Journal: Molecular Therapy

Article Title: Development and Validation of a Small Single-domain Antibody That Effectively Inhibits Matrix Metalloproteinase 8

doi: 10.1038/mt.2016.2

Figure Lengend Snippet: Analysis of binding and inhibitory constants of the different monomeric nanobodies (Nbs) for the catalytic domain of mouse matrix metalloproteinase 8 (mMMP8_CD). (a) Affinity of the anti-MMP8 Nbs for mMMP8_CD was determined by ELISA (KD Nb14, 1.3 nmol/l; KD Nb44, >100 nmol/l). (b) To determine whether the primary structure or the 3D conformation is recognized by the Nbs, binding analysis of the Nbs for both native (full line) and denatured (dotted line) mMMP8_CD was done by ELISA. (c) Cross-reactivity of Nb14 for the catalytic domain of human MMP8 (hMMP8_CD) was determined via ELISA (KD, 158 nmol/l). (d) Binding of Nb14 with mMMP8_CD was also determined by SPR. The full line shows the actual measurement, while the dotted line depicts the best theoretical fit. The best fit to determine the KD value for Nb14 is the two-state binding curve (KD, 228 nmol/l). (e,f) The EnzCheck gelatinase/collagenase assay was used to determine inhibitory capacity of the different Nbs for mMMP8_CD with DQ gelatin as substrate (IC50 Nb14 (e), 4.127 µmol/l; IC50 Nb44 (f), > 100 µmol/l). (g) The inhibitory capacity of Nb14 was also determined with DQ collagen type I as substrate and gave an IC50 value of 19.5 µmol/l. (n = 2). CD, catalytic domain; FL, full-length; h, human; IC50, half maximal inhibitory concentration; KD, binding affinity constant; m, mouse; MMP, matrix metalloproteinase; Nb, nanobody; SPR, surface plasmon resonance.

Article Snippet: Binding affinity was determined for the following substrates: mMMP8_CD (own production, as described above), full-length mouse MMP8 (mMMP8_FL; R&D systems, 2904-MP-010), full-length human MMP8 (hMMP8_FL; Calbiochem, 444229-5UG), and mouse albumin (Sigma, A3559).

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Concentration Assay, SPR Assay

Journal: Molecular Therapy

Article Title: Development and Validation of a Small Single-domain Antibody That Effectively Inhibits Matrix Metalloproteinase 8

doi: 10.1038/mt.2016.2

Figure Lengend Snippet: Pharmacokinetic properties of the MMP8-binding Nb14 and binding capacity of the modified multivalent Nb14. (a) Serum Nb levels at different time points after intraperitoneal injection (i.p.) injection of 100 µg of the monovalent Nb14 in mice (n = 10) (T1/2 Nb14, 2 hours). (b) A schematic overview of the different Nb14 constructs shows that all constructs contain a C-terminal His6 tag for purification and detection purposes and an N-terminal signal sequence (ss) that is removed after secretion. (1) The monovalent Nb14 is cloned into the pHEN6C vector and contains a pelB signal sequence for Nb protein secretion in the periplasmic space of bacteria. (2) The bispecific Nb14_NbAlb contains an anti-albumin binding Nb connected to Nb14 via a flexible linker ([G4S]3). It was cloned in the pHEN6C (pelB) for protein production in bacteria and in the pCAGGs vector for electroporation as cDNA. The pCAGGs vector contains a sigal sequence that leads to secretion of the Nb out of the electroporated cells. (3) The trispecific Nb14_NbAlb_Nb14 contains two flexible hinges to connect an anti-albumin Nb with two Nb14 domains. This construct was cloned in the pAOXZalfa vector, containing the α-mating factor pre-pro-signal sequence as a signal sequence, for the production of Nb proteins in yeast. (c) Affinity of Nb14, Nb14_NbAlb and Nb14_NbAlb_Nb14 (black) and their control Nb counterparts (gray) for mMMP8_CD was determined by ELISA (KD Nb14, 0.24 nmol/l; KD Nb14_NbAlb, 0.1 nmol/l; Nb14_NbAlb_Nb14, 0.016 nmol/l). (d) ELISA was performed to determine binding of the Nbs for mMMP8_FL (black) and results in KD values of 1.69, 0.63, and 0.057 nmol/l for Nb14, Nb14_NbAlb and Nb14_NbAlb_Nb14, respectively. The affinity of all Nbs was compared to mMMP8_CD in the same test (gray). (e) Binding capacity of all Nb conjugates for hMMP8_CD (gray) compared to mMMP8_CD (black) gave KD values of 158.4, 40.4, and 0.158 nmol/l for Nb14, Nb14_NbAlb and Nb14_NbAlb_Nb14, respectively. (n = 2) (f) Binding for albumin was determined via ELISA with Nb14 and Nbctrl as negative controls. Affinity of the monovalent NbAlb and the modified Nbs gives the following KD values: NbAlb, 0.32; Nb14_NbAlb, 8.14 nmol/l; Nb14_NbAlb_Nb14, 1.48 nmol/l; Nbctrl_NbAlb, 39.61 nmol/l; NbAlb_Nbctrl_Nbctrl, 2.013 nmol/l. (g,h) Binding affinities of the bispecific (n = 2) (g) and trispecific Nb14 (h) for mMMP8_CD were determined with surface plasmon resonance (SPR, Biacore). SPR analysis shows the on-rate, when the Nb binds the chip, in the first part of the graph, while the second phase represents the off-rate, when the Nb dissociates from its substrate (arrows). The full line shows the actual measurement, while the dotted line depicts the best theoretical fit. The best fit to determine KD values for Nb14_NbAlb is the two-state binding curve, while the best fit for Nb14_NbAlb_Nb14 is the bivalent binding model. This bivalent binding model describes the interaction of a monovalent ligand with a molecule carrying two identical and independent binding sites (KD Nb14_NbAlb, 240 nmol/l; KD Nb14_NbAlb_Nb14, 3.7 nmol/l). CD, catalytic domain; FL, full-length; h, human; KD, binding affinity constant; m, mouse; MMP, matrix metalloproteinase; Nb, Nanobody; SPR, surface plasmon resonance; T1/2, half-life.

Article Snippet: Binding affinity was determined for the following substrates: mMMP8_CD (own production, as described above), full-length mouse MMP8 (mMMP8_FL; R&D systems, 2904-MP-010), full-length human MMP8 (hMMP8_FL; Calbiochem, 444229-5UG), and mouse albumin (Sigma, A3559).

Techniques: Binding Assay, Modification, Injection, Construct, Purification, Sequencing, Clone Assay, Plasmid Preparation, Bacteria, Electroporation, Control, Enzyme-linked Immunosorbent Assay, SPR Assay

Journal: Molecular Therapy

Article Title: Development and Validation of a Small Single-domain Antibody That Effectively Inhibits Matrix Metalloproteinase 8

doi: 10.1038/mt.2016.2

Figure Lengend Snippet: Inhibitory and pharmacokinetic properties of the modified bispecific and trispecific anti-MMP8 Nb. (a,b) Inhibitory capacity of Nb14_NbAlb (a) and Nb14_NbAlb_Nb14 (b) for mMMP8_CD with gelatin as substrate gives IC50 values of 4.8 and 0.4 µmol/l, respectively. (n = 2) (c,d) Inhibitory capacity with DQ collagen type I as substrate for mMMP8_CD gives 13.8 and 7 µmol/l as IC50 values for Nb14_NbAlb and Nb14_NbAlb_Nb14, respectively. (n = 2) (e) Nb levels in the serum of mice (n = 10) after a single i.p. injection of 100 µg Nb14_NbAlb_Nb14, were determined by ELISA and gives a T1/2 value of 28 hours. IC50, half maximal inhibitory concentration; i.p., intraperitoneal; Nb, nanobody; T1/2, half-life.

Article Snippet: Binding affinity was determined for the following substrates: mMMP8_CD (own production, as described above), full-length mouse MMP8 (mMMP8_FL; R&D systems, 2904-MP-010), full-length human MMP8 (hMMP8_FL; Calbiochem, 444229-5UG), and mouse albumin (Sigma, A3559).

Techniques: Modification, Injection, Enzyme-linked Immunosorbent Assay, Concentration Assay

Journal: Molecular Therapy

Article Title: Development and Validation of a Small Single-domain Antibody That Effectively Inhibits Matrix Metalloproteinase 8

doi: 10.1038/mt.2016.2

Figure Lengend Snippet: In silico docking models of the interaction between mMMP8_CD and hMMP8_CD with Nb14. Docking models were obtained for the best Nb14 models (Nb14_m1 and Nb14_m2, in color) in combination with hMMP8_CD (gray) on one hand and the two best models for mMMP8_CD (mMMP8_CD_m1 and mMMP8_CD_m2, gray) on the other hand. These simulations show three possible binding sites (1–3). Two (1 and 3) of which are present outside the active site and one (2) at the active site. Modeling of hMMP8_CD was based on the experimental structure, while homology models (mMMP8m1 and Swissmodel mMMP8) were built for mMMP8_CD. Note the extra linker for mMMP8m1 (dark gray). Multiple templates (PDB: 4LAJ, 3EZJ, 3TPK and 4M3J) were used to construct Nb14, depicted as MMP8_Nbm1 and MMP8_Nbm9. All models were validated by RAMPAGE46 and the best models were used for docking by ClusPro47 to predict binding of the MMP8_Nb to MMP8. Homology models and docking results were analyzed and figures rendered using PyMOL. CD, catalytic domain; h, human; m, mouse; MMP, matrix metalloproteinase; Nb, nanobody.

Article Snippet: Binding affinity was determined for the following substrates: mMMP8_CD (own production, as described above), full-length mouse MMP8 (mMMP8_FL; R&D systems, 2904-MP-010), full-length human MMP8 (hMMP8_FL; Calbiochem, 444229-5UG), and mouse albumin (Sigma, A3559).

Techniques: In Silico, Binding Assay, Construct

Journal: Molecular Therapy

Article Title: Development and Validation of a Small Single-domain Antibody That Effectively Inhibits Matrix Metalloproteinase 8

doi: 10.1038/mt.2016.2

Figure Lengend Snippet: Therapeutic potential of in vivo electroporation of an MMP8-inhibiting Nb in systemic inflammation. (a) The OD values (raw data) normalized to baseline (no electroporation) are representative for the serum levels of Nb14_NbAlb_Nb14 after in vivo electroporation of the muscles in mice (n = 8). (b) The survival curve of mice electroporated with a plasmid containing Nb14_NbAlb or Nbctrl_NbAlb (PBS-injected group as control) when challenged with LPS. (n = 17) (c) Survival of mice electroporated with the Nb constructs or PBS after kidney I/R, a sterile model for SIRS. (n = 17) (d) The survival curve of mice electroporated with Nb14_NbAlb_Nb14, Nbctrl_NbAlb_Nbctrl or PBS followed by a challenge with LPS. (n = 6) (e) Survival of mice electroporated with the trispecific Nb constructs or PBS after kidney I/R. (n = 6) (f) Total protease/MMP activity in the serum of mice electroporated with Nb14_NbAlb_Nb14 or PBS. (n = 6) (ctrl, control; I/R, ischemia/reperfusion; LPS, lipopolysaccharide; MMP, matrix metalloproteinase; Nb, Nanobody; OD; optic density; SIRS, systemic inflammatory response syndrome; WT, wild type).

Article Snippet: Binding affinity was determined for the following substrates: mMMP8_CD (own production, as described above), full-length mouse MMP8 (mMMP8_FL; R&D systems, 2904-MP-010), full-length human MMP8 (hMMP8_FL; Calbiochem, 444229-5UG), and mouse albumin (Sigma, A3559).

Techniques: In Vivo, Electroporation, Muscles, Plasmid Preparation, Injection, Control, Construct, Sterility, Activity Assay