Preclinical Safety Evaluation Of Adenovirus Injection TC007

Objective:TC007 is a recombinant Hepatitis B therapeutic injection based on adenovirus type 5 as a vector.It can stimulate the immune system repeatedly for a long time by mimic the pathogenic replication process in vivo to a certain extent and induce a strong,long-lasting and comprehensive immune response.It is intended for the treatment of Hepatitis B.In this study,by conduction of general toxicology test in mice and cynomolgus monkey with TC007,safe pharmacological test in cynomolgus monkeys,mouse bone marrow cell micronucleus test,special toxicology test to provide evidence to support for TC007 clinical trials application.Method:1)Single dose toxicity test in C57BL/6 mice:total 4 groups with half male and female mice at vehicle,1.0×109VP/animal,1.0×1011VP/animal,Ad5-null control group,6 animals/group,single subcutaneous dose at dose volume 0.1mL/animal with 14days recovery period.Evaluation measurements include clinical observation,bodyweight,IFN-?ELISPOT and gross findings.2)Single dose toxicity test in cynomolgus monkey:total 4 groups with male and female monkey at vehicle,1.0×109VP/animal,1.0×1011VP/animal,Ad5-null control group,2 animals/group except 4 animals in 1.0×1011VP/animal,single subcutaneous dose at dose volume 0.1mL/animal with 14 days recovery period.Evaluation measurements include clinical observation,bodyweight,ECG,hematology,serum chemistry,IFN-? ELISPOT at d7,biodistribution post-dose 48 h and 15 days,gross findings and tissue microscopic examination.3)Repeated-dose toxicity test in cynomolgus monkey:TC007 were administerd S.c.into total 5 groups to male and female monkey once a week for 7 weeks at vehicle,1.0×109 VP/animal,1.0×1010VP/animal,1.0×1011VP/animal,Ad5-null control group with 4-week recovery period,10 animals/group.Evaluation measurements include clinical observation,bodyweight,food consumption,ophthalmology examination,body temperature,ECG,hematology,serum chemistry,serum electrolyte,coagulation,urine analysis,lymphocyte clustering(CD3+CD8+T cell,CD4+/CD8+,CD3-CD56+NKcell,CD3+CD56+NKTcell,CD45+CD14+macrophage cell),IFN-? ELISPOT test(d7,d35,d45,d68),liver immunohistochemistry of IFN-?/TNF-?/IL-2(d46,d71),immnunogenicity and immunotoxicity testing,biodistribution(d46,d71),gross findings,organ weight,bone marrow examination and tissue microscopic examination.4)Safety pharmacology in cynomolgus monkey:total 5 groups to male and female monkey with vehicle,1.0×109VP/animal,1.0×1010VP/animal,1.0×1011VP/animal,Ad5-null control group,6 animals/group,single subcutaneous dose at dose volume lmL/animal.To collect relevant parameters from cardiovascular respiratory system at least 3 minutes or more at first pre-dose and 2 h,4 h,24 h,72 h,120 h,168 h post the first dose.Evaluation measurements include blood pressure(systolic blood pressure,diastolic blood pressure,mean arterial pressure),electrocardiogram(Heart rate,PR interval,QRS interval,QT interval,QT calibration),respiratory(breath frequency,tidal volume)5)Bone marrow micronucleus test in C57BL/6 mice:TC007 were administerd S.c into total 5 groups to male mice for single dose at vehicle,1.0×109VP/animal,1.0×1011VP/animal,Ad5-null control group and positive control goup,6 animals/group.To necropsy half mouse post 24 hours and 48 hours to prepare smear,then count 2000 polychromatic erythro-cytes to calculate the polychromatic erythro-cyte with micronuleus cells,meanwhile to count 200 PCEs with mature erythrocyte presentation,calculate the PCE ratio.6)Formulation Safety Test:Passive skin allergy test:set TC007 low-dose group(2×109VP/mL,0.5 mL/animal),high-dose group(2×1010 VP/mL,0.5 mL/animal),negative control group(0.9%sodium chloride Injections(0.5mL/animal)and positive control(Ovalbumin 8 mg/mL,0.5 mL/animal)were used in 4 groups.When sensitized serum was prepared,16 guinea pigs(half male and half female)were used,and 4 mice in each group were sensitized by subcutaneous injection.The sensitized serum was prepared once every other day for 3 times and 14 days after the last sensitization.When sensitized and stimulated intradermally,another 24 guinea pigs(half male and half female)were used.Each group had 6 mice.In the case of intradermal sensitization,the previously prepared sensitized serum was treated with 0.9%sodium chloride injection at 1:2,1:4,1:8 dilutions were performed.After guinea pigs were depilated,0.1 mL of sensitized serum was injected intradermally with the appropriate dilution at 3 points.After 48 h,the same dose of test substance as the sensitization dose was added.A total of 1 mL of 0.5%Evans blue dye was used.Guinea pigs were euthanized by CO2 approximately 30 minutes after challenge and the back skin was measured for the size of the medial spot.Active whole body allergy test:36 guinea pigs(half male and half female)were used,Set the low-dose group for the test(2×109 VP/mL,0.5 mL/animal),high-dose group(2×1010 VP/mL,0.5mL/animal),negative control group(0.9%sodium chloride injection,0.5 mL/body)and positive control group(Ovalbumin 4mg/mL,0.5mL/animal)4 groups,8 in each group.The sensitization was administered subcutaneously once every other day for a total of 3 times.On the 14 th and 21st day after the last sensitization,the sensitization dose of the test article or the reference substance was injected intravenously to observe the active systemic anaphylaxis of guinea pigs.Subcutaneous skin irritation test:6?8? New Zealand rabbits,for the test article group and Ad5-null control group,the doses were 1.0×1011VP/animal,the right abdominal subcutaneous injection of test articles,abdominal subcutaneous injection.Dosage volume was 0.5 mL/dose,single dose,2 rabbits were taken for dissection at 72 h after drug administration;continuous 3 d administration,once daily dosing,72 h after the last dosing,each three rabbits from the two groups were taken for dissection;the remaining animals continued to observe for 14 days to understand the reversible degree of the stimuli response.After the recovery period,the left and right sides local tissue need histopathological examination.TC007 hemolysis test in vitro:New Zealand rabbit hearts were collected and prepared as a 2%red cell suspension.After preparation,they were added to a 0.9%sodium chloride solution and ultrapure water in a glass tube according to a certain ratio.After mixing,the mixture was incubated at 37oC.Put in the biochemical incubator for 30 min,then add 5×108VP/mL,5×109VP/mL,and 5×1010VP/mL concentration of the test sample in the corresponding test tube,mix well and place at 37?.In the biochemical incubator,naked eyes were observed for the presence or absence of hemolysis or agglomeration of erythrocytes in rabbits.No hemolysis and condensation were observed in the test tubes of the test samples.Result:1)Single subcutaneous dose toxicity test of TC007 in C57BL/6 mice:No abnormalities of clinical symptoms,body weight,and gross anatomy were observed in all groups;specific IFN-?-secreting T cells against the three HBV antigens(Core,Env,Pol)were detected in the 1.0×109VP and 1.0×1011VP dose groups.2)Single subcutaneous dose toxicity test of TC007 in cynomolgus monkey:In TC007-administered group,Ad5 was mainly distributed locally,and there was a slight distribution in mesenteric lymph nodes.At 48 h after administration,Ad5 was mainly localized in the high-dose group,and Ad5 biodistribution showed a clear elimination trend.The IFN-? ELISpot assay showed that 1.0×109VP and 1.0×1011VP groups were able to detect specific T cells secreting IFN-? against three HBV antigens(Core,Env,Pol).The weight,electrocardiogram,blood routine,and serum biochemical indicators showed no drug-related abnormalities in all dose groups.Clinical observation showed erythema of the skin and visible restoration.Histopathological examination revealed localized slight epidermal scab,dermal layer,and inflammatory cell infiltration in subcutaneous tissue.3)Repeated dose toxicity test by subcutaneous injection of TC007 in Cynomolgus monkeyAfter repeated administration of TC007 to cynomolgus monkeys,biodistribution test results showed that Ad5 was mainly localized in the administration area,with a small distribution in the axillary lymph nodes,and no distribution was found in the heart,liver,spleen,reproductive system,and kidneys.Ad5 biodistribution showed a clear trend.The results of IFN-? ELISpot assay showed that no specific T cells secreting IFN-? against three HBV antigens(Core,Env,Pol)were detected in the vehicle control group and the Ad5-null control group.At the end of administration and 4 weeks after discontinuation of drug withdrawal,the IFN-?/TNF-?/IL-2 positive levels in liver tissue of cynomolgus monkeys in each dose group and Ad5-null control group were significantly higher than those in the vehicle control group.The positive degree increased with the increase of dose;however,there was no significant difference in the positive degree between 1.0×1011VPand Ad5-null control group.Immunogenicity in each dose group and Ad5-null control group all animals produced antibodies,titer increased with increasing dose;withdrawal of 4 weeks recovery,each dose group animal antibody titer is still high.Neutralizing antibody activity test showed that the positive antibodies produced from animals all had neutralizing activity;Ad5-null control group and the high dose group had the neutralizing effect of the serum with the same dilution and the positive antibody titer in the serum was similar.Neutralization effects have no difference.Body weight,food intake,electrocardiogram,ophthalmologic examination,hemotology,serum biochemistry,serum electrolytes,coagulation,urine analysis,bone marrow smear,lymphocyte clustering,complement C3,C4,etc.,compared with vehicle control group,there were no drug-related abnormalities in the indicators in each dose group.The erythema of the skin was observed after clinical observation in each dose group of TC007.The body temperature of TC007 dose group and Ad5-null control group was slightly higher during administration.TC007 high-dose group of circulating immune complex(CIC)compared with pre-dose and vehicle control group showed a slight increase(P?0.05).At the end of administration,histopathological examination showed that local inflammatory cell infiltration(plasma cells,lymphocytes)was observed in each subcutaneous tissue.At the end of the recovery period,the scheduled anatomy and histopathological examination showed local inflammatory cell infiltration(plasma cells,lymphocytes)in the subcutaneous tissue,but the lesions showed obvious recovery compared with the end of administration.4)Safety pharmacological test of TC007 by subcutaneous injection in cynomolgus monkey and mice.The spontaneous activity test and rotating rod test in mice show that there were no drug related effect regarding the balance ability and the nervous system.There were no drug-related abnormalities of systolic blood pressure,diastolic blood pressure,mean arterial pressure at 1.0×109VP,1.0×1010VP and 1.0×1011VP dose groups,and Ad5-null control group compared to vehicle control and pre-dosing.Also,there were no any significant change regarding to respiratory system,heart rate HR,PR interval,QRS,QT interval,and QTcf ECG indicators.5)Bone marrow micronucleus test by subcutaneous injection of TC007 in C57BL/6 mice.In the mice dissected 24 h after administration,the MNPCE ratio in the low-dose group,the high-dose group,and the Ads-null control group did not exceed 2-fold compared with the vehicle control group and no dose-related correlation was observed.The ratio of mouse MNPEC dissected at 48 h after administration was lower than that of the vehicle control group with low dose group,high dose group and Ad5-null control group,and the difference was statistically significant(P<0.05),and has a certain dose-related,positive results.The rate of MNPCE in the positive control group was significantly higher than that in the vehicle control group,and the difference was statistically significant(P<0.05).6)TC007 Formulation Safety Test:Passive skin anaphylaxis test:There were no spots in the three serun injection sites of the low-dose group,high-dose group,and negative control group of the test animals.Spot formation was observed in all the three serum injection sites of the animals of the positive control group and the spot diameter was>5 mo.No passive skin anaphylaxis was observed in TC007 under the test conditions.Active whole body allergy test:No allergic reactions were found in animals of the low-dose group,high-dose group and negative control group;allergic symptoms occurred in about 17 to 40 seconds after challenge in the animals of the positive control group,and the death took about 3 to 6 minutes..Under the experimental conditions,no active whole body allergic reaction was observed in guinea pigs with TC007.Subcutaneous irritation test in rabbits:combined with clinical observation and histopathological examination showed that under the conditions of this test,the test article and Ad5-null at dose of 1.0×1011VP can be applied to the skin of New Zealand rabbits.A significant stimulus response was produced,and after 14 days of recovery,there was a clear recovery trend.Vitro TC007 hemolysis test:The results showed that TC007 did not induce hemolysis and aggregation of New Zealand rabbit red blood cells in vit:ro within 3 h.Conclusion:Via scientific design of the preclinical safety evaluation of adenovirus injection TC007,relevant studies were carried out under the GLP conditions.The results showed that the NOAEL obtained from single test in C57BL/6 mice and repeated tests in single and repeated cynomolgus monkeys was 1.0×1011VP/animal;IFN-?-specific T cells can be found against the three antigens of HBV Core,Env,Pol.The safety dose has been confirmed.No significant target organ toxicity was found in the evaluated species.The immunogenicity did not affect the pharmacological effects.The tissue distribution investigation did not show unexpected distribution.The main and passive allergic reactions were negative,and no hemolysis was found in vitro test.No adverse reactions were observed in the general pharmacological studies of the nervous system,respiration and cardiovascular.Although the micronucleus test results proved to be positive,the null group also showed same results,considering that the clinical extrapolation has certain limitations,and this type of drug is less likely to cause chromosome damage.In summary,the preclinical toxicology test shows that it has some safety window.By following the registration requirements of therapeutic biological products,the preclinical evaluation can support the biopharnaceutical entry into the clinic,and can be submitted for application to investigate the safety and effeciency in clinical trials,meanwhile monitoring the possible adverse reactions.