Anti cd4 and anti cd8 microbeads

IL23 specific antibody and PSMA specific antibody were isolated by phage display, and experiment was done by Y clone LLC and novel generation LLC. All CARs are designed basing on lentiviral vector. IL23mAb was first cloned into pELNS-tetR-T2A-Zeocin to replace the tetR cistron to form pELNS-IL23mAb-T2A-Zeocin. Next, the pELNS-IL23mAb-T2A-Zeocin cassette was cloned in the lentiviral expression plasmid from the Translational Research Program (pTRPE) lentiviral vector. A CAR was then designed and synthesized by Thermo Fisher Scientific. Lentiviral vectors were used to transduce human T cells from normal donor isolated from PBMCs by anti-CD4 and anti-CD8 microbeads (Miltenyi Biotec) and activated for 24 h with anti-CD3/CD28 Dynabeads (Thermo Fisher Scientific).The PC3 prostate cancer cell line (ATCC, CRL-1435) grow to confluency in vitro within 3–4 days of being seeded at 1 × 10⁴ cells/cm² and cultured in D10 media consisting of DMEM with 10% FBS, HEPES, penicillin, and streptomycin. We used pTRPE lentiviral vector to introduce the PSMA protein and sorted the PMSA positive cells by BD FACS Aria III.

Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood samples acquired from healthy volunteer donors at the University Children’s Hospital Tuebingen, by Ficoll centrifugation (Biocoll, Biochrom, Berlin, Germany). T cells were isolated by magnetic separation using anti-CD4 and anti-CD8 microbeads (Miltenyi Biotec, Bergisch-Gladbach, Germany) simultaneously. Isolated T cells were activated with TransAct^TM (Miltenyi Biotec) and cultivated in TexMACS media supplemented with 10 ng/mL IL7 and 5 ng/mL IL15 (Miltenyi Biotec, Bergisch-Gladbach, Germany). TransAct^TM-activated T cells were transduced at a multiplicity of infection (MOI) of 3 after 36 h. Transduced T cells were maintained at 0.5–2 × 10⁶ cells/mL in IL7/IL15-supplemented TexMACS^® media. On Days 7+, CAR transduction efficiency and the CD4/CD8 ratio were determined by flow cytometry.

Fresh peripheral blood mononuclear cells (PBMCs) from healthy donors were provided by the First Affiliated Hospital, College of Medicine, Zhejiang University and Shanghai SAILY Biological Technology Co., Ltd. Recruitments of healthy human blood donors was approved by the Clinical Research Ethics Committee of the First Affiliated Hospital, College of Medicine, Zhejiang University and by Shanghai Zhaxin Traditional Chinese Ethics Committee and Western Medicine Hospital. All donors signed an informed consent form. Fresh PBMCs from patients were collected by apheresis. PBMCs were isolated by density gradient centrifugation using Ficoll (Sigma-Aldrich). T cells were enriched through magnetic separation using anti-CD4 and anti-CD8 microbeads (Miltenyi Biotec) and activated with T Cell TransAct (Miltenyi Biotec). T cells were cultured in X-VIVO 15 medium (Lonza) supplemented with 2% human AB serum or CTS Immune Cell Serum Replacement (Thermo Fisher) and recombinant human IL-2 (100 U ml^–1), IL-7 (5 ng ml^–1) and IL-15 (5 ng ml^–1). Cells were collected once cell number reached the requirement for administration and then washed, formulated and cryopreserved.

BM was prepared from the femurs and tibiae of mice, and depleted of mature T cells using anti-CD4 and anti-CD8 microbeads (Miltenyi). Recipient mice were lethally irradiated (400 rad X 2) and received 5 × 10⁶ donor BM cells. Chimeras were analyzed 9–10 weeks after transplantation.