Anti-EGFR (Nimotuzumab) h(CD28-CD3ζ) CAR, pCDCAR1 vector

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

Details

Target
EGFR

Targeting Cell Type
T cell

Targeting Diseases
Human primary glioblastoma

Generation
Second

Vector Name
pCDCAR1

Vector Length
~8kb

Vector Type
Lentiviral

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 co-stimulatory 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 SPR

Fig.1 SPR/Biacore experiments showing the inhibition of EGF binding to eEGFR with increasing amounts of Nimotuzumab.

CAR Construction :

Fig.1 SPR/Biacore experiments showing the inhibition of EGF binding to eEGFR with increasing amounts of Nimotuzumab.

Each antibody was added in molar excess to a constant concentration of 600 nmol/L of eEGFR and flowed over the EGF molecules immobilized to a level of 500 response units (RU) on a CM5 chip.

Talavera, A., Friemann, R., Gómez-Puerta, S., Martinez-Fleites, C., Garrido, G., Rabasa, A., ... & Moreno, E. (2009). Nimotuzumab, an antitumor antibody that targets the epidermal growth factor receptor, blocks ligand binding while permitting the active receptor conformation. Cancer research, 69(14), 5851-5859.

CAR scFv data FCM

Fig.2 Recognition of the EGFR chimera by Nimotuzumab, measured by FACS.

CAR Construction :

Fig.2 Recognition of the EGFR chimera by Nimotuzumab, measured by FACS.

In the dot plots shown in the first three panels, the Y axis corresponds to size scattering.

Complete CAR data FuncS

Fig.3 Numeric expansion of Cetux-CAR+ and Nimo-CAR+ T cells was determined by calculating fold expansion of CD3+CAR+ T cells, determined by flow cytometry, during each stimulation cycle.

CAR Construction : Nimotuzumab scFv-28ζ

Fig.3 Numeric expansion of Cetux-CAR+ and Nimo-CAR+ T cells was determined by calculating fold expansion of CD3+CAR+ T cells, determined by flow cytometry, during each stimulation cycle.

Data represented as mean ± SD, n=5. Statistical analysis by two-tailed student's t-test.

Caruso, H. G., Hurton, L. V., Najjar, A., Rushworth, D., Ang, S., Olivares, S., ... & Cooper, L. J. (2015). Tuning sensitivity of CAR to EGFR density limits recognition of normal tissue while maintaining potent antitumor activity. Cancer research, 75(17), 3505-3518.

Complete CAR data FuncS

Fig.4 Representative histograms of expression of tEGFR on EL4 cells relative to cell lines negative for EGFR.

CAR Construction : Nimotuzumab scFv-28ζ

Fig.4 Representative histograms of expression of tEGFR on EL4 cells relative to cell lines negative for EGFR.

Nimo-CAR+ T cells have impaired response to low density of EGFR.

Complete CAR data FCM

Fig.5 Representative histograms of Intracellular and surface expression of CAR determined by flow cytometry.

CAR Construction : Nimotuzumab scFv-28ζ

Fig.5 Representative histograms of Intracellular and surface expression of CAR determined by flow cytometry.

Data representative of three independent donors.

Complete CAR data FuncS

Fig.6 Relative tumor growth as assessed by serial BLI of tumor. Significant difference in BLI between mice with no treatment vs. treatment (n=6) with Cetux-CAR+ T cells (n=6, p < 0.01) reached at day 25, two-way ANOVA (Sidak’s post-test).

CAR Construction : Nimotuzumab scFv-28ζ

Nimo-CAR+ T cells exhibit impaired targeting of low-density EGFR cells in vivo.

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.