Anti-HER2 (4D5) 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
HER2

Targeting Cell Type
T cell

Targeting Diseases
Breast 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.

Target

Customize Your CAR Products

Published Data

Complete CAR data ELISA

Fig.1 Cytokine release assay.

CAR Construction : 4D5-4-1BB-CD3ζ

Fig.1 Cytokine release assay.

HER2-directed CAR-T cells specifically secreted IFN-γ and TNF-α when cultured with HER2+ tumor cells.

Zhang, P. F., Huang, Y., Liang, X., Li, D., Jiang, L., Yang, X., ... & Wang, W. (2020). Enhancement of the antitumor effect of HER2‐directed CAR‐T cells through blocking epithelial‐mesenchymal transition in tumor cells. The FASEB Journal, 34(8), 11185-11199.

Complete CAR data BI

Fig.2 Antitumor efficacy of HER2-directed CAR-T cells in vivo.

CAR Construction : 4D5-4-1BB-CD3ζ

Fig.2 Antitumor efficacy of HER2-directed CAR-T cells in vivo.

In vivo live imaging of the metastatic ovarian cancer model. The bioluminescent signals were lower in the CAR-T cells combined with A83-01 group than in other groups.

Complete CAR data FuncS

Fig.3 Kaplan-Meier analysis of the overall survival for each group.

CAR Construction : 4D5-4-1BB-CD3ζ

Fig.3 Kaplan-Meier analysis of the overall survival for each group.

CAR-T cells combined with A83-01 significantly prolonged the survival time of mice in the CAR T+ A83-01 group and significantly prolonged survival compared with all other treatment group.

Complete CAR data ELISA

Fig.4 Cytokine production of 4D5 CAR-transduced T cells.

CAR Construction : 4D5-CD28-CD3ζ

Fig.4 Cytokine production of 4D5 CAR-transduced T cells.

IFN-γ secretion of 4D5-28Z-transduced T cells following coculture with the indicated tumor lines.

Zhao, Y., Wang, Q. J., Yang, S., Kochenderfer, J. N., Zheng, Z., Zhong, X., ... & Morgan, R. A. (2009). A herceptin-based chimeric antigen receptor with modified signaling domains leads to enhanced survival of transduced T lymphocytes and antitumor activity. The Journal of Immunology, 183(9), 5563-5574.

Complete CAR data FuncS

Fig.5 Specific tumor cell lysis by 4D5 CAR-transduced T cells.

CAR Construction : 4D5-CD28-CD3ζ

Fig.5 Specific tumor cell lysis by 4D5 CAR-transduced T cells.

4D5 CAR-transduced PBLs (4D5-28Z) were tested in a 51Cr release assay, where nontransduced PBLs (NV) served as a control.

Complete CAR data FuncS

Fig.6 Treatment of a breast cancer xenograft by ErbB2-CAR-transduced PBLs.

CAR Construction : 4D5-CD28-CD3ζ, 4D5-CD28-4-1BB-CD3ζ

Fig.6 Treatment of a breast cancer xenograft by ErbB2-CAR-transduced PBLs.

Adoptively transferred (i.v.) 4D5-28Z or 4D5-CD8-28BBZ- or SP6-28Z-transduced PBLs were administered to animals bearing human breast tumor.

Complete CAR data ELISA

Fig.7 HER2-CAR T cells administered intrathecally to non-human primates (NHP) did not cause toxicity.

CAR Construction : 4D5-4-1BB-CD3ζ

Fig.7 HER2-CAR T cells administered intrathecally to non-human primates (NHP) did not cause toxicity.

Summary of CSF cytokine levels after infusion of T-HER2 target cells and HER2 CAR T cells.

Nellan, A., Rota, C., Majzner, R., Lester-McCully, C. M., Griesinger, A. M., Mulcahy Levy, J. M., ... & Lee, D. W. (2018). Durable regression of Medulloblastoma after regional and intravenous delivery of anti-HER2 chimeric antigen receptor T cells. Journal for immunotherapy of cancer, 6(1), 1-14.

Complete CAR data FCM

Fig.8 Interferon gamma (IFN-γ) release.

CAR Construction : 4D5-CD28-4-1BB-CD3ζ

Fig.8 Interferon gamma (IFN-γ) release.

CAR-T cells were co-cultured with MDA.MB.468, MDA.MB.231, SKOV3, or MDA.MB.468.Her2 cells with Brefeldin A protein transport
inhibitor for 6 h to detect interferon gamma (IFN-c) release.

Siegler, E., Li, S., Kim, Y. J., & Wang, P. (2017). Designed ankyrin repeat proteins as Her2 targeting domains in chimeric antigen receptor-engineered T cells. Human gene therapy, 28(9), 726-736.

Complete CAR data FCM

Fig.9 Cytotoxicity assay.

CAR Construction : 4D5-CD28-4-1BB-CD3ζ

Fig.9 Cytotoxicity assay.

CAR-T cells were co-cultured for 24 h with MDA.MB.468, MDA.MB.231,SKOV3, or MDA.MB.468.Her2 target cells at 1:1, 3:1, 5:1, or 10:1 effector-to-target ratios.

Siegler, E., Li, S., Kim, Y. J., & Wang, P. (2017). Designed ankyrin repeat proteins as Her2 targeting domains in chimeric antigen receptor-engineered T cells. Human gene therapy, 28(9), 726-736.

Complete CAR data FuncS

Fig.10 4D5 CAR-T cells inhibit tumor growth in vivo.

CAR Construction : 4D5-CD28-4-1BB-CD3ζ

Fig.10 4D5 CAR-T cells inhibit tumor growth in vivo.

Mice bearing SKOV3 cells were treated with 10 million NT, 4D5, G3, or 929 CAR-T cells on day 14 via tail-vein injection.

Siegler, E., Li, S., Kim, Y. J., & Wang, P. (2017). Designed ankyrin repeat proteins as Her2 targeting domains in chimeric antigen receptor-engineered T cells. Human gene therapy, 28(9), 726-736.

CAR scFv data SPR

Fig.11 Binding kinetics of the 4D5 antibodies.

CAR Construction :

Fig.11 Binding kinetics of the 4D5 antibodies.

The increase of functional affinity obtained by multimerization was measured by sur-face plasmon resonance (BIAcore) on recombinant p185HER-2-ECD.

Willuda, J., Kubetzko, S., Waibel, R., Schubiger, P. A., Zangemeister-Wittke, U., & Plückthun, A. (2001). Tumor targeting of mono-, di-, and tetravalent anti-p185HER-2 miniantibodies multimerized by self-associating peptides. Journal of Biological Chemistry, 276(17), 14385-14392.

CAR scFv data SPR

Fig.12 Binding kinetics of the 4D5 antibodies.

CAR Construction :

Fig.12 Binding kinetics of the 4D5 antibodies.

The increase of functional affinity obtained by multimerization was measured on living SK-OV-3 cells overexpressing p185HER-2.

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.