2015;20:1164C1178. in triple-negative breast cancer. and displayed low nM affinity. Treatment of TNBC cells with Fab impaired ECM degradation and cell invasion. In mammary tumor models, treatment with IgG 3369 led to suppression of hypoxia and a skewed TME that limited tumor progression and metastasis. RESULTS Selection of synthetic MMP-14 antibodies To develop improved tools for the blockade of MMP-14 activity, we produced a construct expressing the extracellular domain name (ECD) of MMP-14 fused to the human IgG1 Fc domain name (Physique ?(Figure1A).1A). Upon transient transfection in HEK293T cells, the construct was effectively captured from conditioned media using GammaBind sepharose beads and detected by immunoblot (Physique ?(Figure1B).1B). Following large level expression and affinity purification with Protein A sepharose beads, the Fc-ECD construct was used as bait protein for screening of a phage-displayed synthetic humanized Fab library [21]. Following 4 rounds of sequential negative and positive selection using Fc and Fc-ECD, respectively, DNA sequencing of ELISA positive clones revealed 12 unique clones. These 12 clones were subcloned for expression as free Fab proteins. We detected selective binding of all Fab’s to MMP-14 Fc-ECD construct relative to Fc in an ELISA format (data not shown), and performed surface plasmon resonance assays to define the binding constants (KD) of the selected Fabs. These assays revealed several Fabs that experienced low nM affinity (Physique ?(Physique1C1C). Open in a separate window Physique 1 Anti-MMP-14 synthetic antibody identification and profiling(A) Schematic of MMP-14 extracellular domain name (ECD) fusion with Fc and IL-2 transmission sequence (ss) encoding amino acids (aa) 23-534 of human MMP-14 spanning the prodomain, catalytic domain name (CD) and hemopexin (Hpx) domain name (but not the transmembrane (TM) domain name). (B) Test expression of Fc-ECD in HEK293T cells using lysates and conditioned media (CM). MMP-14 ECD-Fc fusion protein was captured using GammaBind sepharose and detected by IB with anti-MMP14 antibody. (C) The affinity constants (KD) for purified anti-MMP-14 Fabs binding to the MMP-14 ECD-Fc construct were determined by surface plasmon resonance. (D) Inhibition of MMP-14 protease activity (initial rate) was measured using a commercial quenched fluorogenic MMP substrate incubated with MMP-14 catalytic domain name with or without the indicated anti-MMP14 Fabs (800 nM). The small molecule inhibitor NNGH (1.3 M) was used as a control. (E) The dose dependence of Fab 3369 on MMP-14 activity (initial Benzoylhypaconitine rate) was measured as above (IC50 = 62 nM). (F) Trypsin-activated MMP-14 ECD-Fc fusion protein (185 nM) was used as the enzyme incubated with quenched fluorogenic MMP substrate in the absence or presence of the indicated anti-MMP14 Fabs (1 M). The changes in the initial rate relative to no Fab control are shown. To screen for inhibitors, Fab proteins were tested for effects on MMP-14 protease activity towards a quenched fluorogenic substrate. When screened against the purified catalytic domain name of MMP-14, only one Fab (denoted 3369) showed a Benzoylhypaconitine significant suppression of activity that was comparable to that of a small molecule pan MMP inhibitor NNGH (Physique ?(Figure1D).1D). Further titration of Fab 3369 in the Rabbit polyclonal to alpha 1 IL13 Receptor activity assays using the MMP-14 catalytic domain name Benzoylhypaconitine revealed an IC50 value of 62 nM (Physique ?(Figure1E).1E). We also tested the Fab’s using the larger MMP-14 Fc-ECD Benzoylhypaconitine construct, and this further validated Fab 3369 as an inhibitor, but also expanded the number of inhibitory Fab’s to several other clones that may inhibit by binding to epitopes outside of the catalytic domain name (Physique ?(Figure1F).1F). Together, these results identify Fab 3369 as a lead inhibitory synthetic antibody that targets the catalytic domain name of MMP-14. MMP-14 blockade by Fab 3369 inhibits ECM degradation and TNBC cell invasion To test the selectivity of our lead inhibitory Fab 3369 for endogenous MMP-14, we used lentiviral shRNA to select for stable MMP-14 knock-down (KD) in MDA-MB-231 cells, and in a derivative cell collection expressing constitutively active Src (MDA-Src) [22]. Lysates were subjected to immunoblot with MMP-14 antisera, which revealed an almost total silencing of MMP-14 in both cell lines (Physique ?(Physique2A;2A; actin served as a loading control). Using MDA-Src cells that express high levels of MMP-14 around the cell surface [22], we tested binding of Fab 3369 to MDA-Src vector control and MMP-14 KD cells. Flow cytometry analysis indicated that all vector.