A comparison of flexible and constrained haptens in eliciting antibody catalysts for paraoxon hydrolysis.

A new amine-oxide hapten was employed as an antigen, producing seven monoclonal antibodies (mAbs) from a panel of 20 that catalyzed paraoxon hydrolysis. The current hapten design differs from that previously described in that the molecule is inherently more flexible than its constrained predecessor. One of the seven antibody catalysts, mAb 1H9, showed the highest activity and was selected for detailed study. At pH = 8.77, the catalytic hydrolysis of paraoxon by mAb 1H9 followed Michaelis Menten kinetics affording a k(cat) = 3.73 x 10(-4) min(-1) and a Km = 1.12 mM with a rate acceleration k(cat)/k(uncat) = 56. The hapten was found to be a competitive inhibitor of antibody-catalyzed paraoxon hydrolysis with a Ki = 0.54 mM. A comparison of both the number and proficiency of antibody catalysts obtained when utilizing a flexible versus constrained hapten indicates that, for paraoxon hydrolysis, constrained haptens elicit superior catalysts, suggesting that further development should begin with the use of constrained haptens in producing more proficient antibody catalysts for paraoxon hydrolysis.     

An investigation of antibody acyl hydrolysis catalysis using a large set of related haptens

An aspect of catalytic antibody research that receives little attention in the literature involves hapten systems that fail to elicit antibody catalysts despite a high affinity immune response and hapten designs that resemble those known to elicit catalysts. We have investigated a series of 12 phosphate and phosphonate haptens in a total of three animal systems. Dramatic and reproducible differences were observed in the catalytic activities of polyclonal antibodies elicited by the different haptens. A phosphate hapten with a phenyl ring on the side of the hapten opposite the linker elicited reproducibly high levels of polyclonal antibody catalytic activity. The other 11 haptens, most with benzyl groups on the side of the hapten opposite the linker, elicited immune responses in which catalytic activity was significantly weaker in terms of the level of observed catalytic activity, as well as frequency of elicited catalysts. Our results indicate that subtle features of transition state analogue hapten structure can have a dramatic and reproducible influence over the catalytic activity of elicited antibodies in related haptens. Whatever the explanation, subtle changes in mechanistic features due to altered leaving group ability/location or overall hapten flexibility, the comprehensive data presented here indicate that phenyl or 4-nitrophenyl leaving groups located opposite the hapten linker are to be preferred in order to elicit highly active antibody catalysts for acyl hydrolysis reactions.     

天然产物莪术醇单克隆抗体的制备

为制备小分子化合物莪术醇的单克隆抗体,先将莪术醇(curcumol)与载体蛋白牛血清蛋白(BSA)偶联形成完全抗原,用基质辅助激光解吸飞行时间质谱法(MALDI-TOF-MS)鉴定莪术醇人工抗原的偶联率,然后采用杂交瘤技术获得杂交瘤株,并对其进行小鼠腹水的制备与纯化.结果表明:莪术醇半抗原与载体的偶联比为19.6,单克...     

Evidence that the mechanism of antibody-catalysed hydrolysis of arylcarbamates can be determined by the structure of the immunogen used to elicit the catalytic antibody

A kinetically homogeneous anti-phosphate catalytic antibody preparation was shown to catalyse the hydrolysis of a series of O-aryl N-methyl carbamates containing various substituents in the 4-position of the O-phenyl group. The specific nature of the antibody catalysis was demonstrated by the adherence of these reactions to the Michaelis-Menten equation, the complete inhibition by a hapten analogue, and the failure of the antibody to catalyse the hydrolysis of the 2-nitrophenyl analogue of the 4-nitrophenylcarbamate substrate. Hammett sigma-rho analysis suggests that both the non-catalysed and antibody-catalysed reactions proceed by mechanisms in which development of the aryloxyanion of the leaving group is well advanced in the transition state of the rate-determining step. This is probably the ElcB (elimination-addition) mechanism for the non-catalysed reaction, but for the antibody-catalysed reaction might be either ElcB or B(Ac)2 (addition-elimination), in which the elimination of the aryloxy group from the tetrahedral intermediate has become rate-determining. This result provides evidence of the dominance of recognition of phenolate ion character in the phosphate hapten in the elicitation process, and is discussed in connection with data from the literature that suggest a B(Ac)2 mechanism, with rate-determining formation of the tetrahedral intermediate for the hydrolysis of carbamate substrates catalysed by an antibody elicited by a phosphonamidate hapten in which phenolate anion character is minimized. The present paper contributes to the growing awareness that small differences in the structure of haptens can produce large differences in catalytic characteristics.     

催化性抗体及其研究进展

根据过渡态理论,按特定的化学反应机制确定反应中的可能过渡态结构,选择和该过渡态结构类似的化合物作为半抗原,可诱导机体产生具有催化活性的催化性抗体.文章对诱导催化性抗体中半抗原的选择原则、催化性抗体和非催化性抗体间的联系、催化性抗体和酶促催化反应的比较等方面进行了较为全面的综述,并对催化性抗体在医药科学中的应用前景及限制因素进行了讨论.     

Recurrent somatic mutations in mouse antibodies to p-azophenylarsonate increase affinity for hapten

Two mouse mAb specific for the hapten p-azophenylarsonate and encoded by the same combination of germ-line V, D, and J genes differ 200-fold in affinity for hapten. We determined the amino acid sequences of the V regions of the high affinity antibody and compared them to the published sequences of the low affinity antibody which is not somatically mutated. Of 19 amino acid substitutions, two, Ile57 and Thr58 in the H chain, also occur, either alone or together, in other somatically mutated antibodies specific for p-azophenylarsonate; these antibodies have been independently isolated. Introduction of either one of these mutations alone into the low affinity antibody by oligonucleotide-directed mutagenesis increased the antibody affinity for hapten three- to fourfold, whereas introduction of both mutations together conferred an eightfold increase in affinity. These results support the hypothesis that somatic mutations are selected on the basis of the affinity for antigen that they confer, and suggest that even relatively small increases in affinity may be selected, probably in a sequential manner.     

Selection of phage-displayed peptides that bind to a particular ligand-bound antibody

Phage-displayed peptides that selectively bind to aldolase catalytic antibody 93F3 when bound to a particular 1,3-diketone hapten derivative have been developed using designed selection strategies with libraries containing 7-12 randomized amino acid residues. These phage-displayed peptides discriminated the particular 93F3-diketone complex from ligand-free 93F3 and from 93F3 bound to other 1,3-diketone hapten derivatives. By altering the selection procedures, phage-displayed peptides that bind to antibody 93F3 in the absence of 1,3-diketone hapten derivatives have also been developed. With using these phage-displayed peptides, ligand-bound states of the antibody were distinguished from each other. A docking model of one of the peptides bound to the antibody 93F3-diketone complex was created using a sequential divide-and-conquer peptide docking strategy; the model suggests that the peptide interacts with both the antibody and the ligand through a delicate hydrogen bonding network.     

Catalytic features of monoclonal antibody i41SL1-2 subunits

A monoclonal antibody (mAb), i41SL1-2, was obtained by immunizing the peptide of complementarity-determining region-1 (CDRL-1: RSSKSLLYSNGNTYLY) of a super catalytic antibody light chain, 41S-2-L, capable of enzymatically destroying the gp41 molecule of the HIV-1 envelope. From the DNA and the deduced amino acid sequences of the light and heavy chain of i41SL1-2 mAb, molecular modeling was conducted that suggested that both subunits of i41SL1-2 mAb possess catalytic triads in their structures. Especially the light chain of i41SL1-2 mAb possesses a characteristic catalytic triad composed of Asp(1), Ser(27A), and His(93), whose positions are identical to the catalytic antibody light chain, VIPase, of S. Paul and colleagues (see text). The antibody gene of i41SL1-2 light chain and VIPase belong to the same germline, bd2, suggesting that the discrete germline inherently possesses catalytic activity. Both light and heavy chains of i41SL1-2 mAb degraded the antigenic peptide CDRL-1 within 47 and 57 h, respectively. The catalytic reaction constant (kcat) of the light and heavy chain was 6.1 x 10(-1) and 6.2 x 10(-1) min(-1), respectively. These are high values for the natural catalytic antibodies reported so far. The catalytic efficiency (kcat/Km) of the light and heavy chain was 3.1 x 10(5) and 4.9 x 10(4) M(-1) min(-1), respectively. The first cleaved bond of the antigenic peptide by subunits of i41SL1-2 mAb was between Arg(1) and Ser(2) in the sequence of CDRL-1, suggesting a serine protease character.     

Identification of functionally important residues in the pyridoxal-5'-phosphate-dependent catalytic antibody 15A9

Antibody 15A9 is unique in its ability to catalyze the transamination reaction of hydrophobic D-amino acids with pyridoxal-5'-phosphate (PLP). Both previous chemical modification studies and a three dimensional (3-D) homology model indicated the presence of functionally important tyrosine residues in the antigen-binding cavity of antibody 15A9. To gain further insight into the hapten, ligand binding, and catalytic mechanism of 15A9, all tyrosine residues in the complementarity-determining regions (CDRs) and the single arginine residue in CDR3 of the light chain were subject to an alanine scan. Substitution of Tyr(H33), Tyr(L94), or Arg(L91) abolished the catalytic activity and reduced the affinity for PLP and N(a)-(5'-phosphopyridoxyl)-amino acids, which are close analogues of covalent PLP-substrate adducts. The Tyr(H100b)Ala mutant possessed no detectable catalytic activity, while its affinity for each ligand was essentially the same as that of the wild-type antibody. The binding affinity for the hapten was drastically reduced by a Tyr(L32)Ala mutation, suggesting that the hydroxyphenyl group of Tyr(L32) participates in the binding of the extended side chain of the hapten. The other Tyr --> Ala substitutions affected both binding and catalytic activity only to a minor degree. On the basis of the information obtained from the mutagenesis study, we docked N(alpha)-(5'-phosphopyridoxyl)-D-alanine into the antigen-binding site. According to this model, Arg(L91) binds the alpha-carboxylate group of the amino acid substrate and Tyr(H100b) plays an essential role in the catalytic mechanism of antibody 15A9 by facilitating the Calpha/C4' prototropic shift. In addition, the catalytic apparatus of antibody 15A9 revealed several mechanistic features that overlap with those of PLP-dependent enzymes.     

A comparative analysis of the immunological evolution of antibody 28B4

In an effort to gain greater insight into the evolution of the redox active, catalytic antibody 28B4, the germline genes used by the mouse to generate this antibody were cloned and expressed, and the X-ray crystal structures of the unliganded and hapten-bound germline Fab of antibody 28B4 were determined. Comparison with the previously determined structures of the unliganded and hapten-bound affinity-matured Fab [Hsieh-Wilson, L. C., Schultz, P. G., and Stevens, R. C. (1996) Proc. Natl. Acad. Sci. U.S.A. 93, 5363] shows that the germline antibody binds the p-nitrophenyl ring of hapten 3 in an orientation significantly different from that seen in the affinity-matured antibody, whereas the phosphonate moiety is bound in a similar mode by both antibodies. The affinity-matured antibody 28B4 has more electrostatic and hydrophobic interactions with hapten 3 than the germline antibody and binds the hapten in a lock-and-key fashion. In contrast, significant conformational changes occur in the loops of CDR H3 and CDR L1 upon hapten binding to the germline antibody, consistent with the notion of structural plasticity in the germline antibody-combining site [Wedemayer, G. J., Patten, P. A., Wang, L. H., Schultz, P. G., and Stevens, R. C. (1997) Science 276, 1665]. The structural differences are reflected in the differential binding affinities of the germline Fab (K(d) = 25 microM) and 28B4 Fab (K(d) = 37 nM) to hapten 3. Nine replacement mutations were found to accumulate in the affinity-matured antibody 28B4 compared to its germline precursor. The effects of each mutation on the binding affinity of the antibody to hapten 3 were characterized in detail in the contexts of both the germline and the affinity-matured antibodies. One of the mutations, Asp95(H)Trp, leads to a change in the orientation of the bound hapten, and its presence is a prerequisite for other somatic mutations to enhance the binding affinity of the germline antibody for hapten 3. Thus, the germline antibody of 28B4 acquired functionally important mutations in a stepwise manner, which fits into a multicycle mutation, affinity selection, and clonal expansion model for germline antibody evolution. Two other antibodies, 20-1 and NZA6, with very different antigen specificities were found to be highly homologous to the germline antibody of 28B4, consistent with the notion that certain germline variable-region gene combinations can give rise to polyspecific hapten binding sites [Romesberg, F. E., Spiller, B., Schultz, P. G., and Stevens, R. C. (1998) Science 279, 1929]. The ultimate specificity of the polyspecific germline antibody appears to be defined by CDR H3 variability and subsequent somatic mutation. Insights into the evolution of antibody-combining sites provided by this and other structural studies are discussed.     

Diverse structural solutions to catalysis in a family of antibodies

BACKGROUND: Small organic molecules coupled to a carrier protein elicit an antibody response on immunisation. The diversity of this response has been found to be very narrow in several cases. Some antibodies also catalyse chemical reactions. Such catalytic antibodies are usually identified among those that bind tightly to an analogue of the transition state (TSA) of the relevant reaction; therefore, catalytic antibodies are also thought to have restricted diversity. To further characterise this diversity, we investigated the structure and biochemistry of the catalytic antibody 7C8, one of the most efficient of those which enhance the hydrolysis of chloramphenicol esters, and compared it to the other catalytic antibodies elicited in the same immunisation. RESULTS: The structure of a complex of the 7C8 antibody Fab fragment with the hapten TSA used to elicit it was determined at 2.2 A resolution. Structural comparison with another catalytic antibody (6D9) raised against the same hapten revealed that the two antibodies use different binding modes. Furthermore, whereas 6D9 catalyses hydrolysis solely by transition-state stabilisation, data on 7C8 show that the two antibodies use mechanisms where the catalytic residue, substrate specificity and rate-limiting step differ. CONCLUSIONS: Our results demonstrate that substantial diversity may be present among antibodies catalysing the same reaction. Therefore, some of these antibodies represent different starting points for mutagenesis aimed at boosting their activity. This increases the chance of obtaining more proficient catalysts and provides opportunities for tailoring catalysts with different specificities.     

Augmenting the efficacy of anti-cocaine catalytic antibodies through chimeric hapten design and combinatorial vaccination

Given the need for further improvements in anti-cocaine vaccination strategies, a chimeric hapten (GNET) was developed that combines chemically-stable structural features from steady-state haptens with the hydrolytic functionality present in transition-state mimetic haptens. Additionally, as a further investigation into the generation of an improved bifunctional antibody pool, sequential vaccination with steady-state and transition-state mimetic haptens was undertaken. While GNET induced the formation of catalytically-active antibodies, it did not improve overall behavioral efficacy. In contrast, the resulting pool of antibodies from GNE/GNT co-administration demonstrated intermediate efficacy as compared to antibodies developed from either hapten alone. Overall, improved antibody catalytic efficiency appears necessary to achieve the synergistic benefits of combining cocaine hydrolysis with peripheral sequestration.     

Simple method for selecting catalytic monoclonal antibodies that exhibit turnover and specificity

Monoclonal antibodies were raised against a mono-p-nitrophenyl phosphonate ester to elicit catalytic antibodies capable of hydrolyzing the analogous p-nitrophenyl ester or carbonate. Potential catalytic antibody producing clones were selected, by use of a competitive inhibition assay, on the basis of their affinity for a "short" transition-state analogue, a truncated hapten which maximizes the relative contribution of the transition-state structural elements to binding. Of 30-40 clones that would have been examined on the basis of hapten binding alone, 7 were selected and 4 of these catalyzed the hydrolysis of the relevant p-nitrophenyl ester. This competitive inhibition technique represents a general approach for selecting potential catalytic antibodies and significantly increases the probability of obtaining efficient catalytic monoclonal antibodies. Further study of the catalytic antibodies revealed significant rate enhancement (kcat/kuncat approximately 10(4)) and substrate specificity for the hydrolysis of the analogous ester and, for three of the antibodies, of the analogous carbonate. The antibodies displayed turnover, an essential feature of enzymes. Evidence that catalysis occurred at the antibody combining sites was provided by the identity of the binding and the catalysis-inhibition specificity patterns.     

Immunochemical study of polysaccharide antigen in Streptococcus sobrinus and Streptococcus downei with a cross-reactive monoclonal antibody

Abstract A monoclonal antibody (mAb h-448) was prepared after cell fusion of mouse myeloma cells(SP2/0-Ag-14) to the spleen cells of mice immunised with serotype h strain (MF25) of Streptococcus downei . The antibody (IgM class) reacted in enzyme immunoassay only with whole cells as well as purified polysaccharide (PS) antigen of Streptococcus sobrinus (types d and g) and Streptococcus downei (serotypy h), but not with cells or purified PS antigen from any other serotypes of the mutants group of streptococci. mAb h-448 also quantitatively precipitated in solution with the purified antigens. Competitive hapten inhibition tests demonstrated that β-methylgalactopyranoside inhibited the reaction most strongly. Although rhamnose also showed a substantial inhibitory effect, the results of this study indicate that the antigenic determinant of the PS antigen has a structure similar to the β-methylgalactopyranoside molecule.     

Double-modified albumins as a tool for antibody purification

A general approach for anti-hapten antibody purification utilizing double-modified albumins is presented. Purification is based on simultaneous modification of an albumin with a hapten (e.g. fluorescein) and desthiobiotin. Three distinct albumins (BSA, HSA and ovalbumin) were modified accordingly and evaluated for their ability to purify the anti-fluorescein mAb from a mixture of commercial preparation and an E. coli cell lysate. The recovered mAb was obtained at relatively high purity (88-95%), in a wide range of target concentrations (0.66-0.02 mg/ml) within a total purification time of approximately 20 min. Substantial increase in the contamination background did not affect purity.     

Cocaine catalytic antibodies: the primary importance of linker effects

Current treatments for cocaine addiction are not effective. The development of a catalytic monoclonal antibody (mAb) provides a strategy for not only binding, but also degrading cocaine, which offers a broad-based therapy. Hapten design is the central element for programming antibody catalysis. The characteristics of the linker used in classic transition-state analogue phosphonate haptens were shown to be important for obtaining mAbs that hydrolyze the benzoate ester of cocaine.     

Characterization of a Monoclonal Antibody Specific for Lipoteichoic Acid from Various Gram-Positive Bacteria

A hybrid cell line, 3G6, producing monoclonal antibody (mAb) against the polyglycerophosphate (PGP) backbone of lipoteichoic acids has been derived by the polyethylene glycol-induced fusion of mouse myeloma cells and spleen cells from mice immunized with partially purified glucosyltransferase from culture supernatant of Streptococcus mutans strain 6715. Immunodiffusion tests and ELISA revealed that the antibody reacted with purified PGP from group A Streptococcus pyogenes strain Sv as well as crude phenol-water and saline extracts of various gram-positive bacteria except for a few species such as biotype B S. sanguis, Micrococcus sp., and Actinomyces viscosus. Whole cells of serotype b S. mutans and Staphylococcus epidermidis were agglutinated upon addition of 3G6 mAb, while those of most other species were not significantly affected by this procedure. A hapten inhibition study showed that glycerophosphate was only a potent inhibitor of passive hemagglutination reactions between LTA coated sheep erythrocytes and 3G6 mAb.     

Monoclonal antipeptide antibodies as tools to dissect closely related gene products. A model using peptides encoded by the calcitonin gene

We have explored the possibility of using mAb as tools for distinguishing between closely related gene products. We utilized calcitonin (CT) gene products as a model, because this 32-amino-acid-amidated hormone is biosynthesized by post-translational processing of a larger precursor. By using CT as a hapten, we had previously identified a mAb (CT07) with restricted specificity to mature CT, and had shown that another mAb (CT08) directed to a different epitope bound to both CT and the CT precursor. In this study, we used synthetic peptides analogous to various regions of biosynthetic intermediates of CT as haptens, and generated a library of mAb which define distinct epitopes. First, we identified two separate epitopes located in either the 1-11 or the 12-21 region of the C-terminal flanking peptide of CT (katacalcin, KC), and which were recognized by mAb KC01 and KC04, respectively. Second, we identified a conformational epitope in the C-terminal region of the putative glycine-extended form of CT (CT-Gly). This epitope was recognized by mAb CT19 and was shared by mature CT but not by CT precursors. Third, we identified an epitope restricted to CT-Gly and recognized by mAb CT20. For dissecting between related products of the CT gene, we designed different monoclonal immunoradiometric assays (m-IRMA) based on CT08 as the radiolabeled indicator antibody. A first m-IRMA based on CT07 as the capture antibody specifically recognized mature CT and did not cross-react with CT precursors. Conversely, another m-IRMA with KC01 as the capture antibody was specific for CT precursors and did not cross-react with either mature CT or CT-Gly. A third assay based on CT20 as the capture mAb was specific for CT-Gly and was not affected by the presence of either CT precursors or mature CT. We also used these antibodies to demonstrate that neoplastic C cells incompletely released processed CT precursors in serum, in addition to mature CT. This study demonstrates that mAb can be used as tools to selectively recognize closely related gene products. These findings might be applied to the study of other molecules biosynthesized by enzymatic modifications of a larger precursor.     

The in vivo elimination of CD4+ T cells prevents the induction but not the expression of carrier-induced epitopic suppression.

Injection of mice with an immunogenic dose of carrier (keyhole limpet hemocyanin (KLH)) followed by immunization with hapten-carrier conjugate (TNP-KLH) selectively suppresses anti-hapten antibody response. In this study, the cellular basis of this epitopic suppression and also of the suppression induced by a high dose of carrier were analyzed by in vivo depletion of CD4+ or CD8+ T cell subsets by using mAb. The mAb treatments were performed either at the time of carrier priming or at the time of hapten-carrier immunization. The elimination of CD8+ T cells has not modified the anti-carrier antibody response, whether this treatment was performed at the time of KLH-priming or during TNP-KLH immunization. Moreover, the in vivo treatment with the anti-CD8 mAb did not modify the carrier-induced epitopic suppression induced either by a low immunogenic dose of KLH or by a high dose of this Ag. The elimination of CD4+ T cells at the time of KLH immunization has prevented the induction of a memory response to KLH, clearly establishing that CD4+ T cells are essential in memory B cell development to T-dependent Ag. Moreover, this treatment has totally abrogated the epitopic suppression induced either by low or high dosages of KLH. In contrast, the in vivo elimination of CD4+ T cells after carrier immunization did not abolish the secondary anti-carrier antibody response and did not prevent the expression of epitopic suppression. These data indicate that primed CD4+ T cells are required neither for memory B cell expression nor for the expression of suppression. Finally, once induced, the suppression can be evidenced after in vivo depletion of both primed CD4+ and CD8+ T cells. These data support the view that epitopic suppression is induced through the expansion of carrier-specific B cells and resulted from intramolecular antigenic competition between hapten and carrier epitopes.     

A synthetic peptide corresponding to the phosphorylcholine (PC)-binding region of human C-reactive protein possesses the TEPC-15 myeloma PC-idiotype.

C-reactive protein (CRP) is a major acute phase reactant in most mammalian species. CRP molecules from all species display Ca2(+)-dependent binding to phosphorylcholine (PC). The conserved PC-binding region of CRP corresponds to amino acids 51-66 within the human CRP sequence. A synthetic peptide composed of residues 47-63 of human CRP was previously shown to possess PC binding activity. The charged amino acids at positions 57, 58, 60, and 62 of this synthetic peptide were critical for PC-binding based on lower binding activity of synthetic peptides containing uncharged residues at these positions. The PC-binding peptide was used to generate mouse mAb that were tested for reactivity with intact CRP and with the TEPC-15 (T-15) mouse myeloma protein that also binds PC. The PC-binding peptide of CRP was recognized by two mAb specific for the T-15 Id. One of the mAb generated against the PC-binding peptide of CRP (IID6.2) recognized an epitope on the T-15 protein that was also recognized by the near-binding site-specific mAb (F6) to the T-15 PC-Id. Binding of IID6.2 to T-15 myeloma protein was not inhibited by PC and did not require Ca2+; however, binding was inhibited by the synthetic PC-binding peptide itself. Recognition of synthetic peptides containing uncharged amino acid substitutions by mAb F6 and IID6.2 was greatly reduced indicating that the shared epitope on T-15 and CRP was composed of similar charged residues. Therefore, CRP displays the same idiotope as an antibody that shares its specificity for the hapten, PC.