Chemicals and Reagents
Recombinant humanized anti PD-1 monoclonal antibody injection (TA1718-B, 25 mg/ml; purity, 99.7%, determined by molecular exclusion chromatography) was obtained from National Engineering Laboratory of High Level Expression in Mammalian Cells (Lunan Pharmaceutical Group Co. Ltd, Linyi, Shan Dong province, China). 0.9% sodium chloride injection (vehicle) was from Shandong Kelun Pharmaceutical Co. Ltd. (Binzhou, Shandong, China).125I-TA1718-B was supplied by MITRO Biotech Co. Ltd. (Nanjing, Jiangsu Province, China).
All animal experiments were complied with the ARRIVE guidelines and carried out in accordance with the National Institutes of Health guide for the care and use of Laboratory animals (AARIVE guidelines checklist). All the animal including rats, mice and monkeys experiments were approved by the Institutional Animal Care and Use Committees (IACUC) of MITRO Biotech Co. Ltd., Shanghai Inno Star Bio-Tech Co. Ltd. in the contracts, respectively, and followed the “3Rs” rule (Reduction, Refinement, and Replacement).
For PK study, 24 cynomolgus monkeys (12 male/12 female, 2.4-3.1 kg, 2-4.5 years old) were supplied by Guangxi Guidong primate development experiment Co. Ltd. (Wuzhou, Guangxi Zhuang Autonomous Region, China) randomly divided into 4 groups. The animals were weighed before eating during the adaptation period. The animals were divided into two groups according to sex, and then the same sex animals were randomly assigned to four groups according to body weight. During the whole experiment period, all animals received clinical observation including injection site, respiratory, exercise, urinary and behavioral changes. To minimize the harm monkeys, noise in monkey room was controlled and environmental factors were according to guidelines described in the guide to the care and use of experimental animals (Canadian Council on Animal Care, 1993).
For single administration study, animals were intravenously injected with 1, 3, and 10 mg/kg TA1718-B. Before (0 h) and after drug injection (0.25, 0.5, 2, 8, 24, 72, 120, 168, 240, 336, 504, 672, 840, and 1008 hrs), 1 ml of the inguinal vein blood were harvested and stored at -80 °C.
For repeated administration study, all animals were underwent 3 mg/kg i.v injection of TA1718-B, once a week, for 4 weeks. Before (0 h) and after first drug injection (0.25, 0.5, 2, 8, 24, 72, 120, 168 hrs), 0.5 h before and after the second and third drug injection, before and 0.25, 0.5, 2, 8, 24, 72, 120, 168, and 240 hrs after the last (fourth) drug injection, 1 ml of the inguinal vein blood were obtained and stored at -80 °C.
The plasma samples were centrifuged at 4000 rpm for 10 min at 4 °C to obtain the supernatant. The concentrations of TA1718-B in plasma were detected by ELISA.
To verify whether single or repeat injection resulted in immunogenicity reaction, before drug administration (0 h), and 336, 504, 672, 840,1008hrs after single injection, and 336 hrs (before the third drug injection), before and 336 hrs after the last drug delivery, 1 ml of the inguinal vein blood were obtained and centrifuged at 4000 rpm for 10 min at 4 °C to obtain the supernatant. Immunogenicity assay was performed by ELISA.
Tissue distribution study
24 MC38 colon cancer humanized mice (12 males/12 females) were purchased from Beijing Biocytogen Co. Ltd. (Beijing, China) for tissue distribution study. All animals were housed in SPF level barrier animal room of MITRO Biotech Co., Ltd. (Nanjing, Jiangsu Province, China) for 3 days’ quarantine and then entering the project test. Animals were acclimatized to a controlled temperature (23 ± 2˚C) and maintained under a 12/12-h light/dark cycle. The animals were supplied with pellet chow and water ad libitum. Before drug delivery, mice were intragastric injected (i.g) with 1 % potassium iodide (KI) for blocking thyroid, and 0.01% KI solution was drunk during the whole experiment. All mice were i.v injected with 3 mg/kg 125I-TA1718-B (20 ± 10 μCi peer mouse). 2, 96, 168, and 240 hrs after drug injection, tissues including heart, liver, spleen, lung, kidney, stomach, intestine, gonad (testis and ovary), brain, fat, skeletal muscle, femur, tumor, mesenteric lymph node, thyroid, urine and serum were collected. All samples’ total and sedimentary radioactivity were detected by γ-ray counter and converted to specific activity to calculate the concentration of radiopharmaceutical in tissues. To minimize suffering, mice were anaesthetized by intravenous injection of 1%sodium pentobarbital for tissue collection and euthanized by blood letting after anaesthesia .
6 SD rats (3 male/3 female, 5-7 weeks, 200-250 g) were purchased from JOINN Laboratories (Suzhou, Jiangsu Province, China) for urine and feces excretion experiments. 6 bile duct intubation model rats (3 male/3 female, 180-200 g) were purchased from Beijing Vital River Laboratory Animal Technology Co. Ltd. (Beijing, China) for bile excretion experiment. All animals were housed in SPF level barrier animal room of MITRO Biotech Co. Ltd. (Nanjing, Jiangsu Province, China) for 3 days’ quarantine and then entering the project test. Animals were acclimatized to a controlled temperature (23 ± 2˚C) and maintained under a 12/12-h light/dark cycle. The animals were supplied with pellet chow and water ad libitum. Before drug delivery, mice were intragastric injected (i.g) with 1% KI to block thyroid, and 0.01% KI solution was drunk during the whole experiment. All rats were i.v injected with 3 mg/kg 125I-TA1718-B (20 ± 10 μCi per rat). After drug injection, urine and faces of SD rats were collected from 0-8, 8-24, 24-48, 48-96, 96-168, 168-336, 336-504, 504-672, 672-840 hrs, bile of bile duct intubation model rats were collected from 0-4, 4-8, 8-24, 24-48 hrs. Serum and urine samples of experimental animals with tissue distribution (1 male and 1 female at each time point) were prepared and separated for molecular exclusion HPLC detection. All samples’ radioactivity was detected by γ-ray counter and the cumulative excretion rates were calculated. To minimize suffering, rats were anaesthetized by intravenous injection of 2% sodium pentobarbital (Sigma, Louis, MO, USA) for animal operation throughout the experiment process.
Preparation of Standard and QC Solutions
TA1718-B was dissolved in cynomolgus monkey’s serum in a series concentration (8.000, 4.444, 2.469, 1.372, 0.762, 0.423, 0.235, 0.131, 0.073 and 0 μg/ml) as standard solutions. The QC solutions were prepared by cynomolgus monkey’s serum and dissolved in 0.25 μg/ml (LQC), 0.70 μg/ml (MQC), and 3.5 μg/ml (HQC), respectively.
Linearity, Accuracy, and Precision
The calibration curves were established from the peak area of each standard solution against the nominal concentrations using eight level non-zero standards and a linearly weighed (1/x) least squares regression model. The calibration curve required a correlation coefficient (R2) of 0.99 or better.
Method accuracy was estimated by calculating the percent deviation observed in the analysis of quality control (QC) samples and expressed as relative error. Intraday precision was estimated by analyzing QC samples at three concentrations within 24 hrs (n = 5). Inter-day precision was estimated by repeating analysis of QC samples over three consecutive days (n = 15). The variability in determination was expressed as the relative standard deviation (RSD, %) and the accuracy was expressed as the relative error (RE, %). The lower limit of quantification (LLOQ) was defined as the lowest concentration that could be determined with both RE and RSD within 20% (Lee et al., 2011).
125I- TA1718-B Quality Control
The samples of 125I- TA1718-B were separated and purified on Radio-iTLC paper, the samples were spread up in 85% methanol system and scanned by thin-layer chromatography (TLC) scanner. The samples of Na125I solution were placed on Radio-iTLC paper and then spread up in 85% methanol system as control group. The retention factor (Rf) value was calculated by the formula as follows.
Rf= Retention Time (RT, min)-0.15 (min)/0.78 (min)-0.15 (min).
0.15 min is sampling origin, 0.78 min is the final solvent front. RT < 0.15 is defined that Rf is 0, RT > 0.78 is defined that Rf is 1. In this experiment, RT of 125I- TA1718-B should be among 0-0.1, RT of Na125I should be among 0.8-1.0.
125I- TA1718-B Concentration and Specific activity assay
The protein concentrations of 125I- TA1718-B solutions were detected by UV method and calculated according to the specific activity of 125I-TA1718-B solutions.
Specific Activity (SA) = Radioactivity concentration (mCi/ml)/Protein concentration (mg/ml).
Linear log Trapezoidal method of WinNonlin Phoenix Soft (v6.4, Pharsight, Beijing, China) was used to calculated the pharmakinetic parameters such as T1/2, Vd, AUC, CL, Cmax, Tmax, MRT, AUC(0~t), and AUC(0~inf) in non compartment model manners. Data were calculated and analyzed with Excel 2010 (Microsoft, Redmond, WA, United States) and SPSS V19.0 (SPSS, Inc., Chicago, IL, United States). All values were presented as mean + SD, using an unpaired t-test by SPSS Statistics V19.0.