Acta Scientific Pharmaceutical Sciences (ASPS)(ISSN: 2581-5423)

Research Article Volume 7 Issue 4

WIL2-S and an Engineered Jurkat Cell Line as a Model for Assessment of Apoptotic and Phagocytic Activity Upon Treatment with Anti-CD20

Sinosha Paralikar, Keyuri Mokashi, Sheetal Raut and Sanjeev Gupta

Advanced Biotech Lab, Ipca Laboratories Ltd., India

*Corresponding Author: Sanjeev Gupta, Advanced Biotech Lab, Ipca Laboratories Ltd., India.

Received: March 14, 2023; Published: April 11, 2023


I-RmAb is an anti-CD20 monoclonal antibody, that we have developed as a therapeutic to treat B-cell malignancies. It induces cell death via various mechanisms such as complement dependent cytotoxicity (CDC), antibody dependent cell mediated cytotoxicity (ADCC), antibody dependent cell mediated phagocytosis (ADCP), apoptosis and sensitization of cancer cells to immunotherapy.

While other in-vitro assays are well-established, in this research, we focus on comparing Fab mediated apoptosis induced by I-RmAb in two CD20 positive human lymphoblast cell lines - WIL2-S and Raji, using flow cytometry, thus identifying a time window of peak apoptosis which has not been explored in depth before. This method is difficult to develop because I-RmAb elicits a very low apoptotic response and varies within different cell lines and patient derived cells such as B-CLL. We have also explored a luminescence and fluorescence method, and concluded that flow cytometry is a more reliable technique to assess apoptosis. Through this study, we hope to further narrow down the exact time window of apoptotic activity and a cell line sensitive enough to detect the same for developing a robust assay.

Alongside, we also outline a developed method that demonstrates I-RmAb’s activation of the phagocytic pathway as a function of the FcγRIIa receptor in commercially available Jurkat cells harboring the NFAT reporter. Both apoptosis and ADCP assays are an important quality aspect for biosimilarity assessment with limited availability of experimental data in the literature. Hence, research under this wing will help improve the process of designing functional assays.

 Keywords: Apoptosis; ADCP; Anti-CD20; WIL2-S


  1. Alas S and Bonavida B. “Rituximab inactivates signal transducer and activation of transcription 3 (STAT3) activity in B-non-Hodgkin's lymphoma through inhibition of the interleukin 10 autocrine/paracrine loop and results in down-regulation of Bcl-2 and sensitization to cytotoxic drugs”. Cancer Research13 (2001): 5137-5144.
  2. Boross P and Leusen J H W. “Mechanisms of action of CD20 antibodies”. American Journal of Cancer Research6 (2012): 676-690.
  3. Brandsma A M., et al. “Fc receptor inside-out signaling and possible impact on antibody therapy”. Immunological Reviews1 (2015): 74-87.
  4. Byrd, J C., et al. “The mechanism of tumor cell clearance by rituximab in vivo in patients with B-cell chronic lymphocytic leukemia: evidence of caspase activation and apoptosis induction”. Blood 3 (2002): 1038-1043.
  5. Chaperot L., et al. “Differentiation of antigen-presenting cells (dendritic cells and macrophages) for therapeutic application in patients with lymphoma”. Leukemia9 (2000): 1667-1677.
  6. Deans J P., et al. “Association of 75/80-kDa phosphoproteins and the tyrosine kinases Lyn, Fyn, and Lck with the B cell molecule CD20. Evidence against involvement of the cytoplasmic regions of CD20”. The Journal of Biological Chemistry38 (1995): 22632-22638.
  7. Deans J P., et al. “Association of tyrosine and serine kinases with the B cell surface antigen CD20. Induction via CD20 of tyrosine phosphorylation and activation of phospholipase C-gamma 1 and PLC phospholipase C-gamma 2”. Journal of Immunology (Baltimore, Md. : 1950)9 (1993): 4494-4504.
  8. Demidem, A., et al. “Chimeric anti-CD20 (IDEC-C2B8) monoclonal antibody sensitizes a B cell lymphoma cell line to cell killing by cytotoxic drugs”. Cancer Biotherapy and Radiopharmaceuticals3 (1997): 177-186.
  9. Emer J J and Wolinsky, C. “Rituximab: a review of dermatological applications”. The Journal of Clinical and Aesthetic Dermatology5 (2009): 29-37.
  10. Ghetie M A., et al. “Homodimers but not monomers of Rituxan (chimeric anti-CD20) induce apoptosis in human B-lymphoma cells and synergize with a chemotherapeutic agent and an immunotoxin”. Blood97.5 (2001): 1392-1398.
  11. Gogesch , et al. “The Role of Fc Receptors on the Effectiveness of Therapeutic Monoclonal Antibodies”. International Journal of Molecular Sciences 22.16 (2021): 8947. 
  12. Golay J., et al. “Biologic response of B lymphoma cells to anti-CD20 monoclonal antibody rituximab in vitro: CD55 and CD59 regulate complement-mediated cell lysis”. Blood 12 (2000): 3900-3908.
  13. Herbrand U. “Antibody-dependent cellular phagocytosis: the mechanism of action that gets no respect — A discussion about improving bioassay reproducibility”. BioProcessing Journal1 (2016): 26-29.
  14. Hofmeister J K., et al. “Clustered CD20 induced apoptosis: src-family kinase, the proximal regulator of tyrosine phosphorylation, calcium influx, and caspase 3-dependent apoptosis”. Blood Cells, Molecules and Diseases 2 (2000): 133-143.
  15. Jazirehi A R., et al. “Rituximab (anti-CD20) selectively modifies Bcl-xL and apoptosis protease activating factor-1 (Apaf-1) expression and sensitizes human non-Hodgkin's lymphoma B cell lines to paclitaxel-induced apoptosis”. Molecular Cancer Therapeutics11 (2003): 1183-1193.
  16. Kamen L., et al. “A novel method for determining antibody-dependent cellular phagocytosis”. Journal of Immunological Methods 468 (2019): 55-60.
  17. Maloney D G., et al. “IDEC-C2B8 (Rituximab) anti-CD20 monoclonal antibody therapy in patients with relapsed low-grade non-Hodgkin's lymphoma”. Blood6 (1997): 2188-2195.
  18. Manches O., et al. “In vitro mechanisms of action of rituximab on primary non-Hodgkin lymphomas”. Blood3 (2003): 949-954.
  19. Mathas S., et al. “Anti-CD20- and B-cell receptor-mediated apoptosis: evidence for shared intracellular signaling pathways”. Cancer Research24 (2000): 7170-7176.
  20. Miller K., et al. “Design, construction, and in vitro analyses of multivalent antibodies”. Journal of Immunology (Baltimore, Md. : 1950)9 (2003): 4854-4861.
  21. Oflazoglu E and Laurent P A. “Evolution of anti-CD20 monoclonal antibody therapeutics in oncology”. mAbs1 (2010): 14-19.
  22. Pavlasova G and Marek M. “The regulation and function of CD20: an "enigma" of B-cell biology and targeted therapy”. Haematologica6 (2020): 1494-1506.
  23. Pedersen I M., et al. “The chimeric anti-CD20 antibody rituximab induces apoptosis in B-cell chronic lymphocytic leukemia cells through a p38 mitogen activated protein-kinase-dependent mechanism”. Blood 4 (2002): 1314-1319.
  24. Reff M E., et al. “Depletion of B cells in vivo by a chimeric mouse human monoclonal antibody to CD20”. Blood2 (1994): 435-445.
  25. Rose A L., et al. “Glucocorticoids and rituximab in vitro: synergistic direct antiproliferative and apoptotic effects”. Blood 5 (2002): 1765-1773.
  26. Shan D., et al. “Apoptosis of malignant human B cells by ligation of CD20 with monoclonal antibodies”. Blood 5 (1998): 1644-1652.
  27. Shan D., et al. “Signaling events involved in anti-CD20-induced apoptosis of malignant human B cells”. Cancer Immunology, Immunotherapy: CII12 (2000): 673-683.
  28. Simpson T R and Allison J Fc Gamma R. In: Marshall, J. (eds) Cancer Therapeutic Targets (2017): 209-228.
  29. Stewart R.,et al. The role of Fc gamma receptors in the activity of immunomodulatory antibodies for cancer. Journal for ImmunoTherapy of Cancer 29 (2014).
  30. Tay M Z., et al. “Antibody-Dependent Cellular Phagocytosis in Antiviral Immune Responses”. Frontiers in Immunology332 (2019).
  31. van der Kolk L E., et al. “CD20-induced B cell death can bypass mitochondria and caspase activation”. Leukemia9 (2002): 1735-1744.
  32. Vega M I., et al. “Rituximab inhibits p38 MAPK activity in 2F7 B NHL and decreases IL-10 transcription: pivotal role of p38 MAPK in drug resistance”. Oncogene20 (2004): 3530-3540.


Citation: Sanjeev Gupta., et al. “WIL2-S and an Engineered Jurkat Cell Line as a Model for Assessment of Apoptotic and Phagocytic Activity Upon Treatment with Anti-CD20". Acta Scientific Pharmaceutical Sciences 7.5 (2023): 04-12.


Copyright: © 2023 Sanjeev Gupta., et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.


Acceptance rate32%
Acceptance to publication20-30 days

Indexed In

News and Events

  • Certification for Review
    Acta Scientific certifies the Editors/reviewers for their review done towards the assigned articles of the respective journals.
  • Submission Timeline for Upcoming Issue
    The last date for submission of articles for regular Issues is May 30, 2024.
  • Publication Certificate
    Authors will be issued a "Publication Certificate" as a mark of appreciation for publishing their work.
  • Best Article of the Issue
    The Editors will elect one Best Article after each issue release. The authors of this article will be provided with a certificate of "Best Article of the Issue"
  • Welcoming Article Submission
    Acta Scientific delightfully welcomes active researchers for submission of articles towards the upcoming issue of respective journals.

Contact US