ENGINEERING BISPECIFIC MT1-MMP AND αvβ3 INTEGRIN RECEPTOR ANTAGONISTS FOR CANCER THERAPY

Bispecific proteins that bind to two different targets have the benefits of improved affinity and reduced costs of production, toxicity and approval time. Notably, bispecific proteins play an important role in cancer inhibition, as they can engage multiple target receptors that are over-expressed in cancers. A good example for these receptors is the matrix-metalloproteinase-14 (MMP-14) and α_vβ₃ integrin that play significant role in extracellular matrix remodeling and angiogenesis, respectively. Moreover, there is cross-talk between these receptors as they have a cooperative role in activating pro-MMP-2. In this study, we used the N-terminal region of tissue-inhibitor-of-matrix-metalloproteinase-2 (N-TIMP2), a natural inhibitor of MMP-14, as a template for generating bispecific high affinity antagonists against both MMP-14 and α_vβ₃ integrin. We hypothesize that these N-TIMP2 variants will have preferable antagonistic effects compared to ligands that target only one receptor. In order to introduce new binding epitope for α_vβ₃ integrin we created N-TIMP2 library, in which we inserted near the C-terminus of N-TIMP2, a RGD sequence (α_vβ₃ integrin binding motif) with three random amino acids flanking it from each side. This library was screened using yeast surface display and the high α_vβ₃ integrin N-TIMP2-binding populations were enriched using affinity maturation and FACS. Three unique bispecific clones were identified and chosen for follow-up purification. These clones will be characterized for their binding to both receptors, when the latter are in their soluble or membrane bound forms. We believe that these bispecific antagonists are extremely important as they hold the key for better understanding the cross-talk between MMP-14 and α_vβ₃ integrin along with enormous potential to serve as key agents in cancer therapy.