B 세포 림프종 (BCL) 세포주에 대한 NK-92 세포, αβ T세포 및 γδ T세포에서 CD19/CD22 이중 특이적 키메라 항원 수용체 (CAR)의 효용성 연구

Abstract

Background: Anti-CD19 chimeric antigen receptor (CAR) T cell therapy is effective in the treatment of relapsed or refractory aggressive B-cell lymphomas, but it can induce antigen-escape such as CD19low or CD19- malignant B cells and relapse. By developing bispecific CARs for CD22 and CD19, it is intended to prevent relapse through antigen escape of tumor cells and to reduce complications such as GVHD by using an NK-92 cell line and γδ T cells as immune effector cells.

Methods: Seven CAR-expressing vectors were produced using anti-CD19 (clone FMC63) and a novel anti-CD22 monoclonal antibody by modifying the backbone structures. The efficacy of the CAR structures was evaluated in both in vitro and in vivo experiments, using OCI-Ly7 cells, a human B cell lymphoma cell line. OCI-Ly7 cells were labeled with CFSE to distinguish from CAR-NK-92, CAR-αβ T, and CAR-γδ T cells as effector cells. Cell death was assessed with annexin V and 7-aminoactinomycin D in vitro. For in vivo analysis, OCI-Ly7 cells and effector cells were subsequently injected intraperitoneally in NSG mice and collected in the peritoneal lavage 48 hours later.

Results: The decrease of OCI-Ly7 cells was statistically significant in FMC63_CD8-NK-92 (CD19 CAR-NK-92) cells, CI4-47_FMC63-NK-92, and FMC63_2-3-14-NK-92 (CD19/CD22 CAR-NK-92) cells compared with NK-92 cells alone in vitro. CI4-47_FMC63-NK-92 and LOOPCAR6_FMC63_CI4-47-NK-92 (CD19/CD22 CAR-NK-92) cells had higher cytotoxicity in vivo. Several CD19/CD22 CAR-αβ T cells increased apoptosis of OCI-Ly7 cells although it was not statistically significant.

Conclusion: CD19/CD22 bispecific CAR-NK-92 cells had higher cytotoxicity against B cell lymphoma cells than CD19 CAR-NK-92 cells. The efficacy of the CARs could differ, depending on the secondary structure of the protein, the sequence of the antibody domains, and the linker structures.