Co-expression of recombinant immune checkpoint receptor and immune checkpoint inhibiting molecules and application
A technology of checkpoints and receptors, applied in the field of co-expression and application of recombinant immune checkpoint receptors and immune checkpoint inhibitory molecules, to achieve the effect of improving targeted killing effect
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Embodiment 1
[0277] Example 1 Construction of vectors for co-expression of non-functional EGFR receptors, PD1-silencing shRNA (shPD1, iPD1) and PD1-CD28-CD3zeta recombinant receptors
[0278] In this example, the inventors cloned the ζ-chain sequence encoding human PD1 extracellular fragment sequence, CD28 transmembrane and intracellular fragment and T cell receptor into a lentiviral vector containing EF-1 promoter Above, during the cloning process, the selected restriction enzymes are XbaI and NotI double enzyme digestion, and NotI and XhoI double enzyme digestion, through enzyme digestion, ligation, screening and amplification of the target plasmid, a lentivirus expressing the recombinant receptor is generated Plasmid (LV-PD1-CD28-CD3ζ(LV-PD1-CD28z). Sequences containing the IRES and expressing the non-functional EGFR receptor were cloned into the LV-PD1-CD28-CD3ζ vector plasmid, constructed as LV-PD1-CD28-CD3ζ -tEGFR(M)(LV-PD1-CD28z / M). The sequence of shRNA containing U6 and silencing ...
Embodiment 2
[0280] Example 2 Anti-EGFR antibodies can effectively kill and clear T lymphocytes co-expressing non-functional EGFR receptors, recombinant receptors and shRNA (shPD1, iPD1) that silences PD1
[0281] In this example, peripheral blood lymphocytes were obtained from anonymous blood donors. Peripheral blood lymphocytes were separated by gradient centrifugation using Ficoll-Hypaque. T lymphocytes and T cell activator magnetic beads CD3 / CD28 (purchased from Invitrogen, Carlsbad, CA) in 5% CO 2 , Incubated at 37 degrees Celsius for 72 hours, the medium was added with 2mmol / L glutamine, 10% high temperature inactivated fetal calf serum (FCS) (purchased from Sigma-Aldrich Co.) and 100U / ml of penicillin / chain RPMI medium 1640 (purchased from Invitrogen Gibco Cat. no. 12633-012) with antimycin double antibody. After activating and culturing for 72 hours, the cells were rinsed with washing solution to wash away the magnetic beads. The T cells were planted on a cell culture dish lined...
Embodiment 3
[0282] Example 3 Cytolytic Ability of T Lymphocyte Tumor Cells Co-expressing Non-functional EGFR Receptor, shPD1 and PD1-CD28-CD3ζ Recombinant Receptor
[0283] In this example, peripheral blood lymphocytes were separated by gradient centrifugation, and the gradient centrifuge was Ficoll-Hypaque. T lymphocytes and T cell activator magnetic beads CD3 / CD28 (purchased from Invitrogen, Carlsbad, CA) were incubated at 5% CO2, 37 degrees Celsius for 72 hours, the medium was added with 2mmol / L glutamine, 10% high temperature Inactivated fetal calf serum (FCS) (purchased from Sigma-Aldrich Co.) and 100 U / ml penicillin / streptomycin double antibody RPMI medium 1640 (purchased from Invitrogen Gibco Cat. no. 12633-012). After activating and culturing for 72 hours, the cells were rinsed with washing solution to wash away the magnetic beads. The T cells were planted on a cell culture dish lined with recombinant fibronectin fragments (FN ch-296; Retronectin), and transduced with lentiviruse...
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