Human 4-1BB-expressing Raji Cells

Human 4-1BB-expressing B cells

Raji-h4-1BB cells were developed from the human Raji cell line, a human B lymphocyte-derived cell line, and engineered to stably overexpress the human 4‑1BB gene. Raji cells have been successfully used as target cells in antibody effector studies such as antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) assays with peripheral T cell-derived Jurkat reporter cells.

4-1BB (also known as CD137 or TNFSF9) is a co-stimulatory immune checkpoint (IC) receptor that belongs to the tumor necrosis factor receptor (TNFR)/TNF superfamily. 4-1BB is expressed on a multitude of cells including activated CD8+ and CD4+ T cells [1]. Upon 4-1BB binding to its ligand, 4-1BBL (CD137L), expressed on antigen-presenting cells (APCs), NF‑κB‑dependent signaling is activated within the T cell, powerfully augmenting T cell activation, proliferation, and survival [1]. Notably, activation of 4-1BB has the capacity to mediate strong effector T cell immune responses and thus, makes it an attractive target for cancer immunotherapy [1,2]. Despite 4-1BB agonistic antibodies eliciting potent anti-tumor responses, monotherapy progress has been limited in the clinic  [2]. However, combinational therapies may be promising with a significant synergistic effect observed when used in combination with IC inhibitory antibodies (e.g. Anti-CTLA-4 and Anti-PD-1) [1,2]. 

Features of Raji-h4-1BB cells:

Surface expressed markers and ICs in Raji-h4-1BB cells

• Stable overexpression of the human 4-1BB 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-h4-1BB cells:

• Target cell line for ADCC and ADCP assays using InvivoGen's Jurkat-Lucia™ NFAT-CD16 cells and Jurkat-Lucia™ NFAT-CD32 cells, respectively.
• Target cell line for cell toxicity assays using NK or CAR-T cells
• For use in the development novel human 4-1BB mAbs

Validation of Raji-h4-1BB cells:

• Overexpression of 4-1BB verified by flow cytometry
• Functionally tested as target cells in ADCC assays using anti-human 4-1BB mAbs and Jurkat-Lucia™ NFAT-CD16 cells
• Guaranteed mycoplasma-free

​References:

1. Bartkowiak, T. & Curran, M.A. 2015. 4-1BB Agonists: Multi-Potent Potentiators of Tumor Immunity. Front Oncol 5, 117.
2. Ascierto, P.A. et al. 2010. Clinical experiences with anti-CD137 and anti-PD1 therapeutic antibodies. Semin Oncol 37, 508-516.

Figures

Validation of the expression of human 4-1BB by Raji-h4-1BB cells. Raji-Null (A) and Raji‑h4-1BB (B) cells were incubated with a PE-conjugated Anti-h4-1BB mAb for 30 minutes. The binding affinity was then measured using flow cytometry.

Comparison of ADCC potency for native and engineered anti-human 4-1BB antibody isotypes. Raji-h4-1BB cells were incubated with gradient concentrations of Anti-h4-1BB 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:

• Expression of human 4-1BB has been verified by flow-cytometry.
• Induction of antibody-dependent cellular cytotoxicity (ADCC) has been validated using anti-h41BB antibodies and InvivoGen's Jurkat-Lucia™ NFAT-CD16 cells reporter cell line.
• The stability for 20 passages following thawing has been verified.
• Raji-h4-1BB cells are guaranteed mycoplasma-free.

Contents

• 3-7 x 10⁶ Raji-h4-1BB 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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