Anti-PSCA (2B3) h(CD28-41BB-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
PSCA

Targeting Cell Type
T cell

Targeting Diseases
Prostate cancer

Generation
Third

Vector Name
pSBCAR1

Vector Length
~6kb

Vector Type
Sleeping Beauty (SB) transposon

Receptor Construction
scFv-CD28-41BB-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.
41BB
CD137 (also known as 4-1BB) is a surface co-stimulatory glycoprotein originally described as present on activated T lymphocytes, which belongs to the tumor necrosis factor (TNF) receptor superfamily. It is expressed mainly on activated CD4+ and CD8+ T cells, and binds to a high-affinity ligand (4-1BBL) expressed on several antigen-presenting cells such as macrophages and activated B cells. On the basis of preclinical observation, this molecule can promote the persistence of antigen-specific and antigennonspecific chimeric antigen receptor T-cells to significantly increases antitumor 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 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

CAR scFv data ELISA

Fig.1 Competition ELISA binding assay against recombinant PSCA.

CAR Construction :

Fig.1 Competition ELISA binding assay against recombinant PSCA.

All measurements were performed in triplicate.

Leyton, J. V., Olafsen, T., Lepin, E. J., Hahm, S., Bauer, K. B., Reiter, R. E., & Wu, A. M. (2008). Humanized radioiodinated minibody for imaging of prostate stem cell antigen–expressing tumors. Clinical Cancer Research, 14(22), 7488-7496.

CAR scFv data FCM

Fig.2 Flow cytometry a) LNCaP and c) LNCaP-PSCA cells incubated with secondary anti-human (Fc specific) PE-F(ab’)2 only
or b) LNCaP or d) LNCAP-PSCA cells incubated with purified 2B3 minibody (0.1 μg/μl).

CAR Construction :

Fig.2 Flow cytometry a) LNCaP and c) LNCaP-PSCA cells incubated with secondary anti-human (Fc specific) PE-F(ab’)2 only or b) LNCaP or d) LNCAP-PSCA cells incubated with purified 2B3 minibody (0.1 μg/μl).

All measurements were performed in triplicate.

CAR scFv data FCM

Fig.3 Flow cytometry analysis comparing binding of Cy5-cDb to 22Rv1 cells that over express PSCA (left) and the parental line (right) that expresses very low level of PSCA.

CAR Construction :

Fig.3 Flow cytometry analysis comparing binding of Cy5-cDb to 22Rv1 cells that over express PSCA (left) and the parental line (right) that expresses very low level of PSCA.

Strong binding of Cy5-cDb to PSCA (orange line) was evident compared to the positive control.

Sonn, G. A., Behesnilian, A. S., Jiang, Z. K., Zettlitz, K. A., Lepin, E. J., Bentolila, L. A., ... & Reiter, R. E. (2016). Fluorescent Image–Guided Surgery with an Anti-Prostate Stem Cell Antigen (PSCA) Diabody Enables Targeted Resection of Mouse Prostate Cancer Xenografts in Real TimeFluorescent Image–Guided Surgery with an Anti-PSCA Diabody. Clinical Cancer Research, 22(6), 1403-1412.

CAR scFv data SPR

Fig.4 Quartz crystal microbalance (QCM) measurements of the interaction between mock labeled cDb or Cy5-cDb and recombinant PSCA.

CAR Construction :

Fig.4 Quartz crystal microbalance (QCM) measurements of the interaction between mock labeled cDb or Cy5-cDb and recombinant PSCA.

Mock-treated cDb had an apparent affinity of 1.96 nM and Cy5-cDb 0.42 nM, both in the low nanomolar range

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