How are human monoclonal pemphigus antibodies cloned?

Pemphigus is a disease that is caused by autoantibodies that bind to a family of molecules called desmogleins (Dsgs). In mucocutaneous PV, Dsg3 and Dsg1 are "attacked" by the autoantibodies. We know from previous work that these anti-Dsg antibodies (also called immunoglobulins) cause the blisters characteristic of pemphigus.

A relatively new molecular biologic technique called phage display was used to clone these autoantibodies. In this technique the DNA encoding all antibodies contained in a PV patient’s serum was amplified from the patient’s peripheral blood lymphocytes. The DNA was cloned into phage, which are viruses that infect bacteria. The cloning is done so that each phage (i.e. viral) particle contains the DNA for a single immunoglobulin molecule, and that actual antibody molecule is expressed on the surface of the phage particle. Thus, outside each phage is a monoclonal antibody, and inside is the DNA encoding that antibody.

The pool of phage can be considered a "library" that contains all the monoclonal antibodies that make up the total immunoglobulin pool in the patient. However most of these antibodies have nothing to do with pemphigus, but are immunoglobulins for other purposes (mostly the prevention of infectious disease). How, then, do we select the specific anti-Dsg antibodies from this library of antibodies? The answer is to take the library of phage particles and "pan" them on a plate that contains Dsg3 or Dsg1. The phage that have antibodies against the particular Dsg stick to the plate, and the other phage (that are not related to disease) are washed away.

These anti-Dsg phage can then be grown as individual colonies (i.e. clones) and the DNA inside can be sequenced to determine the gene families from which the immunoglobulins are derived. (Immunoglobulin molecules are derived in a complicated way from scores of individual gene families). Furthermore the cloned phage can be grown in bacteria in such a way that they produce essentially unlimited amounts of the monoclonal antibodies that can be characterized as pathogenic or non-pathogenic in cell culture or mouse models of disease.

In this way, Dr. Payne and her colleagues have produced pathogenic and non-pathogenic PV monoclonal antibodies and shown that they are derived from a very limited number of immunoglobulin gene families.

Why is the cloning of human monoclonal antibodies potentially important?

We know that patients’ sera contain a mixture of antibodies to different parts of the Dsgs, and that not all the antibodies are pathogenic (i.e. that antibodies to some parts of the Dsg molecule do not cause disease). In addition, most antibodies in a patient are not in any way related to pemphigus (i.e. are not directed again Dsgs), but are present to protect the patient from infectious disease.

Currently, therapy for pemphigus depends on generalized immunosuppression to lower total antibodies in blood, but antibodies are necessary to prevent infection, therefore, current therapy is a balance between enough immunosuppression to control disease but not too much immunosuppression which would cause susceptibility to infectious disease. In other words, the current therapy is not targeted in any way to immunoglobulins that are pathogenic compared to all the immunoglobulins (most of which are not at all related to pemphigus) in an individual.

By cloning human monoclonal PV autoantibodies, we can determine: a) which specific antibodies are pathogenic and which are not; b) what immunoglobulin gene families are used in anti-Dsg antibodies and anti-Dsg pathogenic antibodies.

These discoveries, then, may allow us to direct therapy just at the immunoglobulin gene families that are used by the pathogenic antibodies (as opposed to all immunoglobulins). In addition, we can use pathogenic monoclonal anti-Dsg antibodies to screen large libraries of peptides to find those that bind the pathogenic antibodies, thereby identifying specific peptides that might block disease-causing immunoglobulins.

Hope for the future

Characterizing pemphigus autoantibodies at the genetic and functional levels will hopefully be a step towards finding more specifically targeted therapies for these severe diseases.