HK1142331A - Antibodies and pharmaceutical compositions containing same useful for inhibiting activity of metalloproteins - Google Patents

Description

Antibodies for inhibiting metalloprotein activity and pharmaceutical compositions containing the same

Field and background of the invention

The present invention relates to hapten molecules and antibodies directed against the haptens that can be used to inhibit the activity of metalloproteins (e.g., metalloproteases), and methods of using the antibodies to treat diseases associated with aberrant activity of metalloproteins (e.g., metastatic cancer).

Matrix Metalloproteins (MMPs) are key enzymes involved in extracellular matrix (ECM) remodeling. These enzymes are capable of destroying various connective tissue components or basement membranes of articular cartilage.

The human MMP gene family consists of at least 28 structurally related proteins (see fig. 1), which share a similar overall spherical topology (fig. 2 and Borkakoti, 1998). Each MMP is secreted as an inactive potential zymogen. The catalytic zinc domain consists of approximately 180 amino acids, with the highly conserved sequence HE-GH-LGL-H providing 3 histidine (i.e., H) residues that bind to the metal Zn (2+) ion. The fourth binding site of the catalytic zinc ion in the proenzyme binds to a cysteine residue (Morganova et al, 1999), which dissociates from the active site upon enzyme activation (Van Wart and Birkedal-Hansen, 1990). As a result, the fourth binding site in activated MMPs is occupied by a water molecule that is also hydrogen bonded to the conserved glutamate residue. This process causes the activated water molecule to hydrolyze the peptide bond of the target substrate.

Uncontrolled breakdown of connective tissue by metalloproteinases is characteristic of many pathological conditions, perhaps caused by excessive MMP activity, or by an imbalance in the ratio of natural MMP Tissue Inhibitors (TIMPs) to MMPs. TIMP inhibits MMPs by forming a stoichiometric complex with the active zinc binding site of the MMP (Gomez et al, 1997; Henriet et al, 1999; Bode et al, 1999; Will et al, 1996). When TIMP levels are insufficient, progressive slow degradation of ECM can lead to loss of cartilage matrix in rheumatoid arthritis (Walakovits et al, arthritis Rheum, 35: 35-42, 1992) and osteoarthritis (Dean et al, J.Clin.invest.84: 678-. In other cases, such as congestive heart failure, rapid degradation of cardiac ECM may occur (Armstrong et al, Canadian J. Cardiol.10: 214-220, 1994).

In addition, MMPs are known to play a role in, for example, cytokine and chemokine maturation: galectin-3 (Ochieng J., Biochemistry, 199433 (47): 14109-14), plasminogen (Patterson, BC., JBC, 1997272 (46): 28823-5), interleukin-8, connective tissue activating peptide III, platelet factor-4 (Van den Steen, 2000blood.2000, 10/15 days; 96 (8): 2673-81), interleukin-1 beta precursor (pro-interleukin-1 beta) (Schonbeck, 1998), interleukin-2 receptor alpha chain [ Sheu, B.C., Huang, S.M., Ho, H., Lien, H.C., Huang, S.C., Lin, R.H.A. novelole of metalloproteinase in 2001.C., Lin, R.H.A. novelole of tumor-mediated growth of tumor-mediated by TGF-growth promoter (TGF- β proteinase) and growth factor for inhibiting the growth of tumor-beta-growth in Cancer cells [ 61, tumor growth factor, 61, Cancer growth factor, beta.),237, stamenkovic, I.cell surface-localized matrix metalloproteinases-9 proteolytic activities TGF-beta and proteins tumor invasion and angiogenesis (cell surface localized matrix metalloproteinase-9proteolytically activates TGF-beta and promotes tumor invasion and angiogenesis) Genes Dev (2000)14, 163-176 ].

Other pathological conditions that are also associated with uncontrolled MMP activity include rapid remodeling of the ECM of metastatic tumor cells. In this case, the activated MMPs are expressed by cancer cells or surrounding tissues. There is considerable evidence that MMPs are involved in tumor growth and spread (see, e.g., Davidson et al, Chemistry & Industry, 258-261, 1997, and references therein). During tumor metastasis, MMPs are used to break down the ECM, allowing primary tumor cancer cells to invade nearby blood vessels, be transported in the blood vessels to different organs, and establish secondary tumors. Invasive growth at these secondary sites is mediated by MMPs, which can destroy tissues. In addition, MMP activity has an effect on the invasive ingrowth of new blood vessels (also known as angiogenesis), which is required for tumor growth beyond a certain size. Studies have shown that one of the members of the MMP family, secreted human MMP-9 (also known as gelatinase B), plays a key role not only in extracellular matrix (ECM) catabolism, but also in the processing of protein substrates associated with neurological diseases such as Multiple Sclerosis (MS) (optenakker, 2003). Recent studies have shown that MMP-9 plays a crucial role in promoting autoimmune diseases by cleaving pre-processed type II collagen (Van den Steen, 2004). The cleavage products are collagen type II fragments which are generally considered to be residual epitopes for the development of autoimmune diseases.

Given the wide role of MMPs in human physiology and pathology, it is not surprising that much effort has been expended in designing drugs that inhibit the hyperactivity of MMPs.

Efforts in drug development have focused on the class of inhibitors containing functional groups that coordinate to zinc ions, thereby inactivating target MMPs. One such inhibitor is the hydroxamate inhibitor (hydroxamate inhibitor), a small peptide analog of fibrous collagen that specifically interacts with zinc ions at the catalytic site in a bidentate fashion through the hydroxyl and carbonyl oxygen groups on the hydroxamate group [ grasss et al (1995), biochem.34: 14012-; bode et al (1994), EMBO j., 13: 1263-1269].

Hydroxamic acid-based MMP inhibitors typically include a carbon backbone (WO 95/29892, WO 97/24117, WO 97/49679, and EP 0780386), a peptidyl backbone (WO 90/05719, WO 93/20047, WO 95/09841, and WO 96/06074), or a peptidomimetic backbone (peptidomimetic back-bone) [ Schwartz et al, progr. 271-334 (1992); rasmussen et al, pharmacol. ther., 75: 69-75 (1997); denis et al, invest. new Drugs, 15: 175-185(1997)]. Alternatively, they contain a sulfonamidosulfonyl group, one side of which is attached to the benzene ring and the nitrogen of the sulfonamido group is attached to the hydroxamic acid group through a chain of 1 to 4 carbon atoms (EP 0757984 a 1).

Other peptide-based MMP inhibitors are mercaptoamides having collagenase inhibitory activity (U.S. patent No. 4,595,700); n-carboxyalkyl derivatives containing diphenylethylglycine (biphenylethylglycine) which inhibit MMP-3, MMP-2 and collagenase (Durette et al, WO-9529689); lactam derivatives that inhibit MMPs, TNF-alpha and aggrecanases (see US6,495,699) and tricyclic sulfonamide compounds (see US6,492,422).

Although peptide-based MMP inhibitors have clear therapeutic potential, their use in clinical therapy is limited. Peptide-based hydroxamic acids are expensive to produce and have poor metabolic stability and oral bioavailability [ e.g., batimastat (batimastat) (BB-94) ]. These compounds are rapidly glucuronidated, oxidized to carboxylic acids and secreted in bile [ Singh et al, bioorg.med.chem.lett.5: 337, 342, 1995; hodgson, "Remodelling MMPIs (Remodelling of MMPI)", Biotechnology 13: 554-557, 1995)]. In addition, peptide-based MMP inhibitors often have the same or similar inhibitory effect on each MMP enzyme. For example, batimastat is reported to have an IC50 value of about 1nM to about 20nM for each of MMP-1, MMP-2, MMP-3, MMP-7, and MMP-9[ Rasmussen et al, pharmacol. ther., 75 (1): 69-75(1997)]. In addition, the use of several hydroxamate inhibitors has been associated with severe side effects, such as the musculoskeletal problems associated with marimastat (BB-2516), generalized maculopapular rash (widespead maculopapular rash) and CGS27023A (Novartis) [ Levitt et al, 2001, clin. 1912-1922] and liver abnormalities, anemia, shoulder and back pain, thrombocytopenia, nausea, fatigue, diarrhea, and deep vein thrombosis and BAY12-9566(Bayer) [ Heath et al, 2001, Cancer Chemother. Pharmacol.48: 269-274] correlation. In addition, phase III clinical trials with marimastat, prinomastat (AG 3340, Agouron) and Bay12-9566 in patients with advanced cancer demonstrated no clinical efficacy in inhibiting metastasis (Zucker et al, 2000, Oncogene 19: 6642-50).

Other MMP inhibitors are chemically modified non-microbial tetracyclines (CMTs) which have been shown to block the expression of several MMPs in vitro. However, the in vivo efficacy of these compounds has been found to be limited, e.g., the CMT inhibitor doxycycline reduces tissue levels of MMP-1, but not MMP-2, MMP-3 or MMP-9 in atherosclerotic plaques in human patients (Axisa et al, 2002, Stroke 33: 2858-2864).

Recently, mechanism-based MMP inhibitors (mechanism-based MMP inhibitors), namely SB-3CT (Brown et al, 2000), have been designed based on X-ray crystallographic information of the MMP active site. X-ray absorption studies show that binding of this molecule to catalytic zinc reconfigures the conformational environment around the active site metal ion back to that of the proenzyme [ Kleifeld et al, 2001, J biol. chem.276: 17125-31]. However, the therapeutic efficacy obtained with this drug remains to be verified.

Another class of natural inhibitors are monoclonal antibodies. Several antibodies have been produced against specific peptide sequences within the MMP-1 catalytic domain (Galvez et al, 2001, J.biol.chem., 276: 37491-. However, although these antibodies can inhibit MMP activity in vitro, the results indicate that the in vivo efficacy of such antibodies has not been demonstrated.

As described above, the catalytic site of MMPs includes a coordinating metal ion that is available for substrate binding after enzymatic activation (see fig. 2 a-c). It will therefore be appreciated that conventional antibodies directed against the primary amino acid sequence of an enzyme cannot distinguish the active form from the inactive form of the enzyme and therefore cannot be used as effective inhibitors of such enzymes.

The present inventors have previously proposed that antibodies which recognize both an electronic determinant and a structural determinant of the catalytic site of MMPs (determinants) are potent inhibitors thereof and thus may be useful in the treatment of diseases associated with dysregulated MMP activity (see PCT publication WO 2004/087042).

Therefore, there is a generally recognized need for specific hapten compounds that mimic the electron determinants and structural determinants of the metalloprotein catalytic site, as well as specific antibodies to the hapten compounds, and it is highly desirable to obtain the specific hapten compounds and their specific antibodies.

Summary of The Invention

According to one aspect of the present invention, there is provided a compound having the following general formula (I):

wherein:

m and n are each independently an integer from 1 to 6;

X₁-X₃and Y₁-Y₃Each independently is O or S;

R₁-R₃each independently selected from hydrogen, alkyl, and cycloalkyl; and

r is (CH)₂)x-C(＝O)NR′-(CH₂)y-NR′R″

Wherein:

x and y are each independently an integer from 1 to 6; and

r 'and R' are each independently selected from hydrogen, alkyl, and cycloalkyl.

According to still further features in preferred embodiments of the invention described below, the compounds of the invention have the following formula (II):

wherein R is-CH₂-C(＝O)NH-CH₂-CH₂-NH₂。

According to another aspect of the present invention, there is provided a compound having the following formula (II):

wherein R is-CH₂-C(＝O)NH-CH₂-CH₂-NH₂。

According to a further aspect of the present invention, there is provided an antibody comprising an antigen recognition region capable of specifically binding to the above compound.

According to still further features in the described preferred embodiments the antigen recognition region comprises a sequence selected from the group consisting of SEQ ID NOs: 7. 8, 9, 10, 11 and 12.

According to still further features in the described preferred embodiments the CDR amino acid sequence consists of a sequence selected from the group consisting of SEQ ID NO: 13. 14, 15, 16, 17 and 18.

According to still further features in the described preferred embodiments the antibody is capable of inhibiting the activity of a metalloprotein.

According to still further features in the described preferred embodiments the metalloprotein is a matrix metalloproteinase.

According to still further features in the described preferred embodiments the matrix metalloproteinase is gelatinase.

According to still further features in the described preferred embodiments the gelatinase is selected from the group consisting of MMP-2 and MMP-9.

According to a further aspect of the present invention, there is provided a method of producing a metalloprotein inhibitor, the method comprising producing an antibody against the above compound, thereby producing the metalloprotein inhibitor.

According to still further features in the described preferred embodiments the antibody is a polyclonal antibody.

According to still further features in the described preferred embodiments the antibody is a monoclonal antibody.

According to yet another aspect of the invention, there is provided a pharmaceutical composition comprising the antibody and a pharmaceutically acceptable carrier.

According to a further aspect of the invention there is provided a method of treating a disease associated with a deregulated or abnormal activity of a metalloprotein in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the antibody of any one of claims 4 to 10, thereby treating the disease associated with a deregulated or abnormal activity of a metalloprotein in the subject.

According to still further features in the described preferred embodiments the disease is inflammatory bowel disease (inflammatory bowel disease).

According to a further aspect of the invention there is provided a method of inhibiting matrix metalloproteinase activity of a cell, the method comprising contacting the cell with an antibody of any one of claims 4 to 10, thereby inhibiting matrix metalloproteinase activity of the cell.

The present invention successfully overcomes the disadvantages of the prior known configurations by providing novel hapten compositions which can be used to generate antibodies which simultaneously recognize both the electron determinants and the structural determinants of the catalytic site of metalloproteins.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. In case of conflict, the present patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

Drawings

The invention is herein described, by way of example only, with reference to the accompanying drawings. It is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of the preferred embodiments of the present invention only, and are presented in the cause of providing what is believed to be the most useful and readily understood description of the principles and conceptual aspects of the invention. In this regard, no attempt is made to show structural details of the present invention in more detail than is necessary for the fundamental understanding of the present invention, the description taken with the drawings making apparent to those skilled in the art how the several forms of the present invention may be embodied in practice.

In the drawings:

FIGS. 1A-D are schematic diagrams of the following molecular structures: Co/ZnTCPP- [ m-Tetrakis (4-carboxyphenyl) -porphyrin ] cobalt/zinc (II) ([ meso-Tetrakis (4-carboxyphenyl) -porphinato ] cobalt/zinc (II)) (FIGS. 1A-B), Imisdp- [2- (2-aminoethylcarbamoyl) -ethoxymethyl ] -tris- [2- (N- (3-imidazol-1-yl-propyl)) -ethoxymethyl ] methane ([2- (2-aminoethylcarbamoyl) -ethoxymethyl ] -tris- [2- (N- (3-imidozol-1-yl-propyl)) -ethoxymethyl ] methane) and a conserved zinc-protein linkage to the catalytic zinc site of MMP.

FIGS. 1E-H are three-dimensional schematic diagrams of the structures shown in FIGS. 1A-D. Note that ZnTCPP maintains a planar conformation, whereas CoTCPP has a distorted micro-periodic conformation (distorstedmicrocycle conformation). Apparently, the Imisdp structure closely resembles the closest environment for the catalytic zinc ion of MMP-9 shown in FIG. 1G.

FIG. 2A is a structural overlay (overlay) between the three-dimensional computational structure of Imisdp (green carbon atom) and the 3 conserved histidines of the MMP-9 active site (PDB code 1GKC, grey carbon atom). Catalytic zinc ions are depicted as orange spheres, water molecules as blue spheres, nitrogen as blue and oxygen as red.

Figure 2B is a structural overlay between the ZnTCPP porphyrin ring (CSD code AKICOM) (green carbon atom) and the MMP-9 active site 3 conserved histidines (gray carbon atom PDB code 1GKC), with catalytic zinc ions depicted as orange spheres and nitrogen colored blue.

FIGS. 3A-C Western blot images show the ability of mouse IgG-Agarose (IgG-Agarose) immobilized mAbs to precipitate recombinant MMP-2 catalytic domain (MMP-2cat) or Pro-MMP-2 and Pro-MMP-9 from solution (pull down). The antibodies used in each experiment were 6C6, 13E11, and 13E 15. FIG. 3A-incubation of MMP-2cat (2. mu.g) with anti-mouse IgG-agarose (cntl, lane 1) or anti-CoTCPP, anti-ZnTCPP and anti-Imisdp mAb (10. mu.g) -anti-mouse IgG-agarose for 2 hours at 20 ℃ immunoprecipitates (lanes 2, 3, 5) were washed three times after centrifugation, separated on SDS/PAGE gels, and visualized by Coomassie staining. FIG. 3B-Pro-MMP-2, Pro-MMP-9 were incubated with mAb-anti-mouse IgG-agarose in the same manner as A. Immunoprecipitates (lanes 2,4, 6 left and lanes 1, 3,5 right) and unbound fraction (lanes 1, 3,5 left and 2,4, 6 right) were separated on SDS/PAGE gels and visualized by Coomassie staining. Figure 3C-conditioned medium of HT1080 cells activated (left) or not activated (right) with APMA was immunoprecipitated with anti-CoTCPP mAb and analyzed by western blotting with specific antibody against MMP-2.

FIGS. 4A-B are Lineweaver-Burk plots of anti-CoTCPP mAb inhibition of MMP-2(A) and MMP-9 (B). The unit of speed is mu mol/second^-1Substrate unit is μ M^-1. FIG. 4A-MAb concentrations were 6 μ M (filled triangles), 18 μ M (filled squares), 24 μ M (open circles) and 0 μ M (open squares). MMP-2cat concentration was 200 nM. FIG. 4B-MMP-9 inhibitory full-length APMA activation, mAb concentrations 6 μ M (open squares), 12 μ M (closed triangles), 24 μ M (open squares) and 0 μ M (closed squares). MMP-9 concentration was 20 nM. Inhibition patterns indicate anti-CoTCPP mAbAct as competitive inhibitors of MMP-2 and MMP-9.

FIG. 5 is a graph showing that anti-Imisdp mAb inhibits MMP-2 and MMP-9. MMP-9 catalytic domain (20nM) (filled circles) or full-length APMA-activated MMP-2 (filled triangles, 5nM) was added to the fluorescent substrate OCAcPLGLA2pr (Dnp) -AR-NH₂(10. mu.M) with buffer R containing increasing concentrations of mAb.

The curve represents a non-linear least squares fit to the following equation using the Origin program: vi/vo ═ (Km + [ S ])/(Km (1+ [ I ]/Ki) + [ S ]).

FIG. 6A is a spectrum of zinc k-edge (k-edge) spectra showing the active form of MMP-2cat and the anti-CoTCPPmAb-inhibiting form of MMP-2 cat. Normalized raw XAS data for the zinc K-border region of the active MMP-2cat (dashed line) and MMP-2cat-mAb complex (solid line) are shown.

FIG. 6B shows that the MMP-2cat-mAb complex (solid line) moves to a higher energy marginal position relative to the active MMP-2cat (dashed line).

FIG. 6C shows the EXAFS results in the form of MMP-2cat activity (black) and inhibition (green) shown. The results are represented by R-space (R-space) and inverse transform to k-space.

FIGS. 7A-B are photographs showing the ability of anti-CoTCPP mAb to inhibit cell surface gelatinase activity. Representative fluorescence micrographs of HT1080 cells plated on cover slips coated with DQ-gelatin in the presence or absence of 1 μ M13E11 mAb. Cell surface gelatin lytic activity was analyzed as a measure of the fluorescence emitted by degraded gelatin. Untreated cells had significant cell surface gelatinase activity, which was significantly inhibited in the presence of 1 μ M anti-CoTCPP mAb. Blue staining of 4', 6-diamidino-2-phenylindole (DAPI) indicates the location of the nucleus.

Fig. 8 is a schematic showing the configuration of the different MMP active sites (S1 pocket).

FIG. 9 is a flow chart of the synthesis of Imisdp.

FIG. 10 shows the amino acid sequence of an antibody of the invention, with the CDR regions highlighted.

FIGS. 11A-D are photographs and models illustrating 6C6 bound only to the active conformation of MMP9 and MMP 2. FIG. 11A: detection of active MMP9 co-purified with 6C6 obtained from ascites in mice. Western Blot (WB) analysis was performed on MAb (10 μ g) purified from ascites of mice containing MMP9 using a commercially available anti-MMP 9 antibody. Non-related IgG mAbs (Non-related IgG mAbs) purified in the same manner were used as negative controls (MAb controls). Human ProMMP9 purified from Hilla transfected cells was used as a molecular weight marker to distinguish active species. Purification was performed by affinity chromatography using protein G microbeads conjugated to the mAb by their constant regions, freeing the antigen binding site for interaction with the antigen. Fig. 11B and 11C: the ability of 6C6mAb immobilized on protein a microbeads to precipitate ProMMP2, ProMMP9, or MMP2 catalytic fragments (lacking hemopexin and prodomain) from solution was analyzed. MAb 6C6 (10. mu.g) immobilized on protein A agarose microbeads was incubated with MMP2 catalytic fragment (1. mu.g) (FIG. 11B), ProMMP9 (FIG. 11C top panel) or ProMMP2 (2. mu.g) (FIG. 11C bottom panel) at 20 ℃ for 2 hours. The microbead-bound mAb complexes were centrifuged and washed three times, separated on SDS/PAGE gels, and visualized by Coomassie staining. Immunoprecipitates (6C6) and unbound fractions were separated on SDS/PAGE gels and visualized by Coomassie staining. Negative controls, which were non-specific uptake enzyme only, were incubated with protein a agarose microbeads. FIG. 11D: the three-dimensional structure of MMP2 lacking the hemopexin domain with (pro-domain) (bottom panel) and without (top panel) the pro domain (PDB ID: 1CK7) is shown in the surface image. The catalytic domain and fibronectin domain are shown in blue-green and the pro-peptide (pro-peptide) in red. The catalytic zinc ion is depicted as an orange sphere, binding 3 conserved histidines as indicated by the yellow bar. As shown, the propeptide domain sterically blocks the active site.

FIGS. 12A-B are graphs and data relating to the mechanism of MMP-9 inhibition by the 6C6 mAb. FIG. 12A: MMP-9 recombinant catalytic fragment (no heme-binding protein and prodomain) was preincubated with varying amounts of mAb. Adding fluorescent peptideThe residual enzyme activity was measured after 10. mu.M. By fitting to the competitive inhibition equation (vi/vo ═ Km + [ S ]]/(Km(1+I/Ki)+[S]) Ki (inset) was calculated when Km was 9.14 ± 0.8. Active MMP-9 (fixed concentration 2nM) in the absence of mAb (●), or in the presence of 0.7. mu.M (■) mAb or 2. mu.M (. smallcircle.) mAb, in 100mM NaCl, 10mM CaCl₂100mM Tris (pH 7.5) at 37 ℃ for 60 minutes. The fluorescent peptide substrate (Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH) was then added₂) To achieve the desired final concentration (S) in the range of 0-30. mu.M, the initial rate of substrate hydrolysis is determined by measuring the increase in fluorescence. The apparent Km and Vmax values were derived by fitting experimental data to the Michaelis-Menten equation. The derived values were used to redraw a biperireciprocal Linweaver-Burk plot, with the cross-over point indicating 6C6 competitively inhibits MMP-9. FIG. 12B: different MMPs were preincubated with varying amounts of mAb. After addition of the fluorescent peptide substrate (10. mu.M), the residual enzyme activity was measured. By fitting to the competitive inhibition equation (vi/vo ═ Km + [ S ]]/(Km(1+I/Ki)+[S]) Ki was determined and Km was 2.46 ± 0.34 for full-length MMP2 purified from Hila cells and 16 ± 1 for the catalytic domain of MT 1-MMP. Effective inhibition of 6C6 was also tested using full-length MMP-2 and MMP-9 (data not shown).

FIG. 13 is a structural overlay of different MMPs showing the overall active site-conserved topology with changes mainly within the peripheral loops (peripheral loops). MMP9(PDB1GKC) -blue-green, MMP2(PDB 1QIB) -magenta, MT1-MMP (PDB1BUV) -orange, MMP7(PDB 1MMQ) -red, TACE (PDB 2I47) -yellow. Conserved histidines are represented by bars and catalytic zinc ions are depicted as orange spheres. Apparently, the overall topology of the peripheral rings of MMP-2 and MMP-9 are similar. This may explain the selectivity of 6C6 for MMP-2 and MMP-9 in the enzymes tested.

FIGS. 14A-C are fluorescence micrographs illustrating 6C6 inhibiting cell surface gelatinase activity. Representative fluorescence micrographs of HT1080 cells (generated by in situ zymography assay) placed on DQ-gelatin coated coverslips in the absence of 5 μ M mAb (FIG. 14A) or in the presence of 5 μ M mAb (FIG. 14B), or in the presence of 15 μ M of the mechanism-based nanomolar gelatinase inhibitor SB-3CT (FIG. 14C). Cell surface gelatin-degrading activity was analyzed as a measure of fluorescence emitted by degraded gelatin. Untreated cells had significant cell surface gelatinase activity (green), which was significantly inhibited in the presence of mAb.

FIGS. 15A-C are graphs showing the effect of 6C6 treatment on various manifestations of acute DSS colitis in C57BL/6 mice. Disease was induced by 2% DSS for 5 days, and mice were given 6C6 daily by intraperitoneal injection of 5 or 1.5mg/kg, starting on day 0. FIG. 15A: DAI was monitored daily (this is a composite score of body weight, rectal bleeding and stool consistency, with a numerical range of 0-4) to find clinical scores. Data are presented as a distribution of points averaged for each animal from day 6 to day 10. FIG. 15B: the length of the colon. FIG. 15C: the mortality rate. The data presented are the combined results of two experiments, 15 mice per group, with a significant effect (p < 0.05) relative to untreated colitis mice.

FIG. 16 is a graph of results from X-ray absorption spectroscopy on the zinc K side of active MMP9 (black) and the inhibitory MMP9-6C6 complex (red). The results are expressed as a radial distribution of zinc ions. The position of the MMP-9 catalytic domain-mAb complex (red) shifts to higher energies relative to active MMP-9 (inset), indicating binding to the catalytic zinc ion. Structural analysis of the X-ray spectroscopic data indicated that 6C6 bound directly to zinc ions and formed a pentacoordinate zinc-protein complex. Apparently, this mode of binding is similar to that of binding to TIMPs at the MMP active site.

Description of the preferred embodiments

The present invention relates to antibodies and fragments thereof that are useful for inhibiting the activity of metalloproteins. Specifically, the antibodies of the invention may be used to treat diseases associated with dysregulated matrix metalloproteinase activity, such as multiple sclerosis, autoimmune diseases and metastatic cancer.

The principles and operation of the present invention may be better understood with reference to the drawings and the accompanying description.

Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not limited in its application to the details set forth in the following description or exemplified in the examples. The invention is capable of other embodiments or of being practiced or carried out in various ways. Also, it is to be understood that the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting.

Matrix metalloproteinases are involved in many biological processes, ranging from cell proliferation, differentiation and extracellular matrix remodelling (ECM) to angiogenesis and cell migration. These processes require a delicate balance between the function of Matrix Metalloproteinases (MMPs) and their natural Tissue Inhibitors (TIMPs). Loss of this balance is a hallmark of a variety of pathological conditions including metastatic tumors, neurodegenerative diseases, and osteoarthritis.

A variety of MMP inhibitors are known in the art, including small peptide inhibitors such as hydroxamic acids, non-microbial tetracycline, and monoclonal antibodies. While the former is limited by high growth cost, strong degradability, low oral bioavailability and lack of specificity, none of the latter demonstrates therapeutic efficacy in vivo.

The present inventors have previously proposed that antibodies that recognize both an electron determinant and a structural determinant of a metalloenzyme catalytic site can be used as potent inhibitors of metalloenzymes. The use of haptens to mimic metalloenzyme metal binding catalytic sites as immunogens allows the generation of highly effective therapeutic antibodies which can be used to treat clinical conditions characterised by an elevated activity of metalloproteins (see WO2004/087042 to the present inventors).

While reducing the present invention to practice, the present inventors have devised novel hapten compounds that accurately mimic the local structure and conformation of the reactive zinc site in MMPs. The compound [2- (2-aminoethylcarbamoyl) -ethoxymethyl ] -tris- [2- (N- (3-imidazol-1-yl-propyl)) -ethoxymethyl ] methane, abbreviated as Imisdp (see figure 1), can mimic the 4-coordination geometry and similar force fields caused by zinc ions aligned with 3 histidines and coordinated with water. The approximate tetrahedral conformation is formed by the 3 imidazole bases and the water molecule as the 4 th ligand. Figure 2A shows a 3D model overlay of the construction of an Imisdp compound constructed with a MMP-9(PDB 1GKC) catalytic site, which has been modified to represent the tetrahedral geometry of the zinc ligand. The modification consists in replacing the ligand present in the X-ray structure (hydroxamate inhibitor) with a water molecule and in using the multilayer QM/MM method (see materials and methods) to optimize the whole enzyme locally to a minimum. A high similarity exists between the zinc histidine motif and Imisdp in MMP-9, calculated from the distances of the epsilon-nitrogen of histidine from the zinc ion (2.04. + -. 0.06 and 2.02, respectively) and the relative orientation of 3 histidines to the metal.

As described below and in the examples section that follows, the inventors of the present invention immunized mice with Imisdp and screened for MMP antibodies that cross-react with MMP-2 and MMP-9. This antibody was designated 6C6 (see figure 10 and examples 1-2 of the examples section that follows). 6C6 was found to bind to MMP-2/9 and competitively inhibit the activity of MMP-9, MMP-2(Ki ranging from 1 μ M to 5 μ M) and MT1-MMP (Ki of 15 μ M, see Table 4 below). Binding and inhibition of MMP-9 and MMP-2 was demonstrated in vitro and in situ by various biochemical and biophysical tools (see examples 4-7 and example 9). Importantly, 6C6 binds only to the activated forms of MMP-9 and MMP-2 (see example 3 and example 8). The enzyme form lacks a prodomain that protects the catalytic zinc complex present within the enzyme moiety. The inventors of the present invention have shown that antibodies generated according to the methods of the present invention are capable of binding to MMP-9 in vivo (FIG. 11A). Furthermore, the inventors of the present invention indicated that the antibody of the present invention has therapeutic potential for the treatment of inflammatory bowel disease (example 10).

In summary, the results of the present invention support the use of Imisdp as an important agent (platform) for the production of metalloprotein inhibitors, and 6C6 and derived and mimetic peptides (peptidomimetics) as valuable therapeutic tools.

These results demonstrate the potential to use these antibodies as a platform for designing selective peptide inhibitors of individual MMPs through phage display and point mutation of mabs or fragments thereof.

Thus, according to one aspect of the present invention, there is provided a compound having the following general formula (I):

wherein:

m and n are each independently an integer from 1 to 6;

X₁-X₃and Y₁-Y₃Each independently is O or S;

R₁-R₃each independently selected from hydrogen, alkyl, and cycloalkyl; and

r is (CH)₂)x-C(＝O)NR′-(CH₂)y-NR′R″

Wherein:

x and y are each independently an integer from 1 to 6; and

r 'and R' are each independently selected from hydrogen, alkyl, and cycloalkyl.

According to a preferred embodiment of this aspect of the invention, the compound is [2- (2-aminoethylcarbamoyl) -ethoxymethyl ] -tris- [2- (N- (3-imidazol-1-yl-propyl)) -ethoxymethyl ] methane, described briefly as Imisdp, having the following general formula (II):

wherein R is-CH₂-C(＝O)NH-CH₂-CH₂-NH₂。

The synthesis of Imisdp is described in example 7 of the examples section that follows.

Since Imisdp mimics the local structure and transient conformation of the reactive zinc site in MMP-9 and MMP-2, it can be used to generate metalloproteinase inhibitors.

Thus, according to one aspect of the present invention, there is provided a method of producing a metalloprotein inhibitor.

This method is accomplished by generating antibodies or antibody fragments against the above-described compound (i.e., Imisdp). See examples 1-2 and the materials and methods section of the examples section that follows.

"metalloprotein" in the context of the present invention refers to a metal-binding protein in which the metal binding site forms part of the enzymatic domain, which is similar in both electron and structure to Imisdp.

The metalloprotein of this aspect of the invention is preferably a metalloprotease-MMP (e.g.gelatinases, such as MMP-2 and MMP-9).

It is understood that all members of the MMP family are translated into potential enzymes which upon activation are converted into active enzymes, wherein the metal ions of the active site readily achieve substrate binding. For example, the previously proposed "cysteine switch model" to explain MMP activation in vitro. The cysteine conversion model suggests that upon activation, the potential zinc binding site is converted to a catalytic zinc binding site by dissociation of a thiol (Cys) -bearing propeptide from the zinc atom. Cleavage of the propeptide results in hydrolysis of the enzyme's prodomain structure, and the shielding of the catalytic zinc ion is removed. Thus, the metal ion and active site pocket are accessible for substrate binding and hydrolysis [ Van Wart and Birkedal-Hansen (1990) Proc. Natl. Acad. Sci. USA 87, 5578-5582 ].

Antibodies and antibody fragments produced according to the teachings of the present invention are useful as potent inhibitors of MMPs due to their ability to bind both metal ions and coordinating amino acids within the catalytic zinc site, thereby specifically inhibiting the active conformation of these enzymes directly involved in the pathological process described above.

As used herein, the term "antibody" refers to intact antibody molecules and the term "antibody fragment" refers to functional fragments thereof, such as Fab, F (ab') capable of binding to macrophages₂And Fv. These functional antibody fragments are defined as follows: (i) fab, which contains a monovalent antigen-binding fragment of an antibody molecule, and which can be produced by digestion of the intact antibody with an enzyme, papain, to yield an intact light chain and a portion of one heavy chain; (ii) fab' can be reduced by pepsin treatment of the intact antibody to obtain this fragment of the antibody molecule, resulting in a portion of the intact light and heavy chains; 2 Fab' fragments were obtained per antibody molecule; (iii) (Fab')₂The antibody fragment can be obtained by treating the whole antibody with the enzyme pepsin without subsequent reduction; f (ab')₂Is a dimer of 2 Fab' fragments linked together by 2 disulfide bonds; (iv) fv, defined as a genetically engineered fragment containing the variable regions of the light and heavy chains expressed as 2 chains; (v) single chain antibody ("SCA"), a genetically engineered molecule comprising a light chain variable region and a heavy chain variable region linked by a suitable polypeptide linker as a genetically fused single chain molecule; and (vi) a peptide encoding a single Complementarity Determining Region (CDR).

Methods for the preparation of antibodies (i.e., monoclonal and polyclonal antibodies) are well known in the art. Antibodies can be prepared by any of several methods known in the art, which can employ the induction of in vivo production of antibody molecules, screening of a library or panel of immunoglobulins with highly specific binding partners as disclosed in the literature [ Orlandi d.r. et al (1989) proc.natl.acad.sci.86: 3833-3837, Winter G et al (1991) Nature 349: 293-299], or by continuous cell line culture. These include, but are not limited to, hybridoma technology, human B-cell hybridoma technology, and epstein-barr virus (EBV) hybridoma technology [ Kohler g, et al (1975) Nature 256: 495-497, Kozbor D. et al (1985) J.Immunol.methods 81: 31-42, Cote R.J. et al (1983) Proc.Natl.Acad.Sci.80: 2026-: 109-120].

If the compounds of the invention are too small to elicit strong immunogens(ii) a sexual response (immunogenicsesponse) whereby such antigen (hapten) can be contacted with an antigen neutral carrier (e.g., keyhole limpet)Hemocyanin (KLH) or serum albumin [ e.g. Bovine Serum Albumin (BSA) ]]Carrier coupling (see us patent numbers 5,189,178 and 5,239,078 and example 2 of the examples section). Coupling to the support may be carried out using methods well known in the art; for example, direct coupling to an amino group can be achieved, optionally followed by reduction of the imino bond formed. Alternatively, the support may be coupled using a condensing agent (e.g., dicyclohexylcarbodiimide) or other carbodiimide dehydrating agent. Linker compounds may also be used to effect coupling; homobifunctional and heterobifunctional linkers are available from Pierce Chemical Company, Rockford, Ill. The resulting immunogenic complex can then be injected into a suitable mammalian subject, such as a mouse, rabbit, or the like. Suitable protocols include repeated injections of the immunogen in the presence of an adjuvant according to a schedule that enhances antibody production in serum. The titer of the immune serum can be readily determined using immunoassays well known in the art.

The antiserum obtained can be used as such or monoclonal antibodies can be obtained as described above.

Antibody fragments can be obtained using methods well known in the art (see, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring harbor Laboratory, New York, 1988, incorporated herein by reference). For example, antibody fragments of the invention can be prepared by proteolysis of the antibody, or by expression of DNA encoding the fragment in e.coli (e.coli) or mammalian cells (e.g., chinese hamster ovary cell cultures or other protein expression systems).

Alternatively, antibody fragments may be obtained by pepsin or papain digestion of whole antibodies by conventional methods. For example, the antibody can be cleaved enzymatically by pepsinFor expression as F (ab')₂To produce antibody fragments. This fragment can also be further cleaved using a thiol reducing agent, optionally with a blocking group for the thiol group (resulting from cleavage of the disulfide bond) to produce a 3.5S Fab' monovalent fragment. Alternatively, enzymatic cleavage using pepsin directly produces 2 monovalent Fab' fragments and an Fc fragment. See, e.g., golden berg, U.S. patent nos. 4,036,945 and 4,331,647, and references included therein, which are all incorporated herein by reference. See also Porter, r.r., biochem.j., 73: 119-126, 1959. Other methods of cleaving antibodies, such as isolating the heavy chain to form monovalent light-heavy chain fragments, further cleaving the fragments, or other enzymatic, chemical or genetic techniques may also be used, so long as the fragments bind to the antigen recognized by the intact antibody.

The Fv fragment comprises V_HChain and V_LAn association of chains. The association may be non-covalent, see Inbar et al, proc.nat' l acad.sci.usa 69: 2659-62, 1972. Alternatively, the variable chains may be linked by intermolecular disulfide bonds, or crosslinked by chemical agents (e.g., glutaraldehyde). Preferably, the Fv fragment comprises V joined by a peptide linker_HAnd V_LAnd (3) a chain. These single-chain antigen binding proteins (sFv) are prepared by constructing a structural gene comprising a sequence encoding V linked by oligonucleotides_HAnd V_LDNA sequence of the region. The structural gene is inserted into an expression vector, which is then introduced into a host cell, for example, Escherichia coli. The recombinant host cell synthesizes a single polypeptide chain with a linker peptide bridging the 2V regions. See, e.g., Whitlow and Filpula, Methods, 2: 97-105, 1991; bird et al, Science 242: 423-426, 1988; pack et al, Bio/Technology 11: 1271-77, 1993; and Ladner et al, U.S. patent No. 4,946,778.

CDR peptides ("minimal recognition units") can be obtained by encoding genes that construct the CDRs of the antibody of interest. Such genes are prepared, for example, by using the polymerase chain reaction to synthesize the variable regions of antibody-producing cellular RNA. See, e.g., Larrick and Fry, Methods, 2: 106-10, 1991.

It will be appreciated that for use in human therapy or diagnostic agents, it is preferred to use humanized antibodies. Humanized forms of non-human (e.g., murine) antibodies are chimeric molecules of immunoglobulins, immunoglobulin chains or fragments thereof (e.g., Fv, Fab ', F (ab')₂Or other antigen-binding subsequences of antibodies) that contain minimal sequences derived from non-human immunoglobulins. Humanized antibodies include human immunoglobulins (recipient antibody) in which residues from a Complementarity Determining Region (CDR) of the recipient are replaced by residues from a CDR of a non-human species, such as mouse, rat or rabbit (donor antibody), having the desired specificity, affinity and performance. In some cases, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies may also comprise residues that are not present in the acceptor antibody, the introduced CDR, or the framework sequence. In general, a humanized antibody will comprise substantially all of at least one, and typically 2, variable regions, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody may also optimally comprise at least a portion of an immunoglobulin constant region (Fc), typically a portion of a human immunoglobulin constant region [ Jones et al, Nature, 321: 522-525 (1986); riechmann et al, Nature, 332: 323-329 (1988); and Presta, curr, op.struct.biol., 2: 593-596(1992)]。

Methods for humanizing non-human antibodies are well known in the art. Humanized antibodies typically have one or more amino acid residues introduced into them from a source other than a human source. These non-human amino acid residues are often referred to as import residues (import residues), and are typically derived from an import variable region. The method can be basically followed by the method of Winter and coworkers [ Jones et al, Nature, 321: 522-525 (1986); riechmann et al, Nature 332: 323-327 (1988); verhoeyen et al, Science, 239: 1534-1536(1988) ], humanization is carried out by replacing the corresponding human antibody sequences with one or more of the rodent CDR sequences. Thus, such humanized antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567) in which substantially no more than one entire human variable region is replaced by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.

Human antibodies can also be made using various techniques known in the art, including phage display libraries [ Hoogenboom and Winter, j.mol.biol., 227: 381 (1991); marks et al, j.mol.biol., 222: 581(1991)]. The techniques of Cole et al and Boerner et al can also be used to prepare human Monoclonal Antibodies (Cole et al, Monoclonal Antibodies and cancer Therapy, Alan R.Liss, p.77 (1985) and Boerner et al, J.Immunol., 147 (1): 86-95 (1991); likewise, human Antibodies can be prepared by introducing the human immunoglobulin locus into transgenic animals, such as mice in which the endogenous immunoglobulin genes have been partially or completely inactivated, after challenge, human antibody production is observed, which is quite similar to that observed in humans in all respects, including gene rearrangement, assembly, and antibody repertoire, see, e.g., U.S. Pat. Nos. 5,545,807, 5,545,806, 5,569,825, 5,625,126, 5,633,425, 5,661,016, and the following publications: Marks et al, Biotechnology/7710, 1992 789,859, 859, 368812, Fisherd et al, (Fisherd et al), nature Biotechnology 14, 845-51 (1996); neuberger, Nature Biotechnology 14, 826 (1996); lonberg and Huszar, Intern.Rev.Immunol.1365-93 (1995).

Once the antibodies are obtained, their metalloprotein inhibitory activity can be determined. Suitable assay conditions for metalloprotein inhibitory activity are described in Knight et al, FEBS Letters 296 (3): 263-266(1992), Cawston et al, anal. biochem, 99: 340-: 771 et seq (1981); cawston et al, biochem.j., 195: 159-165(1981), Weingarten et al, biochem. biophysis. res. comm., 139: 1184-.

As described above, using the above method, the present inventors were able to prepare Matrix Metalloproteinase (MMP) inhibitory antibodies to MMP-2 and MMP-9, abbreviated as 6C6, which are seq id NO: 1. SEQ ID NO: 7. CDR sequences are provided in 8, 9, 10, 11 and 12.

Thus, the present invention provides any (poly) peptide sequence comprising at least one of the above-described CDR sequences and homologues and fragments thereof, as long as it retains metalloprotein inhibitory activity (specifically inhibits the catalytic activity of metalloprotein). Examples of such polypeptides are antibodies (see above).

The term "polypeptide" as used herein includes both natural peptides (either degradation products, or synthetic or recombinant peptides) and mimetic peptides (typically synthetic peptides), as well as peptidomimetics and semi-peptidomimetics (semipeptidomimetics) which are peptide analogs having such modifications as to render the peptides more stable in vivo or more permeable to cells. Such modifications include, but are not limited to, N-terminal modifications, C-terminal modifications, peptide bond modifications (including, but not limited to, CH)₂-NH、CH₂-S、CH₂-S＝O、O＝C-NH、CH₂-O、CH₂-CH₂S ═ C-NH, CH ═ CH, or CF ═ CH), backbone modifications, and residue modifications. Methods for preparing peptidomimetic compounds are well known in the art, see, for example, Quantitative Drug Design, c.a. ramsden Gd., chapter 17.2, f.choplin permamon Press (1992), which is incorporated by reference as if fully published herein. More details of this aspect will be provided below.

Peptide bonds (-CO-NH-) within peptides may be replaced by, for example, the following chemical bonds and derivatives: n-methylated bond (-N (CH)₃) -CO-, ester linkage (-C (R) H-C-O-O-C (R) -N-), ketomethylene linkage (-CO-CH)₂-), α -aza bonds (-NH-N (R) -CO-) (wherein R is any alkyl group such as methyl), carbon (carba) bonds (-CH)₂-NH-, hydroxyethylidene (-CH (OH) -CH)₂-), thioamide bond (-CS-NH-), olefinic double bond (-CH-), retro-amide bond (-NH-CO-), peptide derivative (-N (R) -CH-), and peptide derivative (-NH-CO-)₂-CO-), wherein R is "normal"Side chains, naturally occurring on carbon atoms.

These modifications can occur at any bond along the peptide chain, even at several bonds at the same time (2-3).

The natural aromatic amino acids Trp, Tyr and Phe can also be substituted with, for example, the following non-natural synthetic acids: phenylglycine, Tic, naphthylglycine (naphtylalanine, Nal), phenylisoserine, ketothreoninol (threoninol), cyclomethylated derivatives of Phe, halogenated derivatives of Phe or o-methyl-Tyr.

In addition to the above, the peptides of the invention may also include one or more modified amino acids or one or more non-amino acid monomers (e.g., fatty acids, complex sugars, etc.).

It is to be understood that, in the present description and in the following claims section, the term "amino acid" includes the 20 naturally occurring amino acids; amino acids that are often modified post-translationally in vivo, including, for example, hydroxyproline, phosphoserine, and phosphothreonine; and other unusual amino acids including, but not limited to, 2-aminoadipic acid, hydroxylysine, isodesmosine, norvaline, norleucine, and ornithine. Furthermore, the term "amino acid" includes both D-amino acids and L-amino acids.

Tables 1 and 2 below list naturally occurring amino acids (table 1) and less commonly used or modified amino acids (e.g., synthetic amino acids, table 2) that can be used in the present invention.

TABLE 1

Amino acids	Three letter abbreviation	One letter symbol
			Alanine	Ala	A
Arginine	Arg	R
			Asparagine	Asn	N
Aspartic acid	Asp	D
			Cysteine	Cys	C
Glutamine	Gln	Q
			Glutamic acid	Glu	E
Glycine	Gly	G
			Histidine	His	H
Isoleucine	Iie	I
			Leucine	Leu	L
Lysine	Lys	K
			Methionine	Met	M
Phenylalanine	Phe	F
			Proline	Pro	P
Serine	Ser	S
			Threonine	Thr	T

Amino acids	Three letter abbreviation	One letter symbol
			Tryptophan	Trp	W
Tyrosine	Tyr	Y
			Valine	Val	V
Any of the above amino acids	Xaa	X

TABLE 2

Unusual amino acids	Code	Unusual amino acids	Code
				Alpha-aminobutyric acid	Abu	L-N-methylalanine	Nmala
Alpha-amino-alpha-methylbutyric acid	Mgabu	L-N-methyl arginine	Nmarg
				Aminocyclopropane-carboxylic acid	Cpro	L-N-methyl asparagine	Nmasn
		L-N-methyl aspartic acid	Nmasp
				Aminoisobutyric acid	Aib	L-N-methyl cysteine	Nmcys
Amino norbornyl-carboxylic acids	Norb	L-N-methylglutamine	Nmgin
						L-N-methyl glutamic acid	Nmglu
Cyclohexylalanine	Chexa	L-N-methylhistidine	Nmhis
				Cyclopentylalanine	Cpen	L-N-methylisoleucine	Nmile
D-alanine	Dal	L-N-methylleucine	Nmleu
				D-arginine	Darg	L-N-methyl lysine	Nmlys
D-aspartic acid	Dasp	L-N-methylmethionine	Nmmet
				D-cysteine	Dcys	L-N-Methylnorleucine	Nmnle

Unusual amino acids	Code	Unusual amino acids	Code
				D-glutamine	Dgln	L-N-methylnorvaline	Nmnva
D-glutamic acid	Dglu	L-N-methylornithine	Nmorn
				D-histidine	Dhis	L-N-Methylphenylalanine	Nmphe
D-isoleucine	Dile	L-N-methylproline	Nmpro
				D-leucine	Dleu	L-N-methylserine	Nmser
D-lysine	Dlys	L-N-methyl threonine	Nmthr
				D-methionine	Dmet	L-N-methyltryptophan	Nmtrp
D-ornithine	Dorn	L-N-methyl tyrosine	Nmtyr
				D-phenylalanine	Dphe	L-N-methylvaline	Nmval
D-proline	Dpro	L-N-methylethylglycine	Nmetg
				D-serine	Dser	L-N-methyl-tert-butylglycine	Nmtbug
D-threonine	Dthr	L-norleucine	Nle
				D-tryptophan	Dtrp	L-norvaline	Nva
D-tyrosine	Dtyr	Alpha-methyl-aminoisobutyric acid	Maib
				D-valine	Dval	Alpha-methyl-gamma-aminobutyric acid	Mgabu
D-alpha-methylalanine	Dmala	Alpha-methylcyclohexylalanine	Mchexa
				D-alpha-methyl arginine	Dmarg	Alpha-methylcyclopentylalanine	Mcpen
D-alpha-methyl asparagine	Dmasn	Alpha-methyl-alpha-naphthylalanine	Manap
				D-alpha-methyl aspartic acid	Dmasp	Alpha-methyl penicillamine	Mpen
D-alpha-methyl cysteine	Dmcys	N- (4-aminobutyl) glycine	Nglu

Unusual amino acids	Code	Unusual amino acids	Code
				D-alpha-methylglutamine	Dmgln	N- (2-aminoethyl) glycine	Naeg
D-alpha-methylhistidine	Dmhis	N- (3-aminopropyl) glycine	Norn
				D-alpha-methylisoleucine	Dmile	N-amino-alpha-methylbutyric acid	Nmaabu
D-alpha-methylleucine	Dmleu	Alpha-naphthylalanine	Anap
				D-alpha-methyl lysine	Dmlys	N-benzylglycine	Nphe
D-alpha-methyl methionine	Dmmet	N- (2-carbamoylethyl) glycine	Ngln
				D-alpha-methyl ornithine	Dmorn	N- (carbamoylmethyl) glycine	Nasn
D-alpha-methyl phenylalanine	Dmphe	N- (2-carboxyethyl) glycine	Nglu
				D-alpha-methylproline	Dmpro	N- (carboxymethyl) glycine	Nasp
D-alpha-methylserine	Dmser	N-Cyclobutylglycine	Ncbut
				D-alpha-methyl threonine	Dmthr	N-cycloheptylglycine	Nchep
D-alpha-methyltryptophan	Dmtrp	N-cyclohexyl glycine	Nchex
				D-alpha-methyl tyrosine	Dmty	N-cyclodecylglycine	Ncdec
D-alpha-methylvaline	Dmval	N-cyclododecylglycine	Ncdod
				D-alpha-methylalanine	Dnmala	N-Cyclooctylglycine	Ncoct
D-alpha-methyl arginine	Dnmarg	N-Cyclopropylglycine	Ncpro
				D-alpha-methyl asparagine	Dnmasn	N-cycloundecylglycine	Ncund
D-alpha-methyl aspartic acid	Dnmasp	N- (2, 2-diphenylethyl) glycine	Nbhm
				D-alpha-methyl cysteine	Dnmcys	N- (3, 3-diphenylpropyl) glycine	Nbhe
D-N-methylleucine	Dnmleu	N- (3-indolylethyl) glycine	Nhtrp

Unusual amino acids	Code	Unusual amino acids	Code
				D-N-methyl lysine	Dnmlys	N-methyl-gamma-aminobutyric acid	Nmgabu
N-methylcyclohexylalanine	Nmchexa	D-N-methylmethionine	Dnmmet
				D-N-methylornithine	Dnmorn	N-methylcyclopentylalanine	Nmcpen
N-methylglycine	Nala	D-N-Methylphenylalanine	Dnmphe
				N-methylaminoisobutyric acid	Nmaib	D-N-methylproline	Dnmpro
N- (1-methylpropyl) glycine	Nile	D-N-methylserine	Dnmser
				N- (2-methylpropyl) glycine	Nile	D-N-methylserine	Dnmser
N- (2-methylpropyl) glycine	Nleu	D-N-methyl threonine	Dnmthr
				D-N-methyltryptophan	Dnmtrp	N- (1-methylethyl) glycine	Nva
D-N-methyl tyrosine	Dnmtyr	N-methyl-alpha-naphthylalanine	Nmanap
				D-N-methylvaline	Dnmval	N-methyl penicillamine	Nmpen
Gamma-aminobutyric acid	Gabu	N- (p-hydroxyphenyl) glycine	Nhtyr
				L-tert-butylglycine	Tbug	N- (thiomethyl) glycine	Ncys
L-ethylglycine	Etg	Penicillin amines	Pen
				L-homophenylalanine	Hphe	L-alpha-methylalanine	Mala
L-alpha-methyl arginine	Marg	L-alpha-methyl asparagine	Masn
				L-alpha-methyl aspartic acid	Masp	L-alpha-methyl-Tert-butyl glycine	Mtbug
L-alpha-methyl cysteine	Mcys	L-methyl ethyl glycine	Metg
				L-alpha-methylglutamine	Mgln	L-alpha-methyl glutamic acid	Mglu
L-alpha-methylhistidine	Mhis	L-alpha-Methylperhydrophenylalanine	Mhphe

Unusual amino acids	Code	Unusual amino acids	Code
				L-alpha-methylisoleucine	Mile	N- (2-methylthioethyl) glycine	Nmet
D-N-methylglutamine	Dnmgln	N- (3-guanidinopropyl) glycine	Narg
				D-N-methyl glutamic acid	Dnmglu	N- (1-hydroxyethyl) glycine	Nthr
D-N-methylhistidine	Dnmhis	N- (hydroxyethyl) glycine	Nser
				D-N-methylisoleucine	Dnmile	N- (Imidazoylethyl) glycine	Nhis
D-N-methylleucine	Dnmleu	N- (3-indolylethyl) glycine	Nhtrp
				D-N-methyl lysine	Dnmlys	N-methyl-gamma-aminobutyric acid	Nmgabu
N-methylcyclohexylalanine	Nmchexa	D-N-methylmethionine	Dnmmet
				D-N-methylornithine	Dnmorn	N-methylcyclopentylalanine	Nmcpen
N-methylglycine	Nala	D-N-Methylphenylalanine	Dnmphe
				N-methylaminoisobutyric acid	Nmaib	D-N-methylproline	Dnmpro
N- (1-methylpropyl) glycine	Nile	D-N-methylserine	Dnmser
				N- (2-methylpropyl) glycine	Nleu	D-N-methyl threonine	Dnmthr
D-N-methyltryptophan	Dnmtrp	N- (1-methylethyl) glycine	Nval
				D-N-methyl tyrosine	Dnmtyr	N-methyl-a-naphthylalanine	Nmanap
D-N-methylvaline	Dnmval	N-methyl penicillamine	Nmpen
				Gamma-aminobutyric acid	Gabu	N- (p-hydroxyphenyl) glycine	Nhtyr
L-tert-butylglycine	Tbug	N- (thiomethyl) glycine	Ncys
				L-ethylglycine	Etg	Penicillin amines	Pen
L-homophenylalanine	Hphe	L-alpha-methylalanine	Mala

Unusual amino acids	Code	Unusual amino acids	Code
				L-alpha-methyl arginine	Marg	L-alpha-methyl asparagine	Masn
L-alpha-methyl aspartic acid	Masp	L-alpha-methyl-tert-butylglycine	Mtbug
				L-alpha-methyl cysteine	Mcys	L-methyl ethyl glycine	Metg
L-alpha-methylglutamine	Mgln	L-alpha-methyl glutamic acid	Mglu
				L-alpha-methylhistidine	Mhis	L-alpha-Methylperhydrophenylalanine	Mhphe
L-alpha-methylisoleucine	Mile	N- (2-methylthioethyl) glycine	Nmet
				L-alpha-methylleucine	Mleu	L-alpha-methyl lysine	Mlys
L-alpha-methyl methionine	Mmet	L-alpha-methyl norleucine	Mnle
				L-alpha-methylnorvaline	Mnva	L-alpha-methyl ornithine	Morn
L-alpha-methyl phenylalanine	Mphe	L-alpha-methylproline	Mpro
				L-alpha-methylserine	mser	L-alpha-methyl threonine	Mthr
L-alpha-methylvaline	Mtrp	L-alpha-methyl tyrosine	Mtyr
				L-alpha-methylleucine	Mval	L-N-Methylperhydrophenylalanine	Nmhphe
N- (N- (2, 2-diphenylethyl)	Nnbhm	N- (N- (3, 3-diphenylpropyl)
				Carbamoylmethyl-glycine	Nnbhm	Carbamoylmethyl (1) glycine	Nnbhe
1-carboxy-1- (2, 2-diphenylethylamino) cyclopropane	Nmbc

Peptides with improved affinity or increased biological activity for the metalloprotease of interest can be produced by methods well known in the art, including phage display and computational biology.

The peptides of the invention may be synthesized by any technique known to those skilled in the art of peptide synthesis. For solid phase peptide synthesis, a summary of various techniques can be found in: stewart, j.m. and Young, J.D. (1963), "Solid Phase Peptide Synthesis", w.h.freeman Co. (San Francisco); and Meienhofer, J (1973), "hormonal proteins and Peptides" Vol.2, p.46, Academic Press (New York). For a review of classical liquid phase synthesis see Schroder, g. and Lupke, k. (1965). The Peptides, volume 1, Academic Press (New York). For recombinant techniques, see also the following further references.

The invention also includes nucleic acid sequences encoding the above polypeptide sequences (see SEQ ID NOS: 13, 14, 15, 16, 17 and 18).

As indicated above, one particular use of the antibodies of the invention is in the prevention or treatment of diseases associated with dysregulated or abnormal activity of metalloproteins (e.g., metalloproteases).

Examples of such diseases include, but are not limited to, arthritic diseases such as Osteoarthritis (OA), Rheumatoid Arthritis (RA), septic arthritis, soft tissue rheumatism, polychondritis, and tendonitis; metastatic tumors; periodontal disease; corneal ulcers, such as those caused by alkali or other burns, corneal ulcers caused by radiation, corneal ulcers caused by vitamin E or retinoid deficiencies; glomerulopathy, such as proteinuria; dystrophic epidermolysis bullosa (dystrophobic epidermolysis bullosa); bone resorption diseases (bone resorptiondisorders), such as osteoporosis; paget's disease; hyperparathyroidism and cholesteatoma; birth control by prevention of ovulation or implantation; angiogenesis associated with tumor growth or with neovascularization associated with diabetic retinopathy and macular degeneration; coronary thrombosis with rupture of atherosclerotic plaques; emphysema; wound healing and HIV infection.

As described in example 10, the inventors of the present invention confirmed that the antibody of the present invention can be used for the treatment of irritable bowel disease (irritable bowel disease).

Inflammatory Bowel Disease (IBD) is a severe gastrointestinal disease characterized by enteritis and tissue remodeling, with high frequency of morbidity and potential disabling outcome for patients. The major forms of IBD, Ulcerative Colitis (UC) and Crohn's disease, are chronic, relapsing diseases that are clinically characterized by abdominal pain, diarrhea, rectal bleeding, and fever.

Thus, according to another aspect of the present invention, there is provided a method of inhibiting matrix metalloproteinase activity in a subject in need thereof.

Preferred individual subjects of the invention are animals, e.g., mammals (e.g., dogs, cats, sheep, pigs, horses, cows, primates), preferably humans.

The method comprises providing to the subject a therapeutically effective amount of an MMP inhibitor of the present invention (i.e., an antibody or antibody fragment as described above).

As described in further detail below, the MMP inhibitor can be provided by direct administration (e.g., oral administration or injection) or can be expressed from a polynucleotide construct administered to the individual target cell.

The MMP inhibitors of the present invention are provided to the subject by themselves, or may be provided to the subject as part of a pharmaceutical composition in which they are admixed with a pharmaceutically acceptable carrier.

As used herein, "pharmaceutical composition" refers to a formulation of one or more of the active ingredients described herein with other chemical components such as physiologically suitable carriers and excipients. The purpose of the pharmaceutical composition is to facilitate administration of the compound to the organism.

The term "active ingredient" herein refers to an antibody preparation that is responsible for a biological effect.

The terms "physiologically acceptable carrier" and "pharmaceutically acceptable carrier" are used interchangeably hereinafter and refer to a carrier or diluent that does not significantly irritate the organism and does not impair the biological activity and performance of the administered compound. Adjuvants are included within these terms. One of the components included in a pharmaceutically acceptable carrier can be, for example, polyethylene glycol (PEG), a variety of biocompatible polymers that are soluble in both organic and aqueous media (Mutter et al (1979)).

The term "excipient" herein refers to an inert substance added to a pharmaceutical composition to further facilitate administration of the active ingredient. Examples of excipients include, but are not limited to, calcium carbonate, calcium phosphate, various sugar and starch types, cellulose derivatives, gelatin, vegetable oils, and polyethylene glycols.

Techniques for the preparation and administration of drugs are described in Remington's pharmaceutical sciences, Mack Publishing Co., Easton, Pa., latest edition, which is incorporated herein by reference.

For example, suitable routes of administration may include oral, rectal, transmucosal, especially nasal, intestinal or parenteral delivery, including intramuscular, subcutaneous and intramedullary injections as well as intrathecal, direct intracerebroventricular, intravenous, intraperitoneal, intranasal or intraocular injections.

Alternatively, the formulation may be administered locally rather than systemically, for example by direct injection of the formulation to a particular site in the patient.

The pharmaceutical compositions of the present invention may be prepared by methods well known in the art, for example, by conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.

The pharmaceutical compositions of the present invention may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active ingredients into preparations which can be used pharmaceutically. The appropriate dosage form depends on the chosen route of administration.

For injection, the active ingredients of the invention can be formulated in aqueous solutions, preferably in physiologically compatible buffers, such as Hank's solution, Ringer's solution or physiological salt buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the dosage form. Such penetrants are generally known in the art.

For oral administration, the compounds can be readily formulated by mixing the active compounds with pharmaceutically acceptable carriers well known in the art. Such carriers enable the compounds of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient. Pharmaceutical preparations for oral use can be prepared using solid excipients, optionally grinding the resulting mixture, and processing the mixture of granules, if desired after addition of suitable auxiliaries, to give tablets or dragee cores. Suitable excipients are, in particular, fillers, for example sugars, including lactose, sucrose, mannitol or sorbitol; cellulose preparations, such as corn starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose; and/or physiologically acceptable polymers, such as polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as cross-linked polyvinylpyrrolidone, agar or alginic acid or a salt thereof, such as sodium alginate.

The tablet cores are coated with a suitable coating. For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gum, polyethylene glycol, titanium dioxide, varnish solutions (lacquer dissolution) and suitable organic solvents or solvent mixtures. Dyes or pigments may also be added to the tablets or dragee coatings to identify or characterize different combinations of active compound doses.

Pharmaceutical compositions that can be used orally include push-fit capsules (push-fit capsules) made of gelatin, as well as sealed capsules made of gelatin and a plasticizer (e.g., glycerol or sorbitol). Push-fit capsules may contain the active ingredients in admixture with: fillers such as lactose, binders such as starches, lubricants such as talc or magnesium stearate, and optionally stabilizers. In soft capsules, the active ingredient may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. All dosage forms for oral administration should be in dosages suitable for the chosen route of administration.

For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.

For administration by nasal inhalation, the active ingredients used according to the present invention are conveniently delivered in the form of an aerosol spray presentation from a compressed pack or a nebulizer using a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, difluorotetrafluoroethane or carbon dioxide. In the case of a compressed aerosol, the dosage unit may be determined by providing a valve for delivery in a metered amount. Capsules and cartridges of, for example, gelatin for use in dispensers may be prepared as a powder mix containing the compound and a suitable powder base such as lactose or starch.

The formulations described herein may be formulated for parenteral administration, for example by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an optional addition of a preservative. The compositions may be suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.

Pharmaceutical compositions for parenteral administration include aqueous solutions of the active agents in water-soluble form. Additionally, suspensions of the active ingredients may be formulated as suitable oil-based or water-based injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils (e.g. sesame oil) or synthetic fatty acid esters (e.g. ethyl oleate, triglycerides) or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, for example sodium carboxymethyl cellulose, sorbitol or dextran. Optionally, the suspensions may also contain suitable stabilizers or agents which increase the solubility of the active ingredient to produce highly concentrated solutions.

Alternatively, the active ingredient may be in the form of a powder for injection in a suitable vehicle (e.g. a pyrogen-free sterile aqueous solution) before use.

The formulations of the invention may also be formulated in rectal compositions such as suppositories or retention enemas, using, for example, conventional suppository bases such as cocoa butter or other glycerides.

Pharmaceutical compositions suitable for use in this aspect of the invention include compositions wherein the active ingredient is contained in an amount effective to achieve the intended purpose. More specifically, a therapeutically effective amount refers to an amount of active ingredient effective to prevent, alleviate or ameliorate symptoms of disease or prolong the survival of the subject to be treated.

Determination of a therapeutically effective amount is well within the skill of the art.

For any formulation used in the methods of the invention, a therapeutically effective amount or dose can be estimated initially from in vitro experiments. For example, dosages that can be formulated in animal models and such information can be used to more accurately determine beneficial dosages in humans.

Toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in vitro, in cell cultures or experimental animals. The data obtained from these in vitro and cell culture experiments, as well as animal studies, can be used to formulate various dosages for use in humans. The dosage may vary depending on the dosage form employed and the route of administration employed. The exact dosage form, route of administration and dosage can be selected by The individual physician in view of The patient's condition [ see, e.g., Fingl et al, (1975) "The Pharmacological Basis of Therapeutics", Chapter 1, page 1 ].

Depending on the severity and responsiveness of the disease to be treated, administration may be a single administration or multiple administrations, wherein the course of treatment lasts from several days to several weeks, or until a cure is reached, or a state of remission is reached.

The amount of the composition to be administered will of course depend on the subject to be treated, the severity of the disease, the mode of administration, the judgment of the prescribing physician, and the like.

Compositions comprising the preparations of the invention formulated in a pharmaceutical carrier compatible therewith may also be prepared, filled into suitable containers, and labeled for treatment of a given disease.

If desired, the compositions of the present invention may be presented in a packaging or dispensing device, such as an FDA approved kit, which may contain one or more unit dosage forms containing the active ingredient. For example, the packaging may comprise a metal or plastic film (blister pack), for example. The packaging or dispensing device may be accompanied by instructions for administration. The package or dispenser may also be accompanied by a notice in a format prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice indicates to the agency that the composition is in a form or for human or veterinary administration is approved. Such notice may be, for example, a prescription drug label or an approved drug insert approved by the U.S. food and drug Administration.

As described above, the antibody inhibitor of the present invention may be expressed from a nucleic acid construct.

It will be appreciated that the polynucleotide encoding the antibody of the invention preferably also encodes a signal peptide which allows secretion or transport of the antibody to the subcellular or extracellular location of interest. For example, when the target metalloprotein is an MMP, the secretory signal peptide is preferably conjugated in-frame to the polynucleotide encoding the antibody segment.

It will be further appreciated that recombinant single chain fv (scfv) fragments may be preferentially expressed because their structural complexity is significantly reduced compared to intact antibody molecules. As described above, ScFv consists of V_LAnd V_HProtein composed of antibody polypeptide chain, wherein V_LThe carboxyl terminal of (a) is linked to V via a peptide bridge_HThe amino terminal of (b) is linked to synthesize a single strand. Methods for recombinant production of these peptides are well known in the art [ see Bird et al Science 242: 423-426 (1988); huston et al, Proc.nat' l Acad.Sci.USA 85: 5879-5883 (1988); and de Kruif et al, J.mol.biol.248: 97-105(1995)]. According to an embodiment of this aspect of the invention, spleen mRNA is harvested from the immunized animal after immunization with the compound of the invention and used to generate a cDNA library in phage displaying ScFv fragments. The phage particles are then screened to determine the specificity and preferably which interact with the activated form of the metalloprotein of interest. ScFv segments were recovered from these phage particles and cloned into expression constructs (see U.S. patent No. 5,800,814).

The nucleic acid construct of this aspect of the invention may be administered to target cells of an individual subject (i.e., in vivo gene therapy).

Alternatively, the nucleic acid construct is introduced into a suitable cell by a suitable gene delivery vehicle/method (transfection, transduction, homologous recombination, etc.) and expression system as required, and the modified cell is then propagated in culture and returned to the individual (i.e., ex vivo gene therapy).

In order to enable cellular expression of the antibodies or antibody fragments of the invention, the nucleic acid constructs of the invention further comprise at least one cis-acting regulatory element. The term "cis-acting regulatory element" as used herein refers to a polynucleotide sequence, preferably a promoter, which binds to a trans-acting regulator and regulates the transcription of a coding sequence located downstream thereof.

Any available promoter may be used in the methods of the invention. In a preferred embodiment of the invention, the promoter used in the nucleic acid construct of the invention is active in a specific population of transformed cells. Examples of cell-type specific and/or tissue specific promoters include, for example, the following promoters: liver-specific albumin [ Pinkert et al (1987) Genes dev.1: 268-277), a lymphoid specific promoter [ Calame et al (1988) adv. Immunol.43: 235-275 ]; in particular, the promoter of the T cell receptor [ Winto et al (1989) EMBO J.8: 729-733] and the immunoglobulin promoter [ Banerji et al (1983) Cell 33729-740 ]; neuronal specific promoters, such as the neurofilament promoter [ Byrne et al (1989) proc.natl.acad.sci.usa 86: 5473-5477 ]; pancreas-specific promoters [ Edlunch et al (1985) Science 230: 912-916] or a mammary gland-specific promoter, such as the whey promoter (U.S. Pat. No. 4,873,316 and European application publication No. 264,166). The nucleic acid construct of the invention may further comprise an enhancer, which may be located close to or remote from the promoter sequence and may play a role in up-regulating the transcription initiated thereby.

The constructs of the methods of the invention preferably further comprise a suitable selectable marker and/or an origin of replication. Preferably, the construct used is a shuttle vector which is either amplifiable in E.coli (where the construct contains the appropriate selectable marker and origin of replication) or is compatible with cell proliferation or integrated into the selected gene and tissue. For example, the construct of the invention may be a plasmid, bacmid, phagemid, cosmid, phage, virus or artificial chromosome.

Currently preferred in vivo nucleic acid transfer techniques include transfection with viral or non-viral constructs such as adenovirus, lentivirus, herpes simplex I virus or adeno-associated virus (AAV) and liposome-based systems. Useful lipids for lipid-mediated gene transfer are, for example, DOTMA, DOPE and DC-Chol [ Tonkinson et al, Cancer Investigation, 14 (1): 54-65(1996)]. The most preferred constructs for gene therapy are viruses, most preferably adenoviruses, AAV, lentiviruses or retroviruses. Viral constructs (e.g., retroviral constructs) include at least one transcriptional promoter/enhancer or locus-defining element(s) or other element(s) that control gene expression by other means (e.g., alternative splicing, nuclear RNA export, or post-translational modification of messengers). Such vector constructs also include a packaging signal, Long Terminal Repeats (LTRs) or portions thereof, and plus and minus strand primer binding sites appropriate for the virus used, unless it is already present in the viral construct. In addition, such constructs typically include a signal sequence for secretion of the peptide or antibody from the host cell in which it is placed. Preferred signal sequences for this purpose are mammalian signal sequences. Optionally, the construct may further comprise a signal to direct polyadenylation, as well as one or more restriction sites and translation termination sequences. For example, such constructs may typically include a 5 'LTR, a tRNA binding site, a packaging signal, a DNA second strand synthesis origin, and a 3' LTR or portion thereof. Other vectors that are non-viral, such as cationic lipids, polylysines, and dendrimers, may be used.

Preferred models for the implementation of gene therapy protocols can be found in Somia and Verma [ (2000) Nature Reviews 1: 91-99), Isner (2002) myographic gene therapy (Myocardial gene therapy) Nature 415: 234, 239; high (2001) Gene therapy: a 2001 hyperspectric (gene therapy: 2001 looking forward.) Haemophilia 7: 23-27; and Hammond and McKirnan (2001) endogenous gene therapy for heart disease: a review of animal students and clinical trials (angiogenic gene therapy for heart disease: animal research and clinical trial review). 49: 561-567.

Due to the ability of the antibodies of the invention to differentially recognize activated forms of metalloproteins (see example 3 of the examples section), they can be used as effective diagnostic and prognostic tools, for example by monitoring MMP activity in a biological sample [ i.e. any body sample, such as blood (serum or plasma), saliva, ascites fluid, pleural effusion, urine, biopsy samples, isolated cells and/or cell membrane preparations ]. This becomes particularly important when assessing metastatic characteristics of cancer cells in which deregulation of MMP activation contributes to tumor invasion. Likewise, the antibodies of the invention can be used to monitor therapeutic dosages of MMP inhibitors. For such applications, the antibodies of the invention are preferably labeled with any of the radioactive, fluorescent, biological or enzymatic labels or tags that are of standard use in the art. U.S. patents relating to the use of such labels include U.S. Pat. nos. 3,817,837, 3,850,752, 3,939,350, 3,996,345, 4,277,437, 4,275,149, and 4,366,241.

It will be appreciated that such detection methods may also be useful for high throughput screening of new MMPs. Briefly, a plurality of biological samples can be contacted with an antibody of the invention, wherein an activated MMP is linked to the antibody. Measures are taken to use biological samples comprising activated MMPs, e.g. from tumor cell lines. Typically, radiolabelling is used to reduce the volume of the experiment.

Alternatively, the antibodies of the invention can be used to purify active metalloenzymes in biological samples.

Various protein purification methods are known in the art. For example, the antibodies or antibody fragments of the invention can be used in affinity chromatography to isolate metalloenzymes. Columns may be prepared in which the antibody is attached to a solid substrate, such as particles of agarose, cross-linked dextran (Sephadex), etc., and pure metalloenzyme may be released by passing a biological sample such as cell lysate through the column, washing the column, and passing through a mild denaturing agent at increasing concentrations.

Antibodies or fragments thereof produced according to the teachings of the present invention may be included in a diagnostic or therapeutic kit. The antibody or antibody fragment may be packaged in one or more containers with appropriate buffers and preservatives for diagnostic or for immediate therapeutic treatment.

Thus, each of the antibodies or fragments thereof may be mixed in a single container or placed in separate containers. Preferably the container comprises a packaging label. Suitable containers include, for example, bottles, vials, syringes, and test tubes. The container may be made of a variety of materials, such as glass or plastic.

In addition, other additives such as stabilizers, buffers, blockers, etc. may also be added. The antibodies in such kits may also be linked to a solid support (e.g., a microbead, an array substrate (e.g., a chip), etc.) and used for diagnostic purposes. The kit can further include instructions for determining whether the subject has, or is at risk for having, a condition, disorder or disease associated with expression of a target MMP.

Other objects, advantages and novel features of the present invention will be apparent to one of ordinary skill in the art upon examination of the following examples, which are not intended to be limiting. In addition, experimental support may be found in the following examples of the various embodiments and aspects of the present invention described above and claimed in the appended claims section.

Examples of the experiments

The invention is illustrated in a non-limiting manner by reference to the following examples in combination with the above description.

Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. The literature contains detailed descriptions of such techniques. See, e.g., "Molecular Cloning: a laboratory Manual "Sambrook et al, (1989); "Current Protocols in molecular biology (guide to modern molecular biology), Vol.I-III, Ausubel, R.M. (1994); ausubel et al, "Current Protocols in Molecular Biology (guide to modern Molecular Biology experiments)", John Wiley and Sons, Baltimore, Maryland (1989); perbal, "A Practical Guide to Molecular Cloning (Molecular Cloning Guide)", John Wiley & Sons, New York (1988); watson et al, "Recombinant DNA (recombinant DNA)", Scientific American Books, New York; birren et al (eds) "Genome Analysis: a Laboratory Manual Series (genome analysis: A Laboratory Manual Series) ", volumes 1-4, Cold Spring Harbor Laboratory Press, New York (1998); U.S. patent nos. 4,666,828; 4,683,202, 4,801,531, 5,192,659, and 5,272,057; "Cell Biology: a Laboratory Handbook, Vol.I-III, Cellis, J.E. (1994); "Current protocols in Immunology" Vol.I-III, Coligan J.E. (1994); stits et al (eds.), "Basic and Clinical Immunology" (8 th edition), apple & Lange, Norwalk, CT (1994); MishellShiigi (eds), "Selected Methods in Cellular Immunology" (Methods for Cellular Immunology), w.h.freeman and co., New York (1980); a number of immunoassays are disclosed in the patent and scientific literature as being applicable, see, e.g., U.S. Pat. nos. 3,791,932, 3,839,153, 3,850,752, 3,850,578, 3,853,987, 3,867,517, 3,879,262, 3,901,654, 3,935,074, 3,984,533, 3,996,345, 4,034,074, 4,098,876, 4,879,219, 5,011,771, and 5,281,521; "oligonucleotide synthesis" Gait, m.j. (1984); "Nucleic acid hybridization" Hames, b.d. and Higgins s.j. eds (1985); "Transcription and Translation" Hames, b.d. and Higgins s.j. eds (1984); "Animal Cell Culture" Freshney, r.i. eds (1986); "Immobilized Cells and Enzymes" IRLPress, (1986); "A Practical Guide to Molecular Cloning" (Guide for Molecular Cloning practice) "Perbal, B., (1984) and" Methods in Enzymology "Vol.1-317, Academic Press; "PCR Protocols: a Guide To Methods and applications (PCR protocols: Guide for Methods and applications) ", Academic Press, San Diego, CA (1990); marshak et al, "protocols for Protein Purification and characterization-A Laboratory Course Manual" CSHL Press (1996); all documents are incorporated herein by reference as if fully set forth herein. Other general references are also provided throughout this document. The procedures therein are believed to be well known in the art and are provided herein for the convenience of the reader only. All material included therein is incorporated herein by reference.

Materials and methods

Recombinase-the catalytic domain of MMP-2 (amino acid 110-467 of GenBank accession NP 032636.1) is expressed in BL-21 cells under the control of the T7 promoter. Cells were induced with 1mM isopropyl- β -D-thiogalactopyranoside for 5 hours. The cell pellet was resuspended in 50mM Tris (pH 8.0), 0.5mM EDTA, 50mM NaCl, 5% glycerol and 1% Triton X-100 at a ratio of buffer to initial culture volume of 1: 25. The suspension was centrifuged at 15,000rpm for 10 minutes, and the pellet was dissolved in 50mM Tris (pH 8.0), 0.5mM EDTA, 50mM NaCl, 5% glycerol and 0.2% sarcosyl, and then incubated on ice for 30 minutes. The supernatant fraction was applied to a 5ml gelatin-agarose column (preloaded, Amersham biosciences), pre-equilibrated and dialyzed against dialysis buffer (50mM Tris (pH 8.0), 50mM NaCl, 5mM CaCl₂、10μM ZnCl₂0.02% Brij) wash. The protein was purified with 50mM Tris (pH 8.0), 1M NaCl, 5mM CaCl₂、10μM ZnCl₂0.02% benzylzeum and 15% Me₂SO elution [ Rosen, O., Inhibition of MMPs by monoclonal antibodies ] 2001]Catalytic activity was determined by fluorescent peptide degradation as determined by SDS-PAGE [ Knight, c.g., f.willenbrock and g.murphy, a novel coumarin-layered peptide for sensitive conjugation assays [ FEBS Lett, 1992.296 (3): pages 263 to 6]。

Pro-MMP-9[ lacking the hinge region and hemopexin domain, Ala1-Gly424| P14780| MMP9 human matrix metalloproteinase-9 precursor (MMP-9) (EC 3.4.24.35) ] in the pTWIN expression vector was expressed in e.coli ER2566 and purified from inclusion bodies to homogeneity as described in earlier literature [ bjorklung, m., p.heikkila and e.koivunen, Peptide inhibition of catalytic and non-catalytic activity of matrix metalloproteinase-9 tumor cell migration and invasion ] J Biol Chem, 2004.279 (28): pages 29589-97 ]. Pro-MMP-9 was activated with 1mM Mercury p-aminophenylacetate (APMA, ICN Biomedicals Inc., Ohio, USA) and dissolved in 200mM Tris for 30 min at 37 ℃.

Human recombinant pro-MMP-2 and pro-MMP-9 were expressed in HeLa S3 cells infected with the corresponding recombinant vaccinia virus and purified to homogeneity as described previously [ Olson, M.W., Gervasi, D.C., Mobasherery, S. and Fridman, R. (1997) J.biol.chem.272, 29975-; fridman, R., Fuerst, T.R., Bird, R.E., Hoyhttya, M., Oelkuct, T.M., Kraus, S., Komarek, D., Liotta, L.A., Berman, M.L., and Stetler-Stevenson, W.G. (1992) J.biol.chem.267, 15398-15405 ].

Tetracarboxyphenylporphyrins Co (II)/Zn (II) (CoTCPP/ZnTCPP) -prepared by reacting ZnCl as described in the literature₂ZnTCPP was synthesized by reaction with N, N-Dimethylformamide (DMF) solution of TCPP [ Harada, a. et al, Control of photoinduced electron transfer from zinc-porphyrin to methyl virology by supra molecular formation of supramolecules between monoclonal antibody and zinc-porphyrin ] Photochem photocheiol, 1999.70 (3): 298 th and 302 th pages]. According to the method described in the literature, Co (OAc)₂·4H₂Reaction of O with DMF solution of TCPP to synthesize CoTCPP [ Harada, A. et al, Control of photoinduced electron transfer from zinc-porphyrin to methyl virology by supermolecule formation between monoclonal antibody and zinc-porphyrin ] PhotochemPhotoBiol, 1999.70 (3): 298 th and 302 th pages]And purifying.

Synthesis of Imisdp-see example 7 below.

Hapten conjugation to protein-hapten (4mg) was activated for conjugation by addition of 1, 1' -carbonyldiimidazole in DMF (molar ratio 1: 1) and incubation for 1 hour. Adding 1-50 μmoles of activated hapten to 20mg/mL BSA or keyhole limpetHemocyanin (KLH) in 0.1M carbonate buffer (pH 8). After the solution was stirred at room temperature for 3 hours, it was dialyzed against PBS sufficiently.

Immunization and fusion-BALB/c mice were immunized with each adjuvant (KLH) conjugated to CoTCPP, ZnTCPP or Imisdp. Immunization was performed according to standard procedures followed by fusion with an NSO myeloma cell line [ Harlow, e, and Lane, d., Using Antibodies: ALABORT Manual Portable Protocol No. I.1998 ].

Antibody screening

ELISA-direct ELISA was used in which the corresponding hapten-BSA (3. mu.g/ml/PBS) was coated on Nunc maxisorp plates and the grown hybridoma supernatants were screened for antibodies reactive with ZnTCPP, CoTCPP or Imisdp. The coating was carried out overnight at 4 ℃ and incubated with the antibody for 1 hour at 20 ℃. HRP-conjugated anti-mouse mab (Sigma) was used as secondary antibody, 2, 2' -azino-bis (3-ethylbenzothiazoline-6-sulfonic acid, ABTS, Sigma) was used as substrate. PBS (PBST) containing 0.005% (v/v) Tween 20(Tween 20) was used as a detergent. The dilution buffer was PBS. A602 was recorded with a microplate reader in a SPECTRAFluor Plus spectrometer (Tecan, Austria). In the same manner, control supernatants were incubated with BSA-coated plates. Absorbance values above 0.5 millioptical density (millioptical density) were considered positive.

Competition ELISA-different dilutions of hybridoma supernatants were incubated with hapten-BSA coated plates according to the procedure described earlier. Titration curves were plotted and the titer dilution (titer dilution) was determined at 50% binding. Supernatants diluted to titre were pre-incubated with soluble ZnTCPP, CoTCPP or Imisdp compounds for 30 minutes according to the procedure described previously, and transferred to hapten-coated microtiter plates. The estimated dissociation constant is the concentration of soluble hapten required to achieve 50% binding.

Preparation and purification-selected hybridomas were prepared on a large scale by subcloning limiting dilutions twice, followed by injection of pristine (2, 6, 10, 14-tetramethylpentadecane) pretreated ascites tumors into BALB/c mice. MAb was purified by protein G Sepharose (Sepharose)4 Fast Flow (Amersham Biosciences) affinity chromatography. Ascites fluid was centrifuged at 12,000g for 15 minutes to remove insoluble particles and lipids. 1mL of ascites was diluted to 5 volumes with PBS and added to 5mL of column volume of protein G agarose. The eluted peaks were analyzed by SDS-PAGE.

Isotype determination-culture supernatants obtained from cloned hybridomas grown in culture flasks were used as the mAb source. The isotype of each Antibody was determined by Mouse Monoclonal Antibody isotype assay Kit (HyCult biotechnology b.v., The Netherlands).

Immunoblot analysis of purified antibodies-purified antibodies were separated on an 8% SDS-polyacrylamide gel, transferred to NC membranes (Bio-Rad) and subsequently immunoblot analyzed using anti-MMP-9 antibody (Sigma). Goat anti-mouse IgG conjugated to horseradish peroxidase (Sigma) was used as the secondary antibody. Ecl (pierce) was applied to detect the signal.

Binding assay with purified protein-MAb (10 μ g) was incubated with anti-mouse IgG agarose microbeads (Sigma) in PBS overnight at 4 ℃. After washing the unbound antibody, after incubation for 2 hours at room temperature, purified Pro-MMP-2, Pro-MMP-9, MMP-2 catalytic domain, MT1 catalytic domain, or TACE (2 μ g) was added. The microbeads were collected by centrifugation and washed three times with PBS. Proteins that remain bound to the beads were eluted with SDS sample buffer, fractionated by SDS-PAGE, and then stained with Coomassie blue for detection.

Immunoprecipitation and western blotting-HT 1080 cells were plated into culture dishes. After reaching 80% confluence, the medium (DMEM supplemented with 10% FCS, non-essential amino acids, penicillin, streptomycin, sodium pyruvate and L-glutamine) was changed to serum-free medium (no FCS). After a further 24 h incubation, Conditioned Medium (CM) was harvested from adherent cells and concentrated using Millipore Centricon-10(Bedford, MA). The concentrated supernatant was used for immunoprecipitation. CM was incubated with anti-1 (CoTCPP) mAb (15. mu.g/ml) overnight at 4 ℃. Protein A agarose (CL-4B Amersham Biosciences) was added to the samples and mixed at room temperature for 2 hours. After washing the beads 3 times with PBS, they were suspended in SDS sample buffer and heated to 95 ℃ for 3 minutes. The immunoprecipitates were recovered by centrifugation and subjected to SDS/PAGE. After separation, the proteins were transferred to Nitrocellulose (NC) membranes and probed with anti-MMP-2 antibodies.

To activate ProMMP-2 produced by HT1080 cells, 1mM mercuric 4-aminophenylacetate (APMA) was added to the concentrated CM and incubated at 37 ℃ for 6 hours. After activation, CM was dialyzed against PBS (3 times) at 4 ℃ to remove APMA. Immunoprecipitation was performed with activation medium as described above.

Binding to active MMP-9 by direct ELISA-the MMP-9 catalytic domain (2. mu.g/ml) was immobilized in each well of the microtiter plate. mAb (1mg/ml) was added to each well according to the same method as described in ELISA screening. anti-MMP-9 antibody (Sigma) was used as a positive control, and affinity of non-relevant mouse IgG purified from ascites was used as a negative control.

Kinetic analysis-enzymatic MMP activity was measured as described previously [ Solomon, a. et al, expressed two-dimensional in electronic and chemical properties beta catalytic sites of tumor necrosis factor-alpha-converting enzyme and matrix metalloproteinases, although structural similarity was high, electrochemical properties were still significantly diverse between tumor necrosis factor-alpha-converting enzyme and matrix metalloproteinases catalytic zinc sites ] J Biol Chem, 2004.279 (30): pages 31646 to 54]. As described by Knight et al [ FEBS Lett, 1992.296 (3): pages 263 to 6]By applying at λ_ex340nm and λ_emFluorescent peptide Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH was monitored at 390nm₂(purchased from Calbiochem-Novabiochem AG) to determine the activity of MMP-9, MMP-2 and MT 1-MMP. The standard assay mixture contained 50mM Tris buffer (pH 7.5), 200mM NaCl, 5mM CaCl₂、20μM ZnCl₂And 0.05% bezoar. By monitoring the fluorescent peptide QF-45(Mca-Ser-Pro-Leu-Ala-Gln-Ala-Val-Arg-Ser-Ser-Ser-Arg-Lys (dinitrophenyl) -NH)₂) (purchased from Calbiochem-Novabiochem AG) to determine the enzymatic activity of TACE.

In situ zymography-in order to determine the location of the net gelatin-degrading activity of MMPs by in situ zymography, intramolecular quenched fluorescein isothiocyanate labeled DQ gelatin (molecular probes) was used as a substrate for degradation by gelatinases. Cleaved fluorescein isothiocyanate-gelatin peptide was obtained by proteolytic cleavage with gelatinase, and the fluorescence localized the site indicative of net gelatin decomposition activity. Briefly, human fibrosarcoma HT1080 cells, which produce MMP-2, MMP-9, and MT1-MMP, were mounted on 12mm coverslips. After 24 hours incubation, cells were treated with 1 μ M13E11 mAb for 30 minutes at 37 ℃. Untreated cells were used as a negative control for this experiment. After washing the cells with PBS, the cells were reacted with an enzymatic reaction buffer (0.05M Tris-HCl, 0.15M NaCl, 5mM CaCl) containing 60. mu.g/ml DQ gelatin₂And 0.2mM NaN₃(pH 7.6) incubation together at 37 ℃ overnight, high concentrations of azide prevent gelatin phagocytosis and thus allow cell surface gelatin lytic activity to occur). For the treated cells, the zymography buffer contained 1 μ M CoTCPP mAb. At the end of the incubation period, the position of the gelatinolytic activity of the MMP was determined without fixation or further washing and photographed with a fluorescence microscope and imaged by a Spot digital camera.

Example 1

Conformational simulation of zinc active sites by small organometallic compounds

The zinc ion of the MMP active site is uniformly coordinated by 3 conserved histidine residues. During zymogen activation and substrate proteolysis, the Zinc coordination changes from a 4-coordinating tetrahedral geometry in the non-catalytic phase to a 5-coordinating trigonal bipyramid in the catalytic phase [ Auld, d.s., Zinc coordination sphere in biochemical Zinc sites ], Biometals, 2001.14 (3-4): page 271-313 ]. Thus, the conserved histidines may be of different geometries relative to the zinc ion. To achieve these conformations, 2 compounds were selected as models simulating the Imisdp and Co/ZnTCPP zinc environments (FIG. 1). The Imisdp (synthesized as provided in example 7 below) compound can mimic the 4-coordinate geometry. In this case, a roughly tetrahedral conformation is constituted by 3 imidazole bases and water molecules as a fourth ligand.

FIG. 2A shows a superposition of the constructed Imisdp molecular 3D model with the MMP-9(PDB 1GKC) catalytic site [ Rowsell, S. et al, Crystal structure of human MMP9in complex with a reverse hydroxamate inhibitor (Crystal structure of human MMP9 complexed with a reverse hydroxamic acid inhibitor) ].J Mol Biol, 2002.319 (1): pages 173-81 ], which have been modified to represent the tetrahedral geometry of the zinc ligand. The modification consists in replacing the ligand present in the X-ray structure (hydroxamate inhibitor) with a water molecule and in optimizing the holoenzyme to a local minimum by means of a multi-layer QM/MM process (see materials and methods). A high similarity exists between the zinc histidine motif and Imisdp for MMP-9 calculated from the distances of the epsilon-nitrogen of histidine from the zinc ion (2.04. + -. 0.06 and 2.02, respectively) and the relative orientation of 3 histidines to the metal. The second molecule, Zn/CoTCPP, has 4 imidazolium bases [ Stevens, E.D., Electronic Structure of Metalloporphyrin.1. Experimental Electron Density Distribution of (meso-tetraphenylporphyrinato) cobalt (II) (Experimental Electron Density Distribution of the Electronic Structure of metalloporphyrin 1: (meta-tetraphenylporphyrin) cobalt (II) ], J.Am.chem.SOC, 1981.103 (17): 5087 page 5095 ]. This configuration mimics the conformation of 2 of the 3 histidines in a 5-coordinate trigonal bipyramidal geometry, where the metal is nearly coplanar with the 2 histidines that make up the pyramid base. FIG. 2B shows the crystal structure of MMP-9(PDB 1GKC) in which zinc is coordinated by 5 ligands (2 additional ligands are provided by hydroxamic acid inhibitors), the orientation of the 2 histidines of the pyramid base and their distance from the zinc ion (2.2. + -. 0.02, 2.03. + -. 0.04, and 1.95 for MMP-9, ZnTCPP, and CoTCPP, respectively) being comparable to that of the Co/ZnTCPP molecule.

Example 2

Monoclonal antibody production and screening

Monoclonal antibodies against CoTCPP, ZnTCPP and Imisdp were generated by selecting specific antibodies by ELISA screening using the corresponding compounds as coating antigens after immunizing mice (fig. 1). Three antibodies were selected for in-depth study. It should be noted that these clones were selected because they each showed the best affinity for their immunizing hapten based on competition ELISA screening. Their binding constants ranging from 0.01-0.09. mu.M (Table 3 below) are characteristic of high affinity mAbs. MAbs were propagated in mouse ascites and purified using protein G beads.

TABLE 3 isotypes and ELISA of anti-CoTCPP, ZnTCPP and Imisdp monoclonal antibodies

Competition analysis overview

Immune hapten antibody isotype Kd [ mu.M ]]^*Name of

CoTCPP IgG2b 0.09 13E11

ZnTCPP IgG2a 0.01 15E12

Imisdp IgG2a 0.09 6C6

^*Determination of the binding affinity (Kd) of an antibody for its immunological hapten by competition ELISA (see materials and methods for details).

Example 3

Monoclonal antibodies cross-react with MMP-2 and MMP-9

To determine whether mAbs generated against synthetic compounds that mimic the zinc histidine conformation of the MMP catalytic site cross-react with the exposed zinc histidine motifs in the MMP-2 and MMP-9 active sites, monoclonal antibodies that bind MMP-9 were first screened by direct ELISA.

3 mAbs that bind to the catalytic domain of MMP-9 were adsorbed directly to wells of a microtiter plate (commercially available anti-MMP-9 antibody was used as a positive control and non-related IgG was used as a negative control). Interestingly, mAbs propagated in ascites in mice were co-purified with active MMP-9 present in the mouse ascites. Western blot analysis of only the purified antibody with the anti-MMP-9 antibody as the first antibody showed a clear band, corresponding to the expected molecular weight of about 82KDa for active MMP-9. Thus, mabs form complexes with native enzymes in vivo.

Monoclonal antibodies that bind MMP-2 are then screened using an immunoaffinity-based assay. Antibodies were incubated with MMP-2 catalytic domain (MMP-2cat) in vitro and then precipitated out with anti-mouse IgG agarose microbeads. As shown in FIG. 3A, all mAbs bound to MMP-2 cat.

To determine that binding occurs throughout the direct interaction with the active site, the ability of the mAb to bind Pro-MMP-2 and Pro-MMP-9 was analyzed. Among the potential enzymes, the pro-domain structure protects the catalytic cleft (catalytic cleft). Thus, blocking the active site by the prodomain structure prevents mAb binding, provided that the prodomain structure recognizes the zinc histidine motif within the active site. Under the same conditions, no binding to the zymogen was detected (FIG. 3B). This pattern of binding to active MMP-2, but not to Pro-MMP-2, was further carefully studied in an in vivo environment similar to that of human fibrosarcoma (HT1080) cell cultures secreting full-length native MMP-2. Western blot analysis of HT1080 conditioned media following immunoprecipitation with anti-CoTCPP antibody showed binding to active MMP-2 but not to Pro-MMP-2 (FIG. 3C). These results indicate that all 3 antibodies cross-react with MMP-2 and MMP-9. Exposure of the active site cleft is essential for antibody binding, confirming that mabs interact directly with the active sites of MMP-2 and MMP-9.

Example 4

anti-CoTCPP and anti-Imisdp mAbs inhibit MMP-2 and MMP-9 in vitro

anti-Imisdp and anti-CoTCPP mabs inhibited proteolytic activity of MMP-2 and MMP-9 in the micromolar range (fig. 5). Kinetic analysis of mAb inhibition of MMPs was performed in a continuous fluorometric assay with quenched fluorescent peptide substrate. Unexpectedly, the anti-ZnTCPP mAb showed no inhibitory effect.

To determine the inhibitory properties by anti-CoTCPP mAb, the experiment was performed using enzymes with varying concentrations of fluorescent peptide substrate in the presence or absence of mAb. The data in the Lineweaver-Burk plots shown in FIGS. 4A-B are characteristic competitive inhibition curves with Ki values for MMP-9 and MMP-2 of 13 μ M and 24 μ M, respectively. Competitive inhibition curves indicate that the mAb binds to the same site as the peptide substrate. This inhibition pattern further confirms the direct interaction with the active site. Apparently, the anti-Imisdp mAb showed concentration-dependent inhibition of MMP-2 and MMP-9, showing competitive inhibition, with calculated Ki values for MMP-9 and MMP-2 of 5.8 μ M and 3 μ M, respectively (figure 5). Because mAbs recognize the binding sites for MMP-2 and MMP-9, optimization of the interfacial complementarity between mAb and MMP can be further achieved both structurally and electrostatically by affinity maturation (Paul J. Carter Nature Reviews Immun. Vol.6, 2006343-. In this way highly specific inhibitors can be obtained which will simultaneously exploit the specific characteristics inside or outside the active site.

Example 5

In situ zymography

To confirm the inhibitory activity of anti-CoTCPP mAb at the cellular level, the effect of the antibody on the gelatinolytic activity of human fibrosarcoma HT1080 cells constitutively secreting MMP-2 and MMP-9 was investigated by in situ zymography. To determine the location of the gelatin-degrading activity of MMPs by in situ zymography, intramolecular quenched fluorescein isothiocyanate labeled gelatin (DQ-gelatin) was used as a substrate. The cleavage of fluorescein isothiocyanate-gelatin peptide was obtained by proteolytic cleavage with gelatinase, and the localization of this fluorescence indicated the site of net gelatin decomposition activity.

Untreated human fibrosarcoma HT1080 cells (fig. 7A) had significant cell surface gelatin lytic activity. In the presence of 1 μ M mAb (fig. 7B), gelatinase activity was reduced compared to that observed in control cells. These results indicate that anti-CoTCPP mAb inhibits MMP-2 and MMP-9 at the cellular level.

Example 6

Selectivity of mAbs of the invention

Antibody selectivity was determined by studying the binding and inhibitory effects of anti-CoTCPP and anti-Imisdp mabs against MMP-14(MT 1mmP) and TNF- α -converting enzyme (TACE), a zinc-dependent metalloprotease (ADAM-17) belonging to the relevant ADAM (integrin and metalloprotease) family. Inhibition against MT1MMP and TACE was determined by in vitro luciferase activity assays with appropriate peptide substrates. The anti-CoTCPP mAb showed no inhibitory effect on MT1MMP or TACE. To determine whether it binds TACE and MT1MMP without a corresponding inhibition, an immunoaffinity-based experiment was performed with the purified enzyme and no binding was detected. In contrast to the anti-CoTCPP mAb, the anti-Imisdp mAb inhibited MT1MMP with a Ki value of 10 μ M, but showed no inhibitory effect on TACE. The results are shown in Table 4 below.

TABLE 4

MMP 6C6(IC₅₀μM) 13E11(IC50μM) 15E12(IC50μM)

MMP-2 3±0.2 24±1 NI

MMP-9 4.5±0.2 15±0.8 NI

MT1-MMP 14.4±0.7 NI NI

TACE NI NI NI

NI, not "inhibited" at concentrations up to 30. mu.M "

There is a high degree of structural similarity between members of the MMP family and TACE at the active site, specifically, the three-dimensional structural elements around the zinc binding site are nearly identical due to the need to accommodate the substrate peptide backbone and the presence of the conserved zinc-binding motif EXXHXXGXXH [ Solomon, A. et al, non-conserved in electronic and chemical properties between the catalytic sites of matrix metalloproteinases-alpha-converting enzyme and matrix metalloproteinases with significant diversity in their electrochemical properties despite the high structural similarity between tumor necrosis factor-alpha-converting enzyme and matrix metalloproteinases zinc site ], J Biol Chem, 2004.279 (30): pages 31646-54; lukacova, V.et al, A composition of the binding sites of matrix reactants and gum or fresh factor-alpha phd converting enzyme: interactions for selectivity (selectivity is involved in comparison of matrix metalloprotease and tumor necrosis factor- α convertase binding sites.) J Med Chem, 2005.48 (7): pages 2361-70 ]. Therefore, it is not expected that the inter-MMP mAb selectivity would be based solely on the recognition of the conserved histidine zinc motif. However, unlike small molecular weight synthesis inhibitors, antibodies that are large protein molecules must limit the accessibility to active site clefts embedded within the protein framework. In particular, because mabs have specific interactions with catalytic zinc ions, the extent of exposure of the zinc ions to solution is necessarily critical for antibody binding. MT1MMP is distinguished to a large extent from TACE by the deep S1 pocket (as shown by its crystal structure) associated with the entrapped catalytic zinc ion. This difference in active site depth may explain why antibodies lack inhibitory effects on TACE. These results indicate that selectivity can be achieved depending on the degree of exposure of the catalytic zinc ions. Another important factor that should be considered when comparing MMPs and TACEs is the difference in chemical properties of the active site pocket, such as hydrophobicity and polarity (see figure 8). For example, the active site of TACE is more polar than the active site of most MMPs. Solomon et al demonstrated that such changes in active site polarity directly affect the orientation of the active site histidine imidazole ring to the catalytic zinc ion [ Solomon, A. et al, expressed differential electrochemical and chemical properties between the catalytic zinc ions or new cross factor-alpha-converting enzyme and matrix metalloproteinases, while having high structural similarity between the tumor necrosis factor-alpha-converting enzyme and matrix metalloproteinases catalytic zinc site ] J Biol Chem, 2004.279 (30): pages 31646-54 ].

The selectivity against CoTCPP and against imispdp was further investigated carefully by determining the cross-reactivity of anti-CoTCPP and anti-imispdp with the non-related zinc dependent enzyme Carbonic Anhydrase (CA) and thermoanaerobium brucei (brockii) alcohol dehydrogenase (TbADH). Similar to MMPs, CA contains zinc ions tetrahedrally coordinated with 3 histidine residues and 1 water molecule, and TbADH contains catalytic zinc ions tetrahedrally coordinated with 4 different amino acid residues (histidine, cysteine, aspartic acid, and glutamic acid). Appropriate in vitro functional inhibition experiments, as well as similar immunoaffinity-based experiments, were performed to investigate cross-reactivity with these enzymes, however no binding or inhibition was observed. Cross-reactivity of anti-CoTCPP mAb with related physiological porphyrins (e.g., myoglobin and heme groups within hemoglobin and vitamins, etc.) was also determined. No cross-reactions were detected in the competition ELISA as well as in the immunoaffinity assay.

Both carbonic anhydrase and alcohol dehydrogenase have a significant degree of embedded active sites, and likewise the porphyrin portion of myoglobin and hemoglobin is not exposed. Vitamin B12 contains a metal in the center of the planar imidazole structure, but axial ligands may interfere with mAb binding. Taken together, these results demonstrate that the anti-CoTCPP mAb recognizes a relatively exposed metal-imidazole configuration without interference of axial metal-coordinating residues.

Example 7

-tris- [2- (N- (3-imidazol-1-yl-propane)

Yl)) -ethoxymethyl ] zinc (II) methanesulfonate, FIG. 9(3)

(i) Synthesis of tetrakis (2-pentachloro-phenoxycarbonyl-ethoxymethyl) methane: synthesis of pentachlorophenol-substituted tetra-active ester (pentachlorophenol-inhibition tetra-active ester) was carried out according to the method of HaimWeizmann et al (JACS 1996, 118, 12368-12375).

(a) Preparation of monosubstituted tri-active esters: the tetraactive ester (1) (1g, 0.69mmol) and BocNHCH were added₂CH₂NH₂(100mg, 0.62mmol) was dissolved in 20ml of anhydrous dichloromethane. The solution was stirred overnight while maintaining the pH-8 with triethylamine. The solution was concentrated and purified by flash chromatography (CHCl)₃Ethyl acetate (90: 10)), to give (152mg, yield 15%).¹H NMR 250MHz(CDCl₃)δ：1.4(s，9H，Boc)；2.4(t，2H，J＝6Hz，-CH₂-CH₂-CONH)；2.9(t，6H，J＝6Hz，-CH₂-CH₂-COOPCP)；3.2(q，2H，J＝6Hz，-CONH-CH₂-CH₂-NHBoc)；3.31(t，2H，J＝6Hz，-CONH-CH₂-CH₂-NHBoc)；3.38(s，2H，-C-CH₂-O-CH₂-CH₂-CONH-)；3.42(s，6H，-C-CH₂-O-CH₂-CH₂-COOPCP)；3.61(t，2H，J＝6Hz，-C-CH₂-O-CH₂-CH₂-CONH-)；3.78(t，6H，J＝6Hz，-C-CH₂-O-CH₂-CH₂-CONH-)；5.03(t，1H，NH)；6.7(t，1H，NH)。

(b) III (Mi)Oxazole) preparation: the mono-substituted tri-active ester (150mg, 0.11mmol) and 1- (3-aminopropyl) -imidazole (33. mu. lt, 0.39mmol) were dissolved in anhydrous THF and stirred at room temperature overnight. The white solution was concentrated and purified by column chromatography on silica gel (0.063-0.200mm) using CHCl₃Methanol (50-90%)) to yield (45mg, 44% yield).¹HNMR 250MHz(CDCl₃/MeOD)δ：1.45(s，9H，Boc)；2.0(m，6H，J＝6Hz，-CONH-CH₂-CH₂-CH₂-imi)；2.4(t，6H，J＝6Hz，-O-CH₂-CH₂-CONH-)；2.5(t，2H，J＝6Hz，-CH₂-CH₂-CONH-CH₂-CH₂-NHBoc)；3.0(m，8H，J＝6Hz，-CONH-CH₂-CH₂-CH₂-imi，-CH₂-CH₂-CONH-CH₂-CH₂-NHBoc)；3.1(t，2H，J＝6Hz，-CONH-CH₂-CH₂-NHBoc)；3.4(b，8H，-C-CH₂-O-CH₂-CH₂-CONH-CH₂-CH₂-NHBoc，-C-CH₂-O-CH₂-CH₂-CONH-)；3.6(m，8H，J＝6Hz，-C-CH₂-O-CH₂-CH₂-CONH-，-C-CH₂-O-CH₂-CH₂-CONH-CH₂-CH₂-NHBoc，)；4.0(t，6H，J＝6Hz，-CONH-CH₂-CH₂-CH₂-imi)；5.5(t，1H，NH)；6.98(s，3H，Imi)；7.06(s，3H，Imi)7.32(t，3H，NH)；7.57(s，3H，Imi)。ESI-MS：910.87[M+Na]⁺，925.98[M+K]⁺。

(c) Preparation of tris (imidazole) (2) with free amine: tris (imidazole) (40mg, 0.045mmol) was dissolved in 6ml of a mixture of dichloromethane and trifluoroacetic acid (2: 1) and stirred for 1 hour. The reaction mixture was concentrated, evaporated several times with carbon tetrachloride and dried under high vacuum to remove TFA from the mixture to give (30mg, yield 85%, b).¹H NMR 250MHz(CDCl₃/MeOD)δ：1.9(m，6H，J＝6Hz，-CONH-CH₂-CH₂-CH₂-imi)；2.3(m，8H，J＝6Hz，-O-CH₂-CH₂-CONH-，-CH₂-CH₂-CONH-CH₂-CH₂-NH₂)；2.9(t，2H，J＝6Hz，-CONH-CH₂-CH₂-CH₂-imi)；3.0(t，2H，J＝14Hz，-CONH-CH₂-CH₂-NH₂)；3.31(t，2H，J＝6Hz，-CH₂-CH₂-CONH-CH₂-CH₂-NH₂)；3.4(b，8H，-C-CH₂-O-CH₂-CH₂-CONH-CH₂-CH₂-NH₂，-C-CH₂-O-CH₂-CH₂-CONH-)；3.6(m，8H，J＝6Hz，-C-CH₂-O-CH₂-CH₂-CONH-，-C-CH₂-O-CH₂-CH₂-CONH-CH₂-CH₂-NH₂)；4.0(t，6H，J＝6Hz，-CONH-CH₂-CH₂-CH₂-imi)；7.26(s，3H，Imi)；7.32(s，3H，Imi)；8.82(s，3H，Imi)。

3. Preparation of tris (imidazole) -zn (ii) complex (3): compound 2(30mg, 0.038mmol) was dissolved in 1ml methanol. To this solution were added 2-3 drops of 1N NaOH solution and ZnCl₂(5mg, 0.04mmol) and stirred for half an hour. A white precipitate was obtained by filtration (12mg, yield 37%).¹H NMR 250MHz(MeOD/D₂O)δ：1.8(m，6H，J＝6Hz，-CONH-CH₂-CH₂-CH₂-imi)；2.4(m，8H，J＝6Hz，-O-CH₂-CH₂-CONH-，-CH₂-CH₂-CONH-CH₂-CH₂-NH₂)；3.0(t，2H，J＝6Hz，-CONH-CH₂-CH₂-CH₂-imi)；3.0(t，2H，J＝6Hz，-CONH-CH₂-CH₂-NH₂)；3.31(b，2H，-CH₂-CH₂-CONH-CH₂-CH₂-NH₂)；3.4(b，8H，-C-CH₂-O-CH₂-CH₂-CONH-CH₂-CH₂-NH₂，-C-CH₂-O-CH₂-CH₂-CONH-)；3.6(m，8H，-C-CH₂-O-CH₂-CH₂-CONH-，-C-CH₂-O-CH₂-CH₂-CONH-CH₂-CH₂-NH₂)；4.2(b，6H，-CONH-CH₂-CH₂-CH₂-imi)；7.19(s，3H，Imi)；7.28(s，3H，Imi)；8.55(s，3H，Imi).ESI-MS：852.09[M+1]⁺。

R＝O-CH₂-CH₂-CONH-CH₂-CH₂-NH₂

-tris- [2- (N- (3-imidazol-1-yl-propyl)) -ethoxymethyl ] methane.

Example 8

Cross-reaction of 6C6 with the catalytic site of gelatinase

It has now been found that a certain amount of 6C6 is co-purified with active MMP9 obtained from ascites fluid. Western blot and gelatin zymography (data not shown) showed that detectable amounts of MMP9 were present in ascites tumors, inducing proliferation of mouse mabs. MMP 9-antibody complex was purified from mouse ascites using protein G affinity chromatography (protein G bound to antibody constant regions, leaving free variable regions to interact with antigen). As shown in fig. 11A, co-purified MMP9 was detected using a commercially available anti-MMP 9 antibody, 6C6MMP9 complex purified by western blotting. The band with a molecular weight of-82 kDa corresponding to active MMP9 was identified to lack the pro domain. This band was not detected in the irrelevant mouse mAb control, which was purified and analyzed in the same manner. These results indicate that 6C6 forms a specific in vivo complex with endogenous active mouse MMP 9.

To further examine binding to the active form of the highly homologous MMP2 enzyme, a similar immunoprecipitation experiment was performed in vitro. 6C6 was incubated with purified MMP2 catalytic fragment in a 3: 1 molar ratio. SDS-PAGE analysis of the protein a agarose immunoprecipitates showed that 6C6 formed a specific complex with the catalytic fragment of active MMP2 (fig. 11B). Protein a microbeads alone do not immunoprecipitate MMP 2. Second, binding to inactive zymogen (potential enzyme) forms of MMP2 and MMP9 was determined. Since all MMPs are produced as inactive zymogens, they have an N-terminal propeptide of about 80-90 amino acids that blocks the active site [ Bode, w. and k. maskos, Biol Chem, 2003.384 (6): pages 863-72 (FIG. 11D). In the same manner, immunoprecipitation experiments were performed with pro MMP2 and pro MMP 9. Importantly, the antibody did not bind to the potential enzyme (fig. 11C). Remarkably, 6C6 only bound to the active enzyme conformation in which the active site zinc protein complex was exposed to solution.

These results demonstrate that 6C6 antibody generated and screened against a bio-inorganic chemical hapten mimicking the active site cross-reacts with the protein active sites of MMP2 and MMP 9. Clearly, the zinc tripod hapten is able to mimic the three-dimensional structure of the corresponding zinc-histidine epitope in the native protein. Clearly, recognition of this minimal metal-protein structural epitope is sufficient to cause cross-reactivity with the native enzyme. Binding to the activating enzyme only and not to their potential forms where the pro-domain blocks access to the catalytic zinc protein epitope (fig. 11D) suggests a direct interaction of 6C6 with the zinc catalytic site. It is noted that 6C6 binds native MMP9in vivo, demonstrating that antibodies can form specific complexes with enzymes in complex protein environments.

Distinguishing the class of activating enzymes from the potential forms is a unique and valuable functional property of 6C 6. This activity is unique to 6C6, unlike other antibodies raised against MMP 9. This is because immunization with proteins usually results in epitopes directed against surface loops, whereas the catalytic amino acids are mostly embedded inside the clefts on the enzyme surface. This component of the molecule is considered to be of low immunogenicity. Thus, neutralizing monoclonal antibodies (anti-native proteins or protein fragments) produced by conventional methods typically interact with adjacent or neighboring regions of the active site rather than with the active site catalytic residues and are inhibited by steric hindrance mechanisms. Such antibodies, in addition to binding to the active form, are usually also bound to inactive precursors. The unique active site mimetic hapten immunization approach of the present invention enables the generation of antibodies that recognize catalytic metalloprotein residues in MMPs, which are not available by conventional protein immunization approaches.

Example 9

6C6 selectively inhibits gelatinase in vitro and in situ

To determine the enzyme inhibitory ability of 6C6 against MMP9 and MMP2, inhibition experiments were performed using a small fluorescent peptide substrate (7 amino acids) spanning the gelatinase active site cleft. The initial reaction rates were determined for several concentrations of mAb. 6C6 inhibited the catalytic activity of both enzymes (FIGS. 12A-B). The competitive inhibition mechanism was determined by assaying MMP9 activity as a function of substrate concentration in the presence of varying concentrations of inhibitory antibody. Data in the form of a penultimate Linweaver-Burk plot shown in FIG. 12A, exhibited competitive inhibition curves. Fitting the inhibition data to the competitive inhibition system equation yields Ki of 1 ± 0.1 μ M and 1.4 ± 0.16 μ M for MMP9 and MMP2, respectively. It was also determined that 6C6 was not cleaved by MMP-9 after overnight incubation with high concentrations (30 μ M) of MMP9, indicating that the observed inhibition of MMP9 by 6C6 was not due to cleavage of the competitor substrate. Kinetic analysis of MMP9 was considered a representative 6C6 inhibition mechanism, as it was designed to recognize the same epitope in different MMPs. Inhibition was consistent for both the catalytic fragment classes of MMP2 and MMP9, as well as the full-length enzyme form of gelatinase. Specifically, recombinant MMPs 9 and MMP2 catalytic fragments containing a catalytic domain and a fibronectin domain but no hemopexin domain; and MMP9 recombinant minimal catalytic unit, which contained only a catalytic domain and lacked both fibronectin and hemopexin domains, inhibited full-length (mercury-p-aminobenzoacetate (APMA)) gelatinase as well, gelatinase was purified from the culture medium of HeLa S3 cells infected with recombinant vaccinia virus encoding the full-length cdnas of human proMMP2 and pro MMP 9[ Olson, m.w., et al, J Biol Chem, 2000.275 (4): pages 2661-8 ]. These results demonstrate that inhibition is mediated by direct interaction with the catalytic domain, independent of interaction with the hemopexin or fibronectin domain. Competitive inhibition curves provide yet another basis for direct interaction with catalytic zinc sites. Non-related mabs prepared in the same manner do not interfere with the photolytic activity of the enzyme. Thus, the observed inhibition was not due to traces of co-purified contaminants. The antibodies of these experiments were purified from tissue culture and contained no detectable amounts of active MMP9in the purified antibody fraction.

To examine the selectivity of 6C6, its reactivity was determined against different subsets of matrix metalloproteinases, including stromelysin (MMP7), membrane-type MMP (MT1MMP) and related disintegrins (ADAM), tumor necrosis factor- α -converting enzyme (TACE). The core structures of these enzymes are very similar, with changes mostly occurring within peripheral loops. The specific zinc-histidine scaffold is well conserved, showing that one common helix is followed by a loop that serves as the scaffold for the 3 histidine residues that coordinate to the catalytic zinc ion (fig. 13).

Similar inhibition assays were performed with appropriate fluorescent peptide substrates. Interestingly, neither MMP-7 nor TACE was inhibited to any measurable extent upon incubation with 6C6 at concentrations as high as 30 μ M, indicating a substantial level of selectivity for gelatinase. MT1MMP was inhibited by 6C6 with lower potency, Ki of 14.4. + -. 0.75. mu.M. Interestingly, the origin of this selectivity cannot be explained solely by the design of antibodies recognizing the conserved zinc-histidine scaffold, since the core structure, especially the active sites, are very similar. Sequence changes mostly within the peripheral loop dictate the degree of exposure of the zinc-histidine motif, the shape of the active site and the differences in its surface electrostatics, which may explain this selective inhibition pattern.

The cross-reactivity of 6C6 with different zinc-dependent metalloproteinases, namely carbonic anhydrase and alcohol dehydrogenase, was also determined. Like MMPs, Carbonic Anhydrase (CA) has a catalytic zinc ion tetrahedrally coordinated with 3 histidine ligands and 1 water molecule. Thus, several potent small molecule MMP inhibitors (of the sulfonylated amino acid hydroxamic acid type)MMP inhibitors) may also be used as potent CA inhibitors, and vice versa. Some N-hydroxysulfonamides previously studied as CA inhibitors also have inhibitory properties against MMPs [ Scozzafava, a. and c.t.supuran, J Med Chem, 2000.43 (20): pages 3677-87]. The active site from thermophilic bacteria (TbADH) alcohol dehydrogenase includes various zinc-protein moieties in which zinc binds to histidine, cysteine, aspartic acid and glutamic acid within the cleft. In the presence of mAb at concentrations up to 30. mu.M, appropriate functional inhibition experiments showed no inhibition against both enzymes. It is noted that the active site of CA, located in the central region of the 10-strand, twisted beta sheet, is composed of tapered clefts, deepWherein the tetrahedron Zn²⁺The ions are located at the bottom of the crevice. Unlike small molecule inhibitors, zinc ions must be embedded so deeply that they do not interact with the antibody. Importantly, these experiments further exhibited a selective inhibition curve of 6C 6.

The inhibitory effect of 6C6 against gelatinase was further determined by in situ zymography in the cellular environment using the natural substrate of gelatinase, gelatin. Human fibrosarcoma HT1080 cells expressing membrane-bound MT1MMP and secreting MMP-2 and MMP-9 grown in culture medium [ Giambernardi, t.a. et al, Matrix Biol, 1998.16 (8): pages 483-96 ] are covered with fluorescein-conjugated gelatin (DQ gelatin). As shown in fig. 14A-C, untreated HT1080 cells had significant cell surface gelatin lytic activity. Treatment with 5 μ M mAb significantly reduced surface gelatinolytic activity, similar to that observed for the mechanism-based gelatinase inhibitor, i.e., SB-3 CT. SB-3CT has a similar inhibition curve because it inhibits both gelatinase and MT1MMP (Ki values of 28nM, 400nM and 110nM for MMP2, MMP9 and MT1MMP, respectively). In summary, 6C6 inhibited both synthetic peptide cleavage in vitro and natural macromolecular substrates in situ. 6C6 showed a competitive inhibition pattern against MMP9, similar to the inhibited TIMP mechanism. The competitive inhibition curve is yet another basis for direct interaction with the catalytic zinc moiety. Importantly, 6C6 shows a selective inhibition curve for gelatinase. The origin of this selectivity cannot be explained by antibody targeting of the conserved zinc-histidine motif. These results indicate that the antibodies

Interaction with additional enzyme surface determinants may explain the observed specificity.

Example 10

Effect of 6C6MAb treatment on DSS-induced colitis in mice

There is increasing evidence that MMPs are involved in tissue remodeling and destruction associated with several inflammatory diseases, including Inflammatory Bowel Disease (IBD) [ Baugh, m.d. et al, Gastroenterology, 1999.117 (4): pages 814-22; heuschkel, r.b. et al, Gut, 2000.47 (1): pages 57-62; von Lampe, b. et al, Gut, 2000.47 (1): pages 63-73; kirkegaard, t, et al, Gut, 2004.53 (5): pages 701-9 ].

Thus, the inventors of the present invention investigated the in vivo anti-gelatinase inhibitory effect of 6C6 in a mouse experimental model of inflammatory bowel disease.

To probe the inhibitory activity of 6C6, the ability of mAb treatment to ameliorate DSS-induced acute colitis was investigated. Specifically, the highly susceptible mouse strain C57BL/6 was provided with 2% DSS for 5 days. Treatment with 6C6 was given daily from the day of induction by intraperitoneal injection, 1.5 or 5mg/kg mice. Mice exposed to 2% DSS developed symptoms of acute colitis with diarrhea, rectal bleeding and severe weight loss.

The effect of mAb treatment on daily monitoring of Disease Activity Index (DAI) (combined scores of body weight, bleeding and stool consistency) is shown in figure 15A. MAb treated mice had reduced disease activity (evident from day 6) compared to controls. Another macroscopic manifestation of DSS-induced colitis is a shortening of colon length (fig. 15B). Thus, a 30% reduction in colon length was found in untreated mice 11 days after DSS induction compared to naive mice. In contrast, 6C 6-treated mice given 1.5 and 5mg/kg mice, respectively, were only shortened by an average of 22% or 16%. The protective effect of 6C6 was also confirmed by the mortality rate of the disease. The mortality rate was found to be 60% in untreated mice 11 days after disease induction, while only 33% was observed in 6C6 treated mice (fig. 15C). Thus, treatment of C57BL/6 mice with 6C6 resulted in improved survival in addition to alleviating the symptoms of DSS-induced colitis.

Overall, these results indicate the therapeutic potential of 6C6 as a gelatinase inhibitor.

Example 11

Characterization of MMP9-6C6mAb complexes by X-ray absorption spectroscopy

To further investigate the difference between the active MMP9 and the inhibited MMP9-6C6 complex, X-ray absorption spectroscopy was performed. Fig. 16 shows acquired fluorescent XAS data. The data is presented in the form of Fourier Transform (FT) spectra to provide radial distribution of individual atoms within the first and second coordination shells in the catalytic zinc ion of MMP 9. Apparent changes in the radial distribution spectra of free and inhibited enzymes can be observed above the noise level. These spectral changes indicate that the local environment of the catalytic zinc ion undergoes a structural change when combined with 6C 6. The differences in both spatial distribution and peak intensity of FT spectral features observed between active and inhibited enzymes clearly indicate that the local structure of the catalytic zinc is altered upon mAb complex formation.

It is to be understood that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination.

While the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, the present invention is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims. All publications, patents, and patent applications mentioned in this specification are herein incorporated in their entirety by reference into the specification, to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention.

List of references

(additional references are incorporated herein by reference)

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Claims (17)

1. A compound having the following general formula (I):

wherein:

m and n are each independently an integer from 1 to 6;

X₁-X₃and Y₁-Y₃Each independently is O or S;

R₁-R₃each independently selected from hydrogen, alkyl, and cycloalkyl; and

r is (CH)₂)x-C(＝O)NR′-(CH₂)y-NR′R″

Wherein:

x and y are each independently an integer from 1 to 6; and

r 'and R' are each independently selected from hydrogen, alkyl, and cycloalkyl.

2. The compound of claim 1, having the following formula (II):

wherein R is-CH₂-C(＝O)NH-CH₂-CH₂-NH₂。

3. A compound having the following formula (II):

wherein R is-CH₂-C(＝O)NH-CH₂-CH₂-NH₂。

4. An antibody comprising an antigen recognition region capable of specifically binding to a compound of claim 1, 2 or 3.

5. An antibody comprising an antigen recognition region comprising the amino acid sequence of SEQ ID NO: 7. 8, 9, 10, 11 and 12.

6. The antibody of claim 5, wherein the CDR amino acid sequences consist of amino acid sequences selected from the group consisting of SEQ ID NOs: 13. 14, 15, 16, 17 and 18.

7. The antibody of claim 4, which is capable of inhibiting the activity of a metalloprotein.

8. The antibody of claim 7, wherein the metalloprotein is a matrix metalloproteinase.

9. The antibody of claim 7, wherein the matrix metalloproteinase is gelatinase.

10. The antibody of claim 9, wherein the gelatinase is selected from the group consisting of MMP-2 and MMP-9.

11. A method of producing a metalloprotein inhibitor, the method comprising preparing an antibody against a compound of claim 1, 2 or 3, thereby producing the metalloprotein inhibitor.

12. The method of claim 11, wherein the antibody is a polyclonal antibody.

13. The method of claim 11, wherein the antibody is a monoclonal antibody.

14. A pharmaceutical composition comprising the antibody of claim 4 and a pharmaceutically acceptable carrier.

15. A method of treating a disease associated with a deregulated or abnormal activity of a metalloprotein in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the antibody of any one of claims 4-10, thereby treating the disease associated with the deregulated or abnormal activity of the metalloprotein in the subject.

16. The method of claim 15, wherein the disease is inflammatory bowel disease.

17. A method of inhibiting matrix metalloproteinase activity of a cell, the method comprising contacting a cell with the antibody of any one of claims 4-10, thereby inhibiting matrix metalloproteinase activity of the cell.