Anti-HER2 (FRP5) h(CD28-CD3ζ) CAR, pMMLV vector

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

Details

Target
HER2

Targeting Cell Type
T cell

Targeting Diseases
Breast cancer

Generation
Second

Vector Name
pMMLV

Vector Length
~6kb

Vector Type
Recombinant Moloney murine leukemia virus (MMLV) retroviral vector

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 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 ELISA

Fig.1 Cytokine release assay.

CAR Construction : FRP5-CD28-CD3ζ

Fig.1 Cytokine release assay.

HER2-specific T cells were stimulated with HER2-positive (U2-OS, Sa-OS, SK-ES-1, MNNG, MG-63, HOS, Hs 894, Hs 899, and LM7) or HER2-negative (MDA-MB-468) cells. 24-48-hours poststimulation the IFN-γ and IL-2 concentration was determined.

Ahmed, N., Salsman, V. S., Yvon, E., Louis, C. U., Perlaky, L., Wels, W. S., ... & Gottschalk, S. (2009). Immunotherapy for osteosarcoma: genetic modification of T cells overcomes low levels of tumor antigen expression. Molecular Therapy, 17(10), 1779-1787.

Complete CAR data Cyt

Fig.2 Cytotoxicity assay in vitro.

CAR Construction : FRP5-CD28-CD3ζ

Fig.2 Cytotoxicity assay in vitro.

Only HER2-specific T cells killed HER2 positive osteosarcoma cells, in a 4 hour 51Cr-release cytotoxicity assay; nontransduced (NT) T cells did not.

Complete CAR data BI

Fig.3 Adoptively transferred human HER2-specific T cells induce regression of intraperitoneal osteosarcoma xenografts.

CAR Construction : FRP5-CD28-CD3ζ

Fig.3 Adoptively transferred human HER2-specific T cells induce regression of intraperitoneal osteosarcoma xenografts.

Representative images of LM7.eGFP.FFLuc cells injected animals after CAR-T treatment.

Complete CAR data FuncS

Fig.4 Cytokine secretion assay.

CAR Construction : FRP5-41BB-CD3ζ

Fig.4 Cytokine secretion assay.

Secretion of TNF-α and IFN-γ by CAR T cells assessed by cytometric bead array after 24 h exposure to synovial sarcoma cells.

Murayama, Y., Kawashima, H., Kubo, N., Shin, C., Kasahara, Y., Imamura, M., ... & Imai, C. (2021). Effectiveness of 4-1BB-costimulated HER2-targeted chimeric antigen receptor T cell therapy for synovial sarcoma. Translational Oncology, 14(12), 101227.

Complete CAR data FuncS

Fig.5 Evaluation of the antitumor effect.

CAR Construction : FRP5-41BB-CD3ζ

Fig.5 Evaluation of the antitumor effect.

Quantitative evaluation of the antitumor effect of HER2-targeted CAR T cells on synovial sarcoma cells using real-time cell analysis.

Complete CAR data BI

Fig.6 Scheme of tumor cell spheroid forming and killing.

CAR Construction : FRP5-41BB-CD3ζ, FRP5-CD28-CD3ζ

Fig.6 Scheme of tumor cell spheroid forming and killing.

The loss of GFP can be measured for cytotoxicity readout FRP5 and MGA271 CAR T cells can effectively kill canine cancer spheroids.

Zhang, S., Kohli, K., Black, R. G., Hayes, B., Miller, C., Maeda-Whitaker, M., ... & Pollack, S. (2020). 139 Establishment of canine CAR T cells treatment model for solid tumor immunotherapy development.

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