Anti-CD20 (1F5) 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
CD20

Targeting Cell Type
T cell

Targeting Diseases
Acute lymphoblastic leukemia (ALL)

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

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Published Data

Complete CAR data Cyt

Fig.1 Transduced T cells exhibit CD20-specific cytolytic activity in vitro.

CAR Construction : 1F5-CD28-41BB-CD3ζ

Fig.1 Transduced T cells exhibit CD20-specific cytolytic activity in vitro.

iC9-CD20CAR-Δ19 T cells transduced with CD20-CAR (CD20-CAR T), or non-transduced (mock) T cells were co-cultured with EL4, Daudi, and Granta in various effector to target (E:T) ratios.

Budde L E, Berger C, Lin Y, et al. Combining a CD20 chimeric antigen receptor and an inducible caspase 9 suicide switch to improve the efficacy and safety of T cell adoptive immunotherapy for lymphoma[J]. PloS one, 2013, 8(12): e82742.

Complete CAR data FCM

Fig.2 Anti-tumor effect of the transduced T cells.

CAR Construction : 1F5-CD28-41BB-CD3ζ

Fig.2 Anti-tumor effect of the transduced T cells.

Mock or iC9-CD20CAR-Δ19 transduced T cells were co-cultured with CD20+ Ramos cells in various Effector to Target ratios (E:T) for 48 hours. Cells were then stained with antibodies recognizing CD22 and CD3. Flow cytometric analysis was used to determine the presence of Ramos cells (CD22+CD3-) and T cells (CD22-CD3+).

Complete CAR data ELISA

Fig.3 Cytokine production.

CAR Construction : 1F5-CD28-41BB-CD3ζ

Fig.3 Cytokine production.

Expanded T cells secreted IL2, IFN-γ and TNF-α after co-culture with CD20-expressing Ramos target cells for 48 hours.

Complete CAR data BI

Fig.4 Anti-tumor activity of transduced T cells in vivo.

CAR Construction : 1F5-CD28-41BB-CD3ζ

Fig.4 Anti-tumor activity of transduced T cells in vivo.

Eradication of CD20+ Raji tumors in NOD-SCID mice by CD20-CAR T cells but not non-transduced T cells (Mock T) as measured by in vivo bioluminescent imaging.

Complete CAR data FuncS

Fig.5 Anti-tumor activity of transduced T cells in vivo.

CAR Construction : 1F5-CD28-41BB-CD3ζ

Fig.5 Anti-tumor activity of transduced T cells in vivo.

Kaplan-Meier survival curves of mice receiving iC9-CD20CAR-Δ19 T treatment (CD20-CAR T) or non-transduced mock T cell treatment (NT).

CAR scFv data IF

Fig.6 CD20 binding assay.

CAR Construction :

Fig.6 CD20 binding assay.

Binding of 1F5-IgG1 or 1F5-IgA2 and respective control antibodies to human CD20 transfected CHO-K1 cells was analysed by indirect immunofluorescence using a fluorescein isothicyanate (FITC)-labelled human jlight chain antibody.

Lohse S, Loew S, Kretschmer A, et al. Effector mechanisms of IgA antibodies against CD20 include recruitment of myeloid cells for antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity[J]. British journal of haematology, 2018, 181(3): 413-417.

CAR scFv data ELISA

Fig.7 Binding activity of 1F5 scFv-Ig compared with 1F5.

CAR Construction :

Fig.7 Binding activity of 1F5 scFv-Ig compared with 1F5.

A total of 10^5 Ramos cells were incubated with various concentrations of 1F5 scFv-Ig (GS1) or 1F5, followed by incubation with a peroxidase-conjugated GAH or GAM secondary Ab.

Shan, D., Press, O. W., Tsu, T. T., Hayden, M. S., & Ledbetter, J. A. (1999). Characterization of scFv-Ig constructs generated from the anti-CD20 mAb 1F5 using linker peptides of varying lengths. The Journal of Immunology, 162(11), 6589-6595.

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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.