Fibroblast growth factors (FGFs) represent a family structurally related growth factors that promote a wide range of biological effects by interacting with different FGF receptors (FGFRs) in target cells. Among the different FGFRs, FGFR1 is expressed by various tumor cell types as well as by tumor endothelial cells and plays a prominent role in both tumor cell and stromal cell compartments of the neoplastic tissue by mediating tumor angiogenesis, growth and invasiveness. Humanized antibodies in single-chain fragment variable format (scFv) are antibody fragments with improved pharmacokinetics for tissue penetration and can be easily conjugated allowing their use as cytokine, radionuclide, or drug carriers. Our group identified scFv_RR-C2 as a neutralizing recombinant scFv antibody specific for the extracellular domain of the FGFR1. We screened a human single-chain fragment variable (scFv) antibody phage display library against the extracellular domain of the FGFR-1-IIIc isoform that harbors the FGF binding site. Several phages were isolated and tested for specificity and sensitivity, and the most promising antibody fragment RR-C2 was characterized for its biochemical and biological properties. ScFv RR-C2 specifically recognizes FGFR-1 with a Kd value of ~150 nM. The antibody fragment also recognizes FGFR-1 when the receptor is exposed on the cell surface, thus preventing the formation of the ternary complex among FGFR-1, its ligand FGF2, and cell surface heparan sulfate proteoglycans. Accordingly, scFv RR-C2 specifically inhibits FGF2-mediated mitogenic activity in endothelial cells of human, bovine, and murine origin in a nanomolar range of concentrations. Also, the antibody fragment prevents FGF2-triggered sprouting of both human umbilical vein endothelial cell spheroids and of murine endothelium from aortic rings, and hampers the angiogenic activity exerted both by FGF2 in the chick embryo chorioallantoic membrane assay and by S115 mouse mammary tumor cells in the Matrigel plug assay. Indeed, anti-FGFR1 scFv fragment specifically impairs the proliferation triggered by FGF/FGFR1 autocrine and paracrine loops of stimulation in murine models of prostate (TRAMPC2), melanoma (B16-F10 ) and lung cancer (3LL). The antibody-mediated anti-FGFR1 therapy will target both tumor endothelium, overcoming resistance to anti-VEGF-targeting therapy, and FGFR1-expressing cancer cells. Forthcoming experiments will provide novel scFv antibodies endowed with more potent targeting and neutralizing activity against FGFR1.
Targeting FGF/FGFR1 system for an integrated anti-tumor/anti-angiogenic therapy of cancer
RONCA, Roberto;DELL'ERA, Patrizia;PRESTA, Marco
2014-01-01
Abstract
Fibroblast growth factors (FGFs) represent a family structurally related growth factors that promote a wide range of biological effects by interacting with different FGF receptors (FGFRs) in target cells. Among the different FGFRs, FGFR1 is expressed by various tumor cell types as well as by tumor endothelial cells and plays a prominent role in both tumor cell and stromal cell compartments of the neoplastic tissue by mediating tumor angiogenesis, growth and invasiveness. Humanized antibodies in single-chain fragment variable format (scFv) are antibody fragments with improved pharmacokinetics for tissue penetration and can be easily conjugated allowing their use as cytokine, radionuclide, or drug carriers. Our group identified scFv_RR-C2 as a neutralizing recombinant scFv antibody specific for the extracellular domain of the FGFR1. We screened a human single-chain fragment variable (scFv) antibody phage display library against the extracellular domain of the FGFR-1-IIIc isoform that harbors the FGF binding site. Several phages were isolated and tested for specificity and sensitivity, and the most promising antibody fragment RR-C2 was characterized for its biochemical and biological properties. ScFv RR-C2 specifically recognizes FGFR-1 with a Kd value of ~150 nM. The antibody fragment also recognizes FGFR-1 when the receptor is exposed on the cell surface, thus preventing the formation of the ternary complex among FGFR-1, its ligand FGF2, and cell surface heparan sulfate proteoglycans. Accordingly, scFv RR-C2 specifically inhibits FGF2-mediated mitogenic activity in endothelial cells of human, bovine, and murine origin in a nanomolar range of concentrations. Also, the antibody fragment prevents FGF2-triggered sprouting of both human umbilical vein endothelial cell spheroids and of murine endothelium from aortic rings, and hampers the angiogenic activity exerted both by FGF2 in the chick embryo chorioallantoic membrane assay and by S115 mouse mammary tumor cells in the Matrigel plug assay. Indeed, anti-FGFR1 scFv fragment specifically impairs the proliferation triggered by FGF/FGFR1 autocrine and paracrine loops of stimulation in murine models of prostate (TRAMPC2), melanoma (B16-F10 ) and lung cancer (3LL). The antibody-mediated anti-FGFR1 therapy will target both tumor endothelium, overcoming resistance to anti-VEGF-targeting therapy, and FGFR1-expressing cancer cells. Forthcoming experiments will provide novel scFv antibodies endowed with more potent targeting and neutralizing activity against FGFR1.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
