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Cell-based Screening Kit for Anti-human 4-1BB Antibody (Human CD64 Low Expression)

For research use only.

IDComponentsSize
CHEK-ATF073Human 4-1BB (Luc) HEK293 Reporter Cell2 Vials
SCCHO-ATP062LCHO/Human CD64 Stable Cell Line (Low Expression) Development Service2 Vials
  1. Genetically modified cell lines best reflect MOA (Mechanism of Action)
  2. Higher activity and larger assay window for robust and reproducible cell-based bioassay
  3. Comprehensive application data to support assay development and validation
  4. Full tracible record, stringent quality control and validated cell passage stability
  5. Parental cell line legally obtained from internationally recognized cell resource bank and commercially licensed
  6. Global commercial license assistance whenever regulatory filing is required

描述(Description)

The Cell-based Kit consists of two engineered cell lines, Human 4-1BB (Luc) HEK293 Reporter Cell (Cat. No. CHEK-ATF073) and CHO/Human CD64 Stable Cell Line (Low Expression) (Cat. No. SCCHO-ATP062L). The Human 4-1BB (Luc) HEK293 Reporter Cell was engineered to not only express NF-κB signaling response element, but also express the receptor full length human 4-1BB (Gene ID: 3604), which can drive luciferase expressing systems by 4-1BB ligand/ agonist antibody stimulation. The CHO/Human CD64 Stable Cell Line was engineered to express full length human CD64 receptor (Gene ID: 2209), with different levels of CD64 expression (High, Medium, Low), which can be used to test agonist antibody whether in a CD64-dependent manner to strengthen the agonistic activity. When co-cultured with Human 4-1BB (Luc) HEK293 Reporter Cell and anti-4-1BB agonist antibody, the anti-4-1BB antibody can be crosslinked, thereby strengthening 4-1BB pathway-activated luminescence.

应用说明(Application)

• Screen for Anti-human 4-1BB antibodies whether in a CD64-dependent manner to strengthen the agonistic activity

存储(Storage)

Frozen in liquid nitrogen.

支原体检测(Mycoplasma Testing)

Negative

无菌检测(Sterility Testing)

Negative

使用说明(Instructions for Use)

See data sheet for detailed culturing and assay protocol.

质量管理控制体系(QMS)

  1. 质量管理体系(ISO, GMP)
  2. 质量优势
  3. 质控流程
 

活性(Bioactivity)

4-1BB & CD64 FACS

Expression analysis of human 4-1BB on Human 4-1BB (Luc) HEK293 Reporter Cell by FACS.
Cell surface staining was performed on Human 4-1BB (Luc) HEK293 Reporter Cell or negative control cell using PE-labeled anti-human 4-1BB antibody.

Protocol

4-1BB & CD64 FACS

Expression analysis of human CD64 on CHO/Human CD64 Stable Cell Line by FACS.
Cell surface staining using PE-labeled anti-human CD64 antibody was performed on CHO/Human CD64 Stable Cell Line with different expression levels: CHO/Human CD64 Stable Cell Line (Low Expression); CHO/Human CD64 Stable Cell Line (Medium Expression); CHO/Human CD64 Stable Cell Line (High Expression).

Protocol

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ACRO质量管理体系
 
 

背景(Background)

4-1BB is also known as CD137, tumor necrosis factor receptor superfamily member 9 (TNFRSF9), induced by lymphocyte activation (ILA), is a co-stimulatory molecule of the tumor necrosis factor (TNF) receptor superfamily. CD137 can be expressed by activated T cells, but to a larger extent on CD8 than on CD4 T cells. In addition, CD137 expression is found on dendritic cells, follicular dendritic cells, natural killer cells, granulocytes and cells of blood vessel walls at sites of inflammation. The best characterized activity of CD137 is its costimulatory activity for activated T cells. Crosslinking of CD137 enhances T cell proliferation, IL-2 secretion survival and cytolytic activity. Further, it can enhance immune activity to eliminate tumors in mice. CD137 can enhance activation-induced T cell apoptosis when triggered by engagement of the TCR/CD3 complex. In addition, 4-1BB/4-1BBL co-stimulatory pathway has been shown to augment secondary CTL responses to several viruses, and meanwhile augment anti-tumor immunity. 4-1BB thus is a promising candidate for immunotherapy of human cancer. CD137 has been shown to interact with TRAF2.

Limited Use&License Disclosure

BY USE OF THIS PRODUCT, RESEARCHER AGREES TO BE BOUND BY THE FOLLOWING TERMS OF LIMITED USE OF THIS CELL LINE PRODUCT.

  1. If the researcher is not willing to accept the terms of limited use of this cell line product, and the product is unused, ACRO will accept return of the unused product.
  2. Researchers may use this product for research use only, no commercial use is allowed. "Commercial use" means any and all uses of this product and derivatives by a party for profit or other consideration and may include but is not limited to use in: (1) product manufacture; and (2) to provide a service, information or data; and/or resale of the product or its derivatives, whether or not such product or derivatives are resold for use in research.
  3. This cell line is neither intended for any animal or human therapeutic purposes nor for any direct human in vivo use . You have no right to share, modify, transfer, distribute, sell, sublicense, or otherwise make the cell line available for use to other researchers, laboratories, research institutions, hospitals, universities, or service organizations.
  4. ACROBIOSYSTEMS MAKES NO WARRANTIES OR REPRESENTATIONS OF ANY KIND, EITHER EXPRESSED OR IMPLIED, WITH RESPECT TO THE SUITABILITY OF THE CELL LINE FOR ANY PARTICULAR USE.
  5. ACROBIOSYSTEMS ACCEPTS NO LIABILITY IN CONNECTION WITH THE HANDLING OR USE OF THE CELL LINE.
  6. Modifications of the cell line, transfer to a third party, or commercial use of the cell line may require a separate license and additional fees. Please contact order.cn@acrobiosystems.com for further details.

 

前沿进展

T cells with split CARs specific for NKG2D ligands and PD-L1 exhibit improved selectivity towards monocyte-derived cells while effective in eliminating acute myeloid leukaemia in vivo
Sun, Jiang, Ng et al
J Cancer Res Clin Oncol (2023) 149 (12), 10189-10201
Abstract: The expression of NKG2D ligands and PD-L1 has been detected on acute myeloid leukaemia (AML) cells, as well as normal cells of the myeloid lineage. To target leukemic cells while minimizing collateral damage to normal cells, we constructed a split dual CAR system based on the AND-gate logic.The NKG2D extracellular domain linked with DAP12 without a co-stimulatory signal was used for the basal activation of T cells, and used together with the PD-L1-specific chimeric costimulatory receptor containing the 4-1BB activating domain for co-stimulatory signal 2 input. This dual CAR displayed cell-type specificity and activity similar as a 2nd generation NKG2D ligand-specific CAR.When compared to CD64 and PD-L1-specific 2nd generation CARs, we observed that the split dual CAR offered an improved myeloid cell type selectivity. For example, PD-L1-specific CAR-T cells lysed all tested myeloid cell types that expressed PD-L1, including M0 macrophages (Mø0), LPS-polarized Mø1, IFN-γ polarized Mø1, IL-4 polarized Mø2, monocytes, immature dendritic cells (imDCs), mature DCs, as well as KG-1 AML cells, while the dual CAR-T cells displaying killing activity only towards LPS polarized Mø1, mature DCs and KG-1 cells that expressed both NKG2D ligands and PD-L1. In a mouse liquid tumor model, the dual CAR-T cells were effective in eradicating established KG-1 AML xenografts.The improved cell type specificity offered by our split dual CAR-T cell system targeting paired antigens would favour the reduction of the on-target off-tumor toxicity towards normal myeloid cells during the treatment of myeloid leukaemia.© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.
Evaluation of piggyBac-mediated anti-CD19 CAR-T cells after ex vivo expansion with aAPCs or magnetic beads
Yang, Li, Meng et al
J Cell Mol Med (2021) 25 (2), 686-700
Abstract: Adoptive immunotherapy is a new potential method of tumour therapy, among which anti-CD19 chimeric antigen receptor T-cell therapy (CAR-T cell), is a typical treatment agent for haematological malignancies. Previous clinical trials showed that the quality and phenotype of CAR-T cells expanded ex vivo would seriously affect the tumour treatment efficacy. Although magnetic beads are currently widely used to expand CAR-T cells, the optimal expansion steps and methods have not been completely established. In this study, the differences between CAR-T cells expanded with anti-CD3/CD28 mAb-coated beads and those expanded with cell-based aAPCs expressing CD19/CD64/CD86/CD137L/mIL-15 counter-receptors were compared. The results showed that the number of CD19-specific CAR-T cells with a 4-1BB and CD28 co-stimulatory domain was much greater with stimulation by aAPCs than that with beads. In addition, the expression of memory marker CD45RO was higher, whereas expression of exhausted molecules was lower in CAR-T cells expanded with aAPCs comparing with the beads. Both CAR-T cells showed significant targeted tumoricidal effects. The CAR-T cells stimulated with aAPCs secreted apoptosis-related cytokines. Moreover, they also possessed marked anti-tumour effect on NAMALWA xenograft mouse model. The present findings provided evidence on the safety and advantage of two expansion methods for CAR-T cells genetically modified by piggyBac transposon system.© 2020 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.
Gene-modified NK-92MI cells expressing a chimeric CD16-BB-ζ or CD64-BB-ζ receptor exhibit enhanced cancer-killing ability in combination with therapeutic antibody
Chen, You, Jiang et al
Oncotarget (2017) 8 (23), 37128-37139
Abstract: Natural killer (NK) cells play a pivotal role in monoclonal antibody-mediated immunotherapy through the antibody-dependent cell-mediated cytotoxicity (ADCC) mechanism. NK-92MI is an interleukin-2 (IL-2)-independent cell line, which was derived from NK-92 cells with superior cytotoxicity toward a wide range of tumor cells in vitro and in vivo. Nonetheless, the Fc-receptor (CD16) that usually mediates ADCC is absent in NK-92 and NK-92MI cells. To apply NK-92MI cell-based immunotherapy to cancer treatment, we designed and generated two chimeric receptors in NK-92MI cells that can bind the Fc portion of human immunoglobulins. The construct includes the low-affinity Fc receptor CD16 (158F) or the high-affinity Fc receptor CD64, with the addition of the CD8a extracellular domain, CD28 transmembrane domains, two costimulatory domains (CD28 and 4-1BB), and the signaling domain from CD3ζ. The resulting chimeric receptors, termed CD16-BB-ζ and CD64-BB-ζ, were used to generate modified NK-92MI cells expressing the chimeric receptor, which were named NK-92MIhCD16 and NK-92MIhCD64 cells, respectively. We found that NK-92MIhCD16 and NK-92MIhCD64 cells significantly improved cytotoxicity against CD20-positive non-Hodgkin's lymphoma cells in the presence of rituximab. These results suggest that the chimeric receptor-expressing NK-92MI cells may enhance the clinical responses to currently available anticancer monoclonal antibodies.
Showing 1-3 of 3 papers.
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