啮齿动物,非人灵长类动物 z-bo 2021-11-01 389

　　Introduction: Fibroblast growth factor receptor 1 (FGFR1) belongs to the cell surface FGF receptor family expressed by a certain number of normal tissues, but it is highly amplified and overexpressed in some solid tumors (such as lung cancer and breast cancer). In the process of developing bivalent anti-FGFR1 antibodies, both rodents and monkeys have been reported to have effective but reversible phagocytosis and weight loss. FGFR1 signals FGFR1/β-Klotho in the nervous system through FGF19/21, and plays a role in the regulation of body weight and blood sugar, which may explain these clinical effects. Buss reported that two FGFR1c selective monoclonal antibodies (mAbs) and a FGFR1c/FGFR4 bispecific monoclonal antibody not only induced weight loss in rats, but also further induced morphological changes in bone and heart valves. In bones, the increase in the thickness of the cortical bone in the long bone backbone is probably mediated by the down-regulation of FGFR1c-mediated osteoblast function. In heart valves, the valvular disease of extracellular matrix and mesenchymal cell proliferation is considered to be FGFR1c mediated. Based on the specificity of monoclonal antibodies and the expression of fgfr1mrna in heart valves, this is also considered an agonistic effect. The bivalent nature of anti-FGFR1c mAb can lead to dimerization and agonism rather than antagonism. M6123 has high affinity and selectivity for FGFR1, but has no affinity and selectivity for other FGFR family members such as FGFR2, 3, and 4. Early data from the original A08 bivalent FGFR1 antibody from a set of overexpressed receptors confirmed the lack of cross-reactivity with any other receptor. This also indicates that M6123 is unlikely to interfere with other tyrosine kinase receptors. The binding of M6123 to FGFR1 will block receptor dimerization and ligand-mediated activation, and subsequently inhibit downstream signal transduction, such as receptor autophosphorylation and ERK1/2 activation. This signal inhibition results in the inhibition of FGFR1-dependent tumor growth. The monovalent form of M6123 retains a strong FGFR1 inhibitory effect in human tumor cells grown in a mouse xenograft model, and is well tolerated with no signs of weight loss. In a 4-week repeated dose toxicity study, weekly intravenous infusion was used to evaluate the preclinical safety of M6123 in mice, rats and monkeys to support potential clinical trials. Mice, rats and cynomolgus monkeys have been identified as relevant species for M6123 preclinical toxicity studies. "Mice, rats and cynomolgus monkeys were selected as related species for preclinical toxicity studies of M6123. The sequence and function of FGFR1 are very conserved among these species. In addition, in in vitro pharmacological studies using cell lines from human and animal species, M6123 showed comparable "targeting" activity. Since different toxicity profiles were observed in mice and rats during the relevant dose range study, two rodents were used. The cynomolgus monkey is considered a related non-rodent species and is a suitable model for measuring the effects of M6123 in relation to ADCC, as this species shows effector activity similar to that of humans. In contrast, only limited ADCC activity may originate from effector cells in mice.

　　Mice experimental study design: A 4-week repeated dose key toxicity study was conducted in mice. During the entire study, the temperature in the animal room was controlled between 20 and 24°C, the relative humidity was between 30 and 70%, and the number of air changes was between 15 and 20 times/h. With 0 (vehicle group), 50, 100 or 200 mg/kg M6123 dose group (n = 16/sex/group), intravenous infusion, once a week for 4 consecutive weeks, the remaining 6 animals in each group are observed for 4 weeks during the recovery period . The dose is selected according to the dose exploration study on mice. Based on the protein concentration of the test product in this batch and the maximum dose of intravenous injection, a high dose of 200 mg/kg is considered the maximum feasible dose. All animals were infused caudally by using an electric infusion pump at a rate of approximately 0.6 ml/min and an administration volume of 20 ml/kg. Based on daily clinical observation, weekly recording of body weight and food intake, eye examination, clinicopathological investigation (including hematology, clinical chemistry and immunophenotype), flow cytometry is used to determine absolute and relative peripheral blood lymphocyte subsets Number of life assessments. A standard urinalysis test was performed, and urine calcium (Ca++) and inorganic phosphorus (Pi) levels were measured at the same time. Mice collected TK samples at 2, 24, 48, 96, and 168 hours after administration on the 1st and 22nd days for a study to clarify the changes in serum calcium (Ca++) and inorganic phosphorus (Pi) levels. Anatomy and pathology: On the 30th day or at the end of the recovery period, mice were sacrificed by intraperitoneal injection of excessive sodium pentobarbital and autopsy was performed. After visual inspection, the standard tissue is weighed and histopathological examination is performed. After completing the corresponding blood sampling, overdose of isoflurane was used to kill the satellite group animals for toxicokinetics (TK) and immunophenotype study. Rat study design: The safety of M6123 was evaluated in two 4-week repeated dose toxicity studies in rats: rats (for DRF study, n=5/sex/group, in the 4-week In the follow-up study, n = 10/male/group) M6123 was intravenously infused at a dose of 0 (vehicle), 50, 100, or 200 mg/kg once a week for 4 weeks (DRF study) or 0 (vehicle), 10, 25 or 50mg/kg intravenous infusion of M6123 for 4 weeks (follow-up study). In the follow-up study, only males were used because males were found to be more sensitive to the toxic effects of M6123 than females. Based on the protein concentration of the test product in this batch and the maximum dose for intravenous application, a high dose of 200 mg/kg is considered the maximum feasible dose. All animals were infused through the lateral tail vein at a rate of about 20ml/kg/15min and a volume of 10ml/kg through an infusion pump. Based on daily clinical observation, weight and food intake, eye examination and clinical pathological examination were recorded weekly for evaluation. In the DRF study, hematology, coagulation and serum chemistry analyses were performed approximately 24 hours after the last dose on day 30. On the same day, overnight urine samples of rats in each group were collected and routine urinalysis was performed. TK samples were collected at different time points, and the serum Ca ++ and Pi concentrations were further analyzed. The rats were sacrificed with an overdose of sodium pentobarbital and an autopsy was performed. For DRF studies, a final autopsy was performed 24h after the last dose on day 30; for follow-up studies, a final autopsy was performed 24h after the first, second, or last (fifth) dose. After visual inspection, the standard tissue is weighed and histopathological examination is performed. After completing the corresponding blood sampling, the toxic generation group animals were sacrificed with an overdose of isoflurane.

　　Monkey trial study design: The safety of M6123 was evaluated in a 4-week repeated dose toxicity study in monkeys. Four groups of monkeys (n=5/sex/group), with doses of 0 (vehicle), 50, 100, or 150 mg/kg, intravenously injected M6123 once a week for 4 consecutive weeks, followed by a 4-week recovery period for each group The remaining n=2/gender. Use an infusion pump to infuse the animal intravenously at a rate of approximately 10 mL/kg/h and a volume of 15.4 mL/kg. According to the protein concentration of the test product in this batch and the maximum dose of short-term intravenous infusion, the high dose of 150 mg/kg is regarded as the maximum feasible dose. Evaluation is based on daily clinical observation, weekly recording of body weight and food intake, eye examination, and routine clinical pathology. TK blood samples were collected at various time points after administration and the potential changes in serum and urine Ca ++ and Pi levels were investigated, and the peripheral blood lymphocyte subsets were evaluated for immunophenotype by flow cytometry. Before the test (week 1) and 1h (CNS) or 2h (other parameters) after the fourth infusion (day 22), the monkeys in the control group and the high-dose group were tested for safety pharmacology, including ECG and heart rate , Arterial blood pressure, respiratory rate, body temperature and central nervous system (CNS) function. The monkey was killed with an overdose of sodium pentobarbital and an autopsy was performed. On the 30th day or at the end of the recovery period, a final autopsy will be performed approximately 24 hours after the last dose. After visual inspection, the standard tissue is weighed and histopathological examination is performed.

　　Toxicokinetic analysis and anti-drug antibody (ADA) determination: During the M6123 toxicity study, blood samples were collected from animals at different time points. The anti-M6123 antibody (ADA) assay was performed in rats and monkeys. The validated immunoassay method can be used to detect anti-M6123 antibodies in serum. The samples from the control group and the treatment group were mixed with dissociation buffer (acid buffer) after the minimum required dilution (MRD) and incubated on a polypropylene multiwell plate. The acid pretreatment induces the destruction of the potential ADA-drug complex in the sample, thereby improving the tolerance of the assay drug. A pre-neutralized mixture of capture/detection reagents is added to the plate, allowing binding between ADA and labeled drug. When the binding equilibrium is reached, the sample is transferred to the MSD 96-well L55SA-1 plate pre-coated with streptavidin to capture the formed complex. After the washing step, MSD reading buffer is added and the signal is collected.

　　Result: Safety study of M6123 in mice: M6123 was intravenously injected into mice every week for 4 weeks. The clinical tolerance was good. The maximum feasible dose was 200 mg/kg. There was no M6123 related mortality, clinical abnormalities or ophthalmological changes. The clinicopathological analysis performed on the 9th day (about 24 hours after the second M6123 drug) showed that serum Pi increased in all dose groups (up to about +56% in males and up to about +25% in females), and serum Ca++ The increase is small (up to about +6% in males and females). This effect was accompanied by a dose-dependent increase in the level of Ca++ in the urine of all administered female mice (ranging from +87% to +153%, the value of male mice changed greatly), while urine Pi was not affected . About 24 hours after the last administration of M6123 on the 30th day, the female and male serum Pi of all dose groups were still significantly increased (up to +39% for males, and up to +42% for females compared with the control group); Male serum Ca++ increased the least (up to +7%). These changes are reversible and return to baseline levels at the end of the 4-week recovery period. At the end of the treatment or recovery period, no effect on organ weight was observed and no macroscopic or microscopic findings caused by M6123 administration were recorded. To further clarify the changes in serum Ca++ and Pi levels, another study was conducted using serum samples from the Dose Exploration Study, which used the same dosage and treatment regimen, after the first and fourth infusion2 Samples were collected for TK analysis at 24, 48, 96 and 168h. M6123 caused a slight increase in Ca++ and a moderate increase in Pi, especially after the first dose, which peaked mainly between 24 and 48 hours. On the first day, the exposure dose of M6123 increased from 50 to 100mg/kg roughly proportionally, from 100mg/kg to 200mg/kg, this ratio decreased slightly, and this effect was more pronounced for both sexes on the 22nd day . The accumulation of repeated doses of 50 and 100 mg/kg was observed to be greater than 200 mg/kg. No gender difference was observed at 50 and 200 mg/kg, while the accumulation of males was slightly larger than that of females at 100 mg/kg.

　　Safety study of M6123 in rats: In the DRF study, intravenous infusion of M6123 within the maximum feasible dose of 200mg/kg for 4 weeks in rats did not induce death. A short burst of hair was observed in 200 mg/kg animals after the first treatment. No abnormalities were observed in animals treated at lower doses. By day 7, the weight gain of males who received 200 mg/kg treatment decreased (mean: -8.8% relative to the control group). On the 14th day of the administration period, the animal's body weight recovered significantly. During the first week of the study, the effects of all M6123-treated male groups on body weight were accompanied by a dose-dependent reduction in food intake (about 12% to 18% lower than the control group). In addition, in ophthalmological examinations performed at the end of the treatment period, most animals in all M6123 treatment groups found dose-dependent, focal or diffuse corneal opacities. In addition, diffuse unilateral lens opacity was observed in 2 male rats treated with a dose of 100 mg/kg. In some animals, it was found to be related to the mineralization of the corneal basal layer during histopathological examination. A control female developed bilateral focal opacity of the cornea at the same examination time, and no related histological findings. In a follow-up study, except for a male treated with 25mg/kg M6123 showing unilateral multifocal corneal spots without related histomorphological changes, rats treated with lower doses (10 and 25mg/kg) No clinical abnormalities were shown, and no effect on weight gain, food consumption or eye diseases. In the clinicopathological examination performed at the end of the treatment period of the DRF study, moderate, dose-dependent elevation of serum protein and slight elevation of calcium ions were observed in all M6123 treatment groups. In follow-up studies, clinicopathological examinations performed before each sacrifice (after the first, second or last M6123 administration) showed that the serum Pi levels of animals given all doses at all recorded time points increased (relative to For the control group, increase from +30% to +50%). At different time points on day 1 and day 22 (mainly after the first treatment), a similar increase in Pi levels was observed in serum samples collected from TK animals, reaching a peak 24 hours after treatment. In animals treated with 25 and 50 mg/kg doses, the increase in serum Pi levels was maintained up to 96 hours, while the 10 mg/kg treated animals began to decline after reaching a peak 24 hours. In addition, in these TK samples, serum calcium increased slightly over a similar time frame. Only on day 9, urinalysis of all M6123 treatment groups showed a dose-dependent increase in urinary calcium and a slight decrease in urinary phosphorus, but not on day 30. In addition, only after the second week of treatment (day 9), the lymphocytes of animals treated with doses of 10, 25, and 50 mg/kg increased, but during the first or last treatment (days 2 and 30) After ), no increase in lymphocytes was observed. Neither study found any changes in gross pathology or organ weight associated with M6123. In histopathological examination, mineralization was observed in various organs of rats at all doses studied in DRF. Male rats were significantly more affected than female rats, and there was no obvious dose dependence. Metastatic mineralization was found in the intima, subintima and muscular layer of the aorta, and various other arteries/arterioles, especially in the heart, kidney, stomach and skeletal muscle (calcification confirmed by von Kossa staining). Tissues that further show mineralization include lungs (alveolar ducts), kidneys (kidney tubular cells), stomach (basic mucosa and muscle layer)

　　), corneal basal layer, spinal membrane, lamina propria of trachea and tibia and/or femur periosteum. In bone marrow, male rats in all dose groups showed bone formation or fibrosis, while female rats were not affected. In subsequent studies of male rats treated with 10, 25, or 50 mg/kg M6123, histopathology showed soft tissue mineralization and braided bone formation as observed in previous studies. When using lower doses (10 to 50 mg/kg), it is possible to determine the number of organs involved and find a significant dose and time dependence of severity. 24 hours after the first treatment with the highest dose (50 mg/kg), mineralization was first observed in the periosteum. After the second administration, the aorta, bone, spinal membrane and gastric fundus mucosa of animals in the 25 and 50 mg/kg dose groups showed mineralization. One rat in the 10 mg/kg dose group also found mineralization. After the last dose, mineralization was also found in the kidneys, lungs, eyes, and heart, and there was a significant dose-dependence again in terms of morbidity and severity. In addition, moderate to significant woven bone formation and fibrosis were found in each rat in each dose group. One of the rats receiving 50mg/kg treatment developed subepiphyseal fibrosis on the second day. In addition to tissue mineralization, Kupffer cell activation and an increase in the number of mitotic maps were observed in rat livers at low doses of 10 and 25 mg/kg only on day 9. The exposure of M6123 (peak concentration and AUC168) is roughly proportional to the increase in dose. On day 15 (5 minutes after the second dose), the serum concentration was comparable to that obtained at the same time point on day 1 and day 22. Slight accumulation was observed in all dose groups, and no gender difference was observed at any dose level. For the dose group 10 to 50 mg/kg, ADAs of all animals were negative for M6123.

　　 M6123 safety study in monkeys: weekly intravenous infusion of M6123, repeated treatment for 4 weeks, the maximum feasible dose is 150 mg/kg, well tolerated. No mortality was found directly related to M6123 treatment. A female who received low-dose (50 mg/kg) treatment was euthanized on day 22 due to a thromboembolic infection at the site of administration and deterioration of her health. Although no similar findings were found in all other treated animals, the neutropenia in M6123 treated animals was related to the test article. Except for the prematurely euthanized monkeys described previously, there were no abnormal clinical observations, no eye changes or effects on cardiovascular function, arterial blood pressure, respiratory rate or rectal temperature. Immunophenotyping analysis of blood lymphocytes showed a significant decrease in the natural killer (NK) cell count, which was below the detectable limit of most animals at the end of the treatment period and a significant decrease in the neutrophil count (up to -80%). In all dose groups, large unstained cells (LUC) and gamma globulin increased in a dose-dependent manner. After a 4-week recovery period, these changes showed partial or complete recovery. No effect on serum Ca++/Pi levels was observed, but an increase in urinary calcium (up to 4-fold) was recorded in the monkey groups given 100 and 150 mg/kg M6123 after the second and last dose. At all doses, an increase in the weight of the liver and spleen of both sexes was observed, and was associated with slight to moderate microscopic changes in these organs. In the spleen, red pulp proliferation is associated with significant infiltration of neutrophils. The liver showed Kupffer cell hypertrophy and hyperplasia, accompanied by a significant increase in the number of neutrophils in the sinuses and mild to moderate diffuse lymphoplasmacytic infiltration. Although no obvious dose-dependence of these microscopic changes was found, the degree/number of affected animals tended to increase at a dose of 150 mg/kg. Some medium and high dose animals showed a slight increase in bone marrow production. No toxicological related changes were observed at the end of the recovery period. At the infusion site, inflammatory changes caused by the administration technique were found. After intravenous infusion, the peak concentration of M6123 is usually reached at the end of the infusion (1.5h after the start of dosing), and the average value of tmax is in the range of 1.5-6h. The peak concentration is roughly proportional to the increase in dose. On day 1, the increase in M6123 exposure (AUC0–168) was roughly proportional to the dose, while on day 22, due to the decrease in exposure levels observed in most animals with repeated doses of 50 mg/kg, the exposure The amount (AUC0–168) increased slightly. A slight accumulation was observed in the 100 and 150 mg/kg groups, while in the 50 mg/kg group, all but one animal had reduced exposure. No decrease in peak concentration was observed in these animals, but the rate of decrease in serum concentration was faster than before. There is usually a good correlation between the reduction in exposure observed on day 22 and the development of ADAs, which can explain the reduction in exposure levels after repeated doses. At a dose of 50 mg/kg, ADA-positive samples were observed from day 15 onwards, of which 1 out of 5 females was positive on day 15, 3 out of 4 females and 2 out of 5 males It was positive on the 29th day, and one of the two males was positive during the recovery period. No ADA positive samples were found in the 100mg/kg dose group. At a dose of 150 mg/kg, one of the five males and one female was positive on the 29th day, and two males were positive during the recovery period. No gender difference was observed in the exposure of M6123.