ABZYMES AND ITS

APPLICATIONS
Presented by- Kritika Verma
B.Sc. (Part III) Applied Science
 Introduction
 An abzyme (from antibody and enzyme) is a
monoclonal antibody with catalytic activity.
 Also called catmab (catalytic monoclonal antibody).
 Usually artificial constructs but are also found in
normal humans (anti-vasoactive intestinal peptide
autoantibody) and in patients with autoimmune
diseases such as systematic lupus erythematosus.
 Potential tools in biotechnology.
 Could be of great use in practical medicine and
industries.
 Antibodies
 Also known as immunoglobulins.
 Antibodies are proteins produced by white bloods cells as part
of the immune response against any foreign antigen.
 Produced in all vertebrates.
 Very diverse and specific.
 Structure of antibodies
 Antibodies are large globin proteins assembled in a
crosslinked 4 chain structure.
 Composed of 2 identical heavy chains (approx. 220 amino
acids) and 2 identical heavy chains (approx. 450-575 amino
acids).
 Highly symmetrical and Y-shaped molecule.
 Light chains and heavy chains held together by strong non-
covalent forces and disulphide bonds.
 Ab has 2 fragments- Ag binding fragment (Fab), Constant
(Fc).
 Ag binds to first 100 aa of N-terminus of both light and heavy
chain.This portion is called Ag binding site.
 It is highly variable and called as variable domain.Denoted by VL
for light chain variable domain and as VH for heavy chain variable
domain.
 Remaining part referred to as constant.
 Fc is C-terminal portion of Ig heavy chain.
 Human Ig divided in 5 major classes- IgM,IgA,IgG,IgD,IgE.
 Forces responsible for Ag-Ab binding – Van der Walls
interaction,H-bonding and Coulomb forces.
 Ab characterised as either monoclonal or polyclonal.
 Monoclonal Ab - Ig raised against a particular portion of Ag called
epitope.
 Polyclonal Ab – Collection of Ab elicited against all epitopes of an
Ag elicited by any immune response.
Figure 1. The general structure of an antibody
 Enzymes
 In a living cell, many reactions take place that would not
normally happen if the reactants were simply mixed together.
 Many of reactions proceed too slowly on their own to sustain
life in the cell.
 Therefore, nature has designed biological protein catalysts
which we refer to as enzymes.
 In order for a reaction to occur, reactant molecules must have
sufficient energy to cross a potential activation energy barrier.
 One way in which catalysis occurs is through the enzyme
stabilization of the transition state, a high energy intermediate
substrate configuration.
 An enzyme binds the substrate close to catalytic groups in the
active site and to other substrates in the correct orientation to
react.
 This strong binding interaction contributes to a reduced
activation energy of the catalyzed reaction allowing for the
reaction to proceed at accelerated rates.
 Figure 2. Free energy diagram of a general catalytic
reaction. Catalysis is achieved by lowering the activation
energy for a process.
 History of Abzymology
 In 1946,Pauling proposed theory of enzyme
function,stated that enzyme and Ab have similar
structures. However enzyme active site doesn’t match
native configuration of substrate molecule (as in Ag
and Ab) but strained state or transition state
configuration of substrate molecule.
 Inspired by Pauling’s theory, Jencks proposed a
theory that amalgamated specific properties of Ab
and catalytic properties of enzymes in 1 molecule- the
abzyme (antibody+enzyme).
 In 1966,Slobin reported the first Ab with enzymatic
capabilities.
 Tramontano et al(1986) and Pollock et al(1986) produced the
first monoclonal Ab with catalytic properties.
 To date more than 100 artificially generated monoclonal
abzymes have been produced.They have been found to
catalyze hydrolysis of amides,esters,reactions of cyclisation,
decarboxylation and reactions not known to be catalyzed by
any known enzymes.
 Disadvantages- Slow catalysts,Kcat values 106 less than
common enzymes.
 Advantages- High affinity binding for preselected substrate,
Ability to carryout predetermined reactions with efficiencies
useful in medical applications
 Principle
 Enzymes function by lowering the activation energy of the
transition state, thereby catalyzing the formation of an
otherwise less-favourable molecular intermediate between
reactants and products. If an antibody is developed to a stable
molecule that's similar to an unstable intermediate of another
(potentially unrelated) reaction, the developed antibody will
enzymatically bind to and stabilize the intermediate state, thus
catalyzing the reaction. A new and unique type of enzymes are
produced
 Different approaches for abzyme
production
 Ligand-based abzyme production
 Anti-idiotypic abzyme production
 Natural abzymes
 Ligand-based Abzyme Production
 Monoclonal abzymes were first generated against transition
state analogues by Pollock and Tramontano.
 These antibodies were generated against antigen analogues of
the transition state for the ester hydrolysis of the p –
nitrophenyl phosphorylcholine and monoaryl phosphate esters
respectively.
 In both experiments, a phosphorus atom was used to model the
unstable tetrahedral carbon atom of the proposed transition
state generating a chemically stable transition state analogue
(Figure 2).
 When immunized with an accurate transition state analogue,
antibodies will be produced against the molecule which will
confer the ability to catalyze the desired reaction.
Figure 3. Ligand-based approach of abzyme synthesis. The structure of the
transition state of an ester bond hydrolysis can be mimicked by a stable
phosphonate analogue that is used to elicit catalytic antibodies.
 Natural abzymes
 Ab acquire catalytic activity by natural means..
 In 1989, Paul et al discovered first natural catalytic antibody in
the serum of bronchial asthma patients. It was found that the
isolated antibody, immunoglobin G (IgG), could hydrolyze
vasoactive intestinal peptide .
 In 1992, Shuster et al. detected both DNA and RNA
hydrolyzing antibodies in the sera of patients with systemic
autoimmune disorders such as systemic lupus erythematosus,
scleroderma and rheumatoid arthritis
 Catalytic antibodies have also been isolated from the milk of
healthy women.
 As recent as 2003, the antibodies IgM and IgG displaying
amylolytic activity were detected in the serum of patients with
multiple sclerosis .
 In patients with multiple myeloma, Bence Jones proteins, were
isolated from urine and were shown to exhibit many catalytic
abilities.
 Anti-idiotypic abzyme production
 This approach is an alternative to naturally occurring
abzymes and the anti-transition state analogue
approach for abzyme production.
 This approach was developed in an effort to conserve
some part of the enzyme active site catalytic
machinery in the antigen-binding portion of an
antibody .
 The first antibody (Ab1) is raised against the enzyme
active site creating structural features complementary
to the binding pocket. A second set of antibodies
(Ab2) are produced against the antigen-binding site
of Ab1.
 Some of the second generation anti-idiotypic antibodies (Ab2)
will be a high-order imprint of the enzyme active site where
others will mimic the catalytic function of the enzyme .
 Pillet et al have shown that an Ab3 antibody generated against
the Ab2 recognizes the original antigen. These results indicate
that the idiotypic network makes the transfer of functional
information from the enzyme active site to its antibody
counterpart possible.
 The anti-idiotypic approach to abzyme production was
pioneered by the work of Izadyar et al (1993).
 An interesting discovery was made by Pillet et al (2002) ,they
compared the variable regions of their amidase Ab2 9G4H9
abzyme to the GeneBank database sequences,found that these
highly specific regions of the antibody were homologous to
gene sequences coding for antibodies involved in natural
autoimmune processes.
 This suggests that the idiotypic approach to abzyme
production may be similar to the mysterious immune system
production of naturally occurring abzymes observed in
patients with several autoimmune and viral diseases .
Figure 4. Anti-Idiotypic approach for the generation of abzymes
 Applications of abzymes
 Therapeutic potential
 HIV treatment
 Antibody-Directed Enzyme Prodrug Therapy
(ADEPT)
 Antibody-Directed Abzyme Prodrug Therapy
(ADAPT)
 Industrial and medical applications
 Therapeutic potential
 Abzymes can be engineered to destroy specific viral pathogens
or tumour cells, to scavenge bodies from autoimmune
metabolites, to protect normal cells from cytotoxicity in drug
therapy, to design catalysts suitable for passive
immunotherapy of major disease or to elicit catalytic
immunity via vaccination.
 Abzymes are catalytic,biocomaptible,have turnover
capabilities and long serum half-lives.May be useful in lower
doses as compared to other drugs.
 May be used to destroy peptides or carbohydrates associated
with viral and tumour cells .
 A genetic deficiency of an extracellular enzyme could
be cured by immunization with an appropriate
antigen to elicit the production of an abzymes whose
function would replace that of the missing enzyme.
 Studies have demonstrated that the cocaine-degrading
abzyme 15A10 is capable of protecting rats that have
overdosed on cocaine from seizures and sudden death
and cocaine reinforcing toxic effects in rats.
 Abzymes may be useful as a new approach to prevent
cocaine addiction and overdose.
 It has been found that antibodies selectively bind cocaine and
are effective in preventing cocaine toxicity in animals.These
abzymes conferred the ability to degrade cocaine into the non-
toxic ecgonine methyl ester and bezoic acid byproducts before
it reaches the central nervous system.
 HIV Treatment
 Researches have engineered an abzyme that degrade the
superantigenic region of the gp120 CD4 binding site.This is
the one part of the HIV virus outer coating that does not
change, because it is the attachment point to T-lymphocytes,
the key cell in cell-mediated immunity. Once compromised,
patients produce antibodies to the more changeable parts of
the viral coat. The antibodies are ineffective given the virus'
ability to change that coat rapidly. Because this protein,
gp120, is necessary for the HIV virus to attach, it does not
change, and is vulnerable across the entire range of the HIV
variant population.
 The abzyme binds to the site, rendering the HIV virus inert. It
actually destroys the site, then can attach to other viruses.
 A single abzyme can destroy thousands of HIV viruses.
 Human clinical trials will be the next step in producing
treatment and perhaps even preventative vaccines and
microbicides.
Antibody-Directed Abzyme Prodrug
Therapy (ADAPT)
 ADEPT has been developed with the aim to lessen drug
damage to normal tissues during chemotherapy cancer
treatment.
 ADEPT is a currently practiced tumour drug therapy in which
a prodrug is activated by enzymes conjugated with antibodies
specific to tumour antigens.
 The antibody enzyme conjugate is administered first to allow
for tumour localization.The prodrug is subsequently
administered.
 The prodrug is less active and is selectively cleaved by the
enzyme-antibody conjugate at the tumour site converting it to
the parent cytotoxic drug.The use of prodrugs allows for the
effective tumour cell damage without causing peripheral
cytoxicity of normal cells.
 Drawback- The enzyme component of the antibody enzyme
conjugate is normally bacterial in origin and is designed as
such to avoid activation by any circulating endogenous
enzymes and to be specific for prodrugs . However, the
bacterial origin of the enzyme creates a problem when injected
into the patient because non-human antibodies elicit host
immune responses.This limits the repetition of ADEPT
treatments.
 Prevention- The concomitant administration of
immunosuppressants allow for slightly more repetitions of the
therapy, but still only allowing two to three cycles of
treatments
Antibody-Directed Abzyme Prodrug
Therapy (ADAPT)
 ADAPT been proposed to correct In an attempt to correct the
immunostimulatory effects of ADEPT’s bacterial enzyme
component.
 This therapy strategy employs catalytic antibodies instead of
the antibody-enzyme conjugate.
 ADAPT offers the similar advantage that there is no
equivalent molecule in the organism thus avoiding incidental
activation by circulating endogenous enzymes.
 Additionally, humanized prodrug-activating abzymes can be
prepared to lessen the immunogenic response.Therefore,
allowing for the repeated administration of the drug without
the need for concomitant administration of
immunosuppressant drugs
 Several abzymes have been found to catalyze reactions not
known to be carried out in natural systems. It would be
medicinally beneficial if a highly specific drug-releasing
abzyme could be prepared to exploit this unique ability.
 To date, a complete ADAPT method has not been fully realized
for therapeutic usage. However, there have been promising
results reported from successful animal trials of ADAPT.
 Application of commercially available abzyme 38C2 to the
anticancer drugs oloxrubicin and camptothecin demonstrated
that weakly toxic or nontoxic concentrations of the
corresponding prodrugs can be activated by therapeutically
relevant concentrations of the 38C2 abzymes to kill colon
cancer cell lines .
Figure 5. Selective activation of prodrugs at tumor site
by the Antibody Directed Abzyme Prodrug Therapy
(ADAPT)
 Industrial and medical applications
 Catalytic Ab have just started to achieve commercialization.
The first abzyme to be commercialized is the abzyme with
aldolase activity.
 The use of abzymes at industrial scale for specific synthesis of
molecules is still at the laboratory step.
 Several firms are showing their interest for using the aldolase
abzyme for synthesis of Epothilone A, a new anti-cancer
compound.
 Different laboratories have also proposed to use catalytic
antibodies for medical applications.
          
 One application could consider the use of hydrolytic properties
of abzymes to activate prodrugs. By targetting this activity in
the vicinity to tumour cells, prodrugs could be transformed
into cytotoxic compounds directly on tumour cells.
 Summary
 Growing evidence in the field of abzymology
suggests that catalytic antibodies may be important
mediators of immunological defense, regulation and
the autoimmune dysfunction. At this time further
studies on natural and artificial abzymes are needed
with the goal to gain improved understanding of their
structure-function relationship. This will allow for the
production of tailor-made abzymes for use in drug
therapy and other areas of practical medicine.