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.

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.

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.

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.

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.