ABZYMES AND ITS ABZYMES AND ITS APPLICATIONSAPPLICATIONS

Presented by- Kritika VermaPresented by- Kritika Verma

B.Sc. (Part III) Applied B.Sc. (Part III) Applied ScienceScience

IntroductionIntroduction

An abzyme (from antibody and enzyme) is a An abzyme (from antibody and enzyme) is a monoclonal antibody with catalytic activity.monoclonal antibody with catalytic activity.

Also called catmab (catalytic monoclonal antibody).Also called catmab (catalytic monoclonal antibody). Usually artificial constructs but are also found in Usually artificial constructs but are also found in

normal humans (anti-vasoactive intestinal peptide normal humans (anti-vasoactive intestinal peptide autoantibody) and in patients with autoimmune autoantibody) and in patients with autoimmune diseases such as systematic lupus erythematosus.diseases such as systematic lupus erythematosus.

Potential tools in biotechnology.Potential tools in biotechnology. Could be of great use in practical medicine and Could be of great use in practical medicine and

industries.industries.

AntibodiesAntibodies

Also known as immunoglobulins.Also known as immunoglobulins. Antibodies are proteins produced by white bloods cells as part Antibodies are proteins produced by white bloods cells as part

of the immune response against any foreign antigen.of the immune response against any foreign antigen. Produced in all vertebrates.Produced in all vertebrates. Very diverse and specificVery diverse and specific..

Structure of antibodiesStructure of antibodies Antibodies are large globin proteins assembled in a Antibodies are large globin proteins assembled in a

crosslinked 4 chain structure.crosslinked 4 chain structure. Composed of 2 identical heavy chains (approx. 220 amino Composed of 2 identical heavy chains (approx. 220 amino

acids) and 2 identical heavy chains (approx. 450-575 amino acids) and 2 identical heavy chains (approx. 450-575 amino acids).acids).

Highly symmetrical and Y-shaped molecule.Highly symmetrical and Y-shaped molecule. Light chains and heavy chains held together by strong non-Light chains and heavy chains held together by strong non-

covalent forces and disulphide bonds.covalent forces and disulphide bonds. Ab has 2 fragments- Ag binding fragment (Fab), Constant Ab has 2 fragments- Ag binding fragment (Fab), Constant

(Fc).(Fc). Ag binds to first 100 aa of N-terminus of both light and heavy Ag binds to first 100 aa of N-terminus of both light and heavy

chain.This portion is called Ag binding site.chain.This portion is called Ag binding site.

It is highly variable and called as variable domain.Denoted by It is highly variable and called as variable domain.Denoted by VL for light chain variable domain and as VH for heavy chain VL for light chain variable domain and as VH for heavy chain variable domain.variable domain.

Remaining part referred to as constant.Remaining part referred to as constant. Fc is C-terminal portion of Ig heavy chain.Fc is C-terminal portion of Ig heavy chain. Human Ig divided in 5 major classes- IgM,IgA,IgG,IgD,IgE.Human Ig divided in 5 major classes- IgM,IgA,IgG,IgD,IgE. Forces responsible for Ag-Ab binding – Van der Walls Forces responsible for Ag-Ab binding – Van der Walls

interaction,H-bonding and Coulomb forces.interaction,H-bonding and Coulomb forces. Ab characterised as either monoclonal or polyclonal.Ab characterised as either monoclonal or polyclonal. Monoclonal Ab - Ig raised against a particular portion of Ag Monoclonal Ab - Ig raised against a particular portion of Ag

called epitope.called epitope. Polyclonal Ab – Collection of Ab elicited against all epitopes of Polyclonal Ab – Collection of Ab elicited against all epitopes of

an Ag elicited by any immune response.an Ag elicited by any immune response.

Figure 1Figure 1. The general structure of an antibody. The general structure of an antibody

EnzymesEnzymes

In a living cell, many reactions take place that would not In a living cell, many reactions take place that would not normally happen if the reactants were simply mixed together. normally happen if the reactants were simply mixed together.

Many of reactions proceed too slowly on their own to sustain Many of reactions proceed too slowly on their own to sustain life in the cell.life in the cell.

Therefore, nature has designed biological protein catalysts Therefore, nature has designed biological protein catalysts which we refer to as enzymes. which we refer to as enzymes.

In order for a reaction to occur, reactant molecules must have In order for a reaction to occur, reactant molecules must have sufficient energy to cross a potential activation energy barrier.sufficient energy to cross a potential activation energy barrier.

One way in which catalysis occurs is through the enzyme One way in which catalysis occurs is through the enzyme stabilization of the transition state, a high energy intermediate stabilization of the transition state, a high energy intermediate substrate configuration.substrate configuration.

An enzyme binds the substrate close to catalytic groups in the An enzyme binds the substrate close to catalytic groups in the active site and to other substrates in the correct orientation to active site and to other substrates in the correct orientation to react. react.

This strong binding interaction contributes to a reduced This strong binding interaction contributes to a reduced activation energy of the catalyzed reaction allowing for the activation energy of the catalyzed reaction allowing for the reaction to proceed at accelerated rates.reaction to proceed at accelerated rates.

Figure 2Figure 2. Free energy diagram of a general catalytic . Free energy diagram of a general catalytic reaction. Catalysis is achieved by lowering the activation reaction. Catalysis is achieved by lowering the activation

energy for a process.energy for a process.

History of AbzymologyHistory of Abzymology In 1946,Pauling proposed theory of enzyme In 1946,Pauling proposed theory of enzyme

function,stated that enzyme and Ab have similar function,stated that enzyme and Ab have similar structures. However enzyme active site doesn’t match structures. However enzyme active site doesn’t match native configuration of substrate molecule (as in Ag native configuration of substrate molecule (as in Ag and Ab) but strained state or transition state and Ab) but strained state or transition state configuration of substrate molecule.configuration of substrate molecule.

Inspired by Pauling’s theory, Jencks proposed a Inspired by Pauling’s theory, Jencks proposed a theory that amalgamated specific properties of Ab theory that amalgamated specific properties of Ab and catalytic properties of enzymes in 1 molecule- the and catalytic properties of enzymes in 1 molecule- the abzyme (abzyme (aantintibbody+enody+enzymezyme). ).

In 1966,Slobin reported the first Ab with enzymatic In 1966,Slobin reported the first Ab with enzymatic capabilities.capabilities.

Tramontano et al(1986) and Pollock et al(1986) produced the Tramontano et al(1986) and Pollock et al(1986) produced the first monoclonal Ab with catalytic properties.first monoclonal Ab with catalytic properties.

To date more than 100 artificially generated monoclonal To date more than 100 artificially generated monoclonal abzymes have been produced.They have been found to abzymes have been produced.They have been found to catalyze hydrolysis of amides,esters,reactions of cyclisation, catalyze hydrolysis of amides,esters,reactions of cyclisation, decarboxylation and reactions not known to be catalyzed by decarboxylation and reactions not known to be catalyzed by any known enzymes.any known enzymes.

DisadvantagesDisadvantages- Slow catalysts,Kcat values 106 less than - Slow catalysts,Kcat values 106 less than common enzymes.common enzymes.

AdvantagesAdvantages- High affinity binding for preselected substrate, - High affinity binding for preselected substrate, Ability to carryout predetermined reactions with efficiencies Ability to carryout predetermined reactions with efficiencies useful in medical applicationsuseful in medical applications

PrinciplePrinciple Enzymes function by lowering the activation energy of the Enzymes function by lowering the activation energy of the

transition state, thereby catalyzing the formation of an transition state, thereby catalyzing the formation of an otherwise less-favourable molecular intermediate between otherwise less-favourable molecular intermediate between reactants and products. If an antibody is developed to a stable reactants and products. If an antibody is developed to a stable molecule that's similar to an unstable intermediate of another molecule that's similar to an unstable intermediate of another (potentially unrelated) reaction, the developed antibody will (potentially unrelated) reaction, the developed antibody will enzymatically bind to and stabilize the intermediate state, thus enzymatically bind to and stabilize the intermediate state, thus catalyzing the reaction. A new and unique type of enzymes are catalyzing the reaction. A new and unique type of enzymes are producedproduced

Different approaches for abzyme Different approaches for abzyme productionproduction

Ligand-based abzyme productionLigand-based abzyme production Anti-idiotypic abzyme productionAnti-idiotypic abzyme production Natural abzymesNatural abzymes

Ligand-based Abzyme ProductionLigand-based Abzyme Production

Monoclonal abzymes were first generated against transition Monoclonal abzymes were first generated against transition state analogues by Pollock and Tramontano.state analogues by Pollock and Tramontano.

These antibodies were generated against antigen analogues of These antibodies were generated against antigen analogues of the transition state for the ester hydrolysis of the p –the transition state for the ester hydrolysis of the p –nitrophenyl phosphorylcholine and monoaryl phosphate esters nitrophenyl phosphorylcholine and monoaryl phosphate esters respectively. respectively.

In both experiments, a phosphorus atom was used to model the In both experiments, a phosphorus atom was used to model the unstable tetrahedral carbon atom of the proposed transition unstable tetrahedral carbon atom of the proposed transition state generating a chemically stable transition state analogue state generating a chemically stable transition state analogue (Figure 2). (Figure 2).

When immunized with an accurate transition state analogue, When immunized with an accurate transition state analogue, antibodies will be produced against the molecule which will antibodies will be produced against the molecule which will confer the ability to catalyze the desired reaction.confer the ability to catalyze the desired reaction.

Figure 3Figure 3. Ligand-based approach of abzyme synthesis. The structure of the . Ligand-based approach of abzyme synthesis. The structure of the transition state of antransition state of an ester bond hydrolysis can be mimicked by a stable ester bond hydrolysis can be mimicked by a stable phosphonate analogue that is used to elicit catalytic antibodies.phosphonate analogue that is used to elicit catalytic antibodies.

Natural abzymesNatural abzymes Ab acquire catalytic activity by natural means.. Ab acquire catalytic activity by natural means.. In 1989, Paul et al discovered first natural catalytic antibody in In 1989, Paul et al discovered first natural catalytic antibody in

the serum of bronchial asthma patients. It was found that the the serum of bronchial asthma patients. It was found that the isolated antibody, immunoglobin G (IgG), could hydrolyze isolated antibody, immunoglobin G (IgG), could hydrolyze vasoactive intestinal peptide . vasoactive intestinal peptide .

In 1992, Shuster et al. detected both DNA and RNA In 1992, Shuster et al. detected both DNA and RNA hydrolyzing antibodies in the sera of patients with systemic hydrolyzing antibodies in the sera of patients with systemic autoimmune disorders such as systemic lupus erythematosus, autoimmune disorders such as systemic lupus erythematosus, scleroderma and rheumatoid arthritisscleroderma and rheumatoid arthritis

Catalytic antibodies have also been isolated from the milk of Catalytic antibodies have also been isolated from the milk of healthy women.healthy women.

As recent as 2003, the antibodies IgM and IgG displaying As recent as 2003, the antibodies IgM and IgG displaying amylolytic activity were detected in the serum of patients with amylolytic activity were detected in the serum of patients with multiple sclerosis . multiple sclerosis .

In patients with multiple myeloma, Bence Jones proteins, were In patients with multiple myeloma, Bence Jones proteins, were isolated from urine and were shown to exhibit many catalytic isolated from urine and were shown to exhibit many catalytic abilities.abilities.

Anti-idiotypic abzyme productionAnti-idiotypic abzyme production

This approach is an alternative to naturally occurring This approach is an alternative to naturally occurring abzymes and the anti-transition state analogue abzymes and the anti-transition state analogue approach for abzyme production. approach for abzyme production.

This approach was developed in an effort to conserve This approach was developed in an effort to conserve some part of the enzyme active site catalytic some part of the enzyme active site catalytic machinery in the antigen-binding portion of an machinery in the antigen-binding portion of an antibody . antibody .

The first antibody (Ab1) is raised against the enzyme The first antibody (Ab1) is raised against the enzyme active site creating structural features complementary active site creating structural features complementary to the binding pocket. A second set of antibodies to the binding pocket. A second set of antibodies (Ab2) are produced against the antigen-binding site (Ab2) are produced against the antigen-binding site of Ab1.of Ab1.

Some of the second generation anti-idiotypic antibodies (Ab2) Some of the second generation anti-idiotypic antibodies (Ab2) will be a high-order imprint of the enzyme active site where will be a high-order imprint of the enzyme active site where others will mimic the catalytic function of the enzyme .others will mimic the catalytic function of the enzyme .

Pillet et al have shown that an Ab3 antibody generated against Pillet et al have shown that an Ab3 antibody generated against the Ab2 recognizes the original antigen. These results indicate the Ab2 recognizes the original antigen. These results indicate that the idiotypic network makes the transfer of functional that the idiotypic network makes the transfer of functional information from the enzyme active site to its antibody information from the enzyme active site to its antibody counterpart possible.counterpart possible.

The anti-idiotypic approach to abzyme production was The anti-idiotypic approach to abzyme production was pioneered by the work of Izadyar et al (1993). pioneered by the work of Izadyar et al (1993).

An interesting discovery was made by Pillet et al (2002) ,they An interesting discovery was made by Pillet et al (2002) ,they compared the variable regions of their amidase Ab2 9G4H9 compared the variable regions of their amidase Ab2 9G4H9 abzyme to the GeneBank database sequences,found that these abzyme to the GeneBank database sequences,found that these highly specific regions of the antibody were homologous to highly specific regions of the antibody were homologous to gene sequences coding for antibodies involved in natural gene sequences coding for antibodies involved in natural autoimmune processes. autoimmune processes.

This suggests that the idiotypic approach to abzyme This suggests that the idiotypic approach to abzyme production may be similar to the mysterious immune system production may be similar to the mysterious immune system production of naturally occurring abzymes observed in production of naturally occurring abzymes observed in patients with several autoimmune and viral diseases .patients with several autoimmune and viral diseases .

Figure 4Figure 4. Anti-Idiotypic approach for the generation of abzymes. Anti-Idiotypic approach for the generation of abzymes

Applications of abzymesApplications of abzymes

Therapeutic potentialTherapeutic potential HIV treatmentHIV treatment Antibody-Directed Enzyme Prodrug Therapy Antibody-Directed Enzyme Prodrug Therapy

(ADEPT)(ADEPT) Antibody-Directed Abzyme Prodrug Therapy Antibody-Directed Abzyme Prodrug Therapy

(ADAPT)(ADAPT) Industrial and medical applicationsIndustrial and medical applications

Therapeutic potentialTherapeutic potential Abzymes can be engineered to destroy specific viral pathogens Abzymes can be engineered to destroy specific viral pathogens

or tumour cells, to scavenge bodies from autoimmune or tumour cells, to scavenge bodies from autoimmune metabolites, to protect normal cells from cytotoxicity in drug metabolites, to protect normal cells from cytotoxicity in drug therapy, to design catalysts suitable for passive therapy, to design catalysts suitable for passive immunotherapy of major disease or to elicit catalytic immunotherapy of major disease or to elicit catalytic immunity via vaccination. immunity via vaccination.

Abzymes are catalytic,biocomaptible,have turnover Abzymes are catalytic,biocomaptible,have turnover capabilities and long serum half-lives.May be useful in lower capabilities and long serum half-lives.May be useful in lower doses as compared to other drugs. doses as compared to other drugs.

May be used to destroy peptides or carbohydrates associated May be used to destroy peptides or carbohydrates associated with viral and tumour cells . with viral and tumour cells .

A genetic deficiency of an extracellular enzyme could A genetic deficiency of an extracellular enzyme could be cured by immunization with an appropriate be cured by immunization with an appropriate antigen to elicit the production of an abzymes whose antigen to elicit the production of an abzymes whose function would replace that of the missing enzyme.function would replace that of the missing enzyme.

Studies have demonstrated that the cocaine-degrading Studies have demonstrated that the cocaine-degrading abzyme 15A10 is capable of protecting rats that have abzyme 15A10 is capable of protecting rats that have overdosed on cocaine from seizures and sudden death overdosed on cocaine from seizures and sudden death and cocaine reinforcing toxic effects in rats.and cocaine reinforcing toxic effects in rats.

Abzymes may be useful as a new approach to prevent Abzymes may be useful as a new approach to prevent cocaine addiction and overdose.cocaine addiction and overdose.

It has been found that antibodies selectively bind cocaine and It has been found that antibodies selectively bind cocaine and are effective in preventing cocaine toxicity in animals.These are effective in preventing cocaine toxicity in animals.These abzymes conferred the ability to degrade cocaine into the non-abzymes conferred the ability to degrade cocaine into the non-toxic ecgonine methyl ester and bezoic acid byproducts before toxic ecgonine methyl ester and bezoic acid byproducts before it reaches the central nervous system. it reaches the central nervous system.

HIV TreatmentHIV Treatment Researches have engineered an abzyme that degrade the Researches have engineered an abzyme that degrade the

superantigenic region of the superantigenic region of the gp120gp120 CD4CD4 binding site.This is binding site.This is the one part of the the one part of the HIVHIV virusvirus outer coating that does not outer coating that does not change, because it is the attachment point to T-lymphocytes, change, because it is the attachment point to T-lymphocytes, the key cell in cell-mediated immunity. Once compromised, the key cell in cell-mediated immunity. Once compromised, patients produce antibodies to the more changeable parts of patients produce antibodies to the more changeable parts of the viral coat. The antibodies are ineffective given the virus' the viral coat. The antibodies are ineffective given the virus' ability to change that coat rapidly. Because this protein, ability to change that coat rapidly. Because this protein, gp120, is necessary for the HIV virus to attach, it does not gp120, is necessary for the HIV virus to attach, it does not change, and is vulnerable across the entire range of the HIV change, and is vulnerable across the entire range of the HIV variant population.variant population.

The abzyme binds to the site, rendering the HIV virus inert. It The abzyme binds to the site, rendering the HIV virus inert. It actually destroys the site, then can attach to other viruses. actually destroys the site, then can attach to other viruses.

A single abzyme can destroy thousands of HIV viruses. A single abzyme can destroy thousands of HIV viruses. Human clinical trials will be the next step in producing Human clinical trials will be the next step in producing

treatment and perhaps even preventative vaccines and treatment and perhaps even preventative vaccines and microbicides.microbicides.

Antibody-Directed Abzyme Prodrug Antibody-Directed Abzyme Prodrug Therapy (ADAPT)Therapy (ADAPT) ADEPT has been developed with the aim to lessen drug ADEPT has been developed with the aim to lessen drug

damage to normal tissues during chemotherapy cancer damage to normal tissues during chemotherapy cancer treatment.treatment.

ADEPT is a currently practiced tumour drug therapy in which ADEPT is a currently practiced tumour drug therapy in which a prodrug is activated by enzymes conjugated with antibodies a prodrug is activated by enzymes conjugated with antibodies specific to tumour antigens.specific to tumour antigens.

The antibody enzyme conjugate is administered first to allow The antibody enzyme conjugate is administered first to allow for tumour localization.The prodrug is subsequently for tumour localization.The prodrug is subsequently administered. administered.

The prodrug is less active and is selectively cleaved by the The prodrug is less active and is selectively cleaved by the enzyme-antibody conjugate at the tumour site converting it to enzyme-antibody conjugate at the tumour site converting it to the parent cytotoxic drug.The use of prodrugs allows for the the parent cytotoxic drug.The use of prodrugs allows for the effective tumour cell damage without causing peripheral effective tumour cell damage without causing peripheral cytoxicity of normal cells.cytoxicity of normal cells.

Drawback- Drawback- The enzyme component of the antibody enzyme The enzyme component of the antibody enzyme conjugate is normally bacterial in origin and is designed as conjugate is normally bacterial in origin and is designed as such to avoid activation by any circulating endogenous such to avoid activation by any circulating endogenous enzymes and to be specific for prodrugs . However, the enzymes and to be specific for prodrugs . However, the bacterial origin of the enzyme creates a problem when injected bacterial origin of the enzyme creates a problem when injected into the patient because non-human antibodies elicit host into the patient because non-human antibodies elicit host immune responses.This limits the repetition of ADEPT immune responses.This limits the repetition of ADEPT treatments.treatments.

PreventionPrevention- The concomitant administration of - The concomitant administration of immunosuppressants allow for slightly more repetitions of the immunosuppressants allow for slightly more repetitions of the therapy, but still only allowing two to three cycles of therapy, but still only allowing two to three cycles of treatmentstreatments

Antibody-Directed Abzyme Prodrug Antibody-Directed Abzyme Prodrug Therapy (ADAPT)Therapy (ADAPT)

ADAPT been proposed to correct In an attempt to correct the ADAPT been proposed to correct In an attempt to correct the immunostimulatory effects of ADEPT’s bacterial enzyme immunostimulatory effects of ADEPT’s bacterial enzyme component.component.

This therapy strategy employs catalytic antibodies instead of This therapy strategy employs catalytic antibodies instead of the antibody-enzyme conjugate. the antibody-enzyme conjugate.

ADAPT offers the similar advantage that there is no ADAPT offers the similar advantage that there is no equivalent molecule in the organism thus avoiding incidental equivalent molecule in the organism thus avoiding incidental activation by circulating endogenous enzymes.activation by circulating endogenous enzymes.

Additionally, humanized prodrug-activating abzymes can be Additionally, humanized prodrug-activating abzymes can be prepared to lessen the immunogenic response.Therefore, prepared to lessen the immunogenic response.Therefore, allowing for the repeated administration of the drug without allowing for the repeated administration of the drug without the need for concomitant administration of the need for concomitant administration of immunosuppressant drugs immunosuppressant drugs

Several abzymes have been found to catalyze reactions not Several abzymes have been found to catalyze reactions not known to be carried out in natural systems. It would be known to be carried out in natural systems. It would be medicinally beneficial if a highly specific drug-releasing medicinally beneficial if a highly specific drug-releasing abzyme could be prepared to exploit this unique ability. abzyme could be prepared to exploit this unique ability.

To date, a complete ADAPT method has not been fully realized To date, a complete ADAPT method has not been fully realized for therapeutic usage. However, there have been promising for therapeutic usage. However, there have been promising results reported from successful animal trials of ADAPT. results reported from successful animal trials of ADAPT.

Application of commercially available abzyme 38C2 to the Application of commercially available abzyme 38C2 to the anticancer drugs oloxrubicin and camptothecin demonstrated anticancer drugs oloxrubicin and camptothecin demonstrated that weakly toxic or nontoxic concentrations of the that weakly toxic or nontoxic concentrations of the corresponding prodrugs can be activated by therapeutically corresponding prodrugs can be activated by therapeutically relevant concentrations of the 38C2 abzymes to kill colon relevant concentrations of the 38C2 abzymes to kill colon cancer cell lines .cancer cell lines .

Figure 5Figure 5. Selective activation of prodrugs at tumor site . Selective activation of prodrugs at tumor site by the Antibody Directed Abzyme Prodrug Therapy by the Antibody Directed Abzyme Prodrug Therapy (ADAPT)(ADAPT)

Industrial and medical applicationsIndustrial and medical applications

Catalytic Ab have just started to achieve commercialization. Catalytic Ab have just started to achieve commercialization. The first abzyme to be commercialized is the abzyme with The first abzyme to be commercialized is the abzyme with aldolase activity.aldolase activity.

The use of abzymes at industrial scale for specific synthesis of The use of abzymes at industrial scale for specific synthesis of molecules is still at the laboratory step.molecules is still at the laboratory step.

Several firms are showing their interest for using the aldolase Several firms are showing their interest for using the aldolase abzyme for synthesis of Epothilone A, a new anti-cancer abzyme for synthesis of Epothilone A, a new anti-cancer compound.compound.

Different laboratories have also proposed to use catalytic Different laboratories have also proposed to use catalytic antibodies for medical applications. antibodies for medical applications.

                     

One application could consider the use of hydrolytic properties One application could consider the use of hydrolytic properties of abzymes to activate prodrugs. By targetting this activity in of abzymes to activate prodrugs. By targetting this activity in the vicinity to tumour cells, prodrugs could be transformed the vicinity to tumour cells, prodrugs could be transformed into cytotoxic compounds directly on tumour cells.into cytotoxic compounds directly on tumour cells.

SummarySummary

Growing evidence in the field of abzymology Growing evidence in the field of abzymology suggests that catalytic antibodies may be important suggests that catalytic antibodies may be important mediators of immunological defense, regulation and mediators of immunological defense, regulation and the autoimmune dysfunction. At this time further the autoimmune dysfunction. At this time further studies on natural and artificial abzymes are needed studies on natural and artificial abzymes are needed with the goal to gain improved understanding of their with the goal to gain improved understanding of their structure-function relationship. This will allow for the structure-function relationship. This will allow for the production of tailor-made abzymes for use in drug production of tailor-made abzymes for use in drug therapy and other areas of practical medicine. therapy and other areas of practical medicine.