Construction Of Phage Library Based On Shark Antibody Variable Region(V-NAR) And The Screening,Identification Of PD-L1 Nanobody And Preliminary Evaluation Of Activity

The special class of antibody IgNAR in the shark,and its variable region V-NAR is currently known as the smallest molecular weight antibody fragment,named as nanobody.V-NAR has the advantages of high affinity,strong stability,good solubility,easy coupling modification and good tissue penetration ability.Therefore,it has broad application prospects in the biomedical industry.The synthetic phage antibody library has the advantages of large storage capacity,rich diversity and low production cost,which can be used to screen a variety of antigens.It is an important source for obtaining high-affinity antibodies and has significant significance for the development of V-NAR drugs.In this study,a new shark V-NAR framework was designed using bioinformatics technology,and introduced mutations in the CDR3 region using the NNK method to construct a shark V-NAR synthetic library with a capacity of 1.9 × 109 cfu,indicating that the framework can be used to construct V-NAR nanobody library.In addition,five anti-PD-L1 nanobodies including NbP1,NbP2,NbP3,NbP4 and NbP5 were obtained from the phage library.These five nanobodies specifically bind to PD-L1 and have excellent stability.Among them,the affinity of NbP4 reaches 107 M-1,and can block the binding of PD-1 and PD-L1.Meanwhile,it is also found that the nanobody has good heat resistance,and still has more than 50%activity at 60℃,which confirms that the constructed V-NAR phage library has biological activity and can be used to screen other antigens.In summary,the shark antibody-based V-NAR phage library constructed in this study can be used as a screening platform for different antigens to obtain nanobodies with multiple targets,which has extremely high biomedical application value.Moreover,the new V-NAR gene framework can be used as a general framework for the shark V-NAR antibody library,which provides a new theoretical basis for the construction of antibody libraries.Furthermore,the Anti-PD-Ll nanobody screened in this study has potential application values and can provide new varieties for the development and acquisition of new anti-tumor drugs.