Anti-CEA (BW431/26) h(CD28-OX40-CD3ζ) CAR, pSBCAR1 (CAR-SB-02LX360)

CAR Construction : BW431/26 scFv-28ζ

Fig.2 Serial dilutions of the anti-idiotypic mAb BW2064, which is directed to the CAR anti-CEA scFv domain, or an isotype control mAb IgG1 were coated onto microtiter plates (0.01–10 μg/ml).

CD4+ T cells were engineered with the respective CEA specific CAR and adjusted to equal numbers of CAR expressing cells by adding nonmodified T cells of the same donor.

Hombach, A. A., & Abken, H. (2011). Costimulation by chimeric antigen receptors revisited the T cell antitumor response benefits from combined CD28‐OX40 signalling. International journal of cancer, 129(12), 2935-2944.

CAR Construction : BW431/26 scFv-28ζ

Fig.3 CD4+ and CD8+ T cells were engineered to express anti-CEA CARs with different endodomains domains and coincubated.

The absence of IL-2 reveals different impact of costimulation on redirected cytolysis by CD4 and CD8 T cells.

Hombach, A. A., & Abken, H. (2011). Costimulation by chimeric antigen receptors revisited the T cell antitumor response benefits from combined CD28‐OX40 signalling. International journal of cancer, 129(12), 2935-2944.

CAR Construction :

Fig.4 The humBW431/26-CH2/CH3-g receptor triggers IL-2 secretion after binding to immobilized CEA or anti-idiotypic mAb.

The supernatants were analyzed for IL-2 content by ELISA utilizing a solid phase rat mAb (1 mg/ml) specific for murine IL-2 and a biotinylated rat anti-mouse IL-2 mAb (0.25 mg/ml).

Hombach, A., Koch, D., Sircar, R., Heuser, C., Diehl, V., Kruis, W., ... & Abken, H. (1999). A chimeric receptor that selectively targets membrane-bound carcinoembryonic antigen (mCEA) in the presence of soluble CEA. Gene therapy, 6(2), 300-304.

CAR Construction :

Fig.5 FACS analysis of humBW431/26-CH2/CH3-g receptor grafted MD45 T cells.

Transfected cells were incubated for 20 min with 5-10 mg/ml of different Abs.

Hombach, A., Koch, D., Sircar, R., Heuser, C., Diehl, V., Kruis, W., ... & Abken, H. (1999). A chimeric receptor that selectively targets membrane-bound carcinoembryonic antigen (mCEA) in the presence of soluble CEA. Gene therapy, 6(2), 300-304.

CAR Construction :

Fig.6 Specific cytotoxicity of MD45 cells grafted with the humBW431/26-CH2/CH3-g receptor.

Cytotoxicity of transfected and non-transfected MD45 T cells to target cells was measured by time resolved fluorometry.

Hombach, A., Koch, D., Sircar, R., Heuser, C., Diehl, V., Kruis, W., ... & Abken, H. (1999). A chimeric receptor that selectively targets membrane-bound carcinoembryonic antigen (mCEA) in the presence of soluble CEA. Gene therapy, 6(2), 300-304.

CAR Construction :

Fig.7 Two-color immunofluorescence of anti-CEA receptor-grafted peripheral blood T cells.

Non transduced peripheral blood T cells (A) and T cells grafted with the BW431/26-scFv-Fc- (438) (B), BW431/26-scFv-Fc- (439) receptor were simultaneously incubated with a PE-conjugated anti-CD3 mAb and a FITC-conjugated anti-human IgG1 Ab and analyzed by flow cytometry.

Hombach, A., Wieczarkowiecz, A., Marquardt, T., Heuser, C., Usai, L., Pohl, C., ... & Abken, H. (2001). Tumor-specific T cell activation by recombinant immunoreceptors: CD3ζ signaling and CD28 costimulation are simultaneously required for efficient IL-2 secretion and can be integrated into one combined CD28/CD3ζ signaling receptor molecule. The Journal of Immunology, 167(11), 6123-6131.

CAR Construction :

Fig.1 Peripheral blood T cells were grafted by retroviral gene transfer with the CEA specific immunoreceptor BW431/26-scFv-Fc-ζ.

To monitor receptor expression, T cells were simultaneously incubated with a FITC-conjugated anti-CD3 mAb and a PE-conjugated anti-human IgG1 Ab directed against the extracellular Fc domain of the receptor. Cells were analyzed by flow cytometry.

Hombach, A. A., Schildgen, V., Heuser, C., Finnern, R., Gilham, D. E., & Abken, H. (2007). T cell activation by antibody-like immunoreceptors: the position of the binding epitope within the target molecule determines the efficiency of activation of redirected T cells. The Journal of Immunology, 178(7), 4650-4657.