Recombinant human monoclonal antibodies. Basic principles of the immune system transferred to E. coli.
To produce human monoclonal antibodies in bacteria, a gene repertoire of IgM variable regions was isolated from human peripheral B lymphocytes by the polymerase chain reaction. Alternatively, synthetic antibody genes with random hypervariable regions are being generated that may provide libraries of even higher complexity. For the selection of specific monoclonal antibodies from these libraries, we have developed two E. coli vector systems that facilitate the surface display of an antibody physically linked to its own gene. The phagemid pSEX encodes a fusion protein of an antigen binding domain (Fv-antibody) with the docking protein (pIII) of filamentous phages. Specific antibody genes can therefore be enriched by antigen affinity chromatography. The plasmid pAP1 encodes a fusion protein of an Fv-antibody with a bacterial cell-wall protein. Bacteria carrying this plasmid express functional Fv-antibodies tightly bound to their surface. This should enable the selection of single cells with a fluorescence-assisted cell sorter (FACS) using labeled antigen or by adsorption to immobilized antigen. These vectors permit three major principles of the antibody response to be mimicked in E. coli: 1. Generation of a highly complex antibody repertoire; 2. Clonal selection procedures for library screening; and 3. The possibility of increasing a given affinity by repeated rounds of mutation and selection.
Fuchs
P
P
Recombinant Antibody Group, German Cancer Research Center, Heidelberg.
Dübel
S
S
Breitling
F
F
Braunagel
M
M
Klewinghaus
I
I
Little
M
M
D000911:Antibodies, Monoclonal / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000276:immunology / Q000378:metabolism; D005822:Genetic Vectors; D006801:Humans; D007107:Immune System / Q000276:immunology*; D000073718:Pancreatitis-Associated Proteins; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism
D000911:Antibodies, Monoclonal; D000073718:Pancreatitis-Associated Proteins; C000612141:REG3A protein, human; D011994:Recombinant Proteins