Anti-CEA (C2-45) h(CD28-OX40-CD3ζ) CAR, pSBCAR1 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
Third

Vector Name
pSBCAR1

Vector Length
~6kb

Vector Type
Sleeping Beauty (SB) transposon

Receptor Construction
scFv-CD28-OX40-CD3ζ

Discription of Signaling Cassetes
CD28
CD28 (Cluster of Differentiation 28) is one of the proteins expressed on T cells that provide co-stimulatorysignals 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.
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.
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

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Published Data

CAR scFv data ELISA

Fig.3 Reactivity of C2-45(cIgG1) with purified CEA in ELISA.

CAR Construction :

Fig.3 Reactivity of C2-45(cIgG1) with purified CEA in ELISA.

After washing, the antibodies bound to CEA were detected with
biotinylated goat anti-human IgG.

Huang, J., Shibaguchi, H., Zhao, J., Luo, N., Kuroki, M., Kinugasa, T., ... & Kuroki, M. (2006). IgG isotype conversion of a novel human anti-carcinoembryonic antigen antibody to increase its biological activity. Anticancer research, 26(2A), 1057-1063.

CAR scFv data FCM

Fig.4 Reactivity of C2-45(cIgG1) with CEA-expressing cells in flow cytometric analysis.

CAR Construction :

Fig.4 Reactivity of C2-45(cIgG1) with CEA-expressing cells in flow cytometric analysis.

The cells used were incubated with C2-45(cIgG1),C2-45(IgG4) or control human IgG for 1 hour on ice. After washing, the cells were stained with fluorescein-conjugated rabbit anti-human IgG.

CAR scFv data FCM

Fig.5 Binding of C2-45(IgG1) with CEA-expressing cells in flow cytometric analysis.

CAR Construction :

Fig.5 Binding of C2-45(IgG1) with CEA-expressing cells in flow cytometric analysis.

All measurements were performed in triplicate.

Huang, J., Shibaguchi, H., Kuroki, M., Hirose, Y., Yamada, H., Zhao, J., ... & Kuroki, M. (2006). Subclass Conversion from IgG4 to IgG1 of a Novel Human Anti-CEA Antibody for Increasing Its Biological Activity. Japan Journal of Molecular Tumor Marker Research, 21, 53-54.

CAR scFv data FCM

Fig.6 Specificity of anti-CEA mAb-mediated targeting.

CAR Construction :

Fig.6 Specificity of anti-CEA mAb-mediated targeting.

Transduction efficiency of CEACAM-transfected CHO cells evaluated by flow cytometry.

Tanaka, T., Huang, J., Hirai, S., Kuroki, M., Kuroki, M., Watanabe, N., ... & Hamada, H. (2006). Carcinoembryonic Antigen–Targeted Selective Gene Therapy for Gastric Cancer through FZ33 Fiber-Modified Adenovirus Vectors. Clinical cancer research, 12(12), 3803-3813.

CAR scFv data Inhibit

Fig.7 Competitive inhibition assay. CEA-CHO cells were transduced with Ax3CAZ3-FZ33 at 1,000 VP/cell after preincubation with C2-45.

CAR Construction :

Fig.7 Competitive inhibition assay. CEA-CHO cells were transduced with Ax3CAZ3-FZ33 at 1,000 VP/cell after preincubation with C2-45.

All measurements were performed in triplicate.

CAR scFv data FCM

Fig.8 The L45scFv-CIR expression on human T-cells. Primary human T-cells (5x106 cells) were transfected with 2 Ìg of the L45scFv-CIR gene.

CAR Construction :

Fig.8 The L45scFv-CIR expression on human T-cells. Primary human T-cells (5x106 cells) were transfected with 2 Ìg of the L45scFv-CIR gene.

Transfectants (1x106 cells) were stained with 1 Ìg/ml of APC-labeled BSA or CEA for 1 h at 4ÆC 24 hrs after nucleofection.

Shibaguchi, H., Luo, N. X., Kuroki, M., Zhao, J., Huang, J., Hachimine, K., ... & Kuroki, M. (2006). A fully human chimeric immune receptor for retargeting T-cells to CEA-expressing tumor cells. Anticancer research, 26(6A), 4067-4072.

CAR scFv data IHC

Fig.9 Binding of L45 scFv-CIR transfected primary human T-cells to CEA-expressing tumor cells.

CAR Construction :

Fig.9 Binding of L45 scFv-CIR transfected primary human T-cells to CEA-expressing tumor cells.

The CEA expressing MKN-45 cells were mixed with parental T-cells or transfectants at the ratio of 1:5 and incubated for rossette formation for 1 h at 37C.

CAR scFv data ELISA

Fig.10 The CEA binding activity of serially diluted 45scFvLCHα was evaluated by ELISA.

CAR Construction :

Fig.10 The CEA binding activity of serially diluted 45scFvLCHα was evaluated by ELISA.

Antigen binding activity of isolated 45scFvLCHα.

Zhao, J., Kuroki, M., Shibaguchi, H., Wang, L., Huo, Q., Takami, N., ... & Kuroki, M. (2008). Recombinant human monoclonal igA antibody against CEA to recruit neutrophils to CEA-expressing cells. Oncology Research Featuring Preclinical and Clinical Cancer Therapeutics, 17(5), 217-222.

Complete CAR data WB

Fig.1 The analysis of CAR protein expression.

CAR Construction : C2-45 scFv-28ζ

Fig.1 The analysis of CAR protein expression.

Lysates were prepared from wild-type and CAR-expressing Jurkat cells, and immunoreactive bands were detected by Western blotting using an anti-CD3ζ.

Shirasu, N., Shibaguci, H., Kuroki, M., Yamada, H., & Kuroki, M. (2010). Construction and molecular characterization of human chimeric T-cell antigen receptors specific for carcinoembryonic antigen. Anticancer research, 30(7), 2731-2738.

Complete CAR data FuncS

Fig.2 The formation of the signaling clusters in CAR-expressing cells by antigen stimulation.

CAR Construction : C2-45 scFv-28ζ

Fig.2 The formation of the signaling clusters in CAR-expressing cells by antigen stimulation.

Jurkat cells co-expressing ZAP-70-AcGFP and CAR45-28ζ were plated onto a CEA-coated coverslips, fixed and stained with Alexa-labelled anti-CD3ζ and anti-myc antibodies.

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 (C2-45) h(CD28-OX40-CD3ζ) CAR, pSBCAR1 (CAR-SB-02LX367)

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