Kbm581

The anti-human CD22 scFv and anti-human CD19 scFv were derived from hybridoma clone HIB22 and HI19a respectively, established at the Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College (CAMS & PUMC). Anti-human CD22 scFv or anti-human CD19 scFv was linked with the CD8α signal peptide, CD8α transmembrane domain, 4-1BB costimulatory and CD3ζ cytoplasmic region and assembled into lentiviral pCDH plasmids with GFP as the tag protein. The lentiviruses were produced by transfecting HEK293T cells with CAR plasmids and packaging plasmids including PMD2.g (Invitrogen, USA), pMDLg/pRRE and pRSV-Rev (Biovector Science Lab, China). After isolation from the peripheral blood of healthy donors, T cells were cocultured with CD3/CD28 human T-activator Dynabeads (1 × 10⁶/ml) (Gibco, USA) and recombinant human IL-2 (100–200 U/ml) (R&D, USA) in lymphocyte serum-free medium KBM581 (Corning, USA). T cells were transduced with lentiviruses for 24 h and incubated in medium changed every 2 days, as previously described [27 (link), 28 (link)].

Nalm6, K562, Namalwa cell lines used in this study were purchased from American Type Culture Collection (ATCC). CD19 knockout Nalm6 cell line (Nalm6^CD19KO), luciferase transfected Nalm6 cell line (Nalm6^luciferase) and CD19 overexpression K562 (K562^CD19) cell line were constructed previously and preserved in our lab. All the tumor cell lines were grown in RPMI-1640 with 10% fetal bovine serum (FBS) at 37 °C with 5% CO₂.
Blood samples of B-ALL patients admitted to Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College (CAMS & PUMC), were collected and cultured in IMDM supplemented with 15% FBS, 100 ng/ml rhFLT3-L, 100 ng/ml rhSCF and 50 ng/ml rhTPO (PeproTech, USA).
Human T cells from healthy donors were isolated using Human T Cells Enrichment Cocktail (STEMCELL, USA) according to the manufacturer’s protocol. Patient derived T cells were isolated from B-ALL patients and cultured in lymphocyte medium KBM581 (Corning, USA) supplemented with 10% FBS and 50 IU/mL human interleukin-2 (IL-2) (R&D systems, USA).

The human cell lines CNE-1, CNE-2, 5-8F, and C666.1 were all maintained in RPMI 1640 medium (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (Gibco, Australia). Peripheral blood mononuclear cells (PBMCs) were isolated from the whole blood of healthy individuals by Ficoll density gradient centrifugation, then NK cells were cultured in lymphocyte serum-free medium KBM 581 (Corning, NY, USA) and were amplified by a combination of IL-2 (500IU/mL), IL-12 (2 ug/mL), IL-15 (2 ug/mL), and IL-18 (10 ug/mL) every other day for a total of 14-17 days. Ultimately, highly pure NK cells were isolated by CD56 MicroBeads (Miltenyi Biotec Inc, Auburn, CA, USA) with VarioMACS system. The purity CD3-/CD56+ NK cells was assessed to be 90%-95% on flow cytemerty.