Anti-CEA (BW431/26) h(CD28-CD3ζ) CAR, pMMLV vector

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

Details

Target
CEA

Targeting Cell Type
T cell

Targeting Diseases
Pancreatic cancer

Generation
Second

Vector Name
pMMLV

Vector Length
~6kb

Vector Type
Recombinant Moloney murine leukemia virus (MMLV) retroviral 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 4-1BB and OX40 can be used with CD3-zeta to transmit a proliferative/survival signal, or all three can be used together.

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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

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

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.

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 :

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 :

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.

CAR scFv data Cyt

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

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.

CAR scFv data FCM

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

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 :

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

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.

Anti-CEA (BW431/26) h(CD28-CD3ζ) CAR, pMMLV (CAR-MV-01LX121)

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