Transgenic tumor infiltrating lymphocyte and application thereof
A lymphocyte and tumor infiltration technology, applied in the field of cell therapy, can solve the problems of TILs cells that are difficult to produce clinical curative effect and cell death
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Embodiment 1
[0087] Example 1: Preparation of lentiviruses comprising anti-EGFR scFv-CD28 and anti-HER2 scFv-CD28 genes
[0088] 1. According to the reference (Journal of Immunological Methods. Volume 248, Issues 1–2, 1 February 2001, Pages 17-30. Selection of cell binding and internalizing epidermal growth factor receptor antibodies from a phage display library. MAbs. 2015; 7(6) :1058-71. Stability engineering of anti-EGFR scFvantibodies by rational design of a lambda-to-kappa swap of the VL framework using a structure-guided approach. Andreas Lehmann, Josephine H F Wixted, MaximV Shapovalov, Heinrich Roder, Roland L Dunbrack Jr , Matthew K Robinson.) selected the scFv sequence of EGFR antibody 2224 (see SEQ ID NO.: 1 for the nucleic acid sequence, and SEQ ID NO.: 2 for the amino acid sequence). According to references (Affinity-Tuned ErbB2 or EGFR Chimeric Antigen Receptor T Cells Exhibitan Increased Therapeutic Index against Tumors in Mice. Xiaojun Liu, ShuguangJiang, Chongyun Fang, Shi...
Embodiment 2
[0098] Example 2: Rapid Expansion of TILs
[0099] Select EGFR or HER2 positive tumors for TILs preparation. The basic steps are as follows:
[0100] The surgically obtained tumor tissue was cut into pieces of 1-2 mm in size, and transferred to a 24-well plate with 2 mL of complete medium, one tissue block per well. Complete medium components: RPMI-1640+10% human serum+2mM L-glutamine+25mM HEPES+10ug / mL gentamicin. It is also necessary to add 5000IU / mL IL-2 to the culture medium. Cultured in a cell culture incubator for 2-3 weeks, the TILs cells amplified from each well of a 24-well plate constituted a group. The expanded TIL cells were mixed. Rapid and massive expansion of TIL cells using irradiated allogeneic PBMCs. The irradiated PBMCs (340Gy) and TILs cells were obtained by centrifugation, and the cells were resuspended in 50 / 50 medium according to the ratio of 100:1, and cultured in the incubator. The final concentration of PBMCs was 1e6 / mL. The culture period is ab...
Embodiment 3
[0101] Example 3: Lentiviral transfection of TILs
[0102] Three days after TILs cells were reactivated, T cells were counted. Then adjust its cell concentration to 1×10 6 / ml, transferred to a six-well plate, and lentiviruses containing anti-EGFR scFv-CD28 and anti-HER2 scFv-CD28 DNA sequences were added to the culture wells of EGFR-positive tumor-derived TILs cells and HER2-positive tumor-derived TILs cells After transfection and expansion, the prepared TILs cells were named: anti-EGFR-CD28-TILs and anti-HER2-CD28-TILs. Untransfected TILs cells served as a negative control (control TILs). After transfection, the transfected and non-transfected TILs cells were cultured to a quiescent state.
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