Development and characterization of human monoclonal antibodies that neutralize multiple TGFβ isoforms

Transforming growth factor (TGF)beta levels are elevated in, and drive the progression of, numerous disease states such as advanced metastatic cancer and systemic and ocular fibrosis. There are 3 main isoforms, TGF beta 1, 2, and 3. As multiple TGF beta isoforms are involved in disease processes, maximal therapeutic efficacy may require neutralization of 2 or more of the TGF beta isoforms. Fully human antibody phage display libraries were used to discover a number of antibodies that bind and neutralize various combinations of TGF beta 1, 2 or 3. The primary panning did not yield any uniformly potent pan-isoform neutralizing antibodies; therefore, an antibody that displayed potent TGF beta 1, 2 inhibition, but more modest affinity versus TGF beta 3, was affinity matured by shuffling with a light chain sub-library and further screening. This process yielded a high affinity pan-isoform neutralizing clone. Antibodies were analyzed and compared by binding affinity, as well as receptor and epitope competition by surface plasmon resonance methods. The antibodies were also shown to neutralize TGF beta effects in vitro in 3 assays: 1) interleukin (IL)-4 induced HT-2 cell proliferation; 2) TGF beta-mediated IL-11 release by A549 cells; and 3) decreasing SMAD2 phosphorylation in Detroit 562 cells. The antibodies' potency in these in vitro assays correlated well with their isoform-specific affinities. Furthermore, the ability of the affinity-matured clone to decrease tumor burden in a Detroit 562 xenograft study was superior to that of the parent clone. This affinity-matured antibody acts as a very potent inhibitor of all 3 main isoforms of TGF beta and may have utility for therapeutic intervention in human disease.