Antibodies hapten design

Xu Y, Yamamoto N, Janda KD. Catalytic antibodies hapten design strategies and screening methods. Biooig. Med. Chem. 2004 12(20) 5247-5268. 

Catalytic antibodies hapten design strategies and screening methods. 

While the presentation thus far has emphasized the programmed relationship of hapten design and consequent antibody catalytic activity, there is a growing number of examples where the detailed examination of catalysis reveals mechanistic features that were not evidently design features of the system at the outset. Such discoveries are clearly a strength rather than a weakness of... 

From the results reported to date, it seems that the manner in which haptens are attached to carrier proteins leads to significant differences in certain cases. Clearly, haptens designed with aromatic moieties between the linkage to the immunogenic carrier protein and the TSA motif often have better antibody recognition. Recently, Hilvert pointed out that on both micro and macro levels, mechanistic improvements arise as a function of time. The differences in time scales for the evolution of natural enzymes and antibodies — millions of years versus weeks or months — also appear to be an explanation of the low efficiency of antibody catalysts. He also highlighted that the unique immunoglobulin fold has not been adopted by nature as one of the common scaffolds on which to build enzyme catalytic machinery. Therefore, antibody structure itself places limitations on the kind of reactions amenable to catalysis. 

Hapten design strategy for generation of an active site with a suitable catalytic residue has been further demonstrated. Thus, catalytic antibody 43D4-3D12, which was generated against the tertiary amine (61), has been found to catalyse the selective... 

Naturally occurring redox enzymes have been successfully exploited for asymmetric synthesis for some years.1 Although impressive chemo-, regio-, and enantioselectivities have been achieved in some cases, these biocatalysts have prescribed selectivity and often require expensive cofactors that must be recycled for preparative work. Catalytic antibodies offer an attractive alternative, since they are not limited a priori by Nature s choices. Thus the need for cofactor recycling can be circumvented through the use of inexpensive oxidants and reductants, and, as we have seen above, selectivity can be tailored through appropriate hapten design. 

In our original hapten design for aldolase antibodies, the /3-diketone functionality of hapten 4 was used as a reactive immunogen to trap a chemically reactive lysine residue in the active site of an antibody as a stable enaminone. The chemical mechanism leading up to the stabilized enaminone should match that of Class I aldolases over this portion of the reaction coordinate. 

Success in the synthesis of new catalytic antibodies (CAs) depends on the efficiency of each of the following steps 1) hapten design, 2) immunogen synthesis, 3) preparation of the enzymatic tracer 4) generation and purification of antibodies and 5) kinetic assays. 

DeSilva, B. S., Orosz, G., Egodage, K. L., Carlson, R. G., Schowen, R. L., Wilson, G. S. (2000) Catalytic antibodies for complex reactions hapten design and the importance of screening for catalysis in the generation of catalytic antibodies for the NDA/CN reaction, Appl. Biochem. andBiotechn. 83, 195-208. 

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... 

A merging of chemistry and biology is essential to effectively probe the immune system for catalytic antibodies (Fig. 3). Haptens that are successful in eliciting catalytic antibodies are variations of the central theme that transition state stabilization in the antibody combining site will yield functional catalysts for a desired chemical reaction. The evolution of hapten design will be discussed further in subsequent sections. Once the hapten is selected and synthesized, it is attached to an immunogenic carrier protein, usually via an amide bond, for hyperimmunization. A preliminary screen for antibodies that bind the hapten using an enzyme-linked immunosorbent assay (ELISA) is followed by another screen for catalysis of the reaction for which the hapten... 

The conformational changes and charge distribution along the reaction coordinate of a chemical transformation are fundamental to hapten design. Catalytic antibodies are designed to mimic the catalytic power of an enzyme, which, in part, stems from the stabilization of the high energy transition state. 

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