Anti-CEA (hPR1A3) h(41BB-CD3ζ) CAR, pCDCAR1 (CAR-ZP4934)

CAR Construction :

Fig.1 Time-course FACS analysis of hPR1A3 binding to CEA-expressing MKN45 cells.

Analysis over time suggests that the CEA-PR1A3 complex is not internalised within 3 h of binding to CEA.

Conaghan, P. J., Ashraf, S. Q., Tytherleigh, M. G., Wilding, J. L., Tchilian, E., Bicknell, D., ... & Bodmer, W. F. (2008). Targeted killing of colorectal cancer cell lines by a humanised IgG1 monoclonal antibody that binds to membrane-bound carcinoembryonic antigen. British journal of cancer, 98(7), 1217-1225.

CAR Construction :

Fig.2 Purified NK cells are much more effective killers than unfractionated PBMC.

The flouresencebased EuTDA assay was used with effector:target ratios ranging from 10 : 1 to 50 : 1.

Conaghan, P. J., Ashraf, S. Q., Tytherleigh, M. G., Wilding, J. L., Tchilian, E., Bicknell, D., ... & Bodmer, W. F. (2008). Targeted killing of colorectal cancer cell lines by a humanised IgG1 monoclonal antibody that binds to membrane-bound carcinoembryonic antigen. British journal of cancer, 98(7), 1217-1225.

CAR Construction :

Fig.3 Binding activity of anti-carcinoembryonic antigen (CEA) antibodies to membrane-bound CEA on MKN-45 under the presence or absence of soluble CEA.

All measurements were performed in triplicate.

Shinmi, D., Nakano, R., Mitamura, K., Suzuki‐Imaizumi, M., Iwano, J., Isoda, Y., ... & Masuda, K. (2017). Novel anticarcinoembryonic antigen antibody–drug conjugate has antitumor activity in the existence of soluble antigen. Cancer Medicine, 6(4), 798-808.

CAR Construction :

Fig.4 mPR1A3 binding to cell-surface CEA was determined by flow cytometry.

C15 cells were stained with either mPR1A3, anti-CEA polyclonal serum, or IC mAb.

Wilkinson, R. W., Ross, E. L., Ellison, D., Zimmermann, W., Snary, D., & Mather, S. J. (2002). Evaluation of a transgenic mouse model for anti-human CEA radioimmunotherapeutics. Journal of Nuclear Medicine, 43(10), 1368-1376.

CAR Construction :

Fig.5 mPR1A3 binding to soluble CEA was determined by ELISA.

All measurements were performed in triplicate.

Wilkinson, R. W., Ross, E. L., Ellison, D., Zimmermann, W., Snary, D., & Mather, S. J. (2002). Evaluation of a transgenic mouse model for anti-human CEA radioimmunotherapeutics. Journal of Nuclear Medicine, 43(10), 1368-1376.

CAR Construction :

Fig.6 Comparison of the binding of unmodified humanised IgG1 PR1A3 (uhPR1A3) and glycoengineered IgG1 PR1A3 (ghPR1A3) to the high CEA expressing cell line SKCO-1.

Mean fluorescent intensities, based on flow cytometric analysis, of uhPR1A3 and ghPR1A3 at different antibody concentrations.

Wilkinson, R. W., Ross, E. L., Ellison, D., Zimmermann, W., Snary, D., & Mather, S. J. (2002). Evaluation of a transgenic mouse model for anti-human CEA radioimmunotherapeutics. Journal of Nuclear Medicine, 43(10), 1368-1376.

CAR Construction : PR1A3 scFv-CD28-4-1BB-CD3ζ

Fig.7 Expression of CEA chimeric antigen receptor (CAR) activator and Tmod blocker in Jurkat cells.

Jurkat cells that contain an NFAT-luciferase reporter were engineered to stably express the activator and blocker from two separate constructs.

Sandberg, M. L., Wang, X., Martin, A. D., Nampe, D. P., Gabrelow, G. B., Li, C. Z., ... & Kamb, A. (2022). A carcinoembryonic antigen-specific cell therapy selectively targets tumor cells with HLA loss of heterozygosity in vitro and in vivo. Science Translational Medicine, 14(634), eabm0306.

CAR Construction : PR1A3 scFv-CD28-4-1BB-CD3ζ

Fig.8 Selective cytotoxicity of CEA Tmod and CEA CAR cells co-cultured with A*02+ and A*02- H508 target cells mixed at 1:1 ratios.

Images show representative examples of selectivity of CEA Tmod compared to the TCR in mixed-culture cytotoxicity assays.

Sandberg, M. L., Wang, X., Martin, A. D., Nampe, D. P., Gabrelow, G. B., Li, C. Z., ... & Kamb, A. (2022). A carcinoembryonic antigen-specific cell therapy selectively targets tumor cells with HLA loss of heterozygosity in vitro and in vivo. Science Translational Medicine, 14(634), eabm0306.

CAR Construction : PR1A3 scFv-CD28-4-1BB-CD3ζ

Fig.9 Bioluminescence intensity (BLI) changes of mice dosed at 7x106 T cells per mouse.

Five mice per group were analyzed for BLI.

Sandberg, M. L., Wang, X., Martin, A. D., Nampe, D. P., Gabrelow, G. B., Li, C. Z., ... & Kamb, A. (2022). A carcinoembryonic antigen-specific cell therapy selectively targets tumor cells with HLA loss of heterozygosity in vitro and in vivo. Science Translational Medicine, 14(634), eabm0306.

CAR Construction : PR1A3 scFv-CD28-4-1BB-CD3ζ

Fig.10 T cells from the same donor (D12333) used in vivo were transduced with CEA Tmod, CAR, or TCR lentiviral vectors and cultured with 300 IU/ml IL-2 for 17 days.

Acute cytotoxicity at different E:T ratios (left panel) and cytokine (IFN-γ, IL-2, and TNF-α; right panel) concentrations were determined.

Sandberg, M. L., Wang, X., Martin, A. D., Nampe, D. P., Gabrelow, G. B., Li, C. Z., ... & Kamb, A. (2022). A carcinoembryonic antigen-specific cell therapy selectively targets tumor cells with HLA loss of heterozygosity in vitro and in vivo. Science Translational Medicine, 14(634), eabm0306.

CAR Construction : PR1A3 scFv-CD28-4-1BB-CD3ζ

Fig.11 Surface expression of CEA and A*02 on H508 and SW1463 cell lines and variants.

These lines have antigen numbers and A*02:CEA expression ratios similar to normal colon tissue.

Sandberg, M. L., Wang, X., Martin, A. D., Nampe, D. P., Gabrelow, G. B., Li, C. Z., ... & Kamb, A. (2022). A carcinoembryonic antigen-specific cell therapy selectively targets tumor cells with HLA loss of heterozygosity in vitro and in vivo. Science Translational Medicine, 14(634), eabm0306.