Large-scale Preparation For Allogeneic GVAX Vaccines And Preliminary Evaluation On The Safety In Advanced Lung Cancers

[Objective] To explore large-scale preparation for allogeneic GVAX vaccine and thepreliminary evaluation on the safety of clinical application in advanced lung cancers.[Method] The experiment was conducted from May 2010 to February 2011 in the biotherapycenter of Tianjin 254 Hospital of PLA. In the GMP conditions, the large-scale adenoviral vectorswere produced by 293 cells, with step by step enlargement from a culture flask to 4 or 5 of10-layer cell factories seeded by the adenovirus, cesium chloride gradient centrifugation, anddialysis purification steps. Allogeneic GVAX vaccines was prepared with A549 lung cancer celllines, the culture procedure of which were similar to those of 293 cells, infected by AdhGM-CSF/IL-2 and Ad hCD40L, irradiated by 10,000 rad ray, subpacked into each 2×10⁷ cells/per tube, and kept in liquid nitrogen for use. The efficiency of adenovirus infection to the A549cells, cytokines in supernatants, tumorigenicity in nude mice, endotoxin, and pathogen weredetected. After that, 10 patients with advanced non-small cell lung cancer, who were diagnosedby pathology, invalid or unable to accept conventional treatments, were rolled up for the treatmentwith the allogeneic GVAX vaccine. All of the patients did not receive surgery, chemotherapy,radiotherapy, or immunogene therapy in a month and signed the informed consent. Immediatelyafter the recovery from frozen condition, the GVAX vaccines were intradermally given multipointinjections to the sites rich in lymphatic return, such as the triceps, groin area, with each siteconsisting of 0.2 0.3 ml, a time per week, and a totally for 6 times. Detail data were recordedduring the treatment including the injection site and systemic reactions, and all of adversereactions were assessed by the America NCI adverse reaction/event classification (CTCAE).[Results] In GMP condition, large-scale production of highly purified adenoviral vector wascarried out by tep by step enlargement from a culture flask to 4 or 5 of 10-layer cell factoriesseeded by the adenovirus, cesium chloride gradient centrifugation, and dialysis purification, withthe virus titer up to 4 to 8×10¹¹ pfu per production procedure. Similar methods to culture A549cells, a yield of 1 to 2×10⁹ A549 cells was obtained. Infection efficiency to A549 cells byadenovirus was 91.2%, an amount of GM-CSF and IL-2 in the supernatants was (1680±358) ng/24h/1×10⁶ and (644±102) ng/24 h/1×10⁶, respectively. The detection of tumorigenicity, endotoxin,and pathogen were negative. For the satety evaluation, main adverse reactions in the 10 patientsreceived by the injections of the GVAX vaccines in patients were mild to moderate flu-likesymptoms and local reactions, including fever with 2/10 cases, lower than 38.5°C of bodytemperature, acratia with 3 in10 patients, fatigue with 1 /10, body muscular soreness with 4/10, local injection site pruritus with 3/10, and swelling induration with 1/10. None of the adverse reactionsassociated to heart, liver, kidney and other vital organs were found. A little increase in life qualitywas found by KPS score for 4 patients 2 weeks after the end of treatment.[Conclusion] In GMP condition, a large amount of the adenoviral vector and A549 cells forGVAX vaccines are obtained by step by step enlargement of culture finally to several 10-layercell factories. By strict quality control, the allogeneic the preparation of GVAX vaccines showsthat tumorigenicity, endotoxin and pathogen are negative, a higher secretion of cytokines isproducted. Application of the allogeneic GVAX vaccines to the patients with advanced lungcancer has better safety and lower toxicity, mainly mild to moderate flu-like symptoms anddiscomfort of injection site reactions, all of the patient can be well tolerance.