Study Of The In Vitro Antileukemic Effects By Human T-lymphocytes Transduced With CD19-chimeric Antigen Receptors(CAR)

Background and objectiveWith increasing advances in recombinant DNA technology, cellular immunotherapies of cancer treatment are considered to be the most promising therapies.Among these therapies involve the T lymphocytes transduced with tumor antigen specific chimeric antigen receptors (CAR) without the restriction of MHC. In this study, we used a retroviral vector(MigRl) to construct the second generation MigRl-CD19-CAR that contains three parts:a single chain fragment of variable region (scFv) which targeted to CD19, CD28 costimulatory molecule and TCR-ζ cytoplasmic signaling chain. And to establish a CD19-K562 cell line overexpressing CD19 gene stably and its subcutaneous xenograft model in NOD-SCID mouse. To improve the MigRl-CD19-CAR vector transduction efficiency of human T-lymphocytes and discuss its antileukemic effects.MethodsCD28 costimulatory molecule and TCR-ζ cytoplasmic signaling chain was acquired by overlapping PCR. Insert the recombinant fragments into the retroviral vector (MigRl) through by enzyme digestion and ligation reaction, then transforme DH5a competent cells. Colonies were sequenced after positive clones were picked and analysis the sequencing results. Insert the CD 19 gene into the retroviral vector (MigRl) similarly, after transfecting Plat-A packaging cell lines, viral supernatant was collected to transduce K562 cell line repeatly. We ues flow cytometry to determine the transduction efficiency and RT-PCR to confirm CD 19 gene was transcripted. Cell proliferation and apoptosis were detected by cell count and Annexin V/PI respectively. Construct its subcutaneous xenografting subtype CD19-K562-a cell line later through subcutaneous inoculation and culture in vitro. Thus establish its subcutaneous xenograft model in NOD-SCID mouse and confriming CD19 gene was translated in tumor cell by immunohistochemistry. Peripheral blood mononuclear cells(PBMCs)were activated with CD3/CD28 beads combined with rhIL-2. Viral supernatant was collected to transduce activated human T-lymphocytes. The ability of the transduced T cells to produce IFN-γ and TNF-α in a CD19-specific manner was measured in an enzyme-linked immunosorbent assay.Results1. Sequences of second-generation MigRl-CD19-CAR and MigRl-CD19 recombinant vector were 100% and 99.85%(0.15% was nonsense mutation) similar with target gene sequences.2. The CD19 positive efficiency of K562 cell line was (99.80±0.17)% through retro virus centrifugation transduction repeatly. CD 19 gene was transcripted with high efficience than K562 cell by RT-PCR (P<0.01). And cell proliferation and apoptosis were not influenced by transduction.3. The CD19 positive efficiency of its xenograft subtype CD19-K562-a cell line was (99.78±0.04)%. CD 19 gene and bcr-abl (210) gene were both transcripted with high efficience. Wright’s staining showed CD19-K562-a, CD19-K562, K562cells were in undifferentiated mrophlogy similarly. NOD-SCID subcutaneous xenografts were established through subcutaneous inoculation. The tumors from CD19-K562-a subcutaneous xenografts were CD19 positive, while its K562 counterparts were CD19 negative.4. Using MigRl-CD 19-C AR vector to produce the high titer retrovirus;Transduction efficiency of peripheral blood mononuclear cell (5 health donors and 5 patients) activated by CD3/CD28 beads combined with rhIL-2 was (96.1±4.8)%.5. MigRl-CD 19-C AR transduction efficiency of K562 cell was significantly higher than human T-lymphocytes (80.05±4.35)% VS(25.1±5.77)% (P<0.01);120min centrifugation can significantly improve transduction efficiency of T-lymphocytes to (54.5 ±14.62)%; Transduction efficiency can be improved by deciding transduction time according to T-lymphocytes proliferation fold in vitro individually. The highest transfection efficiency of healthy donors was (68.7±0.6)%, and that of patients samples was(59.8±0.5)%.6. The CD19-CAR gene sequence was transcripted specificly. CAR-T cells were confirmed by RT-PCR through three pairs of primers which are for scFv sequence, scFv to CD28 sequence, CD28 to TCR sequence,whose 2-ΔΔCT is (2057±549)%, (3737± 1101)%, (1001±110)% respectively.7. IFN-γ and TNF-γ released by CD19-CAR transduced T-lymphocytes significantly increased to(13229.93±1542.99) pg/ml and (4217.07±210.77) pg/ml when coculture with CD19-K562 cells.Conlusion1. MigRl-CD19-CAR recombinant vector and CD19-MigRl recombinant vector were successfully constructed.2. CD19-K562 cell line overexpressing CD 19 gene was stably established.3. CD19-K562 subcutaneous xenograft model in NOD-SCID mouse were successfully established.4. Deciding transduction time according to T-lymphocytes proliferation folds in vitro individually can improve the CAR transduction efficiency of T-lymphocytes.5. IFN-y and TNF-a released by CD19-CAR transduced T-lymphocytes significantily increased when actived by target cells.