11 Catalytic Antibodies

GEORGE MICHAEL BLACKBURNARNAUD GARÇON

Sheffield, UK

1 Introduction 4041.1 Antibody Structure and Function 4041.2 The Search of a New Class of Biocatalyst 4041.3 Early Examples for Catalytic Antibodies 4061.4 Methods for Generating Catalytic Antibodies 407

2 Approaches to Hapten Design 4102.1 Transition State Analogs 4102.2 Bait and Switch 4122.3 Entropic Trapping 4152.4 Substrate Desolvation 4192.5 Supplementation of Chemical Functionality 420

3 Spontaneous Features of Antibody Catalysis 4213.1 Spontaneous Covalent Catalysis 4213.2 Spontaneous Metal Ion Catalysis 422

4 How Good are Catalytic Antibodies? 4235 Changing the Regio- and Stereochemistry of Reactions 426

5.1 Diels-Alder Cycloadditions 4265.2 Disfavored Regio- and Stereoselectivity 4275.3 Carbocation Cyclizations 430

6 Difficult Processes 4316.1 Resolution of Diastereoisomers 4326.2 Cleavage of Acetals and Glycosides 4326.3 Phosphate Ester Cleavage 4356.4 Amide Hydrolysis 438

7 Reactive Immunization 4398 Potential Medical Applications 441

8.1 Detoxification 4418.2 Prodrug Activation 4428.3 Abzyme Screening via Cell Growth 445

9 Industrial Future of Abzymes 44610 Conclusions 44611 Glossary 44712 Appendix 451

12.1 Catalog to Antibody Catalyzed Processes 45112.2 Key to Bibliography 479

13 References 481

404 11 Catalytic Antibodies

1 Introduction

This review seeks to deal with most of theimportant developments in the field of catalyt-ic antibodies since the initial successes wereannounced over a dozen years ago (POLLACK

et al., 1986; TRAMONTANO et al., 1986). The de-velopment of catalytic antibodies has requiredcontributions from a number of scientific dis-ciplines, which traditionally have not workedin concert. Thus, while this chapter describescatalytic antibodies, their reactions, and theirmechanisms from a biotransformations view-point, it will also provide a review that de-mands only a basic biochemical knowledge ofantibody structure, function, and production.Sufficient details of these matters have beensupplied to meet the needs of expert and non-expert readers alike. In Sect. 11, there is a glos-sary of most of the immunological terms usedin this review, in language familiar to chemists.While this survey is not fully comprehensive, itseeks to focus on the most significant parts ofa subject which, in a little over a decade, hasachieved much more than most critics expect-ed at the outset. A moderately complete sur-vey of the literature is presented in the form ofan appendix (Sect. 12), which lists over 100 ex-amples of reactions catalyzed by antibodies,the haptens employed, and their kinetic pa-rameters.

1.1 Antibody Structure andFunction

One of the most important biological de-fense mechanisms for higher organisms is theimmune response. It relies on the rapid gener-ation of structurally novel proteins that canidentify and bind tightly to foreign substancesthat would otherwise be damaging to the par-ent organism. These proteins are called immu-noglobulins and constitute a protein super-family. In their simplest form they are made upof four polypeptide chains: one pair of identi-cal short chains and one pair of identical longchains, interconnected by disulfide bridges.The two light and two identical heavy chainscontain repeated homologous sequences ofabout 110 amino acids which fold individually

into similar structural domains, essentially abilayer of antiparallel b-pleated sheets. Thisgives the structure of an IgG immunoglobulinmolecule whose core is formed from twelve,similar structural domains: eight from the twoheavy chains and four from the two lightchains (Fig. 1) (BURTON, 1990). Notwithstand-ing this apparent homogeneity, the N-terminalregions of antibody light and heavy chainsvary greatly in the variety and number of theirconstituent amino acids and thereby providebinding regions of great diversity called hyper-variable regions. The variety of proteins sogenerated approaches 1010 in higher mam-mals.

The essential property of the immunesystem is its ability to respond to single or mul-tiple foreign molecular species (antigens)through rapid diversification of the sequencesof these hypervariable regions by processes in-volving mutation, gene splicing, and RNAsplicing. This initially provides a vast numberof different antibodies which subsequently areselected and amplified in favor of those struc-tures with the strongest affinity for one partic-ular antigen.

1.2 The Search for a New Class ofBiocatalyst

Fifty years ago, LINUS PAULING clearly setout his theory that enzymes achieve catalysisbecause of their complementarity to the tran-sition state for the reaction being catalyzed(PAULING, 1948). With hindsight, this conceptcould be seen as a logical extension of the newtransition state theory that had been recentlydeveloped to explain chemical catalysis(EVANS and POLANYI, 1935; EYRING, 1935).The basic proposition was that the rate of a re-action is related to the difference in Gibbs freeenergy (DG0) between the ground state of re-actant(s) and the transition state for that reac-tion. For catalysis to be manifest, either the en-ergy of the transition state has to be lowered(transition state stabilization) or the energy ofthe substrate has to be elevated (substrate de-stabilization). PAULING applied this concept toenzyme catalysis by stating that an enzymepreferentially binds to and thereby stabilizesthe transition state for a reaction relative to

1 Introduction 405

the ground state of substrate(s) (Fig. 2). Thishas become a classical theory in enzymologyand is widely used to explain the way in whichsuch biocatalysts are able to enhance specificprocesses with rate accelerations of up to 1017

over background (ALBERY and KNOWLES,1976, 1977; ALBERY, 1993 for a review).

PAULING apparently did not bring ideasabout antibodies into his concept of enzymecatalysis, although there is a tantalizing photo-graph in a volume of PAULING’s lectures ca.1948 which shows on a single blackboard a car-toon of an energy profile diagram for the low-ering of a transition state energy profile and al-so reference to an immunoglobulin (PAULING,1947).And so it fell to BILL JENCKS in his mag-isterial work 1969 on catalysis to bring togeth-er the opportunity for synthesis of an enzymeby the use of antibodies that had been engi-

neered by manipulation of the immune system(JENCKS, 1969).

“One way to do this (i.e., synthesize an en-zyme) is to prepare an antibody to a haptenicgroup which resembles the transition state ofa given reaction”.1

The practical achievement of this goal washeld up for 18 years, primarily because of thegreat difficulty in isolation and purification ofsingle-species proteins from the immune rep-ertoire. During that time, many attempts todemonstrate catalysis by inhomogeneous (i.e.,polyclonal) mixtures of antibodies were madeand failed (e.g., RASO and STOLLAR, 1975;

Fig. 1. Scheme to show the structure of the peptide components of an IgG immunoglobulin:the two light (L) and two heavy (H) polypeptide chains; the disulfide bridges (–S–S–)connecting them; four variable regions of the light (VL) and heavy (VH) chains; and the eight“constant” regions of the light (C L) and heavy (CH1, CH2, CH3) chains (shaded rectangle).The hypervariable regions that achieve antigen recognition and binding are located withinsix polypeptide loops, three in the VL and three in the VH sections (shaded circle, top left).These can be excised by protease cleavage to give a fragment antibody, Fab (shaded lobe, top right).

1 In making this statement, JENCKS was apparentlynot aware of PAULING’s idea (JENCKS, personal com-munication).

SUMMERS, 1983). The problem was resolved in1976 by KÖHLER and MILSTEIN’s developmentof hybridoma technology, which has made itpossible both to screen rapidly the “complete”immune repertoire and to produce relativelylarge amounts of one specific monoclonal anti-body species in vitro (KÖHLER and MILSTEIN,1975; KÖHLER et al., 1976).

While transition states have been discussedin terms of their free energies, there have beenrelatively few attempts to describe their struc-ture at atomic resolution for most catalyzedreactions. Transition states are high energyspecies, often involving incompletely formedbonds, and this makes their specification verydifficult. In some cases these transient specieshave been studied using laser femtochemistry(ZEWAIL and BERNSTEIN, 1988; ZEWAIL, 1997),and predictions of some of their geometrieshave been made using molecular orbital calcu-lations (HOUK et al., 1995). Intermediatesalong the reaction coordinate are also often ofvery short lifetime, though some of their struc-tures have been studied under stabilising con-ditions while their existence and general na-ture can often be established using spectro-scopic techniques or trapping experiments(MARCH, 1992a).

The Hammond postulate predicts that if ahigh energy intermediate occurs along a reac-tion pathway, it will resemble the transitionstate nearest to it in energy (HAMMOND, 1955).Conversely, if the transition state is flanked bytwo such intermediates, the one of higher ener-gy will provide a closer approximation to thetransition state structure.This assumption pro-vides a strong basis for the use of mimics of un-stable reaction intermediates as transitionstate analogs (BARTLETT and LAMDEN, 1986;ALBERG et al., 1992).

1.3 Early Examples of CatalyticAntibodies

In 1986, RICHARD LERNER and PETER

SCHULTZ independently reported antibody ca-talysis of the hydrolysis of aryl esters and ofcarbonates respectively (POLLACK et al., 1986;TRAMONTANO et al., 1986). Such reactions arewell-known to involve the formation andbreakdown of an unstable tetrahedral inter-mediate (2), and so this can be deemed to beclosely related to the transition state (TS

cc) of

the reaction (Fig. 3).

406 11 Catalytic Antibodies

Fig. 2. Catalysis is achieved by lowering the free energy of activation for a process, i.e., the catalyst must bindmore strongly to the transition state (TS

cc) of the reaction than to either reactants or products. Thus:

DDGccpDDGCat:S and DDGCat:P.

1 Introduction 407

Transition states of this tetrahedral naturehave now been effectively mimicked by arange of stable analogs, including phosphonicacids, phosphonate esters, a-difluoroketones,and hydroxymethylene functional groups (JA-COBS, 1991). LERNER’s group elicited antibod-ies to a tetrahedral anionic phosphonate hap-ten2 (3) (AE3 2.9) while SCHULTZ’s group iso-lated a protein with high affinity for p-nitro-phenyl cholyl phosphate (5) (Fig. 4, AE 3.2).

1.4 Methods for GeneratingCatalytic Antibodies

It is appropriate at this stage in the review toconsider the stages in production of a catalyticantibody and to put in focus the relative rolesof chemistry, biochemistry, immunology, andmolecular biology. Nothing less than the fullintegration of these cognate sciences is neededfor the fullest realization of the most difficultobjectives in the field of catalytic antibodies. Inbroad terms, the top section of the flow dia-gram for abzyme production (Fig. 5) involveschemistry, the right hand side is immunology,the bottom sector is biochemistry, and molecu-lar biology completes the core of the scheme.

ChemistryAt the outset, chemistry dominates the selec-tion of the process to be investigated. The tar-geted reaction should meet most if not all offollowing criteria:

(1) have a slow but measurable spontane-ous rate under ambient conditions;

(2) be well analyzed in mechanistic terms;(3) be as simple as possible in number of

reaction steps;(4) be easy to monitor;(5) lead to the design of a synthetically ac-

cessible transition state analog (TSA)of adequate stability.

As we shall see later, many catalytic antibodiesachieve rate accelerations in the range 103 to106. It follows that for a very slow reaction, e.g.,the alkaline hydrolysis of a phosphate diesterwith kOH ca. 10P11 MP1 sP1, direct observationof the reaction is going to be experimentallyproblematic. Given that concentrations of cat-alytic antibodies employed are usually in the1–10 µM range, it has been far more realistic totarget the hydrolysis of an aliphatic ester, withkOH ca. 0.1 MP1 sP1 under ambient conditions.

The need for a good understanding of themechanism of the reaction is well illustratedby the case of amide hydrolysis. Many earlyenterprises sought to employ TSAs that werebased on a stable anionic tetrahedral interme-diate, as had been successful for ester hydroly-sis. Such approaches identified catalytic anti-

Fig. 3. The hydrolysis of an aryl ester (1) (XpCH2)or a carbonate (1) (XpO) proceeds through a tetra-hedral intermediate (2) which is a close model of thetransition state for the reaction. It differs substanti-ally in geometry and charge from both reactants andproducts.

2 It might be helpful to the reader to indicate thatthe pyridine-2-6-dicarboxylate component of (3)was designed for a further purpose, neither used norneeded for the activity described in the presentscheme.3 AEpAppendix Entry

408 11 Catalytic Antibodies

Fig. 4. LERNER’s group used phosphonate (3) as the hapten to raise an antibody which was capable of hy-drolyzing the ester (4). SCHULTZ found that naturally occurring antibodies using phosphate (5) as their anti-gen could hydrolyze the corresponding p-nitrophenyl choline carbonate (6). (Parts of haptens (3) and (5) re-quired for antibody recognition have been emphasized in bold).

Fig. 5. Stages in the production of a catalytic antibody.

1 Introduction 409

bodies capable of ester hydrolysis but not ofamide cleavage! There is good evidence thatfor aliphatic amides, breakdown of the tetra-hedral intermediate is the rate determiningstep. This step is catalyzed by protonation ofthe nitrogen leaving group and hence this fea-ture must be part of the TSA design.

The importance of minimizing the numberof covalent steps in the process to be catalyzedis rather obvious. Single- and two-step process-es dominate the abzyme scene. However, thereis substantial evidence that some acyl transferreactions involve covalent antibody intermedi-ates and so must proceed by up to four cova-lent steps. Nonetheless, such antibodies werenot elicited by intentional design but ratherdiscovered as a consequence of efficientscreening for reactivity.

Direct monitoring of the catalyzed reactionhas most usually been carried out in real timeby light absorption of fluorescent emissionanalysis and some initial progress has beenmade with light emission detection. The lowquantities of abzymes usually available at thescreening stage put a premium on the sensitiv-ity of such methods. However, some work hasbeen carried out of necessity using indirectanalysis, e.g., by HPLC or NMR.

Finally, this area of research might well havesupported a Journal of Unsuccessful Abzymes.It is common experience in the field that somethree out of four enterprises fail, and for noobvious reason. It is therefore imperative thatchemical synthesis of a TSA should not be therate determining step of an abzyme project.The average performance target is to achievehapten synthesis within a year: one or two ex-amples have employed TSAs that could befound in a chemical catalog, the most syntheti-cally-demanding cases have perforce em-ployed multi-step routes of considerable so-phistication (e.g.,AE 13.2). Lastly, the TSA hasto survive in vivo for at least two days to elicitthe necessary antigenic response.

ImmunologyThe interface of chemistry and immunologyrequires conjugation of multiple copies of theTSA to a carrier protein for production ofantibodies by standard monoclonal technolo-gy (KÖHLER and MILSTEIN, 1976). One suchconjugate is used for mouse immunization and

a second one for ELISA screening purposes.The carrier proteins selected for this purposeare bovine serum albumin (BSA, RMM67,000), keyhole limpet hemocyanin (KLH,RMM 4 · 106), and chicken ovalbumin (RMM32,000). All of these are basic proteins of highimmunogenicity and with multiple surface ly-sine residues that are widely used as sites forcovalent attachment of hapten. Successfulantibody production can take some threemonths and should deliver from 20 to 200monoclonal antibody lines for screening, pref-erably of IgG isotype.

Screening in early work sought to identifyhigh affinity of the antibody for the TSA, usinga process known as ELISA. This search cannow be performed more quantitatively by BIAcore analysis, based on surface plasmonresonance methodology (LÖFÅS and JOHNS-SON, 1990). A subsequent development is thecatELISA assay (TAWFIK et al., 1993) whichsearches for product formation and hence theidentification of abzymes that can generateproduct.

Methods of this nature are adequate forscreening sets of hybridomas but not for selec-tion from much larger libraries of antibodies.So, most recently, selection methods employ-ing suicide substrates (Sect. 6.2) (JANDA et al.,1997) or DNA amplification methodology(FENNIRI et al., 1995) have been brought intothe repertoire of techniques for the directidentification of antibodies that can turnovertheir substrate. However, the time-consumingscreening of hybridomas remains the mainstayof abzyme identification.

BiochemistryA family of 100 hybridoma antibodies can typ-ically provide 20 tight binders and these needto be assayed for catalysis. At this stage in theproduction of an abzyme, the benefit of a sen-sitive, direct screen for product formationcomes into its own. Following identification ofa successful catalyst, the antibody-producingcell line is usually recloned to ensure purityand stabilization of the clone, then protein isproduced in larger amount (ca. 10 mg) andused for determination of the kinetics andmechanism of the catalyzed process by classi-cal biochemistry. Digestion of such proteinwith trypsin or papain provides fragment anti-

bodies (Fabs), that contain only the attenuatedupper limbs of the intact IgG (Fig. 1). It isthese components that have been crystallized,in many cases with the substrate analog, prod-uct, or TSA bound in the combining site, andtheir structures determined by X-ray diffrac-tion.

Molecular BiologyOnly a few abzymes have reached the stagewhere mutagenesis is being employed in orderto improve their performance (MILLER et al.,1997). Likewise, HILVERT is the first to havereached the stage of redesign of the hapten toattempt the production of antibodies with en-hanced performance (KAST et al., 1996). So,the circle of production has now been com-pleted for at least one example, and chemistrycan start again with a revised synthetic target.

2 Approaches to HaptenDesign

One can now recognize a variety of strate-gies in addition to the earliest ones deployedfor hapten design. Some of these were present-ed originally as discrete solutions of the prob-lem of abzyme generation, but it is now recog-nized that they need not be mutually exclusiveeither in design or in application. Indeed, morerecent work often brings two or more of themtogether interactively. They can be classifiedbroadly into five categories for the purposes ofanalysis of their principal design elements.Thesequence of presentation of these here is inpart related to the chronology of their appear-ance on the abzyme scene.

(1) Transition state analogs.(2) Bait and switch.(3) Entropy traps.(4) Desolvation.(5) Supplementation of chemical function-

ality.

2.1 Transition State AnalogsAs has clearly been shown by the majority

of all published work on catalytic antibodies,the original guided methodology, i.e., the de-sign of stable transition state analogs (TSAs)for use as haptens to induce the generation ofcatalytic antibodies, has served as the bedrockof abzyme research. Most work has been di-rected at hydrolytic reactions of acyl species,perhaps because of the broad knowledge ofthe nature of reaction mechanisms for such re-actions and the wide experience of deployingphosphoryl species as stable mimics of un-stable tetrahedral intermediates. More than 80examples of hydrolytic antibodies have beenreported, including the 47 examples of acylgroup transfer to water (entries under 1–5 ofthe Appendix).

Most such acyl transfer reactions involvestepwise addition of the nucleophile followedby expulsion of the leaving group with a tran-sient, high energy, tetrahedral intermediate

410 11 Catalytic Antibodies

2 Approaches to Hapten Design 411

separating these processes. The fastest of suchreactions generally involve good leavinggroups and the addition of the nucleophile isthe rate determining step. This broad conclu-sion from much detailed kinetic analysis hasbeen endorsed by computation for the hydrol-ysis of methyl acetate (TERAISHI et al., 1994).This places the energy for product formationfrom an anionic tetrahedral intermediate some7.6 kcal molP1 lower than for its reversion toreactants. So, for the generation of antibodiesfor the hydrolysis of aryl esters, alkyl esters,carbonates, and activated anilides, the designof hapten has focused on facilitating nucleo-philic attack, and with considerable success.

The tetrahedral intermediates used for thispurpose initially deployed phosphorus (V)systems, relying on the strong polarization ofthe PuO bond (arguably more accuratelyrepresented as Pc–OP). The range has includ-ed many of the possible species containing anionized P–OH group (Fig. 6). One particularlygood feature of such systems is that thePc–OP bond is intermediate in length(1.521 Å) between the C–OP bond calculatedfor an anionic tetrahedral intermediate(0.2–0.3 Å shorter) and for the C· · ·O break-ing bond in the transition state (some 0.6 Ålonger) (TERAISHI et al., 1994). Other tetrahe-dral systems used have included sulfonamides(SHEN, 1995) and sulfones (BENEDETTI et al.,1996), secondary alcohols (SHOKAT et al.,1990), and a-fluoroketone hydrates (KITAZU-ME et al., 1994).

It is clear that phosphorus-based transitionstates have had the greatest success, as shownby the many entries under 1–5 of the Appen-dix. This may be a direct result of their anionicor partial anionic character, a feature not gen-erally available for the other species illustratedin Fig. 6, though a-difluorosulfonamides mightreasonably also share this feature as a result oftheir enhanced acidity.

Not surprisingly, most of the catalytic anti-body binding sites examined in structural de-tail have been found to contain a basic residuethat provides a coulombic interaction withthese TSAs, for which the prototype is the nat-ural antibody McPC603 to phosphoryl choline,where the phosphate anion is stabilized bycoulombic interaction with ArgH52 (PADLAN etal., 1985). In particular, X-ray structures ana-

lyzed by FUJII (FUJII et al., 1995) have shownthat the protonated HisH27d in catalytic anti-bodies 6D9, 4B5, 8D11, and 9C10 (AE 1.8) iscapable of forming a hydrogen bond to theoxyanion in the transition state for ester hy-drolysis.

In similar vein, KNOSSOW has identifiedHisH35 located proximate to the oxyanion ofp-nitrophenyl methanephosphonate in thecrystalline binary complex of antibodyCNJ206 and TSA, a system designed to hydro-lyze p-nitrophenyl acetate (c.f.AE 2.7) (CHAR-BONNIER et al., 1995). A third example is seenin SCHULTZ’s structure of antibody 48G7,which hydrolyzes methyl p-nitrophenyl car-bonate (AE 3.1c).The hapten p-nitrophenyl 4-carboxybutane-phosphonate is proximate toArgL96 and also forms hydrogen bonds toHisH35, TyrH33, and TyrL94 (Fig. 7) (PATTEN etal., 1996).

Clearly, the oxyanion hole is now as signifi-cant a feature of the binding site of such acyltransfer abzymes as it is already for esterasesand peptidases – and not without good reason.

Fig. 6. Transition state analogs for acyl cleavage re-action via tetrahedral transitions states.

KNOSSOW has analyzed the structures of threeesterase-like catalytic antibodies, each elicitedin response to the same phosphonate TSAhapten (CHARBONNIER et al., 1997). Catalysisfor all three is accounted for by transition statestabilization and in each case there is an oxy-anion hole involving a tyrosine residue. Thisstrongly suggests that evolution of immuno-globulins for binding to a single TSA haptenfollowed by selection from a large hybridomarepertoire by screening for catalysis leads toantibodies with structural convergence. Fur-thermore, the juxtaposition of X-ray structuresof the unliganded esterase mAb D2.3 and itscomplexes with a substrate analog and withone of the products provides a complete de-scription of the reaction pathway. D2.3 acts athigh pH by attack of hydroxide on the sub-strate with preferential stabilization of theoxyanion anionic tetrahedral intermediate, in-volving one tyrosine and one arginine residue.Water readily diffuses to the reaction centerthrough a canal that is buried in the proteinstructure (GIGANT et al., 1997). Such a clearpicture of catalysis now opens the way for site-directed mutagenesis to improve the perform-ance of this antibody.

2.2 Bait and Switch

Charge–charge complementarity is an im-portant feature involved in the specific andtight binding of antibodies to their respectiveantigens. It is the amino acid sequence andconformation of the hypervariable (or comple-mentarity determining regions, CDRs) in theantibody combining site which determine theinteractions between antigen and antibody.This has been exploited in a strategy dubbed“bait and switch” for the induction of antibodycatalysts which perform b-elimination reac-tions (SHOKAT et al., 1989; THORN et al., 1995),acyl-transfer processes (JANDA et al., 1990b,1991b; SUGA et al., 1994a; LI and JANDA, 1995),cis–trans alkene isomerizations (JACKSON andSCHULTZ, 1991), and dehydration reactions(UNO and SCHULTZ, 1992).

The bait and switch methodology deploys ahapten to act as a “bait”.This bait is a modifiedsubstrate that incorporates ionic functions in-tended to represent the coulombic distributionexpected in the transition state. It is therebydesigned to induce complementary, oppositelycharged residues in the combining site of anti-bodies produced by the response of the im-mune system to this hapten. The catalytic abil-ity of these antibodies is then sought by a sub-sequent “switch” to the real substrate andscreening for product formation, as describedabove.

The nature of the combining site of an anti-body responding to charged haptens was firstelucidated by GROSSBERG and PRESSMAN

(GROSSBERG and PRESSMAN, 1960), who used acationic hapten containing a p-azophenyltri-methylammonium ion to make antibodies witha combining site carboxyl group, essential forsubstrate binding (as shown by diazoacet-amide treatment).

The first example of “bait and switch” forcatalytic antibodies was provided by SHOKAT

(SHOKAT et al., 1989), whose antibody 43D4-3D12 raised to hapten (7) was able to catalyzethe b-elimination of (8) to give the trans-enone (9) with a rate acceleration of 8.8 · 104

above background (Fig. 8;AE 8.2). Subsequentanalysis has identified a carboxylate residue,Glu46H as the catalytic function induced by thecationic charge in (7) (Fig. 6) (SHOKAT et al.,1994).

412 11 Catalytic Antibodies

Fig. 7. Binding site details for antibody 48G7 com-plexed with hapten p-nitrophenyl 4-carboxybuta-nephosphonate (PATTEN et al., 1996). NB Aminoacid residues in antibodies are identified by theirpresence in the light (L) or heavy (H) chains with anumber denoting their sequence position from theN-terminus of the chain.

2 Approaches to Hapten Design 413

A similar “bait and switch” approach hasbeen exploited for acyl-transfer reactions(JANDA et al., 1990b, 1991b).The design of hap-ten (10) incorporates both a transition statemimic and the cationic pyridinium moiety, de-signed to induce the presence of a potentialgeneral acid/base or nucleophilic amino acidresidue in the combining site, able to assist incatalysis of the hydrolysis of substrate (11)(Fig. 9, AE 2.6).

Some 30% of all of the monoclonal antibod-ies obtained using hapten (10) were catalytic,and so the work was expanded to survey threeother antigens based on the original TSA de-sign (JANDA et al., 1991b). The carboxylate an-ion in (12) was designed to induce a cationic

combining site residue, while the quaternaryammonium species (13) combines both tetra-hedral mimicry and positive charge in thesame locus. Finally, the hydroxyl group in (14)was designed to explore the effects of a neutralantigen.

Fig. 8. Using the “bait and switch” principle, hapten(7) elicited an antibody, 43D4-3D12, which catalyzedthe b-elimination of (8) to a trans-enone (9). Thecarboxyl function in (7) is necessary for its attach-ment to the carrier protein.

Fig. 9. The original hapten (10) demonstrated theutility of the “bait and switch” strategy in the gener-ation of antibodies to hydrolyze the ester substrate(11). Three haptens (12), (13), (14) were designed toexamine further the effectiveness of point charges inamino acid induction. Both charged haptens (12),(13) produced antibodies that catalyzed the hydroly-sis of (11) whereas the neutral hapten (14) generat-ed antibodies which bound the substrate unproduct-ively.

Abzyme Identity Conditions

43D4-3D12 pH 6; 37 °C

Km 8 kcat 8 Ki 7

182 µM 0.003 sP1 0.29 µM

Three important conclusions arose from thiswork.

(1) A charged functionality is crucial forcatalysis.

(2) Catalytic antibodies are produced fromtargeting different regions of the bind-ing site with positive and negative hap-tens (although more were generated inthe case of the cationic hapten usedoriginally).

(3) The combination of charge plus mimic-ry of the transition state is required toinduce hydrolytic esterases.

Esterolytic antibodies have also been pro-duced by SUGA using an alternative “bait andswitch” strategy (AE 2.1) (SUGA et al., 1994a).A 1,2-aminoalcohol function was designed forgenerating not only esterases but also amidas-es. In order to elicit an anionic combining sitefor covalent catalysis, three haptens were syn-thesized, one contained a protonated amine(15) and two featured trimethylammoniumcations (16), (17) (Fig. 10). The outcome wasinterpreted as suggesting that haptens contain-ing a trimethylammonium group were toosterically demanding, so that the induced an-ionic amino acid residues in the antibody bind-ing pocket were too distant to provide nucleo-philic attack at the carbonyl carbon of sub-strate (18). An alternative explanation may bethat coulombic interactions lacking any hydro-gen bonding capability will not be sufficientlyshort-range for the purpose intended.

The use of secondary hydroxyl groups in thehaptens (15) and (16) was designed to mimicthe tetrahedral geometry of the transitionstate (as in JANDA’s work), while the third hap-ten (17) replaced the neutral OH with an an-ionic phosphate group, designed to elicit a cat-ionic combining site residue to stabilize thetransition state oxyanion. However, this func-tion in (17) may have proved too large to in-duce a catalytic residue close enough to the de-veloping oxyanion, since weaker catalysis wasobserved relative to haptens (15) and (16)(kcat/kuncatp2.4 · 103, 3.3 · 103, and F1 ·103 for(15), (16), and (17), respectively) (Fig. 10).

In order to achieve catalysis employing bothacid and basic functions, an alternative zwitter-ionic hapten was proposed in which the anion-

414 11 Catalytic Antibodies

Fig. 10. Three haptens (15), (16), (17) containing 1,2-aminoalcohol functionality were investigated as al-ternatives for esterase and amidase induction. Halfof the antibodies raised against hapten (15) wereshown to catalyze the hydrolysis of ester (18), there-by establishing the necessity for a compact haptenicstructure. Hapten (19) along with (16) was employedin a heterologous immunization program to elicitboth a general and acid base function in the anti-body binding site.

2 Approaches to Hapten Design 415

ic phosphoryl core is incorporated alongsidethe cationic trimethylammonium moiety (cf.17) (SUGA et al., 1994b). The difficulty in syn-thesizing such a target hapten can be over-come by stimulating the immune system firstwith the cationic and then with the anionicpoint charges using the two structurally relat-ed haptens (16) and (19), respectively. Such asequential strategy has been dubbed “heterol-ogous immunization” (Fig. 10) and this resultsof this strategy were compared with thosefrom the individual use of haptens (16) and(19) in a “homologous immunization” routine.Of 48 clones produced as a result of the ho-mologous protocols, 7 were found to be cata-lytic, giving rate enhancements up to 3 ·103.By contrast, 19 of the 50 clones obtained usingthe heterologous strategy displayed catalysis,the best being up to two orders of magnitudebetter.

A final example of the bait and switch strat-egy (THORN et al., 1995) focuses on the base-promoted decomposition of substituted benzi-soxazole (20) to give cyanophenol (21) (Fig 11,AE 8.4). A cationic hapten (22) was used tomimic the transition state geometry of allreacting bonds. It was anticipated that if thebenzimidazole hapten (22) induced the pres-ence of a carboxylate in the binding site, itwould be ideally positioned to make a hydro-gen bond to the N-3 proton of the substrate.The resultant abzymes would thus have gener-al base capability for abstracting the H-3 in thesubstrate.

Two monoclonals, 34E4 and 35F10, werefound to catalyze the reaction with a rate ac-celeration greater than 108, while the presenceof a carboxylate-containing binding site resi-due was confirmed by pH-rate profiles and co-valent modification by a carbodiimide, whichreduced catalysis by 84%.

The bait and switch tactic clearly illustratesthat antibodies are capable of a coulombic re-sponse that is potentially orthogonal to the useof transition state analogs in engendering ca-talysis. By variations in the hapten employed,it is possible to fashion antibody combiningsites that contain individual residues to deliverintricate mechanisms of catalysis.

2.3 Entropic Trapping

Rotational EntropyAn important component of enzyme catalysisis the control of translational and rotationalentropy in the transition state (PAGE andJENCKS, 1971). This is well exemplified for uni-molecular processes by the enzyme chorismatemutase, which catalyzes the rearrangement ofchorismic acid (23) into prephenic acid (24)

Fig. 11. The use of a cationic hapten (22) mimics thetransition state of the base-promoted decompositionof substituted benzisoxazole (20) to cyanophenol(21) and also acts as a “bait” to induce the presenceof an anion in the combining site that may act as ageneral base.