To prepare distinct human McAbs to TNF-alpha with high neutralizing potency using ribosome display technology.
The immunoglobulin heavy and light chain variable (VH, VL) genes were prepared from the peripheral blood lymphocytes in three arthritis patients by PCR. The genes encoding VH/K fragments were prepared by randomly combining VH and VL genes by SOE PCR. TNF-alpha binding fragments were selected over three cycles of ribosome display and the selected VH/Ks genes were cloned into pET22b(+)/BL21(DE3), from which soluble VH/K fragments were prepared. The expressed products of selected clones were analyzed by ELISA. Then the positive clones were characterized.
A human VH/K gene library with 6.7x10(12) numbers used for ribosome display was constructed. Among the 180 selected clones, two clones TRB21 and TRB409 exhibiting the highest ELISA signals were isolated. The analysis of the sequence of TRB21 and TRB409 showed that they were new human immunoglobulin V genes to TNF-alpha and they recognize TNF-alpha specifically and antagonize the cytolytic effect of TNF-alpha on 1929 cell.
The selected VH/Ks to TNF-alpha will be useful for constructing engineering antibodies with high affinity against arthritis. Ribosome display is a rapid means of generating fully human antibody fragments in vitro.