Assessment of CTLA-4 and Fc receptors affinity of JS007

JS007, which was produced as previously described [22], was captured by CM5 chips that were previously coupled with 40 µg/mL of goat anti-human Fc fragment antibody (ImmunoResearch, 109–001–008), followed by binding with different concentrations of His-tagged recombinant human CTLA-4 extracellular domain (ECD). Ipilimumab, which was manufactured in-house according to the published patent, was used as a reference anti-CTLA-4 mAb in preclinical experiments. Anti-KLH IgG1 manufactured in-house was used as a negative control. Consecutive dynamic binding signals were obtained. The affinity was calculated by fitting the data using the kinetic model of Biacore T200 Evaluation Software 3.0 (GE Healthcare Life Sciences).

JS007 belongs to the human IgG1/κ subtype. The affinity of JS007 for Fc receptors was determined using Biacore (GE Healthcare Life Sciences), in which anti-His tag antibody was coupled to a CM5 chip surface, followed by capturing the His-tagged recombinant human FcγRIIIa (CD16a) V176 (Junmeng Biomedical, 20200421), FcγRIIIa (CD16a) F176 (Junmeng Biomedical, 20200421), FcγRIIa (CD32a) R167 (Sino Biological, 10734-H08C), FcγRI (CD64) (Sino Biological, 10256-H08S), and FcRn (Sino Biological, CT009-H08H). The test antibody was then gradient-diluted, and binding signals were detected.

Direct cytotoxicity test

The direct cytotoxicity of JS007 mediated peripheral blood mononuclear cells (PBMCs, MiaoTong Biotech Company, Donor No. P122030210C) on CHO-hCTLA-4 cells was tested. Serial concentrations of JS007 were incubated with PBMCs and CHO-hCTLA-4 coculture system at a ratio of 25:1 for 4 h. Then, cell supernatants were collected, and the release of lactate dehydrogenase (LDH) in the supernatants was detected using an LDH assay kit (BioVision, K311-400).

JS007 induced T cell activation test

T cell activation induced by JS007 was detected by Staphylococcal enterotoxin B (SEB) model. SEB was a superantigen that could activate T cells by binding to specific TCR Vβ regions and MHC-II molecules, then inducing the release of various cytokines. PBMCs from different donors (ALLCELLS, LP200520, LP200610, and LP200603) were incubated with JS007, PC-mAb, toripalimab (anti-PD-1 antibody), and hIgG1 control, followed by the addition of SEB (Toxin Technology, BT202red). The supernatant was collected and mixed with P-phycoerythrin (BD CBA human IL2 Flex set, 558270). Then the mixture was loaded and analyzed in the flow cytometer (BD. Canto) to determine the MFI-PE intensity of IL-2 bead, and then the IL-2 concentration was calculated based on the standard curve.

Antibody-dependent cellular cytotoxicity (ADCC) activation test

An ADCC reporter gene system was constructed to assess JS007-mediated ADCC activity. The Jurkat-ADCC cells as effector cells with overexpression of FcγRIIIa and downstream fluorescent reporter genes, and CHO-hCTLA-4 cells as target cells were both in-house manufactured. Upon receptor engagement, expression of fluorescent reporter genes in activated effector cells was monitored by adding the fluorescent substrate.

In vivo antitumor effect of JS007 in humanized mice MC38 xenograft model

Dual hPD-1/hCTLA-4 humanized mice were obtained from Biocytogen Co. Ltd (strain: C57BL/6-Pdcd1tm1(PDCD1)BcgenCtla4tm1(CTLA4)Bcgen/Bcgen, Animal Production License No. SCXK (Su) 2016-0062). All mice were kept in specific pathogen-free conditions, and the study was conducted in compliance with applicable regulations for care and use of laboratory animals at Immune Technology (Suzhou) Corp. All procedures were approved by the local Institutional Animal Care and Use Committee (IACUC) of the company lab.

Mouse colon cancer MC38 cells (1 × 106) were inoculated subcutaneously into the right posterior back of the humanized mice. When the tumor size reached about 90 mm3, the mice were randomly divided into six groups and intraperitoneally injected with JS007 (0.3 mg/kg), ipilimumab (0.3 mg/kg), toripalimab (0.3 mg/kg), JS007 + toripalimab (0.3 mg/kg + 0.3 mg/kg), or ipilimumab + toripalimab (0.3 mg/kg + 0.3 mg/kg) on Day 0, Day 3 and Day 7. Antibody of hIgG1 (0.3 mg/kg) was used as negative control. The tumor growth was monitored twice a week, and the tumor volume (Tv) was calculated by the formula: ½length × width. Tumor growth inhibition (TGI) ratio was calculated by the formula: 1-(Ti-T0)/(Vi-V0) × 100% (Ti: Tv of the treatment group on day i of drug administration; T0: Tv of the treatment group on day 0 of drug administration; Vi: Tv of the NC group on day i; V0: Tv of the NC group on day 0).

At necropsy, tumor tissues of individual animals were harvested and cut into pieces of about 1–3 mm, then dissociated with a tumor tissue dissociation kit (MACS, Cas No. 130-096-730) according to manufacturer’s instruction before being filtered with a 40 μm cell filter. About two million cells were resuspended and transferred into Fluorescence-activated cell sorting tubes, then incubated with APC anti-mouse/Rat Foxp3 (Invitrogen, Cas No. 17-5773-82), FITC anti-mouse CD4 (Biolegend, Cas No. 100406), and PE-CY7 anti-mouse CD3e (BD, Cas No. 552774). A flow cytometry (BD, LSR Fortessa) was applied to analyze the tumor-infiltrated cells (TILs).

Pharmacokinetics (PK) study of JS007 in non-human primate (NHP) model

Experimental animals (Rhesus monkey) for single-dose pharmacokinetic study and repeat-dose pharmacokinetic study were purchased from Sichuan Hengshu Biotechnology Co. Ltd with Certificate No. SYXK (Shanghai) 2019-0009. All procedures in compliance with all applicable guidelines were approved and monitored by the local IACUC following the protocol of “Pharmacokinetic Study of JS007 Injection after Single Intravenous Infusion in Rhesus Monkeys” (Study No. S19052PK1) and “A 4-week Repeated Dose Toxicity Study of JS007 Injection via Intravenous Injection in Rhesus Monkeys with a 4-week Recovery Period” (Study No. 19052RD01). The single dose PK of JS007 was assessed in experimental animals after administration of a single intravenous infusion at dose levels of 0.3, 1, or 3 mg/kg, respectively. Each dose group consisted of 6 animals (3 males and 3 females). The blood samples were collected on 0.25 h, 0.5 h, 2 h, 4 h, 8 h, 24 h, 48 h, 96 h, 168 h, 240 h, 336 h, 504 h, 672 h, 840 h, and 1008 h following the dose and processed to serum samples after coagulation and centrifugation. The samples were stored at -80 °C before assay.

As a part of the Good Laboratory Practice toxicity study, the repeat-dose pharmacokinetic study (also known as toxicokinetic study) was conducted in compliance with US FDA CFR Part 58, Good Laboratory Practice for non-clinical studies. A standard panel of toxicological and pathological parameters had been evaluated according to ICH S9 “Guidance for Industry S9 Nonclinical Evaluation for Anticancer Pharmaceuticals” and FDA Redbook 2000: IV.C.3b “Short-Term Toxicity Studies with Non-Rodents, 2003”. The execution and interpretation of the study were done by the qualified study director, quality control inspectors, pathologist, and contributed scientist, in order to determine the highest non-severely toxic dose (HNSTD). During the repeated-dose PK analysis, experimental animals were divided into 4 groups with 5 animals per gender in each group and were treated with JS007 intravenously once weekly five times at dose levels of 3, 10, and 30 mg/kg. Blood samples were collected on 0.083 h, 2 h, 6 h, 24 h, 72 h, 120 h, and 168 h following the 1st and 4th dosing. The concentration of JS007 in serum samples of experimental animals was measured by Enzyme-linked Immunosorbent Assay (ELISA) with a validated methodology. The principle of the method involves coating the enzyme plate with HXT hCTLA-4 ECD his (Suzhou Junmeng Biomedical Technology Co. Batch: No. 20181224), adding the test samples, followed by addition of Human IgG-heavy and light chain monkey-adsorbed Antibody Goat Polyclonal Conjugate HRP (BETHYL. Batch: A80-319P-24) as the detection antibody. The PK curve fitting and parameters calculation including maximum observed concentration (Cmax), area under the plasma drug concentration-time curve (AUC), Clearance (CL), and elimination half-life (T1/2), were performed by using Phoenix WinNonlin (Certara, Version 8.1).

Phase 1a study design

This was a single-arm, multicenter, open-label, multiple-dose trial conducted in 4 centers around China. The study consisted of a dose escalation phase and a dose expansion phase. This study was performed in compliance with the Declaration of Helsinki and Good Clinical Practice guidelines. The written informed consents were provided by all patients before enrollment. Study procedures were approved by the independent ethics committee at each participating site.

Patients

Patients aged 18–75 years, with histologically or cytologically confirmed advanced or recurrent solid tumors and failed with previous standard treatment, or those who had no available standard treatment or refused to receive standard treatment; had at least one measurable lesion according to Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1 [23]; had an Eastern Cooperative Oncology Group performance status (ECOG PS) score of 0 or 1; with a life expectancy of ≥ 3 months; with no prior exposure to anti-CTLA-4 drugs; had adequate organ function were eligible to be enrolled.

Key exclusion criteria included central nervous system metastases; had received immunosuppressive agents within 2 weeks before the first dose of study drug including glucocorticoids at dose equivalent to prednisone > 10 mg/day; systemic anticancer therapy (including radiotherapy, chemotherapy, hormone therapy, surgery, or molecular-targeted therapy) within 4 weeks before study drug was firstly administrated, or unrecovered toxicities from prior anticancer treatment, defined as having not resolved to National Cancer Institute Common Terminology Criteria for Adverse Events (NCI-CTCAE) Grade 1 or below except for alopecia.

Endpoints

The primary endpoints were the maximum tolerated dose (MTD), the incidence and severity of dose-limiting toxicities (DLTs) events, adverse events (AEs), serious adverse events (SAEs), and immune-related adverse events (irAEs). The secondary endpoints were the PK parameters including Cmax, time to maximum observed concentration, AUC0 − t, AUC0−∞, T1/2, CL, volume of distribution, and anti-drug antibody (ADA). The exploratory endpoints were objective response rate (ORR), duration of response (DoR), disease control rate (DCR), progression-free survival (PFS), and overall survival (OS). ORR was defined as the proportion of patients with a confirmed best overall response of complete response (CR) or partial response (PR). DoR was defined as the time from the initial response (CR or PR, which was confirmed later) to the time of disease progression or death of any cause, whichever came first. DCR was defined as the proportion of patients with the best overall response of CR or PR or stable disease (SD). PFS was defined as the time from the first dose of the study drug to the time of documented radiological disease progression or death of any cause, whichever came first. OS was defined as the time from the first dose of the study drug to the time of death of any cause.

Procedures

The study was initiated with an accelerated titration scheme in which one patient was enrolled at the 0.03 mg/kg dose level. If no DLT was observed, the study proceeded following a standard 3 + 3 design with sequential enrollment of three patients at three dose levels of 0.3, 1, and 3 mg/kg. The dose level of 10 mg/kg was added per the suggestion of the Safety Monitoring Committee. JS007 was administered intravenously every 3 weeks. DLT was evaluated within 21 days after the first dose of JS007. If no DLT was reported by the first 3 patients in one dose level, the study proceeded to the next dose level. If 1 patient had DLT, 3 more patients would be enrolled at the current dose level. If one or more of the added 3 patients had DLTs, the dose escalation would be terminated, and the previous dose was considered as the MTD. If the additional 3 patients had no DLT, the dose escalation would be forwarded to the next dose level. If ≥ 2 out of 3 patients in a certain dose level had DLTs, the dose escalation would be terminated, or an intermediate dose would be determined by the investigator and sponsor for continued observation. Then this dose was the intolerable dose, and the previous dose was MTD. In the dose expansion phase, two dose levels were selected based on the results of the dose escalation phase, including safety, efficacy, and pharmacological data. Each dose level would enroll an additional 6–9 patients.

Clinical assessments

The severity of AEs was assessed per NCI-CTCAE version 5.0. AEs were recorded from the first dose of the study drug until 90 days after the last dose of the study drug or initiation of new antitumor therapy. Laboratory examinations included hematology tests, blood chemistry tests, cardiac function tests, urinalysis, coagulation tests, and thyroid function tests. Efficacy assessments according to RECIST version 1.1 were performed at baseline, every 6 weeks within 36 weeks after initial administration of the study drugs, then every 9 weeks until disease progression or initiation of subsequent antitumor therapy. Magnetic resonance imaging or 18 F-fluorodeoxyglucose positron emission tomography could be used when enhanced contrast computed tomography (CT) examination was not feasible.

PK and immunogenicity analysis in human

To evaluate PK parameters, serum concentrations of JS007 were measured using a validated ELISA method. Samples were collected on Cycle 1 and Cycle 4 before infusion, then at 1 h (± 5 min), 6 h (± 15 min), 24 h (± 1 h), 48 h (± 2 h), 168 h (± 8 h), 336 h (± 12 h), and 504 h (± 12 h) after treatment. PK parameter values of an individual patient were derived by non-compartmental methods using a validated PK analysis program (Phoenix WinNonlin version 8.4, Certara, Princeton, NJ, USA). Anti-JS007 antibodies in human serum were detected utilizing a rigorously validated Bridging Electrochemiluminescence Immunoassay methodology. Samples were obtained within an hour before the administration of Cycle 1 through Cycle 4, 6, 8, 12, and 16, and then before the administration of every eight subsequent cycles. Post-treatment sample collection was planned for the end of the treatment course, with optional collections at 30 (± 7) days and 90 (± 7) days after the final treatment.

Statistical analysis

The conventional 3 + 3 design was employed to determine the number of patients enrolled at 5 dose levels in the dose escalation phase, and 6–9 patients were enrolled at each dose level in the dose expansion phase. The full analysis set comprised all patients who received at least one dose of JS007 and had follow-up data after administration. The response evaluable analysis set comprised all patients who received at least one dose of JS007 with an adequate baseline tumor assessment and had undergone at least 1 appropriate tumor response assessment. The safety analysis set comprised all patients who received at least one dose of JS007. DLT analysis set comprised all patients who received at least 80% of the planned dose of JS007 and completed DLT observation, or patients who experienced DLT in the DLT observation window after administration. PK analysis set comprised all patients who received JS007 and had at least one PK evaluable data after the administration of JS007. The ADA analysis set comprised all patients who received at least one dose of JS007 and had evaluable ADA data after the administration of JS007. A two-sided 95% confidence interval (CI) of ORR and DoR was computed using the Exact (Clopper-Pearson) method by dose group. PFS and OS were estimated by the Kaplan-Meier method with 95% CIs calculated based on log-log transformation.