Anti-PSMA (HuJ591) 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
PSMA

Targeting Cell Type
T cell

Targeting Diseases
Prostate 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

Customize Your CAR Products

Published Data

CAR scFv data FCM

Fig.1 Binding of BiJ591, scFvJ591, huJ591, and OKT-3 to human PSMA and CD3 receptors by flow cytometry.

CAR Construction :

Fig.1 Binding of BiJ591, scFvJ591, huJ591, and OKT-3 to human PSMA and CD3 receptors by flow cytometry.

LNCaP (PSMAþ) and Jurkat (CD3þ) cells were incubated with various concentrations (0.001-300 mg/mL) of antibodies and stained with a fluorescent secondary antibody.

Leconet, W., Liu, H., Guo, M., Le Lamer-Déchamps, S., Molinier, C., Kim, S., ... & Bander, N. H. (2018). Anti-PSMA/CD3 bispecific antibody delivery and antitumor activity using a polymeric depot formulation. Molecular Cancer Therapeutics, 17(9), 1927-1940.

CAR scFv data FuncS

Fig.2 Cytokine release of purified T cell in presence or not of LNCaP cells and increasing concentrations of BiJ591.

CAR Construction :

Fig.2 Cytokine release of purified T cell in presence or not of LNCaP cells and increasing concentrations of BiJ591.

Cytokine concentration was determined by cytometry-based bead assay.

CAR scFv data Cyt

Fig.3 BiJ591-dependent cell lysis on PCa cell lines coincubated with human PBMCs (whole WBC population, including lymphocytes and monocytes) or purified T cells.

CAR Construction :

Fig.3 BiJ591-dependent cell lysis on PCa cell lines coincubated with human PBMCs (whole WBC population, including lymphocytes and monocytes) or purified T cells.

All measurements were performed in triplicate.

CAR scFv data FCM

Fig.4 Binding of the anti-PSMA mAbs 3/A12,3/E7,3/F11,and J591 to C4-2 cells.

CAR Construction :

Fig.4 Binding of the anti-PSMA mAbs 3/A12,3/E7,3/F11,and J591 to C4-2 cells.

Cells were incubated with the mAbs,washed to eliminate unbound antibody, stained with goat anti-mouse Ig-RPE as secondary antibody, andanalyzed by flow cytometry.

Wolf, P., Freudenberg, N., Bühler, P., Alt, K., Schultze‐Seemann, W., Wetterauer, U., & Elsässer‐Beile, U. (2010). Three conformational antibodies specific for different PSMA epitopes are promising diagnostic and therapeutic tools for prostate cancer. The Prostate, 70(5), 562-569.

CAR scFv data FCM

Fig.5 Nanoscale flow cytometry of various calibration sizing beads and in vitro generated prostate microparticles.

CAR Construction :

Fig.5 Nanoscale flow cytometry of various calibration sizing beads and in vitro generated prostate microparticles.

Data are represented as mean±s.d.

Biggs, C. N., Siddiqui, K. M., Al-Zahrani, A. A., Pardhan, S., Brett, S. I., Guo, Q. Q., ... & Leong, H. S. (2016). Prostate extracellular vesicles in patient plasma as a liquid biopsy platform for prostate cancer using nanoscale flow cytometry. Oncotarget, 7(8).

CAR scFv data FCM

Fig.6 Specific binding of J591 to PSMA and activation of fluorescence signal after binding.

CAR Construction :

Fig.6 Specific binding of J591 to PSMA and activation of fluorescence signal after binding.

Scale bar indicates 20 μm.

Nakajima, T., Mitsunaga, M., Bander, N. H., Heston, W. D., Choyke, P. L., & Kobayashi, H. (2011). Targeted, activatable, in vivo fluorescence imaging of prostate-specific membrane antigen (PSMA) positive tumors using the quenched humanized J591 antibody–indocyanine green (ICG) conjugate. Bioconjugate chemistry, 22(8), 1700-1705.

Complete CAR data FCM

Fig.7 Evaluation of CAR expression.

CAR Construction : J591 scFv-28ζ

Fig.7 Evaluation of CAR expression.

Activated T cells were transduced with g-retroviral vector and stained with anti-human IgG antibody (CAR) and CD3.

Alzubi, J., Dettmer-Monaco, V., Kuehle, J., Thorausch, N., Seidl, M., Taromi, S., ... & Wolf, P. (2020). PSMA-directed CAR T cells combined with low-dose docetaxel treatment induce tumor regression in a prostate cancer xenograft model. Molecular Therapy-Oncolytics, 18, 226-235.

Complete CAR data FuncS

Fig.8 Cytolytic activity. CAR T cells were co-cultured at the indicated E:T ratios with C4-2 cells.

CAR Construction : J591 scFv-28ζ

Fig.8 Cytolytic activity. CAR T cells were co-cultured at the indicated E:T ratios with C4-2 cells.

Cytotoxicity was determined using a cell viability assay n=6.

Complete CAR data FuncS

Fig.9 Quantification of tumor size. Tumor volumes were determined on days 1, 3, 8, 15, and 22 of treatment.

CAR Construction : J591 scFv-28ζ

Fig.9 Quantification of tumor size. Tumor volumes were determined on days 1, 3, 8, 15, and 22 of treatment.

Shown is antitumor response in
different treatment groups

Complete CAR data FuncS

Fig.10 Cytokine release. CAR T cells were co-cultured with PSMA+ C4-2 or PSMA Du145 cells, respectively.

CAR Construction : J591 scFv-28ζ

Fig.10 Cytokine release. CAR T cells were co-cultured with PSMA+ C4-2 or PSMA Du145 cells, respectively.

The concentration of IFN-g was determined in the supernatant (n = 3).

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.