EP1644031A1 - Inhibitors of the leukocyte prommp-9/beta(2) integrin complex - Google Patents

Abstract

The present invention concerns peptide compounds, which are inhibitors of integrin-MMP complex. The compounds comprise the peptide motif, D/E-/D/E-G/L-W, and they specifically bind to the αM integrin I domain and inhibit its complex formation with proMMP-9. The compounds thus prevent neutrophil migration, as well as leukocyte migration. The compounds can be used in the treatment of inflammatory conditions and leukaemia.

Description

Inhibitors of the leukocyte proMMP-9/beta(2) integrin complex

Field of the Invention

The present invention concerns peptide compounds, which are inhibitors of integrin- MMP complex. In specific, the compounds bind to the CC integrin I domain and inhibit its complex formation with proMMP-9. The compounds thus prevent neutrophil migration, as well as leukocyte migration. The compounds can be used in the treatment of inflammatory conditions, and leukaemia.

Background of the Invention

The leukocyte integrin family consists of four heterodimeric glycoproteins with specific α-chains (OCL, OC_M, ocχ, or OC_D) and a common β₂-chain (CD 18). They play an essential role in mediating adhesion of cells in the immune system (1). The major ligand-binding site locates to an 200 amino acid long sequence within the α-chain called I or inserted domain, which is homologous to the A domains of von Willebrand factor, repeats of cartilage matrix protein and collagen (2).

Among the β₂ integrins, ( _Mβ₂ s the most promiscuous binder being able to interact with a multitude of unrelated ligands. These include ICAM 1 to 5, complement fragment iC3b, fibrinogen, uPAR, E-selectin and various extracellular matrix proteins (see (3) and references therein). The integrin has also been shown to have a capacity to bind certain enzymes, but whether this is important for leukocyte adhesion or immune reactions is unclear. Such enzymes showing integrin-binding activity are catalase (4), myeloperoxidase (5) and the proteinases elastase (6) and urokinase (7).

Extensive work has been done to identify the ligand binding sites in β₂ integrin I- domains, but less is known about the interacting ligand regions (8, 9). Recently, the structure of an α I domain/ICAM-1 complex was reported (10). Low molecular weight peptides binding to the β₂ integrins are useful reagents to study integrin function, and such peptides have been derived from ICAM-2 (11), fibrinogen (12), and Cyrόl (13). We have used phage display libraries to study the peptide binding specificity of integrins and to develop potential drug leads. In our previous study, we isolated the biscyclic peptide CPCFLLGCC (LLG-C4)(unpublished observations of the present inventors) as the most active binder to the purified 0C_Mβ2 integrin (14). Leukocytes can efficiently adhere to the immobilized LLG-C4 peptide via the oc_Mβ₂ and αχβ integrins.

Summary of the Invention

We have now extended phage display screenings to the purified CC_M I domain. This has resulted in the identification of a novel I domain-binding tetrapeptide motif D/E-D/E- G/L-W, which is found on some of the known β integrin ligands and interestingly also on the catalytic domain of MMPs. We show that the D/E-D/E-G/L-W motif mediates binding between an MMP and β integrin, and proMMP-9 gelatinase, which is known to be the major MMP of leukocytes, occurs in complex with the α_Mβ₂ and αιβ₂ integrins in leukaemic cell lines following cellular activation. The peptide inhibitors of the integrin-MMP complex prevent leukaemia cell migration, suggesting a role for the complex in cell motility. The compounds of the invention also attenuated PMN migration in vitro and in vivo, suggesting a role for the MMP-integrin complex in PMN motility.

Detailed description of the Invention

Analysis of < _M I domain-binding peptides led to the finding that MMPs, particularly the MMP-9 a nd M MP-2 p rogelatinases, a re p otent β ₂ i ntegrin 1 igands. O ur s tudies s how that proMMP-9, the major MMP of activated leukocytes, is co-localized with the β ₂ integrin on the cell surface. Cell surface labelling and co-immunoprecipitation further demonstrates the occurrence of the complex in 1 eukemic cell lines. Finally, we have found e vidence t hat t his p roteinase-integrin c omplex p lays a r ole i n m igration o f t he leukemic cells.

Although phage display has been extensively used with whole integrins, to the best of our knowledge, this is the first successful phage display selection on an isolated integrin I domain. We could enrich only one binding motif even though the OC_M I domain can bind a variety of ligands. The peptide motif we isolated could not compete with the ICAM-1, fibrinogen, or LLG-C4 ligands. The success of phage display depends on the libraries used and the bioparming conditions. Our method favoured "high affinity" interactions with the cyclic peptides yielding the D/E-D/E-G/L-W motif. Interestingly, this motif shows a high degree of similarity to the CWDD(G/L)WLC peptide isolated by phage display as an RGD sequence-binding peptide (15). By recognizing the RGD ligand sequence, CWDDGWLC structurally and functionally behaves like a minimal integrin. Here, we have identified the DDGW peptide in a reverse situation, as a ligand to integrin. However, the RGD sequence does not compete with the OCM I domain as the GRGDSP peptide at a 1 mM concentration was unable to inhibit proMMP-9 binding to the I domain (unpublished observations of the present inventors).

Some hints for the binding site of DDGW come from the interaction of iC3b with α_Mβ₂ integrin. Our pepspot analysis showed that the iC3b peptide ARSNLDEDIIAEENI, but not the control peptide ARSNLDAAIIAEENI, bound the OC I domain and the DDGW peptide blocked this binding. The DEDIIAEENI sequence with multiple adjacent negative charges is required for efficient binding of iC3b to α_Mβ₂ integrin. The binding site of complement protein iC3b in the I domain has been mapped indicating a role for the positively charged amino acid residue K²⁴⁵ for iC3b binding. Mutation of this residue does not affect the binding of the fibrinogen recognition peptide. This may account for the inability of the DDGW peptide to inhibit ICAM-1 and fibrinogen- mediated cell adhesion. These findings suggest that the K²⁴⁵ residue is the positively charged contact site for the DDGW peptide and the D/E-D/E-G/L-W motif as well.

The pepspot analysis indicates that a class of β₂ integrin ligands contains an active D/E- D/E-G/L-W motif. These include the previously identified α_Mβ₂ ligands iC3b, thrombospondin-1, and the enzymes myeloperoxidase and catalase. In our experiments, the peptides derived from several secreted MMPs, but not membrane-bound MT1- MMP, were also active. It is notable that the D/E-D/E-G/L-W motif is relatively conserved in the secreted members of the MMP family.

Finding of a dominant integrin-binding site in the catalytic domain of proMMP-9 was unexpected, because previous studies suggested an essential role for another MMP domain, the hemopexin domain, in integrin binding. The hemopexin domains mediated MMP-2 binding to the vβ₃ integrin and MMP-1 binding to the α₂βi integrin. The cleaved hemopexin domain of MMP-2 has also been shown to occur in vivo and to inhibit angiogenesis. Understandably, phage peptide display and pepspot techniques have limitations and only linear peptide sequences can be analysed, not protein conformations. Thus, in the present study we cannot make conclusions of the function of separate MMP-9 domains in integrin binding and it remains to be seen whether cleavage products of MMP-9, if present in vivo, can act as β₂ integrin ligands. Our studies suggest that the peptide sequence from the catalytic domain is essential for the binding o f full-length p roMMP-91 o t he β ₂ i ntegrin, a s t he s ynthetic D DGW p eptide could completely inhibit the integrin binding. It was important to use natural proMMP-9 because dMβ₂ is known to bind to denaturated proteins and this may sometimes be the case for bacterially expressed proteins. Furthermore, we did not observe binding of an active MMP-2 or MMP-9 to the integrin, although the DELW(T/S)LG sequence should remain unchanged in the active enzyme. We rather found that AMPA and trypsin, activators of proMMP-9, released MMP-9 from THP-1 cells, apparently affecting the integrin complex (unpublished observations of the present inventors). These results suggest that the proenzyme presents the integrin binding site more efficiently than the active enzyme and the β₂ integrin may even control the activation of the proenzyme.

In the three-dimensional structures of proMMP-2 and -9, the I domain binding site is located in the vicinity of the zinc-binding catalytic sequence HEFGHALGLDH between the catalytic domain and the fibronectin type II repeats. This location suggests a mechanism for evading proMMP-9 inhibition by tissue inhibitors of MMPs (TIMPs) or α -macroglobulin. In the absence of inhibitors, the cell surface-localized proMMP-9 would be readily susceptible for activation and substrate hydrolysis, which may also occur in the presence of intact propeptide. On the other hand, because the binding site of the I domain is located in the vicinity of the catalytic groove, it also suggests an explanation for the blocking of MMP-9/β₂ integrin interaction by the small molecule MMP inhibitors such as CTT and frihl.

The activity of the DDGW peptide in the THP-1 cell migration assay suggests an important function for the integrin-progelatinase complex in leukocyte migration. Obviously, we cannot exclude the possibility that the DDGW peptide blocks binding of other ligands than gelatinases and in this way inhibits the leukocyte migration. However, as the specific gelatinase inhibitor CTT also blocks the THP-1 cell migration, these results strongly suggest that the proMMP-9/β₂ integrin complex is the main target for DDGW. Interestingly, the DDGW peptide blocked THP-1 cell migration although it increased the level of proMMP-9 in the medium, which suggests that cell-surface bound rather than total MMP-9 level is a critical factor in cell migration.

DDGW and HFDDDE (see below) had potent activities in vivo in the mouse peritonitis model, but it is unclear to what extent this was due to inhibition of proMMP-9 as both peptides can potentially inhibit other β₂ integrin ligands as well. A subset of β₂ integrin ligands have a DDGW-like sequence and these include, in addition to MMPs, at least complement iC3b and thrombospondin-1. The excellent in vivo activity of DDGW makes it a useful tool to study the components involved in leukocyte migration and the peptide may be considered as a lead to develop anti-inflammatory compounds. Our results suggest that the proMMP-9/oiMβ₂ complex may be part of the neutrophil's machinery for a specific β₂ integrin-directed movement.

The present invention is thus directed to new peptide compounds, in specific to peptide compounds comprising the tetrapeptide motif D/E-D/E-G/L-W. Said compounds can be used as pharmaceuticals, which inhibit leukocyte migration, as well as neutrophil migration. The inhibitory activity was shown both in in vitro and in vivo experiments.

Consequently, the compounds can be used to treat leukaemia and prevent and treat inflammatory conditions.

One embodiment of the invention is the use of the compounds of the invention for the manufacture of a pharmaceutical composition for prophylaxis and treatment of conditions dependent on neutrophil migration.

Another embodiment of the invention is the use of the compounds of the invention for the manufacture of a pharmaceutical composition for the treatment of conditions dependent on leukocyte migration.

A further embodiment of the invention is a pharmaceutical composition comprising as an active ingredient a compound of the invention, and a pharmaceutically acceptable carrier.

A still further embodiment of the invention is a method for therapeutic or prophylactic treatment of conditions dependent on leukocyte or neutrophil migration, comprising administering to a mammal in need of such treatment a leukocyte or neutrophil migration inhibiting compound of the invention in an amount which is effective in inhibiting migration of leukocytes or neutrophils. Specific embodiments of the invention include methods for treatment of leukaemia and inflammatory conditions.

Abbreviations used

APMA, aminophenylmercuric acetate; α_Mβ₂, CD lib/CD 18, Mac-1 integrin; CTT, CTTHWGFTLC peptide;

DDGW, ADGACILWMDDGWCGAAG peptide;

GPA, glycolphorin A;

GST, glutathione-S-transferase;

HMEC, human microvascular endothelial cell; ICAM, intercellular adhesion molecule;

Inhl, matrix metalloproteinase inhibitor 1;

KKGW, ADGACILWMKKGWCGAAG peptide;

LLG-C4, CPCFLLGCC peptide;

MMP, matrix metalloproteinase; NGAL, neutrophil gelatinase-associated lipocalin;

PMN, polymorphonuclear neutrophil;

RGD-C4, ACDCRGDCFCG peptide;

STT, STTHWGFTLC peptide;

TAT-2: tumor-associated trypsinogen-2; W→A CTT, CTTHAGFTLC peptide.

Description of the Drawings

FIG. 1A to IE. Identification of an I domain binding site in progelatinase. FIG. 1A, Phage display peptide sequences specifically bound to the αM I domain. The consensus motif is shown in bold. Peptides with the strongest binding (CILWMDDGW) and the highest similarity (CPEELWWLC) are aligned with human MMPs (accession numbers shown in parenthesis).

FIG. IB, Phages bearing the CILWMDDGWC peptide or a control peptide were allowed to bind to immobilized CI_M I domain-GST fusion protein (20 ng/well) in the absence or presence of 15 μM DDGW peptide or LLG-C4 peptide. Bound phages were detected using a monoclonal anti-M13 phage antibody. Mean absorbance of triplicate samples ± SD is shown.

FIG. IC, α , _M, or αx I domain-GST fusions were coated on microtiter wells as in B, and the binding of CILWMDDGWC peptide bearing phage or a control phage was measured.

FIG. ID, Peptides covering the complete sequence of proMMP-9 were synthesized as overlapping peptides on a pepspot membrane. The αM I domain (0.5 μg/ml) was allowed to bind to the peptides followed by immunodetection using anti-α_M I domain antibody LM2/1. The α_M I domain-binding peptide 13 (arrow) is shown in boldface and the zinc binding catalytic sequence is underlined. The prodomain (Pro), catalytic domain containing the fibronectin type II repeats (Cat) and hemopexin domain (Pex) are marked to illustrate the domain structure of proMMP-9. FIG. IE, Alanine-mutated and truncated peptides were synthesized on a pepspot filter and probed with the recombinant α_M I domain (5 μg/ml). Bound I domain was measured using mAb MEM- 170 (5 μg/ml) followed by HRP-conjugated anti-mouse secondary antibody and ECL detection. The binding was quantified by densitometric scanning. The bars show α_M I domain binding to single peptide spots as arbitrary optical density units/mm². Similar results were obtained in three independent experiments.

FIG. 2A to 2C. Binding of progelatinases to purified integrins and I domains. FIG. 2A, proMMP-9, proMMP-2 or their trypsin-activated forms, at the concentrations indicated, were allowed to bind to α_Mβ₂ integrin-coated wells. Appropriate MMP antibodies were used to determine binding. The results in this and other figures are represented as the means ± SD from triplicate wells.

FIG. 2B, binding of proMMP-9 (80 ng/well) was examined on microtiter wells coated with an integrin (αιβ2, 0 Mβ₂, αiβi, α₃β or an I domain (α_L, αM, αx). The binding was determined using anti-MMP-9 antibody. FIG. 2C, proMMP-2 (80 ng/well) was allowed to bind to α_Lβ2, 0CMβ2, αiβi or the I domains α_L, α_M or αx. The binding was determined using an anti-MMP-2 antibody.

FIG. 3A to 3D. Inhibitors of the proMMP-9 / β₂ integrin complex.

FIG. 3A, α_M and α I domain-GST fusions were immobilized on microtiter wells. ProMMP-9 (100 ng/well) was added in the presence or absence of various peptides (200 μM) or lovastatin (100 μM) in a buffer containing 0.5% BSA. ProMMP-9 binding was detected using a monoclonal antibody against MMP-9. The results are shown as percent binding c ompared t o b inding i n t he absence o f i nhibitors ( 100%) a nd no p roMMP-9 added (0%). FIG. 3B, DDGW peptide blocks proMMP-9 binding to α_M in a dose dependent manner.

The assay was done similarly as in (A), except various concentrations of peptides were added to compete for binding. All samples were assayed as triplicates and results shown are means ± SD from a representative experiment.

FIG. 3C, proMMP-9 binding to α_Mβ₂ and αLβ2 was examined in the absence and presence of EDTA (5 mM), MMP inhibitor-1 (100 μM), CTT (200 μM), STT (200 μM), MEM170 (40 μg/ml), or control TL3 antibody (40 μg/ml). The background of primary and secondary antibodies was measured by omitting proMMP-9 from the wells or by coating with ICAM-1.

FIG. 3D, proMMP-9 binding to purified α_M and α_L I domain GST fusion proteins or wild type GST was studied in the absence or presence of competitors as indicated.

Control shows the background when proMMP-9 was omitted.

FIG. 4A and 4B. Coprecipitation of progelatinase with β₂ integrin.

FIG. 4A, α_Mβ₂ integrin (3 μg) was incubated with a 500 μl sample of HT1080 medium containing proMMP-9 and proMMP-2 in the absence or presence of CTT or STT (200 μM) for 2 h. The integrin was immunoprecipitated with the OKM10 antibody, and the immunoprecipitates were analyzed by gelatin zymography. In control experiments, integrin was omitted from the medium and ICAM-1 was added instead.

FIG. 4B, a 500 μl sample of HT1080 medium containing proMMP-9 and proMMP-2 was incubated with the α_M I domain GST (3μg) or LLG-C4-GST control. ICAM-1,

LM2/1, CTT, STT, or LLG-C4 were used as competitors. GST was pulled down with glutathione beads, and bound proteins were analyzed by zymography. The lane 1 in the figure insert: the proMMP-2 and proMMP-9 zymogens present in non-treated HT1080 medium, lane 2: lack of gelatinases pulled down with control LLG-C4-GST, lane 3: proMMP-9 and proMMP-2 coprecipitated by α_M I domain GST fusion protein.

FIG. 5A and 5B. CTT peptide binds to both latent and active MMP-9.

FIG. 5A, Binding of proMMP-9 or APMA-activated MMP-9 to CTT-GST was examined in the absence or presence of competitors CTT (100 μM), W→A mutant CTT (100 μM), and Inhl (100 μM). GST control was LLG-C4-GST. Binding was determined as in Figs. 2 and 3. The background in the absence of proMMP-9 is shown. FIG. 5B, THP-1 cells were incubated in serum-free medium containing CTT, Inhl or W→A CTT at 200 μM concentration. Samples from the media were collected at the time points indicated and analyzed by zymography (panels 1, 3, and 4) or Western blotting (panel 2).

FIG. 6 A to 6 C. Progelatinases o ccur i n c omplex w ith α_M β₂ a nd α_L β₂ i n P DBu- activated THP-1 and Jurkat cells. FIG. 6A, THP-1 cell surface proteins were [³H]-labelled using periodate-tritiated borohydride and analyzed by immunoprecipitation. CTT was used as a competitor (200 μM). The immunoprecipitated samples were resolved on a 8-16% polyacrylamide gel, and the film was exposed for 3 days. Lanes 1-4 are from non-activated cells and lanes 6- 10 from PDBu-activated cells. Lane 5 shows molecular weight markers. FIG. 6B, lysates from PDBu-activated THP-1 cells were immunoprecipitated with integrin or MMP antibodies followed by Western blotting with α_M (OKM10), α_L (TS2/4) or MMP-9 antibodies. Preclearings of the cell lysates were done using α_M (lane 6) and α_L (lane 7) antibodies. FIG. 6C, lysates from PDBu-activated Jurkat cells were subjected to immuno- precipitation followed by blotting with the α_L (MEM83) and MMP-9 antibodies.

FIG. 7A and 7B. PDBu-induced colocalization of α_Mβ2 and proMMP-9 in THP-1 cells. Cells were preincubated for 30 min at +37°C with 50 nM PDBu.

FIG. 7A, cells were treated with anti-αM OKM10 and anti-MMP-9 antibodies followed by FITC-labeled (green fluorescence) and TRITC-labeled (red fluorescence) secondary antibodies. Yellow color indicates colocalization of α_Mβ₂ and proMMP-9. Bars, 8.5 μm. FIG. 7B, immunofluorescence staining shows intense colocalization of MMP-9 (polyclonal antibody) and αMβ2 integrin (OKM-10) on the surface of PDBu-activated THP-1 c ells at higher magnification as visualized by e onfocal microscopy (Bars, 2.5 μm). FIG. 8 A t o 8 D. The D DGW p eptide s upports T HP-1 c ell a dhesion a nd i nduces proMMP-9 release, but does not block adhesion to the major β₂ integrin ligands fibrinogen and ICAM-1.

FIG. 8 A, THP-1 cells were allowed to bind to immobilized, glutaraldehyde polymerized peptides with or without phorbol ester activation (50 nM) and the adherent cells were quantitated by phosphatase assay. THP-1 cells were allowed to bind to immobilized fibrinogen (in FIG. 8B), or recombinant ICAM-1 -Fc (in FIG. 8C), in the presence or absence of 200 μM soluble peptides. All samples were assayed as triplicates and results show means ± SD. Identical results were obtained in two other independent experiments.

FIG. 8D, THP-1 cells were incubated in the presence or absence of peptides at 200 μM concentration for 48 hours. Aliquots of conditioned medium were analyzed by gelatin zymography. Arrows show the 92 kDa proMMP-9 and 220 kDa proMMP-9 dimer.

FIG. 9A to 9C. Peptide inhibition of THP-1 cell migration. THP-1 cells were preincubated with the peptide as indicated at a 200 μM concentration for 1 h at room temperature and applied to transwells in the absence (FIG. 9A), or presence (FIG. 9B), of LLG-C4-GST coating. Cells were allowed to migrate for 16 hours at +37°C. Cells migrated to the lower surface of the filter were stained and counted microscopically. FIG. 9C, HT1080 fibrosarcoma cell migration was similarly assayed in the absence of LLG-C4-coating. The bars show means ± SD from triplicate wells.

FIG. 10A to 10D. α_M-I domain binding to recombinant MMP-9 domains.

FIG. 10A, Schematic representation of MMP-9 and its recombinant forms produced in E. coli.

FIG. 10B, ProMMP-9, its recombinant forms or BSA were coated on microtiter wells

(80μg/well) and soluble GST-α_M I domain was allowed to bind at the concentrations indicated. The binding was determined by anti-GST monoclonal antibody. The results are means ± SD from triplicate wells in this and other figures. FIG. 10C, Binding of proMMP-9 to the immobilized GST-α_M I domain was studied in the presence of each peptide at the concentrations indicated. The binding was determined with the anti-MMP-9 antibody GΕ-213.

FIG. 10D, Binding of GST-α_M I domain to the immobilised proMMP-8, proMMP-9,

ICAM-1, and fibrinogen was studied with ICAM-1, DDGW or KKGW (50 μM) as competitors. In control wells, GST was added instead of GST-α_M I domain. The experiment was repeated three times with similar results.

FIG. 11A to 11D. Recognition of recombinant MMP-9 domains by a_Mp₂ integrin- expressing cells. The studied cells were PMNs (11A, 11B, 11C), a_Mβ₂ L-cell transfectants (11D), non-transfectants (11D), and LAD-1 cells (11D). PMNs were in resting state or stimulated with PMA (11A, 11C) or C5a or TNFα (11B) before the binding experiment to proMMP-9 or its domains. Cells were also pretreated with each peptide (50 μM), antibody (20μg/ml) or the αM I domain as indicated. Unbound cells were removed by washing and the number of adherent cells was quantitated by a phosphatase assay. The experiment was repeated three times with similar results.

FIG. 12A to 12 D. Blockage of PMN and THP-1 cell migration in vitro by gelatin- ase and β₂ integrin inhibitors. PMNs (lxlO⁵ in 100 μl) were applied on the LLG-C4- GST or GST coated surface (12A) or HMEC monolayer (12B) in the absence or presence of peptides (200 μM) or antibodies (20μg/ml) as indicated. PMNs were stimulated with 20 nM PMA (12A), HMECs with 50 μM C5a or lOng/ml TNFα or left untreated (12B). THP-1 cells (5xl0⁴ in lOOμl) were stimulated with 50 nM PMA and applied on the coated surfaces together with each peptide (200 μM) (12C). The cells migrated through transwell filters were stained and counted microscopically. All experiments were repeated at least twice. (12D) Phorbol ester-activated THP-1 cells (5xl0⁴ in lOOμl) were incubated for 16 h at +37°C in the presence or absence of peptides as indicated. The conditioned medium was analyzed by gelatin zymography.

FIG. 13A to 13D. Inhibition of neutrophil migration to an inflammatory tissue. FIG. 13A, Mice were injected with thioglycolate or PBS intraperitoneally. The peptides were applied intravenously at the amounts indicated. After 3 h, the intraperitoneal leukocytes were harvested and counted. The results show means ± SD of 2-4 mice in a group. (*) indicates statistical significant difference (pO.OOl). The experiment was repeated at least 3 times. The infiltrated neutrophils of mice treated with thioglycolate (13B) or PBS (13C) were stained with anti-MMP-9 and anti-α_M by incubating the cells with the antibodies for 3 h. Fluorescence w as studied by confocal microscopy. Bars: 9.1μm and 4.8μm, respectively. FIG. 13D, Gelatinolytic activity of the supernatants from the peritoneal cavities of mice collected as in (13A). Lanes 1-4: samples are from thioglycolate-treated mice; lane 5: a sample from PBS-treated mouse. DDGW, HFDDDE, and DFEDHD were injected intravenously at doses of 0.1, 0.2 and 0.2 mg per mouse. The arrows show proMMP-9 dimer, proMMP-9 and proMMP-2. The experiment was repeated three times with similar results.

The publications and other materials referred to or used herein to illuminate the background of the invention, and in particular, to provide additional details with respect to its practice, are incorporated herein by reference. The invention will be described in more detail in the following Experimental Section.

EXPERIMENTAL

Antibodies and Reagents

The antibodies MEM170 and LM2/1 were against the α_M and the MEM-83 and TS2/4 antibodies against the α_L integrin subunit (19, 20). The monoclonal antibody 7E4 (21) reacted with the common β₂-chain of the leukocyte integrins. The α_M antibody OKM10 was obtained from the American Type Culture Collection, ATCC, Rockville, MD (22). A monoclonal antibody against ICAM-5 (TL3) (23) was used as an antibody control. The monoclonal anti-MMP-9 antibody (GE-213) and anti-MMP-2 antibody (Ab-3) were obtained from Lab Vision Corporation (Fremont CA) and from Oncogene™ research products, respectively. Affinity purified rabbit anti-MMP-9 polyclonal antibodies were from the Borregaard laboratory (24). As monoclonal antibody controls, we used a mouse I gG (Silenius, Hawthorn, Australia) and anti-glycophorin A (GPA) (ATCC). Anti-trypsinogen-2 (TAT-2) antibody was a rabbit polyclonal antibody control (32). The peroxidase-conjugated anti-GST mAb was from Santa Cruz Biotechnology.The rabbit anti-mouse horseradish peroxidase-conjugated secondary antibody was from Dakopatts a/s (Copenhagen, Denmark). Inhl (2R-2-(4- biphenylylsulfonyl)amino-N-hydroxy-propionamide) was purchased from Calbiochem, La Jo 11a, CA. Human recombinant ICAM-1 was obtained commercially by R&D systems (Minneapolis, MN). ICAM-1 -Fc fusion protein was expressed in Chinese hamster ovary cells and purified as described (14). The synthetic peptides CTT, STT, LLG-C4 and RGD-4C were obtained as previously described (14, 25). W→A CTT was ordered from Neosystem, Strasbourg, France. ProMMP-2 and proMMP-9 were obtained commercially (Roche). In zymography, the commercial proMMP-9 showed the 92kDa monomer, 200 kDa homodimer, and 120 kDa NGAL complex bands. The integrins α₁β₁ and α₃βi were purchased from Chemicon International (Temecula, CA). Human plasma fibrinogen and lovastatin were from Calbiochem.

Phage display

Phage display selections were made using a pool of random peptides CX_7-10C and X_9-1o, where C is a cysteine and X is any amino acid (14, 25). Briefly, α_M I domain-GST or GST fusion protein was immobilized on microtiter wells at 20 μg/ml concentrations and the wells were blocked with BSA. The phage library pool was first subtracted on wells coated with GST and then unbound phage was transferred to α_M I domain-GST-coated wells in 50 mM Hepes/5 mM CaCl₂/l μM ZnCl₂/150 mM NaCl/2% BSA (pH 7.5). After three rounds of subtraction and selection, individual phage clones were tested for binding specificity and the sequences of the phage that specifically bound to the I domain were determined (14).

Peptide biosynthesis and chemical synthesis

The phage peptides were initially prepared biosynthetically as intein fusions. The DNA sequences encoding the peptides were PCR cloned from 1 μl aliquots of the phage- containing bacterial colonies that were stored at -20 C. The forward primer was 5'-

CCTTTCTGCTCTTCCAACGCCGACGGGGCT-3' and the reverse primer 5'-

ACTTTCAACCTGCAGTTACCCAGCGGCCCC-3'. The PCR conditions included initial denaturation at 94°C for 2 min followed by 30 cycles of 94°C 30 sec, 55°C 30 sec, and 72°C 30 sec. The PCR products were purified using QIAGEN Nucleotide removal kit. They were then digested with Sapl and Pstl restriction enzymes and ligated to a similarly digested and phosphatase treated pTWTN vector (New England Biolabs).

Correct insertions were verified by DNA sequencing. Intein fusion proteins were produced in E. coli strain ER2566 and affinity purified on a chitin column essentially as described (26). The peptide was cleaved on the column, eluted and finally purified by

HPLC. Chemical peptide synthesis was done using Fmoc-chemistry as described and the sequences were verified by mass spectroscopy (26). Phage binding assay

Phage ( 10⁸ infective p articles/well) in 50 mM Hepes/5 mM C aCl₂/l μM ZnCl₂/0.5% BSA (pH 7.5) were added to microtiter wells coated with I domain-GST fusion or GST (20 ng/well). The phages were allowed to bind in the absence or presence of a competitor peptide (15 μM) for one hour followed by washings with PBS containing 0.05% Tween 20. The bound phage was detected using 1 :3000 dilution of a peroxidase- labelled monoclonal anti-phage antibody (Amersham Biosciences) and o- phenylenediamine dihydrochloride as a substrate. The reactions were stopped by addition of 10% H₂SO₄ and the absorbance was read at 492 nm using a microplate reader.

Pepspot

The peptides were synthesized on cellulose membranes as described (27). The membrane was blocked with 3% BSA in TBS containing 0.05% Tween 20, and incubated with 0.5-5 μg/ml α_M I domain for 2 h at room temperature. The DDGW peptide was used as a competitor at a 50 μM concentration. Bound α_M I domain was detected using the monoclonal antibody LM2/1 (1 μg/ml) or MEM-170 (5 μg/ml) and peroxidase-conjugated rabbit anti-mouse antibody (1:5000 dilution) followed by chemiluminescence detection.

Cell culture

The human HT1080 fibrosarcoma and THP-1 and Jurkat leukemic lines were obtained from ATCC and maintained as described previously (20, 25, 28). OCI/AML-3, derived from the primary blasts of an AML patient (29) was maintained in 10% FBS/RPMI supplemented with L-glutamine, penicillin and streptomycin. Cell viability was assessed with a MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) assay according to the instructions of the manufacturer (Roche).

Purification of integrins α_Lβ₂ (CDlla/CD18, LFA-1), α_Mβ₂ (CDllb/CD18, Mac-1) and α_xβ₂ (CDllc/CD18) integrins were purified from human blood buffy coat cell lysates by adsorption to the anti-CDl la (TS 2/4), anti-CDllb (MEM170), or anti-CDllc (3.9) antibodies linked to protein A-Sepharose CL 4B. The integrins were eluted at pH 11.5 in the presence of 2 mM MgCl₂, and 1% n-octyl glucoside as described previously (28). Expression and purification of GST fusion proteins

The α_L, α_M, and α I domains were produced as GST fusion proteins in E. coli strains BL 21 or JM109 and purified by affinity chromatography on glutathione-coupled beads (30, 31). GST containing CTT in the C-terminus was constructed using the protocols described for LLG-C4-GST (14) and glutathione-coupled beads were employed for purification. The purity of the GST-fusion proteins was confirmed by SDS-PAGE with Coomassie Blue staining and Western blot analysis. For pepspot analysis, GST was cleaved from the α I domain with thrombin.

Binding of MMPs to purified integrins

The purified I domains (GST-αM, GST-αL, GST-αχ), or integrins (αMβ2, αLβ₂, ocχβ₂, αiβ (1 μg/well) were immobilized in 20 mM Tris, 150 mM NaCl, 1 mM CaCl₂, 1 mM MgCl₂, and 1 mM MnCl₂, pH 7.4. The wells were washed with PBST (10 mM phosphate, 140 mM NaCl, pH 7.4, containing 0.05% Tween20) and blocked with 3% BSA in PBST. ProMMP-2, proMMP-9, or the p-aminophenyl mercuric acetate (APMA) or trypsin-activated forms (32) were incubated for 2 h at room temperature. In the inhibition experiments, CTT and Inhl were first preincubated with the proMMPs for 30 minutes at room temperature. The wells were washed three times and incubated with anti-MMP-9 (GE-213) or anti-MMP-2 (Ab-3) antibody at a 2 μg/ml concentration in PBST for 1 h. Bound antibodies were detected using peroxidase-conjugated rabbit anti- mouse IgG (DAKO, Glostrup, Denmark) and o-phenylenediamine dihydrochloride as a substrate.

Coprecipitation of β₂ integrin and progelatinases

Serum-free conditioned m edium containing proMMP-2 and proMMP-9 was collected from human HT-1080 fibrosarcoma cells grown in the presence of 100 nM phorbol ester 4β-Phorbol 12,13-dibutyrate (PDBu) (Sigma-Aldrich, St. Louis, MO) overnight at +37°C. A 500 μ l volume of the supernatant was incubated with 1 00 ng of GST-α_M, GST-α_L, or GST-α_x I domain or α_Mβ2 integrin for 3 h at 25 °C. GST and GST-LLG-C4 were used to determine non-specific binding. CTT, STT, LLG-C4, and ICAM-1 were used as competitors at a 200 μg/ml concentration, and the antibodies LM2/1 and TL3 at 40 μg/ml. After an hour incubation at +4°C, complexes of I domain and gelatinases were pelleted with Glutathione Sepharose. Integrin complexes were captured by incubating first with the OKM10 antibody for 3 h at +4°C and then with protein G Sepharose for 1 h. After centrifugation and washing, samples were analyzed by gelatin zymography on 8% SDS-polyacrylamide gels containing 0.2 % gelatin (32).

Effect of peptides on proMMP-9 release from cells

THP-1 cells (40 000/100 μl) were incubated in serum-free RPMI medium for 48 h in the absence or presence of 200 μM p eptide as described in the text. Aliquots o f the conditioned media were analyzed by gelatin zymography.

Interaction between CTT and proMMP-9

CTT-GST and GST control (5 μg/well) were coated overnight on 96-well microtiter plates in 50 μl TBS followed by blocking of the wells by BSA. proMMP-9 or APMA- activated form (80 ng/well) was incubated in the absence or presence of competitors for 2 h in 50 μM Hepes buffer containing 1 % BSA, 5 mM CaCl₂, and 1 μM ZnCl₂ (pH 7.5). After washing, bound MMP-9 was determined with anti-MMP-9 and HRP- conjugated anti-mouse IgG as described above. To examine complexing of CTT with proMMP-9 in cell culture, THP-1 cells were activated with PDBu for 30 min and then incubated with CTT, W→A CTT, or Inhl (each 200 μM) at +37°C in serum-free medium. Samples were taken from the media at 0, 1, 2, 3, 4, and 5 h time points and analyzed by zymography and Western blotting with polyclonal anti-MMP-9 antibodies. Experiments with HT-1080 cells were performed similarly except that the medium samples were collected after 6 h.

Cell surface labelling, immunoprecipitation and immunoblotting Non-activated or PDBu-activated THP-1 cells (1 x 10⁷) were subjected to surface labelling using periodate tritiated sodium borohydride (33). The [³H]-labelled cells were lysed w ith 1 % ( v/v) Triton X-100 in PBS, c larified b y centrifugation a nd p recleared with protein G-Sepharose. The lysate was immunoprecipitated with polyclonal anti- MMP-9, α_M (OKM-10) or β₂ (7E4) antibodies. After one-hour incubation at +4°C together with protein G-Sepharose, immunocomplexes were pelleted, washed and resolved on 8-16% SDS-PAGE gels (Bio-Rad, Hercules CA). The gels were treated with an enhancer (Amplify, Amersham Biosciences), dried and exposed. Non-labelled THP-1 cells (1 x 10⁷) were similarly lysed and immunoprecipitated as above. The samples were resolved on 4—15% SDS-PAGE gels and transferred to nitrocellulose membranes. Immunodetection was performed with α_M (MEM 170) antibody (10 μg/ml) followed b y p eroxidase-conjugated anti-mouse IgG and chemiluminescence d etection (Amersham Biosciences). The membranes were stripped of bound antibodies and reprobed with monoclonal α_L chain (TS2/4) or polyclonal anti-MMP-9 antibodies.

Immunofluorescence

Immunofluorescence was performed on resting cells or the cells activated with PDBu for 30 min. A portion of the cells was treated with ICAM-1 or CTT to block β₂ integrins or gelatinases, respectively. Cells were bound to poly-L-Lysine coated cover slips, fixed with methanol for 10 min at -20°C or with 4% paraformaldehyde for 15 min at +4°C, and permeabilized with 0.1% Triton X-100 in PBS at room temperature for 10 min followed by several washings. The cover slips were incubated with rabbit anti-MMP-9 polyclonal and mouse anti-αM (OKM-10) antibodies diluted 1:500. After washing with PBS, the secondary antibodies, rhodamine ( TRITC)-conjugated p orcine anti-rabbit or FITC-conjugated goat anti-mouse (Fab')₂ (Dakopatts a/s, Copenhagen, Denmark) were incubated at a 1:1000 dilution for 30 min at room temperature. The samples were mounted with moviol, incubated in the dark for 2 days, and examined by a confocal microscope (Leica multi band confocal imagine spectrophotometer) at a 400x magnification or a fluorescence microscope (Olympus Provis 70) at a 60x magnification.

Neutrophil preparations and cell lines

PMNs were isolated from peripheral blood anticoagulated in acid-citrate dextrose. Erythrocytes were sedimented by centrifugation on 2% Dextran T-500, and the leukocyte-rich supernatant was pelleted, resuspended in saline and centrifuged on a Lymphoprep (Nyegaard, Oslo, Norway) at 400g for 30 minutes to separate polymorphonuclear c ells from p latelets a nd m ononuclear c ells ( 16). P MN p urity was >95% with typically <2% eosinophils. Cell viability was measured using an MTT (3- [4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium) bromide assay as instructed by the manufacturer (Roche).

Human microvascular endothelial cells (HMEC-1) (17), kindly provided by S. Mustjoki (Haartman Institute, University of Helsinki), were grown in RPMI 1640 in the presence of 10% FBS containing 2 mM glutamine, 100 IU/ml penicillin and 100 μg/ml streptomycin. Human m onocytic T HP- 1 c ells w ere m aintained a s d escribed ( 14, 25). Leukocyte adhesion deficiency type-1 (LAD-1) cells, wild type and αMβ₂-transfected L929 mouse fibroblastic cells were generous gifts from Dr. Jean-Pierre Cartron (INSERM, Paris, France). These cells were maintained as described previously (18) and the α_Mβ₂ expression was examined by fluorescence-activated cell sorting (FACS, Becton Dickinson, San Jose, CA).

Cell adhesion and migration

Fibrinogen and ICAM-1 -Fc were coated at 40 μg/ml in TBS at +4°C. Peptides (2 μg/well) were coated in TBS containing 0.25% glutaraldehyde at +37°C. The wells were blocked with 1% BSA in PBS. THP-1 cells (50 000/well) with or without PDBu activation were added in 0.1% BSA-RPMI medium in the presence or absence of 200 μM peptides or monoclonal antibodies at 50 μg/ml. After 30-35 minutes the wells were washed with PBS to remove non-adherent cells and the adhesive cells were quantitated by a phosphatase assay. The cell migration assay was conducted using transwell migration chambers (8 μm pore size, Costar) in serum-containing medium as described (14). Briefly, the membranes were coated on the upper and lower surface with 40 μg/ml GST, LLG-C4-GST, or left uncoated. The wells were blocked with 10% serum- containing medium for 2 h. THP-1 cells (50 000/100 μl) or HT1080 (20 000/100 μl) were preincubated with the peptides for 1 h before transfer to the upper chamber. The lower chamber contained 500 μl of the medium without the peptides. The cells were allowed to migrate to the lower surface of the membrane for 16 h and then stained with crystal violet and counted.

MMP-9 proteins (200 nM in PBS) were coated at +4°C for 16 h and the microtiter wells were blocked with 3 % BSA in PBS for 1 h at room temperature. The α_Mβ₂-integrin L- cell transfectants and PMNs (lxl 0⁵ cells/well) were suspended in RPMI medium supplemented with 2mM MgCl₂ and 0.1% BSA and activated with PMA (20nM) for 20 min, or with C5a (50nM) or TNF-α (lOnM) for 4h at +37°C. The L926 wild type and LAD-1 cells were used as controls. The cells were treated with the indicated antibody (20 μg/ml) or peptide (50 μM) at +37°C for 30 min, washed twice with serum-free medium and incubated in the microtiter wells at +37°C for 30 min. The wells were washed with PBS, and the number of adherent cells was quantitated by a phosphatase assay (14). Cell migration was conducted using Costar 24-transwell migration chambers with a 3 μm pore size for PMNs and 8 μm for THP-1 cells. To study β₂ integrin-directed migration, the chamber membrane was coated on both sides with LLG-C4-GST integrin ligand (40μg/ml) or GST as a control and blocked with 10% serum-containing medium as described above. To study transendothelial migration, confluent HMECs (4xl0⁵ cells/well) were grown on the upper side of the gelatin-coated membrane for 5 days. Culture medium was changed after 3 days. After washing the HMEC layers twice with PBS, chemotactic activation was carried out by adding C5a (50nM), TNF-α (lOng/ml), or medium alone to the lower compartment at +37°C for 4 h. Cultures were then washed again twice to remove all agents. PMNs or THP-1 cells were preincubated with the peptide inhibitor or antibody studied for 1 h before transfer to the upper compartment (lxlO⁵ cells in lOOμl RPMI/0.1 % BSA or the complete 10 % FCS-containing medium). PMNs were allowed to migrate for 2 h through the LLG-C4-GST coated membrane and for 30 min through the HMEC monolayer. THP-1 cells were allowed to migrate for 16 h. The non-migrated cells were removed from the upper surface by a cotton swab and the cells that had traversed the filters were stained with crystal violet and counted.

Mouse inflammation model Balb/c mice at the age of 31-32 weeks were injected intraperitoneally with 3% (w/v) thioglycolate in sterile saline (36). Peptides (5-500μg in lOOμl) were introduced intravenously through the tail vein. Animals were euthanized after 3 h and the peritoneal cells were harvested by injecting 10 ml of sterile PBS through the peritoneal wall. Red blood cells present in the lavage fluid were removed by hypotonic lysis. Cells were centrifuged and resuspended in 1 ml of sterile 0.25% BSA/Krebs-Ringer. The supernatants were also collected and analysed by gelatin zymography. The number of neutrophils was determined following staining with 0.1% crystal violet and using a light microscope equipped with a x 1 00 objective. F or immunofluorescence staining, c ells were allowed to bind to poly-L-lysine coated cover slips, fixed with 2.5% paraformaldehyde in PBS at +4°C for 30 min followed by several w ashings. The Fc receptors were blocked in the presence of 20% of rabbit serum and 3% BSA in PBS. The cells were then incubated with anti-MMP-9 polyclonal and U_M monoclonal (MCA74) antibodies for 30 min. After washing with PBS, the secondary antibodies, rhodamine (TRITC)-conjugated anti-rabbit or FITC-conjugated anti-rat (Fab')₂ were incubated for another 30 min. The samples were examined with a confocal microscope. The animal studies were approved by an ethical committee of Helsinki University.

RESULTS

Identification of the α_M I domain-binding peptide motif D/E-/D/E-G L-W

Using phage peptide display libraries, we selected peptides that interact with the α_M I domain. GST-binding phage were first eliminated on GST-coated wells and the unbound phage preparations were incubated on α_M I domain GST fusion protein-coated wells. The α_M I domain-binding phage were enriched by three rounds of panning and the peptide sequences were determined. With the exception of one linear peptide, the peptides were derived from the cyclic CX₇C and CX₈C libraries. The I domain-binding sequences showed only one conserved motif, a somewhat unexpected finding in terms of the known ligand binding promiscuity of the I domain. The bound peptides contained two consecutive negatively charged amino acids, i.e. glutamatic and/or aspartatic acids, followed by glycine and tryptophan residues (Fig. 1A). The consensus D/E-/D/E-G/L- W determined by this approach was clearly different from LLG-C4 and other β₂ integrin-binding peptides reported so far.

We first prepared the phage display peptides as intein fusion proteins, from which the peptides were cleaved. This allowed us to rapidly test the peptide solubility and the binding specificity before large-scale chemical peptide synthesis. The peptides were cloned using oligonucleotide primers that amplify the peptide library insert from the phage vector. Consequently, all the peptides prepared contain the vector-derived sequences ADGA and GAAG in the NH₂- and COOH- termini, respectively. Phage binding experiments using soluble peptides as competitors indicated that the peptides bearing the two adjacent negative charges bound to a common site (not shown). We chose the p eptide ADGA-CILWMDDGWC-GAAG (DDGW) for further experiments as this peptide showed strong binding and was highly soluble in aqueous buffers (soluble in 50 mM NaOH at >10 mM concentrations). The peptide was also prepared by chemical synthesis. The phage bearing the DDGW sequence avidly bound to the α_M I domain and this was readily inhibited by low concentrations of the DDGW peptide, but only marginally affected by the LLG-C4 peptide, indicating different binding sites for DDGW and LLG-C4 (Fig. IB). Control phage bearing other peptide sequences did not bind. The DDGW-bearing phage also showed also specific binding to the α_L I domain that was inhibitable by DDGW but the interaction was weaker than with the α_M I domain (Fig. IC and data not shown). No binding was observed with the αx I domain or GST used as a control (Fig. IC).

Characterization of DELW sequence on the catalytic domain of gelatinases that mediates interaction with the β₂ integrin I domains

We searched protein databases for matches to the novel D/E-/D/E-G/L-W motif. One of the phage library-derived peptides, CPEELWWLC, was highly similar to the DELW(S/T)LG sequence present on the catalytic domain of MMP-2 and MMP-9 gelatinases (Fig. 1A). DELW-like sequences with double negative charges are also present in other secreted MMPs but not in the membrane-type MMPs such as MMP- 14.

No MMP has been reported to bind to the leukocyte β₂ integrins. We therefore set out to study whether MMP-9 in particular could be a ligand of the β integrins as MMP-9 gelatinase is the major leukocyte MMP and is induced during β₂ integrin activation. As a first step, we synthesized the whole proMMP-9 sequence as overlapping 20-mer peptides on a pepspot membrane. Binding assays with the α_M I domain revealed a single active peptide that located to the MMP-9 catalytic domain (Fig. ID). No binding was observed, when the I domain was omitted and the membrane was probed with antibodies only. The sequence of the I domain-binding peptide was QGDAHFDDDELWSLGKGVVV and it contained the binding motif identified by phage display (Fig. ID).

The active MMP-9 peptide contained four consecutive amino acids with negative charges, DDDE. To study the importance of these residues, the aspartic and glutamic acid residues that were closest to the tryptophan were replaced by alanines. At the same time the peptide length was shortened to 15-mer. The alanine mutagenesis significantly abrogated I domain binding on the pepspot filter; the OD value dropped from 2010 to 476 (Table I). To study whether the negatively charged peptide from other MMPs is also active, we synthesized the corresponding 15-mers and the double alanine mutations. Sequences from MMP-1, 2, 3, 7, 8, 9 and 13, but not the membrane- anchored MMP- 14 (MTl-MMP), could bind the α_M I domain. Alanine mutations always decreased the binding. We did similar pepspot analysis for some of the known I domain ligands, which contain D/E-/D/E-G/L-W like sequences. Peptides derived from myeloperoxidase, catalase, thrombospondin-1 and complement protein iC3b strongly bound the I domain in this assay and the double alanine mutation caused a loss of binding (see Table I). Of the three iC3b peptide permutations tested, ARSNLDEDIIAEENI was the active one. The acidic residues were followed by a hydrophobic isoleucine cluster in this peptide. The soluble DDGW peptide efficiently inhibited the binding of this peptide to the I domain. Weaker I domain binding was observed with one complement factor H-derived peptide and one fibronectin-derived peptide. Peptides derived from ICAMs-1, 2 and 3, neutrophil inhibitory factor, Cyr61, fibrinogen, GPlb, factor X, or E-selectin lacked activity.

Alanine scanning mutagenesis of the DDGW peptide with the pepspot system similarly indicated the importance of the glutamic acid residues for I domain binding (Fig. IE).

Alanine mutations of the glycine or either one of the tryptophan residues also inactivated the peptide. Mutations of the isoleucine, leucine or methionine residues were tolerated. Deletion of the ADGA sequence from the N terminus had no effect on I domain binding, but removal of the C terminal GAAG sequence abolished the binding. As the peptides were immobilized via the C terminus on the filter, a sufficient linker sequence such as GAAG seemed important. We also tested a series of truncated cyclic peptides to identify the shortest active sequence. This analysis showed that ADGA-

CEDGWC-GAAG, but not ADGA-CDDGWC-GAAG was the minimal peptide that supported α_M I domain binding. The longer side chain of glutamate compared to aspartate is probably required to bring the negatively charged carboxyl group in the correct position for I domain binding.

Progelatinases bind to purified αjvιβ2 and α β₂ integrins and their I domains

We next used a microtiter well-based sandwich assay to study gelatinase binding to purified integrins. Progelatinases bound in a concentration-dependent manner to coated α_Mβ₂ integrin (Fig. 2A). Curiously, MMP-2 and MMP-9 lost the integrin binding ability after activation by trypsin or APMA. The binding of proMMP-2 and proMMP-9 was observed with both α_Mβ₂ and αLβ2 integrins and their corresponding I domains (Fig. 2B and 2C). No binding was detected on the α I domain or the α^i and α₃βϊ integrins. The DDGW peptide was an efficient inhibitor and it inhibited proMMP-9 binding to the α_M I domain with an IC50 of 20 μM (Fig. 3 A and 3B). To demonstrate that the negative charges of aspartic acids are essential for the peptide activity, the peptide ADGACILWMKKGWCGAAG (KKGW) containing lysines in place of aspartic acids was prepared. As expected, the KKWG peptide was inactive and did not compete with proMMP-9 binding. We were also interested in testing lovastatin, as its binding site in the α_L I domain is known (34, 35). Lovastatin was not able to compete with proMMP-9 even at a high concentration.

ProMMP-9 bound like a true integrin ligand, as the cation chelator EDTA (5 mM) nearly completely prevented the binding (Fig. 3C and 3D). For background measurement in the sandwich assay, we used antibodies alone (control), or coating with ICAM-1 or wild type GST. The gelatinase-binding peptide CTT (200 μM) inhibited proMMP-9-integrin interaction with the same efficiency as EDTA did. The control peptides STTHWGFTLS (STT) and CTTHAGFTLC (W→A CTT), which lack gelatinase inhibitory activity (26), were without effect. A non-peptide chemical MMP inhibitor (Inhl) also prevented proMMP-9 binding. As EDTA inhibits both the gelatinase and the integrin, we used integrin blocking antibodies and ligand peptides to demonstrate the specific binding activity of β₂ integrin. The known ligand-binding blocking antibodies MEM 170, MEM 83, and LM2/1 inhibited proMMP-9 binding. A control antibody TL3 had no effect. The I domain binding peptide LLG-C4 showed a partial inhibitory effect. RGD-4C, a ligand of αv integrins, served as control peptide and had no effect on proMMP-9 binding. The purity of the integrins was typically more than 90% and that of I domains 95%, making it unlikely that progelatinases would bind to impurities in the preparations.

Progelatinase-integrin complexes were also obtained by co-precipitation experiments using HT1080 conditioned medium as a source of proMMP-9 and proMMP-2, which were analyzed by zymography. The progelatinases co-precipitated with _Mβ₂ integrin or α_M I domain GST fusion protein when these were used as a bait. The integrin added to the medium was immunoprecipitated with the α_M antibody OKM10 (Fig. 4A). The α_M I domain GST protein was pulled down with glutathione-beads (Fig. 4B). CTT but not STT had an inhibitory effect. Inhibition of the I domain by LM2/1, ICAM-1 or LLG-C4 also affected the pull-down of progelatinases. GST control did not coprecipitate the gelatinases. No active forms of gelatinases were found to coprecipitate with α_Mβ₂ or the I domain, when APMA-treated HT-1080 medium or APMA-activated MMP-9 was used (not shown).

As the gelatinase inhibitors CTT and Inhl prevented the binding of proMMP-9 to the integrin, it can be anticipated that CTT and Inhl avidly bind to proMMP-9. To gain more insight into this, we examined binding of proMMP-9 to immobilized CTT peptide. ProMMP-9 specifically bound to the CTT-GST fusion protein (Fig. 5A) but not to LLG-C4-GST. CTT and Inhl at 100 μM concentrations effectively competed in binding but W→A CTT did not. The proMMP-9 preparation did not contain detectable amounts of active MMP-9 on zymography analysis, and after proMMP-9 activation with APMA, the CTT-GST binding increased. CTT and Inhl could also bind to proMMP-9 secreted into the medium of PDBu-activated THP-1 leukemic cells (Fig. 5B) or HT1080 fibrosarcoma cells (not shown). A time-dependent reduction in the gelatinolytic activity of proMMP-9 was observed with CTT (panel 1) and Inhl (panel 3), but not with the W→A CTT peptide (panel 4). Western blot analysis indicated that CTT does not decrease the secretion of proMMP-9 by the cells (panel 2). Furthermore, the CTT complex was reversible and disappeared after repeated freezing and thawing of the samples.

Demonstration of a cell-surface complex between progelatinases and β₂ integrins

To study whether the progelatinases occur in a complex with the β₂ integrins on the leukocyte surface, we performed immunoprecipitation and co-localization studies. First, we examined THP-1 monocytic leukemia cells in the resting state and after induction by PDBu, which mimics leukocyte activation in vivo. THP-1 cell-stimulation with PDBu led to upregulation of MMP-9 (data not shown). The cell surface glycoproteins of THP- 1 cells were labelled with tritium [ H] followed by immunoprecipitation with β₂ integrin and MMP-9 antibodies. In the PDBu-activated cells, the α_M chain antibody OKM10 and β₂ chain antibody 7E4 immunoprecipitated two [³H] -labelled proteins corresponding to the integrin α chain (165 kDa) and β₂ chain (95 kDa) (Fig. 6A, lanes 9-10). Importantly, polyclonal MMP-9 antibodies immunoprecipitated the same two integrin chains (lane 7). In non-activated cells, essentially no co-precipitation of α_M and β₂ were observed with MMP-9 antibodies, although the α_M and β₂ chains were present. The co- precipitation o f t he i ntegrin c hains b y M MP-9 a ntibodies w as p revented b y t he C TT peptide (lane 8). The control antibody (TL3) did not precipitate any proteins.

With the [³H]-labelled cells, we did not observe any band corresponding to proMMP-9, perhaps because the carbohydrates of proMMP-9 are poorly labelled. We therefore analyzed t he P DBu-activated T HP- 1 c ells b y Western b lotting (Fig. 6 B). P roMMP-9 was readily immunoprecipitated with antibodies against MMP-9, α_M or _L, but not by the control antibody. MMP-9 antibodies in turn were able to immunoprecipitate the α_M but not the α_L chain. MMP-2 antibodies similarly co-precipitated α_M but not α_L. When the cell lysate was precleared with the α_M antibody OKM-10, the amount of immunoprecipitated α_M and proMMP-9 clearly decreased (lane 6). Preclearing with the α_L antibody TS2/4 did not significantly remove α_M or proMMP-9, but abolished the α_L precipitation (lane 7).

As THP-1 cells do not express high amounts of the α_L chain (20), we examined the Jurkat T cell line, which expresses more α_L than α_M (28). We observed a significant immunoprecipitation of α_L by MMP-9 and MMP-2 antibodies after PDBu activation (Fig. 6C). Furthermore, the α_L antibody co-precipitated more proMMP-9 in comparison to the α_M antibody. No proMMP-9 co-precipitated with MMP-2 antibodies in Jurkat or THP-1 cells.

ProMMP-9 and α_Mβ₂ were found to co-localize on the cell surface following PDBu- activation of THP-1 cells as studied by fluorescence and confocal microscopy (Figs. 7A and 7B, respectively). Using a higher magnification, colocalization was primarily seen in cell surface clusters (Fig. 7B), and to a lesser extend on areas where cells contacted each other (not shown). We believe that the MMP-9 co-localizing with α_Mβ₂ is the proMMP-9, as the activated MMP-9 did not bind to α β₂- Without PDBu activation, there was hardly any co-localization of proMMP-9 and α_Mβ₂- The secondary antibodies did not stain the cells when the primary antibodies were omitted (data not shown). When the cells were preincubated with the CTT peptide or recombinant soluble ICAM- 1 to block proMMP-9 or α_Mβ2, the cell surface clusters did not form and the proMMP- 9-α_Mβ₂ colocalization as not observed (not shown). P roMMP-9-αMβ₂ colocalization was also observed on Jurkat cells following phorbol ester stimulation (not shown). Blocking the progelatinase/β₂ integrin complex with DDGW releases cell-bound proMMP-9 and inhibits cell migration but not adhesion

As the DDGW peptide is an integrin ligand, one of the questions was whether it can support adhesion of leukocytes. We studied adhesion of human myelomonocytic THP-1 cells on immobilized glutaraldehyde-polymerized peptide. Phorbol-ester activated cells efficiently bound to the DDGW peptide, whereas there was no binding in the absence of cell activation (Fig. 8 A). As a positive control, the recombinant intein-produced ADGA-CPCFLLGCC-GAAG peptide supported adhesion, but unlike the DDGW peptide, it also supported adhesion in the absence of integrin activation. The acute myeloid leukemic cell line OCI/AML-3 also avidly adhered to DDGW, whereas human fibrosarcoma HT1080 cells which lack β integrins did not (not shown). As THP-1 cells were able to adhere on DDGW, we next studied the effect of the peptide on β₂ integrin dependent adhesion to fibrinogen and ICAM-1. Interestingly, DDGW did not block cell adhesion to fibrinogen, whereas the LLG-C4 peptide blocked the adhesion as previously reported (Fig. 8B). Similarly, DDGW did not block the binding of recombinant α_M I domain to immobilized fibrinogen (not shown). DDGW did not either block cell adhesion on ICAM-1 -Fc fusion protein. As a control, the blocking antibody 7E4 against β₂ integrins prevented the ICAM-1 binding, indicating that the THP-1 cells bound in a β₂ integrin dependent manner (Fig. 8C). We also found no blocking effect of DDGW on THP-1 adhesion to LLG-C4-GST fusion protein (not shown).

The second question raised by these studies was whether the DDGW peptide can release cell-bound proMMP-9. When THP-1 cells were cultured for 48 h in the presence of DDGW, an increase of proMMP-9 level was observed in the conditioned medium as studied by gelatin zymography (Fig. 8D). The peptide increased both monomeric and dimeric proMMP-9 in the culture medium. In contrast, CTT slightly decreased or inhibited active proMMP-9. KKGW and W→A CTT had no effect.

We also studied the role of the progelatinase/β₂ integrin complex in leukocyte migration using a transwell assay in which leukocyte migration can be adjusted by the choice of coated matrix or ligand protein. We tested that the CTT, LLG and DDGW peptides are not toxic to the THP-1 cells in a 48 h time frame at >200 μM concentrations using an MTT assay. Using transwells coated with 10% serum in cell culture medium, we first studied the effect of peptides on the basal migration of THP-1 cells in the absence of any stimulus by phorbol ester or an adhesive matrix. Under such conditions, CTT, LLG- C4 or the gelatinase inhibitor Inhl at a 200 μM concentration had no effect on THP-1 migration indicating no active involvement of gelatinases or β₂ integrins (Fig. 9A). We have previously shown that when the transwells are coated with LLG-C4-GST fusion protein, THP-1 cells adhere and migrate in a β₂ integrin dependent manner (14). Thus, transwells were coated with LLG-C4-GST fusion protein or GST alone. Both the DDGW and CTT peptide, but not KKGW, inhibited the migration of THP-1 cells on the LLG-C4-GST substratum (Fig. 9B). The soluble LLG-C4 peptide also blocked the migration. In the presence of GST coating, cell migration was neglible. To verify that the effect of DDGW peptide was β₂ integrin dependent, HT1080 fibrosarcoma cells lacking these integrins were allowed to migrate in the presence of CTT, DDGW, KKGW or LLG-C4. Of these peptides, only CTT was capable of inhibiting cell migration (Fig. 9C).

Peptide inhibitors of the proMMP-9/α_Mβ₂ complex prevent neutrophil migration As shown above, pepspot analysis located the integrin interactive site of proMMP-9 to a 20-amino acid long sequence present in the catalytic domain, QGDAHFDDDE- LWSLGKGVVV. Further screening by the pepspot system has indicated that sufficient integrin binding activity is achieved by truncating this sequence to a hexapeptide, HFDDDE (data not shown). To confirm that such a short sequence is the bioactive site of proMMP-9, we first prepared bacterially expressed recombinant domains of MMP-9 (Fig. 10A). ΔMMP-9 is composed of the prodomain (Pro) and the catalytic domain but lacks the hemopexin domain. The fibronectin type II repeats (Fnll) were also produced as a separate recombinant protein as this is an important substrate-binding region. The procatalytic domain construct ΔMMP-9 bound the α_M I domain nearly as efficiently as the wild type proMMP-9 (Fig. 10B). Fnll protein almost lacked activity. The HFDDDE peptide identified by the solid-phase pepspot analysis was highly active when made by peptide synthesis and inhibited proMMP-9 binding to the α_M I domain with an IC₅₀ of 20 μM (Fig. 10C). The bound proMMP-9 was determined with the GE-213 antibody, which recognizes an epitope of the Fnll domain (data not shown). A scrambled peptide DFEDHD with the same set of negatively charged amino acids was inactive. HFDDDE was equally potent as DDGW, the α_M I domain-binding peptide discovered by phage display and described above. KKGW, the control peptide for DDGW, was without effect. As the HFDDDE sequence is highly conserved in the members of the MMP family, we also examined the α I domain binding to human neutrophil collagenase, MMP-8. I domain showed a similar DDGW-inhibitable binding to proMMP-8 as to proMMP-9 (Fig. 10D). ICAM-1 and fibrinogen did not compete with either proMMP, implying different binding sites for the matrix proteins and proMMPs in the I domain.

After integrin activation, PMNs exhibited an ability to adhere on proMMP-9. PMA- stimulated PMNs bound to microtiter well-coated ΔMMP-9 nearly as strongly as to proMMP-9 (Fig. 11 A). Stimulation of PMNs with C5a or TNF-α gave similar results PMN adherence increasing by 3-fold (Fig. 1 IB). The Fnll domain did not support PMN adhesion. PMN adherence was inhibited by HFDDDE (50μM), DDGW (50μM), the soluble α_M I domain and the MEM 170 antibody (Fig. 11C), indicating β₂ integrin- directed binding. The control peptides (DFEDHD, KKGW) and an irrelevant monoclonal antibody (anti-GPA) had no effect. The CTT peptide, but not the W→A CTT control peptide lacking MMP inhibitory activity, binds to the MMP-9 catalytic domain (unpublished results) and also inhibited the PMN adherence. MMP-9 antibodies inhibited partially.

We also examined α_Mβ₂-transfected L cells. The α_Mβ₂ L-cell transfectants bound to proMMP-9 and ΔMMP-9 similarly as PMNs did and the I domain ligands and MMP-9 inhibitors attenuated the binding (Fig. 1 ID). The transfected cells also showed a weak adherence to Fnll domain, but the studied peptides and antibodies did not inhibit this binding. Wild type L cells or LAD-1 cells showed no binding to proMMP-9 or its domains.

The in vitro migration of PMNs was studied on transwell filter assays. Coating with the artificial β₂ integrin ligand LLG-C4-GST renders cell migration dependent on the β₂ integrins (14). The migration of PMA-activated PMNs was 5-fold in the LLG-C4-GST substratum in comparison to GST substratum (Fig. 12A). HFDDDE (200μM) inhibited the migration of PMA-stimulated cells but not the basal migration of non-activated cells. DDGW, CTT, MEM170 (20μg/ml) and polyclonal anti-MMP-9 (20μg/ml) worked similarly, affecting the migration of the PMA-activated cells only. Control peptides and an antibody control (anti-TAT-2) had no effect. Similar results were obtained in a transendothelial migration assay (Fig. 12B). Chemotaxis with C5a or TNF-α increased PMN transmigration by 5-10 fold and inhibition was obtained by DDGW, HFDDDE, and CTT but not with the control peptides. Similarly, α_M and MMP- 9 antibodies inhibited but an antibody control (anti-GPA) did not. We also examined the effects of peptides on THP-1 leukemia cell migration through the LLG-C4-GST coated transwell filters. The results were the same as for PMNs. HFDDDE, DDGW, and CTT inhibited THP-1 migration and the control peptides did not (Fig. 12C).

We show above that the DDGW peptide can release proMMP-9 from THP-1 cells. We found that the HFDDDE peptide also released proMMP-9 but was less effective than DDGW (Fig. 12D). The scrambled peptide did not induce the release of proMMP-9.

Under the 16 h incubation time, the peptides had no effect on the secretion of proMMP-

2.

To study neutrophil migration in vivo, we used a mouse model of thioglycolate-induced peritonitis. The cells that infiltrated into the peritoneal cavity within 3 h after thioglycolate irritant were judged to be predominantly PMNs by crystal violet staining. The DDGW and HFDDDE peptides had potent in vivo activities in this inflammation model (Fig. 13A). An intravenous tail injection of DDGW or HFDDDE inhibited the intraperitoneal accumulation of PMNs. The KKGW and DFEDHD peptides used as controls had no effect. The effects of DDGW and HFDDDE were concentration- dependent and up to 90 % inhibition w as o btained b y doses of 5 Oμg and 5 OOμg er mouse, respectively. DDGW was active even at 5μg given per mouse corresponding to an effective dose of 0.1 mg/kg mouse tissue. Approximately 20-fold more PMNs were present intraperitoneally after thioglycolate-stimulus in comparison to the PBS control. The collected inflammatory PMNs stained positively for the proMMP-9/αMβ₂ complex by double immunofluorescence (Fig. 13B). The cells collected after PBS injection lacked the complex; they expressed the integrin but had no cell-surface MMP-9 (Fig. 13C). Zymography analysis of the supernatants from the collected intraperitoneal fluid showed that thioglycolate induced elevated levels of gelatinases in comparison to PBS (Fig. 13D). DDGW and HFDDDE, but not the scrambled peptide, prevented the increase in gelatinase levels in accordance with the inhibition of cell migration. REFERENCES

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Table I: Pepspot analysis of peptides derived from MMPs and αιvιβ₂ integrin ligands

Protein Peptide OD/mm² Binding positivity

MMP-1 DAHFDEDERWTNNFR 1792 +

DAHFDEAARWTNNFR 1417 -

MMP-2 DSHFDDDELWTLGEG 4687 +++

DSHFDDAALWTLGEG 3334 ++

MMP-3 DAHFDDDEQWTKDTT 4993 +++

DAHFDDAAQWTKDTT 2188 +

MMP-7 DAHFDEDERWTDGSS 4043 ++

DAHFDEAARWTDGSS 2065 +

MMP-8 DAHFDAEETWTNTSA 4295 ++

DAHFDAAATWTNTSA 1258 -

MMP-9 DAHFDDDELWSLGKG 2010 +

DAHFDDAALWSLGKG 476 -

MMP-13 DAHFDDDETWTSSSK 4324 ++

DAHFDDAATWTSSSK 1732 +

MMP-14 DTHFDSAEPWTVRNE 1264 -

DTHFDSAAPWTVRNE 1198 -

MMP-1 SGDVQLDDIDGIQAI 484 -

SGDVQLAAIDGIQAI 441 -

MMP-3 RFRLSQDDINGIQSL 810 -

RFRLSQAAINGIQSL 1541 +

MMP-8 NYSLPQDDIDGIQAI 3348 ++

NYSLPQAAIDGIQAI 505 +

MMP-13 HFMLPDDDVQGIQSL 542 -

HFMLPDAAVQGIQSL 384 -

MMP-14 NFVLPDDDRRGIQQL 518 -

NFVLPDAARRGIQQL 609 -

Fibronectin HEATCYDDGKTYHVG 1271 -

HEATCYAAGKTYHVG 596 -

ICAM-3 LNATESDDGRSFFCS 369 -

LNATESAAGRSFFCS 277 -

Complement EEMHCSDDGFWSKEK 2972 + factor H

EEMHCSAAGFWSKEK 321 -

TSP-1 WPSDSADDGWSPWSE 4655 +++

WPSDSAAAGWSPWSE 1543 +

NIF DPVCIPDDGVCFIGS 221 -

DPVCIPAAGVCFIGS 114 -

ICAM-2 NSTADREDGHRNFSC 75 -

NSTADRAAGHRNFSC 73 -

Fibronectin NVYQISEDGEQSLIL 1260 -

NVYQISAAGEQSLIL 453 -

Fibronectin VTYSSPEDGIHELFP 301 -

VTYSSPAAGIHELFP 212 -

Cyr61 KMRFRCEDGETFSKN 317 -

KMRFRCAAGETFSKN 386 -

Myeloperoxid WLPAEYEDGFSLPYG 4603 +++ ase

WLPAEYAAGFSLPYG 924-

Catalase AVKFYTEDGNWDLVG 5045 +++

AVKFYTAAG NWDLVG 690-

Fibrinogen KEVVTSEDGSDCPEA 124- alpha

KEVVTSAAGSDCPEA 225-

Fibrinogen RKQCSKEDGGGWWYN 483- beta

RKQCSKAAGGGWWY 325-

N

Fibrinogen GFGSLNDEGEGEFWL 732- alpha

GFGSLNAAGEGEFWL 397-

GP1b GCPTLGDEGDTDLYD 544-

GCPTLGAAGDTDLYD 238-

ICAM-1 VSVTAEDEGTQRLTC 51-

VSVTAEAAGTQRLTC 68-

Factor X DRNTEQEEGGEAVHE 379-

DRNTEQAAGGEAVHE 236-

E-selectin TCTFDCEEGFELMGA 309-

TCTFDCAAGFELMGA 70-

E-selectin SCNFTCEEGFMLQGP 138-

SCNFTCAAGFMLQGP 54-

E-selectin SCAFSCEEGFELHGS 236-

SCAFSCAAGFELHGS 74-

Fibronectin TFHKRHEEGHMLNCT 39-

TFHKRHAAGHMLNCT 12-

Fibronectin VEYELSEEGDEPQYL 3676 ++

VEYELSAAGDEPQYL 2682 +

Range: 0-1499 OD/mm2 = -, 1500-2999 = = +, 3000-4499 = ++, ^■ 4^nn = -M-4-

Peptide No Soluble competitor DDGW

DDGW ADGACILWMDDGWC 10859 0 peptide GAAG iC3b ARSNLDEDIIAEENI 13265 409

ARSNLDAAIIAEENI 0 0 iC3b EDIIAEENIVSRSEF 0 0

EDIIAAANIVSRSEF 0 0 iC3b EGVQKEDIPPADLSD 0 0

EGVQKAAIPPADLSD 0 0

Claims

1. A pharmaceutical composition comprising a compound comprising the tetrapeptide motif D/E-D/E-G/K-W, and a pharmaceutically acceptable carrier.

2. The pharmaceutical composition according to claim 1, wherein the tetrapeptide motif is DDGW.

3. Use of a compound comprising the tetrapeptide motif D/E-D/E-G/K-W for the manufacture of a pharmaceutical composition for the treatment of conditions dependent on leukocyte migration.

4. Use according to claim 3 wherein the condition dependent on leukocyte migration is leukaemia.

5. Use according to claim 3 for the manufacture of a pharmaceutical composition for inhibiting the adhesion of progelatinases to β₂-integrins.

6. Use of the compound comprising the tetrapeptide motif D/E-D/E-G/K-W for the manufacture of a pharmaceutical composition for prophylaxis and treatment of conditions dependent on neutrophil migration.

7. Use according to claim 6 for the manufacture of a pharmaceutical composition for prophylaxis and treatment of inflammatory conditions.

8. Use according to any one of claims 3 to 7, wherein the tetrapeptide motif is DDGW.

9. A method for therapeutic or prophylactic treatment of conditions dependent on leukocyte migration, comprising administering to a mammal in need of such treatment a compound comprising the tetrapeptide motif D/E-D/E-G/K-W in an amount which is effective in inhibiting leukocyte migration.

10. A method for therapeutic or prophylactic treatment of conditions dependent on neutrophil migration, comprising administering to a mammal in need of such treatment a compound comprising the tetrapeptide motif D/E-D/E-G/K-W in an amount which is effective in inhibiting neutrophil migration.

11. A method for therapeutic or prophylactic treatment of leukaemia, comprising administering to a mammal in need of such treatment a compound comprising the tetrapeptide motif D/E-D/E-G/K-W in an amount which is effective in inhibiting leukaemia cell migration.

12. A method for therapeutic or prophylactic treatment of inflammatory conditions, comprising administering to a mammal in need of such treatment a compound comprising the tetrapeptide motif D/E-D/E-G/K-W in an amount which is effective in inhibiting neutrophil migration.

13. The method according any one of claims 9 to 12, wherein the tetrapeptide motif is DDGW.