Monoclonal Antibodies to Human MMP‐9

Abstract

The degradation of extracellular matrix (ECM) components in normal and pathologic processes has been linked to the presence of a family of hydrolytic enzymes known as matrix metalloproteinases (MMPs).' Two members of this family, the 72-kDa (MMP-2) and the 92-kDa (MMP-9) enzyme, are potent gelatinases. MMP-2 and MMP-9 are highly expressed in invasive tumors, suggesting a role in the degradation of ECM and basement membrane during tumor cell invasion and metastasis.' Studies with natural inhibitors, such as the tissue inhibitor of metalloproteinases (TIMP-1 and TIMP-2), have demonstrated the importance of MMPs in cancer in that they inhibit tumor cell invasion in vitro and reduce metastasis in in vivo animal models.Monoclonal antibodies (mAbs) are useful reagents in assessing the role(s) of specific MMPs in tumor cell invasion and metastasis. Antihuman MMP-1 and MMP-2 mAbs neutralized enzymatic activity in solution using radiolabeled collagens type I and IV, respectively.' The physiologic importance of matrix degradation by MMP-2 during tumor cell invasion in vitro was demonstrated using anti-MMP-2 mAbs.2 Similar studies with anti-MMP-9 mAbs have not been reported. Monoclonal antibodies generated against amino-and carboy-terminal peptides of MMP-9 have been described, but their ability to inhibit enzyme activity has not been tested?To study the role of MMP-9 in tumor cell invasion we generated mAbs against partially purified preparations of MMP-2 and MMP-9 from human fibrosarcoma cell line HT1080.4 Three IgGl mAbs designated 6-6B, 7-llC, and 8-3H were identified and an IgGzb-producing switch variant of 6-6B was isolated. The mAbs are specific for MMP-9 and do not react with MMP-2 by ELISA! All four mAbs are reactive by immunoblot to MMP-9 and not to MMP-2, as shown by French et al. (this volume). In addition to the immunoreactivity analyses, the 6-6B mAb neutralizes the in vitro activation of During in vitro activation of proMMP-9, products with apparent molecular weights of 82-83,000 and 67-75,000 are generated.3,5,6 The formation of the lowest molecular weight species has been correlated with an increase in catalytic activity3z6 and may be generated following cleavage of a carboxyterminal ~e p t i d e .~ To determine if 6-6B and 8-3H mAbs react with this low molecular form of MMP-9, immunoprecipitation analyses were performed. With a highly purified preparation of MMP-9, conversion of the enzyme to the lower molecular species after activation with APMA was assessed by silver-stained SDS-PAGE and gelatin zymography (FIG. 1). The starting material contained predominantly the 92-kDa enzyme and a small amount of the 83-kDa form of the enzyme.Over a 6-h period with APMA, most of the enzyme converted to the 83-kDa form and a small amount of the 67-kDa form was visible by silver staining. With gelatin zymography, the lowest molecular species was apparent by 3 h and had increased by 6-h. Immunoreactivity of mAbs with various molecular weight species of MMP-9 was assessed by immunoprecipitation followed by gelatin zymog...

Interferon β‐1b decreases the migration of T lymphocytes in vitro: Effects on matrix metalloproteinase‐9

“…Culture medium was incubated for I hour at room temperature with 2 pg each of monoclonal antibodies to MMP-2, MMP-9 (Ab-l), or MMP-9 (Ab-2). These antibodies have been described by Ramos-De Simone and French [23] and were purchased from Oncogene Science (Cambridge, MA). Protein G-Sepharose beads were then added to samples for 1 hour at room temperature to bind the excess antibody or antibody-antigen complex.…”

Glioma invasionin vitro: regulation by matrix metalloprotease-2 and protein kinase C

“…Immunoprecipitation was performed following the method described by Ramos-DeSimone and French [33]. In brief, equal volumes of the concentrated supernatant from U563 were incubated for 1 h in primary antibody (2 p.g/tube) at room temperature with gentle agitation.…”

Elevated levels of 92‐kd type IV collagenase (matrix metalloproteinase 9) in giant cell arteritis

“…A double-sandwich immunoassay was developed by using 2 different isotypes of monoclonal antibodies (IgG2b and IgG1) generated against the 92-kd gelatinase as previously described (33,34). IgG2b (designated 6-6B) can recognize the zymogen form as well as the 83-kd activated form of the gelatinase and was used as the capture antibody by coating 96-well flat-bottomed microtiter plates at 1 p.g/ml in bicarbonate buffer, pH 9.6.…”

“…The samples were subjected to a double-sandwich immunoassay utilizing 2 different monoclonal antibodies against the 92-kd gelatinase (MMP-9). The specificity of these antibodies has been described previously (33)(34)(35). The mean -+ SEM level of MMP-9 in sera of the patients with GCA was found to be 3005.4 -C 900.6 ng/ml, compared with 31.6 ?…”