Anti-CD20 (Ofatumumab) h(CD28-41BB-CD3ζ) CAR, pCDCAR1 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
B-Cell Malignancies

Generation
Third

Vector Name
pCDCAR1

Vector Length
~8kb

Vector Type
Lentiviral vector

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 co-stimulatory 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 cell 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 antigen-nonspecific 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 CD28 and OX40 can be used with CD4-zeta to transmit a proliferative/survival signal, or all three can be used together.

Target

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Published Data

Complete CAR data BI

Fig.1 Tumor progression after the CD20-targeting CAR-T cells treatment.

CAR Construction : Ofatumumab-CD28-CD3ζ

Fig.1 Tumor progression after the CD20-targeting CAR-T cells treatment.

NSG mice were injected intravenously (i.v.) with 0.5 × 10^6 firefly-luciferase-expressing Raji cells 6 days prior to treatment with 5 × 10^6 CD8+ CD20-targeting CAR-T cells delivered i.v. Tumor progression was monitored by bioluminescence imaging.

Chen, X., Khericha, M., Lakhani, A., Meng, X., Salvestrini, E., Chen, L. C., ... & Chen, Y. Y. (2020). Rational tuning of CAR tonic signaling yields superior T-cell therapy for cancer. bioRxiv.

Complete CAR data ELISA

Fig.2 Evualution of tumor activation and exhaustion.

CAR Construction : Ofatumumab-CD28-CD3ζ

Fig.2 Evualution of tumor activation and exhaustion.

Activation and exhaustion maker expression on CAR+ T cells were evaluated 11 days post Dynabead removal, without CD20 antigen stimulation.

Chen, X., Khericha, M., Lakhani, A., Meng, X., Salvestrini, E., Chen, L. C., ... & Chen, Y. Y. (2020). Rational tuning of CAR tonic signaling yields superior T-cell therapy for cancer. bioRxiv.

Complete CAR data FCM

Fig.3 T-cell proliferation assay

CAR Construction : Ofatumumab-CD28-CD3ζ

Fig.3 T-cell proliferation assay

A 4-day T-cell proliferation assay on CAR+T cells with CellTrace Violet (CTV) dye in the absence or presence of target cells (on-target, CD19+/CD20+K562 cells; off-target, parental K562 cells) at 2:1 effector-to-target (E:T) ratio.

Chen, X., Khericha, M., Lakhani, A., Meng, X., Salvestrini, E., Chen, L. C., ... & Chen, Y. Y. (2020). Rational tuning of CAR tonic signaling yields superior T-cell therapy for cancer. bioRxiv.

CAR scFv data IF

Fig.4 The binding of CHO-ofatumumab to Ramos cells showed typical type I CD20 antibody characteristics, in contrast to the case for CHO-Obinutuzumab.

CAR Construction :

Fig.4 The binding of CHO-ofatumumab to Ramos cells showed typical type I CD20 antibody characteristics, in contrast to the case for CHO-Obinutuzumab.

Each panel is a photomicrograph showing the binding of the antibodies to CD20 localized on the surface of Ramos cells. The binding of each antibody to CD20 was visualized using FITC-conjugated human Fc-specific secondary antibodies.

Jin, N., Lee, J. W., Heo, W., Ryu, M. Y., So, M. K., Ko, B. J., ... & Kim, W. T. (2019). Low binding affinity and reduced complement-dependent cell death efficacy of ofatumumab produced using a plant system (Nicotiana benthamiana L.). Protein expression and purification, 159, 34-41.

CAR scFv data FCM

Fig.5 The binding affinity of plant-ofatumumab-HDEL and plant-ofatumumab compared to CHO-ofatumumab.

CAR Construction :

Fig.5 The binding affinity of plant-ofatumumab-HDEL and plant-ofatumumab compared to CHO-ofatumumab.

More Published Data More Published Data

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