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Anti-CEA (BW431/26) h(CD3ζ-OX40) CAR, pCDCAR1 (CAR-MZ042)


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 retroviral vector expressing scFv of anti-CEA antibody linked to CD3ζ and OX40 signaling domains. And the vector product was designed for the treatment of Colon carcinoma.

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Details

  • Target
  • CEA
  • Targeting Cell Type
  • T cell
  • Targeting Diseases
  • Colon carcinoma
  • Generation
  • Second
  • Vector Name
  • pCDCAR1
  • Vector Length
  • ~8kb
  • Vector Type
  • Retroviral
  • Receptor Construction
  • scFv-CD3ζ-OX40
  • Discription of Signaling Cassetes
  • 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.
    OX40
    OX40, also known as CD134 or TNFRSF4 is a member of the TNFR-superfamily of receptors which is not constitutively expressed on resting naïve T cells, unlike CD28. OX40 is a secondary co-stimulatory immune checkpoint molecule, expressed after 24 to 72 hours following activation. The interaction between OX40 and its binding partner, OX40L (CD252) plays an important role in antigen-specific T-cell expansion and survival. OX40 and OX40L also regulate cytokine production from T cells and modulate cytokine receptor signaling. OX40 cosignaling in CAR improve redirected T-cell effector functions and enhance anti-tumor activity.

Target

  • Clone
  • BW431/26
  • Host
  • Human
  • Target Species
  • Human
  • Gene Name
  • CEA
  • Synonyms
  • CEA; CD66e;

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  • Published Data
Complete CAR data FuncS

Fig.2 Serial dilutions of the anti-idiotypic mAb BW2064, which is directed to the CAR anti-CEA scFv domain, or an isotype control mAb IgG1 were coated onto microtiter plates (0.01–10 μg/ml).

CAR Construction : BW431/26 scFv-28ζ Latest CAR Construction

Fig.2 Serial dilutions of the anti-idiotypic mAb BW2064, which is directed to the CAR anti-CEA scFv domain, or an isotype control mAb IgG1 were coated onto microtiter plates (0.01–10 μg/ml).

CD4+ T cells were engineered with the respective CEA specific CAR and adjusted to equal numbers of CAR expressing cells by adding nonmodified T cells of the same donor.

Hombach, A. A., & Abken, H. (2011). Costimulation by chimeric antigen receptors revisited the T cell antitumor response benefits from combined CD28‐OX40 signalling. International journal of cancer, 129(12), 2935-2944.

Complete CAR data FCM

Fig.3 CD4+ and CD8+ T cells were engineered to express anti-CEA CARs with different endodomains domains and coincubated.

CAR Construction : BW431/26 scFv-28ζ Latest CAR Construction

Fig.3 CD4+ and CD8+ T cells were engineered to express anti-CEA CARs with different endodomains domains and coincubated.

The absence of IL-2 reveals different impact of costimulation on redirected cytolysis by CD4 and CD8 T cells.

Hombach, A. A., & Abken, H. (2011). Costimulation by chimeric antigen receptors revisited the T cell antitumor response benefits from combined CD28‐OX40 signalling. International journal of cancer, 129(12), 2935-2944.

CAR scFv data ELISA

Fig.4 The humBW431/26-CH2/CH3-g receptor triggers IL-2 secretion after binding to immobilized CEA or anti-idiotypic mAb.

CAR Construction : Latest CAR Construction

Fig.4 The humBW431/26-CH2/CH3-g receptor triggers IL-2 secretion after binding to immobilized CEA or anti-idiotypic mAb.

The supernatants were analyzed for IL-2 content by ELISA utilizing a solid phase rat mAb (1 mg/ml) specific for murine IL-2 and a biotinylated rat anti-mouse IL-2 mAb (0.25 mg/ml).

Hombach, A., Koch, D., Sircar, R., Heuser, C., Diehl, V., Kruis, W., ... & Abken, H. (1999). A chimeric receptor that selectively targets membrane-bound carcinoembryonic antigen (mCEA) in the presence of soluble CEA. Gene therapy, 6(2), 300-304.

CAR scFv data FCM

Fig.5 FACS analysis of humBW431/26-CH2/CH3-g receptor grafted MD45 T cells.

CAR Construction : Latest CAR Construction

Fig.5 FACS analysis of humBW431/26-CH2/CH3-g receptor grafted MD45 T cells.

Transfected cells were incubated for 20 min with 5-10 mg/ml of different Abs.

Hombach, A., Koch, D., Sircar, R., Heuser, C., Diehl, V., Kruis, W., ... & Abken, H. (1999). A chimeric receptor that selectively targets membrane-bound carcinoembryonic antigen (mCEA) in the presence of soluble CEA. Gene therapy, 6(2), 300-304.

CAR scFv data Cyt

Fig.6 Specific cytotoxicity of MD45 cells grafted with the humBW431/26-CH2/CH3-g receptor.

CAR Construction : Latest CAR Construction

Fig.6 Specific cytotoxicity of MD45 cells grafted with the humBW431/26-CH2/CH3-g receptor.

Cytotoxicity of transfected and non-transfected MD45 T cells to target cells was measured by time resolved fluorometry.

Hombach, A., Koch, D., Sircar, R., Heuser, C., Diehl, V., Kruis, W., ... & Abken, H. (1999). A chimeric receptor that selectively targets membrane-bound carcinoembryonic antigen (mCEA) in the presence of soluble CEA. Gene therapy, 6(2), 300-304.

CAR scFv data FCM

Fig.7 Two-color immunofluorescence of anti-CEA receptor-grafted peripheral blood T cells.

CAR Construction : Latest CAR Construction

Fig.7 Two-color immunofluorescence of anti-CEA receptor-grafted peripheral blood T cells.

Non transduced peripheral blood T cells (A) and T cells grafted with the BW431/26-scFv-Fc- (438) (B), BW431/26-scFv-Fc- (439) receptor were simultaneously incubated with a PE-conjugated anti-CD3 mAb and a FITC-conjugated anti-human IgG1 Ab and analyzed by flow cytometry.

Hombach, A., Wieczarkowiecz, A., Marquardt, T., Heuser, C., Usai, L., Pohl, C., ... & Abken, H. (2001). Tumor-specific T cell activation by recombinant immunoreceptors: CD3ζ signaling and CD28 costimulation are simultaneously required for efficient IL-2 secretion and can be integrated into one combined CD28/CD3ζ signaling receptor molecule. The Journal of Immunology, 167(11), 6123-6131.

CAR scFv data FCM

Fig.1 Peripheral blood T cells were grafted by retroviral gene transfer with the CEA specific immunoreceptor BW431/26-scFv-Fc-ζ.

CAR Construction : Latest CAR Construction

Fig.1 Peripheral blood T cells were grafted by retroviral gene transfer with the CEA specific immunoreceptor BW431/26-scFv-Fc-ζ.

To monitor receptor expression, T cells were simultaneously incubated with a FITC-conjugated anti-CD3 mAb and a PE-conjugated anti-human IgG1 Ab directed against the extracellular Fc domain of the receptor. Cells were analyzed by flow cytometry.

Hombach, A. A., Schildgen, V., Heuser, C., Finnern, R., Gilham, D. E., & Abken, H. (2007). T cell activation by antibody-like immunoreceptors: the position of the binding epitope within the target molecule determines the efficiency of activation of redirected T cells. The Journal of Immunology, 178(7), 4650-4657.

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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