Mice, cell lines and reagents
Reagents, cell lines and viral strains used in this study are listed in Supplementary Table 1.
Generation of CD25 antibodies
The mice used for immunization are human antibody transgenic mice that generated by our company. They were bred and kept under specific-pathogen free conditions. All animal experiments were complied with relevant ethical regulations regarding animal research. Protocols of mice experiments for immunization were approved by LUYE PHARMA Animal Experimentation and Ethics Committee. Human antibody transgenic mice were sequentially immunized with CD25. In the meantime, titers of antibodies in the mice serum were tested by ELISA. Three days after the last immunization, the spleen was harvested for library construction. The construct of the phage library was carried out according to the method described in Phage Display: A laboratory manual29. Plates coated with CD25 or streptavidin- magnetic beads binding biotin-CD25 were used to capture interest phages. Enriched phages were used to infect E. coli TG1 for expressing single-chain variable fragments (scFvs) and the binding and blocking activity were tested by ELISA. Positive clones were obtained and sequenced.
Heavy chain variable region and light chain variable region were amplified (2 × Phanta Max Master Mix, Vazyme, P515-01) using the positive clones screened from the library as templates. Overlap PCR was conducted to assemble variable region and signal peptide. Purified gene fragments were separately fused (ClonExpress II One Step Cloning Kit, Vazyme, C112-01) into the linearized pcDNA3.4 vectors with constant regions. The recombinant plasmids were prepared for production. Antibodies were expressed with Expi-CHO Expression system (A29133, Gibco) for 12days and the supernatants were harvested and purified by a AT Protein A Diamond (AA0272, Bestchrom).
ELISA - based binding assay
CD25 protein (10165-H08H, Sino Biological) was coated on high binding ELISA plates with 0.2μg/mL at 4°C overnight, and then the plates were blocked with 3% skim milk powder in PBST (PBS containing 0.05% Tween-20) at 37°C for 1 hour. After washing two times with PBST, serially diluted antibodies were added to each well, incubated at 37°C for 1h. Plates were washed two times and then HRP-goat anti-human IgG mAb (474-1006, KPL) was used to detect antibodies binding to CD25. Experiments were performed in triplicate, value=Mean ± SEM.
Cell based binding assay
SU-DHL-1(CRL-2955, ATCC) or HEK293T-CD25 cells were harvested and washed by FACS buffer (0.2% BSA in PBS) two times. Serially diluted antibodies were mixed with 1×106 cells at 4°C for 1 hour. After washing two times by FACS buffer, cells were incubated in dark with Goat Anti-Human IgG-PE (2040-09, Southern Biotech) at 4 °C for 30 min and then analyzed by NovoCyte 2060R flow cytometry. Experiments were performed in triplicate, value=Mean ± SEM.
ADCC reporter bioassay
ADCC reporter bioassay was conducted with SU-DHL-1 cells, HEK293T-CD25 cells or CD8+ T cells as target cells and Jurkat cells (G7011, Promega) as effector cells. Activated CD8+ T cells were got by stimulating CD8+ T cells for 72 hours with OKT3, CD28 antibody and IL-2. 25 μL target cells at 1.2E6/mL, serially diluted antibodies and Jurkat cells at 2.4E6/mL were sequentially added to a White Tissue Culture treated 96 well plate in a cell incubator for 5 hours. Bio-Glo Luciferase Assay Buffer/Substrate (G7940, Promega) was added to react for 15min and then the value of Luminescence was read on Tecan microplate reader. Experiments were performed in duplicate, value=Mean ± SEM.
In vitro ADCC assay
Antibody-dependent cell-mediated cytotoxicity was conducted with SU-DHL-1 cells, inactivated CD8+ T (PB009-3-C, ALLCELLS) cells or activated CD8+T cells as target cells and PBMCs as effector cells. Activated CD8+ T cells were got by stimulating CD8+ T cells for 72 hours with OKT3, CD28 antibody and IL-2. Serially diluted antibodies were mixed with target cells in 96-well plates at 37°C for 15-30 minutes. The effector cells PBMCs (PB003F-C, ALLCELLS) were added and the mixture was incubated at 37°C for 5 hours. After substrate was added, OD490 value was read on the microplate reader. Percent cytotoxicity was computed using following formula：100 ×（OD490 of Experimental wells–OD490 of Experimental Wells Without Antibody）/ (OD490 of Target Cells Maximum LDH Release wells- OD490 of Target Cells Spontaneous LDH Release wells). Experiments were performed in duplicate, value=Mean ± SEM.
Affinity analysis by Surface Plasmon Resonance
SPR measurements were performed at room temperature using a BIAcore 8K system with CM5 chip, which was amino coupled by human antibody capture kit. HBS-EP+ buffer (150mM NaCl, 10mM HEPES, 3mM EDTA and 0.05% (v/v) surfactant P20 pH 7.4) was used as running buffer. The blank channel of the chip served as the negative control. The antibodies were captured on the chip at 400-500 response units. Serial dilutions of CD25 (from 50 nM to 3.125 nM with 2-fold dilution) were applied to flow over the chip surface, which was regenerated with 3 M MgCl2 after each cycle. The affinity was calculated using a 1:1 (Langmuir) binding fit model or two state reaction model with BIA evaluation software. Experiments were performed in triplicate, value=Mean ± standard error.
ELISA -based IL2-binding inhibition assay
CD25 protein (10165-H08H, Sino Biological) was coated on high binding ELISA plates with 0.5μg/mL at 4°C overnight. Plates were blocked with 3% skim milk powder in PBST (PBS containing 0.05% Tween-20) at 37°C for 1 hour. The mixture of IL2-biotin (0.03μg/ml) and serially diluted CD25 antibodies were added to the blocked ELISA plate and incubated at 37°C for 1 hour. After washing, the biotinylated IL2 binding to coated CD25 was detected by HRP-conjugated Streptomycin. Experiments were performed in duplicate, value=Mean ± SEM.
In vitro IL-2 signaling by STAT5 phosphorylation assay
PBMCs were co-cultured with 10μg/mL CD25 antibody in a 96-U bottom plate for 30 minutes, then 1U/mL IL2 was added and cultured for 10 minutes (working medium: 1640+ 10% FBS, containing 2mM L-Glutamine). Cell suspension was prepared as follows. 200 µL Foxp3 fixation/breaking membrane working solution was added to cell pellet in each well and incubated for 30-60 minutes at 2-8°C or room temperature in the dark. After the sample was centrifuged at 400-600g for 5 minutes at room temperature, the supernatant was discarded and plates were washed twice with 200 µL/well 1X rupture solution and once with PBS at room temperature. Ice-cold Phosflow™ Perm Buffer III was slowly added and then incubated on ice for 30 minutes. After washing two times by FACS buffer, cells were stained with fluorescent-labeled antibody at 4 oC for 30 min and then analyzed by NovoCyte 2060R flow cytometry. Experiments were performed in duplicate, value=Mean ± SEM.
Epitope binning of the antibodies was performed on a ForteBio Octet Red96 system (Pall Forte BioCorporation, Menlo Park, CA) using in-tandem format binning assay. CD25-biotin was loaded onto SA sensors (18-5019, fortebio). The sensors were then exposed to the first antibody with 50µg/mL or PBST for 300s, then to the second antibody with 50µg/mL for 300s. Data was processed using ForteBio’s Data Analysis Software 9.0.
Syngeneic mouse models
All animal works were carried out in compliance with ARRIVE guidelines (https://arriveguidelines.org). These experiments were carried out at Beijing Biocytogen Co. Ltd.
For early phase of tumor development, mice were randomized into three groups, with 8 mice per group based on their body weight at day -1(the day before tumor inoculation). Then BA9 and BT942 were dosed at 10 mg/kg intraperitoneally twice a week with vehicle group as the control. 5×105 MC38 cells in PBS were inoculated subcutaneously in the flank of mice at the next day (day 0). On day 16, three mice in each group were sacrificed to test the proportion of CD45+ T, CD3+T, CD4+T, CD8+T, CD25+Foxp3+, Foxp3+(Treg) cells in tumor by FACS. Animals were euthanized by CO2 asphyxiation when the mean tumor volume reached about 1300 mm3 (day 21).
For late phase of tumor development, B-hIL2RA humanized mice were implanted with 5×105 cells MC38 cells subcutaneously in the flank (day 0). Mice were distributed into three groups (n=8) with group mean starting volumes of 50 to 60 mm3 (day 5). Then BA9 and BT942 were also dosed at 10 mg/kg intraperitoneally twice a week with vehicle group as the control. Animals were euthanized by CO2 asphyxiation when the mean tumor volume reached about 2000 mm3 (day 19). On day 19, four mice in each group were sacrificed to test the proportion of CD45+ T, CD3+T, CD4+T, CD8+T, CD25+Foxp3+, Foxp3+(Treg) cells in tumor and peripheral blood by FACS.
For the synergistic effect of BT942 and anti-mouse PD1 antibody at late phase of tumor development, B-hIL2RA humanized mice were implanted with 5×105 MC38 cells subcutaneously in the flank (day 0). Mice were distributed into four groups with 5 mice per group and treated with BT942, anti-mouse PD1 (BioXCell), their combination or vehicle when mean tumor volumes reached 50 to 60 mm3 at day 4. Animals were euthanized by CO2 asphyxiation when mean tumor volume reached about 2000 mm3 (day 19).
Tumor size was measured twice a week by caliper. Tumor volumes were calculated as volume = (w2×l)/2 where w is the tumor width and l is the tumor length in millimeters. For more information please see Methods in Supplementary Information.
Pharmacodynamic and pharmacokinetics experiments
All animal care and experimental procedures were complied with relevant ethical regulations regarding animal research. Pharmacokinetics and pharmacodynamic study protocols in monkeys were approved by Institutional Animal Care and Use Committee(IACUC) and the Approval Number was UPP-IACUC-2020-00000. The BT942 antibody was administered intravenously to cynomolgus monkeys (N = 2, 2 males, body weight 3~5kg) at a dose of 10 mg/kg. Peripheral bloods were collected at predose and 1min, 30 min, 1h, 3h, 6h, 24h, 48h, 96h, 168h, 240h, 336h, 504h post-dose for PK study. ELISA (enzyme-linked immunosorbent assay) was used to determine the concentration of BT942 in serum. In this method, CD25 protein was used as the capture reagent, and goat anti-human IgG, monkey ads-HRP was detecting agent. Results are shown as mean ± SEM. The main PK kinetic parameters were calculated using Phoenix WinNonlin.
Peripheral bloods were collected at pre-dose and 1 min, 3h, 6 h, 24h, 48h, 72h, 168h, 336h post-dose to determine CD4+CD25+FoxP3+ percentage in CD4+ T cells with flow cytometry (CytomicsTM FC500).
Statistical analysis was performed using SPSS 21.0. The tumor volumes were compared between treatment and control groups and the differences were assessed for significance using One-way ANOVA. Inter-group differences for percentages of different cell populations in tumor were assessed using Dunnett-t test (two-tailed). Data were presented as Mean ± SEM. The threshold of significance was set at p<0.05.
The variable region sequences of BT942 MAb have been deposited in GenBank with the accession codes MW251337 for heavy chain and MW251338 for light chain. The variable region sequences of BA9 MAb have been deposited in GenBank with the accession codes MW251339 for heavy chain and MW251340 for light chain.