Anti-CEA (SCA431) h(CD28-41BB-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-CD28-41BB-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.
41BB
CD137 (also known as 4-1BB) is a surface co-stimulatory glycoprotein originally described as present on activated T lymphocytes, which belongs to the tumor necrosis factor (TNF) receptor superfamily. It is expressed mainly on activated CD4+ and CD8+ T cells, and binds to a high-affinity ligand (4-1BBL) expressed on several antigen-presenting cells such as macrophages and activated B cells. On the basis of preclinical observation, this molecule can promote the persistence of antigen-specific and antigennonspecific chimeric antigen receptor T-cells to significantly increases antitumor 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 4-1BB 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

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

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

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).

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

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.

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

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.

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

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.

Complete CAR data FuncS

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

CAR Construction : SCA431 scFv-28ζ

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.

Complete CAR data FCM

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

CAR Construction : SCA431 scFv-28ζ

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

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.

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

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.

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

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

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.

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 (SCA431) h(CD28-41BB-CD3ζ) CAR, pSBCAR1 (CAR-SB-02LX126)

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