Anti-CD19 scFv h(CD28-FcεRIγ) CARNK, pCDCAR1 (CAR-NK-2-M328-BG)

CAR Construction : FMC63 scFv-BBζ; FMC63 scFv-28ζ

Fig.1 Leukaemic response and relapse patterns associated with FMC63 scFv-based CAR.

Kaplan-Meier survival analysis of mice bearing NALM6 cells treated with 0.2 × 10^6 transduced CAR T cells. n = 5-7 mice per group pooled from two independent experiments.

Hamieh, M., Dobrin, A., Cabriolu, A., van der Stegen, S. J., Giavridis, T., Mansilla-Soto, J., ... & Sadelain, M. (2019). CAR T cell trogocytosis and cooperative killing regulate tumour antigen escape. Nature, 568(7750), 112-116.

CAR Construction : FMC63 scFv-BBζ; FMC63 scFv-28ζ

Fig.2 Leukaemic response and relapse patterns associated with FMC63 scFv-based CAR.

Representative FACS profile of PD-1, LAG-3 and TIM-3 surface expression on BBζ CAR T cells isolated from relapsed mice. (data are representative of n = 3 mice per group).

Hamieh, M., Dobrin, A., Cabriolu, A., van der Stegen, S. J., Giavridis, T., Mansilla-Soto, J., ... & Sadelain, M. (2019). CAR T cell trogocytosis and cooperative killing regulate tumour antigen escape. Nature, 568(7750), 112-116.

CAR Construction : FMC63 scFv-28ζ

Fig.4 FMC63 and 4G7 CAR T cells effectively responded to CD19‐positive NALM‐6 cells.

Tumor-killing ability of FMC63, 4G7, and FITC CAR T cells against luciferase-expressing NALM-6 and U937. FMC63, 4G7, and FITC CAR T cells (2 × 10^5 cells) were co-cultured with luciferase-expressing NALM-6 (2 × 10^4 cells) or U937 (2 × 10^4 cells) for indicated incubation times at the E:T ratio 10:1. Then, luminescence values were measured.

Kang, C. H., Kim, Y., Lee, H. K., Lee, S. M., Jeong, H. G., Choi, S. U., & Park, C. H. (2020). Identification of potent CD19 scFv for CAR T cells through scFv screening with NK/T-cell line. International Journal of Molecular Sciences, 21(23), 9163.

CAR Construction :

Fig.5 Kinetic binding properties of CD19 scFv derived from CD19 hybridomas.

Anti-CD19 scFv was fused to mouse IgG2a-Fc in a SFG-eBFP γ-retroviral expression vector and HEK293T cells were transfected to generate secreted scFv, which were then purified
on a protein A column.

Ghorashian, S., Kramer, A. M., Onuoha, S., Wright, G., Bartram, J., Richardson, R., ... & Amrolia, P. J. (2019). Enhanced CAR T cell expansion and prolonged persistence in pediatric patients with ALL treated with a low-affinity CD19 CAR. Nature medicine, 25(9), 1408-1414.

CAR Construction : FMC63 scFv-BBζ

Fig.6 Antigen-specific killing of CD19+ tumor cells by CD19 CAR+ T cells (FMC63 or CAT) or non-transduced (NT) T cells.

CAR+ T cell cytotoxic activity against SupT1 cells that are engineered to express CD19 (SupT1CD19, left) as well as target-antigen-negative non-transduced SupT1NT (right), as measured by standard 4-h 51Chromium-release assay.

Ghorashian, S., Kramer, A. M., Onuoha, S., Wright, G., Bartram, J., Richardson, R., ... & Amrolia, P. J. (2019). Enhanced CAR T cell expansion and prolonged persistence in pediatric patients with ALL treated with a low-affinity CD19 CAR. Nature medicine, 25(9), 1408-1414.

CAR Construction : FMC63 scFv-BBζ

Fig.7 Cytokine production in response to 1:1 coculture of FMC63-CAR cells with irradiated Raji cells.

Production of cytokines in response to 1:1 coculture of CAR cells with irradiated Raji cells, measured by a cytokine bead array of culture supernatants
taken at 48 h.

Ghorashian, S., Kramer, A. M., Onuoha, S., Wright, G., Bartram, J., Richardson, R., ... & Amrolia, P. J. (2019). Enhanced CAR T cell expansion and prolonged persistence in pediatric patients with ALL treated with a low-affinity CD19 CAR. Nature medicine, 25(9), 1408-1414.

CAR Construction : FMC63 scFv-BBζ

Fig.8 FMC63 CAR-T cells show effector-specific function with wildtype CD19+ targeted cells in vitro.

Tumor cells were incubated with CAR-T cells for 6 hours. Then the tumor cells were collected and stained with Annexin-V/PI to determine specific lysis.

Zhang, Z., Chen, X., Tian, Y., Li, F., Zhao, X., Liu, J., ... & Zhang, Y. (2020). Point mutation in CD19 facilitates immune escape of B cell lymphoma from CAR-T cell therapy. Journal for immunotherapy of cancer, 8(2).

CAR Construction : FMC63 scFv-BBζ

Fig.9 FMC63 CAR-T cells show effector-specific function with mutant CD19+ targeted cells in vitro.

Tumor cells were incubated with CAR-T cells for 6 hours. Then the tumor cells were collected and stained with Annexin-V/PI to determine specific lysis.

Zhang, Z., Chen, X., Tian, Y., Li, F., Zhao, X., Liu, J., ... & Zhang, Y. (2020). Point mutation in CD19 facilitates immune escape of B cell lymphoma from CAR-T cell therapy. Journal for immunotherapy of cancer, 8(2).

CAR Construction : FMC63 scFv-BBζ

Fig.10 Bioluminescent imaging of antitumor ability of CD19 CAR-T cells in vivo.

1e6 luciferase-transgenic Raji cells were inoculated into SCID-beige mice via tail on day 7. The 1e6 wild type or mutated CD19 positive H322/Luc cells were subcutaneously injected into the mice. On day 0, 1e7 CAR-Τ cells were injected intravenously (intravenous). Then, tumor growth was monitored via bioluminescent imaging test.

Zhang, Z., Chen, X., Tian, Y., Li, F., Zhao, X., Liu, J., ... & Zhang, Y. (2020). Point mutation in CD19 facilitates immune escape of B cell lymphoma from CAR-T cell therapy. Journal for immunotherapy of cancer, 8(2).

CAR Construction : FMC63 scFv-BBζ

Fig.11 Bioluminescent imaging of antitumor ability of CD19 CAR-T cells in vivo.

1e6 luciferase-transgenic Raji cells were inoculated into SCID-beige mice via tail on day 7. The 1e6 wild type or mutated CD19 positive H322/Luc cells were subcutaneously injected into the mice. On day 0, 1e7 CAR-Τ cells were injected intravenously (intravenous). Then, tumor growth was monitored via bioluminescent imaging test.

Zhang, Z., Chen, X., Tian, Y., Li, F., Zhao, X., Liu, J., ... & Zhang, Y. (2020). Point mutation in CD19 facilitates immune escape of B cell lymphoma from CAR-T cell therapy. Journal for immunotherapy of cancer, 8(2).

CAR Construction : FMC63 scFv-BBζ

Fig.12 Data summary of antitumor ability of CD19 CAR-T cells in vivo.

1e6 luciferase-transgenic Raji cells were inoculated into SCID-beige mice via tail on day 7. The 1e6 wild type or mutated CD19 positive H322/Luc cells were subcutaneously injected into the mice. On day 0, 1e7 CAR-Τ cells were injected intravenously (intravenous). Then, tumor growth was monitored via bioluminescent imaging test.

Zhang, Z., Chen, X., Tian, Y., Li, F., Zhao, X., Liu, J., ... & Zhang, Y. (2020). Point mutation in CD19 facilitates immune escape of B cell lymphoma from CAR-T cell therapy. Journal for immunotherapy of cancer, 8(2).

CAR Construction : FMC63 scFv-BBζ; FMC63 scFv-28BBζ

Fig.13 Anti-tumor activities of mFMC63-CAR T cells.

Fluorescence images of HeLa-CD19+ expressing mCherry co-cultured with CAR T cells at an E:T ratio of 10:1 as analyzed by fluorescence microscopy.

Wutti-In, Y., Sujjitjoon, J., Sawasdee, N., Panya, A., Kongkla, K., Yuti, P., ... & Yenchitsomanus, P. T. (2021). Development of a Novel Anti-CD19 CAR Containing a Fully Human scFv and Three Costimulatory Domains. Frontiers in oncology, 11.

CAR Construction : FMC63 scFv-BBζ; FMC63 scFv-28BBζ

Fig.14 Anti-tumor activities of mFMC63-CAR T cells.

Bar graphs represent the percentages of cytotoxicity against HeLa-CD19+ target cells by CAR T cells as measured by crystal violet staining assay.

Wutti-In, Y., Sujjitjoon, J., Sawasdee, N., Panya, A., Kongkla, K., Yuti, P., ... & Yenchitsomanus, P. T. (2021). Development of a Novel Anti-CD19 CAR Containing a Fully Human scFv and Three Costimulatory Domains. Frontiers in oncology, 11.

CAR Construction : FMC63 scFv-BBζ; FMC63 scFv-28BBζ

Fig.15 Anti-tumor activities of mFMC63-CAR T cells.

Cytotoxicity of Raji cells expressing CD19 antigen by CAR T cells after co-culturing at an E:T ratio of 1:1 for 24 and 48 h.

Wutti-In, Y., Sujjitjoon, J., Sawasdee, N., Panya, A., Kongkla, K., Yuti, P., ... & Yenchitsomanus, P. T. (2021). Development of a Novel Anti-CD19 CAR Containing a Fully Human scFv and Three Costimulatory Domains. Frontiers in oncology, 11.

CAR Construction : FMC63 scFv-BBζ; FMC63 scFv-28BBζ

Fig.16 IL-2 production in response to coculture of FMC63-CAR cells with Raji cells.

Levels of secreted IL-2 cytokines in the culture supernatants of co-cultured CAR T cells and Raji cells.

Wutti-In, Y., Sujjitjoon, J., Sawasdee, N., Panya, A., Kongkla, K., Yuti, P., ... & Yenchitsomanus, P. T. (2021). Development of a Novel Anti-CD19 CAR Containing a Fully Human scFv and Three Costimulatory Domains. Frontiers in oncology, 11.

CAR Construction : FMC63 scFv-BBζ; FMC63 scFv-28BBζ

Fig.17 IFN-γ production in response to coculture of FMC63-CAR cells with Raji cells.

Levels of secreted IFN-γ cytokines in the culture supernatants of co-cultured CAR T cells and Raji cells.

Wutti-In, Y., Sujjitjoon, J., Sawasdee, N., Panya, A., Kongkla, K., Yuti, P., ... & Yenchitsomanus, P. T. (2021). Development of a Novel Anti-CD19 CAR Containing a Fully Human scFv and Three Costimulatory Domains. Frontiers in oncology, 11.

CAR Construction : FMC63 scFv-BBζ; FMC63 scFv-28BBζ

Fig.18 TNF-α production in response to coculture of FMC63-CAR cells with Raji cells.

Levels of secreted TNF-α cytokines in the culture supernatants of co-cultured CAR T cells and Raji cells.

Wutti-In, Y., Sujjitjoon, J., Sawasdee, N., Panya, A., Kongkla, K., Yuti, P., ... & Yenchitsomanus, P. T. (2021). Development of a Novel Anti-CD19 CAR Containing a Fully Human scFv and Three Costimulatory Domains. Frontiers in oncology, 11.

CAR Construction : FMC63 scFv-28BBζ

Fig.19 The killing activities of mFMC63-CAR4 T cells against Raji cell line.

The killing activities of mFMC63-CAR4 T cells against Raji (CD19+) cells after co-culturing at effector to target (E:T) ratios of 1:1, 2.5:1, and 5:1 for 4 hours.

Wutti-In, Y., Sujjitjoon, J., Sawasdee, N., Panya, A., Kongkla, K., Yuti, P., ... & Yenchitsomanus, P. T. (2021). Development of a Novel Anti-CD19 CAR Containing a Fully Human scFv and Three Costimulatory Domains. Frontiers in oncology, 11.

CAR Construction : FMC63 scFv-28BBζ

Fig.20 The killing activities of mFMC63-CAR4 T cells against K562-CD19.

The killing activities of mFMC63-CAR4 T cells against K562-CD19+ cells after co-culturing at effector to target (E:T) ratios of 1:1, 2.5:1, and 5:1 for 4 hours.

Wutti-In, Y., Sujjitjoon, J., Sawasdee, N., Panya, A., Kongkla, K., Yuti, P., ... & Yenchitsomanus, P. T. (2021). Development of a Novel Anti-CD19 CAR Containing a Fully Human scFv and Three Costimulatory Domains. Frontiers in oncology, 11.

CAR Construction : FMC63 scFv-28BBζ

Fig.21 IFN-γ production in the cell culture supernatants of anti-CD19 CAR T cells after activation by culturing with Raji (CD19+) cells.

The levels of IFN-γ production in the cell culture supernatants of anti-CD19 CAR T cells after activation by culturing with Raji (CD19+) cells at an E:T ratio of 5:1 for 24 h were analyzed by enzyme-linked immunosorbent assay (ELISA).

Wutti-In, Y., Sujjitjoon, J., Sawasdee, N., Panya, A., Kongkla, K., Yuti, P., ... & Yenchitsomanus, P. T. (2021). Development of a Novel Anti-CD19 CAR Containing a Fully Human scFv and Three Costimulatory Domains. Frontiers in oncology, 11.

CAR Construction : FMC63 scFv-28BBζ

Fig.22 TNF-α production in the cell culture supernatants of anti-CD19 CAR T cells after activation by culturing with Raji (CD19+) cells.

The levels of TNF-α production in the cell culture supernatants of anti-CD19 CAR T cells after activation by culturing with Raji (CD19+) cells at an E:T ratio of 5:1 for 24 h were analyzed by enzyme-linked immunosorbent assay (ELISA).

Wutti-In, Y., Sujjitjoon, J., Sawasdee, N., Panya, A., Kongkla, K., Yuti, P., ... & Yenchitsomanus, P. T. (2021). Development of a Novel Anti-CD19 CAR Containing a Fully Human scFv and Three Costimulatory Domains. Frontiers in oncology, 11.