Selection, Expression And Characterization Of A Human Neutralizing Antibody To Human Fibroblast Growth Factor-2

BACKGROUND AND OBJECTIVEFibroblast growth factors (FGFs), in particular FGF-2, have long been recognized as highly potent endothelial-cell mitogens and angiogenesis-inducing agents (1). FGF-2 is produced by a wide variety of cell types and accumulates in the extracellular matrix of most tissues where it remains sequestered in an inactive form. FGF-2 activity is thought to be regulated by enzymes that degrade extracellular matrix components, thus liberating active growth factor. FGF-2 predominantly activates the FGF-receptor type 1 (FGFR1) present on endothelial cells and most other cell types, including most tumor cells, which commonly results in either increased cell proliferation, migration or survival(2). There is an abundance of evidence indicating that increased FGF-2 activity is correlated with increased angiogenesis, both physiological and pathological, as well as tumor growth.Antagonism of FGF-2 has proven efficacious in proof-of-principle studies in many animal models of cancer (3,4). In particular, monoclonal and polyclonal antibodies which are specific for FGF-2 have been employed (5,6). However, these antibodies have been raised in animals and thus their therapeutic benefit in man may be limited because of their potential to induce immunogenic responses, and their rapid pharmacokinetic clearance. Entirely human antibodies would be much more desirable for therapeutic intervention in FGF-2-stimulated angiogenesis and tumor growth. METHODS1. The antibody library was panned with human recombinant FGF-2 for 4 rounds. The antigen binding activities of random clones were tested by ELISA in order to select specific antibodies, which were then examined by DNA sequence analysis.2. For obtaining soluble scFv proteins, phagemid DNA was isolated from positive clones and transformed into E. coli HB2151. After induced by addition of isopropyl-b-D-thiogalactopyranoside (IPTG), the bacteria were harvested and released the scFvs in the periplasmic compartment of the bacteria by ultrasonication, scFvs were further assayed for binding to FGF-2 by ELISA, SDS-PSGE, Western-blot.3. scFv 44 was converted into a full-length human IgG1 antibody. were sequentially inserted into the expression vector pIgG1 containing the gene for IgG1 CH1-hinge-CH2-CH3 from the Scripps research institute (La Jolla, California). A recombinant plasmid encoding the full length human IgG1 anti-FGF-2 antibody (pIgG1-44) was obtained. For the expression of soluble antibodies in mammalian cells, pIgG1-44 was transiently transfected into human 293T cells with FuGene HD, IgG-44 fusion proteins secreted into the medium were purified with Protein G affinity chromatography (Pharmacia). Purified hIgG1-44 was concentrated to 1 mg/ml, sterile filtered, and characterized by SDS-PAGE, ELISA and Western-blot.4. To test the neutralizing activity of hIgG1-44, purified hIgG1-44 was added into the HUVECs, to test whether it was able to inhibit the proliferation, migration and tube formation of the cells that was induced by FGF-2. then the purified hIgG1-44 was added to glioma cells, to examine if hIgG1-44 could inhibit tumor cell proliferation.5. In the present study, we constructed murtation phage-display libraries by mutagenizing germline hot spots in heavy chain CDR1 of the clone 44 Fv portion. clone 44 Fv was a first generation mutant of RFB4 obtained by phage-display targeting the antibody heavy chain CDR3 hot spots (27). DNA oligomers were designed to generate a library randomizing 9 nucleotides (3 consecutive amino acids). Degenerate oligomers with the sequence NNS were used (N randomizing with all four nucleotides and S introducing only C or G). The phagemid pComb3X-CDR1 for display of clone 44 single chain Fv (scFv) on the surface of bacteriophage M13 was made. after three rounds of panning, the specific phage antibodies against FGF-2 were highly enriched. The high affinity phage antibodies against FGF-2 were identified by phage-ELISA.RESULTS1. After 4 rounds of "adhesion-elution-amplification" panning,39 clones expressing anti-FGF-2 scFv were obtained, though DNA fingerprint and sequence analysis showed that they had diferent antibody V region encoding gene. After expression and purification in bacteria, three scFv, named clone 44, binds to FGF-2 with a high affinity and specificity, and completes with FGF-2 binding to its receptor.2. Thorugh optimize transfection reagen, transfection methodes, culture temperature, and added histone deacetylase inhibitors, the expression of human anti-FGF-2 antibody in 293T cells was increased ten-fold from 1.5mg/L to 15.1mg/L.after purified by protein G, we could obtain 12 mg purified antibody from 1L cell culture medium.3. In a set of vitro assays, hIgG1-44 inhibited various biological activities of FGF-2 such as the proliferation, migration and tube formation of human umbilical vein endothelial cells. More importantly, hIgG1-44 antibody also efficiently blocked the growth while inducing apoptosis of glioma cells.4. The results showed that we build a mutational library contained 2.5×106clones Under the super-infection of helper phage VCSM13, After three rounds of "adsorption-elution-amplification", phage antibodies against FGF-2 have been enriched effectively.50 phage antibody clones were rescued and 4 scFv phage antibody clones with high affinity and specificity to FGF-2 were obtained by phage-ELISA, when measure the affinity constant, we found the best one's affinity was increased 4 fold compared with hIgGl-44.CONCLUSIONS1. through screening a large single-chain phage antibody library of 6×1010 in size, we isolated seven scFv clones that bind to FGF-2. Among them,1A2 showed a high affinity and specificity. It does not bind to FGF-1, the closest member of FGF-2, but competes the binding of FGF-2 to FGFR1βⅢc. This suggests that 1A2 scFv and FGF-1βⅢc may bind to the same sites on FGF-2. After reformatted into a full length human IgG1 molecule, the hIgG1-1A2 also showed a high affinity to rhFGF-2, The full length hIgG1-1A2 antibody inhibited various biological activities of FGF-2 in vitro, including the proliferation, migration and tube formation of human umbilical vein endothelial cells. More importantly, it also inhibited proliferation of tumor cells by promoting their apoptosis. Our in vitro findings suggest the potential utility of hIgG1-1A2 anti-FGF-2 antibody as an anti-tumor agent.2. The results presented here demonstrate that an evolutionstrategy using intrinsic mutational hot spots as targets canbe used for antibody affinity maturation in vitro and therefore improve neutralizing activity.