Rational Construction Of Nanobody Immunotoxin Targeting BCMA In The Treatment Of Multiple Myeloma

Multiple myeloma（MM）is a malignant hematological tumor derived from bone marrow plasma cells.The incidence of MM accounts for 1%of all tumors and accounting for 10%of hematological tumors,which ranks second in hematological tumors.The death rate of MM is 2.1%of all tumors.According to the data from the US SEER（Tumor Surveillance,Epidemic,and End-Occurrence）database,the incidence of MM increased year by year,and the incidence was 1.8%in 2019.After receiving traditional chemotherapy,the median survival time of MM patients was about 3 years.In recent years,although immunotherapy has made great progress in MM therapy,MM is still an incurable disease accompanied by serious side effects,poor prognosis and high recurrence rate.After recurrence,it often turns into refractory MM.Therefore,it is particularly important to explore novel treatment methods and targets of MM.B cell maturation antigen（BCMA）is a protein specifically expressed in MM and mature plasma cells.At present,BCMA has attracted attention as a new target for the treatment of MM.In this study,alpaca was immunized with BCMA protein,the total RNA of peripheral blood lymphocytes of alpaca was extracted,and a 2.8×10⁷ cfu nanobody library was constructed.Based on this,a 2.25×10¹³ cfu/m L phage display library was obtained,and seven candidate nanobodies targeting BCMA were screened.The expression plasmid was constructed by genetic engineering,and the seven candidate nanobodies were ligated with PE38 toxin through connecting peptide（GGGGS）to construct the anti BCMA-PE38 immunotoxin.E.coli BL21 expression system was used for the expression of the anti BCMA-PE38 immunotoxin.Through the inclusion body renaturation,His tag binding and AKTA system,the anti BCMA-PE38 immunotoxin were purified with purity≥85%.Nb BCMA-PE38-1 and Nb BCMA-PE38-3 were identified by cell binding assay and affinity determination experiment.The affinity dissociation constant（K_d）of Nb BCMA-PE38-1 and Nb BCMA-PE38-3 were4.09 n M and 4.17 n M,respectively.In the cytotoxicity-test of U266 cell line,the half maximal inhibitory concentration(IC₅₀)of Nb BCMA-PE38-1 and Nb BCMA-PE38-3 were 1252 ng/m L and 7251 ng/m L,respectively.