Human OX40-expressing Raji Cells

Human OX40-expressing B cells

Raji-hOX40 cells were developed from the human Raji cell line, a human B lymphocyte-derived cell line, and engineered to stably overexpress the human OX40 gene. Raji cells have been successfully used as target cells in human effector studies such as antibody-dependent cellular cytotoxicity (ADCC), either with peripheral blood mononuclear cells, natural killer (NK) cells, or T cell-derived Jurkat reporter cells.

OX40 (also known as CD134, TNFRSF4) is a co-stimulatory immune checkpoint (IC) and a member of the tumor necrosis factor receptor superfamily (TNFRSF). OX40 is transiently expressed on activated CD4+ and CD8+ T-cells, while its ligand, OX40 ligand (OX40L, TNFSF4, CD252), is predominantly expressed on activated antigen-presenting cells [1]. The engagement of OX40 with OX40L ultimately leads to the activation of the NF-κB pathway [1,2]. The OX40-OX40L axis promotes the survival of effector T cells and the generation of T cell memory, while also being associated with local inflammation and auto-immune disease (e.g. SLE) [1]. Specifically for inflammatory diseases, novel therapeutics that aim to antagonize this interaction are of interest in the clinic [3]. On the other hand, treatment with agonistic anti-OX40 mAbs has been shown to augment T cell differentiation and cytolytic function leading to enhanced anti-tumor immunity in a variety of tumor models [2]. Thus, combination therapies with blockade IC mAbs (i.e. anti-CTLA-4 and anti-PD-1) are being explored [2,4].

Features of Raji-hOX40 cells:

Surface expressed markers and ICs in Raji-hOX40 cells

• Stable overexpression of the human OX40 gene
• Characterized by a number of cell-surface expressed markers including the B cell receptor (BCR), CD19, and CD20
• Constitutive expression of various immune checkpoints (ICs) such as CD27, CD70, CD80, PD-L1, and 4-1BBL

Applications for Raji-hOX40 cells:

• Target cell line for ADCC assays using InvivoGen's Jurkat-Lucia™ NFAT-CD16 cells
• Target cell line for cell toxicity assays using NK or CAR-T cells
• For use in the development of novel OX40 agonistic and antagonistic mAbs 

Validation of Raji-hOX40 cells:

• Overexpression of OX40 verified by flow cytometry
• Functionally tested as target cells in ADCC assays using anti-human OX40 mAbs and Jurkat-Lucia™ NFAT-CD16 cells
• Guaranteed mycoplasma-free

​References:

1. Gough, M.J. et al. 2009. OX40 (CD134) and OX40L. Adv Exp Med Biol 647, 94-107.
2. Alves Costa Silva, C. et al. 2020. New pathways in immune stimulation: targeting OX40. ESMO Open, 5.
3. Webb, G.J. et al. 2016. OX40, OX40L and Autoimmunity: a Comprehensive Review. Clin Rev Allergy Immunol 50, 312-332.
4. Linch, S.N. et al. 2015. OX40 Agonists and Combination Immunotherapy: Putting the Pedal to the Metal. Front Oncol 5, 34.

Figures

Validation of the expression of human OX40 by Raji-hOX40 cells. Raji-Null (A) and Raji‑hOX40 (B) cells were incubated with a PE-conjugated Anti-hOX40 mAb for 30 minutes. The binding affinity was then measured using flow cytometry.

Comparison of ADCC potency for native and engineered anti-human OX40 antibody isotypes. Raji-hOX40 cells were incubated with gradient concentrations of Anti-hOX40 or Anti-β-galactosidase (β-Gal) mAbs for 1 hour. Jurkat-Lucia™ NFAT-CD16 effector cells were then co-incubated with target cells for 6 hours. NFAT activation, reflecting the induced ADCC response, was assessed by determining Lucia luciferase activity in the supernatant using QUANTI-Luc™. Percentages of the maximal response normalized to the IgG1 isotype are shown.

Specifications

Antibiotic resistance: Blasticidin

Growth medium: IMDM, 2 mM L-glutamine, 25 mM HEPES, 10% heat-inactivated fetal bovine serum (FBS; 30 min at 56 °C), Pen-Strep (100 U/ml-100 µg/ml), 100 µg/ml Normocin™

Test medium: IMDM, 2 mM L-glutamine, 25 mM HEPES, 10% heat-inactivated FBS, Pen-Strep (100 U/ml-100 µg/ml)

Quality control:

• Human OX40 expression has been verified by flow cytometry.
• Induction of antibody-dependent cellular cytotoxicity (ADCC) has been validated using anti-hOX40 antibodies and InvivoGen's Jurkat-NFAT Lucia™ CD16 reporter cell line.
• The stability for 20 passages following thawing has been verified.
• Raji-hOX40 cells are guaranteed mycoplasma-free.

Contents

• 3-7 x 10⁶ Raji-hOX40 cells in a cryovial or shipping flask.
• 1 ml of Blasticidin (10 mg/ml). Store at 4 °C or at -20 °C.
• 1 ml of Normocin™ (50 mg/ml). Normocin™ is a formulation of three antibiotics active against mycoplasmas, bacteria, and fungi. Store at -20 °C.

IMPORTANT: If cells are shipped frozen (i.e. in a cryovial) and are not frozen upon arrival, contact InvivoGen immediately.

Shipped on dry ice (Europe, USA & Canada)

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