Figure 2.

High-throughput CAR activity screening for sdAbs

(A) CD22-specific sdAbs were transferred directly from phagemid-VHH vectors into pSLCAR screening backbone via PCR and tested for CAR-Jurkat assay. Jurkat cells were electroporated with various CD22-sdCAR, CD22-scFv-CAR, or control scFv-CAR plasmids and immediately mixed at 1:1 E:T with various red fluorescent protein-marked target cells. (B) Ramos or Ramos-CD22KO cells. (C and D) Raji or Raji-CD22KO cells (C), or (D) K562-WT, K562-CD22-overexpressing, or irrelevant SKOV3 cells. After overnight coculture, cells were stained with anti-human CD69 antibody and assessed via flow cytometry. (E) The mean CD69 expression of all CD22sdCARs versus on-rate (k_on), off-rate (k_off), or affinity (K_D) derived from SPR measurements is shown. (F) CAR-Jurkat cell target-cell doublet formation was examined using coculture with CD22⁺ Ramos cells or CD22KO cells for 30 min, followed by flow cytometry. Results show the mean of 3 experiments performed in duplicate ± SEM.