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Anti-CEA (SCA431) h(CD28-CD3ζ) CAR, Pcdcar1 (CAR-MZ088)


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 mouse CEA. The T cells are genetically modified through transduction with a retroviral vector expressing scFv of anti-CEA antibody linked to CD28 and CD3ζ signaling domains. And the vector product was designed for the treatment of Pancreatic carcinoma.

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Details

  • Target
  • CEA
  • Targeting Cell Type
  • T cell
  • Targeting Diseases
  • Pancreatic carcinoma
  • Generation
  • Second
  • Vector Name
  • pCDCAR1
  • Vector Length
  • ~8kb
  • Vector Type
  • Retroviral
  • 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 co-stimulatory 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
  • SCA431
  • Host
  • Mouse
  • Target Species
  • Mouse
  • Gene Name
  • CEA
  • Synonyms
  • CEA; CD66e;

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  • Published Data
Complete CAR data FCM

Fig.3 CD4 T cells were modified with the CAR and used for adoptive transfer in this study.

CAR Construction : SCA431 scFv-28ζ Latest CAR Construction

Fig.3 CD4 T cells were modified with the CAR and used for adoptive transfer in this study.

CAR expression was monitored by flow cytometry using a phycoerythrin-labeled anti-mouse IgG1 antibody and an allophycocyanin-conjugated antibody directed against CD4.

Chmielewski, M., Hahn, O., Rappl, G., Nowak, M., Schmidt–Wolf, I. H., Hombach, A. A., & Abken, H. (2012). T cells that target carcinoembryonic antigen eradicate orthotopic pancreatic carcinomas without inducing autoimmune colitis in mice. Gastroenterology, 143(4), 1095-1107.

Complete CAR data ELISA

Fig.4 T cells with anti-CEA CAR (7.8 1021.0 105 cells/well) were coincubated at the indicated effector-to-tumor cell ratios with Panc02 cells with or without CEA expression for 48 hours.

CAR Construction : SCA431 scFv-28ζ Latest CAR Construction

Fig.4 T cells with anti-CEA CAR (7.8 1021.0 105 cells/well) were coincubated at the indicated effector-to-tumor cell ratios with Panc02 cells with or without CEA expression for 48 hours.

Interferon-gamma in the culture supernatant as marker for T-cell activation was determined by enzyme-linked immunosorbent assay (ELISA).

Chmielewski, M., Hahn, O., Rappl, G., Nowak, M., Schmidt–Wolf, I. H., Hombach, A. A., & Abken, H. (2012). T cells that target carcinoembryonic antigen eradicate orthotopic pancreatic carcinomas without inducing autoimmune colitis in mice. Gastroenterology, 143(4), 1095-1107.

Complete CAR data FCM

Fig.5 Lung epithelia cells were isolated from the lung of CEAtg mice. Cells were stained for CEA and flow sorted before use in the study.

CAR Construction : SCA431 scFv-28ζ Latest CAR Construction

Fig.5 Lung epithelia cells were isolated from the lung of CEAtg mice. Cells were stained for CEA and flow sorted before use in the study.

Mouse CEA Panc02 pancreatic carcinoma cells and fibrosarcoma CEA C15A3 cells were used for comparison.

Chmielewski, M., Hahn, O., Rappl, G., Nowak, M., Schmidt–Wolf, I. H., Hombach, A. A., & Abken, H. (2012). T cells that target carcinoembryonic antigen eradicate orthotopic pancreatic carcinomas without inducing autoimmune colitis in mice. Gastroenterology, 143(4), 1095-1107.

Complete CAR data FuncS

Fig.6 CBLuc-marked Panc02 tumor cells and GLuc-marked T cells were recorded by bioluminescence imaging in the same mouse at the indicated days before and after T-cell transfer.

CAR Construction : SCA431 scFv-28ζ Latest CAR Construction

Fig.6 CBLuc-marked Panc02 tumor cells and GLuc-marked T cells were recorded by bioluminescence imaging in the same mouse at the indicated days before and after T-cell transfer.

Data of bioluminescence signals were quantified.

Chmielewski, M., Hahn, O., Rappl, G., Nowak, M., Schmidt–Wolf, I. H., Hombach, A. A., & Abken, H. (2012). T cells that target carcinoembryonic antigen eradicate orthotopic pancreatic carcinomas without inducing autoimmune colitis in mice. Gastroenterology, 143(4), 1095-1107.

Complete CAR data IHC

Fig.7 Tissues from CEAtg mice treated with CAR-engineered or nonmodified T cells were stained with H&E and recorded for CEA by antibody staining.

CAR Construction : SCA431 scFv-28ζ Latest CAR Construction

Fig.7 Tissues from CEAtg mice treated with CAR-engineered or nonmodified T cells were stained with H&E and recorded for CEA by antibody staining.

Wild-type (wt) mice treated with CAR T cells were analyzed for comparison.

Chmielewski, M., Hahn, O., Rappl, G., Nowak, M., Schmidt–Wolf, I. H., Hombach, A. A., & Abken, H. (2012). T cells that target carcinoembryonic antigen eradicate orthotopic pancreatic carcinomas without inducing autoimmune colitis in mice. Gastroenterology, 143(4), 1095-1107.

Complete CAR data FuncS

Fig.8 Lymphodepletion before therapy did not favor autoimmune colitis upon CAR T-cell infusion.

CAR Construction : SCA431 scFv-28ζ Latest CAR Construction

Fig.8 Lymphodepletion before therapy did not favor autoimmune colitis upon CAR T-cell infusion.

Body weight of mice did not substantially alter during treatment.

Chmielewski, M., Hahn, O., Rappl, G., Nowak, M., Schmidt–Wolf, I. H., Hombach, A. A., & Abken, H. (2012). T cells that target carcinoembryonic antigen eradicate orthotopic pancreatic carcinomas without inducing autoimmune colitis in mice. Gastroenterology, 143(4), 1095-1107.

Complete CAR data FCM

Fig.9 Transduction efficiency of CARS into Teffs and Tregs.

CAR Construction : SCA431 scFv-28ζ Latest CAR Construction

Fig.9 Transduction efficiency of CARS into Teffs and Tregs.

Expression of the CEA-specific CAR was detected by incubating the cells with soluble CEA followed by staining with an anti-CEA antibody.

Blat, D., Zigmond, E., Alteber, Z., Waks, T., & Eshhar, Z. (2014). Suppression of murine colitis and its associated cancer by carcinoembryonic antigen-specific regulatory T cells. Molecular Therapy, 22(5), 1018-1028.

Complete CAR data FuncS

Fig.10 In vitro stimulation and cytotoxicity of CEA-specific CAR T cells.

CAR Construction : SCA431 scFv-28ζ Latest CAR Construction

Fig.10 In vitro stimulation and cytotoxicity of CEA-specific CAR T cells.

Colorectal tumors were extracted from CEABAC-2 mice and dissociated to serve as target cells for CD4+ CAR Teff stimulation.

Blat, D., Zigmond, E., Alteber, Z., Waks, T., & Eshhar, Z. (2014). Suppression of murine colitis and its associated cancer by carcinoembryonic antigen-specific regulatory T cells. Molecular Therapy, 22(5), 1018-1028.

Complete CAR data Cyt

Fig.11 Cytotoxicity of CEA-specific CAR CD4+ Teffs, CD8+ Teffs, and Tregs towards CEA-expressing Capan-1 cells
transfected with firefly luciferase, as measured by bioluminescence signal intensity.

CAR Construction : SCA431 scFv-28ζ Latest CAR Construction

Fig.11 Cytotoxicity of CEA-specific CAR CD4+ Teffs, CD8+ Teffs, and Tregs towards CEA-expressing Capan-1 cells transfected with firefly luciferase, as measured by bioluminescence signal intensity.

Control: Irrelevant CAR Teffs.

Blat, D., Zigmond, E., Alteber, Z., Waks, T., & Eshhar, Z. (2014). Suppression of murine colitis and its associated cancer by carcinoembryonic antigen-specific regulatory T cells. Molecular Therapy, 22(5), 1018-1028.

Complete CAR data FuncS

Fig.1 Severe weight loss and the eventual death of CEA-Tg mice transferred with CEA-specific CAR-T cells.

CAR Construction : SCA431 scFv-28ζ Latest CAR Construction

Fig.1 Severe weight loss and the eventual death of CEA-Tg mice transferred with CEA-specific CAR-T cells.

Cryostat sections of organs collected from mice at 17 day after tumor inoculation.

Wang, L., Ma, N., Okamoto, S., Amaishi, Y., Sato, E., Seo, N., ... & Shiku, H. (2016). Efficient tumor regression by adoptively transferred CEA-specific CAR-T cells associated with symptoms of mild cytokine release syndrome. Oncoimmunology, 5(9), e1211218.

Complete CAR data FuncS

Fig.2 Increased efficacy of tumor growth suppression by CEA-specific CAR-T cells in preconditioned mice is associated with severe weight loss and eventual death of CEA-Tg mice, but not WT mice

CAR Construction : SCA431 scFv-28ζ Latest CAR Construction

Fig.2 Increased efficacy of tumor growth suppression by CEA-specific CAR-T cells in preconditioned mice is associated with severe weight loss and eventual death of CEA-Tg mice, but not WT mice

CAR-T cells prepared from CEA-Tg and WT mice and those mock-transduced T cells from WT mice were transferred into MC32a tumor-bearing CEA-Tg mice that also received fludarabine,cyclophosphamide, and total body irradiation. Body weight changes of CEA-Tg were analyzed at indicated time points.

Wang, L., Ma, N., Okamoto, S., Amaishi, Y., Sato, E., Seo, N., ... & Shiku, H. (2016). Efficient tumor regression by adoptively transferred CEA-specific CAR-T cells associated with symptoms of mild cytokine release syndrome. Oncoimmunology, 5(9), e1211218.

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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