Generation of nanobodies (Nbs)
The anti-human CD19 and CD20 nanobodies were selected from the bactrian camel naïve phage display library. First, 500ul phage and 500ul prepared biotinylated CD19 or CD20 antigen(Shanghai Anyan, China) using EZ-Link Sulfo-NHS-LC-Biotin (Thermo) were put into 1.5 ml centrifuge tube together, rotated for 1h at room temperature. Then added 50ul streptavidin magnetic beads (invitrogen) and incubated for 30 min, phage were eluted with 800ul pH 2.7 Tris-HCl after several times washing. Finally, the neutralizated eluent were added to TG1 bacteria and incubated at 37℃ for 30min, then plated on 2YT plates supplemented with 2% glucose, 100ug/ml ampicillin, 50ug/ml and cultured overnight at 37℃. Collect the colonies, named the first round of panning library and stored in glycerine for later use. Apart of the colonies were retained to precipitated phage, the second round of panning is carried out with the same methods as first, repeated for 4 times. Several single colonies from 3 and 4 round of panning plates were picked and identified. The identified Nb sequence linked with IgG Fc, His-tags, and then recombinant to pCZN1 vector, the purified Nbs were analysed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).
Fluorescent semiconductor quantum dots (QDs) immunofluorescence
Daudi cells were mounted on the cell slide by centrifuge with 1000rpm, 15min, and fixed with 4% paraformaldehyde for 10 minutes, washed the cells with TBS and permeabilized with 0.1% Triton X-100 for 20 minutes, blocked by 5% bovine serum albumin for 30 min. Then stained with a 1:250 dilution of Nbs in TBS or TBS alone at 4℃ for 12h, next day, incubated with a 1:200 dilution of biotinylated anti-his-tag secondary antibody at RT for 1 h, blocked again and incubated with QD-streptavidin for 30min, followed by imaging under a fluorescence microscope.
Surface Plasmon Resonance (SPR)
To detect the affinity of the obtained nanoantibodies, SPR were used. The experiment was operated using a Biacore T200 system (Healthcare Life Sciences, GE). Mouse anti-human IgG (FC) antibodies were immobilized on a sensor chip (GE Healthcare) to capture the nanobody. Next, the nanobody was injected into the experimental channel at a flow rate of 10μL/min. Finally, 7 groups of serial twofold dilutions of 10μg/mL human CD19/CD20 antigen were successively injected into the test channel and reference channel at the flow rate of 30μL/min, the association time was 120s, and the separation time was 300s. Kd values were calculated using Biacore T200 analysis software.
Cell lines, patient samples
The following cell lines were used: K562 (a chronic myelogenous leukemia line), Daudi, Raji (burkitt lymphoma cell line), and 293T (an embryonic kidney cell line). All cell lines were purchased from ATCC and maintained in RPMI 1640 supplemented with 10% fetal bovine serum (BI) and 1% penicillin-streptomycin (Solarbio), while 293T cells were cultured in DMEM. To generated the luciferase-expressing cell lines, wild-type tumor lines were transduced with lentiviral vector encoding firefly luciferase, then followed by puromycin selection of luciferase-positive cells up to 14 days. ALL cells were obtained by gradient centrifugation using Lymphocyte Separation Medium (Solarbio), cultured in RPMI 1640 supplemented with 10% FBS.
CARs construction
For CD19 Nb CAR and CD20 Nb CAR, nanobodies were incorporated into basic CARs that were composed of IgG4 hinge, CD8 transmembrane domains, 4-1BB and CD3ζ signaling domain. For Bispecific Nb CAR, the anti-CD20 and anti-CD19 nanobodies were linked by (EAAAK)3, then combined with basic CARs. All CARs included a fluorescent protein tracer linked to a P2A sequence. Then complete CARs sequences were cloned into a lentiviral plasmid backbone under the regulation of a human EF-1α promoter. The lentiviruses were produced by transfecting 293 T cells. concentrated by ultracentrifuging, concentrated CAR lenti-virus was immediately stored at -80℃.
Western blot
Proteins of 293T cells were harvested after 72h of transduction, then western blots were performed using mouse anti-human CD3ζ primary antibody at 500:1 (Thermo).
Primary T cells isolation, expansion and transduction
Peripheral blood that collected using Heparin or Ethylene Diamine Tetraacetic Acid (EDTA) anticoagulant blood vessel were purified by density gradient centrifugation (TBD), then CD3 positive T cell were enriched by positive selection using the magnetic bead separation (Miltenyi Biotec).
For optimize T cell expand conditions, isolated T cells were cultured in X-VIVO15 medium (Lonza) supplemented with 200 or 400U/ml IL-2, with or without 10% FBS or autologous plasma, at a density of 1×106 cells/ml, then activated with immobilized 2ug/ml anti-human CD3/CD28 antibody (Peprotech) or together with additional 5ug/ml RetroNectin for 24h, 48h, 72h, respectively.
For transduction, a 48-well cell culture plate that coated with 20ug/ml RetroNectin (Takara Bio) were used. First, according to multiplicity of infection (MOI) of 3, calculated relevant volume of virus, virus were added to prepared 48-well plate and centrifuged at 32℃ (2000g, 2h). Then, T cells were collected from the plates that post 48h of activation, centrifuged with viral supernatants at 32℃ (1000g, 1h) in the presence of 8ug/ml polybrene (Solarbio), 200 U/ml IL-2, 1% penicillin-streptomycin, followed by incubated overnight at 37℃, 5% CO2. After 24h, add equal volume of medium, then T cells were expanded at a density of 0.7-1×106 cells/ml, cell numbers were counted every 2 days. CAR expression was detected by flow cytometry, expand cells were used for in vitro assay on day 10.
Flow cytometry
For tumor cells expressing CD19 and CD20, 5×105 tumor cells were harvested and washed twice with PBS. Then, tumor cells were stained with 20ul of APC-conjugated mouse anti-human CD19 (BD) and 20 ul of PE-conjugated mouse anti-human CD20 (BD) at room temperature and protected from light for 30 min, washed with PBS once, and resuspended in FACS buffer for assessment. For CAR expression, T cells were stained with PE-conjugated mouse anti-human CD3, CAR-T cells were gated on FITC+PE+. For viability and CD8/CD4 ratio, T cells were stained with 7-AAD, APC-Cy7TM-conjugated mouse anti-humanCD8, BV510-conjugated mouse anti-human CD4 (BD). For T cell activation, after overnight incubation at the E: T of 2:1, the CD69 were detected using the PE-conjugated CD69 antibody (Biolegend). All data were recorded using a FACSCelestaTM Flow cytometer and analysed by Flowjo 10.
CAR-T cell proliferation
For proliferation assays, tumor cells were treated with 20μg/mL mitomycin C (MCE) for 12h. CAR-T cells were labeled with 1µM CellTrace Far Red (Life Technologies) in PBS at 37°C for 20min, then 4-5fold complete medium were added, some CAR-T cells were collected for flow cytometry analysis after 1 hours incubated. Then, labeled T cells were co-cultured with treated target cells at a 2:1 effector/target ratio in X-VIVO15 medium without IL-2 for 5days, followed by flow cytometry analysis, CAR T cells were gated on GFP signals.
ELISA
CAR-T cells and tumor cells were co-cultured at ratio of 2:1 in X-VIVO medium without additional cytokines in 24-well plates, after 24 hours, the supernatant was collected and used for IL-2 ELISA measurements (Biolegend).
Cytotoxicity assay
For tumor cell lines, cytotoxicity was evaluated by luciferase report assay. CAR-T cells and tumor cells were co-cultured at ratio of 2:1 for 24 h in 96-well plates. The same volume of assay buffer was added into the wells and incubated for 10 min at room temperature, then luminescence values were obtained by Microporous Plate Detector. Percentage of specific lysis was calculated by the following formula:
For tumor cells derives from ALL patient, cytotoxicity was evaluated by lactate dehydrogenase (LDH, Dojindo). Primary ALL cells were isolated through gradient centrifugation and then some cells were used to test the expression of CD19 and CD20 by Flow cytometry. Primary ALL cells were incubated with CAR-T cells for 18h, experiment stetting was followed to instruction of test kits. Before added assay buffer, lysis buffer was put to Target Maximum Release and Volume Correction Control wells, after 30min incubated at room, the OD490nm were obtained by Thermo Scientific Microplate Reader. Percentage of specific lysis was calculated by the following formula:
Statistical analysis
All statistical analyses were performed using SPSS 26.0 and GraphPad Prism version 7. Data variance were analyzed by one-way ANOVA. *p<0.05, **p<0.01, ***p<0.001, ns, not significant (P>0.05). All data were shown as means ± SD with three independent replicates.