Study On The Anti-human-IL-13Rα2 Single Domain Antibody Of Chiloscyllium Plagiosum

Background: Glioma is a common and fatal primary tumor with high invasiveness,poor prognosis with conventional treatment,and low survival rate.Studies have found that IL-13Rα2 is highly expressed on the surface of cancer cells such as gliomas,but not expressed in normal cells or expressed in trace amounts.It is a good tumor target.However,in targeted therapy,conventional drugs cannot pass the blood-brain-tumor barrier and cannot be used in the treatment of brain tumors.Therefore,the development of a class of small molecule antibodies with high affinity,high stability and high tissue penetration is an urgent need for clinical treatment of brain tumors.Studies have shown that single-domain antibodies derived from cartilaginous fish have a molecular weight of only 12 k Da and can cross the blood-brain barrier.They are potential therapeutic drugs for brain diseases and have attracted much attention in recent years.Purpose: In this study,the shark specific monoclonal antibody against IL-13Rα2was obtained by immunoscreening using the Chiloscyllium plagiosum as the model organism.The recombinant monoclonal antibody with target activity was quickly obtained by recombinant expression,which provided a new idea and experimental basis for the targeted therapy of clinical glioma.Methods: After one week of laboratory culture system,Chiloscyllium plagiosum was randomly divided into two groups.The experimental group was immunized with IL-13Rα2 and Freund’s incomplete adjuvant,while the control group was immunized with PBS instead of IL-13Rα2.Other conditions were controlled the same,and regular immunization stimulated the shark to produce specific antibody against IL-13Rα2.Whole blood and spleen samples were collected after immunization.Genome,transcriptome and proteome libraries were constructed to obtain specific VNAR through comprehensive screening.The recombinant p ET-32a-VNAR vector was constructed by whole gene synthesis.The recombinant monoclonal antibody was expressed by prokaryotic expression.The affinity of the recombinant monoclonal antibody was detected by ELISA.Results: 1)The transcriptome library was successfully constructed,and several immune-related gene information were obtained after enrichment of differential genes;Genomic libraries composed of repeated VNAR sequences in parallel experimental groups were successfully obtained.2)The immune serum was successfully purified by antigen affinity column.After purification,1484 proteins were quantified by mass spectrometry,and all quantified proteins were corresponding quantified in the transcriptome library.3)Twelve pre-selected antibody sequences were successfully screened and all of them were soluble expressed in the prokaryotic expression system.High purity recombinant monoclonal antibodies were obtained after purification.4)The in vitro binding activity of the recombinant antibodies was preliminarily detected by ELISA,and 9 of the recombinant antibodies showed high binding ability.5)Three recombinant proteins were used in cell experiments.The results showed that the optimized antibody had significant cytokilling effect in a dose-dependent and concentration-dependent manner.At the same time,it can inhibit the migration of glioma cells,and the inhibition effect is significant.Conclusion: By immunizing the Chiloscyllium plagiosum,the immune system can be successfully stimulated and specific antibodies can be induced.And a series of immune responses can be characterized and detected by the spleen and serum.The successful construction of genome,transcriptome and proteome library by using spleen tissue and serum not only fills the gap in the study of striped bamboo shark,but also lays a foundation for subsequent screening.The recombinant VNAR sequences obtained by multi-omics combined screening showed high affinity after expression,which indicated that the screening method was reliable and effective,and could provide a reference for subsequent similar experiments.The specific monodomain antibody can be highly expressed in the prokaryotic system with the p ET-32 a vector.The recombinant monodomain antibody has high affinity activity and in vitro activity,which provides a new idea and experimental basis for the research and development of drug candidates for the targeted therapy of clinical glioma.