Phase â…  Clinical Trial Of Rh-Apo2L And Apo2L-Related Experimental Study

Apo-2 ligand (Apo2L), also designated as tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), which is a new typical member of tumor necrosis factor superfamily (TNFSF) found in the recent years, can priming apoptosis signal conduction and selectively induce tumor cells to apoptosis without obvious harm to normal tissues by binding with its corresponding death receptor on cell surface, and now it becomes a research hot spot on the anticancer drugs in the field of apoptosis. Basic on the special mechanism of action of Apo2L/TRAIL, the broad-spectrum antitumor activity and superordinary safety of recombinant human soluble Apo2L/TRAIL in preclinical researches, and the progression of the antitumor study on endogenous antiangiogenic factors, two studies were involved in this paper, including phase I clinical trial of rh-Apo2L and the study on expression of the fusion proteins of human Apo2L and endogenous antiangiogenic factors.SectionⅠ: PhaseⅠclinical trial of rh-Apo2LObjective: To assess the feasibility and toleration of intravenous rh-Apo2L in patients with advanced cancer, perform the pharmacokinetic study and investigate the pharmacokinetic parameters, initially observe its antitumor activity, and recommend the reasonable dose and regimen in phaseⅡclinical trial. Methods: Volunteers were patients with advanced cancer who were consistent with recruited criteria and signed informed consent. They all received intradermal allergy test of rh-Apo2L firstly. Only these patients who were negative to intradermal allergy test were permitted to treat with rh-Apo2L. The dose levels were gradually escalated from low to high according to "3 plus 3 principle of dose escalation". Each dose level also each group included 3～6 patients. The regimen was the study drug was dissolved in 250ml normal sodium and administered by intravenous injection over 2 hours, daily for 14 days every 28 days for one cycle. The single dose or multiple doses pharmacokinetic study was performed by "Sandwich ELISA" using the pretreated blood serum of selected patients according to the toleration study result. Results: Total twenty two patients were recruited, but two patients exited due to positive to intradermal allergy test. The other twenty patients finished the toleration study and dose escalation. They received escalating dose of rh-Apo2L from 10μg/kg/d to 300μg/kg/d grouped 6 dose levels as 10μg/kg/d, 30μg/kg/d, 100μg/kg/d, 150μg/kg/d, 200μg/kg/d, and 300μg/kg/d. The results revealed the adverse reactions of rh-Apo2L were mild and tolerated. All but gradeⅢsystem inflammatory reaction symptoms at 300μg/kg/d dose level were gradeⅠ/Ⅱadverse reactions including fever, fatigue, debility, skin or mucosa alter, GI dysfunction, cardiovascular system toxicity, myelosuppression, hepatic or renal dysfunction, and so on. But all adverse reactions could recovery in 2 weeks after last dose. No allergic shock and drug-related death occurred. Therapeutic effect could be evaluated in 19 patients, 13 cases were evaluated as stable disease (SD), 6 cases were evaluated as progress disease (PD). Eighteen cases treated with four different dose-level (30μg/kg/d,100μg/kg/d,150μg/kg/d,200μg/kg/d) took part in the single dose or multiple doses pharmacokinetic study. Conclusion: The intravenous rh-Apo2L is safety. Dose limiting toxicity (DLT) is system inflammatory reaction symptoms. Maximally tolerated dose (MTD) is defined as 200μg/kg/d. The pharmacokinetic parameters of intravenous rh-Apo2L are consistent with two compartment model and linear kinetics. The recommended regimen of phaseⅡclinical trial is 150μg/kg, daily for 14 days, every 28 days for one cycle.SectionⅡ: The study on expression of the fusion proteins of human Apo2L and endogenous antiangiogenic factorsObjective: To clone and express the fusion proteins of human Apo2L and three endogenous antiangiogenic factors of different tissues origin respectively, including kininostatin of blood clotting system origin, vasostatin of intracellular protein origin, and canstatin of typeⅣcollagen origin; to seek the bifunctional proteins with anti-angiogenesis and tumor cell toxicity from these fusion proteins through the initial biological activity observation. Methods: The corresponding primers were designed in the study according to the encoding sequence of human kininostatin, vasostatin, canstatin, and Apo2L. The plasmids containing the encoding sequences of these molecules as templates, the fusion genes linked by linker (which encoding GGGSGGSG) were amplified by splicing by overlap extension (SOEing). The fusion genes were inserted into the prokaryotic expression vector pBV220 and the recombinant plasmids were constructed and sequenced. The transformants were selected out and the recombinant peptides were expressed in BL21 after 42℃thermal induction. Thus a series of fusion proteins, such as A-K (Apo2L-Kininostatin), K-A (Kininostatin-Apo2L), A-V (Apo2L-Vasostatin), V-A (Vasostatin-Apo2L), A-C (Apo2L-Canstatin), C-A (Canstatin-Apo2L), were produced. The biological activities of these fusion proteins were assessed by the inhibition of tumor cell lines (SW1990 and NCI-H460), endothelial cell proliferation and tube formation (ECV304) in vitro. Results: All six fusion proteins were expressed in the prokaryotic expression system. Two fusion proteins K-A (kininostatin-Apo2L) and V-A (vasostatin-Apo2L) closely maintained the tumor cell toxicity of Apo2L/TRAIL, and also enhanced ECV304 cell toxicity of Apo2L/TRAIL so that the antiangiogenic activity of the fusion proteins were identified. It was confirmed the inhibition of neovascularization in the study on the inhibition of ECV304 tube formation. Conclusion: In this study, a prokaryotic expression system was used to produce a series of fusion proteins of Apo2L/TRAIL, part of these fusion proteins showed the inhibition of tumor cell and endothelial cell proliferation, growth and angiogenesis. All of these maybe contribute the bifunctional fusion protein combined antiangiogenic factor and apoptosis-inducing factor to exploration as the new anticancer drug.