Development And Biological Characterization Of Targeted Immune Checkpoint LAG-3 Full Human Antibody

Background: In recent years,tumor immunotherapy has been at the forefront of research.Targeting immune checkpoints,especially programmed cell death protein-1(PD-1)/ programmed cell death ligand protein 1(PD-L1),a breakthrough has been made in the treatment of advanced malignancies.Therefore,it is particularly important to find new immune checkpoints to deeply explore the therapeutic effects of potential therapeutic targets.Suppressive immune checkpoint Lymphocyte-activation gene 3(LAG-3)has huge therapeutic potential.LAG-3 can inhibit T cell activation and cytokine secretion,ensuring immune homeostasis.It has different inhibitory effects on various types of lymphocytes and has synergistic effects with PD-1.Immunotherapy targeting LAG-3 is being actively carried out in clinical trials.The combined immunotherapy of anti-LAG-3 and anti-PD-1 has shown good clinical effects in PD-1 resistant circumstances.In this paper,we successfully prepared a novel fully human monoclonal antibody targeting LAG-3,and identified its biological activity,laying a foundation for the subsequent research.Objective: To screened specific single chain(sc Fv)antibodies against LAG-3 from a large-capacity fully human phage antibody library constructed by the laboratory and to use homologous recombination technology to obtain the fully human anti-LAG-3 antibody and to identify it's biological activity.Method: The fully human phage antibody library constructed and optimized by our laboratory was used to screen fully human anti-LAG-3 single-chain phage antibodies after multiple rounds of "adsorption-elution-amplification" process.ELISA experiments and DNA sequences analysis were carried out to identify the obtained Sc Fvs.And surface plasmon resonance technology was used to determine the affinity constant of anti-LAG-3 human single-chain antibodies.After analysis and preliminary identification.PCR technology,homologous recombination technology and eukaryotic expression system were used to obtain the fully human anti-LAG-3 antibody.The SDS-PAGE electrophoresis was used to identify the purity of the purified fully antibodies,and ELISA experiments were used to confirm the specificity of anti-LAG-3 fully human antibodies The ability of anti-LAG-3 fully human antibodies binding to activated T cells was identified by flow cytometry.Results: 1.Through 3 rounds of screening and enrichment,29 positive clones were obtained that bind to human LAG-3 recombinant protein,of which 16 clones were specific and there were 4 genotypes in 16 positive clones.The results of affinity detection were ranked from No.2,No.8,No.14 to No.13 in descending order.Among them,the single-chain antibody No.2 has the best affinity constant,being 3.48×10-10.2.The expression vector of anti-LAG-3 fully human monoclonal antibodies were successfully constructed.After eukaryotic transfection and affinity purification,the purity of anti-LAG-3 fully human monoclonal antibodies were above 90%.3.The specificity identification showed that LAG-3-2,LAG-3-8,and LAG-3-13 fully human antibodies were specific.Flow cytometry showed that LAG-3-2.LAG-3-8 and LAG-3-13 fully human antibodies can bind to activated T cells,and LAG-3-2 has the best binding activity.Conclusion: With a large-capacity fully human phage antibody library,4 genotypes of specific human single-chain antibodies against LAG-3 were screened out.Subsequently,fully human anti-LAG-3 antibody were constructed by the eukaryotic expression system.Biological activity identification indicated LAG-3-2,LAG-3-8,LAG-3-13 fully human antibodies have high specificity,and all can bind to activated T cells.