Improving Pharmacokinetic Properties Of CD47 Nanobody By Fusion To Albumin Binding And FcRn Binding Domains

Monoclonal antibodies(Mc Abs)are becoming the drug of choice for the treatment of cancer and autoimmune diseases due to their high selectivity for targets.However,due to its complex multi domain characteristics and macromolecular properties,the application of Mc Abs are limited.Nanobodies are single-domain antibodies derived from the variable region of camel heavy chain,and are currently known as the smallest molecular weight antibody fragments with complete antigen-binding ability,and can be used as an ideal replacement for Mc Abs.However,due to the small size of the nanobody and the lack of Fc structure,the half-life in vivo is too short to obtain an effective in vivo therapeutic effect.Therefore,there is a need to develop strategies to extend the half-life of Nanobodies to improve their metabolic properties in vivo..Currently,strategies such as pegylation,fusion of Fc fragments,binding to albumin,and targeting of FcRn are used to extend the plasma half-life of small molecule drugs.But which method is more effective for prolonging the half-life of nanobodies has not been reported.Therefore,in this study,we chose the albumin binding domain(ABD)and neonatal Fc receptor binding domain(Z_FcRn)to fuse them with a CD47 specific nanobody3D3-2,which was obtained in our earlier stage.The effects of two fusion strategies on improving the half-life of nanobody were evaluated and compared in vivo in mice.The content of this research mainly includes the following two aspects:1.Preparation and identification of recombinant CD47 Nanobody fused with ABD and ZFcRnUsing DNA recombination technology,the DNA sequences of the synthesized ABD and Z_FcRn were fused with nanobody 3D3-2 at the carboxyl end through a linker(G4S)₃and connected to the expression vector to construct the recombinant plasmid p ET22b-3D3-2-ABD and p ET22b-3D3-2-Z_FcRn were transformed into E.coli BL21(DE3)and expressed by IPTG induction.The expressed fusion protein was purified by Ni ion affinity chromatography.The results showed that the expression bacteria transformed with recombinant plasmids were induced by IPTG and showed obvious expression of recombinant ABD and Z_FcRn fusion proteins on SDS-PAGE gel,with molecular weights of 27 k Da and 28 k Da,respectively,in line with expectations.After affinity purification of the expressed nanobodies,purified antibodies with a purity of more than 90%were obtained with yields of 5.5 mg/L and 6.5 mg/L,respectively.Western blot shows that it can bind to specific antibodies.2.Recombinant nanobody 3D3-2-ABD and 3D3-2-Z_FcRn antigen binding and half-life analysis in vivoTo understand the effect of the fusion of ABD and Z_FcRn on the binding of 3D3-2 with CD47 and serum albumin,indirect ELISA was used to detect the binding of 3D3-22,3D3-2-ABD and 3D3-2-Z_FcRn with human CD47,human serum albumin(HSA)and mouse serum albumin(MSA).The results show that the three nanobodies have a high specific binding activity between CD47 and a significant concentration dependence.Compared with 3D3-2,the binding ability of 3D3-2-ABD,3D3-2-ZFcRn and CD47 was reduced after fusion,but not significant(p>0.05).Nanobodies fused to ABD domain have obtained strong specific binding ability to HSA and MSA,and have obvious concentration dependence.Nanobodies fused with Z_FcRn domain did not show the binding activity with recombinant protein FcRn.To understand the effect of fusion of ABD and Z_FcRn on nanobody 3D3-2 in prolonging the plasma half-life,we injected three recombinant nanobodies into ICR mice through the tail vein,and collected serum samples at different time points after the injection.Double antibody sandwich ELISA method was used to determine the concentration of nanobody in serum at various time points,and the data processing software Graphpad prism 5 was used to draw the drug time curve and calculate the terminal half-life.The results showed that the plasma half-life of the nanobody 3D3-2-ABD fused with the ABD domain was about 14 times longer than that of the unfused nanobody 3D3-2.The plasma half-life before and after fusion was 35.82 min and 501.52 min,respectively.The half-life of 3D3-2-Z_FcRn was 71.65 min,which was not significant compared with that of unfused antibody.In addition,it should be noted that the antibody responses of the three kinds of nanobodies were detected 7 days after injection,indicating that they all have immunogenicity.Summarizing the above results,in this study,two new recombinant CD47 nanobodies were obtained by fusing ABD and Z_FcRndomains.Fusion of ABD and Z_FcRn did not affect the binding of nanobodies to CD4 7 antigen,but after fusion of ABD CD47 nanobodies could be combined with HSA and MSA specific binding.In vivo metabolism experiments showed that nanobody can significantly increase its plasma half-life after fusion with ABD.The results of this study provide a feasible strategy for prolonging the in vivo metabolism of nanobodies and have a certain reference value for improving the in vivo efficacy of nanobodies.