The Humanization Of PD1 Murine Antibody And Verification Of Activity

As an important checkpoint,PD1 involved in development and function of T cells.PDL1,the ligand of PD1,expressed on the cell surface of numerous cancers.PD-1-mediated signaling inhibits T lymphocyte glucose consumption,cytokine production,proliferation,and survival.Inhibition of PD1 interacted with PDL1 can enhance the activity of T cells which increases immune response.The antibody humanization is usually replacing murine genes with human genes through the gene modification.With keeping its affinity and biological activity,it gets a great decrease of immunogenicity and also improves the safety in medical treatment.We have already got the hybridomas of PD1 murine monoclonal antibody and purified monoclonal antibodies.First of all,we put effort on its subtype identification and found it had two Kappa chains and Ig G chains.Then we transformed RNA into c DNA and cloned variable gene regions,connected these variable genes to the human Kappa chain and Ig G chain with the same eukaryotic expression vector.We compared the affinities between PD1 murine monoclonal antibody and PD1 murine chimeric antibody after cell expression by flow cytometry.We found that PD1 murine chimeric antibody had a decreased ability in antigen-binding.We humanized four framework regions(FRs)which belongs to variable gene regions and got VK1-39 and VK2-28 as two humanized light chains,VH3-23,VH3-23-M,VH4-34 and VH4-34-M as four humanized heavy chains.We tried all eight combinations but it seemed that chimeric antibodies with VK2-28 heavy chain either had very low expression or poor affinities.Thus,we put effort on both VK1-39 &VH3-23-M and VK1-39 & VH4-34-M.After purifying these two kinds of PD1 humanized chimeric antibodies,we used some DTT as a reducing agent to check thespacial conformation in good condition.To rule out the effects from antibody purification,we test their affinities again.We found that VK1-39 & VH3-23-M showed the minimum difference with PD1 murine chimeric antibody and it has a higher purification than VK1-39 & VH4-34-M.Finally,we performed mixed lymphocyte reaction(MLR)to detect the biological activity both of VK1-39 & VH3-23-M and murine chimeric antibody.When in a high concentration dose,VK1-39 & VH3-23-M showed a better activity.In conclusion,we got PD1 murine chimeric antibody and PD1 humanized chimeric antibody successfully,which were humanized in constant regions or FRs of variable regions,and demonstrated that they all have good affinities and biological activity.