Fig 2: In vitro evaluation of anti-CD19/CD22 bispecific CAR-NK-92 cells. (A) In vitro experimental timeline. CAR-NK-92 cells were transduced with lentiviral vectors expressing CAR structures twice and Myc+ cells were sorted after 48 h. CAR-NK-92 cells were cryopreserved until use. For in vitro evaluation of anti-CD19/CD22 bispecific CAR-NK-92 cells, CAR-NK-92 cells were co-cultured with CFSE-labeled OCI-Ly7 cells for 4 h and measured for AV/7-AAD by flow cytometry. (B) Summary of AV+ 7-AAD- early and AV+ 7-AAD+ late apoptotic CFSE+ target cells. (C) Numbers of CFSE+ target cells (mean ± SEM; n=4). (D) Representative flow cytometry of AV/7-AAD cell death assay. *P<0.05. CAR, chimeric antigen receptor; NK, natural killer; CFSE, carboxyfluorescein diacetate succinimidyl ester; 7-AAD, 7-amino-actinomycin D; AV, Annexin V.

Fig 3: Representative structures of anti-CD19 and anti-CD22 specific and bispecific CAR structures. (A) A total of four bispecific CAR structures were produced using anti-CD22 antibody clones, C14-47, CI3-23, 2-3-14, and 2-3-16. (B) Anti-CD19/CD22 bispecific CAR structures were produced using CI4-47 clone into the backbone structure. (C) Anti-CD19/CD22 bispecific CAR structures were produced using CI4-47 clone into the backbone structure with a loop to optimize the CAR structures for natural killer cells. (D) Anti-CD19 (FMC63) were integrated to the backbone structure. (E) Anti-CD22 antigen-binding domains were integrated as well. FMC63, anti-CD19 antigen-binding domain; CI4-47, anti-CD22 antigen-binding domain; CAR, chimeric antigen receptor; scFv, single chain variable fragment.

Fig 4: Binding assay of anti-CD22 antibody. Recombinant anti-CD22 scFv-C?-HA fusion protein was subjected to (A) ELISA and (B) flow cytometry. Grey, secondary antibody only; red, anti-CD22 scFv-C?-HA fusion protein tested. Enzyme immunoassay was performed in triplicate and data are presented as the mean ± SD. scFv, single chain variable fragment.