anti-CTGF Antibody Research Articles

A virus-like particle-based connective tissue growth factor vaccine suppresses carbon tetrachloride-induced hepatic fibrosis in mice.

Connective tissue growth factor (CTGF) has been recognized as a central mediator and promising therapeutic target in hepatic fibrosis. In this study, we generated a novel virus-like particle (VLP) CTGF vaccine by inserting the 138–159 amino acid (aa) fragment of CTGF into the central c/e1 epitope of C-terminus truncated hepatitis B virus core antigen (HBc, aa 1–149) using a prokaryotic expression system. Immunization of BALB/c mice with the VLP vaccine efficiently elicited the production of anti-CTGF neutralizing antibodies. Vaccination with this CTGF vaccine significantly protected BALB/c mice from carbon tetrachloride (CCl4)-induced hepatic fibrosis, as indicated by decreased hepatic hydroxyproline content and lower fibrotic score. CCl4 intoxication-induced hepatic stellate cell activation was inhibited by the vaccination, as indicated by decreased α-smooth muscle actin expression and Smad2 phosphorylation. Vaccination against CTGF also attenuated the over-expression of some profibrogenic factors, such as CTGF, transforming growth factor-β1, platelet-derived growth factor-B and tissue inhibitor of metalloproteinase-1 in the fibrotic mouse livers, decreased hepatocyte apoptosis and accelerated hepatocyte proliferation in the fibrotic mouse livers. Our results clearly indicate that vaccination against CTGF inhibits fibrogenesis, alleviates hepatocyte apoptosis and facilitate hepatic regeneration. We suggest that the vaccine should be developed into an effective therapeutic measure for hepatic fibrosis.

CTGF mediates high-glucose induced epithelial–mesenchymal transition through activation of β-catenin in podocytes

Objective: It is known that connective tissue growth factor (CTGF) and β-catenin are involved in DN; however, the underlying molecular mechanisms remain unknown. Here we hypothesized that podocytes undergo epithelial–mesenchymal transition (EMT) in high-glucose condition and CTGF mediates high-glucose induced EMT by activating β-catenin in podocytes.Methods: The differentiated podocytes were cultured and divided into three groups: the normal glucose group (5 mmol/L glucose), the high-glucose group (30 mmol/L glucose), and the osmotic control group (5 mmol/L glucose supplemented with 25 mmol/L mannitol). The morphology of cultured podocytes was observed under phase contrast microscopy. To study the relevant markers of EMT, as well as CTGF and β-catenin, the mRNA and protein expressions were analyzed by real-time PCR and western blotting, respectively. In addition, the effects of inhibition CTGF by anti-CTGF antibody on high-glucose-induced EMT and β-catenin expression in podocytes were studied.Results: High glucose not only induced phenotypic transition of podocytes but also increased the expression of CTGF and β-catenin. Under high-glucose condition, podocytes underwent EMT, which were demonstrated by downregulation of nephrin and upregulation of desmin. Moreover, high-glucose-induced EMT and β-catenin overexpression in podocytes were attenuated by anti-CTGF antibody.Conclusion: CTGF and β-catenin are involved in the EMT of podocytes in diabetes. CTGF mediates high-glucose induced EMT through activation of β-catenin in podocytes. CTGF inhibition may protect podocytes from EMT in diabetes.

Endothelin-1 induces connective tissue growth factor expression in human lung fibroblasts by ETAR-dependent JNK/AP-1 pathway

Endothelin-1 (ET-1) acts as a key mediator of vasoconstriction and tissue repair. Overproduction of connective tissue growth factor (CTGF) underlies the development of lung fibrosis. ET-1 induces expression of matrix-associated genes in lung fibroblasts, however, little is known about the signaling pathway of CTGF expression caused by ET-1. In this study, we found that ET-1 caused concentration- and time-dependently increases in CTGF expression in human embryonic lung fibroblast cell line (WI-38). ET-1-induced CTGF expression was inhibited by BQ123 (ETAR antagonist), but not BQ788 (ETBR antagonist). Moreover, ET-1-induced CTGF expression was significantly reduced by JNK inhibitor (SP600125), the dominant-negative mutants of JNK1/2 (JNK1/2 DN), and AP-1 inhibitor (curcumin). ET-1 induced phosphorylations of JNK and c-Jun in time-dependent manners. AP-1 luciferase activity was concentration-dependently increased by ET-1, and this effect was attenuated by SP600125. We also found that ET-1-induced CTGF expression was most controlled by the AP-1 binding region of CTGF promoter. ET-1-indiced CTGF luciferase activity was predominately controlled by the sequence −747 to −408bp upstream of the transcription start site on the human CTGF promoter. Furthermore, ET-1 caused the formation of AP-1-specific DNA-protein complex and the recruitment of c-Jun to the CTGF promoter. Moreover, we found that ET-1 induced α-smooth muscle actin (α-SMA) expression, which was inhibited by BQ123, SP600125, curcumin, and anti-CTGF antibody. These results suggest that ET-1 stimulates expressions of CTGF and α-SMA through ETAR/JNK/AP-1 signaling pathway, and CTGF is required for ET-1-induced α-SMA expression in human lung fibroblasts.

Anti-Connective Tissue Growth Factor Antibody Treatment Reduces Extracellular Matrix Production in Trabecular Meshwork and Lamina Cribrosa Cells

We have previously demonstrated elevated levels of connective tissue growth factor (CTGF/CCN2) in the aqueous humor (AqH) of pseudoexfoliation glaucoma (PXFG) patients when compared with cataract controls. Furthermore, there is a significant trabecular meshwork (TM) and lamina cribrosa (LC) fibrotic phenotype associated with glaucoma, possibly driven by CTGF. The purpose of this study was to investigate the potential of anti-CTGF immunotherapy in glaucoma. Primary TM and LC cells were cultured from human donors with (GTM/GLC) and without (NTM/NLC) primary open angle glaucoma (POAG). Aqueous humor samples from PXFG, POAG, and control cataract patients were applied to N/GTM and N/GLC cells in the presence or absence of a therapeutic, humanized monoclonal anti-CTGF antibody FG-3019 (10 μg/mL). Hydrogen peroxide (H2O2) was also used as a stimulus. Expression of fibrotic genes (fibronectin-1, fibrillin-1, CTGF, collagen type I α1, and α-smooth muscle actin) was assessed by q-PCR. Protein expression of collagen 1A1 and α-smooth muscle actin was examined in N/G TM cells by SDS-PAGE. The modulatory effect of FG-3019 (10 μg/mL) and IgG (10 μg/mL) were also assessed. Treatment of cells with AqH from PXFG and POAG patients and H2O2 induced a significant (P < 0.05) increase in expression of profibrotic genes, which was significantly reduced by pretreatment with FG-3019 (P < 0.05). FG-3019 also reduced expression of α-smooth muscle actin and collagen 1A1 protein expression in N/GTM cells. FG-3019 is effective in blocking extracellular matrix production in TM and LC cells, thus supporting a role for the use of anti-CTGF immunotherapy in the treatment of glaucoma.

Targeting connective tissue growth factor (CTGF) in acute lymphoblastic leukemia preclinical models: anti-CTGF monoclonal antibody attenuates leukemia growth

Connective tissue growth factor (CTGF/CCN2) is involved in extracellular matrix production, tumor cell proliferation, adhesion, migration, and metastasis. Recent studies have shown that CTGF expression is elevated in precursor B-acute lymphoblastic leukemia (ALL) and that increased expression of CTGF is associated with inferior outcome in B-ALL. In this study, we characterized the functional role and downstream signaling pathways of CTGF in ALL cells. First, we utilized lentiviral shRNA to knockdown CTGF in RS4;11 and REH ALL cells expressing high levels of CTGF mRNA. Silencing of CTGF resulted in significant suppression of leukemia cell growth compared to control vector, which was associated with AKT/mTOR inactivation and increased levels of cyclin-dependent kinase inhibitor p27. CTGF knockdown sensitized ALL cells to vincristine and methotrexate. Treatment with an anti-CTGF monoclonal antibody, FG-3019, significantly prolonged survival of mice injected with primary xenograft B-ALL cells when co-treated with conventional chemotherapy (vincristine, L-asparaginase and dexamethasone). Data suggest that CTGF represents a targetable molecular aberration in B-ALL, and blocking CTGF signaling in conjunction with administration of chemotherapy may represent a novel therapeutic approach for ALL patients.

Abstract B58: Connective tissue growth factor (CTGF) as a novel therapeutic target in high grade serous ovarian cancer

Abstract The tumor microenvironment plays an active role in promoting proliferation, invasion and metastasis, thereby making soluble factors secreted by cancer-associated fibroblasts promising therapeutic targets. As a result, secreted tumor stromal factors are increasingly being targeted by newly developed anti-cancer therapies. To identify such stromal factors in ovarian cancer, we performed gene expression profiling on microdissected fibroblasts from normal ovary and from high-grade, late stage serous ovarian tumors. We identified a signature of TGFb-regulated genes, over-expressed in cancer-associated fibroblasts, which encode for secreted factors that may serve as potential therapeutic targets. One of these genes, connective tissue growth factor (CTGF), is a secreted protein that promotes proliferation, motility and extracellular matrix production in a number of cancer types. CTGF is currently being pursued as a therapeutic target by FibroGen Inc., who has developed a therapeutic anti-CTGF monoclonal antibody FG-3019. FG-3019 is currently under clinical investigation in pancreatic cancer and fibrotic diseases. To determine the role of CTGF and its potential as a therapeutic target in high-grade serous ovarian cancer (HGSOC), we examined its effect on tumor promotion in ovarian cancer cell lines and associations between CTGF expression and clinico-pathologic characteristics in HGSOC patients. We performed functional studies in SKOV3 and OVCAR3 cell lines using recombinant protein, demonstrating that CTGF promotes anchorage-independent proliferation and migration. These effects of CTGF are blocked by FG-3019. During cancer progression, HGSOC cells from the ovary disseminate and adhere throughout the peritoneal cavity, posing a major challenge in treatment. We and others have shown that mesothelial cells in peritoneal tissue, peritoneal fibroblasts and ovarian cancer omental metastases express CTGF, suggesting that it may be targeted to help prevent recurrence following debulking surgery. To address this, we performed ex-vivo peritoneal adhesion assays, and demonstrate that recombinant CTGF increases a5b1 integrin-mediated adhesion of ovarian cancer cells to peritoneal tissue. In addition, enhancement of peritoneal adhesion by CTGF was blocked upon addition of FG-3019, further supporting its potential as a therapeutic target. In investigating clinico-pathologic characteristics in HGSOC patients, we confirmed over-expression of CTGF in cancer-associated fibroblasts by immunohistochemistry. Furthermore, we found that survival was correlated with CTGF expression, with HGSOC patients expressing the highest levels of tumor stromal CTGF harboring the poorest prognosis. Together, our findings identify and characterize CTGF as a promoter of peritoneal adhesion, likely to mediate metastasis, and a potential therapeutic target in HGSOC. Further studies should investigate the therapeutic efficacy of FG-3019 in combination with platinum/taxane, in HGSOC. Citation Format: Kim Moran-Jones, Rajmohan Murali, David K. Chang, Carol W. Wong, Suzanne M. Spong, Neville F. Hacker, Samuel C. Mok, Michael J. Birrer, Goli Samimi. Connective tissue growth factor (CTGF) as a novel therapeutic target in high grade serous ovarian cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr B58.

The Endothelin A Receptor Mediates Fibrocyte Differentiation in Chronic Obstructive Asthma. The Involvement of Connective Tissue Growth Factor

Fibrocytes possess increased differentiability into α-smooth muscle actin (α-SMA)(+) myofibroblasts in chronic obstructive asthma (COA) and contribute to pulmonary fibrosis. Endothelin-1 (ET-1) induces matrix-associated gene expression through the ETA receptor (ETAR) and promotes fibroblast differentiation. However, the mechanism of fibrocyte differentiation remains unclear. To define the roles of the ETAR and connective tissue growth factor (CTGF) expression in fibrocytes in the development of fibrosis in COA. Blood nonadherent non-T (NANT) cells were isolated, and fibrocytes expressing CD45, collagen I, CTGF, ETAR, or α-SMA were identified by flow cytometry. We showed the accumulation of fibrocytes in bronchial walls and overexpression of CTGF in fibrocytes from patients with COA. After being cultured, CTGF was increased in fibrocytes from patients with COA, but not from those of normal participants or patients with asthma without obstruction. Serum levels of ET-1 and the expression of the ETAR in fibrocytes were significantly higher in patients with COA compared with normal participants and patients with asthma without obstruction. Treatment with the ETAR antagonist (BQ123), but not ETBR antagonist (BQ788), reduced the expression of CTGF and α-SMA in fibrocytes and fibrocyte differentiation in patients with COA. Furthermore, treatment with BQ123 or an anti-CTGF antibody attenuated α-SMA expression induced by ET-1 in fibrocytes from normal participants. Our findings demonstrate for the first time that the ETAR pathway is vital for CTGF expression, which results in fibrocyte differentiation in COA, and suggests that an ETAR antagonist may be a potential antifibrotic agent in preventing the development of fibrosis in patients with COA.

Reducing CTGF/CCN2 slows down mdx muscle dystrophy and improves cell therapy

In Duchenne muscular dystrophy (DMD) and the mdx mouse model, the absence of the cytoskeletal protein dystrophin causes defective anchoring of myofibres to the basal lamina. The resultant myofibre degeneration and necrosis lead to a progressive loss of muscle mass, increased fibrosis and ultimately fatal weakness. Connective tissue growth factor (CTGF/CCN-2) is critically involved in several chronic fibro-degenerative diseases. In DMD, the role of CTGF might extend well beyond replacement fibrosis secondary to loss of muscle fibres, since its overexpression in skeletal muscle could by itself induce a dystrophic phenotype. Using two independent approaches, we here show that mdx mice with reduced CTGF availability do indeed have less severe muscular dystrophy. Mdx mice with hemizygous CTGF deletion (mdx-Ctgf+/-), and mdx mice treated with a neutralizing anti-CTGF monoclonal antibody (FG-3019), performed better in an exercise endurance test, had better muscle strength in isolated muscles and reduced skeletal muscle impairment, apoptotic damage and fibrosis. Transforming growth factor type-β (TGF-β), pERK1/2 and p38 signalling remained unaffected during CTGF suppression. Moreover, both mdx-Ctgf+/- and FG-3019 treated mdx mice had improved grafting upon intramuscular injection of dystrophin-positive satellite cells. These findings reveal the potential of targeting CTGF to reduce disease progression and to improve cell therapy in DMD.

SAT0062 Blockade of connective tissue growth factor (CTGF) ameliorates murine model of rheumatoid arthritis

BackgroundOur previous study demonstrated changes in the profiles of serum protein biomarkers in infliximab-treated rheumatoid arthritis (RA) patients using a novel approach to proteomic research (1). Several proteins exhibited vast...

Inhibition of Connective Tissue Growth Factor Ameliorates Disease in a Murine Model of Rheumatoid Arthritis

We have shown that connective tissue growth factor (CTGF) plays an important role in the pathogenesis of rheumatoid arthritis (RA). This study was undertaken to evaluate the effects of blockade of the CTGF pathway on the development of collagen-induced arthritis (CIA) in mice. Arthritis was induced in DBA/1J mice by immunization with a combination of type II collagen (CII) and Freund's complete adjuvant. We evaluated the development of arthritis in mice with CIA left untreated versus treated with neutralizing anti-CTGF monoclonal antibody (mAb). Inhibition of CTGF in mice treated with neutralizing anti-CTGF mAb significantly ameliorated arthritis compared to the untreated mice with CIA. Serum levels of matrix metalloproteinase 3 were reduced by anti-CTGF mAb treatment. Moreover, blockade of CTGF decreased interleukin-17 expression on purified CD4+ T lymphocytes. Although the expression of the retinoic acid receptor-related orphan receptor γt gene was not suppressed by anti-CTGF mAb treatment, that of interferon regulatory factor 4 (IRF-4) and IκBζ (Nfkbiz), which are other important molecules for the differentiation of Th17 cells, was suppressed. In addition, blockade of CTGF inhibited pathologic proliferation of T lymphocytes in response to CII restimulation in vitro. Moreover, aberrant osteoclastogenesis in mice with CIA was restored by anti-CTGF mAb treatment. Our findings indicate that blockade of CTGF prevents the progression of arthritis in mice with CIA. Anti-CTGF mAb treatment suppresses pathologic T cell function and restores aberrant osteoclastogenesis in mice with CIA. CTGF may become a new target for the treatment of RA.

CTGF is a therapeutic target for metastatic melanoma

Metastatic melanoma remains a devastating disease with a 5-year survival rate of less than five percent. Despite recent advances in targeted therapies for melanoma, only a small percentage of melanoma patients experience durable remissions. Therefore, it is critical to identify new therapies for the treatment of advanced melanoma. Here, we define connective tissue growth factor (CTGF) as a therapeutic target for metastatic melanoma. Clinically, CTGF expression correlates with tumor progression and is strongly induced by hypoxia through HIF-1 and HIF-2-dependent mechanisms. Genetic inhibition of CTGF in human melanoma cells is sufficient to significantly reduce orthotopic tumor growth, as well as metastatic tumor growth in the lung of severe combined immunodeficient (SCID) mice. Mechanistically, inhibition of CTGF decreased invasion and migration associated with reduced matrix metalloproteinase-9 expression. Most importantly, the anti-CTGF antibody, FG-3019, had a profound inhibitory effect on the progression of established metastatic melanoma. These results offer the first preclinical validation of anti-CTGF therapy for the treatment of advanced melanoma and underscore the importance of tumor hypoxia in melanoma progression.

EFFECT OF HIGH GLUCOSE ON THE EXPRESSION OF CONNECTIVE TISSUE GROWTH FACTOR IN THE CULTURED CARDIOMYOCYTES OF NEONATAL RATS

ObjectivesTo investigate the effect of high glucose on the expression of connective tissue growth factor (CTGF) in the cultured cardiomyocytes of neonatal rats and the role of CTGF in the...

The roles of connective tissue growth factor and integrin‐linked kinase in high glucose‐induced phenotypic alterations of podocytes

Emerging evidence has suggested that podocytes undergo epithelial-mesenchymal transition (EMT) in diabetic nephropathy (DN). Connective tissue growth factor (CTGF) and integrin-linked kinase (ILK) are involved in the progression of DN. However, the underlying mechanisms of EMT are not well understood. The study aimed to investigate the roles of CTGF and ILK in high glucose-induced phenotypic alterations of podocytes and determine whether ILK signaling is downstream of CTGF. The epithelial marker of nephrin and the mesenchymal marker of desmin were investigated by real-time RT-PCR and Western blotting. The results demonstrated that podocytes displayed a spreading, arborized morphology in normal glucose, whereas they had a cobblestone morphology in high glucose conditions, accompanied by decreased nephrin expression and increased desmin expression, suggesting podocytes underwent EMT. In response to high glucose, CTGF and ILK expression in podocytes were increased in a dose- and time-dependent manner, whereas the increase did not occur in the osmotic control. Furthermore, the inhibition of CTGF with anti-CTGF antibody prevented the phenotypic transition, as demonstrated by the preservation of epithelial morphology, the suppression of high glucose-induced desmin overexpression and the restoration of nephrin. Of note, the upregulation of ILK induced by high glucose was partially blocked by the inhibition of CTGF. In summary, these findings suggested that CTGF and ILK were involved in high glucose-induced phenotypic alterations of podocytes. ILK acted as a downstream kinase of CTGF and high glucose-induced ILK expression might occur through CTGF-dependent and -independent pathways.

Role of Connective Tissue Growth Factor (CTGF) in Survival and Chemosensitivity of Acute Lymphoblastic Leukemia

Abstract 2593Connective tissue growth factor (CTGF/CCN2) is a member of the CCN family of proteins involved in extracellular matrix production, tumor cell proliferation, adhesion, migration, and metastasis. Recent studies have shown that CTGF expression is elevated in 75% of acute lymphoblastic leukemia (Br J Haematol, 2007; 138(6):740–8), and that increased expression of CTGF is associated with inferior outcome in B-ALL (Blood, 2007; 109(7):3080–3). In this study, we characterized the functional role and downstream signaling pathways of CTGF in ALL cells. First, we utilized lentiviral shRNA to knock-down CTGF in RS4;11 and REH ALL cells expressing high levels of CTGF mRNA (479.3±37.2 and 57.3±5.9 copies per 100 copies of ABL1, respectively). Silencing of CTGF (CTGF-knockdown, CTGF-kd) resulted in significant suppression of leukemia cell growth (57% in RS4;11 and by 70% in REH) compared to control vector. CTGF knockdown moderately reduced adhesion of RS4;11 to fibronectin (27%±0.1%). In the in vitro culture system, CTGF knockdown significantly enhanced growth inhibition and apoptosis induction after 48 hour exposure to chemotherapy agents (annexinV(+): Vincristine 25.8±3.5%, Vincristine/CTGF-kd 42.6±2.8%; Dexamethasone 66.3±1.8%, Dexamethasone/CTGF-kd 99.3±0.6%; Methotrexate, 17.4±0.6%, Methotrexate/CTGF-kd 39.5±3.9). Analysis of signaling pathways showed that CTGF down-regulation inhibits Src phosphorylation at Tyr416. Remarkably, phosphorylation of AKT at Ser473, and of mTOR downstream targets S6 Ribosomal Protein and 4E-BP1 were significantly inhibited in CTGF-knockdown RS4;11 cells, concomitantly with upregulation of expression of AKT targets Bim and p27. No changes in the levels of apoptotic regulators cIAP1 and Bcl-xL were found. This data suggest that CTGF regulates growth and chemosensitivity of ALL cells through Src and AKT/mTOR signaling. We previously reported that an anti-CTGF monoclonal antibody significantly extended median survival of mice implanted with xenografts derived from a primary CTGF expressing ALL sample in NOD/SCID mice. We are now investigating the effects of combining anti-CTGF treatment with cytotoxic chemotherapy in this model. Blocking CTGF signaling may represent a useful adjunct to cytotoxic therapies in acute lymphoblastic leukemia. Disclosures:Spong:Fibrogen: Employment, Equity Ownership.

Abstract B206: FG-3019, an anti-CTGF antibody, attenuates tumor invasion, delays tumor growth, and prolongs survival alone and in combination with radiation in an orthotopic glioma model.

Abstract Objective: Despite progress in understanding molecular changes in glioblastoma multiforme (GBM), it remains a cancer with poor prognosis in need of better therapy. Pathological features of GBM are explosive growth, angiogenesis and its unique diffuse, infiltrative growth pattern. There is a consensus that cell-matrix-interactions play a pivotal role for infiltrative growth. Connective tissue growth factor (CTGF) is a matricellular protein that is involved in many pathological processes including tumorigenesis and invasion. CTGF modulates cell-matrix-interactions that may allow glioma cells to invade brain parenchyma, degrade the extracellular matrix (ECM), and enhance proliferation and angiogenesis in GBM. High CTGF-expression levels have been reported to correlate with tumor grade, invasiveness and poor patient survival. Here the effects of a human monoclonal anti-CTGF-antibody (FG-3019) were investigated alone and in combination with irradiation in vitro and in vivo in an orthotopic glioma model in mice. Methods and materials: In vitro studies were performed with two established human glioma cell lines, U87MG and T98G, as well as with primary isolated human glioblastoma cancer stem-like cells (CSLCs) cells. The effects of FG-3019 alone and in combination with photon irradiation (6 MV Linac) on clonogenic survival, proliferation, migration, neurosphere formation, and limiting dilution were determined. RNA was isolated from CSLCs and gene expression changes were examined after different treatments using micro-arrays and RT-PCR. In vivo, CSLCs were subcultured as neurospheres and stereotactically implanted into brains of SCID-beige mice. Mice brains were treated with 7Gy single dose radiation +/− FG-3019. The treatment effects were assessed by MRI, survival time and histology. Results: Tumor growth delay in the orthotopic tumor model resulted from treatment with radiation or FG-3019 alone, which was significantly enhanced by combined treatment. Similarly, animal survival was prolonged by each monotherapy and significantly enhanced by the combination. Histology showed that FG-3019 significantly reduced constitutive and radiation-induced tumor invasion into the brain parenchyma. FG-3019 also reduced intracellular CTGF expression, glioma cell proliferation, deposition of different types of collagen and reduced microvessel density. FG-3019 also reduced expression of Nestin and the stem cell marker SOX-2. In vitro, FG-3019 and irradiation reduced clonogenicity, proliferation and migration of U87MG, T98G and CSLCs, which was enhanced by the combination. Gene expression analysis revealed that FG-3019 down regulated tumorigenesis- and tumor-invasion-related genes. Conclusions: The human monoclonal CTGF antibody, FG-3019, shows remarkable anti-tumorigenic and anti-invasive effects in vitro and in vivo in a primary human glioblastoma model growing orthotopically in mice. Our results indicate that FG-3019 alone and in combination with radiotherapy is a promising approach for clinical translation in humans. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B206.

Establishment of the biotin-streptavidin ELISA for detection of serum connective tissue growth factor and its preliminary clinical application

Objective To establish a biotin-streptavidin ELISA method to measure CTGF,and evaluate the clinical value of CTGF for the diagnosis of liver fibrosis in chronic hepatitis B (CHB) patients.Methods Biotinylated anti-CTGF polyclonal antibody and monoclonal antibodies were prepared for the establishment of this biotin-streptavidin ELISA method.Two hundreds and sixty-four CHB patients were subjected into non-or mild liver fibrosis group (S0-S1,108 cases) and severe liver fibrosis group (S2-S4,156 cases),according to the liver biopsy pathological diagnosis.The CTGF assay's diagnostic capacity for CHB was assessed by comparing the area under ROC (AUC) with that of a panel of hepatic fibrosis markers ( HA,PC Ⅲ,C Ⅳ,LN and APRI).Analysis of variance and rank sum test were performed to carry out comparisons between multiple groups.Student's t-test and Mann-Whitney U test were performed for the pairwise comparison between multiple samples.Spearman rank correlation test was performed to analyze the correlation between different hepatic fibrosis stages.Results The minimum detectable dose and detection rang of the ELISA was 0.2 μg/L and 0-64 μg/L respectively.The intra-assay and inter-assay CV at high and low level were 4.5％,9.8％ and 10.1％,12.8％ respectively.Serum CTGF concentrations in S0-S1 group and S2-S4 group were 6.7(3.1 - 10.1 ) μg/L and 16.1 ( 11.8 -27.2) μg/L,with a statistically significant difference (U =1 217,P ＜0.001 ).There was a significant correlation between the levels of serum CTGF and fibrosis stages ( r =0.689,P ＜ 0.001 ),AUC of CTGF was 0.841 (95％ CI:0.762 - 0.920) in distinguishing mild fibrosis from significant fibrosis.When the cut-off value of CTGF was 10.3 μg/L,the sensitivity and specification was 70.5％ and 82.4％ respectively.The sensitivity of parallel combination test of CTGF and APRI was 96.1％,which was higher than that of HA (75.6％),PC Ⅲ (70.5％ ),C Ⅳ(63.6％),LN(79.5％ ),APRI(86.3％ ).The specificity of the combination test was 65.5％,which was lower than of above liver fibrosis markers [HA ( 72.5％ ),PC Ⅲ ( 76.5％ ),C Ⅳ ( 78.4％ )].The specificity of serial combination test of CTGF and PC Ⅲ was 95.9％,which was higher than that of HA,PC Ⅲ,CⅣ,LN(64.7％ ),APRI(66.1％ ),however,the sensitivity of the combination test was 67.7％,which was lower than that of above HA,PC Ⅲ,and APRI.Conclusions The biotin-streptavidin ELISA method measuring serum CTGF has a high minimu detectable dose sensitivity,and specificity.Serum CTGF level is significantly correlated with fibrosis stage,and CTGF maybe a valuable marker for liver fibrosis assessment.The paralledl combination of CTGF and APRI could be used as screening for significant liver fibrosis markers.The serial combination of CTGF and PC Ⅲ may be considered as a confirmatory diagnostic marker for liver fibrosis. Key words: Connective tissue growth factor; Liver cirrhosis; Hepatitis B,chronic; Enzymelinked immunosorbent assay

Pulmonary microvascular endothelial cells from bleomycin‐induced rats promote the transformation and collagen synthesis of fibroblasts

Accumulation and activation of myofibroblasts are the hallmark of progressive pulmonary fibrosis, and the resident fibroblasts are the major source of myofibroblasts. However, the key factors involved in the transformation of fibroblasts are unknown. Pulmonary microvascular endothelial cells (PMVECs), major effector cells against pathogenesis in early stages of the disease, can secrete cytokines to induce the differentiation of mesenchymal cells. We speculated that PMVECs could secrete pro-fibrotic cytokines and promote the transformation of fibroblasts into myofibroblasts. Accordingly, we established a co-culture system with PMVECs and fibroblasts to examine the specific transformation and collagen synthesis of the co-cultured fibroblasts by FACS and Western blot, prior to and after treatment with neutralizing antibodies against transforming growth factor-beta1 (TGF-β1) and connective tissue growth factor (CTGF). We also analyzed expression of TGF-β1 and CTGF in PMVECs. The synthesis and secretion of TGF-β1 and CTGF protein were up-regulated in PMVECs isolated from bleomycin (BLM)-treated rats, most prominently at 7 days post-instillation. We showed that the PMVECs isolated from BLM-induced rats could induce the transformation of normal fibroblasts and their secretion of collagen I, which was inhibited by both neutralizing anti-TGF-β1 and anti-CTGF antibodies. Therefore, up-regulation of TGF-β1 and CTGF in PMVECs plays an important role in activation, transformation, and collagen synthesis of fibroblasts; in particular, these effects in PMVECs are likely to be the key factors for activation and stimulation of static fibroblasts in lung interstitium in early stages of pulmonary fibrosis disease.

Cooperative interaction of CTGF and TGF-β in animal models of fibrotic disease

BackgroundConnective tissue growth factor (CTGF) is widely thought to promote the development of fibrosis in collaboration with transforming growth factor (TGF)-β; however, most of the evidence for its involvement comes from correlative and culture-based studies. In this study, the importance of CTGF in tissue fibrosis was directly examined in three murine models of fibrotic disease: a novel model of multiorgan fibrosis induced by repeated intraperitoneal injections of CTGF and TGF-β2; the unilateral ureteral obstruction (UUO) renal fibrosis model; and an intratracheal bleomycin instillation model of pulmonary fibrosis.ResultsIntraperitoneal coadministration of CTGF and TGF-β2 elicited a profound fibrotic response that was inhibited by the human anti-CTGF antibody FG-3019, as indicated by the ability of FG-3019 to ameliorate the histologic signs of fibrosis and reduce the otherwise increased hydroxyproline:proline (Hyp:Pro) ratios by 25% in kidney (P < 0.05), 30% in liver (P < 0.01) and 63% in lung (P < 0.05). Moreover, administration of either cytokine alone failed to elicit a fibrotic response, thus demonstrating that CTGF is both necessary and sufficient to initiate fibrosis in the presence of TGF-β and vice versa. In keeping with this requirement for CTGF function in fibrosis, FG-3019 also reduced the renal Hyp:Pro response up to 20% after UUO (P < 0.05). In bleomycin-injured animals, a similar trend towards a FG-3019 treatment effect was observed (38% reduction in total lung Hyp, P = 0.056). Thus, FG-3019 antibody treatment consistently reduced excessive collagen deposition and the pathologic severity of fibrosis in all models.ConclusionCooperative interactions between CTGF and TGF-β signaling are required to elicit overt tissue fibrosis. This interdependence and the observed anti-fibrotic effects of FG-3019 indicate that anti-CTGF therapy may provide therapeutic benefit in different forms of fibroproliferative disease.

Targeting Connective Tissue Growth Factor (CTGF) In Acute Lymphoblastic Leukemia Pre-Clinical Models: Anti-CTGF Monoclonal Antibody Attenuates Tumor Growth In Mouse Models of ALL

AbstractAbstract 3257Connective tissue growth factor (CTGF/CCN2) is a member of the CCN family of proteins involved in extracellular matrix production, tumor cell proliferation, adhesion, migration, and metastasis. In pancreatic cancer, CTGF derived from tumor cells is a critical regulator of tumor growth, and CTGF-specific antibody attenuates tumor growth and metastases in vivo (Cancer Res., 2006; 66(11):5816-27). Elevated CTGF levels have been detected in a number of malignancies, including in lymphoblasts from patients with acute lymphoblastic leukaemia (ALL), while no expression of CTGF was detected in normal peripheral blood mononuclear cells. Most importantly, recent studies with microarrays of B-ALL cells showed evidence of increased expression of CTGF, which was significantly associated with inferior outcome (Blood, 2007; 109(7):3080-3). In this study, we characterized mRNA expression and function of CTGF in ALL cell lines (Jurkat, REH, RS4;11, Nalm6) and in samples from primary ALL samples. RS4;11 and REH expressed high levels of CTGF mRNA (479.26±37.17 and 57.31±5.87 per 100 copies of ABL1, respectively), and CTGF secretion into culture supernatants was confirmed by immunoblotting with anti-CTGF antibody. In eleven primary pre-B-ALL patient samples, CTGF mRNA levels varied from 68.68±2.1 to 7030.2±170.47 per 100 copies of ABL1. In contrast, two T-ALL samples expressed very low levels of CTGF (4.7±0.19 and 5.2±0.12, respectively). To determine the functional role of CTGF in proliferation and survival of ALL cells, we utilized lentiviral shRNA to knock-down CTGF in RS4;11 cells. Cells stably infected with shRNA showed significant repression of CTGF levels (75.15%±0.98). Remarkably, CTGF knock down resulted in significant suppression of leukemia cell proliferation (by 57%) compared to control vector, but not in apoptosis induction. This data indicate that CTGF is critically involved in ALL cell proliferation.We next investigated the anti-leukemia efficacy of the fully human anti-CTGF monoclonal antibody FG-3019 (FibroGen, Inc.) using CTGF-expressing A20-luc/YFP leukemia cells in NOD/SCID/IL2 receptor gamma KO (NOG) mice. FG-3019 was administered intraperitoneally at 10 mg/kg/day twice a week starting on day 7 after leukemia cell injection. Compared to control human IgG, FG-3019 significantly inhibited leukemia growth as determined by bioluminescence imaging (38.3%±1.25) (Figure 1A) and significantly prolonged overall survival from 21 days to 31 days (p=0.01) (Figure 1B). In a subsequent in vivo experiment in NOD/Scid mice, xenografts derived from a primary CTGF-expressing ALL sample were treated with FG-3019 or control human IgG at 10 mg/kg/day twice a week starting at 7 days after cell injection. Similar to the cell line experiment, FG-3019 significantly prolonged the overall survival of animals otherwise dying from leukemia (median survival, control - 24 days, FG-3019- 31 days, p<0.01)(Figure 1C). Altogether, this data demonstrate that autocrine CTGF is highly expressed in pre B-ALL cell lines and primary ALL samples and represents a potential target for therapies blocking CTGF signaling. This is of particular importance given our recent findings of critical role of CTGF in the mesenchymal cells of the bone marrow microenvironment (Battula, ASH 2010). Mechanistic studies of the growth-promoting effects of CTGF in ALL are currently ongoing and will be presented. [Display omitted] (A) Serial bioluminescence images of mice injected with A20-luc/YFP leukemia in groups receiving FG3019 or hIgG. Bioluminescence imaging results were averaged from the peak light-emitting exposure from each group and displayed as photons per second. Error bars represent the SEM of each group.(B) Overall survival of control (hIgG-treated) and FG3019-treated mice in the A20-luc/YFP leukemia murine model. Statistical significance was calculated using the log-rank test.(C) In the NOD/Scid mice xenograft model of ALL leukemia, the overall survival in two different groups was estimated by Kaplan-Meier method. Statistical significance was calculated using the log-rank test. Disclosures:Off Label Use: use of FG-3019 for treatment of ALL. Spong: FibroGen.Inc.: Employment.

Attenuation and Reversal of Radiation-induced Pulmonary Fibrosis in a Murine Model by an Anti-CTGF Monoclonal Antibody

Purpose/Objective(s): Connective tissue growth factor (CTGF) is a matricellular protein that is a central mediator of tissue remodeling. CTGF expression is elevated in patients with idiopathic pulmonary fibrosis. Several studies in different organs have suggested that CTGF is essential for sustained fibrosis. Therefore, inhibition of CTGF may be therapeutically beneficial to fibrosis patients. Radiation induced lung fibrosis is a dose limiting side effect. Here we investigated this concept in a murine model of radiation induced lung fibrosis.