Epitope mapping via phage display random peptide library
Bio-panning of a 12-mer phage display random peptide library (SUT12) against the anti-CD147 mAb (clone M6-1B9) was performed as noted in previous research36,41. Briefly, three rounds of biopanning were performed by gradually reducing the amount of the M6-1B9 antibody, ranging from 10 to 5 and 2 µg for each consecutive round of affinity selection. After three rounds of biopanning, 16 individual phage clones were randomly chosen and amplified to assess their binding activity against the M6-1B9 mAb by phage ELISA, as previously reported42. The phagemid DNA from positive phage clones was prepared for nucleotide sequencing through automated DNA sequencing services using the -96gII primer (5′-CCC TCA TAG TTA GCG TAA CG-3′). The amino acid sequences were analyzed using SnapGene software.
Humanization of ScFvM61B9 and property validation
The suitable frameworks in the variable domain of HuScFvM61B9 were assigned from parental ScFvM61B9 using the antibody humanization process of the BioLuminate 4.0 demo software (Schrödinger, LLC, USA)27. Upon submitting the amino acid sequence of ScFvM61B9 heavy and light chain variable domains, a PDB number 6N4Q crystal structure containing a mouse Fab template was selected for conforming to the possible structure. Subsequently, the human frameworks of PDB number 5HYS were automatically retrieved to substitute the frameworks of the modelled ScFvM61B9 structure to retain the canonical shape of antibody CDR loops. The structure comparison and RMSD calculation of the designed HuScFvM61B9 versus parental ScFvM61B9 were performed using UCSF Chimera 1.15 software. The CDR regions of the generated HuScFvM61B9 structure were deduced using the PyIgClassify Web server43. The humanness of HuScFvM61B9 was calculated using the T20 score analyzer tool35.
Molecular model of HuScFvM61B9 against CD147 domain 1
The generated structure of HuScFvM61B9 from BioLuminate was submitted to the ClusPro 2.0 Web server along with the CD147 domain 1 extracted from PDB number 5X0T44. The antibody mode option was selected to automatically mask non-CDR regions in this in silico protein–protein docking process.
Construction of plasmid expressing HuScFvM61B9
The amino acid sequence of modified ScFvM61B9 was reverse transcribed and optimized using the GenScript web service for proper expression in E. coli. The HuScFvM61B9 coding sequence containing 5′ NheI and 3′ HindIII restriction sites was synthesized (GenScript, USA). The synthesized polynucleotide was subsequently digested with NheI and HindIII and cloned into the NheI and HindIII sites of the pET-21a plasmid vector to generate the pET-21a-HuScFvM61B9(HIS6X) plasmid.
Expression and purification of HuScFvM61B9
The pET-21a-HuScFvM61B9(HIS6X) plasmid was transformed into competent E. coli Origami B (DE3) cells to produce a humanized single-chain variable fragment of M6-1B9 containing a His6x tag (HuScFvM61B9). A single colony was picked and grown in a 5 ml super broth medium starter culture overnight at 37 °C. Then, the culture was inoculated into a 500 mL SB medium containing 0.05% glucose and 100 µg/mL ampicillin at 37 °C until an OD600 of 0.8 was reached. Protein expression was induced by adding 50 µM IPTG, and this process continued for 16–18 h at 20 °C. The induced bacteria expressing HuScFvM61B9 were washed twice with PBS and lysed via three sonication times of 5 min each at 0.5 cycles with 80% amplitude on ice. The lysed bacteria were subjected to freeze–thaw cycling, followed by centrifugation at 4000g for 30 min at 4 °C. The cell lysate was collected, and HuScFvM61B9 was purified via affinity chromatography on a HisTrap HP column (GE Healthcare) using ÄKTA Prime plus. The collected fractions were analyzed via SDS-PAGE on a 15% gel with PageBlue staining to determine the purity of the HuScFvM61B9 proteins. The collected fractions were subjected to Western blot analysis. Proteins were separated by SDS-PAGE, transferred onto a PVDF membrane, and subsequently probed with an HRP-conjugated anti-His-tag antibody (BioLegend, 652504) at 1:3000 dilution. The reaction was developed using a chemiluminescent substrate detection system.
Affinity determination of HuScFvM61B9 via biolayer interferometry
Biolayer interferometry was used to analyze the binding affinity of HuScFvM61B9 to human CD147-BCCP and compared to its parental mouse mAb, M6-1B9. The CD147-BCCP was in situ biotinylated in E. coli strain Origami B harboring pAK400cb-CD147-BCCP as described previously27,45. The CD147-BCCP was validated by Western immunoblotting using either M6-1B9 mAb and followed by HRP-conjugated anti-mouse Igs antibody or HRP-conjugated Streptavidin, and stored at − 20 °C for further analysis. Two hundred microliters of 2 mg/ml crude CD147-BCCP were immobilized on Streptavidin biosensor tip (Sartorius FortéBio) and then placed to buffer (0.05% Tween-20 in PBS) to generate the baseline. To determine the association signal, the tips were subsequently placed into either mouse anti-CD147 mAb clone M6-1B9 or HuScFvM61B9 at 20, 10, and 5 μg/ml. After that, the tips were placed into the buffer to generate the dissociation signal. The constants of association (ka) and dissociation (kd) were analyzed, and the KD was calculated via the same formular by BlitZPro 1 program.
Specificity of HuScFvM61B9 against the wild-type CD147 and mutant CD147
To assess the specificity of HuScFvM61B9 by Western immunoblotting, wild-type CD147-BCCP (CD147WT-BCCP) and mutant CD147-BCCP (CD147Δ32DL33-BCCP) were generated. Briefly, deletion of 32DL33 in EDLGS of pAK400cb-CD147-BCCP was performed using QuickChange Lightning Multi Site-Directed Mutagenesis kit (Agilent) following the manufacturer’s instruction. Mutated primer DNA sequences are listed as follows.
The pAK400cb-CD147-BCCP-del32DL33 was subsequently transformed into competent E. coli DH5α, and the clones were selected on Luria–Bertani (LB) agar containing 25 μg/ml of chloramphenicol. The plasmid from transformed E. coli DH5α was prepared by QIAGEN Miniprep kit (QIAGEN) and sequenced. The corrected pAK400cb-CD147-BCCP-del32DL33 was transformed to E. coli strain Origami B to produce CD147Δ32DL33-BCCP, and validated as described previously28,45. Crude lysate protein at 1:2 dilution of both CD147WT-BCCP and CD147Δ32DL33-BCCP were subsequently separated by SDS-PAGE, transferred onto a PVDF membrane, and subsequently probed with HuScFvM61B9 (1 μg/ml) followed by HRP-conjugated anti-His-tag mAb at 1:1000 dilution, M6-1B9 mAb (0.5 μg/ml) followed by HRP-conjugated anti-mouse Igs (Dako, P0260) at 1:2000 dilution and HRP-conjugated Streptavidin (Seracare, 5270-0029) at 1:500 dilution. The reaction was developed using a chemiluminescent substrate detection system.
Binding assay of HuScFvM61B9 by indirect ELISA
To investigate the binding activity of HuScFvM61B9 to CD147, the indirect ELISA was performed. The microtiter plates were immobilized with 50 µL of 10 µg/mL CD147Rg and incubated overnight at 4 °C in a moist chamber. The other steps were performed at ambient temperature in a moist chamber. The coated wells were washed three times with a washing buffer (0.05% Tween 20 in PBS), followed by blocking with 2% skim milk in PBS for 1 h. After washing, purified HuScFvM61B9 protein was added at various concentrations to the wells and incubated for 1 h. After washing three times, 50 µL of HRP-conjugated anti-His-tag mAb at 1:5000 dilution was added and incubated for 1 h. The reaction was developed using a TMB substrate, and then the reaction was ceased with 1 N HCl. Absorbance at 450 nm was measured using an ELISA reader.
Inhibition binding analysis of HuScFvM61B9 with anti-CD147 mAbs via inhibition ELISA
Inhibition binding activity of purified HuScFvM61B9 was assessed using the inhibition ELISA. Fifty microliters of 10 µg/mL CD147Rg were immobilized on a microtiter plate and incubated overnight at 4 °C in a moist chamber. The other steps were performed at ambient temperature in a moist chamber. Non-specific protein binding was blocked using 2% skim milk in PBS. Fifty microliters of 1 and 5 µg/mL mouse anti-CD147 mAb (M6-1B9 and M6-1E9) were added and incubated for 1 h. The microtiter plate was washed three times with 0.05% Tween20 in PBS and 50 μL of 10 µg/mL purified HuScFvM61B9 was added to the well. After 1 h of incubation, the microtiter plate was washed three times with 0.05% Tween20 in PBS. HRP-conjugated anti-His-tag mAb at 1:5000 dilution was subsequently added to the well and incubated for 1 h. TMB substrate was then added to develop the reaction. The absorbance at 450 nm was measured using a microplate reader after adding 1 N HCl.
Immunofluorescence analysis of the reactivity of HuScFvM61B9 to CD147 on SupT1 cells
Flow cytometric analysis was performed to evaluate the functional reactivity of HuScFvM61B9 against CD147 on a human T-cell lymphoblastic lymphoma cell line, SupT1, compared to its parental M6-1B9 mAb25. SupT1 cells were washed three times with a FACS buffer and adjusted to 107 cells/mL with 10% human AB serum for 30 min on ice to block the Fc receptor. Fifty microliters of 10 µg/mL HuScFvM61B9 or M6-1B9 mAb was added into 50 μL of blocked cells and incubated on ice for 30 min. The cells were washed twice with 1% FBS in PBS. After washing, a PE-conjugated anti-His-tag antibody (BioLegend, 362603) at 1:20 dilution or conjugated anti-mouse Igs was added and incubated on ice for another 30 min. Finally, the cells were washed three times and fixed with 1% paraformaldehyde in PBS. The fluorescence intensity of the stained cells was analyzed using a BD FACSCelesta instrument and FlowJo software.
Generation of CD147 knockout Jurkat cells for analysis the specificity of HuScFvM61B9
For the generation of CD147 knockout Jurkat cells, the sgRNA targeting CD147 molecule34 was generated using the GeneArt Precision gRNA Synthesis kit (Invitrogen, A29377). The RNP complex was prepared by mixing 150 μg/mL of SpCas9 (Integrated DNA Technologies) and 90 μg/mL of sgRNA at a 1:3 molar ratio, then incubated for 10 min at room temperature. 1 × 106 Jurkat cells were nucleofected with RNP using the Cell Line Nucleofector Solution V (Lonza, VCA-1003) and the program X-005. On day 11, the edited cell pool (CD147 knockout Jurkat cells) was used to analyze the specificity of HuScFvM61B9 by immunofluorescence analysis using the protocol described above except 40 μg/ml concentration was used. Untransfected Jurkat cells were used as a positive control.
Effect of HuScFvM61B9 on T-cell proliferation in PBMC after anti-CD3 antibody activation
T cell proliferation was assessed using 5-carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling. PBMCs were washed with PBS 3 times and adjusted to 107 PBMCs/ml with PBS. CFSE (Sigma-Aldrich) at a final concentration of 1 μM was added to PBMCs for 10 min at 37 °C. Excess CFSE was quenched with cold 10% FBS-RPMI. CFSE-labelled PBMCs were washed twice with RPMI. To determine the effect of HuScFvM61B9 on T-cell proliferation in PBMC after anti-CD3 activation, triplicate aliquots of 105 CFSE-labeled PBMCs were cultured with immobilized CD3 mAb OKT3 (12.5 ng/mL) with various concentrations of purified HuScFvM61B9. Mouse M6-1B9 mAb was used as a control. The culture was incubated for 5 days in a 5% CO2 incubator at 37 °C. Cells from each treatment were then washed twice with PBS, fixed with 1% formaldehyde in PBS and analyzed using BD Accuri C6 flow cytometer and FlowJo software.
Ethical declarations
The study protocol was approved by the Ethics Committee, Faculty of Associated Medical Sciences, Chiang Mai University, Thailand (AMSEC-64EX-016).
