Anti-CEA (MFE-23) h(CD28-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
Colon Cancer

Generation
Second

Vector Name
pSBCAR1

Vector Length
~6kb

Vector Type
Sleeping Beauty (SB) transposon

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 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 FCM

Fig.1 Binding capacity of ssSM3E for CEA using flow cytometry.

CAR Construction :

Fig.1 Binding capacity of ssSM3E for CEA using flow cytometry.

As positive control, the anti-human CEA polyclonal antibody was used confirming the high and low expression levels of CEA on the tumor cells.

Boonstra, M. C., Tolner, B., Schaafsma, B. E., Boogerd, L. S., Prevoo, H. A., Bhavsar, G., ... & Vahrmeijer, A. L. (2015). Preclinical evaluation of a novel CEA‐targeting near‐infrared fluorescent tracer delineating colorectal and pancreatic tumors. International journal of cancer, 137(8), 1910-1920.

CAR scFv data IF

Fig.2 High and low CEA-expressing tumor cells were incubated with fluorescein-labeled ssSM3E or fluorescein-labeled polyclonal antibody.

CAR Construction :

Fig.2 High and low CEA-expressing tumor cells were incubated with fluorescein-labeled ssSM3E or fluorescein-labeled polyclonal antibody.

An increase in concentration showed higher tumor-specific signal while no signal was observed on the negative cell line.

CAR scFv data IHC

Fig.3 Validating ssSM3E/800CW in orthotopic mice models and histological validation.

CAR Construction :

Fig.3 Validating ssSM3E/800CW in orthotopic mice models and histological validation.

Tumor specificity of the signals was confirmed using additional immunohistochemical stainings for human-cytokeratin and human-CEA. Magnification: 320.

CAR scFv data FCM

Fig.6 The effect of IFN-g as a single agent and in combination with IL-6 on CEA-expression on HT29-19A tumour cells.

CAR Construction :

Fig.6 The effect of IFN-g as a single agent and in combination with IL-6 on CEA-expression on HT29-19A tumour cells.

All incubation times are 48 h. Mean ¯uorescence intensities are given in the upper right corner.

Verhaar, M. J., Damen, C. A., Zonnenberg, B. A., & Blijham, G. H. (1999). In vitro upregulation of carcinoembryonic antigen expression by combinations of cytokines. Cancer letters, 139(1), 67-73.

CAR scFv data ELISA

Fig.7 Titration curves comparing the functionality of purified MFE23-GRNLY and MFE23 by ELISA against plastic immobilized CEA.

CAR Construction :

Fig.7 Titration curves comparing the functionality of purified MFE23-GRNLY and MFE23 by ELISA against plastic immobilized CEA.

Characterization of purified MFE23-GRNLY immunotoxin.

Ibáñez-Pérez, R., Guerrero-Ochoa, P., Al-Wasaby, S., Navarro, R., Tapia-Galisteo, A., De Miguel, D., ... & Anel, A. (2019). Anti-tumoral potential of a human granulysin-based, CEA-targeted cytolytic immunotoxin. Oncoimmunology, 8(11), 1641392.

CAR scFv data FCM

Fig.8 MFE23-GRNLY binding to CEA expressed on the cell surface.

CAR Construction :

Fig.8 MFE23-GRNLY binding to CEA expressed on the cell surface.

Dark-gray histograms correspond to the negative controls.

CAR scFv data Cyt

Fig.9 In vitro cytotoxicity of GRNLY and MFE23-GRNLY on HT-29 and HeLa-CEA cells.

CAR Construction :

Fig.9 In vitro cytotoxicity of GRNLY and MFE23-GRNLY on HT-29 and HeLa-CEA cells.

HT-29 or HeLa-CEA cells were incubated with increasing concentrations of recombinant granulysin (blackbars) or of the chimeric MFE23-GRNLY (whitebars) during 24h.

CAR scFv data FCM

Fig.4 Flow cytometry analyses show concentration-dependent binding of B6-1 to αvβ6 on cells.

CAR Construction :

Fig.4 Flow cytometry analyses show concentration-dependent binding of B6-1 to αvβ6 on cells.

MFE-23 or B6-1 was allowed to bind to αvβ6-expressing (A375Pβ6) and non-expressing (A375Ppuro) cells.

Kogelberg, H., Tolner, B., Thomas, G. J., Di Cara, D., Minogue, S., Ramesh, B., ... & Chester, K. (2008). Engineering a single-chain fv antibody to αvβ6 integrin using the specificity-determining loop of a foot-and-mouth disease virus. Journal of molecular biology, 382(2), 385-401.

CAR scFv data Inhibit

Fig.5 Inhibition of cell migration by B6-1 and B6-2.

CAR Construction :

Fig.5 Inhibition of cell migration by B6-1 and B6-2.

Charts showing that B6-1 and B6-2 blocked the migration of αvβ6-expressing cells towards LAP.

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 (MFE-23) h(CD28-CD3ζ) CAR, pSBCAR1 (CAR-SB-LX0158)

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