Anti-GD2 (3F8) h(CD28-CD3ζ) CAR, pCDCAR1 (CAR-YF204)

CAR Construction : 3F8 scfv-CD28-41BB-CD3ζ-icasp9

Fig.5 Anti-GD2 CART cells are depleted upon stimulation with GD2(C) target cells in vitro.

T cells that were transduced with the hu3F8CAR (anti-GD2) and expanded for 8 d thereafter were stimulated with irradiated IMR32luc (GD2+) or THP1 (GD2-) cells. After 1, 2, and 4 d, cells were counted and CAR expression was assessed by anti-idiotype antibody (A1G4). Dead cells were excluded by Annexin-V staining. Absolute cell number was calculated based on flow cytometry.

Hoseini, Sayed Shahabuddin, et al. "Bispecific antibody does not induce T-cell death mediated by chimeric antigen receptor against disialoganglioside GD2." Oncoimmunology 6.6 (2017): e1320625. Distributed under Open Access license CC BY 4.0, without modification.

CAR Construction : 3F8 scfv-CD28-41BB-CD3ζ-icasp9

Fig.6 Anti-GD2 CART cells are depleted upon stimulation with GD2(C) target cells in vitro.

T cells that were transduced with the hu3F8CAR (anti-GD2) and expanded for 8 d thereafter were stimulated with irradiated IMR32luc (GD2+) or THP1 (GD2-) cells. After 1, 2, and 4 d, cells were counted and CAR expression was assessed by anti-idiotype antibody (A1G4). Dead cells were excluded by Annexin-V staining. Absolute cell number was calculated based on cell count.

Hoseini, Sayed Shahabuddin, et al. "Bispecific antibody does not induce T-cell death mediated by chimeric antigen receptor against disialoganglioside GD2." Oncoimmunology 6.6 (2017): e1320625. Distributed under Open Access license CC BY 4.0, without modification.

CAR Construction : 3F8 scfv-CD28-41BB-CD3ζ-icasp9

Fig.7 Lowering CAR affinity cannot prevent CART cell depletion upon antigen exposure.

T cells that were transduced with the 2nd-generation hu3F8 (loweraffinity) or hu3F8(D32H-E1K) (higher-affinity) CARs, and expanded thereafter for 8 d were stimulated with irradiated IMR32luc (GD2+) or THP1 (GD2-) cells. One day later, cells were assessed by flow cytometry for CAR expression.

Hoseini, Sayed Shahabuddin, et al. "Bispecific antibody does not induce T-cell death mediated by chimeric antigen receptor against disialoganglioside GD2." Oncoimmunology 6.6 (2017): e1320625. Distributed under Open Access license CC BY 4.0, without modification.

CAR Construction : 3F8 scfv-CD28-41BB-CD3ζ-icasp9

Fig.8 Lowering CAR affinity cannot prevent CART cell depletion upon antigen exposure.

T cells that were transduced with the 1st-generation or 2nd-generation hu3F8 CARs, and expanded thereafter for 8 d were stimulated with irradiated IMR32luc (GD2+) or THP1 (GD2-) cells. One day later, cells were assessed by flow cytometry for CAR expression.

Hoseini, Sayed Shahabuddin, et al. "Bispecific antibody does not induce T-cell death mediated by chimeric antigen receptor against disialoganglioside GD2." Oncoimmunology 6.6 (2017): e1320625. Distributed under Open Access license CC BY 4.0, without modification.

CAR Construction : 3F8 scfv-CD28-41BB-CD3ζ-icasp9

Fig.9 GD2-specific CART cell depletion severely compromises their function in short-term killing assays.

T cells that were transduced with the hu3F8CAR and expanded thereafter for 8 d were stimulated with irradiated IMR32luc (GD2+) or THP1 (GD2-) cells. Four days later, hu3F8CART cells were tested against IMR32luc targets in a 4-h 51Cr-release assay.

Hoseini, Sayed Shahabuddin, et al. "Bispecific antibody does not induce T-cell death mediated by chimeric antigen receptor against disialoganglioside GD2." Oncoimmunology 6.6 (2017): e1320625. Distributed under Open Access license CC BY 4.0, without modification.

CAR Construction : 3F8 scfv-CD28-41BB-CD3ζ-icasp9

Fig.10 Surviving CART cells retain a significant antitumor cytotoxicity in long-term killing assays.

Hu3F8CART cells were exposed to GD2(+) IMR32luc cells in triplicates for 3 d at an E:T=1. On day 3, cells were harvested, counted, and mixed with fresh IMR32luc targets (E:T=1). After 4 more days, cells were counted and assessed by flow cytometry. Number of live target cells was calculated based on cell count.

Hoseini, Sayed Shahabuddin, et al. "Bispecific antibody does not induce T-cell death mediated by chimeric antigen receptor against disialoganglioside GD2." Oncoimmunology 6.6 (2017): e1320625. Distributed under Open Access license CC BY 4.0, without modification.

CAR Construction : 3F8 scfv-CD28-41BB-CD3ζ-icasp9

Fig.11 Surviving CART cells retain a significant antitumor cytotoxicity in long-term killing assays.

Hu3F8CART cells were exposed to GD2(+) IMR32luc cells in triplicates for 3 d at an E:T=1. On day 3, cells were harvested, counted, and mixed with fresh IMR32luc targets (E:T=1). After 4 more days, cells were counted and assessed by flow cytometry. Number of live target cells was calculated based on flow cytometry.

Hoseini, Sayed Shahabuddin, et al. "Bispecific antibody does not induce T-cell death mediated by chimeric antigen receptor against disialoganglioside GD2." Oncoimmunology 6.6 (2017): e1320625. Distributed under Open Access license CC BY 4.0, without modification.

CAR Construction : 3F8 scfv-CD28-41BB-CD3ζ-icasp9

Fig.12 Anti-GD2 CAR-T cells cure melanoma tumors in vivo.

Immunodeficient DKO mice were subcutaneously inoculated with GD2(+) M14luc human melanoma xenografts (4E6 cells). CART Therapy was started after 7 d. Tumor volume was measured weekly.

Hoseini, Sayed Shahabuddin, et al. "Bispecific antibody does not induce T-cell death mediated by chimeric antigen receptor against disialoganglioside GD2." Oncoimmunology 6.6 (2017): e1320625. Distributed under Open Access license CC BY 4.0, without modification.