Efficient and selective p-nitrophenyl-ester-hydrolyzing antibodies elicited by a p-nitrobenzyl phosphonate hapten.

A number of monoclonal antibodies elicited against a nitrobenzyl (Nbzl)-phosphonate transition-state analogue (TSA), and which were selected for the hydrolysis of the corresponding Nbzl-ester, were also found to catalyze the hydrolysis of the analogous p-nitrophenyl(Np) ester with notable efficiency and specificity. The activity towards the Np-ester is higher in terms of rates (k(cat); as expected from the higher intrinsic reactivity of Np-esters); however, the rate acceleration (k(cat)/k(uncat)) is close to or lower than that observed with the Nbzl-ester. Unexpectedly, the affinity to the Np-ester substrate (1/K(M)) and therefore k(cat)/K(M) are significantly higher. The best example is antibody D2.4 having a k(cat)/K(M) value of 64 s(-1) x M(-1) with the Nbzl-ester and 9400 s(-1) x M(-1) with the Np-ester. Moreover, due to a lower product inhibition by p-nitrophenol relative to p-nitrobenzyl alcohol, these antibodies exhibit more than 1000 turnovers with the Np-ester. The differential affinity of these antibodies to the Nbzl-phosphonate TSA versus the Nbzl-ester substrate (K(S)/K(TSA) or K(M)/K(i)) correlates well with the observed rate enhancement (k(cat)/k(uncat)). For the Np-ester, however, stabilisation of the transition state (as reflected by K(S)/K(TSA) and by the catalytic proficiencies, k(cat)/K(M)/k(uncat)) does not fully account for the catalytic power (k(cat)/k(uncat)), indicating a more complex catalytic mechanism than simply transition-state stabilization. A comparison of the kinetic parameters of D2.4 with other Np-ester-hydrolyzing antibodies raised against Np-phosphonate haptens emphasizes the marked advantage of this antibody which was elicited against an Nbzl-phosphonate hapten. These results appear to be general: anti-(Nbzl-phosphonate TSA) antibodies obtained from other mouse strains and using different immunization protocols are also efficient Np-esterases. They demonstrate the use of an expanded TSA-hapten, where a spacer (a methylene group) mimics bonds that are partially cleaved in the transition state of the catalyzed reaction.

Structural convergence in the active sites of a family of catalytic antibodies.

The x-ray structures of three esterase-like catalytic antibodies identified by screening for catalytic activity the entire hybridoma repertoire, elicited in response to a phosphonate transition state analog (TSA) hapten, were analyzed. The high resolution structures account for catalysis by transition state stabilization, and in all three antibodies a tyrosine residue participates in the oxyanion hole. Despite significant conformational differences in their combining sites, the three antibodies, which are the most efficient among those elicited, achieve catalysis in essentially the same mode, suggesting that evolution for binding to a single TSA followed by screening for catalysis lead to antibodies with structural convergence.

Unexpectedly high occurrence of catalytic antibodies in MRL/lpr and SJL mice immunized with a transition-state analog: is there a linkage to autoimmunity?

Upon testing the ability of several strains of mice to elicit esterolytic antibodies after immunization with a p-nitrobenzyl phosphonate hapten, we have found that the occurrence of catalytic antibodies in SJL and MRL/lpr autoimmune mice is dramatically higher than in normal mouse strains (e.g., the wild-type MRL/++ or BALB/c). Fewer than 10 catalytic clones are usually obtained from a single fusion of lymphocytes taken from normal mice, whereas several hundred catalytic clones are obtained in SJL or MRL/lpr mice. Differences in the numbers of hapten-binding clones do not account for the high occurrences of catalytic clones in these strains. This phenomenon prevailed in the early responses; in both SJL and MRL/lpr mice a significant decline in the appearance of catalytic clones was observed after multiple immunizations. Esterolytic antibodies were not found in MRL/lpr mice immunized with haptens that do not mimic the transition state for the hydrolysis of the ester substrate (e.g., with a substrate analog). The catalytic antibodies manifest high specificity to the antigen and variability in their binding and catalytic properties. The use of autoimmunity-prone mice may greatly expand the repertoire of catalytic clones elicited against a transition-state analog hapten. More intriguing is the possible linkage between autoimmunity and the appearance of catalytic antibodies. These results suggest that there is normally a selection against the expression of certain variable genes encoding antibodies with catalytic activity.

pH on-off switching of antibody-hapten binding by site-specific chemical modification of tyrosine.

Tetranitromethane (TNM) chemically mutates the binding sites of antibodies so that the nitrated antibodies exhibit pH-dependent binding near physiological pH. Three monoclonal antibodies were selectively modified, each under different conditions, with the resultant loss of binding activity at pH > 8 which is recovered at pH < 6. Recovery and loss of binding are ascribed to the protonation and deprotonation, respectively, of the hydroxyl group of the resulting 3-nitrotyrosine side chain (pKa approximately 7) at the binding site of these antibodies. pH on-off dependency of binding activity, common to all TNM-modified antibodies studied by us so far, may find use in a variety of applications in which controlled modulation under mild conditions is required.

catELISA: a facile general route to catalytic antibodies.

The low abundance and activity of catalytic antibodies are major obstacles to their selection from the virtually unlimited repertoire of antibody binding sites. The requirement for new screening methodologies is further emphasized by the availability of combinatorial libraries, in which a functional polypeptide has to be selected out of millions of possibilities. We present a simple and sensitive screening approach (termed catELISA) based on immobilized substrates and immunodetection of the end product of the catalyzed reaction. The feasibility of catELISA is demonstrated here by the generation of potent ester-hydrolyzing antibodies by direct screening of hybridoma supernatants. We show that this approach is not only facile but general: it is not limited by type of reaction, substrate, or catalyst (enzymes, catalytic antibodies, chemical catalysts). catELISA opens a route to catalytic antibodies that replaces existing lengthy and arduous methods, thus allowing us to expand their number and improve their quality and to address questions that would otherwise be difficult to answer.