Catalytic Antibodies

The ability to directly design proteins that efficiently perform predefined tasks would have a profound impact on science and on the everyday life of human beings. Designer proteins might enable us to dissect biological pathways and mechanisms, rapidly create and synthesize new drugs, use natural resources efficiently, and create new agricultural products. These proteins might help explain our past and define our future.

Two problems that thwart the realization of this goal are the protein-folding problem and the chemistry of catalysis. An alternative approach to the production of designer protein catalysts was developed in 1986 by the laboratories of Lerner and Schultz. This work gave rise to a new area of investigation: catalytic antibodies. A large part of this work is built on the Haldane-Pauling hypothesis of transition-state stabilization as a primary effector of catalysis.

In our laboratory, we are extending and refining approaches to catalytic antibodies by using novel recombinant strategies coupled with reactive immunization and chemical-event selections. We are happy to acknowledge our long-term collaborator in this endeavor, Prof. Richard A. Lerner. Together we are developing in vitro selection and evolutionary strategies as a route for obtaining antibodies of defined biological and chemical activity. This strategy involves the directed evolution of human as well as rodent antibodies and synthetic antibodies. Essentially, we are evolving proteins to function as efficient catalysts, a task that nature has performed in eons, we aim to complete in weeks. The approach is a blend of chemistry, enzymology, and molecular biology.

A major focus of our work is the development of strategies to produce antibodies that efficiently form and break carbon-carbon bonds. Much of this work centers on the chemistry of enamines and the development of antibodies that use covalent catalysis. The specific reactions we are examining are the aldol, the Michael, the Diels-Alder, and a variety of decarboxylation reactions.

To develop novel catalysts of the aldol reaction (aldolases), we developed the approach of reactive immunization for the aldol reaction and applied it towards programming a multi-step covalent reaction coordinate into antibodies. In this study a 1,3-dicarbonyl hapten was designed to act as a chemical and entropic trap to select for antibodies with a reactive lysine residue in their active site. Antibodies displaying the appropriate chemical reactivity, transform the hapten to form a covalently bound enaminone. The reaction mechanism that results in the formation of the hapten-antibody complex can then be recruited to catalyze aldol reactions. In this way, we can program the mechanism defined for natural class I aldolase enzymes into antibodies.

A product of these studies is antibody 38C2, the world's first commercially available catalytic antibody.

38C2 has been shown to catalyze the aldol addition of a wide variety of aliphatic open chain and aliphatic cyclic ketones to various aromatic and aliphatic aldehydes. More than 100 different substrate combinations - cross aldol and also intramolecular aldol reactions - have been identified. Solution of the X-ray crystal structure of the antibody with the Wilson laboratory has confirmed our approach to mechanistic programming of biocatalysts.

We have demonstrated the use of 38C2 as an efficient catalyst for the retro-aldol reaction, allowing for the kinetic resolution of racemic secondary aldols. By using both the forward (or synthetic) aldol and retro-aldol reactions, both aldol enantiomers become accessible (Scheme 1).

 Beyond nature's enzymes and traditional organic chemistry: tertiary aldols

Tertiary aldols contain a heteroatom-substituted quaternary carbon stereocenter, which constitutes one of the most demanding challenges in synthetic chemistry. General methods, either chemical or enzymatic, for the preparation of enantiomerically enriched tertiary aldols have not been developed. This is particularly true when this problem is approached through aldol chemistry. Nonetheless, tertiary aldols proved to be very good substrates for antibody catalyzed retro-aldol reactions with catalytic proficiencies, (k_cat/K_m)/k_un, in the range of 10¹⁰ M^-1. We have demonstrated that the antibody catalyzed approach provides a rapid entry to structurally varied and highly enantiomerically enriched tertiary aldols. This result highlights the potential synthetic utility of catalytic antibodies as artificial enzymes in addressing problems in organic chemistry that are not solved by natural enzymes or more traditional synthetic methods.

We hope that the catalysts we prepare will become commercially viable in the synthesis of enantiomerically pure drugs. With novel catalytic antibodies we have demonstrated the efficient asymmetric synthesis and resolution of a variety of molecules including tertiary and fluorinated aldols. Using an antibody-based synthon approach we have synthesized carbohydrates, insect pheromones, and derivatives of the epothilone anti-cancer drugs.

With improved hapten/reaction design, we have now created aldolase catalysts that are faster than 38C2 and as fast as nature's aldolases. We intend to go even further.

Catalytic Antibodies: Targeting Cancer with Prodrugs

Not only can catalytic antibodies be used to synthesize anti-cancer drugs, they can be used to deliver them in a highly specific fashion to the cancer itself. Chemotherapeutic regimes are typically limited by nonspecific toxicity. To address this problem we have developed a novel and broadly applicable drug masking chemistry that operates in conjunction with our unique broad scope catalytic antibody 38C2. This masking chemistry is applicable to a wide range of drugs since it is compatible with virtually any heteroatom. We have demonstrated that generic drug masking groups can be selectively removed by sequential retro-aldol-retro-Michael reactions catalyzed by antibody 38C2 (Fig. 1).

 
Fig. 1. Doxorubicin prodrug activation via a tandem retro-aldol-retro-Michael reaction catalyzed by antibody 38C2.

Fig. 2. Growth inhibition of LIM1215 human colon carcinoma cells in vitro by doxorubicin (closed squares) and prodoxorubicin (open squares)(Bars indicate SD; n = 4). Note the reduced capacity of prodoxorubicin for cell growth inhibition.

This reaction cascade is not catalyzed by any known natural enzyme. Application of this masking chemistry to the anticancer drugs doxorubicin and camptothecin produced prodrugs with substantially reduced toxicity (Fig. 2). These prodrugs are selectively unmasked by the catalytic antibody when it is applied at therapeutically relevant concentrations. We have demonstrated the efficacy of this approach using human colon and prostate cancer cell lines. The antibody demonstrated a long in vivo half-life described here has the potential to become a key tool in selective chemotherapeutic strategies. We hope to advance to animal models of cancer in the next year. If you would like to see catalytic antibodies in action check out our antibody/prodrug movie.

For more information, see our Aldolase Antibody Lab Manual.