Section 7.4  

Catalytic Antibodies

Imagine being able to design a chemical reaction in much the same way that a computer programmer designs a software program. Perhaps you want to create an enzyme that detoxifies a drug in the bloodstream, destroys a virus, or targets tumor cells. These custom-made enzymes are becoming a reality with recent advances in the production of catalytic antibodies, also called "abzymes."

Antibodies molecules are produced by the immune system to bind and neutralize foreign substances called antigens. Foreign proteins of bacteria and viruses, as well as some small chemical molecules called haptens, act as antigens, and elicit the production of antibodies to protect the host from harm. In fact, the human body is capable of producing antibodies to virtually any encountered antigen. Each antibody binds its own unique target similar to a key fitting in a lock.

Animation 1: antibody structure and antigen

Structurally, antibodies (immunoglobulin G-type) are "Y-shaped" molecules (see animation 1). They consist of two identical heterodimers joined together by disulfide bonds. Each heterodimer consists of a short peptide called the light chain and a longer peptide called the heavy chain. The heavy and light chains are also joined together by disulfide bonds. One end of the antibody contains conserved regions, called constant domains (Fc, CH 1-3), that are formed by the interface of two of the two heavy chains. CH domains have similar amino acid compositions in most IgG’s, despite the antigen to which the antibody binds. The opposite end of the molecule (Fv) is variable in structure and amino acid sequence, however. These variable domains are responsible for specifically binding antigen. The interface of two of the heavy and light chain variable regions (VL and VH) forms a single deep pocket (antigen binding site) that molds to the shape of the antigen. Notice that each IgG molecule contains two identical antigen binding sites because of the "Y-like" shape. "Hot spots" within the variable domains are called complementarity determining regions (CDRs). Amino acids within the CDRs specifically contact the antigen via non-covalent interactions to mediate binding to the foreign particles.

In most cases, antibodies tightly bind the antigen, but do not specifically alter its chemical nature. Natural enzymes within the body, on the other hand, bind biomolecules and subsequently catalyze their conversion to new products. According to "transition state theory," enzymes catalyze a reaction by stabilizing the chemical intermediate, or transition state, between substrates and products <link to enzyme unit>. Formation of this transition state geometry is energetically unfavorable in the absence of enzyme. However, enzymes provide the chemical momentum (activation energy) to push a reaction through its transition state. The net result of enzyme catalysis is the acceleration of the reaction rate.

Theoretically, if an antibody binds to a transition-state molecule, it may be expected to catalyze a corresponding chemical reaction by forcing substrates into transition-state geometry. But how can an antibody be raised against such a fleetingly unstable chemical? The answer lies in the synthesis of "look-alikes" called transition-state analogs. These molecules are more stable than the transition state itself, but mimic its three-dimensional structure. If injected into the bloodstream of an animal, transition state analogs act as haptens, and elicit antibody production. Antibodies are isolated from the serum of the animal, and then screened by experimental assays to determine which catalyze the selected reaction.

In 1986, Peter Schultz and Richard Lerner demonstrated the feasibility of this proposal by generating abzymes that catalyze ester hydrolysis, the breakage of an ester bond through the addition of water. Animation 2 illustrates the hydrolysis reaction of p-nitrobenzoate. The mechanism involves the nucleophilic attack of the oxygen atom of water on the carbonyl atom in p-nitrobenzoate. This interaction produces a transition state with a tetrahedral geometry. Organic chemists have synthesized an analog of the proposed intermediate, also shown in Figure 2. By replacing the carbon at the center of the tetrahedron with phosphate, the chemical is stable for synthesis and injection into laboratory animals. The rates of reactions catalyzed with abzymes, as measured by kinetic parameters such as KM and Vmax, are up to a million-fold greater than the corresponding uncatalyzed reactions; however, in many cases, catalytic antibodies have not yet approached the rates of reactions catalyzed by natural enzymes. Since the experiments of Schultz and Lerner, however, over 100 different abzymes have been generated that catalyze a wide variety of reactions.

Animation 2: geometry of the hydrolysis of p-nitrobenzoate

Through the use of protein engineering, abzyme catalysis can be improved even further, perhaps even to surpass the activity of natural enzymes. Molecular biologists have developed methods to clone the array of genes that encode IgG molecules. The millions of gene products from an immunized animal are screened for the production of antibodies with desirable catalytic activites. Once candidates are isolated, these so-called "recombinant antibodies" can be produced in bacteria in large amounts. In this way, an antibody gene can be "immortalized" for unlimited study.

Figure 3: cocaine detoxification

The gene encoding a selected recombinant antibody can be subsequently altered (mutated), selectively or randomly, in attempts to improve the activity of the original catalytic antibody. Scientists often target the CDR’s for mutational analysis since these regions contain amino acids that directly contact the antigen. The resulting protein mutants are once again screened for improvement in function. In this way, a starting abzyme with moderate catalytic activity can be vastly improved.

Catalytic antibodies have great potential in the pharmaceutical industries. Abzymes have been implicated for use in the detoxification of cocaine. Catalytic antibodies have been generated that cleave the cocaine molecule at specific bonds (Figure 3), thereby eliminating the toxic effect of the drug. Notice the similarity between the hydrolysis of cocaine and the hydrolysis of p-nitrobenzoate in Figure 2. Both reactions proceed through tetrahedral intermediates and the transition-state analogs mimic this geometry. As a pharmaceutical reagent, anti-cocaine abzymes could treat patients who are addicted to cocaine, or reverse the lethal effects of a cocaine overdose.

Figure 4: Anti-cancer tumer-targetting abzyme

Perhaps the most exciting application of abyzme technology is the specific targeting of cancer cells for destruction. Cancer cells contain unique determinants, called tumor cell antigens, on their surface that are lacking in normal cells. By utilizing antibodies that specifically bind these tumor cell antigens, cancer drugs can be delivered directly to the tumor. In the case of abzymes, scientists have envisioned antibodies with two distinct antigen binding sites (Figure 4): one site binds with high affinity to a tumor cell antigen, while the second site catalyzes the cleavage of a prodrug. The prodrug is a non-toxic precursor of a cytotoxic drug. First, the antibody is administered to patients, and it binds the tumor cells with high affinity. Secondly, the prodrug is introduced into the bloodstream, but only becomes activated in the vicinity of the targeted antibody. By this technique, tumors are selectively destroyed while healthy cells are spared from the toxic affect of cancer drugs.

While still in the early stages, other reports have indicated possible uses of abzymes to inactivate viruses. For instance, abzymes have been isolated that cleave viral coat proteins of human immunodeficiency virus (HIV). Researchers have also developed abzymes that catalyze the specific destruction of viral genes. Perhaps in the future, we will have the tools to treat a wide variety of diseases through the use of catalytic antibody technology.

Copyright 2002, John Wiley & Sons Publishers, Inc.