Determination of the starting dose of a bispecific T-cell redirected antibody ERY974 targeting glycan-3 in the first human clinical trial.

Ethics statement

The protocols for all animal studies were approved by the Institutional Animal Care and Use Committee (IACUC) of Chugai Pharmaceutical Co., Ltd. Ltd. All animal studies were conducted in an AAALAC-accredited animal facility. All studies using human samples, such as PBMCs, were conducted in accordance with the Chugai Ethical Committee policy. All studies were performed in accordance with relevant guidelines and regulations.

reagents

ERY974 has been prepared by Chugai Pharmaceutical., Co., Ltd. (Tokyo, Japan).

cell lines

huH-1, a human hepatocellular carcinoma cell line, was purchased from the Japanese Research Biosource Group (JCRB) (Osaka, Japan). HepG2, a human hepatoblastoma cell line, and SK-HEP-1, a human hepatoma cell line, were purchased from the American Type Culture Collection (ATCC) (Manassas, VA). SK-pca31a SK-pca13a and SK-pca60 are derivatives of SK-HEP-1 expressing GPC3 slightly, moderately or strongly, respectively. PC-10, a human lung cancer cell line, was purchased from Immunology Biological Laboratories (IBL) (Gunma, Japan). All cell strains were cultured according to the manufacturer’s instructions. We did not validate these cell lines ourselves.

PBMC . preparation

All studies using human PBMCs were approved by the Chugai Ethical Committee, and the study was conducted in accordance with its policy. Informed consent was obtained from all donors through Chugai Pharmaceutical Co Ltd, or Cellular Technology Ltd. For various in vitro assays with huH-1, frozen human cryopreserved PBMCs were purchased from 10 different donors from Cellular Technology Ltd. (Shaker Heights, OH). For species comparison studies, human PBMCs were isolated from heparinized blood obtained from healthy volunteer donors by Ficoll–Paque PLUS density gradient centrifugation (GE Healthcare, Chicago, IL), following the manufacturer’s instructions. Briefly, heparin blood was diluted twice with PBS and transferred to Leucosep (Greiner Bio-One, Frickenhausen, Germany). After centrifugation (2300 rpm, 10 min) at room temperature, PBMCs were washed twice with PBS (1100 rpm, 10 min) at room temperature, and cells were counted. Cynomolgus monkey PBMCs were also isolated by Ficoll–Paque PLUS by density gradient centrifugation without Leucosep. After centrifugation (2300 rpm, 30 min) at room temperature, the PBMCs were collected as described above.

ABC of GPC3

The ABC of GPC3 in each cell line was determined using QIFIKIT (DAKO, Glostrup, Denmark) according to the manufacturer’s instructions. Cells were incubated with 20 μg/ml murine anti-GPC3 mAb (internal preparation) for 30 min at 4 °C. After washing, cells were incubated with the secondary antibody (included in QIFIKIT) for 30 min at 4 °C. After further washing, cells were analyzed with FACSlytic (BD, Franklin Lakes, NJ). ABC was calculated using a calibration curve.

Cytotoxicity assay

Cancer cells (1 x 10 .)4/96 well), frozen PBMCs (2 × 10 .).5/96 well), and ERY974 at different concentrations were in a round-bottom 96-well plate and incubated for 24 h at 37 °C with 5% CO2. LDH derived from lysed tumor cells was measured using an LDH Cytotoxicity Detection Kit (TAKARA Bio, Shiga, Japan). The cytotoxicity of ERY974 was calculated according to the manufacturer’s instructions.

T cell activation assay

After cytotoxicity examination, residual CD3+ Cells were collected and used to measure the expression of CD69 and CD25. Cells were washed with 0.5 w/v% bovine serum albumin (BSA)/CellWASH (FACS/PBS) (BD, Franklin Lakes, NJ), and then diluted Fc blocking reagent (Miltenyi, Gladbach, Germany) was added. Samples were incubated at room temperature for 10 minutes. Next, antibodies against CD3 (APC-conjugated) (BD), CD25 (PE-conjugated) (BD), and CD69 (FITC-conjugated) (BD) were added, and samples were incubated on ice for 30 min. After washing the cells with PBS, samples were analyzed with FACSVerse (BD). Live/dead cell staining (Fixable Viability Dye eFluor 780; Thermo Fisher Scientific, Waltham, MA) was used to quantify the number of live cells. Data were analyzed using FlowJo ver software. 7.6.5 (Digital Biology Tomy) to calculate CD25+ or CD69+ The ratio in CD3+ Population.

Cytokine measurement

After performing the cytotoxicity assay described above, the remaining culture medium was collected and used to measure the levels of the cytokines IL-2, IL-4, IL-6, IL-10, TNFα and IFNγ using the human cytokine CBA Th1/Th2 group II (BD) with FACSVerse . According to the manufacturer’s instructions. Data were analyzed using FCAP Array ver software. 3.0 (Bahraini Dinar).

Omani Rial account

RO was calculated using the following formula:

$$ {\text{RO}}\; \left(\% \right) = \left[ {{\text{mAb}}} \right]{/}\left left[ {K_{{\text{D}}} } \right] + \ left[ {{\text{mAb}}} \right]} \ right) \ 100 times. $$

The KDr The values ​​of ERY974 to GPC3 and CD3ε measured by Surface i resonance where different concentrations of ERY974 were applied to GPC3 and/or CD3 inactivated peptide were 1.46 × 10–9 and 2.07 x 10-7 mol / l, respectively8.

Tissue reactivity study

All studies were conducted at Charles River Laboratories, Inc. (Frederick, MD) as per IACUC policy. All studies using human samples were conducted in accordance with the policy of the Chugai Ethical Committee, and informed consent for all human samples was obtained through each company or institute described as follows. Tissue samples were collected as surgical or autopsy specimens from humans, obtained from Charles River Laboratories, Human Tissue Network Collaborative (Nashville, TN), National Disease Research Exchange (Philadelphia, PA), Corelin (Brisbane, CA), Poetics (Basel). , Switzerland)), Biological Analytical Services (Wilmington, DE), Frederick Memorial Hospital (Frederick, MD), or National Children’s Hospital (Columbus, OH), and autopsy specimens from cynomolgus monkeys, obtained from Charles River Laboratories, Covence (Burlington, NY). , NC), Bioqual, Inc. (Rockville, MD), or Alpha Genesis (Yemassee, SC), infused with Tissue-Tek OCT (Sakura Finetek, Street Torrance, CA), and stored in a refrigerator at -85 °C to -70 °C. Tissue was cut into sections approximately 5 μm thick and fixed in acetone. For control samples, 5-week-old NOD/ShiJic-scidJcl (NOD/SCID) mice were purchased from CLEA Japan, Inc (Tokyo, Japan). After acclimatization for 1~3 weeks, 1 x 107 SK-HEP-1, SK-pca31a and SK-pca13a cells were inoculated subcutaneously into the right flank of mice. The stable tumor was taken and mobilized with Tek OCT compound. For immunohistochemistry (IHC), the methods of Tuson et al. and Fung et al. and Hierck et al. used33,34,35. Briefly, ERY974 or a control antibody homologously was mixed with anti-human F(ab)2 from F(ab)2, and an antibody specific to the Fcγ (DkαHuIgG) fragment (Jackson ImmunoResearch Laboratories, West Grove, PA), and incubated overnight at 4 degrees Celsius. Slides were then pretreated with ABC Elite reagent (Vector Laboratories, Burlingame CA) for 30 min, rinsed with TBS, and then treated with DAB (Sigma-Aldrich, St Louis, MO) for 4 min as peroxidase substrate.

A single dose study in cynomolgus . monkeys

Study in Cynomolgus monkeys (Macaca’s footnote) conducted at Covance Inc. According to ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines.36. All procedures have been approved by the IACUC, and the facility is accredited by the AAALAC. Male and female cynomolgus monkeys were classified into five groups. Each group consists of three males and three females. Monkeys were injected once with ERY974 or with vehicle by intravenous infusion via the saphenous vein for approximately 30 min on the first day. Dose levels were 0, 0.1, 1, 10, and 10 mcg/kg for groups 1, 2, 3, 4, and 5, respectively. Animals in groups 1 and 5 were sacrificed on day 8 of the dosing phase, while animals in groups 2, 3 and 4 were sacrificed on day 22 of the dosing phase. Blood samples were collected via the femoral vein in the pre-dose phase and in the dosing phase at approximately 2, 8, 24, 48, 72 and 168 h for cytokine measurement using a non-human CBA cytokine kit from Th1/Th2 primates (BD). On the eighth day of the dosing phase, all animals in groups 1 and 5 were fasted overnight, anesthetized with sodium pentobarbital, drained of blood, and dissected. On day 22 of the dosing phase, all animals in groups 2, 3 and 4 were treated as described above. Most tissues from each animal were preserved in 10% neutral buffered formalin, embedded in paraffin, sectioned, sliced ​​and stained with hematoxylin and eosin for macroscopic examinations.

Calculation of FIH dose based on MABEL and NOAEL نهج approaches

In the MABEL approach, the dose of FIH, which corresponds to the plasma concentration in patients 4 hours after intravenous infusion of ERY974 and EC10 The value obtained was determined in the cytotoxicity assay (0.0767 ng/ml). The human PK profile was predicted using the differential scaling method in cynomolgus monkeys. Furthermore, a traditional approach based on a no-observable adverse effect level (NOAEL) using toxicity data in cynomolgus monkeys was adopted to calculate the FIH dose using body surface area correction, taking into account differences between species.

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