﹛﹛HEK293FT cells (Thermo Fisher Scientific, R70007) were cultured in Dulbecco＊s modified Eagle＊s medium (DMEM) (Thermo Fisher Scientific). Jurkat cells [American Type Culture Collection (ATCC), TIB-152] were cultured in RPMI 1640 medium (Thermo Fisher Scientific). SK-BR-3 cells (ATCC, HTB-30) were cultured in McCoy＊s 5A (modified) medium (Biological Industries). All the culture medium was supplemented with 10% fetal bovine serum (FBS; Biological Industries), 1℅ nonessential amino acids, penicillin (100 U/ml), streptomycin (100 米g/ml), and 12.5 mM Hepes (Thermo Fisher Scientific). HEK293F cells (Thermo Fisher Scientific, R79007) were suspended and cultured in FreeStyle 293 Expression Medium (Thermo Fisher Scientific). All cells were maintained in a CO2 incubator at 37～C.
﹛﹛Jurkat/pIL2-eGFP was developed to monitor the activation of T cells. The Jurkat cell line was transfected with a vector carrying the eGFP reporter gene under the control of the full-length IL-2 promoter (the region from ?648 to ?1 upstream of the translation initiation codon of IL2). After stimulation with anti-CD3 and anti-CD28 antibodies, cells expressing high levels of GFP were sorted into wells of a 96-well plate using fluorescence-activated cell sorter (FACS), and individual clones of the Jurkat/pIL2-eGFP cell line were characterized.
﹛﹛Jurkat/NF-百B-GFP-hCD40 reporter cells were developed to monitor the activation of CD40. Jurkat cells were transfected with a vector carrying the eGFP reporter gene under the control of the NF-百B response element. After stimulation with TNF汐 (10 ng/ml; Sino Biological), cells expressing a high level of eGFP were sorted by FACS, and the Jurkat/NF-百B-eGFP cell line was characterized. The resulting Jurkat/NF-百B-eGFP cell line was infected with lentivirus expressing full-length human CD40. After stimulation with 100 nM hexameric CD40L Fc fusion protein, individual cells with a high GFP signal level were sorted into wells of a 96-well plate by FACS, and individual clones of Jurkat/NF-百B-GFP-hCD40 cells were characterized.
﹛﹛All microfluidic chips were fabricated in polydimethylsiloxane (PDMS) polymers (Sylgard 184 Elastomer Kit; Dow Corning Corp.) using standard soft lithography as previously described (41). Masters were made using one layer of SU-8 photoresist (MicroChem). The depth of the two devices is 40 ㊣ 1 米m to allow the droplet to generate or flow in a monolayer format. Device i is composed of one oil inlet and two aqueous phase inlets where reporter cells, infected cells, and bioassay reagents are added. For device ii, PDMS is bound to a piezoelectric substrate (Y128-cut lithium niobate wafer), where a gold interdigital electrode is patterned with standard lift-off technology and aligned with the fluidic channel above. Microfluidic devices were treated before use with 1% (v/v) 1H,1H,2H,2H-perfluorodecyltrichlorosilane (Alfa Aesar) in Novec HFE7500 fluorinated oil (3M) to prevent droplets from wetting the channel walls.
﹛﹛Aqueous phases containing infected cells and reporter cells were coflowed and partitioned into droplets with hydrodynamic flow focusing in dripping mode on a microfluidic chip (Fig. 2A). The nozzle is 15 米m wide, 40 米m deep, and 10 米m long. The continuous phase was Novec HFE7500 fluorinated oil (3M) containing 2% (w/w) 008-FluoroSurfactant (RAN Biotechnologies). Pressure pumps (Fluigent) were used to generate monodispersed droplets of 100 ㊣ 10 pl at 5000 Hz. The droplets were collected into a 10-ml tube and incubated at 37～C in 5% CO2 to allow antibody secretion and subsequent activity to occur within each droplet before screening.
﹛﹛Droplet fluorescence analysis and sorting operations were performed on a dedicated droplet microfluidic station, similar to that described by Mazutis et al. (15). Pressure pumps (Fluigent) were used to inject the collected droplets into the sorter device (Fig. 2C) at a frequency of 1000 to 3000 Hz. The sorter device was mounted on an inverted microscope (Applied Scientific Instrumentation Microscope) equipped with a 940-nm light-emitting diode illumination source (Thorlabs, M940L3) and a fixed focus laser line (solid-state laser with a wavelength of 405, 488, 561, or 635 nm; Omicron) with photomultiplier tube bandpass filters of 440/40 to 25, 525/40 to 25, 593/46 to 25, and 708/75 to 25 nm (Hamamatsu).
﹛﹛The fluorescence of each droplet was measured as the droplet flowed past an observation constraint in the microfluidic channel where the laser line was positioned. The emitted fluorescence was detected with photomultiplier tubes, converted into corresponding signal output voltages, and recorded by the data acquisition card (FPGA PCIe-7842R). These voltages were then processed by the card and custom LabVIEW software to identify droplets according to their fluorescence intensity and size. These characteristics were used to determine whether each droplet should be sorted.
﹛﹛Droplets were sorted on the basis of the surface acoustic wave deflection as described by Franke et al. (21, 42) with a gigahertz signal generator (Wavetek, model 3010). Sorted droplets were collected in a 1.5-ml Eppendorf tube. Cells were recovered by adding 100 米l of DMEM culture medium, followed by 100 米l of 1H,1H,2H,2H-perfluoro-1-octanol (Sigma-Aldrich, 37053), and then cells were pooled and centrifuged at 400g for 5 min at 4～C for subsequent steps, such as subculture or DNA sequencing.
﹛﹛A human na?ve scFv library was constructed using PBMCs from 30 healthy donors with standard protocols. The phage library was incubated with biotinylated CD40-Fc fusion protein or Her2 recombinant protein (ACROBiosystems) for 2 hours at room temperature, and the phage-antigen complex was captured by Dynabeads M280 (Life Technologies). Bound phages were eluted using glycine-HCl (pH 2.2) for 10 min at room temperature and neutralized with tris-HCl (pH 8.0) to adjust the pH to 7.5. The phagemid DNA was isolated using a plasmid miniprep kit (QIAGEN).
﹛﹛Both phagemids and the lentiviral vector pLV-ef1汐-scFv-Fc were digested with the Sfi I enzyme. The lentiviral vector and scFv genes were isolated after electrophoresis. ScFv genes were ligated into a lentiviral vector. The product of the ligation reaction was transformed into XL1-Blue competent cells by the electroporation transformation technique, and most of the transformed bacteria were plated on LB agar plates. The remaining bacteria were serially diluted and plated to estimate the size of the library. The lentiviral plasmid was prepared using a plasmid midiprep kit (QIAGEN) for lentivirus preparation.
﹛﹛When cell confluence reached 80%, HEK293FT cells were transfected with the lentiviral backbone plasmid and packaging plasmids using polyethylenimine (PEI; Polysciences) transfection reagent. The medium was changed to fresh complete culture medium 6 hours after transfection. The supernatant containing lentivirus was harvested after 48 hours, centrifuged at 300g for 5 min at 4～C, and filtered through a 0.45-米m filter to remove cell debris. Viral titer was measured using a P24 ELISA kit (Clontech). The virus was aliquoted and stored at ?80～C.
﹛﹛For aqueous phase 1, Jurkat/pIL2-eGFP reporter cells were washed with phosphate-buffered saline (PBS) and stained with 1 米M CellTrace Yellow dye (Thermo Fisher Scientific, C34573) for 10 min at 37～C. The stained Jurkat/pIL2-eGFP reporter cells were washed two times with RPMI 1640 and resuspended in cell culture medium (RPMI 1640, 5% FBS, 25 mM Hepes, and 0.1% Pluronic F-68) containing anti-CD28 antibody (1 米g/ml; Invitrogen), which was added here to enhance T cell activation (fig. S1).
﹛﹛For aqueous phase 2, stable Her2-expressing K562 cells were infected with a lentiviral antibody library. The resulting antibody-secreting K562-Her2 cells were washed with PBS and stained with 1 米M CellTrace Violet dye (Thermo Fisher Scientific, C34571) for 10 min at 37～C. The stained cells were washed twice with RPMI 1640 and resuspended in cell culture medium containing 200 nM DY647 (Dyomics, 647-00). K562-Her2 cells infected with positive control lentivirus were resuspended in cell culture medium containing 1500 nM DY647.
﹛﹛Aqueous phases 1 and 2 were injected into the droplet generation chip from different inlets and used as the dispersion phase. Novec HFE7500 fluorinated oil (3M) containing 2% (w/w) fluorosurfactant (RAN Biotechnologies) was used as the continuous phase to produce droplets with an average size of 100 pl. The flow rates of aqueous phase 1, aqueous phase 2, and oil phase were adjusted so that 1 reporter cell and 0.5 antibody-secreting cells were coencapsulated per droplet on average. During droplet production, the cell suspension was cooled using ice water to inhibit antibody secretion. The droplets were collected and incubated at 37～C for 16 hours.
﹛﹛The droplets were first gated to eliminate coalesced droplets and retain only droplets of the desired size. The positive control and the screening population droplets were distinguished on the basis of the different intensities of fluorescence of DY647. For the screening population, droplets were selected for the presence of Jurkat/pIL2-eGFP reporter cells based on CellTrace Yellow signal and K562-Her2 cells based on CellTrace Violet signal. FADS was performed to sort the droplets containing Jurkat/pIL2-eGFP emitting GFP fluorescence. Last, we gated and sorted the droplets with GFP signal colocalized with reporter cell staining signal but not with K562-Her2 signal.
﹛﹛The cells were recovered from the sorted droplets by adding 200 米l of RPMI 1640 medium supplemented with 10% FBS and 24% Nycodenz (Serva, 31000.01), followed by the addition of 50 米l of 1H,1H,2H,2H-perfluoro-1-octanol (Sigma-Aldrich, 370533). After mixing thoroughly and centrifuging at 300g for 5 min at 4～C, the aqueous layer was completely separated and washed with RPMI 1640 medium. The recovered cells were lysed, and antibody genes were amplified from the cells.
﹛﹛To determine the activity of anti-Her2 ℅ anti-CD3 antibody candidates, Jurkat/pIL2-eGFP reporter cells were stimulated with different concentrations of anti-Her2/anti-CD3 antibodies and anti-CD28 antibody (1 米g/ml) in the presence of K562 cells or K562-Her2 cells. After 16 hours of incubation, GFP expression in reporter cells was detected by flow cytometry.
﹛﹛A total of 1 ℅ 105 PBMC cells were cocultured with SK-BR-3 cells at a 1:1 ratio. Different concentrations of anti-Her2 ℅ anti-CD3 antibody or control antibody together with anti-CD28 antibody (1 米g/ml) were added. After 48 hours of incubation, cells were collected and stained with anti每CD3-FITC (BioLegend, 300406) and anti每CD69-APC (BioLegend, 310910) for 30 min at 4～C. T cell activation was determined by flow cytometry. Flow cytometry results were analyzed with FlowJo X software.
﹛﹛The cell supernatant was collected to quantify cytokine release and cytotoxicity. IL-2 (BD, 550611) and IFN-污 (R&D Systems, VAL104) were measured with ELISA kits according to the manufacturer＊s instructions. Cytotoxicity was analyzed by measuring the levels of released lactate dehydrogenase (LDH) using the CytoTox 96 Non-radioactive Cytotoxicity Assay protocol (Promega).
﹛﹛For aqueous phase 1, Jurkat/NF-百B-GFP-hCD40 reporter cells were washed with PBS and stained with 1 米M CellTrace Yellow dye for 10 min at 37～C. The stained Jurkat/NF-百B-GFP-hCD40 reporter cells were washed two times with DMEM and resuspended at 20 million cells/ml in cell culture medium (DMEM, 5% FBS, 25 mM Hepes, and 0.1% Pluronic F-68) containing 16.67 nM DyLight 650每conjugated goat anti-human Fc IgG (Abcam, ab98622) and 24% Nycodenz. The secondary antibody DyLight 650每conjugated goat anti-human Fc IgG was used to mimic the cross-linking action by the Fc receptor (43).
﹛﹛For aqueous phase 2, for the screening population, HEK293FT cells were infected with a lentiviral antibody library and resuspended in cell culture medium containing 500 nM DY405 (Dyomics, 405-00). For positive control droplets, HEK293FT cells were resuspended in cell culture medium containing soluble hexameric CD40L protein and 1500 nM DY405. For negative control droplets, HEK293FT cells were resuspended in cell culture medium containing anti-HEL antibody and 2500 nM DY405.
﹛﹛Aqueous phases 1 and 2 were injected into the droplet generation chip from different inlets and used as the dispersion phase. Novec HFE7500 fluorinated oil (3M) containing 2% (w/w) fluorosurfactant (RAN Biotechnologies, 008-FluoroSurfactant) was used as the continuous phase to produce droplets with an average size of 100 pl. The flow rates of aqueous phase 1, aqueous phase 2, and oil phase were adjusted so, on average, 1 reporter cell and 0.5 antibody-secreting cell were coencapsulated per droplet. During droplet production, the cell suspension was cooled using ice water to inhibit antibody secretion. The droplets were collected and incubated at 37～C for 16 hours.
﹛﹛The droplets were first gated to eliminate coalesced droplets and retain only droplets of the desired size. Negative control droplets, positive control droplets, and screening droplets were distinguished on the basis of the different intensities of blue fluorescence of DY405. For the screening population, droplets were selected for the presence of Jurkat/NF-百B-GFP-hCD40 reporter cells in the droplet based on the yellow fluorescence of CellTrace Yellow dye. Last, FADS was performed to sort droplets containing Jurkat/NF-百B-GFP-hCD40 emitting DyLight 650 and GFP fluorescence. The cells were recovered from the sorted droplets by adding 200 米l of DMEM supplemented with 10% FBS and 24% Nycodenz, followed by adding 50 米l of 1H,1H,2H,2H-perfluoro-1-octanol (Sigma-Aldrich, 370533). After mixing thoroughly and centrifuging at 300g for 5 min at 4～C, the aqueous layer was completely separated and washed with DMEM. The cells were resuspended in DMEM containing 10% FBS and 1% penicillin and streptomycin and then cultured for 1 to 2 weeks.
﹛﹛A single-molecule real-time (SMRT) sequencing platform (Pacific Biosciences) generated long sequencing reads with an average read length of ~20 kb, which could sequence a scFv (subreads) more than 10 times. The circular consensus sequencing program from PacBio SMRT portal software (version 4.1.0) can take multiple subreads of the same SMRTbell sequence and combine them, using a statistical model, to produce one high-quality CCS. After CCS from long sequencing reads was generated, scFv flanking sequences were trimmed, scFv DNA sequences were translated into proteins using the CLC Genomics Workbench (version 11.0.1), and CDR1 to CDR3 regions for heavy and light chains were identified using IgBLAST (version 1.15.0). CD-hit (version 4.8.1) was used to group scFvs with a protein similarity of 95%. The frequency of scFvs for each round was calculated in the Java program. ScFvs that appeared in only one sample were removed because they were likely PCR artifact products. The bar plot was drawn using the R package ggplot2 (version 3.2.1).
﹛﹛Equal amounts of the heavy chain and light chain expression plasmids were cotransfected into HEK293F cells. Five days after transfection, cells were centrifuged at 3000 rpm for 10 min at 4～C, and supernatants were harvested and passed through a 0.45-米m filter. Antibodies were purified with a HiTrap Protein A column (GE) using an ?KTA purifier chromatography system.
﹛﹛Species cross-reactivity of antibody. Cross-reactivity of C04 was assessed by flow cytometry analysis. Briefly, HEK293FT cells were transiently transfected with human CD40 (hCD40)每 or rhesus macaque CD40 (rCD40)每expressing vector using PEI. After 48 hours, HEK293FT-hCD40 or HEK293FT-rCD40 cells were incubated with different concentrations of antibody for 30 min at room temperature. Then, cells were stained with Alexa Fluor 488每conjugated goat anti-human Fc (Life Technologies) at room temperature for 30 min and analyzed by flow cytometry. The fluorescence intensity is equal to the percentage of GFP-positive cells multiplied by the median fluorescence intensity (MFI). The fluorescence intensity was plotted against the antibody concentrations using GraphPad Prism software.
﹛﹛SPR analysis. SPR experiments were performed with a Biacore T200 SPR system (GE Healthcare). In brief, experiments were performed at 20～C in HBS-P+ buffer [0.01 M Hepes, 0.15 M NaCl, and 0.05% (v/v) Surfactant P20]. Anti-his antibody was immobilized on a Series S CM5 chip by amine coupling, and his-tagged cynomolgus monkey CD40 was captured by immobilized anti-his antibody at a flow rate of 10 米l/min for 60 s. Twofold serially diluted CD40 antibodies were injected through flow cells for 120 s followed by a 130-s dissociation phase at a flow rate of 30 米l/min. Before the next assay cycle, the sensor surface was regenerated with glycine-HCl (pH 1.5) for 30 s at a flow rate of 30 米l/min. Background binding to blank immobilized flow cells was subtracted, and equilibrium dissociation constant (KD) values were calculated using the 1:1 binding kinetics model built in Biacore T200 Evaluation Software (version 3.2).
﹛﹛CD40 antibody selectivity. Human CD40 (ACROBiosystems), GITR (ACROBiosystems), OX40 (ACROBiosystems), 4-1BB (ACROBiosystems), or bovine serum albumin (BSA) (Solarbio) was plated onto a microtiter plate at 4～C overnight. The coated wells were blocked with 0.5% BSA in PBS at 37～C for 1 hour. Serially diluted antibodies were added and incubated at 37～C for 1 hour and washed eight times, followed by the addition of goat anti-human IgG每horseradish peroxidase (SouthernBiotech) and incubation at 37～C for 30 min. After eight washes, 2,2＊-azinobis [3-ethylbenzthiazoline-6-sulfonic acid] diammonium salt (ABTS) substrate solution (Thermo Fisher Scientific) was added, and the optical density at 405 nm was measured with a plate reader.
﹛﹛Jurkat/NF-百B-GFP-hCD40 reporter cell assays. For CD40 agonist activity detection, Jurkat/NF-百B-GFP-hCD40 reporter cells were incubated with different concentrations of CD40 agonist antibodies with or without goat anti-human Fc antibody (SouthernBiotech) for 24 hours. GFP expression was detected by flow cytometry.
﹛﹛For the Fc污RIIB dependency experiment, HEK293FT cells were transiently transfected with the Fc污RIIB-expressing vector using PEI. After 36 hours, HEK293FT-Fc污RIIB cells were plated in 48-well plates and cultured overnight at 37～C. Then, Jurkat/NF-百B-GFP-hCD40 reporter cells and different concentrations of C04 or anti-HEL were cocultured with HEK293FT-Fc污RIIB cells for 24 hours. GFP expression was detected by flow cytometry.
﹛﹛For data analysis of Jurkat/NF-百B-GFP-hCD40 reporter cell assays, flow cytometry results were analyzed with FlowJo X software, and the fluorescence intensity was equal to the percentage of GFP-positive cells multiplied by MFI. The fluorescence intensity of cells was plotted against the antibody concentrations using GraphPad Prism software.
﹛﹛Following thawing and recovery of human PBMCs, monocytes were selected by adhering to plastic and then cultured for 8 days in RPMI containing 10% FBS (Gibco), granulocyte-macrophage colony-stimulating factor (100 ng/ml; R&D Systems), and IL-4 (10 ng/ml; R&D Systems). Suspended cells were harvested and confirmed to be DCs by CD11c expression.
﹛﹛B cells were isolated from PBMCs by magnetic selection using CD19 beads (Miltenyi). A total of 1 ℅ 105 DCs or B cells were incubated with different concentrations of C04 with or without goat anti-human Fc antibody (SouthernBiotech) for 48 hours. Up-regulation of the activation marker CD86 was analyzed by flow cytometry (BioLegend). Flow cytometry results were analyzed with FlowJo X software. The MFI of cells was plotted against the antibody concentrations using GraphPad Prism software.
﹛﹛Fc污R/CD40-humanized mice have been described previously and were provided by J. Ravetch (Rockefeller University). Mice were bred and maintained in a specific pathogen每free environment at the Department of Laboratory of Animal Science, Shanghai Jiao Tong University School of Medicine (SJTUSM). All animal care and study were performed in compliance with institutional and National Institutes of Health guidelines and were approved by the SJTUSM Institutional Animal Care and Use Committee (protocol registry number: A-2015-014).
﹛﹛Fc污R/CD40-humanized mice were adoptively transferred with CD45.1+ splenic OT-I cells (2 ℅ 106 cells in 200 米l of PBS per mouse) via tail vein injection 1 day before immunization with 2 米g of CD205 (DEC)-OVA antibody in the presence of the CD40 agonist antibody or the isotype control by intraperitoneal injection. On day 6, spleen cells were harvested. After red blood cell lysis, the single-cell suspension was stained with anti-CD4 (clone RM4-5), anti-CD8 (clone 53-6.7), anti-CD45.1 (A20), and anti每TCR-V汐2 (B20.1) to quantify OVA-specific OT-I CD8+ T cells. OT-I CD8+ T cells were defined as CD45.1+CD8+TCR-V汐2+ cells.
﹛﹛Fc污R/CD40-humanized mice were subcutaneously inoculated with 2 ℅ 106 MC38 cells. When tumor volumes reached 50 to 100 mm3, mice were randomly assigned to different groups (n=5). MC38-bearing Fc污R/CD40-humanized mice were intraperitoneally treated with C04, CP-870,893, or anti-HEL (3 mg/kg, q3d ℅ 2). Tumor growth was monitored every 3 days by measuring the length (L) and width (W) with calipers, and tumor volume was calculated using the formula (L ℅ W2)/2.