Table 1.
Characterization of the affinity, species specificity, and potency of anti-MMP9 antibodies.
Table 2.
Characterization of the affinity, species specificity, and potency of anti-MMP9 antibodies.
Fig 1.
Characterization of the mode of inhibition and binding epitopes for anti-MMP9 antibodies.
(A) AB0041, AB0046, and GS-5745 demonstrate noncompetitive inhibition of MMP9: Antibody-mediated inhibition of MMP9 activity was assessed at multiple concentrations of substrate (a fluorogenic peptide). (B) AB0041 inhibition of human DQ-gelatin was evaluated over a dilution series of antibody concentration. Shown are a representative curve and the average ± standard deviation of three independent experiments. (C) AB0041 inhibition of human DQ-collagen IV was evaluated over a dilution series of antibody concentration. Shown are a representative curve and the average ± standard deviation of three independent experiments. (D) Structural model of MMP9. Residues identified as involved in AB0041 binding to MMP9 are shown in pink (including the key residue R162) and are distinct from residues engaging the active site Zn++ (shown in purple).
Fig 2.
Effects of AB0041 on rat joints.
(A) Assessment of AB0041 (50 mg/kg, twice daily) and marimastat (5.5–7 mg/kg/day) in an MSS model: Mean total disease scores (+/- SD) of rats treated with AB0041, marimastat, or vehicle are shown. Marimastat and AB0041 groups each have a matched vehicle group. (B) Representative 50x and 100x images of H&E-stained sections of joints from AB0041- or marimastat-treated rats demonstrate evidence of joint disease with marimastat treatment but not with AB0041 treatment. Significance was assessed with a Mann-Whitney test. P value designations are as follows: * < 0.05, ** < 0.01, *** <0.001, **** < 0.0001.
Fig 3.
MMP9 expression and association with disease in ulcerative colitis and in DSS-induced colitis.
IHC was conducted on serial sections of frozen human non-diseased colon tissue (from healthy individuals, or in non-diseased colonic crypts found adjacent to diseased regions in UC patient samples, n = 12 samples; representative images shown) (A) or of mouse non-diseased colon tissue (n = 2) (B). MMP9 immunoreactivity was limited, and consisted primarily of cytoplasmic staining of a subset of immune cells such as macrophages/histiocytes, lymphocytes, and neutrophils within the lamina propria and submucosal regions of the colon (black arrow). (C) IHC was conducted on serial sections of frozen human UC patient colon tissue (n = 7 patients; representative images shown) and demonstrated MMP9 induction at a disease focus (top left image, black arrow) surrounding an abscessed epithelial crypt (red arrow), and MMP9 expression coincident with regions of neutrophil (MPO, middle left image, yellow arrow) and macrophage (PM2K, bottom left image, black arrow) infiltration/expansion, as well as with regions of disrupted epithelial basement membrane (COLIV, bottom right image, black arrow). MMP9 induction in colonic epithelium was also observed (top right image, blue arrow, 400x). (D) IHC conducted on serial sections of frozen mouse DSS colitis tissue (n = 9) showed an MMP9 expression pattern similar to human UC, with MMP9 induction in a region of inflammation surrounding diseased epithelial crypts (top left image, black arrow), MMP9 induction in colonic epithelium (top right image, blue arrow, 400x), neutrophil infiltration at the active disease site (MPO, bottom left image, yellow arrow) and disruption of the epithelial basement membrane in a disease area (COLIV, bottom right image, black arrow). All images are at 200X magnification unless otherwise noted. NPC = no primary antibody control.
Fig 4.
Efficacy of MMP9-targeting antibody in mouse DSS model of colitis.
Treatment was initiated after establishment of colitis (Day 6): Vehicle control, IgG control (30 mg/kg), and AB0046 (30 mg/kg) were dosed every three days, and entanercept (10 mg/kg) was dosed every two days (A) The area under the curve (AUC) was calculated for daily body weight changes in each animal by the trapezoidal rule method;. (B) The incidence of diarrhea was recorded daily and the AUC calculation was performed as above. (C) Endoscopic evaluation was performed on all groups at study termination. Scoring was based on the single most severe lesion observed in the distal 5 cm of colon. (D) Blinded histopathological analysis was performed on colons excised at study termination. The degree of inflammation (primarily macrophages and neutrophils), edema, and necrosis was scored. (E) Images representative of study groups (40X magnification) were taken by a pathologist and highlight areas of inflammation/mucosal necrosis (black arrows) and edema (blue arrows), which are reduced in AB0046 and etanercept-treated animals. Statistical significance was assessed by one-way ANOVA with Dunnett’s Multiple Comparison post-test. P value designations are as follows: * < 0.05, ** < 0.01, *** <0.001, **** < 0.0001.
Fig 5.
Anti-MMP9 antibody reduces soluble TNF-α generation.
To assess cleavage, 2 μg of pro-TNF-α was incubated with either 2 μg ADAM17 or 2 μg MMP9 in the presence or absence of inhibitor. Cleavage reactions were allowed to incubate overnight at 37°C and were then analyzed by immunoblotting. Lanes are as follows: 1. Molecular weight markers, 2. Pro-TNF-α alone, 3. Pro-TNF-α + ADAM17, 4. pro-TNF-α + ADAM17 + BB-94 (10 μM), 5. Pro-TNF-α + MMP9, 6. Pro-TNF-α + MMP9 + AB0041 (10 μM), 7. Pro-TNF-α + MMP9 + BB-94 (10 μM), 8. Soluble TNF-α.
Fig 6.
MMP9 expression in human CRC and in an orthotopic xenograft mouse model of CRC.
IHC analysis of HCT116-derived xenograft tumors (A, B) or of human CRC tumors (D, E). MMP9 staining from various cellular sources is highlighted as follows: blue arrows, tumor cells; yellow arrows, inflammatory cells; white arrows, stromal cells such as fibroblasts or smooth muscle cells. (A, B) Immunohistochemical staining for MMP9 in HCT116-derived tumors at 200x (A) or 400x (B) magnification. (D, E) Immunohistochemical staining for MMP9 in a human colorectal carcinoma at 200x (D) or 400x (E) magnification. Panels C (HCT116-derived tumors) and F (human CRC) show tissue sections that were incubated with secondary antibody only and demonstrate the absence of non-specific secondary antibody binding (200x magnification).
Fig 7.
Efficacy of MMP9 inhibition in an orthotopic xenograft model of CRC.
Tumors in the HCT116 model were allowed to reach a volume of 70–100 mm3 before treatment with a control IgG or with antibodies targeting mouse MMP9 (AB0046; anti-MMP9 [m]), human MMP9 (AB0041; anti-MMP9 [h]), or both (anti-MMP9 [m+h]). Each antibody was dosed twice a week at 15 mg/kg, whether singly or in combination, and study groups containing AB0046 received a loading dose of 50 mg/kg on the day prior to initiation of treatment. Three separate studies are shown; Study 3 (panels A, D, G); Study 2 (panels B, E, H), and Study 1 (panels C, F, I). (A-C) Change in tumor growth: (A) Study 3, (B) Study 2, (C) Study 1. For a given mouse, raw tumor volume measurements (by caliper) were normalized to the corresponding initial tumor volume (prior to the start of treatment initiation). Normalized volumes for individual mice were then averaged for each timepoint;plots show group mean +/- SEM; standard error of the mean. Significance was assessed by Kruskal-Wallis analysis (A, B) or by Mann-Whitney analysis (C). Treatment was initiated 16 days after surgical implantation of tumor fragments for Studies 1 and 3, and 17 days after implantation for Study 2. The last measurement of tumor volume was 35 days post-implantation for Study 3, 34 days post-implantation for Study 2, and 44 days post-implantation for Study 1. (D-F) Final tumor weight: (D) Study 3, (E) Study 2, (F) Study 1. Plots show mean tumor weight +/- SEM. Significance was assessed by Kruskal-Wallis analysis (D,E) or by Mann-Whitney analysis (F). Mice were terminated at 20 days (Study 3), 18 days (Study 2), or 32 days (Study 1) after treatment initiation, which corresponds to 36 days (Study 3), 35 days (Study 2), or 48 days (Study 1) after tumor implantation. (G-I) Studies 1–3; metastases incidence at study termination: (G) Study 3, (H) Study 2, (I) Study 1. Metastases were scored as present or absent, based on open-imaging visualization of the GFP-labeled HCT116 tumor cells at areas distal to the primary tumor mass. Plots show the percentage of mice displaying metastases per group. Significance was assessed by Fisher’s exact test. For all panels in Fig 7, P value designations are as follows: * < 0.05, ** < 0.01, *** <0.001, **** < 0.0001.