Preclinical safety assessment of toxicity and biodistribution of oncolytic virus HSV-1 expressing human PD-1 antibody in mice.

HSV-1/hPD-1 is composed of engineered herpes simplex virus type-1 and two inserted copies of the human PD-1 antibody sequence. It is a novel oncolytic virus product designed to cure malignancies. The objective of this study was to estimate its toxicity in mice. In the single-dose toxicity study, no mortality and abnormal symptoms were observed in animals injected with 4.0 × 107 pfu/mouse dose. In the repeat-dose toxicity study, HSV-1/hPD-1 in animals intramuscularly treated with 1.0 × 107, 2.0 × 107, or 4.0 × 107 pfu/mouse doses was well tolerated in terms of clinical observation, body weight, food consumption, hematology and biochemistry indexes, T lymphocyte counting, immune reaction, and organ weight, except for some histopathological changes, such as the irreversible degeneration of the sciatic nerve, which was considered related to the adopted administration route. Synchronously, a biodistribution study in mice was performed to examine whether HSV-1/hPD-1 could spread to the injection site, gonads, liver, lung, heart, mesenteric and inguinal lymph nodes, skin, dorsal root ganglia, and blood, and then be gradually eliminated. Thus, two safety dose levels-the maximum tolerance dose of 4.0 × 107 pfu/mouse and the no-observed-adverse-effect-level dose of 1.0 × 107 pfu/mouse-were determined to help design patients' dose regimens. Our research data have been successfully accepted for investigational new drug (IND) application in China.

Regulatory landscape of dietary supplements and herbal medicines from a global perspective.

The number of Individuals that use dietary supplements and herbal medicine products are continuous to increase in many countries. The context of usage of a dietary supplement varies widely from country-to-country; in some countries supplement use is just limited to general health and well-being while others permit use for medicinal purposes. To date, there is little consensus from country to country on the scope, requirements, definition, or even the terminology in which dietary supplement and herbal medicines categories could be classified. Transparent science-based quality standards for the ingredients across these regulatory frameworks/definitions becomes even more important given the international supply chain. Meanwhile, there has been a rapid advancement in emerging technologies and data science applied to the field. This review was conceived at the Global Summit on Regulatory Sciences that took place in Beijing on September 2018 (GSRS2018) which is organized by Global Coalition for Regulatory Science Research (GCRSR) that consists of the global regulatory agencies from over ten countries including the European Union. This review summarizes a significant portion of discussions relating to a longitudinal comparison of the status for dietary supplements and herbal medicines among the different national jurisdictions and to the extent of how new tools and methodologies can improve the regulatory application.

Quality, bioactivity study, and preclinical acute toxicity, safety pharmacology evaluation of PEGylated recombinant human endostatin (M2 ES).

Endostar, a potent endogenous antiangiogenic factor, is wildly used in clinics. However, it was easily degraded by enzymes and rapidly cleared by the kidneys. To overcome these shortcomings, PEGylated recombinant human endostatin was developed. In this study, the purity of M2 ES was evaluated by silver stain and reversed-phase high-performance liquid chromatography. Ultraviolet spectrum was used to examine the structural of M2 ES and endostar. The bioactivity and antitumor efficacy of M2 ES were evaluated using an in vitro endothelial cell migration model and athymic nude mouse xenograft model of a heterogeneous lung adenocarcinoma, respectively. A preclinical study was performed to evaluate the acute toxicity and safety pharmacology in rhesus monkeys. The purity of M2 ES was more than 98%; PEG modification has no effect on endostatin structure. Compared with the control group, M2 ES dramatically retards endothelial cell migration and tumor growth. After intravenous (IV) infusions of M2 ES at a dose level of three and 75 mg/kg in rhesus monkeys, there was no observable serious adverse event in both acute toxicity and safety pharmacology study. On the basis of the quality and bioactivity study data of M2 ES and the absence of serious side effect in rhesus monkeys, M2 ES was authorized to initiate a phase I clinical trial.

A case report of spontaneous staphylococcal meningitis in a cynomolgus monkey.

This report describes a suppurative meningitis in a young cynomolgus. The animal had neutrophil aggregation in the subarachnoid space and hemorrhage in bilateral adrenal glands. Staphylococcus was identified by FISH in brain. To our knowledge, this is the first case of staphylococcal meningitis with Waterhouse-Friderichsen syndrome in a cynomolgus monkey.

A pre-clinical safety study of PEGylated recombinant human endostatin (M2ES) in Sprague Dawley rats.

PEGylated recombinant human endostatin (M2ES) exhibited prolonged serum half-life and enhanced antitumor activity when compared with endostatin. A pre-clinical study was performed to evaluate the safety of M2ES in rats. After intravenous (IV) infusions of M2ES at a dose level of 3, 15 and 75 mg/kg in Sprague Dawley (SD) rats, M2ES was well tolerated in animals, with no observable changes in clinical observation, body weight, food consumption, urine analysis, hematology and serum biochemical analysis. The increase of kidney weights, and slight to severe vacuolation and necrosis of proximal tubule epithelial cells in kidney were observed in 15 and 75 mg/kg M2ES groups, but this adverse-effect was reversible. In summary, the major toxicity target organ of M2ES might be kidney, and the no observed adverse effect level (NOAEL) of M2ES in rats was 3 mg/kg in this study. These pre-clinical safety data contribute to the initiation of the ongoing clinical study.

The HepaRG cell line, a superior in vitro model to L-02, HepG2 and hiHeps cell lines for assessing drug-induced liver injury.

Drug-induced liver injury (DILI) is a leading cause of discontinuation of new drug approval or withdrawal of marketed medicine based on safety due to organ vulnerability. The aim of this research is to investigate the potential abilities of four different in vitro cell models (L-02, HepG2, HepaRG, and hiHeps cell lines) in assessing marketed drugs labeled with apparently different types of liver injury. A total of 17 drugs with versatile pharmacological profiles were chosen, of which, 14 drugs are recognized as DILI agents and 3 drugs are DILI irrelevant. Preliminary cellular screening assays indicated that the HepaRG cell line had an advantage over other cell lines in predicting drugs associated with DILI in vitro as it had the highest Youden's index (71.4%). A multi-parametric screening assay showed that oxidative stress, mitochondrial damage, and disorders of neutral lipid metabolism were changed notably in the HepaRG cell line after DILI-related drugs exposure, accounting for its high sensitivity in comparison with other three cell lines. In addition, aspartate aminotransferase (AST), lactate dehydrogenase (LDH), and malate dehydrogenase (MDH) all correlated with the cytotoxic effects of diclofenac sodium (p < 0.05), buspirone hydrochloride (p < 0.01), and danazol (p < 0.01) in the HepaRG cell line. We conclude that the HepaRG cell line is a superior in vitro cell model to other three cell lines for evaluating drugs with DILI potential.

Polygonumnolides C1-C4; minor dianthrone glycosides from the roots of Polygonum multiflorum Thunb.

Four new dianthrone glycosides, named polygonumnolides C1-C4 (1-4), were isolated from the dried roots of Polygonum multiflorum Thunb, together with two known emodin dianthrones (5-6). Their hepatotoxicities were evaluated against L-02 cell lines. Compounds 1-4 showed weak hepatotoxicity against L-02 cell lines with IC50 values of 313.05, 205.20, 294.20, and 207.35 µM, respectively.

Hep-2 cell based indirect immunofluorescence assay for antinuclear antibodies as a potential diagnosis of drug-induced autoimmunity in nonclinical toxicity testing.

Antinuclear antibodies (ANAs) are important biomarkers in the diagnosis of autoimmune diseases in humans; however, the diagnostic performance of ANA in nonclinical safety studies are not well understood. Here, we studied the use of ANAs as potential nonclinical biomarkers for drug-induced autoimmunity (DIA) using a Hep-2 based indirect immunofluorescence assay (IFA). Initially, MRL-fas(lpr)/J mice and HgCl2-treated rats were used as SLE-positive models. Serum samples obtained from 94 normal mice or 204 normal rats aged one to four months served as the negative control. The IFA effectively distinguished ANAs-positive samples in both species with a cut-off titer of 1:100. Brown Norway rats were treated with 450 mg/kg D-penicillamine for 30 consecutive days. ANAs were generated and corresponded with DIA development. Human Hep-2 cells, mice Neuro 2A cells, and Chinese Hamster Lung cells served as antigen from different species, which were found cross-reactive with ANA-positive serum samples from mice, rats, and humans without any differences in diagnosis. This methodology showed no species-specificity for ANA detection. Furthermore, we found approximately 20 percentage of the mice aged seven to eight months demonstrated age-related ANAs, which was consistent with humans. Overall, our findings demonstrated the use of ANA detection using IFA in the nonclinical diagnosis of murine drug-induced autoimmunity, and age-related ANAs should be considered when aged animals are used.

High daily dose and being a substrate of cytochrome P450 enzymes are two important predictors of drug-induced liver injury.

Drug-induced liver injury (DILI) is complicated and difficult to predict. It has been observed that drugs with extensive hepatic metabolism have a higher likelihood of causing DILI. Cytochrome P450 (P450) enzymes are primarily involved in hepatic metabolism. Identifying the associations of DILI with drugs that are P450 substrates, inhibitors, or inducers will be extremely helpful to clinicians during the decision-making process of caring for a patient suspected of having DILI. We collected metabolism data on P450 enzymes for 254 orally administered drugs in the Liver Toxicity Knowledge Base Benchmark Dataset with a known daily dose, and applied logistic regression to identify these associations. We revealed that drugs that are substrates of P450 enzymes have a higher likelihood of causing DILI [odds ratio (OR), 3.99; 95% confidence interval (95% CI), 2.07-7.67; P < 0.0001], which is dose-independent, and drugs that are P450 inhibitors have a higher likelihood of generating DILI only when they are administered at high daily doses (OR, 6.03; 95% CI, 1.32-27.5; P = 0.0098). However, drugs that are P450 inducers are not observed to be associated with DILI (OR, 1.55; 95% CI, 0.65-3.68; P = 0.3246). Our findings will be useful in identifying the suspected medication as a cause of liver injury in clinical settings.

Pre-clinical toxicokinetics and safety study of M2ES, a PEGylated recombinant human endostatin, in rhesus monkeys.

PEGylated recombinant human endostatin (M2ES) exhibited prolonged serum half-life and enhanced antitumor activity when compared with endostatin. A non-clinical study was performed to evaluate the toxicokinetics and safety of M2ES in rhesus monkeys. After intravenous (IV) infusions of M2ES at a dose level of 3, 10, and 30mg/kg in rhesus monkeys, the concentration-time curves of M2ES were best fitted to a non-compartment model, and area under the curve (AUC) was positively correlated with the dosage. M2ES had a tendency to accumulate in vivo following successive IV infusions. Serum anti-M2ES IgG antibodies were generated quickly during IV administration, and the antibody level in serum did not significantly decrease after four-week recovery period. Animals administered IV infusions twice weekly (M2ES at 10 or 30mg/kg body weight per day) for 3months developed mild or moderate vacuolation of proximal tubule epithelial cell in proximal convoluted tubule of kidney, but this adverse-effect was reversible. In summary, M2ES was well tolerated and did not cause any serious toxicity. These pre-clinical safety data contribute to the initiation of the ongoing clinical study.

A 3D spheroid model of quadruple cell co-culture with improved liver functions for hepatotoxicity prediction.

Drug-induced liver injury (DILI) is one of the major concerns during drug development. Wide acceptance of the 3 R principles and the innovation of in-vitro techniques have introduced various novel model options, among which the three-dimensional (3D) cell spheroid cultures have shown a promising prospect in DILI prediction. The present study developed a 3D quadruple cell co-culture liver spheroid model for DILI prediction via self-assembly. Induction by phorbol 12-myristate 13-acetate at the concentration of 15.42 ng/mL for 48 hours with a following 24-hour rest period was used for THP-1 cell differentiation, resulting in credible macrophagic phenotypes. HepG2 cells, PUMC-HUVEC-T1 cells, THP-1-originated macrophages, and human hepatic stellate cells were selected as the components, which exhibited adaptability in the designated spheroid culture conditions. Following establishment, the characterization demonstrated the competence of the model in long-term stability reflected by the maintenance of morphology, viability, cellular integration, and cell-cell junctions for at least six days, as well as the reliable liver-specific functions including superior albumin and urea secretion, improved drug metabolic enzyme expression and CYP3A4 activity, and the expression of MRP2, BSEP, and P-GP accompanied by the bile acid efflux transport function. In the comparative testing using 22 DILI-positive and 5 DILI-negative compounds among the novel 3D co-culture model, 3D HepG2 spheroids, and 2D HepG2 monolayers, the 3D culture method significantly enhanced the model sensitivity to compound cytotoxicity compared to the 2D form. The novel co-culture liver spheroid model exhibited higher overall predictive power with margin of safety as the classifying tool. In addition, the non-parenchymal cell components could amplify the toxicity of isoniazid in the 3D model, suggesting their potential mediating role in immune-mediated toxicity. The proof-of-concept experiments demonstrated the capability of the model in replicating drug-induced lipid dysregulation, bile acid efflux inhibition, and α-SMA upregulation, which are the key features of liver steatosis and phospholipidosis, cholestasis, and fibrosis, respectively. Overall, the novel 3D quadruple cell co-culture spheroid model is a reliable and readily available option for DILI prediction.

Genotoxicity assessments of N-nitrosoethylisopropylamine (NEIPA) and N-nitrosodiisopropylamine (NDIPA) in the C57BL/6J mouse.

N-Nitrosamines, known as drug impurities and suspected carcinogens, have drawn significant public concern. In response to drug regulatory needs, the European Medicines Agency (EMA) has previously proposed a carcinogenic potency categorization approach based on the N-nitrosamine α-hydroxylation hypothesis, i.e., that N-nitrosamine mutagenicity increases with the number of α-hydrogen atoms. However, this structure-activity relationship has not been fully tested in vivo. NEIPA (N-nitrosoethylisopropylamine) and NDIPA (N-nitrosodiisopropylamine) are small N-Nitrosamines with similar structures, differing in that the former compound has an additional α-hydrogen atom. In this study, NEIPA and NEIPA doses, 25-100 mg/kg, were administered orally to C57BL/6 J mice for seven consecutive days, and their mutation and DNA damage effects were compared. Compared with NDIPA, the mutagenicity and DNA damage potencies of NEIPA (which contains one more α-hydrogen) were much greater. These differences may be related to their distinct metabolic pathways and target organs. This case study confirms the role of α-hydroxyl modification in the mutagenicity of nitrosamines, with oxidation at the α-hydrogen being a crucial step in the formation of mutagens from N-Nitrosamines, and can inform mutagenicity risk assessment and the formulation of regulatory standards for N-nitrosamine impurities.

Bio-distribution and toxicity potential of human umbilical cord mesenchymal stem cells in cynomolgus monkeys.

Mesenchymal stem cells (MSCs) have demonstrated promising advantages in the therapies of many diseases, while its multi-directional differentiation potential and immunotoxicity are the major concerns hindered their clinical translation. In this study, human umbilical Mesenchymal stem cell (hUC-MSCs) were labeled with a near-infrared fluorescent dye DiR before infused into cynomolgus monkeys, and the amount of hUC-MSCs in the peripheral blood were dynamically estimated from 5 min to 28 days post a single administration at 3 × 106 cells/kg and 2 × 107 cells/kg intravenously. As results, some hUC-MSCs distributed to the whole body within 5 min, while most of the cells accumulate in the lungs along with the systemic blood circulation, and subsequently released into the blood. The toxicity potentials of hUC-MSCs were investigated in another 30 cynomolgus monkeys, and the cells were repeatedly administrated at doses of 3 × 106 cells/kg and 2 × 107 cells/kg for 5 times on a weekly basis, with a recovery period of 1 months. hUC-MSCs showed no obvious toxic effects in cynomolgus monkeys, except xenogeneic immune rejection to human stem cells. Low levels of the hUC-MSC gene were detected in the peripheral blood of a few animals administered 2 × 107 cells/kg at 30 min subsequent to the first and last administration, and there was no significant difference in the copy number of the hUC-MSC gene in the blood samples compared with the first and last administration, indicating that the hUC-MSC was not significantly amplified in vivo, and it its safe in non-human primates. Our study for the first time verified the safety of long-term use of hUC-MSCs in primates. We have pioneered a technology for the real-time detection of hUC-MSCs in peripheral blood and provide dynamicand rapid monitoring of the distribution characteristics of hUC-MSCs in vivo. Here, we provide data supporting the application of such products for clinical treatment and the application of stem cells in major refractory diseases and regenerative medicine.

Safety evaluation of PEGylated MNPs and p-PEGylated MNPs in SD rats.

Polyethylene glycol-coated magnetic nanoparticles (PEGylated MNPs) have demonstrated prominent advantages in cancer diagnosis and hyperthermia therapy. However, there is currently lack of standard mode and sufficient toxicity data for determining the delayed risk of PEGylated MNPs. Nevertheless, the toxicity potentials, especially those associated with the oxidative stress, were ubiquitously reported. In this study, PEGylated MNPs and p-PEGylated MNPs were administrated to SD (Sprague Dawley) rats by single intravenously injection, and various toxicity indicators were monitored till 56 days post-administration for a comprehensive toxicity evaluation. We revealed that both nanoparticles could be rapidly cleared from plasma and enter tissues, such as, liver, kidneys and spleen, and p-PEGylated MNP is less prone to be accumulated in the tissues, indicating a lower toxicity risk. PEGylated MNPs were more likely to up-regulate the expression levels of Th2 type cytokines and trigger inflammatory pathways, but no related pathological change was found. Both MNPs are not mutagenic, while recoverable mild DNA damage associated with the presence of nanoparticles might also be observed. This study demonstrated a research approach for the non-clinical safety evaluation of nanoparticles. It also provided comprehensive valuable safety data for PEGylated and p-PEGylated MNPs, for promoting the clinical application and bio-medical translation of such MNPs with PEG modifications in the cancer diagnosis and therapy.

Tocilizumab treatment in neuromyelitis optica spectrum disorders: Updated meta-analysis of efficacy and safety.

This systematic review and meta-analysis summarize the efficacy and safety of Tocilizumab (TCZ) in treating NMOSD and investigates the factors that affect its efficacy. TCZ is the first monoclonal antibody against the IL-6 receptor for treating NMOSD, and its efficacy and safety vary in different studies. We collected English-language research literature until January 1, 2023, by searching databases such as PubMed, MEDLINE, Embase, Cochrane Library, and clinicaltrials.gov, and identified 9 studies involving 153 patients (139 female and 14 male) that met our inclusion criteria. In these studies, the average ARR ratio and EDSS score reduction values in the TCZ treatment group were -1.34 (95 % CI, -1.60 to -1.09) and -0.81 (95 % CI, -1.04 to -0.58), respectively. Based on the data we have collected, compared to the AQP4-IgG negative NMOSD patients, TCZ demonstrates a more pronounced effectiveness in AQP4-IgG positive NMOSD patients. The study also found that the effectiveness of TCZ in reducing NMOSD patients' ARR ratio was related to gender, race, and TCZ dosage, while the effectiveness of reducing EDSS score was not related to these factors. Among the 153 patients receiving TCZ treatment, 101 (66 %) experienced mild adverse reactions, and one patient experienced a severe adverse reaction (facial cellulitis). The comprehensive data indicate that TCZ treatment can reduce the frequency of NMOSD relapses, improve patients' neurological function, and have good safety. The effectiveness of TCZ in reducing NMOSD patients' ARR ratio is related to multiple factors.

Targeting chemoattractant chemokine (C-C motif) ligand 2 derived from astrocytes is a promising therapeutic approach in the treatment of neuromyelitis optica spectrum disorders.

Introduction: Aquaporin-4 immunoglobulin G (AQP4-IgG)-induced astrocytes injury is a key mechanism in the pathogenesis of neuromyelitis spectrum disorder (NMOSD), and although CCL2 is involved, its specific role has not been reported. We aimed to further investigate the role and potential mechanisms of CCL2 in AQP4-IgG-induced astrocyte injury. Methods: First, we evaluated CCL2 levels in paired samples of subject patients by automated microfluidic platform, Ella®. Second, we knock down astrocyte's CCL2 gene in vitro and in vivo to define the function of CCL2 in AQP4-IgG-induced astrocyte injury. Third, astrocyte injury and brain injury in live mice were assessed by immunofluorescence staining and 7.0T MRI, respectively. Western blotting and high-content screening were conducted to clarify the activation of inflammatory signaling pathways, and changes in CCL2 mRNA and cytokine/chemokines were measured by qPCR technique and flow cytometry, respectively. Results: There were greatly higher CSF-CCL2 levels in NMOSD patients than that in other non-inflammatory neurological diseases (OND) groups. Blocking astrocyte CCL2 gene expression can efficiently mitigate AQP4-IgG-induced damage in vitro and in vivo. Interestingly, prevention of CCL2 expression could decrease other inflammatory cytokines released, including IL-6 and IL-1ß. Our data suggest that CCL2 involves in the initiation and plays a pivotal role in AQP4-IgG-damaged astrocytes. Discussion: Our results indicate that CCL2 may serve as a promising candidate target for inflammatory disorder therapy, including NMOSD.

Corn oil as a vehicle in drug development exerts a dose-dependent effect on gene expression profiles in rat thymus.

The purpose of this study was to investigate the effects of corn oil (CO), which is widely used as a vehicle for water-insoluble agents in drug development, on the gene expression profiles in rat thymus with microarray technique. Female Wistar rats were administered daily with normal saline (NS) and CO 2, 5 and 10 ml kg⁻¹ per day for 14 days, respectively. Then, the thymus samples of rats were collected for microarray test and histopathology examination. CD4⁺ and CD8⁺ lymphocytes in peripheral blood were also numerated to assess the effects on lymphocyte subpopulations. The microarray data showed that the numbers of differentially expressed genes in the 2, 5 and 10 ml kg⁻¹ CO groups were 0, 40 and 458, respectively, compared with the NS control group. The altered genes were mainly associated with immune response, cellular response to organic cyclic substance and regulation of fatty acid ß-oxidation. However, no abnormal changes in thymus weight, CD4⁺ and CD8⁺ lymphocytes counts and histopathological examination were observed in the three CO groups. These data showed that 10 ml kg⁻¹ CO, the usually recommended dosing volume as a vehicle in drug safety assessment, caused obvious dysregulated genes in rat thymus. Our study suggests that the appropriate dosing volume of CO gavage as a vehicle for water-insoluble agents in drug development should be 2 ml kg⁻¹ per day, if agent effects on thymus will be assessed in gene levels.

Market entry system considering the biosafety of nanomedical devices in China.

Our current knowledge on nanomaterials is mostly built on data from basic studies, and the application and developmental potentials of nanomaterials are emphasized. On the other hand, standard evaluation methods, models, exposure methods, standards, and guidelines for biological effect evaluation are inadequate. In response to the bottlenecks of supervision, scientific research institutes and regulatory organizations in China have cooperated closely to research and establish an evaluation system for nanomedical devices, and silver-containing nanomaterials have been adopted as an example. In such a context, reference materials, characterization strategies, in vitro and in vivo distribution and toxicity evaluation standards have been established. This article highlights research on the risk assessment of nanomedical device products (taking silver-containing nanomedical device products as an example) in China, including the characterization and release determination strategies, determination of nanosilver in tissues, applications of three-dimensional skin models and in vitro and toxicity evaluation standards have been established. This article highlights research on technical standards. As a consequence, the "Guidelines for the safety and effectiveness evaluation of nanomedical devices" were published in 2021, and a market entry framework for nanomedical devices has been preliminarily formed as a significant component in scientific supervision. This Guideline supervises the review and supervision of nanomedical devices and, therefore, provides a guarantee for the market access of nanomedical devices in China. This article is categorized under: Toxicology and Regulatory Issues in Nanomedicine > Toxicology of Nanomaterials Toxicology and Regulatory Issues in Nanomedicine > Regulatory and Policy Issues in Nanomedicine.

Pre-clinical efficacy of CD20-targeted chimeric antigen receptor T cells for non-Hodgkin's lymphoma.

BACKGROUND: A 4-1BB/CD3-ζ-costimulated CAR-T against CD20 (CAR-T20) was subjected to a systemic efficacy evaluation in a cell co-culture model, and NOD-SCID IL-2 receptor gamma null mice (short for NSG mice) were xenografted with human Burkitt's lymphoma Raji cells. METHODS: CAR-T20 cells were incubated with target cells (K562, K562 CD20 or Raji cells) at ratios of 10:1 and 5:1 for 24 h, and the killing rate was estimated by an LDH cytotoxicity assay. To evaluate the effect of CAR-T20 on the survival time of tumor-bearing animals, 30 NSG mice were employed, and Raji-Luc cells (5 × 105 cells per mouse) were administered prior to CAR-T20 administration. The survival time, optical intensity of Raji-Luc cells, clinical symptoms, and body mass of the animals were observed. Another 144 male NSG mice were employed to investigate the proliferation and antitumor effects of CAR-T20. Human cytokine and murine cytokines were detected at 1, 7, 14, 21, 28, 42, 56 and 90 days post-CAR-T administration, while biochemistry index analysis, T-cell and CAR-T-cell detection in peripheral blood, and histopathological examination were performed at 14, 28, 56 and 90 days post-administration. RESULTS: CAR-T20 cells had a specific killing effect on CD20-expressing cells in vitro. At a dose of 1 × 106 per mouse or above, CAR-T20 prolonged the median survival time from 14 days to more than 3 months, inhibited the proliferation of Raji cells in mice, and alleviated the clinical manifestations and weight loss caused by the Raji-Luc cell load. CAR-T20 at a dose of 2 × 106 per mouse or above inhibited the proliferation of Raji cells in mice for up to 111 days post-administration without recurrence. The numbers of T cells and CAR-T cells in the animals administered CAR-T20 increased significantly when Raji cells were markedly proliferated and subsequently decreased when Raji cells were predominantly inhibited. CAR-T20 increased human IFN-γ, murine TNF and murine IL-6 levels and decreased human IL-10 levels in tumor-bearing mice. The incidences of xenografted tumors in organs/tissues were also reduced effectively by CAR-T20. CONCLUSION: The effective dose of CAR-T20 in mice starts from 1 × 106 per mouse, equivalent to a clinical dose of 5 × 106/kg. Together, our data support the clinical translation of CAR-T20 for R/R B-cell NHL patients.

Functional differences and similarities in activated peripheral blood mononuclear cells by lipopolysaccharide or phytohemagglutinin stimulation between human and cynomolgus monkeys.

BACKGROUND: The monkey is a primary species used in toxicological research. However, the failures of preclinical studies to predict a life-threatening "cytokine storm", which, for instance, rapidly occurred in six healthy volunteers with the CD28 superagonist monoclonal antibody (mAb) TGN1412 in the first-in-human phase I clinical trial, have emphasized a need to clarify the differences between human and monkey immune systems. METHODS: In the present study, we analyzed and compared the lymphocyte proliferation, cytokine secretion, and gene expression profiles after phytohemagglutinin (PHA) and lipopolysaccharide (LPS) stimulation of peripheral blood mononuclear cells (PBMCs) from three healthy humans and cynomolgus monkeys (Macaca fascicularis). RESULTS: The results derived from comparison with the corresponding control groups showed that PHA in humans induced a stronger proliferation and wider range of cytokine secretion, along with a greater number of differently expressed genes (DEGs), than when PHA was applied in cynomolgus monkeys. The significant upregulation of genes involved in the mitotic cell cycle, including cyclin B2, TOP2A, TYMS, and CEP55, was observed in human PBMCs with PHA stimulation, while only infrequent or slight upregulation occurred in cynomolgus monkey PBMCs, which may be one of the reasons for a stronger response to PHA in humans. In contrast to PHA, LPS in both species induced a similar proliferation ratio, cytokine profile, and DEG count, suggesting that human and cynomolgus monkeys have a similar response intensity for innate immune responses. Furthermore, 38 and 20 overlapped genes under PHA and LPS stimulation, respectively, were found in both species. These overlapped DEGs were associated with the same biological functions, including DNA replication, mitosis, immune response, chemotaxis, and inflammatory response. Thus, these results might reflect the highly conserved signatures of immune responses to PHA/LPS stimulation across the primates. Moreover, there were some differences in antigen processing and presentation, and the interferon gamma (INF-γ)-mediated signaling pathway in these species detected by gene expression profile study. CONCLUSIONS: In conclusion, this is the first study to compare data on the responses of PBMCs to PHA and LPS in humans versus cynomolgus monkeys, and these findings may provide crucial insights into translating non-human primate (NHP) studies into human trials.