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pSBCAR1 CD20 (Ofatumumab) h(28ζ) (CAR-SB-LX0075)

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All products and services are For Research Use Only and CANNOT be used in the treatment or diagnosis of disease.

Sleeping Beauty (SB) transposon, a type of nonviral integrative vectors, provides an alternative to modify primary T cells. Creative biolabs has developed SB transposon CAR vector pSBCAR1 CD20 (Ofatumumab) h(28ζ), which is constructed for the engineering of T cells to target human CD20. The T cells are genetically modified through transduction with a nonviral vector expressing scFv of anti-CD20 antibody linked to CD28 and CD3ζ signaling domains. And the vector product was designed for the treatment of Acute lymphoblastic leukemia (ALL).

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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

  • Clone
  • Ofatumumab
  • Host
  • Human
  • Target Species
  • Human
  • Gene Name
  • membrane spanning 4-domains A1
  • Synonyms
  • B1; S7; Bp35; CD20; CVID5; MS4A2; LEU-16; BA0185

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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 Latest CAR Construction

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 Latest CAR Construction

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 Latest CAR Construction

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 : Latest 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 : Latest CAR Construction

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

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.

More Published Data More Published Data

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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.

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