Methods and compositions for t-cell immunotherapy
A cell and genome technology, applied in drug combinations, animal cells, immunoglobulins, etc.
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Embodiment 1
[0258] Generation and functional characterization of CEA-specific CAR T cells
[0259] This example describes the generation of a CEA-specific CAR and its functional expression in human T cells. In particular, a CEA-specific CAR carrying the CD28 / CD3ζ signaling domain was designed and functionally evaluated.
[0260] vector design. Ad5[E1-, E2b-] was constructed and prepared as previously described. CEA hybridomas were generated from BALB / c mice immunized with cultured human colon cancer cells. The scFv CEA was isolated from a hybridoma and then cloned in frame with the human IgG1-CH2CH3 domain, CD28 co-stimulatory intracellular domain and CD3ζ chain into the Ad5[E1-, E2b-] backbone (Ad5[E1-, E2b- ]-CEA.CAR).
[0261] Generation of viral supernatants. For the production of adenovirus, appropriate packaging cells (E.C7 cells) were used per dish at 1.2×10 6 cells were seeded into 10-cm culture dishes. After 24 hours, cells were transfected with 10 μg of adenoviral vector ...
Embodiment 2
[0266] Functional characterization of T cells expressing Ad5[E1-,E2b-]-CEA.CAR
[0267] Intracellular IFN-γ staining
[0268] This example describes the functional characterization of Ad5[E1-,E2b-]-CEA.CAR expressing T cells by intracellular IFN-γ staining. To assess the potential cytotoxic effect of transduced T cells, different cytotoxicity assays were performed. The ability of T cells expressing Ad5[E1-,E2b-]-CEA.CAR to recognize human colon cancer cells and their subsequent activation was tested. Typically, activation of CAR-expressing T cells can be measured by IFN-γ (or equivalent cytokine) production following stimulation with a cognate antigen (eg, CEA). 1×10 5 1 x 10 CAR-transduced T cells with cognate antigen expressing CAR 5 CEA+ tumor cells were incubated together (in this example, CEA+ tumor cells together with anti-CEA specific CAR). In 1 μl ml at 37 °C - 1 After 16 hours of incubation in the presence of Golgiplug (BD Biosciences), cells were washed and st...
Embodiment 3
[0277] Clinical expansion of CEA-specific CAR T cells
[0278] This example describes the clinical expansion of CEA-specific CAR T cells. To generate large numbers of transduced T cells, cell proliferation was induced using the Rapid Expansion Protocol (REP). T cells were initiated in culture with anti-CD3, anti-CD28, and IL-2 prior to use in REP and transduced the day after initiation of culture, as described above. Place the cells at 75cm 2 flask at 37 °C and 5% CO 2 under cultivation. Cells were counted every two days and counted at 0.5 × 10 6 Cells / mL were suspended in fresh T cell medium containing 300 IU / mL IL-2 and kept in culture for the rest of the time.
[0279] Costimulatory domains, including any of the molecules shown in Table 2, were included in the CEA vectors described above to enhance the immunogenicity of the resulting CEA-specific CAR T cells.
[0280] Table 2 co-stimulatory domain
[0281]
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