The articles in this supplement were presented at the “12th Conference on Cancer Therapy with Antibodies and Immunoconjugates,” in Parsippany, New Jersey, October 16-18, 2008.
Significant antitumor effects were previously observed with radioimmunotherapy (RIT) using an anti-carcinoembryonic antigen (CEA) monoclonal antibody (F6) labeled with iodine-131 in medullary thyroid cancer (MTC)-bearing nude mice. Nevertheless, no complete response was achieved. Because angiogenesis is critical for tumor growth, bevacizumab is used to treat solid tumor in clinical practice. The present pilot study evaluated toxicity and efficacy of RIT combined with bevacizumab in mice subcutaneously grafted with TT MTC cells.
Groups of 4-6 nude mice were treated with 5 μg/g bevacizumab twice weekly during 4 weeks and/or 100 MBq of 131I-F6. For combined therapy, bevacizumab was given at Day 0 followed by 131I-F6 at Day 30. The control group received no treatment. Animal weight, hematological toxicity, tumor volume, and serum calcitonin were monitored for 2 or 4 months.
Bevacizumab alone induced no cytopenia and no significant weight loss. A weight loss of 12 ± 1% and 15 ± 2% was observed in mice treated by RIT alone or bevacizumab + RIT, respectively. RIT alone and combined treatment induced leukopenia and anemia. RIT alone and RIT plus bevacizumab induced tumor responses with minimum relative tumor volume of 0.38 ± 0.24 and 0.15 ± 0.07%, respectively, and time to progression of 35 ± 5 and 56 ± 11 days, respectively.
Radioimmunotherapy (RIT) is a validated therapeutic modality for the treatment of non-Hodgkin lymphoma.1, 2 For more radiation-resistant solid tumors requiring higher tumor absorbed dose, RIT efficacy is less evident, with responses observed mostly in residual disease, indolent types of carcinoma, with repeated courses of RIT, or with therapy combining RIT and radiosensitizing chemotherapeutic agents.3-6 Angiogenesis is critical to sustain the nutrient and oxygen supply during tumor growth and metastatic spread of most malignant neoplasms.7, 8 In its absence, tumors cannot grow beyond 1 to 2 mm. Direct correlations between density of microvessels, metastases and survival of patients have been shown.9-11 Different antiangiogenic agents, like bevacizumab (Avastin) or thalidomide, showed efficacy in solid tumors. Pharmacological inhibition of tumor endothelium growth combined with RIT against tumor cells will target different cellular components and may show benefit in the treatment of solid tumors.
Medullary thyroid carcinoma (MTC) is a neuroendocrine tumor representing about 10% of all thyroid cancers. Because MTC expresses and secretes carcinoembryonic antigen (CEA), it constitutes a potential application for RIT with anti-CEA antibodies.13 Our group demonstrated the efficacy of RIT in the treatment of MTC in animal models and in phase 1 and 2 clinical trials.14-17 Nevertheless, RIT efficacy must be improved, in particular, in cases of aggressive forms or large tumor masses. We showed, in an animal model, that RIT efficacy could be improved using a radiosensitizing agent like paclitaxel.18 Moreover, MTC appeared to be a good model to study combinations of antiangiogenic drugs and RIT, because of the sensitivity of neuroendocrine tumors to antiangiogenic treatments.19
The purpose of the current pilot study was to assess the toxicity and efficacy of combined antiangiogenic therapy using a humanized antivascular endothelial growth factor (VEGF) monoclonal antibody, bevacizumab, and anti-CEA RIT in nude mice grafted with a human MTC cell line.
MATERIALS AND METHODS
The TT line of human MTC was obtained from the ATCC (Rockville, Md) and expresses CEA on its cell membrane and secretes calcitonin (Ct). It was cultured as adherent-cell monolayers in Gibco RPMI Medium 1640 (Gibco BRL, Cergy-Pontoise, France) supplemented with 10% fetal calf serum (Gibco BRL, Cergy-Pontoise, France), 1% glutamine (L-glutamine 200 MM; Gibco BRL), and 1% antibiotic (penicillin 100 U/mL, streptomycin 100 U/mL; Gibco BRL, Cergy-Pontoise, France).
Female nude mice over 10 weeks of age were purchased from Janvier (L'arbresle, France). Mice were housed at the animal core facility of the Cancer Research Center. This facility is approved by the French Association for Accreditation of Laboratory Animal Care and is maintained in accordance with the regulations and standards of Inserm and the French Department of Agriculture. Mice were grafted subcutaneously in the right flank with 106 TT cells in 0.3 mL of sterile normal saline solution. The animals were housed under aseptic conditions. Lugol's (KI) solution 0.1% was added to drinking water (1 mL/100 mL) the week before and 2 weeks after injection of the radioiodinated monoclonal antibody. Mice were sacrificed if tumor size exceeded 1000 mm3.
Antibody, Labeling, and Controls
The antibody was the F(ab′)2 fragment of the F6 anti-CEA antibody. This mouse IgG1 antibody was purchased from Immunotech SA (Marseille, France). The antibody was labeled using iodogen, as described by Fraker et al.20 The specific activity of the 131I-F6, measured using an ionization chamber (Medi-202; Medisystème; France), ranged from 180 to 350 MBq/mg for the different preparations. The radiochemical purity of 131I-F6, as determined by thin-layer chromatography on ITLC-SG chromatography paper (Gelman; Ann Arbor, Mich), was always above 98%. The immunoreactivity, assessed using anti-idiotype antibody 44-12-13 (kindly provided by Immunotech SA), was 85 to 96%.
Experimental RIT and Antiangiogenic Treatment
Four groups of 4-6 mice each were studied. The different treatment procedures are summarized in Figure 1. One group was injected intravenously into the lateral tail vein with 100 MBq (100 μL) of 131I-F6. The maximal tolerated dose of RIT, determined in a preliminary study, was 100 MBq (unpublished results). The second group was injected with bevacizumab (Avastin, Genentech, South San Francisco, Calif) intraperitoneally, 5 μg/1g, twice weekly during 4 weeks. The third group received combined therapy that comprised bevacizumab given at Day 0 followed by 131I-F6 at Day 30. The last group received no treatment.
Tumor length (L), width (W), and thickness (T) were measured with a sliding caliper every 5 days for 100 days. Tumor volume (V) was calculated according to the formula: V = L × W × T × π/6. All animals were weighed on the day of injection and then every 5 days for 100 days. Biological monitoring was performed on a blood sample drawn from the anterior chamber of the eye. The parameters used to evaluate the toxicity of each type of treatment were maximal weight loss and variation in the number of red blood cells, leukocytes, and platelets measured on Days 0, 15, 35, 45, 60, and 75. Efficacy was evaluated according size tumor and Ct level. The maximum objective response was determined as the ratio of initial volume to the minimum volume measured during the follow-up period. Tumor volume doubling time was also assessed. Variation in Ct concentration was measured by radioimmunoassay on Days 0, 30, 60, 90, and 120 (calcitonin immunoradiometric assay, CIS Bio International, Gif sur Yvette, France) expressed as the ratio between Ct serum level at a given time after treatment initiation and the value measured just prior treatment and time to progression (time between treatment and tumor volume progression).
Because of the limited number of animals, the means for the quantitative variables of the different groups were compared using nonparametric tests (the Mann-Whitney U test for comparison of 2 groups and the Kruskall-Wallis test for comparison of more than 2 groups). P values ≤.05 were considered significant. BMDP Statistical Software, Version 7.0 (Cork, Ireland) was used for the analysis.
Weight loss was observed in mice treated with RIT and bevacizumab followed by RIT of 12 ± 1% at 7 ± 5 days and 15 ± 2% at 37 ± 5 days (7 ± 5 days from RIT injection), respectively. No significant weight loss was observed in mice treated with bevacizumab alone, and in the control group, weight increase was proportional to tumor growth.
In untreated controls, the mean leukocyte count was 3320 ± 100/mm3, the mean hemoglobin concentration, 8.1 ± 0.5 g/dL, and the mean platelet count, 1.2 × 106 ± 0.5 106/mm3. Toxicity of blood cells was expressed as the percentage of the counts relative to the baseline value (Table 1).
Day 0 represents the beginning of the first treatment.
Leukocytes expressed as the percentage ± SD of the counts relative to baseline value
26 ± 12
75 ± 43
99 ± 44
96 ± 48
101 ± 44
89 ± 37
175 ± 53
162 ± 57
83 ± 13
74 ± 34
15 ± 13
111 ± 61
Platelets expressed as the percentage ± SD of the counts relative to baseline value
139 ± 69
80 ± 33
104 ± 40
82 ± 22
126 ± 19
100 ± 7
88 ± 24
74 ± 20
123 ± 40
106 ± 28
74 ± 34
97 ± 23
Hemoglobin expressed as the percentage ± SD of the concentration relative to baseline value
89 ± 11
87 ± 30
97 ± 11
84 ± 25
102 ± 3
88 ± 16
91 ± 8
93 ± 5
102 ± 6
91 ± 9
60 ± 13
97 ± 3
No leukopenia was observed in the group treated by bevacizumab alone, but. leukopenia was observed in the group treated with RIT alone, with a nadir at Day 15 (26 ± 12 % the initial count) and with the cell count was restored at Day 45. In the group treated with bevacizumab + RIT, leukopenia and anemia were observed with a nadir obtained 15 days after RIT (15 ± 13 % and 60 ± 13% of initial leukocyte count and hemoglobin concentration, respectively). These reductions were restored at Day 30.
No thrombocytopenia or animal deaths were observed in any groups during the monitoring period.
Between the control group and the group treated by bevacizumab alone, no statistical difference was observed for tumor volume doubling time, at 16 ± 4 days and 21 ± 14 days, respectively (P = .59). Tumor volume doubling time of mice treated by RIT alone (87 ± 25 days) was significantly longer than that of mice treated by bevacizumab alone (P = .006), and tumor volume doubling time of mice treated by bevacizumab followed by RIT (127 ± 5 days) was significantly longer than that of mice treated by RIT alone (P = .013). Tumor volume doubling time was not statistically different between the group treated by RIT alone and bevacizumab followed by RIT but did appear longer in the group pretreated by bevacizumab, 97 ± 5 days versus 87 ± 25 days (P = .52).
Figure 2 shows relative tumor volume of each group. Change in Ct levels confirmed the morphological tumor response (Fig. 3). Ratios at Day 60 were 8.3 ± 2.2 in the control group, 6.0 ± 8.0 in the group treated by bevacuzimab, 0.5 ± 0.4 in the group treated by RIT and bevacuzimab, and 0.3 ± 0.4 in the group treated by bevacuzimab + RIT.
No decrease of tumor volume was observed in the control group and in the group treated by bevacizumab alone. The minimum relative volume between was 0.22 ± 0.13 and 0.19 ± 0.08 (P = .99), in the group treated by RIT alone and bevacizumab + RIT, respectively. Figure 4 shows the time to progression for the group treated by RIT alone and by RIT + bevacizumab. Time to progression was significantly longer in group treated by bevacizumab + RIT than by RIT alone (80 ± 15 days and 50 ± 18 days, respectively; P = .033).
Angiogenesis is a complex and coordinated process, requiring several signaling steps. VEGFs and their receptors are key molecules in the regulation of vessel growth.21 Bevacizumab, a monoclonal antibody targeting VEGF is indicated, in combination with chemotherapy, for first-line or second-line treatment of patients with metastatic carcinoma of the colon or rectum, for first-line treatment of patients with unresectable, locally advanced, recurrent or metastatic nonsquamous, nonsmall cell lung cancer, and for the treatment of patients who have not received chemotherapy for metastatic HER2-negative breast cancer. Neuroendocrine tumors are characterized by abundant vasculature and high levels of VEGF expression and are, therefore, potentially susceptible to therapeutic strategies targeting angiogenic pathways.22 Bevacizumab, SU11248, a small molecular weight tyrosine kinase inhibitor targeting the VEGF receptor and thalidomide have recently been shown to have single-agent activity in neuroendocrine tumors.23-25 Angiogenesis probably plays an important prognostic role in MTC. Indeed, a pathology study assessing 53 MTC patients observed that the number of newly formed vessels was significantly associated with poor prognosis.26 All patients who died of the disease showed a microvessel count higher than 30, which resulted in a high statistical difference compared with living patients (P < .001). Moreover, encouraging evidence of antitumor activity has been reported in patients with metastatic hereditary MTC given vandetanib, a multitargeted kinase inhibitor exhibiting potent activity against VEGF receptor-2 kinase insert domain-containing receptor and, to a lesser extent, epidermal growth factor receptor, and RET kinase.27
Combination of an antiangiogenic agent and RIT appeared interesting to test because they target different cells involved in tumor survival. Tonami and coworkers reported benefits of the combination of RIT with antiangiogenic agents in human colon cancer xenografts.28-30 Thalidomide associated with antiglycoprotein monoclonal antibody A7 RIT showed higher efficacy than the monotherapy.28 Immunohistochemistry revealed a decrease in the microvessel number within tumors treated with thalidomide, and that combined therapy further reduced the microvessel number. Moreover, in an animal model of liver metastases, this group assessed antiangiogenic therapy with 2 methoxyestradiol (2-ME) and RIT using the same monoclonal antibody.30 Monotherapy comprising 2-ME treatment resulted in slight survival improvement over the controls, but RIT displayed a marked therapeutic effect. The combined regimen demonstrated superior survival in comparison to monotherapy. Another group assessed the cyclic Arg-Gly-Asp peptide, cilengitide (EMD 121,974), which targets the alpha(v)beta3 integrin receptor expressed by the neovasculature, combined with systemic RIT, in an aggressive human breast cancer model having mutant p53 and expressing bcl-2.31 Cilengitide combined with RIT significantly increased therapeutic efficacy and apoptosis when compared with single-modality therapy.
The present study suggests benefits of a pretreatment with bevacizumab before RIT in MTC. As reported for combination of RIT and chemotherapy,32 the timing of the combination appeared to be critical. We showed, in a previous study performed in the same MTC animal model that thalidomide improved the efficacy of RIT when administered before or at the same time as RIT, and that no benefit was observed when the antiangiogenic agent was given after RIT.33 One explanation is that antiangiogenic agents could improve tumor uptake of the monoclonal antibody. A second hypothesis is that the antiangiogenic agents normalize vascularization, increase tumor perfusion, reduce hypoxia, and increase radiosensitivity.34 Moreover, VEGF and bFGF are cytokines known to optimize the survival of endothelial cells under stress. By inhibiting these endothelial survival pathways, antiangiogenic agents could sensitize endothelial cells to radiation delivered by RIT, increase death of endothelial cells, and, in turn, reduce tumor growth.35
The clinical application of combination of RIT and antiangiogenic therapy might be promising only if acceptable toxicity is observed. The most serious adverse reactions in patients receiving bevacizumab are gastrointestinal perforation, nongastrointestinal fistula formation, wound-healing complications, hemorrhage, arterial thromboembolic events, hypertensive crises, reversible posterior leukoencephalopathy syndrome, neutropenia and infection, nephrotic syndrome, and congestive heart failure. Because RIT can induce myelosuppression, it is of concern whether such adverse side effects could be exaggerated. However, our study showed that the combined regimens did not delay recovery of the leukocyte count or the hemoglobin concentration, as compared with RIT alone.
In conclusion, pretreatment with bevacizumab may improve RIT efficacy in MTC, with acceptable toxicity. This pilot study needs to be expanded in other tumor models and under different experimental protocols and with different RIT and antiangiogenic agents. Future investigations also will be performed to understand how antiangiogenic agents enhance RIT efficacy.
CONFLICT OF INTEREST DISCLOSURES
The articles in this supplement represent proceedings of the “12th Conference on Cancer Therapy with Antibodies and Immunoconjugates,” in Parsippany, New Jersey, October 16-18, 2008. Unrestricted grant support for the conference was provided by Actinium Pharmaceuticals, Inc., Bayer Schering Pharma, Center for Molecular Medicine and Immunology, ImClone Systems Corporation, MDS Nordion, National Cancer Institute, NIH, New Jersey Commission on Cancer Research, and PerkinElmer Life and Analytical Sciences. The supplement was supported by an unrestricted educational grant from ImClone Systems Corporation, a wholly-owned subsidiary of Eli Lilly and Company, and by page charges to the authors.