ABZYMES (Catalytic Antibodies)

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By-Ruchika Annie O’Niel (MB0715)Atul Kotian (MB0215)Akansha Ganguly (MB0415)

MBT 223 EnzymologyDepartment of Biotechnology

Goa University2nd April ‘16(2015-2016)

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

INTRODUCTIONAntibody 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.

CATEGORIES OF REACTIONS CATALYSEDcategories 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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HAPTEN RECOGNITION STRATERGIES1. 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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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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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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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 ANTIBODIES12

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MEANS OF PRODUCTION

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REFERENCESAdvanced 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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Abzymes II – Polyclonal Abzymes, Photoabzymes and Screening

Atul KotianMBT 1

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Polyclonal Catalytic AntibodiesPolyclonal 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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(a) Demonstration of catalytic assayBrocklehurst 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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(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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PhotoabzymesVery 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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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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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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Screening of AbzymesOne 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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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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HPLC

ELISA and Catalysis

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• Hilvert, D., Hill, K. W., Nared, K. D., Auditor, M.-T. M., J. Am. Chem. Soc. 111 (1989), p.9261–2

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• Bensel, N., Reymond, M. T., Reymond, J.-L., Chem. Eur. J. 7 (2001), p. 4604–4612

Research on catalytic antibodies

Akansha GangulyMB0415

Department of Biotechnology Goa University

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Therapeutic tools

Source: Se´verine et al, Biotechnol Lett (2014)

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• 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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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