Anti-HER2 (4D5) h(CD28-CD3ζ) CAR, pMMLV (CAR-MV-01LX022)

CAR Construction : 4D5-4-1BB-CD3ζ

Fig.1 Cytokine release assay.

HER2-directed CAR-T cells specifically secreted IFN-γ and TNF-α when cultured with HER2+ tumor cells.

Zhang, P. F., Huang, Y., Liang, X., Li, D., Jiang, L., Yang, X., ... & Wang, W. (2020). Enhancement of the antitumor effect of HER2‐directed CAR‐T cells through blocking epithelial‐mesenchymal transition in tumor cells. The FASEB Journal, 34(8), 11185-11199.

CAR Construction : 4D5-4-1BB-CD3ζ

Fig.2 Antitumor efficacy of HER2-directed CAR-T cells in vivo.

In vivo live imaging of the metastatic ovarian cancer model. The bioluminescent signals were lower in the CAR-T cells combined with A83-01 group than in other groups.

Zhang, P. F., Huang, Y., Liang, X., Li, D., Jiang, L., Yang, X., ... & Wang, W. (2020). Enhancement of the antitumor effect of HER2‐directed CAR‐T cells through blocking epithelial‐mesenchymal transition in tumor cells. The FASEB Journal, 34(8), 11185-11199.

CAR Construction : 4D5-4-1BB-CD3ζ

Fig.3 Kaplan-Meier analysis of the overall survival for each group.

CAR-T cells combined with A83-01 significantly prolonged the survival time of mice in the CAR T+ A83-01 group and significantly prolonged survival compared with all other treatment group.

Zhang, P. F., Huang, Y., Liang, X., Li, D., Jiang, L., Yang, X., ... & Wang, W. (2020). Enhancement of the antitumor effect of HER2‐directed CAR‐T cells through blocking epithelial‐mesenchymal transition in tumor cells. The FASEB Journal, 34(8), 11185-11199.

CAR Construction : 4D5-CD28-CD3ζ

Fig.4 Cytokine production of 4D5 CAR-transduced T cells.

IFN-γ secretion of 4D5-28Z-transduced T cells following coculture with the indicated tumor lines.

Zhao, Y., Wang, Q. J., Yang, S., Kochenderfer, J. N., Zheng, Z., Zhong, X., ... & Morgan, R. A. (2009). A herceptin-based chimeric antigen receptor with modified signaling domains leads to enhanced survival of transduced T lymphocytes and antitumor activity. The Journal of Immunology, 183(9), 5563-5574.

CAR Construction : 4D5-CD28-CD3ζ

Fig.5 Specific tumor cell lysis by 4D5 CAR-transduced T cells.

4D5 CAR-transduced PBLs (4D5-28Z) were tested in a 51Cr release assay, where nontransduced PBLs (NV) served as a control.

Zhao, Y., Wang, Q. J., Yang, S., Kochenderfer, J. N., Zheng, Z., Zhong, X., ... & Morgan, R. A. (2009). A herceptin-based chimeric antigen receptor with modified signaling domains leads to enhanced survival of transduced T lymphocytes and antitumor activity. The Journal of Immunology, 183(9), 5563-5574.

CAR Construction : 4D5-CD28-CD3ζ, 4D5-CD28-4-1BB-CD3ζ

Fig.6 Treatment of a breast cancer xenograft by ErbB2-CAR-transduced PBLs.

Adoptively transferred (i.v.) 4D5-28Z or 4D5-CD8-28BBZ- or SP6-28Z-transduced PBLs were administered to animals bearing human breast tumor.

Zhao, Y., Wang, Q. J., Yang, S., Kochenderfer, J. N., Zheng, Z., Zhong, X., ... & Morgan, R. A. (2009). A herceptin-based chimeric antigen receptor with modified signaling domains leads to enhanced survival of transduced T lymphocytes and antitumor activity. The Journal of Immunology, 183(9), 5563-5574.

CAR Construction : 4D5-4-1BB-CD3ζ

Fig.7 HER2-CAR T cells administered intrathecally to non-human primates (NHP) did not cause toxicity.

Summary of CSF cytokine levels after infusion of T-HER2 target cells and HER2 CAR T cells.

Nellan, A., Rota, C., Majzner, R., Lester-McCully, C. M., Griesinger, A. M., Mulcahy Levy, J. M., ... & Lee, D. W. (2018). Durable regression of Medulloblastoma after regional and intravenous delivery of anti-HER2 chimeric antigen receptor T cells. Journal for immunotherapy of cancer, 6(1), 1-14.

CAR Construction : 4D5-CD28-4-1BB-CD3ζ

Fig.8 Interferon gamma (IFN-γ) release.

CAR-T cells were co-cultured with MDA.MB.468, MDA.MB.231, SKOV3, or MDA.MB.468.Her2 cells with Brefeldin A protein transport
inhibitor for 6 h to detect interferon gamma (IFN-c) release.

Siegler, E., Li, S., Kim, Y. J., & Wang, P. (2017). Designed ankyrin repeat proteins as Her2 targeting domains in chimeric antigen receptor-engineered T cells. Human gene therapy, 28(9), 726-736.

CAR Construction : 4D5-CD28-4-1BB-CD3ζ

Fig.9 Cytotoxicity assay.

CAR-T cells were co-cultured for 24 h with MDA.MB.468, MDA.MB.231,SKOV3, or MDA.MB.468.Her2 target cells at 1:1, 3:1, 5:1, or 10:1 effector-to-target ratios.

Siegler, E., Li, S., Kim, Y. J., & Wang, P. (2017). Designed ankyrin repeat proteins as Her2 targeting domains in chimeric antigen receptor-engineered T cells. Human gene therapy, 28(9), 726-736.

CAR Construction : 4D5-CD28-4-1BB-CD3ζ

Fig.10 4D5 CAR-T cells inhibit tumor growth in vivo.

Mice bearing SKOV3 cells were treated with 10 million NT, 4D5, G3, or 929 CAR-T cells on day 14 via tail-vein injection.

Siegler, E., Li, S., Kim, Y. J., & Wang, P. (2017). Designed ankyrin repeat proteins as Her2 targeting domains in chimeric antigen receptor-engineered T cells. Human gene therapy, 28(9), 726-736.

CAR Construction :

Fig.11 Binding kinetics of the 4D5 antibodies.

The increase of functional affinity obtained by multimerization was measured by sur-face plasmon resonance (BIAcore) on recombinant p185HER-2-ECD.

Willuda, J., Kubetzko, S., Waibel, R., Schubiger, P. A., Zangemeister-Wittke, U., & Plückthun, A. (2001). Tumor targeting of mono-, di-, and tetravalent anti-p185HER-2 miniantibodies multimerized by self-associating peptides. Journal of Biological Chemistry, 276(17), 14385-14392.

CAR Construction :

Fig.12 Binding kinetics of the 4D5 antibodies.

The increase of functional affinity obtained by multimerization was measured on living SK-OV-3 cells overexpressing p185HER-2.

Willuda, J., Kubetzko, S., Waibel, R., Schubiger, P. A., Zangemeister-Wittke, U., & Plückthun, A. (2001). Tumor targeting of mono-, di-, and tetravalent anti-p185HER-2 miniantibodies multimerized by self-associating peptides. Journal of Biological Chemistry, 276(17), 14385-14392.