Changes in cell differentiation and proliferation lead to production of abzymes in EAE mice treated with DNA-Histone complexes.

Experimental autoimmune encephalomyelitis (EAE)-prone C57BL/6 mice are used as a model of human multiple sclerosis. We immunize mice with myelin oligodendrocyte glycoprotein (MOG), DNA-histone and DNA-methylated bovine serum albumin (met-BSA) complexes to reveal different characteristics of EAE development including bone marrow lymphocyte proliferation and differentiation profiles of hematopoietic stem cells. Immunization of C57BL/6 mice with MOG35-55 results in the acceleration of EAE development. Anti-DNA antibodies are usually directed against DNA-histone complexes resulting from cell apoptosis. During the acute EAE phase (7-20 days after immunization), catalytic antibodies efficiently hydrolysing myelin basic protein (MBP), MOG and DNA are produced with parallel suppression of antibodies hydrolysing histones. We could show that in contrast to MOG, immunization with histone-DNA results in a reduction of proteinuria, a significant increase in anti-DNA, anti-MBP and anti-MOG antibody titres, as well as an increase in their catalytic activities for antigen hydrolysis, but slightly changes the concentration of cytokines. Contrary to MOG, DNA-histone and DNA-met-BSA only stimulated the formation of anti-DNA antibodies hydrolysing DNA with a long delay (15-20 days after immunization). Our data indicate that for C57BL/6 mice immunization with DNA-met-BSA and DNA-histone complexes may have opposing effects compared to MOG. DNA-histone stimulates the appearance of histone-hydrolysing abzymes in the acute EAE phase, while abzymes with DNase activity appear at significantly later time-points. We conclude that MOG, DNA-histone and DNA-met-BSA have different effects on numerous bone marrow, cellular, immunological and biochemical parameters of immunized mice, but all antigens finally significantly stimulate the development of the EAE.

Antibodies against H3 and H4 histones from the sera of HIV-infected patients catalyze site-specific degradation of these histones.

Histones and their posttranslational modified forms play pivotal roles in chromatin functioning and gene transcription. Also, histones are harmful when they enter the intercellular space; their administration to animals results in systemic inflammatory and toxic responses. Autoantibodies having enzymatic activities (abzymes) are the specific feature of several autoimmune and viral diseases. Electrophoretically homogeneous IgGs containing no canonical proteases were purified from sera of HIV-infected patients by using several affinity chromatographies. In contrast to known canonical proteases, Abs from HIV-infected patients hydrolyzed exclusively only histones but no other control globular proteins. The H3 and H4 histone cleavage sites by antihistone IgGs were determined by matrix-assisted laser desorption/ionization mass spectrometry for the first time. Two clusters of H3 hydrolysis contain major (↕) and minor (*) cleavage sites: 18-K*Q*LA↕TK*A↕AR*KS↕A*P-30 and 34-G*VK*KPHR*YRPGTVA*L*R-50. H4 histone has only 1 cluster of cleavage sites containing additionally moderate (↓) cleavage sites: 15-A↕KR↕HR↕KVLR↓D*NIQ↓GIT*K-31. Sites of these histones cleavage correspond mainly to their known epitopes. It was surprising that most of the cleavage sites of histones are involved in the interaction with DNA of nucleosome core. Because histones act as damage-associated molecules, abzymes against H3 and H4 can play important role in pathogenesis of AIDs and probably other viral and immune diseases.

Changes in haematopoietic progenitor colony differentiation and proliferation and the production of different abzymes in EAE mice treated with DNA.

Immunization of experimental autoimmune encephalomyelitis (EAE)-prone C57BL/6 mice with MOG35-55 (a model used to study aspects of human multiple sclerosis) is known to lead to the production of various abzymes. The production of catalytic IgGs that can efficiently hydrolyse myelin basic protein (MBP), MOG and DNA is associated with changes in the profile of differentiation and level of proliferation of mice bone marrow haematopoietic stem cells (HSCs). As MOG simulates the production of abzymes with high DNase activity, we compared the effects of DNA and MOG immunization on EAE-prone mice. In contrast to MOG, immunization with DNA leads to a suppression of proteinuria, a decrease in the concentrations of antibodies to MOG and DNA and a reduction in abzyme production. Immunization with DNA only resulted in a significant increase in DNase activity over 40 days where it became 122-fold higher than before immunization, and fivefold higher when comparing to the maximal activity obtained after MOG treatment. DNA and MOG immunization had different effects on the differentiation profiles of HSCs, lymphocyte proliferation, and the level of apoptosis in bone marrow and other organs of mice. The data indicate that for C57BL/6 mice, DNA may have antagonistic effects with respect to MOG immunization. The usually fast immune response following MOG injection in C57BL/6 mice is strongly delayed after immunization with DNA, which is probably due to a rearrangement of the immune system following the response to DNA.

Multitarget selection of catalytic antibodies with ß-lactamase activity using phage display.

ß-lactamase enzymes responsible for bacterial resistance to antibiotics are among the most important health threats to the human population today. Understanding the increasingly vast structural motifs responsible for the catalytic mechanism of ß-lactamases will help improve the future design of new generation antibiotics and mechanism-based inhibitors of these enzymes. Here we report the construction of a large murine single chain fragment variable (scFv) phage display library of size 2.7 × 109 with extended diversity by combining different mouse models. We have used two molecularly different inhibitors of the R-TEM ß-lactamase as targets for selection of catalytic antibodies with ß-lactamase activity. This novel methodology has led to the isolation of five antibody fragments, which are all capable of hydrolyzing the ß-lactam ring. Structural modeling of the selected scFv has revealed the presence of different motifs in each of the antibody fragments potentially responsible for their catalytic activity. Our results confirm (a) the validity of using our two target inhibitors for the in vitro selection of catalytic antibodies endowed with ß-lactamase activity, and (b) the plasticity of the ß-lactamase active site responsible for the wide resistance of these enzymes to clinically available inhibitors and antibiotics.

New Genetic Constructs for Generation of Stable Therapeutic Antibodies to Organophosphorus Toxins in Methylotrophic Yeasts Pichia Pastoris.

We propose a new method of obtaining of stable Fab-fragments of antibodies in Pichia pastoris expression system. Recently, we obtained Fab-fragments of antibodies neutralizing organophosphorus toxins. However, high yield of the target products was not attained because of high level of proteolytic degradation. In the present study, we identified sites of proteolytic degradation in Fab-fragments and endogenous proteases performing degradation, which allowed obtaining optimized genetic constructs for expression of antibody heavy chains (IgGγ1) and kappa and lambda isotypes of light chains. Co-transformation of these vectors allowed obtaining Fab-fragments of antibodies to organophosphorus toxins without proteolytic degradation of the product.

Characterization of fragmented 3-phosphoinsitide-dependent protein kinase-1 (PDK1) by phosphosite-specific antibodies.

AIMS: The 3-phosphoinositide-dependent protein kinase-1 (PDK1) activates a number of protein kinases of the AGC subfamily, including protein kinase B and ribosomal S6 protein kinase by phosphorylating these kinases at the activation-loop. PDK1 activity is regulated by auto-phosphorylation and is further increased by stimulation of cells. PDK1 has been shown to have several phosphorylation sites including 5 serine and 3 tyrosine residues. However, Ser241 and Tyr373/376 are only involved in the regulation of PDK1 activity. MAIN METHODS: In this study, we found the putative fragments of PDK1 by using anti-Myc and anti-PDK1 antibodies. Furthermore, the existence of four different sizes of PDK1 were confirmed with other phosphosite specific antibodies. KEY FINDINGS: Taken together, the catalytic domain of PDK1 (42 kDa and 37 kDa) is separately existed in the cells and might be important for the regulation of subset of PDK1 substrate. Because the crystal structural studies suggested that PIF-pocket is located at the catalytic domain and plays a critical role on substrate recognition. SIGNIFICANCE: These suggested importance and roles of this fragment are needed to be determined. Further study on these fragments of PDK1 will provide new insight on the regulatory mechanism of PDK1 in patho-physiological condition.

Antibody-mediated catalysis: induction and therapeutic relevance.

Abzymes are immunoglobulins endowed with enzymatic activities. The catalytic activity of an abzyme resides in the variable domain of the antibody, which is constituted by the close spatial arrangement of amino acid residues involved in catalysis. The origin of abzymes is conferred by the innate diversity of the immunoglobulin gene repertoire. Under deregulated immune conditions, as in autoimmune diseases, the generation of abzymes to self-antigens could be deleterious. Technical advancement in the ability to generate monoclonal antibodies has been exploited in the generation of abzymes with defined specificities and activities. Therapeutic applications of abzymes are being investigated with the generation of monoclonal abzymes against several pathogenesis-associated antigens. Here, we review the different contexts in which abzymes are generated, and we discuss the relevance of monoclonal abzymes for the treatment of human diseases.

[Expression of the catalytic antibodies in eukaryotic systems].

Expression of recombinant antibodies in mammalian cells is one of key problems in immunobiotechnology. Alternatively, expression of a broad panel of antibodies and of their fragments may be effectively done in yeast cells. We obtained expression strains of the methylotrophic beast Pichia pastoris producing single chain human catalytic antibody A17 (A.17scFv), Fab-fragment (A.17Fab) and full-size light chain (A.17Lch). These antibodies were characterized in terms of functional activity. The capacity to specifically bind and transform organophosphorus compounds has been demonstrated for A.17scFv and A.17Fab. The loss of activity of the antibody light chain when expressed alone indicates that the active site is formed by both heavy and light chains of the antibody. We determined the reversible constant Kd and the first order constant (k2) of the reaction of the covalent modification of A.17scFv and A.17Fab by irreversible inhibitor of the serine proteases p-nitrophenyl 8-methyl-8-azobicyclo[3.2.1]phosphonate (Phosphonate X). Calculated values indicate that activity of the antibodies expressed in yeast is similar to the full-size antibody A17 and single chain antibody A.17 expressed in CHO and E. coli cells respectively.

Strategies for induction of catalytic antibodies toward HIV-1 glycoprotein gp120 in autoimmune prone mice.

Tremendous efforts to produce an efficient vaccine for HIV infection have been unsuccessful. The ability of HIV to utilize sophisticated mechanisms to escape killing by host immune system rises dramatic problems in the development of antiviral therapeutics. The HIV infection proceeds by interaction of coat viral glycoprotein gp120 trimer with CD4(+) receptor of the lymphocyte. Thus this surface antigen may be regarded as a favorable target for immunotherapy. In the present study, we have developed three different strategies to produce gp120-specific response in autoimmune prone mice (SJL strain) as potential tools for production "catalytic vaccine". Therefore (i) reactive immunization by peptidylphosphonate, structural part of the coat glycoprotein, (ii) immunization by engineered fused epitopes of gp120 and encephalogenic peptide, a part of myelin basic protein, and (iii) combined vaccination by DNA and corresponding gp120 fragments incorporated into liposomes were investigated. In the first two cases monoclonal antibodies and their recombinant fragments with amidolytic and gp120-specific proteolytic activities were characterized. In the last case, catalytic antibodies with virus neutralizing activity proved in cell line models were harvested.

Immunization of rabbits with DNase II leads to formation of polyclonal antibodies with DNase and RNase activities.

The serum of patients with many autoimmune (AI) diseases contains small fractions of antibodies possessing both DNase and RNase activities. It was shown that immunization of rabbits with DNA, RNA, DNase I and RNase leads to production of antibodies with DNase and RNase activities. It is not known whether anti-idiotypic antibodies against DNase II can possess DNase or RNase activity. Electrophoretically and immunologically homogeneous polyclonal IgGs (pIgGs) from the sera of rabbits immunized with DNase II were obtained by sequential chromatography of the serum proteins on Protein A-Sepharose and gel filtration. It was shown for the first time that immunization of healthy rabbits with bovine DNase II produces IgGs with intrinsic DNase and RNase activities. IgGs from rabbits immunized with BSA or non-immunized animals were catalytically inactive. It was shown that approximately 10% of the total IgG DNase and RNase activities belong to anti-idiotypic antibodies to DNase II ( approximately 0.1% of total pIgGs), while 90% of the activities did not interact with Sepharose bearing antibodies against DNase II and might be antibodies to nucleic acids bound to DNase II. Affinity chromatography on DNA-cellulose using elution of antibodies with different concentration of NaCl and an acidic buffer separated catalytic IgGs into five antibody subfractions, three of which hydrolyzed RNA faster than DNA while two subfractions demonstrated only DNase activity. Our data suggest that a fraction of abzymes from AI patients hydrolyzing both DNA and RNA can contain subfraction of antibodies against DNase II.

Generation of humanized anti-DNA hydrolyzing catalytic antibodies by complementarity determining region grafting.

Humanization of nonhuman antibodies (Abs) has been carried out mainly for Abs which bind to antigen without catalytic activity. Here we report humanization of mouse-originated 3D8 (m3D8) mAbs (scFv, VH, and VL) with DNA hydrolyzing catalytic activity by grafting the complementarity determining regions (CDRs) into the corresponding regions of a fixed human framework scaffold, generating humanized 3D8 (h3D8) Abs in the respective format of scFv, VH, and VL. h3D8 Abs retained comparable DNA binding and hydrolyzing activities to those of the corresponding m3D8 Abs. Our results suggest that CDRs of anti-DNA hydrolyzing Abs might possess the intrinsic properties of DNA binding and hydrolyzing activities.

Antibodies against pancreatic ribonuclease A hydrolyze RNA and DNA.

The sera of patients with autoimmune (AI) diseases contain antibodies with DNase and RNase activities. We have shown for the first time that immunization of healthy rabbits with RNase A conjugated with BSA produces a better immune response than immunization with pure RNase and induced IgGs with RNase and DNase activities, which were intrinsic properties of IgGs, while polyclonal IgGs (pIgGs) from non-immunized rabbits and animals immunized with BSA were catalytically inactive. It was shown that 74-85% of the total IgG DNase and RNase activities belongs to anti-idiotypic antibodies to RNase A (0.6-0.8% of total pIgGs), while 15-26% of the activities cannot interact with Sepharose-bearing antibodies against RNase A and may be antibodies to nucleic acids bound to RNase. Affinity chromatography on DNA-cellulose separated catalytic IgGs into several antibody subfractions demonstrating only DNase or RNase activity or hydrolyzing RNA faster than DNA. Our data suggest that a fraction of abzymes (or catalytically active antibodies) from AI patients hydrolyzing both DNA and RNA may be antibodies against RNase or its complexes with other proteins.

Immunization of rabbits with DNase I produces polyclonal antibodies with DNase and RNase activities.

Immunization of rabbits with DNase I leads to the production of antiidiotypic Abs with DNase activity. It is not known at present whether antiidiotypic Abs against DNA-hydrolyzing enzymes can possess RNase activity. Here we show that immunization of healthy rabbits with bovine DNase I produces IgGs with intrinsic DNase and RNase activities. Electrophoretically and immunologically homogeneous polyclonal IgGs were obtained by sequential chromatography of the immune sera on Protein A-Sepharose and gel filtration. Affinity chromatography on DNA cellulose using elution of Abs with different concentrations of NaCl and an acidic buffer separated catalytic IgGs into four Ab subfractions, three of which demonstrated only DNase activity while one subfraction hydrolyzed RNA faster than DNA. The serum of patients with many different autoimmune (AI) diseases contains small fractions of antibodies (Abs) interacting with immobilized DNA, which possess both DNase and RNase activities. Our data suggest that a fraction of abzymes from AI patients hydrolyzing both DNA and RNA can contain a subfraction of Abs against DNase I.

[Catalytically active antibodies (abzymes) in human milk].

The review is focused on the analysis of published data and the results obtained by the authors about the catalytic activity of antibodies (abzymes) of human colostrum and milk. Possible mechanisms of origination of these abzymes and their potential role in the regulation of biological activity of human milk compounds are considered. A hypothesis about the role of secretoty abzymes in non-specific humoral defense for the epithelial cells against viral infections is proposed.

Catalytic IgG from patients with hemophilia A inactivate therapeutic factor VIII.

Factor VIII (FVIII) inhibitors are anti-FVIII IgG that arise in up to 50% of the patients with hemophilia A, upon therapeutic administration of exogenous FVIII. Factor VIII inhibitors neutralize the activity of the administered FVIII by sterically hindering its interaction with molecules of the coagulation cascade, or by forming immune complexes with FVIII and accelerating its clearance from the circulation. We have shown previously that a subset of anti-factor VIII IgG hydrolyzes FVIII. FVIII-hydrolyzing IgG are detected in over 50% of inhibitor-positive patients with severe hemophilia A, and are not found in inhibitor-negative patients. Although human proficient catalytic Abs have been described in a number of inflammatory and autoimmune disorders, their pathological relevance remains elusive. We demonstrate here that the kinetics of FVIII degradation by FVIII-hydrolyzing IgG are compatible with a pathogenic role for IgG catalysts. We also report that FVIII-hydrolyzing IgG from each patient exhibit multiple cleavage sites on FVIII and that, while the specificity of cleavage varies from one patient to another, catalytic IgG preferentially hydrolyze peptide bonds containing basic amino acids.

Toward bifunctional antibody catalysis.

Antibodies that catalyze the deprotonation of unactivated benzisoxazoles to give the corresponding salicylonitriles were prepared using as antigen a 2-aminobenzimidazolium derivative coupled to a carrier protein via its benzene ring. The hapten was designed to induce an antibody binding site with both a base and an acid, in position to initiate proton transfer and stabilize developing negative charge at the phenoxide leaving group, respectively. Consistent with this design, the catalysts exhibit bell-shaped pH-rate profiles, while chemical modification identified several functional groups that could participate in bifunctional catalysis. One of the antibodies, 13G5, is particularly notable in catalyzing the elimination of 6-glutaramidebenzisoxazole with a > 10(5)-fold rate acceleration over background and an effective molarity of > 10(4) M for its catalytic base. These properties compare favorably to the efficiencies achieved by the best previously characterized antibodies with substantially more reactive substrates.

Squaric monoamide monoester as a new class of reactive immunization hapten for catalytic antibodies.

A squaric monoester monoamide motif was employed as an effective reactive immunogen for the discovery of monoclonal antibodies with reactive residue(s) in their combining sites. Two antibodies, 2D4 and 3C8, were uncovered that enhance paraoxon hydrolysis over background. Kinetic analysis of these antibodies was performed and interestingly both undergo a single turnover event due to covalent modification within the antibody combining site. Because antibodies 2D4 and 3C8 result in covalent attachment and thus inactivation of paraoxon, they could be useful probes for investigating paraoxon intoxication.

Positional ordering of reacting groups contributes significantly to the efficiency of proton transfer at an antibody active site.

Catalytic antibody 34E4 accelerates the conversion of benzisoxazoles to salicylonitriles with surprising efficiency, exploiting a carboxylate base with an elevated pKa for proton abstraction. Mutagenesis of this antibody, produced as a chimeric Fab, confirms the prediction of a homology model that GluH50 is the essential catalytic residue. Replacement of this residue by glutamine, alanine, or glycine reduces catalytic activity by more than 2.6 x 10(4)-fold. By comparing the chemical proficiencies of the parent antibody with the chemical proficiencies of acetate and the mutants, the effective concentration of the catalytic side chain was estimated to be >51 000 M. The 2.1 kcal/mol destabilization of the transition state observed when GluH50 is replaced by aspartate suggests that positional ordering imposed by the antibody active site contributes significantly to the efficiency of proton transfer. The observation that the GluH50Ala and GluH50Gly variants could not be chemically rescued by exogenous addition of high concentrations of formate or acetate further underscores the advantage the antibody derives from covalently fixing its base at the active site. Although medium effects also play an important role in 34E4, for example in enhancing the reactivity of the carboxylate side chain through desolvation, comparison of 34E4 with less proficient antibodies shows that positioning a carboxylate in a hydrophobic binding pocket alone is insufficient for efficient general base catalysis. Our results demonstrate that structural complementarity between the antibody and its substrate in the transition state is an important and necessary component of 34E4's high activity. By harnessing an additional catalytic group that could serve as a general acid to stabilize developing negative charge in the leaving group, overall efficiencies rivaling those of highly evolved enzymes should be accessible.

A catalytic antibody against a tocopherol cyclase inhibitor.

The cyclic ammonium cation 5 and its guanidinium analogue 4 are inhibitors of tocopherol cyclase. Monoclonal antibodies were raised against protein conjugates of the haptens 1-3 and screened for catalytic reactions with alkene 8, a short chain analogue of the natural substrate phytyl-hydroquinone 6, and its enol ether analogues 10a,b. Antibody 16E7 raised against hapten 3 was found to catalyze the hydrolysis of Z enol ether 10a to form hemiacetal 12 with an apparent rate acceleration of k(cat)/k(uncat)=1400. Antibody 16E7 also catalyzed the elimination of Kemp's benzisoxazole 59. The absence of cyclization in the reaction of enol ether 10a was attributed to the competition of water molecules for the oxocarbonium cation intermediate within the antibody binding pocket. Hapten and reaction design features contributing to this outcome are discussed. Antibody 16E7 provides the first example of a carboxyl group acting both as an acid in an intrinsically acid-catalyzed process and as a base in an intrinsically base-catalyzed process, as expected from first principles. In contrast to the many examples of general-acid-catalyzed processes known to be catalyzed by catalytic antibodies, the specific-acid-catalyzed cyclization of phytyl-hydroquinone 6 or its analogue 8 still eludes antibody catalysis.