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pCDCAR1 ROR1 h(BBζ) (CAR-LC216)


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

The vector of anti-ROR1 chimeric antigen receptor (CAR) is constructed for the engineering of T cells to target human ROR1. The T cells are genetically modified through transduction with a lentiviral vector expressing scFv of anti-ROR1 antibody linked to 4-1BB (CD137) and CD3ζ signaling domains. And the vector product was designed for the treatment of B cell-chronic lymphocytic leukemia.

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Details

  • Target
  • ROR1
  • Targeting Cell Type
  • T cell
  • Targeting Diseases
  • B cell-chronic lymphocytic leukemia
  • Generation
  • Second
  • Vector Name
  • pCDCAR1
  • Vector Length
  • ~8kb
  • Vector Type
  • Lentiviral
  • Receptor Construction
  • scFv-41BB-CD3ζ
  • Discription of Signaling Cassetes
  • 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

  • Clone
  • R12
  • Host
  • Rabbit
  • Target Species
  • Human
  • Gene Name
  • Receptor tyrosine kinase like orphan receptor 1
  • Synonyms
  • ROR1; NTRKR1; dJ537F10.1

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  • Published Data
CAR scFv data ELISA

Fig.1 Specificity and epitope mapping studies by ELISA.

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Fig.1 Specificity and epitope mapping studies by ELISA.

The binding of chimeric rabbit/human IgG1 R11, R12, Y31, and P14 to immobilized human ROR1 (Fc-hROR1), mouse ROR1 (Fc-mROR1), and human ROR2 (hROR2-Fc) was analyzed by ELISA.

Yang, J., Baskar, S., Kwong, K. Y., Kennedy, M. G., Wiestner, A., & Rader, C. (2011). Therapeutic potential and challenges of targeting receptor tyrosine kinase ROR1 with monoclonal antibodies in B-cell malignancies. PloS one, 6(6), e21018.

CAR scFv data SPR

Fig.2 Epitope mapping studies by surface plasmon resonance.

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Fig.2 Epitope mapping studies by surface plasmon resonance.

Shown are Biacore 6100 sensorgrams obtained for the binding of IgG1 R11, R12, and Y31 to immobilized Fc-hROR1.

Yang, J., Baskar, S., Kwong, K. Y., Kennedy, M. G., Wiestner, A., & Rader, C. (2011). Therapeutic potential and challenges of targeting receptor tyrosine kinase ROR1 with monoclonal antibodies in B-cell malignancies. PloS one, 6(6), e21018.

CAR scFv data SPR

Fig.3 Affinity measurements of R12 by surface plasmon resonance.

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Fig.3 Affinity measurements of R12 by surface plasmon resonance.

The mAbs were injected at five or six different concentrations (shown in different colors) ranging from 1.5 to 100 nM. Each concentration was tested in duplicates.

Yang, J., Baskar, S., Kwong, K. Y., Kennedy, M. G., Wiestner, A., & Rader, C. (2011). Therapeutic potential and challenges of targeting receptor tyrosine kinase ROR1 with monoclonal antibodies in B-cell malignancies. PloS one, 6(6), e21018.

CAR scFv data FCM

Fig.4 Flow cytometry was used to analyze the binding of IgG1 R11 (green line; 5 mg/mL), R12 (blue line; 1 mg/mL), and Y31 (red line; 5 mg/mL) to (A) the surface of JeKo-1 (top) and HBL-2 cells (bottom).

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Fig.4 Flow cytometry was used to analyze the binding of IgG1 R11 (green line; 5 mg/mL), R12 (blue line; 1 mg/mL), and Y31 (red line; 5 mg/mL) to (A) the surface of JeKo-1 (top) and HBL-2 cells (bottom).

Biotinylated IgG1 in combination with FITC-CD19/APC-CD5 were detected with PE-streptavidin. The y axis depicts the number of events, the x axis the fluorescence intensity.

Yang, J., Baskar, S., Kwong, K. Y., Kennedy, M. G., Wiestner, A., & Rader, C. (2011). Therapeutic potential and challenges of targeting receptor tyrosine kinase ROR1 with monoclonal antibodies in B-cell malignancies. PloS one, 6(6), e21018.

CAR scFv data FCM

Fig.5 PI staining followed by flow cytometry was used to analyze the CDC potency of IgG1 R11, R12, and Y31 in comparison to IgG1 P14 (negative control), and rituximab.

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Fig.5 PI staining followed by flow cytometry was used to analyze the CDC potency of IgG1 R11, R12, and Y31 in comparison to IgG1 P14 (negative control), and rituximab.

The percentage of dead cells (PI-positive) after RTX-mediated CDC was 70.7% (JeKo-1), 54.4% (HBL-2), and 12.7% (CLL).

Yang, J., Baskar, S., Kwong, K. Y., Kennedy, M. G., Wiestner, A., & Rader, C. (2011). Therapeutic potential and challenges of targeting receptor tyrosine kinase ROR1 with monoclonal antibodies in B-cell malignancies. PloS one, 6(6), e21018.

Complete CAR data FCM

Fig.6 Flow cytometric analysis of purity and phenotype of ROR1 CAR-modified CD4+ and CD8+ T cells showing expression of tCD19 in CD4+ (top panels) or CD8+ (bottom panels) T cells either mocktransduced, after transduction with the ROR1 CAR (pre-selection).

CAR Construction : R12 scFv-BBζ Latest CAR Construction

Fig.6 Flow cytometric analysis of purity and phenotype of ROR1 CAR-modified CD4+ and CD8+ T cells showing expression of tCD19 in CD4+ (top panels) or CD8+ (bottom panels) T cells either mocktransduced, after transduction with the ROR1 CAR (pre-selection).

All samples are gated on CD3+ cells. D, cytolytic activity of
CD4+ and CD8+ ROR1 CAR-T cells against 51Cr-labeled K562/ROR1 or K562 cells.

Berger, C., Sommermeyer, D., Hudecek, M., Berger, M., Balakrishnan, A., Paszkiewicz, P. J., ... & Riddell, S. R. (2015). Safety of Targeting ROR1 in Primates with Chimeric Antigen Receptor–Modified T CellsSafety of ROR1 CAR-T Cells in Macaques. Cancer immunology research, 3(2), 206-216.

Complete CAR data FuncS

Fig.7 Luminex cytokine assay of supernatants obtained after 24 hours from triplicate co-cultures of 5×104 CD4+ or
CD8+ROR1 CAR-T cells with K562/ROR1 cells or media alone.

CAR Construction : R12 scFv-BBζ Latest CAR Construction

Fig.7 Luminex cytokine assay of supernatants obtained after 24 hours from triplicate co-cultures of 5×104 CD4+ or CD8+ROR1 CAR-T cells with K562/ROR1 cells or media alone.

Cytokine levels in media controls were below detection level. Data in C-F is shown for macaque A13002 and representative of results in 3 animals.

Berger, C., Sommermeyer, D., Hudecek, M., Berger, M., Balakrishnan, A., Paszkiewicz, P. J., ... & Riddell, S. R. (2015). Safety of Targeting ROR1 in Primates with Chimeric Antigen Receptor–Modified T CellsSafety of ROR1 CAR-T Cells in Macaques. Cancer immunology research, 3(2), 206-216.

Complete CAR data FuncS

Fig.8 Body weight and serum chemistry before and at the indicated days after the T-cell infusion.

CAR Construction : R12 scFv-BBζ Latest CAR Construction

Fig.8 Body weight and serum chemistry before and at the indicated days after the T-cell infusion.

The grey shaded area demarks the normal range for each parameter in rhesus macaques.

Berger, C., Sommermeyer, D., Hudecek, M., Berger, M., Balakrishnan, A., Paszkiewicz, P. J., ... & Riddell, S. R. (2015). Safety of Targeting ROR1 in Primates with Chimeric Antigen Receptor–Modified T CellsSafety of ROR1 CAR-T Cells in Macaques. Cancer immunology research, 3(2), 206-216.

Complete CAR data Cyt

Fig.9 Detection of a transgene product-specific T-cell response after transfer of ROR1 CAR-T cells.

CAR Construction : R12 scFv-BBζ Latest CAR Construction

Fig.9 Detection of a transgene product-specific T-cell response after transfer of ROR1 CAR-T cells.

Aliquots of these cultures were evaluated in a cytotoxicity assay for recognition of autologous 51Cr-labeled ROR1 CAR-T cells or unmodified T cells.

Berger, C., Sommermeyer, D., Hudecek, M., Berger, M., Balakrishnan, A., Paszkiewicz, P. J., ... & Riddell, S. R. (2015). Safety of Targeting ROR1 in Primates with Chimeric Antigen Receptor–Modified T CellsSafety of ROR1 CAR-T Cells in Macaques. Cancer immunology research, 3(2), 206-216.

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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