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Preclinical Development of Recombinant Human Tissue Inhibitor of Metalloprotease‐2 as a Novel Biologic Therapy for Cancer
Author(s) -
Chowdhury Ananda,
Wei Beiyang,
Noonan Silvia,
StetlerStevenson William
Publication year - 2015
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.29.1_supplement.945.16
Subject(s) - recombinant dna , chemistry , metalloproteinase , cell culture , blot , cancer cell , matrix metalloproteinase , cancer , microbiology and biotechnology , biochemistry , biology , genetics , gene
Tissue Inhibitor of Metalloprotease‐2 (TIMP‐2), a 21.75 KDa protein, has a unique dual activity and mediates its anti‐cancer properties by metalloprotease (MMP) inhibition and as an anti‐ angiogenic agent with direct anti‐proliferative effects on tumor cells. TIMP‐2 is a promising candidate for further pre‐clinical studies and clinical development. Recombinant human TIMP‐2‐His tagged protein (rh‐TIMP‐2‐6XHis) was expressed in solution culture adapted HEK 293 cells in sequentially expanding culture volumes up to 5 liters in wave bioreactor and purified in two steps by affinity and revered phase chromatography. rh‐TIMP‐2‐6XHis can be obtained to up to 97% purity with a maximum associated loss of about 25% and final yields of >25mg/liter. Purification steps and final TIMP‐2 protein were characterized by SDS‐PAGE, Western Blotting as well as ESI‐TOF mass spectrometry, and quantified by Sandwich ELISA assay. These analyses revealed an efficient purification procedure and high degree of monodispersity of the final product. Circular Dichorism spectroscopy exhibited alpha‐helical content of ~33% for TIMP‐2 at pH‐7 and overall pH dependent change of secondary structure. Purified rh‐TIMP‐2 demonstrates inhibition of MMP‐2 activity and inhibition of EGF stimulated proliferation of A549 human lung cancer cell line. Peptide gel encapsulated rh‐TIMP‐2 dosage forms are being tested for loading capacity and release rates. These results demonstrates a straightforward and robust development route for a novel biologic therapeutic for cancer treatment.