Removal of the Tag from His-tagged ILYd4, a Human CD59 Inhibitor, Significantly Improves its Physical Properties and its Activity

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 three-fold 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.

How Does Complement Affect Hematological Malignancies: From Basic Mechanisms to Clinical Application

“…Recombinant ILYd4, a novel CD59 inhibitor, effectively enhances the RTX-mediated CDC effect on RTX-sensitive RL-7 lymphoma cells and RTX-induced resistant RR51.2 cells. Meanwhile, recombinant ILYd4 also enhances the effect of RTX and anti-CD24 mAb on the refractory MM cell line ARH-77 (104,105). Similarly, sorafenib potentiates RTX and ofatumumab efficacy in CLL and HL patients by decreasing the expression of complement regulatory proteins.…”

Modulation of Endolysin LysECD7 Bactericidal Activity by Different Peptide Tag Fusion

“…It is known that almost all protein molecules in the research phase are first expressed with a histidine tag [10]. However, there is evidence that even relatively small histidine tags, in some cases, can decrease [11], increase [12], or modify the functional activity of target molecules after fusion [10,13]. Nevertheless, researchers rarely try several tag modifications when purifying the target protein and look for alternative purification methods only if no (or few) biological effects are observed [14,15].…”

Engineering Self-Assembling Protein Nanoparticles for Therapeutic Delivery

“…Process development for manufacturing large-scale protein nanoparticle therapeutics must adhere to production efficiency requirements and current good manufacturing process (cGMP) standards. , Fortunately, the ability to perform in vitro nanoparticle assembly offers several advantages for manufacturing multicomponent protein nanoparticles. In vitro assembly enables two (or more) standard recombinant biologics to be separately purified before nanoparticle assembly, benefiting from decades of research and development in manufacturing recombinant protein biologics like monoclonal antibodies. , Assembling protein nanoparticles in vitro increases sample purity and gives engineers precise control over subunit composition and cargo encapsulation.…”