Antibodies reactive immunization

Wirsching, P, et al., 1995. Reactive immunization. Science 270 1775-1783. Description of reactive immunization, in which a highly reactive compound is used as antigen. Antibodies raised against such an antigen show catalytic activity for tlie chemical reaction that the antigen undergoes. 

Like many other antibodies, the activity of antibody 14D9 is sufficient for preparative application, yet it remains modest when compared to that of enzymes. The protein is relatively difficult to produce, although a recombinant format as a fusion vdth the NusA protein was found to provide the antibody in soluble form with good activity [20]. It should be mentioned that aldolase catalytic antibodies operating by an enamine mechanism, obtained by the principle of reactive immunization mentioned above [15], represent another example of enantioselective antibodies, which have proven to be preparatively useful in organic synthesis [21]. One such aldolase antibody, antibody 38C2, is commercially available and provides a useful alternative to natural aldolases to prepare a variety of enantiomerically pure aldol products, which are otherwise difficult to prepare, allovdng applications in natural product synthesis [22]. 

Figure 17 Generation, by the reactive immunization strategy, of antibody SP049H that catalyzes the hydrolysis of phosphonate diester 24 and that of ester 25.

Figure 19 A success of the reactive immunization strategy. Aldolization reaction catalyzed by antibody 38C2 raised against a /3-, 3-diketone hapten.

In order to improve aldolase antibodies, Zong et al employed reactive immunization in combination with transition state theory. Based on hapten 30, a hybrid, hapten 31, was designed, recruiting not only a sulfone... 

Primary antibody reactive against the desired antigen from the same species as that used for the PAP immune complex see Note 2). 

Antibody Catalysis. Recent advances in biocatalysis have led to the generation of catalytic antibodies exhibiting aldolase activity by Lemer and Barbas. The antibody-catalyzed aldol addition reactions display remarkable enantioselectivity and substrate scope [18]. The requisite antibodies were produced through the process of reactive immunization wherein antibodies were raised against a [Tdiketone hapten. During the selection process, the presence of a suitably oriented lysine leads to the condensation of the -amine with the hapten. The formation of enaminone at the active site results in a molecular imprint that leads to the production of antibodies that function as aldol catalysts via a lysine-dependent class I aldolase mechanism (Eq. 8B2.12). 

G. Zhong, R. A. Lerner, and C. F. Barbas III, Enhancement of the repertoire of catalytic antibodies with aldolase activity by combination of reactive immunization and transition state theory, Angew. Chem. Int. 

Viruses. Rabbits have been induced to produce anti-RNP antibodies by immunization with the p30 gag protein, a protein of several mammalian C-type viruses (Ql). Blomberg et al. found an increased frequency of antibodies that were cross-reactive with baboon endogenous retrovirus and murine leukemia virus among 72 SLE patients compared with 88 controls (B17). Plotz found that autoantibodies in patients with lupus are antiidiotype antibodies to antiviral antibodies (P4). 

The use of reactive immunization to generate catalytic antibodies (or abzymes) that catalyze aldolase reactions has been described, offering additional utility for this synthetically useful transformation.260 Two such abzymes, 38C2 and 84G3, are available commercially and their respective, diverse activities have been described.261-262... 

Second, methods for the characterization of complex antisera are difficult. Antisera to E. coli protein mixtures have been developed with impressive spectra of reactivity using conventional immunization methods (6,22-23). An exact assessment of the spectrum of antibody reactivity is often limited, however, by the resolution of the analytical methods used. Counter immunoelectrophoresis is limited by the relatively low sensitivity of detection and resolution for complex mixtures of reacting species. One dimensional silver stained SDS-PAGE and immunoblotting provides sensitive detection limits but lacks resolution. Therefore, methods which have a high degree of resolution and sensitivity are required to best compare potential improvements in the production of antibodies to minor components in the mixture. 

The best method to determine the success of antibody production to the minor components was made by two dimensional SDS-PAGE and immunoblotting (24). A comparison of the antisera (day 112 antisera) from the three groups demonstrated that the cascade immunization antisera detected a number of minor components (Figure 4C, arrows) which were not observed with the conventional or passive antisera (Figure 4B and D). It was clear from these results that the cascade antisera was far superior in its spectrum of antibody reactivity and, in fact, was comparable or superior in detection of ECPs to silver stain (Figure 4A). Although silver stain appeared to have an improved detection of certain low MW or basic proteins,... 

These data suggested that a mechanism of early priming of the immune response though the cascade procedure resulted in a broader spectrum of antibody reactivity. This improvement also required additional time (56 days) and/or subsequent injections of the total antigen mixture because similar experiments with day 56 antisera demonstrated equivalent antisera reactivity (24). 

The antigenicity, rather than the immunogenicity, should drive the research and development of new VLP-based vaccines and thus antibody reactivity tests should be performed as early in the process as possible. For example, a porcine parvovirus vaccine is composed of a single viral protein (VP2), which represents 95% of the native virus total protein and is able do induce antibody production in immunized animals (Rueda et al., 1999). In contrast, the human parvovirus B19 contains the exact same proportion of VP2 in the native virus but VLPs made solely of VP2 are unable to induce neutralizing antibodies (Brown et al., 1991 Tsao et al., 1996). In this case even a VLP containing VP1 and VP2 at a ratio of 1 24, respectively, which is very similar to that of the native virus, was not... 

In contrast to human antibodies derived from large naive or synthetic human antibody libraries, antibodies from immune animals were subjected to in vivo selection and are therefore more likely to recognize a given antigen selectively (i.e., without cross-reactivity to another antigen). 

Reactive immunization is a fundamentally different approach to selecting antibody pockets that contain functional groups. This method employs mechanism-based inhibitors as haptens these molecules react covalently with appropriately functionalized antibodies, allowing direct selection of active clones from large pools of inactive variants. When a suitable substrate is used in place of the inhibitor, reactive residues in the selected antibodies can often mediate its conversion into product. 

Aldolase antibodies obtained by reactive immunization are notable for high activity, broad substrate specificity, and high selectivities [53]. Rate accelerations are typically in the range 105 to 107-fold over background. Although the k /K values are 102 to 104 lower than those of aldolase enzymes, these are among the most efficient antibody catalysts described to date. Their efficacy is all the more notable in light of the inherently complex, multistep process they catalyze. 

Scheme 4.8 Hapten 17, designed to combine transition state mimicry and reactive immunization strategies, produced an aldolase antibody (84C3) that promotes aldol reactions with typically higher rates and selectivitiesthan antibodies raised against 15. The retro-aldol reaction of 18 is catalyzed with notable efficiency by this antibody.

Current and potential applications of catalytic antibodies in reactive immunization, therapy, biochemical analysis and biotechnology have been discussed (Schultz and Lemer, 1995 Rader and List, 2000 Blackburn and Garcon, 2000 Hilvert, 2000 Rader and List, 2000 Blackburn and Garcon, 2000 Vayron, 2000a,b). 

The development of the concept of reactive immunization yielded more effective antibody aldolases.119-120 In this new approach, rather than raise antibodies against an unreactive hapten designed to mimic the transition state, the antibodies were raised against a reactive moiety. Specifically, a p-diketone that serves as a chemical trap to imprint a lysine residue in the active site of the Ab (Scheme 5.65) was used.340 A reactive lysine is a requirement of the type I aldolase mechanism. By this method two aldolase catalytic antibodies, 38C2 and 33F12 were identified.119... 

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