Cell lines
For the HUVEC proliferation, apoptosis, and cell cycle analysis assays, HUVECs (Sciencell Research Laboratories, Carlsbad, CA, USA, #8000) were cultured in ECM medium (ScienCell, #1001) containing 5% FBS (ScienCell, #0025), 1% penicillin/streptomycin (GIBCO, Invitrogen Inc., Carlsbad, CA, USA, #15140–122) and 1% ECGS (ScienCell, #1052). For HUVEC Western blot, migration inhibition, and scratch assays, HUVECs (AllCells, Alameda, CA, USA) were maintained in HUVEC medium (Allcells, #H004B) with 10% FBS (Allcells, #H005) and HUVEC growth factors (Allcells, #H005). HUVECs at passages 4–8 were used in the experiments. The human non-small cell lung cancer cell line PC9 was purchased from Riken BioResource Research Center (Ibaraki, Japan, #RCB4455). The mouse lung cancer cell line 3LL was purchased from JCRB Cell Bank of the National Institutes of Biomedical Innovation, Health, and Nutrition (Tokyo, Japan, #JCRB-1348). The mouse colorectal cell line CT26 was purchased from the American Type Culture Collection (ATCC) (Manassas, VA, USA, #CRL-2638). The PC9, 3LL, and CT26 cells were cultured in RPMI-1640 medium (Gibco, #22400–089) with 10% FBS (Hyclone, Thermo Fisher Scientific, Waltham, MA, USA, #SV30087.03), 100 U/ml penicillin and 100 μg/ml streptomycin (Gibco, #15240–062).
ELISA-based assays
VEGF/VEGFR2 binding blocking assay
Blank ELISA plates were coated with VEGFR2-his (Sino Biuological Inc., Beijing, China, #HPLC-10012-H08H) and blocked with 1% BSA in PBS. Different concentrations of BD0801 (Jiangsu Simcere Pharmaceutical Co., Ltd., Nanjing, China) or bevacizumab (Genentech/Roche, San Francisco, CA, USA) were incubated with human VEGF165 (PrimeGene, Shanghai, China, #105–05) at 37 °C for 1 h before adding into the plates coated with VEGFR2-his for incubation. Unbounded VEGF165 was washed off, and the primary antibody anti-VEGF rabbit mAb (Sino Biological, #11066-R105) was added for incubation. After washing the plates, the secondary antibody Peroxidase AffiniPure Donkey Anti-Rabbit IgG Jackson (Jackson ImmunoResearch, West Grove, PA, USA, #711–035-152) was added. After incubation, the redundant HRP complex was washed off. Finally, peroxidase (HRP) substrate TMB (Thermo Fisher Scientific, Waltham, MA, USA, #34029) was added, and the OD value was measured by SpectraMax I3X (Molecular Devices, LLC, Sunnyvale, CA, USA). The detection wavelength was 450 nm, and the reference wavelength was 630 nm. The IC50 was calculated based on the OD value at different concentrations by GraphPad Prism 8.0 (GraphPad Software Inc., San Diego, CA, USA). Each concentration was measured in duplicates in each experiment, and the experiment was repeated three times.
ELISA binding assay
Blank ELISA plates were coated with human VEGF165 (PrimeGene, Shanghai, China, #105–05), rat VEGF164 (PrimeGene, #145–07), or mouse VEGF164 (PrimeGene, #125–07) and blocked with 1% BSA in PBS. The different BD0801 or bevacizumab concentrations were added to the plates and unbound BD0801 or bevacizumab was washed off. The peroxidase affiniPure donkey anti-human IgG (Jackson ImmunoResearch, West Grove, PA, USA, #709–035-149) was added. After incubation, the redundant HRP complex was washed off. Finally, HRP substrate TMB (Thermo Fisher Scientific, Waltham, MA, USA, #34029) was added, and the OD value was measured by SpectraMax I3X. The detection wavelength was 450 nm, and the reference wavelength was 630 nm. The EC50 was calculated based on the OD value at different concentrations by GraphPad Prism 8.0. Each concentration was measured in triplicates.
ELISA analysis for BD0801 concentration in mouse serum
Blank ELISA plates were coated with VEGF165 (Sino Biuological Inc., Beijing, China, #11066-HNAB), and the serum samples were added. BD0801 in the serum samples were bound to VEGF, and the unbound BD0801 was washed off. Peroxidase AffiniPure donkey anti-human IgG (Jackson ImmunoResearch, West Grove, PA, USA, #709–035-149) was added. After incubation, redundant HRP complexes were washed off. HRP substrate TMB (R&D Systems, Minneapolis, MN, USA, #BMS258/2) was added. The OD value was measured using a microplate reader (TECAN, Männedorf, Switzerland). The detection wavelength was 450 nm, and the reference wavelength was 620 nm. Each sample was measured in duplicates. The concentration calculation of BD0801 for standard curve, quality control, and serum samples was performed by Microsoft Excel 2010 and SigmaPlot10.0. The standard curve fitting equation:
$$ \mathrm{y}=\mathrm{D}+\left(\mathrm{A}\hbox{-} \mathrm{D}\right)/\left(1+10\hat{\mkern6mu} \left(\left(\mathrm{x}\hbox{-} \mathrm{logC}\right)\ast \mathrm{B}\right)\right),\left(\mathrm{Weight}=1/\mathrm{y}\hat{\mkern6mu} 2\right) $$
x, Log [BD0801]; y, OD value.
HUVEC proliferation inhibition assay
Inhibition of HUVEC proliferation was detected by CellTiter Glo staining (Promega, Madison, WI, USA). HUVECs in the logarithmic growth phase were plated into 96-well plates in ECM medium (ScienCell, #1001) containing 1% FBS (ScienCell, #0025) and 1% penicillin/streptomycin (GIBCO, #15140–122). After 18–20 h, different concentrations of BD0801 (Jiangsu Simcere Pharmaceutical Co., Ltd., Nanjing, China) or bevacizumab (Genentech/Roche, San Francisco, CA, USA) were incubated with 50 ng/ml VEGF (PrimeGene, Shanghai, China, #105–05) in ECM medium containing 0.5% FBS and 1% penicillin/streptomycin for 2 h at 37 °C before they were added into the HUVEC culture. The HUVECs were cultured at 37 °C, 5% CO2 for 72 h. CellTiter Glo working solution was added to the cells at room temperature according to the manufacturer’s manual. Then, the luminescence was measured by PheraStar FS (BMG Labtech, Offenburg, Germany). The IC50 was calculated based on the luminescence at different concentrations by GraphPad Prism 8.0 (GraphPad Software, San Diego, CA, USA). Each concentration was measured in triplicates in each experiment, and the experiment was repeated three times.
HUVEC apoptosis assay
HUVECs in the logarithmic growth phase were plated into 24-well plates in ECM medium (ScienCell, #1001) containing 1% FBS (ScienCell, #0025) and 1% penicillin/streptomycin (GIBCO, #15140–122). After 18–20 h, different concentrations of BD0801 (Jiangsu Simcere Pharmaceutical Co., Ltd., Nanjing, China) or bevacizumab (Genentech/Roche, San Francisco, CA, USA) were incubated with 25 ng/ml VEGF (PrimeGene, Shanghai, China, #105–05) in ECM medium containing 0.5% FBS and 1% penicillin/streptomycin for 2 h at 37 °C before they were added into the HUVEC culture. The HUVECs were cultured at 37 °C, 5% CO2 for 48 h. The HUVECs were fixed by 4% paraformaldehyde and ethanol and stained by DAPI (Abcam, #Ab104139) to reveal the chromosome status. The apoptotic cells were identified as cells with altered nuclei, such as condensed chromosomes or disintegrated nuclei. Each condition was performed in triplicates.
HUVEC cell cycle analysis
HUVECs in the logarithmic growth phase were plated into 6-well plates in ECM medium (ScienCell, #1001) containing 1% FBS (ScienCell, #0025) and 1% penicillin/streptomycin (GIBCO, #15140–122). After 18–20 h, different concentrations of BD0801 (Jiangsu Simcere Pharmaceutical Co., Ltd., Nanjing, China) or bevacizumab (Genentech/Roche, San Francisco, CA, USA) were incubated with 50 ng/ml VEGF (PrimeGene, Shanghai, China, #105–05) in ECM medium containing 0.5% FBS and 1% penicillin/streptomycin for 2 h at 37 °C before they were added into the HUVEC culture. The HUVECs were cultured at 37 °C, 5% CO2 for 48 h. The HUVECs were digested with 0.25% trypsin-EDTA (GIBCO, #25200–056), fixed by 75% ethanol, and stained by PI (Invitrogen, #P3566) to reveal the cell cycle distribution by flow cytometry analysis using BD FACSCanto II (BD, Franklin Lakes, NJ, USA).
HUVEC migration inhibition assay
Cell migration assay was conducted using the Boyden Chamber Transwell method. The inner chamber with an 8.0-μm pores polycarbonate membrane was used. HUVECs suspended in HUVEC complete medium without serum were cultured in the inner chamber. Different concentrations of BD0801 (Jiangsu Simcere Pharmaceutical Co., Ltd., Nanjing, China) or bevacizumab (Genentech/Roche, San Francisco, CA, USA) were incubated with 20 ng/ml VEGF (Peprotech, Cranbury, NJ, USA, #100–20-10 μg) for 30 min at 37 °C before they were added into the outer chamber. The inner chamber was placed into the outer chamber. After incubation at 37 °C for 16 h, the medium in the inner chamber was removed, and the cells within the inner chamber were wiped off using cotton swabs. Then, the medium in the outer chamber was removed, and cells in the outer chamber were washed with PBS, fixed with ethanol, and washed with PBS again. The migrated cells were stained by crystal violet at room temperature for 20 min and washed with PBS to remove the residual dye. The effect of the test articles on the migration of HUVECs was observed under the microscope. Each condition was performed in triplicates.
HUVEC scratch assay
HUVECs in the logarithmic growth phase were plated into 96-well plates and cultured overnight. The cells were starved in the standard medium without FBS at 37 °C for 16 h, and then wounds were created simultaneously in all wells. The cells were washed with PBS. Different concentrations of BD0801 (Jiangsu Simcere Pharmaceutical Co., Ltd., Nanjing, China) or bevacizumab (Genentech/Roche, San Francisco, CA, USA) were incubated with 20 ng/ml VEGF (Peprotech, Cranbury, NJ, USA, #100–20-10 μg) for 30 min at 37 °C before they were added into the wells. The plate was incubated at 37 °C, and the images of the wells were taken for 24 h. Each condition was performed in triplicates. The relative wound density was calculated as (Original wound area at 0 h-Wound area following treatment at different timepoint)/Original wound area at 0 h × 100%.
Western blot
HUVECs were cultured in T25 culture flasks without FBS for 24 h. Different concentrations of BD0801 (Jiangsu Simcere Pharmaceutical Co., Ltd., Nanjing, China) or bevacizumab (Genentech/Roche, San Francisco, CA, USA) were incubated with 50 ng/ml VEGF (Peprotech, Cranbury, NJ, USA, #100–20-10 μg) for 30 min before they were added to HUVEC culture for 3 min. After incubation, the cells were harvested and lysed. The supernatant of cell lysate was heated at 100 °C for 10 min to be denatured and used in the SDS-PAGE, and the protein was transferred onto a PVDF membrane using a wet transfer system. The membrane was incubated with the primary antibody overnight, washed with Tris-buffered saline Tween (TBST), incubated with the secondary antibody for 1 h at room temperature, washed with TBST buffer, and then developed by ECL kit or luminescence. As for detailed antibody information, please see Supplementary Table S1. The Western blot analysis was repeated using the same protocol three times.
Flow cytometry analysis (FACS)
For flow cytometry, tumors were disassociated with digestion enzyme mixture in GentleMACS Octo Dissociator with Heaters (Miltenyi Biotec, Bergisch Gladbach, Germany). The cells were suspended in FACS staining buffer as 1 × 106 cells/100 μl in 96-well and stained with Fc Block for 5 min and other antibodies for 30 min, away from light at 4 °C. The cells were washed, fixed, and analyzed by FACS LSR Fortessa X20 (BD, Franklin Lakes, NJ, USA). Purified Rat Anti-Mouse CD16/CD32 (Mouse BD Fc Block™), FITC-CD45, PerCP-Cy5.5-CD4, and APC-CD8 antibodies were purchased from BD, and the Catalog numbers are 553,142, 553,080, 550,954, and 553,035, respectively. BV421-Live/Dead was purchased from Thermo Fisher (Catalog No. L34964). APC-Cy7-CD3 antibody was purchased from Biolegend (Catalog No. 100222).
Immunohistochemistry (IHC) and immunofluorescence (IF) staining
The tumor samples harvested at the end of the study were fixed in Zn fixing buffer for about 36 h at room temperature, changed to ddH2O for 5 min, dehydrated, and embedded in paraffin using the Leica ASP300S system and EG 1150H + C system (Leica, Wetzlar, Germany). For double staining of PD-1 and CD8, the paraffin slides were stained with Leica BOND-III using the double staining program with rabbit anti-mouse CD8 (Abcam, Cambridge, United Kingdom, #ab237723) labeled by HRP (Bond Polymer Refine Detection, Leica, #DS9800) and rabbit anti-mouse PD-1 (Abcam, #ab214421) labeled by AP (Bond Polymer Refine Red Detection, Leica, #DS9390). The stained slides were scanned using Leica Aperio CS2. Six fields were viewed and analyzed for each slide/sample using ImageScope (Leica). The percentage of PD-1+ cells within the CD8+ cells was analyzed by ImageJ (NIH, Bethesda, MD, USA), and the average percentage of the six fields was calculated for each sample. For single staining of PD-L1, the paraffin slides were stained with Leica BOND-III using the DAB staining program with rabbit anti-mouse PD-L1 (Cell Signaling Technology, Boston, MA, USA, #64988 s) labeled by HRP (Bond Polymer Refine Detection, Leica, #DS9800). For single staining of CD31, the slides were dewaxed, and the endogenous peroxidase was quenched using 3% hydrogen peroxide. The slides were blocked, stained by CD31 primary antibody (Pharmingen, BD Biosciences, Franklin Lake, NJ, USA, #550274), HRP-conjugated secondary antibody, and DAB (Beijing Zhongshan Jinqiao Biotechnology Co., Ltd., Beijing, China, #PV-9004). The stained slides were scanned using Leica Aeprio VERSA, and the images were analyzed by HALO (Indica Labs, Albuquerque, NM, USA). The microvessels were counted based on CD31 staining, and the MVD was calculated by the total microvessel count divided by the tumor area.
For IF, the tumor samples were harvested and embedded in paraffin, as described in the above section. For staining of PD-1 and CD8 using IF, the paraffin slides were dewaxed with Leica Autostainer XL. EDTA was used as the antigen retrieval reagent. The slides were stained sequentially with rabbit anti-mouse CD8 (Abcam, Cambridge, United Kingdom, #ab237723), goat anti-rabbit IgG H&L Alexa Fluor 594 (Abcam, #ab150080), rabbit anti-mouse PD-1 (Abcam, #ab214421), goat anti-rabbit IgG H&L Alexa Fluor 488 (Abcam, #ab150081) and DAPI (Abcam, #ab104139). The stained slides were viewed by Nikon ECLIPSE Ni-U (Nikon, Tokyo, Japan). Five fields were viewed and analyzed for each slide/sample in red, green, and blue channels. The percentage of PD-1+ cells within the CD8+ cells was analyzed by ImageJ (NIH, Bethesda, MD, USA). The average percentage of the five fields was calculated for each sample.
Animals
All experimental procedures involving animals and their care were conducted in conformity with the State Council Regulations for Laboratory Animal Management (Enacted in 1988) and were approved by the Institutional Animal Care and Use Committee of the WuXi AppTec, People’s Republic of China. Female Balb/c nude mice at 6–8 weeks of age were purchased from Shanghai Sippr-BK Laboratory Animal Co., Ltd. (Shanghai, China). Female C57BL/6 and Balb/c mice of 6–8 weeks of age were purchased from Shanghai Lingchang Biotechnology Co., Ltd. (Shanghai, China).
PC9 xenograft mouse model
To establish the PC9 xenograft mouse model, 5 × 106 PC9 cells per mouse were injected subcutaneously into female Balb/c nude mice’s right flank. As the average tumor volume reached 100–150 mm3, the mice were randomly assigned to experimental groups and treated with vehicle, AZD9291, or BD0801 (Jiangsu Simcere Pharmaceutical Co., Ltd., Nanjing, China), respectively.
PK and efficacy studies in the PC9 mouse model
For the PK study, each dosing group contained nine mice, and three subgroups were formed for sampling at different time points. On day 0, a single dose of different concentrations of BD0801 was injected i.v. into PC9 tumor-bearing mice. The mice were sampled at various time points, and 40 μl serum was collected for each mouse at each time point.
For the efficacy study, tumor-bearing mice were treated with vehicle control, intravenously (i.v.) twice a week, AZD9291 (5 mg/kg dissolved in 0.5% methylcellulose and 0.1% Tween80 in water), orally every day, or different concentration of BD0801 (dissolved in saline), i.v. twice a week. Treatment started on day 0. The tumor volume and the bodyweight of the animals were measured twice a week. On day 24, just before the last dose of BD0801, mice treated with vehicle and BD0801 were sampled for drug exposure analysis. On day 27, mice from the vehicle- and BD0801-treated groups were sampled again before all of the animals were sacrificed. The long diameter (a) and the short diameter (b) of the tumor were measured using a caliper, and the tumor volume was calculated using the following formula: V = 0.5 x a x b2. T/C (%) = TRTV/CRTV × 100%. RTV, relative tumor volume; RTV = Vt/V0; V0 is the tumor volume of the animal when treatment starts; Vt is the tumor volume of the animal someday after treatment; TRTV: the mean RTV of the treatment group; CRTV: the mean RTV of the control group.
Efficacy studies in the 3LL and CT26 syngeneic mouse models
To establish the 3LL syngeneic mouse model, 2 × 106 3LL cells per mouse were injected subcutaneously into female C57BL/6 mice’s right flank. To establish the CT26 syngeneic mouse model, 3 × 105 CT26 cells per mouse were injected subcutaneously into female Balb/c mice’s right flank. When the average tumor volume reached 50–80 mm3, the mice were randomly assigned to each group according to the tumor volume and treated with BD0801, anti-PD-1 antibody (BioXcell, Lebanon, NH, USA, clone #RMP1–14, #BE0146), anti-PD-L1 antibody (BioXcell, Lebanon, NH, USA, clone #10F.9G2, #BE0101), or combinations. Antibodies were injected on the same day sequentially. The tumor volume and the bodyweight of the animals were measured three times a week. Tumor samples harvested at the end of the study were divided into two parts: one part was digested for flow cytometry analysis, and the other part was fixed in Zn fixing buffer for immunohistochemistry.
Statistical analysis
The comparison between BD0801 and bevacizumab in the VEGF/VEGFR2 binding blocking assay and HUVEC proliferation inhibition assay was analyzed by t TEST. The statistics of HUVEC migration inhibition assay, HUVEC apoptosis assay, HUVEC Western blot quantification, FACS, IHC, and IF analysis were analyzed by one-way ANOVA followed by uncorrected Fisher’s LSD. The statistics of scratch assay and tumor volume were analyzed by two-way ANOVA followed by Tukey’s multiple comparisons test. All analyses were performed using GraphPad Prism 8.0 (GraphPad Software Inc., San Diego, CA, USA). P-values < 0.05 were considered statistically significant.