| Research Background and Purpose:Chimeric Antigen Receptor T-cell(CAR-T)therapy is a new type of precise targeted therapy,which has made a breakthrough in the treatment of hematologic tumors.Antigen recognition domains are the basis of specific binding of tumor antigens to CAR-T cells,and their recognition of target antigens and binding ability directly affect the tumor killing effect of CAR-T cells.Therefore,new antigen recognition domains are the key to the optimization of CAR-T cells.Antigen recognition domains are usually composed of a single chain antibody fragment(sc Fv).Nanoantibodies with smaller molecular weight,more stable structure,and easier modification are considered to be sc Fv alternatives with greater potential for CAR-T applications.CD22 is also an ideal therapeutic target for B-cell malignancies because of its B-cell specificity and ubiquity in B-cell malignancies.In this study,a new nanoantibody sequence targeting CD22 was obtained by immunizing allamas,and CD22(Nb)-CAR-T cells were prepared to screen out the sequence with anti-tumor activity in vitro,which is expected to be further developed as a cell therapy product for CD22 target diseases.Materials and Methods:In this study,CD22 protein was first used to immunize alpaca,isolate the peripheral blood B lymphocytes of alpaca,extract RNA,reverse transcription to obtain c DNA,using c DNA as substrate PCR to obtain a variety of nano antibody gene fragments,and construct bacterial library and Phage library.After three rounds of panning,phage-ELISA monoclonal verification was performed.All the positive clones were sequenced,and several different CD22 nanoantibody sequences were finally obtained.CD22-CAR T cells were prepared by constructing plasmids with different nanosequences,packaging CD22-CAR lentivirus and infecting human T lymphocytes.To verify the killing ability of CD22-CAR T cells constructed with different sequences:Firstly,Flow Cyto Metry(FCM)was used to detect the expression of CD22 antigen on Raji and Namalwa cells.After the first round of preliminary screening,the killing effect of CD22-CAR-T cells on tumor cells was observed under the condition of 1:1effect-target ratio.The groups with better killing effect in the first round of screening were repeated in the second round of verification.The killing effect of CD22-CAR-T cells on tumor cells,the expansion of CAR-T cells,the expression of CD62L molecules and the secretion of cytokines after killing target cells were observed under the condition of the effector-target ratio of 1:3.Results:1.After immunization of alpaca with CD22 protein antigen for 5 times,the plasma titer reached 10~5level,and the phage library was successfully constructed.The library capacity was 2.1×10~8cfu,the recombination rate was 94%,and the diversity was 100%.2.After three rounds of Phage library panning with CD22 protein as the target antigen,the positive clones identified by phage-ELISA were sequenced,and 46different CD22 nanoantibody sequences were obtained.3.Fourteen sequences screened from 46 CD22 nanoantibody sequences were successfully constructed to package CD22-CAR lentivirus,and CD22-CAR-T cells were successfully prepared.4.In the first round of in vitro killing experiments,9 sequences with better killing effect were selected under the condition of Raji and Namalwa cells with E:T=1:1.5.In the second round of in vitro killing experiments,when the tumor cells were Raji and Namalwa cells,and the E:T ratio was 1:3,the results of repeated verification showed that all the 9 sequences had good killing effect,and one sequence had the best killing effect.Conclusions:1.A phage library with large capacity and good diversity was successfully constructed.After screening and enrichment,46 different sequences were finally obtained.2.Nine nanoantibody sequences with good anti-tumor activity in vitro were successfully screened,and one of them was the preferred sequence.All the 9nanoantibody sequences could be used in the subsequent research related to CAR-T cell therapy. |
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