ABSTRACT
@#[Abstract] Objective: To construct and purify the recombinant bispecific antibody (BsAb) targeting PD-1 and CD19 and evaluate its activity. Methods: With pCAR1 plasmid as the vector, the eukaryotic expression vector of anti-PD-1/CD19 BsAb was constructed by molecular cloning technology, and then transfected into mammalian cell line CHO-S by PEI reagent for transiently expressing antibody. The BsAb was purified by Affinity chromatography and then identified by SDS-PAGE and WB. The blocking activity of BsAb on PD-1/PD-L1 in vitro was detected by Luciferase reporter gene assay. The activity of antibody (BsAb)-dependent cell (PBMC)-mediated cytotoxicity (ADCC) in vitro was evaluated by lactate dehydrogenase (LDH) cytotoxicity assay. Results: The double plasmid eukaryotic expression vector pCAR1-19X3 was successfully constructed, and anti-PD-1/CD19 BsAb was successfully expressed in CHO-S cells, named pCAR1-19X3-TY. pCAR1-19X3-TY could effectively block the binding of PD-1 to its ligand PD-L1 in vitro, and the EC50 based on the dose-response curve was 0.306 μg/ml. ADCC results showed that pCAR1-19X3-TY could mediate the cytotoxicity of PBMC against Raji cells, and the curve showed a linear upward trend; when the effect/target ratio was 50∶1, the target cell lysis rate of pCAR1-19X3-TY was (38.9±0.3)%, which was not significantly different from that of the positive treatment group (46.7±4.9)% (P>0.05), but significantly higher than that of the negative control group (1.2±0.1)% (P<0.05). Conclusion: The recombinant anti-PD-1/CD19 BsAb can effectively block the binding of PD-1 and PD-L1 and activate PBMC mediated cytotoxicity against Raji cells. pCAR1-19X3-TY has the potential application value in the treatment of B-cell malignant tumor.
ABSTRACT
Antibody-based PD-1/PD-L1 blockade therapies have taken center stage in immunotherapies for cancer, with multiple clinical successes. PD-1 signaling plays pivotal roles in tumor-driven T-cell dysfunction. In contrast to prior approaches to generate or boost tumor-specific T-cell responses, antibody-based PD-1/PD-L1 blockade targets tumor-induced T-cell defects and restores pre-existing T-cell function to modulate antitumor immunity. In this review, the fundamental knowledge on the expression regulations and inhibitory functions of PD-1 and the present understanding of antibody-based PD-1/PD-L1 blockade therapies are briefly summarized. We then focus on the recent breakthrough work concerning the structural basis of the PD-1/PD-Ls interaction and how therapeutic antibodies, pembrolizumab targeting PD-1 and avelumab targeting PD-L1, compete with the binding of PD-1/PD-L1 to interrupt the PD-1/PD-L1 interaction. We believe that this structural information will benefit the design and improvement of therapeutic antibodies targeting PD-1 signaling.