This document provides an overview of abzymes (catalytic antibodies). It discusses how abzymes can catalyze a variety of reaction types through shape-based recognition of transition states or developing charges. Hapten design strategies include using transition state analogs, bait-and-switch approaches, and supplementing chemical functionality. Polyclonal abzymes have benefits like representing the full immune response and being easily extracted. Photoabzymes allow photocatalysis and screening methods for abzymes include HPLC, ELISA, and TLC. Research applications of abzymes include detoxification, biosensing, and antibody-directed enzyme prodrug therapy.

1. ABZYMES (Catalytic Antibodies)
1
By-
Ruchika Annie O’Niel (MB0715)
Atul Kotian (MB0215)
Akansha Ganguly (MB0415)
MBT 223 Enzymology
Department of Biotechnology
Goa University
2nd April ‘16
(2015-2016)

2. Contents
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Ruchika
• Introduction
• Categories of reactions catalysed
• Hapten recognition strategies
• Hapten design
• Means of production
• References
Atul
• Polyclonal catalytic antibodies
• Photo abzymes
• Screening methods
• Applications
• References
Akansha
• Research on catalytic antibodies
• References

3. INTRODUCTION
Antibody structure and function: large, homodimeric protein,
bearing two identical binding sites and consisting of six β-loops.
Antibodies recognize their antigens with high affinity and extreme selectivity.
rapid diversification of the sequences of these hypervariable regions
by processes involving mutation, gene splicing, and RNA splicing
New Class of Biocatalyst - enzyme mimics.
● potential for unique substrate selectivity by catalytic antibodies
(abzymes), based on molecular recognition
● Use transition state analogs (TSA) as haptens for obtaining
catalytic antibodies, support the idea that enzymes operate by
stabilizing the reaction's transition state, (Pauling).
● SLE (systemic lupus erythematosis) - IgG, RNAase and DNAase
● Mothers milk
● Eliciting abzymes for specific reactions. Practical, tailor-made catalysts
and for fundamental insights that could lead to greater understanding
and control of biological catalysis.
● Use of abzymes for nonbiological processes that demand regiochemical
or stereochemical specificity.

4. CATEGORIES OF REACTIONS CATALYSED
categories include olefin isomerization, reduction-oxidations, electrocyclic reactions, and addition-eliminations.
common feature: low activation energies. hence, readily measured rates of the uncatalyzed reactions. not a criterion for
catalysis, reference frame for low to moderate activities that could be expected under specific conditions..
Simple unimolecular processes, (lactonization, decarboxylation, olefin isomerization, and the Claisen rearrangement),
have modest requirements for catalysis and benefit from the shape recognition provided by the antibody-binding site
in guiding the substrate toward a productive conformation or straining it in favor of the transition- state geometry.
Processes that introduce a stereochemical center in the product, utilize the potential for the antibody to act as a
stereospecific catalyst (Diels-Alder reaction).
Limitation: turnover numbers and limited substrate conversion may restrict practical application.
Advantage: appreciable specificities and rate accelerations that are attainable. broad range of reactions and substrate
structures amenable to the technique, niche for practical use.
Antigenicity: substrates and haptens are hydrophobic, aromatic compounds that also make good antigens. very polar or
hydrophilic compounds, such as saccharides, tend to be poor haptens. Indirectly affect catalysis.poor antigenicity could
deter the targeting of certain transformations in which polar groups surround the site of chemical action. e.g. glycosidase
activities and endonuclease activities
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5. HAPTEN RECOGNITION STRATERGIES
1. Recognition of topology (transition state)
Shape Recognition uses highly structured transition states and highly specific binding site based on
molecular "shape", best for "no mechanism" reactions (sigma- tropic rearrangements, cycloadditions, and
pericyclic) reactions. e.g. Classical examples of such reactions are the Claisen re - arrangement, the
Diels-Alder process.)
Entropic factor is a principal component to the activation energy, reduce the rotational entropy.
Bimolecular processes depend mainly on translational entropy
Molecular recognition and steareospecific reaction transition states. Basically what the substrate must
assume in order to undergo bond reorganization
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8. 2. Recognition of developing charge (electrostatics)
Making/breaking of bonds: development of charged intermediates or charge separation in the transition
state.
Examples: charged intermediates or transition states are modeled by ionic analogs as haptens. (catalysis
of ester, amide bond hydrolysis, elicited against negatively charged haptens.)
Generally optimal activity in alkaline pH - hydroxide, or its equivalent ion, reacts with the ester or amide
substrate generating a full negative charge on the transition state.
At neutral pH, for hydrolysis, neutral nucleophiles or water participate in the reaction, is best described by
charge separation in the transition state. In that case, a zwitterionic hapten. (bait and switch example).
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9. 3. Participation of chemical groups in catalysis.
Molecular recognition: ion pairing, hydrogen bonding, and hydrophobic interactions
between ligands.
Polar or ionized groups elicit antibodies whose combining site residues can act as lures
( properly orientation to bound substrate)
"bait and switch''- hapten has unique structural components that represent substrate
features or a desired complementary chemical group.
. This evidence generally amounts to a pH dependence and protein chemical group
modification of the abzyme that are consistent with the predicted mechanism involving general acid or base catalysis.
Types of anti- bodies are selected on the basis of their high affinity for a hapten presenting an ionic group
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12. HAPTEN DESIGN
(1) Transition state analogs
(2) Bait and switch
(3) Entropy traps
(4) Desolvation
(5) Supplementation of chemical functionality (cofactors)
REACTIVE IMMUNIZATION
ANTI IDIOTYPIC ANTIBODIES
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15. 15
MEANS OF PRODUCTION

16. 16

17. REFERENCES
Advanced Dairy Chemistry: Volume 1: Proteins, Parts A&B - P. F. Fox, Paul L. H. McSweeney
A New Generation of Artificial Enzymes: Catalytic Antibodies or ‘Abzymes’ Re´ my Ricoux and Jean-Pierre Mahy,
Universite´ de Paris-sud 11, Orsay Cedex, France.
Antibody-antigen pair probed by combinatorial approach and rational design: Bringing together structural insights,
directed evolution, and novel functionality. - Alexey Belogurov Jr., Ivan Smirnov, Natalya Ponomarenko, Alexander
Gabibov (www.FEBSLetters.org)
Antibody-mediated catalysis: Induction and therapeutic relevance - Ankit Mahendra , Desirazu N. Rao, Ivan Peyron, Cyril
Planchais, Jordan D. Dimitrov, Srini V. Kaveri, Sébastien Lacroix-Desmazes. (Elsevier)
Catalytic antibodies and their applications in biotechnology: state of the art - Severine Padiolleau-Lefevre, Raouia Ben
Naya, Melody A., Shahsavarian, Alain Friboulet, Berangere Avalle. (Biotechnology letters)
Catalytic Antibodies George Micheal Blackburn, Arnuad Garcon, Sheffield
Immune Recognition, Antigen Design, and Catalytic Antibody Production Alfonso Tramontano,IGEN Research Institute,
Seattle, WA 98109.
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18. Abzymes II – Polyclonal
Abzymes, Photoabzymes and
Screening
Atul Kotian
MBT 1

19. Polyclonal Catalytic Antibodies
Polyclonal antibodies can be beneficial as they represent the entirety of the immune
response as no IgG are lost in isolation
Can be extracted without sacrificing host
When extracted at regular intervals after immunisation, maturity of catalytic activity can
be monitored
Cheaper to produce as compared to monoclonal antibodies
If sheep antibodies are generated, Large quantities of IgG can be extracted out
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20. (a) Demonstration of catalytic assay
Brocklehurst and Gallacher was the first to describe the generation and characterization of polyclonal
catalytic antibodies. A preparation of sheep IgG was obtained by immunization with phosphate (1) and
shown to catalyze the hydrolysis of the mixed carbonate (2). The reaction was chosen because the
products, two alcohols and carbon dioxide, are less likely to give product inhibition.
Iverson et al. in 1993 reported the production and characterization of rabbit polyclonal antibodies that
catalyze the hydrolysis of the triphenylmethyl ether substrate
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21. (b) Therapeutic applications
Some benefits are (i) catalytic antibodies having turnover capabilities could be used in
much lower doses (ii) antibodies are biocompatible and have long half lives in serum; (iii)
the progress in antibody engineering led to a reduction of the immunogenicity of xeno-
antibodies
Immunisation can be active or passive
Once immunised, patient can produce his own catalytic antibodies
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22. Photoabzymes
Very few reported till date
Antibodies generally known to be quite robust under UV radiation and capable of
handling reactive intermediates, including free radicals, carbonium ions and reactive
oxygen species as such species are commonly intermediates in photo catalysis
Allows carrying out photocatalysis in more efficient way
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23. The first attempt to generate a photocatalytic antibody, which was one of the
very first attempts to create any catalytic antibody, targeted the
photodimerization of methyl p-nitrocinnamate
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24. More research needed to study Photocatalysis by abzymes and their possible
applications in therapy
Strong potential for use in organic synthesis
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25. Screening of Abzymes
One of the critical steps in the process of preparing catalytic antibodies concerns the
detection of catalytic activity in the antibody-containing samples
Reverse phase HPLC is most commonly employed technique for checking presence of
compounds
As a result, most antigens and model substrates, were designed with strongly
immunogenic aromatic groups attached to relatively polar groups such as amides or
nitro groups to provide aqueous solubility and easy visualization
Certain unexpected compounds can also be visualized
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26. If the substrate lacks chromophores for HPLC detection, detection can be done by GC
Second most popular method is by Chromogenic or Fluorogenic reactions and
visualisation by uV/Vis spectroscopy
Enzyme coupled assays can also be performed
The secondary enzyme is most often a dehydrogenase, consumes the reaction product
to convert nicotinamide adenine dinucleotide (NAD) from its reduced (NADH) to its
oxidized (NAD+) state. uV spectra of NADH and NAD are different thereby allowing
visualization
cat-ELISA also performed by generating secondary antibodies against the abzymes
TLC can also be used
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27. 27
HPLC
ELISA and Catalysis

28. References
• Cochran, A. G., Pham, T., Sugasawara, R., Schultz, P. G., J. Am. Chem. Soc. 113 (1991), p. 6670–2
• Uno, T., Gong, B., Schultz, P. G., J. Am. Chem. Soc. 116 (1994), p. 1145–5
• Taran, F., Renard, P. Y., Bernard, H., Mioskowski, C., Frobert, Y., Pradelles, P., Grassi, J., J. Am. Chem. Soc. 120 (1998), p. 3332–9
• Jackson, D. Y., Jacobs, J. W., Sugasawara, R., Reich, S. H., Bartlett, P. A., Schultz, P. G., J. Am. Chem. Soc. 110 (1988), p. 4841–2
• Hilvert, D., Carpenter, S. H., Nared, K. D., Auditor, M. T. M., Proc. Natl. Acad. Sci. USA 85 (1988), p. 4953–5
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29. • Hilvert, D., Hill, K. W., Nared, K. D., Auditor, M.-T. M., J. Am. Chem. Soc. 111 (1989),
p.9261–2
• Meekel, A. A. P., Resmini, M., Pandit, U. K., Bioorg. Med. Chem. Lett. 4 (1996)
• Bahr, N., Güller, R., Reymond, J.-L., Lerner, R. A., J. Am. Chem. Soc. 118 (1996), p. 3550–
3555
• Bensel, N., Bahr, N., Reymond, M. T., Schenkels, C., Reymond, J.-L., Helv. Chim. Acta 82
(1999), p. 44–52
• Hugot, M., Bensel, N., Vogel, M., Reymond, M. T., Stadler, B., Reymond, J.-L., Baumann,
U., Proc. Natl. Acad. Sci. USA 99 (2002), p. 9674–8
• Bensel, N., Reymond, M. T., Reymond, J.-L., Chem. Eur. J. 7 (2001), p. 4604–4612
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30. Research on catalytic
antibodies
Akansha Ganguly
MB0415
Department of Biotechnology
Goa University

31. Therapeutic tools
Source: Se´verine et al,
Biotechnol Lett (2014)
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32. • Detoxification of organophosphate compounds
Smirnov et al. (2011): scFv A17 antibody, metabolizes organophosphate
substrate, prevents binding with acetylcholinesterases
• Biosensors
Iwai et al (2016) – Cbody-cpBLA fusion protein, antigen-dependent catalytic activity
at suboptimal reaction conditions
• Abzyme prodrug therapy (ADEPT)
Goswami et al. (2009) – 38C2 antibody variants found to inhibit primary and
metastatic tumours, activate prodrug form of the anti-cancer drugs doxorubicin and
camptothecin
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33. References
• Goswami RK, Huang ZZ, Forsyth JS, Felding-Habermann B, Sinha SC (2009). Multiple catalytic aldolase
antibodies suitable for chemical programming. Bioorg Med Chem Lett 19:3821–3824
• Smirnov I, Carletti E, Kurkova I, Nachon F, Nicolet Y, Mitkevich VA, De´bat H, Avalle B, Belogurov AA,
Kuznetsov N, Reshetnyak A, Masson P, Tonevitsky AG, Ponomarenko N, Makarov AA, Friboulet A,
Tramontano A, Gabibov A (2011). Reactibodies generated by kinetic selection couple chemical
reactivity with favorable protein dynamics. Proc Natl Acad Sci USA 108:15954–15959
• Iwai H, Kojima-Misaizu M, Dong J, Ueda H (2016). Creation of a ligand-dependent enzyme by fusing
circularly permuted antibody variable region domains. Bioconjug Chem
DOI: 10.1021/acs.bioconjchem.6b00040
• Padiolleau-Lefe`vre S, Naya BR, Shahsavarian MA, Friboulet A, Avalle B(2014). Catalytic antibodies and
their applications in biotechnology: state of the art. Biotechnol Lett 36:1369–1379
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