Selection And Characterization Of Single-chain Antibodies Against IL1RAP

The interleukin-1 receptor accessory protein(IL1RAP)is a co-receptor involved in many signal transduction pathways of interleukin-1 family factors.IL1 RAP expresses on the surface of stem cells of chronic myelogenous leukemia,but not in normal hematopoietic stem cells.The expression of IL1 RAP increases significantly in the primordial cells of patients with acute myelogenous leukemia or myelodysplastic syndrome.It turns to be a cell surface marker of various tumors,especially blood cancer.Therefore,development of anti-leukemia drugs targeting IL1 RAP is a new trend of optimistic prospects.Variable regions of heavy chain(VH)and light chain(VL)linked by a flexible polypeptide chain constitutes a single chain antibody(scFv)that retains the smallest structural units with specific affinity to antigens and greatly reduces immune rejection.Threfore scFv is widely used in the research and clinlic development of antibody drugs.In this study,we used human IL1 RAP protein as the antigen and screened for single chain antibodies against IL1 RAP from humanized scFv library,taking advantage of phage display and yeast surface display technology,and analyzed their characteristics.Firstly,the human scFv library was screened by two rounds of phage display screening based on the principle of the combination of streptavidin and biotin.ELISA was carried out and determined enrichment efficiency after phage display selection.The enriched phage vector was digested and purified to obtain scFv fragments,which was re-constructed as yeast display sublibrary.The yeast display system Aga1-Aga2 was then used to enrich scFv pool with higher affinity through three rounds selection by FACS.The DNA sequence of enriched scFv against IL1 RAP was then confirmed by DNA sequencing.IL1RAP scFv sequence was further subcloned into pFuse vector and transfected into 293 F cells.scFv protein containing Fc segment was expressed and purified by protein A column affinity chromatography.In addition,IL1 RAP knock-out 293 F cell line was generated by CRISPR-Cas9 editing technology and was used for specificity analysis.Finally,two scFv sequences with high affinity against IL1 RAP were obtained.In summary,the single-chain antibody sequences targeting IL1 RAP were obtained by phage and yeast display screening.Two scFv sequnces were further characterized and showed high affinity to IL1 RAP antigen.Therefore our work laid a good foundation for IL1 RAP targeted immunotherapy of leukemia and other diseases.