Increased production of matrix metalloproteinases (MMPs) has been associated with increases in invasive and metastatic potential in many types of human carcinoma. Tissue inhibitors of metalloproteinase (TIMP)-1 inhibits most interstitial collagenases and MMP-9. TIMP-2 binds specifically and noncovalently to the pro-form of MMP-2 and inhibits its enzyme activity. In this study, we examined TIMP-1 and TIMP-2 expressions in relation to clinicopathological variables in colorectal carcinoma with in situ hybridization and immunohistochemistry. TIMP-1 and TIMP-2 expressions were localized overwhelmingly to pericancer stromal cells, while malignant and normal mucosal cells were weak or negative. Strong stromal TIMP-1 immunoreactivity correlated with Dukes' stage (p=0.022), status of lymph node metastasis (p=0.044) and poor survival (p= 0.005). The degree of immunohistochemical staining of TIMP-2 did not correlate with all clinicopathological variables. The correlation between enhanced TIMP-1 expression and advanced stage and poor survival suggest a growth promoting activity of TIMP-1 in colorectal carcinoma.
Adenocarcinoma/pathology ; Adenocarcinoma/mortality ; Adenocarcinoma/enzymology* ; Adult ; Aged ; Aged, 80 and over ; Antibodies ; Collagenases/immunology ; Collagenases/genetics* ; Collagenases/analysis ; Colorectal Neoplasms/pathology ; Colorectal Neoplasms/mortality ; Colorectal Neoplasms/enzymology* ; DNA Probes ; Female ; Gelatinase A ; Gelatinase B ; Gelatinases/immunology ; Gelatinases/genetics* ; Gelatinases/analysis ; Gene Expression Regulation, Enzymologic ; Gene Expression Regulation, Neoplastic ; Human ; In Situ Hybridization ; Male ; Metalloendopeptidases/immunology ; Metalloendopeptidases/genetics* ; Metalloendopeptidases/analysis ; Middle Age ; Predictive Value of Tests ; RNA, Messenger/analysis ; Stromal Cells/pathology ; Stromal Cells/enzymology ; Survival Analysis ; Tissue Inhibitor-of Metalloproteinase-2/immunology ; Tissue Inhibitor-of Metalloproteinase-2/genetics* ; Tissue Inhibitor-of Metalloproteinase-2/analysis ; Tissue-Inhibitor of Metalloproteinase-1/immunology ; Tissue-Inhibitor of Metalloproteinase-1/genetics* ; Tissue-Inhibitor of Metalloproteinase-1/analysis

To investigate the antitumor activities of the immunoconjugates composed of anti-type IV collagenase monoclonal antibody Fab' fragment and lidamycin (LDM) prepared with different linkers. The immunoconjugates were prepared by linking Fab' to lysine-69 of LDM apoprotein by SPDP, LCSPDP, SMBS or SSMPB as the intermediate drug linkers. Immunoreactivities of the conjugates were determined by ELISA. The cytotoxicities of the conjugates were examined by clonogenic assay. In vivo antitumor effects of the conjugates were evaluated in nude mice bearing subcutaneously implanted HT-1080 tumor. ELISA assay showed that the conjugates retained part of the immunoreactivity of 3G11 against the antigen. The cytotoxicities of the Fab'-SMBS-LDM and Fab'-SSMPB-LDM to HT-1080 cells were significantly potent, compared with Fab'-SPDP-LDM, Fab'-LCSPDP-LDM and free LDM. In animal models at the same condition, free LDM, Fab'-SPDP-LDM and Fab'-LCSPDP-LDM inhibited the growth of HT-1080 tumor by 70.9%, 74.8% and 72.3%, while Fab'-SMBS-LDM and Fab'-SSMPB-LDM reached 78.0% and 87.7%, respectively. The median survival time of the mice treated with free LDM, Fab'-SPDP-LDM and Fab'-LCSPDP-LDM were prolonged by 71.9%, 82.2% and 107.5%, respectively, compared with that of untreated group. Whereas, the median survival time of Fab'-SMBS-LDM and Fab'-SSMPB-LDM were prolonged by 145.2% and 165.8%, respectively, indicating that Fab'-SSMPB-LDM was more effective than Fab'-SMBS-LDM in tumor suppression and life span prolongation. Fab'-SSMPB-LDM has more marked selective antitumor efficacy and lower toxicity, and might be a novel candidate for cancer therapy.
Aminoglycosides ; pharmacology ; Animals ; Antibiotics, Antineoplastic ; pharmacology ; Antibodies, Monoclonal ; immunology ; Cell Line, Tumor ; drug effects ; Cell Proliferation ; drug effects ; Collagenases ; immunology ; Enediynes ; pharmacology ; Fibrosarcoma ; pathology ; Humans ; Immunoconjugates ; pharmacology ; Immunoglobulin Fab Fragments ; immunology ; Matrix Metalloproteinase Inhibitors ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Neoplasm Transplantation ; Tumor Burden ; drug effects

This study is to investigate the antitumor activities of the immunoconjugates composed of anti-type IV collagenase monoclonal antibody 3G11 and lidamycin (LDM) prepared by different methods. The immunoconjugates were prepared by linking 2-iminothiolane modified 3G11 to lysine-69 of LDM apoprotein by SPDP and SMBS as the intermediate drug linker. Immunoreactivity of the conjugates was determined by ELISA. The cytotoxicity of the conjugates was examined by clonogenic assay. Antitumor effects of the conjugates in vivo were evaluated in nude mice bearing subcutaneously implanted HT-1080 tumor. ELISA assay showed that the immunoconjugates retained the immunoreactivity of 3G11 against type IV collagenase. The cytotoxicity of the 3G11-SMBS-LDM to HT-1080 cells was significantly more potent than that of free LDM and 3G11-SPDP-LDM. In animal model at the same condition, free LDM inhibited the growth of HT-1080 tumor by 71.2%, while 3G11-SPDP-LDM and 3Gl1-SMBS-LDM reached 77.1% and 86.1%, respectively. The median survival time of the mice treated with free LDM was prolonged by 71.9% compared with that of untreated group. Whereas, the median survival time of 3G11-SPDP-LDM and 3G11-SMBS-LDM was prolonged by 125.3% and 163.7%, respectively, indicating that 3G11-SMBS-LDM was more effective than 3G11-SPDP-LDM in tumor suppression and life span prolongation. 3Gll-SMBS-LDM has more selective antitumor efficacy and lower toxicity, and might be a novel candidate for cancer therapy. LDM was more effective than 3G11-SPDP-LDM in tumor suppression and life span prolongation. 3Gll-SMBS-LDM has more selective antitumor efficacy and lower toxicity, and might be a novel candidate for cancer therapy.
Aminoglycosides ; therapeutic use ; Animals ; Antibiotics, Antineoplastic ; therapeutic use ; Antibodies, Monoclonal ; immunology ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Collagenases ; immunology ; Enediynes ; therapeutic use ; Fibrosarcoma ; pathology ; therapy ; Humans ; Immunoconjugates ; therapeutic use ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Neoplasm Transplantation ; Tumor Burden ; drug effects

This study is to optimize the preparation process of fusion protein Fv-LDP which was expressed in the form of inclusion body and consisted of lidamycin apoprotein LDP and single-chain Fv antibody (scFv) directed against type IV collagenase. The preparation and the dissolution of inclusion body, the immobilized metal affinity chromatography of the target protein and the renaturization by stepwise dialysis were optimized by single-factor analysis or orthogonal design. In addition, the refolded fusion protein Fv-LDP was refined by Sephadex G-75 chromatography followed by fluorescence-activated cell sorter (FACS)-based saturation binding assay to measure its antigen-binding activity. After optimization of the process, the purity of fusion protein Fv-LDP existed in the inclusion body was 63.9% and the corresponding solubility was 95.7%; Under denaturing conditions, the purity of fusion protein Fv-LDP was more than 95% after the purification process. The percentage of monomeric fusion protein Fv-LDP was 60% after the refolding process, while it was further refined to 85% which was 5.6-fold higher than that of the initial refolding condition. The refined fusion protein Fv-LDP could bind to human lung adenocarcinoma PAa cells and human hepatoma BEL-7402 cells with the dissociation constants (Kd) of 0.176 micromol x L(-1) and 0.904 micromol x L(-1), respectively. The preparation process of fusion protein Fv-LDP has been successfully optimized, which provides the experimental basis for the production and future development of fusion protein Fv-LDP, and might serve as a relatively practical system for the preparation of other scFv-based proteins expressed in the form of inclusion body.
Adenocarcinoma ; metabolism ; pathology ; Aminoglycosides ; chemistry ; metabolism ; Antibiotics, Antineoplastic ; chemistry ; metabolism ; Apoproteins ; chemistry ; metabolism ; Carcinoma, Hepatocellular ; metabolism ; pathology ; Cell Line, Tumor ; Collagenases ; immunology ; Enediynes ; chemistry ; metabolism ; Escherichia coli ; chemistry ; metabolism ; Humans ; Inclusion Bodies ; chemistry ; metabolism ; Liver Neoplasms ; metabolism ; pathology ; Lung Neoplasms ; metabolism ; pathology ; Protein Binding ; Recombinant Fusion Proteins ; chemistry ; metabolism ; Single-Chain Antibodies ; chemistry ; metabolism