Investigational New Drugs

, Volume 32, Issue 1, pp 75–86 | Cite as

Preclinical studies of targeted therapies for CD20-positive B lymphoid malignancies by Ofatumumab conjugated with auristatin

  • Zhao Hui Li
  • Qian Zhang
  • Hai Bin Wang
  • Ya Nan Zhang
  • Ding Ding
  • Li Qiang Pan
  • David Miao
  • Shi Xu
  • Chen Zhang
  • Pei Hua Luo
  • Hua NaranmanduraEmail author
  • Shu Qing ChenEmail author


Utilization of antibodies to deliver highly potent cytotoxic agents to corresponding antigen-overexpressed tumor cells is a clinically validated therapeutic strategy. Ofatumumab (OFA, trade name Arzerra) is a fully human CD20-specific antibody that is active against CD20-positive B-cell lymphoma/chronic lymphocytic leukemia cells. In order to further enhance the anticancer effect of OFA, anti-CD20 OFA has been conjugated with highly cytotoxic monomethyl auristatin E (MMAE) through a cathepsin-B-cleavable valine-citrulline (vc) dipeptide linkage to form OFA-vcMMAE and the anti-tumor activity of OFA-vcMMAE against CD20-positive B lymphoma cells are then evaluated in vitro and in vivo. As a result, conjugation of OFA with MMAE has kept the initial effector functional activities of OFA such as binding affinity, complement-dependent cytotoxicity (CDC) as well as antibody-dependent cell-mediated cytotoxicity (ADCC). In addition, the conjugation of MMAE significantly improved the cytotoxic activity of OFA against CD20-positive cells (i.e., Raji, Daudi and WIL2-S cells) but not against CD20-negative K562 cells. On the other hand, OFA-vcMMAE was modulated from the CD20-positive cell surface and then entered the lysosomes by receptor-mediated endocytosis, underwent proteolytic degradation and released active drug MMAE to induce apoptotic cell death through a caspase-3-like protease-dependent pathway. Surprisingly, OFA-vcMMAE completely inhibited the growth of CD20-positive Daudi and Ramos lymphoma xenografts in vivo, and exhibited greater anti-tumor activity than unconjugated OFA, suggesting that the anti-tumor activity of anti-CD20 antibody can be enhanced by conjugation with MMAE. In the near future, this new approach might be used as a clinical treatment of CD20-positive B lymphoid malignancies.


Ofatumumab MMAE Conjugate CD20 Targeted therapy 





Antibody-drug conjugate


Antibody-dependent cell-mediated cytotoxicity


Cell Counting Kit-8


Complement-dependent cytotoxicity


Monoclonal antibody


Geometric mean fluorescence intensity ratio


Monomethyl auristatin E




Poly (ADP-ribose) polymerase


Propidium iodide





The authors wish to acknowledge the National Natural Science Foundation of China (No. 81001477, 81274138), the Science and Technology Department of Zhejiang Province (No. 2009C13034), Zhejiang Provincial Natural Science Foundation of China (No.R2110231), the Key Science and Technology Innovation Team of Zhejiang Province (No. 2010R50047).

Competing interests

The authors declare that there are no conflicts of interest.


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Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • Zhao Hui Li
    • 1
  • Qian Zhang
    • 1
  • Hai Bin Wang
    • 2
  • Ya Nan Zhang
    • 1
  • Ding Ding
    • 1
  • Li Qiang Pan
    • 1
  • David Miao
    • 3
  • Shi Xu
    • 1
  • Chen Zhang
    • 1
  • Pei Hua Luo
    • 1
  • Hua Naranmandura
    • 1
    Email author
  • Shu Qing Chen
    • 1
    Email author
  1. 1.Department of Pharmacology, Toxicology and Biochemical Pharmaceutics, College of Pharmaceutical SciencesZhejiang UniversityHangzhouChina
  2. 2.Biotechnology R&D DepartmentZhejiang Hisun Pharmaceutical Co. Ltd.TaizhouChina
  3. 3.Concortis Biosystems, Corp.San DiegoUSA

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