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Chapter 32. Catalytic Antibodies: A New Class of Designer Enzymes
Chapter 32: Catalytic Antibodies: A New Class of Designer Enzymes Grant A. Krafft and Gary T. Wang Abbott Laboratories Abbott Diagnostics Division Abbott Park, Illinois 60064 lntroduct i o n - Catalysis of chemical reactions is an enduring goal of science. However, the unlimited ability to select or program specific catalysis for any desired chemical transformation of any particular reactant has not yet been achieved. Recently, dramatic progress has been made toward this goal, through the development of a new class of designer enzymes, the catalytic antibodies. The vast repository of potential "catalytic active sites" available via the immune system and clonal selection presents a tantalizing picture. Initial advances resulted from the pivotal integration of the chemists' molecular design ingenuity and the immunologists hybridoma technology. In less than five years, catalytic antibodies have evolved from the idea stage to demonstrated practice, though the full potential of these new catalysts remains largely untapped. This chapter will describe some of the catalytic processes mediated by antibodies, and will discuss the potential impact that their unique features may bring to bear in the biomedical arena. For a comprehensive survey of this field, the interested reader should consult one of several excellent reviews that have been published recently (1-6).
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Racka round Antibodies are proteins generated by the immune system to recognize foreign entities and initiate their neutralization or destruction. They are capable of recognizing with high affinity (Ka's as high as 1014 M-l), a diverse spectrum of molecules, ranging in size from 6 to 35 A. A typical IgG antibody (Fig. 1) has a molecular weight of 150,000kD, with two ligand combining regions. The combining sites are defined by the hypervariable regions of light and heavy polypeptide chains (VL and VH respectively), and located in the first 1 1 0 amino terminal residues of these polypeptides. More than 108 different antibody molecules are available through recombination of VL and VH genes, making the antibody pool a particularly rich and attractive source of molecular specificities (7). The conceptual foundation for catalytic antibodies dates back to Pauling's conjecture that antibodies raised against transition state-like haptens should lower the activation free energy along the reaction coordinate leading from reactant(s) to product(s) (8). Jencks elaborated on this concept, coining the term "abzyme" to describe these antibody-enzyme hybrids (9). It was not until 1975,however, that the first experimental attempts to raise catalytic antibodies were described by Raso and Stollar. They immunized rabbits with a stable Schiff's base intermediate of a tyrosine-pyridoxal transamination reaction, but could detect no catalytic activity among affinity-purified fractions of the polyclonal sera (10,ll).This failure was attributed, in large part, to difficulties associated with isolation and purification of their polyclonal antibody preparations.
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Copyright 0 1989 by Academic Press, h c . All rights of reproduction in any form reserved.
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In 1975, Kohler and Milstein provided an elegant solution to this problem with their pioneering development of immortalized immune cell hybrids that produced monoclonal antibodies (7,12). Since this discovery, hybridoma technology has been used to generate hundreds of thousands of antibodies with distinct epitopic binding specificities. More than a decade passed, however, before the incubation of ideas and technology culminated in successful generation of catalytic antibodies by Schultz and coworkers (13), and Tramantano and coworkers (14). Independently and simultaneously, these two groups reported that mouse monoclonal antibodies raised against tetrahedral phosphate or phosphonate haptens could catalyze the hydrolysis of carbonates (13-15) and esters (16-20). Since these initial studies, reports on amide hydrolysis (21,22), lactonization (23), aminolysis (24,25), lipase activity (26), concerted rearrangements (27-29), peliminations (30), cycloadditions (31), photo-cycloreversions (32),and redox reactions (33,34) have appeared. Fig. 1. Schematic diagram of an IgG molecule. I
s-s
s-s
Hinge Region
CATALYTIC ANTIBODIES Catalvtic Antibodv Desian -- Princides and Tools - Antibody catalysis of chemical reactions entails two elements - recognition and catalytic capability. Recognition allows the antibody to select one or a small subset of molecules from all molecules functionally capable of participating in the catalyzed chemical reaction, and this selectable recognition/specificity is a unique attribute of catalytic antibodies. Catalytic capability is the sum of several components that result in a lowered activation energy for the rate determining step of a reaction. As suggested by Pauling (a), one of these components is favorable binding interactions between antibody and reactant to stabilize a high energy conformation approximating that of the reaction transition state. A second component is minimization of unfavorable entropy associated with pre-organization or orientation of reactants. A third aspect of catalysis is the precise placement of essential catalytic functional groups or cofactors to execute the chemical transformation.
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Several tools are available in the design of catalytic antibodies, the most important of which is the hapten molecule used to elicit the desired immune specificity (1-6). Hapten design is critical, since hapten structure and functionality will define or dictate the steric and electronic topology of the antibody combining site(s) ultimately selected from the immune reservoir. Thus, effective hapten design involves careful approximation of the transition state geometry of the intended reactant, and incorporation of specific functionality that will result in suitable placement of necessary catalytic amino acid residues at the combining site. The second important design tool is the direct introduction of catalytic functional groups into antibody combining sites, by directed chemical modification. After a desired specificity, perhaps with some level of catalytic capability has been elicited in a particular antibody, ligand-directed chemical modification can be used to incorporate additional catalytic groups or cofactors. A third design tool is sitedirected mutagenesis. This technique, widely used for modification of enzyme active sites, will be used extensively in the future to enhance the catalytic capability of antibody combining sites, after analysis of the combining site-ligand structure. Generation of Cata lvtic Antibodies - Most catalytic antibodies described to date have been generated by classical hybridoma techniques (?,I 2), which typically involve four steps: 1) immunization of mice with a hapten-protein carrier conjugate; 2) generation of immortalized hybrid clones by fusion of murine spleen cells with murine myeloma cells; 3) screening of individual clones for specific antibody binding to hapten, and 4) screening of hapten-specific antibodies for desired catalytic activity. This technique works reasonably well, though it is essential to completely purify the catalytic antibodies from endogenous murine enzymes that might be capable of catalyzing the same type of reaction. Recombinant DNA methodology is emerging as an effective technology for rapid and efficient generation of catalytic Fab fragments consisting of VL and VH chains. The first major advance in this area involved the cloning of the immunological repertoire into E. Coli by construction of a cDNA library specific for the heavy chain variable region (35). In an important follow-up to this work, libraries of cDNA for both the light and heavy variable chains were generated from harvested spleen RNA using the polymerase chain reaction (PCR), and inserted into phage h expression vectors containing specially tailored restriction sites (36). These restriction sites were designed such that when the digested DNA was recombined, the only viable vectors were those capable of expressing combinations of VH and VL chains, i.e., Fabs. Using this technique it is possible to screen large numbers of phage to identify those clones that bind antigen. Similar techniques also should be applicable to generating fully intact antibodies. Antibodv Cata Iv7ed React'ions - Hvdro lvtic and Transacvlat ion Reactions Hydrolysis of ester or carbonate bonds represented an excellent test of Pauling's transition state argument for catalytic antibodies, since formation of a negatively charged tetrahedral intermediate had been well documented as the rate determining step for the uncatalyzed reaction. Phosphate I or phosphonate 2 was used as a hapten to obtain monoclonal antibodies that catalyzed the hydrolysis of carbonates 9 and 4, respectively (1 3, 14). Several bis- arylphosphonate haptens were similarly employed to obtain hydrolytic antibodies. Immunization with phosphonate 5 led to an antibody that catalyzed the hydrolysis of ester 6 (14). The catalysis by these antibodies represented rate accelerations of 770, 16,000, and 960, respectively, over the background hydrolysis for these reactants. For each of these antibodies, good discrimination against dissimilar substrate structures was exhibited, and competitive inhibition of hydrolysis by the hapten was observed.
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2
1
6
z
R=NHCO(CHz), COOH
Since these initial reports, faster and more specific hydrolytic antibodies have been described. The most efficient catalytic antibody was capable of a 6.25~106 rate enhancement for hydrolysis of the ester Z (17). Stereospecificity greater than 200/1 was observed in the hydrolysis of the two diastereomers of 8, by several antibodies (20). These antibodies did not exhibit large turnover numbers; less of the binding energy is derived from interactions with the phosphonate transition state mimic, than from interactions with other substituents along the tripeptide backbone. A stereospecific lipase antibody, catalyzing only the hydrolysis of (R)-9, also has been described (26). It is noteworthy that the diastereomers of 8 also are fluorogenic substrates, due to quenching of the anthranilamide fluorophore by the nitroaromatic group via resonant energy transfer. These substrates permit direct measurement of hydrolysis kinetics, obviating the need for tedious HPLC quantitation of reactants and products. This fluorescence energy transfer assay technique is emerging as a powerful method for evaluation of a broad spectrum of cleavage reactions, and in this context, will facilitate rapid screening and evaluation of large numbers of catalytic antibodies (37-39).
Amide hydrolysis, particularly as it relates to peptide bond cleavage, is an important challenge for catalytic antibodies, since peptidases with tuned sequence specificity would be extremely valuable. However, the inherent stability of amides ( t 1 =~ 7 yrs) suggests that efficient hydrolysis by antibodies would be difficult. Phosphonamidate 1p,was used to generate an antibody that hydrolyzed JJ with a rate acceleration of 250,000, relative to the background hydrolysis. Inhibition and binding studies with a hapten analog suggested that catalysis by this antibody involved more than simple transition state stabilization: some type of acid-base assistance by residues at the combining site was suspected (21). In an attempt to further accelerate amide hydrolysis, antibodies were raised to hapten 12, containing a metal-trien cofactor. One of the antibodies obtained catalyzed the hydrolysis of peptides n o r 14 (3.6~10-2min-I), but only in the presence of the metal-trien cofactor (22). These turnovers are relatively low, and improvements in catalytic efficiency will be a continuing goal.
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It also has been possible to generate antibodies that catalyze the conversion of esters to amides via transacylation. In these instances, the antibodies were raised using phosphonamidate haptens resembling the transacylation transition state (24,25).
Recently, a different strategy for generating hydrolytic antibodies has been described, in which a charged amino acid was induced at the combining site using the cationic hapten This strategy, employed initially by Schultz to generate an eliminative antibody (vide infra), resulted in generation of seven antibodies that catalyzed the hydrolysis of B. None of the antibodies generated by the uncharged hapten 17 hydrolyzed U, indicating an essential role for a charged binding site group in the catalyzed hydrolysis. The pH dependence of katfor hydrolysis of J.6 indicated participation of a dissociable group with a measured pKa of 6.26 (40).
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15
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O 0 Q
’ R
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/
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Unimolecular Cvclization Reactions - Unfavorable entropic factors associated with highly ordered transition states can be overcome by favorable binding interactions at an antibody combining site. Antibody catalysis of two different types of unimolecular reactions, a Claisen rearrangement (27-29) and lactone-forming transacylation reaction (23) has relied on antibody binding energy. The bicyclic and 1p, induced antibodies that catalyzed the chorismate to haptens, prephenate Claisen rearrangement One of these antibodies accelerated the rearrangement by 10,000 fold over the uncatalyzed rate, comparing reasonably to a rate of 3 . 6 ~ 1 0 6for chorismate mutase from E. coli. Formation of a lactone was catalyzed by an antibody raised against the cyclic phosphonate ester 24. This represented an acceleration of 167 over the uncatalyzed solution rate. The cyclization was stereoselective, generating a 94:6 ratio of enantiomeric lactones from racemic starting material. These two reactions illustrate the powerful capability of antibody combining sites to restrict the degrees of freedom of conformationally mobile reactants, resulting in lowered entropic requirements in the rate determining steps of these unirnolecular reactions.
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(=+a)
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22
&
0 NHAc
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N ~ C 0 2 S " C H
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A P-Elimination Reaction - The ability to orchestrate placement of specific catalytic functional groups at an antibody combining site was first demonstrated by the generation of antibodies that catalyzed a p-elimination reaction. The tertiary amine hapten protonated under immunization conditions, induced antibodies that catalyzed the elimination of HF from 26. (30). Studies in which antibody catalytic activity was measured as a function of pH indicated that a glutamate or aspartate residue was situated at the combining site in a position to abstract an a-proton from the substrate B.The 88,000 fold rate enhancement attributable to the combining site carboxylate for this reaction is comparable to accelerations mediated by carboxylate bases in several other enzymes. This tactic of inducing specific, catalytic functional groups is quite effective in generating catalytic antibodies that cannot be induced simply by imprinting transition state conformations onto the antibody combining site.
a,
P h w c l o r e v e r s i o n Reaction - Another elegant example of catalysis brought about by specific induction of a catalytic amino acid residue at the combining site was recently described (32). It was postulated that a tryptophan residue could be induced at an antibody combining site via n-stacking interactions with a hapten containing a polarized K system. The tryptophan would then be capable of sensitizing a [2+2] photo-cycloreversion reaction. Immunization with hapten 27 resulted in generation of antibodies that bound thymine dimer 28,and catalyzed the photo-reversion to when irradiated with UV light (>300 nm).
a
-tor M e d i w Reacti o G - Induction of cofactor binding sites and modulation of cofactor reactivity represent other strategies for the the generation of catalytic antibodies (33, 34). The flavin hapten induced antibodies that lowered the reduction potential of by almost 40% (33). These antibodies bound to more weakly than to but enhanced the reducing capability of upon binding. Thus, normally unable to reduce compounds with reduction potentials lower than -206 mV, was capable of reducing Safranine T (reduction potential of -289 mV). In this instance, antibodies altered the thermodynamics of a redox process, facilitating a reaction not accessible to the free flavin. In another study, fluorescein was
a,
a,
a
a
a
a,
(a)
(a)
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used as a hapten to generate antibodies that accelerated the bimolecular reduction of resazurin by sulfite (34). In this instance, two reactant binding sites were generated by fluorescein -- one for a xanthenoid type ring, the other for the negatively charge sulfite. The binding affinities of 34 and to the antibody were similar, indicating that the rate acceleration was not due to alteration of the redox potentials. The high avidity of antibody for the product limited the turnover for this reaction, since product dissociation was slow.
P
L C 0 2 H
\
33
34
H
35
Bimolecular Cvcloadditions - A Diels-Alder Reaction - Another bimolecular reaction catalyzed by an antibody was recently reported (31). Using hapten which resembled a late transition state for the [4+2] cycloaddition of and 38,several antibodies that catalyzed this Diels-Alder reaction were obtained. In this study, the key factor was selection of a cycloaddition reaction that ultimately generated, after loss of S02, a product structurally dissimilar from the hapten or the initial transition state. This strategy provides an excellent solution to the problem of product inhibition, which has limited the turnover capabilities of many catalytic antibodies. The generation of catalysts for multi-substrate reactions will become an important goal, since catalysts or enzymes simply do not exist for many important birnolecular processes.
a,
Modification of Cataly-tic Ant ibodies - The introduction of functional groups at the combining site of antibodies is not limited to induction by specifically designed haptens, but can also be accomplished by specific chemical modification or site directed mutagenesis. Schultz described a technique in which thiol groups could be introduced by antigen directed affinity labeling in the vicinity of the combining site (41). These thiol groups were available for subsequent modification by a variety of functional groups (41) or spectroscopic probes (42). In one instance, a
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histidine group was introduced at the periphery of the combining site of the MOPC315 Fab fragment specific for 2,4-dinitrophenyl groups. This functionalization imparted hydrolytic capability to the Fab for the coumarin ester 39 (31). Sitedirected mutagenesis (Tyr34L -+ His34L) of MOPC315 Fab also generated a catalytic antibody that hydrolyzed coumarin ester 4 !2 (43).
o
./ \”
p
NO2
39 JblFnICAl
Jy-oyyy
\ /q \ 0
0
0
0
02N
/
/
NO2
/
a2
AND DIAGNOSTIC APPl ICATIONS OF CATALYTIC A N T I B O D E
The major promise of catalytic antibodies lies in medical and diagnostic applications that capitalize on the combination of the inherent properties of antibodies and specifically tailored catalytic properties. It has been proposed that antibodies can be generated to recognize tumor-specific antigens and then carry out catalytic reactions to mediate the destruction of those tumor cells (44). This is similar in concept to approaches involving antibody-enzyme conjugates as described by several groups (45). However, catalytic antibodies offer a number of advantages. The pharmacokinetics and distribution of antibodies is better than that of large enzyme-antibody conjugates, and catalytic antibodies are much less immunogenic than the conjugates. Many other therapeutic applications can be envisioned, in which the gross antibody structure simply provides a serum stable framework for a catalytic entity, or in which the specific antigen targeting ability of antibodies is combined with a catalytic function to carry out a desired chemical reaction. An obvious example would be a hydrolytic antibody specific for plasminogen or fibrin, to replace the use of tissue plasminogen activator in the treatment of myocardial infarction. Enzymes have been used extensively in diagnostic applications ranging from immunoassays to biosensors. It has been proposed that the use of catalytic antibodies or catalytic Fab fragments could be used for a broad range of diagnostic applications, essentially replacing the function of enzymes in these applications (46). The realization of some of these ideas may come to pass, though the catalytic efficiencies of antibodies described to date are too poor to function effectively in these applications. The real benefit of catalytic antibodies in diagnostic applications may arise in situations where a desired catalytic reaction simply cannot be carried out by known enzymes, but can be accomplished by a specifically tailored catalytic antibody. Results obtained during the next several years will begin to establish the scope and limitations of catalytic antibodies in the diagnostic arena. CATALYTIC ANTIBODIES - FUTURE CHAl I FNGES The major challenge for scientists designing catalytic antibodies is the improvement of catalytic efficiency. Ultimately, this aspect of the problem will critically affect the extent to which this methodology is applied to medical and diagnostic problems. Although it was the similarities between antibody combining sites and enzyme active sites that stimulated the development of catalytic antibodies with a broad spectrum of catalytic capabilities, the fundamental differences between antibodies and enzymes must now be overcome. Antibodies
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are relatively rigid structures, capable of binding with high affinity to antigens, while enzymes are inherently flexible molecules that permit the facile access of substrates to the active site, and the efficient dissociation of products. Scientists will need to learn how to manipulate antibody structure to impart greater flexibility and higher catalytic efficiency. Many of the tools needed to accomplish this are in hand, including site-directed mutagenesis and selective chemical modification, but an even greater emphasis will be placed on understanding and manipulating antibody structure in order to generate highly efficient catalytic antibodies. A second challenge is to broaden the scope of reactions that can be catalyzed by antibodies. Based on the progress of the past several years, it is likely that many antibodies with novel catalytic capabilities will be developed in the near future.
A third challenge is the development of more efficient, streamlined methods for generation and production of catalytic antibodies or antibody fragments. This and other challenges presented by catalytic antibodies will demand further creative assimilation of ideas in chemistry, immunology, protein biochemistry and molecular biology. Pefer1. 2. 3. 4. 5. 6. 7.
8. 9. 10. 11.
12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33.
a,
P. G. Schultz, Science, 426 (1988). R. A. Lerner, S J. Benkovic, Bioessays, 9, 107 (1988). P. G. Schultz, Am. Chem. Res., 2 , 2 8 7 (1989). P. G. Schultz, Angew. Chem. Int. Ed. Eng., 1283 (1989).
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K. M. Shokat, P. G. Schultz, Ann. Rev. Immunol., in press. 6.S. Green, Adv. Biotechnol. Processes, 11,359 (1989). F. R. Seiler, P. Gronski, R. Kurrle, G. Luben, H. P. Hartbus, W. Ax, K. Bosslet, H. G. Schwick, Angew. Chem. Int. Ed. Engl., 24, 139 (1985). L. Pauling, Am. Sci., 2, 519 (1948). W. Jencks, "Catalysis in Chemistry and Enzymology", McGraw-Hill, New York, NY, 1969. V. Raso, B. D. Stollar, Biochemistry, 14,584 (1975). V Raso, B. 0. Stollar, Biochemistry, 14, 591 (1975). G. Kohler, C. Milstein, Nature, 5517 (1975). S. J. Pollack, J. W. Jacobs, P. G. Schultz, Science, 1570 (1986). A. Tramontano, K. D. Janda, R . A. Lerner, Science, 1566 (1986). J. Jacobs, P. G. Schultz, J. Am. Chem. Soc.,1119,2174 (1987). J. Jacobs, P. G. Schultz, R. Sugasawara, M. Powell, J. Am. Chem. SOC.,XU,1570 (1987). A. Tramontano, A. A. Ammann, R. A. Lerner, J. Am. Chem. SOC.,IlQ,2282 (1988). C. N. Durfor, R. J. Bolin, R. J. Sugasawara, R. J. Massey, J. W. Jacobs, P. G. Schultz, J. Am. Chem. SOC., 8713 (1988). K. D. Janda. S. J. Benkovic, R. A. Lerner, Science, 244,437 (1989). S. J. Pollack, P. Husiun, P. G. Schultz, J. Am. Chem. SOC.,111,5961 (1989). K. D. Janda, D. Schloeder, S. J. Benkovic, R. A. Lerner, Science, 24.L1188 (1988). B. L. Iverson, R. A. Lerner, Science, 1184 (1989). A. D. Napper, S. J. Benkovic, A. Tramontano, R. A. Lerner, Science, 1041 (1987). S. J. Benkovic, A. D. Napper, R. A.Lerner, Proc. Natl. Acad. Sci. USA., &%, 5355 (1988). K. D. Janda, R. A. Lerner, A. Tramontano, J. Am. Chem. SOC.1111,4835 (1988). K. D. Janda, S. J. Benkovic, R. A. Lerner, Science, 244,437 (1989). D. Hilvert, K. 0. Nared, J. Am. Chem. SOC., 5593 (1988). D. Hilvert, S. H. Carpenter, K. D. Nared, M. T. M. Auditor, Proc. Natl. Acad. Sci. USA, &, 4953 ( 1988). D. Y. Jackson, J. W. Jacobs, R. Sugasawara, S. H. Reich, P. Bartlett, P. G. Schultz, J. Am. Chem. SOC., 4841 (1988). K. M. Shokat, C. J. Leumann, R. Sugasawara, P. G. Schultz, Nature, 269 (1989). D. Hilvert, K. W. Hill, K. D. Nared, M. T. M. Auditor, J. Am. Chem. Soc., 111, 9262 (1989). A. G. Cochran. R. Sugasawara, P. G. Schultz, J. Am. Chem. SOC.JJ-Q, 7888 (1988). K. M. Shokat, C. H. Leumann, R. Sugasawara, P. G. Schultz, Angew. Chern. I.@, 1227 (1988).
m,
m, m,
m,
a,
m,
m,
m,
m,
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34.
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K. M. Shokat, C. H. Leumann, R. Sugasawara, P. G. Schultz, Angew. Chem. Int. Ed. Engl.,
22,1172 (1988).
L. Sastry, M. Alting-Mees, W. D. Huse, J. M. Short, J. A. Sorge, B. N. Hay, K. D.Janda., S. J. Benkovic, R. N. Lerner, Proc. Natl. Acad. Sci. USA, 86, 5728 (1989). 36. W. D. Huse, A. Sastry, S. A. Iverson. A. S. Kang, M. Aiting-Mees, D. R. Burton, S. J. Benkovic, R. A. Lerner, Science, 1275 (1989). 37. A. Yaron, A. Carmel, E. Katchalski-Katzir, Anal. Biochem., 95,228 (1979). (1990). 38. E. D. Matayoshi, G. T. Wang, G. A. Krafft, J. W. Erickson, Science,=7,954 39. R. T. Cummings, G. A. Krafft, Tetrahedron Lett., 29, 65 (1988). 40. K. D. Janda, M. I. Weinhouse, D. M. Schloeder, R. A. Lerner, S. J. Benkovic, J. Am. Chem. SOC. 1274 (1990). 41. S. J. Pollack, G. R. Nakayam, P. G. Schultz, Science, 242, 1038, (1988). 1929 (1989). 42. S. J. Pollack, P. G. Schultz, J. Am. Chem. SOC. 1104, (1989). 43 E. Baldwin, P. G. Schultz, Science, 592 (1989). 44. R. 0. Dillman, Ann. Int. Med., 111, 45. P. D. Senter, FASEB J., 4, 188 (1990). 46. G. E. Blackbum, C. Durfor, M. J. Powell, R. J. Massey, Int. Patent Appl. WO 89/05977, 1989. 35.
a,
u,
I
w,
m,
-
Racka round Antibodies are proteins generated by the immune system to recognize foreign entities and initiate their neutralization or destruction. They are capable of recognizing with high affinity (Ka's as high as 1014 M-l), a diverse spectrum of molecules, ranging in size from 6 to 35 A. A typical IgG antibody (Fig. 1) has a molecular weight of 150,000kD, with two ligand combining regions. The combining sites are defined by the hypervariable regions of light and heavy polypeptide chains (VL and VH respectively), and located in the first 1 1 0 amino terminal residues of these polypeptides. More than 108 different antibody molecules are available through recombination of VL and VH genes, making the antibody pool a particularly rich and attractive source of molecular specificities (7). The conceptual foundation for catalytic antibodies dates back to Pauling's conjecture that antibodies raised against transition state-like haptens should lower the activation free energy along the reaction coordinate leading from reactant(s) to product(s) (8). Jencks elaborated on this concept, coining the term "abzyme" to describe these antibody-enzyme hybrids (9). It was not until 1975,however, that the first experimental attempts to raise catalytic antibodies were described by Raso and Stollar. They immunized rabbits with a stable Schiff's base intermediate of a tyrosine-pyridoxal transamination reaction, but could detect no catalytic activity among affinity-purified fractions of the polyclonal sera (10,ll).This failure was attributed, in large part, to difficulties associated with isolation and purification of their polyclonal antibody preparations.
ANNUAL REPORTS IN MEDICINAL CHEMISTRY-25
Copyright 0 1989 by Academic Press, h c . All rights of reproduction in any form reserved.
300
Section VI-’Ibpics in Chemistry and Drug Design
Vinick, Ed.
In 1975, Kohler and Milstein provided an elegant solution to this problem with their pioneering development of immortalized immune cell hybrids that produced monoclonal antibodies (7,12). Since this discovery, hybridoma technology has been used to generate hundreds of thousands of antibodies with distinct epitopic binding specificities. More than a decade passed, however, before the incubation of ideas and technology culminated in successful generation of catalytic antibodies by Schultz and coworkers (13), and Tramantano and coworkers (14). Independently and simultaneously, these two groups reported that mouse monoclonal antibodies raised against tetrahedral phosphate or phosphonate haptens could catalyze the hydrolysis of carbonates (13-15) and esters (16-20). Since these initial studies, reports on amide hydrolysis (21,22), lactonization (23), aminolysis (24,25), lipase activity (26), concerted rearrangements (27-29), peliminations (30), cycloadditions (31), photo-cycloreversions (32),and redox reactions (33,34) have appeared. Fig. 1. Schematic diagram of an IgG molecule. I
s-s
s-s
Hinge Region
CATALYTIC ANTIBODIES Catalvtic Antibodv Desian -- Princides and Tools - Antibody catalysis of chemical reactions entails two elements - recognition and catalytic capability. Recognition allows the antibody to select one or a small subset of molecules from all molecules functionally capable of participating in the catalyzed chemical reaction, and this selectable recognition/specificity is a unique attribute of catalytic antibodies. Catalytic capability is the sum of several components that result in a lowered activation energy for the rate determining step of a reaction. As suggested by Pauling (a), one of these components is favorable binding interactions between antibody and reactant to stabilize a high energy conformation approximating that of the reaction transition state. A second component is minimization of unfavorable entropy associated with pre-organization or orientation of reactants. A third aspect of catalysis is the precise placement of essential catalytic functional groups or cofactors to execute the chemical transformation.
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Several tools are available in the design of catalytic antibodies, the most important of which is the hapten molecule used to elicit the desired immune specificity (1-6). Hapten design is critical, since hapten structure and functionality will define or dictate the steric and electronic topology of the antibody combining site(s) ultimately selected from the immune reservoir. Thus, effective hapten design involves careful approximation of the transition state geometry of the intended reactant, and incorporation of specific functionality that will result in suitable placement of necessary catalytic amino acid residues at the combining site. The second important design tool is the direct introduction of catalytic functional groups into antibody combining sites, by directed chemical modification. After a desired specificity, perhaps with some level of catalytic capability has been elicited in a particular antibody, ligand-directed chemical modification can be used to incorporate additional catalytic groups or cofactors. A third design tool is sitedirected mutagenesis. This technique, widely used for modification of enzyme active sites, will be used extensively in the future to enhance the catalytic capability of antibody combining sites, after analysis of the combining site-ligand structure. Generation of Cata lvtic Antibodies - Most catalytic antibodies described to date have been generated by classical hybridoma techniques (?,I 2), which typically involve four steps: 1) immunization of mice with a hapten-protein carrier conjugate; 2) generation of immortalized hybrid clones by fusion of murine spleen cells with murine myeloma cells; 3) screening of individual clones for specific antibody binding to hapten, and 4) screening of hapten-specific antibodies for desired catalytic activity. This technique works reasonably well, though it is essential to completely purify the catalytic antibodies from endogenous murine enzymes that might be capable of catalyzing the same type of reaction. Recombinant DNA methodology is emerging as an effective technology for rapid and efficient generation of catalytic Fab fragments consisting of VL and VH chains. The first major advance in this area involved the cloning of the immunological repertoire into E. Coli by construction of a cDNA library specific for the heavy chain variable region (35). In an important follow-up to this work, libraries of cDNA for both the light and heavy variable chains were generated from harvested spleen RNA using the polymerase chain reaction (PCR), and inserted into phage h expression vectors containing specially tailored restriction sites (36). These restriction sites were designed such that when the digested DNA was recombined, the only viable vectors were those capable of expressing combinations of VH and VL chains, i.e., Fabs. Using this technique it is possible to screen large numbers of phage to identify those clones that bind antigen. Similar techniques also should be applicable to generating fully intact antibodies. Antibodv Cata Iv7ed React'ions - Hvdro lvtic and Transacvlat ion Reactions Hydrolysis of ester or carbonate bonds represented an excellent test of Pauling's transition state argument for catalytic antibodies, since formation of a negatively charged tetrahedral intermediate had been well documented as the rate determining step for the uncatalyzed reaction. Phosphate I or phosphonate 2 was used as a hapten to obtain monoclonal antibodies that catalyzed the hydrolysis of carbonates 9 and 4, respectively (1 3, 14). Several bis- arylphosphonate haptens were similarly employed to obtain hydrolytic antibodies. Immunization with phosphonate 5 led to an antibody that catalyzed the hydrolysis of ester 6 (14). The catalysis by these antibodies represented rate accelerations of 770, 16,000, and 960, respectively, over the background hydrolysis for these reactants. For each of these antibodies, good discrimination against dissimilar substrate structures was exhibited, and competitive inhibition of hydrolysis by the hapten was observed.
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2
1
6
z
R=NHCO(CHz), COOH
Since these initial reports, faster and more specific hydrolytic antibodies have been described. The most efficient catalytic antibody was capable of a 6.25~106 rate enhancement for hydrolysis of the ester Z (17). Stereospecificity greater than 200/1 was observed in the hydrolysis of the two diastereomers of 8, by several antibodies (20). These antibodies did not exhibit large turnover numbers; less of the binding energy is derived from interactions with the phosphonate transition state mimic, than from interactions with other substituents along the tripeptide backbone. A stereospecific lipase antibody, catalyzing only the hydrolysis of (R)-9, also has been described (26). It is noteworthy that the diastereomers of 8 also are fluorogenic substrates, due to quenching of the anthranilamide fluorophore by the nitroaromatic group via resonant energy transfer. These substrates permit direct measurement of hydrolysis kinetics, obviating the need for tedious HPLC quantitation of reactants and products. This fluorescence energy transfer assay technique is emerging as a powerful method for evaluation of a broad spectrum of cleavage reactions, and in this context, will facilitate rapid screening and evaluation of large numbers of catalytic antibodies (37-39).
Amide hydrolysis, particularly as it relates to peptide bond cleavage, is an important challenge for catalytic antibodies, since peptidases with tuned sequence specificity would be extremely valuable. However, the inherent stability of amides ( t 1 =~ 7 yrs) suggests that efficient hydrolysis by antibodies would be difficult. Phosphonamidate 1p,was used to generate an antibody that hydrolyzed JJ with a rate acceleration of 250,000, relative to the background hydrolysis. Inhibition and binding studies with a hapten analog suggested that catalysis by this antibody involved more than simple transition state stabilization: some type of acid-base assistance by residues at the combining site was suspected (21). In an attempt to further accelerate amide hydrolysis, antibodies were raised to hapten 12, containing a metal-trien cofactor. One of the antibodies obtained catalyzed the hydrolysis of peptides n o r 14 (3.6~10-2min-I), but only in the presence of the metal-trien cofactor (22). These turnovers are relatively low, and improvements in catalytic efficiency will be a continuing goal.
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It also has been possible to generate antibodies that catalyze the conversion of esters to amides via transacylation. In these instances, the antibodies were raised using phosphonamidate haptens resembling the transacylation transition state (24,25).
Recently, a different strategy for generating hydrolytic antibodies has been described, in which a charged amino acid was induced at the combining site using the cationic hapten This strategy, employed initially by Schultz to generate an eliminative antibody (vide infra), resulted in generation of seven antibodies that catalyzed the hydrolysis of B. None of the antibodies generated by the uncharged hapten 17 hydrolyzed U, indicating an essential role for a charged binding site group in the catalyzed hydrolysis. The pH dependence of katfor hydrolysis of J.6 indicated participation of a dissociable group with a measured pKa of 6.26 (40).
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Unimolecular Cvclization Reactions - Unfavorable entropic factors associated with highly ordered transition states can be overcome by favorable binding interactions at an antibody combining site. Antibody catalysis of two different types of unimolecular reactions, a Claisen rearrangement (27-29) and lactone-forming transacylation reaction (23) has relied on antibody binding energy. The bicyclic and 1p, induced antibodies that catalyzed the chorismate to haptens, prephenate Claisen rearrangement One of these antibodies accelerated the rearrangement by 10,000 fold over the uncatalyzed rate, comparing reasonably to a rate of 3 . 6 ~ 1 0 6for chorismate mutase from E. coli. Formation of a lactone was catalyzed by an antibody raised against the cyclic phosphonate ester 24. This represented an acceleration of 167 over the uncatalyzed solution rate. The cyclization was stereoselective, generating a 94:6 ratio of enantiomeric lactones from racemic starting material. These two reactions illustrate the powerful capability of antibody combining sites to restrict the degrees of freedom of conformationally mobile reactants, resulting in lowered entropic requirements in the rate determining steps of these unirnolecular reactions.
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A P-Elimination Reaction - The ability to orchestrate placement of specific catalytic functional groups at an antibody combining site was first demonstrated by the generation of antibodies that catalyzed a p-elimination reaction. The tertiary amine hapten protonated under immunization conditions, induced antibodies that catalyzed the elimination of HF from 26. (30). Studies in which antibody catalytic activity was measured as a function of pH indicated that a glutamate or aspartate residue was situated at the combining site in a position to abstract an a-proton from the substrate B.The 88,000 fold rate enhancement attributable to the combining site carboxylate for this reaction is comparable to accelerations mediated by carboxylate bases in several other enzymes. This tactic of inducing specific, catalytic functional groups is quite effective in generating catalytic antibodies that cannot be induced simply by imprinting transition state conformations onto the antibody combining site.
a,
P h w c l o r e v e r s i o n Reaction - Another elegant example of catalysis brought about by specific induction of a catalytic amino acid residue at the combining site was recently described (32). It was postulated that a tryptophan residue could be induced at an antibody combining site via n-stacking interactions with a hapten containing a polarized K system. The tryptophan would then be capable of sensitizing a [2+2] photo-cycloreversion reaction. Immunization with hapten 27 resulted in generation of antibodies that bound thymine dimer 28,and catalyzed the photo-reversion to when irradiated with UV light (>300 nm).
a
-tor M e d i w Reacti o G - Induction of cofactor binding sites and modulation of cofactor reactivity represent other strategies for the the generation of catalytic antibodies (33, 34). The flavin hapten induced antibodies that lowered the reduction potential of by almost 40% (33). These antibodies bound to more weakly than to but enhanced the reducing capability of upon binding. Thus, normally unable to reduce compounds with reduction potentials lower than -206 mV, was capable of reducing Safranine T (reduction potential of -289 mV). In this instance, antibodies altered the thermodynamics of a redox process, facilitating a reaction not accessible to the free flavin. In another study, fluorescein was
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a,
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(a)
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used as a hapten to generate antibodies that accelerated the bimolecular reduction of resazurin by sulfite (34). In this instance, two reactant binding sites were generated by fluorescein -- one for a xanthenoid type ring, the other for the negatively charge sulfite. The binding affinities of 34 and to the antibody were similar, indicating that the rate acceleration was not due to alteration of the redox potentials. The high avidity of antibody for the product limited the turnover for this reaction, since product dissociation was slow.
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33
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H
35
Bimolecular Cvcloadditions - A Diels-Alder Reaction - Another bimolecular reaction catalyzed by an antibody was recently reported (31). Using hapten which resembled a late transition state for the [4+2] cycloaddition of and 38,several antibodies that catalyzed this Diels-Alder reaction were obtained. In this study, the key factor was selection of a cycloaddition reaction that ultimately generated, after loss of S02, a product structurally dissimilar from the hapten or the initial transition state. This strategy provides an excellent solution to the problem of product inhibition, which has limited the turnover capabilities of many catalytic antibodies. The generation of catalysts for multi-substrate reactions will become an important goal, since catalysts or enzymes simply do not exist for many important birnolecular processes.
a,
Modification of Cataly-tic Ant ibodies - The introduction of functional groups at the combining site of antibodies is not limited to induction by specifically designed haptens, but can also be accomplished by specific chemical modification or site directed mutagenesis. Schultz described a technique in which thiol groups could be introduced by antigen directed affinity labeling in the vicinity of the combining site (41). These thiol groups were available for subsequent modification by a variety of functional groups (41) or spectroscopic probes (42). In one instance, a
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histidine group was introduced at the periphery of the combining site of the MOPC315 Fab fragment specific for 2,4-dinitrophenyl groups. This functionalization imparted hydrolytic capability to the Fab for the coumarin ester 39 (31). Sitedirected mutagenesis (Tyr34L -+ His34L) of MOPC315 Fab also generated a catalytic antibody that hydrolyzed coumarin ester 4 !2 (43).
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AND DIAGNOSTIC APPl ICATIONS OF CATALYTIC A N T I B O D E
The major promise of catalytic antibodies lies in medical and diagnostic applications that capitalize on the combination of the inherent properties of antibodies and specifically tailored catalytic properties. It has been proposed that antibodies can be generated to recognize tumor-specific antigens and then carry out catalytic reactions to mediate the destruction of those tumor cells (44). This is similar in concept to approaches involving antibody-enzyme conjugates as described by several groups (45). However, catalytic antibodies offer a number of advantages. The pharmacokinetics and distribution of antibodies is better than that of large enzyme-antibody conjugates, and catalytic antibodies are much less immunogenic than the conjugates. Many other therapeutic applications can be envisioned, in which the gross antibody structure simply provides a serum stable framework for a catalytic entity, or in which the specific antigen targeting ability of antibodies is combined with a catalytic function to carry out a desired chemical reaction. An obvious example would be a hydrolytic antibody specific for plasminogen or fibrin, to replace the use of tissue plasminogen activator in the treatment of myocardial infarction. Enzymes have been used extensively in diagnostic applications ranging from immunoassays to biosensors. It has been proposed that the use of catalytic antibodies or catalytic Fab fragments could be used for a broad range of diagnostic applications, essentially replacing the function of enzymes in these applications (46). The realization of some of these ideas may come to pass, though the catalytic efficiencies of antibodies described to date are too poor to function effectively in these applications. The real benefit of catalytic antibodies in diagnostic applications may arise in situations where a desired catalytic reaction simply cannot be carried out by known enzymes, but can be accomplished by a specifically tailored catalytic antibody. Results obtained during the next several years will begin to establish the scope and limitations of catalytic antibodies in the diagnostic arena. CATALYTIC ANTIBODIES - FUTURE CHAl I FNGES The major challenge for scientists designing catalytic antibodies is the improvement of catalytic efficiency. Ultimately, this aspect of the problem will critically affect the extent to which this methodology is applied to medical and diagnostic problems. Although it was the similarities between antibody combining sites and enzyme active sites that stimulated the development of catalytic antibodies with a broad spectrum of catalytic capabilities, the fundamental differences between antibodies and enzymes must now be overcome. Antibodies
Catalytic Antibodies
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are relatively rigid structures, capable of binding with high affinity to antigens, while enzymes are inherently flexible molecules that permit the facile access of substrates to the active site, and the efficient dissociation of products. Scientists will need to learn how to manipulate antibody structure to impart greater flexibility and higher catalytic efficiency. Many of the tools needed to accomplish this are in hand, including site-directed mutagenesis and selective chemical modification, but an even greater emphasis will be placed on understanding and manipulating antibody structure in order to generate highly efficient catalytic antibodies. A second challenge is to broaden the scope of reactions that can be catalyzed by antibodies. Based on the progress of the past several years, it is likely that many antibodies with novel catalytic capabilities will be developed in the near future.
A third challenge is the development of more efficient, streamlined methods for generation and production of catalytic antibodies or antibody fragments. This and other challenges presented by catalytic antibodies will demand further creative assimilation of ideas in chemistry, immunology, protein biochemistry and molecular biology. Pefer1. 2. 3. 4. 5. 6. 7.
8. 9. 10. 11.
12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33.
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