Anti-CEA (H10) 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.

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Published Data

CAR scFv data FuncS

Fig.2 Monitoring of the formation of multimeric biotinylated scFv using streptavidin molecules.

CAR Construction :

Fig.2 Monitoring of the formation of multimeric biotinylated scFv using streptavidin molecules.

The percentage specific binding of 125I-labeled streptavidin to purified CEA-sepharose was obtained by subtracting the percentages detected on control BSA-Sepharose from those measured on CEA-Sepharose.

Cloutier, S. M., Couty, S., Terskikh, A., Marguerat, L., Crivelli, V., Pugnieres, M., ... & Deperthes, D. (2000). Streptabody, a high avidity molecule made by tetramerization of in vivo biotinylated, phage display-selected scFv fragments on streptavidin. Molecular immunology, 37(17), 1067-1077.

CAR scFv data Inhibit

Fig.3 Competitive inhibition of binding of 125I-labeled mAb 35A7 to CEA–Sepharose in the presence of different scFv and streptabody concentrations.

CAR Construction :

Fig.3 Competitive inhibition of binding of 125I-labeled mAb 35A7 to CEA–Sepharose in the presence of different scFv and streptabody concentrations.

All measurements were performed in triplicate.

CAR scFv data SPR

Fig.4 Analysis of the CEA-binding behavior of the scFvs (BAD) and Streptabodies with surface plasmon resonance on a Biacore biosensor instrument.

CAR Construction :

Fig.4 Analysis of the CEA-binding behavior of the scFvs (BAD) and Streptabodies with surface plasmon resonance on a Biacore biosensor instrument.

All measurements were performed in triplicate.

CAR scFv data SPR

Fig.5 Binding of antibodies to captured cell membrane vesicles. Anti-CD30 Ki-4 diabody and Ki-4 Moab, as well as PBS and anti-CEA H10 scFv were sequentially passed over flow cell 1 and flow cell 2.

CAR Construction :

Fig.5 Binding of antibodies to captured cell membrane vesicles. Anti-CD30 Ki-4 diabody and Ki-4 Moab, as well as PBS and anti-CEA H10 scFv were sequentially passed over flow cell 1 and flow cell 2.

The absolute response signals on both flow cells were subtracted from each other in real time (flow cell 2 - flow cell 1) and subsequently X- and Y-transformed using BIAeval 3.1 program.

Klimka, A., Tur, M. K., Huhn, M., Bierfreund, U., Terrada, E., Fischer, R., ... & Barth, S. (2004). Measurement of antibody-membrane interactions by surface plasmon resonance. International journal of molecular medicine, 14(4), 765-773.

CAR scFv data FCM

Fig.1 Peripheral blood T cells were grafted by retroviral gene transfer with the CEA specific immunoreceptor H10-scFv-Fc-ζ.

CAR Construction :

Fig.1 Peripheral blood T cells were grafted by retroviral gene transfer with the CEA specific immunoreceptor H10-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 (H10) h(CD28-OX40-CD3ζ) CAR, pSBCAR1 (CAR-SB-02LX359)

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