Anti-CD20 scFv h(CD28-41BB-CD3ζ) CARNK, 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
NK cell

Targeting Diseases
Chronic lymphocytic leukemia (CLL)

Generation
Third

Vector Name
pCDCAR1

Vector Length
8kb

Vector Type
Lentiviral

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-stimulatorysignals 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 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 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 FCM

Fig.1 Rituximab blocks antigen binding of Ab used to derive CAR scFv.

CAR Construction : rituximab scFv-41BB-CD28ζ

Fig.1 Rituximab blocks antigen binding of Ab used to derive CAR scFv.

Ramos cells (CD20þ) were incubated with the indicated rituximab concentrations (conc.) for 30 minutes, followed by incubation with PE-labeled anti-CD20 Ab (clone Leu16) or isotype control at either 4C or 37C for 30 minutes. Cells were washed and analyzed by flow cytometry to determine available CD20 binding sites as measured by PE fluorescence intensity. The panel summarizes the geometric mean fluorescence intensity (MFI) at either 4C or 37C as a function of rituximab concentration.

Rufener, G. A., Press, O. W., Olsen, P., Lee, S. Y., Jensen, M. C., Gopal, A. K., Pender, B., Budde, L. E., Rossow, J. K., Green, D. J., Maloney, D. G., Riddell, S. R., & Till, B. G. (2016). Preserved Activity of CD20-Specific Chimeric Antigen Receptor-Expressing T Cells in the Presence of Rituximab. Cancer immunology research, 4(6), 509–519.

Complete CAR data FuncS

Fig.2 Effect of rituximab on CAR T-cell function in vitro.

CAR Construction : rituximab scFv-41BB-CD28ζ

Fig.2 Effect of rituximab on CAR T-cell function in vitro.

The indicated B-cell NHL cell lines were irradiated and incubated for 30 minutes at room temperature with varying rituximab concentrations (at two times the concentrations during incubation to yield the indicated final concentrations after addition of T cells).

Complete CAR data Cyt

Fig.3 Effect of rituximab on CAR T-cell– mediated cytotoxicity.

CAR Construction : rituximab scFv-41BB-CD28ζ

Fig.3 Effect of rituximab on CAR T-cell– mediated cytotoxicity.

The indicated 51Cr-labeled target cells were preincubated for 30 minutes with rituximab (at two times the concentrations during incubation
to yield the indicated final concentrations after the addition of T cells), and then CD8þ T cells expressing the Leu16-28-z CAR were added at the E:T ratios shown in a standard 5-hour 51Cr-release assay. Mock-transduced T cells and samples with rituximab and target cells only ("0:1") were used as negative controls.

Complete CAR data FuncS

Fig.4 In vivo effect of rituximab on CD20 CAR T-cell function.

CAR Construction : rituximab scFv-41BB-CD28ζ

Fig.4 In vivo effect of rituximab on CD20 CAR T-cell function.

NSG mice were injected i.v. with 5 105 rituximab-refractory Raji-ffLuc lymphoma cells, followed by one of the following treatments: no treatment, rituximab only (25 mg or 200 mg) i.p. 5 days later, 107 1.5.3-NQ-28-BBz CAR T cells only 6 days after tumor,
or rituximab 25 mg or 200 mg i.p. at 5 days followed by 107 CAR T cells at 6 days after tumor. Mice were imaged twice weekly for bioluminescence.

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