Anti-CD20 (Leu16) h(CD28-CD3ζ) CAR, pMMLV vector

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Details

Target
CD20

Targeting Cell Type
T cell

Targeting Diseases
Acute lymphoblastic leukemia (ALL)

Generation
Second

Vector Name
pMMLV

Vector Length
~6kb

Vector Type
Recombinant Moloney murine leukemia virus (MMLV) retroviral vector

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 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

Complete CAR data FuncS

Fig.1 Specific cytotoxicity assay.

CAR Construction : leu-16-CD28-41BB-CD3ζ

Fig.1 Specific cytotoxicity assay.

CD20-specific cytotoxicity of G418-selected autologous patient T cells was assessed with 5-hour chromium-release assays using the following 51Cr-labeled target cells: Granta cells (MCL), Daudi cells (Burkitt NHL), EL4 cells transfected to express CD20, or the parental EL4 cell line lacking CD20 expression.

Till, B. G., Jensen, M. C., Wang, J., Qian, X., Gopal, A. K., Maloney, D. G., ... & Press, O. W. (2012). CD20-specific adoptive immunotherapy for lymphoma using a chimeric antigen receptor with both CD28 and 4-1BB domains: pilot clinical trial results. Blood, The Journal of the American Society of Hematology, 119(17), 3940-3950.

Complete CAR data FuncS

Fig.2 Change in tumor volume over time.

CAR Construction : leu-16-CD28-41BB-CD3ζ

Fig.2 Change in tumor volume over time.

The sum of the products of the diameters of the 7 largest lymph nodes seen on the baseline and 1-, 6-, and 12-month CT scans.

Complete CAR data FuncS

Fig.3 Immune responses against modified cells.

CAR Construction : leu-16-CD28-41BB-CD3ζ

Fig.3 Immune responses against modified cells.

An ELISA testing Ab binding to the murine Leu16 from which the αCD20-28-BB-ζ scFv is derived (Figure A); and a flow cytometry-based assay using HEK-293 cells transfected with the L29.19.1 plasmid (Figure B). No convincing evidence indicated humoral immune responses to the infused cells. Cellular immune responses were tested (Figure C). No evidence indicated cellular immune responses against the modified T cells

Complete CAR data FuncS

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

CAR Construction : leu-16-CD28-41BB-CD3ζ

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

NSG mice were inoculated i.v. with Raji-ffLuc tumors followed 2 days later by infusion of 5 × 106^6 CD20-targeted CAR+T cells, with or without i.p. injection of 30 mg i.v immune globulin (IVIG) per mouse 4 hours prior to T cell infusion. Survival curves is compared using a log-rank (Mantel-Cox) test.

Lee, S. Y., Olsen, P., Lee, D. H., Kenoyer, A. L., Budde, L. E., O’Steen, S., ... & Till, B. G. (2018). Preclinical optimization of a CD20-specific chimeric antigen receptor vector and culture conditions. Journal of immunotherapy (Hagerstown, Md.: 1997), 41(1), 19.

Complete CAR data BI

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

CAR Construction : leu-16-CD28-CD3ζ

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

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. Black dotted line in the right data box denotes day 12 post T-cell injection, the time at which the CAR-T cell group began to rapidly lose tumor control.

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.6 Evualution of tumor activation and exhaustion.

CAR Construction : leu-16-CD28-CD3ζ

Fig.6 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.

CAR scFv data FACS

Fig.7 Competition cell-binding assay.

CAR Construction :

Fig.7 Competition cell-binding assay.

Binding to CD20-positive Daudi cells was evaluated by competition of parental Leu-16 or scFv-Fc with FITC-Leu-16 by FACS. (A) GS18/C233S scFv-Fc. (B) GS8/nat scFv-Fc.

Wu A M, Tan G J, Sherman M A, et al. Multimerization of a chimeric anti-CD20 single-chain Fv-Fc fusion protein is mediated through variable domain exchange[J]. Protein engineering, 2001, 14(12): 1025-1033.

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