Antiangiogenic And Antitumor Activities Of Anti-bFGF Monoclonal Antibodies

BACKGROUND & OBJECTIVEBasic fibroblast growth fator (bFGF, FGF-2) belongs to the family of heparin-binding growth factors. Acting as a broad-spectrum mitogen and potent angiogenic agent, bFGF mediates a variety of cellular responses including cell proliferation and differentiation. bFGF functions mainly by interacting with high affinity tyrosine kinase FGF receptors (FGFRs) on the surface of target cells. There are four Fgf receptor genes in mammals, FgfR1-FgfR4, encoding several splice variants that increase the signaling repertoire. bFGF could bind to all FGFR including FGFRlb, FGFR1c, FGFR2c, FGFR3b, FGFR3c, FGR4 except for FGFR2b. Upon binding to bFGF, FGFRs dimerize and are tyrosine phosphorylated, leading to the activation of downstream signaling pathways including MAPKs (mitogen-activated protein kinases)/ERKs (extracellular signal-regulated kinases), PI3K (phosphoinositide 3-kinase)/AKT (protein kinase B), and PLCy(Phospholipase C gamma)/PKC(protein kinase).bFGF and FGFR are highly expressed in many tumor tissue including melanoma and lung cancer, which play important role in tumor development and progression. All of these reports suggest the potential of bFGF as a target for tumor therapy.Angiogenesis is the process of new blood vessel formation from pre-existing ones, which play a key role in various physiological and pathological conditions, including embryonic development, wound repair, inflammation, and tumor growth. Tumor growth is absolutely dependent on the induction of a neovascular network.Tumor cells recruit new blood vessels that serve as conduits for delivery of nutrients and for tumor metastsis. Typically, angiogenesis occurs through endothelial cell sprouting. The first stage involves the degradation of the basement membrane around a blood vessel and of the interstitial matrix. Endothelial cells migrate in response to bFGF and proliferate along a leading migratory edge.Canalization or lumen formation, as well as branch and loop formation, lead to anastamosis of these spouts allowing blood flow.The final stage involve structural inclusion of pericytes and formation of a new basement membrane surrounding the new vessel.Malignant melanoma is one of the most aggressive Human tumors. In recent decades, the incidence of malignant melanoma has steadily increased. A particularly worrying feature of the tumor is its increasing incidence and its capacity for rapid metastatic spread. Because of their resistance to current therapies, such as surgical excision, systemic chemotherapy with dacarbazine, and immunotherapy with vaccines, melanomas remain a significant cause of mortality. To achieve improvement in overall survival, it is important to develop effective new therapies. The expression of bFGF is absent in normal melanocytes, which require exogenous bFGF to maintain the growth. bFGF transduced normal melanocytes exhibit transformed phenotype resembling early-stage melanoma, indicating a critical role of bFGF in the transformation of melanocytes to melanoma Most melanomas express high levels of bFGF and FGFRs, which form an autocrine loop and are critical for the survival and growth of melanoma cells; melanoma growth can be arrested by interfering with the production or biological activity of bFGF alone. bFGF has long been regarded as an important tumor angiogenic factor, and the specific roles of bFGF in angiogenesis and spontaneous metastasis of melanoma have been revealed too. All of these reports suggest the potential of bFGF as a target for melanoma therapy. Antisense targeting of bFGF/FGFR-1 in primary and metastatic melanoma cells blocks the tumor growth by inducing apoptosis without activation or increased production of VEGF. In addition, bFGF peptide vaccines also show significant inhibition on angiogenesis and tumor growth.Lung cancer is the major cause of death from all Human malignancies in CHINA and the other countries, and is frequently associated with poor prognosis. Overall 5-year relative survival rate of lung cancer for all stage is very low. This is mainly due to ineffective detection of lung cancer at its early stage. A particularly worrying feature of the tumor is its drug resistance and its capacity for rapid metastatic spread. bFGF is one of the epigenetic mechanisms explaining the multidrug resistance (MDR) of tumors. Some previous studies have shown that bFGF prevents chemotherapy induced apoptosis, which result in chemoresistance in many cancers such as small cell lung cancer. This anti-apoptotic effect is mediated by MAPK/ERKs, PI3K/AKT, and/or PKC that increase the expression and activation of several anti-apoptotic proteins including Bcl-2, Bcl-XL, and XIAP. All of these reports suggest the potential of bFGF as a target for lung cancer therapy.In this study, HUVECs, melanoma cells, and lung caner cells were used as targeting cells for anti-angiogenesis and anti-tumor therapy by anti-bFGF monoclonal antibodies.METHODS1. Anti-angiogenesis activity by anti-bFGF mAbs1) The hybridomas cells secreting monoclonal antibodies against bFGF were cloning. Ascites were produced by injecting hybridomas cells into Balb/c mouse. The antibodies were purified by a protein G column, and the isotyping of antibodies was carried out using ELISA. Anti-bFGF mAbs were incubated with rhFGF-2, rhFGF-1or rhVEGF165 immobilized onto 96-well plates and bound proteins were detected with a HRP-conjugated second antibody. To determine whether the anti-bFGF mAbs recognize conformational or linear epitopes of bFGF molecule, native or heat-denured bFGF was used in ELISA.2) In the competitive FGFR1/FGF-2 blocking assay,96-well plate was coated with 0.5μg/ml bFGF. Then FGFR-1βⅢC-Fc at a fixed amount (50 ng) was mixed with anti-bFGF mAbs at increasing concentrations and onto the coated plate. After incubation at 37℃for 1h, bound FGFR-1βⅢC-Fc were detected. In the competitive heparin/FGF-2 blocking assay,96-well plate was coated with 0.5μg/ml bFGF. Then anti-bFGF mAbs at a fixed amount was mixed with heparin at increasing concentrations and onto the coated plate. After incubation at 37℃for 1h, bound anti-bFGF mAbs were detected.3) HUVECs were seeded in 96-well plates. After overnight culture at 37℃under 5% CO2 in a humidified incubator, serial dilutions of purified anti-bFGF mAbs with lOng/ml rhFGF-2 were added to the wells. After the cells were incubated at 37℃for 4 more days,10μl CCK-8 reagent was added into each well.4) ECM gel-induced capillary tube formation assay was used as an in vitro measurement of angiogenesis. HUVECs were added onto each well in the presence of anti-bFGF mAbs with or without bFGF.5) HUVECs were harvested from culture flasks by trypsinization; Aliquot of 100μl cell suspension was added to each transwell insert of 8-μm-pore membrane. The inserts were then placed into the wells of the 24-well plates containing 0.6 ml of M199 with or without rhFGF-2. After incubation for 20 h at 37℃, cells remaining at the upper surface of the membrane were removed using a cotton swab, while the cells that migrated across the membrane to the lower surface were fixed with ethanol and stained with Giemsa solution. The number of stained cells was counted under several high-power microscopic fields.6) HUVECs were treated with anti-bFGF mAbs for 48h. B16F10 cells were added to HUVECs. After 15min incubation, the B16F10 cells adhering to HUVECs were counted.7) The 7-day-old embryos with intact yolks were placed in a bowl and incubated at 37℃with 3% CO2. Anti-bFGF mAbs or control mIgG were separately applied to the CAM of individual embryos. After 24 hours of incubation, neovascularization in chicken CAMs was observed with a stereomicroscope.2. Anti-melanoma growth activity by anti-bFGF mAbs1) Expression of bFGF, FGFR, Glypicanl, Glypican5, Syndecanl, Syndecan4 were detected by RT-PCR.2) Inhibition of B16 cells growth were assayed by CCK-8 kit.3) B16 cells were incubated for 96 h with anti-bFGF mAbs, or mouse normal IgG. The fraction of apoptotic cells were marked with FITC-conjugated Annexin-V and propodium iodide (PI). After 15 min of incubation at room temperature, the samples were analyzed by flow cytometry.4) B16F10 cells migration was assayed using a Transwell chamber as preciously described.5) Six-to seven-week-old male and female C57 BL/6 mice were subcutaneously inoculated in the right flank with 1×105 B16 cells; One week later, when palpable tumors (≥5 mm in diameter) developed, the mice were subcutaneously (around tumors) injected with anti-bFGF mAbs every 3 days for a total of five injections. Tumor size was measured every 3 days in two dimensions using a vernier caliper. Mice were sacrificed 3 weeks after tumor cell inoculation, and tumor weights were measured. The expression of CD31 in tumor tissues was analyzed to determine the intratumoral microvessel density. Apoptotic cells in tumor sections were detected using TUNEL assay kit.6) C57 BL/6 mice were injected with B16 cells as before mentioned. Mouse survival was monitored from the day of tumor inoculation until the day of animal death.3. Anti-lung cancer growth activity by anti-bFGF mAbsThe mouse lung cancer graft model was established by injection of Lewis lung carcinoma cells subcutaneously into the the right flank of C57BL/6 mice. When palpable tumors (≥5mm in diameter) developed, the mice were subcutaneously (around tumors) injected with anti-bFGF mAbs in 200μl PBS. Control mice received equal amounts of control IgG or PBS. Tumor volume and metastases of Lewis lung carcinoma were counted. The microvessel density (MVD) in tumor mass were measured by imunohistochemistry staining (IHCS). RESULTS1. Anti-angiogenesis activity by anti-bFGF mAbs1) No binding of purified anti-bFGF antibodies to aFGF and VEGF were detected. The result of MabF7 binding to both native and denatured bFGF implies that MabF7 recognizes a continuous epitope. MabF10 and MabF12 do not recognize the heat-denatured bFGF suggesting that they both recognize conformation-dependent epitopes on bFGF.2) Binding of bFGF to FGFR-1 was inhibited by anti-bFGF mAbs. IC50 of MabF7, MabF10 and MabF12 were 7.5μg/ml,65μg/ml,50μg/ml respectively. However anti-bFGF mAbs could not inhibit bFGF binding to heparin.3) Anti-bFGF mAbs could inhibit HUVECs proliferation induced by bFGF. IC50 of MabF7, MabF10 and MabF12 were 50μg/ml,100μg/ml和150μg/ml respectively.4) The tube formation in groups with no IgG, control IgG, MabF7, MabF10, and MabF12 are 100%,105.93±3.85%,56.53±4.35%,29.23±6.45%,12.77±2.67%, respectively. It indicates that anti-bFGF mAbs significantly inhibit the tube formation of HUVECs; however, control IgG shows no effect.5) The number of migrating cells in the presence of mAbs reduces significantly compared to the control group (treatment without supplement), and the migration ratio in groups treated with no IgG, control IgG, MabF7, MabF10, and MabF12 are100%,106.25±7.89%,69.50±6.86%,74.00±4.16%,67.75±3.30%, respectively.6) Anti-bFGF mAbs could not inhibit attaching of B16F10 to HUVECs.7)The blood vessel of CAM in groups treated with bFGF, bFGF+MabF7, bFGF+MabF 10, and bFGF+MabF 12 are 15±0.82,7.5±1.29,13.5±3.10,8.5±0.58 respectively.2. Anti-melanoma growth activity by anti-bFGF mAbs1) Anti-bFGF mAbs could inhibit B16 cells proliferation. Inhibition of MabF7, MabF10 and MabF12 were 42.37±2.38%,40.71±3.90%,41.48±2.76% respectively.2) Apoptosis was analyzed by flow cytometry with Annexin V/PI staining. With mAbs at a concentration of 200μg/ml,73.20±10% of B16 cells show apoptosis after 96 h of incubation, whereas IgG control group shows no effect compared with medium control3) The number of migrating cells in the presence of mAbs reduces significantly compared to the control group (treatment without supplement), and the migration ratio in groups treated with no IgG, control IgG, MabF7, MabF10, and MabF12 are100%,109.00±9.56%,72.14±13.57%,34.68±14.37%, and 36.56±13.60%, respectively.4) MabF7 remarkably reduces tumor burdens and suppresses tumor growth by 46.40% at the concentration of 5 mg/ml. However, the inhibitions by MabF10 and MabF12 are 23.24% and 19.89%.5) The microvessel density in tumor is determined by staining the tumor section with anti-CD31 antibody, and the numbers of microvessel density in tumor reduces significantly in MabF7 group (24.67±5.20) in comparison with that in IgG control group (45.38±12.59).6) More TUNEL-positive cells with deep brown stained nuclei are detected in MabF7 treated tumors than that in control IgG treated group.7) To investigate whether the inhibition of tumor growth by MabF7 impacts the survival of animals, a survival assay has been performed as shown in the Kaolan-Meier survival curve in Fig.8. The median survival time of mice treated with MabF7 (32 days) is longer than that of mice treated with PBS (24 days) or control IgG (26 days)3. Anti-Lewis lung cancer growth activity by anti-bFGF mAbsAnti-bFGF mAb could inhibit tumor growth in C57BL/6 mice Lewis lung cancer model. Tumor weight in MabF7 group is 2.64±1.05 g which is lower than that of PBS group (5.15±1.31 g) (P<0.05). The mean number of metastatic lung nodules in MabF7 group is 3.00±2.10 which is lower than that of PBS group (12.29±2.63) (P<0.05). Moreover MabF7 reduce the expression of MVD in the tumor tissue. CONCLUSIONS1. Binding of bFGF to FGFR-1 was inhibited by anti-bFGF mAbs MabF7, MabF10, MabF12. All of them could also inhibit HUVECs proliferation, tube formation, and migration. However in CAM assay, only MabF7 and MabF12 could inhibit angiogenesis.2. MabF7, MabF10, MabF12 could inhibit melanoma cells proliferation, migration, and induce apoptosis in melanoma cells in vitro. However only MabF7 could inhibit melanoma growth in vivo through inhibition of the angiogenesis and induction of the apoptosis in tumor.3. Anti-bFGF mAb could inhibit tumor growth and pulmonary metastasis in C57BL/6 mice Lewis lung cancer model, and suppress angiogenesis of tumor tissue.