Preparation Of Humanized PD-L1 MAb And Construction Of EGFR×PD-L1 Dual-target Chimeric Antigen Receptor

Objective:To generate a humanized monoclonal antibody?m Ab?against human programmed death ligand-1?PD-L1?that is suitable for potential application in clinical detection and treatment,and to build a specific chimeric antigen receptor?CAR?targeting both tumor-associated antigen epidermal growth factor receptor?EGFR?and PD-L1 by lentiviral vector expression system.Methods:?1?Obtaining PD-L1 antibody by hybridoma technology.Five BALB/c mice were immunized abdominally three times each with a recombinant human PD-L1 extracellular domain–Fc fusion protein,the resulting titer was determined,and two mice with high titers were selected.After the selected mice were given a shock immunization,their spleen cells were fused with mouse myeloma cells?SP2/0?.Positive clones were screened by indirect enzyme-linked immunosorbent assays?ELISAs?.The selected hybridomas were injected into the abdominal cavities of mice to obtain ascites,and antibodies were purified from these ascites by saturated ammonium sulfate precipitation and protein G affinity chromatography,then lyophilized for use.Regarding the anti-PD-L1 antibody,its binding to 293T cell transfected by PD-L1 expression vector was assessed thro?gh Western blot,its blocking effect on the binding of PD-1 with PD-L1 was detected by Fluorescence activated cell sorting?FACS?in transfected 293T cells and by competitive ELISA.Their m Ab isotypes were analyzed by ELISA.Affinity was measured by Fortebio Octet96.Clones with highly secretory antibodies were selected for further subcloning to establish stable lines.The binding activity of engineered PD-L1-sc Fv to PD-L1protein was further measured by ELISA.?2?Humanization of the murine antibodies conducted by the transplantation of complementarity-determining regions?CDRs?.Full-length murine anti-PD-L1 antibody was sequenced to obtain the sequences of the heavy chain variable region?VH?and light chain variable region?VL?;these sequences were compared with an immunogenetics database?IMGT?to select the humanized antibody framework region?FR?containing the highest similarity to murine antibodies.The mouse CDR region was then implanted into the humanized antibody FR,and the CDRs of the parental antibody were grafted into the human receptor vector to obtain five humanized light chains and five humanized heavy chains of the parent antibody,resulting in the expression of 25 humanized antibodies in HEK293T cells.Affinity determination was performed at different concentrations to rank these antibodies.The top three purified antibodies from the affinity ranking were selected.Finally,one humanized antibody?VH5+VL5?with a binding affinity similar to that of the chimeric antibody was purified for further study.?3?Construction and expression of the EGFR×PD-L1 dual-targeting CAR.The light chain and heavy chain variable region?VL,VH?genes from the anti-human EGFR m Ab hybridoma were amplified by 5'RACE technology to construct sc Fv,which was then cloned into the eukaryotic vector pc DNA3.1 for expression identification.To establish the EGFR×PD-L1 dual-targeting CAR structure,the sequences of EGFR-specific CAR?introduction of CD137 synergistic signal intracellular domain?and humanized PD-L1-sc Fv linked by 2A sequence were synthesized and cloned into the lentiviral p LVX-EF1a-IRES-Zs Green1 expression vector.Lenti-X Packaging Single Shots?VSV-G?were used to co-transfect 293T cells for obtaining packaging virus.Fluorescence-activated cell sorting?FACS?was used to determine CAR membrane expression,and ELISA was performed to detect PD-L1-sc Fv expression in the 293V cell supernatant after infection.With the same protocol,the above CAR construction was also cloned into the GV401 vector to prepare infectious lentivirus.The ability of the resulting membrane-expressing lentivirus?KL46407-1?to infect and activate human peripheral blood T cells was assessed.Results:?1?One monoclonal antibody?11E3,subclass Ig G2a?was identified from the 10 selected hybridomas,which bound PD-L1 with high affinity(K_D1.66×10^-10mol/L)and high ligand-blocking performance.?2?The humanized transformation of11E3 showed stable affinity(K_D2.67×10^-10mol/L).?3?Based on the expression of EGFR-sc Fv,we further constructed EGFR-CAR and PD-L1-sc Fv double-modified lentivirus vectors which containing secreted PD-L1-sc Fv?CTC0537-1?or membrane-expressed PD-L1-sc Fv?CTC0537-2?.The infectious rate in 293V cells by both lentivirus were 10%.In lentiviras?CTZ0431-1?infected 293V cells,EGFR-sc Fv was effectively expressed on the cell membrane and PD-L1-sc Fv was detected in the culture supernatant,while both EGFR-sc Fv and PD-L1-sc Fv were effectively expressed on the cell membrane in lentiviras?CTZ0431-2?infected 293V cells.The CAR expression rate of activated T cells infected with membrane-expressing lentivirus?KL46407-1?was 39.3%.Conclusion:We prerared a group of mouse anti-human PD-L1 hybridomas,from which a clone with high-affinity binding to human PD-L1 as well as high ligand receptor-blocking performance was obtained,thus supporting its potential application in PD-L1 detection and anti-PD-1 effectiveness evaluation.Lentiviral vectors with dual expression of EGFR-CAR and PD-L1-sc Fv were successfully constructed,among which the EGFR-CAR had a moderate binding affinity;therefore,it has potential to become a key tool for studying solid tumor treatment in EGFR-targeting and anti-PD-L1 antibody double-modified CAR-T cells.