Humanization Of PD-1 Nanobodies And Its Recombinant Expression In E.coli

Objective:Programmed cell death protein-1?pd-1?is an inhibitory receptor protein located on the surface of T cells and regulates the immune response together with its ligand PD-L1.Tumor cells can upregulate expression of PD-L1 on cell surface and activate PD-1/PD-L1 signaling pathway of T cells by ligation with PD-1 to escape from immune system attack.Therefore,the development of antibodies targeting PD-1 protein has garnered a great deal of attention around the world.In the previous work,a camel-derived nanobody with high affinity to PD-1 protein was screened out in our laboratory.In this study,we intend to humanize this camel-derived nanobody to reduce immunogenicity to develop a potential therapeutic anti-PD1 antibody.Methods and Results:1.In this study,framework regions?FR?and complementary determining regions?CDR?of this camel-derived nanobody were numbered according to the IMGT Scientific chart for the V-domain.Two amino acid sequences of humanized antibodies,Hu1C6 and Hu2C6,were designed based on two different humanized templates respectively.Based on the sequence of Hu2C6,another amino acid sequence,hAC6,was designed by back mutation at Trp36^th to Ala50^th.Then,hBC6 was designed by back mutation at Val5^th,Leu11^th,Ala88^th and Leu125^th based on hAC6.2.These four humanized nanobodies were expressed in Escherichia coli by combination of multiple plasmids and host.Three of the humanized nanobodies were successfully expressed using plasmid pET-28a but accumulated as inclusion bodies,while Hu1C6 was not expressed in E.coli.Lower culture temperatures didn't increase the yields of soluble protein effectively.Also,the mammalian cells,HEK293F cells and CHO cells were tried to express these humanized nanobodies and were not the proper choice,either.3.Next we tried to express camel-derived nanobody C6 in E.coli fused with solubility enhancing tag,including MBP,SUMO and GST.It was showed that MBP protein was the best to promotesoluble expression,followed by SUMO protein,and GST protein had nearly no solublity enhancing effect.4.MBP and SUMO protein were fused with humanized nanobody hBC6 respectively to increase the expression of soluble protein in E.coli.We also tried to fused double tags?MBP and SUMO?with hBC6 to express the fusion protein MBP-SUMO-hBC6.Both fusion protein MBP-hBC6 and MBP-SUMO-hBC6 were expressed at high soluble levels.Then these fusion proteins were digested to obtain the target humanized nanobodies.5.Three humanized nanobodies Hu2C6,hAC6 and hBC6 were proved to have the ability of binding to PD-1 protien by Elisa,of which hBC6 had the highest affinity.Compared with hBC6 whichwas non-fused expressed in E.coli,MS-Cut-hBC6,digestion products of MBP-SUMO-hBC6,had a higher affinity with PD-1 protein,whose EC50 and affinity constants value were 0.997 mg/L and 7.52×10⁶ L/mol respectively.6.It was proved that humanized nanobody hBC6 and camel-derived nanobody C6 could bind to PD-1 proteins recombinant expressed on 293T cells and on Jurkat cells which was activated by PHA.Both nanobodies could increase the secretion of IL-2 and TNF-?by activated Jurkat cells when co-incubated with Raji-PDL1 cells,indicating that PD-1 nanobodies could block the ligation of PD-1 on Jurkat cells with PD-L1 on Raji cells while there were no significant difference between two nanobodies?p>0.05?.It was also found that both camel-derived nanobody C6 and humanized nanobody hBC6 could promote the proliferation of activated Jurkat cells.Conclusion:In this study,camel-derived anti-PD1 nanobody C6 was humanized and expressed in E.coli.The humanized nanobody hBC6 could bind to PD-1 protein in vitro and block the ligation of PD-1 of Jurkat cells and PD-L1 on Raji cells to increase the secretion of IL-2 and TNF-?.However,humanized nanobody hBC6 had a lower affinity to PD-1 protein than camel-derived nanobody C6,which we'll study and improve the humanization in the future.