Removal of the tag from His-tagged ILYd4, a human CD59 inhibitor, significantly improves its physical properties and its activity.
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.
Department of Hematology, Shanghai Jiaotong University Affiliated First People's Hospital, China.
Lin
L
Wu
Sanbao
S
Su
Fengming
F
Liu
Tao
T
Xu
Xiaoxiao
X
Wang
Yan
Y
Huang
Xinlu
X
Sun
Xiaowen
X
Ge
Ting
T
Chen
Huixia
H
Liu
Chun
C
Wang
Michael
M
Chorev
Ting
T
Xu
Xuebin
X
Qin
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D061067:Antibodies, Monoclonal, Humanized; D058846:Antibodies, Monoclonal, Murine-Derived / Q000494:pharmacology; D000970:Antineoplastic Agents / Q000494:pharmacology*; D001430:Bacteriocins / Q000008:administration & dosage / Q000737:chemistry / Q000494:pharmacology*; D018957:CD59 Antigens / Q000276:immunology*; D017024:Chemotherapy, Adjuvant / Q000379:methods; D015195:Drug Design; D019008:Drug Resistance, Neoplasm; D004355:Drug Stability; D006639:Histidine / Q000737:chemistry; D006801:Humans; D015451:Leukemia, Lymphocytic, Chronic, B-Cell / Q000188:drug therapy / Q000276:immunology / Q000473:pathology; D016393:Lymphoma, B-Cell / Q000188:drug therapy / Q000276:immunology / Q000473:pathology; D000069283:Rituximab; D012995:Solubility
D000911:Antibodies, Monoclonal; D061067:Antibodies, Monoclonal, Humanized; D058846:Antibodies, Monoclonal, Murine-Derived; D000970:Antineoplastic Agents; D001430:Bacteriocins; D018957:CD59 Antigens; C403910:intermedilysin protein, Streptococcus intermedius; D000069283:Rituximab; D006639:Histidine; C527517:ofatumumab