Preclinical Elucidation of Recombinant Humanized Anti-PD-1 Monoclonal Antibody (TA1718-B) Pharmacokinetics in Cynomolgus Monkeys, Rats, and Colon Cancer Humanized Mice CURRENT STATUS: POSTED

EXSCINDED Abstract Background PD-1 monoclonal antibodies (mAbs) are widely applicated in clinical as therapeutic strategy for multiple types of cancer. TA1718-B, a novel PD-1 mAb, is now entering clinical phase I.Methods In present study, the preclinical pharmacokinetic (PK) properties of TA1718-B were investigatedviavarious experiments in animal species. In plasma kinetic studies, cynomolgus monkeys underwent either single (1, 3, and 10 mg/kg) or repeat (4-times, 3 mg/kg) intravenous injection and plasma were collected in different time points and detected by ELISA. Tissue distributions were performed in MC38 colon cancer humanizedmice. The excretion of TA1718-B was evaluated in rats.Results TA1718-B exhibited long T1/2 (from 97 to 136 hrs) and low clearance (Cl) (from 0.25 to 0.38 ml/h/kg) after intravenous single injection in cynomolgus monkeys. And 4-time continuous injections were not resulted in significant drug accumulation. However, both two drug delivery ways resulted in high anti-drug antibody (ADA) positive rates. The tissue distributions of TA1718-B were widely (almost in all tissues) and higher in serum, ovary, lung, tumorthan the other tissues in MC38 colon cancer humanized mice. The excretion of TA1718-B in rats were mainly metabolized and eliminated through proteolytic degradation.Radiopharmaceuticals in urine mainly existed in the form of proteolytic degradationfrom the data.Conclusion This systematic investigation of pharmacokinetic profiles of TA1718-B supplies basis for clinical trials and need to be further studied in Phase I clinical trial.


Background
In human, tumors occurrence commonly caused by genetic and epigenetic aberrations and produced altered antigenic profiles which can be selectively recognized by the adaptive immune response [1]. Between host and tumor, a dynamic interaction existed and evasion 3 of immune recognition ability of the tumor directly determines the clinical course of the disease [2,3]. Cancer immunotherapy, also called immuno-oncology, is an anti-tumor therapeutic strategy which could improve the organic natural ability to fight cancer via using artificial agents (such as monoclonal antibodies, lymphocytes and cytokines) to stimulate the immune system [4][5][6]. The objective of tumor immunotherapy is to increase the specific immune response of tumor specific CD4 + and CD8 + T cell. And effective immunotherapeutic strategies are designed to enhance endogenous anti-tumor responses, such as cancer vaccines, which are far less successful [7,8]. In recent years, checkpoint blockade-based cancer immunotherapy is a research hotspot [9][10][11]. Immune checkpoints consist with a group of molecules which are targets for cancer immunotherapy because of their potential protective effects on tumors to get rid of immune system's attacks [12].
The initial clinical trial results of first PD-1 antibody (Nivolumab, Bristol-Myers Squibb Co., Inc.) were published in 2010 [12] and approved in 2014 for treating melanoma, lung cancer, kidney cancer, bladder cancer, head and neck cancer, and Hodgkin's lymphoma.
Subsequently, another PD-1 inhibitor (Pembrolizumab, Merck & Co., Inc.) was approved to treat melanoma and lung cancer by the FDA in 2014 [17]. Although several PD-1 monoclonal antibodies were approved by FDA and related pharmacokinetics studies are researched well, TA1718-B is a novel PD-1 mAb, lacking of drug metabolism and tissue distribution data. Therefore, to supply basis for clinical trials, PK parameters and biodistribution are evaluated.
Although numerous studies have discovered multiple pharmacological and extensively therapeutic activities of PD-1 monoclonal antibody, systematic evaluation of its pharmacokinetic characteristics are still limited. TA1718-B, a novel recombinant 4 humanized anti-PD-1 monoclonal antibody was researched and developed independently by Lunan Pharmaceutical Group Co. Ltd.. The preclinical pharmacological studies of TA1718-B have been performed and applied for clinical trials. In current report, the plasma pharmacokinetic parameters, tissue distributions and excretion of TA1718-B were investigated in cynomolgus monkeys, MC38 colon cancer humanized mice and SD rats.

Ethics
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).

5
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).
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.

Immunogenicity assay
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 6 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 125 I-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 .  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 (R 2 ) 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).

I-TA1718-B Quality Control
The samples of 125 I-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 Na 125 I 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.

Precision and Accuracy
The concentration, stability and homogeneity of the TA1718-B solutions were determined by UV-V is spectrophotometer (UV-6300 PC, MAPADA, Shanghai, China). For concentration of the upper, middle, and lower solutions determination was conducted in triplicate, respectively. The RSD (%) between the measured concentrations and theoretical values should be from 90.77 to 95.32%, and the RE (%) were lower than 5%. ELISA was used to detect the protein concentrations in cynomolgus monkeys' serum. After the standard curve fitting, the correlation coefficient of R 2 was above 0.99.

I-TA1718-B Quality Control
125 I-TA1718-B quality control assay demonstrated that the radiochemical purity (RCP) in 3 batches were 100%, meet the standard (RCP > 90%), and the specific activity (SA) were among 0.03-0.33. Radio-iTLC was used to verify the stability of 125 I-TA1718-B. The results showed that either in 3.5 or 23 hrs, the RCP were 100%, which meet the testing requirement.

Cynomolgus monkeys' Plasma Pharmacokinetics Study
To determine the PK characteristics, the plasma concentration-time profiles of TA1718-B were detected following single (1, 3, and 10 mg/kg) and repeat (3 mg/kg) i.v injection and the results were showed in Figure 1. The PK parameters were calculated and showed in Table 1 and Table 2. Compared the individual data in male and female monkeys, all PK parameters showed no significant differences in TA1718-B 1, 10 mg/kg single and 3 mg/kg repeat injected groups, only C m ax in TA1718-B-3 mg/kg single injection group exhibited significant difference, but the ratio was above 1.5, which indicated that there were no significant sexual differences (data was not shown) between male and female animals.
In additional, the fitting results of exposure parameters (C m ax and AUC) in 3-dosage groups were showed and the slope (β-value) and 90% confidence interval (Cl) were calculated. As showed in Table S1, the β-value and 90% Cl of C m ax were 0.856 and 0.787-0.926, respectively, which excessed 0.8-1.25, as well as AUC INF_obs (the β-value and 90% Cl were 0.947 and 0.798-1,097, respectively). However, the β-value and 90% Cl of AUC last were within the range (β-value was 1.039, 90% Cl was 0.887-1.191). All these results indicated that the increase of AUC last was proportional to the dosage increasement (from 1 to 10 mg/kg), but not in C m ax and AUC INF_obs. There was no difference in dose groups and also no difference between male and female monkeys.

Immunogenicity Assay
To verify whether cynomolgus monkeys developed anti drug antibody (ADA) resulted from single/repeat TA1718-B injection, serum of animals at different time points after single (from 0 to 1008 hrs) or repeat (from 0 to 1512 hrs) drug injection were collected and detected by ELISA. As results showed in Figure S1A, the ADA positive rates were 100% (6/6), 100% (6/6), and 66.7% (4/6) when monkeys underwent 1, 3, and 10 mg/kg i.v injection, respectively. In additional, 3 mg/kg TA1718-B repeated injection resulted in100% (6/6) ADA positive rate (Figure S1B). There was no difference in dose groups and also no difference between male and female monkeys.

MC38 Colon Cancer Humanized Mice Tissue Distribution Study
To investigate the tissue distribution of TA1718-B, 125 I labeled TA1718-B was used to i.v inject into MC38 colon cancer humanized mice. Tissues, plasma, and urine were collected at 2, 96, 168, and 240 hrs after drug delivery, and the gamma counting results of different fractions after HPLC separation were detected and calculated.
The HPLC showed that the retention time of 125 I-TA1718-B was 10.7 min (Figure S2A). At 2, and 96 hrs after i.v injection, prototype drug could be detected but not at 168 and 240 hrs in plasma (Figure S2B-E). In contrast, prototype 125 I-TA1718-B were not detected in urine during the whole experiment (Figure S2F-I), which indicated that metabolites of 125 I-TA1718-B were not mainly by urine but by proteolytic degradation. Meanwhile, in order to avoid the influence of thyroid on drug distribution, the drug total radioactivity in thyroid were 0.09 ± 0.02% (2 hrs), 0.05 ± 0.01% (96 hrs), 0.05 ± 0.01% (168 hrs), and 0.06 ± 0.03% (240 hrs), respectively, which indicated that the reliability of tissue distributions of 125 I-TA1718-B was not interfered by thyroid (Table S2).
To verify the trichloroacetic acid (TCA) precipitation method, all mice tissues/body fluid 13 and vehicle were added 125 I-TA1718-B in a series of concentrations (3 μg, 0.3μg, 30 ng, 15 ng, and 3 ng), total and precipitated radioactivity were calculated and the result was showed in Table S3 and Figure S3. All results demonstrated that there were linear correlations between the total radioactivity and precipitated radio activity. The correlation coefficients were above 0.999, and the acid precipitation rates were more than 94%.
The tissue distributions of 125 I-TA1718-B in mice were performed and showed in Figure 2 and Table S2. The data suggested that the 125 I-TA1718-B was widely distributed throughout all tissues and even the brain, testis (or uterus) and bone (Figure 2A). At 2 hrs after 125 I-TA1718-B injection, the drug concentrations in ovary and lung were peaked (14.00 ± 1.91 and 12.14 ± 2.45 μgEqu/g, respectively); the kidney, tumor, mesenteric nodes, spleen, heart, liver, bone, intestine, testis, and stomach contained moderate amounts of 125 I-TA1718-B (from 2.44 to 8.40 μgEqu/g); and tissue extracts from fat, brain, and muscle contained low concentrations (below 2.00 μgEqu/g). At 96 hrs after 125 I-TA1718-B administration, tissue extracts from tumor, mesenteric nodes, and lung still had comparatively high concentrations of 125 I-TA1718-B (4.47 ± 1.58, 4.09 ± 1.09, and 3.56 ± 0.84 μgEqu/g, respectively). In additional, at 240 hrs after administration, 125 I-TA1718-B was undetected in ovary, liver, fat, brain, and muscle, and other tissues ( Figure 2B). The AUC from 0 to 240 hrs in different tissues after drug injection was plasma > ovary > lung > tumor > mesenteric nodes > kidney > spleen > liver > heart > fat > stomach > bone > intestine > testis > muscle > brain (Figure 3). In addition to lung, ovary and serum, tumors had the highest exposure to radio pharmaceutical compared with other tissues, indicating that tumors had specific uptake of 125 I-TA1718-B. There was no difference in different groups.

The Excretion of 125 I-TA1718-B in Rats
To detect the excretion characteristics of 125 I-TA1718-B, BDC and SD rats were i.v injected with 125 I-TA1718-B. Urine, feces and bile (BDC rats) were collected at different time points and detected by HPLC and γ-ray counter. As results showed in Figure 4A and Table 3, the accumulated excretion ratios of 125 I-TA1718-B from 0 h to 4, 8, 24, and 48 hrs in BDC rats' bile were 0.2 ± 0.1%, 0.4 ± 0.1%, 1.6 ± 0.4%, and 3.9 ± 0.9%, respectively. After drug injection, the accumulative excretion rates (from 0 to 840 hrs) in urine, feces, and total (urine plus feces) were 69.2% ± 5.9%, 17.2% ± 11.7%, and 86.4% ± 9.3%, respectively ( Table 4 and Figure 4B). However, no radioactivity peak was observed in urine samples at the corresponding time of the prototype drug, which indicated that the radiopharmaceuticals in urine mainly existed in the form of proteolytic degradation. Early PK liabilities of mAb drugs were often usually identified using rodent models. Pilot PK/PD and safety studies are in appropriate species (NHP and rodent) [18]. So we evaluated the PK of TA1718-B using rats, mice and monkeys.

Discussion
The cynomolgus monkeys were underwent single (1, 3, and 10 mg/kg) or repeat (4 times, showed that 1, 3, and 10 mg/kg single injection did not result in significantly differences between male and female monkeys. And from 1 to 10 mg/kg drug delivery dose, the increase of AUCl ast was proportional to the dosage increasement (The AUC last were 3160 ± 583, 7000 ± 1920, and 36400 ± 16300 h*μg/ml, respectively. The AUC last and dosage ratios were 1 : 2.2 : 11.5 and 1 : 3 : 10, respectively.). However, the C m ax increases were indirectly correlated with the dosage (The C m ax were 27 ± 2.5, 64.4 ± 13.5, and 194 ± 28.9 μg/ml, respectively. The ratio was 1 : 2.38 : 7.18), and the growth rates were slower than the increase of dose. In additional, C m ax ratio between the first and the last drug exposure after 4-times continuous injection (3 mg/kg) was 1.32-fold (the C m ax-first and C m ax-last were 83.6 ± 16.0 and 110 ± 33.8 μg/ml, respectively). The ratio of AUC (0-168hrs) was 1.0-fold (AUC (0-168hrs)-first and AUC (0-168hrs)-last were 6610 ± 1530 and 6640 ± 5860 h*μg/ml, respectively). In additional, the C through before each injection were 0.00, 28.1, 40.8, and 31.1 μg/ml. All these results demonstrated that 4-times continuous TA1718-B i.v injection could not result insignificant drug accumulation. 3 mg dose was used for repeat administration due to previous pharmacodynamics study we also referred to the clinical doses of Nivolumab and Keytruda (2-3 mg/kg) [19]. It is a dose determined to use for human body.
With the clinical applications of more and more protein medicines to address unmet medical needs such as cancer, autoimmune disease and other diseases, their safety and efficacy should be ensured (FDA, 2014). As with all therapeutic proteins, there is a potential for immunogenicity. The emergence of ADA (anti-drug antibodies) can potentially lead to loss of efficacy and/or adverse effects because of protein medicines' structure containing potential B-and T-cell epitopes, which resulted in the production of T-cell epitope peptides [20]. Therefore, evaluating the potential immunogenicity risk should be considered during the development of therapeutic protein products [21]. The immunogenicity of TA1718-B single or repeat injected in cynomolgus monkeys were detected by ELISA. The positive rates of ADA after 1, 3, and 10 mg/kg single administration were 100% (6/6), 100% (6/6), and 66.7% (4/6). In contrast, 3 mg/kg TA1718-B repeat injection resulted in 100% (6/6) of ADA positive rate. These results suggested that the ADA may neutralized the activity of TA1718-B and influenced drug's elimination, plasma T 1/2 , tissue distributions, and even PD/PK features. To cynomolgus monkeys, this recombined mAb is a heterologous protein, so ADA rate is high. And only part of the ADA induced by PD-1 mAb is neutralizing antibody. And it is reported that, in most cases, ADA occurs mainly in animals exposed to lower doses rather than higher doses. This may be associated with high dose induced tolerance (high zone tolerance) [22]. Immunogenicity or ADA response found in preclinical species does not predict ADA response in human. Keytruda is a good example that is quite immunogenic in cynomolgus monkeys with a high incidence of ADA but only with low ADA incidence (1.7%) (http://www.accessdata.fda.gov/drugsatfda_docs/nda/2014/125514Orig1s000PharmR.pdf)). So here we speculate the incidence of ADA and neutralizing antibody may be low in human.
Once a drug enters into systemic circulation by absorption or direct administration, it must be distributed into different organs or tissues [23] and its metabolites need to be removed from the body via excretion through the kidneys (urine) or the feces [24].
Although there are differences between small molecules and monoclonal antibodies, mAbs also can arrive at different tissues by convection. And in general, antibody concentrations in tissue interstitial fluid are substantially lower than antibody concentrations in plasma because of the differences in the rate of convective uptake (which is slow) and elimination of antibody from tissue (which could be fast) [22]. Otherwise, antibodies are mainly eliminated by excretion or catabolism. Although mAb is too large to be eliminated by urine excretion, but proteolytic fragments can be filtered by kidney when mAb is catabolic in vivo. TCA precipitability indicated that all of the radioactivity in urine was represented by proteolyzed/degraded mAb. The distributive and excretory capacity of a drug is strongly associated with its efficacy and/or adverse events [25]. In this study, tissue distributions of TA1718-B were detected in MC38 colon cancer humanized mice by i.v injection with3 mg/kg 125 I-TA1718-B. MC38 colon cancer humanized mice is a general model to evaluate biodistribution or intratumoral distribution of PD1/PDL1 mAb [26]. All tissues were collected from 2 hrs to 240 hrs after drug delivery and the total radioactivity of 125 I-TA1718-B were detected. Results demonstrated that the AUC from 0 to 240 hrs in different tissues after drug injection were plasma > ovary > lung > tumor > mesenteric nodes > kidney > spleen > liver > heart > fat > stomach > bone > intestine > testis > muscle > brain. In addition to serum, ovaries and lungs, tumors had the highest exposure to radiopharmaceuticals compared with other tissues, indicating that tumors had specific uptake of 125 I-TA1718-B.The mAB tissue distribution is affected by various factors such as isoelectric point (PI), protein size and affinity to target of mAb [27]. Although antibodies developed against an antigen are highly specific to that antigen, some mAbs can have multi-specificity binding capability for irrelevant antigens. It is reported that small proteins could be eliminated rapidly by kidney filtration and a better affinity could improve tumor accumulation. But impact of large mAb protein on tumor accumulation is dismissed [22]. Our data could not exhibit that the antibody is nonspecific.
Our results displayed that TA1718-B concentration in tumor tissue is lower than that in ovary, lung, kidney, liver and so on. There is a theory "binding site barrier" to explain it, which means a mAb with a moderate affinity was associated with the highest tumor accumulation, however, mAb with high affinity produces low tumor accumulation [28,29].
And TA1718-B is a PD-1 mAb with a high affinity, so it exhibited a low accumulation in tumor tissue.
Subsequently, we investigated the urine, feces and bile excretions of TA1718-B in SD or BDC rats after i.v of 125 I-TA1718-B (3 mg/kg). Within 840 hrs after drug delivery, the urine accumulative excretion ratio was 69.2% ± 5.9%, and the ratio reached a platform after 336 hrs, probably resulting from its high lipophilic and plasma protein binding capacity. Shanghai InnoStar Bio-Tech Co. Ltd. in the contracts, respectively, and followed the "3Rs" rule (Reduction, Refinement, and Replacement).

Consent for publication
Not applicable.

Availability of data and materials
The datasets used and/or analyzed during the current study are available from the 20 corresponding author on reasonable request.

Competing interest
The authors declare that they have no competing interests.