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Anti-CEA (PR1A3) h(CD28-CD3ζ) CAR, pCDCAR1 (CAR-ZP4931)


All products and services are For Research Use Only and CANNOT be used in the treatment or diagnosis of disease.

The vector of anti-CEA chimeric antigen receptor (CAR) is constructed for the engineering of T cells to target Human CEA. The T cells are genetically modified through transduction with a lentiviral vector expressing scFv of anti-CEA antibody linked to CD28 and CD3ζ signaling domains. And the vector product was designed for the treatment of Colorectal cancer.

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Details

  • Target
  • CEA
  • Targeting Cell Type
  • T cell
  • Targeting Diseases
  • Colorectal cancer
  • Generation
  • Second
  • Vector Name
  • pCDCAR1
  • Vector Length
  • ~8kb
  • Vector Type
  • Lentiviral vector
  • Receptor Construction
  • scFv-CD28-CD3ζ
  • Discription of Signaling Cassetes
  • CD28
    CD28 (Cluster of Differentiation 28) is one of the proteins expressed on T cells that provide costimulatory signals required for T cell activation and survival. CD28 is the receptor for CD80 (B7.1) and CD86 (B7.2) proteins which are expressed on antigen-presenting cells (APC). CD28 modulates the primary TCR/CD3ζ signal in a different fashion than the late costimulatory elements OX40 and 4-1BB. CD28 enhances the expression of downstream regulators that impact on T-cell proliferation, death, differentiation, and effector functions. CAR+ T cells containing the CD28 endodomain showed strikingly enhanced sustained T cell activation, growth, survival. And CD28 results in a brightly expressed, stable receptor as the transmembrane domain. Including CD28 costimulatory domains in CARs led to enhanced anti-malignancy efficacy.
    CD3ζ
    CD3ζ, also known as T-cell receptor zeta, which together with T-cell receptor and CD3γ, δ , ε chain, forms the TCR-CD3 complex. ζ was expressed independently from the complex. The zeta chain plays an important role in coupling antigen recognition to several intracellular signal-transduction pathways. CD3-zeta, which contains 3 ITAMs, is the most commonly used endodomain component of CARs. It transmits an activation signal to the T cell after antigen is bound. CD3-zeta may not provide a fully competent activation signal and additional co-stimulatory signaling is needed. For example, chimeric CD28 and OX40 can be used with CD3-zeta to transmit a proliferative/survival signal, or all three can be used together.

Target

  • Clone
  • PR1A3
  • Host
  • Mouse
  • Target Species
  • Human
  • Gene Name
  • CEA
  • Synonyms
  • CEA; CD66e;

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  • Published Data
CAR scFv data FCM

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

CAR Construction : Latest 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 scFv data FuncS

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

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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 scFv data FuncS

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.

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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 scFv data FCM

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

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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 scFv data ELISA

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

CAR Construction : Latest 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 scFv data FCM

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.

CAR Construction : Latest 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.

Complete CAR data FCM

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

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

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.

Complete CAR data Cyt

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.

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

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.

Complete CAR data BLI

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

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

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.

Complete CAR data FuncS

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.

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

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.

Complete CAR data FCM

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

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

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.

More Published Data More Published Data

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For research use only. Not intended for any clinical use. No products from Creative Biolabs may be resold, modified for resale or used to manufacture commercial products without prior written approval from Creative Biolabs.

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