Angiogenesis plays a pivotal role in tumor growth, tissue invasion and metastasis. Endostatin is an angiogenesis inhibitor and has been shown to reduce tumor growth in animal models. However, therapy with recombinant endostatin protein was hampered by its short half-life and very-low yield of bioactive protein. We performed a phase I dose–escalation clinical trial using intratumoral injection of an adenovirus containing human endostatin gene (Ad-rhE; E10A; 1010–1012 virus particles (vp)) in 15 patients with advanced solid tumors. We observed intratumoral injections of E10A without dose-limiting toxicity. Most frequently reported E10A-related adverse events were transient fever and local response. Distribution studies revealed that the vector was detected in the blood, throat and injection site, but rarely in the urine and stool. An increased endostatin expression was detected using enzyme immunoassay in serum in 13 of 14 treated patients throughout the period of treatment despite the presence of neutralizing antiadenovirus antibody. Median serum basic fibroblast growth factor levels decreased from 32.4 pg ml−1 at baseline to 24.9 pg ml−1 after 28 days of first treatment. Thus, direct intratumoral injection of up to 1012 vp of E10A to patients is well tolerated and further studies are necessary to define and increase clinical efficacy.
Accumulating evidence suggests that angiogenesis plays a pivotal role in tumor growth, tissue invasion and metastasis.1, 2, 3 Tumor angiogenesis is a complex and multi-step process in which new blood vessels are formed in response to interactions between tumor cells and endothelial cells, growth factors and extracellular matrix components.4, 5 This process is regulated precisely under the control of both proangiogenic and antiangiogenic factors.1, 3 The abnormal features of the tumor vasculature perhaps represent an imbalanced expression of proangiogenic factors and antiangiogenic factors.1, 6 An encouraging progress has been achieved over the past few years, and the first FDA-approved antiangiogenesis agents (Avastin) demonstrated significant prolongation of survival in colon and lung cancer.7, 8 However, advanced stages of cancer cells secrete several proangiogenic factors, including vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), IL-8, PDGF and so on.9, 10 Therefore, combinations of single-targeted angiogenesis inhibitor or broad-spectrum angiogenesis inhibitor could be a trend in anticancer agent research and development in the future.
Of the endogenous antiangiogenic factors in the body, endostatin has the broadest anticancer spectrum and less toxicity in mice and human.7 Endostatin, a 20 kDa C-terminal fragment of the C terminus of collagen XVIII, has been shown to block endothelial cell proliferation, survival and migration, in part through downregulation of proangiogenic factor and upregulation of antiangiogenic factors.11, 12, 13 However, therapy with recombinant endostatin protein was hampered by its short half-life and very-low yields of bioactive protein.7 Furthermore, the inhibition of tumor angiogenesis is a long-term process of treatment. Gene therapy may overcome these difficulties by introducing human endostatin cDNA into the host and using the body as an endogenous factory to generate highly bioactive gene product. We and others have shown previously that the expression of endostatin by adenoviral gene transfer generates a strong therapeutic effect in several models of solid tumors in mice.14, 15, 16, 17 In preclinical studies, intratumoral injections of E10A into subcutaneous xenografts of hepatocellular carcinoma, nasopharyngeal carcinoma, tongue cancer in nude mice demonstrated a significant inhibition to tumor growth, reduced angiogenesis in tumors and no toxic effects. On the basis of promising preclinical results, we undertook a phase I trial of E10A in the treatment of patients with advanced solid tumors. The purpose of this trial was not only to determine the clinical toxicity, but also to include the degree of expression of endostatin, virus pharmacokinetic and immune response.
Between April 2005 and January 2006, 15 patients (nine men and six women) were enrolled onto this clinical trial (Table 1). Median age was 45 years (range: 33–59 years). Nine patients had head and neck cancer, three had sarcoma, one of each had colon, cervix, cutaneous cancer and all patients had metastatic disease. All patients had received prior therapy: surgery (73%), chemotherapy (100%) and/or radiotherapy (67%).
Intratumoral injection of E10A was feasible in 100% of cases with virus dose ranged from 1010 to 1012 virus particles (vp). As presented in Table 1, 29 injections were administered to 15 patients directly (n=25) or guided by ultrasonography (n=4). Fourteen patients underwent all of the two-cycle planned doses, with one patient receiving only one injection.
Toxicities and clinical response
No dose-limiting toxicity has been observed up to and including the maximum dose of 1012 vp. None of the patients withdrew from the study because of adverse events. Adverse events were mostly grade 1 or grade 2. Most frequently reported adverse events were moderate fever, injection-site pain and erythema being noted locally in some patients. No hepatic, renal or hematologic toxicity has been observed at any dose level.
Although clinical response was not a primary end point of this phase I study, the objective response (MR) was observed in one patient with NPC, about 37% reduction of the target tumor. Others, 12 patients on study had stable disease and two had progression of disease.
Vector shedding and biodistribution
Quantitative PCR using E10A-specific primer and probe combination demonstrated that vector DNA in the injection-site swab, throat swab, urine, stool and plasma samples were examined for virus shedding and kinetics. Twelve of 15 patients had injection-site swab and throat swab sampled for the detection of shedding E10A vector (Table 2). Vector DNA was detectable in the injection-site swab in all patients immediately following intratumoral injection, declining to non-detectable levels after 24–48 h for most patients. In all the 24 injections of 12 patients tested, 20 (83%) throat swab samples were positive for vector DNA after E10A administration. DNA vector was also detected in urine (14 of 15 patients) and stool (7 of 15 patients) samples at a low level and found non-detectable in any dose at 24 h following intratumoral injection of E10A (data not shown).
Fourteen of 15 patients had plasma sampling for the detection of circulating E10A DNA. Vector DNA was detectable in the blood within 4 h and peaked around 8 h following intratumoral injection of E10A, declining to non-detectable levels after 24–48 h for most patients (Figure 1). No clear correlation between dose of E10A and level and duration of vector DNA detectable in blood was observed, although there appears to be a general trend toward more circulating DNA and longer clearance time with higher doses.
Serum levels of endostatin assay
One of the major end points of this trial was to define the transgene expression of endostatin that might be evident at the systematic level. We measured serum concentrations of endostatin after intratumoral injection of E10A. A total of 322 serum samples for serum levels of endostatin analysis were available from 14 of 15 patients enrolled according to the protocol. In these patients, the serum endostatin levels have been detected increasing in 11 and 13 of 14 patients after first and second treatments, respectively. Pre-existing immunoreactive endostatin was detectable in all patients with a mean value at 8, 14 and 22 on the day just before the study. Serum levels of endostatin were corrected for the endogenous endostatin by subtracting the mean values. Serum endostatin levels showed a dynamic range of changes following intratumoral injection of E10A (Figure 2). We also detected intratumoral expression of endostatin in two patients at day 7 after second E10A injection. A representative example is shown in Figure 3, which illustrates the high level of endostatin immunostaining after E10A injection. Serum concentrations peaked on average 2.5–5.4 days after first treatment and 10.4–13.3 days at the second treatment after the beginning of study. In patients 6, 8, 9 and 13, serum endostatin levels were higher and the expression durations were longer than those of others. We noted that these patients did not receive radiotherapy before enrolling in this study, although there were no differences between all patients with radiotherapy and without radiotherapy.
Immune responses to E10A
Besides no significant changes in mean frequency of CD3+, CD4+ and CD8+ of peripheral blood T cells after E10A intratumoral injection was seen, a significant elevation in percentage of CD8+ T cells at day 21 post-injection in six cases is shown (Figure 3e). The results were in line with the tumor infiltration by CD8+ T cells (Figures 3c and d). Meanwhile, in these patients, CD4:CD8 ratio reduced after E10A treatment (Figure 3f).
To explore the possibility that E10A may immunize against viral antigens, we measured antibodies against adenovirus. Before injection of E10A, many patients had detectable antibodies to adenovirus as measured in group-specific enzyme-linked immunosorbent assay (ELISA) tests for IgG and IgM. All but three of the patients showed an increase in titer of IgG between 7 and 28 days post-treatment (Figure 4b). However, only three of the 14 patients have an increase in antibody titer of IgM after the administration of E10A (Figure 4a). There was no clear seroconversion from an IgM response to IgG in any patient.
Neutralization antibody tests using Ad5 β-galactosidase showed that many patients had specific activity against Ad5 before receiving E10A. Eleven of 14 patients showed enhanced neutralization at 14–28 days following treatment (Figure 4c). There was no correlation between E10A dose or level of virus DNA in blood and extent of antibody response, or between the level of pre-existing antibody and the magnitude of the response.
Assays were conducted to assess the effect of E10A on the levels of four different serum proteins thought to be important mediators of angiogenesis and metastasis. The levels were measured pre-injection and on days 7, 14, 21 and 28 after the first treatment. Serum bFGF, VEGF, soluble vascular cell adhesion molecule (sVCAM-1) and E-selectin results are presented in Table 3 and Figure 5. There was considerable variation in the baseline levels of all four factors. Median serum bFGF levels decreased from 32.4 (range: 21.0–55.9 pg ml−1) at baseline to 24.9 pg ml−1 (range: 12.4–36.6 pg ml−1) after 28 days of first treatment (P=0.038). There were no significant differences between cohorts with different dose. Although no overall statistically significant trend in plasma VEGF, sVCAM-1 and E-selectin was observed, several patients seemed to have depressed E-selectin levels while receiving E10A (Figure 5).
Extensive research has emphasized the importance of continuously elevated circulating levels of endostatin to achieve optimum inhibition and regression of tumors.18, 19 Clinical development of recombinant endostatin proteins as an anticancer agent was hindered by its short half-life and difficulties in protein production.20, 21, 22 Theoretically, endostatin gene transfer to the tumor may result in high local production and prolongs its half-life time that would in turn facilitate the induction of antitumor effects while minimizing systemic toxicity. In this report, we demonstrate for the first time that intratumoral administration of adenoviral-mediated transfer of endostatin genes (E10A) is feasible, safe and well tolerated in patients with advanced solid tumors.
Up to a maximum dose of 1012 vp, no dose-limiting toxicity has been observed. Most frequently reported adverse events were moderate fever, injection-site pain and erythema being noted locally in some patients. No changes in the biochemical assays of hepatic and renal function were observed, which suggested that the direct local injection of E10A may minimize potential adenovirus-mediated liver or renal toxicity.
Virus DNA was detectable in the blood of patients as soon as 4–8 h after intratumoral injection of E10A, indicating some vector systemic dissemination from injection site. E10A DNA was undetectable 48–72 h later in the blood of all patients. In most cases, the vector was shed into injection site and throat, whereas vector was rarely detectable in urine and stool samples despite the systemic spread of the vector.
A significant increase in the CD3+CD8+ T cells in many patients at day 21 post-treatment is further indication of the immune activation mediated by E10A. The antitumor activity of E10A coupled with the potential for bystander effects mediated by antiangiogenic and immunostimulatory mechanisms. This immune response should be advantageous to improve antitumor effects. Antibody titers of IgG against adenovirus were increased in the patients after injection of E10A. Most of these antibodies were able to neutralize Ad5 in vitro. Despite the presence of neutralizing antibodies for adenovirus, we found that endostatin transgene expression was detectable in most patients throughout the period of treatment.
This study has demonstrated the successful application of the E10A vector to express endostatin in 13 of 14 treated patients. Prior radiotherapy might be associated with a decreasing trend in endostatin levels and duration in serum. A possible explanation might be that the local tissue fibrosis induced by radiotherapy could affect expression of endostatin by E10A. However, there were no differences between patients with radiotherapy and without radiotherapy in our study and further studies are needed in more patients in future. In this study, plasma concentration of endostatin did not exceed 200 ng ml−1 in any patient at any time point (normal 67.4±14.0 ng ml−1), which were significantly below the peak plasma concentrations of endostatin (approximately 9280–12 520 ng ml−1) observed in clinical studies using infusions of the recombinant protein.20, 21, 22 However, serum endostatin levels have been continuously elevated throughout the period in this trial in some patients after two cycles of treatment with E10A, which is well in line with several studies that have emphasized the importance of continuously elevated circulating levels of endostatin to achieve optimum inhibition of tumor growth and endothelial cell migration, vascular morphogenesis in vivo.19, 23, 24 The previous study of dynamic endostatin levels in mice after a single intratumoral administration of E10A indicated a concurrent increase of endostatin in serum and tumor tissue with a 4.8-fold higher concentration in tumor as that in blood.25 Furthermore, further analyses are needed to compare serum and tumor tissue concentration of endostatin in human after intratumoral injection of E10A.
The effect of E10A administration on serum markers of angiogenesis, such as bFGF, VEGF, E-selectin and sVCAM-1, were performed in this phase I study strictly as an exploratory exercise to investigate the value as potential biomarkers for indication of antiangiogenesis cancer therapy. The evaluation of serum angiogenesis markers identified a significant decrease in median serum bFGF levels on day 28 compared with baseline. Although no statistical differences in other cytokines before and after E10A administration were observed for the group as a whole, or for any of the dose levels. Several individual patients demonstrated declines in E-selectin levels when enrolled in the study. The cause of the observed bFGF decrease remains unclear. It could be related to direct effects of endostatin and/or changes in tumor status. Some recent research reported that endostatin could downregulate proangiogenic factors.7, 13, 26 However, the clinical significance of circulating bFGF in relation to tumor status is still controversial.21, 22, 27
In summary, this study demonstrates that weekly intratumoral injections of E10A to patients with advanced solid tumors at doses up to 1012 vp for 2 weeks to patients are feasible and well tolerated. Although undergoing only two treatment cycles in this study, most of the patients experienced decrease in serum markers of angiogenesis. It seems to be reasonable to perform phase II and pharmacodynamic studies to identify the therapeutic benefit of E10A in patients with advanced solid tumors, and also to evaluate whether the combination of E10A-administered intratumoral injections with cytotoxic agents enhances antitumor effect.
Patients and methods
Permission for this clinical trial was obtained from the Local Institutional Ethical Committee and China's State Food and Drug Administration. All patients provided signed informed consent. Inclusion criteria included age between 18 and 65 years; an Eastern Cooperative Oncology Group performance status of 1 or 0; pathologically confirmed malignancy and at least one tumor or metastasis mass accessible for needle injection; refractory to standard therapy; and life expectancy of more than 3 months. Exclusion criteria included pregnancy or lactation; adequate organ function was required (including hematopoietic, hepatic and renal); potential immuno-incompetence (including chemotherapy or radiotherapy within 4 weeks, or known HIV positivity); with primary brain tumors or brain metastases.
This was an open-label, non-randomized, dose–escalation phase I clinical study in which groups of three to six patients were to receive intratumoral administration E10A until dose-limiting toxicity was seen in at least two of six patients. Patients received direct intratumoral E10A injections into the preselected tumor mass once per week for 2 continuous weeks. Virus solution was taken suction to the appropriate dose corresponding to each cohort and was then further diluted to a final volume equivalent to 30% of the tumor volume to be injected. Patients were enrolled consecutively in three cohorts with the following plan: cohort 1, 1011 viral particles; cohort 2, 5 × 1011 viral particles; cohort 3, 1012 viral particles. Five additional patients were included in the last cohort for pharmacokinetics study because of the modification of the protocol, meeting the specifications agreed with the China's State Food and Drug Administration regulatory authorities. Before cohort 1 began to investigate, a patient was intratumorally injected with E10A of 1010 viral particles as a preliminary study.
E10A and manufacture
E10A is a second-generation, replication-defective adenoviral vector that expresses the human endostatin under the control of the cytomegalovirus immediate-early promoter. E10A was expanded in 293 cells, purified by cesium chloride density gradient and dialyzed. Clinical-grade E10A with a viral particle plaque-forming unit ratio of 25–30:1 was prepared under current Good Manufacturing Practices. E10A was provided as a frozen vial suspension at a concentration of 1 × 1012 vp per ml in a buffer containing saline and 10% glycerol. The biologic activity of human endostatin secreted on HepG2 cells transfected with E10A was confirmed by an ELISA and western blot assay, respectively.
Vector dissemination and biodistribution
Peripheral venous blood, injection-site swab, throat swab, urine and stool samples in relation to treatment were analyzed for the presence of vector DNA at the following time intervals: pretreatment, immediately at the completion of virus injection, and 2, 4, 8, 12, 24, 48, 72, 96, 120, 144 and 168 h after injection. DNA was isolated from samples using the QIamp DNA Mini Kit (Qiagen, Hilden, Germany) and analyzed by a quantitative PCR. The primers and probe specific for the amplicon overlaps the E1B region deletion were used to allow specific detection of E10A DNA, and the quantitation was performed over 45 cycles of amplification using a standard curve of heat-inactivated E10A.
Serum levels of endostatin measurement
Endostatin was measured in serum after intratumoral injection of E10A. Blood samples were collected in sodium heparin tubes preinjection, at the end of the injection and then 2, 4, 8, 12, 16, 24, 72, 120 and 168 h post-injection. Blood samples were also drawn before and at the end of injection on 2, 4, 8, 15, 22 and 28 days after first injection. Serum was separated and stored at −80 °C until analysis. Serum endostatin levels were measured using the commercially available Human Endostatin Immunoassay kit (R&D Systems, Minneapolis, MN, USA). This assay had a detection limit ranging from 3.9 to 500 ng ml−1.
Flow cytometric immunophenotype analysis
Peripheral blood immunophenotype analysis was carried out by a two-color immunofluorescence reaction and flow cytometric analysis as described previously.28
Briefly, patients’ peripheral blood was collected by venipuncture. One hundred microliters of whole blood was treated with 20 μl of the following reactant mixtures to determine the frequency distribution of CD3, CD4 and CD8 (all from BD Biosciences, CA, USA). The reactants were fixed with 1% paraformaldehyde before flow cytometric analysis (BD Biosciences) with the cellQuest software (BD Biosciences).
Immunohistochemical detection of endostatin and lymphocytes infiltration
Tumor samples were fixed in 10% buffered formalin, sections were incubated with antibodies against endostatin (Santa Cruz Biotechnology, dilution 1:150), CD4 (dilution 1:30) and CD8 (both from DAKO, Hamburg, Germany, dilution 1:40). After blocking of endogenous peroxide using peroxidase-blocking agent, slides were incubated with primary antibody overnight at 4°C. Then, the sections were washed and followed by horse radish peroxidase-conjugated secondary antibody (DAKO, dilution 1:60). Position staining was detected using the DAB substrate (Vector Laboratories, Burlingame, CA, USA), and sections were counterstained with hematoxylin. Then stained slides were washed in running distilled water, dehydrated, mounted and examined by light microscopy.
Plasma samples were taken at pretreatment day, days 7, 14, 21 and 28 after treatment, heat-inactivated at 56 °C for 30 min and analyzed by ELISA to quantify IgM and IgG response against adenovirus. Neutralizing antibodies against Ad5 were tested using an E1-deleted replication-defective Ad5 virus encoding β-galactosidase (Ad-laz) in 293 cells, as described previously.29 Briefly, the mixture of an Ad5-laz suspension and diluted plasma was applied to 293 cells in 96-well plates. At 24 h post-infection, cell lysates were analyzed for β-galactosidase activity using a Multifunction plate reader (GENios, Tecan Group Ltd, Switzerland). Neutralization antibody titers were defined as the plasma titer giving 50% reduction in infectivity relative to a positive control of virus-infected cells.
Proangiogenic factors assays
Blood samples (3 ml) for determination of VEGF, bFGF, VCAM-1 and E-selectin levels were taken pretreatment and on days 7, 14, 21 and 28 after the first treatment. Serums were separated, and analysis carried out using Quantikine ELISA kit (R & D Systems, Minneapolis, MN, USA).20 The concentration of these proangiogenic factors was determined by comparing their absorbance against a standard curve. The lower limit of quantitation for VEGF, bFGF, VCAM-1 and E-selectin were 31.2, 10 pg ml−1, 6.25 and 0.47 ng ml−1, respectively.
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Supported by the National High Technology Research and Development Program of China, Grant no. 2006AA02Z489; the National Basic Research Program of China, Grant no.: 2004CB518801; the Research and Development Grand of Guangdong Province, Grant no.: 2003A10902; and The CMB-SUMS Scholar Program, Grant no.: 98-677.
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