| PD-1(programmed cell death 1)is expressed as an immunosuppressive receptor on the surface of activated T,B,NK cells and macrophages.The use of antibodies to block the binding of PD-1 has become a very effective immunotherapy method for cancer in recent years.However,the immune clearance effect mediated by antibody Fc and Fc receptors on the surface of immune cells will seriously interfere with the therapeutic effect of anti-PD-1 antibodies.Therefore,the development of antibodies or antibody fragments lacking Fc fragments or Fc functions has become one of the research directions.Nanobodies are single-domain antibodies derived from the variable region of camel heavy chain antibodies.They do not have Fc fragments and have certain advantages as potential PD-1 blockers.At the same time,as a small molecule antibody,Nanobodies can be efficiently expressed in prokaryotic cells,which provides a simple and cheap source for the production of Nanobodies.However,because Nanobodies have conserved inter-chain and intra-chain disulfide bonds,they need to be expressed in a soluble form and under reducing conditions to maintain their antibody activity to the greatest extent.Therefore,it is necessary to select an appropriate expression host and expression conditions,and select an appropriate expression system to obtain nanobodies with high activity and good yield.In addition,in order to overcome the shortcomings of fast metabolism of nanobodies in vivo,strategies need to be adopted to extend their plasma half-life.In this study,an anti-PD-1 nanobody(code: C3)selected from the PD-1immunized phage display library was screened by the research group in the early stage to carry out prokaryotic expression and blocking function analysis and experimental work to extend the half-life.Provide materials for the further development of the anti-tumor effect of the nanobody in the future.This research mainly includes the following three parts:1.Analysis and optimization of PD-1 nanobody C3 expression conditions in E.coliThe anti-PD-1 nanobody(C3)target fragment was constructed into pET22b-HA and pMECS vectors,and transformed into BL21,TG1,WK6,respectively,to obtain C3-pET22b-BL21,C3-pMECS-WK6 and C3-pMECS-TG1.In the three expression systems,it was found that C3-pMECS-WK6 expression was in the middle but the soluble expression was the most.The binding activity of C3-pMECS-WK6 and C3-pMECS-TG1 was significantly higher than that of C3-pET22b-HA-BL21.Therefore,in order to obtain more active soluble proteins,C3-pMECS-WK6 is the best expression system.Protein expression under the C3-pMECS-WK6 system,through different processing methods such as ultrasonic disruption and high and low osmotic repeated freezing and thawing methods,to obtain three relatively single expression proteins,of which the expression of stromal protein is the least,and the expression of cytoplasmic protein most.After ELISA analysis,it was found that the binding activity of the inclusion bodies was the lowest,and the cytoplasmic binding activity was the highest,but the binding activity of the three proteins was similar.Since both the stromal and cytoplasmic expressed proteins are soluble proteins,and the expression of inclusion bodies needs to be refolded to regain the activity and the activity is unstable,so cytoplasmic expression should be selected to obtain the target protein.The target protein was obtained by cytoplasmic expression under the C3-pMECS-WK6 system.By comparing the expression level and antigen binding activity at the three expression temperatures of 16°C,28°C and 37°C,it was found that the expression level at 16°C was not the same as that at 37°C.It is comparable,but the soluble expression at 16°C is the highest and accounts for the highest proportion of the total expression.The C3 protein and PD-1 protein at the three temperatures have a certain binding,and there is little difference.Therefore,16°C was chosen as the optimal expression temperature for anti-PD-1 Nanobody(C3).2.Analysis of the blocking effect of PD-1 Nanobody C3 on the binding of PD-1/PD-L1Under optimal conditions,a large amount of anti-PD-1 nanobody C3 protein was expressed.ELISA method was used to verify that anti-PD-1 nanobody can effectively prevent the binding of PD-1 and PD-L1 protein,and the PD-1/PD-L1 pathway has a significant blocking effect.3.The effect of fusion anti-albumin nanobody Alb on prolonging the half-life of PD-1 nanobody C3 in vivoThe recombinant plasmid C3-Alb-pET22 b was successfully constructed,and C3-Alb protein was normally expressed in E.coli.It was identified by SDS-PAGE and ELISA that it had a certain degree of binding with PD-1 protein and HSA protein,which was the subsequent half-life extended measurement experiment provides the basis.In summary,this experiment compared the expression level and binding activity of C3 protein under different expression systems,different expression sites and different expression temperatures in E.coli,and finally determined that pMECS-WK6 system,cytoplasmic expression,16°C is the optimal expression system,the optimal expression site and the optimal expression temperature of nanobody(C3)soluble protein.Through the verification of the blocking effect of the anti-PD-1 nanobody in the binding of PD-1and PD-L1 proteins,it was found that it can effectively block the PD-1/PD-L1 pathway,which is a future study on the soluble expression of nanobodies and PD-L1.Nanobody drug development provides expression basis and experimental basis.The successful construction of the fusion protein C3-Alb provides a new way and direction for future antibody research and development,provides new ideas and experimental accumulation for the extension of the half-life of nanobody drugs,and provides a research basis for the later production of new drugs. |
