Stabilized-Angiostatin Gene Transfer: Inhibition of Metastasis Development in an Eye-tumor Transgenic Mouse Model

Abstract

Abstract: : Purpose: : Angiostatin can inhibit growth of different types of tumors . In order to increase the half-life of this anti-angiogenic factor in vivo, we have fused it to HSA (human serum albumin) and studied the biological properties of the chimeric molecule Methods: A chimeric gene coding for a conjugate between angiostatin and HSA (K1-3 – HSA) was inserted under the control of the early promoter of CMV within an E1/E3-deleted recombinant adenovirus (AdK3-HSA). AdCO1 were used as controls. Activity of the K1-3 - HSA conjugate: human endothelial HMEC-1 cells were infected with the virus to perform an in vitro proliferation assay.Nude mice were injected intravenously with 5 10⁹ pfu of AdCO1, AdK3 or AdK3-HSA. Expression levels of K3 and K3-HSA were monitored by ELISA over 91 days after virus injection.MDA-MB-231 human breast carcinoma were grown on the dorsa of female nude mice. When tumor volumes reached 30 mm³, mice were injected intravenously with 5. 10⁹ pfu of AdK3-HSA, AdK3, or AdCO1. The anti-metastatic effect of high serum K3-HSA levels was further characterized in a transgenic mouse model (TRP1) which spontaneously develop tumors of the retinal pigment epithelium with brain metastases .A dose of 10⁹ pfu of AdK3-HSA, AdK3, or AdCO1 was injected into the temporal vein of 3 day-old TRP-1 male mice, a second i.v. injection was administered 12 days later.Blood was collected weekly until the animals were sacrified on day 62 after birth. The quantity of K3-HSA was determined by sandwich ELISA. We have evaluate the survival of AdK3-HSA injected mice. Results: Systemic injection of AdK3-HSA in immunodeficient mice led to robust and long-lasting expression of the chimeric antagonist as compared to its unfused counterpart, together with a more pronounced effect on the growth of MDA-MB-231 grafted tumors (79% inhibition with AdK3-HSA versus 44% with AdK3), Antitumoral effects associated with systemic injection of AdK3 virus in TRP-1 transgenic mice: Two months after virus injections, only 46% of AdK3-HSA treated animals developed metastases in the brain ( 0.95 mm3 ± 1.87 mm3) whereas 94% of the AdCO1-injected group displayed numerous and larger metastases ( 4.33 mm3 ± 6.6 mm3) These antitumoral effects were associated with an extended life span in the AdK3-HSA group (4 weeks over AdCO1-injected animals) Conclusions: Our data demonstrate that the use of HSA-stabilized anti-angiogenic factors delivered by gene transfer can block primary tumor growth and can prevent metastasis.

Keywords: adenovirus • gene transfer/gene therapy • oncology