Chimeric antigen receptor with cytokine receptor activating or blocking domain
A chimeric antigen receptor, activation domain technology, used in cytokines/lymphokines/interferons, receptors/cell surface antigens/cell surface determinants, targeting specific cell fusion, etc., can solve the problem of IL -12 Issues such as limited release
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Embodiment 1
[0197] Example 1: Expression of CAR with IL-12 receptor activation domain (hereinafter referred to as: IL12-CAR) in T cell subsets.
[0198] Transplantation of peripheral blood T cells with IL12-CAR.
[0199] T cells were isolated from peripheral blood by Ficoll and activated in the presence of agonistic anti-CD3 and anti-CD28 antibodies (100 ng / ml each) and 500 U / ml IL2, respectively. Activated T cells were retrovirally transduced with a vector encoding IL12-CAR or a CAR without the IL12 receptor activation domain ( figure 1 ). The antibody-derived antigen-binding domains of both CARs are directed against the CEA tumor antigen. Transduced T cells were stained with anti-CD3 mAb and anti-human IgG antibody to monitor CAR expression ( figure 2 ). IL12-CAR was expressed in peripheral T cells with similar efficiency as CAR without the IL12 receptor activation domain.
[0200] IL12-CAR is expressed in different T cell subsets.
[0201] Peripheral T cells were transduced with...
Embodiment 2
[0204] Example 2: IL12-CAR induces CD56+CD62L+ phenotype of engineered T cells.
[0205] T cells were isolated from peripheral blood and activated in the presence of agonistic anti-CD3 and anti-CD28 mAbs (100 ng / ml each) and 500 U / ml IL, respectively. Activated T cells were retrovirally transduced according to standard protocols with a vector encoding IL12-CAR or, as a control, a CAR without the cytokine receptor activation domain. Transduced T cells were cultured for 9 days and stained with anti-CD3, anti-CD56 and anti-CD62L antibodies ( Figure 4 ). IL12-CAR preferentially induces the CD56+CD62L+ phenotype of IL12-CAR T cells.
[0206] in conclusion:
[0207] The phenotype of IL12-CAR-transduced T cells was similar to that of IL12-matured NK / NKT cells reported to acquire improved therapeutic potential (Lehman Dd et al., PLoS One 9, e87131, 2014).
Embodiment 3
[0208] Example 3: IL12-CAR signaling leads to improved IFN-γ secretion.
[0209] IFN-γ secretion is important for effective antitumor responses of adoptively transferred CAR T cells. Therefore, CARs with high IFN-γ inducibility would be valuable for clinical applications. To test the IFN-γ secretory properties of IL12-CAR, T cells from peripheral blood were isolated and transduced as described above to express IL12-CAR and, for comparison, a CAR without the cytokine receptor binding domain. CAR T cells were co-cultured with CEA+LS174T and CEA-Colo320 tumor cells for 48 hours, and IFN-γ secretion was recorded by ELISA. T cells expressing IL12-CAR secrete substantially more IFN-γ upon antigen encounter than T cells with CARs without the IL12 receptor activation domain ( Figure 5 ). Furthermore, even in the absence of antigen, IL12-CAR T cells secrete IFN-γ that would modulate the tumor environment to become more susceptible to cellular immune attack.
[0210] in conclusion:...
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