An Antibody Discovery Platform Based On Next-generation Sequencing And Mass Spectrometry Combined With Bioinformatics

According to the structure,therapeutic antibody drugs can be classified into monoclonal antibodies,antibody fragments and antibody conjugates,etc.among which monoclonal antibody drugs occupied the largest market share.Since the first antibody drug Orthoclone OKT3 was approved in 1986,the US Food and Drug Administration(FDA)has approved more than 70 antibody drugs.Although monoclonal antibody drugs have achieved great success in clinical and commercial,there are still many challenges in their research and development.In terms of pre-development work of antibody drugs,the current mainstream technology is based on experimental means,and the core is screening for high specificity,high affinity monoclonal antibodies.There are two major technology platforms for mainstream library screening: phage/yeast display library screening and hybridoma cell screening,but these platforms have limitations such as long cycle,high cost,complicated experimental process and difficult to mass production.Faced with these challenges,related research and technicians are actively developing new methods to find new technological breakthroughs.With advances in technology,the costs of sequencing are decrease significantly,and Next Generation Sequencing(NGS)technology is widely used in many fields of biology and translational medicine research.In recent years,the scope of NGS applications has been extended to the field of B/T cell immunology library sequencing.It has been reported that the combination of immune repertoire sequencing and proteomics techniques is applied to the screening of high-affinity antibodies,However,this report does not make full use of the information from the antibody(immune)repertoire to improve the screening efficiency.Based on a thorough understanding of NGS technology and mass spectrometry technology,this study developed a bioinformatics analysis platform for rapid and high-throughput screening antigen-specific antibody sequences.After multiple immunizations for the antibody repertoire,our platform comprehensively analyzed the relative abundance and dynamics of antibodies in repertoire.Based on quality assessment and alignment analysis of NGS data,the structural information,such as the nucleic acid and amino acid sequence information in framework region and complementarity determining region in different immunization batches were determined,relative abundance of CDR3 sequences were counted,and the abundance dynamics of the same CDR3 sequence in different immunization batches were explored.Antibody sequences obtained by NGS analysis were used as reference database for mass spectrometry data analysis,the analysis result of the mass spectrometry data was integrated to determining the sequence information of the identified peptides and corresponding antibody proteins,location and coverage information of the identified peptide were acquired based on above result.Further,candidate antibodies were ranked by those features.Finally,we selected 10 of the candidate antibodies for synthesis and experimental verification.The results showed that 5 of 10 were positive antibodies,with the positive rate reached 50%.Compared with the traditional antibody drug pre-development platform,our platform is easy to operate,faster and more efficient.The whole data analysis process takes only one week,plus the time of previous animal immunization and post-experimental verification,the whole process takes only three months,and the currently widely used phage display platform takes about half a year.Therefore,this platform has greatly shortened the antibody development cycle and has a strong market application value.