SPECIAL REPORT
CATALYTIC
ANTIBODIES Peter G. Schultz, University of California, Berkeley; Richard A. Lerner, Research Institute of Scripps Clinic; and Stephen J. Benkovic, Pennsylvania State University
The immune system produces a vast repertoire—numbering in the hundreds of billions—of exquisitely specific antibodies that protect vertebrates from "foreign" invaders, such as pathogenic bacteria and viruses, parasites, and cancer cells. Hybridoma technology, which makes it possible to generate in vitro large amounts of homogeneous antibody molecules, has dramatically expanded the role of antibodies in biology and medicine. Antibodies have become invaluable tools in the detection, isolation, and analysis of biological materials and are key elements in many devices used to diagnose infectious diseases. They also hold considerable promise as highly selective therapeutic and imaging agents. But the potential of antibodies is not limited to biology and medicine; they offer exciting possibilities for chemists as well. In the past three years the diversity and specificity of the immune system have been merged with our understanding of chemical reactivity to generate a new class of antibody molecules—catalytic antibodies. This new technology makes possible the generation of antibodies that not only bind but also chemically transform a target molecule. Because antibodies can be elicited to a huge array of biopolymèrs, natural products, or synthetic molecules, catalytic antibodies offer a unique approach for generating tailormade, enzymelike catalysts. Catalytic antibodies might be used, for example, to develop a family of catalysts analogous to restriction enzymes that cleave proteins or sugars at a particular bond. Such antibodies would be invaluable reagents in biology and might find use as therapeutic agents to selectively hydrolyze protein or carbohydrate coats of viruses, cancer cells, or other physiological targets. Catalytic antibodies also could serve as selective catalysts for the synthesis of pharmaceuticals, fine chemicals, and novel materials. At the same time, the characterization of catalytic antibodies provides fundamental insight into important aspects of biological catalysis, including the importance of transition-state stabilization, proximity effects, general acid and base catalysts, electrophilic and nucleophilic catalysis, and strain. 26
May 28, 1990 C&EN
A number of approaches have been used to generate catalytic antibodies. The complementarity of an antibody to its corresponding hapten (the ligand against which the antibody is elicited) has been exploited to generate combining sites that are complementary to the rate-determining transition state, that act to overcome the entropy requirements involved in orienting reaction partners, or that contain an appropriately positioned catalytic amino-acid side chain or cofactor. Cata-
Antibody binding involves variable region of both light and heavy chains
Hypervariabli loops
The antibody binding site is encoded by the VH> D, and J H genes for the heavy chain and the VL and J L genes for the light chain, as shown on the right arm of this schematic representation of a typical antibody molecule. Within the variable regions are six hypervariable loops, three each In the light and heavy chain, as shown on the left arm of the diagram. The constant regions consist of CL for the light chain and C H 1, CH2, and CH3 for the heavy chain as well as the hinge region. The heavy and light chains of the antibody molecule are held together by a series of disulfide bonds.
Antibody combining sites can contain catalytic side chains lytic groups have also been intro duced directly into the combining site of an antibody by chemical modification and by site-directed mutagenesis. These strategies have led to the generation of antibodies that catalyze a wide array of chem ical and biological reactions, in cluding selective peptide bond cleavage, stereospecific ester hy drolysis, thymine dimer photocleavage, concerted Claisen rear rangements and Diels-Alder reac tions, elimination reactions, redox reactions, transacylation and lactonization reactions, and porphy rin metallation.
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Raising antibodies to the alkylammonium ion shown at left produces an anti body (red) with a negatively charged carboxylate in its combining site. When the antibody binds a β-fluoroketone, as shown below, the carboxylate is cor rectly aligned to abstract an α-carbon proton, thereby catalyzing the elimina tion of HF from the substrate.
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Immune system and antibodies Antibodies are large proteins (about 150,000 daltons in the case of an immunoglobulin G monomer) that consist of four polypeptide chains: two identical heavy chains of about 50,000 daltons and two identical light chains of about 25,000 daltons, crosslinked by disulfide bonds. The light chains are divided into two domains, VL (variable) and CL (constant), while the heavy chains consist of V H , C H 1, CH2, and CH3 domains. VH and VL (located in roughly the first 110 amino acids of the heavy and light chains) are highly polymorphic and change with each antigen (molecules recognized by antibodies are termed antigens) whereas the constant re gions are relatively invariant. The three-dimensional structures of at least a dozen antibody molecules, some in combination with their antigens, have been solved by x-ray crystallography. The basic fold of the variable region is that of an eightstranded β-barrel onto which six loops of extended chain have been grafted; three each from the light- and heavy-chain variable domains. These loop segments, which are often called hypervariable, display a high degree of sequence variability and provide the basis for the diversity of the antibody molecule. A number of gene segments encode both the light and the heavy chains, and different combinations of these segments— as well as different associations of heavy and light chains—generate a minimum of a 100 million different possible antibody molecules. The variable regions are also subject to mutations, which expands the baseline repertoire of combining sites still further. The specificity of antibodies was first documented in the 1940s by Karl Landsteiner of Rockefeller University in studies of polyclonal antibody sera. (Polyclonal anti bodies are heterogeneous mixtures of antibodies formed from a large number of antibody-producing cells). The high specificity of antibody-antigen interac tion is illustrated by the following examples: antibodies generated against ds-N-phenylmaleic acid monoamide bind the trans isomer with 1000 times lower affinity; antibodies against 3,17-androstanedione bind 3a,17-dihydroxyandrostane with 1000 times lower affinity; and antibodies against the tetrapeptide (L-alanine)4 bind glycine-(L-alanine)3 with 30-fold lower affinity.
Antibodies bind to molecules ranging in size from about 6 to 34 À with association constants that range from 104 to 1014 M"1. Crystal structures of antigen-antibody complexes reveal that when antigens are small molecules the molecule is typically bound in a cleft, but for large molecules the binding site is an extended surface that can cover 600 to 800 square angstrom. Water molecules are effectively excluded from the interacting surfaces, and binding occurs by van der Waals, hydrophobic, electrostatic, and hydrogen bonding interactions. How does the immune system evolve specific receptors on such a short time scale when enzymes acquired their specificity over millions of years of evolution? The mechanism of antibody diversity is clonal—each antibody molecule of the primary immunological repertoire is stored on the surface of cells called lymphocytes. When a cell—with a unique antibody expressed on its surface—encounters an antigen, it undergoes division and differentiation and starts to produce soluble antibody molecules. At the same time, the genes encoding the binding site of the antibody undergo mutation, leading to an increase in affinity for the hapten.
Making antibodies catalytic The specificity of antibodies makes them ideal starting points for developing selective catalysts for reactions that involve complex multifunctional substrates. For example, the design and synthesis of even small quantities of a combining site capable of discriminating between the tripeptides phenylalanine-leucine-alanine and phenylalanine-isoleucine-alanine or between lactose and glucose could take years. In contrast, hybridoma technology makes it possible to generate gram quantities of an antibody with just such a specificity in a few months. The challenge, then, is to develop general strategies for introducing catalytic activity into antibody combining sites. Many enzyme active sites contain nucleophilic, electrophilic, basic, or acidic amino acid side chains that are precisely positioned to react with a bound substrate. For example, serine 195 in chymotrypsin is acylated by May 28, 1990 C&EN
27
Special Report
Hybridoma technology is key to making and characterizing catalytic antibodies
Spleen
Spleen cells
nature of the myeloma parent and the crucial gene from the spleen parent. The hybridomas are then cloned, or separated into colonies, each of which produces a single, homogeneous antibody. The colonies are screened using an enzyme-linked immunosorbent assay (ELISA) for their ability to generate antibodies that bind selectively to the original antigen and not to the carrier protein.
Myeloma cells Fuse
Culture plate 21 Days Ε
:c
Keep hapten-specific hybrids Discard nonhaptenspecific hybrids
A major advance in immunology was the development of hybridoma technol ogy in 1975 by Cesar Milstein of the British Medical Research Council Labo ratories at Cambridge and Georges Kôhler, now at Max Planck Institute of Immunology in Freiburg, West Germany. In essence, individual clones of antibody-producing cells are given immortality, thereby allowing them to produce large quantities—grams or more—of homogeneous antibodies. The first step in this process involves immunization with a particular antigen, thereby causing the host organism to produce antibodies to the antigen. In order to generate antibodies to small molecules, the molecules must, in general, be linked to "carrier proteins" such as keyhole limpet hemocyanin. Couplings generally involve amide bond formation between carboxyl groups on haptens and e-amino groups
Screen (ELISA) hybrids for hapten ^^ifi^itw
specificity
of surface lysine residues on carrier proteins. Some haptens have been coupled via diazo linkages to surface tyrosine residues, disulfide exchange reactions, and reductive amination. Typically, a spacer between the hapten and carrier protein avoids any steric interference from the carrier protein. Antibody-producing spleen cells cannot be cultured in vitro and are therefore fused with myeloma cells, which grow avidly in vitro. The resulting cells—termed hybridomas—both produce antibodies and can be grown in cell culture. Hybridoma cells are selected by growing them on a medium that contains hypoxanthine, aminopterin, and thymine. Unfused myeloma cells, which lack a key enzyme that would allow them to use hypoxanthine as a nucleotide source, cannot live in this medium. Hybrid cells survive because they have both the neoplastic
the substrate, and glutamate 43 of staphylococcal nuclease is positioned to activate a water molecule for attack on substrate. The introduction of such amino acid side chains into an antibody combining site should be an effective method of catalyzing a variety of reactions including condensation, isomerization, and hydrolytic reactions. The high effective concentration of the catalytic group in the antibody combining site as well as favorable orbital alignment of reactants should lower considerably the entropy (S*) and enthalpy (Η φ ) of ac tivation for reaction. In the 1950s several investigators showed that elec trostatic interactions play an important role in the rec 28 May 28, 1990 C&EN
Selection
One form of ELISA, shown in the insert at lower left of the diagram, involves first binding the antigen to a solid support. Antibody-containing media are added, antigen-antibody complex forms, and the solid support is washed repeatedly to remove any unbound antibody. The complex is then assayed with a second antibody-enzyme complex that binds specifically to the constant region of the first antibody. Nanogram quantities of the antigen-antibody complex can be detected in this fashion. The ability to produce homogeneous, monoclonal antibodies in large amounts via hybridoma technology is necessary to characterize meaningfully and reproducibly the properties of a catalytic antibody. Purity is especially important if a natural enzyme also catalyzes the reaction of interest. For example, for an antibody with a catalytic turnover rate of about one turnover per minute and an enzyme with a turnover rate of about 30,000 per minute, contamination of the antibody with only one enzyme molecule per 60,000 molecules of antibody would suggest that the antibody was catalytic when, in fact, all observed activity was coming from the contaminating enzyme.
ognition of charged haptens by antibodies. They found that negatively charged aspartate or glutamate residues were present in the combining sites of antibodies raised toward p-azobenzenetrimethylammonium cation, and, conversely, positively charged arginine and lysine resi dues were present in the combining sites of antibodies elicited against negatively charged p-azobenzoate. One of us (Schultz), along with Kevan M. Shokat and coworkers, used this idea of electrostatic complementa rity to generate a carboxylate in an antibody combining site. The carboxylate is appropriately aligned to cata lyze the elimination of hydrogen fluoride from a β-fluoroketone by abstraction of an α-carbon proton. Six an-
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Special Report tibodies were generated to a hapten in which an ammo nium ion replaces the abstractable proton of the substrate. Four of the antibodies catalyzed the elimina tion reaction. As expected, the positively charged anti gen induced a carboxylate in the antibody within bonding distance of the abstractable proton. This car boxylate is a good base (pKa 6.2) at physiological pH, and isotope effect studies demonstrate that the rate-de termining step in the elimination reaction is deprotonation of the /3-fluoroketone. Antibody-catalyzed reactions, just as those catalyzed by enzymes, involve noncovalent complexation of sub strate by antibody, followed by conversion of substrate to product. These reactions demonstrate saturation ki netics and are in most cases characterized by the simpli fied kinetic scheme: A+S
*1
->
AS
A+P
*-1
V =
*cat[A T ][S] KM + [S]
KM =
+ fr_ *1
KM, the Michaelis constant, is equal to the concentra tion of substrate (S), which affords one half the maxi mal catalyzed rate (fccat[AT]); AT is the total antibody concentration; and kcat is the unimolecular rate constant for the catalytic step. Comparison of kcat/KM (the appar ent second order rate constant) for the above antibodycatalyzed reaction with that of the acetate-catalyzed elimination reaction affords a rate acceleration of about 100,000. This value reflects the contribution of proxim ity of substrate and a catalytic group in a protein bind ing site to rate enhancement. It compares with the rate acceleration of about 10,000 attributable to glutamate at position 43 in staphylococcal nuclease or aspartate at position 102 in trypsin. A second example in which hapten structure was used to induce a catalytic side chain in an antibody combining site involves the generation of antibodies that catalyze the light-dependent [2 + 2] cycloreversion reaction of a thymine dimer, the predominant DNA photolesion produced by ultraviolet light. Model stud ies have shown that photosensitizers such as indoles, quinones, or flavins can reversibly transfer an electron to or from a thymine dimer, resulting in facile cleavage of the intermediate thymine-dimer radical. These re sults suggest that an antibody combining site specific for a thymine dimer and containing an appropriately positioned sensitizer should act as a photorepair en zyme. It therefore seemed reasonable that antibodies generated against the polarized π system of a pyrimidine dimer might contain a complementary tryptophan residue in the combining site. Andrea G. Cochran, Schultz, and coworkers have shown that antibodies generated to a thymine-dimer derivative accelerate the conversion of thymine dimer to thymine in the presence of 300-nm light. Five of six antibodies isolated were active. The kcat of one antibody was 1.2 per minute, close to the kcat of 3.4 per minute for thymine-dimer cleavage by the repair enzyme, £s30
May 28, 1990 C&EN
Thymine dimer
For substrate: R = OH For antigen: R = NHCH2COOH
cherichia coli DNA photolyase. The quantum yield of the photocleavage reaction was greater than 0.75 (more than 3 out of 4 photons resulted in cleavage), compared with the quantum yield of approximately 1.0 for DNA photolyase. Further mechanistic experiments on this antibody should shed important insight into the mech anisms of DNA photorepair. These successes suggest that hapten-antibody com plementarity should prove to be a general strategy for generating catalytic antibodies. For example, antibodies to the appropriate diamines might generate two active site carboxylates that could catalyze glycosyl transfer reactions (analogous to the lysozyme mechanism) or amide bond hydrolysis (much like the aspartyl proteas es). Antibodies generated to aminocyclohexanes might catalyze an oxy-Cope rearrangement (via an anionic substituent effect) and antibodies generated to 2'aminouridine analogs might catalyze RNA hydrolysis (as does staphylococcal nuclease). Another important approach to the generation of cat alytic antibodies exploits the notion of transition-state stabilization. Linus Pauling first proposed more than 40 years ago that the fundamental difference between en zymes and antibodies is that enzymes have evolved to
Catalytic antibodies lower free energy of transition states Free energy
Rate-determining transition state
Uncatalyzed reaction
Reactants Products Reaction coordinate
Antibodies can catalyze reactions by stabilizing transition states. In the ideal case, the antibody selectively binds only the transition-state configuration. The difference in binding affinity of the antibody for the reactants and transition state is reflected in a lowered free energy of activation, A G * .
Antibodies raised to stable transition-state analogs atalyze hydrolysis reactions
V— Substrate
Π * Β
Υ°~
OADH
|_ S~
+ HOR'
Ο Products
5-
Transition stats
Transition-state analog
The hydrolysis of esters and carbonates involves formation of a tetrahedral, negatively charged tran sition state, as shown in the generalized reaction scheme above. The corresponding phosphonate is a stable transition-state analog, which can be used to produce antibodies that bind specifically to that transition-state configuration. The technique has been applied in several labs to produce antibodies that catalyze the hydrolysis of esters and carbonates. Several of these substrates, along with the corresponding transition-state analogs, are shown below.
CH3
F3C
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3
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