Immunoreactivity of humanized single-chain variable fragment against its functional epitope on domain 1 of CD147

Abstract

Domain 1 of CD147 participates in matrix metalloproteinase (MMP) production and is a candidate for targeted therapy to prevent cancer invasion and metastasis. A functional mouse anti-CD147 monoclonal antibody, M6-1B9, was found to recognize domain 1 of CD147, and its respective mouse single-chain variable fragment (ScFvM61B9) was subsequently generated. The EDLGS epitope candidate for M6-1B9 was identified using the phage display peptide technique in this study. For future clinical applications, humanized ScFv specific to domain 1 of CD147 (HuScFvM61B9) was partially adopted from the hypervariable sequences of parental mouse ScFvM61B9 and grafted onto suitable human immunoglobulin frameworks. Molecular modelling and simulation were performed in silico to generate the conformational structure of HuScFvM61B9. These results elucidated the amino acid residues that contributed to the interactions between CDRs and the epitope motif. The expressed HuScFvM61B9 specifically interacted with CD147 at the same epitope as the original mAb, M6-1B9, and retained immunoreactivity against CD147 in SupT1 cells. The reactivity of HuScFvM61B9 was confirmed using CD147 knockout Jurkat cells. In addition, the inhibitory effect of HuScFvM61B9 on OKT3-induced T-cell proliferation as M6-1B9 mAb was preserved. As domain 1 is responsible for cancer invasion and metastasis, HuScFvM61B9 would be a candidate for cancer targeted therapy in the future.

Engineered CD147-Deficient THP-1 Impairs Monocytic Myeloid-Derived Suppressor Cell Differentiation but Maintains Antibody-Dependent Cellular Phagocytosis Function for Jurkat T-ALL Cells with Humanized Anti-CD147 Antibody

“…The K D value of Takatamab (2.05 ± 0.30 × 10 −9 M) demonstrated a comparable degree to its parental antibody, M6-1B9 (2.17 ± 0.55 × 10 −9 M). Additionally, the K D of Takatamab was higher than that of the HuScFv form, as reported in our previous study [ 16 ]. In addition, the epitope recognized by Takatamab was proven to overlap with parental mAb M6-1B9 by competitive ELISA.…”

“…The pVITRO1-HuM6-1B9-IgG1/κ plasmid was constructed through subcloning of the V L C L and V H C H regions of HuScFvM6-1B9 [ 16 ] into a pVITRO1-Trastuzumab-IgG1/κ plasmid. This plasmid was designed to produce the Fc portion of the recombinant antibody in the human IgG1 isotype, known for its ability to bind to all subclasses of human FcγR receptors [ 35 ] and its potential to stimulate immune-effector functions, including ADCP [ 35 ].…”

“…Jurkat T cells, clone E6-1 (ATCC, Manassas, VA, USA), CD147 KO Jurkat [ 16 ], WT THP-1 (ATCC, Manassas, VA, USA), and CD147 KO THP-1 were cultured in the Roswell Park Memorial Institute 1640 (RPMI-1640) medium supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 U/mL penicillin, 100 μg/mL streptomycin, and 2 mM L-glutamine. CHO-K1 cells were purchased from ATCC (ATCC, Manassas, VA, USA) and maintained in the Iscove’s modified Dulbecco’s medium (IMDM), supplemented with 10% FBS, 100 U/mL penicillin, 100 μg/mL streptomycin, and 2 mM L-glutamine.…”

“…To establish CD147 KO THP-1 cells, the formation of ribonucleoprotein (RNP) complex, including spCas9 (Integrated DNA Technologies, Coralville, IA, USA) and single guide RNA (sgRNA) targeting CD147 was prepared as previously reported [ 16 ]. The RNA complex was then transfected into 5 × 10 5 THP-1 cells using 4D-Nucleofector TM X Kit S (Lonza, Basel, Switzerland) with the FF100 program of the 4D-Nucleofector TM X unit.…”

“…Hence, ADCP can enhance the clearance of living T-ALL. Recently, a humanized single chain variable fragment (scFv) called HuScFvM6-1B9 specific to CD147 has been generated and characterized [ 16 ]. In this study, we further synthesized a fully humanized antibody called HuM6-1B9 or Takatamab for equipping with M-LPS to target Jurkat cells with ADCP function.…”