WORKSHOP
Advanced Capabilities for Ocular Drug Development Using NHP Models
West Concourse of the Orange County Convention Center
Room W2088
Session Description:
Non-human primates (NHPs) are widely used in the preclinical development of ocular drugs due to their unique anatomy and physiology. During this presentation, we will highlight several key concepts for using NHPs in preclinical drug development and compare the ocular anatomy and physiology of NHPs to human and other preclinical models (rodents and rabbits). We will share the major findings in ocular toxicology studies performed in multiple species using different types of test articles including biologics and gene therapies. Immunogenicity findings in the eye will also be presented.
Contact bd@biomere.com if you would like us to share the logistics with you directly, or send a poster reprint post meeting.
Analysis of Toxicity Characteristics of GLP-1 Receptor Agonists in Non-clinical Safety Evaluation
Weiqing Kong, Yanlin Zhang, Jinling Ma
JOINN Laboratories, Beijing
Glucagon-like peptide-1 receptor agonist (GLP-1RA) is a new hypoglycemic drug class that can lower blood sugar and help with weight loss and functions by activating the GLP-1 receptor. GLP-1RA also has multiple clinical benefits, including controlling body mass and improving non-alcoholic fatty liver disease and is gradually becoming the primary prescription drug for diabetes. This abstract summarizes the results of the toxicity tests of GLP-1RA conducted in our institution, combined with the toxicity characteristics of similar products on the market, to provide a reference for non-clinical safety evaluation and toxicity analysis of such products.
The first step in safety evaluation is to select the appropriate animal species for testing. It is necessary to understand GLP-1 receptor binding and in vitro pharmacological activity in different species. For chemically synthesized peptides such as GLP-1, it is also important to select the species that has similar metabolic characteristics as humans. Rat and cynomolgus monkeys are the commonly selected animal species. The toxicity endpoints in rats typically include measurement of weight loss and decrease in food intake and the clinical pathology endpoints includes reduced Retic count, reduced concentrations of triglycerides, creatinine, total protein, albumin and α1-globulin, and a slight increase in urea concentration along with measuring weight reduction of various organs. Histopathology analysis includes duodenal intestinal gland hypertrophy, thymic atrophy, and mild pancreatic acinar cell atrophy. There is also pancreatic zymogen depletion and persistent lobular atrophy related to sustained reduction of amylase. With analysis of the abnormal changes mentioned above, it is believed that they are mainly caused by the pharmacological effects of the test substance and the secondary changes, as well as the distribution of GLP-1 receptors on the surface of various organs.
The toxicity endpoints of cynomolgus monkeys include gastrointestinal reactions, dehydration, reduced activity, weight loss and reduced food intake. Clinical pathology endpoints include reduced indicators related to red blood cells, increased/decreased Retic count, slight decrease in blood sugar, increased bilirubin, increased weight of multiple organs, reduced weight of thymus, increased heart rate, left bundle branch block; and right ventricular epicardial paleness. Histopathology analysis includes thymic atrophy, decreased pancreatic zymogen granules, mild multifocal myocardial vacuolization and degeneration, inflammatory cell infiltration in the kidney or gastric pylorus, minor gastrointestinal bleeding, and local irritation from injection. Based on the identification of the abnormal changes mentioned above, it is believed that they are mainly caused by the pharmacological effects of the test substance and the secondary changes, as well as the distribution of GLP-1 receptors visible on the organs. Among them, heart-related changes are considered possible toxic reactions.
Fertility and early embryonic development toxicity in endpoints in rats include weight loss/slow growth, reduced food intake, prolonged female estrous cycle, reduced corpora luteum, implantation and live fetuses. No abnormality in male fertility is detected except for slightly reduced sperm motility, reduced reproductive organ weight. However, there was no histopathological abnormalities in males. In Sprague Dawley (SD) rats, there is maternal body weight decrease/slow growth, decreased food intake, decreased number of live fetuses, increased post-implantation loss, decreased fetal weight, decreased body length and tail length, skeletal development retardation, significant increase in bone and visceral mutations or malformations. In New Zealand rabbits, there is significantly decreased body weight and food intake (even death); miscarriage or premature birth, fewer live fetuses, and increased post-implantation losses; decreased fetal weight; retarded skeletal development, and slight increase in bone and visceral variations or deformities. However, in cynomolgus monkeys, there is maternal body weight loss/slow growth, decreased food intake, normal fetal development, and no obvious deformities. Perinatal developmental toxicity in rats includes reduced weight/slow growth in the F0 generation, reduced food intake, slightly delayed delivery or normal delivery, reduced birth weight in F1 generation, which may not recover until adulthood, some abnormal developmental indicators of rats, and possible abnormal behavioral functions. No abnormality in reproductive ability was found. Based on the identification of the above reproductive toxicity changes, it is believed that they are mainly caused by the pharmacological effects of the test substance, fetal effects secondary to impaired maternal weight gain during pregnancy, and effects that may be directly related to drug exposure.
The 2-year carcinogenicity test in rats showed thyroid C-cell adenomas or cancers, and hyperplasia and the carcinogenicity test in transgenic mice showed thyroid C-cell adenomas or cancers, or no increase in tumors. It is currently believed that the above-mentioned changes are species-specific. GLP-1R is highly expressed on the thyroid gland in rodents and stimulates C cell proliferation. However, the expression of GLP-1R is low on C-cells in humans and monkeys so the correlation of the rodent data with humans is uncertain. A black box warning is included in the instructions for commercially available products. The European Medicines Agency (EMA) announced on October 27, 2023 that, after a comprehensive and in-depth review, its Safety Committee found no conclusive evidence that there is a direct cause-and-effect relationship between Novo Nordisk’s Semaglutide or other GLP-1 receptor agonists and thyroid cance. However, the EMA still recommends continuing long-term safety and effectiveness monitoring of GLP-1 drugs to ensure patient safety.
Immunogenicity Concerns in Preclinical Safety Evaluation of Biologics in Non-human Primates
Luke Zhang, Sucai Zhang, Yanlin Zhang, Yongbin Zhang
JOINN Laboratories (Suzhou) Co., Ltd
No.1 Zhaoxi Road, Biomedical Industrial Park, Taicang, Jiangsu 215421, P. R. China
Background and Purpose: Biological drugs such as monoclonal antibodies (mAbs), bispecific antibodies, and fusion proteins directed towards soluble biological targets make a great improvement in medical therapy of difficult to treat or rare diseases. Non-human Primates (NHPs) have been demonstrated to be a valuable animal species for preclinical safety evaluation of biologics, as they have good predictive value for pharmacodynamics, toxicological impacts, and physiological effects, However, immunogenicity is still a big concern for safety evaluation of biologics conducted in NHPs that can may compromise the predictive value for human adverse effects. This study aims to present specific parameters or methods for immunogenicity recognition and intervention regime in preclinical safety evaluation studies of biological products.
Methods: To explore and assess immunogenicity of biologics in preclinical safety evaluation performed in NHPs, data from 245 preclinical toxicity studies conducted in the past 5 years to evaluate biologics safety were analyzed. A series of parameters including animal information, clinical signs, immunogenicity, and related-laboratory indexes such as clinical pathology, cytokines, circulating immune complex (CIC) and antidrug antibodies (ADAs), as well as medical interventions were analyzed.
Results: The data analysis revealed that biologics safety studies were mostly performed at dose levels of 5 to 300 mg/kg in NHPs with animal ages ranging from 1.2 to 5 years. Immunogenicity was not dose-dependent and/or age-related in the commonly used animal groups for safety evaluation and was not associated with body weight. However, animal response to immunogenicity was dose-related and varied largely based upon the biological targets. Immunological response (anaphylactic reactions) mostly occurred about 2 weeks (>95%) after dose initiation and persisted throughout the dosing duration with certain biologics being adapted after repeated exposure. The anaphylactic reactions were mostly acute and transient abnormal reactions occurred within 30 minutes after the start of dosing with incidences observed between 20% to 100% of animals, once the immunogenic response was triggered. Commonly observed abnormal clinical symptoms included pale cheek and gingiva, asthenia, lethargy, prostration, tachypnea, gasping, mouth breathing, coughing, retching, red nasal discharge, red emesis and red spots on skin, etc. Some animals died rapidly (up to 19.2%) due to acute pulmonary failure or shock, which were induced by drug-ADAs-mediated hypersensitivity and/or cytokine storm. The animal deaths were considered to be biologics-induced hypersensitivity reactions, a phenomenon that does not translation to humans who are exposed to antibodies. When immunogenicity was triggered, biologics-related clinical pathology findings were commonly observed including mild changes in red blood cell (RBC)-related parameters with decreased RBC counts, hemoglobin, hematocrit ranging from 5% to 20% and increased Retic counts, decreased lymphocyte counts ranging from about 10% to 55 %. increases in PT, APTT and FIB (ranged from 7% to 28%). Additionally, clinical chemistry changes of decreased Albumin (ranged from 13% to 27%) and A/G (ranged from 13% to 27%), increased globulin (ranged from 10% to 69%), were observed among the biologics-treated animals. Cytokine detections as a sensitive indicator of immunogenicity were analyzed, although they are not solely attributed to anaphylactic reactions. Cytokine changes which correlated to immunogenicity included increased IL-6, IL-10, and IL-12 p40 levels (ranged from 6% to 52%), as well as decreased complement protein (C3 and C4, ranged from 3% to 32%). Meanwhile, increased total immunoglobulin G, and CIC were detected. Most animals had detectable positive ADAs (ranged from 70% to 100%), suggesting biologics were immunogenic in NHPs, however, the immunogenic reactions in this species are not predictive of similar human risk for humanized biologics like antibodies. Additionally, the results indicated that generation of ADAs might result in decreases in systemic exposure of biologics. Collectively, the above information provide reference for biologics safety evaluation in preclinical toxicity studies in NHPs.
Additionally, the data analyses showed that anti-anaphylactic treatment on dosing day shortly after dose administration significantly reduced symptoms of immunological responses, indicating the anaphylactic reactions of animals were effectively inhibited or alleviated. However, the treatment might not be effective in some cases with severe hypersensitive reactions, especially during late stage (> 3weeks) of dose administration, which mostly required anti-anaphylactic pretreatment on the dosing days.
Conclusion: The results of this review showed that although non-human primates have good predictive value for human pharmacodynamics, toxicological impacts, and physiological effects of biologics, but no evidence indicate that NHPs have superior predictive value for human adverse immunological effects.
Immunogenicity Concerns in Preclinical Safety Evaluation of Biologics in Sprague-Dawley Rats
Sucai Zhang, Yanlin Zhang, Yongbin Zhang, Luke Zhang
JOINN Laboratories (Suzhou) Co., Ltd
No.1 Zhaoxi Road, Biomedical Industrial Park, Taicang, Jiangsu 215421, P. R. China
Background and Purpose: The development of biologics such as monoclonal antibodies (mAbs), bispecific antibodies, fusion proteins etc., have recently contributed significantly to the improvement of medical treatment of various diseases relating to cardiovascular system, respiratory system, cancer, hematopoietic disease, autoimmune disease etc. Animal studies with the Sprague-Dawley (SD) rat is a common and valuable tool for safety evaluation of the biologics. However, immunogenicity is still a concern and/or challenge when a safety evaluation study for biologics is conducted in rats which may compromise the predictive value for human adverse effects. However, rats do have good predictive value for human pharmacodynamics, and toxicological impacts. The purpose of this review was to assess the preclinical data of biologics toxicity studies to investigate immunogenicity of biological products.
Methods: To verify and support this recent assessment of immunogenicity in rats due to biologics exposure, we performed a retrospective evaluation of 18 toxicity rat studies conducted with biologics at the JOINN Laboratories site in Suzhou China during the last 5 years. The main endpoints analyzed included in-life animal information, biological treatments, clinical signs, body weight changes, duration of immunogenicity, and related-laboratory indexes such as clinical pathology, cytokines, and antidrug antibodies (ADAs), as well as medical interventions.
Results: The data analyses revealed that biologics safety studies were mostly performed at dose levels of 0.1 to 200 mg/kg in SD rats with animal ages ranging from 7 to 9 weeks. Immunogenicity was not dose-dependent and/or age-related in commonly used animal groups for safety evaluation. However, animal response to immunogenicity was dose-related and varied largely based upon the biological targets. Immunological response (anaphylactic reactions) mostly occurred about 2-3 weeks (>65%) after dose initiation and persisted throughout the dosing duration with certain biologics being adapted after repeated exposure. The anaphylactic reactions were observed from immediately post-dose through days before next dose with incidences observed between 10% to 75% of animals, once immunogenicity was triggered. Commonly observed abnormal clinical symptoms included pale face and gingiva, decreased body weight, asthenia, hunched back, tachypnea, gasping, mouth breathing, and red nasal discharge, etc. Occasional animal death observed (up to 8.3%) due to acute anaphylactic reactions and/or body condition decline, which were likely induced by drug-ADAs-mediated hypersensitivity and/or cytokine release. When immunogenicity was triggered, biologics-related clinical pathology findings were commonly observed including decreased hemoglobin and hematocrit ranging from 3% to 26%, decreased lymphocyte counts ranging from about 8% to 26 % and increases in PT, APTT and FIB (ranged from 6% to 37%). Additionally, clinical chemistry changes of decreased albumin (ranged from 8.2% to 32%) and total protein (ranged from 8.3% to 18.6%), increased LDH, AST, ALT, ALP and/or CHO (ranged from 18.6% to 138%), were observed among the biologics-treated animals. Cytokine detections as a sensitive indicator of immunogenicity were analyzed, although they are not solely attributed to anaphylactic reactions. Cytokine changes which correlated to immunogenicity included increased IL-6 level (ranged from 2% to 36%). Meanwhile, ADA detected positive in relatively low population (ranged from 8% to 26%), suggesting biologics were immunogenic in SD rats at low levels. However, the immunogenic reactions in this species are not predictive of similar human risk for humanized biologics like antibodies. Additionally, the results indicated that generation of ADAs might result in decreases in systemic exposure of biologics. As anaphylactic reactions observed in rats were mostly delayed post-dose and persisted over time, anti-anaphylactic treatment seems unnecessary or not very meaningful for this type of studies.
Conclusion: The results of this review showed that although SD rats have good predictive value for human pharmacodynamics and toxicological impacts of biologics in safety evaluation, the animal responses of immunogenicity may impact toxicity assessment due to similarity in clinical signs and laboratory indexes of toxicity.
Therapeutic effects of anti-TNF-α monoclonal antibody on cytokine release induced by CAR T-cells targeting CD22
Yang Cao, Sha Li, Yang Chen, Li Sun, Yasuhiko Hirouchi
JOINN Laboratories (Suzhou) Co., Ltd.
Background and Purpose: Although cytokine release syndrome (CRS) is the most common clinical side effect of CAR T-cell therapies, it is rarely seen in preclinical studies using tumor-bearing immune deficient mouse models. When we evaluated the efficacy and safety of a CD22-specific CAR T-cell product in a NOG-Raji mouse model, CAR T-cell associated toxic reactions were found, including transient weight loss and death. In subsequent data analyses, abnormally elevated levels of TNF-α were considered to be associated with such toxic reactions in mice. Treatment with anti-TNF-α monoclonal antibody (Adalimumab) antagonized weight loss and reduced mortality rate in mice without affecting the antitumor effects of CAR-T cells. Our results suggest that elevated peripheral blood TNF-α level may be a future clinical toxicity marker for this CAR T-cell therapy, and TNF-α neutralization therapies maybe the feasible scheme to prevent and treat potential clinical CRS.
Methods: Female, 8-week old NOD-Cg.PrkdcSCID IL-2rgtm1sug/JicCrl (NOG) mice were intravenously inoculated with 1M Burkitt’s lymphoma Raji-Luc-GFP cells per animal under aseptic conditions. Five days after tumor cell inoculation, the tumor-bearing mice were assigned to vehicle group, mock T-cells group (the dose is equal to that in the high dose group), low (0.1e7 cells/animal), middle (0.3e7 cells/animal) and high doses (1e7 cells/animal) of CD22-specific CAR-T cell groups and CD22 CAR T-cells(1e7 cells/animal) in combination with low and high dose of Adalimumab groups (10 and 30 mg/kg). Clinical observations including food consumption and discharge were performed daily and body weight was monitored twice a week. Bioluminescent signals were assessed with a Bruker in vivo imaging system weekly after CAR T-cell administration and blood samples were collected on days 3, 5, 7 after drug administration to measure cytokine levels.
Results: The survival time of vehicle treated mice was less than 2 weeks due to tumor burden, and most of the animals showed rough coat, bowed back, limb weakness, lethargy and other symptoms before death. The symptoms listed above did not occur after treatment with 0.1~1e7 CD22 specific CAR T-cells/animal and body weight increased steadily, but the high dose group showed transient weight loss and even animal death. Serum hIFN-γ levels increased in a dose-dependent manner in the CD22 CAR T-cell treatment groups due to T cell activation. It is worth noting that, when detecting cytokines, we found that serum levels of hTNF-α in the mice treated with high-dose CD22 CAR T-cells were significantly increased, and we speculated that this was the cause of weight loss and death of mice in the high-dose group. To verify this hypothesis, we investigated the efficacy and safety of the anti-TNF-α monoclonal antibody, Adalimumab, in combination with CD22 CAR T-cells in NOG mice. Consistent with the previous study, all mice in the vehicle treated group died within 2 weeks after tumor cell inoculation. Although the survival rate of mice could be improved after administration of CD22 specific CAR T-cells, some mice still died, while no death or abnormal clinical manifestations were observed after treatment of Adalimumab at the dose of 10 and 30 mg/kg. Consistent with previous study, the body weight of mice treated with CD22-specific CAR T-cells continued to decrease within 10 days after treatment, but CD22 specific CAR T-cells combined with any dose of Adalimumab showed no weight loss. The bioluminescence intensity in tumor cells was detected by an in vivo imaging system to evaluate the tumor burden in mice. The bioluminescence intensity decreased significantly after CD22 specific CAR T-cells administration. Importantly, coadministration with Adalimumab did not affect the efficacy of CD22 CAR T-cells and actually induced a more intense and rapid anti-tumor effect. Consistent with previous study, the level of hTNF-α in CD22-specific CAR T-cells treated only group drastically increased to 800-900 pg/mL in D5, but the level of hTNF-α was no higher than 5 pg/mL after 10 or 30 mg/kg Adalimumab administration. In conclusion, anti-TNF-α monoclonal antibody can antagonize the elevated levels of hTNF-α induced by CD22-specific CAR-T cells.
Conclusion: In some CD22-specific CAR T-cell treatments, increase of TNF-α level was the root cause of animal death, and which also suggested that elevated peripheral blood TNF-α level may be a future clinical toxicity marker of this CAR-T cell product. TNF-α neutralization therapies may be a feasible regimen for the prevention and treatment of potential clinical CRS.
Keywords:
Keyword 1: CAR-T cell
Keyword 2: anti-TNF-α monoclonal antibody
Keyword 3: cytokine release
Comparison of Cytokine Release Syndrome induced by Chimeric Antigen Receptor T-Cells in Humanized NOG-EXL mice and NOG mice
Yang Cao, Yang Chen, Jinzhou Yan, Yunxia Sun
JOINN Laboratories (Suzhou) Co., Ltd; Beijing Vital River Laboratory Animal Technology Co., Ltd; JOINN Laboratories (Beijing) Inc., Beijing Key Laboratory for Biological Products Safety Evaluation.
Background and Purpose: T-cell mediated cancer immunotherapies, including chimeric antigen receptor (CAR) T-cell therapies and bispecific antibodies that recruit cytotoxic T-lymphocytes (CTLs) to cancer cells, directly recognize surface antigens of cancer cells regardless of MHC restriction. Despite the remarkable efficacy against malignancies in clinical applications, CAR T-cell therapies are frequently accompanied with severe cytokine release syndrome (CRS), which is one of the drawbacks. We previously reported activation of infused CAR T-cells and regression of tumor burden in tumor bearing NOG mice, in which human cytokine release in serum was subtle. A variety of human immune cells contribute to CRS, so we evaluated CRS in NOG-EXL mice transplanted with human hematopoietic stem cell (HSC) followed by tumor engraftment. In the humanized NOG-EXL model, we found expansions of human T-, B- and myeloid cells, as well as elevated human cytokine levels after CAR T-cell injection in peripheral blood.
Methods: Female NOD-Cg.PrkdcSCID IL-2rgtm1sug/JicCrl (NOG) mice and NOD.Cg-PrkdcSCIDIL2rgtm1SugTg(SV40/HTLV-IL3,CSF2)10-7Jic/JicCrl (NOG-EXL) mice were obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd. Twenty-four hours after irradiation, NOG-EXL mice were intravenously injected with human hematopoietic stem cells (HSCs), and humanized animals with the proportion of hCD45+ cells greater than 25% were used in this study. NOG mice and humanized NOG-EXL mice were inoculated with 1M Raji cells per animal intravenously. Five days later, animals were randomly divided into vehicle and treatment groups, according to the tumor burden. Animals in treatment group were injected with 5E6 CD19-CD22 CAR-T cells per animal. Clinical observation was performed daily. Body weight was monitored twice a week and body temperature was continuously monitored until 7 days after CAR-T cells treatment. Bioluminescence intensity was assessed with a Bruker in vivo imaging system weekly after CAR T-cell administration. Immune cell population was analyzed with flow cytometry (FCM). The levels of human cytokines such as IL-2, IL-4, IL-10, IL-6, TNF-α, IFN-γ were measured with the BD cytometric bead array (CBA) kit.
Results: The FCM results indicated that NOG-EXL mice can support human hematopoietic stem cell (HSC) differentiation into hCD45+ hCD3+ T cells and hCD45+ hCD33+ myeloid cells
at 11 weeks after transplantation and no treatment related toxic reactions such as CRS and ICANS (immune effector cell-associated neurotoxicity syndrome) were observed. In this study, the body weight of humanized NOG-EXL mice treated with vehicle decreased gradually. The body weight of animals in the CD19-CD22 CAR T-cell group decreased gradually before Day 8 then increased gradually from Day 8 to Day 15. Compared with the vehicle group, the body temperature of animals in the CD19-CD22 CAR T-cells group was only slightly increased at 2-8 h after drug administration. In humanized NOG-EXL mice, the growth of Raji cells was relatively slower than that in NOG mice. However, CD19-CD22 CAR T-cells injection could significantly inhibit tumor growth in both NOG mice and humanized NOG-EXL mice models. After CAR T-cell injection, there was significant increase in IL-2 and IFN-γ, slight increase in IL-6 and TNF-α, and decrease in IL-10 level in NOG mice. In humanized NOG-EXL mouse model, the highest IL-6 level in CD19-CD22 CAR T-cells treated group could rise up to 5857.75 pg/mL, and the average IL-6 level was up to1583.06±2123.12 pg/mL at 48 h after CAR-T cell injection, then decreased to 117.96±120.64 pg/mL by Day 7. Humanized NOG-EXL mice had perivascular mononuclear cell infiltration in liver, perivascular mononuclear cell infiltration in lungs, and increased white pulp cellularity in spleen. There was no difference in other histopathological findings between the treatment group and vehicle group, except for the degree of tumor cell infiltration.
Conclusion: In summary, humanized NOG-EXL mice with multiple human immune cells may be a better model to predict the intensity of potential cytokine release syndrome than the currently routinely used animal models.
Keyword 1: Cytokine Release Syndrome
Keyword 2: Humanized NOG-EXL mice
Keyword 3: CAR T-cell
Pharmacodynamic Effects and Toxicity Study of Multispecific Antibodies in a Humanized Mouse Model Bearing Tumors.
Yang Cao, Yang Chen, Li Sun, Yasuhiko Hirouchi.
JOINN Laboratories (Suzhou) Co., Ltd.
Background and Purpose: T-cell engagers (bispecific antibodies) can bind both tumor cells and immune cells and direct immune cell mediated tumor cell killing. However, bispecific antibodies can be easily depleted causing activated T-cells to become unresponsive, shortly after treatment, due to lack of costimulatory molecules. To overcome this challenge, a tri-specific antibody was developed that includes a costimulatory molecule to increase the efficacy of T-cell mediate tumor cell killing. In this study, we evaluated the anti-tumor efficacy and potential toxicity of a CD3/CD20/CD28 antibody and a CD3/CD19 T cell engager (Blinatumomab) on Raji tumor-bearing NPG immune-deficient mice that were transplanted with human hematopoietic stem cells (HSCs), as well as their in vitro effect on activation of human peripheral blood mononuclear cells (PBMCs).
Methods: NPG mice were transplanted with human hematopoietic stem cells (HSCs) by intravenous injection at 4-5 weeks of age after irradiation, and the proportion of CD45+ cells in the peripheral blood of mice was measured 12 weeks after transplantation. Animals with greater than 25% of CD45+ cells were used in the study. For the in vitro cytokine release assay, vehicle, Blinatumomab and multiple doses (low, medium and high) of the tri-specific antibody test article were plated overnight before the addition of PBMCs. The plates were incubated for 48 hours before the supernatants were collected for analysis. The cytokine release assay was performed in solid phase and solution phase.
Results: By Day 28 after treatment, 3/10 and 1/10 animals in the tri-specific antibody middle and high dose groups died, with pale limbs and ears, emaciation and other symptoms observed before death. Combined with lymphocyte population data, it was speculated that the animal death may be related to graft-versus-host disease (GvHD) caused by the high number of lymphocytes in the animals. Due to the proliferation of human lymphocytes in HSC-NPG mice, the tumor volume in vehicle group decreased since Day 10 and tumor growth was significantly inhibited by Blinatumomab and all doses of the tri-specific antibody. During the study, the proportion and absolute number of CD45+CD20+ B cells were decreased after administration of Blinatumomab and the tri-specific antibody while the cytokine levels in each dose group of tri-specific antibody were higher than those in the Blinatumomab group. The results of the in vitro cytokine release assay (solid phase) indicated that the cytokine levels in each dose of the test article were higher than those of Blinatumomab and the results of solution phase assay were similar to those of the solid phase. On Day 28, compared with vehicle group, animals in the Blinatumomab group and tri-specific antibody groups did not show abnormalities in organs except the spleen. Compared with the vehicle group, organ weight and organ-to-brain weight ratio were decreased in the Blinatumomab group, low and middle dose groups of the tri-specific antibody, and increased in the high dose group of tri-specific antibody. Gross observation of the four dead animals from the middle and high dose tri-specific antibody groups did not reveal significant changes, so the cause of death was unclear. In this study, dead animals and animals euthanized on Day 28 were found with pathological changes related to Blinatumomab or tri-specific antibody in spleen by microscopic observation, mainly manifested as decreased lymphocytes in white pulp.
Conclusion: The results indicated that both the tri-specific antibody and Blinatumomab could induce T-cell activation, significantly inhibit tumor growth, induce cytokine release, deplete T-cells, and induce target-related histopathological changes in vivo. Since CD28 costimulatory factor is added to the tri-specific antibody molecule, its killing effect on tumor cells may be higher than that of Blinatumomab, but the risk of causing cytokine release may also be increased.
Keyword 1: Pharmacodynamic Effect/Toxicity Study
Keyword 2: T-cell engagers
Keyword 3: Humanized Immune-deficient Mouse Model
In vivo Imaging and Quantification of Retinal and Choroidal Vasculature in Cynomolgus Monkeys using Optical Coherence Tomography Angiography
Jin Cai, Qin Liao, Yanlin Zhang, Sucai Zhang, Yongbin Zhang, Wankun Xie
JOINN Laboratories (Suzhou) Co., Ltd.
Abstract will be posted soon