"...

If we wish to catch up with Nature, we shall need to use the same methods as she
does, and I can foresee a time in which physiological chemistry will not only make
greater use of natural enzymes but will actually resort to creating synthetic ones."

Emil Fischer, 1902, Nobel Lecture

Emil Fischer,   German Chemist
was known for his works on
the structure of simple oses,
on proteins,   amino-acids
and for the synthese of numerous
chemical substances.
 

 Nobel Price in Chemistry, 1902

Schematic representation of an Antibody

 The concept that antibodies may catalyze chemical reactions was first
proposed by Linus Pauling in the 40's. He suggested that an enzyme lowers
the energy barriers of a reaction by stabilizing preferentially the transition
state of the substrate during the reaction rather than the substrate in its
ground state.

Linus Carl Pauling,   American

chemist,   was   known  for
his   works   on   organic
macromolecules   and   on
chemical   links.
 
 
 Nobel Price in Chemistry, 1954
 Nobel Price for Peace, 1962

           Twenty years later, William Jencks has proposed a strategy to prepare

new biocatalysts. This strategy was based on the production of antibodies
directed against stable molecules resembling the transition state structure of a
specific chemical transformation.

           A breakdown in the field came from the development of the

monoclonal antibody technique by Köhler and Milstein that has provided the
means for producing antibodies with a single antigenic defined specificity.

           If the first report suggesting that antibodies may act as catalysts was
published by the group of Bernard Green, the demonstration that antibodies
may be tailor-made to catalyze specifically chemical reactions was
simultaneously brought by the Californian laboratories of Richard
Lerner and Peter Schultz.

           Since these reports, strategies aimed to improve the efficiency of

abzymes were developed, including hapten design, immunization strategies,
or methods for screenig and selection (see for review Stevenson and Thomas-
2000).
   Antibodies directed againt transition state analogs  
  The anti-idiotypic approach: from enzymes to abzymes
  Naturally occurring abzymes
  Metallo- and Hemoabzymes

Antibodies directed againt transition state analogs: the

substrate-based approach
           Historically, the first approach for producing abzymes mimicking
enzymatic reactions, based on the concept proposed by L. Pauling and
developed by W. Jencks, consisted in immunizing mice with stable molecules
resembling the transition state structure of the chemical reaction that was
expected to be catalyzed.
The first successful results were obtained for hydrolysis of ester and carbonate
bonds by the groups of R. Lerner and P. Schultz. They used as immunogens
organophosphate compounds that were thought to mimic the tetrahedral
structure of the carbon atom during the hydrolytic process (cf. figure n°1).

 
 
Figure n°1
 
The Hydrolytic
Process
 

           Since that time, more than 70 different chemical reactions were
described to be catalyzed by antibodies. These reactions include hydrolysis of
chemical bonds (esters, carbonates, amides, phosphates,…), stereospecific
synthesis of compounds (esters, amides, Diels-Alder addition, …), as well as
reactions of isomerization, decarboxylation, oxidation and reduction, ….

           If the first obtained abzymes were characterized by a rather low

efficiency, the improvements in hapten design (bait and switch), in the
strategies used for immunization (heterologous immunization, reactive
immunization), in the site-directed modification of antibodies (mutagenesis)
or in the methods developed for screening (catELISA) and selection (phage
display) allowed to obtain catalysts with efficiency sufficient for an industrial
or a medical application. 

           On the other hand the resolution of an increasing number of

tridimensional structures of abzymes brings a better understanding of the
appearance and evolution of the catalytic function, not only in antibody
binding sites but also in enzyme active sites.

The anti-idiotypic approach: from enzymes to abzymes

           In 1974, Niels Jerne advanced the theory that regarded the immune
system as a network of interacting idiotypes. A major postulate of the
idiotypic network was that for each immunoglobulin (Ab1) generated against
an antigenic determinant there existed a complementary antibody (Ab2)
directed against the idiotypic determinants of Ab1.

           When the idiotypic determinant superimpose with the binding site of
Ab1, some of the Ab2 may mimic the antigen's determinants and are designed
as "internal images" of the original antigen. 

           Figure n°2 shows the experimental process which is used to product
abzymes.

 
 
Figure n°2
  
Experimental
Process
to obtain
Abzymes
 

For producing catalytic antibodies, a first antibody Ab1 is raised that

recognizes the active site of an enzyme so that the combining site of Ab1 has
structural features complementary to those of the enzyme.
A second set of antibodies (Ab2) is produced against the Ab1 combining site.
Among these second-generation, or anti-idiotypic antibodies, some of them
may represent a structural internal image of the original enzymatic site, and
in some cases may exhibit a catalytic activity.

           Using this approach, antibodies with esterase and amidase activities

were characterized. These abzymes usually bear efficient catalytic activities,
with a relaxed specificity when compared with the model enzyme.

Naturally occurring abzymes

           Antibodies with catalytic activities were isolated from sera of patients
with different diseases. The first natural abzymes were obtained by means of
antibody purification from human serum.
           Antibodies with protease activity against vasoactif intestinal peptide
(VIP) were first isolated in the serum of patients with asthma. Surprisingly,
antibodies exhibiting the same catalytic activity were obtained by immunizing
mice with VIP in its ground state. These monoclonal antibodies have allowed
to demonstrate that the catalytic activity is borne by the isolated light chain of
the antibody molecule.

           This VIP-ase activity was also shown to be present on Bence-Jones

proteins, that are monoclonal human light chains found in urine of patients
with multiple myeloma.

           Other protease activities were charaterized for cleaving of throglobulin

in the serum of patients with Hashimoto thyroiditis, or for hydrolyzing factor
VIII in hemophilia patients infused with homologous factor VIII. DNA
hydrolyzing autoantibodies were also isolated from the sera of patients with
systemic lupus erythematosus or rheumatoid arthritis. The DNA-hydrolyzing
activity could be correlated to the presence of high levels of anti-
topoisomerase I antibodies in the sera of patients.

Metallo- and Hemoabzymes

           The idea to produce antibodies that mimic metalloenzymes or
hemoenzymes was early advanced to generate antibodies able to cleave stable
chemical bonds or to catalyze peroxidase-like reactions.

           In 1989, an antibody catalyzing the hydrolysis of a Glycine-

Phenylalanine bond was obtained by using as the antigen an hapten that not
only induce the selective recognition of the Gly-Phe sequence, but also
induce the generation of residues able to complex a metal ion. When
complexed with zinc, this antibody exhibits a good catalytic activity.

           The generation of antibodies directed against different

metalloporphyrins has allowed to demonstrate that antibodies may mimic
metalloproteins. The Hemoabzymes obtained are able to catalyze peroxidase-
like activities and could be very interesting tools for the enantioselective
oxidation of molecules.
 Industrial and medical applications
           13 years after catalytic antibodies were introduced simultaneously
by Richard Lerner and Peter Schultz, they are just beginning to achieve
commercialization. The first antibody to be commercialized is the abzyme
with an aldolase activity developed in the Lerner's group and sold by Aldrich
Chemical Group.
A handful of firms, notably Med-Immune (Gaithersburg, MD, USA),
Advanced Biotech Ltd. (Ariel, Israel) and Prolifaron (San Diego, USA) are
developing catalytic antibodies for commercialization, along with researchers
at The Scripps Research Institute, Hebrew University of Jerusalem (Israel) and
Columbia University (New York City).

           The use of abzymes at industrial scale for specific synthesis of

molecules is still at the laboratory step, even if some firms like Novartis
(Switzerland) or Bristol-Myer Squibb (USA) have shown their interest for
using the aldolase abzyme, produced in the Lerner's group, 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 concern the use of hydrolytic
properties of abzymes to activate prodrugs. By targeting this activity in the
vicinity to tumor cells, prodrugs could be transformed into cytotoxic
compounds directly on tumor cells.
This anti-cancer therapy is designed as Antibody-Directed Abzyme Prodrug
Therapy (ADAPT): cf. figure n° 4. 
  
Figure n°4
 
Antibody-
Directed
Abzyme  
Prodrug
Therapy
 
(click on the picture
to see it on full screen).

           Another example of medical application concerns antibodies that

specifically hydrolyze cocaine. A commercialization agreement between
Columbia University and Ixsys Inc. could allow to use abzymes for treating
cocaine overdose and addiction. A third example is developed in Israel where
Advanced Biotech is producing catalytic antibodies for the treatment of gram-
negative sepsis, targeting the endotoxin LPS.

           Finally, all researches aimed to produce antibodies with a sequence-

specific protease activity could open new ways for anti-virus therapy and for
the conception of new vaccines.