New Technology: Production of Stabilized Antibodies and Related Proteins

A new business opportunity is emerging where one can create stable and optimized antibodies, the proof of concept exists and collaboration with a established lab is possible.  In addition to the enhancement of existing and new antibodies used in the multi-billion dollar immunodiagnostic and immunotherapeutic industries, innovative potential applications of highly stable antibodies include:

It should be noted that the non-medical applications that are created by improved antibody stability can get to the market in one-tenth to one-fifth the time required for the FDA pipeline.   The non-medical field is not crowded with competitors

Antibodies are the basis of the adaptive immune system, which provides a natural response against infection by viruses, bacteria, and fungi.  Antibodies can also be evoked by vaccines, resulting in immunization against diseases such as polio.  Similarly, antisera that contain antibodies that recognize particular molecules of interest can be generated by inoculating animals with molecules for which a valuable detection requirement exists.  This capability is the basis of the multibillion dollar immunodiagnostics industry and the emerging immunotherapeutics field that provides treatments for diseases such as rheumatoid arthritis and some cancers.

However, commercial applications of antibodies have not significantly developed beyond extensive biomedical and limited biological research uses.  Antibodies, being proteins that evolved in animals, exhibit only moderate levels of stability.  Without gentle handling, which is acceptable for biomedical and bioresearch applications, antibodies tend to lose functional capabilities.  This characteristic has proven to be an essentially impenetrable roadblock to development of non-biomedical products that exploit the molecular recognition potential of antibodies. 

Antibodies are the ultimate example of combinatorial biochemistry.  Each human is thought to be capable of producing on the order of one billion different antibodies, generating a library that exceeds the diversity of any that has been produced by combinatorial chemistry efforts.   The binding site of an antibody is formed at the junction of two protein domains or modules.  Thus, different combinations of these domains lead to different combinations of amino acids in the binding site.  Different patterns of amino acids result in different binding specificity.  Thus, an antibody that interacts with a particular hormone will not interact with an explosive molecule, eg. TNT, and vice versa.

The modules that make up the binding site are known as variable domains; “variable” indicates differences in the amino acid sequences generated by the several genes that provide alternative amino acid sequences for each module.  One of the modules is known as the heavy chain variable domain and the other as the light chain variable domain, referring to the two types of polypeptide chain from which antibodies are assembled.  Humans have about 50 and 40 light and heavy chain variable domain genes, respectively.   This basis set would yield only 2000 different combinations or 2000 different binding sites.  However, as the cells that produce antibodies mature, additional mechanisms, largely mutation, result in several billion different combinations.  Many of these potential binding sites are filtered out of the collection if a tendency to react with molecules in the body is detected.

To generate a monoclonal antibody it is necessary to construct a clone of cells, all of which produce the same antibody.  To accomplish this, mice are first immunized with the molecule of interest.  After some time, a large number of cells that produce antibodies can be found in the spleen.  When spleen cells are fused to cells of an antibody-producing type of laboratory cancer cell line, some hybrid cells result that yield the antibody of interest and that can grow and divide indefinitely.  Thus, monoclonal antibodies can be produced against essentially any potential subject of analysis but almost all are suited only for laboratory applications.

Another contemporary strategy for acquiring antibody-type reagents is to collect, usually from human antibody-producing cells, the pieces of RNA that contain the information for the amino acid sequences of light and heavy chain variable domains.  These pieces are linked together and the DNA is inserted into the gene for a protein that is exposed on the surface of a virus that attacks bacteria.  This results in a library of viruses known as phage that exhibit or display a large number of different combinations of light and heavy chain variable domains on their surfaces.  When exposed to immobilized target molecules, some of the viruses are likely to stick to the target.  When removed and used to infect bacteria, a large quantity of viruses that generate antibody-type particles result.  In principle, the DNA that encodes the antibody-type particle can be transferred to E. coli or other organisms and the protein produced.  In practice, most of the time this procedure results in a very unstable construct that is not useful.  The tremendous potential of this technology, known as phage display, to produce scFv constructs (single chain antibody variable fragments), cannot be achieved if the instability problem is not resolved.
New studies have been directed towards identification of the structural basis of stability of human antibody light chains by combination of several stabilizing amino acid substitutions.  The resulting construct was 100,000 times more stable than the original variable domain in terms of the improved ratio of normal structure to unfolded.  This result strongly indicated that major improvements in the stability of antibodies can be achieved. 

Results
To test our strategy, we stabilized an antibody without impairing function. To examine this, two anti-laminin scFv constructs were modified, with different amino acid replacements, to 100-fold improvements in stability. The activity of the constructs improved by approximately 50-fold by minimizing scFv precipitation --- indicate that improved stability can be achieved without sacrificing performance. 

There is little doubt that antibodies can be stabilized in a routine manner. All of the opportunities and potential applications are made possible by the feasibility of engineering antibodies by limited amino acid replacements such that they can be virtually thought of as “devices” rather than fragile objects of biological origin.  As devices, applications can be taken to the field rather than be trapped in the laboratory.

Other novel applications are also possible.  It is well known that antibodies are members of the so-called immunoglobulin family, a group of more than 400 proteins in humans that are evolutionarily related to antibodies.   We now believe that at least three dozen other groups of families, not previously considered members of the immunoglobulin superfamily, also share a common, more distant, evolutionary origin.  Several of these proteins, such as tumor necrosis factor, are members of the innate immune system, while antibodies are the active component of the adaptive immune system. We anticipate that the stability of all of these classes of protein can be substantially improved, resulting in many potential therapeutic products as well as other commercial applications, such as heat-stable delta toxins for more robust agricultural use.  In addition, with a little imagination, novel, hybrid proteins can be contemplated.  For instance, “cuprebodies” could merge the electron transfer capabilities of cupredoxins with the specific binding properties of antibodies, effectively resulting in a sensor with built in signally capacity.  “Immunodismutases” designates any member of the immunoglobulin superfamily that is modified to add dismutase activity to its natural functions. The many members of the immunoglobulin superfamily, as well as the more distantly related molecules, potentially represent a new frontier for therapy.