Title:Removal of the Tag from His-tagged ILYd4, a Human CD59 Inhibitor, Significantly Improves its Physical Properties and its Activity
Volume: 18
Issue: 27
Author(s): Lin Wu, Sanbao Su, Fengming Liu, Tao Xu, Xiaoxiao Wang, Yan Huang, Xinlu Sun, Xiaowen Ge, Ting Chen, Huixia Liu, Chun Wang, Michael Chorev, Ting Xu and Xuebin Qin
Affiliation:
Keywords:
Rituximab, complement, CD59, intermedilysin, and his-tag, Complement dependent cytotoxicity (CDC), Ofatumumab, B-cell non-Hodgkin’s lymphoma (NHL), chronic lymphocytic leukemia (CLL), thermal stability.
Abstract: Complement dependent cytotoxicity (CDC) significantly contributes to Rituximab (RTX) and Ofatumumab (OFA) efficacies
in the treatment of B-cell non-Hodgkin’s lymphoma (NHL) and chronic lymphocytic leukemia (CLL). Human CD59 (hCD59) is a key
complement regulatory protein that restricts the formation of the membrane attack complex and thereby inhibits CDC. hCD59 is an important
determinant of the sensitivity of NHL and CLL to RTX and OFA treatment. Recently, we developed a specific and potent hCD59
inhibitor, His-tagged ILYd4, which consists of 30 amino acid sequences extending from the N-terminus of ILYd4. Our previously published
results indicate that His-tagged ILYd4 can be used as a lead candidate to further develop a potential therapeutic adjuvant for RTX
and OFA treatment of RTX-resistant NHL and CLL. However, these studies were conducted using ILYd4 tagged on the N-terminus with
30 additional amino acids (AA) containing 6 X His used for immobilized metal affinity chromatograph. As a further step towards the development
of ILYd4-based therapeutics, we investigated the impact of the removal of this extraneous sequence on the anti-hCD59 activity.
In this paper, we report the generation and characterization of tag-free ILYd4. We demonstrate that tag-free ILYd4 has over threefold
higher anti-hCD59 activities than the His-tagged ILYd4. The enhanced RTX-mediated CDC effect on B-cell malignant cells comes
from tag-free ILYd4’s improved functionality and physical properties including better solubility, reduced tendency to aggregation, and
greater thermal stability. Therefore, tag-free ILYd4 is a better candidate for the further development for the clinical application.
