-electrophilic phosphonate inhibitor adducts was unaffected by EDTA, suggesting that the metal exerts a favorable effect on a catalytic step after the initial catabody nucleophilic attack on Aß. The EDTA inactivated catabody failed to disaggregate fibrillar Aß, indicating the functional importance of the Aß hydrolytic activity. Treating the EDTA-inactivated catabody with Zn(2+) or Co(2+) restored the Aß hydrolytic activity, and Zn(2+)-induced catabody conformational transitions were evident by fluorescence emission spectroscopy. The studies reveal the absolute catabody dependence on a metal cofactor.

Physiological IgM class catalytic antibodies selective for transthyretin amyloid.

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 ß-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 tetrameric TTR (phyTTR). IgM class B cell 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 ß 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.

Transthyretin aggregate-specific antibodies recognize cryptic epitopes on patient-derived amyloid fibrils.

Amyloidosis involves the extracellular deposition of proteinaceous amyloid fibrils and accessory molecules in organ(s) and/or tissue(s), and is associated with a host of human diseases, including Alzheimer disease, diabetes, and heart disease. Unfortunately, the amyloidoses are currently incurable, and there is an urgent need for less invasive diagnostics. To address this, we have generated 22 monoclonal antibodies (mAbs) against aggregates formed by a blood transport protein, transthyretin (TTR), which primarily forms amyloid fibrils in a patient's heart and/or peripheral nerves. Four of the mAbs, 2T5C9, 2G9C, T1F11, and TB2H7, demonstrated diagnostic potential in enzyme-linked immunosorbent assays (ELISA) by their low to sub-nanomolar cross-reactivity with recombinant wild-type (WT) and mutant TTR aggregates and lack of binding to native TTR or amyloid fibrils formed by other peptides or proteins. Notably, in the presence of normal human sera, three of the four mAbs, 2T5C9, 2G9C, and T1F11, retained low nM binding to TTR amyloid fibrils derived from two patients with familial amyloidotic polyneuropathy (FAP). The two most promising mAbs, 2T5C9 and 2G9C, were also shown by immunohistochemistry to have low nM binding to TTR amyloid deposits in cardiac tissue sections from two FAP patients. Taken together, these findings strongly support further investigations on the diagnostic utility of TTR aggregate specific mAbs for patients with TTR amyloidoses.

CD4 binding determinant mimicry for HIV vaccine design.

The immunodominant epitopes expressed by the HIV-1 envelope protein gp120 are hypermutable, defeating attempts to develop an effective HIV vaccine. Targeting the structurally conserved gp120 determinant that binds host CD4 receptors (CD4BD) and initiates infection is a more promising route to vaccination, but this has proved difficult because of the conformational flexibility of gp120 and immune evasion mechanisms used by the virus. Mimicking the outer CD4BD conformational epitopes is difficult because of their discontinuous nature. The CD4BD region composed of residues 421-433 (CD4BD(core)) is a linear epitope, but this region possesses B cell superantigenic character. While superantigen epitopes are vulnerable to a small subset of spontaneously produced neutralizing antibodies present in humans without infection (innate antibodies), their non-covalent binding to B cell receptors (BCRs) does not stimulate an effective adaptive response from B cells. Covalent binding at naturally occurring nucleophilic sites of the BCRs by an electrophilic gp120 (E-gp120) analog is a promising solution. E-gp120 induces the synthesis of neutralizing antibodies the CD4BD(core). The highly energetic covalent reaction is hypothesized to convert the abortive superantigens-BCR interaction into a stimulatory signal, and the binding of a spatially distinct epitope at the traditional combining site of the BCRs may furnish a second stimulatory signal. Flexible synthetic peptides can detect pre-existing CD4BD(core)-specific neutralizing antibodies. However, induced-fit conformational transitions of the peptides dictated by the antibody combining site structure may induce the synthesis of non-neutralizing antibodies. Successful vaccine targeting of the CD4BD will require a sufficiently rigid immunogen that mimics the native epitope conformation and bypasses B cell checkpoints restricting synthesis of the neutralizing antibodies.

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 (CD4BD(core)) to identify Abs produced without prior exposure to HIV (constitutive Abs). The CD4BD(core) 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 V(H)4-family single-chain Fv with the corresponding V(H)3-family FRs from single-chain Fv JL427 improved the CD4BD(core) peptide-binding activity, suggesting a CD4BD(core) 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 CD4BD(core)-specific serum IgAs from noninfected humans without autoimmune disease isolated by epitope-specific chromatography neutralized the virus potently. A CD4BD(core)-specific, HIV neutralizing murine IgM with H and L chain V regions (V(H) and V(L) regions) free of immunogen-driven somatic mutations was induced by immunization with a CD4BD(core) 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 CD4BD(core) epitope that is available for amplification for vaccination against HIV.

Constant domain-regulated antibody catalysis.

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.

Nature and nurture of catalytic antibodies.

Immunoglobulins (antibodies) frequently express constitutive functions. Two such functions are nucleophilic catalysis and the reversible binding to B-cell superantigens. Constitutive or "naturally-occurring" antibodies are produced spontaneously from germline genetic information. The antibody structural elements mediating the constitutive functions have originated over millions of years of phylogenic evolution, contrasting with antigen-driven, somatic sequence diversification of the complementarity determining regions (CDR) that underlies the better-known high affinity antigen binding function of antibodies. Often, the framework regions (FRs) play a dominant role in antibody constitutive functions. Catalytic antibody subsets with promiscuous, autoantigen-directed and microbe-directed specificities have been identified. Mucosal antibodies may be specialized to express high-level catalytic activity against microbes transmitted by the mucosal route, exemplified by constitutive production of IgA class antibodies in mucosal secretions that catalyze the cleavage of HIV gp120. Catalytic specificity can be gained by constitutive noncovalent superantigen binding at the FRs and by adaptive development of noncovalent classical antigen or superantigen binding, respectively, at the CDRs and FRs. Growing evidence suggests important functional roles for catalytic antibodies in homeostasis, autoimmune disease and protection against infection. Adaptive antibody responses to microbial superantigens are proscribed underphysiological circumstances. Covalent electrophilic immunogen binding to constitutively expressed nucleophilic sites in B-cell receptors bypasses the restriction on adaptive antibody production, and simultaneous occupancy of the CDR binding site by a stimulatory antigenic epitope can also overcome the downregulatory effect of superantigen binding at the FRs. These concepts may be useful for developing novel vaccines that capitalize and improve on constitutive antibody functions for protection against microbes.

Constitutive production of catalytic antibodies to a Staphylococcus aureus virulence factor and effect of infection.

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.

Back to the future: covalent epitope-based HIV vaccine development.

Traditional HIV vaccine approaches have proved ineffective because the immunodominant viral epitopes are mutable and the conserved epitopes necessary for infection are not sufficiently immunogenic. The CD4 binding site expressed by the HIV envelope protein of glycoprotein 120 is essential for viral entry into host cells. In this article, we review the B-cell superantigenic character of the CD4 binding site as the cause of its poor immunogenicity. We summarize evidence supporting development of covalent immunization as the first vaccine strategy with the potential to induce an antibody response to a conserved HIV epitope that neutralizes genetically divergent HIV strains.

Immunological origin and functional properties of catalytic autoantibodies to amyloid beta peptide.

OBJECTIVES: Objectives The objectives of this study are to (1) evaluate the ability of the immune system to synthesize specific antibodies that catalyze the degradation of amyloid beta peptide (Abeta) and to (2) evaluate the prospect of developing a catalytic IVIG (CIVIG) formulation for therapy of Alzheimer's disease (AD). CONCLUSIONS: Polyclonal autoantibodies from humans without dementia hydrolyzed Abeta specifically. The catalytic activity improved as a function of age. Patients with AD produced catalytic antibodies at increased levels. IgM-class antibodies expressed the activity at levels superior to IgGs. Production of catalytic autoantibodies appears to be an innate immunity function with adaptive improvements occurring upon Abeta overexpression, which suggests a beneficial function of the catalytic activity. The catalytic autoantibodies impeded Abeta aggregation, dissolved preformed Abeta aggregates, and inhibited Abeta cytotoxicity in tissue culture. Recombinant catalytic antibodies from a human library have been identified, validating the phenomenon of antibody-catalyzed Abeta cleavage. As a single catalyst molecule inactivates multiple Abeta molecules, catalytic antibodies may clear Abeta efficiently. IVIG did not cleave Abeta, indicating the importance of purification procedures that maintain catalytic site integrity. Traditional Abeta-binding antibodies form immune complexes that can induce inflammatory reaction and vascular dysfunction. Catalysts do not form stable immune complexes, minimizing these risks. Criteria appropriate for developing a CIVIG formulation with potential therapeutic utility are discussed, including isolation of the Abeta-specific catalytic subsets present in IgM and IgG from human blood.

Beneficial catalytic immunity to abeta peptide.

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.

Neutralization of genetically diverse HIV-1 strains by IgA antibodies to the gp120-CD4-binding site from long-term survivors of HIV infection.

OBJECTIVE: To identify an HIV epitope suitable for vaccine development. DESIGN: Diverse HIV-1 strains express few structurally constant regions on their surface vulnerable to neutralizing antibodies. The mostly conserved CD4-binding site (CD4BS) of gp120 is essential for host cell binding and infection by the virus. Antibodies that recognize the CD4BS are rare, and one component of the CD4BS, the 421-433 peptide region, expresses B-cell superantigenic character, a property predicted to impair the anti-CD4BS adaptive immune response. METHODS: IgA samples purified from the plasma of patients with HIV infection were analyzed for the ability to bind synthetic mimetics containing the 416-433 gp120 region and full-length gp120. Infection of peripheral blood mononuclear cells by clinical HIV isolates was measured by p24 ELISA. RESULTS: IgA preparations from three patients with subtype B infection for 19-21 years neutralized heterologous, coreceptor CCR5-dependent subtype A, B, C, D, and AE strains with exceptional potency. The IgAs displayed specific binding of a synthetic 416-433 peptide mimetic dependent on recognition of the CD4-binding residues located in this region. Immunoadsorption, affinity chromatography, and mutation procedures indicated that HIV neutralization occurred by IgA recognition of the CD4BS. CONCLUSION: These observations identify the 421-433 peptide region as a vulnerable HIV site to which survivors of infection can produce powerful neutralizing antibodies. This indicates that the human immune system can bypass restrictions on the adaptive B cell response to the CD4BS, opening the route to targeting the 421-433 region for attaining control of HIV infection.

Toward effective HIV vaccination: induction of binary epitope reactive antibodies with broad HIV neutralizing activity.

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 (V(H)) domain framework (FR) residues. Substitution of the FR cavity V(H) Lys-19 residue by an Ala residue resulted in attenuated binding of the 421-433 region peptide probe. The CDRs and V(H) 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 V(H)1 family genes, suggesting biased recruitment of the V gene germ line repertoire by E-gp120. 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.

Antigen-specific proteolysis by hybrid antibodies containing promiscuous proteolytic light chains paired with an antigen-binding heavy chain.

The antigen recognition site of antibodies consists of the heavy and light chain variable domains (V(L) and V(H) domains). V(L) domains catalyze peptide bond hydrolysis independent of V(H) domains (Mei, S., Mody, B., Eklund, S. H., and Paul, S. (1991) J. Biol. Chem. 266, 15571-15574). V(H) domains bind antigens noncovalently independent of V(L) 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 V(H) domain of an anti-E2 IgG paired with promiscuously catalytic V(L) 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 approximately 11 kDa from the N terminus. GST-E2 was hydrolyzed by the hybrid IgG 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.