Chimeric antigen receptors targeting cd37 and cd19
A chimeric antigen receptor, CD37 technology, applied in the direction of targeting specific cell fusions, antibodies, polypeptides containing localization/targeting motifs, etc., can solve poor treatment, PTCL clinical problems, treatment of T cell malignant Problems such as unproven tumor
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Embodiment 1
[0219] Example 1. Primary human T cell culture
[0220] For primary T lymphocyte expansion, bulk human T cells (day 0) were activated using anti-CD3 / CD28 Dynabeads (Life Technologies) and subsequently transduced 24 hours later with a CAR-encoding lentiviral vector. From day 0 of culture, culture T cells in medium supplemented with 20 IU / ml rhIL-2 and count them by counting every 2-3 days at 0.5x10 6 T cells were maintained at a constant cell concentration per mL. For functional assays, CAR T cells were cryopreserved on days 8–10 in culture and stimulated with antigen or injected into mice immediately after thawing.
Embodiment 2
[0221] Example 2. Cell lines and culture conditions
[0222] JEKO-1, RAJI and wild-type parental K562 cells were purchased from American Type Culture Collection (ATCC). K562 cells were engineered to express CD37 and CD19 (K562-CD37-CD19). For some assays, cell lines were engineered to constitutively express click beetle green (CBG) luciferase / enhanced GFP (eGFP), and then expressed in a FACSAria (BD ) to obtain a population with a purity ≥ 99% (CBG-GFP+). Cell lines were grown in RPMI medium containing 10% fetal bovine serum (FBS), penicillin and streptomycin.
Embodiment 3
[0223] Example 3. Flow Cytometry
[0224]The following antibody was used: CD37-APC (clone MB-1 ), CD37-BV711 (clone MB-371, BD ), CD19-Pacific Blue (clone HIB19, ), CD19-FITC (clone 4G7, BD), CD5-BUV737 (clone UCHT2, BD), CD20-APC Cy7 (clone 2H7, ), CD79b-PE (clone CB3-1, ), CD3-BV786 (clone SK7, BD), CD3-BV605 (clone OKT3, ), CD45-PeCy7 (clone HI30, ), CD16-PE (clone B73.1, ), CD14-PacificBlue (clone HCD14, ), CD56-APC (clone HCD56, ), CD33-BV510 (clone P67.6, ), CD107a-AF700 (clone H4A3, BD ), CD69-APC (clone FN50, ) and IFNγ-FITC (clone GZ-4, ). Cells were stained for 30 min at 4°C in the dark and washed twice in PBS containing 2% FBS. DAPI was added to gate live cells prior to acquisition. Measured using antibody bound per cell (ABC) and using Quantum TM Antigen density was calculated by Simply Cellular (Bangs Laboratories).
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