Production of catalytic

antibody

The year is 2050 - Drugs are being designed

with built-in homing devices that seek out their
targets like microscopic missiles.
Once the target is found, the drug molecules
fire until the target is effectively destroyed,
without creating so much as a ripple of side
effects to the patient.
interesting isnt it?

The Abzyme or catalytic antibody technology is the

offspring of the Monoclonal antibody technology that
came up in the 70s. The basic outline of abzyme action
came up 28 years back with the proof of concept
coming in 1986 with the first descriptions of active
abzymes capable of catalyzing ester hydrolysis
reactions.

The basics:
Enzymes are biological catalysts that speed up biochemical reactions
without undergoing any change themselves.
The mode of action of enzymes is that enzymes stabilize the transition state
by bringing down the activation energy (G) thus speeding up the reaction
converting it to product.
Thus they drive reactions that are, otherwise impossible at biological
temperatures.
Enzymes have active site that is complimentary and chemically to the
substrate, making them specific (hand in glove hypothesis).
Antibodies are specific substances that are produced by the body in
response to the presence of any foreign body, the Antigen.
The antigen-antibody specificity causes them to bind which then signal the
immune system to send in the troops i.e. the Leucocytes that destroy them.

WhyAbzymes?

The mechanism of antibody action is incomplete as it only targets

and catches hold of the intruder, while enzymes actually drive a reaction.
So why not combine their actions and make a substance that catches
hold of the antigen (analogous to function of antibody) and neutralizes it
(analogous to function of enzymes making it inactive by certain chemical
reactions) thus was born the idea of combining the functions of antibody
and enzymes to create abzymes.

Production of catalytic
antibody

Abzyme =

The principle:
The generation of abzymes is based on the Transition-state theory
of catalysis.
Catalysts work by stabilizing the transient form of a molecule (decrease the
activation energy G, less energy more stable), known as the transition state,
which occurs as the original molecule changes its shape during a chemical
reaction.
For example, a molecule will bend and strain just prior to being broken into two
pieces. If a mimic of the transition state, i.e. the strained version of the
molecule could be generated, then an antibody that would tightly bind that
mimic should be able to catalyze the reaction of the original molecule, by
causing it to bend and strain and ultimately break.
Therefore, by designing a good transition state mimic (Transition State Analog
TSA), novel catalysts can be created by harnessing the power of the immune
system and directing it towards a new function.

The production technique:

This hypothesis is made reality by the fact that antibody to almost any molecule
can be produced by the use of the immune system. The process of making
abzyme goes like this:
Step 1: the transition state of the specific enzyme reaction is studied.
Step 2: suitable transition state analogs (TSA) or HAPTENs that mimic the
transition states are created.
Step 3: introduce these HAPTENs into the organism and let the immune
system do the rest.
Step 4: the antibodies are raised against the TSA and are recovered.
These when in action bind to the specific antigen (because they are
antibodies) and also destroy them or whatever they are meant to do (because
they resemble the more stable transition state they drive the reaction to
completion).

The improvements in production:

Although abzymes have been shown to be able to catalyze a wide range
of different reactions, the catalytic efficiency of the ones prepared to date is
usually low (catalysis rateskcat>105) when compared with their natural,
enzyme counterparts.
This has led to efforts to improve the efficiency of abzymes by random
mutagenesis methods, such as phage display. In this method, hypervariable
regions of the abzyme's binding site are mutated and variants screened for
improved binding to substrate and catalytic activity. Previously hybridoma cells
of hyper-immunizedmice were used to synthesize antibodyin vivowhich took
a longer time (4-6 months) while advances in cell-culture techniques has made
it possible to carry out splenocyte immunizationsin vitroby incubating the
naive spleen cells in medium containing the antigen.
This process typically takes 3-5 days, at which stage a cell fusion can be used
to obtain hybridomas thus reducing abzyme production time.

Applications:
Targeting Device: In treatment of cancer to enhance drug
delivery
Abzymes have been used as tools to function as homing devices for the
site-specific delivery of the prodrug and activators of the prodrug into the
cytotoxic form. In cancer treatment one of the major hurdles is that the cytotoxic
chemicals destroy both the normal and tumor cells. Hence there is a need for
specific targeting of tumor cells and subsequent activation of the prodrug which
then destroy the oncogenic cells.
This is where the abzymes fit into picture. Prodrug is a pharmacologically
inactive compound that converts to the active form of the drug by endogenous
enzymes or metabolism. It is generally designed to overcome problems
associated with stability, toxicity, lack of specificity, or limited bioavailability.

The prodrug is comprised of the active drug compound itself and a

chemical masking group that temporarily suppresses activity and appreciably
reduces toxicity.
The first approach for site specific targeting is called as ADEPT (Antibody
Directed Enzyme Prodrug Therapy). It involves an antibody-enzyme conjugate
and prodrug mixture that is injected into the patient. The antibody binds to the
specific tumor antigens present on the surface of tumor cells. The antibodyenzyme complex on binding the cell releases the free enzyme which cleaves
the prodrug into the drug (active form) and the co-enzyme that suppresses the
drug of its function. The released drug then kills the tumor cell by its toxicity. But
the problem is that this enzyme should not be present in humans. Also foreign
enzymes from bacteria cannot be used due to immunogenicity problems
leading to low clinical value.

So abzymes are used in the modified ADAPT (Antibody Directed Abzyme

Prodrug Therapy). In this approach, antibodies are raised against the
appropriate transition state analogues (TSAs) to enable them to catalyze
prodrug activation.
The main drawback is that catalytic rate is slow and cell kill is only 75 -80% as
against 90% required to treat tumor else the escaped cells may develop
resistance.

New Targets:

Abzyme-targeted prodrug activation can be used to target HIV-infected

cytotoxic T-cells. This method can also be used for other chronic viral diseases,
if infected cells display viral protein that can be targeted like in Hepatitis C.

Correction of inborn metabolic errors, such as those that occur in patients

with severe combined immune deficiency. These individuals are highly
susceptible to infectious agents because their immune system is unable to
produce antibodies. The disease could be managed with a long-lived catalytic
antibody.

In military to destroy chemical and biological weapons (with extensive

modifications, of course)

Future promise:
The growing field of catalytic enzymes or abzymes holds great promise in therapies and if everything goes
well the futuristic therapy quoted in the beginning may become a reality soon and revolutionize the way drugs
target diseases.